MI I l 'HIIHHII il §§ IIIWIHHIHHWNHIHHWHIIW'IWIJ THS ulImumfulfillmummmlull, 3 1293 10585 3802 “3338? Michigan State University * This is to certify that the thesis entitled AN ANALYSIS OF FACTORS RESPONSIBLE FOR RESORPTION OF FETUSES IN CISPLATIN TREATED RATS presented by Mary Lynn Bajt has been accepted towards fulfillment of the requirements for Masters degree in Zoology 61¢ng Major professor Date September 7, 1982 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution _.._.__. - .._,. MSU LIBRARIES \. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. «lfi'fiééfl AN ANALYSIS OF FACTORS RESPONSIBLE FOR RESORPTION OF FETUSES IN CISPLATIN TREATED RATS By Mary Lynn Bajt A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology I982 ABSTRACT AN ANALYSIS OF FACTORS RESPONSIBLE FOR RESORPTION 0F FETUSES IN CISPLATIN-TREATED RATS By Mary Lynn Bajt Pregnant rats were injected i.p. with 7 mg/kg cisplatin on day 6 of gestation to study its effect on fetal resorption. Serum con- centrations of prolactin, luteinizing hormone (LH), and progesterone were determined by radioimmunoassay in pregnant rats, and related to the effects of cisplatin on the maintenance of pregnancy. The noctur- nal prolactin surge on day 9 of gestation was abolished in cisplatin- treated rats. Within 3 days after drug injection, LH concentrations decreased 39%, while serum progesterone levels decreased 63% by day IO. A histochemical study of 20a-hydroxysteroid dehydrogenase acti- vity revealed no enzyme activity by day 10. It is proposed that the cause of cisplatin—related fetal resorption in rats is due to decreases in pituitary hormones observed after drug treatment. To my parents for their constant love and support ii ACKNOWLEDGEMENTS I wish to thank Dr. S.K. Aggarwal for serving as my major professor. Appreciation is also due to Drs. J. Meites and L. Clemens who served as guidance committee members. I wish to especially thank Dr. J. Meites for his generosity with regards to laboratory facili- ties and research materials provided. I would also like to express appreciation to Bill Sonnteg for his technical help and friendship, and to "grumpy" Paul Sylvester and Vince Hylka. A special thanks goes to Jim San Antonio for the technical help and advice but mostly for our eternal friendship. TABLE OF CONTENTS DEDICATION ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTI N MATERIALS AND METHODS Animals Tissue Handling Procedur-. Histochemistry Radioimmunoassays (RIA) Statistics RESULTS Maternal Height Gain/Loss Due to Cisplatin ------------------ Embryolethality Effects of Cisplatin on Pituitary Luteotropic Hormone ------- Serum Concentrations of Prolactin Serum Concentrations of Luteinizing Hormone (LH) ------- Effects of Cisplatin on Ovarian Function Ovarian weights Serum Concentrations of Progestc.V”- ZOa-Hydroxysteroid Dehydrogenase (ZOa-OHSD) Activity--- Histology of Rat Corpora Lutea and Fetuscs DISCUSSION BIBLIOGRAPHY Page ii dO‘LDU'I-D -b .—l 12 12 18 I8 21 2T 2] 25 28 34 39 Table LIST OF TABLES Page Effects of cisplatin (CDDP) given i.p. to pregnant rats on day 6 of gestation ---------------------------------- l7 Effects of cisplatin (CDDP) on ovarian weights during gestation .............................................. 24 ~t -:n~"wf __, . Figure 1 LIST OF FIGURES Standard curves of Prolactin RP-l and serum from an animal treated with CDDP on day 6 of gestation --------- Maternal body weight changes for female Wistar rats following i.p. injection of saline or cisplatin (CDDP) on day 6 of gestation Isolated uteri of control and cisplatin-treated preg- nant rats Serum concentrations of prolactin at 0800 h and 1350 h on day 9 of gestation following injection of saline or 7 mg/kg cisplatin (CDDP) on day 6 Serum concentrations of luteinizing hormone (LH) at l350 h on day 9 of gestation following injection of saline or 7 mg/kg cisplatin (CDDP) on day 6 ------------ Serum concentrations of progesterone at 1350 h on days 9, 10, and 12 of gestation following cisplatin (CDDP) treatment on day 5 Light micrographs of corpora lutea on day 9 of gesta- tion from rats treated with saline or 7 mg/kg cisplatin on day 6 of gestation Light micrographs of longitudinal sections through embryos 9 days old from rats treated with saline or 7 mg/kg cisplatin on day 6 of gestation vi Page 22 26 29 32 INTRODUCTION Cis-diamminedichloroplatinum II (CDDP or cisplatin) is the first member of a family of platinum coordination complexes to be used in cancer chemotherapy (41). Cisplatin use as a potent anticancer drug was first demonstrated by Rosenberg et a1. (42). Since its introduc- tion into clinical trials in 1972 by the National Cancer Institute (41), cisplatin has been approved primarily for clinical use against testicular and ovarian tumors (20, 25, 40). Cisplatin, when used in combination with other antitumor agents, has proven to be effective against cancers of the head, neck, bladder, prostate, lung, and cervix (20, 25, 41). The biological activity of cisplatin on inhibition of tumor growth is still questionable (41). The primary mechanism of cisplat- in at the cellular level appears to be the inhibition of DNA synthe- sis through intra- (19, 23) and interstrand DNA crosslinks (53). Cisplatin may interfere with division of tumor cells by causing de- polymerization of microfilaments and preventing polar migration of the centrioles (1). Therapeutic usage of cisplatin is hampered by severe dose- limiting toxic side effects including renal toxicity, nausea and vomiting, myelosuppression, and decreases in serum electrolytes (25, 41, 45). Slow intravenous infusion of cisplatin, intravenous saline 2 hydration, and diuresis have helped decrease the cisplatin-induced nephrotoxicity (25, 41, 51) which is no longer considered to be a dose-limiting factor (41). Antiemetic treatment slightly decreases the drug-induced nausea and vomiting (41). Cisplatin distribution is initially highest in the excretory organs, gonads, and spleen (27, 28, 30), but after 4 days, cisplatin is concentrated mainly in the kidney, uterus, and ovaries (30). In male mice and monkeys, cisplatin treatment has been demonstrated to kill differentiated spermatogonia (35, 45). Furthermore, it has been shown to be embryotoxic in rats and mice (24, 29) and teratogenic in mice (29). The cause of cisplatin-induced fetal resorption in the rat is unknown. Since cisplatin is used in the treatment of women of child- bearing age, it is important to study the effects of the drug on the embryo and the maintenance of pregnancy. Hormones play a major role in maintenance of pregnancy, primarily those secreted by the pituitary, ovary, and placenta (34). Progester- one, mainly from the corpora lutea of ovaries, is one of the basic factors responsible for maintenance of pregnancy (9, 44). It is therefore essential that regression of the corpora lutea be prevented for a continual secretion of progesterone. The secretion of a luteotropic complex is largely responsible for the prolongation of the life span of the corpora lutea (21, 34). During the first half of gestation, the luteotropic complex in rats consists of pituitary hormones luteinizing hormone (LH) and prolactin (PRL) (21). LH and PRL play an indespensable role during gestation, since hypophysectomy before day 12 terminates pregnancy (39). 3 The soluble enzyme 20u-hydroxysteroid dehydrogenase (20a-OHSD) in corpora lutea is responsible for the conversion of progesterone to the hormonally inactive derivative, 20a-hydroxypregn-4-en-3-one (26). The induction of 20a-0HSD has been used as an index of luteolysis in the rat (21, 26). The aim of this study was to determine the effects of cisplatin on serum concentrations of PRL, LH, and progesterone because of the importance of these hormones in the maintenance of pregnancy. The appearance of histochemically demonstrable ZOa-OHSD enzyme activity was studied because of its importance in steroidogenesis. MATERIALS AND METHODS Animals Laboratory bred Nistar virgin female rats (Charles River Breed- ing Lab, Nillington, MA) weighing between 250-300 grams and approxi- mately 3 months of age were used in these experiments. Rats were maintained under a controlled 12:12 h light/dark schedule (lights on 0900 h to 2100 h) and temperature (23°C), and were given Wayne laboratory animal food (Allied Mills, Inc., Chicago) and tap water ag_1ibitum. During pair-fed control experiments, intake of food by the control animals was limited to that consumed by drug-treated animals. This was done because drug treatment is known to decrease food intake in rats (24). Estrous cycles were monitored by daily vaginal smears between 1000 h and 1300 h and only rats showing regu- lar 4 day cycles were included in experiments. Rats in proestrus were placed with males overnight; if spermatozoa were found in vaginal smears on the following day, this was considered day 1 of pregnancy. Animals were weighed daily during the length of the experiment. Rats exhibiting daily diestrous smears 6 days after mating were assigned to control or drug-treated groups so that equal numbers of animals from each group were sacrificed on the same day. Control and drug-treated animals were housed separately with 4 animals per cage. 5 Cisplatin (Johnson Matthey Research Laboratories, Sonning, U.K.) in powder form was dissolved in physiological saline prepared just before use. Rats received a single i.p. injection of 4 or 7 mg/kg of cisplatin in saline on day 6 of gestation and control animals re- ceived saline only. Animals were sacrificed on day 9 at 0800 h and 1350 h, while on day 10 or 12 they were sacrificed at 1350 h. Rats were decapitated within 20 seconds after removal from their cages. Trunk blood was collected and allowed to clot overnight at 4°C. The following day clots were removed and samples were centrifuged at 5000 rpm for 10 minutes. Serum was stored frozen at -20°C until assayed. Tissue Handling Procedures Uteri and ovaries were dissected from one third of the rats and fixed in Bouin's fluid for routine histological examination. Tissue samples were serially sectioned at 8 pm and stained with hematoxylin and eosin. In sections of ovaries, luteal cells were measured by use of an ocular micrometer. The uteri and left ovaries from the rest of the animals were placed in 25 ml of saline and widths of embryonic swellings were recorded. The ovaries were blotted on a filter paper and weighed to nearest 0.001 g. The right ovaries were removed and prepared for histochemical evaluation of 20a-hydroxysteroid dehydro- genase activity. Histochemistry Ovaries were placed on a microtome stub covered with 0.C.T. com- p0und (Miles Laboratories, I11.) and immediately frozen by immersion 6 for 20 seconds in liquid nitrogen. Frozen sections were cut at 10 um in a freezing microtome. Sections were placed on coverslips and thawed at room temperature for 30 minutes. Sections were treated according to a modified procedure of Balagh (6). Coverslips were transferred by forceps into the incubation medium. The incubation medium was composed of 5 mg of nitro-blue tetrazolium, 5 mg B-nico- tinamide adenine dinucleotide phosphate (TPN), and 10 mg of disodium ethylenediaminetetraacetate dissolved in 2 m1 of tris HCl at pH 9.0. Two m1 of 50% polyvinylpyrrolidine K-30 (PV) solution (in tris-HCl buffer pH 9.0) and 1 m1 of N,N-dimethylformamide containing 5 mg of 20a-hydroxypregn4-en-3-one was then pipetted into the medium. Control incubation medium was prepared identically with the exclusion of the substrate, 20u-hydroxypregn—4-en-3-one. Tissues were incu- bated at 37°C for 60 minutes. Sections were then fixed for 1 hour in neutral 10% formalin at room temperature, rinsed in saline, and mounted on microscope slides with glycerin jelly. All compounds were obtained from Sigma (St. Louis, Mo.) and solutions were mixed fresh for each experiment. Slides were examined by light microscopy. Radioimmgpoassays (RIA) Serum levels of PRL and LH were measured using the RIA method originally developed by Niswender et a1. (37, 38). Material for RIA were provided by NIAMDD (Bethesda, Md.). A non-equilibrium assay was used for PRL and LH RIA and the protocols were essentially the same. The reference preparation was serially diluted and the various con- centrations were used to determine a standard curve. Antiserum raised in rabbits was incubated with the standard or serum sample at 7 4°C for approximately 24 hours. Hormone iodinated by chloramine T (17) was then added to all tubes, and tubes were allowed to incubate for another 72 hours at 4°C. Total count tubes (iodinated hormone only), total bound tubes (no cold hormone added), and nonspecific bound tubes (excess amount of cold hormone added) were also included in the assay. Protein A (196 SORB) of 2.0% cell suspension was then added to separate antibody-bound hormone from free hormone. After 30 minutes incubation at 4°C, 2 m1 of ice cold saline was added and tubes were centrifuged at 25,000 rpm for 20 minutes. The supernatants were decanted and the precipitates counted on a tracer analytic gamma spectrometer. For prolactin RIA, reference preparation Rat Prolactin RP—l and antiserum Anti-Rat Prolactin #4-10 (gift from Dr. D. Chen) was used. The minimum detectable dose was calculated to be 0.05 ng/ tube and 50% inhibition of tracer binding was achieved at 0.7 ng/ tube. The intra-assay coefficient of variation was 4%. For LH RIA, reference preparation NIAMDD Rat LH RP-l and anti- serum NIAMDD Anti-Rat LH S—5 was used. The minimum detectable dose was calculated to be 0.8 ng/tube and 50% inhibition of tracer binding was achieved at 3.95 ng/tube. The intra- and inter-assay coeffi- cients of variation were 4% and 7%. Since cisplatin within serum samples might interfere with RIA, serum samples from rats treated on day 6 of gestation with CDDP and Prolactin RP-l were quantitated for standard curves (47). The slopes of the linear curves were not significantly different (Figure 1). Therefore, no interference due to cisplatin within serum samples was observed. Serum progesterone levels were determined by a non-chromato- graphic radioimmunoassay method. Serum samples were diluted 1:20 in assay buffer in duplicate. Radiorecovery solution, 20% of radio- active 1, 2, 6, 7, 3H-progesterone (New England Nuclear, Mass.) was added to one set of duplicates to correct for extraction procedural losses. Serum was extracted twice with distilled petroleum ether and dried extracts were resuspended in assay buffer. Reference preparation, 4-pregnene-3,20-dione (Sigma, St. Louis, Mo.), from a stock solution in ethanol was added to standard tubes at various concentrations to determine a standard curve. Serum samples which contained radiorecovery 1, 2, 6, 7 3 H-progesterone were used to calculate percent recovery of progesterone extracted. Hormone specific antibody raised in rabbits (gift from Dr. Niswender) and radioactive 1, 2, 6, 7 3 H-progesterone were added to all tubes. A reference preparation or serum sample was then added to tubes and tubes were incubated at 4°C for approximately 24 hours. Total count, total bound, nonspecific bound, and radiore- covery tubes were included in the assay. A suspension of activated charcoal (Matheson Coleman and Bell, Norwood, Ohio) coated dextran T—70 (Pharmacia, Uppsala, Sweden) was added to separate free hormone from the soluble antibody hormone complex for 10 minutes at 4°C. Tubes were spun down at 3000 rpm for 10 minutes in a refrigerated centrifuge. The supernatant was de- canted into scintillation vials and 10 m1 of Formula 963, aqueous .msa—o> Lo cowumepcwocou mcoseo; mo mop ecu pmcwmmo umppomg Aucmmwea mv about» apco ems: ccaon among» we a. cowpumem on“ m_m=cm om use m\u==om n > msmczw > pvmo_ mo meemp cw noupopa mm>L=u .m xmu :o nmumeeuem use cowuepmm wo m Ann :0 :vuepamwu mx\ms A cow: umpmmep Fmevcm cm sot» Eaemm use Flam :wuumpogm oo mm>e=u cemecmpm ._ wgzmwd 10 Oi Em 2.559: 4 _ . 22mm . as E: 2.5505 60.. 3. o :3 «Scum 00.. mi 0... m6- 0...- (°e/e) 1.1901 D 11 counting cocktail, was added. Sample radioactivity was determined by use of a scintillation counter. Correction for procedural losses during extraction was accomplished by calculation of the percent recovery of l, 2, 6, 7 3 H-progesterone added to each serum sample. Minimum detectable dose was calculated to be 0.88 pg/tube and 50% inhibition of tracer binding was achieved at 51.3 pg/tube. The intra-assay coefficient of variation was 2%. Serum concentrations of PRL, LH, and progesterone were deter- mined by comparison of the amount of labeled hormone precipitated in the presence of the serum sample to that observed in a standard curve prepared with the appropriate reference preparation. A compu- ter program using logit and log transformations and linear regres- sion analysis was used for calculation of hormone levels in the sample. Serum from normal females, castrated males, and charcoal washed serum were included in the assays to serve as controls. Statistics All data were statistically analyzed by use of the student's "t" test when a comparison between control and CDDP treatment was made. Data from experiments including controls, pair-fed controls, and CDDP-treated animals was analyzed by use of analysis of variance followed by Duncan's New multiple range test. RESULTS Maternal Weight Gain/Loss Due to Cisplatin Changes in the maternal weight of cisplatin-treated rats were compared to that of controls in order to determine if the appetite- suppressant effects of cisplatin could be partly responsible for the drug-related maternal toxicity. Drug-treated and control dams were injected on day 6 of gestation (Figure 2). A 19% weight loss occurred within three days following treatment with 7 mg/kg cisplatin. Pair-fed controls with the 7 mg/kg cisplatin-treated dams exhibited a 13% weight reduction by day 9 of gestation. Maternal weight of dams treated with 4 mg/kg cisplatin decreased 12% by day 9 and 24% by day 11 compared to controls. By day 12, however, maternal weight increased 4% from day 11. Cisplatin caused a decrease in maternal weight which can be partly attributed to decreased food intake as indicated by pair-fed controls. With a lower dosage of cisplatin, 4 mg/kg, maternal weight decreased during the first five days following treatment but an in- crease in weight was apparent by the sixth day. Embryolethality, Cisplatin was highly embryolethal in rats, causing 100% resorp- tion when administered on day 6 of gestation (Figure 3). Table l 12 13 .m—asvcm umummgu -cwpmpamvu mx\ms o.~ an nmssmcou was» on umpwswp mo: mxmucw uoom mmocz mfimspcm umpmep mcwpmm .mum .mfims_:m w mo omegm>m cm co women ucwoa seam .No.n A pcwoq sumo psonm emumem cam: .cowpmummm mo m zen :o Amooov cwprQmwo Lo wcwpmm we cowuuwnco .a.w mcrzoppom mum; mem_3 «Poem; Low momcwgo ucmwoz Avon Facempwz .N mezmwm 14 N .F F >Oz<20mmm ".0 >wm mpwzmeme we; ee..H «.4 am a N_ acne o.¢ c m..u 0.x _m N NF ace_am we; ee..H o.m er m a some 0.“ e e..H m.m em a m eu-m : e..H e.m on m m ecwwmm Ages zececmwwm mmch—ezm www%»weam mFeEwc< eeewwwgeem Amx\mev awe euepm emcezgesm we we seamem we gossaz :ewpepmmw we zen weesuemww mzuewz emeem>< .cewueumem we 0 Ace co mum; pcecmege op .e.w ce>wm AQQQUV :wpewemwe we mpeewwm .H eweew summarizes the effects of a single i.p. injection of cisplatin on fetal destruction in pregnant rats. Cisplatin (7 mg/kg) caused a significant reduction in widths of embryonic swellings by day 9 of gestation (p<0.01). Pair-fed control dams did not demonstrate any significant difference from the controls. Dams treated with 4 mg/kg cisplatin showed a significant (p<0.01) reduction in widths of em- bryonic swellings by day 12 of gestation compared to controls. The appetite-suppressant effects of cisplatin alone cannot account for the resorption of fetuses, since pair-fed control dams did not demonstrate the same embryolethal effects as cisplatin- treated dams. Effects of Cisplatin on Pituitary Luteotropic Hormone Serum concentrations ofgprolactin: To investigate the possi— bility that cisplatin affects surges of prolactin during pregnancy, rats were decapitated on day 9 of gestation to obtain blood for pro— lactin analysis (Figure 4). Rats were sacrificed at 0800 h to obtain concentrations of prolactin during the nocturnal surge and at 1350 h for baseline levels of prolactin. Mean concentrations of prolactin in control animals at 0800 h indicated the presence of the nocturnal surge of prolactin with a 9-fold increase above baseline levels ob- tained at 1350 h. Cisplatin treatment (7 mg/kg) on day 6 of gestation abolished the nocturnal surge of prolactin on day 9. Baseline levels of prolactin at 1350 h were not affected by cisplatin treatment as compared to control prolactin concentrations. 19 .3228 22w 29931295.,wa awuceewwwcmwm .* .mwmezpceeee cw ewe: mum; we sense: now: .n.m + sees use mpwzmmm .m Ame :e Aeoouv :wpewemwe mx\mE A we ecwwem we :ewuomncw mewZewwew eewpepmem we m mew :e ; ommw ece ; come we ewpeeweee we meewueeuceecee Eagem .e onemww EnoEmoEES‘m to ma: . one. . come i. on 20 GOP 09. 88 7% .6528 D 21 Serum concentrations of luteinizing,hormonei(LH): In order to determine if cisplatin affects LH levels during pregnancy, trunk blood was collected from pregnant rats at 1350 h on day 9 of gesta- tion (Figure 5). Dams treated with cisplatin (7 mg/kg) showed a significant decrease (p<0.05) in circulating LH levels as compared to controls. To assure that the decrease in concentration of LH was not due to the decreased food intake caused by cisplatin treatment, LH con- centration in pair-fed control animals was determined. Pair-fed controls had no significant difference in serum LH concentrations as compared to controls, indicating that the decrease in LH levels observed in cisplatin-treated dams was due to the effect of the drug alone. Effects of Cisplatin on Ovarian Function Ovarian weights: Heights of the left ovaries from control and drug-treated animals were recorded to determine if cisplatin affects ovarian luteinization (Table 2). Heights of ovaries from cisplatin- treated (7 mg/kg) dams were significantly lower (p<0.05) than ovaries from saline-treated animals, on day 9 of gestation. Ovaries from pair-fed control dams did not show any significant difference com- pared with the ovaries of control dams fed ad libitum. Ovaries ob- tained from cisplatin-treated (7 mg/kg) dams on day 10 of gestation showed decreased ovarian weights as compared with controls, but the decrease was not statistically significant. Rats treated with 4 mg/kg cisplatin and sacrificed on day 12 of gestation demonstrated a 22 .muee eeueeeu-=wue_emwe mx\ms w an easemeee “esp op empwsw— we: exeucw eeew ewes: mweewce eeuemgu ezwwem .w-m .wespcee Eesw Amp.ovev peeeewwwe zwuceewwwcmwm .a .mwmezuzegee cw ewe: mum; we geese: cow: .o.m + sees one muwsmem .0 see :e Agency :wuewemwu mx\ms N no eewwem we :ewpeenew mcwzew—ew cewpeumem we a xee :e ; omm_ we Azov eceELe; mewchwepzw we mcewpesuceucee Eagem .m eesmww 23 gene % “7w I .6528 _H_ menace wzmsEqu O Elm/5“] H1 INHHES 24 Table II. Effects of cisplatin (CDDP) on ovarian weights during gestation. Day of Average Weighs a Treatment . Number of Gestation of Left Ovary (mg/k9) Sacrificed Dams (mg) Saline 9 5 48 1.5 7.0 CDDP 9 9 40‘_ s-F° 9 4 45 : 5d Saline 10 45 8 7.0 CDDP 10 37 2 Saline 12 52 :_3 4.0 coop 12 41 : 4d aInjected i.p. on day 6 of gestation. bResults given as mean :_S.D. cSaline treated animals whose food intake was limited to that consumed by 7.0 mg/kg CDDP-treated animals. dSignificantly different (p<0.05) from control. 25 significant decrease (p<0.05) in ovarian weights as compared to ovaries from controls. In general, a decrease in ovarian weights was demonstrated following cisplatin treatment. Serum concentrations of progesterone: To determine the effects of cisplatin on ovarian secretion of progesterone, trunk blood was analyzed for progesterone following cisplatin treatment on day 6 of gestation (Figure 6). There was no difference in progesterone levels between cisplatin-treated, pair-fed controls, and saline-treated rats on day 9 of gestation. Four days after cisplatin treatment a signi- ficant decrease (p<0.01) in serum progesterone occurred in 7 mg/kg cisplatin-treated dams as compared with controls. Cisplatin dosage was lowered from 7 to 4 mg/kg to obtain a greater percent survival of cisplatin-treated dams, and rats were sacrificed on day 12 of gestation. A decrease in concentration of progesterone was apparent in 4 mg/kg cisplatin-treated animals, as compared to controls although it was not a significant difference. In summary, serum pro- gesterone decreased by day 10 of gestation, 4 days after 7 mg/kg cisplatin treatment. 20a-Hydroxysteroid dehydrogenase (20a-0HSD) activity: Histo- chemical appearance of 20a-0HSD activity in the corpus luteum was studied as an index of luteolysis (21, 26) in drug-treated animals. Histochemical reaction was taken to be dark blue tissue staining as confirmed by controls (no substrate). Results from this experi- ment indicated that cisplatin treatment did not induce 20a0HSD enzyme activity in corpora lutea by day 9, 10, or 12 of gestation. .mweswee empemgpucwuewemwe mx\ms w xe easemcee pen» on emuweww we: exepcw eeew ewes: mweewce eeeeeep ecwwem .wum .weeucee Eeww Awo.o~mv pceemwwwe zwpceewwwcmwm .w .mwmezpceeee cw emu: mum; we Lease: new; .o.m + coma ewe mpwamma .o wee :e “seaweeep Agency cwpewemwe mcwzewwew :ewpeumem we NF ece .ow .m exec :e ; omm— we eceweummmeee we mcewpeeuceuceu Esswm .m ewamww 27 >Uz