DIFFERENTIAL EXPRESSION OF SUPEROXIDE DISMUT ASES (SODS) IN

BOVINE CORPUS LUTEUM DURING ESTROUS CYCLE AND PREGNANCY.

A THESIS SUBMITIED TO THE GRADUATE DMSION OF THE

UNIVERSITY OF HA WAI"I IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

ANIMAL SCIENCES

DECEMBER 2006

By

Ravi K Putluru

Thesis Committee:

Chin N. Lee, Chairperson

Yong Soo Kim

Michael A. Dunn

We certilY that we have read this thesis and that, in our opinion, it is satisfactory in

scope and quality as a thesis for the degree of Mater of Science in Animal Sciences.

THESIS COMMITTEE

~$ Chin N. Lee, Chairperson

YongSooKim

Michael A. Dunn

ACKNOWLEDGMENT

I never thought that 101b January 2003 would make a big difference in my

life. That is the day I got a reply from Dr. Chin N Lee that he would consider my

application for masters program in University of Hawaii at Manoa. From then onwards

Dr. Lee has been a continuous inspiration in my life not only in achieving my career

goals but also in my personal development I am very thankful to Dr. Lee for helping me

to come to United States and pursue my dreams. I am greatly indebted to him for his

moral support throughout the period of my masters program and to the rest of my life.

I am very thankful to Dr. Yong Soo Kim for allowing me to use his lab for

my research and teaching me the research techniques. I enjoyed his classes and I learned

a lot from his classes. I am thankful to Dr. Michael A. Dunn for his advise on right

approach to the research. I like Dr. Dunn's in depth scientific analysis and his patience

while doing any experiment. I am fortunate to have friends like Naveen K Bobbili,

Rosalin Pattnaik and Simon K Lee, who were always with me and supported me during

my happiness and sadness. I am thankful to the local slaughter house staff, Mountain

View dairy and Pacific dairy for providing animals and facilities for my research.

I am thankful to my loving wife Sreevani for her support and understanding

while writing my thesis. My sincere gratitude goes to my mother Laksbmi Devi Putiuru,

my father Lakshmi Narayana Putluru, and my family who supported my idea of coming

to a foreign country for my higher studies, which means sending their loving son far

away from them. I am very very thankful to their support and understanding.

II

TABLE OF CONTENTS

ACKN'OWLEDGEMENT .......................................................•................. 1

~~c:;]l • •••••••••••••••••••••••••••••••••••••••••••••••••••••• •••••••••••••••••••••••••••••• u.~

~1r OJr Jrl4[;~~ .......................................................••••••••••••••••••••••• ~

CHAPTERl: LlTERA~REVIEW.

1.1 BOVINE REPRODUCTION ................................................................. 1

1.1.1 Estrous cycle ............................................................................... 1

1.1.2 Bovine estrous cycle ......................................................................... 1

1.1.3 Corpus luteum ............................................................................. 2

1.1.4 Stages of the corpu luteum ............................................................... 3

1.1.5 Regression of corpus luteum (Luteolysis) ............................................... 4

1.2 REACTIVE OXYGEN SPECIES AND THEIR TYPES ................................ 7

1.2.1 Reactive oxygen species (ROS) ......................................................... 7

1.2.2 Superoxides (02·-) or superoxide radicals (SOR) .................................... 7

1.2.3 Hydroxyl radical (0H") ................................................................... 9

[.2.4 Hydrogen peroxides: (H2~) ..... " ..... " ..... ""'''''''''''''''''''''''''''''''' ..... 1 0

III

1.3 EFFECTS OF ROS AND DEFENCES AGAINST THEM ......................... 11

1.3.1 Biological effects ofROS ........................................................... 11

1.3.2 Defenses against ROS ............................................................... 12

1.4 ROS IN FEMALE REPRODUCTION AND DEFENCES AGAINST THEM ... 13

1.4.1 Role of ROS in female reproduction ............................................... 13

1.4.2 Role of ROS in oocyte maturation and ovulation ................................ 14

1.4.3 Role of ROS in regulation ofluteal function .............•....................... 15

1.4.4 Expression of superoxides in corpus luteum ..................................... 17

1.4.5 Expression of superoxide dismutases in corpus luteum...................... 17

1.4.6 Differential expression of superoxide dismutases ............................... 18

1.5 OXIDATIVE STRESS AND ITS MARKERS ........................................ 19

1.5.1 Oxidative stress ...................................................................... 19

1.5.2 Biomarkers of oxidative stress.................................................... 19

1.5.3 Formation ofF2- isoprostanes ...........................................•.......... 20

1.5.4 Measurement ofF2-isoprostanes •.................................................. 21

1.5.5 Advantages ofF2-isoprostanes measurement .................................... 21

1.5.6 Disadvantages ofF2-isoprostanes measurement ................................. 21

IV

CHAPTER 2: DIFFERENTIAL EXPRESSION OF SUPEROXIDE DISMUTASES

(SODS) IN BOVINE CORPUS LUTEUM

2.1 INTRODUCTION ......................................................................... 23

2.2 MATERIALS AND METHODS....................................... .............. 25

2.2.1 Sample collection......................................................... 25

2.2.2 Different stages of CLs in estrous cycle...... ............ ...... ........ 26

2.2.3 Sample preparation....................................... ................ 30

2.2.4 Protein assay.. . . . . . .. .... .. .. . . . . . .. .. . . . . . . . . .. . .. .. . ... .. .... ... . .. . . . .... 30

2.2.5 Sodium dodecyl sulphate and plyacrylamide gel electrophoresis

(SDS-PAGE) analysis................................................. 30

2.2.6 Western blot analysis ....................................................... 30

2.2.7 Isolation of Mn-SOD by electro elution......... ...................... 31

2.2.8 Obtaining Mn-SOD standard curve.................................... 31

2.2.9 Quantification of Mn-SOD in samples ................................... 32

2.2.10 Isolation of CulZn-SOD by electro elution........................... 32

2.2.11 Obtaining CulZn-SOD standard curve.................................. 33

2.2.12 Quantification ofCulZn-SOD in samples .............................. 33

2.2.13 Assay ofMn-SOD activity............ .................................. 34

2.2.14 Assay of CulZn-SOD activity..................... ...................... 35

2.2.15 Progesterone concentration measurement.............................. 36

2.2.16 8-isoprostane measurement............................................. 37

v

2.3 STATISTICAL ANALySIS............... .......................................... 38

2.4. RESULTS............ .................. ............................................. 39

2.4.1 Progesterone concentration in serum at different stages of

estrous cycle during pregnancy......... ......... ... ...•..•........ 39

2.4.2 Differential expression of 23 kD protein at different stages

ofCL during estrous cycle and during pregnancy............. 39

2.4.3 Identification of 23 kD protein.. .................................... 39

2.4.4 Measurement of Mn-SOD concentration

in CL samples at different stages of CL.. . . . . .. . . . . . . . .. . . . . .. . . . . 40

2.4.5 Mn-SOD concentration in different stages

of CL during estrous cycle and during pregnancy.. . ... .. .... .. . 40

2.4.6 Identification of 15.6 kD protein.. ................................ 41

2.4.7 Measurement ofCulZn-SOD concentration in

different stages of CL.... . . . . . . . .. . . . .... . . ... . .. . . .... . .. . . . ... . . . ... ... 41

2.4.8 CulZn-SOD concentration in different stages

of CL during estrous cycle and during pregnancy. . .. . . ..• . •... . ... 41

2.4.9 Activity ofMn-SOD in different stages

of CL during estrous cycle and during pregnancy. . . ... . . . .. . . . . . ... 42

2.4.10 Activity of CulZn-SOD in different stages

of CL during estrous cycle and during pregnancy............... 43

2.4.11 Levels of 8-Isoprostanes in different stages

of CL during estrous cycle and during pregnancy............... 43

VI

2.5 DISCUSSION....................................... .................................... 44

REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 65

APPENDIX............ ...................................................... .................. 79

VII

List of Figures:

Figure Page No.

1 Progesterone concentration in serum at different stages

of estrous cycle and during pregnancy............................................... 50

2 SDS-PAGE of CL samples during estrous cycle

and during pregnancy. . . . . . . .. . . . . . . . . . . . . ... .. . .. .... . .. . . . .. . .... ... .. ... . ...... .. . .. 51

3 Western Blot analysis of binding characteristics of

Mn-SOD antibody to 23 kD protein in different stages of CL. ................ 52

4 DensitometIy Analysis of Mn-SOD bands..................................... ... 53

5 Generation of Mn-SOD Standard Curve ............................................ 54

6 Mn-SOD Standard Curve .............................................................. 55

7 Levels ofMn-SOD in different stages ofCL during

estrous cycle and during pregnancy .................................................. 56

8 Western blot analysis of binding characteristics of CulZn-SOD antibody

to 15.6 kD protein in different stages of CL......................... ............... 57

9 DensitometJ:y analysis of CulZn-SOD bands ........................................ 58

10 Generation of CulZn-SOD Standard Curve........................................ 59

11 CulZn-SOD Standard Curve.......................................................... 60

VIII

12 Levels of CulZn-SOD in different stages of CL during

estrous cycle and during pregnancy ........ " . .. .. .. . .. .. . .. . .. ... .. . .. .. .. . .. .... 61

13 Activity of Mn-SOD in different stages of CL during

estrous cycle and during pregnancy.. . . .. .... . . . .. .. . . ... . .. .. . ... .. .. . .... . .. .. . 62

14 Activity ofCulZn-SOD in different stages ofCL

during estrous cycle and during pregnancy. .. . . . . .. . ... . . . .. ... . . . .. . ... . . . . . . ..... 63

15 Levels of 8-Isoprostanes in different stages of CL

during estrous cycle and during pregnancy.. . . .. . . . . . . .. . ... . . .. .. . .. . .. .. . . . . . ... 64

IX

ABSTRACT

While many factors contribute to a successful pregnancy, an important

ingredient is a healthy functional corpus luteum (CL) for the maintenance of pregnancy.

Studies in rats have shown that hyperthennia induces an increase in the production of free

radicals (FR) and studies in rabbits showed that FR, including superoxides, may playa

role in the regression of CL and subsequent luteolysis. The up-regulation of superoxide

dismutase (SOD), a free radical scavenging enzyme in pregnant rats, is involved in the

rescue of the CL from luteolysis. The objectives of this study were to investigate the

presence and expression of different types of SODs in the bovine CLs at different stages

of the estrous cycle (1", 2nd, 3rd, 4th and Corpus A1bicans) and pregnancy. CL samples

were collected from a local slaughterhouse and were classified into different stages based

on morphological classification. CL samples (250 mgs) were homogenized and the

protein concentration of each CL was measured. Equal amount of protein from each

sample was subjected to SDS-PAGE and Western Blotting using commercially available

anti-Mn-SOD and anti-CulZn-SOD antibodies. The Mn-SOD and CulZn-SOD were

quantified using densitometry analysis. Mn-SOD expression increased from 1" stage CL

to 4th stage CL and dropped in corpus albicans (CA). The CulZn-SOD expression

increased from 1" stage to 2nd stage and remained elevated in all the stages of estrous

cycle including CA. Expressions of both Mn-SOD and CulZn-SOD were high in a

pregnant CL. Variations in the enzyme activities of both Mn-SOD and CuJZn-SOD

coincided with their levels of expression. Levels of 8-Isoprostanes, stable biomarkers of

oxidative stress in vivo, were highest in the CA and they were lowest in the pregnant CL.

Present results suggest that the Mn-SOD and CulZn-SOD are probably involved in the

x

maintenance of bovine pregnant corpus luteum and Mn-SOD may be involved in the

maintenance of corpus luteum during the estrous cycle.

XI

Literature Review.

1.1 Bovine Reproduetion.

1.1.1 Estrous eyele:

Estrous cycle is an important aspect of the female reproduction. It provides repeated

opportunities for females to become pregnant and produce offsprings. The estrous cycle

consists of follicular phase and luteal phase. The follicular phase is relatively of short

duration; it begins with the regression of the previous corpus luteum (CL) and ends with

the ovulation of dominant follicle. The luteal phase is relatively of longer duration; it

begins after the ovulation and ends with the regression of the CL. The follicular phase is

dominated by preovulatory follicles, which produce a hormone called estradiol. Estradiol

is responsible for changes in the reproductive tract, which will initiate the sexual

receptivity of the female animal to be mounted by male animal. The luteal phase is

dominated by a structure called CL, which produces a hormone called progesterone that

prepares reproductive tract for pregnancy and makes female avoid sexual receptivity if

the animal conceives. If the animal does not conceive, another follicular phase begins and

give the animal another chance to become pregnant

1.1.2 Bovine estrous eyde:

The bovine estrous cycle typically is 21 days long. This can be divided into four stages;

proestrous, estrus, metestrous and diestrous. Proestrous and estrous together constitute

follicular phase, metestrous and diestrous together constitute luteal phase.

1

Proestrous begins after the regression of the previous corpus luteum and ends after the

onset of estrus. 1bis is the period in which the progesterone concentration decreases and

estrogen concentration increases.

Estrus is the stage of estrous cycle in which the animaI is receptive for the copuIation.

This stage ranges for about 6-24 hours. Estrogen hormone dominates in this stage.

Prominent behavioral changes such as the nudging, licking and sniffing of

the genital area of other cows, making repeated attempts to mount other cows

can be noticed in this stage.

Metestrons is the stage of estrous cycle in which the progesterone production starts. 1bis

begins after the ovulation and formation of corpus luteum by a process ca1Ied

lutenization.

Diestrus is the stage of estrous cycle in which the progesterone concentration dominateR.

It starts after the formation of corpns luteum and ends after the regression of corpus

luteum. This is the longest stage of the estrous cycle.

1.1.3 Corpns luteum:

The CL is a transient reproductive gland, which is formed after ovulation (after the

transformation of follicular cells from estrogen production to progesterone production)

and regresses at the end of estrous cycle. The CL was first named by Marcello Malpighi

(Review by Niswender et al., 2000) and was first accurately described by Regnier de

2

Graaf (Review by Niswender et aI., 2000). CL is formed from the theca interna and

granulosa! cells of the ovulated follicle by a process ca1led luteinization. Luteinization

shifts the steroid production by the ovary from estradiol to progesterone (Juengel and

Niswender, 1999). Progesterone is essential for the establishment and maintenance of

pregnancy. The CL is a heterogeneous tissue consisting of steroidogenic large and smaIl

luteal cells (Oshea et aI., 1989). Large luteal cells are responsible for the production of

80% of progesterone (Niswender and Hoyer, 1985). Luteinizing hormone (LH) and

Growth hormone (OH) are the primary luteotropic hormones. LH controls the production

ofP4 by the smaIl luteal cells (Niswender et aI., 2000), and studies have shown that most

of the LH receptors are present on the smaIl luteal cells and most of the OH receptors are

located on the larger luteal cells (Koelle et aI. 1998). CL is produced in the early luteal

phase, which is metesturs, and it grows in size in mid luteal phase, which is diestrus.

During diestrus it produces large quantities of P4. The P4 levels decrease towards the end

of late luteal phase where CL regresses by a process ca1led luteolysis. After luteolysis the

CL loses its shape and function and remains as corpus aIbicans (CA). The CA is non­

functional remnant of the CL that appears like a white scar and it can be seen for a long

time after luteolysis.

1.1.4 Stages ofthe CL:

CL in cattle can be divided into 4 stages based on the morphology (Ireland et aI. 1980).

Stage I of the CL is from days 1-4 after ovulation that results from the ruptureofblood

vessels within the follicular wall. This results in a bloody structure ca1led corpus

hemorrhagicum. Stage II of the CL is from days 5-10 after ovulation, at this stage the CL

3

is fully formed and vasuclature can be seen around its periphery. Stage m of the CL is

from days 11-17 after ovulation, this is the largest of all the stages of CL and contains

very well developed vasculature. Stage IV of the CL is from days 18-20 after ovulation

and this is the regressing form of CL. In this stage we can also notice the change in color

of CL from orange to yellow. After day 20 of ovulation we can see the regressed form of

CL, which is CA.

1.1.5 Regression of Corpus Luteum (Luteolysis):

The regression of CL is also called luteolysis in which CL is irreversibly damaged. As a

result, the blood P4 levels will drop drastically. Two main hormones that regulate

luteolysis are prostaglandin F2 alpha (pGF2a) and oxytocin. The uterus produces these

two hormones.

It has been proposed that the signal for the release ofPGF2a from the uterus is the

estradiol from the developing preovulatory follicles that stimulates the hypophysial

oxytocin, which in turn stimulates the uterus to produce sma1l quantities of PGF2a

(Fairclough et al.1980). The small quantities of PGF2a produced by a positive feed back

mechanism initiates the release oflarger quantities ofPGF2a and oxytocin by the coIpUS

luteum in sheep (Tsai and Wiltbank, 1997) and by the uterus in heifers (Lafrance and

Goff, 1988).

Two events can be noticed during norma1luteolysis, the first being the loss of CL ability

to produce P4 (McGuire et al.1994) and the second being the decrease in the size of CL

4

as a result of the loss of the cells that form the CL (Knickerbocker et aI. 1988). The

former process is called functional luteolysis and the later process is called as the

structma1luteolysis (Sugino, 2005).

Studies in both normal and PGF2a induced luteolysis have shown that the main eftect of

PGF2a is to cause drastic decrease in blood flow to the CL (Azmi et aI. 1982;

Knickerbocker et aI. 1988), thereby the CL will be deprived of the nutrients and will

eventually regress. Studies in sheep also showed PGF2a cause damage to the endothelial

cells of the capillary blood vessels in the CL and result in the reduced blood supply to the

1utea1 tissue (Azmi and Oshea, 1984; Oshea et aI. I 977).

Studies on PGF2a induced luteolysis in sheep have shown that 24 hrs after the

administration of PGF2a, the number of smalllutea1 cells decrease and the size of the

large 1utea1 cells decrease at 36 hrs after the administration ofPGF2a (Braden et aI.

1988).

Studies have shown that there is a strong evidence of the involvement of immune system

in the luteolysis process. Infiltration ofiarge number ofieukocytes and T-Iymphocytes

was noticed in the CL during luteolysis (Review by Murdoch et aI., 1988). PGF2a­

induced luteolysis in pigs showed that macrophages infiltrate the parenchyma and blood

vessels of the CL (Henke et aI. 1994). In bovine CLs it has been shown that macrophages

produce tumor necrosis factor - a (TNF-a), which inhibit basa1 progesterone secretion

and stimulates PGF2Ja secretion (Benyo and Pate, 1992).

5

Recent studies have shown the involvement of Reactive Oxygen Species (ROS) in the

regression of CL. Superoxide mdica1s (SOR). hydrogen peroxides (H2<h). and hydroxyl

mdica1s (OHj. are the primary reactive oxygen species that are genemted in

steroidogenic cells. Studies in mts showed that the SOR (Sawada and Carlson, 1989) and

H2<h (Behrman and Aten, 1991) levels increase in the CL during regression.

6

1.2 REACfIVE OXYGEN SPECIES AND THEIR TYPES:

1.2.1 Reactive Oxygen Species: (ROS)

It has long been known that oxygen can be toxic. Toxic properties of oxygen were first

clearly explained by Gershman in his free radical theory of oxygen toxicity, which states

that, toxicity of oxygen is due to partially reduced forms of oxygen (Gerschman et aI.,

1954). Reactive oxygen species (ROS) include free radicals, as well as other oxygen­

related reactive compmmds (Halliwell, 1991).

A free radical is defined as any species capable of independent existence that contains

one or more unpaired electrons (Halliwell 1991 and 1994). Among the ROS, the

important ones are superoxide (020-) hydroxyl radical (OHi, nitric oxide (NOe), and

hydrogen peroxide (H20z).

1.2.2 Superoxides (020-) or Superoxide radicals (SOR)

Superoxide radical (SOR) is oxygen centered radical with an unpaired electron residing

on the oxygen. Superoxides are accidentally produced by mitochondria in the electron

transport chain due to auto oxidation reactions and leaking of electrons from the electron

transport chain onto oxygen. This leaking of electrons onto the oxygen occurs during the

passage of electrons from CoQH" (reduced Coenzyme Q) as part of the electron chain.

These superoxides are very reactive and cause damage to the living cells. Superoxide

generated extracellularly by a xanthine oxidase/purine system has been shown to kill the

bacterium Staphylococcus epiderimidis.

7

• Oi e- NADH CoQ

'>,., • ~ ~ 02

NAD+

Production of Superoxide radicals Leakage of electrons from electron chain

(Adopted from Bandyopadhyay et aI., 1999)

Besides mitochondria, superoxides are also shown to be produced in significant quantities

by cell nucleus (Bartoli et aI., 1977), macrophages (Johnston et aI., 1976), microsomes

(Aust, et aI., 1972) and human monocytes (Johnston et aI., 1976).

Recent studies in rats have shown that SORs are produced in the corpus luteum shortly

after PGF2a treatment. Huge amounts ofSORs are produced in the CL before the

reduction in the concentration of progesterone in the circulation, suggesting the role of

SORs in damaging the steroidogenic property ofIuteaI cells during the luteolysis

(Sawada and Carlson, 1989). Superoxide dismutase (SOD), an important free radicaI-

scavenging enzyme, converts SOR to less toxic hydrogen peroxide (H2~)' and water

(H20) (Fridovich, 1995).

8

1.2.3 Hydroxyl radical (OHj:

This is the most reactive radical among all ROS. This is also an oxygen centered

radical and it can be produced when the body is exposed to the gamma radiation

or the low wavelength electro magnetic radiation. The radiation splits oxygen -

hydrogen covalent bonds in water leaving a single electron on hydrogen and one

on oxygen, thus creating two radicals: one hydrogen radical (H.) and one

hydroxyl radical (OH") as shown in the following reaction.

Ir +OIr

(Adopted from 8andyopadhyay et aI., 1999)

Hydroxyl radicals are also produced by a reaction called Haber-Weiss reaction in

which hydrogen peroxide and superoxide radical combine together and give rise

to hydroxyl radical as shown in the following reaction.

---~.~ Oz +Off + oIr

(Adopted from 8andyopadhyay et al., 1999)

Hydroxyl radical is very reactive and can attack any molecule in the living cell.

The rate of reaction is very fast that they cannot persist in the cell even for

microseconds and rapidly combines with the molecules in its immediate vicinity

and causes potent damage to the molecules (Halliwell, 1991). Reactions of

hydroxyl radical include its ability to interact with the purine and pyrimidine

9

bases of DNA, leading to radicals that have a number of possible chemical fates

(Halliwell, 1989). Hydroxyl radical can also abstract hydrogen atoms from many

biologic molecules, including thiols. The resulting sulfur radicals (thiyl radicals)

can combine with oxygen to generate oxysulfur radicals, which damage biologic

molecules (Halliwell, 1989).

1.2.4 Hydrogen peroxides: (H202)

Even though hydrogen peroxide is not a radical as it lacks unpaired electrons, it is

considered as reactive oxygen species as it easily diffuses through the cells and

cause damage to the cells. Hydrogen peroxide is generated from superoxide

radicals by an enzyme called superoxide dismutase (SOD) as shown in the

following reaction.

SOD

020- + 020

- + 2W -----1.~ 02 +

(Adopted from Bandyopadhyay et al., 1999).

Hydrogen peroxide itself can be quite toxic to cells. For example, incubation of

cells with H202 causes deoxyribonucleic acid (DNA) damage and membrane

disruption (Halliwell, 1991).

10

1.3 EFFECTS OF ROS AND DEFENCES AGAINST THEM

1.3.1 Biological effects of ROS:

Among many consequences of the generation ofROS in vivo, the well-known ones are

protein oxidation (Pacifici et a1., 1993), lipid peroxidation (Halliwell, 1991), and DNA

strand scission (Brawn and Fridovich, 1981). These reactions will alter intrinsic

membrane properties like fluidity and ion transport. ROS also affect enzyme activity,

protein cross-linking, protein synthesis and cause damage to DNA that will ultimately

result in cell death (Halliwell, 1991).

Generation of higher amounts ofROS in the cells leads to the oxidative damage

of the cells and finally leads to several pathological conditions, the following are

some of the very important disease conditions:

Role ofROS has been well documented in

1. Alzheimer's disease (Hensley et al., 1996; AsIan and Ozben. 2004).

2. Down's syndrome (Buscigilo and Yankner, 1995).

3. Cancer (Yanbo et al., 1998)

4. Parkinson's disease (Winyard et al., 1998).

5. Atherosclerosis (Steinberg et al., 1989).

6. Ischemic reperfusion injury in heart, kidney, liver, gastro-intestinal tract and brain

(McCord, 1987).

7. Aging (Stadtman, 1998).

8. Stress-induced gastric ulcer and inflammatory bowel diseases (Das et al. 1997).

11

9. Progressive loss of T lymphocytes in human immunodeficiency virus infection

(Flores, 1998).

1.3.2 Defenses against ROS:

Aerobic organisms protect the cells from the damaging effects of ROS, by

producing antioxidants (Riley and Behrman, 1991 b), which defend the cells by

detoxifying the ROS or preventing their formation.

These antioxidants include Vitamin C, Vitamin E and enzymes superoxide

dismutase (SOD), catalase and peroxidases. These three enzymes work together to

detoxifY ROS. SOD converts superoxides into peroxides where as peroxidases

and catalases convert peroxides into water and oxygen (Bandyopadhyay et al.,

1999) in the following steps.

epaat&II<oII>

SOD 1 10,- + 2B' .. OA + 0,

GSD - paaIIdIttr ROOH/HaOJ .. ROB/H,O + GSSG

pmqIdaR BA + AlII ~ 2.010 + A

..w-2. BzOz ~ 2.HzO + OJ

(Adopted from Bandyopadhyay et al., 1999)

12

Superoxide dismutase (SOD) catalyzes the reaction of converting the superoxides

into peroxides (Fridovich, 1983). Types of SOD and their distribution will be

discussed under separate subheading.

Glutathione peroxidase catalyses the reaction of hydroperoxides and lipid peroxides. It is

a selenium-containing enzyme present in liver. Two-third of it is present in the cytosol

and one-third in the mitochondria (Freeman and Crapo, 1982).

Catalase is present in almost all the mammalian cells and is localized in the peroxisomes

or the micro peroxisomes. This is an iron containing metalloenzyme that catalyzes the

conversion of hydrogen peroxide into water and oxygen (Chance et al.1979).

Somce of protection from the damaging effects ofROS also comes from vitamin E and

vitamin C. Vitamin E protects the cell membranes from the damaging effects ofROS by

terminating the peroxidative chain reactions of unsaturated lipids, whereas vitamin C

protects aqueous compartments of cells from the damaging effects ofROS. Vitamin C

also recycles the oxidized vitamin E back to reduced state (Riley and Behrman, 1991 a

and b).

1.4 ROS IN FEMALE REPRODUCTION AND DEFENCES AGAINST THEM.

1.4.1 Role of ROS in female reproduction:

Reactive oxygen species plays both physiologic and pathologic actions in female

reproductive tract. The pathologic effects ofROS are caused by various mechanisms

including lipid damage, inhibition of protein synthesis, DNA damage and mitochondrial

13

alterations (Cooke et al., 2003). The physiological functions ofROS on female

reproductive system are evident in different aspects of reproduction, including oocyte

maturation and ovulation, luteolysis, and luteal maintenance in pregnancy.

1.4.2 Role of ROS in ooeyte maturation and ovulation:

In the follicular phase of reproductive cycle, a cohort of primordial follicles are recruited

for the development and maturation. At the end, one or more dominant mature follicles

ovulate, and the other group of follicles regress. Follicular growth and development are

regulated by two honnones namely, estradiol and follicle stimulating honnone (Riley and

Behnnan, 1991a).

The follicular regression is characterized, in part, by loss of sensitivity to the

gonadotropic honnones, particularly follicle stimulating honnone (Riley and Behrman,

199Ia). Margolin et al. (1990) suggested indirect evidence for the involvement ofROS in

rat oocyte regression. They showed that the granulosa cells are extremely sensitive to the

low concentration of H202 that rapidly inhibited FSH, cyclic AMP accumulation and

progesterone production.

Ovulation is typically characterized by luteinizing honnone (LH) surge and generation of

PGE2 and PGF21l by the preovulatory follicles (Riley and Behnnan, 1991a). In vitro

studies by Miyazaki et al. (1991) with rabbit ovary showed that ovulation was markedly

reduced when SOD was added to the incubation medium, suggesting the stimulatory

involvement of ROS, especially superoxides in the process of ovulation.

14

1.4.3 Role of ROS in regulation of luteal function.

Corpus luteum is a transient reproductive gland, which produces progesterone that is

important for the maintenance of pregnancy. The CL forms after ovulation and regresses

at the end of estrous cycle if the animal does not conceive, but grows in size and produces

higher levels of progesterone if the animal conceives. Thus, the strategy of reproduction

in the ovary is rapid rescue of CL when animal conceives and rapid regression and death

of CL when the animal fails to conceive, so that the animal will ovulate further and gain

another chance to conceive (Sugino, 2005).

Studies have shown the involvement of ROS in this process of CL function. Sawada and

Carlson, (1989) showed that in PGF2a. induced functionalluteolysis in pseudo pregnant

rats, ROS, including superoxide radicals and hydrogen peroxides, increase in the corpus

luteum during the regression phase.

It is well known that CL produces progesterone and PGF2a. is involved in the regression

of CL by stopping the production of progesterone but, studies aIso showed that ROS are

generated in the corpus luteum and influence progesterone synthesis (Behrman and Aten,

1991; Gatzuli et aI., 1991; Carlson et aI., 1995). It has proven that superoxide radicals

inhibit progesterone production by rat luteal cells (Sawada and Carlson, 1994; Kato et aI.,

1997 and Sugino et aI., 1999) and hydrogen peroxide inhibits progesterone production by

rat and human luteal cells (Behrman and Preston., 1989; Endo et aI., 1993).

15

Sawada and Carlson (1991) and Wu et aI. (1993) showed that ROS cause several changes

that disrupt the plasma membrane of luteal cells thus involving in the regression of CL.

Sugino et aI. (1996), Aten et aI. (1998) and Minegishi et aI. (2002) all showed that

PGF2a also stimulated ROS production by phagocytic leukocytes such as macrophages

or neutrophils in the corpus luteum of rats. Studies by Pepperell et aI. (1992) showed that

hydrogen peroxide produced by nuetrophils inhibit the progesterone production by

entering into the luteal cells. This suggests the involvement ofPGF2a in increasing ROS

production and causing the luteal regression as well as stopping the production of

progesterone.

There is also evidence that ROS can increase PGF2a synthesis by activating

phospholipase A2 activity and cyclooxygenase-2 expression, which playa key role in

PGF2a synthesis in corpus luteum (WU and Carlson, 1990; Sawada and Carlson., 1991

and Nakamura et aI., 2001). Taken together, the evidences show that there is a close

interrelation between PGF2a and ROS in the regression of CL, as suggested by Sugino

(2005).

Involvement of changes in ovarian blood flow in the regression of CL has been studied

extensively. Azmi et aI. (1982) and Knickerbockers et aI. (1988) showed that PGF2a

cause drastic decrease in blood flow to the CL thereby inhibit nutrients and eventually

cause it to regress. Studies in sheep also showed PGF2a causes damage to the endothelial

16

cells of the capillary blood vessels in the CL and result in the reduced blood supply to the

luteal tissue (Azmi and Oshea 1984; Oshea et al.1977).

In a variety of organs, it is also well known that the decrease in blood flow causes tissue

damage by generation of ROS by a mechanism called ischemia-reperfusion injury

(Sugino et al., 1993). In rats CL. it was proved that experimentally induced ovarian

ischemia-reperfusion caused increase in the production ofROS and decrease in serum

progesterone levels (Sugino, 2005).

Sakka et al. (1997) showed that isolated bovine luteal cell suspensions are capable of

generating a marked acute ROS response triggered by activation of protein kinase C

(PKC) and/or elevation of cytosolic calcium.

1.4.4 Expression of Snperoxides in Corpus Lntenm.

Involvement of superoxides in the regression of corpus luteum has been well documented

in different species of animals. Sawada and Carlson (1991) and Aten et al. (1998) showed

that PGF2a treatment(s) in vivo in rat corpus luteum stimnlate(s) superoxide production

by the nonsteroidogenic cells. Increased production of superoxides during regression of

the bovine corpus luteum was noticed by Sakka et al. (1997).

1.4.5 Expression of Snperoxide Dismntases in Corpus Lntenm.

Superoxide dismutase is the first line of antioxidant defense against superoxides.

It is a metaIloprotein found in both prokaryotic and enkaryotic cells (Fridovich,

1983). Prokaryotes contain iron-containing SOD (Fe-SOD) and manganese-

17

containing SOD (Mn-SOD) (Fridovich, 1983). Eukaryotic cells contain copper

and zinc containing SOD (CulZn-SOD) and manganese containing SOD (Mn­

SOD). CUlZn-SOD is located in the cytosol and Mn-SOD is located in the

mitochondrial matrix (Fridovich, 1983).

Induction of SOD by increased intmcellular fluxes of superoxide radicals has been

observed in numerous microorganisms (Fridovich, 1983), as well as in

higher organisms (Crapo and McCord, 1976).

Presence ofMn-SOD and CulZn-SOD in CL were reported during the bovine estrous

cycle (Rapport et aI. 1998) and also during the pregnancy in pigs (EIiasson et aI. 1999).

Sugino (1993) and Shimamura et aI. (1995) have shown the presence of both Mn-SOD

and CulZn-SOD in pregnant and nonpregnant rat corpus Iuteum.

1.4.6 Differential expression of Superoxide Dismutases

Different types of superoxide disumutases are differentia1ly expressed in different stages

of corpus luteum, in different species of animals_ Sugino (1998) showed that CulZn-SOD

levels during late pregnancy decreased where as Mn-SOD levels remained elevated in

rats CL. Studies with human corpus luteum during the menstrual cycle by Sugino et aI.

(2000) showed that CulZn-SOD expression increased from early to mid 1utea1 phase and

decreased thereafter and the expression was lowest in regressed form of corpus luteum,

which is corpus albicans. However, expression ofMn-SOD was low in mid 1utea1 phase

and increased during the regression phase.

18

1.5 OXIDATIVE STRESS AND ITS MARKERS

1.5.1 Oxidative stress.

One accepted definition for oxidative stress is by Sies (1991). It is defined as .. a

disturbance in the pro-oxidant - antioxidant balance in favor of the former, leading to

potential cellular damage. Oxidative stress develops when there is a depletion of levels of

antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (OP) in the

cells or when there is an increased production of ROS in the cells or combination of both.

1.5.2 Biomarkers of oxidative stress:

A central feature of oxidative stress is peroxidation of lipids. Many methods have been

developed to quantify the products of free radical induced lipid peroxidation as a

potential means to assess oxidant injury. These methods among many others include

measuring lipid hydroperoxides, Malondialdehyde (MDA), conjugated dienes and short

chain alkanes. Many of these methods may provide an accurate index of lipid

peroxidation in vitro. However, inaccuracies have been observed with most of these

methods when used to assess oxidant stress in vivo (Halliwell, 1987).

In 1990, Morrow et al. reported that a series of prostaglandin like compounds are

produced in vivo in humans independent of the cyclooxygenase enzyme by free radical

catalyzed peroxidation of arachidonic acid. Since their discovery, considerable amount of

evidences have been reported to suggest that measurement of these unique products of

19

lipid peroxidation, which are later tenned as F2- isoprostanes, can provide a reliable

measure of oxidant injury not only in vitro, but also in vivo (Morrow et aI. 1990).

1.5.3 Formation ofFz- isoprostanes:

Pryor and Stanley (1975) proposed the fonnation ofF2-Isoprostanes from arachidonic

acid (AA). It was based on the generation ofbicycloendoperoxide intermediates resulting

from the peroxidation of polyunsaturated fatty acids.

Arachidonic acid is the precursor for F2-Isoprostanes and undergoes abstraction of an

allylic hydrogen atom to yield an archidonyl carbon centered radical and in the next step

there is an insertion of oxygen to yield peroxyl radicals. Four different types of peroxyl

radicals are fonned depending on the site of hydrogen abstraction and oxygen insertion.

Further addition of one more molecule of oxygen result in endocycIization of the radicals

and yield four bicycloendoperoxide (pGG2-like) regioisomers. Fonnation of four

regioisomers ofF2-Isoprostanes namely, 5-,12-,8- and 15- series compounds takes place

by the reduction of four bicycloendoperoxides. Among all these four types ofF2-

Isoprostanes, 8-Isoprostanes are extensively studied (Morrow and Roberts, 2003).

It is important to know that these F2-isoprostanes can be detected in the biological

samples both from humans and animals. F2- isoprostanes were detected in measurable

quantities in fresh human plasma from normal volunteers analyzed immediately after

collection (Morrow etal., 1990). A 200 times increase in the levels ofF2-Isoprostanes

were noted in the plasma of rats treated with carbon tetrachloride (CC4) to induce an

oxidant injury (Morrow et aI., 1990).

20

1.5.4 Measuremeut of Frisoprostanes:

Mass spectrometry is the ideal method of quantification ofF2-isoprostanes as it is highly

sensitive and highly accurate with 96% accuracy (Morrow and Roberts, 1999). However,

it is very labor intensive and not widely available. To solve this problem both commercial

enterprises and academic investigators have developed immunoassays for specific F2-

isoprostanes. lmmunoassays are advantageous as they are more economical less labor

intensive and commercial polyclonal antibodies are available for specific isoprostanes.

1.5.5 Advantages of Fz-isoprostanes measurement:

F2-Isoprostanes discovery is important as: 1) they can be generated in the biological

fluids and they can be used as an index of lipid peroxidation or oxidant stress in vivo,

2) it is also one of the most reliable non-invasive methods of assessing the oxidative

stress status in humans, 3) availability of mass spectrometric method of analysis ofF2-

isoprostanes is very advantageous and this method is also very sensitive that can be

quantified in small biopsies ofhwnan tissue, 4) F2-isoprostanes are chemically stable and

are specific products ofperoxidation of lipids, 5) levels ofF2-isoprostanes increase

substantially in animal models of oxidant injury (Morrow et a1., 1990), 6) Their levels are

unaffected by the lipid content in the diet (Montuschi et a1., 2004).

1.5.6 Disadvantages of Frisoprostanes measurement:

The disadvantages ofF2-Isoprostanes are: -

1) F2-isoprostanes represents only one of a myriad of arachidonate oxygenation products.

21

2) Analysis is labor intensive and requires expensive equipment, and

3) There was a 100-fold increase in the levels ofF2-isoprostanes when they were stored at

_200 C (Morrow et aI. 1990). To avoid this either they should be analyzed immediately

after collection of samples or they should be stored at _700 C.

22

Chapter 2

Differential Expression of Super oxide Dismutases (SODs) in Bovine Corpus

Luteum

2.1 Introduction:

The CL is a transient reproductive gland, which is formed after ovulation and regresses at

the end of estrous cycle. Progesterone (P 4), a steroid hormone secreted from CL, is

essentiaI for the maintenance of pregnancy. If pregnancy is not established, the CL

regresses Quteolysis) both structurally and functionally in response to PGF2a, which is

secreted by the endometrium. Luteolysis is important for the ovulation of the remaining

follicles and to control the normaI cycling of estrous cycle.

Studies showed many mechanisms of luteolysis, such as changes in blood flow to the

corpus luteum, involvement of immune system by infiltration ofleukocytes, T­

lymphocytes and macrophages. Recent studies showed the involvement ofROS in the

regression of CL. It is well known that the ROS, including superoxide radicals (SOR),

hydrogen peroxides (H202), and hydroxyl radicals (OR) cause cell damage. The ROS are

known to increase in the CL during regression phase in rats (Aten et aI., 1998; Riley and

Behrman, 1991a; Sawada and Carlson., 1994) and in bovine species (Sakka et aI., 1997),

which suggests the involvement ofROS in the regression ofCL. To counteract the

damaging effects of ROS, the CL has antioxidant enzymes, such as superoxide dismutase

(SOD), catalse and glutathione peroxidase. Rappoport et aI. (1998) with studies in

bovines and A1-Gubory et aI. (2005b) in sheep showed that the expression of these

23

antioxidants change dramatically during estrous cycle. Eliasson et aI. (1999) showed that

these antioxidants change drastically during pregnancy in pigs.

Superoxide dismutase (SOD) forms the first line of defense against superoxide radicals.

Bovine CL has two types of SODs (Nakamura and Sakamoto, 2001). Manganese SOD is

located in the mitochondria and Copper-Zinc SOD is located in the cytoplasm. Sugino et

aI. (2000) reported that these two SODs are differentially expressed during menstrual

cycle in human CL. They showed that CulZn-SOD activity and expression increases from

early to mid luteal phase and gradually decreases during the regression phase. In contrast,

Mn-SOD activity and expression was low in mid luteal phase and increased during the

late luteal phase and regression phase. Sugino et aI. (1998) also showed that both Mn­

SOD and CulZn-SOD are differentially expressed during the different stages of

pregnancy in rats. They also reported that, Mn-SOD, but not the CuIZn-SOD, is highly

induced by the inflammatory cytokines during the regression phase. This differential

expression suggests that SODs may play an important role in the regulation ofCL during

cycling of the ovaries and during the pregnancy. However, in bovines very few studies

examined the expression of SODs during estrous cycle and during pregnancy. Therefore

the present study investigated the changes in activities and expression of Mn-SOD and

CuIZn-SOD, and 8-Isoprostane levels, in different stages ofCL during estrous cycle and

during pregnancy. This information will be helpful in future studies to detect the effect of

heat stress on conception rates and pregnancy rates of dairy cattle in hot climates like in

tropics during hot periods of the year. For example if we notice increased levels of 8-

Isoprostanes in the pregnant CL of dairy cattle due to heat stress during hot periods of the

24

year, we might suggest that oxidative stress due to excessive heat is at least the part of the

reason for increase in the levels of 8-Isoprostanes. Study oflevels and activities ofMn­

SOD and CulZn- SOD during this period in the pregnant CL as a defense mechanism

against oxidative stress will be also very useful.

2.2 Materials and Methods

2.2.1 Sample collection

Corpora lutea (CL) (n=120) were collected from Hostein cows within one hour after

slaughter from a local slaughter house. The estrous stages of these CLs were classified

based on morphology into four different stages, 1st (1-4 days), 2nd stage (5-10 days), 3rd

stage (11-17 days) and 4th stage (18-20 days) (Ireland et al. 1980). The 3n1 stage of the CL

was again divided into early 3n1 stage, mid 3n1 stage and late 3n1 stage based on the

morphology. The regressed form of CL, corpus albicans (CA) and pregnant CLs were

also collected. Blood samples were collected form these animals and their sera were

collected by centrifuging the blood at 3000 rpm for 10 minutes. Both CLs and serum

were stored at -800 C until further analysis.

25

2.2.2 Different stages of CLs in Estrus Cycle.

1" stage: The CL is newly formed with the point of ovulation still open and bloody, very

little amount of tissue can be seen.

2nd stage: Still red in color but, the point of ovulation is closed. Very little amount of

tissue can be seen. Internally also red in color and sometimes contains blood.

26

Early 3'd stage: The amount of tissue is considerably large and sometimes we can see

the point of ovulation but it is closed, inside of it is brown in colour, sometimes with a

cavity.

Mid 3'd stage: It contains the highest amount of tissue in the entire Estrous cycle and

contains vasculature on its surface. Here we cannot see the point of ovulation, internally

it is brown in color with no cavity.

27

Late 3'd stage: From now onwards the amount oftissue starts decreasing and the internal

tissue starts turning into yellow color, we can still see little vasculature.

4th Stage: Here the amount of tissue is low, internal tissue is yellow in color, and we can

see a well-developed follicle on the ovarian surface.

28

Corpus Albicans: This is regressed part ofCL, white in co lour; the amount of tissue is

very very little.

CA

Pregnant Corpus Luteum: Here the amount of tissue is more than the mid 3n1 stage of

CL. Brown in colour internally with no cavity.

29

2.2.3 Sample preparation:

CL tissue (250 mg) was taken from each stage and was homogenized in 10 ml (40

volumes) of phosphate saline buffer (PBS, 10mM sodium phosphate, 0.9% NaCl, pH 7.0)

by using polytron homogenizer at maximum speed for 1 minute. The homogenate was

centrifuged at 1,500 g for 10 minutes at 4°C to remove tissue debris. The supernatant

was collected for protein concentration measurement.

2.2.4 Protein Assay:

After centrifugation, 100 !Jl aliquot of supernatant was used for protein concentration

measurement by Lowry method (1951) with bovine serum albumin (BSA) as a standard.

2.2.5 Sodium dodeeyl sulphate and plyaerylamide gel electrophoresis (SDS-PAGE):

SOS-PAGE was performed on mini gels (9XI0 cm) by the method ofLaemmli (1970)

using 15% Polyacrylamide gels in the presence of 0.1 % SOS under reducing conditions.

Supernatant containing 10 Ilg protein was loaded to each lane. The gels were either

stained with Coomassie blue or subjected to electrophoretic transfer onto a

Polyvinylidene Difluoride (PVDF) membrane for Western blot analysis.

2.2.6 Western blot analysis:

Proteins from Polyacrylamide gel were electrophoretically transferred onto

Polyvinylidene Oifluoride (PVDF) membrane while immersed in Towbin transfer buffer

(25 roM Tris, 192 roM glycine, 20% methanol, 0.1 % SOS). After protein transfer,

30

membranes were blocked with TBS (125 mM NaCI, 25 mM Tris, pH 8.0) buffer

containing 0.5% Tween 20 for 2 hours at room temperature. Membranes were incubated

for I hour at room temperature in TBS buffer containing plyclonal anti-rabbit Mn-SOD

antibody (1 :40,000 times dilution, ROI, Concord MA) and plyclonal anti-rabbit CulZn­

SOD antibody (I :40,000 times dilution, ROI, Concord, MA) separately. The membrane

was washed three times with TBS for 10 minutes each and was reacted with all<JIline

phosphatase conjugated anti-rabbit IgG (1: 1 0,000 times dilution; Sigma, Stlouis, MO)

for one hour at room temperature. The membrane was again washed and developed using

nitroblue tetrazolium and bromo-chloro-indolys phosphate (BCIPINBT).

2.2.7 Isolation of Mn-SOD by electro elution:

CL supernatant was run through the SDS-PAGE to fractionate the proteins by molecular

weight. The protein band corresponding to the molecular weight ofMn-SOD (23 kD) was

excised, and protein from the band was electro eluted by using a 422 Bio-Rad electro

eluter (catalog no. 165-2976) following manufacturers protocol. The eluted protein was

run through the SDS-PAGE and subjected to western blotting to confirm that the eluted

protein is Mn-SOD.

2.2.8 Obtaining Mn-SOD standard curve:

Protein concentration of Mn-SOD protein, isolated by electro elution was measured by

Lowry method. Different concentrations of extracted Mn-SOD (200 ng, lOOng, 50 ng

and 25 ng) were run in a western blot and the band volume (ODU·mm2) of different

concentrations of Mn-SOD was measured by using a Molecular Dynamics laser

31

densitometer (Bio-Rad) with Imagequant software (Sunnyvale, CAl. Mn-SOD standard

curve was plotted by taking protein concentration on X-axis and ODU value of the

corresponding band on the Y-axis.

2.2.9 Quantification of Mn-SOD in samples:

CL samples (n = 120) of different stages were homogenized and the protein concentration

of each homogenate was measured by Lowry method. 1 0 J,tg of protein from sample was

subjected to SDS-PAGE to separate the proteins by molecular weight The separated

proteins were blotted on to a Polyvinylidene Difluoride (PVDF) membrane. The

membrane was incubated with the polyclonal anti-rabbit Mn-SOD antibody and band

volume (ODU*mm2) ofMn-SOD in different stages ofCL was measured by using a

Bio - Rad software (Sunnyvale, CAl.

Mn-SOD concentration in each Mn-SOD band was calculated by using the Mn-SOD

standard curve. Isolated pure Mn-SOD was also assayed in each western blot to make

sure that we were working in a range within the band volume that was proportional to the

concentration ofMn-SOD.

2.2.10 Isolation ofCulZn-SOD by electro elution:

The known volume ofCL homogenate (10!Jls) was run through the SDS-PAGE to

separate the proteins by molecular weight. The protein band corresponding to the

molecular weight ofCulZn-SOD (15 leD) was excised and protein from the band was

electro eluted by using a 422 Bio-Rad electro eluter (catalog no. 1 65-2976) following

32

manufacturers protocol. The eluted protein was again run through the SDS-PAGE and

transferred on to Polyvinylidene Difluoride (PVDF) membrane. The membrane was

incubated with the polyclonal anti-rabbit Cu/Zn- SOD antibody to confirm that the eluted

protein is CU/Zn-SOD.

2.2.11 Obtaining Cu/Zn-SOD standard curve:

Protein concentration ofCu/Zn-SOD protein, isolated by electro elution was measured by

Lowry method. Different concentrations of extracted CulZn-SOD (200 ng, .00 ng, 50 ng

and 25 ng) were taken in a western blot and the band volume (ODU"'mm2) of different

concentrations of CU/Zn-SOD was measured by using a Molecular Dynamics 1aser

densitometer (Bio-Rad) with lmagequant software (Sunnyvale, CA). Cu/Zn-SOD

standard curve was plotted by taking protein concentration on X-axis and OD value of the

corresponding band on the Y-axis.

2.2.12 Quantification ofCulZn-SOD in samples:

CL samples of different stages were homogenized and the protein concentration of each

homogenate was measured by Lowry method .• 0 fJg of protein from sample was

subjected to SDS-PAGE to separate the proteins by molecular weight. The separated

proteins were blotted against a Polyvinylidene Difluoride (PVDF) membrane. The

membrane was incubated with the polyclonal anti-rabbit CU/Zn-SOD antibody and band

volume (ODU*mm2) ofCU/Zn-SOD in different stages ofCL was measured by using a

Molecular Dynamics laser densitometer (Bio-Rad) with lmagequant software

(Sunnyvale, CA).

33

CulZn-SOD concentration in each CulZn-SOD band was calculated by using the CulZn­

SOD standard curve. Isolated pure CulZn-SOD was also assayed in each western blot to

make sure that we were working in a range within the band volume that was proportional

to the concentration ofCulZn-SOD.

2.2.13 Assay of Mn-SOD Activity:

Activity ofMn-SOD was measured by using Superoxide Dismutase Assay Kit from

Cayman (Ann Arbor, MI) (catalog no. 706002). This assay utilizes a tetrazolium salt to

detect the superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit

of SOD is defmed as the amount of SOD required to exhibit 50% dismutation of the

superoxide radicals.

250 mg ofCL sample was homogenized with polytron homogenizer in 10 ml ofHepes

buffer, pH 7.2 containing 1 mM EGTA, 210 mM mannitol and 70 mM sucrose. The

homogenate was centrifuged at 1,500 g for 5 minutes at 4°C, and the supernatant was

separated. The resulting supernatant was further centrifuged at 10,000 g to separate

mitochondrial Mn-SOD from the cytosol CulZn-SOD. The resulting supernatant contains

CulZn-SOD and the pellet contains Mn-SOD. The Mn-SOD pellet was suspended in cold

Hepes buffer.

The assay was performed by taking suspended Mn-SOD pellet as sample. SOD standard

curve was obtained by taking different concentrations of given SOD standard in the assay

34

kit. SOD standard wells in the plate were added with 200 III of diluted radical detector

and 10 III of SOD standard. 200 III of diluted radical detector and 10 III of sample were

added to the sample wells. The reaction was initiated by adding 20 III of xanthine oxidase

to all the wells. After shaking the plate well, it was incubated for 20 minutes at room

temperature and the absorbance of the plates was read at 450 run using a plate reader.

2.2.14 Assay of CulZn-SOD Activity:

Activity of CulZn-SOD was measured by using Superoxide Dismutase Assay Kit from

Cayman (catalog no. 706002). This assay utilizes a tetrazolium salt to detect the

superoxide radicals generated by xanthine oxidase and hypxanthine. One unit of SOD is

defined as the amount of SOD required to exhibit 50% dismutation of the superoxide

radicals.

CL samples from different stages of estrous cycle of cow and pregnant cow collected

from a slaughterhouse were rinsed with the phosphate buffered saline containing 0.16

mg/ml heparin to remove any red blood cells and clots.250 mg of tissue from each CL

sample was homogenized with polytron homogenizer in 10 ml ofHepes buffer, pH 7.2

containing 1 roM EGT A, 210 roM mannitol and 70 roM sucrose. The homogenate was

centrifuged at 1,500 g for 5 minutes at 4 °C and the supernatant was separated. The

resulting supernatant was again centrifuged at 10,000 g to separate Mn-SOD from the

CulZn-SOD. The resulting supernatant contains CulZn-SOD and the pellet contains Mn­

SOD. The Mn-SOD pellet was suspended in cold Hepes buffer.

35

The assay was performed by taking supernatant containing CU/Zn-SOD as sample. SOD

standard curve was obtained by taking different concentrations of given SOD standard in

the assay kit. SOD standard wells in the plate were added with 200 ILl of diluted radical

detector and 10 !J.l of SOD standard Sample wells were added with 200 !J.l of diluted

radical detector and 10 ILl of sample. The reaction was initiated by adding 20 !J.l of

xanthine oxidase to all the wells. After shaking the plate well, it was incubated for 20

minutes at room tempemture and the absorbance of the plates was read at 450 nm using a

plate reader.

2.2.15 Progesterone concentration measurement in serum during different stages of

estrous cycle and during pregnancy:

Progesterone concentmtion in serum samples was measured by nsing progesterone EIA

kit from Cayman (Ann Arbor, MI) (catalog no. 582601). This assay is the competition

between the progesterone and progesterone-acetyl cholinestemse (AChE) conjugate

(progesterone tmcer) for a limited number of progesterone specific mbbit antiserum

binding sites. Becanse concentmtion of the progesterone tmcer is held constant while the

concentration of progesterone varies, the amount of progesterone that is able to bind to

the mbbit antiserum will be inversely proportional to the concentration of progesterone in

the well.

A 50 ILl of serum sample was added to the sample wells in EIA plate and different

concentmtions of progesterone EIA standard was added to the plate to obtain a

progesterone standard curve. Each sample was assayed in two dilutions and each dilution

36

was assayed in duplicates. Equal amount (50 ,.u) of AchE tracer and progesterone EIA

antiserum was added to the wells containing sample and progesterone standard. The plate

was covered with the paraffin film and incubated at room temperature for 1 hour on an

orbital shaker. After 1 hour, the plate was washed five times with the wash buffer and

developed with 200 III of Elman's reagent in each well. The plate was read at 420 run.

2.2.16 II-Isoprostane measurement:

8-isoprostanes from CL samples were measured by using an immuno assay kit from

Cayman (Ann Arbor, MI) (catalog no. 516351), different catalog numbers of contents of

the contents of the kit are mentioned below.

CL samples (250 mg) were homogenized in 10 ml buffer solution on ice by using

polytron homogenizer for one minute at full speed. The homogenized buffer solution was

0.1 M phosphate buffer, pH 7.4, containing 1 mM EDTA, 10 !lM indomethacin (catalog

no. 70270; Cayman), and 0.005% butylated hydroxytoluene (Sigma). The samples were

centrifuged at 1,500 g for 15 min at 40°C. Protein content was measured in 100 III of the

supernatant. 2 ml Supernatant from each sample was diluted with 8 ml of column buffer

(catalog no. 400220; Cayman) (1:5 dilutions) and passed through an 8-isoprostane

affinity column (catalog no. 416358; Cayman). Each column was used for two samples in

order to avoid depletion of binding capacity of the column. The affinity column was

prewashed with 10 ml of column buffer (catalog no. 400220; Cayman). After passing the

sample solution the column was washed with 10 ml of ultra pure water. The 8-

Isoprostanes from the column were eluted by using 5 ml of elution solution, consisting of

95% absolute ethanol and 5% ultra pure water (catalog no. 400230; Cayman). The eluant

37

was collected and brought to dryness by using vacufuge (Eppendorf). The resulting

extracts were immediately dissolved in EIA buffer (catalog no. 516351; Cayman) and

stored at -80cC until further analysis. The 8-isoprostanes concentrations in the sample

were quantified using an 8-Isoprostane Enzyme Immunoassay kit (catalog no. 516358;

Cayman). This assay is based on the competition between 8-isoprostane and an 8-

isoprostane-acetylcholinesterase conjugate (8-isoprostane tracer) for a limited number of

8-isoprostane-specific rabbit antiserum binding sites. The concentration of the 8-

isoprostane tracer was held constant, whereas the concentration of 8-isoprostane varied.

The amount of 8-isoprostane tracer that is bound to the rabbit antiserum will be inversely

proportional to the concentration of 8-isoprostane in the well. The absorbance of the plate

was measured at 420 nm.

2.3 Statistical analysis

Data were analyzed by one-way ANOVA and Duncan's multiple range test with the help

of SAS program. Differences were considered to be significant if P < 0.05. Data were

expressed in mean ± standard error.

38

2.4 Results:

2.4.1 Progesterone Concentration in serum at different stages of estrous cycle and

during pregnancy.

Figure 1 shows the progesterone concentrations in serum samples measured by

progesterone EIA kit from Cayman (catalog no. 582601). Progesterone concentration in

serum samples increased from 1 st stage CL (1.08 ± 0.05 ng/ml) up to the 3n1 stage CL

(6.00 ± 0.28 ng/ml) and decreased from 3n1 stage CL to regressed CL (1.09 ± 0.09ng/ml)

(P ::; 0.05). There was no significant difference in the concentration of progesterone in

different sub stages of 3n1 stage CL (P > 0.05). Progesterone concentration was highest in

pregnant CL (9.52 ng/ml).

2.4.2 Differential expression of 23 kD protein at different stages of CL during

estrous cycle and during pregnancy.

In figure 2, SDS-PAGE shows that, 23kD protein is differentially expressed and the

expression is high in 3n1 and 41h stages of CL during estrous cycle and low in regressed

CL. We also noticed higher expression of this 23 kD protein in pregnant CL.

2.4.3 Identification of 23 kD protein.

By using Rabbit anti- SOD (Mn-SOD) antibody (RDI inc., Concord MA), we noticed a

thick band at the level of23 kD and identified this band as Mn-SOD. Later this band was

electro eluted from the gel as explained in the materials and methods and confirmed that

this 23 kD protein is Mn-SOD, by using Rabbit anti-Mn-SOD in western blot Figure 3

39

shows the Western Blot analysis of a CL homogenates. Increased expression ofMn-SOD

from 1 st stage of CL to 4th stage of CL was noticed. However, the expression of Mn-SOD

was significantly low in CA. Expression ofMn-SOD was highest in pregnant CL.

2.4.4 Measurement of Mn-SOD concentration in CL samples at different stages of

CL.

Figure 4 explains the process of quantifying the Mn-SOD concentration by molecular

dynamics laser densitometer, by using Bio - Rad software, which was discussed in the

materials and methods. Figure 5 and figure 6 explain pictorially the process of obtaining

the Mn-SOD standard curve by using different concentrations of extracted Mn-SOD that

was mentioned earlier in materials and methods.

2.4.5 Mn-SOD concentration in different stages of CL during estrons cycle and

dnring pregnancy.

Figure 7 shows the concentration of Mn-SOD in different stages ofCL during the estrous

cycle and during pregnancy. Mn-SOD concentration increased from 1st stage CL (18.5 ±

0.4 ngllO ng oftota! protein) up to 4th stage CL (220.5 ± 22.3 ngllO J.tg oftota! protein)

(P< 0.01). The increase in concentration was significantly different from early 3n1 stage

to mid 3n1 stage (P < O.OI).There was a sudden decrease of Mn-SOD concentration in CA

(23.5 ± 0.8 ngllO ng oftota! protein) (P <0.01). However, Mn-SOD concentration was

highest in pregnant CL (249.8 ± 24.4 ngll0 J.tg oftota! protein) (P < 0.01).

40

2.4.6 Identification of 15.6 kD protein.

Presence of CulZn-SOD in rat CL was reported by Sugino et al., (1998). The molecular

weight ofCulZn-SOD is 15.6 kD. We could not identitY any band in the SDS-PAGE near

the range of 15.6 kD with the bovine CL in figure 2. However, a thick band at the level of

15.6 kD was observed when Rabbit anti- SOD (CulZn-SOD) antibody (RDI inc.,

Concord MA) was used in Western blot Figure 8 shows the expression of CuIZn-SOD in

different stages of CL

2.4.7 Measurement of CulZo-SOD concentration in different stages of CL.

Figure 9 explains the process of quantifYing the CU/Zn-SOD concentration by molecular

dynamics laser densitometer, by using Bio - Rad software, which was discussed in the

materials and methods. Figure 10 and figure 11 explain the process of obtaining the

Cu/Zn-SOD standard curve by using different concentrations of extracted CU/Zn-SOD

that was mentioned earlier in materials and methods.

2.4.8 CulZn-SOD concentration in different stages of CL during estrous cycle and

during pregnancy.

CU/Zn-SOD concentration was also measured by Molecular Dynamics laser densitometer

with Imagequant software (Sunnyvale, CA) as explained in materials and methods.

Figure 12 shows the concentration ofCU/Zn-SOD in different stages ofCL during estrous

cycle. There was a slight increase in concentration of CU/Zn-SOD from 151 stage (72 ± 7.4

ng /10 ng of total protein) to 2nd stage CL (120.8 ± 16.9 ng/IO ng of total protein),but this

height was not significantly different From 2nd stage it remained same and elevated even

41

in the regressed fonn ofeL that is CA (154.5 ± 19.5 ngll0 IJ.8 oftota! protein). There

was no significant difference in the concentrations ofCuIZn-SOD in 2nd stage CL, Early

3'" stage CL, mid 3rd stage CL, late 3'" stage CL, 4th stage CL and CA (P > 0.05). Highest

concentration ofCulZn-SOD was observed in pregnant CL (196 ± 27.7 ngilO ng oftota!

protein).

2.4.9 Activity of Mn-SOD in different stages ofCL during estrous cycle and during

pregnancy.

Figure 13 shows the activity ofMn-SOD in different stages of CL during estrous cycle.

Activity of Mn-SOD increased from 1st stage (1.48 ± 0.43 Activity unitslml of

homogenate) to 2nd stage (3.21 ± 0.69) Activity unitslml of homogenate). Activity

increased from 2nd stage to early 3'" stage (4.21 ± 0.97 Activity unitslml of homogenate)

but this was not statistically significant (P > 0.05). However, there was a significant

increase in activity of Mn-SOD from early 2nd stage to mid 3'" stage and remained

elevated until 4th stage (5.9 ± 0.61 Activity unitslml of homogenate). There was a sudden

decrease in activity of Mn-SOD from 4th stage to CA (1.63 ± 0.14 Activity unitslml of

homogenate) (P < 0.01) .The activity ofMn-SOD was highest in pregnant CL (7.4 ± 0.39

Activity units/~ of homogenate) when compared to CLs of estrous cycle.

42

2.4.10 Activity of CnlZn-SOD in different stages of CL during estrous cycle and

during pregnancy.

Figure 14 shows the activity of CU/Zn-SOD in different stages of CL during estrous

cycle. Activity ofCU/Zn-SOD increased from 1st stage (10.3 ± 0.56 Activity units/ml of

homogenate) to the 2nd stage (13.2 ± 2.4 Activity units/ml of homogenate) but the

increase was not statistically significant (P > 0.05). However there was a significant

increase in the activity of CU/Zn-SOD from 2nd stage CL (13.2 ± 2.4 Activity units/ml of

homogenate) to early 3rd stage CL (21.6 ± 1.28 Activity units/ml of homogenate)

(P < 0.01) and remained elevated up to 4th stage CL (26.21 ± 0.18 Activity units/ml of

homogenate) and dropped slightly in CA (18.8 ± 1.9Activity units/ml of homogenate).

This drop was not statistically significant from the 4th stage (P> 0.05). Activity of

Cu/Zn-SOD was highest in pregnant CL (28.4 ± 0.79 Activity units/ml of homogenate)

when compared to CLs of estrous cycle.

2.4.11 Levels of 8-Isoprostanes in different stages of CL during estrous cycle and

during pregnancy.

8-isoprostanes, a prostaglandin like compounds are the reliable biomarkers of oxidative

stress. Figure 15 shows the levels of 8- lsoprostanes in the CL samples. These levels

were quantified by using an 8-lsoprostane Enzyme Immunoassay kit (catalog no. 516358;

Cayman). The levels were low in 1st (49.6 ± 1.0 pg/mg of protein) and 2nd stage ofCL

(63.5 ± 1.7 pg/mg of protein). The levels increased slightly from 2nd to early 3rd stage

(87.4 ± 0.8 pg/mg of protein) (P > 0.05) and remained stable up to 4th stage (90.6 ± 1.8

pg/mg of protein). There was no significant difference in the levels of 8-Isoprostanes

43

form early 3rd stage upto 4th stage ( P > O.OS).Interestingly, there was a huge increase in

the levels of8-Isoprostanes in CA (249.5 ± 5.5 pglmg of protein) (P < 0.01). The levels

of8-Isoprostanes were lowest in pregnant CL (53.4 ± 1.3 pglmg of protein) when

compared to CLs of estrous cycle.

2.S Discussion:

Studies showed that ROS cause damage to the CL and increase during the regression of

CL (Aten et aI., 1998; Riley and Behrman., 1991a; Sawada and Carlson.1994). Studies

also showed that CL has antioxidants to scavenge the deleterious effects of these ROS (

Rueda et aI., 1995). Among many antioxidants, superoxide dismutases are the first line of

defense against the superoxides. There are two types of SODs, Mn-SOD, which is present

in the mitochondria of the cells, and CuJZn-SOD, which is present in the cytoplasm of the

cells (Fridovich, 1986). Studies in rats, mice and humans showed that the expression of

these two types of antioxidants differ in different stages of CL during luteal phase and

during pregnancy. Sugino et. aI., (1998) showed that mRNA expression of CuJZn-SOD

levels decreased whereas Mn-SOD levels remained elevated in the CL during late

pregnancy in rats. Studies in human CL during the menstrual cycle by Sugino et aI.

(2000) showed that CU/Zn-SOD expression increased from early to mid luteal phase and

decreased thereafter and the expression of mRNA was lowest in regressed form of corpus

luteum, the corpus albicans. However, expression ofMn-SOD mRNAwas low in mid

luteal phase and increased during the regression phase.

Present study in bovine CL shows that these SOD protein levels are differentially

expressed at different stages of CL during estrous cycle. This study shows that protein

44

levels of Mn-SOD increase (10 fold) from 1st stage ofCL up to 4th stage ofCL and

decreases in corpus albicans (CA). In contrast, protein levels ofCu/Zn-SOD remained

elevated during the entire estrous cycle. even in the CA. Changes in the enzyme activity

of both Mn-SOD and CuJZn-SOD correspond with the protein levels of those enzymes.

Present study also shows that 8-lsoprostanes. stable biommers of oxidative stress in vivo

are increased in the CA and are low in other stages ofCL. Taken together. it is suggested

that expression of Mn-SOD may be involved in the mechanisms leading to the regression

of CL. However. the stimulus for down regulation of Mn-SOD in CA remains to be

determined Other possible mechanisms for luteal regression may include decrease in the

levels of steroidogenic cytochrome P450scc or catalase or ascorbate and decrease in

ovarian blood circulation (Rappoport et al .• 1995).

In contrast to the differential expression during bovine estrous cycle in the current study.

the levels of both Mn-SOD and CU/Zn-SOD were high and the levels of 8-Isoprostanes

were low in the bovine pregnant CL. This may suggest that the pregnant CL is very

efficient in counteracting the oxidative stress by superoxide radicals and up regulation of

the levels of both Mn-SOD and CuJZn-SOD may be helpful in this process. Sugino et al.

(2000) reported that human chorionic gonadotrphin (HCG) stimulated the expression of

CuJZn-SOD in vitro in human pregnant corpus luteum. This group also reported that

placentallactogens up regulated the expression of SOD mRNA in rat luteal cells (Sugino

et al.. 1998). In this context, it will be interesting to investigate the hormonal stimulus for

the increase in expression ofMn-SOD during the regression stage of CL and drop in the

Mn-SOD expression in CA. Regulation ofMn-SOD and CU/Zn-SOD in bovine pregnant

45

CL is also not understood. It will be also interesting to examine the levels of both Mn­

SOD and CulZn-SOD in the aborted bovine CL and to see the difference in their

expression, if such CLs are available.

Recent studies in mts by Yune et aI. (2004) showed that the levels of only Mn-SOD but

not CulZn-SOD increased after spinal cord injury, and external administmtion of tumor

necrosis factor-a (INF-a) increased Mn-SOD expression in uninjured spinal cord.

Sugino et aI., (1998) reported that inflammatory cytokines including lNF-a induces the

expression of Mn-SOD, but not CulZn-SOD in pregnant mt CL. These data suggest that

inflammatory cytokines including lNF-a may serve as stimulus for the higher expression

ofMn-SOD in regressing CL.

Studies showed that heat stress increases the influx of free mdica1s (Flanagan et ai, 1998)

into the living cells. Increased genemtion of oxygen-centered free mdica1s cause

oxidative stress, which may mediate at least in part heat-induced cellular damage. High

levels of free mdicals genemted during summer seasons in hot climates might cause

damage to the CL and subsequent luteolysis, which might result in early abortions in case

of pregnant animals or conception failure in case of repeat breeders. Studies also showed

that the activity of Mn-SOD increases in heat shocked cells (llangovan et aI., 2006). In

this perspective, it will be of interest to study the involvement of free mdica1s and SOD in

reproductive performance of dairy cattle in hot climates.

46

Studies showed that apoptosis might be involved in the regulation of corpus luteum

(Riley and Behrman, 1991b). CL regression involves functionalluteolysis and structural

luteolysis. PGF2a has been implicated in the process of apoptosis in the corpus luteum

(Reviewed in Niswender et al., 2000). PGF2a appears to be involved in increased

generation of reactive oxygen species in rat luteal cells (Sawada and Carlson., 1991) an

event that has been linked to both a loss of progesterone biosynthesis (functional

luteolysis) (Musicki et al., 1994) and the induction of apoptosis cell death (Structural

luteolysis) (Tilly and Tilly., 1995).

PGF2a interacts with its G protein-coupled receptor, which is present predominantly on

large luteal cells of the corpus luteum (McCracken et al., 1999) and activates

Gqlphospholipase C or protein kinase C pathway (McGuire et al., 1994), resulting in

decreased progesterone production, which is called as functionalluteolysis.

The intracellular signaling events that lead to structural regression ofluteal tissue are

now well established. Apoptosis or programmed cell death plays a central role in the

structural regression of luteal tissue during PGF2a induced or spontaneous luteolysis of

several species (Yadav et al., 2005).

The first morphological evidence that a cell is apoptotic is the appearance of nuclear

fragments containing degenerate chromatin, cell shrinkage, and appearance of

membrane-bound cytoplasmic fractions (Sawyer et al., 1990). Another characteristic

feature of apoptosis is intemucleosomal cleavage of genomic DNA into 185-bp

47

fragments (oligonuc1eosomes). This characteristic DNA fragmentation is seen as a ladder

pattern on agarose gels (Arends et al., 1990).

Appearance of oligonucleosomes in response to PGF2a. in cattle CL is the evidence for

the role of apoptosis ofluteal regression (Juengel et al., 1993). Involvement of Bcl-2

family genes that regulates apoptosis, studied extensively in the CL regression.

Membrane- associated Bcl-2 prevents cell death by regulating the maintenance ofea2+

homeostatic mechanisms (Baffy et al., 1993), attenuating oxidative stress (Hockenberry

et al., 1993), and interacting with bax (Korsmeyer, 1995). Bax (Bcl-2-associated-gene-x)

promotes apoptosis (Korsmeyer 1992). The ratio ofBcl-2 and Bax within a cell is related

to that cell's potential to become apoptotic. During luteolysis in cattle, mRNA encoding

bax is elevated while mRNA encoding Bcl-2 remains unchanged (Rueda et al., 1997),

resulting in an increased ratio ofbax to Bcl-2. an event consistent with bax-mediated

apoptosis.

Studies by Buttke in 1994 showed that apoptosis is caused by increase in concentrations

of superoxide radicals and decrease in SOD in the cells. Interestingly, studies by

Greenlund et al. (1995) showed that SOD delays apoptosis in neuronal cells. Taken

together, it may be suggested that, SOD is involved in the rescue of CL from apoptosis

and plays an important role in the maintenance of luteal function in CL.

Recent studies by Al-Gubory (2005a), used fibered confocal fluorescence microscopy

(FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep

48

corpus luteum in the living animal. This technology may also be useful in studying the

apoptosis mechanism and simultaneous SOD expression in bovine CL. This study will be

particularly useful in studying the apoptotic DNA fragmentation in bovine CL during the

regression phase and simultaneous measurement of SOD expression.

In conclusion the present study shows that Mn-SOD and CulZn-SOD are di:fIelentially

expressed in the CL and Mn-SOD may play an impoItant role in regulation of CL during

estrous cycle. High levels of expression of both Mn-SOD and CulZn-SOD in pregnant

CL suggest that these two might be involved in the rescue of CL from superoxides during

pxegnancy.

49

II

12 9.52± 1.74

10

8 b b b

6.00 ± 0.28 E 5.30±0.48 4.73 ± 0.88 - 6 OJ c

e 4 e

e 1.64±0.18 e

2 1.08 ± 0.05

1st 2nd 3E 3M 3L 4th CA Pr p So 0.05

StageofCL

Figure 1. Progesterone concentration in serum at different stages of estrous cycle

and during pregnancy.

Blood samples from the slaughterhouse were collected and the sera were separated by

centrifuging at 3,000g for 10 minutes. Progesterone assay kits (Cayman, Ann Arbor, MI)

were used to measure progesterone concentration in serum samples. Values are mean ±

SEM. Results were analyzed by one-way ANOV A and Duncan's multiple range test with

the help of SAS program. Means of different CL stages with different superscripts differ

(P~ 0.05).

50

25 kD

15 kD

S Mn 1st 2nd 3E 3M 3L 4th CA Pr

Figure 2. SDS-PAGE ofCL samples during estrous cycle and during pregnancy.

250 mg of CL tissue was homogenized in 10 ml of PBS buffer and centrifuged at

1,500 g for 10 minutes at 4 °C to separate the tissue debris. Protein concentration of the

supernatant was measured by the Lowry method and 1 0 ~g of protein from each sample

was loaded in to 15% SDS-gel and electrophorosed at 25 rnA current for 45 minutes. The

gel was stained with comassie blue stain. Higher expression of23 kD band, was observed

in late 3'd stage, 4th stage and pregnant CL. However, 15.6 kD band, was not observed in

any of the stages.

51

23 kD

Figure 3. Western blot analysis of binding characteristics of Mn-SOD antibody to

23 kD protein in different stages of CL.

Proteins from SDS-PAGE were transferred on to a PVDF membrane and were

immunoblotted with the commercially available antibodies against Mo-SOD (Research

Diagnostic Incorporation). Increased expression of Mo-SOD (23 kD) from 151 stage up to

4th stage CL and sudden decrease in Corpus Albicans (CA) was noticed. Mo-SOD was

highly expressed in pregnant CL.

In second lane Mn-SOD represents the purified Mn-SOD protein whose band volume

will be used as a reference for quantifYing Mn-SOD protein concentration in CL samples.

52

Mn-SOD in Different Stages of CL

3M 3L CA PrCL

Index Name Volume Adj. Vol. 0Ilu"mm2

1 Mr>-SOO Standard 27.13491507 10.29842947 2 loin-1st stage 12.22471798 0.03807&188 3 Mn-2nd stage 8.847484992 1.574794540 4 Mn-Ea~y 3rd stage 10.16311871 7.054463639 5 Mn-Mid 3rd stage 8.320729095 5.583885898 6 Mn-Late 3rd " .595n139 9.430S40188 7 Mn-4th stage 18.13881293 13.97312273 8 Mn-CorpusAibicans 4.698095.225 2.5328&1022 9 Mn-Pnlgnant CL 21.81eaage8 19.18092789

Measured by Bio-Rad Quantity-One software

Figure 4. Densitometry analysis of Mn-SOD bands

Bio-Rad Quantity one software was used to measure the band volume of the Mn-SOD on

western blot. I st lane represents the pure Mn-SOD and remaining lanes represent the

different stages of CL.Volume represents the volume in the box, where as Adj .Vol.

represents only the volume of the band inside the box, after subtracting the background.

53

Mn-SOD Standard Curve

200ng 100 ng 50ng 25ng

'1 p t

ng of Mn-SOD Log Con. Adjusted Band Volume Units

200 2.3 18.4

100 2.0 12.0

50 1.7 7.9

25 1.4 2.9

Figure 5. Generation of Mn-SOD Standard Curve

Different concentrations of extracted Mn-SOD (200 ng, 100 ng, 50 ng and 25 ng were

taken in a western blot to get Mn-SOD standard curve.

Con. represents the concentration of extracted Mn-SOD in nanograms, taken in each lane.

Log can. represents the log value of Mn-SOD concentration.

Adjusted Band Volume represents the volume of only band after subtracting the

background in the box.

54

Mn-SOD standard curve

20.0

1 18.0

16.0 0 0 14.0 en .: 12.0 ::; ~ 10.0 0 ~ ~ 8.0 .. > 0 6.0 0

4° L 2.0

0.0 1.0 1.2 1.4 1.6 1.8

Log concentration

Figure 6. Mn-SOD Standard curve

y = 16.753x - 20.667 R' = 0.9922

•

2.0 2.2 2.4

Mn-SOD standard curve was obtained by taking the log value of Mn-SOD

concentration taken in the western blot on X-axis and 00 value of band

volume on Y-axis.

55

300

250

c: 'a:; -e 200 0.

ro -0 - 150 '0 Cl ::J

0 ...... 100 --Cl c:

50 18.5 ± 0.4

1st P S 0.01

C

152.1 ± 12.0

55.7 ± 6.2

27.5 ± 1.7

he

179.4 ± 21.6

220.5 ± 22.3

rl 23.5 ± 0.8

249.8 ± 24.4

2nd 3 Early 3 Middle 3 Late 4th CA Pregnant

Stage ofCL

Figure 7. Levels of Mn-SOD in different stages of CL during estrous cycle and

during pregnancy.

Mn-SOD concentration in each stage of the CL was calculated by converting band

volume into protein concentration based on the Mn-SOD standard curve equation.

All values are expressed in means ± SEM, n = Sample size. Results were analyzed by

one-way ANOV A and Duncan's multiple range test with the help of SAS program.

Means of different CL samples with different superscripts differ (P 5. 0.01).

56

15

Figure 8. Western blot analysis of binding characteristics of Cu/Zn-SOD antibody to

15.6 kD protein in different stages of CL.

Proteins from SDS-PAGE were transferred on to a PYDF membrane and were

irnmunoblotted with the commercially available antibodies against CU/Zn-SOD

(Research Diagnostic Incorporation). Higher expression of CU/Zn-SOD can be noticed in

CA. In second lane CU/Zn-SOD represents the purified CU/Zn-SOD protein whose band

volume will be used as a reference for quantifying CU/Zn-SOD protein concentration in

CL samples.

57

15

Irodeoo Nwnt v"""" At! Vd OIJu"rnm2 OOU'mm2

1 C.an-soo_ 2D I!Ol57649 2.051754184 2 CulZn-lt1 .. 22.1IlIl86451 4.195OeOO3O 3 W2n-2rod thge 26.52065716 7.1116I1l907 4

Cu/Zno&1y 3nS_ 24.37784897 e 252'588300

5 c.th1 Mid 3nS_ 2752447&47 7.58)g662l)2 6 CufZn.loIe 3nS ... 26 257Il53B3 8.~ 7

CU'Zn-<ItI_ 26 4307D)1 77S6838553

8 C\>'lJ>.Corpuo --

36.54625805 7730434187

• ~Cl 4177506522 8._'5786

Measured by Bio-Rad Quantity-One software

Figure 9. Densitometry analysis of Cu/Zn-SOD bands

Bio-Rad Quantity one software was used to measure the band volume of the Cu/Zn-SOD

on western blot. I " lane represents the pure CU/Zn-SOD and remaining lanes represent the

different stages of CL.

Volume represents the volume in the box, where as Adj .Vol. represents only the volume

of the band inside the box, after subtracti ng the background.

58

.... ,,,"u/Zn-SOD Standard Curve

100 ng 50 ng 25 ng

15 kD 1- 11 - 1 1

ng of Cu/Zn-SOD Log Con. Band Densitometrv Units

200 2.3 6.023

100 2 4.07

50 1.7 2.29

25 1.4 0.91

Figure 10. Generation of C u/Zn-SOD Standard C urve

Different concentrations of extracted CU/Zn-SOD (200 ng, 100 ng, 50 ng and 25 ng were

taken in a western blot to get CU/Zn-SOD standard curve.con. represents the

concentration of extracted CU/Zn-SOD in nanograms, taken in each lane. Log con.

represents the log value of CU/Zn-SOD concentration. Adjusted Band Volume represents

the volume of only band after subtracting the background in the box.

59

r

C 1.4 o til 1.2 ~ 1 "3 () 0.8

'0 0.6

" .2 0.4 ~

~ 0.2

o 0 + 2 2.2

Cu/Zn·SOD Standard curve y = O.9600x· 1.9731

~ = 0.9986

2.4 2.6 2.8 3 3.2 3.4

Log Concentration

Figure 11. Cu/Zn-SOD Standard curve

CulZn-SOD standard curve was obtained by taking the log value of CulZn-SOD

concentration taken in the western blot on X-axis and 00 value of band volume on Y-

axis.

60

250

200 c .Qi 15 ~

a. ro 150 15 ..... '0 OJ :::J 100

0 ...... -. OJ c

50

Hh 120.8 ± 16.9

72 ± 7.4

~h

121.6 ± 7

h

h 156.9 ± 17.3

~h 145.2 ± 16.3

133.9 ± 15.6

1st 2nd 3 Early 3 Middle 3 Late 4th

p ~ 0.01 Stage of CL

h 196 ± 27.7

h

--154.5 ± 19.5

CA Pregnant

Figure 12. Levels of Cu/Zn-SOD in different stages of CL during estrous cycle and

during pregnancy.

CU/Zn-SOD concentration in each stage of the CL was calculated by converting band

volume into protein concentration based on the CU/Zn-SOD standard curve equation.

All values are expressed in means ± SEM, n = sample size. Results were analyzed by

one-way ANOY A and Duncan's multiple range test with the help of SAS program.

Means of different CL samples with different superscripts differ (P :::: 0.01).

61

10

a

ah a b a b 7.4 ± 0.39 8

6.4 ± 0.52 5.7 ± 0.67 5.9 ± 0.61

E -- 6

he en ~

4.21 ± 0.97 : c :l de ~ '5

4 :0:> 3.21 ± 0.69

U « d 1.63 ± 0.14 ,

l.4B ± 0.43

2 d

o n=5 n=5 n=5 n=5 n= n=5 n=5 naS , , , , 1st 2nd 3E 3M 3L 4th CA Pr

P .$. 0.01 StageofCL

Figure 13. Activity of Mn-SOD in different stages of CL during estrous cycle and

during pregnancy.

Mn-SOD activity in each stage of the CL was calculated by using commercially available

activity assay kit from Cayman. All values are expressed in means ± SEM, n = sample

size. Results were analyzed by one-way ANOV A and Duncan's multiple range test with

the help of SAS program. Means of different CL samples with different superscripts

differ (P ::: 0.01). One activity unit is defined as the amount of SOD required to exhibit

the di smutation of 50% of superoxide radicals.

62

35 "

~b ab 28.4 ± 0.79

30 :\b 24.5 ± 3.0 26.21 ± 0.18 ab

22.6 ±2.5 ;~

25 r-=-

21.6 ± 1.28 bc k';:!

E 18.8 ± 1.9

--CJ) 20 -'c cd ::::> >-- 15 '5

d 13.2 ± 2.4

.. :;::::;

;J. 10.3 ± 0.56 .;

'e' I · .<

10 .-

5

0=5 0=5 n=5 0=5 n=S n=S naS ~S 0

1 st 2nd 3E 3M 3L 4th CA Pr p ~ 0.01

Stage of CL

Figure 14. Activity of Cu/Zn-SOD in different stages of CL during estrous cycle and during pregnancy.

CulZD-SOD activity in each stage of the CL was calculated by using commercially

available activity assay kit from Cayman. All values are expressed in means ± SEM, n =

sample size. Results were analyzed by one-way ANOVA and Duncan's multiple range

test with the help of SAS program. Means of different CL samples with different

superscripts di ffer (P :::: 0.0 I). One activi ty unit is defined as the amount of SOD required

to exhibit the dismutation of 50% of superoxide radicals.

63

300 249.5 ± 5.5

a

250

c: Q) .... e 200 a. -0 OJ E 150 --<f)

E ro .... OJ 100 0 0 0..

95.1 ± 1.8 90.6 ± 1.8

87.4 ± 0.8 90.3 ± 2.9

b be be 63.5 ± 1.7 be 53.4 ± 1.3

49.6 ± 1.0 r-c-=-- -=- r=-be

c be ~

50 r- r: n=5 n=5 n=5 n=5 n=5 n=5 n- S n-S

o P .'O 0.01 1st 2nd 3E 3M 3L 4th CA Pr

Stage ofCL

Figure 15. Levels of 8-isoprostanes in different stages of CL during estrous cycle and during pregnancy

8-isoprostanes levels in each stage of the CL were measured by using 8-Isoprostane

Enzyme Immunoassay kit (Cayman Chemical). All values are expressed in means ±

SEM, n = sample size. Results were analyzed by one-way ANOV A and Duncan's

mUltiple range test with the help of SAS program. Means of different CL samples with

different superscripts differ (P ~ 0.0 I).

64

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78

Appendix

Table 1. Progesterone Concentration in serum at different stages of estrous cycle and during pregnancy.

istalle of CL Progesterone con. (ng/mll ~. Error

~st 1.08' 0.08

~nd 1.64 • 0.18

~ Early 5.30b

0.49

~ Middle 6.00b

0.29

~Late 4.73 b 0.88

~th 2.41' 0.20

CA 1.09' 0.09

Pregnant 9.52" 1.74

P<O.Ol

Table 2. Levels of Mn-SOD in different stages of CL during estrous cycle and during pregnancy.

Stage of CL nil of Mn-80D Std Error

~st 18.53 d 0.37

~nd 27.57 d 1.74

~ Early 55.71 d 8.19

~ Middle 152.15 • 12.07

3 Late 179.38 b. 21.59

4th 220.55" b 22.33

CA 23.53 d 0.83

Pregnant 249.83 " 24.42

P<O.Ol

79

Table 3. Levels of CulZn-SOD in ditTerent stages of CL during estrous cycle and during pregnancy.

StageofCL na of CuJZn-SOD Std Error

at 71.97 b 7.38

~d 120.818 b 16.89

~ Early 121.668 b 7.03

~ Middle 133.888 b 16.66

,Late 145.20 8 16.26

*" 166.938

17.30

CA 164.498

19.63

Pregnant 196.998

27.73 P < 0.01

Table 4. Activities of Mn-SOD in different stages of CL during estrous cycle and during pregnancy.

ismae ofCL Mn-SOD (Activity of unlts/mn Std. Error

~at 1.49 d 0.44

2nd 3.21 d. 0.69

a Early 4.22 b. 0.97

, Middle 6.418b

0.63

3 Late 6.788b

0.70

4th 6.988b

0.62

CA 1.63 d 0.16

Pregnant 7.448

0.39

P <0.01

80

Table 5. Activities of CuIZn-SOD in different stages of CL during estrous eycle and during pregnaney.

StageofCl CuIZn-50D (Activity units/mil Std.Error

at 10.33 d 0.57

2nd 13.23 d. 2.41

3 Early 21.61 ab 1.29

3 Middle 22.70ab

2.57

3Late 24.51 ab 3.08

4th 26.21 ab 0.18

CA 18.84 be 1.96

!pregnant 28.47a

0.79 P<O.OI

Table 6. Levels of 8-Isoprostanes in different stages of CL during estrous eycle and during pregnaney.

StageofCl 8-lsoprostanes (pg/mg of protein) Std. Error

~at 49.66' 1.04

~nd 63.50 be 1.72

~ Early 87.39 be 0.81

I Middle 95.09b

1.85

~Lste 90.26 be 2.93

4th 90.58 b. 1.90

CA 249.51 a 6.55

Pregnant 63.45 be 1.36

P <0,01

81

Tissue culture Supernatant SOS-PAGE

S 1 2 3 4 5 6

S =STANDARD 1 =MID 3RD CONTROll #6 0 hrs 2 =MID 3RD CONTROll #617 hrs 3 =MID 3RD + 50 uls of lutalyse a hrs 4 =MID 3RD + 50 uls of lutalyse 17 hrs 5 =2 M Pregnant Cl control a hrs 6 =2 M Pregnant Cl control 17 hrs 7 =2 M Pregnant Cl + 50 uls lut a hrs

7

8 =2 M Preganant Cl + 50 uls lut 17 hrs

8

1---- Mn-Sod

CL samples from slaughter house were collected and cut into small slices. The CL slices were placed in a test tube containing phosphate buffered saline ( PBS). The control was only CL slices and PBS where as treatment group received different concentrations of lutalyse (pGF2a).

82

Raw data for levels of Progesterone in serum at different stages of estrous cycle and during pregnancy

Name Stage nalml 1st staae 1 0.9673 1st stage 1 1.1463 1st staae 1 0.9417 1st stage 1 1.2568 1st stage 1 1.0949 2nd staae 2 1.5044 2nd stage 2 1.7083 2nd staae 2 1.7338 2nd stage 2 1.0712 2nd staae 2 2.1906 Early 3rc! stage :l 4.1695 Early 3rc! staae 3 5.8179 Early 3rc! stage 3 4.1807 Early 3rc! staae 3 6.6276 Early 3rc! stege 3 5.7176 Mid 3rc! steae 4 5.3525 Mid 3rc! stege 4 7.0291 Mid 3rc! steae 4 5.6867 Mid 3rc! steae 4 6.1717 Mid 3rc! stage 4 5.7689 Lete 3rc! steae 5 2.3269 Late 3rc! stage 5 2.9574 Lete 3rc! stege 5 6.1971 Lete 3rc! stage 5 6.6965 Lete 3rc! stage 5 5.4961 14th stage 6 3.0536 4th stage 6 1.9935 4th staae 6 2.0347 4th stage 6 2.2744 4th staae 6 2.6822 Corpus Alblcans 7 1.0763 Comus Albicans 7 0.9216 Corpus Alblcans 7 1.0969 Comus Alblcans 7 0.9171 Corpus Alblcans 7 1.4174 Pregnant 8 11.3652

83

Pregnant 8 9.4533 Pregnant 8 8.070 Pregnant 8 6.6194 Pregnant 8 12.0876

Raw data for levels of Mn-SOD in different stages of CL during estrous cycle and during pregnancy

Name Stage ~tllog (ng of Mn-80D) Mn-1st staae 1 17.41 Mn-1 st stage 1 18.95 Mn-1 st stage 1 18.21 Mn-1 st stage 1 18.47 Mn-1st stage 1 19.63 Mn-2nd staae 2 22.02 Mn- 2nd stage 2 27.02 Mn-2nd staae 2 36.85 Mn-2nd stage 2 29.84 Mn-2nd staae 2 31.02 Mn-2nd stage 2 33.80 Mn-2nd stage 2 22.64 Mn-2nd stage ~ 23.49 Mn-2nd stage 2 28.98 Mn-2nd staae 2 20.05 Mn-Earlv 3rd stage 3 52.43 Mn-Earlv 3rd staae 3 45.87 Mn-Earlv 3rd stage 3 58.98 Mn-Earlv 3rd staae 3 70.00 Mn-Earlv 3rd Wlge 3 121.82 Mn-Earlv 3rd staae 3 35.80 Mn-Earlv 3rd s~ge 3 48.14 Mn-Earlv 3rd stage 3 39.04 Mn-Earlv 3rd staae 3 74.32 Mn-Earlv 3rd stage 3 40.79 Mn-Earlv 3rd staae 3 33.39 Mn-Earlv 3rd staae 3 71.23 Mn-Earlv 3rd stage 3 46.03 Mn-Earlv 3rd staae 3 42.30 Mn-Mid 3rd stage 4 82.94 Mn-Mid 3rd staae 4 210.05 Mn-Mld 3rd stage 4 208.39 Mn-Mld 3rd staae 4 134.60 Mn-Mid 3rd stage 4 176.27 Mn-Mid 3rd staae 4 175.84 Mn-Mld 3rd stage 4 187.98 Mn-Mld 3rd staae 4 138.48 Mn-Mid 3rd stage 4 79.06

84

Mn-Mld 3rd stage 4 107.50 Mn-Mld 3rd stage 4 141.30 Mn-Mld 3rd stage 4 114.18 Mn-Late 3rd stage 5 103.16 Mn-Late 3rd stage 5 121.27 Mn-Late 3rd stage 5 258.31 Mn-Late 3rd stage 5 195.87 Mn-Late 3rd staQe 5 284.51 Mn-Late 3rd stage 5 164.72 Mn-Late 3rd stage 5 127.82 Mn-Late 3rd staQe 5 150.39 Mn-Late 3rd stage 5 115.75 Mn-4th stage 6 159.93 Mn-4th stage 6 314.55 Mn- 4th stage 6 222.15 Mn-4th staQe 6 350.85 Mn-4th stage 6 119.84 Mn-4th staQe 6 142.69 Mn-4th stage 6 190.95 Mn-4th stage 6 263.45 Mn-4th stage 6 218.39 Mn-4th stage 6 142.55 Mn-Comus Alblcans 7 25.69 Mn-Corpus Albicans 7 21.65 Mn-Corpus Alblcans 7 22.64 Mn-Corpus Albicans 7 19.47 Mn-Corpus Albicans 7 25.14 Mn-Corpus Albicans 7 23.19 Mn-Corpus Albicans 7 20.87 Mn-Corpus Albicans 7 28.74 Mn-Corpus Albicans 7 23.96 Mn-Corpus Albicans 7 23.92 Mn-pregnant 8 363.17 Mn-Preanant 8 167.62 Mn-4months 8 202.40 Mn-5 Months 8 366.28 Mn-5 Months 8 154.73 Mn-Preanant 8 244.79 Mn-Pregnant 8 191.55 Mn-Pregnant 8 283.13 Mn-Pregnant 8 252.24 Mn-Pregnant 8 174.98

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Raw data for levels of CulZn-SOD in different stages of CL during estrous cycle and during pregnancy

Name Stage Antilog (ng of CuJZn-SODI CuJZn-1 sl staae 1 78.5S CuJZn-1s1 stage 1 79.86 CuJZn-1s1 stage 1 91.67 CuJZn-1s1 stage 1 52.54 CuJZn-1s1 stage 1 57.24 CuJZn-2nd stage 2 157.88 CuJZn-2nd stage 2 115.06 CuJZn-2nd staae 2 101.90 CuJZn-2nd stage 2 111.04 CuJZn-2nd stage 2 61.66 CuJZn 2nd stage 2 177.33 CuJZn-Early 3rd staae 3 121.88 CuJZn Early 3rd stage 3 111.06 CuJZn Early 3rd stage 3 136.69 CuJZn Early 3rd stage 3 121.48 CuJZn Early 3rd stage 3 95.58 CuJZn-Early 3rd stage 3 143.19 CuJZn Mid 3rd stage 4 182.25 CuJZn-Mld 3rd staae 4 124.68 CuJZn-Mld 3rd stage 4 70.36 CuJZn-Mld 3rd staae 4 168.84 CuJZn-Mld 3rd stage 4 103.14 CuJZn-Mld 3rd staae 4 109.76 CuJZn-Mld 3rd stage 4 197.11 CuJZn-Mld 3rd stage 4 114.92 CuJZn-Late 3rd staae 5 212.16 CuJZn-Lale 3rd stage 5 111.93 CuJZn-Lale 3rd staae 5 100.51 CuJZn-Lale 3rd stage 5 162.73 CuJZn-Late 3rd stage 5 139.44 CuJZn-Lale 3rd stage 5 144.45 CuJZn-4th stage 6 193.63 CuJZn-4th stage 6 122.10 CuJZn-4th stage 6 96.69 CuJZn-4th stage 6 148.32 CuJZn-4th stage 6 203.68 CuJZn 4th stage CL 6 179.18 CuJZn-Corpus A1bicans 7 190.25 CuJZn-Corpus A1bicans 7 122.32

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CuJZn-Corpus A1blcans 7 117.63 CuJZn-Corpus A1blcans 7 195.72 CuJZn-COf"DUS Alblcans 7 205.47 CuJZn-Corpus A1blcans 7 95.54 CuJZn-Pl"9!lnant CL 8 236.63 CuJZn-Pregnant CL 8 102.72 CuJZn-Pregnant CL 8 133.50 CuJZn-Preanant CL 8 261.14 CuJZn-Pregnant CL 8 261.72 CuJZn-Pregnant CL 8 180.24

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Raw data for activities ofMn-SOD in different stages of CL during estrous cycle and during pregnancy

Name Staae .. of unltslul

Mn-1st stage 1 1.8919 Mn-1st stage 1 2.4370 Mn-1ststaae 1 2.2343 Mn-1 st stage 1 0.3797 Mn-1st stage 1 0.4848 Mn-2nd stage 2 4.8439 Mn-2nd staae 2 4.1148 Mn-2nd stage 2 3.8382 Mn-2nd staae 2 1.0617 Mn-2nd stage 2 2.2128 Mn-Early 3rd staae 3 1.8547 Mn-Early 3rd stage 3 5.6843 Mn-Early 3rd stage 3 5.1047 Mn-Earlv 3rd staae 3 6.5043 Mn-Early 3rd stage 3 1.9298 Mn-Mld 3rd stalle 4 4.9461 Mn-Mld 3rd stage 4 7.0610 Mn-Mld 3rd stalle 4 7.9838 Mn-Mld 3rd stage 4 6.3567 Mn-Mld 3rd stalle 4 5.6843 Mn-Late 3rd stage 5 6.2138 Mn-Late 3rd stalle 5 5.1047 Mn-Late 3rd stage 5 7.5945 Mn-Late 3rd stage 5 6.5043 Mn-Late 3rd stage 5 3.4798 Mn-4lh stage 6 6.3567 Mn-4lh stalle 6 4.8946 Mn-4lh stage 6 7.5945 Mn-4lh stalle 6 6.8144 Mn-4lh stage 6 4.2406 Mn-CoJ'Pus Albicans 7 1.5416 Mn-Corpus Albicans 7 1.2335 Mn-CoJ'Pus Albicans 7 1.7404 Mn-CoJ'Pus Albicans 7 1.5201 Mn-CoJ'Pus Albicans 7 2.1234 Mn-Prellnant 8 6.9774 Mn-Pregnant 8 7.3207 Mn-PrB!!nant 8 8.4035

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Mn-P nant 8.189~ 6.284

Raw data for activity of CulZn-SOD in different stages of CL during estrous cycle and during pregnancy

Name Stalle unitslml ( Cu) Cu/Zn-1st staae 1 9.33128536 Cu/Zn-1st stage 1 9.842404162 Cu/Zn-1st staae 1 12.48384783 Cu/Zn-1 st stage 1 9.53247042 Cu/Zn-1 st staae 1 10.49343817 Cu/Zn-2nd stage 2 13.16144144 Cu/Zn-2nd staae 2 6.605318418 Cu/Zn-2nd stage 2 15.37242584 Cu/Zn-2nd stage 2 20.87044203 Cu/Zn-2nd stage 2 10.1625555 Cu/Zn-EarJv 3rd staae 3 18.27388331 Cu/Zn-Earlv 3rd stage 3 23.08502766 Cu/Zn-EarJv 3rd staae 3 22.79074713 Cu/Zn-EarJv 3rd stage 3 18.93707363 Cu/Zn-EarJv 3rd staae 3 24.96955485 Cu/Zn-Mld 3rd stage 4 16.84737273 Cu/Zn-Mld 3rd stage 4 22.79074713 Cu/Zn-Mld 3rd stage 4 29.9052213 Cu/Zn-Mld 3rd stage 4 26.71448743 Cu/Zn-Mld 3rd stage 4 17.43616921 Cu/Zn-Late 3rd stage 5 28.84505114 Cu/Zn-Late 3rd staae 5 20.11546966 Cu/Zn-Late 3rd stage 5 31.25131215 Cu/Zn-Late 3rd staae 5 27.84869361 Cu/Zn-Late 3rd stage 5 14.69803278 Cu/Zn-4th stage 6 25.99578252 Cu/Zn-4th staae 6 26.35154145 Cu/Zn-4th stage 6 25.84699926 Cu/Zn-4th staae 6 26.71448743 Cu/Zn-4th stage 6 26.35154145 Cu/Zn-COrDUS A1blcans 7 26.03682563 Cu/Zn-Corpus A1bicans 7 16.95199222 Cu/Zn-COrDUS A1bicans 7 19.47598408 Cu/Zn-Corpus A1bicans 7 17.13437713 Cu/Zn-COrDUS Alblcans 7 14.57841955 Cu/Zn-Preanant CL 8 30.3439472 Cu/Zn-Pregnant CL 8 28.24268102 Cu/Zn-Preanant CL 8 29.47603291

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nantCL 28.64505114 nantCL 25.64699926

Raw data for levels of 8-Isoprostanes in different stages of CL during estrous cycle and during pregnancy

Name Stage lsoprostanes (pg/mll 1st staae 1 12.08809788 1st stage 1 14.37653506 1st stage 1 15.17326217 1st staae 1 9.752098217 1st stage 1 10.68257906 2nd staae 2 13.38613151 2nd stage 2 18.13161555 2nd stage 2 20.99382246 2nd staae 2 11.18687515 2nd stage 2 15.67714313 Earlv 3rd staae 3 21.63266204 Early 3rd stage 3 21.7107385 Earlv 3rd staae 3 21.08297502 Early 3rd stage 3 24.85497164 Early 3rd staae 3 19.95892314 Mid 3rd stage 4 24.35749029 Mid 3rd staae 4 19.23896851 Mid 3rd stage 4 19.85052726 Mid 3rd stage 4 26.95260303 Mid 3rd stage 4 28.46826278 Late 3rd staae 5 24.2051364 Late 3rd stage 5 19.17455487 Late 3rd stage 5 31.57403277 Late 3rd staae 5 23.95507089 Late 3rd stage 5 13.92088679 4th staae 6 23.74300028 14th stage 6 22.76081188 14th staae 6 21.67540793 14th stage 6 28.42646897 14th staae 6 16.61934403 Corpus Albicans 7 60.68799512 Corpus A1bicans 7 52.41860206 Corpus A1bicans 7 82.13880298 Corpus Albicans 7 65.06379807 Corpus Alblcans 7 51.58118618 Pregnant 8 14.53732416 Preanant 8 13.99304849 Pregnant 8 10.48033877

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17.540888751 10.26044483

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