Expression of type I GNRH receptor and in vivo and in vitro GNRH-I effects in corpora lutea of pseudopregnant rabbits

The expression of type I GNRH receptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidenced GNRHR-I and GNRH-I in luteal cells at early (day 4 pseudopregnancy)-, mid (day 9)-, and late (day 13)-luteal stages. Real-time RT-PCR and western blotting revealed GNRHR-I mRNA and protein at the three luteal stages. Buserelin in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24 h respectively. In in vitro cultured CL, buserelin reduced progesterone secretion, increased prostaglandin F(2α) (PGF(2α)) secretion and cyclo-oxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE? at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP?, 2-amino-ethoxydiphenylborate), and diacylglycerol (DAG, 1-hexadecyl-2-acetyl glycerol) or inhibitors for phospholipase C (PLC, compound 48/80), and protein kinase C (PKC, staurosporine) counteracted the buserelin effects. Buserelin co-incubated with COX inhibitor (acetylsalicylic acid) increased progesterone and decreased PGF(2α) and NOS activity at days 9 and 13, whereas co-incubation with NOS inhibitor (N-nitro-l-arginine methyl ester) increased progesterone at the same luteal stages. These results suggest that GNRHR-I is constitutively expressed in rabbit CL independently of luteal stage, whereas GNRH-I down-regulates directly CL progesterone production via PGF(2α) at mid- and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP?, DAG, PKC, COX-2, and NOS.     

Expression patterns of endothelial and inducible nitric oxide synthase isoforms in corpora lutea of pseudopregnant rabbits at different luteal stages

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P相似文献   

Prostaglandin receptors and role of G protein-activated pathways on corpora lutea of pseudopregnant rabbit in vitro

Studies were conducted to characterize receptors for prostaglandin (PG) F(2alpha) (PGF(2alpha)) and PGE(2), and the signalling pathways regulating total nitric oxide synthase activity and progesterone production in rabbit corpora lutea (CL) of different luteal stages. CL were obtained at days 4, 9 and 13 of pseudopregnancy and cultured in vitro for 2 h with PGF(2alpha) or PGE(2) and with activators and inhibitors of G protein (Gp), phospholipase C (PLC), protein kinase C (PKC), adenylate cyclase (AC) and protein kinase A (PKA). High affinity PGF(2alpha) receptor (K(d)=1.9+/-0.6 nM mean+/-s.e.m. ) concentrations increased (P< or =0.01) four- to five-fold from early to mid- and late-luteal phases (50.6+/-8.5, 188.3+/-36.1 and 231.4+/-38.8 fmol/mg protein respectively). By contrast, PGE(2) receptor (K(d)=1.6+/-0.5 nM) concentrations decreased (P< or =0.01) from day 4 to day 9 and 13 (27.5+/-7.7, 12.4+/-2.4 and 16.5+/-3.0 fmol/mg protein respectively). The Gp-dependent AC/PKA pathway was triggered only on day 4 CL, mimicking the PGE(2) treatment and increasing progesterone production. In both day 9 and day 13 CL, the Gp-activated PLC/PKC pathway evoked a luteolytic effect similar to that induced by PGF(2alpha). The time-dependent selective resistance to PGF(2alpha) and PGE(2) by rabbit CL is mediated by factors other than a lack of luteal receptor-ligand interactions.     

Alterations in the cellular membranes of regressing rat corpora lutea

Wide angle x-ray diffraction and fluorescence polarization were used to determine the structural properties of membranes from rat luteal cells. Examination of a plasma membrane fraction by x-ray diffraction revealed a significant increase in the gel phase melting temperature during luteal regression. The membrane fluidity of this fraction as well as that of a preparation of microsomes was also studied by fluorescence polarization. Using a fluorescent probe, membrane fluidity was observed to decrease during luteolysis. The temporal correlation between structural changes in the membrane and decreased progesterone secretion suggests that alterations in the physical properties of cellular membranes may be involved in the process of luteal cell regression.     

The fate of corpora lutea in the cyclic golden hamster

Structural luteolysis shows striking interspecies differences. Morphological changes in the corpus luteum (CL) of the cyclic hamster have been studied alongside the potential involvement of known luteolytic hormones. Ovaries from intact Syrian golden hamsters killed at 1100 h on days 1 and 2 and at 1100 and 1700 h on days 3 and 4 of the estrous cycle were dissected for histological study. The day of ovulation, the day of estrus, was arbitrarily designated day 1 of the estrous cycle. Steroidogenic cells in the CL were scarcely luteinized on day 1 and reached full luteinization on day 2. On the morning of day 3, initial regressive changes (accumulation of lipid droplets, invasion by neutrophils, and accumulation of phagocytic cells) were observed. These regressive changes increased progressively and apoptotic cells as well as phagocytic cells containing phagocytized apoptotic cells were abundant on the evening of day 3. On the morning of day 4, apoptotic cells/bodies and phagocytic cells containing phagocytized material were extremely abundant throughout the CL. However, steroidogenic cells with intact nuclei and well-preserved blood vessels were also found. Surviving cells in the CL showed progressive morphological changes. These cells showed morphological features intermediate between luteal and interstitial cells in the evening of day 4 and were virtually indistinguishable from interstitial cells on day 1 of the following cycle. Additional animals were injected at 1100 h on day 2 with: (a) the dopaminergic agonist CB154 (0.4 mg) to block prolactin secretion, (b) the anti-estrogen LY117018 (1.6 mg) or the anti-androgen Flutamide (3 mg) to block estrogen or androgen receptors, respectively, and (c) progesterone (2 mg) to prevent the fall in serum progesterone concentrations. Ovaries from these animals were collected at 1700 h on day 3 and at 1000 h on day 4. The luteolytic process was not affected by any treatment. These data indicate that, in contrast to its close relatives (e.g., the rat), structural luteolysis in the hamster is independent of the apoptotic inducing luteolytic hormones. In addition, differences in the cellular mechanisms responsible for CL elimination were also present. In the hamster, part of the luteal cells do not undergo apoptosis and seemed to progress through another developmental path giving rise to interstitial-like cells.     

Progesterone synthesis in developing rabbit corpora lutea in the absence of follicular estrogens.

We have investigated the role of 17beta-estradiol in the early development of the rabbit corpus luteum. Ectopic corpora lutea were established in all animals by autotransplanting preovulatory follicles beneath the kidney capsule 6.5 to 8 h after mating (day 0). At this time, the rabbits were bilaterally ovariectomized and in some of these rabbits a Silastic capsule containing crystalline 17beta-estradiol was implanted SC. Mean serum concentration of estradiol in rabbits with an estradiol impant was 16.4 pg/ml. In rabbits without an estradiol implant, the estradiol concentration averaged 1 pg/ml despite the presence of transplanted luteinized follicles. Daily blood samples were analyzed for progesterone by radioimmunoassay. Ectopic corpora lutea developed in rabbits with or without estradiol treatment. Serum progesterone concentrations in the two groups increased above castrate values and were not significantly different from one another through day 5. After day 5, progesterone concentrations steadily increased in rabbits treated with estradiol and reached a value of 4.8 ng/ml by day 10. In contrast serum progesterone steadily decreased after day 5 in rabbits without estradiol treatment to a level of 450 pg/ml by day 10. Hysterectomy on day 0 did not prevent this decline in progesterone, indicating that a uterine luteolytic agent was not involved. Total luteal weight on day 10 was positively correlated with serum progesterone concentration (r equals .92; P greater than 0.01). These results indicate that for a period of approximately 5 days afte r ovulation, the development of the rabbit ectopic corpus luteum and the secretion of progesterone are autonomous from estradiol secreted by ovarian follicles. After this time, there is an absolute requirement for estrogen which permits further development of the corpus luteum and the continuation of progesterone synthesis.     

Inadequate function of corpora lutea following the induction of ovulation with monensin and FSH in seasonally anoestrous ewes

Sixteen ewes in mid-seasonal anoestrus were stimulated to ovulate using sequential injections of FSH (total dose 10 mg) over a 4-day period. Half of the ewes received a dietary growth promotant (monensin) known to enhance the ovarian response to exogenous gonadotrophins. The ewes were ovariectomized on day 5 or 11 (day 0 = the initiation of FSH treatment). Serial blood samples were taken in half of the ewes to determine peripheral concentrations of LH and a single sample of ovarian venous blood was collected before ovariectomy. All luteal structures were dissected from the ovaries, counted and incubated in vitro to determine progesterone production. The luteal structures were then examined histologically for the abundance of luteal cells. The physical appearance of the ovary, along with plasma concentrations of LH and ovarian venous oestradiol indicated that the monensin-treated ewes ovulated before control ewes. The corpora lutea from control ewes produced significantly (P less than 0.05) more progesterone than did the corpora lutea from the monensin-treated group. Furthermore, only 7% of the remaining luteal structures in the monensin-treated group produced significant amounts of progesterone on day 11, whereas 61% of the luteal structures in the control group were actively secreting progesterone. The mean number of granulosa cells in the follicles was similar at ovulation in the two groups, but the mean numbers of large and small luteal cells were significantly (P less than 0.05) lower in luteal structures from the monensin-treated ewes than in those from the control ewes.(ABSTRACT TRUNCATED AT 250 WORDS)