Changes of ovarian interstitial cell hormone receptors and behavior of resident mesenchymal cells in developing and adult rats with steroid-induced sterility

In the present paper, we report that injection of testosterone propionate (500 microg) during the critical window of rat development (postnatal day 5) induces temporary appearance of aged interstitial cells in developing ovaries (days 7 and 10). Aged interstitial cells showed large size (> or = 12 microm), enhanced androgen receptor (AR) and low estrogen (ER) and luteinizing hormone receptor (LHR) expression. Although normal mature interstitial cells (large size and strong ER and LHR expression) appeared later (day 14), and ovaries of androgenized rats were similar to normal ovaries between days 14 and 35, ovaries of adult androgenized females showed only aged and no mature interstitial cells. Androgenization on day 10 caused the development of aged interstitial cells on day 14, but adult ovaries were normal. Long lasting postnatal estrogenization (estradiol dipropionate for four postnatal weeks) caused in developing and adult ovaries a lack of interstitial cell development beyond the immature state. Immature interstitial cells were characterized by a small size (< or = 7 microm) and a lack of AR, ER and LHR expression. Because the critical window for steroid-induced sterility coincides with the termination of immune adaptation, we also investigated distribution of mesenchymal cells (Thy-1 mast cells and pericytes, ED1 monocyte-derived cells, CD8 T cells, and cells expressing OX-62 of dendritic cells) in developing and adult ovaries. Developing ovaries of normal, androgenized and estrogenized females were populated by similar mesenchymal cells, regardless of differences in the state of differentiation of interstitial cells. However, mesenchymal cells in adult ovaries showed distinct behavior. In normal adult ovaries, differentiation of mature interstitial cells was accompanied by differentiation of mesenchymal cells. Aged interstitial cells in ovaries of androgenized rats showed precipitous degeneration of resident mesenchymal cells. Immature interstitial cells in ovaries of estrogenized rats showed a lack of differentiation of resident mesenchymal cells. These observations indicate that an alteration of interstitial cell differentiation during immune adaptation toward the aged phenotype results in precipitous degeneration of resident mesenchymal cells and premature aging of ovaries in adult rats, and alteration toward immature phenotype results in a lack of differentiation of mesenchymal cells and permanent immaturity of ovaries in adult females.     

Effects of thyroid and luteinizing hormones on the onset of precursor cell differentiation into leydig progenitor cells in the prepubertal rat testis

Leydig cells in the adult rat testis differentiate during the neonatal-prepubertal period. However, the stimulus for the initiation of their differentiation is still not clear. In the present study our objectives were to test the effects of thyroid hormone and LH on the initiation of precursor cell differentiation into Leydig cells in the prepubertal rat testis. Four groups of Sprague-Dawley rats were used. All treatments began at postnatal Day 1. Rats in groups I, II, and III received daily s.c. injections of saline (200 microl, controls), triiodothyronine (T(3), 50 microg/kg body weight, hyperthyroid), and LH (ovine LH 10 microg/rat/day), respectively. Rats in group IV were made hypothyroid from postnatal Day 1 by adding 0.1% propylthiouracil (PTU) to their mother's drinking water. Testes of rats were collected at 7, 8, 9, 10, 11, 12, 16, and 21 days of age, fixed in Bouin's solution, and embedded in paraffin for immunocytochemical studies. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and LH receptors (LHR) in testicular interstitial cells (other than the fetal Leydig cells) was observed using the avidin-biotin method. In control rats, out of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for 3beta-HSD, beginning from the postnatal Day 11. However, positive immunolabeling for LHR was first detected in these cells at Day 12, i.e., after acquiring the steroidogenic enzyme activity. In T(3)-treated rats 3beta-HSD positive spindle-shaped cells were first observed at Day 9 (i.e., 2 days earlier than controls), and LHR-positive cells were first observed on Day 11 (2 days later than obtaining 3beta-HSD immunoactivity); they were exclusively the peritubular mesenchymal cells. The 3beta-HSD- and LHR-positive spindle-shaped cells were absent in the testis interstitium of LH-injected rats from Days 7 through 12 but were present at postnatal Day 16. In addition, more fetal Leydig cell clusters and fetal Leydig cells in mitosis were present in LH-treated rats compared to rats in all other treatment groups. Following their first detection, the number of positive cells for each protein continued to increase at each subsequent age in controls, T(3)-, and LH-injected groups. In PTU rats, 3beta-HSD and LHR-positive spindle-shaped cells were absent throughout the experimental period. From these observations, it is possible to suggest the following regarding the developing rat testis interstitium. 1) The precursor cells for the adult generation of Leydig cells in the postnatal rat testis are the peritubular mesenchymal cells. 2) Luteinizing hormone does not initiate the onset of mesenchymal cell differentiation into Leydig cells, instead it delays this process. However, daily LH treatment causes mitosis in fetal Leydig cells and increase in fetal Leydig cell clusters. 3) Thyroid hormone is critical to initiate the onset of mesenchymal cell differentiation into adult Leydig cells.     

Role of Thy-1+ and Ia+ cells in ovarian function

Cryostat sections of anovulatory ovaries from persistent estrous rats following a single postnatal dose of testosterone and from persistent diestrous rats following long-term postnatal estradiol treatment were investigated. The indirect immunoperoxidase technique was used to localize ovarian Thy-1 and Ia glycoproteins, as well as several other cell surface markers, and the results were compared with those obtained in normal ovaries of cycling females. Folliculogenesis in persistent estrous rats proceeded up to cystic antral follicles and was associated with the occurrence of Thy-1+ stromal cells under follicular basal lamina. In contrast to normal ovaries, Thy-1+ material did not invade the basal layers of granulosa cells. There was also no association of Ia+ cells with follicular basal lamina, but Ia+ cells were usually found associated with some thecal vessels. In persistent diestrous rats folliculogenesis was significantly retarded in both advanced antrum formation and thecal development. Thy-1+ cells were usually present in theca. No Thy-1+ material was found among basal layers of granulosa cells and the depletion of thecal Ia+ cells was almost complete. We suggest that normal follicular development may be dependent on the correct effects of Thy-1+ and Ia+ cells in addition to appropriate gonadotropin and steroid stimulation. On the other hand, anovulatory syndromes following postnatal androgen or estrogen treatment might be induced by temporary direct ovarian effects disturbing the establishment of the normal relationship between follicular structures and the immune system.     

Expression of follicle stimulating hormone and luteinizing hormone receptors during follicular growth in the domestic cat ovary

In order to better understand the pituitary regulation of follicular growth in the domestic cat, follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors (R) were localized and quantified in relation to follicle diameter and atresia using in situ ligand binding on ovarian sections. Expression of FSHR was homogeneous and restricted to follicle granulosa cells from the early antral stage onwards, whereas expression of LHR was heterogeneous on theca cells of all follicles from the early antral stage onward, and homogeneous on granulosa cells of healthy follicles larger than 800 microm in diameter and in corpora lutea. LHR were also widely expressed as heterogeneous aggregates in the ovarian interstitial tissue. Atretic follicles exhibited significantly reduced levels of both FSHR and LHR on granulosa cells, compared with healthy follicles whatever the follicular diameter, whereas levels of LHR on theca cells were lower only for atretic follicles larger than 1,600 microm in diameter. In healthy follicles, levels of FSHR and LHR in all follicular compartments increased significantly with diameter. Although generally comparable to that observed in other mammals, the expression pattern of gonadotropin receptors in the cat ovary is characterized by an early acquisition of LHR on granulosa cells of growing follicles and islets of LH binding sites in the ovarian interstitial tissue.     

Oogenesis in cultures derived from adult human ovaries

Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.     

Unilaminar follicular cells transiently express galectin-3 during ovarian folliculogenesis in pigs

The localization of galectin-3, a β-galactoside-binding animal lectin, was immunohistochemically studied in the ovaries of pigs to determine its expression in ovarian folliculogenesis. Various stages of ovarian follicles were identified in the ovaries of adult pigs. Galectin-3 was immunostained in the squamous follicular cells surrounding oocytes in primordial follicles and in the unilaminar granulosa cells of primary follicles, but not in oocytes of multilaminar follicles (including primary, secondary, and tertiary Graafian follicles). As in adult ovaries, galectin-3 immunoreactivity was prominent in the unilaminar follicles in neonatal ovaries. Galectin-3 was also immunolocalized in the luteal cells in the corpus luteum and granulosa cells of atretic follicles as well as in interstitial macrophages in porcine ovaries. Collectively, these results suggest that galectin-3 is transiently expressed in follicular cells in the unilaminar ovarian follicles (primordial and primary) but not in multilaminar ovarian follicles (primary to tertiary), implying that galectin-3 is embryologically involved in ovum generation.     

Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P <0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P < 0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells.     

E-cadherin-catenin complex in the rat ovary: cell-specific expression during folliculogenesis and luteal formation

The cadherins and their cytoplasmic counterparts, the catenins, form the adherens junctions, which are of importance for tissue integrity and barrier functions. The development and maturation of the ovarian follicle is characterized by structural changes, which require altered expression or function of the components involved in cell-cell contacts. The present study examined the cell-specific localization and temporal expression of epithelial cadherin (E-cadherin) and alpha- and beta-catenin during follicular development, ovulation and corpus luteum formation in the immature gonadotrophin- and oestrogen-stimulated rat ovary. Immunohistochemistry and immunoblotting demonstrated the expression of E-cadherin in theca and interstitial cells of immature ovaries before and after injection of equine chorionic gonadotrophin (eCG). E-cadherin was not detected in granulosa cells, except in the preantral follicles located to the inner region of the ovary. The content of E-cadherin in theca and interstitial cells decreased after an ovulatory dose of hCG. Granulosa cells of apoptotic follicles did not express E-cadherin. Oestrogen treatment (diethylstilboestrol) of immature rats for up to 3 days did not result in a measurable expression of E-cadherin in granulosa cells. alpha- and beta-catenin were expressed in all ovarian compartments. The concentration of beta-catenin was constant during the follicular phase, whereas the content of alpha-catenin decreased in granulosa cells after treatment with diethylstilboestrol or hCG. The expression of alpha-catenin was also reduced in theca and interstitial cells after hCG. alpha- and beta-catenin were present in most ovarian cells at all stages of folliculogenesis. Therefore, the catenins have the potential to associate with different members of the cadherin family and to participate in the regulation of cytoskeletal structures and intracellular signalling. The restricted expression of E-cadherin in granulosa cells of preantral follicles indicates a role in the recruitment of these follicles to subsequent cycles. The specific decrease of alpha-catenin in granulosa cells and the reduction of both alpha-catenin and E-cadherin in theca cells of ovulatory follicles might reflect some of the molecular changes in cell-cell adhesion associated with ovulation and luteinization.     

Polycystic ovary syndrome: interaction of follicle stimulating hormone and polypeptide growth factors in oestradiol production by human granulosa cells

The mechanism of the ovarian dysfunction in polycystic ovary syndrome, the most common cause of anovulatory infertility, remains obscure. Clinical data suggest that follicle stimulating hormone (FSH) action may be inhibited at the ovarian level by paracrine factors derived, presumably, from interstitial cells. The greater responsiveness to FSH of granulosa cells isolated from polycystic ovaries (PCO) compared with that seen in cells derived from normal ovaries, provides some support for this hypothesis and we present data which suggests that epidermal growth factor, or more likely transforming growth factor alpha, could be a candidate for this inhibitor. It should be emphasized, however, that the cardinal biochemical feature of the PCO is hypersecretion of androgens by interstitial cells. Stromal tissue from the PCO will secrete significant quantities of androstenedione in response to LH, whereas there is a negligible response in stroma from normal ovaries. It remains to be determined whether androgens have a direct inhibitory effect on FSH-induced oestradiol production in the human follicle, or whether they might exert an indirect effect by activating inhibitory polypeptide growth factors.     

Effects of methoxychlor on IGF-I signaling pathway in rat ovary

Follicular development and other ovarian functions are regulated by growth factors that can be affected by exogenous agents. Methoxychlor (MXC) is an organochloride pesticide that causes female infertility. We investigated how MXC affects the distribution of developing ovarian follicles in adult rats after treatment between embryonic day (E) 18 and postnatal day (PND) 7. We also measured insulin-like growth factor-I (IGF-I) and its receptor, IGF-IR, expressions in ovarian follicles and investigated whether MXC changed the levels of IGF-I and IGF-IR in the ovary. Using immunohistochemical (IHC) staining, we detected IGF-I expression in oocytes and granulosa cells of the follicles, luteal cells, interstitial cells, theca externa and theca interna, and the smooth muscle of ovarian vessels. IGF-IR was co-localized with IGF-I in the ovary except for the theca externa. IGF-I expression was decreased in granulosa cells of preantral and antral follicles after treatment with MXC compared to granulosa cells of preantral and antral follicles of the control group. We also observed that oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the MXC treated groups showed increased IGF-IR expression compared to oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the control group. We also detected more secondary and preantral follicles, and fewer primordial and antral follicles after MXC administration compared to controls. Therefore, the IGF signaling pathway may participate in MXC induced ovary dysfunction and female infertility.     

Immunolocalization of GnRHRI,gonadotropin receptors,PGR, and PGRMCI during follicular development in the rabbit ovary

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.     

Origin of germ cells and formation of new primary follicles in adult human ovaries

Recent reports indicate that functional mouse oocytes and sperm can be derived in vitro from somatic cell lines. We hypothesize that in adult human ovaries, mesenchymal cells in the tunica albuginea (TA) are bipotent progenitors with a commitment for both primitive granulosa and germ cells. We investigated ovaries of twelve adult women (mean age 32.8 ± 4.1 SD, range 27–38 years) by single, double, and triple color immunohistochemistry. We show that cytokeratin (CK)+ mesenchymal cells in ovarian TA differentiate into surface epithelium (SE) cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex are overgrown by TA, forming solid epithelial cords, which fragment into small (20 micron) epithelial nests descending into the lower ovarian cortex, before assembling with zona pellucida (ZP)+ oocytes. Germ cells can originate from SE cells which cover the TA. Small (10 micron) germ-like cells showing PS1 meiotically expressed oocyte carbohydrate protein are derived from SE cells via asymmetric division. They show nuclear MAPK immunoexpression, subsequently divide symmetrically, and enter adjacent cortical vessels. During vascular transport, the putative germ cells increase to oocyte size, and are picked-up by epithelial nests associated with the vessels. During follicle formation, extensions of granulosa cells enter the oocyte cytoplasm, forming a single paranuclear CK+ Balbiani body supplying all the mitochondria of the oocyte. In the ovarian medulla, occasional vessels show an accumulation of ZP+ oocytes (25–30 microns) or their remnants, suggesting that some oocytes degenerate. In contrast to males, adult human female gonads do not preserve germline type stem cells. This study expands our previous observations on the formation of germ cells in adult human ovaries. Differentiation of primitive granulosa and germ cells from the bipotent mesenchymal cell precursors of TA in adult human ovaries represents a most sophisticated adaptive mechanism created during the evolution of female reproduction. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. An essential mission of such follicular turnover might be elimination of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles.     

Studies on the onset of Leydig precursor cell differentiation in the prepubertal rat testis

Leydig cells of the adult rat testis differentiate postnatally from spindle-shaped cells in the testis interstitium during the neonatal-prepubertal period. Which spindle-shaped cell types are the precursor for Leydig cells and the stimulus for initiation of their differentiation are, however, two unresolved issues. In the present study, our objectives were to identify unequivocally which spindle-shaped cells are the precursors to Leydig cells and to test whether the initiation of their differentiation into Leydig cells depends on LH. Testes from fifteen groups of Sprague-Dawley rats (n = 4 per group) from 7-21 days of age were fixed in Bouin solution and embedded in paraffin. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome P450 side-chain cleavage (P450(scc)), 17alpha-hydroxylase cytochrome P450 (P450(c17)), and LH receptors (LHR) in interstitial cells (other than fetal Leydig cells) was observed using the avidin biotin method. Of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for all three steroidogenic enzymes, beginning from the 11th postnatal day. All three enzymes were expressed simultaneously in these cells, and their numbers increased significantly thereafter. Immunoexpression of LHR in a few of these cells was just evident for the first time on postnatal Day 12 (i.e., after acquiring the steroidogenic enzyme activity). Their numbers gradually increased with time. The number of immunolabeled cells per 1000 interstitial cells (excluding fetal Leydig cells and capillary endothelial cells) was not significantly different for the three steroidogenic enzymes tested at all ages; however, a lower value was observed for LHR at each time-point. Based on these observations, we suggest that 1) the precursor cell type for the adult generation of Leydig cells in the postnatal rat testis is the peritubular mesenchymal cells, 2) precursor cells acquire 3beta-HSD, P450(scc), and P450(c17) enzyme activity simultaneously during Leydig cell differentiation, and 3) onset of precursor cell differentiation during Leydig cell development does not depend on LH.     

Direct ovarian effects of a GnRH agonist in hypophysectomized immature rats: inhibition of theca-interstitial luteinizing hormone receptor mRNA expression and induction of interstitial cell differentiation

The effect of a gonadotropin-releasing hormone (GnRH) agonist on luteinizing hormone (LH) receptor mRNA expression was examined histologically in the ovaries of immature hypophysectomized (HPX) rats by in situ hybridization. In the ovaries of HPX rats treated with diethylstilbestrol (DES) and pregnant mare serum gonadotropin (PMSG), LH receptor mRNA was expressed in the granulosa cells of mature follicles as well as the theca-interstitial cells. In DES-primed ovaries of rats treated with both GnRH agonist plus PMSG, many follicles were luteinized without ovulation, and the signal of LH receptor mRNA disappeared completely in the theca-interstitial cells as well as the luteinized cells, but remained in the granulosa cells of unaffected mature follicles. The complete suppression of the theca-interstitial LH receptor expression by GnRH agonist was also observed in HPX rats that received no other treatment. On the other hand, the coadministration of a GnRH antagonist with PMSG resulted in the hyperstimulation of follicular growth, accompanied by very strong expression of LH receptor mRNA in the granulosa cells as well as the thecainterstitial cells. In addition, morphological changes in the ovarian interstitial cells were also induced by the administration of GnRH agonist in HPX rats: loose connective tissue decreased and the interstitial cell mass markedly increased. The increase of the interstitial cells became more prominent when rats were treated with GnRH agonist and testosterone simultaneously. These results suggest that GnRH may be an important factor for modulating the interstitial cell function and differentiation in the rat ovary.     

Immunohistochemical and mRNA localization of the angiotensin II receptor subtype 2 (AT2) in follicular granulosa cells of the rat ovary.

A local renin-angiotensin system (RAS), including specific angiotensin II receptor subtypes, is present in the rat ovary. Immunohistochemistry using a polyclonal antibody and mRNA in situ hybridization were performed on perfusion-fixed, paraffin-embedded ovaries obtained from untreated sexually mature, normally cycling rats. Immunofluorescent staining revealed an exclusive and distinct labeling of follicular granulosa cells showing a plaque-like expression pattern at the cell borders, being detectable in different stages of atretic degeneration. On adjacent sections the expression of the respective mRNA could be shown in granulosa cells of the same follicle. The AT2 receptor may be implicated in the ovarian atretic process by influencing follicular cell-cell communication.     

Evidence for a granulosa cell site of dihydrostestosterone metabolism in the adult rat ovary

The ovarian enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) converts dihydrotestosterone (DHT) to 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), a reduced androgen that does not bind to the granulosa cell androgen receptor. To determine the relative contribution of the granulosa cells to total ovarian 3 alpha-HSD activity, adult rats treated with either medroxyprogesterone acetate (MPA) or vehicle underwent ovarian microdissection. 3 alpha-Hydroxysteroid dehydrogenase is primarily located in excised follicles and corpora lutea, and is inhibited in the follicles but not corpora lutea by MPA (P less than 0.05). Elimination of healthy granulosa cells while maintaining healthy theca cells by irradiation of the exteriorized ovaries with 6000 rads resulted in a marked reduction in 3 alpha-HSD to 19% of control levels on a per-organ basis (P less than 0.01). The granulosa cell is the major ovarian site for 3 alpha-hydroxylation of ring A-reduced C19 steroids in the adult rat.