Bovine theca and granulosa cells interact to promote androgen production

Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

The source of follicular androgens in the hamster follicle

The comparative ability of granulosa cells and theca of the hamster preovulatory follicle to produce androgens in vitro from endogenous and exogenous substrates was assessed. The results indicate that theca are the major source of follicular androstenedione, but that the granulosa cells may be the major source of follicular testosterone. Theca and granulosa cells accumulate comparable amounts of dihydrotestosterone from exogenous androstenedione and testosterone and both may be a significant source of follicular DHT. LH stimulates the conversion of progesterone and 17 alpha-OH progesterone to androstenedione, testosterone and DHT in theca. LH does not stimulate the conversion of androstenedione to testosterone or DHT, and that of testosterone to DHT in either granulosa cells or theca. FSH, in granulosa cells but not in theca, stimulates the conversion of adrostenedione to testosterone but it has no effect in DHT accumulation from exogenous testosterone.     

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.     

Steroid production by isolated theca and granulosa cells after initiation of atresia in the hamster

Hypophysectomized PMSG-primed hamsters were injected with PMSG antiserum and the theca and granulosa cells of the resulting atretic follicles were incubated in vitro. In the absence of added hormone, 17 alpha-hydroxyprogesterone and oestradiol production was not detectable in granulosa cells collected and incubated at 0, 12 and 24 h after antiserum. Progesterone production was not detected in control incubations at 0 h but was measurable with cells collected at 12 h after PMSG antiserum. When incubated with androstenedione or pregnenolone (10 ng/ml for each) 17 alpha-hydroxyprogesterone and progesterone production by granulosa cells were significantly increased at 0, 12 and 24 h after antiserum. Granulosa cells were capable of aromatizing androstenedione to oestradiol at all times examined. At 0 and 12 h after antiserum to PMSG, isolated thecal shells produced androstenedione. LH stimulation caused increased androstenedione production in all thecae at 0 h, in 50% of the thecae at 12 h and in none at 24 h after antiserum. Thecal shells produced 17 alpha-hydroxyprogesterone in response to LH at 0, 12 and 24 h after antiserum, and produced progesterone at all times examined. Thecae also responded to LH with increased progesterone production up to 72 h after antiserum. These experiments demonstrate that one important steroidogenic event in atresia may be the loss of activity of C 17,20 lyase in the theca leading to loss of substrate (androstenedione) for granulosa cell aromatization, although aromatase activity is present until at least 24 h after the induction of atresia.     

Comparative studies of androgen metabolism in theca and granulosa cells of human follicles in vitro

In eight separate experiments, theca and granulosa were isolated from human follicles (5–25 mm in diameter), and their capacities to metabolize radiolabelled testosterone in 24 hour cultures were assessed. Theca metabolized testosterone primarily to androstenedione, however significant aromatization to estradiol-17β and to estrone was also observed. Granulosa metabolized testosterone primarily to estradiol-17β and estrone, while smaller quantities were converted to androstenedione. In seven of these experiments, the intermediate of aromatization, 19-hydroxytestosterone, was identified. In six of these experiments, theca, when compared to granulosa, produced more androstenedione but less estradiol-17β and estrone. 5α-Reduced androgens were non-detectable or produced in small quantities. In a single experiment, metabolism of androstenedione was compared to metabolism of testosterone by both theca and granulosa. Theca metabolized androstenedione to testosterone in smaller quantities than testosterone to androstenedione. Granulosa metabolized androstenedione to testosterone in higher quantities than testosterone to androstenedione. Both theca and granulosa aromatized androstenedione more readily than testosterone.     

Significance of the delta 5 and delta 4 steroidogenic pathways in the hamster preovulatory follicle

Isolated hamster granulosa cells and theca from preovulatory follicles were incubated in vitro for 2 and 6 h in the absence/or presence of LH and steroid substrates. The purpose of the experiments was to determine, in theca, the relative activities of the delta 5 and delta 4 pathways under controlled conditions, and to compare the ability of granulosa cells and theca to form progesterone from exogenous pregnenolone. The results of the experiments show that the delta 5 pathway in theca predominates before and up to 2 h after LH stimulation. The delayed effect of LH after 2 h is a switch from delta 5 to delta 4 as the major metabolic pathway. Progesterone formation from exogenous pregnenolone is 7 to 10 times greater in unstimulated granulosa cells than in theca. Acute effects of LH lead to increased conversion of exogenous pregnenolone to progesterone in granulosa cells but not theca. LH does, however, acutely stimulate the thecal conversion of DHEA to androstenedione. The longer term effect of LH in both cell types is to increase pregnenolone conversion to progesterone.     

Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells: influence of follicle size on responses to GDF9

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.     

Estradiol-17 beta and androgen secretion by isolated porcine ovarian follicular cells in vitro

The cellular sources and gonadotropic regulation of porcine ovarian estrogen and androgen were assessed by culturing isolated granulosa cells and thecal cells from medium size follicles (4-6 mm diameter) separately for 24 h in a chemically defined medium containing gonadotropins and (or) testosterone. At the end of the culture period, estradiol-17 beta (estradiol) and androgens in the media were determined by radioimmunoassays. Production of estradiol by granulosa cells without an exogenous aromatizable androgen was low in the absence or presence of a highly purified preparation of either follicle-stimulating hormone (FSH. 0.25 microgram/mL) or luteinizing hormone (LH. 1 microgram/mL). Addition of testosterone or androstenedione (0.5 microM), but not dihydrotestosterone or pregnenolone, significantly increased estradiol secretion. Additional increases were observed when FSH, LH, prostaglandin E2, or dibutyryl cyclic 3'.5'-adenosine monophosphate was present. Production of estradiol by thecal cells was low in the presence or absence of exogenous testosterone, and was essentially unaffected by the presence of gonadotropins. Thecal cells, however, released large amounts of androstenedione and smaller amounts of testosterone and other androgens during 24-h culture and the production of these androgens was stimulated by LH but not by FSH. Androgen secretion by granulosa cells was negligible when compared with the theca and was unaffected by gonadotropins. It is concluded that the theca is the prime site for follicular androgen biosynthesis by the porcine ovarian follicle, and, upon LH stimulation, may provide androgen precursors for estradiol production by granulosa cells.     

In-vitro effects of angiotensin II on steroid production by hamster ovarian follicles and on ultrastructure of the theca interna

Summary Angiotensin II (AII) is present in the mammalian ovary and has been correlated with atresia in follicles. Since the theca interna may be one site at which atresia is intiated, we wished to determine whether AII exerts an effect on theca interna from explanted ovarian follicles of hamsters. Hamsters were sacrified on the morning of proestrus, and ovaries were removed. Preovulatory follicles were excised from the ovaries, and cultured with one of the following components: medium alone (control); medium plus AII (1x10^-6 M); the AII-receptor antagonist [Sar¹, Ile⁸] AII (1x10^-4 M); or AII plus antagonist. After 72 h, the follicles were processed for transmission electron microscopy (to determine quantities of theca interna organelles involved in the steroid synthetic pathway) or for protein determination (to normalize steroid production rates). The incubation medium was drawn off and analyzed by radioimmunoassay for progesterone, androstenedione, or estradiol-17. There was a significant positive correlation (r=0.92, P<0.01) between follicular androstenedione secretion and area comprising theca interna smooth endoplasmic reticulum. In the theca interna, AII induced a two-fold and 1.6-fold increase in lipid droplet number and area comprising smooth endoplasmic reticulum, respectively (P<0.05). Excess antagonist negated the increase in cell or-ganelles and also reduced androstenedione secretion compared with AII alone (P<0.05). Most importantly, AII significantly augmented the ratio of androstenedione: estradiol-17 secretion by 44% over that of control. The ultrastructural changes observed in this study and the increase in the andostenedione: estradiol-17 production ratio are consistent with atresia-like changes in ovarian follicles. We believe, therefore, that AII is involved, possibly at its membrane receptor, in an aspect of the overall process of follicular atresia, operating in part at the level of the theca interna.     

Distribution of delta-5 -3beta-hydroxysteroid dehydrogenase activity in the Graafian follicle of the sheep.

The localization of delta-5 -3beta-hydroxysteroid dehydrogenase (3 beta-HSD) has been examined in ovarian follicles in vivo and in vitro, and related to oestrogen and progesterone production. In vivo, during the oestrous cycle, enzyme activity was restricted to the theca interna of the one or two most advanced follicles in each animal, but was present only between Day 2 and 5 and between Day 13 and ovulation. High levels of oestrogen were found in the ovarian venous blood only when follicles containing 3 beta-HSD were present. When sheep were injected with PMSG, the theca interna in a number ofsmall follicles acquired 3 beta-HSD activity and began to secrete oestrogen within 12 hr of the injection. The enzyme was not detected in the membrana granulosa of any follicles before ovulation but within a few hours of ovulation, 3 beta-HSD activity was present in the granulosa lutein cells. In vitro, large activated follicles exhibited 3 beta-HSD activity in the theca interna and secreted high levels of oestrogen into the culture medium. When LH was added to the medium oestrogen secretion was inhibited; within 48 hr, the follicles were secreting high levels of progesterone, and 3 beta-HSD activity was present in both the membrana granulosa and the theca interna. Dibutyryl cyclic adenosine monophosphate mimicked the effect of LH in suppressing oestrogen secretiion, but did not induce production of progesterone; the distribution of 3 beta-HSD activity infollicles treated with this nucleotide was the same as in those cultured in control medium.     

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.     

In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of follicle-regulatory protein by porcine granulosa cells

Follicle-regulatory protein (FRP) affects ovarian steroidogenesis and thus follicular maturation. However, secretion of FRP by cells from different-sized follicles as well as the modulation of FRP production by gonadotropins and locally produced steroids are unknown. To evaluate which cell type secretes FRP, theca and granulosa cells were obtained from porcine follicles. In addition, the effects of follicle-stimulating hormone (FSH) and steroids on FRP secretion from granulosa cells of small (less than 3 mm), medium (3-6 mm), and large (greater than 8 mm) porcine follicles and theca cells of large follicles were determined. Granulosa cells were obtained from follicular aspirates, whereas theca cells were recovered after digestion of the stereomicroscopically removed thecal layer. Both were cultured in monolayer in serum-free medium. Granulosa cells were treated as follows: 1) control; 2) FSH (250 ng/ml); 3) progesterone (500 ng/ml, 3 micrograms/ml), or estradiol-17 beta (500 ng/ml, 4 micrograms/ml), or dihydrotestosterone (500 ng/ml, 1 microgram/ml); 4) FSH + progesterone, or estradiol-17 beta, or dihydrotestosterone. Theca cells received the same treatment except that human chorionic gonadotropin (hCG) (5m IU/ml) was used in place of FSH. At 48 or 96 h, media were removed and FRP was quantitated by an Enzyme-Linked Immunosorbent Assay (ELISA). FRP was identified in granulosal medium from follicles of all sizes, but was not present in thecal cultures. At 48 h, granulosa cells from small and medium-sized follicles produced more FRP (20.04 +/- 4.4, 35.42 +/- 4.1 immunoreactive units [IRU]) than cells from large (3.53 +/- 0.97 IRU) follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro steroidogenesis by dissociated rat follicles, primary to antral, before and after injection of equine chorionic gonadotropin.

Prepubertal female rats were injected s.c. with 5.0 IU eCG, and ovaries were collected 24 and 48 h post-eCG, on Day 25, as well as from an untreated group also on Day 25. Large antral follicles were manually dissected, and the ovarian remnants were incubated with collagenase overnight to liberate preantral follicles from adhering stromal cells. The viability of the follicles was established by normal histology and lack of pyknotic granulosa cells (GCs) and by their ability to secrete steroids. After a 1-h baseline incubation, either 10 ng LH or 100 ng FSH was added for an additional hour, and the media-before and after gonadotropin administration-were used to measure progesterone, androstenedione, and estradiol by RIA. A distinct hierarchy existed in steroid synthesis, with the maximal production by the largest (700 microm) antral follicles. The major steroid that had accumulated after addition of LH at 48 h post-eCG was androstenedione (1099 pg/follicle per hour), followed by equal amounts of progesterone (155 pg/follicle per hour) and estradiol (191 pg/follicle per hour). There was a precipitous drop in steroid production by 550-microm and 400-microm antral follicles, especially in estradiol for the latter-sized follicles (0.08 pg/follicle per hour). Preantral follicles also produced progesterone and androstenedione after addition of LH. For example, follicles 222 microm in diameter with 4-5 layers of GCs and well-developed theca responded to LH at 48 h post-eCG by accumulating androstenedione (37 pg/follicle per hour) and progesterone (6 pg/follicle per hour) but negligible estradiol. The smallest follicles secreting steroids, 110-148 microm in diameter, had 2-4 layers of GCs. However, primary follicles (1 layer of GCs and no theca) did not synthesize appreciable amounts of any steroid. Although small preantral follicles were consistently stimulated by LH, FSH was ineffective. This result differs from findings in the hamster showing that intact preantral follicles with 1-4 layers of GCs and no theca respond to FSH by secreting progesterone in vitro (Roy and Greenwald, Biol Reprod 1987; 31:39-46). The technique developed to collect intact rat follicles should be useful for numerous investigations.     

Heme oxygenase in the rat ovary: immunohistochemical localization and possible role in steroidogenesis

The objectives of this study were to determine if heme oxygenase (HO), which catalyzes the degradation of heme and the formation of carbon monoxide (CO), is localized in the rat ovary and, if so, to determine if hemin (a substrate for HO) or chromium mesoporphyrin (CrMP, an inhibitor of HO), alter basal or gonadotropin-induced steroidogenesis. The hypothesis was that CO produced endogenously by HO suppresses steroid hormone production by the ovary similar to the action of nitric oxide. For the histological localization of HO, sections of ovaries obtained from mature Holtzman Sprague-Dawley rats were immunostained for two of the HO isoforms, HO-1 and HO-2. Theca cells and granulosa cells of follicles and luteal cells stained for HO-1, whereas the ovarian stroma showed a low intensity of staining. Theca, granulosa cells, and corpora lutea as well as the ovarian stroma exhibited HO-2 staining. HO-2 immunostaining appeared more intense for theca cells than granulosa cells. In the study of steroidogenesis, three daily injections of hemin stimulated basal- and gonadotropin-induced androstenedione and estradiol secretion from ovaries of pregnant mare serum gonadotropin-treated immature rats in vitro, but had no effect on progesterone production. A similar treatment with CrMP suppressed basal- and gonadotropin-induced secretion of progesterone and androstenedione, but had no effect on estradiol production. These data, taken together, show the existence of HO in the rat ovary and suggest a possible stimulatory role of endogenous CO in the production of ovarian steroids.     

Steroidogenesis by bovine theca interna in an in vitro perifusion system

The aims of these studies were: to examine the steroidogenic responses of perifused bovine theca interna to varying flow rates of media and varying amounts of luteinizing hormone (LH), and to compare the steroidogenic outputs of theca interna from follicles of differing size and health with those of other ovarian tissues. The results showed that the outputs of androstenedione by thecae interna from healthy but not atretic follicles, with or without stimulation by LH, were amplified by the flow rate of media. Steroidogenesis by perifused theca interna was also influenced by the mass and concentration of LH as well as by the duration of exposure to LH. When expressed on a per unit mass basis, the outputs of androstenedione from LH-primed thecae interna from small (2-5.5 mm diameter), medium (6-9.5 mm diameter) and large (greater than or equal to 10 mm diameter) healthy follicles were comparable. But when the above data were expressed per total mass of theca interna, the androstenedione output increased significantly with increasing follicular diameter (P less than 0.01). Under the experimental conditions employed, the fraction of androstenedione produced by thecal tissue as a percentage of the total output of progesterone, androstenedione, testosterone and estradiol was 82%, whereas the progesterone, testosterone and estradiol fractions were 1%, 15% and 2%, respectively. By contrast, the granulosa cell output of progesterone, androstenedione, testosterone and estradiol were 79%, 0%, 0% and 21%, respectively. When this cell type was supplied with saturating amounts of androstenedione, it contributed greater than or equal to 90% of the total quantity of estradiol by the two cell types in isolation.     

Steroid hormone formation in bovine ovarian follicles.

In an attempt to assess histophysiological implication of the follicular compartment of the bovine ovary in steroid hormone formation and the effect of human chorionic gonadotropin (hCG) in vitro on follicular steroidogenesis, minces of follicular tissues from non-gravid bovine ovaries were incubated with radioactive testosterone or acetate in the presence and absence of hCG. Significant amounts of estrone and estradiol-17beta were formed on incubation with testosterone-4-14C; hCG decreased the conversion approximately by 30%. The major radioactive products formed from acetate-l-14C were androstenedione and testosterone with lesser amounts of dehydroepiandrosterone and 17-hydroxyprogesterone. In addition, small amounts of progesterone, 17-hydroxypregnenolone, estrone and estradiol-17beta were formed. Histology of the dissected follicle specimens was characterized by dominant theca cells undergoing luteinization with small amounts of granulosa cells, which showed neither proliferation nor luteinization. The pattern of distribution of radioactivity among the steroids formed from acetate-14C was considered to represent steroidogenic profile of bovine atretic follicles. The addition of hCG in vitro increased the overall incorporation of radioactive acetate into the steroids approximately by 50%, although the range of increase was not uniform in the individual steroids under the exprimental conditions.     

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 5β-pregnandione. DHEA metabolism by granulosa cells yielded mainly A4, and minute quantities of 5β-androstan-3,17-dione (5β-dione) were detected. The only significant metabolite obtained in granulosa cells from A4 was 5β-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, 17α-OHP4, 17α-OHP5, 5β-pregnandione, and DHEA, were found. When DHEA was the precursor A4 was produced in higher amounts than 5β-dione. A4 was mainly transformed into 5β-dione. In similar conditions, T was transformed into A4. These results show that granulosa cells have enzymatic activities of 3β-hydroxysteroid dehydrogenase/5-4 isomerase (3β-HSD from P5 and DHEA), 17β-hydroxysteroid dehydrogenase (17β-HSD from T) and 5β-reductase (from P5, DHEA and A4). Whereas theca interna cells have enzymatic activities of cytochrome P450c17 (from P5 and P4), 3β-HSD (from P5 and DHEA), 17β-HSD (from T) and 5β-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.     

In vitro steroidogenesis by primary to antral follicles in the hamster during the periovulatory period: effects of follicle-stimulating hormone, luteinizing hormone, and prolactin

Hamster ovarian follicles at Stages 1 to 10 (Stages 1-4: follicles with 1-4 layers of granulosa cells (GC); Stages 5-7: 5-10 layers GC plus theca; Stages 8-10: antral follicles) were isolated on the morning of proestrus or estrus and incubated for 2 h in the absence or presence of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (Prl), progesterone (P4), 17 alpha-hydroxyprogesterone (17OHP), or androstenedione (A). Steroid accumulations in the media were measured by radioimmunoassay (RIA). On proestrus, without any hormonal stimulus, consistent accumulation of P4 through estradiol-17 beta (E2) occurred in low amounts only from Stage 6 and on; both FSH (5-25 ng) and LH (1-25 ng) significantly stimulated steroidogenesis by Stage 6-10 follicles, and the effects of FSH, except for Stage 10, were largely attributable to LH contamination. However, 25 ng FSH significantly stimulated A production by Stages 1-4, whereas 1-25 ng LH was ineffective. On estrus, follicles at all stages, especially 1-6, showed significant and dose-dependent increases in P4 production in response to FSH; both FSH and LH significantly stimulated P4 and 17OHP accumulation from Stage 5 onwards; however, there was no increase in A and E2 compared to controls. Even the smallest estrous follicles showed a shift to predominance of P4 accumulation. On proestrus, Prl had a negative influence on LH-induced accumulation of P4 and 17OHP by Stages 7-9 and 6-8, respectively, without affecting A or E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.