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.     

Flow cytometric analysis of steroidogenic organelles in differentiating granulosa cells.

Functional and structural changes accompany the differentiation of granulosa cells during follicular development. We used flow cytometry and fluorescent dyes to characterize two organelles important to the steroidogenic process. Mitochondria, which contain the rate-limiting enzyme responsible for cholesterol conversion to pregnenolone, and lipid droplets, which store cholesterol substrate, were probed in viable hen granulosa cells during differentiation. The fluorescent dye Dio3-C5 (DiO) was used to probe mitochondrial membrane potential, indicative of mitochondrial activity and/or number, during rapid granulosa cell differentiation in a hierarchy of individual developing hen preovulatory follicles (F6, smallest, to F1, largest). Cellular DiO fluorescence, granularity, and cell size were significantly elevated with increasing maturation state. Treatment with LH significantly increased DiO fluorescence in granulosa cells from F1 but not F3. The increased mitochondrial activity/number in granulosa cells that accompanies follicular maturation and is influenced by LH may reflect, at least in part, increased activity or amount of hormone-regulated mitochondrial enzymes controlling steroidogenesis. Flow spectrofluorometry and the metachromatic lipid dye, nile red, were used to probe lipid droplets in differentiating granulosa cells from F6 to F1. There was a dramatic increase in the fluorescence component related to lipid droplets with increasing stages of follicular maturation, suggesting recruitment of lipids into droplets during the differentiation of granulosa cells into hormone-responsive steroidogenic cells. The results demonstrate the dynamic nature of the granulosa cell morphology involved in steroidogenesis during follicular development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Onset of steroidogenic enzyme gene expression during ovarian follicular development in sheep

Steroidogenesis is a major function of the developing follicle. However, little is known about the stage of onset of steroid regulatory proteins during follicular development in sheep. In this study, several steroidogenic enzymes were studied by immunohistochemistry and/or in situ hybridization; cytochrome P450 side chain cleavage (P450(scc)), cytochrome P450 17alpha-hydroxylase (17alphaOH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 aromatase (P450(arom)), steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), and LH receptor (LH-R). To define the stages of follicular growth, ovarian maps were drawn from serial sections of ovine ovaries, and follicles were located and classified at specific stages of growth based on morphological criteria. In this way, the precise onset of gene expression with respect to stages of follicular growth for all these proteins could be observed. The key findings were that ovine oocytes express StAR mRNA at all stages of follicular development and that granulosa cells in follicle types 1-3 express 3beta-HSD and SF-1. Furthermore, the onset of expression in theca cells of StAR, P450(scc), 17alphaOH, 3beta-HSD, and LH-R occurred in large type 4 follicles just before antrum formation. This finding suggests that although the theca interna forms from the type 2 stage, it does not become steroidogenically active until later in development. These studies also confirm that granulosa cells of large type 5 follicles express SF-1, StAR, P450(scc), LH-R, and P450(arom) genes. These findings raise new questions regarding the roles of steroidogenic regulatory factors in early follicular development.     

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)     

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.     

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.     

Prolactin in follicular fluid and intracellular store calcium in follicular cells are related to morphological signs of ovarian follicle atresia in cows: work in progress

It is known that prolactin (PRL) is the third pituitary hormone serving gonadotropic function in mammals. However, its role in the regulation of ovarian folliculogenesis and, in particular, its relationship to follicular atresia as well as the mechanism of its influence on follicular cells are poorly understood. We investigated PRL levels in follicular fluids (FFs) and intracellular store calcium ([Ca2+]is) in cell walls of bovine ovarian follicles with diameters of 10 to 20 mm and their relationship to follicular atresia. Ovarian follicles were categorized on the basis of macroscopic criteria and of microscopic examination of granulosa cell (GC) smears. Prolactin concentrations in FFs were measured by RIA and levels of [Ca2+]is in follicular cells were determined by using the fluorophore chlortetracycline. Compared to atretic follicles, morphologically normal follicles were characterized by higher concentrations of PRL in FFs (P < 0.001) and lower contents of [Ca2+]is in follicular cells (P < 0.01). Furthermore, follicles containing no more than 20% of pycnotic GCs had higher levels of PRL in their fluids than those containing over 40% of pycnotic GCs (P < 0.05). Finally, the direct effect of PRL on [Ca2+]is content in follicular cells was studied in vitro. Compared to control, PRL decreased (P < 0.001) the levels of [Ca2+]is in the cells after 24 h culture of follicular walls from morphologically normal follicles in TCM 199 supplemented by 10% fetal calf serum. Our findings suggest that the decline of PRL concentrations in FFs and the rise of [Ca2+]is contents in follicular cells are related to atresia of large bovine follicles and that there appears to be a relationship between the two biochemical parameters.     

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.     

Regulation of the follicular hierarchy and ovulation

Studies are discussed which investigate the regulation of follicular maturation and the ovulation sequence of the domestic hen. The number of FSH receptors of ovarian granulosa cells decreases as the follicle matures, and this decrease in receptor number is paralleled by a gradual loss of FSH-stimulable adenylyl cyclase (AC) activity. By contrast, LH-stimulable AC activity increases as the follicle progresses through the hierarchy. In addition, FSH stimulates progesterone secretion by granulosa cells of the smaller preovulatory follicles, whereas these cells are only minimally responsive to LH. These data suggest that the maturation of less mature (smaller) follicles is primarily controlled by FSH, while LH may serve primarily as the ovulation-inducing hormone. The ability of LH to stimulate progesterone release and induce premature ovulation is dependent upon the stage of the sequence. Injection of ovine LH 12 hr prior to ovulation of the first (C1) egg of the sequence induces fully potentiated preovulatory plasma progesterone surges and 100% premature ovulation, whereas injection prior to the second (C2) ovulation of the sequence fails to stimulate prolonged progesterone release and induces premature ovulation in less than 50% of injected hens. These results are consistent with data obtained in vitro which suggest that granulosa cells obtained 12 hr prior to a C1 ovulation secrete more progesterone in response to chicken LH compared to those obtained 12 hr prior to the C2 ovulation. These data are discussed in terms of the ovary's ability to act as a regulator of the ovulatory cycle.     

Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin alpha subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin alpha subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F(3) + F(4) granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F(3) + F(4) cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F(1) and F(3) + F(4) granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F(3) + F(4) granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.     

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.     

Influence of follicular maturation on luteinizing hormone-, cyclic 3',5'-adenosine monophosphate-, forskolin- and cholesterol-stimulated progesterone production in hen granulosa cells

The influence of follicular maturation on progesterone production by collagenase-dispersed hen granulosa cells was measured in short-term incubations. Granulosa cells of the largest follicle (F1) produced up to ten times more progesterone than cells from smaller follicles (F3-F5), not only in response to luteinizing hormone (LH), but also when stimulated by exogenous cyclic AMP or forskolin, both of which raise intracellular cyclic AMP levels by nonreceptor-mediated mechanisms. Moreover, when granulosa cell progesterone synthesis was stimulated by incorporating 25-hydroxy-cholesterol into the incubation medium, an identical pattern was obtained. This could be attributed to a corresponding increase in the specific activity of the mitochondrial cholesterol side-chain cleavage enzyme (20,22 desmolase). An increase in the apparent Vmax was observed without a change in the apparent Km values. Pregnenolone substrate at concentrations which raised progesterone production to levels similar to those observed in response to LH stimulation was utilized equally by granulosa cells of mature and developing follicles. However, at high pregnenolone concentrations, granulosa cells of mature follicles converted significantly more of the precursor to progesterone. Assay of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) showed that the enzyme has two Kms: a low Km present in cells of both mature and developing follicles, and a high Km found only in granulosa cells of more mature follicles. It is concluded that LH-promoted progesterone synthesis in granulosa cells of developing chicken follicles is restricted not so much by the availability of receptors and the competence of the adenylate cyclase/cyclic AMP system, but by the activity of key enzymes, principally the cholesterol-20,22 desmolase.     

Identification of calcitonin expression in the chicken ovary: influence of follicular maturation and ovarian steroids

Calcitonin (CALCA), a hormone primarily known for its role in calcium homeostasis, has recently been linked to reproduction, specifically as a marker for embryo implantation in the uterus. Although CALCA expression has been documented in several tissues, there has been no report of production of CALCA in the ovary of any vertebrate species. We hypothesized that the Calca gene is expressed in the chicken ovary, and its expression will be altered by follicular maturation or gonadal steroid administration. Using RT-PCR, we detected Calca mRNA and the calcitonin receptor (Calcr) mRNA in the granulosa and theca layers of preovulatory and prehierarchial follicles. Both CALCA and Calca mRNA were localized in granulosa and thecal cells by confocal microscopy. Using quantitative PCR analysis, F1 follicle granulosa layer was found to contain significantly greater Calca mRNA and Calcr mRNA levels compared with those of any other preovulatory or prehierarchial follicle. The granulosa layer contained relatively greater Calca and Calcr mRNA levels compared with the thecal layer in both prehierarchial and preovulatory follicles. Progesterone (P(4)) treatment of sexually immature chickens resulted in a significantly greater abundance of ovarian Calca mRNA, whereas estradiol (E(2)) or P(4) + E(2) treatment significantly reduced ovarian Calca mRNA quantity. Treatment of prehierarchial follicular granulosa cells in vitro with CALCA significantly decreased FSH-stimulated cellular viability. Collectively, our results indicate that follicular maturation and gonadal steroids influence Calca and Calcr gene expression in the chicken ovary. We conclude that ovarian CALCA is possibly involved in regulating follicular maturation in the chicken ovary.     

Quantitative cytochemistry of 3 beta-hydroxysteroid dehydrogenase activity in avian granulosa cells during follicular maturation

Previous studies have shown that biosynthesis of progesterone, the major steroid product of hen granulosa cells, increases during follicular maturation. However, the contribution of individual granulosa cells to the total progesterone production of each follicle is not known. The objective of the present study was to determine the presence and relative activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in individual granulosa cells isolated from each of the five largest yolk-filled preovulatory follicles of laying hens. 3 beta-HSD cytochemistry in the presence or absence of pregnenolone substrate was performed on digitonin-permeabilized granulosa cells in suspension. The stained cells were fixed in a 70% ethanol solution until 1) the percentage of cells from each follicle that stained dark blue-indicating the presence of 3 beta-HSD activity-was determined by counting under light microscopy, and 2) the intensity of staining-indicating the relative amount of enzyme activity-was quantified using video image analysis. There were three findings. First, 100% of granulosa cells from each of the five largest preovulatory follicles stained positively for the presence of 3 beta-HSD activity. Second, the amount of 3 beta-HSD activity was normally distributed among granulosa cells in the population from each follicle. Third, as follicles matured from the fifth largest to the largest follicle, 3 beta-HSD activity increased steadily in individual cells, as indicated by increased staining intensities. The results indicate uniformity in the steroidogenic capacity of cells in the granulosa layer of hen preovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of dominant versus subordinate follicles in cattle

Selection of a dominant follicle, capable of ovulating, from among a cohort of similarly sized follicles is a critical transition in follicular development. The mechanisms that regulate the selection of a species-specific number of dominant follicles for ovulation are not well understood. Cattle provide a very useful animal model for studies on follicular selection and dominance. During the bovine estrous cycle, two or three sequential waves of follicular development occur, each producing a dominant follicle capable of ovulating if luteal regression occurs. Follicles are large enough to allow analysis of multiple endpoints within a single follicle, and follicular development and regression can be followed via ultrasonographic imaging. Characteristics of recruited and selected follicles, obtained at various times during the first follicular wave, have been determined in some studies, whereas dominant and subordinate follicles have been compared around the time of selection in others. As follicular recruitment proceeds, mRNA for P450 aromatase increases. By the time of morphological selection, the dominant follicle has much higher concentrations of estradiol in follicular fluid, and its granulosa cells produce more estradiol in vitro than cells from subordinate follicles. Shortly after selection, dominant follicles have higher levels of mRNAs for gonadotropin receptors and steroidogenic enzymes. It has been hypothesized that granulosa cells of the selected follicle acquire LH receptors (LHr) to allow them to increase aromatization in response to LH, as well as FSH. However, LH does not appear to stimulate estradiol production by bovine granulosa cells, and the role of LHr acquisition remains to be determined. Recent evidence suggests a key role for changes in the intrafollicular insulin-like growth factor (IGF) system in selection of the dominant follicle. When follicular fluid was sampled in vivo before morphological selection, the lowest concentration of IGF binding protein-4 (IGFBP-4) was more predictive of future dominance than size or estradiol concentration. Consistent with this finding, dominant follicles acquire an FSH-induced IGFBP-4 protease activity. Thus, a decrease in IGFBP-4, which would make more IGF available to interact with its receptors and synergize with FSH to promote follicular growth and aromatization, appears to be a critical determinant of follicular selection for dominance.     

Luteinizing hormone increases inositol trisphosphate and cytosolic free Ca2+ in isolated bovine luteal cells

The present studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases intracellular cAMP, also increases "second messengers" derived from inositol phospholipid hydrolysis in isolated bovine luteal cells. In luteal cells prelabeled with 32PO4, LH provoked increases in labeling of phosphatidic acid, phosphatidylinositol, and polyphosphatidylinositol (PIP). No reductions in 32P-prelabeled PIP and PIP2 were observed in LH-treated cells. In luteal cells prelabeled with myo-[2-3H]inositol, LH provoked rapid (10-30 s) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, and IP3, respectively. IP3 was formed more rapidly than IP2 or IP following LH treatment. In addition, LH increased (50%) levels of [3H]inositol phospholipids in 30-min incubations. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH. Maximal increases in IP3 occurred at 1-10 micrograms/ml of LH. Similar temporal and dose-response relationships were observed for LH-stimulated IP3 and cAMP accumulation. However, exogenous cAMP (8-bromo-cAMP, 5 mM) and forskolin (10 microM) had no effect on inositol phosphate synthesis. The initial (1 min) effects of LH on IP3 and cAMP were independent of extracellular calcium concentrations, whereas the sustained (5 min) effect of LH on IP3, but not cAMP, was dependent on a source of extracellular calcium. LH-stimulated progesterone synthesis was also dependent on the presence of extracellular calcium. LH induced rapid and concentration-dependent increases in [Ca2+]i as measured by Quin 2 fluorescence. The LH-induced increases in [Ca2+]i were maximal within 30 s (approximately 2-fold) and remained elevated for at least 10 min. In Ca2+-free media containing 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, LH was still able to increase [Ca2+]i, but the increase was slightly less in magnitude and of shorter duration (2-4 min). These findings demonstrate that LH can rapidly raise levels of IP3 and [Ca2+]i, as well as, cAMP in bovine luteal cells. These findings suggest that at least two second messenger systems exist to mediate the action of LH in the corpus luteum.     

Dominant bovine ovarian follicular cysts express increased levels of messenger RNAs for luteinizing hormone receptor and 3 beta-hydroxysteroid dehydrogenase delta(4),delta(5) isomerase compared to normal dominant follicles

The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17 alpha-hydroxylase, cytochrome P450 aromatase, 3 beta-hydroxysteroid dehydrogenase Delta(4),Delta(5) isomerase, LH, and FSH receptors and estrogen receptor-beta in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17 beta, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17 beta (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17 beta concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-beta mRNAs than other groups. In summary, increased expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17 beta concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.     

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+.     

Influence of follicular maturation on 3-hydroxy-methylglutaryl coenzyme A reductase activity in hen granulosa cells

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase: EC 1.1.1.34) was measured in a microsomal preparation of the granulosa of rapidly growing ovarian follicles of laying hens in the late preovulatory period (2-3 h before expected ovulation). The specific activity of the enzyme was measured in the five largest (F1-F5) preovulatory follicles, F1 being the follicle destined to ovulate first. Enzyme activity increased concomitantly with follicle size. The apparent Km of the enzyme decreased 60-80% from the smallest to the largest preovulatory follicle. There was no significant change in the Vmax during follicle development. Although our results have demonstrated the presence of HMG/CoA reductase in chicken granulosa cells and the progressive increase of its activity with follicular maturation, the quantitative significance of de-novo synthesized cholesterol as steroid hormone precursor remains to be ascertained.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)