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PubMed1999

Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary.

There is increasing evidence that leptin is a physiological link between obesity and infertility. Although leptin receptors have been demonstrated in human ovaries, there is no information regarding the effects of leptin on cells from developing ovarian follicles. To test the direct effects of leptin on human ovarian cells, granulosa cells (GC) and theca cells were isolated from the ovaries of regularly cycling women. Serum was obtained at the time of surgery, and follicular fluid was aspirated from the follicles before isolation of the ovarian cells. Leptin concentrations were similar in follicular fluid and serum. RT-PCR analysis demonstrated that the long, signaling form of the leptin receptor was expressed in both theca and GC. In cultured GC, leptin had no effect on estradiol production, alone or in the presence of FSH, but caused a concentration-related inhibition of the insulin-like growth factor I (IGF-I) augmentation of FSH-stimulated estradiol production. The effect of leptin was specific, because there was no effect on progesterone production. In cultured theca cells, leptin did not alter androstenedione production, alone or in the presence of LH. Leptin caused a concentration-related inhibition of the IGF-I augmentation of LH-stimulated androstenedione production. These data demonstrate that leptin can directly inhibit IGF-I action in ovarian theca and GC at concentrations commonly present in obese women.

Adult↗
PubMed1983

Partly luteinized theca cell tumor of the ovary.

The partly luteinized theca cell tumor is a variant of the theca cell tumor in which extensive foci of luteinization occur. This neoplasm belongs to a set of tumors, which includes the theca cell tumor, that is believed to be derived from mature ovarian stroma. This tumor can be classified as a tumor of specialized gonadal stroma of "ovarian cell type" intermediate between the theca cell tumor and the stromal luteoma. Two patients were virilized, and one had evidence of endometrial hyperplasia, whereas in the fourth no endocrine function was evident. None of the patients were pregnant at the time of discovery of the tumor. On gross examination these tumors consisted of a mixture of firm gray-white and yellow tissue. The presence of multiple yellow nodules in two tumors distinguished it from the usual theca cell tumor. A third tumor was distinctly lobulated. The tumor in only one of the cases arose in a background of ovarian stromal hyperplasia. Although the number of cases reported up to now is small and variability exists, the tumors tend to occur in patients in the early reproductive age group, often produce significant quantities of steroid hormones, and the clinical course has been benign.

Adolescent↗
PubMed2000

Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture.

The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.

Androstenedione↗
PubMed1997

Direct actions of kit-ligand on theca cell growth and differentiation during follicle development.

The direct actions of kit-ligand/stem cell factor (KL) in developing ovarian follicles were investigated. Previous studies have shown that granulosa cells express KL that can support oocyte development. The current study demonstrates that KL can also act directly on theca cells to promote cellular growth and differentiation. Through RT-PCR analysis it was shown that bovine granulosa cells express KL, and theca cells express the receptor c-kit. Bovine theca interna cells were isolated and cultured in serum-free conditions to study KL actions. KL stimulated theca cell growth in a dose-dependent manner as measured by [3H]thymidine incorporation into DNA when cells were cultured under subconfluent conditions. KL had no effect on theca cell androstenedione or progesterone production under these growth-permissive conditions. In contrast, KL stimulated theca cell androstenedione production but had no effect on progesterone production when theca cells were cultured under confluent (non-growth-permissive) conditions. Estradiol (10(-7) M) and human CG (100 ng/ml) were used as controls and regulated theca cell steroid production at any cell density. These results demonstrate that KL can directly stimulate theca cell growth and steroid production during follicular development. The observation that KL stimulated androstenedione production but not progesterone production suggests that KL promotes a follicular phase differentiated state in theca cells. The potential regulation of KL and c-kit expression during follicular development was studied using a specific quantitative RT-PCR procedure. Total RNA from granulosa cells (for KL) and theca cells (for c-kit) was examined from small (<5 mm), medium (5-10 mm), and large (>10 mm) size follicles. Steady state levels of KL messenger RNA were highest in granulosa cells from large size follicles and lowest in small and medium size follicles. No differences were observed in the steady state levels of c-kit messenger RNA in theca cells from small, medium, or large size follicles. The observation that KL expression is highest in large size follicles suggests that KL may be important for increased growth and steroid production in large and dominant follicles. Observations demonstrate that KL can dramatically alter theca cell function and support the hypothesis that local granulosa-theca cell interactions play an important role in regulating cellular function within ovarian follicles. This study identifies KL as the first granulosa cell-derived growth factor that can directly stimulate theca cell growth and androstenedione production in the absence of gonadotropins.

Androstenedione↗
PubMed1991

Role of calcium in the regulation of theca cell androstenedione production in the domestic hen.

Theca cells were collected from the second largest preovulatory follicle. Chelation of extracellular calcium with EGTA attenuated LH (10 ng)-induced androstenedione production by theca cells, and this effect was more pronounced in calcium-deficient than in calcium-replete incubation medium. Incubation of theca cells with steroidogenic agonists in the presence of the calcium channel blocker verapamil (100 microM) suppressed androstenedione production stimulated by LH (a 57% decrease), the adenylate cyclase activator forskolin (a 59% decrease) and the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP (a 61% decrease). Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a putative inhibitor of intracellular calcium mobilization, suppressed LH-induced androstenedione production in a dose-dependent fashion. The calmodulin inhibitors trifluoperazine (100 microM) and R24571 (50 microM) inhibited androstenedione production stimulated by hormonal (LH) and non-hormonal (forskolin, 8-bromo-cAMP) agonists (decreases ranging from 76 to 98%). While increasing the intracellular calcium ion concentrations with the calcium ionophore A23187 did not affect basal concentrations of androstenedione, treatment of LH-stimulated cells with the ionophore caused dose-dependent inhibition of androstenedione production; these effects were enhanced by coincubation with phorbol 12-myristate 13-acetate (a known activator of protein kinase C). We conclude that the mobilization of calcium is critical for agonist-stimulated steroidogenesis in hen theca cells, apparently requiring the interaction of calcium with its binding protein, calmodulin. Furthermore, increased cytosolic calcium concentrations may be involved in the suppression of androstenedione production, possibly as a result of an interaction with protein kinase C.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

Quantification of insulin-like growth factor binding protein mRNA using real-time PCR in bovine granulosa and theca cells: effect of estradiol, insulin, and gonadotropins.

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1-5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased (P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased (P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased (P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased (P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect (P>0.10). Estradiol (3 and 300 ng/ml) decreased (P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased (P<0.05) IGFBP-4 expression, but had no effect (P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased (P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells.

Animals↗
PubMed1991

Theca cells may be present at the outset of follicular growth.

Opinions differ concerning when theca cells arise during follicular growth. Theca cells cannot be distinguished from ovarian stroma cells on the basis of morphology alone. However, long-term continuous infusion of [3]thymidine (3H-TdR) thoroughly labels theca cells of large and medium-sized follicles whereas most stromal cells remain unlabeled. The objective of this study was to determine when, during the course of follicular development, labeled, squamous cells located just outside the follicular basement membrane (putative theca cells) first become apparent. Rats were given infusions of 3H-TdR for various periods of time. Autoradiographs were prepared from 2-microns-thick plastic sections of ovaries. Morphologically recognizable theca cells of large and medium-sized follicles were sharply delineated from surrounding stroma cells by their labeling pattern. Although unilaminar follicles lacked morphologically distinct theca layers, they all were closely enveloped by labeled cells just outside the basement membrane. Follicles with a few as 4-5 granulosa cells in cross section often had labeled cells on the convex surface of their basement membranes. These observations suggest the possibility that theca cells may already be present at the very outset of follicular growth.

Animals↗
PubMed1992

The regulation of porcine theca cell proliferation in vitro: synergistic actions of epidermal growth factor and platelet-derived growth factor.

An important but poorly understood aspect of mammalian follicle development involves the regulation of theca cell proliferation. To investigate the premise that growth factors regulate theca cell proliferation, porcine theca cells were prepared by collagenase/DN'ase digestion of follicle linings after the removal of the granulosa cells and allowed to attach for 24 h. This method provided a monolayer of theca cells that had little if any granulosa cell contamination and which secreted high levels of androstenedione relative to granulosa cells during moderate-term culture (33-fold difference, P less than 0.01). In medium containing fetal calf serum (10%), theca cells were significantly more responsive to platelet-derived growth factor (PDGF) than epidermal growth factor (EGF) in terms of proliferation (13.4 +/- 0.2-vs. 7.0 +/- 0.1-fold increases relative to the initial cell count, P less than 0.05). This is in contrast to granulosa cells which were significantly more responsive to EGF than PDGF (7.1 +/- 0.1 vs. 4.0 +/- 0.2 fold-increases, P less than 0.05). Since serum has been shown to contain both EGF and PDGF, proliferation studies were performed using plasma-derived serum (PDS) which is growth factor restricted to examine more closely the direct effects of growth factors. In medium containing 0.25% PDS and within experiments, PDGF (1-25 ng/ml) stimulated theca cell proliferation in a dose-dependent manner (2.3-fold increase relative to controls, P less than 0.05) whereas EGF did not. EGF, however, markedly enhanced the proliferative action of PDGF (6.4-fold increase relative to controls, P less than 0.05). Insulin-like growth factor I and low density lipoprotein, factors which enhance markedly the proliferative effects of EGF and PDGF in terms of granulosa cell proliferation, exhibited only a modest synergistic effect with respect to EGF and PDGF upon theca cells (9.5-fold increase vs. a 6.4-fold increase above controls, P less than 0.05). Temporal studies in vitro indicate that theca cell proliferation is low during the first 3-day exposure to growth factors irrespective of treatment (a 2-fold increase over the seeding density). During the second 3-day exposure, however, theca cell proliferation increases 4- to 5-fold. The temporal pattern of theca cell proliferation stimulated by fetal calf serum supplemented with EGF or PDGF and PDS-containing medium supplemented with PDGF, EGF, insulin-like growth factor I, and low density lipoprotein is similar. These results suggest that PDGF is a major mitogen toward porcine theca cells and that EGF greatly enhances its activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2001

Partial characterization of the factor in theca-cell conditioned medium that inhibits the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to the membrana granulosa.

A factor, secreted by theca cells, inhibits FSH induced resumption of meiosis in bovine oocytes that are surrounded by cumulus cells which are attached to a piece of the membrana granulosa (COCGs). In order to characterize this factor, theca cell conditioned medium (CMt) was heat-treated, filtered through a 5 kD spin off filter, charcoal treated, chloroform extracted and protease treated. To investigate whether the meiosis inhibiting factor produced by theca cells was also present in follicular fluid (FF), the same treatments were done with 50% bovine follicular fluid (bFF). COCGs, originating from 2 to 8 mm follicles of bovine ovaries collected at a slaughterhouse, were cultured in groups of 15 per 600 microl medium supplemented with 0.05 IU ml FSH for 22 hr at 39 degrees C in a humidified atmosphere of 5% CO(2). After culture the oocytes were denuded, stained with orcein, and the nuclear status assessed. Heat treatment did not affect the meiosis arresting capacity of CMt since a similar proportion of the oocytes remained at the GV stage after 22 hr of culture in heat treated CMt as compared to the proportion of oocytes in the GV stage after culture in untreated CMt. Filtering through a 5 kD spin-off filter revealed that the meiosis inhibiting action was maintained in the <5 kD fraction, although there was a significant (P < 0.05) loss of inhibiting activity compared to nonfiltered CMt. No significant decrease was observed in the meiosis arresting capacity of the <5 kD fraction after charcoal or protease treatment. Extraction of the <5 kD fraction with chloroform also did not affect the theca cell produced factor. The effect of the theca cell factor on the progression of meiosis of the oocytes that resumed meiosis, as demonstrated by a very low percentage of the oocytes that matured up to the M2 stage, was not affected following any of the treatments. With regard to bFF, the results show a lower percentage of the oocytes in the GV stage after culture in 50% bFF as compared to culture in CMt, but progression of meiosis was clearly inhibited as demonstrated by a significant higher proportion of the oocytes blocked in the M1 stage after resumption of meiosis. In general, with regard to meiotic inhibition, bFF showed the same pattern as CMt following the various treatments. It is concluded that the theca cell secreted factor which inhibits the FSH-induced resumption of meiosis in COCGs is a small, stable, polar molecule which is not a peptide.

Animals↗
PubMed2000

Interactive stimulation by luteinizing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17alpha-hydroxylase/17,20-lyase (CYP17) genes in porcine theca cells.

LH and insulin are postulated to jointly stimulate theca-cell androgen biosynthesis in patients with hyperthecosis or polycystic ovarian syndrome. To explore the mechanisms of putative LH and insulin steroidogenic synergy in primary culture of normal theca cells, we have implemented an in vitro serum-free monolayer culture system of Percoll-purified, porcine theca cells harvested from immature ovaries. Initial dose and time course analyses revealed that a maximally effective concentration of LH (100 ng/ml) or insulin (100 ng/ml) individually will drive androstenedione production (at 6 to 48 h) by 1.5- to 2.6- and 1.1- to 1.7-fold, respectively, while combined agonists act synergistically over the interval 12 to 48 h yielding a 3- to 4-fold joint effect. Coadministration of LH and insulin can augment theca-cell concentrations of CYP17 and StAR messenger RNA (mRNA) resulting in 3.4- to 3.9- and 3.8- to 4.1-fold increases at 24 to 48 h, respectively (P < 0.01). Combined LH and insulin stimulation also amplified the nuclear content of intron-specific heterogeneous nuclear (hn)RNAs encoding CYP17 and StAR. Insulin significantly enhanced LH-driven but not basal cAMP accumulation (14-18 vs. 3-5.5 pmol/microg DNA/12-48 h) (P < 0.01). A stable exogenous analog of cAMP, 8 Br-cAMP, mimicked LH's effect on steroidogenesis and StAR and CYP17 gene expression and with insulin stimulated StAR mRNA and hnRNA accumulation synergistically. However, unlike LH, 8 Br-cAMP did not synergize with insulin on theca-cell androstenedione biosynthesis or CYP17 mRNA and hnRNA expression. In summary, the present in vitro data identify molecular interactions of LH and insulin on StAR and CYP17 gene expression, thus establishing potent signaling interfaces between these distinct hormonal agonists in regulating theca-cell steroidogenesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Effects of ovarian theca cells on apoptosis and proliferation of granulosa cells: changes during bovine follicular maturation.

We have investigated the role of theca cells in the control of apoptosis and proliferation of granulosa cells during bovine ovarian follicular development using a coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. A DNA fluorescence flow cytometry was used to determine the extent of apoptosis and proliferation in populations of granulosa cells. When granulosa cells were isolated from small follicles (3-5 mm), the percentage of apoptotic cells gradually increased by 1.8-fold during the 3 days of culture. This change was reduced (3.1-fold) by the presence of theca cells. When the cells were isolated from large follicles (15-18 mm), the percentage of apoptotic granulosa cells was gradually reduced (3.4-fold) during the 3 days of culture in single-cultured groups. The percentage of apoptosis on Day 1 was reduced (1.6-fold) by the presence of theca cells. However, such an effect was not detected on Days 2 and 3 of the culture. Theca cells did not affect the proliferation of granulosa cells obtained from either small or large follicles. The present study suggests that theca cells regulate the fate of granulosa cells throughout the follicular maturation process by secreting factors that suppress apoptosis.

Animals↗
PubMed1994

Preparation of highly purified porcine theca cells.

A novel method for purifying dispersed porcine theca cells, with less than 3% granulosa cell contamination, was developed by the repeated use of mechanical and enzymatic procedures. The steroidogenic criteria used for the identification and purity evaluation of both theca and granulosa cells were also improved. Purified theca and granulosa cells from medium-sized follicles displayed steroidogenic differences when they were cultured in the presence of 10% fetal bovine serum: (1) the theca cells synthesized oestradiol (239.1 +/- 35.1 pg ml-1 per 2.5 x 10(5) cells in 40 h), but the granulosa cells did not synthesize it unless aromatizable androgens were added; (2) theca cells synthesized androstenedione (73.2 +/- 14.4 ng ml-1 per 2.5 x 10(5) cells in 40 h), but granulosa cells did not; (3) FSH did not affect progesterone production in theca cells; (4) the theca cells secreted androstenedione for up to 48 h; and (5) FSH significantly stimulated progesterone production in granulosa cells during a culture for 40 h (P < 0.05), but not during culture for 12 h. The lack of response to FSH was used as a reliable, functional indicator of the purity of porcine theca cells. However, this criterion proved not to be useful for cells cultured for 12 h; porcine FSH had no effect on the progesterone production of theca cells co-cultured for this time with as many as 20% granulosa cells. However, after co-culturing for 40 h, this criterion resulted in the detection of only 3% granulosa cell contamination.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗
PubMed1999

Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries.

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17alpha-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17beta-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17alpha-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.

17-Hydroxysteroid Dehydrogenases↗
PubMed2006

Role of granulosa and theca cell interactions in ovarian follicular maturation.

We developed a culture system in which two types of ovarian follicular cells were allowed to attach to opposite sides of a collagen membrane. Using this in vitro cell culture system, we studied the effects of granulosa- and theca-cell interaction on the morphology, structure, and function of bovine ovarian follicular cells. In the first part of the study, we explored how the interaction between theca and granulosa cells affects the morphology and structure of the cells. This study was done using follicular cells collected from bovine ovarian follicles at the early developmental stage. Granulosa cells cultured alone were flattened, and formed a monolayer sheet. By contrast, granulosa cells cultured with theca cells were convex, and formed multilayer sheets. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cultured with granulosa cells were also spindle-shaped; however, they appeared convex and more densely packed when compared with theca cells cultured alone. In the second part of the study, the possible role of the cellular interaction in the control of differentiation and growth of granulosa and theca cells was investigated. When follicular cells were isolated from the early stage of follicular development, theca cells reduced progesterone and inhibin production by granulosa cells and augmented the growth of granulosa cells. When the cells were isolated from the late stage of follicular development, by contrast, theca cells augmented hormonal production by granulosa cells, and did not affect the growth of granulosa cells. The growth and androstenedione production by theca cells were increased by the presence of granulosa cells, irrespective of the origin of follicular cells. These results demonstrated that communication between two types of follicular cells results in reciprocal modulation of their morphology, structure, growth, and function. Cellular interactions seem to be one of the major factors controlling the differentiation and growth of the follicular cells during the follicular maturation process. In contrast to granulosa and theca cells cultured alone, cells in the coculture seemed to possess morphological and functional characteristics more similar to those of cells in the growing follicular wall in vivo. Thus, we speculate that the interaction between these two types of follicular cells is essential for the maintenance of original structure and function of the bovine follicular wall.

Animals↗
PubMed1994

The role of granulosa and theca cell interactions in ovarian structure and function.

The term paracrine control has recently become very fashionable in the field of endocrinology: however, no study has yet clarified directly the role of paracrine activity in the functional and morphological development of endocrine organs. We developed a culture system in which two types of cells were allowed to attach to opposite sides of a collagen membrane in order to observe cellular communication directly and used it to explore the role of granulosa and theca cell interactions in ovarian structure and function. In the first series of the study, we explored the interaction between theca and granulosa cells by investigating the morphology, proliferation, and steroidogenesis of the cells. Granulosa cells cultured alone were flattened and polygonal in shape and formed a monolayer sheet. Granulosa cells cocultured with theca cells formed multilayer sheets. The apical surface of each cell appeared convex. Numerous filopodia spread over the cellular surfaces to connect individual cells. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cocultured with granulosa cells were also spindle-shaped; however, the apical surface appeared convex. The cell numbers of both granulosa and theca cells in the cocultured group increased approximately twofold compared to control cells cultured alone. The progesterone producing activity of granulosa cells in the cocultured group was reduced to 40% of that of cells in the control group. In contrast, the androstenedione producing activity of theca cells in the cocultured group increased approximately threefold compared to that of control group cells. In the second series of experiments, effects of cellular communication on cellular response to gonadotropins were investigated. When granulosa cells were cultured with theca cells, FSH treatment (1 microgram/ml) promoted cellular growth: however, LH treatment (1 microgram/ml) suppressed cellular growth and augmented their progesterone production. In contrast, such effects of gonadotropins were not detected when granulosa cells were cultured alone. LH induced estradiol production by granulosa cells both cultured alone and cocultured with theca cells: however, FSH-induced estradiol production was not detected in this experimental condition. LH treatment increased androstenedione production of theca cells cocultured with granulosa cells, but no increase was observed for theca cells cultured alone. These results demonstrate that communication between these two types of follicular cells results in reciprocal modulation of their morphology, structure, growth, and function and that the actions of gonadotropins on target cells in the follicular wall can also be modified by the communication between these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle.

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Cyclic adenosine monophosphate (cAMP) production in avian theca cells during follicular maturation.

LH was used to stimulate cAMP production in theca cells from the 5 largest preovulatory follicles of hens and this was related to LH-stimulated androstenedione production in the same cells. cAMP production was stimulated by LH to the same extent in theca cells from each follicle. However, LH was not effective in stimulating androstenedione production in theca cells from the largest follicle (T1), although androstenedione production was greatly increased by LH in the smaller follicles (T2-T5). Effects similar to those of LH on cAMP production were observed in response to forskolin, indicating that the intrinsic adenylate cyclase activity was similar in theca cells from each follicle. In addition, forskolin was unable to stimulate androstenedione production by T1 cells. Our results provide evidence that the levels of receptor-mediated and non-receptor-mediated cAMP production are similar in theca cells from the 5 largest follicles. We conclude that the step that restricts the ability of T1 cells to produce androgen is distal to cAMP generation.

Androstenedione↗
PubMed2004

Effect of insulin-like growth factors (IGF), FSH, and leptin on IGF-binding-protein mRNA expression in bovine granulosa and theca cells: quantitative detection by real-time PCR.

To determine if insulin-like growth factor (IGF)-1 and -2, FSH, or leptin alter IGF-binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and (or) theca cells, granulosa and theca cells were collected from bovine ovarian follicles, plated for 48 h in 10% FCS and then treated for 24 h in serum-free medium containing various hormone treatments arranged in three different experiments. Amounts of IGFBP-2, -3, -4, and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. Neither 100 ng/ml of IGF-1 nor IGF-2 had an effect (P > 0.10) on IGFBP-2, -3, -4, or -5 mRNA levels in small-follicle (1-5 mm; Experiment 1) granulosa cells. In large-follicle (>7.9 mm; Experiment 2) granulosa cells, 100 ng/ml of IGF-1 increased (P < 0.05) IGFBP-2 mRNA levels above controls and 3 ng/ml of IGF-1; 100 ng/ml of IGF-1 also decreased (P < 0.10) IGFBP-5 mRNA levels compared to 3 ng/ml of IGF-1 or FSH or 100 ng/ml leptin, while 100 ng/ml of IGF-2 had no effect (P > 0.10) on IGFBP-2, -3, -4, and -5 mRNA levels (Experiment 2). At the doses tested, leptin and FSH had no effect (P > 0.10) on IGFBP-2, -3, -4, and -5 mRNA levels in large-follicle granulosa cells. In theca cells, IGF-2 decreased (P < 0.05) IGFBP-2 mRNA levels, but had no effect on IGFBP-3 or -4 mRNA expression (Exp. 3); IGF-1 did not affect (P > 0.10) thecal IGFBP-2, -3 or -4 mRNA levels. In contrast, IGF-1 but not IGF-2 increased (P < 0.01) thecal IGFBP-5 mRNA levels. Ligand blotting revealed that both IGF-1 and -2 increased IGFBP-2 and -5 (protein) and had no effect on IGFBP-3 (protein), whereas IGF-1 (but not IGF-2) increased IGFBP-4 (protein), suggesting IGFBP-2, -4, and -5 are post-transcriptionally regulated. These results suggest that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by IGF-1 and -2, therefore discretely modulating the amount of bio-available IGFs to these cells depending upon the specific hormonal milieu.

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