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.     

Effect of activin-a on goat granulosa cell steroidogenesis

Growth factors are said to play a significant role in the development of ovarian follicles. We wished to measure the content of one growth factor, activin-A in goat ovarian follicles, and study its effect on goat granulosa cells steroidogenesis. The follicular fluid content of activin-A from small, medium and large antral follicles was determined by two-site enzyme immunoassay. The results showed that activin-A concentration in the follicular fluid increased as the size of the follicle increased and, thus, may act as a local regulator of follicle development. To examine this possibility, the effect of increasing concentration of activin-A (0, 1, 10, 100 ng/mL) on differentiated goat granulosa cells steroidogenesis was evaluated in vitro for 48 hours in a chemically defined medium. Activin-A treatment resulted in a significant inhibition of progesterone production concomitant with a significant stimulation of estradiol production. These results were confirmed by time-effect of 50 ng/mL activin-A on goat granulosa cells steroidogenesis for 24, 48 and 72 hours. Granulosa cells displayed differential steroidogenic responses to activin-A, estradiol production becoming enhanced and progesterone production suppressed. Based on these findings, it appears that activin-A is a local regulator of goat granulosa cell steroidogenesis, and may act to promote granulosa cell differentiation and inhibit its luteinization.     

Intrafollicular concentrations of steroids and steroidogenic enzymes in relation to follicular development in the mare

The objective of the present study was to determine the changes in follicular fluid steroid concentrations and in granulosa cell steroidogenic enzyme expression during the follicular phase, in relation to follicular size and physiological status in the mare. Follicular fluid and follicular cells were recovered by ultrasound-guided follicular punctures either around the time of emergence of the dominant follicle, at the end of the dominant follicle growth, or at the preovulatory stage, after injection of gonadotropin to induce ovulation. Cellular relative amounts of steroidogenic acute regulatory protein (StAR), P450-side chain cleavage (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17alpha-hydroxylase, and aromatase were assessed by semiquantitative Western blot and densitometry. Follicular fluid was assayed for cholesterol concentrations by colorimetric assay and for progesterone, testosterone, and estradiol-17beta concentrations by RIA. Intrafollicular concentrations of progesterone and estradiol-17beta significantly increased in the dominant follicle during growth. After injection of gonadotropin, follicular maturation was characterized by a decrease in estradiol-17beta concentrations and a further increase in progesterone concentrations. Granulosa cells from dominant follicles had increased levels of StAR, P450(scc), 3betaHSD, and aromatase during growth, but decreased levels during maturation. Levels of StAR, P450(scc), 3betaHSD, and aromatase, as well as progesterone and estradiol-17beta, were lower in granulosa cells from subordinate than from dominant follicles. We did not observe a relationship between the steroidogenic activity of follicles and the capacity of their enclosed oocytes to complete meiosis in vitro.     

Intrafollicular content of luteinizing hormone receptor, alpha-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare

The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm were collected. The nuclear stage of the oocytes after in vitro culture was determined microscopically. Granulosa and cumulus cell amounts of LH receptor, alpha-inhibin, and aromatase were assessed by the semiquantitative Western blot method and image analysis. Follicular fluids were assayed for progesterone (P4) and estradiol-17beta (E2). The three factors were expressed in mural granulosa and cumulus cells from all follicles from the gonadotropin-independent growth period until the preovulatory stage. Considering all the follicles punctured, the amounts of LH receptor and alpha-inhibin in granulosa cells were not different for the three physiological stages studied. The amounts of aromatase in granulosa cells, as well as the E2:P4 ratios, were higher for follicles punctured during the follicular phase than for the two other groups (p < 0.05). Considering the data from the three groups, the E2:P4 ratio and the LH receptor and aromatase contents, but not alpha-inhibin, in granulosa cells increased with an increase in follicular diameter (p < 0.01). The E2:P4 ratios and the amounts of LH receptor, alpha-inhibin, and aromatase in granulosa cells were lower in follicles 5-9 mm in diameter than in larger ones (p < 0.05). In cumulus cells, the amounts of the three factors were different neither between the three groups nor between the follicular diameters. Although we could not establish any obvious relationship to oocyte competence for in vitro maturation, the influence of the follicle diameter on the content of LH receptors, alpha-inhibin, and aromatase in granulosa cells was similar to the influence of follicle diameter on oocyte competence. Therefore, one can hypothesize that, in the mare, there is a link between the acquisition of oocyte competence and the expression of these factors in the follicular cells.     

Insulin-like growth factor (IGF) 2 stimulates steroidogenesis and mitosis of bovine granulosa cells through the IGF1 receptor: role of follicle-stimulating hormone and IGF2 receptor

Little is known regarding the role of insulin-like growth factor 2 (IGF2) and the regulation of the IGF2 receptor (IGF2R) during follicular development. Granulosa cells were collected from small (1-5 mm) and large (8-22 mm) bovine follicles and were treated with IGF2 for 1-2 days in serum-free medium, and steroid production, cell proliferation, specific (125)I-IGF2 binding, and gene expression were quantified. IGF2 increased both estradiol and progesterone production by granulosa cells, and cells from large follicles were more responsive to the effects of IGF2 than those from small follicles. Abundance of aromatase (CYP19A1) mRNA was stimulated by IGF2 and IGF1. The effective dose (ED(50)) of IGF2 stimulating 50% of the maximal estradiol production was 63 ng/ml for small follicles and 12 ng/ml for large follicles, and these values were not affected by FSH. The ED(50) of IGF2 for progesterone production was 20 ng/ml for both small and large follicles. IGF2 also increased proliferation of granulosa cells by 2- to 3-fold, as determined by increased cell numbers and (3)H-thymidine incorporation into DNA. Treatment with IGF1R antibodies reduced the stimulatory effect of IGF2 and IGF1 on estradiol production and cell proliferation. Specific receptors for (125)I-IGF2 existed in granulosa cells, and 2-day treatment with estradiol, FSH, or cortisol had no significant effect on specific (125)I-IGF2 binding. Also, FSH treatment of small- and large-follicle granulosa cells had no effect on IGF2R mRNA levels, whereas IGF1 decreased IGF2R mRNA and specific (125)I-IGF2 binding. Granulosa cell IGF2R mRNA abundance was 3-fold greater in small than in large follicles. These findings support the hypothesis that both IGF2 and its receptor may play a role in granulosa cell function during follicular development. In particular, increased free IGF1 in developing follicles may decrease synthesis of IGF2R, thereby allowing for more IGF2 to be bioavailable (free) for induction of steroidogenesis and mitogenesis via the IGF1R.     

Local regulation of granulosa cell maturation

Fluid from small antral follicles inhibits several functions of porcine granulosa cells from 3-10-mm follicles in vitro, whereas fluid from large follicles stimulates cells from small follicles. Local factors may be needed in vivo to enable granulosa cells to fully respond to gonadotrophins. Only those follicles containing local stimulators may develop while those containing inhibitors may become arrested in development or become atretic. We have compared the actions of GnRH analogs and chondroitin sulfate (CS) on porcine granulosa cell steroidogenesis with actions of follicular fluids. GnRH agonist mimicked follicular fluid inhibition of progesterone secretion but GnRH antagonist did not antagonize follicular fluid's inhibitory actions. GnRH antagonist mimicked follicular fluid enhancement of basal and LH-stimulated progesterone secretion, but did not mimic follicular fluid enhancement of FSH action or stimulation of estrogen secretion. GnRH agonist blocked the enhancement of LH-stimulated progesterone secretion by both GnRH antagonist and stimulatory follicular fluid. CS inhibited basal and LH-stimulated progesterone secretion but did not inhibit pregnenolone utilization, aromatase activity or estrogen secretion. GnRH-like molecules and CS may be partially responsible for follicular fluid actions on granulosa cells. The actions of other molecules are needed to explain the total effects of follicular fluids on granulosa cells.     

Effect of insulin-like growth factor-1 on steroidogenesis in cultured carp ovarian follicles: interactions with estradiol

The biological action of insulin like growth factor-1 (IGF-1) on follicular steroidogenesis during follicular development in common carp was examined. Studies were carried out by culturing small (1-2 mm diam.) and large (> 2 mm diam.) follicles. IGF-1 (0.3-100 ng/ml) had no effect on progesterone accumulation or aromatase activity during 48 hr culture of small follicles. Progesterone accumulation by large follicles was also unaffected by IGF-1 over the same period, although aromatase activity was stimulated in a dose dependent manner (8-fold increase over basal levels with a maximum stimulatory dose of 30 ng IGF-1/ml). In contrast, small and large follicles responded to IGF-1 in terms of both progesterone accumulation and aromatase activity after longer periods of culture (4 days for progesterone and 6 days for aromatase). Concurrent treatment of small follicles with estradiol (10(-7) M) enhanced the action of IGF-1 on both indices of steroidogenesis and advanced the time at which IGF-1 stimulated activity was first detectable. The effect of estradiol on follicular IGF-1 responsiveness were independent of cell number. In summary, these results demonstrate varied actions of IGF-1 carp ovarian follicular steroidogenesis in vitro. The results indicate that carp follicles acquire responsiveness to IGF-1 in terms of aromatase activity during follicular development in vivo and that estradiol can induce the response in vitro. The results also suggest that estrogen and progesterone biosynthesis by cultured carp ovarian follicles is differentially regulated by IGF-1. Together, these results provide new insights into the biological actions of IGF-1 in fish ovary.     

Decline in circulating estradiol during the periovulatory period is correlated with decreases in estradiol and androgen, and in messenger RNA for p450 aromatase and p450 17alpha-hydroxylase, in bovine preovulatory follicles

The preovulatory surge of gonadotropins induces meiotic maturation of the oocyte, the follicular/luteal phase shift in hormone production, and ovulation. This complex and rapid series of developmental changes is difficult to study in large mammals, such as primates and ruminants, because variability in the length of individual reproductive cycles makes it virtually impossible to predict the time of the LH surge. We have validated an experimental model for inducing the LH surge and ovulation in cattle and used it to study the sequence of changes in hormone secretion and some of the mechanisms of these changes. Luteolysis and a follicular phase were induced by injection of prostaglandin F(2alpha); injection of a GnRH analogue 36 h later induced an LH surge and ovulation. The LH surge peaked 2 h after GnRH and ovulation followed 22-31 h after the surge, consistent with the periovulatory interval in natural cycles. The ensuing luteal phase was normal, both in length and in concentrations of circulating progesterone. In experiment I, the uteroovarian effluent was collected, via cannulation of the vena cava, at frequent intervals relative to GnRH injection. Circulating estradiol declined progressively after GnRH, reaching a nadir by 8-10 h before ovulation, whereas concentrations of androstenedione and testosterone remained constant. In experiment II, preovulatory follicles were obtained at 0, 3.5, 6, 12, 18, or 24 h after GNRH: Concentrations of androgens and estradiol were measured in follicular fluid and medium from cultures of follicle wall (theca + granulosa cells); steady-state levels of mRNA for 17alpha-hydroxylase (17alphaOH) and P450 aromatase were measured in follicular tissue. Shortly after the LH surge (3.5 h post-GnRH) there was an acute increase in the capacity of follicular tissue to secrete androstenedione, but not estradiol, in vitro. Thereafter, both androgens and estradiol declined, both in follicular fluid and in medium collected from cultures of follicle wall. Levels of mRNA for 17alphaOH and aromatase in follicle wall decreased significantly by 6 h after GnRH, suggesting that declining levels of these enzymes underlie the decreases in steroid production by follicular cells. These results show that in cattle the preovulatory decrease in follicular estradiol production is mediated by redundant mechanisms, because androgen production and the capacity of granulosa cells to convert androgens to estradiol decline coordinately, in concert with decreases in mRNA for 17alphaOH and P450 aromatase.     

Granulosa cell steroidogenesis and follicular fluid steroid concentrations after the onset of oestrus in cows

Granulosa cell responsiveness at an early (1-2 h) or late (14-16 h) stage of differentiation following the onset of oestrus [and presumably the LH surge] was studied in 16 cows. Follicular fluid collected at the early stage (8 preovulatory follicles) had a higher concentration of testosterone (P less than 0.05), oestradiol (P less than 0.01) and oestrone (P less than 0.01) than did follicular fluid collected at the late stage of oestrus (8 preovulatory follicles). No difference in follicular fluid progesterone was noted between follicles collected at the early and late stages of oestrus. Granulosa cells collected at the early stage of oestrus had a higher in-vitro response (progesterone production) to LH (P less than 0.05), forskolin (P less than 0.08) and diacylglycerol (P less than 0.05) than did granulosa cells collected at the late stage of oestrus. However, later stage granulosa cells produced more (P less than 0.01) progesterone after culture with prostaglandin E-2 than did earlier stage granulosa cells. These results show that follicular fluid oestrogen decreases, which suggests a loss of aromatase activity as oestrus progresses, and that granulosa cells become refractory (low progesterone production) to in-vitro LH, forskolin, and diacylglycerol challenge, yet acquire responsiveness to prostaglandin E-2 as oestrus progresses.     

Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.     

Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.

Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

Morphological and biochemical characteristics during ovarian follicular development in the pig

Ovaries were recovered from groups of naturally cyclic pigs (N = 5) on each of Days 16, 18, 20 and 21 of the oestrous cycle. Follicular diameter, follicular fluid volume and concentrations of oestradiol, testosterone and progesterone, and granulosa cell number were determined in all follicles greater than or equal to 2 mm in diameter (n = 511). In alternate follicles either granulosa cell aromatase activity and theca testosterone content or 125I-labelled hCG binding to granulosa and theca were determined. The mean total number of follicles recovered per animal decreased as the follicular phase progressed and a strong positive relationship (P less than 0.001) existed between follicular diameter and volume on all days. The number of granulosa cells recovered per follicle was variable, and not related to oestrogenic activity of the follicles. Mean follicular fluid oestradiol, testosterone and 125I-labelled hCG binding all increased until Day 20 and decreased on Day 21, whereas mean theca testosterone content, 125I-labelled hCG binding to theca tissue and aromatase were all maximal on Day 21. On Days 20 and 21 a subset of 14-16 large follicles was readily distinguishable from the remaining smaller, less oestrogenically active population in each animal. Yet, consistently within these subsets there was a difference in follicular diameter of approximately 2.0 mm and also a considerable range of biochemical development even among follicles of equal size. These results indicate asynchrony at the time of recruitment and selection among follicles destined to ovulate and suggest that heterogeneity continues into the immediate preovulatory period.     

The role of ovarian proteases and their inhibitors in ovulation

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.     

Time dependent and cell-specific action of polychlorinated biphenyls (PCB 153 and PCB 126) on steroid secretion by porcine theca and granulosa cells in mono- and co-culture.

To characterize PCB action on follicular cell steroidogenesis two PCB congeners were selected as model substances. PCB 126 because of its dioxin-like configuration and high toxicity and PCB 153 because it is one of the most commonly detected PCB congeners in breast milk. The direct effect of PCBs was investigated using a culture system of porcine theca and granulosa cells collected from porcine preovulatory follicles. Granulosa and theca cells were cultured in M199 medium supplemented with 1, 10 or 100 pg/ml of PCB 126 or 1, 10 and 100 ng/ml of PCB 153. The media were changed after 48, 96 and 144 h and frozen until further estradiol (E2) analysis. Additionally, progesterone (P4) was measured in the granulosa cells culture medium and testosterone (T) in theca cells culture medium. Decrease of testosterone concentration in the theca cells culture medium was found after 96 and 144 hours in culture by both investigated PCB congeners. A decrease in E2 concentration was found after exposure to PCB 153. These findings suggest different actions of two congeners on the steroid synthesis in theca cells. The lack of an increase in E2 secretion after the exposure to PCB 126 could be due to depletion of androgen precursor. In granulosa cell culture PCB153 decreased E2 secretion and increased P4 secretion suggesting luteinization and disruption of aromatization process. PCB 126 in a doses from 1 to 10 pg had no effect on granulosa cells steroidogenesis. However, the highest dose (100 pg) increased concentration of both E2 and P4. This observation suggest that PCB 126 in a pharmacological doses may affect cell membrane permeability, thereby increasing steroid outflow into the medium. These results suggest time dependent and cell-specific differences in PCB 153 and 126 action on follicular cells steroidogenesis. Further studies are required to elucidate the mechanism of PCBs action on ovarian steroidogenesis.     

Effects of Kit Ligand and anti-Kit antibody on growth of cultured mouse preantral follicles

Paracrine regulations between the oocyte and granulosa cells are likely to be key regulators of early folliculogenesis. Evidence obtained from genetic mutants as well as in vivo experiments suggest that Kit and Kit Ligand (KL) may regulate early follicular morphogenesis and function. In this study, we used in vitro culture of intact mouse follicles to confirm and extend these findings. Two concentrations of Kit Ligand (20 and 50 ng/ml) or an antibody blocking the Kit-Kit Ligand interactions (SC1494) were added to preantral follicles grown individually for 12 days and which were finally triggered to ovulate. Effects on follicle and oocyte survival, granulosa cell function (antrum formation, cell numbers, steroidogenesis), and oocyte function (growth, survival, nuclear and/or cytoplasmic maturation) were then analyzed. In optimal culture conditions (presence of 5% fetal calf serum), 50 ng/ml of KL significantly improved cytoplasmic maturation of the oocyte and increased follicular testosterone output, but other parameters were not altered. In serum-free culture conditions, KL was mitogenic for granulosa cells at 50 ng/ml, but could not induce antrum formation and no differences were observed between control and treated groups for steroidogenesis or oocyte growth. Blockade of Kit-Kit Ligand interactions by addition of a blocking antibody decreased oocyte survival 6-9 days after addition of the antibody, but did not upset granulosa cell proliferation. Antrum formation was, however, strongly inhibited. In addition, the blocking antibody markedly reduced aromatase activity of granulosa cells. We conclude that Kit/KL interactions are important for antrum formation and follicular steroidogenesis and regulate survival and cytoplasmic maturation of the oocyte.     

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)     

Interleukin-1 alpha increases thecal progesterone production of preovulatory follicles in cyclic hamsters

Adult cyclic hamsters were used to study the effects of interleukin-1 alpha (IL-1 alpha) on in vitro steroidogenesis in preovulatory follicles. IL-1 alpha increased progesterone secretion by preovulatory follicles during a 24-h incubation in RPMI-1640 medium containing hCG (100 mIU/ml) (progesterone levels: 17.5 +/- 2.2 vs. 10.6 +/- 1.9 ng/follicle/ml, p less than 0.05). IL-1 alpha alone had no effect on follicular steroidogenesis. The source of increased progesterone secretion was the thecae (9.8 +/- 1.0 vs. 5.8 +/- 0.4 ng/2 thecae/ml, p less than 0.01) and not the granulosa cells (6.6 +/- 0.2 vs. 6.8 +/- 0.5 ng/20,000 viable granulosa cells/ml). IL-1 alpha also stimulated production of testosterone in thecae of preovulatory follicles. The follicular progesterone increase was dependent on the time of incubation and dose of IL-1 alpha. IL-1 alpha at 5-50 U/ml maximally stimulated progesterone production in the preovulatory follicles, and no significant effect of IL-1 alpha was observed until the 12th hour of incubation. The effects of IL-1 alpha on in vitro steroidogenesis in preantral follicles, experimentally induced atretic preovulatory follicles, and newly formed corpora lutea were examined. IL-1 alpha in the presence of hCG also significantly increased progesterone secretion by atretic preovulatory follicles. In the incubation of preantral follicles or newly formed corpora lutea, however, IL-1 alpha did not alter steroidogenesis. These results indicate that IL-1 alpha stimulates progesterone secretion by preovulatory follicles and that the target tissue for this effect is the thecal layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroidogenesis in porcine atretic follicles: loss of aromatase activity in isolated granulosa and theca

To evaluate the mechanisms involved in the reduction of estrogen concentrations in porcine follicular fluid during atresia, nonatretic and atretic follicles ranging from 4 to 7 mm in diameter were selected. Follicular fluid estrogen concentrations were 7-16-fold less in the atretic follicles. Isolated granulosa cells from atretic follicles demonstrated a significant reduction in aromatase activity and in follicle-stimulating hormone (FSH)-induced progesterone production in vitro compared to granulosa cells from nonatretic follicles. Isolated theca from atretic follicles also demonstrated a reduction in estrogen production. However, androgen concentrations were equivalent in the follicular fluid of atretic and nonatretic follicles, and theca from atretic follicles maintained testosterone and androstenedione production in vitro. The loss of thecal aromatase activity with atresia is not secondary to a reduction in FSH responsiveness, since FSH did not increase thecal progesterone production in vitro. Cell degeneration also does not account for the reduction in thecal estrogen production, since both androgen output in vitro and follicular fluid androgen concentrations were maintained. These data thus demonstrate that a mechanism other than reduced FSH responsiveness must account for the selective loss of thecal aromatase activity in this stage of atresia.     

Short-term effects of FSH in vitro on granulosa cells of individual sheep follicles

In Romanov ewes at Day 13 or 14 of the cycle, granulosa cells originating from individual follicles were studied in short-term incubations for aromatase activity and thymidine incorporation. The study was performed on 76 follicles of different sizes (2-7 mm diameter) and degree of atresia, as assessed by histological examination of smears of granulosa cells. As atresia progressed, the labelling index and aromatase activity of granulosa cells decreased. In normal follicles, when follicular diameter increased, the labelling index decreased, while aromatase activity of granulosa cells and oestradiol-17 beta concentration in follicular fluid increased. There was a negative relationship between oestradiol concentration in follicular fluid and the labelling index of granulosa cells in vitro (rs = -0.75; P less than 0.01), suggesting an inverse relationship between growth and differentiation of granulosa cells in normal sheep follicles. In normal small and medium-sized follicles (2-6 mm), incubation with FSH (100 ng/ml) for 2 h increased significantly the labelling index of granulosa cells. In normal medium-sized follicles (4-6 mm), incubation with FSH (50 ng/ml) for 1 h decreased the aromatase activity of granulosa cells. From these results, it is suggested that FSH acts mainly on cells in the G1 phase of the cell cycle, which are steroidogenically active, and makes them move into the S phase where their steroidogenic activity is temporarily inhibited.