Rabbit Ovary Research Articles

Expression of the rabbit cytochrome P450 aromatase encoding gene uses alternative tissue-specific promoters.

The aim of the present study was to analyse the tissue-specific expression of various promoter-derived transcripts from the gene encoding rabbit aromatase cytochrome P450. A new promoter, named I.r, was identified, and promoters II and I.r were sequenced. Promoter I.r-derived transcripts were found in preovulatory granulosa cells, corpus luteum, placenta and adipose tissue. An alternative splice variant of this transcript was found with tissue-specific preference. Tissue-specific expression of promoter-derived variants was studied in the ovary before and after ovulation. While the level of promoter II-derived transcript decreased dramatically after ovulation, that of promoter I.r-derived transcript remained unchanged, indicating that promoter II and promoter I.r were not controlled by a single regulation system. The existence of this dual system of regulation suggests that the rabbit ovary could be a useful model to study the promoter-specific regulation of aromatase.

Neutral endopeptidase is expressed on the follicular granulosa cells of rabbit ovaries

Neutral endopeptidase (NEP) is a zinc metallopeptidase ubiquitously distributed in various tissues in mammals. This peptidase is involved in the post-secretory metabolism of various neuropeptides and peptide hormones in vivo, such as enkephalins, bradykinin, atrial natriuretic peptide, substance P and endothelins. In this paper we show that NEP is expressed in ovaries as a 110-kDa glycosylated integral membrane protein with enzymatic properties similar to those of the kidney protein. Using immunohistochemistry, we localize the peptidase in the granulosa cells of follicles at all stages of maturation, with the exception of atretic follicles. We also observe immunoreactive staining in the epithelia that lines the blood vessels in the medulla and the surface of the ovary. The co-localization of NEP and bioactive peptides known to be physiological substrates of NEP in other tissues suggests an important role for this protein in processes such as follicle maturation, ovulation, and/or regulation of ovarian blood flow, by modulating the physiological function of these peptides.

Meloxicam inhibits rabbit ovulation

The nonsteroidal antiinflammatory, selective cyclo-oxygenase-2 (COX-2) inhibitor, meloxicam, was tested to assess its effect on rabbit ovulation. Meloxicam in different doses was administered intraperitoneally (ip) to adult female Californian rabbits at 2, 5, 8, and 24 h postcoitus with sperm-positive rabbits. Rabbits were killed on Day 10 of gestation. Meloxicam produced significant inhibition of ovulation in rabbits. This inhibition of ovulation by meloxicam was dose- and time-dependent. Ovulation in rabbits was completely inhibited by a single ip administration of meloxicam (20 mg/kg) when the drug was administered at 2 and 5 h postcoitus, whereas neither ovulation nor implantation were inhibited (pregnancy rate 75%) by the same dose administered 24 h postcoitus (approximately 14 h post ovulation). Further, ovulation was completely inhibited by 10 mg/kg of meloxicam when the drug was administered at 5 or 8 h postcoitus, but there was less inhibition of ovulation when 10 mg/kg of the drug was administered at 2 or 24 h postcoitus (pregnancy rate 25 and 80%, respectively). Corpora lutea, maternal plasma progesterone, ovary fresh weight, and maternal body weight gain were affected by meloxicam treatment. Histopathological findings observed in the ovaries of treated rabbits included microscopic dilatation of graffian follicles, particularly mature follicles. Some of the follicles were cystically dilated in addition to severe hemorrhage within the follicles which lost ova. These results show that ovulation can be inhibited in rabbits by meloxicam. Further studies are needed to assess the value of selective COX-2 inhibitors as potential nonhormonal contraceptive agents.

Steroidogenic Enzyme Gene Expression in Androgen Induced Polycystic Ovaries in the Rabbit

We investigated ovarian steroidogenic enzyme gene expression in exogenous androgen induced polycystic ovaries (PCO) in rabbits. Twelve 3-month peripubertal, and seven 9-11 month reproductive aged New Zealand White female rabbits were divided into four treatment (Rx) groups: controls receiving saline only, testosterone (T) treated (6-10 mg/Rx), LHRH analog (L) treated (leuprolide depot 1.0-1.2 mg/Rx), and T plus L treated (T+L). Animals in each group were administered the treatment IM every 2 to 3 weeks for 12-14 weeks. Serum T levels (every 1-3 wks) in T (127 ± 29 ng/dl) and T+L treated (144 ± 27 ng/dl) groups during the treatment period were unequivocally higher than the levels in controls (16 ± 6 ng/dl) and L treatment (22 ± 4 ng/dl) groups. Serum E2, LH and FSH levels (every 1-3 wks) showed no discernable differences between the groups. Microscopic examination of the L, T+L and T treated rabbit ovaries revealed a spectrum of PCO changes in comparison to the age-matched control ovaries, depicting marked interstitial hypercellularity and large cystic follicles in all except markedly decreased immature follicles and a few small cystic follicles in T treated peripubertal rabbits. Ovarian P450 cholesterol side-chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA and protein levels in PCOs of T and T + L treated animals were similar to the age-matched control ovaries, while in L treated PCOs both P450scc and 3β-HSD mRNA and protein levels were increased. Thus excess T alone did not alter the ovarian steroidogenic enzyme gene expressions despite the morphology changes. The L treatment alone had a gonadotropin agonistic effect resulting in both PCO and increased ovarian steroidogenic enzyme gene expression. However, excess T abolished the L-mediated increased ovarian steroidogenic enzyme gene expression, indicating the presence of an inhibitory effect of T on L induced ovarian steroidogenic enzyme gene expression. These findings suggest that the pathogenic mechanism by which PCO is induced by extra-ovarian source of T differs from that induced by increased gonadotropic effect alone.

Cloning and expression of cDNA encoding hamster liver 3-hydroxyhexobarbital/17 β(3 α)-hydroxysteroid dehydrogenase 1

Using RACE techniques we have cloned and sequenced one of the hamster liver 3-hydroxy-hexobarbital dehydrogenases which catalyze not only cyclic alcohols but also 17 β-hydroxy-steroids and 3 α-hydroxysteroids. The gene specific primers to 3-hydroxyhexobarbital dehydrogenase 1 (G2) were synthesized on the basis of its partial peptide sequences. The sequence of full length cDNA generated by 3′- and 5′-RACE PCR consisted of 1225 nucleotides including an open reading frame of 972 nucleotides encoding a protein of 323 amino acids. The deduced amino acid sequence matched exactly with the partial peptide sequences of hamster liver 3-hydroxyhexobarbital dehydrogenase 1 (G2). The sequence showed 84.5% identity to mouse liver 17 β-dehydrogenase(A-specific), and 74–76% identity to human liver bile acid binding protein/3 α-hydroxysteroid dehydrogenase (DD2), human liver 3 α-hydroxysteroid dehydrogenase type I (DD4) and type II (DD3), and rabbit ovary 20 α-hydroxysteroid dehydrogenase. The protein contains catalytic residues of aldo-keto reductases, Asp50, Tyr55, Lys84, His117. These results suggest that the hamster liver 3-hydroxyhexobarbital/17 β(3 α)-hydroxysteroid dehydrogenase belongs to aldo-keto reductase superfamily. The insert containing the full-length cDNA of 3-hydroxyhexobarbital dehydrogenase and vector specific overhang produced by PCR was annealed with pET-32 Xa/LIC vector. The plasmid was transformed into BL21 (DE3) cells containing pLysS. The recombinant enzyme was induced 1 mM IPTG. The expressed enzyme was produced as fusion protein and purified by nickel chelating affinity chromatography followed by POROS CM column chromatography and superdex 75 gel filtration. Molecular weight of the recombinant enzyme fused thioredoxin and his•tag was about 55 000 and that was 35 000 after Factor Xa protease treatment. The recombinant enzyme dehydrogenated 3-hydroxy-hexobarbital, 1-acenaphthenol, 2-cyclohexen-1-ol, testosterone, glycolithocholic acid as well as the native enzyme purified from hamster liver.

Effects of castor bean extract and ricin A-chain on ovulation and implantation in rabbits

The anti-implantation and antiovulation effects of castor bean extract (CBE) and ricin A-chain (RAC) were evaluated in rabbits. Both CBE and RAC, administered intraperitoneally on days 5–9 of pregnancy, exhibited a pronounced decrease in maternal body weight gain and in death of all fetuses. A significant (p <0.01) decrease of implantation sites resulted after rabbits were treated with RAC on the first 6 consecutive days of pregnancy. When female rabbits were treated with RAC for 10 consecutive days followed by human chorionic gonadotropin (hCG) (50 IU/kg intravenously), there was a 30% reduction in the number of corpora lutae. These data clearly indicate that CBE and RAC possess potent effects on implantation and ovulation in rabbits. The protein contents of castor bean extract, separated by polyacrylamide gel electrophoresis, revealed the presence of several protein bands, ricin toxin being a major constituent of the extract.

31P NMR studies of phosphate metabolism in the ovulatory process induced by gonadotropins in perfused rabbit ovary

The concentrations of phosphate metabolites were measured in perfused rabbit ovaries before, during and after ischemia, and during the ovulatory process induced by the administration of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) using 31P NMR spectroscopy. A decrease in adenosine triphosphate (ATP) and an increase in inorganic phosphate (Pi) during ischemia were observed. After reperfusion of the ovary, both ATP and Pi levels returned to their initial control levels. These results are in agreement with the results observed in other organ tissues. On the other hand, ATP levels rapidly decreased dose dependently with infusion of HCG during 10 h of perfusion. A decrease in ovarian ATP concentration after infusion of HCG was also confirmed by enzymatic analysis of the extracts from ovarian tissues. On the other hand, significant changes in Pi levels were not observed throughout the perfusion period. Furthermore, significant changes in phosphomonoester (PME) levels, adenosine diphosphate (ADP) levels and intracellular pH were not observed before and during the infusion of HCG. The concentration of cyclic adenosine 3′,5′-monophosphate (cAMP) in the perfusate after the perfusion was also measured during the ovulatory period. Within 30 min cAMP significantly increased dose dependently and reached peak levels within 60 min. The concentration of cAMP then gradually decreased. Ovulatory efficacy during the infusion of HCG was also observed. These results suggest that the decreased ATP in the successive processes leading to ovulation probably reflects protein phosphorylation through the activation of the cAMP-dependent protein kinases prior to induction of various chemical reactions related to the initiation of ovulation. Copyright © 1999 John Wiley & Sons, Ltd.

31P NMR studies of phosphate metabolism in the ovulatory process induced by gonadotropins in perfused rabbit ovary.

The concentrations of phosphate metabolites were measured in perfused rabbit ovaries before, during and after ischemia, and during the ovulatory process induced by the administration of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) using 31P NMR spectroscopy. A decrease in adenosine triphosphate (ATP) and an increase in inorganic phosphate (Pi) during ischemia were observed. After reperfusion of the ovary, both ATP and Pi levels returned to their initial control levels. These results are in agreement with the results observed in other organ tissues. On the other hand, ATP levels rapidly decreased dose dependently with infusion of HCG during 10 h of perfusion. A decrease in ovarian ATP concentration after infusion of HCG was also confirmed by enzymatic analysis of the extracts from ovarian tissues. On the other hand, significant changes in Pi levels were not observed throughout the perfusion period. Furthermore, significant changes in phosphomonoester (PME) levels, adenosine diphosphate (ADP) levels and intracellular pH were not observed before and during the infusion of HCG. The concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the perfusate after the perfusion was also measured during the ovulatory period. Within 30 min cAMP significantly increased dose dependently and reached peak levels within 60 min. The concentration of cAMP then gradually decreased. Ovulatory efficacy during the infusion of HCG was also observed. These results suggest that the decreased ATP in the successive processes leading to ovulation probably reflects protein phosphorylation through the activation of the cAMP-dependent protein kinases prior to induction of various chemical reactions related to the initiation of ovulation.

Effects of nitric oxide on ovulation and ovarian steroidogenesis and prostaglandin production in the rabbit.

Evidence supports the involvement of nitric oxide (NO) in ovarian physiology. The present study was undertaken to investigate the role of the NO/NO synthase (NOS) systems in ovulation, oocyte maturation, ovarian steroidogenesis, and PG production using in vitro perfused rabbit ovaries. The addition of the NOS inhibitors, aminoguanidine hemisulfate salt (AG) and N-omega-nitro-L-arginine methyl ester (L-NAME), to the perfusate inhibited the ovulation induced by hCG in a dose-dependent manner, whereas D-NAME had no significant effect. Neither AG nor L-NAME affected the hCG-induced meiotic maturation of the ovulated ova. The exogenous administration of the NO generator, sodium nitroprusside (NP), induced follicle rupture in the absence of gonadotropin, but did not induce oocyte maturation. Inhibition of endogenous NOS by AG and L-NAME resulted in a significant elevation in the production of estradiol (E2), but not of progesterone, stimulated by hCG. The concomitant administration of NP significantly reduced the AG-stimulated production of E2 by ovaries perfused in the presence of hCG, which suggests that NO down-regulates ovarian E2 synthesis. Ovarian production of PGE2 and PGF2alpha in response to hCG was significantly blocked by L-NAME, and exogenous administration of NP stimulated the production of PGs in the absence of gonadotropin. Significant correlations were observed between the ovulatory efficiencies and the production of PGs by rabbit ovaries perfused with or without L-NAME. In conclusion, the ovarian NO/NOS system is involved in follicle rupture during the ovulatory process. NO may induce follicle rupture in rabbit ovaries at least in part by the stimulation of PG production.

Luteotrophic factors in hyperstimulated pseudopregnant rabbit: I--Evidence for aromatase activity in luteal tissue and luteal cells.

It is well established that the rabbit corpus luteum (CL) function depends upon endogenous oestradiol, the major source of which in the rabbit ovary is considered to be the ovarian follicles. The absence of oestradiol formation by the rabbit CL has been previously reported. In a hyperstimulated pseudopregnant rabbit model used in our laboratory which developed a large number of corpora lutea in response to chorionic gonadotrophin (eCG)/hCG, we observed the survival of corpora lutea in vivo, and normal levels of plasma progesterone throughout pseudopregnancy (PP), despite the scarcity or the absence of follicles as a source of the luteotrophic hormone. Measurement of oestradiol in the plasma indicated that it was at high levels and correlated with the number of corpora lutea. This led us to investigate the luteal origin of oestradiol in this model. PP was induced in rabbits by i.m. injection of 200 IU eCG daily for 2 days followed on day 4 by i.m. injection of 200 IU hCG (day 0 of PP). Luteal tissue obtained at days 5, 9 and 12 of PP and cultured for 24 h synthesized oestradiol and testosterone in addition to progesterone. However, under the same conditions, follicles had limited capacity to secrete oestradiol. The presence of an aromatase activity in luteal tissue was confirmed when exogenous testosterone was added to the culture medium. P450aromatase (P450arom) mRNA was found in luteal tissue at days 5, 9 and 12 of PP. Small or large luteal cells, obtained by enzymatic digestion of the tissue followed by centrifugation in a Percoll density gradient, were cultured during several days with or without gonadotrophin or dibutyryl cAMP (dbcAMP). Both types of cells secreted oestradiol. In small cells and luteal tissue, aromatase activity was stimulated (1.5-2-fold) by hCG and dbcAMP. Large cells exhibited a greater capacity to aromatize testosterone than small cells, but aromatase activity was not modified by hCG or by dbcAMP. FSH had no effect on aromatase activity of either luteal cell type. This intrinsic luteal tissue aromatase capacity and the absence of premature regression of corpora lutea despite the limited support of follicular oestrogen, suggest an autocrine and luteotrophic role for this luteal oestrogen.

Dietary carrot results in diminished ovarian progesterone secretion, whereas a metabolite, retinoic acid, stimulates progesterone secretion in the in vitro perfused rabbit ovary

Objective: To determine the effects of dietary carrots and retinoic, acid on P secretion in the in vitro perfused rabbit ovary. Design: Controlled experiment. Setting: Laboratory. Subjects: Sexually mature New Zealand white rabbits. Intervention(s): Experiments were done in vitro using an isolated rabbit ovarian perfusion system after acute feeding of carrots or with in vitro exposure to retinoic acid. Main Outcome Measure(s): Progesterone and E 2 were measured in aliquots taken from arterial and venous cannulas. Result(s): Progesterone secretion during the in vitro perfusion of six ovaries is significantly diminished after the acute feeding of carrots. Human chorionic gonadotropin-induced P secretion also is significantly reduced. Progesterone secretion during in vitro perfusion on day 1 of pseudopregnancy was significantly increased in the perfusate samples from ovaries taken from rabbits not acutely fed carrots but that were exposed to retinoic acid. Progesterone secretion was increased but only marginally significant on day 11 of pseudopregnancy in perfusate samples from ovaries exposed to retinoic acid. Conclusion(s): Rabbit ovarian P secretion may be modified by carrots and carotene metabolites. This effect on steroid secretion may contribute to the relationship between hypercarotenemia and alterations in menstrual function.

Correlation relationship between cadmium accumulation and histological structures of ovary and uterus in rabbits

Cadmium accumulation in ovaries and uterus after i.p. and p.o. administration are reported. Time and dose dependent increase in cadmium concentration was found. More than 10 times higher accumulation is after i.p. administration in comparison with p.o. administration in ovaries as well as in uterus. There is very high correlation relationship among growing follicles and stroma in general as well as in all groups. So, we can report that with the increasing number of growing follicles there is lower amount of stroma. Generally we have found mean correlation relationship among cadmium content in ovaries and the percentual amount of growing follicles and stroma. We can say that with higher cadmium content in ovaries there is lower amount of primary follicles and stroma, but more growing follicles. On the other hand our previous study have described higher incidence of follicular atresia, which affect mainly growing follicles. In evaluation of numer of ovarian follicles to cadmium content, we report that generally there is mean correlation relationship among cadmium content and follicles with less than 2 layers of granulosa cells. This study also reports correlation relationship between cadmium content in ovary and diameter of follicles. Generally we can conclude, that the correlation relationship is very low and cadmium do not affect the diameter of follicles. Correlation analysis shows that there is mean relationship between cadmium content in uterus and stroma in endometrium. With higher cadmium content there is more stroma, what is a sign of oedematizatization.

The angioarchitecture of estrous, pseudopregnant and pregnant rabbit ovary as seen by scanning electron microscopy of vascular corrosion casts

Ovarian angioarchitecture was studied by scanning electron microscopy of vascular corrosion casts in estrous, pseudopregnant (stimulated with human chorionic gonadotropin) and pregnant rabbits. In all samples, the proper ovarian branch of the ovarian artery (ramus ovaricus) entered the ovarian hilus near the caudal pole of the organ and ran parallel to the major axis of the hilus. The extraovarian venous drainage was formed by several vessels emptying into a distal large vein. The ramus ovaricus exhibited various degrees of coiling and branched in the medulla. The coiling of the ramus ovaricus and its ramifications were maintained in all samples. A venous meshwork and/or flat vein branches closely enveloped the arterial coils found in the hilus and outer medulla. At this level numerous arteriovenous contacts were demonstrated in all samples. The coiled arteries, prior to entering the ovarian cortex, supplied several small peripheral follicles which were drained by the hilar veins. In the cortex the coiled arteries branched in numerous thin, straight or slightly undulated arterioles which supplied developing estrous follicles and pseudopregnant corpora lutea. The arterioles supplying the pregnant corpora lutea were long, large and tightly spiraled. The venous drainage followed the modifications of the arterial supply. These data demonstrate that ovarian cycle and pregnancy induced significant changes in the cortical vessels, which adapted their structure to the temporary functional needs of the recruited follicles or corpora lutea. Hilar and medullary vessels have permanent structures that may represent morphological devices for (a) a continuous control of the blood flow (spiral arteries) and (b) a local recirculation of endocrine products (arteriovenous contacts) comparable to the "countercurrent mechanism" previously shown to operate in ovaries of other species, but not yet found in rabbits.

The role of insulin-like growth factor-I (IGF-I) and estradiol in rabbit corpus luteum progesterone production.

To determine whether insulin-like growth factor-I (IGF-I) plays a role in rabbit corpus luteum (CL) physiology the authors examined: IGF-I expression, the effect of IGF-I on progesterone (P) production in vitro, and the interaction of IGF-I with estradiol, the primary luteotropin in the rabbit. Northern blot analysis revealed that IGF-I mRNA is present in the rabbit CL throughout pseudopregnanacy. An intact ovarian in vitro perfusion model and dispersed luteal cell culture were used to determine effects of IGF-I on P production and interactions with estradiol. IGF-I significantly stimulated P production compared to control medium by the isolated, intact perfused rabbit ovary and by dispersed, cultured luteal cells. Estradiol alone did not stimulate P production in vitro. Estradiol did augment IGF-I stimulated P production in the intact perfused ovary and in luteal cell culture. These findings support a role for IGF-I in rabbit CL P production.

Effects of a gonadotropin-releasing hormone analog on rabbit ovarian function.

This study was undertaken to elucidate the effects of a GnRH analog (GnRH-a) on rabbit ovulation, oocyte maturation, and steroidogenesis, and to verify whether treatment with a GnRH-a interferes with ovarian response to exogenous gonadotropin (hCG), both in vivo and in vitro. Three approaches were used. In the first, adult New Zealand White (NZW) rabbits were divided into two groups. Both received PMSG and hCG administered 72 h after PMSG. In the test group a GnRH-a, leuprolide acetate (LA; 20 microg/kg) was administered s.c. every 24 h. Treated rabbits showed a significant decrease in ovulatory efficiency (control = 88%; treated = 72%), and an increase in degeneration rate of preimplantation embryos (control = 30% vs. treated = 40%). For the second approach, in vitro perfusion experiments were designed to compare the direct effects of LA (10.000 ng/ml) and hCG (50 IU) on ovarian function and to verify whether the presence of a GnRH-a in the perfusate modifies the actions of hCG. LA reduced the ovulatory efficiency of hCG-treated ovaries perfused in vitro (hCG-treated = 87%; hCG-treated LA-perfused = 70%), reduced the potential for preimplantation development (morula stage: hCG-treated = 53%; hCG-treated LA-perfused = 31%; LA-perfused = 12%), and increased the degeneration rate of early embryos (21%, 48%, and 56% respectively). In the third approach, the direct effect of LA (Group I: control, Group II:1.000 ng/ml, and Group III:10.000 ng/ml) on the in vitro maturation of denuded rabbit oocytes was evaluated. LA induced meiotic maturation, but increased oocyte degeneration rate. The potential for preimplantation development was reduced (Morula stage: control = 16%, Group II = 8%, and Group III = 6%), and degeneration rate was increased (38%, 65%, 63% respectively). This study suggests that pharmacological doses of LA may exert a negative effect on oocyte function by direct action on the oocyte, indirectly via alteration of the intrafollicular environment and/or through interference with gonadotropin-induced biological effects within the ovary.

Involvement of angiotensin II in the process of gonadotropin-induced ovulation in rabbits.

In the present study we investigated the role of angiotensin II (Ang II) receptor subtypes in gonadotropin-induced ovulation, oocyte maturation, and ovarian steroidogenesis and prostaglandin (PG) production in in vitro-perfused rabbit ovaries. The addition to the perfusate of PD123319, a nonpeptide Ang II antagonist with a high affinity for AT2 receptors, inhibited hCG-induced ovulation in a dose-dependent manner, whereas CV-11974, a nonpeptide AT1 receptor antagonist, had no effect. The majority of ovulated ova and follicular oocytes resumed meiotic maturation in response to hCG; and PD123319, but not CV-11974, significantly inhibited hCG-induced oocyte maturation. The addition of both Ang II receptor antagonists to the perfusate had no significant effect on the concentration of progesterone in the perfusate of hCG-treated ovaries, whereas PD123319 inhibited the hCG-stimulated production of estradiol. The production of PGE2 and PGF2 alpha was significantly increased at 6 h in hCG-treated ovaries compared with ovaries before hCG administration. PD123319 inhibited the hCG-stimulated production of PGs by perfused rabbit ovaries in a dose-dependent manner, indicating that hCG-induced PG synthesis is mediated, at least in part, via the activation of AT2 receptors. Ovulatory efficiency in ovaries perfused with or without PD123319 in the presence of hCG was significantly correlated with PG production by perfused rabbit ovaries 12 h after exposure to hCG (r = 0.6553 for PGE2, p < 0.001; r = 0.4758 for PGF2 alpha, p < 0.05). In conclusion, Ang II exerts complex and coordinated control on at least two distinct aspects in the normal ovulatory process, ovulation and oocyte maturation. Ang II produced locally by gonadotropin exposure may be a part of a novel intraovarian paracrine or autocrine control mechanism that operates via the AT2 receptor in the ovary.

Localization of laminin proteins during early follicular development in pig and rabbit ovaries.

Laminin, a major component of the basal lamina, is known to be important in the differentiation of epithelial cells. The outer granulosa cell layer of ovarian follicles is attached to a basal lamina surrounding the follicle and it has been demonstrated that proteins of the basal lamina can alter the steroidogenic capacity and cytoskeletal composition of mature granulosa cells. The present studies were carried out to evaluate the developmental expression of laminin proteins in the basal lamina of ovarian follicles during early stages of granulosa cell differentiation. Ovaries from sexually immature pigs (0-20 weeks of age) and rabbits (0-12 weeks of age) were used for this study because formation of primordial follicles and initiation of follicular growth occur partially or completely postnatally. Specific antibodies were made against laminin beta 1/gamma 1 chains using protein purified by high resolution two-dimensional polyacrylmide gel electrophoresis. These antibodies were used in immunohistochemical localization studies to demonstrate that laminin is associated with the basal lamina surrounding egg clusters and their connections to the ovarian surface epithelium and ovarian rete during early ovarian development. In addition, laminin was observed to associate with a continuous matrix that surrounds forming primordial follicles as they are isolated from the egg clusters. Laminin is localized in the basal lamina of primordial, primary, secondary, and tertiary follicles of both pig and rabbit ovaries. Laminin proteins are not only expressed throughout early follicular development in pigs and rabbits, but are also found surrounding the germ cell compartments prior to initiation of meiosis and formation of primordial follicles. These results demonstrate that laminin proteins are deposited in the basal lamina well before granulosa cells undergo morphological differentiation. This pattern of expression suggests that laminin proteins alone do not control changes in granulosa cell morphology during early development but may be required to maintain cell lineage commitment.

Interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system in follicular growth and ovulation.

The interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system (RAS) in follicular growth and ovulation were studied with the use of an isolated perfused rabbit ovary preparation. Ovulation failed to occur in either control ovaries or the experimental ovaries perfused with IGF-I in a concentration of 1, 10, or 100 ng/ml in the absence of gonadotropin. Exposure to IGF-I stimulated the secretion rate of angiotensin II-like immunoreactivity (Ang II-IR) in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries perfused with IGF-I for 12 h was significantly correlated with the secretion rate of Ang II-IR at 12 h after exposure to IGF-I. The addition of IGFBP-3 to the perfusate did not induce ovulation in the absence of gonadotropin, but exposure to IGFBP-3 inhibited hCG-induced ovulation in a dose-dependent manner. In addition, IGFBP-3 significantly reduced the ovarian secretion rate of Ang II-IR and prostaglandins stimulated by hCG administration. Intrafollicular plasminogen activator (PA) activity significantly increased within 4 h after exposure to 100 ng/ml of IGF-I, compared with that in control ovaries perfused with medium alone. The concomitant addition of IGFBP-3 to the perfusate significantly reduced the IGF-I-stimulated PA activity in the preovulatory follicles at 4, 6, and 8 h after exposure to IGF-I. However, IGFBP-3 alone affected neither the ovarian secretion rate of Ang II-IR nor intrafollicular PA activity. Exposure to streptokinase, an exogenous PA, in vitro stimulated both follicular growth and the intrafollicular Ang II-IR content. In conclusion, IGF-I enhances both ovarian Ang II production and follicular development by stimulating intrafollicular PA activity.

Effects of insulin-like growth factor-I on follicle growth, oocyte maturation, and ovarian steroidogenesis and plasminogen activator activity in the rabbit.

We examined the effects of insulin-like growth factor (IGF)-I on follicular growth, oocyte maturation, and ovarian steroidogenesis and plasminogen activator (PA) activity in vitro, using a perfused rabbit ovary preparation in order to determine whether the follicle-stimulating effects of growth hormone (GH) are mediated by IGF-I. The addition of IGF-I to the perfusate stimulated follicular growth and the resumption of meiosis in follicular oocytes in a dose-dependent manner. There was no significant difference in the production of progesterone by perfused rabbit ovaries between IGF-I-treated and control ovaries, whereas IGF-I increased the production of estradiol (E2) by perfused rabbit ovaries in a dose-dependent manner. The concomitant addition of a monoclonal antibody recognizing the type I IGF receptor, alpha IR-3, to the perfusate significantly blocked IGF-I-stimulated follicular growth, oocyte maturation, and E2 production. Intrafollicular PA activity increased significantly 4 h after exposure to 10 or 100 ng/ml of IGF-I and reached maximal levels at 6 h. The percentage increase in follicle diameter at 6 h after exposure to IGF-I was significantly correlated with the intrafollicular PA activity. Treatment with GH resulted in a 2.7-fold increase in intrafollicular levels of IGF-I mRNA. The binding of [125I]-IGF-I to rabbit ovarian membrane preparations was inhibited by unlabeled IGF-I and IGF-II in a concentration-dependent manner. The relative affinity of the IGF-I receptor for IGF-I, IGF-II, and insulin was typical of type I binding (IGF-I > IGF-II > insulin). Affinity cross-linking of ovarian membranes with [125I]-IGF-I revealed a radiolabeled band corresponding to a molecular weight of 135,000, the alpha subunit of the type I IGF receptor. This band was totally displaced by IGF-I and alpha IR-3. It was concluded that IGF-I stimulated follicular development, E2 production, and oocyte maturation by interacting with its specific receptor located in rabbit ovarian membranes.

Angiotensin II induces ovulation and oocyte maturation in rabbit ovaries via the AT2 receptor subtype.

The present study was undertaken to investigate the role of angiotensin II (Ang II) in ovulation and ovarian steroidogenesis and prostaglandin (PG) production via the Ang II receptors in rabbit ovaries. In in vitro perfused rabbit ovaries, PD123319, a selective nonpeptide antagonist for AT2 receptors, reduced the Ang II-induced ovulation in a dose-dependent manner, whereas CV-11974, a selective nonpeptide antagonist for AT1 receptor, did not affect the Ang II-induced ovulation. Ang II also significantly stimulated the meiotic maturation of ovulated ova and follicular oocytes in the absence of gonadotropin. The addition of PD123319 at 10 (-6) M to the perfusate significantly inhibited the Ang II-induced oocyte maturation. Ang II did not stimulate the production of progesterone by perfused rabbit ovaries but significantly stimulated the production of estradiol (E2) and PGs. When PD123319 at 10(-6) M was added to the perfusate 30 min before the onset of Ang II administration, the Ang II-stimulated production of E2 and PGs was significantly blocked. Saralasin, a peptide analog of Ang II, inhibited the specific binding of [125I] iodo-[Sar1, Ile8] Ang II to rabbit ovarian membranes in a concentration-dependent manner, yielding an inhibitory constant (IC50) value of 1.58 x 10(-9) M. PD123319 and CV-11974 also inhibited the binding of [125I]iodo-[Sar1, Ile8] Ang II; however, PD123319 and CV-11974 were 15 and 40 times less potent than saralasin, respectively. Autoradiographic study revealed that an intense localization of Ang II receptors in the rabbit ovaries was present in the granulosa cell layers and the stroma of the preovulatory follicles. AT2 receptors were predominantly located in granulosa cells, whereas AT1 receptors were more concentrated in the stroma and thecal cell layers. In summary, Ang II induced ovulation and oocyte maturation and stimulated the production of E2 and PG by perfused rabbit ovary in vitro via the AT2 receptor. Thus, locally produced Ang II may be part of a novel intraovarian paracrine or autocrine control mechanism during the ovulatory process.