Quantitative electron-microscopic analysis of the in vivo effects of tetrahydrocannabinol on rat preovulatory follicles

The ultrastructural composition of 4 regions of rat preovulatory follicles was examined using stereological techniques following an intraperitoneal injection of tetrahydrocannabinol (THC), once each day, for 3 days prior to sacrifice on proestrus. Control rats were injected with solvent alone. Changes in nuclear, mitochondrial, lipid droplet and dense-body volume density were measured. No differences in volume density were noted in the theca or antral region of the membrana granulosa. Compared to controls, the mitochondrial volume density decreased in the peripheral region and the corona radiata. The volume density of lipid droplets decreased in the peripheral region. These changes, and their relationship to the roles of these regions in follicular steroidogenesis, are discussed.     

Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

A quantitative cytochemical study of glucose-6-phosphate dehydrogenase and Δ 5-3β-hydroxysteroid dehydrogenase activity in the Membrana granulosa of the ovulable type of follicle of the rat

Summary During the last four days of follicular development prior to ovulation, the activities of ⁵-3-hydroxysteroid dehydrogenase (3OHD) and glucose-6-phosphate dehydrogenase (G-6-PD) were quantified in cryostat sections of the rat ovary. The product of the enzyme reactions were measured using a scanning and integrating microdensitometer. The enzyme activity was measured in the peripheral region, the antral region and the cumulus of the membrana granulosa (MG) of these follicles on the morning of each of the four days of the estrous cycle. G-6-PD activity was measured in the presence and absence of an intermediate hydrogen acceptor, phenazine methosulphate, to provide a measure of the quantity of Type I and Type II Hydrogen (H) generated: Type I H is considered to be related to hydroxylating reactions such as those of steroids and Type II H to other general biosynthetic activities of cells.In all three regions of the MG of follicles of the ovulable type, 3OHD activity was lowest in estrus and diestrus-1, increased on diestrus-2 and peaked in proestrus. In estrus and diestrus-1, the level of 3OHD activity in the three regions was comparable. However, by diestrus-2, and even more conspicuously in proestrus, enzyme activity was significantly greater in the peripheral region than in the antral region or in the cumulus. During the same period, the level of enzyme activity remained comparable in the last two regions. Throughout the estrous cycle, both Type I and Type II H generation from G-6-PD was greatest in the peripheral region, less in the antral region and least in the cumulus. In the peripheral region, Type I H generation increased progressively after diestrus-1, to reach a maximum in proestrus. In the antral region, Type I H generation increased between diestrus-1 and diestrus-2 and then remained unchanged through proestrus. In the cumulus, Type I H generation remained at levels seen in estrus throughout the remainder of the cycle. Generation of Type II H, in the peripheral region was constant throughout the estrous cycle. In contrast, in the antral region and cumulus, Type II H generation was greater in diestrus-1 and diestrus-2 than on either proestrus or estrus.This work was supported by research grants from the National Institute of Child Health and Human Development (# HD-12684) and (# HD-09542) and from the Rockefeller Foundation     

Determination of steroidogenic potential of ovarian cells of the brushtail possum (Trichosurus vulpecula)

The ovary of the brushtail possum (Trichosurus vulpecula) secretes steroids; however, little is known about the identity of the steroidogenic cells in the ovary. The aim of the present study was to determine the identity of the ovarian cell types expressing mRNAs encoding proteins important for steroidogenesis and determine at what stage of follicular development they are expressed. The genes examined were those for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome p450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta5,Delta4 isomerase (3betaHSD), cytochrome p45017alphahydroxylase (p45017alphaOH), and p450 aromatase (p450arom). None of the genes examined were expressed in oocytes at any stage of follicular development. SF-1 was expressed in granulosa cells from the type 2 or the primary stage of development and thereafter to the preovulatory stage. In addition, the theca interna of small and medium-size antral but not preovulatory follicles and the interstitial glands and corpora lutea expressed SF-1 mRNA. Granulosa cells of preantral and small to medium-size antral follicles were not capable of synthesizing steroids from cholesterol because they did not contain p450scc mRNA. However, granulosa cells of many of the small to medium-size antral follicles expressed p450arom and 3betaHSD mRNA. The interstitial glands, theca interna, and corpus luteum expressed StAR, p450scc, 3betaHSD, and p45017alphaOH mRNA, suggesting that these tissues are capable of synthesizing progestins and androgens. The corpus luteum expressed p450arom, indicating that this tissue also has the potential to secrete estrogens in this species.     

Inhibition of cytochrome P-450 C17 enzyme by a GnRH agonist in ovarian follicles from gonadotropin-stimulated rats

Our objective was to study the direct action of a GnRH-I agonist, leuprolide acetate (LA), on ovarian steroidogenesis in preovulatory follicles obtained from equine chorionic gonadotropin (eCG)-treated rats. Previously, we have demonstrated an inhibitory effect of LA on steroidogenesis and follicular development. In this study, we tested the hypothesis that gonadotropin-releasing hormone (GnRH) exerts its negative effect on follicular development by inhibiting thecal cytochrome P-450 C17 (P450C17) alpha-hydroxylase expression and, consequently, androgen synthesis. Studies were carried out in prepubertal female rats injected with either eCG (control) or eCG plus LA (LA) and killed at different time points. Immunohistochemical studies indicated that LA induced steroidogenic acute regulatory protein (StAR) expression mainly in theca cells of preantral and antral follicles. In addition, serum progesterone levels increased significantly (P < 0.05), whereas those of androsterone decreased (P < 0.05) after 8 h of LA treatment. This inhibition caused by LA seemed to be a consequence of the decreased expression of follicular P450C17 alpha-hydroxylase, as demonstrated by Western blot and RT-PCR techniques. In vitro studies using follicles isolated from 48-h-eCG-treated rats and cultured with LA showed a significant (P < 0.05) inhibition of FSH-induced androsterone follicular content as well as P450C17 alpha-hydroxylase protein levels, as determined by Western analysis. However, LA increased StAR protein expression in these follicles without significant changes in P450scc enzyme levels. Taking all these findings into account, we suggest that GnRH-I exerts a direct inhibitory action on gonadotropin-induced follicular development by decreasing the temporal expression of the P450C17 enzyme and, consequently, androgen production, thus reducing the supply of estrogens available to developing follicles.     

Localization of preovulatory expression of plasminogen activator inhibitor type-1 and tissue inhibitor of metalloproteinase type-1 mRNAs in the rat ovary.

Proteinases and their inhibitors control follicular connective tissue remodeling associated with follicular rupture. We examined the regulation and cellular localization of plasminogen activator inhibitor type-1 (PAI-1) and tissue inhibitor of metalloproteinase type-1 (TIMP-1) mRNAs by in situ hybridization. [35S]UTP-labeled RNA probes were hybridized to ovarian sections of eCG-primed immature rats treated with hCG. Before hCG stimulation of ovulation, very low expression of PAI-1 mRNA was observed in theca cells. After hCG administration, expression of PAI-1 mRNA was increased in theca cells of most antral follicles, whereas expression in granulosa cells was limited to preovulatory follicles and only to areas where the basal membrane was dissociated. Before hCG treatment, low expression of TIMP-1 mRNA was observed in theca cells, but not in granulosa cells. After hCG treatment, TIMP-1 mRNA was greatly stimulated in theca cells irrespective of follicle size, while the expression in granulosa cells was limited to large antral follicles. The present study demonstrates cell-specific expression of PAI-1 and TIMP-1 mRNAs in the LH/hCG-stimulated ovary, thus confirming the localized control of preovulatory proteolysis by coexpression of both enzymes and their respective inhibitors.     

Evidence for interaction between granulosa cells and theca in early progesterone synthesis

The temporal characteristics of steroidogenesis in vitro by hamster preovulatory follicles, were compared to granulosa cells and theca incubated separately. Gonadotropin-stimulated intact follicles or recombined granulosa cells and theca synthesized increased amounts of progesterone by 30-120 minutes of incubation. The granulosa cells and theca, when incubated separately, did not begin to accumulate progesterone until 4 to 6 hours. The relatively rapid rise in follicular progesterone synthesis after in vitro gonadotropin stimulation follows the same time course as the rapid rise in vivo of hamster and rat preovulatory progesterone after the gonadotropin surge. The sharp differences in the temporal characteristics of progesterone synthesis between follicles and separated follicular cell types suggest an interaction between granulosa cells and theca in at least one phase of progesterone synthesis.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in preovulatory follicles, IGF-I stimulated LHR mRNA expression. These results show that the interaction between ECF and IGF-I may be involved in the regulation of atresia of follicles at different stages of development.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in pr     

Insulin-like growth factor binding protein 4 expression parallels luteinizing hormone receptor expression and follicular luteinization in the primate ovary

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.     

The in vivo effects of tetrahydrocannabinol on the activity of lactate and succinate dehydrogenases in rat preovulatory follicles

Summary The effects of ⁹-tetrahydrocannabinol on the activities of lactate and succinate dehydrogenases in the theca interna and membrana granulosa of rat preovulatory follicles have been analysed microdensitometrically using the same injection regime employed in a previous study on steroidogenic enzymes. A small but statistically significant (18%) decrease in succinate dehydrogenase activity was observed in the theca interna, but none in any region of the membrana granulosa. Lactate dehydrogenase activity was unaffected by THC administration. Thus, a dosage and regimen sufficient to cause significant decreases in the activities of steroidogenic enzymes had little effect on succinate and lactate dehydrogenases in rat preovulatory follicles.     

mRNA expression pattern of gonadotropin receptors in bovine follicular cysts

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.     

Expression of prostasin serine protease and protease nexin-1 (PN-1) in rhesus monkey ovary during menstrual cycle and early pregnancy.

Serine proteases and their cognate serpin-class inhibitors are involved in the controlled proteolytic events during follicular development, ovulation, formation, and maintenance of the corpus luteum (CL). In this study, we investigated the expression patterns of prostasin serine protease and protease nexin-1 (PN-1), a serine protease inhibitor also called serpin-E2, in rhesus monkey ovaries during the menstrual cycle and early pregnancy, by using in situ hybridization and immunohistochemistry. Expression of prostasin was localized in oocyte, granulosa cells, and/or theca cells of early antral follicles and antral follicles, with high levels observed in preovulatory follicles. Prostasin was also localized at high levels of abundance in the CL during the menstrual cycle and early pregnancy. During the menstrual cycle, PN-1 was coordinately localized with prostasin in oocytes, granulosa cells, and theca cells of antral follicles and preovulatory follicles and in the CL. In addition, the PN-1 expression level in macaque CL during early pregnancy increased as pregnancy proceeded. We propose that prostasin may be involved in follicular development, ovulation, and CL formation, whereas PN-1 may be present to regulate the proteolysis in these processes.     

Autoradiographic analysis of follicle-stimulating hormone and human chorionic gonadotropin receptors in the ovary of immature rats treated with equine chorionic gonadotropin.

The gonadotropin-primed immature rat has become the most common model for the study of follicular development and ovulation. In this study, prepubertal female rats, 23 and 24 days old, were injected s. c. with 5 IU eCG, and ovaries were collected for topical autoradiography of FSH and hCG receptors at 48 or 24 h post-eCG, respectively (i.e., Day 25). In a baseline group, on Day 25 (before eCG), even the smallest preantral follicles with 1 layer of granulosa cells (GCs; primary follicles) possessed FSH receptors, but hCG receptors were found only on the theca of follicles with 2 or more layers of GCs. Human CG receptors were especially prominent in the interstitium that intimately surrounds preantral follicles without any distinction between theca and interstitial cells. There was a discrete theca surrounding antral follicles. Occasionally antral follicles had hCG receptors in the interstitium, but the adjacent theca was negative, suggesting that these follicles might be destined for atresia. By 24 h post-eCG, a now-discrete theca layer with hCG receptors surrounded all preantral follicles except for the primary follicles, which never responded to eCG. The interstitium was hypertrophied and epithelioid, as was the theca surrounding nonatretic preantral and antral follicles. Increased mitotic activity characterized the growing preantral follicle, and for the first time, FSH binding in GCs of antral follicles was greater than in the preantral population. By 48 h post-eCG, the primary follicles were still unresponsive to eCG. FSH receptors were even more pronounced in the GCs of large antral follicles, although hCG receptors were present in the GCs of only one third of the antral follicles, reflecting the small dose of eCG administered. By 48 h post-eCG, receptors in the interstitium were barely detectable. Using this model, the following study considers the functional in vitro changes in steroidogenesis in follicles from the smallest preantral follicles to the largest antral follicles.     

A quantitative cytochemical study of glucose-6-phosphate dehydrogenase and delta 5-3 beta-hydroxysteroid dehydrogenase activity in the membrana granulosa of the ovulable type of follicle of the rat

During the last four days of follicular development prior to ovulation, the activities of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta OHD) and glucose-6-phosphate dehydrogenase (G-6-PD) were quantified in cryostat sections of the rat ovary. The product of the enzyme reactions were measured using a scanning and integrating microdensitometer. The enzyme activity was measured in the peripheral region, the antral region and the cumulus of the membrana granulosa (MG) of these follicles on the morning of each of the four days of the estrous cycle. G-6-PD activity was measured in the presence and absence of an intermediate hydrogen acceptor, phenazine methosulphate, to provide a measure of the quantity of Type I and Type II Hydrogen (H) generated: Type I H is considered to be related to hydroxylating reactions such as those of steroids and Type II H to other general biosynthetic activities of cells. In all three regions of the MG of follicles of the ovulable type, 3 beta OHD activity was lowest in estrus and diestrus-1, increased on diestrus-2 and peaked in proestrus. In estrus and diestrus-1, the level of 3 beta OHD activity in the three regions was comparable. However, by diestrus-2, and even more conspicuously in proestrus, enzyme activity was significantly greater in the peripheral region than in the antral region or in the cumulus. During the same period, the level of enzyme activity remained comparable in the last two regions. Throughout the estrous cycle, both Type I and Type II H generation from G-6-PD was greatest in the peripheral region, less in the antral region and least in the cumulus. In the eripheral region, Type I H generation increased progressively after diestrus-1, to reach a maximum in prestrus. In the antral region, Type I H generation increased between diestrus-1 and diestrus-2 and then remained unchanged through proestrus. In the cumulus, Type I H generation remained at levels seen in estrus throughout the remainder of the cycle. Generation of Type II H, in the peripheral region was constant throughout the estrous cycle. In contrast, in the antral region and cumulus, Type II H generation was greater in diestrus-1 and diestrus-2 than on either proestrus or estrus.     

Atresia of large ovarian follicles of the rat

In the rat, at the beginning of pregnancy a cohort of antral follicles develops until the preovulatory stage. However, these follicles, differentiating in the hyperprolactinemic milieu, produce only small amount of estradiol, do not ovulate and undergo rapid degeneration. They constitute an interesting physiological model of atresia. In the present study, we analysed the development and subsequent degeneration of such follicles. The study was performed on Wistar female rats killed in succession between days 1-9 of pregnancy. Excised ovaries were submitted to a routine histological procedure. Paraffin sections were subjected to hematoxylin and eosin staining or in situ DNA labelling. Histological and TUNEL staining revealed that the investigated group of follicles grew slower than that on the corresponding days of the estrous cycle and reached a preovulatory size and morphological appearance on day 5 of pregnancy. They did not ovulate and between days 6 and 9 of pregnancy an increasing number of apoptotic cells appeared within these follicles. They were localized predominantly in the antral granulosa layer, especially near the cumulus oophorus complex (COC) and in the region linking the COC with the follicular wall. The COC and the theca layer were much less affected. In late stages of atresia, also cumulus cells became apoptotic but degenerating oocytes did not exhibit positive TUNEL staining. Only limited number of the theca cells have undergone apoptosis and generally they were not hypertrophied. Our findings indicate that much smaller than normal amount of intrafollicular estradiol was sufficient to support a normal, according to the morphological criteria, although slower development of antral follicles to the late preovulatory stage.     

Permeability of rat ovarian follicles to LH during development and luteinization

A 'double isotope' technique has been used to describe the temporal relationship between plasma and follicular concentrations of LH after injection of 51Cr and 125I-rat LH into immature rats. Radiolabelled LH was detectable in all follicles 1 min after injection. Concentrations in small antral and large preovulatory follicles were not significantly different at any time and reached a maximum of 34.2 +/- 3.0% of plasma concentrations at 40 min. Concentrations of LH in preovulatory follicles exposed to an ovulatory dose of hCG 4 h previously were significantly greater (P less than 0.05) than those in small antral and preovulatory follicles at all times, and reached a maximum of 46.2 +/- 1.7% of plasma concentrations after 1 h. Polyacrylamide gel electrophoresis and immunoprecipitation with an antibody specific for rat LH indicated that radioactivity in plasma and follicular fluid represented radio-iodinated LH. Steroidogenic activities, light microscopy and measurements of follicular volume of each class of follicle confirmed that small antral, preovulatory follicles and preovulatory follicles exposed to an ovulatory dose of hCG in vivo could be isolated specifically. Based on these findings it is possible to calculate that, during an endogenous pulse of LH secretion, follicular concentrations of LH never exceed 20% of peak plasma concentrations. Pronounced increases in functional activities during antral growth were not correlated with increased follicular permeability. Only after acute exposure to an ovulatory dose of hCG in vivo was permeability significantly increased. We conclude that entry of LH into antral follicles is restricted and that exposure to an ovulatory dose of hCG results in greater amounts of LH entering preovulatory follicles.     

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)