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The theca cell layer of the ovarian follicle secretes growth factors that impact the function of granulosa cells. One such factor is fibroblast growth factor 18 (FGF18) that causes apoptosis of granulosa cells, however it is not known if FGF18 induces apoptosis also in theca cells. Addition of recombinant FGF18 to bovine theca cells in vitro inhibited steroidogenesis but, in contrast to previous data in granulosa cells, decreased the incidence of apoptosis. FGF18 activated typical FGF signaling pathways in theca cells, which was not previously observed in granulosa cells. The transcription factor Early Growth Response-1 (EGR1) was a target of FGF18 action; overexpression and knock-down experiments demonstrated that EGR1 is a major upstream component of FGF signaling in theca cells and that it directs cell fate toward proliferation. These data suggest that FGF18 is mitogenic for theca cells while being pro-apoptotic in granulosa 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.

Adiponectin is an adipokine that has been implicated in insulin resistance, a condition associated with polycystic ovarian syndrome in humans, but whether adiponectin can directly affect ovarian theca or granulosa cell function is unknown. Therefore, to determine the effects of adiponectin on proliferation, steroidogenesis and gene expression of large-follicle theca and granulosa cells, experiments were conducted using bovine ovarian cell cultures. RT-PCR was used to elucidate the effects of adiponectin on gene expression of CYP11A1 and LH receptor (LHR) in large-follicle theca and granulosa cells, as well as expression of CYP17A1 in theca cells and CYP19A1 in granulosa cells. Adiponectin decreased (P0.05) insulin-induced progesterone and androstenedione production as well as attenuated IGF-I-induced LHR, CYP11A1, and CYP17A1 gene expression in theca cells. In contrast, adiponectin decreased (P0.05) LHR mRNA abundance in granulosa cells but did not affect steroidogenic enzyme gene expression in granulosa cells. Adiponectin had no effect (P0.10) on proliferation of large-follicle theca cells. RT-PCR also revealed that abundance of mRNA for the adiponectin receptor (ADIPOR2) was greater (P0.05) in large-follicle than in small-follicle theca cells and did not significantly differ between small- and large-follicle granulosa cells. In cultured theca cells, LH increased (P0.05) and IGF-I decreased (P0.05) ADIPOR2 mRNA abundance. These results indicate that the inhibitory effects of adiponectin on steroidogenesis are primarily localized to theca cells and that the response of theca cells to adiponectin (i.e., ADIPOR2) may be regulated by LH and IGF-I.

The ovary is a complex endocrine gland responsible for production of sex steroids and is the source of fertilizable ova for reproduction. It also produces various growth factors, transcription factors and cytokines that assist in the complex signaling pathways of folliculogenesis. The ovary possesses two primary steroidogenic cell types. The theca cells (and to a lesser extent, the stroma) are responsible for androgen synthesis, and the granulosa cells are responsible for conversion of androgens to estrogens, as well as progesterone synthesis. These cells undergo a transformation in the luteal phase of the menstrual cycle, converting them from estrogen producing, to predominantly progesterone producing cells. Understanding the molecular mechanisms regulating these cells is essential in understanding the regulation of steroidogenesis and reproduction. Creation of appropriate in vitro cell model systems can provide important tools for the study of ovarian function. This has led to the development of ovarian steroidogenic cell lines in several laboratories. Developing theca cell lines has met with limited success. Conversely, numerous human and animal granulosa cell lines have been developed. This review will discuss the existing granulosa cell lines and their characteristics.

In the fish ovary, LH is the main factor regulating the production of steroids during the periovulatory period and its effects are believed to be mediated, at least partially, through the cAMP-dependent protein kinase (PKA) signaling pathway. However, there is no direct evidence for the presence of PKA in the fish ovary nor on the regulation of its activity by fish LH. Here, we show the identification of regulatory (R) and catalytic (C) subunits of PKA in trout theca cells by immunoblotting. DEAE-cellulose chromatography of theca cell extracts indicated the presence of PKA type I and II and showed that trout theca cells display PKA-specific phosphotransferase and cAMP-binding activities. Salmon LH (sLH) stimulated PKA activity and increased the levels of immunoreactive RIIalpha, RIIbeta and C subunits in trout theca layers. These observations, coupled with the sLH-dependent decrease in the half-life of the C subunit, as shown by pulse-chase experiments, strongly suggest that sLH activates PKA in trout theca cells. Furthermore, our results suggest that ovarian PKA activity and its regulation by LH has been well conserved from fish to humans.

In a sexually mature female, primordial follicles continuously leave the arrested pool and undergo the primordial to primary follicle transition. The oocytes increase in size and the surrounding squamous pre-granulosa cells become cuboidal and proliferate to form a layer of cuboidal cells around the growing oocyte. This development of the primordial follicle commits the follicle to undergo the process of folliculogenesis. When the available pool of primordial follicles is depleted reproductive function ceases and humans enter menopause. The current study examines whether leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition that initiates follicular development. Ovaries from 4 day-old rats were cultured in the absence or presence of LIF or neutralizing antibody to LIF. LIF treatment increased the proportion of follicles that initiated the primordial to primary follicle transition to 59%, compared to 45% in untreated cultured ovaries. The ability of LIF to induce primordial follicle development was enhanced to greater than 75% by the presence of insulin in the culture medium. Anti-LIF neutralizing antibody reduced the proportion of spontaneous developing primordial follicles. Immunocytochemical studies demonstrated higher levels of LIF protein in the granulosa and surrounding somatic cells of primordial and primary follicles compared to the oocyte. In contrast, later pre-antral and antral stage follicles showed LIF expression primarily in the oocyte. In granulosa and theca cell cultures LIF had no effect on cell proliferation. However, LIF treatment did increase expression of Kit ligand (KL) mRNA in cultured granulosa cells. KL has been shown to promote ovarian cell growth and induce primordial follicle development. LIF induction of KL expression may be involved in the actions of LIF to promote primordial to primary follicle transition. In summary, LIF treatment increased the primordial to primary follicle transition in cultured ovaries and LIF may interact with KL to promote primordial follicle development.

Polycystic ovary syndrome (PCOS) is characterized by increased ovarian androgen production. We have developed a strategy to identify differentially transcribed genes in PCOS theca cells. Using long-term cultures of normal and PCOS theca cells, we are characterizing the complete repertoire of differentially transcribed genes, the common transcription factor mediators, and the signal transduction cascade(s) that are abnormal in PCOS theca cells. The latter will be interrogated for mutations/genetic variants linked to PCOS.

The present study utilizes in situ molecular hybridization and immunocytochemistry to investigate the follicular localization and expression of four thematically related sterol-metabolizing genes; low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (CYP11A) enzyme, and cytochrome P450 17α-hydroxylase/C 17–20 lyase (CYP17). To this end, semiquantitative analyses were applied to individual nonatretic follicles ( N =54) harvested from cycling gilts slaughtered on days 1, 3, 5, and 7 ( N =3 per day) following withdrawal of the progesterone agonist, altrenogest. In situ and immunocytochemical signal intensities were assigned numerical values of 0–3 corresponding to the degree of expression of each mRNA and its corresponding protein. LDL receptor mRNA and protein content was undectable in theca and granulosa cells on days 1, 3, and 5, and then increased to low levels in theca cells on day 7. StAR message rose progressively in theca cells with follicular maturation, reaching a maximum on day 5, and then declining slightly on day 7 after the LH surge. In granulosa cells, small amounts of StAR mRNA and protein were detected on days 5 and 7. The amounts of CYP11A mRNA and protein were high in theca cells, and increased at each time point studied. Granulosa cells exhibited minimal CYP11A message on days 3, 5, and 7, while protein became detectable at low levels on day 7 only. Expression of CYP17 was localized exclusively in theca cells with protein and message content increasing unidirectionally to maxima on days 5 (RNA) and 7 (protein), respectively. Follicular fluid concentrations of androstenedione, and progesterone in contralateral ovaries correlated strongly and positively with accumulation of CYP17, and CYP11A proteins. In summary, these analyses demonstrate that preovulatory follicular development proceeds with the coordinate induction of pivotal genes and proteins that mediate the selective uptake, delivery and utilization of sterol substrate in granulosa and theca-cell steroidogenesis.

Alterations of ovarian follicle morphology and function have been well documented in women with PCOS. These include increased numbers of growing preantral follicles, failure of follicle growth beyond the mid-antral stage, evidence of granulosa call degeneration, and theca cell hyperplasia. Functional abnormalities include paradoxical granulosa cell hyperresponsiveness to FSH which is clinically linked to ovarian hyperstimulation during ovulation induction. In addition, there is likely a primary theca cell defect that accounts for the majority of excess androgen production in this disorder. The precise mechanisms responsible for altered follicle function are not completely clear. However, several factors appear to influence normal advancement of follicle development as well as impair ovarian steroidogenesis. These include intra- as well as extraovarian influences that distort normal ovarian growth and disrupt steroid production by follicle cells.

Orphan nuclear receptor NR4A1, a member of the nuclear receptor superfamily, is widely expressed in different cell types and mediates diverse biological processes. Recent emerging evidence suggests that NR4A1 is involved in the transcriptional regulation of several steroidogenic enzyme genes in gonads and adrenals. However, its function in ovarian theca cells remains to be defined. In the present study, immunohistochemical staining of NR4A1 in healthy human ovaries indicate that it is expressed in theca cells and granulosa cells. In an effort to explore the function of NR4A1 in the transcript regulation of steroidogenic enzyme genes responsible for ovarian theca cell steroidogenesis, we constructed recombinant adenovirus AdCMV-NR4A1 and AdH1-NR4A1 to enhance or knockdown the expression of NR4A1 in theca cells, respectively. The expression patterns of StAR, CYP11A1, CYP17 and HSD3B2 were subsequently analyzed by real-time RT-PCR. Moreover, concentrations of testosterone in the spent medium were measured by radioimmunoassay. Our results show that overexpression of NR4A1 in theca cells stimulates the expression of StAR, CYP11A1, CYP17 and HSD3B2, leading to increased testosterone production. Conversely, knockdown of the endogenous NR4A1 exhibits a significant decrease in StAR, CYP11A1, CYP17 and HSD3B2 expression and testosterone production. Since expression of NR4A1 in the endocrine organs is known to be regulated by both cAMP/PKA mediated hormones, ACTH and LH, forskolin (FSK), an activator of cAMP/PKA pathway, was applied to the cultured follicles. FSK rapidly increases the NR4A1 mRNA levels followed by an increase in StAR, CYP11A1, CYP17 and HSD3B2. Collectively, our results outline a previously unrecognized role for NR4A1 in the transcriptional regulation of StAR, CYP11A1, CYP17 and HSD3B2 in ovarian theca cells. Modulation of these steroidogenic enzymes by NR4A1 could influence the capacity of the ovarian theca cells to produce androgen.

The development of the ovarian follicle, oocyte maturation, and ovulation require a complex set of endocrine, paracrine, and autocrine inputs that are translated into the regulation of cyclic nucleotide levels. Changes in intracellular cAMP mediate the gonadotropin regulation of granulosa and theca cell functions. Likewise, a decrease in cAMP concentration in the oocyte has been associated with the resumption of meiosis. Using pharmacological and molecular approaches, we determined that the expression of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, is compartmentalized in the ovarian follicle of all species studied, with PDE3 present in the oocytes and PDE4s in granulosa cells. The PDE3 expressed in the mouse oocyte was cloned, and the protein expressed in a heterologous system had properties similar to those of a PDE3A derived from somatic cells. Inhibition of the oocyte PDE3 completely blocked oocyte maturation in vitro and in vivo, demonstrating that the activity of this enzyme is essential for oocyte maturation. Heterologous expression of PDE3A in Xenopus oocyte causes morphological changes distinctive of resumption of meiosis (GVBD), as well as activation of mos translation and MAPK phosphorylation. Using mRNA and antibody microinjection in the Xenopus eggs, we have shown that PDE3 is downstream from the kinase PKB/Akt in the pathway that mediates IGF-1 but not progesterone-induced meiotic resumption. The presence of a similar regulatory module in mammalian oocytes is inferred by pharmacological studies with PDE3 inhibitors and measurement of PDE activity. Thus, PDE3 plays an essential role in the signaling pathway that controls resumption of meiosis in amphibians and mammals. Understanding the regulation of this enzyme may shed some light on the signals that trigger oocyte maturation.

Insulin-like growth factor binding proteins (IGFBPs) secreted by bovine granulosa and theca interna cells cultured in the presence of different luteinizing factors--insulin (2 micrograms/ml), forskolin (10 microM), or a combination of the two were examined and characterized. Direct binding of [125I]IGF to the conditioned media was compared to progesterone production under these different treatments. In theca cells, maximal secretion of IGFBPs was achieved using forskolin alone, whereas maximal progesterone production was induced by the insulin+forskolin treatment. In contrast, maximal secretion of both IGFBPs and progesterone in granulosa cells was achieved using forskolin alone. IGFBP species secreted by the two cell types under the different treatments were detected by ligand blotting. Conditioned media from theca cells in serum-free medium collected on the seventh day of culture exhibited three bands of 34, 40 and 44 kDa when treated with insulin or forskolin. The intensity of the 40-44 kDa complex was enhanced and a 21 kDa band appeared when cells were treated with a combination of insulin plus forskolin. Conditioned media of granulosa cells stimulated with insulin or forskolin exhibited 21, 27, 29, 34 and 40-44 kDa bands. Treatment with insulin+forskolin greatly increased the intensity of a 40-44 kDa complex. A similar shift towards high molecular weight binding proteins was observed when these media were analyzed by high-performance liquid chromatography gel filtration. These findings substantiate the secretion of IGFBPs by bovine theca and granulosa cells and show it to be dependent on culture treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

We have recently reported that extracts of the superior ovarian nerve (SON) of rats inhibit porcine theca cell androstenedione production. Theca cells obtained from prepubertal gilts were cultured under serum-free conditions for 48 h. The inhibitory effect of SON extracts occurred within 6 h of treatment, was irreversible and independent of the dose of luteinizing hormone (LH) employed. The SON extracts' actions were exerted at a site(s) distal to the generation of adenosine 3',5'-monophosphate (cyclic AMP), since they did not affect extracellular cyclic AMP accumulation, while causing a significant inhibition of androstenedione production. The SON extract decreased 17α-hydroxyprogesterone, androstenedione, testosterone, estradiol and estrone production while increasing progesterone and pregnenolone production, suggesting that the SON extract causes an inhibition of the 17α-hydroxylase:C17–20 lyase complex. These results indicate that a factor(s) in the SON may play an important role in the regulation of follicular development, since thecal androgens are substrates for granulosa cell estrogen biosynthesis and are also involved in follicular atresia.

Distribution of progesterone receptor (PR) was studied immunohistochemically in rabbit ovary and uterus using a monoclonal anti-receptor antibody. At the subcellular level, PR was located in cell nuclei of prepubertal rabbits, which were either non-treated, primed with estrogen or made pseudopregnant. At the tissue level, germinal epithelium, the external theca cell layer and granulosa cell layer were PR-positive. The internal theca cell layer had no PR immunoreactivity. The corpora lutea of pseudopregnant rabbits contained small amounts of PR in some of the animals. During pseudopregnancy, the distribution and staining intensity of PR-positive cells in the ovary was essentially similar to that in non-treated rabbits except for weak PR immunoreactivity in the internal theca cell layer. This differed significantly from the uterus, in which pseudopregnancy caused a marked decrease in PR immunoreactivity. This implies that receptor downregulation by endogenous progesterone is not the same in different organs and cell types. Immunohistochemical techniques give valuable information as to the steroid hormone target cell types and their distribution which are not available by conventional steroid-binding assays.