[CANCER RESEARCH 49, 1660-1664, April 1. 1989]

Effect of Cyclophosphamide on the Immature Rat Ovary1

Khalid M. .Maya,2 Frederick A. Valeriote, and Alfida J. Raniuhi-Ataya

Department of Obstetrics and Gynecology [K. M. A., A. J. R-A.J and Department of Medicine [F. A. V.¡,Wayne State University, Detroit, Michigan 48201

ABSTRACT

To investigate the early ovarian changes after Cyclophosphamide treatment, immature rats primed for 48 h with pregnant mare serum Donado-tro pin were given injections i.p. of Cyclophosphamide (100 mg/kg) at 1,2, 4, 16, and 24 h before decapitation. Serum estradiol dropped significantly after 24 h of exposure to Cyclophosphamide (/" < 0.001). Following

16 and 24 h of Cyclophosphamide exposure, (a) the number of granulosacells expressed from each ovary decreased (/" < 0.05 and /' < 0.01,

respectively); (b) the number of nucleated bone marrow cells decreased(/' < 0.01 and /' < 0.01), and their median nuclear size was significantlyreduced (P < 0.05 and P < 0.05) as measured by Coulter Counter and C-256 channelyzer (Hialeah, FL); and (c) the mean follicular diameter andthe number of follicles with diameters greater than 300 *imwere significantly lower than in control. After 4, 16, and 24 h of exposure, mediangranulosa cell nuclear size significantly increased (/' < 0.05, /' < 0.01,and P < 0.01, respectively). DNA cross-links in granulosa cells, measuredby alkaline elution, reached a maximum at 2 h of exposure and decreasedthereafter. The above findings demonstrate that Cyclophosphamide hassignificant effects on the rat ovary structure and function and that thegranulosa cell is an important target of cyclophosphamide-induced ovarian toxicity.

INTRODUCTION

Cyclophosphamide is the most commonly used chemothera-peutic agent in the treatment of cancer (1). It is used alone orin combination with other antineoplastic drugs in Hodgkin'sdisease, Burkitt's lymphoma, acute lymphoblastic leukemia of

childhood, and breast cancer (2-5). It is also used in thetreatment of some connective tissue and autoimmune diseases,minimal lesion gloriimi loncphrit is, and for the control of organrejection after transplantation (6-13).

A number of reports described the adverse effects of Cyclophosphamide on fertility in humans and animals of both sexes(14, 15). Men treated with Cyclophosphamide, 1 to 2 mg/kg formore than 4 mo, develop oligospermia or azospermia (16-18).The induced testicular damage appears to be dose dependent( 19). In rodents, Cyclophosphamide results in testicular damage(20,21) and ovarian toxicity (22-26). In women this drug causesovarian failure with associated amenorrhea and infertility (27-

29). Koyama et al. (5) estimated that doses of 10.4, 9.3, or 5.2g of Cyclophosphamide resulted in amenorrhea in patients intheir 20s, 30s, and 40s, respectively. With longer life expectancyin many patients, gonadal toxicity from chemotherapy represents an undesirable complication. Understanding the mechanisms of this toxicity is essential for a rational approach to itsprevention.

The advent of combination chemotherapy has made it moredifficult to evaluate the toxic effects of individual drugs on thegonads and other tissues. Thus, animal models to study eachdrug separately become a necessity in understanding the mech-

Received 5/24/88; revised 10/5/88, 12/29/88; accepted 1/4/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This research was funded by the American Cancer Society (Grant CH-365),Wayne State University Institute of Chemical Toxicology, and Children's Leu

kemia Foundation of Michigan.*To whom requests for reprints should be addressed, at Division of Reproduc

tive Endocrinology and Infertility, Department of Obstetrics and Gynecology,Wayne State University, 275 East Hancock, Detroit, MI 48201.

anisms of gonadal toxicity. In this report, we describe the effectof Cyclophosphamide on ovarian granulosa cells obtained fromimmature rats primed with PMSG.3 PMSG has been shown to

stimulate follicular development and increase granulosa cellproliferation within a short time (48 h). In adult rodents, thetime required for a follicle to reach the preovulatory stage isapproximately 15 to 17 days (30), a rather long time. We havestudied the effects of long-term administration of Cyclophosphamide on adult rat ovaries (22). While those studies demonstrated a reduction in the number of follicles, they do not definethe mechanisms involved at the cellular and subcellular levels.The advantages of using our present model of immature ratsinclude minimal prior exposure of the ovaries to endogenousgonadotropins. Variability in follicular development and ovarian steroid secretion in different days of the estrus cycle characteristic of cycling adult rats is avoided. The time relationshipof serum estradiol to endogenous gonadotropin surges thatresult in ovulation is difficult to control for. In addition, adultrat cycle length can vary unpredictably, thus adding to potentialvariability in reproductive and endocrine parameters (31). Forcomparative purposes the response of the bone marrow, aknown target of cytotoxic agents, was also examined.

MATERIALS AND METHODS

Chemicals. Cyclophosphamide (Cytoxan for injection), donated byBristol-Myers (Syracuse, NY), was dissolved in 0.9% NaCl solution.PMSG was purchased from Sigma (St. Louis, MO) and dissolved in0.9% NaCl solution. Medium M199 and HBSS were purchased from(.¡¡beo(Grand Island, NY). BSA was purchased from Sigma(St. Louis, MO). Two liters of buffer for alkaline elution were preparedby adding distilled water to 16 g of NaCl, 0.8 g of KC1, 2 g of dextrose,0.7 g of NaHCO3, and 3.722 g of disodium EDTA, supplemented with1% BSA and the pH adjusted to 7.4. Zapoglobin and Isotone (CoulterDiagnostics, Hialeah, FL) were used for cell counts using a Model ZMCoulter Counter (Coulter Electronics, Hialeah, FL). Serum estradiolwas measured using radioimmunoassay kits from Radioassay SystemsLaboratories (Carson, CA) after extraction with ether (10:1).

Treatment Regimens. Immature Sprague-Dawley rats (Charles River,Portage, MI) were housed 5 per cage under controlled temperature (20-22°C)and light (14 h of light, 10 h of dark). At 24 to 25 days of age,

they were given injections i.p. of 20 IU of PMSG and sacrificed 48 hlater by decapitation. At 1, 2, 4, 16, and 24 h before sacrifice, 100 mg/kg of Cyclophosphamide were injected i.p. Trunk blood was collectedand allowed to clot. Serum was separated and frozen at —¿�20°Cuntil

assayed for estradiol. The ovaries were excised, cleaned, weighed, andplaced in Medium 199, containing 25 mM 4-(2-hydroxyethyl)-l-piper-azineethanesulfonic acid with 1% bovine serum albumin.

Bone Marrow. Bone marrow from one femur of each rat was preparedas follows. The femur was cleaned of muscle and connective tissue, andthe two ends were removed without actually exposing the marrowcavity. Using a 23-gauge needle, a hole was made at either side, andHBSS with 1% BSA and 0.15% EDTA was used to irrigate the bonemarrow cavity. The solution (1.5 ml) and air (1 ml) were injected fromeither femur side and recovered through the other end into a vial. Ineach experiment, bone marrow of all rats within each treatment groupwas pooled. The marrow was then finely suspended and pushed through

3The abbreviations used are: PMSG, pregnant mare serum gonadotropin;HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; CLF, crosslink factor; FSH, follicle-stimulating hormone; EGTA, ethyleneglycol bis(/3-aminoethyl ether)-/V,JV,W,Ar'-tetraacetic acid.

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a 25-gauge needle. Nucleated cell count, nuclear size distribution, andits median for the different treatment groups were obtained using aModel ZM Coulter Counter with C-256 channelyzer.

Granulosa Cells. The ovaries of each treatment group were pooledtogether and incubated with 6.8 mM EGTA for 15 min in hypertonicsucrose (0.5 M) for 5 min. Granulosa cells were expressed into themedium containing equimolar amounts of EGTA and calcium bypuncturing the follicles with a 25-gauge needle as described by Campbell(32) and modified by Clark et al. (33). The ovaries were then gentlycompressed in a multiwell glass plate using a curved metallic spatula.The cell suspension was then filtered through a 100-^m nylon mesh.The cells were well dispersed as judged by microscopic examination.After washing the cells with Medium 199 (without EGTA), cell viabilitywas evaluated by the trypan blue dye exclusion test. Granulosa cellcounts were obtained by a Model ZM Coulter Counter, and theirnuclear size distribution and median size were determined using a C-

256 channelyzer after cell lysis with Zapoglobin (6 drops/20 ml ofIsotone). Granulosa cell number per ovary was obtained by dividing thetotal cell count by the number of ovaries which varied between 4 and 8ovaries per treatment group in each experiment. Meanwhile, the granulosa cell suspensions were kept on ice until 10 cells were taken foralkaline elution to evaluate the degree of granulosa cell interstrandDNA and DNA-protein cross-links as described by Kohn (34). Thecells were irradiated at 0°Cwith 500 R from a Gammacell mCs

irradiator (Mark I, Model 68; G. L. Shepherd & Associates, Glen-dale, CA). The fraction of their DNA retained on the filter is directlyrelated to the degree of cross-links. A CLF was calculated using theformula

CLF = log (fSOO/fO)log (fx/fO)

where/500 is the fraction of DNA eluted after control cells (not exposedto cyclophosphamide) were irradiated, /0 is the fraction eluted fromcontrol nonirradiated cells, and /.vis the fraction eluted from granulosacells of different treatment groups. DNA was measured fluorometri-cally.

Ovarian Histology. Ovaries were obtained 48 h after PMSG injectionfrom control rats and from those exposed to cyclophosphamide (100mg/kg) for 16 or 24 h. One ovary from each rat was fixed, embeddedin paraffin, serially sectioned, and stained with hematoxylin and eosin.Every eighth section was examined using a light microscope attachedto a BioQuant image analysis computer system (22). Antral follicleswere counted when a nuclear membrane and/or nucleolus of the oocytewas observed. The mean follicle diameter was calculated after measuring the longest and shortest follicle diameters (22).

Statistics. Contrast analysis and the Dunette test were used to evaluate the significance of differences between treatment groups and theircontrols. P < 0.05 was considered to indicate statistical significance.

RESULTS

Effect of Cyclophosphamide on Bone Marrow Nucleated CellCounts. Cyclophosphamide given 16 and 24 h before sacrificeresulted in a significant reduction in bone marrow nucleatedcell counts per femur of 63 ±5% and 79 ±3%, respectively (P< 0.01 for both), in comparison to control. No effect was notedat 1, 2, and 4 h of exposure to cyclophosphamide (Fig. 1.1).The controls had 51 ±4 million cells/femur.

Effect of Cyclophosphamide on Median Bone Marrow CellNuclear Size. Significant reductions in median bone marrowcell nuclear size of 6.2 ±2.6% and 8.0 ±1.6% (P< 0.05) wereobtained when cyclophosphamide was given 16 and 24 h beforesacrifice in comparison to control (Fig. 15). Control rats had amedian bone marrow cell nuclear size of 64 ±2 femtoliters. Nosignificant effect was noted when cyclophosphamide was given1, 2, and 4 h before sacrifice.

Effect of PMSG and Cyclophosphamide on Ovarian Weight.As expected, PMSG significantly increased ovarian weight (P

< 0.001) from 14.1 ±0.4 (for rats not treated with PMSG,mean ±SEM, n = 12) to 68.7 ±4.1 mg (for PMSG-treatedrats, n = 13). Cyclophosphamide did not have a significanteffect on ovarian weight in any of the groups compared tocontrol.

Effect of Cyclophosphamide on Granulosa Cell Number andViability. As shown in Fig. 1C, cyclophosphamide given 16 and24 h before sacrifice reduced granulosa cell number per ovaryby 24 ±5% and 51 ±4% in comparison to control (P < 0.05and P < 0.01, respectively). No significant change in cellnumber was noted when cyclophosphamide was given 1, 2, or4 h before sacrifice. The control group had 4.7 ±0.7 millioncells per ovary. No significant change in the percentage of cellviability was observed in any of the treatment groups comparedto control as evaluated by trypan blue exclusion.

Effect of Cyclophosphamide on Median Granulosa Cell Nuclear Size. Cyclophosphamide given at 4, 16, and 24 h beforesacrifice resulted in 11 ±0.8%, 21 ±4%, and 17 ±4% increasein granulosa cell nuclear size in comparison to control (P <0.05, P < 0.01, and P < 0.01, respectively). No significantchange in granulosa cell nuclear size was noted when cyclophosphamide was given 1 and 2 h before sacrifice (Fig. ID).The control group granulosa cell median nuclear size was 77 ±4 femtoliters.

Effect of Cyclophosphamide on Granulosa Cell DNA Cross-Links. CLF obtained from alkaline elution data reached amaximum at 2 h and decreased thereafter as shown in Fig. 2.The cross-link factor was 1.39 ±0.17 at 1 h, 2.77 ±0.90 at 2h, 1.77 ±0.42 at 4 h, 1.73 ±0.3 at 16 h, and 1.61 ±0.10 at 24h. No statistical comparison was performed, since the formulafor CLF always gives a value of 1 for control.

Effect of Cyclophosphamide on Serum Estradiol. A significantreduction in serum estradici was obtained when cyclophosphamide was injected 24 h before sacrifice (P < 0.005). Serumestradici was 1116 ±83 pg/ml for control and 326 ±70 pg/mlfor the 24-h group. No significant effect was noted when cyclophosphamide was given 1, 2, 4, or 16 h before sacrifice (Fig.3).

Effect of Cyclophosphamide on Antral Follicular Development.The number of mitral follicles counted per ovary was 36 ±3for ovaries exposed to cyclophosphamide for 16 h (n = 7) and27 ±4 for ovaries exposed for 24 h (n = 6). None wassignificantly different from control (32 ±3, n = 10). The meandiameter (/¿m)of antral follicles was lower in the groups exposedfor 16(264±9,/><0.001)and24h(237± 11, P< 0.001) in

comparison to control (317 ±8). The number of large antralfollicles (>300 /¿min diameter) was lower in the groups exposedfor 16 h (11 ±1, P < 0.05) and 24 h (7 ±2, P < 0.01) incomparison to control (17 ±2). No evidence of luteinizationwas observed.

DISCUSSION

Granulosa cell proliferation and ovarian hormone (estradiol)secretion were significantly modified by cyclophosphamide. Astatistically significant decrease in granulosa cell number perovary as well as mean follicular diameter was observed 16 and24 h after cyclophosphamide injection. Alkaline elution studiesshowed maximum DNA cross-linkage in granulosa cells at 2 hafter injection of cyclophosphamide. Alterations in granulosacell structure and function detectable by our methods wererelatively delayed. Four h after cyclophosphamide injection, asignificant increase in median granulosa cell nuclear size wasdetected consistent with a Gz-phase block.

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CVCLOPHOSPHAMIDE AND RAT OVARY

A. EFFECT OF CYCLOPHOSPHAMIDE

ON BONE MARROW NUCLEATED CELL COUNTS

C EFFECT OF CYCLOPHOSPHAMIDE

ON GRANULOSA CELL «/OVARY

Control I 2 4 16 24

Hours of Cyclophosphomida Exposure

Control I 2 4 16 24

Hours of Cyclophosphomid« Exposure

B. EFFECT OF CYCLOPHOSPHAMIDE

ON BONE MARROW NUCLEAR SIZE

D. EFFECT OF CYCLOPHOSPHAMIDE

ON GRANULOSA CELL NUCLEAR SIZE

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Fig. 1. Effect of cyclophosphamide on bone marrow nucleated cell counts (A), bone marrow cell median nuclear size (B), granulosa cell number per ovary (C). andgranulosa cell median nuclear size (D). Control is considered 100%. Other treatment groups are expressed as the percentage of control. *, P < 0.05; **, P < 0.01.Numbers in parentheses, number of experiments. Bars, SE.

Alkylating agents are known to have two important effectson intact living mammalian cells: cell killing and delay in cellcycle progression with accumulation of living cells in the G2phase (35). These effects have been observed in vivo and in vitro.More of the surviving granulosa cells had bigger nuclei as theyaccumulated more DNA in the preceding S phase of the cellcycle. The decrease in nuclear size of bone marrow cells probably reflects killing of the actively dividing giant stem cells ofthe bone marrow. This may have masked the effects of the Gz-phase block which is expected to increase nuclear size. Unlikethe bone marrow, the ovary does not have giant stem cells.

The expected decrease in bone marrow nucleated cell countwas observed. Consistent with the cytotoxic effects of cyclophosphamide on proliferating cells, similar reductions in thenumber of granulosa cells per ovary and mean ovarian fol lieu lardiameter were also observed. Mean follicle diameter is knownto correlate strongly with the number of granulosa cells perfollicle (36), and both are expected to increase following PMSGinjection (37). The decrease in serum estradici obtained 24 hafter injecting cyclophosphamide may represent an interference

2-

Control 1 2 4 16

Hours of Cyclophosphamide Exposure24

Fig. 2.CLFof 1.

Effect of cyclophosphamide on DNA cross-links. The controls have aNumbers in parentheses, number of experiments. Bars, SE.

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CYCLOPHOSPHAMIDE AND RAT OVARY

1500

f 1000

1

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_Control I 3 4 16 24

Hours of Cyclophosphomide ExposureFig. Ì.Effect of cyclophosphamide on serum estradici. ***, P < 0.001.

Numbers in parentheses, total number of rats in each group whose sera wereassayed. Bars, SE.

with granulosa cell function. Granulosa cells are the mainsource of serum estradici which increases with larger folliclediameter and granulosa cell number (38). In fact, serum estradici and follicle diameter measurements are used routinelyduring monitoring of follicle maturation and ovulation inwomen (38). The above changes in granulosa cell nuclear size,number, and serum estradiol may be related to cross-linking ofDNA. On the other hand, cyclophosphamide metabolites canalso alkylate other macromolecules, such as proteins and enzymes, that may be involved in mitosis and steroid synthesisand secretion.

The early events leading to ovarian failure after cyclophosphamide treatment have not been well characterized previously.Most previous studies documented the rather late effects ofcyclophosphamide treatment: accelerated depletion of ovarianfollicles (3, 22-24). Histological sections of ovaries from patients treated with cyclophosphamide were found to be devoidof follicles (3). Significant reductions (P < 0.01) of primordialand maturing follicles as well as corpora lutea have been observed in cyclophosphamide-treated mice compared to control(24). Burkl and Schicchi (23) studied the early disturbances infollicle growth induced by busulphan and cyclophosphamide inrats using histológica! techniques. Secondary and ant ral folliclesdisappeared within a short period of time after treatment.Degenerating cells containing DNA inclusions were frequentlyseen in the fol lieu lar epithelium. The growth of primary oocytesin primary follicles continued with apparently normal cyto-plasmic structures. The growth of the normal oocyte is knownto take place very rapidly in the early stages of follicular growthand is nearly completed in the late secondary follicle (39, 40).The number of layers of granulosa cells in treated rats did notincrease as it should under physiological conditions (23). Thesefindings in granulosa cells are consistent with ours, indicatingthat granulosa cells represent a primary target ovarian compartment involved in the eventual follicle depletion. Otherstudies in our laboratory indicate that oocytes may also beinvolved, however. Oocyte fertilizability and early cleavage weresignificantly impaired by cyclophosphamide metabolites (41).Thus, although the oocyte morphology may appear normal(23), their function may be adversely affected (41).

The dynamics of accelerated follicular atresia during chemotherapy have not been extensively studied. Starting with adefined total number of follicles in the ovary, follicular recruitment and development from the pool of primordial follicles area dynamic continuous process occurring all the time. We haveshown that cyclophosphamide can reduce estradiol levels inimmature PMSG-treated rats. A similar decrease can possiblyoccur in adult rats and in women. This would be consistent

with our findings of decreased follicular diameters in ovaries ofadult (22) and PMSG-treated immature rats (42). Our experiments show that exposure of granulosa cells to the effects ofcyclophosphamide, as measured by DNA cross-links, continuesup to and probably beyond 24 h. A similar situation may alsooccur with other macromolecules (enzymes, proteins) that arealkylated by cyclophosphamide metabolites. Thus, with repeated administration of the drug (sometimes given daily),rather prolonged exposure of granulosa cells to the alkylatingeffects of cyclophosphamide is ensured. The endogenous go-nadotropins, being related to estrogens by a negative feedbackmechanism (38), are expected to increase in response to decreasing estrogen levels. The increased endogenous FSH secretionmay accelerate further follicular recruitment into the developingcyclophosphamide-sensitive pool of follicles (22, 43). The Sphase and other phases of the granulosa cell division cycle havebeen shown to be shortened by FSH (43). This mechanismperpetuates a vicious cycle, whereby cyclophosphamide destroysthe developing follicles by attacking rapidly dividing granulosacells, reducing their steroid secretion, leading to increased pituitary gonadotropin production that enhances further recruitment of follicles into the pool of maturing follicles susceptibleto cyclophosphamide. Thus, follicular recruitment and atresiaare accelerated, resulting in eventual premature depletion ofthe limited number of follicles in the ovaries. A similar mechanism may also apply to humans, where estrogens and FSH arerelated by a negative feedback mechanism (38). In this context,we have shown that cyclophosphamide metabolites can reducesteroidogenesis in human granulosa cells (44).

In conclusion, granulosa cells appear to be important targetsfor toxicity in the ovaries of rats treated with cyclophosphamide. This is further supported by the recent demonstration ofa direct in vitro effect of 4-hydroperoxycyclophosphamide, anactivated form of cyclophosphamide, on rat granulosa cells(from PMSG-primed immature rats) and human granulosa cells(obtained from women undergoing follicle aspiration for invitro fertilization) (44, 45). In those studies, granulosa cellsurvival and progesterone accumulation were significantly reduced in a dose-related manner. Whether these cells are thetargets of damage in women in vivo remains unknown.

ACKNOWLEDGMENTS

We thank Maryann Milewski for secretarial assistance andMagai) Muskus and Carleen Yost for technical assistance.

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