Phosphate metabolism during oogenesis in Rana temporaria,

PHOSPHATE METABOLISM DURING OOGENESIS I N RANA TEMPORARIA z,

PHILIP GRANT Department of Zoology, Columbia University, New Pork City

SEVEN FIGURES

The maturation of the Rana temporaria oocyte extends over a period of three years in the adult and is characterized by two significant phases which reflect the major contributions of the female gamete: (1) a nuclear maturation phenomenon which provides for the genetic contribution of the gamete and future regulation of morphogenesis and (2) a cytoplasmic phenomenon concerned with the elaboration of a “develop- mental matrix” through which the embryo derives its nutri- ent requirements. This latter phase is marked by a period of intense synthesis of yolk, glycogen and lipids and occurs during the third year of maturation (Bleibtreu, ’10 ; Terroine and Barthelemy, ’21).

The pattern of morphogenesis is determined, in part, by the nature of the matrix established during this period. The organization of the matrix influences the future polarity and symmetry of the egg and embryo (Wittek, ’52). It may also influence the distribution of yolk (Sze, unpublished). The pattern of yolk distribution determines the rate and character of early cleavage and affects future morphogenetic movements. Fur- thermore the various constituents of the mature egg provide the mechanisms and materials for future growth and differ-

% Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Faculty of Pure Science, Columbia University, New York City.

This investigation was supported in par t by a research grant from the Division of Research Grants of the National Institutes of Health, Public Health Service, administered by Dr. L. G. Barth.

Fulbright Fellow, 1950-51, at Laboratoire Morphologie Animale, Brussels.

513

514 PHILIP GRANT

entiation. From the accumulated store of metabolites, the developing embryo derives its energy sources.

It has been demonstrated in recent years, that phosphate compounds, known to be involved in energy transformations in animal and plant tissues, participate in the energy metabolism of amphibian morphogenesis (Barth and Jaeger, ’47). Their role in differentiation has been suggested by Barth and Jaeger ( ’50) in studies on a transphosphorylation system in the developing embryo. Kutsky ( ’50), using radioactive phosphorus, has indicated significant changes in P32 distribution associated with gastrulation and neurulation. Brachet ( ’47a and b) has emphasized the possibility of nucleic acids and nucleotides as being the natural organizer. It is suggested that the participation of these biologically important compounds in morphogenesis and differentiation is determined, in part, by their distribution in the “morphogenetic sub- stratum” of the mature egg.

I t is of interest, therefore, to study the manner whereby these substances become incorporated into the organization of the egg. This report results from a series of experiments designed to describe the development of phosphate compounds during oogenesis ; furthermore, P32, tracer methodology has been applied in an attempt to correlate changes in phosphate metabolism with the distinct phases of yolk synthesis and oocyte growth.

MATERIALS AND METHODS

A. Biological

The ovaries of adult frogs of the species Rana temporaria were used in all experiments. These frogs were obtained from Baal, Belgium, during the year 1950-1951. The frogs were weighed and then injected with 0.1ml of a solution of P32 (in the form of H,PO,), the activity of which had previously been determined on a standard solution. The amount injected varied from frog to frog and was approximately equivalent to 0.1 millicurie. The material was introduced intraperito- neally and the frogs were kept at room temperature for 24

P H O S P H A T E M E T A B O L I S M IN OOGENESIS 515

hours at which time they were pithed and the body cavity exposed. The ovaries were removed and placed in ice cold 0.9% NaC1. I n several experiments, blood was drawn from the heart for the determination of plasma inorganic P.

The separation of the oocytes was begun immediately. The ovary was cut into small portions and placed in a Syracuse dish containing ice cold 0.9% NaC1. This was then placed in an ice bath under the binocular microscope and the oocytes were separated from their membranes with watchmaker’s forceps.

The isolation and separation of the oocyte classes generally extended over one to two days since it was necessary to obtain a sufficient number of small oocytes for analysis. The question of the continued viability of the oocytes during the separation procedure arises. The criterion of viability em- ployed was the property of protoplasmic transparency. The smallest oocytes, lacking in pigment or yolk, were transparent when examined in a freshly excised ovary. Throughout the separation, cellular transparency was maintained and it was assumed that the oocytes were still viable when analyzed.

The following table lists the classes of oocytes analyzed with their characteristics :

CHARACTEBJSTICS CLASS SIZE RANQE SYMBOL DIAMETER

mm A 0.05-0.25 Transparent, no yolk, found in small clusters

on ovary membranes.

B 0.05-0.40 A mixed population containing oocytes from class A above and larger oocytes containing small amounts of yolk.

C 0.50-0.90 Large amounts of yolk. Some pigment present.

D1 0.90-1.00 More pigment present.

E 0.90-1.50 Mature oocytes.

One experiment only.

516 PHILIP GRART

Experiments performed from May through July, 1951, involved classes A, B, and C, depending on the conditions of the experiment. One experiment was completed in late summer, involving groups A, B, and D. Experiments on classes B and E were carried out during the period October to Janu- ary, 1951-1952.

After separation into classes, the oocytes were counted and placed in small centrifuge tubes (vol. 3 ml). The number of oocytes analyzed varied with the size of the oocyte and with the experiment.

R. Chemical The application of standard methods for the extraction of

phosphate compounds to tracer studies of phosphate metabolism has led to problems of fraction purity and contamination. This has been particularly important in turnover studies of nucleic acids and phosphoproteins since contamination of these relatively inactive compounds by highly active phosphate fractions, such as inorganic phosphate, leads to incorrect activity values (Jeener, ’49; Davidson et al., ’51). Similarly, contamination interferes with determinations of the more active acid soluble components (Kamen and Spiegelman, ’48 ; Ennor and Rosenberg, ’52). It becomes evident, therefore, that within the reference framework of tracer studies, caution in the interpretation of one’s results is of paramount importance, unless appropriate purification procedures are adopted for the various phosphate compounds.

A modified Schneider technique (’45) has been used in these experiments. An outline of the procedure is shown in figure 1. The extraction was carried out in a 4°C. cold room and all reagents and vessels were chilled in ice water baths. Duplicate analyses were carried out for each oocyte class. The fractions isolated and analyzed were the following:

Total P Total acid soluble P ‘ ‘ Phospholipid P ’ ’

“Total nucleic acid P’’ ‘ ‘ Phosphoprotein P ”

Since the oocytes are generally rich in protein, 75% TCA was used in the initial extraction to insure complete protein precipitation (Barth and Jaeger, ’47).

2 x 0.5 mllO% TCA washes. Chill 10 mins. before centrif uging 9000 g 5 min.

-

' Add 0.2ml ice cold 7576 TCA. Homogenize. Chill 10 mins. Add 1.5 ml ice cold H,O. Continue honiogeniza- tion. Chill 4 hr. Remove 0.2 ml aliquot (Total P). Centri- fuge approx. 9OOOg for 5 mins.

1.0ml 95% ethyl ale. 4 hr. room temp. Centrifuge - 8000 g

3 X 0.5 ml ale-ether ( 3 : 1) boil 3 mins. Centrifuge

Fig. 1 Flow diagram of extraction procedure.

Key: R, = residues; WlnndZ = TCA washes: S = acid soluble supernatant: Ll-3 = lipid supernatants; Nl-3 = nucleie acid supernatants; = protein supernatants; PCA = 70% pcrchlorie acid.

0.5 ml 5% TCA. Centrifuge 8000 g 10 mins.

Evaporate alcohol. Re-extract 3 X 1.0 ml petroleum ether; boil 30 see. Centrifuge

1.0 ml 5% I TCA 15 mins. a t 90°C. Centrifuge

0.5 ml 5% TCA ~

Ce itrif uge

R,---N,

N,-

Residue (Phospholipid P ) (Discard) PCA digestion

followed by magnesia mixture precipitation

Combine W,, N,, N, (Nuclei0 Acid P )

518 PHILIP GRANT

Aliquots of the various extracts, or the total extract, were digested in 10ml Kjeldahl flasks to which 0.25ml of 70% perchloric acid was added. After digestion, the flasks were rinsed with two washings of distilled water and a washing of magnesia mixture. The washings were combined and phosphate precipitated as the magnesium ammonium complex f o r 24 hours in the cold.

To insure equivalent geometries for counting, 1 m l of a kaolin suspension (20 mg/ml) was added to each tube which was then centrifuged. The precipitates were filtered through a stainless steel filtering device (Tracer Lab) on discs of Whatman no. 2 filter paper. The precipitate was washed several times with 3% ammonium hydroxide solution and dried under suction. The precipitate was transferred to a brass holder and counted.

Counting was carried out on a Tracer Lab 100 Scaler and on a Nucleonic Corp. of America RC2 Scaler with a Geiger- Muller tube of 2-3 mg/cm2 window thickness. Appropriate conversion factors were obtained for the tu7o counters by counting identical uranium acetate standards.

The counting periods for each sample varied; however, a sufficient number of counts were obtained to maintain the counting error at 1-2”/, . Coincidence corrections were applied to all counts over 500 per minute.

The instrument was checked daily against a uranium acetate standard prepared by evaporating a known quantity of uranium acetate solution on a brass holder. All counts were corrected to the day of injection using appropriate decay factors.

After counting, each sample with its filter paper was placed in a 15ml centrifuge tube containing 2ml of 1 N sulphuric acid and mixed thoroughly. The tube was allowed to stand at room temperature for one hour during which time it was shaken frequently. The tubes were centrifuged and the supernatants were poured into a calibrated test-tube. Two additional 2ml sulphuric acid washes were collected and all the washings were combined and diluted to 10 ml with 1 N sul-

PHOSPHATE METABOLISM I N OOGENESIS 519

phuric acid. I n several experiments, wherein the amounts of phosphate were known to be small in specific fractions, small amounts of acid were used and the final volume was 5 ml.

Reagent blanks in duplicate were carried through the entire procedure along with the experimental tubes.

A 0.5 ml aliquot was taken from each sample and the phosphate was determined by the micromethod of Berenblum and Chain ( '38) using the vessel described by Wiame ( '47).

C. Errors The immediate question arises as to whether the procedure

outlined above meets with the criteria for the extractions of radioactive compounds mentioned in the preliminary discussion of method. Considering the objections raised by David- son et al. ('51) concerning the reliability of the Schneider method, one is compelled to evaluate the results with extreme caution. An attempt was made to obtain a relatively pure nucleic acid component by the chromatographic method of Szafarz and Paternotte ('52) without success. The amounts of nucleic acid in the extracts were insufficient to allow for accurate separation. This was particularly true for the small oocyte classes.

The recovery of activity and the absolute amounts of phosphorus varied with the size of the oocyte. The mean error for the recovery of total P varied from 17.5% for the smallest oocytes to 5% for the largest. The mean error for activity recovery varied between 10 and 15%.

Undoubtedly, some of this loss resulted from the magnesia mixture precipitation of the orthophosphate, particularly in those fractions with small amounts of phosphorus (Jeener, '49).

Additional sources of variation (seasonal, biological, etc.) contributed to the variability observed in the results. For example, total P for the large oocytes varied from 687 to 1730 micrograms of phosphorus per 100 oocytes. Greater variations were observed in the total P of smaller oocytes.

520 PHILIP GRANT

This source of variation arises from the arbitrary grouping of oocyte classes. As a result of the continuous nature of oocyte growth, oocytes collected in the early spring are generally smaller than oocytes of the same class collected in the late summer or winter. Yet, these oocytes are grouped together in the statistical analysis of the data, since they do represent a similar phase of oocyte growth.

TABLE 1

Distribution of phosphorus (P") in oocyte classes

OOCYTE CLASS FRACTION

A B c D 1 E

Total P 4.87 & 1.17 11.71 * 2.92 49.94 * 13.49 137.30 1036 & 275

Phospho- protein P 0.76 i 0.48 1.87 2 1.65 3.91 & 3.42 23.95 631.3 i 78.8

Nucleic acid P 3.24 i 1.48 5.51 i 2.24 19.60 2 4.49 26.10 217.3 i 98.2

Phospho- lipid P 0.24 zk 0.19 0.71 i 0.61 7.88 * 2.26 22.60 232.8 & 9.1

Acid soluble P 1.23 i 1.14 3.07 i 1.44 8.25 -C- 2.30 24.13 73.9 k 41.0

One experiment only. Values expressed as micrograms P/100 oocytes.

In spite of these difliculties, the trends in growth of the various phosphate fractions and their pattern of activities were not altered to any significant extent since relative values are considered rather than absolute amounts.

RESULTS

A. The patterm of phosphorus distribution

It is important, at first, to describe the pattern of distribution of the phosphate components in the various classes of oocytes. In table 1, the mean values of the absolute amounts of P in each fraction are tabulated for the 5 classes of oocytes analyzed. These values represent the means of 5 to 9 experiments, except where indicated. The large standard deviations

P H O S P H A T E METABOLISM I N OOGENESIS 521

shown in the table indicate a high degree of variability, probably resulting from the arbitrary grouping into classes. However, statistical analysis by the method of individual comparison (Snedicor, '45) reveals that the difference between classes in all fractions is significant (P < 0.05).

0 0

\ a - 0 + 0 +

x

to ta l

a c i d

A

nucle;c

soluble

acid P

P

B C D E

oocyto class I od

Fig. 2 The per cent distribution of phosphorus (P") in all classes of oocytes.

In figure 2, the distribution of phosphorus in the classes of oocytes is illustrated to emphasize the trends. In young oocytes, the dominant fractions are nucleic acid and acid soluble phosphorus which decrease in importance as phosphoprotein and phospholipid accumulate.

522 PHILIP GRANT

3-

2 Y, 2 - x u 0 0

0 0

a - \ C I -

0

u b

._ +

z 2 0- CP

CT 0 -

The changing pattern of phosphate distribution can be shown to be attributable almost entirely to the rapid synthesis of yolk. In figure 3, a log-log plot has been constructed to

/ /i/./

@f/g /

/ H

0 .

0

I I I

I 2 3

Fig. 3 A log-log plot relating the increase in phosphorus (P") in an isolated fraction with the increase in total phosphorus per oocyte.

Key : Closed circles, lipid phosphorus ; open circles, phosphoprotein phosphorus ; half circles, total nucleic acid phosphorus ; squares, total acid soluble phosphorus.

relate the increase of individual phosphate fractions with the increase in total phosphate during oogenesis. These curves appear to exhibit a heterauxetic growth pattern following the equation Y = bXk (Huxley, '32) and suggest that the nueleic

PHOSPHATE METABOLISM IN OOGEXESIS 523

acid and acid soluble phosphorus follow a pattern of growth similar to that of total phosphorus, although increasing at a lower rate. The slopes of these curves (I< values) indicate that the protein phosphorus exhibits a rate of growth practically equal to that of the total phosphorus during the early growth phases (k = 0.93) and then increases markedly with the onset of yolk synthesis (k=1.66). The phospholipid

4

li

I0 20 30 40 h o u r s

Fig. 4 Time course curve fo r the uptake of P3* into inorganic phosphorus fraction of plasma. Specific activity measured as:

cts/min/micrograms Pdl injected activity/wt. gm ___-____

phosphorus shows a continuous rapid rise throughout growth (k = 1.41). The behavior of these two fractions suggests a two-phase growth pattern : (1) an early phase characterized by intense lipid synthesis and (2) a later phase marked by intense protein synthesis. However, because of variations observed, this can only be a tentative conclusion. Further work is necessary to demonstrate this two-phase growth phenomenon.

524 PHILIP GRANT

B. A comparison of activity of plasma inorgarzic P with oocyte wptake of P3=

To determine the relationship between plasma activity and the activity recovered in the oocytes, experiments were performed to compare the time-course uptake of plasma with

0

8 F 0 x 7 1

a-

@--;'.

2

I

!b 30 40 5'0

hours

Fig. 5 Time course curve for the uptake of P3' by various phosphate fractions extracted from mature oocytes (class E). Specific activity measured as:

Key :

cts/min / m i c r o g r a m c l injected aetivity/wt. gm

0 inorganic P 0 total nucleic acid P C, acid soluble P phospholipid P 0 phosphoprotein P @ total P

that of the mature oocytes (class E). Figure 4 represents a time-course curve for the inorganic phosphorus of the plasma taken over a period of two days. It can be seen that the maximum specific activity appears after the first hour and then falls to an equilibrium value after 24 hours. The number of points on the curve are rather few; however, the pattern of the curve is very similar to those reported in the


literature for mammalian blood (Hevesy and Hahn, '40). Hevesy and Rebbe ( '40) have studied the distribution of P32 in Rama esculemta and have found that the maximum activity (measured as per cent injected activity in the total plasma) occurs after one and one-half hours and then proceeds to fall. The results reported here show good agreement.

One hundred mature oocytes were removed from the ovaries of the frogs used above and extracted according to the

TABLE 2

Distribution of P32

OOCYTE CLASS

TRACTION Summer Winter

A B C B E

A / O % A c t . A / O % A c t . A / O % A c t . A / O % A c t . A / O % A c t . Total P 1.62 5.49 18.05 1.85 31.50

Phospho- protein P 0.07 5.49 0.17 3.68 0.45 2.58 0.12 5.70 10.70 34.60

Xucleic acid P 0.14 11.60 0.33 6.56 0.86 5.25 0.21 12.70 3.49 16.60

Phospho- lipid P 0.02 1.67 0.08 1.04 0.21 1.64 0.10 4.09 0.59 2.14

Acid soluble P 0.93 80.60 4.60 88.70 16.58 90.60 1.51 77.70 15.12 46.10

cts/min/100 oocytes injected act/wt gms

'A/O = activity per oocyte = x 10s.

procedures outlined. In addition to the fractions isolated, acid soluble inorganic phosphorus was also obtained. Figure 5 represents a time course uptake curve for the phosphate compounds extracted from the large oocytes.

The maximum specific activity appears at 24 hours for practically all fractions. This corresponds with the equilibrium time for inorganic P of the plasma and suggests that equilibrium between plasma inorganic phosphorus and intracellular inorganic phosphorus of the oocytes is established at 24 hours. In all experiments that followed, 24 hours' exposure

526 PHILIP GRANT

was permitted to obtain the maximum activity values for the intracellular phosphate compounds.

G. Distribution of P32 I n table 2, the mean values of recovered activity per 100

oocytes are summarized for all classes of oocytes. As the oocyte diameter increases, the activity incorporated in all fractions exhibit marked increases. The pattern of distribution is indicated by the per cent values.

Approximately 80-90% of the activity is found in the acid soluble fractions in summer and winter oocytes except for the large mature oocytes (class E). The rise in the early stages is significant (class A-C) (P < 0.05). Nucleic acid activity shows a significant decrease in these same oocytes (P < 0.01). Similarly for the phosphoprotein phosphorus, the decrease in per cent incorporation is also significant (P < 0.05). The lipid P is constant in summer and winter oocytes. It appears, therefore, that there are significant shifts in the pattern of phosphate turnover in young oocytes.

The pattern in the mature oocytes remains to be explained. A sharp fall in the proportion of the active acid soluble phosphorus is accompanied by increases in the proportions of protein P and nucleic acid P. The change is so marked that it is difficult t o attribute it entirely to contamination of these fractions (Davidson et al., '51). These results are quite different from those reported by Kutsky ('50) with respect to distribution of P32 in fertilized eggs of Ruvza p ip i em. Species differences might account for some of this variability ; it is unlikely that it accounts for all. It is possible that tech- nical differences are responsible, since she obtains eggs 48 hours after the simultaneous injection of P32 and pituitary glands. It is conceivable that the period of exposure to P32 under these conditions is less than 24 hours since ovulation may occur before this time. This may account for the relatively higher proportion of activity in the acid soluble fraction which she obtains after 48 hours (see fig. 5 ) . However, as yet a suitable explanation for the difference has not been found.


2.5 -

2.0 - $

4- A

D. Seasonal variation. and P32 uptake

Since class B oocytes represent comparable oocytes under summer and winter conditions, a comparison of the data in this group (table 2) indicates that total activity is approximately three times greater in the summer than in the winter.

a

0

\ A 'c > u

-0

0 u

.- +

1.0

+

2 .- 0.5

/

surface area ( ? x ~ 0 4 )

Fig. 6 Relationship between surface area of oocyte and the incorporation of P3*. Activity measured as : e t s / m i n / l O O ,,ocytes -__

injected activity/wt. gm Key : Open circles, summer oocytes ; closed circles, winter oocytes.

This applies more so to the acid soluble phosphorus than to the other fractions. Unfortunately, no comparisons are possible for other oocyte classes.

During the late spring and summer, the overall activity of the frog is a t a maximum and one would expect metabolism

528 PHILIP GRANT

during this period to be more active than during the winter. Holmes ( '27) has indicated the distinct seasonal changes that occur in liver glycogen metabolism so that it is not surprising that the uptake of P32 by the ovaries is subject to seasonal influences.

E. Surface area of the oocyte aizd P32 uptake

To determine whether oocyte surface area influences the incorporation of P32, the total activity per 100 oocytes has been plotted against the surface area of both summer and

TABLE 3

Influence of surface area of oocyte on incorporation of plz

OOCYTE CLASS

FRACTION Summer Winter

a B C B E ~ ~

Total P 2.29 2.16 1.38 0.73 0.65

Nucleic acid P 0.20 0.13 0.07 0.08 0.07

Acid soluble P 1.32 1.78 1.26 0.59 0.31

Phosphoprotein P 0.10 0.07 0.03 0.05 0.02

Phospholipid P 0.03 0.03 0.02 0.01 0.01

cts/min/100 oocytes Values expressed as surface area (p * ) X lo'.

injected act/wt/of frog (gms) /

winter oocytes (see fig. 6) . Although the number of points is limited, a linear relationship is suggested for both summer and winter oocytes.

I t becomes likely, therefore, that surface area of the oocyte may be a limiting factor for incorporation of P32. This is applicable both to a situation involving simple diffusion of inorganic phosphate across the cell membrane, or to a more active transfer process involving an esterification mechanism at the surface of the cell (Sacks, '44; Kamen and Spiegelman, '48).

One may ask, however, whether each phosphorus component shows a linear relationship with surface area. In table 3, the activity data for each fraction has been corrected for surface.


One would expect that those components which are surface related would exhibit constant values from one class to an- other. One notes, in the table, that total P, acid soluble P, and phospholipid P fractions exhibit a surface relationship, while the nucleic acid and phosphoprotein P show distinct decreases. (P < 0.05, calculated by the method of individual comparison.) This is particularly interesting and suggests that for the latter compounds there are other limiting factors affect- ing P S 2 incorporation. The decrease observed in the acid soluble P of winter oocytes is not significant.

TABLE 4

Relative specific activity

OOCYTE CLASS

FRACTION Summer

A B c D '

Total P 3.33 4.68 3.62 0.85 Phosphoprotein P 0.92 0.91 1.15 0.01 Xucleic acid P 0.43 0.60 0.44 0.20 Phospholipid P 0.83 1.13 0.27 0.09 Acid soluble P 7.56 14.99 20.10 4.34

Winter

B E

1.58 0.34 0.64 0.17 0.38 0.16 1.41 0.03 4.92 2.05

-

/micrograms P/IOO oocytes x 104. cts/min/100 oocytes injected act/wt (gms)

Activity expressed as .

One experiment only.

F. Specific activity

In table 4, the relative specific activities of the phosphorus fractions are summarized for the different classes of oocytes. One notes that in oocytes of classes A through C , the relative specific activity remains relatively constant f o r nucleic acid P and phosphoprotein P. The decrease of phospholipid P in class C is significant and the increases in the acid soluble P are also significant (P < 0.05).

As the amount of inert material (yolk) increases in the oocyte, it is to be expected that the overall specific activity will decrease. This is shown in class D and E oocytes where there is a 10-fold decrease in the turnover of total P, acid

530 PHILIP GRANT

soluble P, and lipid P, whereas protein P and nucleic acid P exhibit 6-fold and two-fold decreases respectively from class C to class D. This decrease is also shown in the winter oocytes in a comparison of class B and class E oocytes.

1.5-

- - E E -

+ 1.0

v

L 8

E - 0 - .- V

as

0.1 -

0.0 yolk platelets z llpochondria -* lipid granules

ik m I to c h o nd r i a * ma. (cytoplasm)

nucleoll

glycogen granules

1st year 2 n d year 3rd vear Fig. 7 The cytological pattern for the seasonal growth of the ooeyte of Rana

temporaria. For explanation, see text.

DISCUSSION

A. The evolution o f the a m ~ h ~ ~ i a , n oocyte

The literature on amphibian oogenesis is quite extensive and it is almost exclusively concerned with the application of cytological and cytochemical methods to problems relating cellular morphological changes with oocyte growth. From this rich literature, it has been possible to reconstruct a general cytological and cytochemical pattern of oocyte development. Figure 7 represents a compilation of some of

PHOSPHATE METABOLISM IN OOGENESIS 531

the cytological evidence of oocyte growth. It has been drawn from several sources : (Marza, '38; Panijel, '51; Wagner, '23 ; Carnoy and Lebrun, 1897-'00 ; Gatenby, '16 ; Konapacka and Konapacki, '26 ; Lams, '07 ; Wittek, '52).

The continuous and synchronous " evolution by group" nature (Marza, '38) of oocyte growth is clearly illustrated and serves as a reference framework for speculation concerning possible mechanisms. although the slopes of the curves are arbitrary, the rhythm and pattern of oocyte development is made graphic.

Physiologically, Panijel ( '51) considers two phases of synthesis. (1) The early period of non-yolk synthesis in which the oocytes increase from 5 0 p to 3 0 0 ~ in diameter (A-E in fig. 7). It is characterized by synthetic activity in the region of the nucleus with the subsequent migration of syn- thesized products to the periphery of the oocyte (lipid granules and mitochondria). (2) The period of growth marked by yolk synthesis results in the major increase in oocyte volume. Syntheses predominate at the periphery and products migrate towards the interior of the oocyte (glycogen granules and yolk platelets). See figure 7 E-G.

Relating these growth phases to the classes of oocytes taken for analysis in this study will emphasize the difficulty of studying a continuous growth process which extends over a period of three years. One notes from figure 7 that oocytes from groups 1, 2, 3, after ovulation in the spring (April and May), are distinctly smaller compared to oocytes from these same groups in the late summer and fall. It is apparent, therefore, that grouping oocytes into arbitrary classes representing three phases of growth would yield overlapping and large size variations within classes. Thus, class A oocytes are a mixture of oocytes (A-D, fig. 7) representing the early phases of oocyte growth. Class B oocytes are also a mixed group. Oocytes of class A as well as oocytes in the early phases of yolk synthesis are included (A-E, fig. 7). It is probable that the larger oocytes of this group make the most significant contributions to the phosphate fractions extracted. Class C

532 PHILIP GRANT

contains oocytes in the early and mid stages of yolk synthesis (F, fig. 7) , while classes D and E represent oocytes in the final stages of yolk synthesis (G, fig. 7) . It is obvious that a well defined distinction between classes does not exist ; rather, it is more likely that the classes used in these experiments represent a continuous spectrum of oocyte growth.

B. Heterogevzeity of phosphate fractiovzs It is particularly important to relate substances extracted

from the oocyte to well defined cytological structures so as to correlate changes in the cytological pattern with observed changes in the pattern of phosphate distribution and activity. A review of the data with reference to previously reported analyses of cytoplasmic inclusions in Rana oocytes (McClen- don, '09 ; Holtfreter, '46 ; Panijel, '51) will provide some indication of the origin of the fractions isolated. Although these studies were performed on mature oocytes, it is possible to extrapolate these results to the younger oocytes. Essen- tially, the results of this analysis demonstrate the heterogeneity of origin for practically all fractions isolated in young and mature oocytes. It is, therefore, impossible to relate any phosphate fraction to a specific cytoplasmic inclusion, except for the phosphoprotein, which can be shown to owe its origin almost exclusively to yolk.

Panijel ('50) separated yolk platelets from mature eggs and determined the phosphorus constituents of the small and large yolk platelets. It is possible to combine Panijel's data and the data reported here, to calculate the contribution of the yolk to phosphate fractions obtained from large oocytes. Table 5 summarizes the results.

The large contribution by yolk to the total phosphorus of the egg was indicated by McClendon's analysis of the yolky layer of centrifuged eggs. He found 94.5% of the total egg phosphorus in the yolky layer. His high value may be attributed to contamination resulting from lipochondria ad- sorbed (Holtfreter, '46) and from difficulties in separation of layers.

P H O S P H A T E M E T A B O L I S M I N O O G E S E S I S 533

The lipid P of the mature oocyte has several origins. More than half can be attributed to yolk, while the remainder probably arises from the lipochondria (Holtfreter, '46). Some contribution to this fraction is made by other formed elements such as the nucleus, mitochondria and microsomes (Claude, '45 ; Schneider, '46).

The nucleic acid phosphorus is also heterogeneous. Ap- proximately 34% arises from yolk; however, this figure seems high, and it may be due to adsorption of nucleic acid on yolk during the extraction procedure (Chantrenne, '47). The other sources of nucleic acid are the nucleus, mitochondria, and the RNA granules (microsomes) .

TABLE 5

Large oocytes - (micrograms P/oocyte)

F R A C T I O N TOTAL YOLK P E R C E N T O F T O T A L

Total P 11.74 8.20 70.00 Phosphoprotein P 6.31 5.79 91.70 Wucleic acid P 2.17 0.74 34.10 Lipid P 2.33 1.23 52.80 Acid soluble P 0.74 0.30 40.55

Organic acid soluble P 0.36 0.11 30.55 Inorganic P 0.12 0.19

One notes that a considerable amount of acid soluble phosphorus arises from yolk. The remainder of this fraction may be assumed to come from the non-granular ground cytoplasm (Bourne, '50). As fo r the phosphoprotein, practically all is derived from yolk.

Since yolk is either lacking or in small amounts in the younger oocytes, it does not represent an important phosphate source in these oocytes. As seen in figure 2, the major phosphate fractions in the younger oocytes are the nucleic acid and acid soluble. The nucleic acid can be attributed, in large measure, to the microsomes which are in high concentration (Brachet, '40 ; Panijel, '51), the mitochondria, and the

534 PHILIP GRANT

nucleus. Furthermore, Brachet has reported a significant quantity of “free” nucleic acid in the cytoplasm of small oocyt e s.

The lipid P extracted from small oocytes is correlated with the early appearance of lipid granules (Konopacka and Kono- packi, ’27). The phospholipid derived from mitochondria, microsomes, and nuclei are also sources of phospholipid P, in these small oocytes.

The phosphoprotein in these oocytes is probably from oocytes in which yolk synthesis has begun, although in class A oocytes this fraction may be derived from non-yolk phosphoprotein.

An isotope analysis of heterogeneous systems is difficult (Barnum and Huseby, ’51). The establishment of precursor relationships becomes extremely complex when dealing with these systems since fractions isolated will represent, in part, the activity of a variety of different cellular structures. Clearly, a study of the problems of phosphate metabolism in cells and tissues by gross extractions of phosphate constituents, provides only a limited approach to the study of cell metabolism.

P. Nuture of oocyte growth

The two phase nature of oocyte growth is suggested by the curves of figure 3. The break in the protein phosphorus curve seems to occur in those oocytes at the beginning of yolk synthesis and the change in slope can be attributed to yolk synthesis.

Before the onset of yolk synthesis, the predominant synthetic activity is the production of lipid phosphorus. This is correlated with the cytological evidence for the early appearance of lipid granules (Konopacka and Konopacki, ’27). Fur- thermore, this may also be related to the rapid increase in mitochondria seen in oocytes less than 2 5 0 ~ in diameter (Panijel, ’51). The increase in phospholipid phosphorus continues a t a constant rate throughout development, possibly reflecting a continuous replacement of the granule system


as well as contributing to the increase in lipochondria. Dur- ing yolk synthesis, large amounts of phospholipid are also incorporated into yolk platelets.

The lower rate of nucleic acid and total acid soluble phosphorus increase results in a definite change in the distribution of phosphorus, as seen in figure 2. The early stages are characterized by a high proportion of nucleic acid phosphorus, which may be correlated with the cytological evidence for high basophilia in the same oocytes (Brachet, '40). This con- dition is continued up to those oocytes in which yolk synthesis has already begun (class C oocytes). The acid soluble fraction exhibits a similar pattern of distribution, high in the early stages and falling off as the proportion of yolk increases.

It is suggested that the high nucleic acid concentration in the early stages, reflects the rapid development of a protein synthesizing system, consisting of microsomes and mitochondria rich in synthesizing enzyme systems. This corresponds to the high proportion of small RNA rich granules obtained by Panijel in these young oocytes. The high value may also be attributed to the fact that the nucleus occupies a large proportion of oocyte volume. The continued increase in the absolute amounts of nucleic acid per oocyte suggest that the protein synthesizing mechanism is being maintained, probably as a result of intense nuclear and nucleolar activity (Caspersson, '50). Duryee ( '50) has reported the extrusion of nucleolar material from the germinal vesicle of the amphibian oocyte.

Brachet ('40) and recently Panijel ('51) have shown a decrease in the amount of basophilia in the cytoplasm as the oocyte increases in volume. It is more likely that the decrease in basophilia observed is due, in part, to a decrease in the concentration of basophilic granules, since the absolute amount of nucleic acid per oocyte increases with increasing growth, although the proportion of nucleic acid phosphorus decreases with yolk synthesis.

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D. Distribution of P32

There is an apparent relationship between the surface area of the oocyte cell membrane and its incorporation of (see fig. 6). Furthermore, in table 3 the data suggests that for some phosphorus fractions there is also a relationship between oocyte surface area and P32 incorporation. This is the case f o r systems associated with the ground cytoplasm, namely the acid soluble fraction. I f we assume that most of the acid soluble components are found in the hyaloplasm (Bourne, '50) it is not surprising to observe the rapidity with which the acid soluble fraction incorporates P32. On the other hand, phosphorus constituents found in the formed elements of the oocyte will exhibit no apparent cell surface relationship ; rather, the incorporation into cellular inclusions is limited by the surface characteristics of organized structures and by the chain of reactions and precursor relations involved in their turnover.

In table 3, one notes that nucleic acid P and phosphoprotein P exhibit no surface relationship, which follows from the fact that these components owe their origin to structural units. The behavior of the phospholipid P indicates a surface relationship and may be explained by assuming that phospholipid may enter the oocyte as phospholipid from the plasma. Hevesy and Hahn ('38) have suggested this possibility in their studies on the origin of phospholipids in hen egg yolk. The low turnover of the phospholipid P in the amphibian mature oocyte agrees with a similar low turnover found by Hevesy and Hahn in the hen's egg.

Thus, it is possible to differentiate three types of metabolic systems available for P32 uptake in the oocyte: (1) a system associated with the ground cytoplasm or hyaloplasm, generally free of known granule-enzyme complexes ; (2 ) a granule system, composed of a variety of well defined organized structures, exhibiting a wide variety of enzyme activities, chemical composition, and turnover rates. It includes mitochondria, microsomes, lipochondria, and yolk platelets ; (3)


a nuclear system composed of structural elements (nucleoli and chromosomes) and a ground nucleoplasm.

The turnover behavior of a system is partly determined by the relative size and complexity of its organization. The evidence from the studies of Jeener and Szafarz ('50) and of Barnum and Huseby ('50), indicates that the incorporation of P3* into a phosphate component of an organized intracellular structure is related to the size of the particle; smaller granules generally exhibiting a higher turnover than larger granules. This suggests the importance of intracellular sur- faces in the turnover of phosphate compounds.

The distribution of activity in the oocyte is, in part, a function of the relative physical states of the systems contributing to a fraction. The acid soluble P exhibits a coii- stantly high proportion of the activity in all oocytes, assuming that it represents the rapidly metabolizing P3* pool of the ground cytoplasm.

The relative specific activities of the other phosphorus components are generally much lower, since they can be shown to be associated with formed elements which are metabolically less active than the acid soluble pool.

I n class A oocytes, the high proportion of phosphorus in the nucleic acid and acid soluble fractions suggest that these two fractions dominate the metabolism of the oocyte. This is born out by the high proportion of Psz in these two fractions (see table 2). The high activity of nucleic acid phosphorus in these oocytes may represent the early stages in the production of metabolically active small RNA granules (Panijel, '51). Furthermore, since the nucleus occupies a significant portion of the volume of the oocyte, it may be the major participant in nucleic acid turnover. Jeener and Xzafarz have reported that the activity of nuclear RNA is approximately 20 times the activity of cytoplasmic RNA iii rat liver and the evidence of Duryee as to the extrusion of nucleolar material from the germinal vesicle further indicates the important role played by the nucleus during oocyte growth.

538 PHILIP GRANT

In class B oocytes, the first evidences of yolk synthesis appear. This is marked by a significant increase in the relative specific activity of the actively metabolizing acid soluble fraction. Since the period of exposure of P32 in the frog is only 24 hours, the distribution of Ps2 is a reflection of the equilibrium state between cells and blood plasma. Since the granules (lipid and RNA), which have increased in the class B oocytes, would turn over only slightly during this period, it is not surprising to observe no change in specific activity of components which are derived from them.

Thus, nucleic acid, protein and lipid phosphorus maintain a constant relative turnover between class A and B oocytes. The increase in acid soluble turnover is further shown in a shift in the proportion of activity (table 2). The increase in acid soluble phosphorus is correlated with a decrease in nucleic acid and protein phosphorus. The phospholipid maintains a constant proportion of activity.

Yolk synthesis predominates in the metabolism of class C oocytes. This is illustrated by a sharp increase in specific activity of the acid soluble phosphorus. The distribution of P32 in these oocytes (table 2) is similar to class B oocytes. The sharp drop in specific activity of lipid phosphorus is a reflection of a sharp increase in the amount of lipid phosphorus (see table 1). 1 (A 10-fold increase compared to a two- fold increase in phosphoprotein phosphorus.) The early stages of oocyte growth, before yolk synthesis, are characterized by an intense synthesis of lipid granules (lipochondria) which contain a central core of lipid surrounded by protein (Holtfreter, '46). These are present before the yolk platelets have appeared.

The winter oocytes (class B and E) suggest a general depressing of the overall metabolism of the oocyte. The proportion of P328 found in the acid soluble fraction is lower in class B winter oocytes. This lowered acid soluble turnover is even more noticeable in the large mature oocytes, which show a 10-fold decrease from the actively metabolizing class C oocytes.


As the amounts of yolk increase, the overall activity of the oocyte decreases. Oxygen consumption reaches a maximum in those oocytes beginning yolk synthesis and proceeds to fall in the later stages (Meshtcherskaja, ' 35 ) . Dipeptidase activity also exhibits a decline in larger oocytes, its maximum having been reached in oocytes beginning yolk synthesis (Duspiva, '42). It appears that the decrease in activity is a function of the quantity of inert material.

The difference in activity between class B and E corresponds with Duryee's results ( '50). He demonstrated a three-fold decrease in activity from small to large oocytes.

The pattern of oocyte growth suggests that the process of egg cell differentiation in R. temporaria is characterized by two distinct phases. Phase 1 involves the differentiation of the cell into a complex synthesizing system, consisting of a complete complement of highly organized enzyme systems, capable of performing a variety of syntheses. This consists of RNA granules (microsomes) and mitochondria. The importance of these structures in the synthesis of proteins has been demonstrated by recent experiments of Siekewitz ('52) and by the review of Brachet ('52). This first phase is concerned in the elaboration by the nucleus of a granule system which becomes oriented in the cytoplasm (A-E, fig. 7) . It is further suggested that the evolution of these cytoplasmic units and their subsequent regulation continues under the influence of the nucleus throughout oogenesis. (See Mazia, '52; Brachet, '52, as to the role of the nucleus in protein synthesis.)

The second stage of oocyte growth is characterized by the metabolism of this system with the resulting synthesis of protein and lipid to form yolk (F-G, fig. 7 ) .

SUMMARY

Using radioactive phosphorus tracer techniques, phosphate metabolism during oogenesis in Rama temporaria has been studied. Acid soluble phosphorus, total nucleic acid phosphorus, phospholipid phosphorus and phosphoprotein phos-

540 PHILIP GRANT

phorus have been extracted from oocytes representing different growth phases. The synthesis of these components during oogenesis suggests a two-phase growth pattern; an early phase characterized by the synthesis of lipid phosphorus compounds and a later phase marked by a period of intense yolk synthesis, shown as an increase in phosphoprotein phosphorus.

Yolk synthesis alters the pattern of phosphorus distribution with increasing age of oocyte. I n young oocytes nucleic acid phosphorus and acid soluble phosphorus predominate. Mature oocytes contain large proportions of protein phosphorus and lipid phosphorus.

The heterogeneous origin of the phosphate fractions makes a turnover analysis extremely difficult. The activity of the fractions reflects, in part, the state of organization of the intracellular systems to which they owe their origin. The surface areas of the oocytes also exhibit an apparent influence on the uptake of P32,.

The pattern of P32 activity as illustrated by per cent distribution and relative specific activity data lend credence to the hypothesis that growth of the oocyte involves the establishment of a synthesizing system in the early stages followed by the turnover of that system to produce yolk.

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr. L. G. Barth for his guidance, criticism and encouragement during the course of this work. I wish also to thank Professor J. Brachet for his kind advice during the course of my stay in his laboratory as a Fulbright fellow.

Thanks are due to Dr. L. C. Sze, Dr. H. Chantrenne, Pro- fessor R. Jeener and Mr. G. G. Berg for their helpful criti- cisms.

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Phosphate metabolism during oogenesis in Rana temporaria,

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