Death Processes in Bovine Theca and Granulosa Cells Modelled and Analysed Using a Systems Biology Approach.

In this paper, newly discovered mechanisms of atresia and cell death processes in bovine ovarian follicles are investigated. For this purpose the mRNA expression of receptor interacting protein kinases 1 and 3 (RIPK1 and RIPK3) of the granulosa and theca cells derived from healthy and atretic follicles are studied. The follicles were assigned as either healthy or atretic based on the estradiol to progesterone ratio. A statistically significant difference was recorded for the mRNA expression of a RIPK1 and RIPK3 between granulosa cells from healthy and atretic follicles. To further investigate this result a systems biology approach was used. The genes playing roles in necroptosis, apoptosis and atresia were chosen and a network was created based on human genes annotated by the IMEx database in Cytoscape to identify hubs and bottle-necks. Moreover, correlation networks were built in the Cluepedia plug-in. The networks were created separately for terms describing apoptosis and programmed cell death. We demonstrate that necroptosis (RIPK-dependent cell death pathway) is an alternative mechanism responsible for death of bovine granulosa and theca cells. We conclude that both apoptosis and necroptosis occur in the granulosa cells of dominant follicles undergoing luteinisation and in the theca cells from newly selected follicles.

Cells responding to hedgehog signaling contribute to the theca of ovarian follicles.

Cell-fate mapping was used to identify cells that respond to the hedgehog (HH) signaling pathway and that are incorporated into the theca cell layer during ovarian follicle development. Expression of Gli1 is increased by HH signaling and can be used as a marker of cells responsive to HH in reporter mice. In transgenic Gli1ERcre/tdT mice, injection of tamoxifen (TAM) induces cre-mediated recombination and expression of td tomato (tdT) which leads to permanent fluorescent marking of cells expressing Gli1 and their progeny. The identity of tdT-positive cells was determined by co-staining ovaries for endothelial cells (CD31), pericytes (CSPG4), vascular smooth muscle cells (VSMC; smooth muscle actin) and steroidogenic cells (cytochrome P450 17A1). Gli1ERcre/tdT mice were injected with TAM on the day of birth. Cells positive for tdT in 2-day-old mice were identified as pericytes, located primarily in the medulla of the ovary in close proximity to endothelial cells. In both prepubertal mice and adult mice treated with equine chorionic gonadotropin to induce the formation of preovulatory follicles, tdT-positive cells were located within the theca cell layer and were identified as pericytes, VSMC and steroidogenic theca cells. Granulosa cells are known to express two HH ligands, Indian HH and desert HH (DHH). In DHHcre/tdT reporter mice, endothelial cells were marked as tdT-positive indicating that endothelial cells, in addition to granulosa cells, express Dhh in the ovary. These findings suggest that HH signaling may stimulate the development of the vasculature along with steroidogenic capacity of the theca layer during follicle development.

Engineering Functional Rat Ovarian Spheroids Using Granulosa and Theca Cells.

Although menopausal hormone therapy (MHT) is the most effective approach to managing the loss of ovarian activity, serious side effects have been reported. Cell-based therapy is a promising alternative for MHT. This study constructed engineered ovarian cell spheroids and investigated their endocrine function. Theca and granulosa cells were isolated from ovaries of 10-week-old rats. Two types of engineered ovarian cell spheroids were fabricated through forced aggregation in microwells, multilayered spheroids with centralized granulosa aggregates surrounded by an outer layer of theca cells and mixed ovarian spheroids lacking spatial rearrangement. The ovarian cell spheroids were encapsulated into a collagen gel. Non-aggregated ovarian cells served as controls. The endocrine function of the engineered ovarian spheroids was assessed over 30 days. The structure of the spheroids was well maintained during culture. The secretion of 17ß-estradiol from both types of engineered ovarian cell spheroids was higher than in the control group and increased continuously in a time-dependent manner. Secretion of 17ß-estradiol in the multi-layered ovarian cell spheroids was higher than in the non-layered constructs. Increased secretion of progesterone was detected in the multi-layered ovarian cell spheroids at day 5 of culture and was sustained during the culture period. The initial secretion level of progesterone in the non-layered ovarian cell spheroids was similar to those from the controls and increased significantly from days 21 to 30. An in vitro rat model of engineered ovarian cell spheroids was developed that was capable of secreting sex steroid hormones, indicating that the hormone secreting function of ovaries can be recapitulated ex vivo and potentially adapted for MHT.

Changes in the localization of ovarian visfatin protein and its possible role during estrous cycle of mice.

Visfatin is a crucial adipokine, which also regulates ovarian functions in many animals. Mice estrous cycle is characterized by a dynamic complex physiological process in the reproductive system. Expression of various factors changes during the estrous cycle in the ovary. To the best of our knowledge, no previous study has been conducted on the expression of visfatin in mice ovaries during the estrous cycle. Therefore, we investigated the localization and expression of visfatin protein in the ovary of mice during the estrous cycle. Western blot analysis showed the elevated expression of visfatin in proestrus and lowest in diestrus. Immunohistochemical localization of visfatin showed intense staining in the corpus luteum of proestrus and diestrus ovaries. Thecal cells, granulosa cells, and oocytes also showed the presence of visfatin. Expression of ovarian visfatin was correlated to BCL2 and active caspase3 expression and exhibited a significant positive correlation. Furthermore, in vivo inhibition of visfatin by FK866 in the proestrus ovary down-regulated active caspase3 and PCNA expression, and up-regulated the BCL2 expression. These results suggest the role of visfatin in the proliferation and apoptosis of the follicles and specific localization of visfatin in the corpus luteum also indicate its role in corpus luteum function, which may be in progesterone biosynthesis and regression of old corpus luteum. However, further study is required to support these findings. In conclusion, visfatin may also be regulating follicular growth during the estrous cycle by regulating proliferation and apoptosis.

The ovarian stroma as a new frontier.

Historically, research in ovarian biology has focused on folliculogenesis, but recently the ovarian stroma has become an exciting new frontier for research, holding critical keys to understanding complex ovarian dynamics. Ovarian follicles, which are the functional units of the ovary, comprise the ovarian parenchyma, while the ovarian stroma thus refers to the inverse or the components of the ovary that are not ovarian follicles. The ovarian stroma includes more general components such as immune cells, blood vessels, nerves, and lymphatic vessels, as well as ovary-specific components including ovarian surface epithelium, tunica albuginea, intraovarian rete ovarii, hilar cells, stem cells, and a majority of incompletely characterized stromal cells including the fibroblast-like, spindle-shaped, and interstitial cells. The stroma also includes ovarian extracellular matrix components. This review combines foundational and emerging scholarship regarding the structures and roles of the different components of the ovarian stroma in normal physiology. This is followed by a discussion of key areas for further research regarding the ovarian stroma, including elucidating theca cell origins, understanding stromal cell hormone production and responsiveness, investigating pathological conditions such as polycystic ovary syndrome (PCOS), developing artificial ovary technology, and using technological advances to further delineate the multiple stromal cell types.

Prenatal programming by testosterone of follicular theca cell functions in ovary.

In mammalian ovaries, the theca layers of growing follicles are critical for maintaining their structural integrity and supporting androgen synthesis. Through combining the postnatal monitoring of ovaries by abdominal magnetic resonance imaging, endocrine profiling, hormonal analysis of the follicular fluid of growing follicles, and transcriptomic analysis of follicular theca cells, we provide evidence that the exposure of ovine fetuses to testosterone excess activates postnatal follicular growth and strongly affects the functions of follicular theca in adulthood. Prenatal exposure to testosterone impaired androgen synthesis in the small antral follicles of adults and affected the expression in their theca cells of a wide array of genes encoding extracellular matrix components, their membrane receptors, and signaling pathways. Most expression changes were uncorrelated with the concentrations of gonadotropins, steroids, and anti-Müllerian hormone in the recent hormonal environment of theca cells, suggesting that these changes rather result from the long-term developmental effects of testosterone on theca cell precursors in fetal ovaries. Disruptions of the extracellular matrix structure and signaling in the follicular theca and ovarian cortex can explain the acceleration of follicle growth through altering the stiffness of ovarian tissue. We propose that these mechanisms participate in the etiology of the polycystic ovarian syndrome, a major reproductive pathology in woman.

Evidence that downregulation of Wilms' tumor 1 (WT1) is involved in cortical stromal cell differentiation into theca cells in adult bovine ovaries.

Bovine theca cells are thought to differentiate from cortical stromal cells, and ovary-derived Wilms' tumor 1+ (WT1+ ) cells are the primary source of mouse theca cells. However, it is not known whether the differentiation of cortical stromal cells is regulated by WT1. Here, we identified WT1 in the cortical stroma and theca layer of the bovine ovary and analyzed the theca cell functional markers in cortical stromal cells and theca cells; in addition, we determined the effects of this gene on the secretion of androstenedione and progesterone by cortical stromal cells and the responsiveness of cortical stromal cells to luteinizing hormone (LH) in vitro. We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR), western blot analysis, and immunohistochemistry to discover that the cortical stroma had higher WT1 expression than the theca layer. We used RT-qPCR and ELISA analyses to determine that the cortical stromal cells had lower levels of androstenedione and progesterone secretion and LHR messenger RNA expression than the levels of the theca cells. In cultured bovine cortical stromal cells, we found that WT1 downregulation increased androstenedione and progesterone secretion but had no effect on the LH responsiveness. Notably, the increase in androstenedione and progesterone secretion was associated with an increase in 3-ß-hydroxysteroid dehydrogenase expression. In conclusion, the results suggest that WT1 is involved in the differentiation of cortical stromal cells into cells with characteristics similar to theca cells of antral follicles in adult bovine ovaries.

Regulation of the transcription factor E2F8 gene expression in bovine ovarian cells.

Overexpression of the transcription factor, E2F8, has been associated with ovarian cancer. Objectives of this study were to determine: 1) if E2F8 gene expression in granulosa cells (GC) and theca cells (TC) change with follicular development, and 2) if E2F8 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F8 mRNA abundance in GC and TC was greater (P < 0.05) in small than large follicles. FGF9 induced an increase (P < 0.05) in E2F8 mRNA abundance by 1.6- to 7-fold in large-follicle (8-20 mm) TC and GC as well as in small-follicle (1-5 mm) GC. Abundance of E2F8 mRNA in TC was increased (P < 0.05) with FGF2, FGF9 or VEGFA treatments alone in vitro, and concomitant treatment of VEGFA with FGF9 increased (P < 0.05) abundance of E2F8 mRNA above any of the singular treatments; BMP4, WNT3A and LH were without effect. IGF1 amplified the stimulatory effect of FGF9 on E2F8 mRNA abundance by 2.7-fold. Collectively, our studies show for the first time that follicular E2F8 is developmentally and hormonally regulated indicating that E2F8 may be involved in follicular development.

Transcriptome analyses of ovarian stroma: tunica albuginea, interstitium and theca interna.

The ovary has specialised stromal compartments, including the tunica albuginea, interstitial stroma and theca interna, which develops concurrently with the follicular antrum. To characterise the molecular determinants of these compartments, stroma adjacent to preantral follicles (pre-theca), interstitium and tunica albuginea were laser microdissected (n = 4 per group) and theca interna was dissected from bovine antral follicles (n = 6). RNA microarray analysis showed minimal differences between interstitial stroma and pre-theca, and these were combined for some analyses and referred to as stroma. Genes significantly upregulated in theca interna compared to stroma included INSL3, LHCGR, HSD3B1, CYP17A1, ALDH1A1, OGN, POSTN and ASPN. Quantitative RT-PCR showed significantly greater expression of OGN and LGALS1 in interstitial stroma and theca interna versus tunica and greater expression of ACD in tunica compared to theca interna. PLN was significantly higher in interstitial stroma compared to tunica and theca. Ingenuity pathway, network and upstream regulator analyses were undertaken. Cell survival was also upregulated in theca interna. The tunica albuginea was associated with GPCR and cAMP signalling, suggesting tunica contractility. It was also associated with TGF-ß signalling and increased fibrous matrix. Western immunoblotting was positive for OGN, LGALS1, ALDH1A1, ACD and PLN with PLN and OGN highly expressed in tunica and interstitial stroma (each n = 6), but not in theca interna from antral follicles (n = 24). Immunohistochemistry localised LGALS1 and POSTN to extracellular matrix and PLN to smooth muscle cells. These results have identified novel differences between the ovarian stromal compartments.

Transcript abundance of stromal and thecal cell related genes during bovine ovarian development.

Movement and expansion of mesonephric-derived stroma appears to be very important in the development of the ovary. Here, we examined the expression of 24 genes associated with stroma in fetal ovaries during gestation (n = 17; days 58-274) from Bos taurus cattle. RNA was isolated from ovaries for quantitative RT-PCR. Expression of the majority of genes in TGFß signalling, stromal transcription factors (NR2F2, AR), and some stromal matrix genes (COL1A1, COL3A1 and FBN1, but not FBN3) showed a positive linear increase with gestational age. Expression of genes associated with follicles (INSL3, CYP17A1, CYP11A1 and HSD3B1), was low until mid-gestation and then increased with gestational age. LHCGR showed an unusual bimodal pattern; high levels in the first and last trimesters. RARRES1 and IGFBP3 also increased with gestational age. To relate changes in gene expression in stromal cells with that in non stromal cells during development of the ovary we combined the data on the stromal genes with another 20 genes from non stromal cells published previously and then performed hierarchical clustering analysis. Three major clusters were identified. Cluster 1 genes (GATA4, FBN3, LHCGR, CYP19A1, ESR2, OCT4, DSG2, TGFB1, CCND2, LGR5, NR5A1) were characterised by high expression only in the first trimester. Cluster 2 genes (FSHR, INSL3, HSD3B1, CYP11A1, CYP17A1, AMH, IGFBP3, INHBA) were highly expressed in the third trimester and largely associated with follicle function. Cluster 3 (COL1A1, COL3A1, FBN1, TGFB2 TGFB3, TGFBR2, TGFBR3, LTBP2, LTBP3, LTBP4, TGFB1I1, ALDH1A1, AR, ESR1, NR2F2) had much low expression in the first trimester rising in the second trimester and remaining at that level during the third trimester. Cluster 3 contained members of two pathways, androgen and TGFß signalling, including a common member of both pathways namely the androgen receptor cofactor TGFß1 induced transcript 1 protein (TGFB1I1; hic5). GATA4, FBN3 and LHCGR, were highly correlated with each other and were expressed highly in the first trimester during stromal expansion before follicle formation, suggesting that this could be a critical phase in the development of the ovarian stroma.

Acquisition of developmental competence and in vitro growth culture of bovine oocytes.

Recently, the demand of transferable embryos in cattle industry is increasing, and the number of embryos produced in vitro is also increasing in the world. Although oocytes are collected from individual elite cattle by ovum-pick up (OPU) and used for in vitro production (IVP) of embryos, the cattle are mono-ovulatory animal. It means that most of oocytes collected from ovaries are destined to degenerate. To improve the IVP efficiency, we should predict the developmental competence of oocytes correctly and culture them by the suitable way. In addition, in vitro production of bovine oocytes by in vitro growth (IVG) culture system will become a candidate of supply source of oocytes for IVP. If we can produce high competent oocytes by IVG, IVP efficiency will be improved and the genetic improvement of cattle will be dramatically accelerated. In the review, I introduce our researches related to oocyte morphology, the developmental competence, and the production of oocytes having high developmental competence by IVG culture.

Morphological rearrangement of the cortical region, in aging ovaries.

The ovary is a structurally dynamic organ that alters with age. Modifications in the paracrine status influence the capacity of aging oocytes to develop normal embryos. Despite the importance of understanding the cellular and molecular mechanism involved in the process of ovarian aging, histological changes remain poorly understood. Correlating the process of folliculogenesis and somatic cell function during ovarian aging is essential to explain the reproductive decline of aged mammalian species, including humans. Here, we performed a morphological and immunohistological study on the ovaries of chinchilla rabbits that varied in age from one to 34-months. The spatiotemporal expression of the cholesterol side-chain cleavage cytochrome P450scc (CYP11A) and the smooth muscle actin (SMA) were analyzed. A significant histological rearrangement of immunodetected cells in theca interna, theca externa and the interstitial tissue around the follicles occurred. The expression of CYP11A1 decreased considerably in antral follicles of aging ovaries. Moreover, we found that the secondary interstitial gland developed extensively, and a remarkable rearrangement of the surface epithelium occurred in aging ovaries. In contrast to ovaries during the reproductive period, the immunohistological changes demonstrate that the interstitial gland became the most abundant tissue during the aging of ovaries. Thus, the current study provides new data for understanding the alteration of somatic cell function in elderly ovaries and how this affects their declined fertility.

Presence of anti-Müllerian hormone (AMH) during follicular development in the porcine ovary.

BACKGROUND: Anti-Müllerian hormone (AMH) is expressed by granulosa cells of developing follicles and plays an inhibiting role in the cyclic process of follicular recruitment by determining follicle-stimulating hormone threshold levels. Knowledge of AMH expression in the porcine ovary is important to understand the reproductive efficiency in female pigs. RESEARCH AIM: In the present study we investigated the expression of AMH during follicular development in prepubertal and adult female pigs by immunohistochemistry, laser capture micro-dissection and RT-qPCR. RESULTS AND CONCLUSION: Although in many aspects the immunohistochemical localization of AMH in the porcine ovary does not differ from other species, there are also some striking differences. As in most species, AMH appears for the first time during porcine follicular development in the fusiform granulosa cells of recruited primordial follicles and continues to be present in granulosa cells up to the antral stage. By the time follicles reach the pre-ovulatory stage, AMH staining intensity increases significantly, and both protein and gene expression is not restricted to granulosa cells; theca cells now also express AMH. AMH continues to be expressed after ovulation in the luteal cells of the corpus luteum, a phenomenon unique to the porcine ovary. The physiological function of AMH in the corpus luteum is at present not clear. One can speculate that it may contribute to the regulation of the cyclic recruitment of small antral follicles. By avoiding premature exhaustion of the ovarian follicular reserve, AMH may contribute to optimization of reproductive performance in female pigs.

Ovine Granulosa Cells Isolation and Culture to Improve Oocyte Quality.

The functional cooperation between the oocyte and its surrounding granulosa cells is mandatory to oocyte growth and acquisition of developmental competence in mammalian species. Availability of in vitro methods for culture granulosa cells is important in understanding the biology of these cells and the response of maturing oocyte to in vitro culture and co-culture. Using the ovine as a model the ensuing chapter describes methods for primary culture of ovine granulosa cells and their co-culture with theca cells or oocytes that allow to mimic the molecular interactions between somatic cells and maturing oocyte and improve oocyte quality.

A transcriptomic comparison of theca and granulosa cells in chicken and cattle follicles reveals ESR2 as a potential regulator of CYP19A1 expression in the theca cells of chicken follicles.

Previous studies have shown that theca and granulosa cell layers in follicles do not play the same roles in mammals and birds, especially regarding the synthesis of estrogen. The functions of these two cell types have been well characterized in cattle, but they remain unclear in chickens. To clarify this issue, a comparison of small yellow follicles (SYFs) in chickens and cattle at different follicular development stages was done by weighted gene co-expression network analysis (WGCNA). The modules obtained from WGCNA were used for further identification of the key genes associated with CYP19A1 expression. Module preservation analysis showed high similarity between cow_D (the follicular phase before the LH surge) and chicken_SYF (small yellow follicle between 6 and 8 mm in diameter) datasets, and 10 top hub genes highly associated with CYP19A1 expression in chicken SYFs were identified in each module. A comparison of the transcriptomes of theca and granulosa cells (TCs and GCs) between chicken SYFs and cattle follicles at the differentiation stage, as well as the aforementioned hub genes, revealed that ESR2 is a potential regulator of CYP19A1 expression in the theca cells of chicken SYFs. Furthermore, 197 cell-specific (179 in theca and 18 in granulosa) and 235 cell-biased expressed genes (196 in theca and 39 in granulosa) in chicken small yellow follicles were also identified by transcriptomic comparison of theca and granulosa cells.

Interaction of Melatonin and BMP-6 in Ovarian Steroidogenesis.

The bone morphogenetic protein (BMP) system in the ovary plays a physiological role as a luteinization inhibitor in growing follicles. BMP-6 secreted from oocytes and granulosa cells can exert an inhibitory effect on follicle-stimulating hormone (FSH) actions by suppressing adenylate cyclase activity downstream of the FSH receptor. The inhibition of FSH-induced progesterone production by BMP-6 is impaired by melatonin treatment in granulosa cells. Intracellular Smad signaling induced by BMP-6 is suppressed by melatonin, suggesting that melatonin has a regulatory role in BMP receptor signaling in granulosa cells. Since the expression of BMP-6 in granulosa cells is increased in patients with polycystic ovary syndrome, melatonin may play an important role in the maintenance of progesterone production by suppressing BMP-6 signaling, leading to the preservation of ovarian function.

Expression and distribution of the zinc finger protein, SNAI3, in mouse ovaries and pre-implantation embryos.

The Snail gene family includes Snai1, Snai2, and Snai3 that encode zinc finger-containing transcriptional repressors in mammals. The expression and localization of SNAI1 and SNAI2 have been studied extensively during folliculogenesis, ovulation, luteinization, and embryogenesis in mice. However, the role of SNAI3 is unknown. In this study, we investigated the expression of SNAI3 during these processes. Our immunohistochemistry data showed that SNAI3 first appeared in oocytes by postnatal day (PD) 9. Following this, SNAI3 was found to be expressed consistently in theca and interstitial cells, along with oocytes. In gonadotropin-treated immature mice, the expression of SNAI3 did not change significantly during follicular development. The expression of SNAI3 was reduced during ovulation, after which it increased gradually during luteinization. Similar results were obtained from western blot analyses. Furthermore, real-time polymerase chain reaction (RT-PCR) analyses revealed varying mRNA levels of different Snail factors at a given time in gonadotropin-induced ovaries. During early embryo cleavage, SNAI3 was localized to the nucleus, except the nucleolus at the germinal vesicle and one-cell stages. From two- to eight-cell stages, SNAI3 was localized only to the nucleolus. Thereafter, SNAI3 was detected only in the cytoplasm, except during the blastocyst stage when it was localized to the nucleus of the trophectoderm and the inner cell mass. RT-PCR results showed that the expression of Snail superfamily genes was decreased during the blastocyst stage. From the eight-cell to morula stage, when compaction occurs that is a prerequisite for blastocyst formation, Snai3 mRNA was expressed at very low levels and was opposite to the highest expression level of the compaction-related gene, E-cadherin, at the eight-cell stage. Taken together, our results suggest that SNAI3 likely plays some roles during folliculogenesis, luteinization, and early embryonic development.

miR-26a-5p Regulates TNRC6A Expression and Facilitates Theca Cell Proliferation in Chicken Ovarian Follicles.

Ovarian theca cells play an indispensable role in ovarian follicular development and hormone secretion. miR-26a-5p was reported to be differentially expressed in mature and immature chicken ovaries in our previous study; however, the role of miR-26a-5p in regulating ovarian follicle function is still unclear. In this study, we demonstrated that the expression dynamics of TNRC6A mRNA in either chicken ovaries or follicles showed an opposite trend compared with that of chicken miR-26a-5p expression. miR-26a-5p inhibited TNRC6A mRNA expression by directly targeting its 3'-untranslated region in cultured chicken theca cells. Overexpression of miR-26a-5p promoted chicken follicular theca cell proliferation in vitro. Furthermore, overexpression of miR-26a-5p and knockdown of TNRC6A significantly upregulated the antiapoptotic BCL-2 gene. Taken together, this study revealed the expression dynamics of miR-26a-5p and TNRC6A in chicken ovaries and ovarian follicles and the relationship between the expression of miR-26a-5p and TNRC6A in chicken ovarian theca cells. These results suggest that miR-26a-5p facilitates chicken ovarian theca cell proliferation by targeting the TNRC6A gene.

Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary.