Effect of genetically defined oocyte depletion on production of androgens and oestrogens by ovaries of suckling mice

Steroid production and histological features of ovaries were compared either among normal +/+ mice of 3-12 days of age or among 12-day old mutant mice with various degrees of oocyte depletion. Whole ovaries were cultured in the medium containing [3H]progesterone and hCG or 4-androstene-3,17-dione and FSH; amounts of [3H]androgens or oestrogens released from the ovaries were assayed. FSH-responsive aromatase activity was detectable in ovaries of +/+ mice on day 3 after birth (2.6 +/- 0.4 pmol/2 ovaries/48 h), but the activity producing androgens from progesterone, under stimulation of hCG, was not detectable even on day 6 after birth (less than 0.1 pmol/2 ovaries/48 h). The androgen-producing activity appeared on day 9 after birth (1.16 +/- 0.25 pmol/2 ovaries/48 h), when follicles with more than two layers of granulosa cells developed. The ovaries of 12-day old Sl/Slt mice contained a considerable number of follicles with a single layer of granulosa cells, but did not contain any follicles with more than two layers of granulosa cells. The ovaries of Sl/Slt mice possessed aromatase activity (3.3 +/- 0.4 pmol/2 ovaries/48 h) but, not androgen-producing activity (less than 0.1 pmol/2 ovaries/48 h). The present results suggest that development of follicles with more than two layers of granulosa cells may induce the activity producing androgens from progesterone under stimulation of LH in suckling mouse ovaries, though the FSH-responsive aromatase activity is present even in follicles with a single layer of granulosa cells.     

Haemopoietic stem cell proliferation in the bone marrow of S1/S1d mice

In marrow from Sl/Sld mice (but not +/+ mice) day 7 and day 8 CFU-S proliferate whilst day 10 and day 12 CFU-S exhibit negligible proliferation. Media conditioned by both +/+ and Sl/Sld marrow contains an inhibitor of CFU-S proliferation but day 8 CFU-S in +/+ and Sl/Sld marrow show marked dose-response differences to this factor. To inhibit the proliferation of Sl/Sld CFU-S required approximately ten times the concentration of inhibitor that inhibited the proliferation of +/+ CFU-S. Thus abnormally responsive day 8-CFU-S were shown to proliferate in an inhibitory environment. Abnormalities in Sl/Sld CFU-S function were also demonstrated in heterotopic transplantation experiments using +/+ and Sl/Sld donors and hosts to obtain ectopic bone marrow with various stromal (donor) and haemopoietic (host) combinations. Day 8 Sl/Sld CFU-S were seen to proliferate, irrespective of whether the stromal environment was derived from Sl/Sld or +/+ marrow. Sl/Sld mice are generally regarded as animals in which there is a genetically determined defect in haemopoiesis due to an abnormality in the haemopoietic environment. It is difficult, however, to attribute the abnormal CFU-S behaviour in these experiments to environmental factors and the results are consistent with mutation at the Sl locus affecting the responses of CFU-S to regulatory signals, i.e. the genetic defect is not confined to the stromal environment.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. I. Different distribution patterns between melanocytes and mast cells in the skin

In spite of their different origin, both melanocytes and mast cells are deficient in the skin of mutant mice of the Sl/Sld genotype. Since the neural crest and the liver of Sl/Sld embryos contain normal precursors of melanocytes and mast cells, respectively, the deficiency is attributed to a defect in tissue environment necessary for migration and/or differentiation of precursor cells. We investigated whether the tissue environment used for differentiation of melanocytes and mast cells was identical by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent pigmented and nonpigmented stripes were obtained. In the nonpigmented stripes of these Sl/Sld in equilibrium with +/+ chimaeras, melanocytes were not detectable in hair follicles but were detectable in the dermis. In contrast, melanocytes were detectable neither in hair follicles nor in the dermis of nonchimaeric Sl/Sld mice. Concentrations of mast cells were comparable in the pigmented and nonpigmented stripes of Sl/Sld in equilibrium with +/+ chimaeras, but the average concentration of mast cells significantly varied in the chimaeras (from 8% to 74% of the value observed in control +/+ mice). The present result suggests that mesodermal cells that support the migration and differentiation of both melanocyte precursors and mast-cell precursors mix homogeneously in the dermis and that ectodermal cells that influence the invasion of differentiating melanocytes into hair follicles make discrete patches.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

Functionally abnormal stromal cells and megakaryocyte size, ploidy, and ultrastructure in Sl/Sld mice

The first goal of the present studies was to determine if Sl/Sld megakaryocytes have features in common with the macrocytic megakaryocytes that genetically normal mice produce in response to acute platelet depletion. The second was to test the hypothesis that megakaryocyte abnormalities in Sl/Sld mice are due to genetically determined hemopoietic stromal cell abnormalities. Sizes and ploidies of mature Sl/Sld megakaryocytes were measured. Macrocytosis and a shift to higher ploidy values were found compared with normal. Within ploidy groups 16N-64N, Sl/Sld megakaryocytes were larger than normal megakaryocytes of the same ploidy. Transmission electron microscopy revealed that Sl/Sld megakaryocyte nuclei contain more and larger nucleoli, and the chromatin was more dispersed than in normal megakaryocyte nuclei of comparable maturity. Asynchronous megakaryocyte cytoplasmic maturation was found. Sl/Sld macrophages were also ultrastructurally abnormal. Megakaryocytic macrocytosis was reproduced in long-term bone marrow cultures in which the adherent layer was formed by Sl/Sld cells. It was the same if cultures were recharged with Sl/Sld or +/+ hemopoietic cells. Previously reported ambiguities in mixed cell cultures were avoided by recharging the adherent layers with only a million cells. These results were correlated with previously published observations. Sl/Sld megakaryocytes have features in common with megakaryocytes from acutely thrombocytopenic animals. One feature, macrocytosis, appears to be due to abnormal Sl/Sld stromal cells that are reproduced as adherent layer cells in long-term cultures. The responsible stromal cells in Sl/Sld mice may be counterparts of megakaryocytopoietic regulatory cells in the marrow stroma of normal animals.     

Sl/Sld mice have an increased number of gap junctions in their bone marrow stromal cells

To study the defect of the hematopoietic inductive microenvironment (HIM) in Sl/Sld mice, femoral bone marrow tissue of 10 of each mutant, (Sl/Sld and W/Wv) their normal littermates (Sl+/Sl+ and W+/W+), and 20 normal C57BL mice were examined by electron microscopy using morphometric and statistical methods. Gap junctions were observed in all strains of mice, in the following stromal cell types: 1) reticular cells, 2) between reticular cells and periarterial adventitial cells, and 3) between periarterial adventitial cells. The frequency of gap junctions in bone marrow stromal cells of Sl/Sld mice (mean = 2.2/9.4 X 10(-3) mm2) was significantly higher than in control mice. It is suggested that there is a relationship between the increased numbers of gap junctions in bone marrow stromal cells of Sl/Sld mice and the defect in HIM function in these genetically anemic animals.     

Haemopoietic stem cell proliferation in the bone marrow of Sl/Sld mice

Abstract. In marrow from Sl/Sl^d mice (but not +/+ mice) day 7 and day 8 CFU-S proliferate whilst day 10 and day 12 CFU-S exhibit negligible proliferation. Media conditioned by both +/+ and Sl/Sl^d marrow contains an inhibitor of CFU-S proliferation but day 8 CFU-S in +/+ and Sl/Sl^d marrow show marked dose-response differences to this factor. To inhibit the proliferation of Sl/Sl^d CFU-S required approximately ten times the concentration of inhibitor that inhibited the proliferation of +/+ CFU-S. Thus abnormally responsive day 8-CFU-S were shown to proliferate in an inhibitory environment.
Abnormalities in Sl/Sl^d CFU-S function were also demonstrated in heterotopic transplantation experiments using +/+ and Sl/Sl^d donors and hosts to obtain ectopic bone marrow with various stromal (donor) and haemopoietic (host) combinations. Day 8 Sl/Sl^d CFU-S were seen to proliferate, irrespective of whether the stromal environment was derived from Sl/Sl^d or +/+ marrow.
Sl/Sl^d mice are generally regarded as animals in which there is a genetically determined defect in haemopoiesis due to an abnormality in the haemopoietic environment. It is difficult, however, to attribute the abnormal CFU-S behaviour in these experiments to environmental factors and the results are consistent with mutation at the Sl locus affecting the responses of CFU-S to regulatory signals, i.e. the genetic defect is not confined to the stromal environment.     

Increased female fertility in aquaporin 8-deficient mice

Aquaporin-8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8(-/-) mothers compared with wild-type mothers in cross-mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8(-/-) ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8(-/-) ovaries, suggesting increased maturation and ovulation of follicles. By RT-PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8(-/-) mice showed 45% of decreased membrane water permeability than wild-type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild-type and AQP8(-/-) mice. The results indicated significantly lower apoptosis rate in AQP8(-/-) granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8(+/+) granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.     

Radiation and haematopoiesis in Harwell steel mice

Haematological information on steel (Sl) mice is limited largely to Sl/Sld mice of Bar Harbor stock (WC.B6 F1). Therefore, two Harwell alleles, SlgbH and Slcon, were investigated. In the steady state both heterozygotes were modestly anaemic, homozygous Slcon and compound Slcon/SlgbH more so. On perturbation by X-irradiation Slcon/SlgbH showed a decrease in median lethal dose (MLD)--6.5 Gy, Slcon/+ and Slcon/Slcon slightly less change (7.5 Gy) compared with +/+, 8 Gy. In recovery from sublethal doses single heterozygotes, double heterozygotes with Wv, and compounds showed no delay in restoration of the count of red blood corpuscles (RBC) such as that seen in typical W mice (e.g. Wv/+, W/Wv). Effects on Slcon/Slcon and Slcon/SlgbH differ from those reported for Sl/Sld in that they show normal growth of spleen colonies when used as lethally irradiated recipients of bone marrow, they support growth of implanted bone marrow to form radiation chimaeras. When Harwell steel mice are donors of bone marrow to lethally irradiated +/+ mice the chimaeras ultimately are not anaemic; when lethally irradiated Harwell steel mice are recipients of +/+ marrow they remain macrocytically anaemic. One deduces that, for normal development and production of normal RBC in the steady state, the erythron requires intrinsic factors determined by wild type alleles at the W locus and extrinsic factors determined by wild type alleles at the Sl locus. Mutant alleles at either locus may determine macrocytosis. Two mutant alleles at either locus are still more deleterious, often lethal. Whereas mutant W alleles may also influence the pluripotent haematopoietic stem cell (HSC) leading to reduced MLD on X-irradiation, a similarly reduced MLD for Sl mutants may represent an increased need for and consumption of products of the haematopoietic stem cells rather than truly increased radiosensitivity, since the Do for spleen colony-forming units is the same for Slcon/SlgbH as +/+ mice.     

Biochemical and functional characterization of proteoglycans produced by Sl/Sld murine bone marrow stromal cell lines

The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (Sl/Sld) animals and two control stromal cell lines, one (+/+2.4) from a non-anemic littermate, and one (GBl/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCl density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co-culture of an IL-3-dependent cell line (FDC-P1) with cell-free ECM preparations from an Sl/Sld and a control (GBl/6) stromal cell line, with and without pre-incubation with IL-3. Cell-free ECM preparations from Sl/Sld and control cell lines supported FDC-P1 growth to an approximately equal extent after pre-incubation with IL-3. FDC-P1 growth support by ECM preparations from both cell lines was also observed without IL-3 pre-incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.     

Characterization of integrin expression in the mouse ovary

Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.     

Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor.

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.     

Adhesion of mouse mast cells to fibroblasts: adverse effects of steel (Sl) mutation

Mouse bone marrow-derived cultured mast cells proliferate on +/+ mouse embryo-derived 3T3 fibroblasts, but not on Sl/Sld mouse embryo-derived 3T3 fibroblasts, in the absence of IL-3 and IL-4 (Fujita et al: Proc. Natl. Acad. Sci. U.S.A. 86:2888-2891, 1989). To further characterize the mast cell-fibroblast interactions and the effects of Sl mutation, we tried to analyze the adhesion of cultured mast cells to 3T3 fibroblasts in vitro. Mast cells plated onto NIH/3T3 fibroblasts showed marked adhesion within 30 min, which reached a plateau after 3 h. The numbers of adhered mast cells were linear over the range of 10(3) to 5 x 10(5) cells inoculated into each (2 cm2) of 24 multiwells. Adhesion required active energy production and the presence of divalent cations. It was not inhibited by an RGD-containing peptide, an anti-LFA-1 antibody, or asialofetuin. Mast cells adhered efficiently to the eight 3T3 cell lines derived from +/+ mouse embryos, but not to the eight 3T3 cell lines derived from Sl/Sld mouse embryos. Adhesion to +/+ mouse spleen-derived fibroblasts lacking mast cell-supporting activity was comparable to that to Sl/Sld/3T3 cells. The failure of mast cells to adhere to fibroblasts with the Sl mutations was not due to a production of a diffusible inhibitor by the latter. These results indicate that production of wild type Sl gene product by fibroblasts is mandatory for adhesion/migration, as well as for proliferation of mast cells on them, and that the coculture system should be useful for the biochemical and molecular analysis of these interactions.     

Levels of alpha-inhibin in aging female mice.

alpha-inhibin was immunocytochemically localized in granulosa cells of different stages of developing follicles, freshly formed corpora lutea, and scattered interstitial cells (pigmented or ceroid cells) in ovaries of 6-, 14-, and 23-25-mo-old C57BL/6NNia mice. Developing follicles exhibited the greatest amount of staining. Quantitation of the stain using an image analysis system indicated the staining intensity within ovarian follicles of 14-mo-old mice was greater than that in 23-25-mo-old mice. The levels of plasma alpha-inhibin and estradiol (E2) decreased with age. The number of follicles present in ovaries of middle-aged mice was comparable to those of 6-mo-old mice, yet plasma levels of FSH were significantly higher than those of 6-mo-old mice. This may be due to an age-related loss in the sensitivity of the hypothalamus and/or pituitary of middle-aged mice to ovarian hormones. In contrast, ovaries of 23-25-mo-old mice contained few antral follicles and consequently produced little alpha-inhibin. There appeared to be little negative feedback regulation of FSH secretion in 23-25-mo-old mice as a result of age-related ovarian impairments. This study supports an earlier hypothesis from our laboratory [Biol Reprod 1985; 32:989-997] that the primary defect(s) limiting age-related reproductive performance in mice appears to reside within the hypothalamo-hypophyseal axis, whereas secondary defects arise from the ovary.     

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.     

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.     

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.     

Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa cells

The nuclear receptor steroidogenic factor 1 (SF-1 [officially designated NR5A1]) is essential for fetal gonadal development, but its roles in postnatal ovarian function are less well defined. Herein, we have extended our analyses of knockout (KO) mice with markedly decreased SF-1 expression in granulosa cells. As described, these SF-1 KO mice had hypoplastic ovaries that contained a decreased number of follicles and lacked corpora lutea. In the present study, we showed that SF-1 KO mice exhibited abnormal estrous cycles, were infertile, and released significantly fewer oocytes in response to a standard superovulation regimen. Moreover, they had blunted induction of plasma estradiol in response to gonadotropins. The granulosa cell-specific SF-1 KO also significantly affected ovarian expression of putative SF-1 target genes. Consistent with their decreased follicle number, these mice had reduced ovarian expression of anti-müllerian hormone (Amh), which correlates with the reserve pool of ovarian follicles, as well as decreased gonadotropin-induced ovarian expression of aromatase (Cyp19a1) and cyclin D2 (Ccnd2). In contrast, perhaps because of their abnormal cyclicity, SF-1 KO ovaries had higher basal expression of inhibin-alpha. They also had decreased immunoreactivity for genes related to proliferation (Ccnd2 and Mki67 [also known as Ki67]) and increased expression of Cdkn1b, also known as p27, which inhibits cyclin-dependent kinases, arguing for a role of SF-1 in granulosa cell proliferation. These findings demonstrate that SF-1 has a key role in female reproduction via essential actions in granulosa cells.