Follicle-stimulating hormone regulation of inhibin alpha- and beta(B)-subunit and follistatin messenger ribonucleic acid in cultured avian granulosa cells

FSH regulation of inhibin alpha-, beta(B)-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F(4) + F(5) follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P: < 0. 05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin alpha-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin alpha-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P: < 0.05) mRNA levels for the inhibin alpha-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F(4) + F(5) granulosa cells. The effects of FSH on follistatin and ss(B)-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin alpha-subunit in small prehierarchical follicles.     

Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation

In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells.     

Vitamin D regulates anti-Mullerian hormone expression in granulosa cells of the hen

Anti-Mullerian hormone (AMH) is involved in the regression of the Mullerian ducts in mammalian and avian male embryos as well as the right oviduct in avian female embryos. AMH is expressed by granulosa cells of adult hens and mammals and is thought to be involved in the recruitment of follicles from the primordial pool as well as in regulating follicle-stimulating hormone (FSH) sensitivity. We have shown that AMH expression by the granulosa layer of hens is high in the small follicles but decreased in the larger hierarchical follicles. The decline in expression of AMH with increasing follicle size is associated with an increase in expression of the receptor for FSH (FSHR) in the granulosa layer, although the mechanism is not known. In this study, we tested whether vitamin D (1,25-dihydroxyvitamin D3) regulates expression of AMH mRNA in granulosa cells of the hen. Granulosa cell layers were removed from follicles 3-5 mm and 6-8 mm in size, dispersed, and cultured for 24 h in Medium 199 + 5% fetal bovine serum (n = 7). The medium was removed and replaced with Medium 199 + 0.1% bovine serum albumin and vitamin D (at doses of 0, 10, and 100 nM) and cultured for 24 h. Cells were harvested and RNA was extracted for use in quantitative PCR. Parallel 96-well plates were set up to examine cell proliferation. AMH and FSHR mRNA expressions were evaluated, and all values were standardized to 18S reactions. There was a significant (P < 0.05) dose-related decrease in the expression of AMH mRNA in granulosa cells of 3- to 5-mm and 6- to 8-mm follicles in response to vitamin D. Additionally, FSHR mRNA and cell proliferation were significantly (P < 0.05) increased by vitamin D in both groups. Western blot analysis for the vitamin D receptor (VDR) showed doublet bands at the expected sizes (58 and 60 kDa) in protein isolated from the chicken granulosa layer. Immunohistochemistry was used to identify VDR within the follicle, and it predominantly localized to the nucleus of granulosa cells. VDR mRNA expression in the granulosa layer, relative to follicle development, was increased (n = 4; P < 0.05) with follicle development, with greatest expression in the F1 follicle. There was no evidence for expression (mRNA or protein) of the calcium-binding protein, calbindin (CALB1), in the ovary or granulosa layer. Overall, these results suggest that vitamin D regulates AMH expression, and thereby may influence follicle selection in the hen.     

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.     

Source of immunoreactive inhibin in the chicken ovary.

High concentrations of immunoreactive inhibin were detected in the plasma of the laying domestic hen using a heterologous RIA validated for use in the chicken. Cessation of egg production induced by restricting the intake of nutrients decreased circulating inhibin to approximately 20% of its original concentration within 8 days, indicating that the ovary is the major source of the measured material. Dissection of ovarian follicles revealed that inhibin is nearly exclusively produced in the granulosa cell layer. When expressed per milligram cell protein the concentration of inhibin decreased significantly in granulosa layers of follicles of succeeding order in the hierarchy (F4 to F1). The concentration of progesterone increased in the granulosa layers of the same follicles whereas oestradiol in the surrounding theca layers decreased. In vitro culture of granulosa cells derived from follicles at different stages of development confirmed the decrease in inhibin secretion as a function of follicular growth observed in vivo. The granulosa cell inhibin secretion is stimulated by LH as well as by FSH, the former being the most effective one. The physiological significance of these changes in inhibin concentration during follicular maturation requires further investigation. It may be concluded, however, that the chicken presents a useful model for the study of the endocrine as well as the paracrine function of ovarian inhibin.     

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.     

Cellular localization of inhibin mRNA in the bovine ovary by in-situ hybridization

Hybridization histochemistry has been used to detect the presence of mRNA for the alpha and beta A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine alpha or beta A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The alpha subunit mRNA was located in the granulosa layer of antral follicles greater than 0.36 mm in diameter while the alpha and beta A subunit mRNA were both present in follicles of greater than 0.8 mm. In these latter follicles, the thecal layer hybridized with only the alpha subunit mRNA. No hybridization of the alpha or beta A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the alpha inhibin subunit can be detected in granulosa and theca cells whereas the beta A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.     

Interrelationship of growth differentiation factor 9 and inhibin in early folliculogenesis and ovarian tumorigenesis in mice

To investigate the interrelationship of inhibin alpha and growth differentiation factor 9 (GDF9) during early folliculogenesis, we generated mice lacking both inhibin alpha and GDF9. Our findings on these Inha Gdf9 double-mutant mice are as follows: 1). females develop ovarian tumors and a cachexia-like wasting syndrome, resembling mice lacking inhibin alpha alone. This indicates that the granulosa cells are competent to proliferate despite the lack of GDF9; 2). follicular development progresses to multiple-layer follicle stages before tumorigenesis. This demonstrates that the up-regulation of inhibin alpha in the Gdf9 knockout ovary directly prevents the proliferation of the granulosa cells at the primary follicle stage, an effect that is released in the absence of inhibin alpha; 3). a morphological theca forms around the preantral follicles with no detectable selective theca markers [i.e. 17alpha-hydroxylase (Cyp17), LH receptor (Lhr), and Kit]. These results indicate that the theca recruitment can occur independently of GDF9, but the differentiation of thecal cells is blocked; and 4). inhibin/activin subunits betaA, betaB, and Kit ligand (Kitl) mRNA are highly up-regulated, suggesting that the increased activins and KITL play functional roles in early folliculogenesis. Thus, GDF9 appears to function indirectly to regulate early granulosa cell proliferation and theca recruitment in vivo.     

Rapid changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in ovarian cell types during the rat estrous cycle

Distributions of inhibin alpha-, beta A-, and beta B-subunits in different ovarian compartments were studied in cycling female rats by in situ hybridization with complementary RNA probes and using immunohistochemical localization with antibodies selective for each inhibin subunit. Consistent with earlier studies showing inhibin production by granulosa cells of maturing follicles, we also detected mRNAs for inhibin alpha-, beta A-, and beta B-subunits in granulosa cells of these follicles. However, based on immunohistochemistry and in situ hybridization, we found that inhibin alpha- is not only expressed in granulosa cells of mature follicles but in follicles at all stages of maturation, including primary to tertiary follicles. A number of primordial follicles also contained alpha mRNA and immunodetectable alpha-subunit. Interestingly, theca interna and interstitial gland cells contained inhibin alpha mRNA and alpha-subunit. Low levels of inhibin alpha immunoreactivity as well as specific hybridization to the complementary inhibin alpha mRNA probe were observed in newly formed luteal tissue. beta-Subunits, on the other hand, were detected exclusively in granulosa cells of healthy tertiary follicles. The changes in expression of inhibin alpha-, beta A-, and beta B-subunits were more pronounced during the follicular phase of the cycle: inhibin alpha reached its highest level in granulosa cells, theca interna, and interstitial gland cells a few hours after the LH/FSH surge, while at the same time the beta-subunits decreased dramatically in granulosa cells of mature follicles. Immediately before ovulation (estrus 0200 h), the alpha-subunit sharply declined in preovulatory follicles and was present mainly in granulosa cells from nonovulatory follicles at various stages of maturation. At that time, the beta A- and beta B-subunits could not be detected in preovulatory follicles but were localized mainly in small tertiary follicles (less than 300 microns). Unlike for the alpha- and beta B-subunits, beta A mRNA and immunoreactivity was present in large tertiary follicles (approximately 600 microns) immediately before ovulation. The present findings support the hypothesis that a decrease in inhibin production could be responsible for the secondary FSH surge observed early on estrus. This could be initiated by a change in the ratios of activin-inhibin production by decreasing first, the levels of beta-subunits, second, the levels of alpha-subunit, and third, by a resurgence of activin A produced mainly by granulosa cells from large tertiary follicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of EGF on initiation of primordial follicle growth in ovary of newborn rat

The present study was designed to look at the effect of epidermal growth factor (EGF) and tomcie-stimulating hormone (FSH) on initiation of primordial follicle growth and differentiation in the ovary of newborn rat with a sensitive marker of proliferating cell nuclear antigen (PCNA). The results showed that more cuboidal granulosa cells (GC) were found in the ovary two days after injection of EGF. More proliferative GC were observed on D4. No such action of FSH on primordial follicles was demonstrated. Using in situ hybridization, inhibin a mRNA expression in GC was detected from D5, while FSH receptor (FSHR) mRNA expression started from D6 after birth. Both mRNAs increased following further development of the follicles. These results suggest that it is EGF, but not FSH, that may play a certain role in initiation of primordial follicle growth. FSH may be involved in further differentiation and growth of the early developmental follicles.     

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.     

Effect of EGF on initiation of primordial follicle growth in ovary of newborn rat

The present study was designed to look at the effect of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) on initiation of primordial follicle growth and differentiation in the ovary of newborn rat with a sensitive marker of proliferating cell nuclear antigen (PCNA). The results showed that more cuboidal granulosa cells (GC) were found in the ovary two days after injection of EGF. More proliferative GC were observed on D4. No such action of FSH on primordial follicles was demonstrated. Using in situ hybridization, inhibin a mRNA expression in GC was detected from D5, while FSH receptor (FSHR) mRNA expression started from D6 after birth. Both mRNAs increased following further development of the follicles. These results suggest that it is EGF, but not FSH, that may play a certain role in initiation of primordial follicle growth. FSH may be involved in further differentiation and growth of the early developmental follicles.     

Catecholamine content of the preovulatory follicles of the domestic hen

The role of catecholamines in ovarian function of the domestic hen has not been examined extensively. The aim of this study was first to determine the location of catecholamines in the preovulatory follicle of the domestic hen. Second, norepinephrine (NE), epinephrine (EPI) and dopamine (DA) were measured in the isolated theca layer of the five largest preovulatory follicles at specific times during the ovulatory cycle and changes in catecholamine content were correlated with ovarian events. The five largest preovulatory follicles were removed from chickens at 24, 18, 12, 6 and 2 h before ovulation of the largest (F1) follicle. Theca and granulosa layers were isolated, frozen, weighed and prepared for measurements of catecholamines by the double isotope radio-enzymatic assay. Catecholamines were localized primarily in the theca layer with only small amounts present in the granulosa layer. Norepinephrine was present in the theca layer in concentrations 6- and 30-fold those of EPI and DA, respectively. The content of NE and EPI in the theca layer of the F1 follicle was significantly (p less than 0.01) higher at 6 h before ovulation than at other times for the F1 follicle. In contrast, NE and EPI content of the theca layer of second (F2) and third (F3) largest follicles did not change during the ovulatory cycle. The content of DA was elevated (p less than 0.05) at 12 h before ovulation in F1 and F2 follicles. There was a significant reduction in NE in the theca layer of the fifth largest (F5) follicle between 24 and 18 h before ovulation of the F1 follicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of estrogen, pituitary gonadotropins and prolactin on immunohistochemical localization of inhibin subunits in the ovary of hypophysectomized female rats.

Effects of estrogen, pituitary gonadotropins and prolactin on immunohistochemical localization of alpha- and beta A-subunits in the ovaries of hypophysectomized female rats were investigated. Hypophysectomy resulted in disappearance of immunoreactive inhibin subunits in the ovary. Administration of DES, FSH and LH to hypophysectomized rats provoked growth of follicles, and resulted in positive immunostaining for inhibin subunits in the granulosa cells. In contrast to follicle-stimulating hormone (FSH) and luteinizing hormone (LH), prolactin administration failed to demonstrate positive staining for inhibin subunits in the ovary. The present in vivo results suggest that several hormones which are known to stimulate granulosa cell growth and maturation, such as estrogen, FSH and LH, enhance inhibin subunit production by the ovary. The morphologic aspect of inhibin subunit production by the ovary in response to several hormones has been demonstrated in the present study.     

Expression and regulation of anti-mullerian hormone in an oviparous species, the hen

Anti-mullerian hormone (AMH) has a critical role in regression of the mullerian duct system during development in male mammalian and avian species and in regression of the right oviduct in female avian species. AMH in adult female birds has not been investigated. Chicken-specific cDNA primers were used to isolate Amh by RT-PCR. This probe was used in Northern blot analysis to identify a 2.8-kb band with expression in total ovarian RNA and in granulosa cell RNA. Quantitative real-time PCR was used to assess Amh expression in follicles of different maturity (1, 3, 5, and 6-12 mm and the largest F1 follicle; n = 4-6 of each size). There was an increased amount of Amh mRNA in the granulosa layer of the smaller follicles and a lower amount in the granulosa layer of the larger follicles (P < 0.01). There was no difference in granulosa Amh expression between the germinal disc and non-germinal disc region of 6- to 12-mm follicles, although expression differed with follicle size (P < 0.01). To examine hormone regulation of Amh, granulosa cells (from 6- to 8-mm follicles) were cultured with various concentrations of estradiol (E(2)) and progesterone (P(4)), and Amh mRNA was assessed. Neither E(2) nor P(4) influenced Amh mRNA accumulation. Granulosa cells were also cultured in the presence of oocyte-conditioned medium (OCM), which decreased Amh mRNA expression in a dose-related manner (P < 0.05); FSH receptor expression was not affected. Heat treatment of OCM abolished the effect, but growth differentiation factor 9 antiserum did not block the suppression. Immunohistochemistry confirmed that the granulosa layer was the predominant source of AMH in the small follicles of the hen and indicated that AMH was present early in follicle development, with expression in very small follicles (approximately 150 mum).     

Gene expression in abnormal ovarian structures of ewes homozygous for the inverdale prolificacy gene

Animals heterozygous (I+) for the Inverdale prolificacy gene (FecX(I)) have an increased ovulation rate whereas those homozygous (II) for FecX(I) are infertile with "streak" ovaries and follicular development arrested at the primary (type 2 follicle) stage. The streak ovaries also contain small oocyte-free nodules with granulosa-like cells and often tumor-like structures. It has been hypothesized that these abnormal structures are of granulosa cell origin, and the aim of this study was to determine whether genes normally expressed in granulosa cells are also expressed in the nodules and tumor-like structures. The mRNAs encoding c-kit and its ligand stem cell factor (SCF), FSH receptor (FSH-R), follistatin, alpha-inhibin subunit, and the beta(A)- and beta(B)-activin/inhibin subunits were localized in ovaries of ewes with 0 (++), 1 (I+), or 2 (II) copies of the FecX(I) gene (n = 4-9 animals per genotype per gene) using in situ hybridization. Ontogeny of expression of all mRNAs examined was similar between ++ and I+ ewes. Expression of c-kit mRNA was observed in the oocyte of all follicular types present in ++, I+, and II ewes. Moreover, granulosa cells of type 2 (II) and type 2 and larger follicles (++, I+) expressed SCF mRNA. The mRNAs encoding FSH-R, follistatin, alpha-inhibin subunit, and beta(B)-activin/inhibin subunit were identified in type 3 and larger follicles of ++ and I+ ewes but not in follicles of II ewes that were only at the type 1, 1a, or 2 stages of development. However, the cells within the oocyte-free nodules of II ewes expressed all of these genes. The mRNAs encoding c-kit and beta(A)-activin/inhibin subunit were not observed in granulosa cells until antrum formation (type 5 follicles) or in the nodules of II ewes. Tumors from 4 ewes were obtained and classified as cystic, semisolid, or solid structures containing granulosa-like cells or as solid structures containing predominately fibroblast- and luteal-like cells. Often, two tumors were present on the same ovary. Tumors containing granulosa-like cells (n = 3-4 per gene) expressed the mRNAs encoding alpha-inhibin subunit, beta(A)-, and beta(B)-activin/inhibin subunits, follistatin, and the FSH-R but did not contain detectable amounts of mRNA for c-kit or SCF. Tumors composed predominately of fibroblast- and luteal-like cells expressed very low levels of SCF mRNA; of the other mRNAs examined, none were detected. Also, none of the genes examined were found to be expressed by the surface epithelium, theca externa, fibroblast, or vascular cells within the ovary of animals of any genotype. These findings are consistent with the hypothesis that the somatic cells in oocyte-free nodules and tumor-like tissue in II ewes originate from the granulosa cells of the small follicles.