Effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation implantation dysfunction.

The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation (COH) implantation dysfunction were studied. The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotrophin (PMSG), followed by 7.5 IU human chorionic gonadotrophin (HCG) 48 h later. Then the female mice were mated with male at a ratio of 2:1 in the same cage at 6:00 p.m. The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time. Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m. of the next day. Fifty successfully pregnant mice were equally randomly divided into 5 groups: normal control pregnant group (NC), COH implantation dysfunction model group (COH), low dosage of Bu-Shen-An-Tai recipe group (LOW), middle dosage of Bu-Shen-An-Tai recipe group (MID) and high dosage of Bu-Shen-An-Tai recipe group (HIGH). Then from day 1, the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m. for 5 consecutive days. The concentrations of 17ß-estradiol (E2) and progesterone (P4) were determined by radioimmunoassay (RIA). The ultrastructural changes of ovarian tissues were observed by transmission electron microscope (TEM). The histopathological changes of ovarian tissues were observed by HE staining. The number of atretic follicles and pregnant corpus luteum were also recorded. TUNEL was applied to measure apoptotic cells of ovarian tissues. Western blotting was used to detect the protein expression of apoptosis- related factors like Bax, Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice. The results showed that ovarian weight, the concentrations of E2 and P4, the number of atretic follicles and pregnant corpus luteum, as well as the apoptosis of granulosa cells were significantly increased in the COH group. The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased, agglutinated, aggregated or crescent-shaped. The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis. After the treatment with different doses of Bu-Shen-An-Tai recipe, the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM. Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced. The lipid dropltes were shown in a circluar or oval shape. The protein expression levels of Bax and cleaved-caspase-3 were decreased, and the expression of Bcl-2 protein was increased after treatment. It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells, probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3, which contributes to the formation and maintenance of ovarian corpus luteum. It's helpful to promote the embryonic implantation, to reduce embryo loss and ultimately to improve the success rate of pregnancy.

Effect of fish meal supplementation on spatial distribution of lipid microdomains and on the lateral mobility of membrane-bound prostaglandin F2α receptors in bovine corpora lutea.

This study examined the effects of fish meal supplementation on spatial distribution of lipid microdomains and lateral mobility of prostaglandin F2α (FP) receptors on cell plasma membranes of the bovine corpus luteum (CL). Beef cows were stratified by BW and randomly assigned to receive a corn gluten meal supplement (n = 4) or fish meal supplement (n = 4) for 60 d to allow incorporation of fish meal-derived omega-3 fatty acids into luteal tissue. Ovaries bearing the CL were surgically removed between days 10 to 12 after estrus corresponding to approximately day 60 of supplementation. A 200-mg sample of luteal tissue was analyzed for fatty acid content using gas-liquid chromatography (GLC). The remaining tissue was enzymatically digested with collagenase to dissociate individual cells from the tissue. Cells were cultured to determine the effects of dietary supplementation on lipid microdomains and lateral mobility of FP receptors. Luteal tissue collected from fish meal-supplemented cows had increased omega-3 fatty acids content (P < 0.05). Lipid microdomain total fluorescent intensity was decreased in dissociated luteal cells from fish meal-supplemented cows (P < 0.05). Micro and macro diffusion coefficients of FP receptors were greater for cells obtained from fish meal-supplemented cows (P < 0.05). In addition, compartment diameter of domains was larger, whereas resident time was shorter for receptors from cells obtained from fish meal-supplemented cows (P < 0.05). Data indicate that dietary supplementation with fish meal increases omega-3 fatty acid content in luteal tissue causing disruption of lipid microdomains. This disruption leads to increased lateral mobility of the FP receptor, increased compartment sizes, and decreased resident time, which may influence prostaglandin signaling in the bovine CL.

Equol and para-ethyl-phenol stimulate prostaglandin F(2alpha) secretion in bovine corpus luteum: intracellular mechanisms of action.

Corpus luteum (CL) is a reproductive gland that plays a crucial endocrine role in the regulation of the estrous cycle, fertility, and pregnancy in cattle. The main function of CL is secretion of progesterone (P4), an important hormone for establishment a successful pregnancy, whereas prostaglandin F(2alpha) (PGF(2alpha)), 17beta-estradiol (E(2)) and testosterone (T) are implicated in the regulation of luteolysis. It has been shown that phytoestrogens may disrupt numerous reproductive functions on several levels of regulation and via different intracellular mechanisms. Using a cell-culture system of steroidogenic cells of the bovine CL, we determined effects of active phytoestrogen metabolites (equol and para-ethyl-phenol) on PGF(2alpha), P4, and T synthesis in steroidogenic CL cells. Moreover, we examined the intracellular mechanisms of phytoestrogen metabolite actions. Phytoestrogen metabolites did not affect P4 production in steroidogenic CL cells. However, PGF(2alpha) and T were significantly stimulated by metabolites of phytoestrogens in the bovine steroidogenic CL cells. To study the intracellular mechanism of endogenous E(2) and phytoestrogen metabolites action, steroidogenic cells were preincubated with a phospholipase C inhibitor (U73122), a protein kinase C inhibitor (staurosporine), an estrogen receptor antagonist (ICI) and a transcription inhibitor (actinomycin D) for 0.5h, and then stimulated with para-ethyl-phenol, equol or E(2). Only U73122 and staurosporine totally reduced the stimulatory effect of E(2) on PGF(2alpha) production by the cells. ICI and actinomycin D only partially reduced E(2) action on CL cells. In contrast, the stimulatory effect of phytoestrogen metabolites was totally inhibited by ICI and actinomycin D. Moreover, in contrast to E(2) action, phytoestrogen metabolites did not cause intracellular calcium mobilization in the cells. The present study demonstrated that phytoestrogen metabolites stimulate PGF(2alpha) secretion in steroidogenic cells of the bovine CL via the estrogen receptor-dependent, genomic pathway.

Expression of phospholipase A2 group IVA (PLA2G4A) is upregulated by human chorionic gonadotropin in bovine granulosa cells of ovulatory follicles.

Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2-4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 microM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.

Effect of electro-acupuncture on ovarian expression of alpha (1)- and beta (2)-adrenoceptors, and p75 neurotrophin receptors in rats with steroid-induced polycystic ovaries.

BACKGROUND: Estradiol valerate (EV)-induced polycystic ovaries (PCO) in rats is associated with an increase in ovarian sympathetic outflow. Low-frequency (2 Hz) electro-acupuncture (EA) has been shown to modulate sympathetic markers as well as ovarian blood flow as a reflex response via the ovarian sympathetic nerves, in rats with EV-induced PCO. METHODS: In the present study, we further tested the hypothesis that repeated 2 Hz EA treatments modulate ovarian sympathetic outflow in rats with PCO, induced by a single i.m. injection of EV, by investigating the mRNA expression, the amount and distribution of proteins of alpha1a-, alpha1b-, alpha1d-, and beta2-adrenoceptors (ARs), as well as the low-affinity neurotrophin receptor (p75NTR). RESULTS: It was found that EV injection results in significantly higher mRNA expression of ovarian alpha1b- and alpha1d-AR in PCO rats compared to control rats. The p75NTR and beta2-ARs mRNA expression were unchanged in the PCO ovary. Low-frequency EA resulted in a significantly lower expression of beta2-ARs mRNA expression in PCO rats. The p75NTR mRNA was unaffected in both PCO and control rats. PCO ovaries displayed significantly higher amount of protein of alpha1a-, alpha1b- and alpha1d-ARs, and of p75NTR, compared to control rats, that were all counteracted by repeated low-frequency EA treatments, except for alpha1b-AR. CONCLUSION: The present study shows that EA normalizes most of the EV-induced changes in ovarian ARs. Furthermore, EA was able to prevent the EV-induced up regulation of p75NTR, probably by normalizing the sympathetic ovarian response to NGF action. Our data indicate a possible role of EA in the regulation of ovarian responsiveness to sympathetic inputs and depict a possible complementary therapeutic approach to overcoming sympathetic-related anovulation in women with PCOS.

Phytoestrogens inhibit aromatase but not 17beta-hydroxysteroid dehydrogenase (HSD) type 1 in human granulosa-luteal cells: evidence for FSH induction of 17beta-HSD.

BACKGROUND: Studies using purified enzyme preparations, placental microsomes or cell lines have shown that certain phytoestrogens can inhibit the enzymes that convert androgens to estrogens, namely aromatase and 17beta-hydroxysteroid dehydrogenase (HSD) type 1 and type 5. The study aim was to investigate the effects of selected phytoestrogens on aromatase and 17beta-HSD type 1 activity in primary cultures of human granulosa-luteal (GL) cells. METHODS AND RESULTS: GL cells, cultured for 48 h in medium containing 5% fetal calf serum and for a further 24 h in serum-free medium with or without hFSH or hCG, were exposed to steroid substrates during the last 1-4 h of the experiment. The production of progesterone in the presence of pregnenolone or estradiol synthesis from androstenedione, estrone or testosterone showed dose- and time-dependent increases. Whilst hCG priming had no effect on progesterone production, FSH priming induced mean 68 and 56% increases in the production of estradiol from androstenedione (A-dione) and estrone respectively, but had no significant effect on the metabolism of testosterone to estradiol. None of the phytoestrogens investigated had any acute effects on enzyme activity. In contrast, when GL cells were exposed to the compounds for 24 h prior to exposure to steroid substrates for 4 h, 10 micro mol/l apigenin and zearalenone significantly inhibited aromatase activity, whilst biochanin A and quercetin had no effect. None of the phytoestrogens inhibited FSH-induced 17beta-HSD type 1 activity, and only quercetin significantly inhibited progesterone production. CONCLUSIONS: The inability of phytoestrogens to acutely inhibit steroidogenic enzymes in human GL cells (as has been shown in cell-free models) suggests that they are either rapidly metabolized to relatively inactive compounds or that the high enzyme activity in human GL cells masks any inhibitory effects of the compounds at the concentration tested.

Transcriptional regulation of the cyclooxygenase-2 gene changes from protein kinase (PK) A- to PKC-dependence after luteinization of granulosa cells.

This study was designed to elucidate the molecular mechanism(s) mediating cyclooxygenase-2 (Cox-2) regulation during differentiation of the granulosa cell. The 5' flanking sequence of the Cox-2 gene was linked to a vector with a luciferase reporter gene, and this vector was transfected into freshly isolated bovine granulosa cells or granulosa cells after culture with or without forskolin to induce luteinization in vitro. The Cox-2 promoter was inducible by 8-bromo cAMP but not by phorbol esters in fresh granulosa cells, and maximal expression by cAMP was delayed until 48 h after treatment. In contrast, after luteinization of granulosa cells by 8-day treatment with forskolin, the Cox-2 promoter was immediately inducible by phorbol esters but not by cAMP. In granulosa cells cultured for 8 days without forskolin, the Cox-2 promoter continued to be inducible only by cAMP and not by phorbol esters. Unexpectedly, no delay was observed in the induction of Cox-2 by cAMP in granulosa cells that were cultured without forskolin, compared with an approximately 1 day delay in Cox-2 induction by cAMP in fresh granulosa cells. Myristoylated protein kinase (PK) A and PKC inhibitory peptides were utilized to further confirm the PKA- or PKC-dependence of Cox-2 induction. Time-course experiments showed that only 2 days of forskolin treatment could induce PKC-responsiveness of the Cox-2 promoter, although maximal responsiveness was not observed until 10 days of luteinization. Promoter activity was also analyzed in a series of deletion mutants as well as site-directed mutants of C/EBP, CRE, and E-box. A 282-base pair sequence in the Cox-2 5' flanking region maintained full inducibility by PKA in granulosa cells and by PKC in luteinized granulosa cells. The E-box element was found to be the critical regulatory element for Cox-2 induction by either PKA in granulosa cells or by PKC in luteinized granulosa cells. Electrophoretic mobility shift assays were performed on nuclear extracts from fresh or luteinized granulosa cells. Upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box of the Cox-2 gene, and binding was similar for nuclear extracts from fresh, cultured, or luteinized granulosa cells. Thus, although luteinization changes transcriptional regulation of Cox-2 from PKA- to PKC-dependence, the crucial role of the E-box element in this transcriptional activation is conserved.

Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis.

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.

Ginseng flowers stimulate progesterone production from bovine luteal cells.

Our previous report first showed evidence that polysaccharides isolated from ginseng leaves obtained from Jilin, China possess luteotropic activities. In this study, we made further investigations on the root and flowers of Korean ginseng by means of the same bioassay system described briefly as follows. Frozen-thawed bovine luteal cells (1 x 10(5) cells/ml/well) in M199 were incubated in 24-well culture plates at 37 degrees C in a 5 % CO2 incubator. Ten microl of tested drugs with 1, 10 and 100 microg/ml were added into each well. After 4- and 24-hr incubation, the media were harvested and assayed for progesterone by an enzyme immunoassay. The production of progesterone from cells is the indicator for evaluating the action of tested drugs. Results showed that hot water extracts ofginseng flowers (GF-1) with 10 to 100 microg/ml significantly increased progesterone production, whereas those from ginseng root (GR-1) could not. Crude polysaccharides (GF-2) isolated from GF-1 is the active component and the small molecules (mw < 10,000 dalton) are excluded, indicating that the ginseng root has no luteotropic activities, but the polysaccharides of ginseng flowers have.

Effect of selenium on cultured bovine luteal cells.

The present study examined the possibility that selenium (Se) directly affects the corpus luteum (CL) and contributes to the maintenance of reproductive function. Primary culture of bovine CL was used to investigate this possibility. Se addition from 5 to 200 ppb elevated progesterone concentration of the culture medium in a dose-dependent manner which was associated with an increase in cell proliferation rather than an increase in progesterone production per cell, and decreased the lipid peroxide content of luteal cells. However, the luteal cells in which hormone production was increased by LH treatment, accumulated more lipid peroxide than in control incubations. The results suggest that Se is involved in the degradation of lipid peroxides which are by-products of active progesterone production by luteal cells. The increase of cell proliferation observed in this experiment was associated with the removal of these toxic peroxides.

Differential gene expression within the ovine corpus luteum: identification of secreted protein acidic and rich in cysteine as a major secretory product of small but not large luteal cells.

Limited information is available regarding secretory proteins of the corpus luteum (CL), and the potential local role these proteins may play in control of luteal function. An ovine small luteal cell complementary DNA library was immunologically screened with a polyclonal antiserum generated against small cell secretory proteins. A relatively abundant complementary DNA (approximately 2.1 kb) encoding a calcium binding glycoprotein Secreted Protein Acidic and Rich in Cysteine (SPARC) was isolated. Production of SPARC protein by ovine luteal cells was confirmed by immunoprecipitating it from labeled culture medium. Expression of SPARC messenger RNA (mRNA) was examined within CL collected on days 3, 7, 10, 13, and 16 post estrus (n = 4, 4, 4, 3, and 4 respectively), and within pools of purified small (n = 3) and large (n = 4) luteal cells by Northern and dot blot analysis. Amounts of SPARC mRNA increased during the early luteal phase, peaked by day 7 (P < 0.05) and subsequently declined on days 10 and 13 (P < 0.05). SPARC mRNA content was significantly higher in the small than in the large cells (P < 0.003). In situ hybridization showed that SPARC mRNA was localized to the thecal layer of Graafian follicles and to day 3 and day 10 CL. Within CL, immunohistochemistry indicated that SPARC protein was associated with small luteal cells (spindle shaped, avg = 17 microM in diameter) but not with large cells. This specific localization to small cells was confirmed by colocalization of SPARC with 3 beta-hydroxysteroid dehydrogenase. We conclude that SPARC is a major secretory product of small steroidogenic luteal cells of the ovine CL. As SPARC is known to modulate many aspects of tissue reorganization, expression by small luteal cells may play a role in regulation of CL maturation.

Stimulation of progesterone production in bovine luteal cells by co-incubation with bovine blastocyst-stage embryos or trophoblastic vesicles.

A study was conducted to determine whether bovine blastocyst-stage embryos and trophoblastic vesicles stimulate the production of progesterone in bovine luteal cells during incubation in vitro. The effects of co-incubation of these embryos and vesicles with uterine endometrial tissue on progesterone production was also investigated. Bovine small and large luteal cells were obtained on day 12 of the oestrous cycle, dispersed by unit gravity sedimentation and recombined to provide preparations free of accessory cells. Blastocyst-stage embryos were obtained on day 7 and trophoblastic vesicles were obtained from bovine embryos on day 12. A uterine endometrial tissue sample was obtained from the same cow from which the corpus luteum was taken. Treatment groups were arranged in 24-well plates as follows: luteal cells alone; luteal cells and one trophoblastic vesicle; luteal cells and one blastocyst embryo; luteal cells and a 10 mg uterine endometrial sample; luteal cells, one trophoblastic vesicle and a uterine endometrial sample; and luteal cells, one blastocyst embryo and a uterine endometrial sample. All treatment groups were incubated (at 37 degrees C under 5% CO2) in Ham's F-12 medium supplemented with antibiotics (100 micrograms penicillin ml-1 and 100 U streptomycin ml-1, L-glutamine (0.29 mg ml-1), insulin (5 micrograms ml-1), transferrin (5 micrograms ml-1) and selenium (5 ng ml-1) for 12 h. Samples of the medium were harvested 10 min (basal concentration) and 2, 6 and 12 h after incubation to determine the concentrations of progesterone and prostaglandin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of antibiotics and medium supplements on steroidogenesis in cultured cow luteal cells.

Corpora lutea were removed from regularly cycling dairy cows, dissociated with collagenase and cultured for 8 or 10 days in Ham's F-12 medium. In Exp. 1 treatment with insulin, or an insulin-transferrin-selenium combination (ITS), increased progesterone production from basal levels on Day 4 of culture to 234% (P less than 0.01) above controls on Day 10. LH alone increased progesterone production 45% above controls on Day 10 (P greater than 0.05). When LH was combined with insulin or ITS, progesterone production was stimulated to an average of 1802% (P less than 0.01) above controls on Day 10 of culture. Transferrin or selenium without insulin did not allow LH to stimulate progesterone synthesis. In Exp. II, LH alone or LH plus gentamicin or penicillin-streptomycin increased progesterone production from basal levels on Day 2 steadily to an average of 468% (P less than 0.01) above controls (no antibiotics) by Day 8 of culture. The addition of amphotericin-B, alone or in combination with the other antibiotics, inhibited all LH-stimulated progesterone synthesis, but did not affect basal progesterone levels. We conclude that insulin is essential for maximal steroidogenesis in a bovine luteal cell culture system, and that LH-stimulated progesterone production is inhibited in the presence of amphotericin-B, but is not inhibited by gentamicin or penicillin-streptomycin. The elimination of amphotericin-B, coupled with the addition of insulin to the cell culture system increased the responsiveness of the cells to LH. These culture conditions represent the first report in which LH increased total progesterone production for 10 days, maintaining luteal function in a chemically-defined culture system.

Induction of persistent estrus by electrolytic lesions placed neonatally in the hypothalamus of the female rat.

Electrolytic lesions were placed in the hypothalamus of two-day-old female rats. Destruction of the mediobasal part of the preoptic area resulted in persistent vaginal estrus starting on the day of vaginal opening, while lesions placed laterally in the preoptic-anterior hypothalamic area did not interfere with normal cycles. Therefore, the mediobasal hypothalamus is capable of undergoing maturation without any postnatal influence from at least the mediobasal part of the anterior hypothalamus. Destruction of the anterior wall of the third ventricle also caused persistent or prolonged vaginal estrus preceded by normal cycles. The relationship between the loci of lesions and the occurrence of sexual cyclicity was discussed.

Masculine-liky hypothalamic-pituitary axis in the androgen-insensitive genetically male rat pseudohermaphrodite.

The hypothalamic-pituitary sex of the androgen insensitive, genetically male rat pseudohermaphrodite was studied by examining its vaginal cytology, response to ovarian transplants and urinary steroidal excretion patterns. More than half the pseudohermaphrodites studied were in constant vaginal estrus, while the remaining rats displayed either persistent diestrus or irregular cyclicity tending towards lengthened estrus. Following gonadectomy and ovarian transplantation, normal females displayed regular 14-day cycles while pseudohermaphrodites remained in constant vaginal estrus. In pseudohermaphrodites with ovarian transplants, only C19 steroids were detected in the urine while females excreted both C21 and C19 steroids. Indicative of the urinary findings, transplants in females had corpora lutea and maturing follicles while grafts from pseudohermaphrodites and males contained follicular cysts and luteinized theca. In addition, distribution and activity of histochemical 3beta-hydroxy-delta5 steroid oxidoreductase were similar in the grafts from pseudohermaphrodites and males, but unlike the females. Although previous reports have shown that much of the sex-dependent differentiation of the genetic male rat pseudohermaphrodite is phenotypically female, our results suggest that the phenotype of the hypothalamic-pituitary axis of this animal is, at least in certain respects, male.