Steroidogenesis in granulosa cells during follicular maturation: evidence for desensitization-resensitization during the ovulation cycle

Output of progesterone, the end product of the steroidogenic pathway, was studied in isolated chicken granulosa cells in relation to follicular maturation and during the ovulation cycle with particular reference to the period between the LH peak and ovulation. The evidence gathered from a series of experiments conducted during the past 2-3 years in the authors' laboratory indicate that the steroidogenic capacity of granulosa cells during follicular maturation is regulated not so much by receptor-coupled adenylate cyclase activity as has been proposed by other investigators, but by the activity of key steroidogenic enzymes, particularly the cholesterol 20,22 desmolase. Furthermore, granulosa cells undergo cyclic sensitization following the endogenous LH surge reaching maximal responsiveness about 1 hr before oviposition. This is followed by a rapid desensitization shortly before ovulation. This desensitization extends to the second and subsequent developing follicles probably in proportion to the evolving LH receptors. It is suggested that granulosa cells remain in such a desensitized state for several hours postovulation, during which time progesterone responses to LH are attenuated and consequently ovulation does not occur prematurely. It is proposed that this intraovarian mechanism is an important component of the physiological events that regulate the ovulation cycle in the domestic fowl.     

Effects of gonadotrophins on progesterone production by isolated granulosa cells of the adenohypophysectomized domestic fowl (Gallus domesticus)

Ovine LH and ovine FSH stimulated progesterone production in granulosa cells isolated from the F1, F2 and F3 follicles of hypophysectomized and control (sham-operation) hens when they were collected 6 h after operation, but the steroidogenic response to LH was greater for granulosa cells from hypophysectomized hens. At 15 h after operation progesterone production by granulosa cells was stimulated by LH in all 3 follicle types of control hens, but only in the F1 follicles of hypophysectomized hens. The response to FSH at 15 h was similar for control and hypophysectomized hens. The time after hypophysectomy therefore appears to affect the LH-stimulated progesterone production by granulosa cells of the F2 and F3 follicles.     

Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone.

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in 'second messengers' derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.     

Effects of restraint stress on plasma LH and testosterone concentrations, Leydig cell LH/hCG receptors, and in vitro testicular steroidogenesis in adult rats

We examined the effect of restraint stress (3 hr) on plasma LH and testosterone levels, on the Leydig cell LH/hCG receptor, and on the activity of enzymes in the testicular steroidogenic pathway of the adult rat. Restraint stress caused a 47% reduction in plasma testosterone concentrations, but had no effect on plasma LH levels. The binding capacity and affinity of Leydig cell LH/hCG receptors were not affected by restraint. Stress did not affect the testicular activity of 20,22 desmolase or 3 beta-hydroxysteroid dehydrogenase, but testicular interstitial cells of stressed rats incubated in vitro with progesterone as a substrate produced more 17 alpha-hydroxyprogesterone but less testosterone than control cells, and when incubated with 17 alpha-hydroxypregnenolone, produced 39% less androstenedione and 40% less testosterone than control cells. These results suggest that restraint stress inhibited 17,20 desmolase but not 17 alpha-hydroxylase activity. When the delta 4 pathway was blocked with cyanoketone (3 beta-HSD inhibitor), stress did not alter the production of pregnenolone or 17 alpha-hydroxypregnenolone, but the production of dehydroepiandrosterone by cells from stressed rats was subnormal, suggesting again a reduction of 17,20 desmolase activity. The data suggest that a major site of the inhibitory action of restraint stress on testicular steroidogenesis is the 17,20 desmolase step. The disruption of androgen production by restraint appears to be LH independent since stress did not affect plasma LH levels, the binding capacity or affinity of LH/hCG receptors, or the activity of 20,22 desmolase.     

Multiple inhibitory effects of a luteinizing hormone-releasing hormone agonist on hCG-dependent steroidogenesis and on FSH-dependent responses in ovarian cells in vivo and in vitro

[125I] labelled [D-Leu6, des-Gly-NH10(2)] LH-RH ethylamide (LH-RHa), when injected into immature female rats, bound specifically not only to the pituitary but also to the ovaries. LH-RHa inhibited hCG-stimulated progesterone production and ovarian weight augmentation in hypophysectomized immature female rats in vivo. FSH-induced ovarian hCG receptors and ovarian weight gain in diethylstilbestrol (DES)-treated hypophysectomized immature female rats were also suppressed by LH-RHa. Progesterone production by rat luteal cells in vitro was inhibited by LH-RHa. LH-RHa did not change the affinity or population of LH/hCG receptor in porcine granulosa cells in short term incubation. However, LH-RHa inhibited induction of LH/hCG receptor stimulated by FSH and insulin in long term culture of porcine granulosa cells. LH-RHa delayed hCG-stimulated cyclic AMP accumulation in porcine granulosa cells. These findings suggest that LH-RHa inhibits hCG-stimulated cyclic AMP accumulation and subsequent progesterone production as well as FSH-stimulated LH/hCG receptor induction by acting directly on ovarian cells.     

Influence of kaurenol on basal, LH- and forskolin-stimulated progesterone and cAMP production in avian granulosa cells

The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 microM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10 microM), kaurenol (10-100 microM) dose-dependently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenol's stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system.     

Ovarian steroidogenesis during follicular maturation in the domestic fowl (Gallus domesticus)

The steroidogenic potential of various physiological compartments within the ovary of the hen were examined using in vitro systems. Three-hour incubations of individual whole small follicles (less than 1 mm-1 cm) or 100,000 collagenase-dispersed theca cells of the five largest ovarian follicles (F1-F5) were conducted in 1 ml of Medium 199 at 37 degrees C in the presence and absence of luteinizing hormone (LH) (0.39, 0.78, 1.56, 3.13 and 6.25 ng), progesterone (5 ng), and dehydroepiandrosterone (DHEA, 5 ng). Steroid output was measured by radioimmunoassay of incubation media. Progesterone was not produced by small follicles although they are a major source of DHEA and estradiol and a significant source of androstenedione. Output of DHEA, androstenedione and estradiol was highly stimulated by LH. The substrate for androstenedione and estradiol in small follicles is probably DHEA. Output of DHEA and androstenedione in theca cells of F2-F5 was stimulated by LH in a dose-related manner. A dose-response relationship between estradiol output and the concentration of LH in media was not apparent in theca cells from F2-F5. Steroidogenesis in theca tissue of large follicles occurs predominantly via the delta 4 pathway. The ability of these theca cells to metabolize progesterone to androstenedione is lost between 36 and 12 h before ovulation. Their ability to metabolize DHEA to androstenedione is still present 12 h before ovulation. Aromatase activity is significantly reduced between 36 and 12 h before ovulation. These data indicate that both large and small follicles can be stimulated by LH. The small follicles are the major source of estrogen. As the large yolky follicles mature, steroidogenesis shifts from the delta 5 to the delta 4 pathway. By 12 h before ovulation, the F1 follicle has lost the ability to convert progesterone to androstenedione. The inability of the largest ovarian follicle to convert progesterone to androstenedione contributes at least in part to the preovulatory increase in the plasma concentration of progesterone that generates the preovulatory LH surge by positive feedback.     

Effects of diacylglycerol and inositol trisphosphate on steroidogenesis by ovarian granulosa from pigs

In addition to increasing cyclic adenosine 3',5'-monophosphate (cAMP) levels, luteinizing hormone (LH) stimulation of granulosa results in phosphoinositide hydrolysis producing inositol trisphosphate (IP3) and diacylglycerol. The roles of these putative second messengers were investigated by measuring production of progesterone and inositol phosphates by granulosa from medium-sized porcine follicles (3-7 mm) after 15 min incubation with or without LH (1 microgram/ml), 5 microM dibutyryl cAMP (dbcAMP), or 5 microM 1-oleoyl,2-acetylglycerol (OAG). Compared to a control rate of 5.4 pmoles/10(7) cells/15 min, LH and dbcAMP stimulated progesterone production to 12.8 and 15.9 pmoles, respectively, and OAG decreased progesterone production to 3.7 pmoles. LH also stimulated inositol phosphate (IP) and bisphosphate (IP2) accumulations by approximately 5-fold and IP3 accumulation by 20-fold. In experiments where granulosa were premeabilized with saponin, LH, dbcAMP, and IP3 stimulated progesterone production from 1.3 pmol in control cells to 5.2, 3.2, and 5.1 pmol, respectively, and OAG decreased progesterone production to 1.0 pmol. LH stimulated accumulation of all inositol phosphates in permeabilized cells, whereas the addition of IP3 only increased IP2 and IP3 accumulations. In granulosa preincubated with 0.9 mM [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, A23187 increased progesterone production from 3.7 to 5.8 pmol. Addition of 1-20 nmoles IP3 to 10(7) granulosa incubated in a Ca2+-free medium increased Ca2+ efflux linearly. These data suggest that IP3 may have a role in regulating steroid production in granulosa by regulating intracellular Ca2+.     

Trifluoperazine inhibits ovarian mitochondrial side-chain cleavage enzyme activity in the absence of calcium

In a previous report we described the inhibitory effect of trifluoperazine (TFP) on steroidogenesis in avian granulosa cells. To clarify the possible site of action of TFP we measured the cholesterol side-chain cleavage enzyme (CSCC) activity in a mitochondrial preparation of granulosa cells isolated from mature and developing ovarian follicles. Using a calcium free medium, TFP inhibited CSCC in a dose related manner with an IC50 of 50 microM. Kinetic parameters (apparent Km and Vmax) obtained in the presence of TFP are indicative of uncompetitive inhibition of CSCC. Moreover, enzyme activity increased during follicular maturation while the efficacy of TFP was similar in both young and mature follicles. Because the inhibitory effects of TFP were manifest in medium from which calcium was omitted, it is suggested that the drug acts independently of the calcium-calmodulin system to suppress CSCC activity.     

Estrogen regulation of progestin biosynthetic enzymes in cultured rat granulosa cells

The mechanism by which estrogens enhance gonadotropin-stimulated ovarian progestin production was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) in cultured rat granulosa cells. Cells from immature hypophysectomized, estrogen-treated rats were cultured for 3 days with follicle-stimulating hormone (FSH) and/or estrogens. Pregnenolone production was measured in the presence of cyanoketone which inhibits 3 beta-HSD activity. Activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. Some cells were also primed with FSH to induce luteinizing hormone (LH) receptors for studies on the effects of estrogens on LH-modulated parameters. Pregnenolone production by cultured granulosa cells was stimulated by FSH, while treatment with diethylstilbestrol (DES) or estradiol further enhanced the gonadotropin action. Treatment with FSH increased 3 beta-HSD activity. Similarly, concomitant treatment with DES further enhanced 3 beta-HSD activity in a dose-dependent manner with an apparent ED50 of 10(-8) M. Also, treatment with estrogens alone increased 3 beta-HSD activity. The increases in enzyme activity induced by estrogen alone or in combination with FSH were not associated with changes in the apparent Km values. FSH also stimulated 20 alpha-HSD activity by 2-fold in these cells, while concomitant treatment with DES did not affect the FSH action. In FSH-primed cells, LH stimulated pregnenolone production while the LH action was enhanced by concomitant treatment with the estrogens. Likewise, LH stimulated the activity of 3 beta-HSD, while concomitant DES treatment further augmented LH action. LH did not stimulate 20 alpha-HSD activity when added alone or in combination with DES. Thus, the estrogen enhancement of the gonadotropin-stimulated progesterone production in cultured rat granulosa cells is associated with increases in pregnenolone biosynthesis and the activity of the 3 beta-HSD enzyme, without affecting the 20 alpha-HSD activity.     

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.     

Effect of basal lamina on progesterone production by chicken granulosa cells in vitro — influence of follicular development

Experiments were conducted in vitro to study the regulation of progesterone production in chicken granulosa cells by homologous basal lamina isolated from preovulatory follicles of chicken ovary. The majority of components of the basal lamina (90–95% by weight) were solubilized with guanidine-HCl (and designated fraction 1); the remaining components were solubilized with β-mercaptoethanol containing guanidine-HCl (and designated fraction 2). The ability of fraction 1 to regulate progesterone production in granulosa cells obtained from the largest (F₁, mature), third largest (F₃, growing), fifth to seventh largest (F_5–7, growing) follicles and a pool of small yellow follicles (SYF, immature) of chicken ovary was assessed. Granulosa cells isolated from SYF follicles were in the least differentiated (undifferentiated) and those obtained from F₁ follicles were in the most differentiated state. The ability of fraction 1 to regulate progesterone production by chicken granulosa cells was influenced both by the state of cell differentiation and the form of the matrix material (whether solid or liquid). When fraction 1 was added as liquid to the incubation mixture, it promoted progesterone production by granulosa cells at all stages of differentiation; however, it caused a greater relative increase in the amount of progesterone produced by undifferentiated (SYF) and differentiating (F₃) granulosa cells than by differentiated (F₁) ones. In the presence of the liquid-form of fraction 1, luteinizing hormone (LH) stimulated progesterone production in differentiated (F₁) and differentiating (F_5–7) granulosa cells. Similarly, follicle-stimulating hormone (FSH) stimulated progesterone production by differentiating (F₃) and undifferentiated (SYF) granulosa cells in the presence of the liquid-form of fraction 1 protein. In culture wells that had been pre-coated with fraction 1 (solid-form), progesterone production by less differentiated (SYF, F_5–7) granulosa cells was enhanced, whereas progesterone production by differentiated (F₁) cells was reduced. The solid-form of fraction 1 augmented LH-stimulated progesterone production by less differentiated (F_5–7) granulosa cells however, it attenuated LH-induced progesterone production in differentiated (F₁) cells. FSH-promoted progesterone production in granulosa cells from immature follicles (SYF) was augmented by solid-form of fraction 1 whereas the effect of FSH on cells obtained from older follicle (F₃) was suppressed by solid-form of fraction 1. In experiments in which gonadotropin action was attenuated by solid-form of fraction 1, the amount of progesterone produced in the presence of maximally inhibiting concentrations of fraction 1 protein was greater than control values (no fraction 1, no gonadotropin). These results show that basal lamina of the ovarian follicle can regulate progesterone production by granulosa cells. The data demonstrate that the interactions between the components of basal lamina and LH or FSH on granulosa cell function were dependent on the stage of follicular development and were influenced by the form of the matrix material. It is concluded that the basal lamina of the chicken ovarian follicle is biologically active and regulates granulosa cell function.     

Effect of TNF-alpha on LH and IGF-I modulated chicken granulosa cell proliferation and progesterone production during follicular development

This study demonstrates the effects of recombinant human tumour necrosis factor a (rhTNF-alpha) and conditioned medium of the HD11-transformed chicken macrophage cell line on cultured chicken granulosa cells. Effects were studied on basal, IGF-I- and LH-stimulated progesterone production and cell proliferation. Recombinant human TNF-alpha stimulated basal progesterone production in a dose-dependent manner in the granulosa cells of the largest follicle but had no effect on cells from the third largest follicle. TNF-alpha stimulated and sometimes inhibited progesterone production stimulated by IGF-I and LH alone or in combination depending on the size of the follicle and the concentration of LH or IGF-I applied. However, the inhibitory effect of TNF-alpha was significantly more pronounced in cells from the third largest follicle when high concentrations of IGF-I, LH or a combination of both were applied. TNF-alpha had no effect on basal cell proliferation in both the largest and the third largest follicles, but regulated responses to IGF-I and a combination IGF-I and LH in the cells of the third largest follicle but not those of the largest follicle. The data indicate that the normal hierarchy of follicles is maintained in the chicken ovary through the regulation of the activity of IGF-I and its interaction with LH. Conditioned medium of LPS-activated HD11 macrophages mimicked the effects of TNF-alpha and its interaction with IGF-I and LH on progesterone production and cell proliferation. The observation that the HD11-conditioned medium contained TNF-alpha indicates that TNF-alpha produced by macrophages found in chicken follicles modulates granulosa cell growth and differentiation.     

Changes in responsiveness of rat granulosa cells to prostaglandin E2 and follicle-stimulating hormone during culture

The changes in responsiveness of granulosa cells to either FSH or prostaglandin E2 (PGE2), during culture of the cells, have been examined. In freshly isolated cells, FSH and PGE2 stimulated both cyclic AMP and progesterone production in a dose-dependent manner. In these cells, FSH stimulated cyclic AMP production to a greater extent than did PGE2. After the cells had been cultured for 2 days, neither FSH nor PGE2 stimulated progesterone production to any detectable extent. In these cells the ability of FSH to stimulate cyclic AMP was decreased, and that of PGE2 was increased markedly, such that PGE2 was far more effective than FSH in stimulating cyclic AMP. After culture of the cells for a further 2 days (4 days total), the FSH stimulation of cyclic AMP returned to that seen in freshly isolated cells, whereas the stimulation by PGE2 remained elevated. The acute stimulation of progesterone production could be restored by chronic exposure of the cells to either FSH or PGE2. These results demonstrate that dramatic changes in responsiveness of granulosa cells take place during culture. The results also suggest that some stimulating factor must be present to maintain the steroidogenic capabilities of the cells. Without this, although the cells are able to produce cyclic AMP in response to FSH, they cannot produce progesterone.     

Role of pituitary adenylate cyclase-activating peptide (PACAP) in the cyclic recruitment of immature follicles in the rat ovary

Following the midcyclic gonadotropin surge, PACAP is transiently expressed for approximately 12 h in the cyclic adult rat ovary. PACAP is observed in granulosa/lutein cells of the large mature follicles destined to ovulate and is believed to be a regulator of acute progesterone production and luteinization in these follicles. PACAP is also observed in solitary theca cells of immature follicles and in interstitial glandular cells intimately surrounding immature follicles. To examine if PACAP could be involved in the process of cyclic recruitment of such immature follicles, we primed immature granulosa cells from prepubertal ovaries with PACAP (1 nM and 100 nM) for 12 h. The treatment significantly stimulated the subsequent 24 h FSH-induced estradiol production (2.2 and 2.4 fold, respectively). The response seemed to be caused by a stimulation of aromatase activity. Estradiol production induced by testosterone was increased 2.4 and 2.6 fold, respectively, whereas functional FSH-receptors (cAMP production following FSH stimulation) or spontaneous apoptosis (immunohistochemical detection of DNA fragments) was unaffected. We conclude that PACAP priming of immature rat granulosa cells for 12 h increases subsequent FSH induced estradiol production and that PACAP could be involved in the cyclic recruitment of immature follicles in the adult rat ovary.     

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.     

Modulation of functional capacity of small ovarian follicles in the post-partum cow by prolactin

This study was undertaken to examine the possibility that the prolonged anovulatory period frequently experienced by the post-partum cow is due to a disruption of function at the ovarian level promoted by the high, suckling-induced, blood prolactin concentrations. Fifteen cows, less than 35 days post partum, were allocated to three groups (1, 3 and 5) and given no hormonal treatment, prostaglandin plus pregnant mare serum gonadotrophin (PMSG) treatment or injected with 2-bromo-alpha-ergocryptine to reduce circulating prolactin levels. Ten synchronized cyclic cows were allocated to two groups (2 and 4) and given prostaglandin or prostaglandin plus PMGS treatment. All cows were ovariectomized 1 or 2 days after treatment of Graafian follicles less than 9 mm in diameter were selected after dissection from the ovaries. The follicles were cultured for 18 h with or without prolactin (1 microgram/ml) and steroid accumulation in the culture medium estimated. The follicles were then separated into theca and granulosa which were incubated for 40 min with LH (1 microgram/ml) or FSH (5 micrograms/ml). Cyclic AMP concentrations were estimated as an indication of tissue responsiveness to gonadotrophins. The secretion of oestradiol-17 beta, progesterone, testosterone or androstenedione during 18 h culture did not differ between follicles isolated from post-partum or cyclic cows. The presence of prolactin in the culture medium had no overall effect on steroid secretion although some specific effects within each group were noticed. Incubation with LH increased cyclic AMP levels in the theca but the granulosa did not respond. Likewise FSH increased cyclic AMP levels in granulosa preparations but not in theca. There were no differences in response between post-partum and cyclic cows, but exposure of the follicles to prolactin in vitro did significantly reduce the LH-induced increase in cyclic AMP levels in isolated theca. We have concluded that endogenous prolactin may modify but does not inhibit the resumption of ovarian function following parturition in the beef cow.     

Adenylyl cyclase system of the small preovulatory follicles of the domestic hen: responsiveness to follicle-stimulating hormone and luteinizing hormone

The purpose of this study was to determine if the granulosa cells of the small preovulatory follicles of the domestic hen are a target tissue for follicle-stimulating hormone (FSH). The third largest (F3), fourth largest (F4), and fifth largest (F5) follicles were removed from hens at 20, 12, 6 and 2 h before ovulation of the F1 follicle. Basal, FSH- and luteinizing hormone (LH)-stimulable adenylyl cyclase (AC) activities were measured in the granulosa cells. Isolated granulosa cells of the F5 follicle, obtained 20 h before ovulation of the F1 follicle, were incubated with ovine (o) or turkey (t) FSH and progesterone (P4) was assayed in the medium. Basal AC activity was similar for F5, F4 and F3 granulosa cells except for an increase (P less than 0.01) in F3 follicles removed 2 h before ovulation of the F1 follicle. The FSH-stimulable AC activity of F5, F4 and F3 granulosa cells was elevated over basal (P less than 0.01). The greatest responsiveness was seen in the F5 follicle and the least in the F3 follicle. LH-stimulable AC activity was absent in the F5 follicle but present in the F4 and F3 follicles with the greater responsiveness in the F3 follicle. Isolated F5 granulosa cells secreted significant amounts of P4 in response to oFSH and tFSH. The data indicate that: 1) FSH stimulates the AC system of granulosa cells of the smaller preovulatory follicles (F5 greater than F4 greater than F3) while LH stimulates the AC system of granulosa cells of the larger follicles (F3 greater than F4), and 2) FSH promotes P4 production by granulosa cells of F5 follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inositol phosphates accumulation in ovarian granulosa after stimulation by luteinizing hormone

Accumulation of inositol phosphates by granulosa cells from medium follicles of porcine ovaries was studied to determine if hydrolysis of phosphoinositides is stimulated by luteinizing hormone (LH). Although follicle-stimulating hormone (FSH), D-alanine-gonadotropin-releasing hormone (D-ala-GnRH), and dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) had no effect, LH increased accumulation of inositol phosphate (IP), -bisphosphate (IP2), and trisphosphate (IP3) by severalfold. Furthermore, 0.01 microgram LH/ml increased IP3 accumulation threefold, while 0.1 microgram/ml stimulated accumulation of all inositol phosphates. Compared to untreated cells, LH-treated granulosa cells produced approximately twice as much progesterone in 30 min. Preincubation of cells with lithium chloride (LiCl) was necessary to measure IP accumulation, but not IP2 and IP3 accumulations. However, IP2 and IP3 accumulations were higher in LH-treated granulosa after pretreatment with LiCl. Maximal increases in IP3 and IP2 accumulations occurred approximately 15 min and 30 min, respectively, after LH stimulation, whereas the effect of LH on IP accumulation continued for at least 60 min. Granulosa, made permeable to IP3 with saponin treatment, did not hydrolyze [3H]IP3 to [3H]IP2 or [3H]IP. Thus, it is hypothesized that LH stimulates phosphoinositide hydrolysis in granulosa cells, thereby generating putative second messengers.     

Influence of follicular health on the steroidogenic and morphological characteristics of bovine granulosa cells in vitro

In 24-h cultures, steroid production by cells from non-atretic follicles increased with increasing follicular diameter. Cells from atretic follicles, of all sizes, produced low amounts of oestradiol-17 beta, but very high amounts of progesterone, relative to cells from non-atretic follicles. Increasing the culture period to 72 h caused little change in daily progesterone and oestradiol-17 beta production by granulosa cells from atretic follicles. In contrast, in cells from non-atretic follicles, daily progesterone production increased and daily oestradiol-17 beta production decreased to the levels observed with cells from atretic follicles. Dibutyryl cyclic AMP (1.0 mM) significantly stimulated progesterone production by cells from atretic, but not from non-atretic, follicles. Testosterone (1 microgram/ml) had no effect on progesterone production by cells from atretic follicles, while oestradiol-17 beta, oestrone, testosterone, androstenedione and 5 alpha-dihydro-testosterone (0-1000 ng/ml) each significantly suppressed progesterone production by cells from non-atretic follicles in a dose-dependent manner. Morphometric analysis revealed few subcellular differences between cells from non-atretic and atretic follicles. Mean cell volume was significantly higher for cells from atretic compared to non-atretic follicles, but the mean volumes of the major subcellular components were not influenced by follicle health. The mean surface area of the plasma and nuclear membrane, and granular endoplasmic reticulum was also significantly higher in cells from atretic compared to non-atretic follicles.