Biomedical subjects

S Gräs

Publications and source records attributed to S Gräs.

5 recordsLinked to original sources

PubMed2000

Pituitary adenylate cyclase-activating peptide stimulates acute progesterone production in rat granulosa/Lutein cells via two receptor subtypes.

Pituitary adenylate cyclase-activating peptide (PACAP) is transiently expressed in ovarian granulosa/lutein cells from eCG/hCG-treated rats, and in vitro immunoneutralization of endogenously released PACAP inhibits acute progesterone secretion and subsequent luteinization in such cells. This suggests that PACAP mediates locally some of the effects of the LH surge, but the putative PACAP receptor(s) involved in such an auto or paracrine activity is presently unknown. Reverse-transcription polymerase chain reaction with specific primers to the three cloned PACAP-binding receptors called PAC(1), VPAC(1), and VPAC(2) demonstrated both PAC(1) and VPAC(2) mRNA in extracts from preovulatory follicular cells. Radioligand-binding assays revealed the presence of high-affinity binding sites with characteristics of these two receptors on the intact cells, and autoradiography demonstrated that the binding was restricted to a minor proportion of the follicular cells as well as the oocytes. Pituitary adenylate cyclase-activating peptide and vasoactive intestinal peptide (VIP) dose-dependently stimulated cAMP accumulation and acute progesterone accumulation. Forskolin and db-cAMP also stimulated acute progesterone accumulation, and the protein kinase A inhibitor H89 dose-dependently inhibited peptide induced acute progesterone accumulation, suggesting involvement of cAMP and the protein kinase A pathway in the process. In conclusion, two of the three PACAP binding receptors are present on preovulatory follicular cells and are involved in the effects of PACAP on acute progesterone production. The data provide further evidence to establish PACAP as an auto- or paracrine regulator of LH-induced acute progesterone production in rat preovulatory follicles.

Animals↗

PubMed1999

Pituitary adenylate cyclase-activating polypeptide is an auto/paracrine stimulator of acute progesterone accumulation and subsequent luteinization in cultured periovulatory granulosa/lutein cells.

Recently, we have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) is transiently expressed in steroidogenic ovarian cells during the periovulatory period. This prompted us to establish an in vitro system in which the potential local regulatory role of PACAP during periovulatory progesterone production could be examined. Granulosa/lutein cells from PMSG- and human CG (hCG)-stimulated immature rats were used. The cells were isolated from preovulatory follicles 4-6 h after the hCG injection, at which time the transient ovarian PACAP expression begins in vivo. By immunocytochemistry on intact cells and RIA on cell extracts and culture medium, granulosa/lutein cells were found to accumulate and secrete PACAP during incubation. Furthermore, the cells responded to exogenous PACAP 38 with a rapid (10(-7) M induced a peak value 20-fold higher than controls at 2 h) and dose-dependent accumulation of progesterone. PACAP 38 (5 x 10(-9) M), in combination with an approximately half-maximal dose of hCG (1 ng/ml), showed an additive effect on progesterone accumulation. Immunoneutralization of endogenously released PACAP was performed using the IgG fraction from a specific PACAP antiserum that dose-dependently inhibits the progesterone accumulating effect of exogenous PACAP 38. The acute effects of endogenously released PACAP were studied during 8 h of incubation of granulosa/lutein cells with anti-PACAP IgG (100 microg/ml). A significant reduction in progesterone accumulation was observed after 4, 6, and 8 h [38.7% (P < 0.05), 41.2% (P < 0.02), and 50% (P < 0.002), respectively], compared with nonimmune IgG (100 microg/ml) treated cultures. The long-term effects on luteinization induced by endogenously released PACAP were studied after incubation of the cells with anti-PACAP IgG or nonimmune IgG for 24 h, followed by incubation for 9 days in serum-containing medium. Under these conditions, nonimmune IgG-treated cells assumed a luteal phenotype, accumulating large and stable amounts of progesterone and acquiring hypertrophic cell bodies with numerous lipid droplets and distinct nucleoli in the large nuclei. Anti-PACAP IgG-treated cells displayed morphological and functional signs of impaired luteinization being smaller and more irregular and with progesterone accumulation being significantly lower throughout the incubation period [56.4% (P < 0.02), 69.2% (P < 0.05), 43.8% (P < 0.02), and 52.2% (P < 0.02) at 1, 4, 7, and 10 days, respectively]. Together, these findings support an auto- or paracrine role for PACAP during gonadotropin-induced acute periovulatory progesterone production and subsequent luteinization in granulosa/lutein cells.

Animals↗

PubMed1996

Transient periovulatory expression of pituitary adenylate cyclase activating peptide in rat ovarian cells.

Pituitary adenylate cyclase activating polypeptide (PACAP), a potent stimulator of cAMP formation, has a widespread distribution in neuronal elements of the central nervous system and a number of peripheral organs. Recently, PACAP has also been shown to be stage specifically expressed in the spermatogenic cells from the rat testis. This prompted us to examine the cell-specific expression of PACAP during the estrous cycle in the rat ovary by in situ hybridization with a digoxiginin UTP-labeled RNA probe and by immunohistochemistry using a specific monoclonal antibody. Ovaries from PMS gonadotropin (PMSG)/human CG (hCG) treated immature animals were examined 0, 12, 24, 48, 54, 57, 58.5, 60, 61.5, 63, 66, 72, and 96 h after PMSG treatment. Ovaries from adult cyclic rats were examined at 1000 h on each day of the estrous cycle and at 0400 h on estrus morning. PACAP positive cells were observed in the preovulatory and ovulatory period, i.e. 6-18 h following hCG injection in the PMSG induced cycle and at 0400 h and 1000 h on estrus morning in adult cyclic animals. Both PACAP messenger RNA (mRNA) and PACAP immunoreactivity were observed in the majority of granulosa and cumulus cells from large preovulatory follicles, in the majority of the cells comprising the interstitial glandular tissue and in solitary theca cells of growing and mature follicles. In PMSG/hCG treated animals, positive signals were also observed in solitary theca and interstitial cells 12 h after PMSG injection. PACAP immunoreactive nerve fibers were observed in all ovaries examined, located in the interstitial glandular tissue or in the loose connective tissue. Northern blotting of ovarian tissue confirmed the transient preovulatory and ovulatory expression of PACAP. Three transcripts were observed. The most predominant band had a size of approximately 1.2 kilobase pairs, corresponding to the transcript observed in the testis. Two bands of 2.4 and 3.0 kilobase pairs, corresponding to the transcripts from neural tissue, were also observed. The complex distribution of PACAP positive cells and nerve fibers suggests that the peptide could be a local regulator of a number of the events that take place in the periovulatory period as well as during early folliculogenesis in the ovary.

Animals↗

PubMed1994

Vasoactive intestinal polypeptide and peptide histidine methionine. Presence in human follicular fluid and effects on DNA synthesis and steroid secretion in cultured human granulosa/lutein cells.

Vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) originate from the same precursor molecule, prepro VIP. In the present study we examined the concentrations of VIP and PHM in human follicular fluid and their effects on cultured human granulosa/lutein cells. Follicular fluid and cells were obtained from patients undergoing in-vitro fertilization for tubal infertility. The concentrations of VIP and PHM in pre-ovulatory human follicular fluid were measured radioimmunochemically. Granulosa/lutein cells isolated from follicular fluid were cultured under serum-free conditions with VIP and PHM in varying concentrations (0.1, 10, 1000 nmol/l). [3H]Thymidine incorporation in the cells and oestradiol as well as progesterone concentrations in the culture medium were measured. The mean (+/- SEM) concentrations of VIP and PHM were 6.8 +/- 0.1 and 7.7 +/- 0.8 pmol/l, respectively. VIP at a concentration of 10 nmol/l caused a significant increase in [3H]thymidine incorporation, and at 1000 nmol/l a significant increase in oestradiol secretion was observed. VIP had no effect on progesterone secretion. PHM at the concentrations tested did not influence any of the activities. We conclude that VIP and PHM are present in human preovulatory follicular fluid and that VIP stimulates DNA synthesis and oestradiol secretion in cultured human granulosa/lutein cells. This indicates that VIP and perhaps PHM participate in the local nervous regulation of human ovarian function.

Cell Division↗

PubMed1988

[An adrenal cyst].

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Adrenal Gland Diseases↗