Activity of an enzyme converting single-chain tissue-type plasminogen activator to the two-chain form in preovulatory human follicular fluid

The biological role of the tissue-type plasminogen activator (tPA)/plasmin system has long been implicated in ovarian function. We have recently shown that the follicular fluid of human ovaries contains an alpha(2)-macroglobulin/protease complex capable of converting single-chain (sc) tPA to the two-chain (tc) enzyme tPA, suggesting the occurrence of its corresponding enzyme in a free form in the fluid. The aim of the current study is therefore to gain further information about the putative sctPA-converting enzyme present in follicular fluid. Incubation of human recombinant sctPA with the fluid brought about the production of tctPA. It was also demonstrated that tctPA production resulted in the activation of endogenous fluid plasminogen. Production of tctPA and plasmin both was strongly inhibited by aprotinin, suggesting that the enzyme is a serine protease. The sctPA-converting enzyme was partially purified from the fluid by column chromatographies. The enzyme preferably hydrolyzed synthetic peptide substrates containing arginine at the P(1) position. The enzyme preparation had a protease inhibitor profile similar to that observed with the crude fluid sample. These results clearly demonstrated that follicular fluid contains an enzyme capable of efficiently converting sctPA to tctPA. Discovery of this sctPA-converting enzyme strongly suggests that the tPA/plasmin system in the preovulatory follicle of human ovaries is operated through the proteolytic conversion of sctPA to tctPA rather than being regulated by a fibrin-dependent mechanism.     

Ovarian sympathectomy in the guinea pig

The influence of ovarian adrenergic nerves on follicular growth during the estrous cycle in the adult guinea pig was ascertained by comparing follicular development in control and chemically sympathectomized ovaries from the same animal. Selective ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian membranous sac (bursa) on day 2 of the cycle (day 1 = day of estrus). The contralateral surgically closed ovarian bursa was injected with solvent used for 6-hydroxydopamine. Animals were laparotomized on days 5, 10 and 14 of the cycle. Blood from the utero-ovarian vein was collected bilaterally for measurement of progesterone and androstenedione. The ovaries were processed for histologic examination, and the number of follicles in each ovary was analyzed morphometrically. Sympathectomy on day 2 caused a decrease in healthy preovulatory follicles (greater than 700 micron diameter) on day 10 of the cycle. There were no differences in ovarian weights or the total number of follicles per ovary at this time. On days 5 and 14 of the cycle, there were no differences in ovarian weights, total number of follicles per ovary or follicles in any size classification. Sympathectomy did not alter progesterone levels in the utero- ovarian vein as compared to contralateral control levels. From control ovaries, there was a significant increase in progesterone in the blood of the utero-ovarian vein on day 10 but venous levels of progesterone from sympathectomized ovaries were not significantly different at any day of the cycle. In the venous effluent from sympathectomized ovaries, androstenedione was elevated at day 5 compared to days 10 and 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrafollicular steroids and anti-mullerian hormone during normal and cystic ovarian follicular development in the cow

Development of follicular cysts is a frequent ovarian dysfunction in cattle. Functional changes that precede cyst formation are unknown, but a role for anti-Müllerian hormone (AMH) in the development of follicular cysts has been suggested in humans. This study aimed to characterize intrafollicular steroids and AMH during follicular growth in a strain of beef cows exhibiting a high incidence of occurrence of follicular cysts. Normal follicular growth and cyst development were assessed by ovarian ultrasonography scanning during the 8 days before slaughtering. Experimental regression of cysts was followed by rapid growth of follicles that reached the size of cysts within 3-5 days. These young cysts exhibited higher intrafollicular concentrations of testosterone, estradiol-17beta, and progesterone than large early dominant follicles did in normal ovaries, but they exhibited similar concentrations of AMH. Later-stage cysts were characterized by hypertrophy of theca interna cells, high intrafollicular progesterone concentration, and high steroidogenic acute regulatory protein mRNA expression in granulosa cells. Progesterone and AMH concentrations in the largest follicles (> or =10 mm) and cysts were negatively correlated (r = -0.45, P < 0.01). Smaller follicles (<10 mm) exhibited higher intrafollicular testosterone and estradiol-17beta concentrations in ovaries with cysts compared to normal ovaries. During follicular growth, AMH concentration dropped in follicles larger than 5 mm in diameter and in a similar way in ovaries with and without cysts. In conclusion, enhanced growth and steroidogenesis in antral follicles <10 mm preceded cyst formation in cow ovaries. Intrafollicular AMH was not a marker of cystic development in the cow, but low AMH concentrations in cysts were associated with luteinization.     

Can ovarian infertility be treated with bone marrow- or ovary-derived germ cells?

A year ago, reproductive biologists and general public were astonished with evidence reported by Johnson et al. in Nature 428:145 that mammalian ovaries possess persisting large germline stem cells, which allegedly enable follicular renewal in adult females. Recently, the same research group declared such view obscure, and reported that mammalian oocytes originate from putative germ cells in bone marrow and are distributed by peripheral blood to the ovaries (Cell 122:303). While neglecting available data on the germ cell origin from the ovarian surface epithelium (OSE) in adult mouse and human females and complexity of follicular renewal in humans, the authors widely extrapolated their observations on formation of allogeneic oocytes after bone marrow (or blood) transplantation in ovaries of adult mice treated with cytostatics to clinical implications in the public media. Yet, the resulting outcome that such allogeneic oocytes may enable the propagation of ovarian cycles is a poor alleviation for the women with ovarian infertility. Women lacking primary follicles, or carrying follicles with low quality eggs persisting in aging ovaries, are not concerned about the lack of menstrual cycles or ovarian steroids, but about virtually no chance of having genetically related children. Johnson et al. also reported that the germ cell formation in bone marrow disappears in ovariectomized mice. Such observation, however, raises solid doubts on the bone marrow origin of oocytes. Since germ cells developing from the OSE cells of adult human ovaries during periodical follicular renewal are known to enter blood vessels in order to enable formation of primary follicles at distant ovarian sites, they also contaminate peripheral blood and hence bone marrow. Better knowledge on the complexity of follicular renewal in humans and exploration of a potential of human OSE cells to produce new oocytes in vitro are essential for novel approaches to the autologous treatment of premature ovarian failure and age induced ovarian infertility.     

Ovarian follicles during infancy in Romanov and Ile-de-France ewe lambs

The ovaries of new born lambs (15 Ile-de-France and 19 Romanov, 34 ovaries) and of 4-week-old lambs (6 Ile-de-France and 12 Romanov, 18 ovaries) were examined histologically to compare ovarian follicular development in infant lambs of breeds differing in their prolificacy. Breed was the major factor affecting follicular population at birth. Ile-de-France lambs had a higher total number of growing follicles (P less than 0.001), and more preantral (P less than 0.001) and antral (P less than 0.005) follicles than did Romanov lambs. Furthermore, the size of the largest follicles was also reduced in Romanov compared to Ile-de-France lambs. At 4 weeks of age, most of the features of the ovarian follicular population except the mean size of the third largest follicle were similar between the two breeds. However, atresia of antral follicles had appeared only in Ile-de-France and not in Romanov lambs. When a challenge with exogenous gonadotrophins (1000 i.u. PMSG followed by 1500 i.u. hCG) was attempted, ovulation was triggered in 2/6 and 0/12 Ile-de-France and Romanov lambs respectively. Massive follicular development was noted in 3/6 Ile-de-France lambs but in none of 12 Romanov lambs. Retardation of follicular development together with retardation in the establishment of ovarian sensitivity to gonadotrophins are therefore features typical of the ovaries of Romanov lambs compared to Ile-de-France lambs during the post-natal period.     

Requirement for ADAMTS-1 in extracellular matrix remodeling during ovarian folliculogenesis and lymphangiogenesis

Murine ovarian folliculogenesis commences after birth involving oocyte growth, somatic cell differentiation and structural remodeling of follicle stromal boundaries. The extracellular metalloproteinase ADAMTS-1 has activity against proteoglycans and collagen and is produced by the granulosa cells of ovarian follicles. Mice with ADAMTS-1 gene disruption are subfertile due to an unknown mechanism resulting in severely reduced ovulation. Here we show that ADAMTS-1 is necessary for structural remodeling during ovarian follicle growth. A significant reduction in the number of healthy growing follicles and corresponding follicle dysmorphogenesis commencing at the stage of antrum formation was identified in ADAMTS-1-/- ovaries. Morphological analysis and immunostaining of basement membrane components identified stages of follicle dysgenesis from focal disruption in ECM integrity to complete loss of follicular structures. Cells expressing the thecal marker Cyp-17 were lost from dysgenic regions, while oocytes and dispersed cells expressing the granulosa cell marker anti-mullerian hormone persisted in ovarian stroma. Furthermore, we found that the ovarian lymphatic system develops coincidentally with follicular development in early postnatal life but is severely delayed in ADAMTS-1-/- ovaries. These novel roles for ADAMTS-1 in structural maintenance of follicular basement membranes and lymphangiogenesis provide new mechanistic understanding of folliculogenesis, fertility and disease.     

A novel serine protease (IRCM-serine protease 1) from porcine neurointermediate and anterior pituitary lobes. Isolation, polypeptide chain structure, inhibitor sensitivity, and substrate specificity with fluorogenic peptide substrates

A novel serine protease, which we have called IRCM-serine protease 1, was purified from both porcine neurointermediate and anterior pituitary lobes. The enzyme was inhibited by soybean trypsin inhibitor, pancreatic trypsin inhibitor, benzamidine, phenylmethyl-sulfonyl fluoride, and thiol reagents including HgCl2, p-chloromercuribenzoate, and 5,5'-dithiobis-(2-nitrobenzoic acid) and was resistant to lima bean trypsin inhibitor, alpha 2-macroglobulin, alpha 1-antitrypsin, and C1-esterase inhibitor. IRCM-serine protease 1 displayed "trypsin-like" specificity toward a number of tripeptide coumarin-containing substrates, with kcat/km values ranging from 10(4) to 10(6) M-1 S-1. The best substrate was benzyloxycarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide with a kcat/Km value of 2.27 X 10(6) M-1 S-1. IRCM-serine protease 1, Mr = 169,000-190,000 determined by gradient gel electrophoresis and gel filtration, respectively, appears to be a homologous dimer. The monomeric subunits of the enzyme are composed of an Mr = 38,000 polypeptide chain which is modifiable by 125I-D-Tyr-Glu-Phe-Lys-Arg-CH2Cl, disulfide-linked to another polypeptide resulting in a subunit molecular weight of 88,000.     

Inhibition of gonadotropin sensitive adenylate cyclase by ovarian follicular fluid.

Follicular fluid obtained from medium or large bovine ovarian follicles inhibited ovarian luteinizing hormone/human chorionic gonadotropin sensitive adenylate cyclase in a dose-dependent manner (I₅₀ = 3 mg follicular fluid protein/ml). The inhibitory activity was excluded by Sephadex G-10 and was fully retained following treatment with charcoal. Fluoride-stimulated enzyme activity was not inhibited. Binding of ¹²⁵I human chorionic gonadotropin to ovarian plasma membranes was only slightly reduced by the follicular fluid. The post-microsomal supernatant of homogenates from ovaries of immature (27-day-old) rats collected 24–36 h after treatment with 15 i.u. of pregnant mare serum gonadotropin also inhibited luteinizing hormone-sensitive adenylate cyclase. The extent of this inhibition seemed to decline with follicular maturation. The possibility is raised that ovarian sulfated glycosaminoglycans are responsible for the observed inhibition of adenylate cyclase.     

Increase in ovarian kallikrein activity during ovulation in the gonadotrophin-primed immature rat

The ovulatory process was initiated in 25-day-old rats by injecting them with hCG (10 i.u., s.c.) 2 days after the animals had been primed with PMSG (10 i.u., s.c.). At 2-h intervals after hCG, the ovaries were extracted and assayed for glandular kallikrein activity by using a chromogenic substrate (H-D-Val-Leu-Arg-p-nitroanilide) which exhibits optical density (at 405 nm) upon hydrolysis. In 0-h control ovaries the activity was 12.5 x 10(-3) kallikrein units (KU)/mg protein and it increased to a peak of 56.6 x 10(-3) KU/mg at 12 h after hCG, when the follicles first began to rupture. The kallikrein activity was distinguishable from ovarian plasminogen activator activity on the basis of pH optima and response to trypsin inhibitor (SBTI). The activity was inhibited by a s.c. dose of indomethacin of 0.3 mg/rat, or higher, and this dosage inhibited ovulation. The results suggest that kallikrein activity contributes to the degradation of Graafian follicles during ovulation in mammals.     

Development of codominant follicles in cattle is associated with a follicle-stimulating hormone-dependent insulin-like growth factor binding protein-4 protease.

Low molecular weight insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-4, are believed to inhibit the actions of insulin-like growth factors (IGFs). We showed previously that ovarian follicular dominance in cattle is associated with the presence of a protease that degrades IGFBP-4. To test the hypothesis that specific IGFBP-4 proteolysis is associated with selection of the dominant follicle, we induced codominant follicles (co-DFs) during the first follicular wave of the estrous cycle. The ovaries of Holstein heifers were examined twice daily by ultrasonography; when the largest follicle reached 6 mm in diameter, saline (control, n = 5) or 2 mg of recombinant bovine (rb) FSH (FSH, n = 5) was injected i.m. every 12 h for 48 h. Follicular fluid was collected by aspiration from the two largest follicles/heifer 12 h after the last injection. IGFBPs in follicular fluid were quantified by Western ligand blotting/phosphorimaging. IGFBP-4 protease activity was measured by incubating follicular fluid with recombinant human (rh) IGFBP-4 substrate, followed by ligand blotting/phosphorimaging to quantify the percent of substrate loss and Western immunoblotting to detect specific proteolytic fragments. Co-DFs of FSH heifers did not differ (P > 0.05) from the single dominant follicle of controls in size, or in concentration of progesterone or level of IGFBP-4 in follicular fluid. In contrast, the largest subordinate follicle of control heifers was smaller, with lower progesterone and higher IGFBP-4 in the follicular fluid (P < 0.05). Concentrations of estradiol in follicular fluid were high in dominant follicles, intermediate in co-DFs, and low in subordinate follicles (P < 0.05). IGFBP-4 protease activity in co-DFs was similar (P > 0.05) to that of dominant follicles, but fourfold higher (P < 0.05) than that of subordinate follicles. The results strongly suggest that an FSH-dependent IGFBP-4 protease is associated with selection of the dominant follicle in cattle.     

Gene expression pattern of adiponectin and adiponectin receptors in dominant and atretic follicles and oocytes screened based on brilliant cresyl blue staining

Adiponectin and its receptors (AdipoR1 and AdipoR2) are novel endocrine systems that act at various levels to control male and female fertility. The aim of this study was to determine whether adiponectin and its receptors gene expression levels differ between dominant follicle (DF) and atretic follicle (AF) and also between oocytes which were stained positively and negatively with brilliant cresyl blue (BCB(+) and BCB(-)). Based on estradiol/progesterone ratio, follicles from ovaries were classified as AFs and DFs. The stages of estrous cycle (follicular or luteal phases) were defined by macroscopic observation of the ovaries and the uterus. Oocytes were stained with BCB for 90 min. The relative expression of adiponectin, AdipoR1 and AdipoR2 mRNA in theca and cumulus cells and oocytes of different follicles were determined by quantitative real time PCR. Adiponectin and its receptors genes were clearly expressed higher (P<0.05) in theca and cumulus cells and oocytes of DFs than those of AFs during the follicular and luteal phases. BCB(+) oocytes showed a higher (P<0.05) expression of adiponectin and its receptors compared with their BCB(-) counterparts. Positive correlation (r>0.725, P<0.001) was observed between adiponectin mRNA level in ovarian cells of DFs and follicular fluid E2 concentration in follicular phase. Adiponectin mRNA abundance in ovarian cells of AFs showed a significant negative correlation with follicular fluid progesterone concentration in follicular and luteal phases (r<-0.731, P<0.001). This work has revealed the novel association of adiponectin and its receptors genes with follicular dominance and oocyte competence, thereby opening several new avenues of research into the mechanisms of dominance and competence in animal and human.     

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.     

Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Biochemical and hormonal characterization of follicles from follicular and luteal phase ovaries of goat and sheep.

Follicles from goat and sheep ovaries were characterized for their biochemical and hormonal parameters to investigate the effect of developmental stage of follicles on ovarian steroidogenesis. The follicles were isolated mechanically from follicular and luteal phase ovaries and divided in 6 morphologically different groups (small, medium and large follicular and small, medium and large luteal). Follicles were characterized for their contents of protein, DNA, estradiol-17 beta and progesterone and the activity of 3 beta-hydroxysteroid dehydrogenase. There was a progressive increase in the contents of all these biomolecules and activity of the enzyme as size of follicles increased in both the follicular and luteal phase ovaries. Follicles from follicular phase ovaries exhibited higher estradiol-17 beta content than those shown by luteal phase follicles. The reverse pattern was obtained for progesterone content. The results provide the basic data on biochemical and hormonal entities at different stages of follicular development in small ruminants which may be useful for in vitro studies on regulation of follicular development and steroidogenesis.     

The role of ovarian proteases and their inhibitors in ovulation

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.     

Ovarian follicular atresia is mediated by heterophagy,autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Ovarian follicular cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol

The occurrence of follicular cell hyperplasia was studied by light and electron microscopy in fetal mouse ovaries exposed to ethinyl estradiol (EE) from day 11 through day 17 of pregnancy. Pregnant mice were given EE in olive oil (0.02, or 0.2 mg/kg of body weight) and were sacrificed on day 18. The female fetuses were examined for ovarian histogenesis. Follicular cell hyperplasia was detected in both of the experimental groups, but the incidence was statistically significant only in fetuses exposed to 0.2 mg/kg of EE. Light and electron microscopic observations of the ovaries showed that the hyperplasia was located in the medullary region, and the follicular cells showed pleomorphism. Accumulation of abundant lipid droplets, enlarged rough endoplasmic reticulum with granular material, dense bodies, and vague masses of fibrous structures were seen in the cytoplasm. These morphological observations indicate that hyperplasia of follicular cells in fetal mouse ovaries at term can be induced by prenatal treatment with EE.