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PubMed2004

The response of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A pathways in porcine theca interna cells to opioid agonist FK 33-824.

Opioids were found as factors affecting porcine ovarian steroidogenesis. The mechanism of opioid action, however, on porcine theca interna cells is completely unknown. Therefore, the present study was designed to investigate the possible involvement of two intracellular pathways, phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A, in opioid signal transduction in porcine theca cells treated with mu opioid receptor agonist, FK 33-824. Incubation of the cells for 4 h with FK 33-824 at the dose 1 nM resulted in decreases in inositol phosphate accumulation as well as androstenedione (A(4)), testosterone (T), and estradiol (E(2)) secretions. Protein kinase C (PKC) inhibitors, staurosporine (1-100 nM), D-sphingosine (10-500 nM), and PKCi (100-2000 nM), both added alone and together with the opioid agonist, depressed release of the steroid hormones. PKC activator, phorbol ester (PMA, 1-100 nM), used alone was without effect on theca cell steroidogenesis, but added in combination with FK 33-824 abolished inhibitory influence of the opioid on A(4), T, and E(2) output. The steroid hormone secretion by PKC-deficient theca cells was inhibited by the opioid agonist. FK 33-824 also suppressed PKC activity reducing [(3)H]PDBu specific binding to theca cells, whereas ionomycin (a positive control) increased labeled phorbol ester binding to the cells. In the next experiment, cAMP release from theca cells during 2 and 4 h incubations with FK 33-824 (1-100 nM), naloxone (10 microM; opioid receptor antagonist), and LH (100 ng/mL; a positive control) was examined. FK 33-824 at the dose 1 nM inhibited cAMP secretion during 2 h incubation, but had no effect during longer incubation. LH in a manner independent on incubation time multiplied cAMP release. Protein kinase A inhibitor, PKAi (100-2000 nM), alone and in combination with FK 33-824 (1 nM), inhibited A(4), T, and E(2) secretions by theca cells. PKA activator, 8BrcAMP (10-1000 microM), stimulated the steroid hormone release, but this stimulatory effect was diminished in the presence of FK 33-824. The results allow to suggest that opioid peptides affect porcine theca cell steroidogenesis and their acute action on the cells is connected with the inhibition of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A signal transduction systems.

Adenylyl Cyclases↗
PubMed1989

Evidence for a switch in the site of relaxin production from small theca-derived cells to large luteal cells during early pregnancy in the pig.

The presence of immunoreactive relaxin was studied in corpora lutea of sows during the oestrous cycle and early pregnancy by immunohistochemistry and radioimmunoassay using three different anti-relaxin sera. Sections were immunostained using the peroxidase-anti-peroxidase or the immunogold-silver technique. Before Day 14, staining in corpora lutea from non-pregnant and pregnant animals was indistinguishable. With all antisera, no immunostaining was seen on Day 3, but was detected on Days 5-7 in cells from the theca interna. In non-pregnant animals, this immunostaining decreased and by Day 15 only an occasional large cell in the centre of the corpus luteum was stained. No staining was seen by Day 22. The relaxin content of corpora lutea measured by radioimmunoassay remained low throughout the luteal phase. In contrast, the amount of immunoreactive relaxin in corpora lutea rose dramatically (140-fold) between Days 11 and 14 of pregnancy and by Day 14 of pregnancy immunostaining was seen in the majority of large luteal cells. By Day 20 of pregnancy the concentrations of immunoreactive relaxin had further increased. Histochemical staining for alkaline phosphatase suggested that, while the relaxin-immunoreactive cells seen in the early luteal phase may be theca-derived, those during early pregnancy may be derived from the granulosa. The results are compatible with the suggestion that relaxin is produced by theca-derived cells during the early luteal phase and that between Days 11 and 14 there is a switch in the site of relaxin synthesis from theca-derived cells to granulosa-derived large luteal cells. In the absence of luteolysis, as during pregnancy, this switch is accompanied by a dramatic increase in relaxin synthesis.

Animals↗
PubMed1996

Comparison of immunocytologic localization of insulin-like growth factor binding protein-4 in normal and polycystic ovary syndrome human ovaries.

The cytologic localization and cellular levels of insulin-like growth factor binding protein-4 (IGFBP-4) in follicular and stromal compartments of normal and polycystic ovary syndrome (PCOS) ovaries during follicular growth and regression were investigated by the avidin/biotin immunoperoxidase method with a polyclonal antibody to human IGFBP-4, and a comparative assessment of IGFBP-4 expression in normal and PCOS ovaries was provided. In normal human ovaries, IGFBP-4 was immunolocalized to the oocyte throughout follicular growth, while the surrounding granulosa and theca cells were negligible for IGFBP-4 immunostaining in primordial, preantral and antral follicles. IGFBP-4 immunostaining became apparent, however, in the lutein cells of corpora lutea and the granulosa and theca cells of atretic follicles. In PCOS ovaries, prominent immunostaining for IGFBP-4 was apparent not only in the oocyte, but also in the surrounding granulosa cells in preantral follicles. In antral follicles from PCOS women without hyperinsulinemia, IGFBP-4 immunostaining was more prominent in the granulosa cells than the theca cells, whereas in antral follicles from PCOS women with hyperinsulinemia IGFBP-4 immunostaining was more prominent in the theca cells than the granulosa cells. Furthermore, in atretic follicles within PCOS ovaries IGFBP-4 immunostaining was prominent in the theca cells, regardless of the association of hyperinsulinemia. These results demonstrate for the first time that there is a great difference in cellular expression of IGFBP-4 between normal and PCOS human ovaries. In light of the high affinity of IGFBP-4 for IGF-1, the abundant expression of IGFBP-4 in granulosa and theca cells of preantral and antral follicles of PCOS ovaries may lead to decreases in the bioavailability of IGF-I in those follicles. The decrease in IGF-I-mediated stimulation of gonadotropin actions on granulosa and theca cells in preantral and antral follicles may impair the induction of aromatase activity, causing an androgenic microenvironment which is characteristic of atretic follicles and PCOS follicles.

Adult↗
PubMed1991

Characterization of dissimilar steroid productions by granulosa, theca interna and theca externa cells during follicular maturation in the turkey (Meleagris gallopavo).

Unlike the established models for steroidogenesis in the rat and human, we have previously demonstrated that in the turkey progesterone (P), androgen (A), and estrodiol (E) are primarily produced by the granulosa, theca interna, and theca externa cells, respectively. In the present study, experiments were conducted to further characterize steroid productions by these cell types during follicular maturation. In Experiments 1 and 2, granulosa cells and theca internal cells, respectively, from the larger (F1) and fifth largest (F5) preovulatory follicles were incubated (1 x 10(5) cells/ml) with ovine luteinizing hormone (oLH) or porcine follicle stimulating hormone (pFSH) for 5 hr. Granulosa production of P from both follicles was stimulated in response to oLH, with both basal and LH-stimulated P production greater in the larger F1 than in the smaller F5 follicle. No A or E production was detected in granulosa cells from either follicle size tested. Theca interna cell productions of P and A were stimulated by oLH in the smaller F5, but not in the larger F1, with both basal and stimulated levels of A greater in F5 than in F1. Medium content of E was non-detectable in cultures of theca interna cells from all follicles tested. In Experiment 3, theca externa cells were incubated (1 x 10(6) cells/ml) from F1 and F5 follicles. The theca interna cells from F5 and the seventh largest follicle (F7) were pooled (1 x 10(5) cells/ml) to provide substrate(s) for theca externa steroidogenesis. Theca externa cells were incubated alone and in combination with the pooled theca interna cells and with or without oLH or pFSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1996

Follicular maturation and atresia--possible role of intraovarian regulatory factors.

In addition to the gonadotrophins, factors known to be present or produced within the ovary are also involved in the regulation of follicular development and atresia. One such modulator of ovarian function is the prorenin-renin-angiotensin system (PRAS) which seems to be associated with atresia. Although it is primarily under gonadotrophic control, the expression of the ovarian PRAS is also shown here to be subject to modulation by intraovarian factors. Addition of granulosa cell conditioned medium to bovine theca cells inhibited the LH-stimulated prorenin secretion in a dose-dependent manner, which leads to the assumption that granulosa cells secrete a factor(s) that, even in the presence of LH, keeps prorenin secretion low. Since growth factors and cytokines are known to be produced by granulosa cells, we have evaluated the effects of tumour necrosis factor alpha, basic fibroblast growth factor and transforming growth factor alpha on the LH- and 8Br-cAMP-induced prorenin secretion, and observed that all agonists inhibited the LH- and 8Br-cAMP-stimulated prorenin secretion in a dose-dependent manner. In contrast, addition of transforming growth factor beta, a secretory product of theca cells, further augmented gonadotrophin-induced prorenin synthesis. In conclusion, we assume that in the presence of healthy granulosa cells, suppression of the ovarian PRAS by granulosa cell derived factors may prevent a follicle from undergoing atresia, whereas in the presence of atretic granulosa cells, theca cell derived factors may enhance the expression of the PRAS, resulting in follicular atresia.

Angiotensin II↗
PubMed1992

Presence of transforming growth factor-beta and their selective cellular localization in human ovarian tissue of various reproductive stages.

Immunohistochemical studies were performed using specific polyclonal antibodies to transforming growth factor (TGF)-beta 1 and TGF-beta 2 to determine their presence and cellular localization in human ovarian tissues of various reproductive states. In the small ovarian follicles, the immunostaining for TGF-beta 1 was present in oocytes, follicle cells, and granulosa and theca cell layers. The level of immunostaining associated with granulosa and theca cell layers intensified as the size of the follicles increased. In the luteal tissue, both the small and large luteal cells immunostained for TGF-beta 1 and their intensities were similar to theca and granulosa cell layers, respectively. The patterns of immunostaining were similar in early (days 14-19), mid (days 22-25), and late (days 26-29) luteal phases; however, the intensity was highest at mid and decreased at late luteal phase. Corpus albicans showed a very weak immunostaining for TGF-beta 1, whereas ectopic pregnancy small luteal cells immunostained relatively intensely. The ovarian stromal, luteal tissue fibroblasts, and arterioles endothelial and smooth muscle cells were also immunostained for TGF-beta 1. The immunostaining of the ovarian tissues for TGF-beta 2 indicated that the theca cell layers were the exclusive cells in the follicles with intense immunostaining, which increased in the larger follicles. A low immunostaining was also observed in granulosa cell of the large follicles. In the luteal tissues, only small luteal cells showed intense immunostaining for TGF-beta 2, which was similar in intensity to that in the theca cells; however, the large luteal cells showed a low level of immunostaining at midluteal phase. The small luteal cells in corpus albicans and ectopic pregnancy luteal tissues retained their immunostaining for TGF-beta 2, but with lower intensity. Endothelial and smooth muscle cells of arterioles also immunostained for TGF-beta 2, but not ovarian stromal cells. Atretic follicles showed very low or no detectable immunostaining for TGF-beta 1 or TGF-beta 2. The results of present studies show that human ovarian tissue at all the reproductive states locally produces TGF-beta 1 and TGF-beta 2, and although TGF-beta 1 is present in most major ovarian cell types, TGF-beta 2 is only produced by theca cells in the follicles and small luteal cells in luteal tissues.

Endothelium, Vascular↗
PubMed2001

Effects of transforming growth factors-alpha and -beta on proliferation and apoptosis of rat theca-interstitial cells.

Ovarian development, follicular growth and atresia require mechanisms regulating proliferation and death of ovarian cells including theca-interstitial (T-I) cells. Transforming growth factors-alpha and -beta (TGF-alpha and TGF-beta) are well recognized local modulators of T-I function. This study was performed to evaluate the effects of TGF-alpha and TGF-beta on ovarian T-I cell proliferation, differentiation and apoptosis. T-I cells from immature Sprague-Dawley rats were purified and incubated in chemically defined media. Proliferation was assessed by [3H]thymidine incorporation assay and by cell counting. Steroidogenically active cells were identified histochemically by detection of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity. DNA was extracted and apoptosis was identified by detection of internucleosomal DNA cleavage producing the characteristic 'ladder pattern' of low-molecular weight (LMW) DNA following agarose gel electrophoresis. Quantification of apoptosis was carried out with the aid of 3'-end labeling of DNA fragments with [32P]-dideoxy-ATP. TGF-alpha and TGF-beta stimulated [3H]thymidine incorporation by 2.2- to 3.1-fold and 1.7- to 3.4-fold respectively (P<0.005). A combination of TGF-alpha and TGF-beta produced a synergistic increase in DNA synthesis by 6.7-fold (at 1 ng/ml of each TGF-alpha and TGF-beta; P<0.001) and tenfold (at 10 ng/ml of each TGF-alpha and TGF-beta; P<0.001). Cell counting revealed that TGF-alpha increased the total number of cells 2.8-fold and TGF-beta 2.8-fold. The combination of TGF-alpha and TGF-beta increased the total cell count 3.2-fold, compared with control (P<0.05). The percentage of the steroidogenically active cells was 37+/-9% (mean+/-s.e.m. ) in the control cultures, 50+/-5% in the presence of TGF-alpha, 42+/-8% in the presence of TGF-beta, and 47+/-13% in the presence of both TGF-alpha and TGF-beta. TGF-alpha decreased apoptosis by 63+/-14% (P=0.02) while TGF-beta had no statistically significant effect. TGF-alpha in combination with TGF-beta produced the greatest inhibition of apoptosis by 73+/-8% (P=0.01). These findings demonstrate that TGF-alpha and -beta stimulate proliferation of both steroidogenically active and inactive T-I cells. Furthermore, TGF-alpha alone and in combination with TGF-beta protects T-I cells from apoptotic death. These effects of TGFs may be important in physiologic maintenance of ovarian mesenchymal growth and homeostasis as well as in pathophysiologic conditions associated with excessive growth of the T-I compartment, such as polycystic ovary syndrome.

3-Hydroxysteroid Dehydrogenases↗
PubMed2005

Keratinocyte growth factor acts as a mesenchymal factor that promotes ovarian primordial to primary follicle transition.

An important but poorly understood process in ovarian biology is the transition of the developmentally arrested primordial follicle to the developing primary follicle. Interactions between the epithelial and mesenchymal cells of the follicle are critical for the coordination of ovarian follicle development. The mesenchymal growth factor keratinocyte growth factor (KGF) (i.e., fibroblast growth factor-7) and the epithelial growth factor kit ligand (KITL) are known to interact to coordinate the growth of later-stage antral follicles. The hypothesis tested in the current study is that KGF acts as a mesenchymal factor to promote the primordial to primary follicle transition. A postnatal 4-day-old rat ovary organ culture system was used to investigate the actions of KGF. KGF treatment promoted 65% of follicles to undergo the primordial to primary follicle transition, but only 45% underwent development in control ovaries. Neutralizing antibody for KGF was found to attenuate the stimulatory action of KITL, but neutralizing antibody for KITL was not able to attenuate the stimulatory action of KGF. Further analysis demonstrated that KGF was found to stimulate the expression of KITL (i.e., mRNA levels) by granulosa cells. KITL in turn was found to stimulate the expression of KGF to create a positive feedback loop. Interestingly, KGF expression was localized to selected mesenchymal cells (i.e., precursor theca cells) surrounding the developing primordial follicle. Observations suggest that developing granulosa cells of the primordial follicles produce KITL, which helps recruit precursor theca cells to the follicle; the thecal cells then produce KGF, which acts on the granulosa to amplify KITL expression and support primordial follicle development. KGF appears to be a mesenchymal factor that promotes the primordial to primary follicle transitions.

Animals↗
PubMed1987

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture.

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.

17-alpha-Hydroxyprogesterone↗
PubMed1985

Differential production of steroids by dispersed granulosa and theca interna cells from developing preovulatory follicles of pigs.

Dispersed granulosa and theca interna cells were recovered from follicles of prepubertal gilts at 36, 72 and 108 h after treatment with 750 i.u. PMSG, followed 72 h later with 500 i.u. hCG to stimulate follicular growth and ovulation. In the absence of aromatizable substrate, theca interna cells produced substantially more oestrogen than did granulosa cells. Oestrogen production was increased markedly in the presence of androstenedione and testosterone in granulosa cells but only to a limited extent in theca interna cells. The ability of both cellular compartments to produce oestrogen increased up to 72 h with androstenedione being the preferred substrate. Oestrogen production by the two cell types incubated together was greater than the sum produced when incubated alone. Theca interna cells were the principal source of androgen, predominantly androstenedione. Thecal androgen production increased with follicular development and was enhanced by addition of pregnenolone or by LH 36 and 72 h after PMSG treatment. The ability of granulosa and thecal cells to produce progesterone increased with follicular development and addition of pregnenolone. After exposure of developing follicles to hCG in vivo, both cell types lost their ability to produce oestrogen. Thecal cells continued to produce androgen and progesterone but no longer responded to LH in vitro. These studies indicate that several functional changes in the steroidogenic abilities of the granulosa and theca interna compartments occur during follicular maturation.

Androstenedione↗
PubMed1979

In vitro studies of gonadal organogenesis in the presence and absence of H-Y antigen.

In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S-F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors-the characteristic of fetal Leydig cells.

Animals↗
PubMed2003

Differential expression of steroidogenic factor-1 and FTF/LRH-1 in the rodent ovary.

Steroidogenic factor-1 (SF-1) (NR5A1) is an orphan nuclear receptor that plays a premier role in ovarian organogenesis. Recent studies document mRNA expression of the structurally related factor NR5A2 (FTF, LRH-1, SF-2) in the adult ovary and more specifically in granulosa cells and luteal cells but not theca cells. Conversely, SF-1 was shown to be expressed at higher levels in theca/interstitial cells. These latter observations raised the possibility that FTF/LRH-1 may control target gene expression in granulosa cells of developing follicles. Using quantitative PCR our results show that FTF/LRH-1 message is expressed at higher levels in the ovary than in liver or other tissues analyzed. We show by in situ hybridization and LacZ expression in ovaries of transgenic mice bearing an FTF-promoter-LacZ fusion gene that FTF/LRH-1 is selectively expressed in granulosa cells of rat and mouse ovaries and is not present in theca cells or interstitial cells. However, by a variety of approaches, we showed that SF-1 mRNA and protein are expressed in greater amounts than FTF/LRH-1 in granulosa cells of follicles at all stages of development. Expression of SF-1 mRNA and protein in granulosa cells was verified by in situ hybridization, immunohistochemistry of ovarian sections, and immunocytochemistry of cultured rat granulosa cells. The significance of SF-1 in regulating target gene activation was supported by EMSA. An abundant granulosa cell protein binding to the SF-1-binding motif (CCAAGGTCA) present in the aromatase promoter and an FTF/LRH-1 motif (TGTCCTTGAACA) in the alpha-fetoprotein promoter was supershifted by two SF-1-specific antibodies but not by an FTF antibody. Conversely, with the same probes, a less abundant protein/DNA complex present in liver and ovarian cell extracts was shifted by an FTF antibody but not by the SF-1 antibodies. SF-1 and FTF/LRH-1 were differentially regulated in vivo by estradiol, FSH and prolactin. Collectively these data indicate that granulosa cells of small and preovulatory follicles express both SF-1 and FTF/LRH-1 and that each orphan receptor may regulate target gene expression in these cells.

Animals↗
PubMed1998

Steroid metabolism in granulosa and theca interna cells from preovulatory follicles of domestic hen (Gallus domesticus).

The capability of granulosa and theca interna cells, from preovulatory follicles of the domestic hen, to metabolize steroid precursors was evaluated. Granulosa and theca interna cells were isolated from ovarian preovulatory follicles at three different developmental stages: F1, F3 and F5. Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4) and testosterone (T) were employed as precursors and their metabolic products were evaluated. The major metabolite of P5 by granulosa cells was P4, but we also observed low amounts of 5beta-pregnandione. DHEA metabolism by granulosa cells yielded mainly A4, and minute quantities of 5beta-androstan-3,17-dione (5beta-dione) were detected. The only significant metabolite obtained in granulosa cells from A4 was 5beta-dione, whereas T was only transformed into A4. On the other hand, P5 metabolism by theca interna cells yielded A4 as the main product, also P4, 17alpha-OHP4, 17alpha-OHP5, 5beta-pregnandione, and DHEA, were found. When DHEA was the precursor A4 was produced in higher amounts than 5beta-dione. A4 was mainly transformed into 5beta-dione. In similar conditions, T was transformed into A4. These results show that granulosa cells have enzymatic activities of 3beta-hydroxysteroid dehydrogenase/5-4 isomerase (3beta-HSD from P5 and DHEA), 17beta-hydroxysteroid dehydrogenase (17beta-HSD from T) and 5beta-reductase (from P5, DHEA and A4). Whereas theca interna cells have enzymatic activities of cytochrome P450c17 (from P5 and P4), 3beta-HSD (from P5 and DHEA), 17beta-HSD (from T) and 5beta-reductase (from P4, DHEA and A4). These data support the concept that theca interna cells have the ability to synthesize androgens from progestins produced in granulosa cells. In addition, since theca interna cells did not show the capacity to aromatize androgens suggests that interaction between theca interna and theca externa cells occurs in vivo, thus confirming the three cell model for estrogen production. Furthermore, the fact that other metabolites were produced both in granulosa and theca interna cells, but in a different extent, suggests that complex mechanisms are participating in the regulation of steroid synthesis in avian ovary follicles.

Androstenedione↗
PubMed1995

Granulosa cell modulation of luteinizing hormone-dependent androgen production by ovarian theca-interstitial cells: a temporal switch from suppression to augmentation stimulated by follicle-stimulating hormone in vitro.

The production of estradiol-17 beta (E2) by granulosa cells (GC) of the dominant follicle is dependent upon LH-stimulated synthesis of androgens by ovarian theca-interstitial cells (TIC). Recent evidence has pointed toward an intrafollicular paracrine system, regulated by FSH and involving GC, that may modulate the LH-dependent production of androgens by TIC. In the present study, the role of GC and FSH in modulating LH-dependent TIC and androsterone production was examined. In cultures of dispersed whole ovarian cells (containing populations of both GC and TIC) from intact immature rats, LH stimulated a 10-fold increase in androsterone production (maximum androsterone = 21.0 +/- 1.1 ng/ml). By comparison, androsterone production was increased 50-fold in LH-stimulated cultures of dispersed whole ovarian cells from hypophysectomized immature rats (108 +/- 18 ng androsterone/ml). The EC50 for LH (0.02 +/- 0.001 ng/ml) was identical in the two cell preparations. We hypothesized that the lesser androgen production by whole ovarian cell cultures from intact rats was due to suppression by the GC. To investigate the role of GC in modulating TIC androgen production, highly purified TIC from immature hypophysectomized rats were cultured in the presence of GC obtained from intact immature rats. Increasing numbers of GC (2.5-100 x 10(3) GC per well) caused a progressive decrease in LH-dependent androsterone production by TIC. Additionally, LH-dependent androsterone production was suppressed by the conditioned medium from recombinant human FSH (rFSH)-stimulated GC (54% of the value for LH-stimulated TIC controls), indicating the involvement of a GC-secreted paracrine factor or factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed2000

Isolation of thecal cells: an assessment of purity and steroidogenic potential.

In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E(2)) and progesterone (P(4)) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E(2) could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P(4) in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E(2) was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P(4) (P<0.05) in the serum-free cell culture media. The stimulation of P(4) production was greater at higher LH concentration (50 ng/ml) (P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P(4) by LH-stimulated thecal cell cultures (P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC(50)<1 ng/ml at LH-20 ng/ml vs. EC(50)>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E(2) production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.

Animals↗
PubMed2001

Regulation of high density lipoprotein receptor messenger ribonucleic acid expression and cholesterol transport in theca-interstitial cells by insulin and human chorionic gonadotropin.

The synthesis of androgens by theca-interstitial cells is stimulated by LH and the insulin/insulin-like growth factor I (IGF-I) system. An essential element of the steroidogenesis is the uptake of plasma cholesterol and transformation to steroid hormones. In the rat, the uptake of cholesterol by the theca-interstitial cells is mediated by the high density lipoprotein receptor. The goal of the present study was to examine whether insulin has any effect on cholesterol delivery into theca-interstitial cells. The effects of insulin and hCG on the expression of the high density lipoprotein receptor (SR-BI) messenger RNA (mRNA) and intracellular cholesterol levels were examined in rat theca-interstitial cells under in vivo and in vitro conditions. Twenty-five-day-old rats were treated with insulin, hCG, or insulin followed by hCG. The expression of SR-BI mRNA was then examined in ovaries enriched in theca-interstitial cell population by Northern blot analysis. Treatment with insulin increased the expression of SR-BI mRNA over that in controls treated with saline. hCG administration also increased the expression of SR-BI mRNA. A combination of insulin followed by hCG produced an even greater increase in SR-BI mRNA expression. Measurements of cellular cholesterol in the ovarian tissue showed an increase in total and free cholesterol levels in response to insulin treatment. As expected, administration of hCG produced a depletion of cellular cholesterol, and the depletion was even more pronounced in response to treatment with insulin and hCG. The effect of insulin and hCG on SR-SBI mRNA expression was then examined under in vitro conditions using primary cultures of theca-interstitial cells. Treatment with insulin produced an increase in SR-BI mRNA expression. As the cultured theca-interstitial cells were not able to maintain hCG receptors, hCG addition produced no increase in SR-BI mRNA expression. However, in the presence of insulin, these cells were able to maintain hCG receptors and readily responded to hCG to increase SR-BI mRNA expression. Although insulin alone produced a modest increase in total and free cholesterol levels, in the presence of insulin, hCG produced the expected depletion of cellular cholesterol content. The present study shows that insulin has a stimulatory effect on the expression of high density lipoprotein receptors in theca-interstitial cells, suggesting that one of the actions of insulin is to increase intracellular cholesterol, which is subsequently mobilized for androgen biosynthesis in theca-interstitial cells.

Animals↗
PubMed1991

In vitro studies on steroidogenesis in the presence of pregnenolone as precursors by the follicular tissue of the domestic fowl (Gallus domesticus).

Chicken granulosa and theca cells were isolated from F1 and F4-6 follicles 2-4 h before ovulation, and the amounts of progesterone, testosterone and oestradiol released in the medium during incubation for 3 h, in the presence or absence of pregnenolone as a percursor and stimulatory drugs or inhibitory drugs, were measured. Progesterone synthesis by granulosa cells was stimulated with oLH or theophylline. Much more progesterone was synthesized when pregnenolone was added to the medium. The amount of testosterone produced by the granulosa cells was similar to that produced by the theca cells. The production of testosterone was increased by the addition of oLH or theophylline. Oestradiol synthesis by F4-6 follicles was higher than by F1 follicles, and it was higher in the theca cells than in the granulosa cells. The addition of oLH or theophylline increased oestradiol synthesis in the theca cells and the granulosa cells of F4-6 follicles. The results indicate that oestradiol can be produced from pregnenolone by the theca cells alone. It is possible, however, that the theca cells also take in the precursors for the production of oestradiol from the granulosa cells.

Animals↗
PubMed2000

Morphological assessment of the effect of growth hormone on preantral follicles from 11-day-old mice in an in vitro culture system.

The present study was carried out to verify that the cells attached to the outside of the basement membrane of mechanically isolated follicles are theca cells and to evaluate the effect of growth hormone (GH) on these cells. Preantral follicles, 100-140 micrometer in diameter, were mechanically isolated from 11-day-old BDF1 hybrid immature mice, divided randomly into two groups, and cultured in vitro. One group was treated with 0.1% collagenase immediately after mechanical isolation in an attempt to remove theca cells attached to the outside of the basement membrane. The other group was untreated. Morphological examination revealed that 86.1% of mechanically isolated follicles before collagenase treatment had at least one theca cell around the basement membrane on the single section. However, after collagenase treatment no theca cells remained on the basement membrane of the follicles. Androstenedione secretion as a result of stimulation by 100 ng/ml hCG was significantly higher in the culture medium of the follicles with theca cells than in those of collagenase-pretreated follicles (p < 0.0001), indicating that the cells attached to the outside of the basement membrane were actually functional theca cells, not interstitial cells. To elucidate the effect of GH on theca cells, preantral follicles cultured in the presence of 1.0 mIU/ml GH were morphologically examined. Preantral follicles mechanically isolated from immature mice showed significant proliferation of not only granulosa cells but also theca cells in the presence of GH. In particular, theca cells, which remained dotted on the basement membrane in a small number just after isolation, proliferated and finally formed complete layers after the culture with GH. This is the first report that GH induced the proliferation of theca cells to form morphologically complete layers around the preantral follicle from 11-day-old mice.

Androstenedione↗