In vivo and in vitro studies of the role of the adrenergic system and follicular wall contractility in the pathogenesis and resolution of bovine follicular cysts

Bovine follicular cysts (FCs) are a common cause of economic loss in modern dairy herds. Their aetiopathogenesis is not completely understood, even though an inadequate hypothalamic release of GnRH at the time of ovulation is considered to be their main cause. Much evidence, however, suggests a role for adrenergic innervation in ovarian functions, such as follicular development, steroid hormone secretion, and follicular contractility, the latter being an event important for ovulation. Moreover, in humans, polycystic ovary syndrome, a disease very similar to bovine follicular cysts, is characterised by increased density of adrenergic nerves. Given these premises, the aim of our study was to evaluate the effectiveness and mode of action of a novel strategy for the treatment of bovine follicular cysts. In the in vivo experiment, 170 Friesian cows diagnosed with follicular cysts were assigned to four groups (groups A, B, C, and D) to assess the effects of epidural administration of a β-adrenergic antagonist (carazolol) alone or in combination with a GnRH analogue (lecirelin). The four groups underwent the following treatments: Group A was administered lecirelin in combination with carazolol; Group B was administered carazolol; Group C was administered lecirelin; and Group D was administered only normal saline solution. In the in vitro experiment, strips of the walls of cystic follicles recovered post-mortem were suspended in an organ bath, connected to an isometric force transducer and exposed to increasing doses of epinephrine or to the same treatment after exposure to carazolol for 15 min (n = 10). The amplitude and frequency of the contractile activity were recorded. None of the control cows was observed in oestrus or was submitted to AI. The combination of lecirelin and carazolol induced a significant increase in the number of cows in oestrus (88%) compared to lecirelin alone or to carazolol alone (P < 0.05 and P < 0.01, respectively). The combination of lecirelin and carazolol and lecirelin alone were significantly more efficacious than carazolol alone (P < 0.01 and P < 0.05, respectively). In the in vitro experiment, epinephrine increased the amplitude of the contractions of the strips in a dose-dependent manner. This response was significantly enhanced in strips pre-treated with carazolol. The treatments had no effect on the frequency of contractions. In conclusion, our work demonstrates that the epidural administration of a GnRH analogue and a β-adrenergic blocker is an effective means of treating cows with cystic ovarian disease. Moreover, it confirms, from a clinical point of view, that alterations of the adrenergic system and of the contractility of the follicular wall can be considered aetiopathogenic factors involved in the development of FCs. The results of this study lay the basis for a new therapeutic approach to FCs.     

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.     

Involvement of voltage-dependent calcium channels (VDCC) in the action of GnRH on GtH release in common carp (Cyprinus carpio L): comparison with K+ action.

The involvement of different types of voltage-dependent calcium channels (VDCC) in the stimulatory action of GnRH (in comparison with K+) on maturational gonadotropin (GtH) release was investigated using superfused carp pituitary cells. The action of these 2 stimulants was not modified either by D600 or nifedipine (drugs blocking L-type of VDCC). Cadmium (Cd2+), which blocks all types of VDCC indifferently, provoked a dose-dependent stimulation of GtH secretion. Cd2+ action was not altered by addition of sGnRH in any of the doses. Similar results were obtained using K+ as a secretagogue, but only the highest dose of Cd2+ (200 mumol/l) was able to completely block K+ action. Low doses (0.1 and 1 mumol/l) of the L-type VDCC activator BAY-K8644 did not change basal GtH secretion and had no effect on sGnRH-stimulated GtH secretion. Surprisingly, doses (10 mumol/l and higher) of BAY-K8644 evoked dose-dependent inhibition of GtH secretion. On the other hand, a higher concentration (20 mumol/l) of nifedipine provoked a stimulation of GtH release. Our results indicate that the stimulatory action of GnRH and K+ involves activation of a certain type of cadmium-sensitive VDCC (probably T- or N-type VDCC) whereas dihydropyridine and diphenylalkylamine sensitive VDCC (L-type VDCC) does not participate in this phenomenon. The inhibitory action of BAY-K8644 and, on the other hand, the stimulatory action of nifedipine indicate that L-type VDCC probably play a role in other physiological pathways regulating GtH release in carp.     

Gonadotropin surge induces two separate increases in messenger RNA for progesterone receptor in bovine preovulatory follicles

In mice deficient in progesterone receptor (PR), follicles of ovulatory size develop but fail to ovulate, providing evidence for an essential role for progesterone and PR in ovulation in mice. However, little is known about the expression and regulation of PR mRNA in preovulatory follicles of ruminant species. One objective of this study was to determine whether and when PR mRNA is expressed in bovine follicular cells during the periovulatory period. Luteolysis and the LH/FSH surge were induced with prostaglandin F(2alpha) and a GnRH analogue, respectively, and the preovulatory follicle was obtained at 0, 3.5, 6, 12, 18, or 24 h after GnRH treatment. RNase protection assays revealed a transient increase in levels of PR mRNA, which peaked at 6 h after GnRH and declined to the time 0 value by 12 h and a second increase at 24 h. The second objective was to investigate the mechanisms that regulate PR mRNA expression through in vitro studies on follicular cells of preovulatory follicles obtained before the LH/FSH surge. Theca and granulosa cells were isolated and cultured with or without a luteinizing dose of LH or FSH, progesterone, LH + progesterone, or LH + antiprogestin (RU486). Levels of PR mRNA increased in a time-dependent manner in granulosa cells cultured with LH or FSH and in theca cells cultured with LH, peaking at 10 h of culture. In contrast, progesterone (200 ng/ml) did not upregulate mRNA for its own receptor, and neither progesterone nor RU486 affected LH-stimulated PR mRNA accumulation. Furthermore, RU486 completely blocked LH-stimulated expression of oxytocin mRNA, indicating that PR induced by LH in vitro is functional. These results show that the gonadotropin surge induces a rapid and transient increase in expression of PR mRNA in both theca and granulosa cells of bovine periovulatory follicles followed by a second rise close to the time of ovulation and that the first increase in PR mRNA can be mimicked in vitro by gonadotropins but not by progesterone. These results suggest multiple and time-dependent roles for progesterone and PR in the regulation of periovulatory events in cattle.     

Gonadotropin surge-induced differential upregulation of collagenase-1 (MMP-1) and collagenase-3 (MMP-13) mRNA and protein in bovine preovulatory follicles

The ovulatory process is characterized by focalized extracellular matrix degradation at the apex of preovulatory follicles. Many studies have implicated the matrix metalloproteinases (MMPs) as potential mediators of follicle rupture. Objectives of this study were to determine localization and effect of the gonadotropin surge on temporal expression of MMP-1 and MMP-13 in bovine preovulatory follicles. Samples were collected at 0, 6, 12, 18, 24, and 48 h (corpora lutea) after GnRH injection (n = 5-6 per time point) and amounts of MMP-1 and MMP-13 mRNA and protein determined using dot blot or semiquantitative RT-PCR and Western blot analyses. Samples were also collected at 0 and 20 h after GnRH injection for immunohistochemical localization of MMP-1 and MMP-13. Results indicate that follicular expression of MMP-1 and MMP-13 increased following the gonadotropin surge. Abundance of MMP-1 mRNA increased at 6, 12, and 48 h post-GnRH injection. Immunoreactive MMP-1 was localized to granulosal and thecal layers of preovulatory follicles. Amounts of MMP-1 protein increased in both the apex and the base of preovulatory follicles. Abundance of MMP-13 mRNA increased at 6, 24, and 48 h post GnRH injection. Amounts of MMP-13 protein also increased in the follicular apex and base. Immunoreactive MMP-13 was localized to granulosal and thecal layers of preovulatory follicles. Results indicate MMP-1 and MMP-13 are increased in bovine preovulatory follicles following the gonadotropin surge but do not support a requirement for differential up-regulation of MMP-1 and MMP-13 (follicular apex vs. base) for the preovulatory collagenolysis required for follicle rupture.     

Epidural vs intramuscular administration of lecirelin, a GnRH analogue, for the resolution of follicular cysts in dairy cows

Bovine follicular cysts are an ovarian disorder of dairy cows associated with abnormal estrous behaviour and infertility. The treatment of choice is intramuscular administration of a GnRH analogue, which acts by triggering pituitary release of LH. However, the presence of GnRH and GnRH receptors on spinal cord and ovary in some species, and the kind of innervation of the ovary, let us hypothesize that GnRH and its analogues may also act when administered by epidural route, as happens for other drugs. Therefore the aim of this study was to compare the effects of epidural vs intramuscular administration of lecirelin (a GnRH analogue) on FC regression, estrus detection and pregnancy outcomes. The study was conducted on 220 Friesian cows affected by follicular cysts, divided among 4 groups: Group L(epid) and Group L(im) received, respectively 50 μg of lecirelin in the epidural space and intramuscular; Group C(epid) and Group C(im) were used as control groups. In Group L(epid), estrus induction and pregnancy rates were significantly higher than in Group L(im). The results of this study show that the epidural administration of lecirelin promoted the remission of follicular cysts and an improvement of reproductive parameters compared to intramuscular administration. Thus, an alternative therapeutical approach is available for FC treatment, in order to obtain an easier restoration of the ovarian activity, especially in those cases refractory to classical therapeutic approaches.     

The effect of GnRH on in vitro bovine myometrial activity

The aim of this study was to evaluate, in vitro, the effects of increasing concentrations of GnRH on spontaneous mechanical activity patterns of uterine smooth muscle preparations of cows during the follicular and the luteal phases of the oestrus cycle. Uterine smooth muscle strips from 14 cows in follicular and 9 in luteal phase were collected immediately after slaughter and processed within 60 min from collection. Two strips of the same uterus were mounted in an isolated organ bath with two chambers to evaluate the role of decapeptide GnRH on spontaneous myometrial contractility. After equilibration period at 20 mN resting tension, the mechanical activity of the uterus was recorded for 10 min and the mean contractile force (MCF) was calculated. Then GnRH antagonist (antide) was added to one chamber at fixed concentration (10(-4)mol) and allowed to diffuse in solution and make contact with the strips. Subsequently, GnRH was added to the two baths at the same time at increasing concentration and MCF was recorded for 10 min. The effect of GnRH on spontaneous myometrial activity was evident only in the strips from subjects in follicular phase. Our results are suggestive of the presence of GnRH receptors in bovine myometrial tissue. The involvement of GnRH on uterine contractions at mating can be postulated.     

Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes

The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3' end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.     

Oxytocin and estradiol concentrations in follicular fluid as a means for the classification of large bovine follicles

Large antral follicles (13 to 20 mm in diameter) were collected from ovaries of 109 cows and 17 heifers that also had a regressed corpus luteum at slaughter. Thirty percent of the animals had been injected once with prostaglandin F(2)alpha 48 hours before slaughter. Follicles were divided into 3 groups based on estradiol and oxytocin concentrations in the follicular fluid: Group I follicles, estradiol>/=100 ng/ml and oxytocin<65 pg/ml (preovulatory and assumed pre-gonadotropin surge); Group II follicles, estradiol<100 ng/ml and oxytocin>/=65 pg/ml (preovulatory and assumed post-gonadotropin surge); and Group III follicles, estradiol<100 ng/ml and oxytocin<65 pg/ml (atretic follicles). Treatment with prostaglandin F(2)alpha significantly increased the number of viable granulosa cells and estradiol content in Group I follicles. The estradiol: progesterone ratio was significantly higher in Group I vs Groups II and III, but it was similar for Group II healthy follicles and Group III atretic follicles. To ascertain the classification of follicles, PGF(2)alpha was administered on Day 6 of the cycle to induce corpus luteum regression, and a GnRH analog was administered 24 hours later. At 23 hours after GnRH analog treatment, follicular oxytocin levels significantly rose to 103 pg/ml. Concomitantly, estradiol concentrations fell to below 100 ng/ml. This response was not evident by 13 h after injection of the GnRH analog. The results indicate that follicular estradiol and oxytocin concentrations may be used as a means for the physiological classification of large bovine follicles.     

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.     

Gonadotropin-releasing hormone antagonist antide inhibits apoptosis of preovulatory follicle cells in rat ovary

Analogs of GnRH, including agonists (GnRH-a) and antagonists (GnRH-ant), have been widely used to inhibit gonadotropin pituitary release. Aside from the effect of GnRH analogs on the pituitary-gonadal axis, studies have shown that GnRH has extrapituitary effects, particularly on rat and human ovaries. In the present study, we evaluated the direct in vivo effects of the GnRH-a, leuprolide acetate (LA), or the GnRH-ant, Antide (Ant), either singly or together, on ovarian follicular development in prepubertal eCG-treated rats. LA significantly decreased ovarian weight, whereas Ant increased ovarian weight compared with controls; however, coinjection of both compounds had no effect. In addition, LA increased the number of preantral follicles (PFs) and atretic follicles, and decreased the number of early antral follicles (EAFs) and preovulatory follicles (POFs). Coinjection of Ant interfered with this LA effect. Ant alone increased the number of POFs compared with that of controls. Analysis of apoptosis has shown that LA increases the percentage of apoptotic cells in PFs, EAFs, and POFs; however, Ant prevented this effect. In addition, Ant alone decreased the percentage of apoptotic cells in EAFs and POFs. Data have shown that Ant per se inhibited BAX translocation from cytosol to mitochondria and retained cytochrome C in the mitochondria, whereas LA induced cytochrome C release. We conclude that Ant inhibits apoptosis in preovulatory follicles through a decrease of BAX translocation to mitochondria, suggesting that GnRH may act as a physiological intraovarian modulator factor that is able to interfere with follicular development through an increase in apoptotic events mediated by an imbalance among the BCL-2 family members.     

Neuroendocrine regulation of ovulation in fishes: basic and applied aspects

This review summarizes the major neuroendocrine mechanisms regulating ovulation, thus providing a basis for understanding the various environmental and hormonal techniques for induction of ovulation of cultured teleosts. The secretion of gonadotrophin-ii(GtH-ii) is stimulated by gonadotrophin-releasing hormone (GnRH), and, although some teleosts have three different forms of GnRH regionally distributed in the brain, in most species investigated, only one form is present in the pituitary and apparently involved in GtH-ii secretion. In nearly all species investigated, dopamine (DA) inhibits GtH-ii secretion by direct actions on gonadotrophs, as well as by inhibition of GnRH release. Sex steroids act at both brain and pituitary levels to regulate GtH-ii secretion through a combination of positive and negative feedback actions; one important positive feedback action is that sex steroids enhance the responsiveness of the pituitary to GnRH and an important negative feedback action is to increase DA turnover, thereby increasing the overall DA inhibitory tone on GtH-ii secretion. The preovulatory surge of release of GtH-ii is stimulated by a surge release of GnRH. A decrease in DA turnover also occurs to disinhibit GnRH and GtH-ii release. Environmental factors including photoperiod, temperature and spawning substrate may cue ovulation and spawning. Social and pheromonal interactions play a very important role in synchronizing preovulatory endocrine changes, ovulation and spawning behaviour in many species. A widely used technique for inducing ovulation of cultured fishes is injection of the combination of a GnRH superactive analogue, to stimulate GtH-ii release, and a DA receptor antagonist, to block the inhibitory actions of DA. This is termed the Linpe technique and has proven particularly useful with those species having synchronous or group synchronous follicular development and a large preovulatory surge of GtH-ii. In other groups of teleosts, particularly those species having asynchronous ovarian development and multiple spawnings over an extended period, treatment with a sustained-release preparation of a GnRH superactive analogue to cause a prolonged, somewhat enhanced release of GtH-ii has proven highly successful in inducing multiple ovulations and spawnings. However, the lack of specific radioimmunoassays for GtH-ii in many of these species has hindered progress, as the precise pattern of GtH-ii release necessary for the recruitment of vitellogenic oocytes into final maturation and ovulation in these multiple spawners remains an intriguing neuroendocrine question     

Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.     

Pattern of follicular development during the estrous cycle of aged rats

Summary The pattern of follicular development during the estrous cycles of aged rats was examined and compared with that of mature rats. In both, preovulatory follicles are derived from a select group of small pre-Graafian follicles which begin to develop at estrus and reach the preovulatory size by the morning of proestrus, but the rate of growth, as judged by an increase in the percentage of granulosa cells incorporating ³H-thymidine, is accelerated in the follicles of aged rats. A second mechanism, which accounts for preovulatory follicles in aged rats, involves the rescue from atresia of pre-Graafian and preovulatory follicles. The existence of this mechanism is supported by the observation that at metestrus in aged rats virtually all follicles, regardless of their state of atresia, possess a high percentage of granulosa cells incorporating ³H-thymidine, indicating that the follicles are growing rapidly. However, some of these rapidly growing follicles show signs of atresia such as pyknotic nuclei within their granulosa cell layers. Since follicles in the initial stage of atresia contain defective oocytes (Peluso et al. 1979b), their rescue and development into preovulatory follicles would result in the ovulation of defective oocytes, a fact which accounts in part of the lower fertility in these older animals.     

Local regulation of primate granulosa cell aromatase activity

Granulosa cells produce inhibin and activin, proteins implicated in the local regulation of preovulatory follicular development. To assess interactions among FSH, LH, inhibin and activin on primate granulosa cell aromatase activity, we studied primary granulosa cell cultures from the ovaries of the common marmoset (Callithrix jacchus), a monkey with an ovarian cycle similar in length to the human cycle. The distinctive action of activin was augmentation of gonadotropin-responsive aromatase activity throughout antral follicular development. FSH-stimulated aromatase activity in granulosa cells from immature follicles was augmented many fold by picomolar amounts of activin. In cell cultures from preovulatory follicles, the presence of activin stimulated basal aromatase activity in the absence of gonadotropin, as well as augmenting the action of LH. Thus, locally produced activin has the potential to modulate aromatase activity in developing ovarian follicles. By contrast, inhibin or inhibin -subunit purified from bovine follicular fluid had minimal effects on aromatase activity. The only significant effect was slight suppression of FSH-inducible aromatase activity in granulosa cells from immature follicles at an inhibin concentration of 100 ng/ml. The finding that inhibin has a negligible effect on aromatase activity in granulosa cells from mature follicles suggests that it is unlikely to exert a physiologically significant influence on aromatase activity in vivo. However, evidence from other studies suggests that inhibin might affect aromatization indirectly through acting locally to modulate thecal androgen (aromatase substrate) production. Therefore, both inhibin and activin have the potential to contribute at different levels to paracrine and autocrine regulation of follicular oestrogen synthesis.     

Calcium ions as a mediator in GnRH action on gonadotropin release in the common carp (Cyprinus carpio L)

Collagenase-dispersed carp pituitary cells in a perifusion system were used to study the role of calcium ions in the mechanism of GnRH action on the release of maturational gonadotropin (GtH) in fish. The specific calcium chelator EGTA and the calcium antagonist manganese (Mn2+) caused a 40% inhibition in the basal GtH release and completely blocked GnRH-stimulated GtH release. Short-term application of graded doses of calcium ionophore A23187 caused a dose-dependent increase in GtH secretion. A23187 failed to stimulate GtH secretion in the presence of EGTA. Depolarization of the membrane by K+ caused a strong stimulation of GtH release similar to the action of GnRH. Stimulatory action of K+ was inhibited by EGTA. These data suggest a role for extracellular calcium as an intracellular mediator in GnRH-stimulated, as well as in basal, GtH release in carp. The stimulation of GtH release by K+ also indicates that voltage-dependent processes could be involved in this phenomenon.     

GnRH actions on rat preovulatory follicles are mediated by paracrine EGF-like factors

Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-alpha-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG.