Acute changes in the concentrations of prostaglandin F2α (PGF) and cortisol in uterine and ovarian venous blood during PGF-induced luteolysis in cows.

Prostaglandin F2α (PGF) is considered to be the main luteolysin in cattle. We have previously demonstrated that cortisol (Cr) suppresses PGF production in non-pregnant bovine endometrium. This study was carried out to test whether exogenous PGF increases ovarian and/or uterine PGF production and to determine the temporal relationship between PGF and Cr in ovarian and uterine circulations during PGF-induced luteolysis in cows. Catheters were inserted into the ovarian vein (OV), uterine vein (UV) and jugular vein (JV) of 10 cows on Day 9 of the oestrous cycle (Ovulation = Day 0) for frequent blood collection. On Day 10, the cows were divided randomly into two groups and treated with a luteolytic dose of a PGF analogue (cloprostenol) or saline solution. Blood samples were collected at -0.25, 0, 0.25, 0.5, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). The basal concentrations of PGF and Cr in OV and UV plasma were not significantly different. Injection of a PGF analogue induced more than twofold increases in the levels of PGF between 0.25 and 1 h in UV plasma, but not in OV plasma. PGF increased (p < 0.05) the concentrations of Cr in OV, UV and JV plasma between 0.5 and 1 h. The Cr levels in OV, UV and JV plasma were similar. The PGF levels in UV plasma decreased after Cr reached its highest levels. The overall results suggest that the uterus rather than the ovary increases PGF production in response to PGF injection. Based on the temporal changes of PGF and Cr in the ovarian and uterine circulations, Cr may act to reduce uterine PGF production in non-pregnant cows in vivo.

Transdermal iontophoretic delivery of triptorelin in vitro.

The feasibility of delivering triptorelin ([D-Trp6]LHRH) by transdermal iontophoresis was evaluated in vitro. Peptide electrotransport at different current densities and donor concentrations was measured across porcine ear skin. The concomitant delivery of an electroosmotic marker enabled calculation of the respective contributions of electromigration (EM) and electroosmosis (EO) to iontophoretic delivery. At a given concentration (3 mM), a threefold increase in current density produced a corresponding increase in the cumulative amount of peptide present in the receptor compartment. Conversely, doubling the concentration to 6 mM produced a twofold reduction in the amount of peptide delivered, partly due to a concentration-dependent inhibition of EO. EM was revealed to be the predominant transport mechanism, accounting for 80% of overall delivery. Finally, despite the inhibition of EO, the results indicate that application of an iontophoretic current of 0.8 mA over a relatively small contact area (4 cm2) would provide a delivery rate of 36 microg/h, largely sufficient for therapeutic requirements.

Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha).

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).

N-cadherin-mediated human granulosa cell adhesion prevents apoptosis: a role in follicular atresia and luteolysis?

Studies suggest that cell-cell interactions may regulate apoptosis, and in particular, the calcium-dependent cell adhesion molecule N-cadherin has been shown to be capable of modulating this process. Rat granulosa cells (GCs) are known to express N-cadherin whereas cAMP is known to induce apoptosis in human and rat GCs. Based on these observations, we hypothesized that N-cadherin regulates human GC apoptosis via a cAMP-dependent mechanism. N-cadherin expression was evaluated in ovarian follicles and corpora lutea utilizing immunohistochemical techniques and in luteinized GCs in culture using immunoblotting, flow cytometric analysis, immunohistochemistry, and indirect immunofluorescence techniques utilizing anti-N-cadherin antibodies directed against both the extracellular and cytoplasmic domains of the molecule. Apoptosis was assessed by TUNEL and DNA fragmentation analysis and confirmed by flow cytometric cell cycle analysis and electron microscopy. The rate of GC apoptosis was found to be two- to three-fold lower among aggregated cells, as compared with single cells. N-cadherin was found to be expressed by aggregating GCs in vitro and GCs cultured in the presence of either N-cadherin function disrupting antibodies or peptides exhibiting enhanced rates of apoptosis. GCs in situ stained intensely for N-cadherin in preantral and normal growing preovulatory follicles as well as early corpora lutea. N-cadherin was weak in atretic follicles and regressing corpora lutea. Exposure of GCs to cAMP increased apoptosis while decreasing N-cadherin protein expression in a dose-dependent manner. Cell culture under serum-free conditions increased apoptosis and decreased N-cadherin expression, in part through cleavage of the extracellular domain of the molecule. The metalloproteinase inhibitor 1-10-phenanthroline inhibited the cleavage of the extracellular domain of N-cadherin and concomitantly inhibited the serum-deprivation-induced apoptosis of aggregated GCs. Collectively, these observations suggest that down-regulation of N-cadherin or the absence of a functional extracellular domain of the molecule prevents cell aggregation and is associated with GC apoptosis. In addition, cAMP induces apoptosis in a dose-dependent manner, and this process is dependent, at least in part, on regulation of the N-cadherin molecule at the surface of the cells. We conclude that N-cadherin-mediated GC signaling plays a central role in follicular and luteal cell survival.

Inhibition of the first ovulation and ovarian prostaglandin F2 alpha metabolism by danazol in rats.

The present study was conducted to investigate the mode of action of danazol by monitoring the first ovulation, serum luteinizing hormone (LH) levels and ovarian prostaglandin (PG) F2 alpha metabolism in pregnant mare serum gonadotropin (PMSG)-primed immature female rats. When danazol (750 mg/kg) was given p.o. once a day for 5 days (day 24-28), the occurrence of the first ovulation, the increase in capacity to form 13,14-dihydro-PGF2 alpha and PGF2 alpha levels induced by PMSG (5 IU) injected on day 26 were clearly inhibited on day 29. Danazol also markedly suppressed the LH surge occurring on day 28. Although the danazol-induced blockage of ovulation was restored by injection of human chorionic gonadotropin, the number of oocytes was significantly decreased as compared with that of controls. The present data indicate that the inhibitory actions of danazol on ovulation and ovarian PGF2 alpha metabolism may occur via some direct effects on the ovary in addition to the suppression of gonadotropin release from the pituitary gland.

Immunohistochemical localization and physiological regulation of carbonyl reductase in immature rat ovary.

The present study was performed to clarify the role of the ovarian carbonyl reductase (OCR) in ovarian function in immature rats. The OCR activities towards three specific substrates, 13,14-dihydro-PGF2 alpha, 4-benzoylpyridine and menadione, were photometrically and radiochemically determined in the 9,000 x g supernatants of ovaries, and OCR content was measured by Western-blot-peroxidase anti-peroxidase (PAP) analysis. Immunohistochemical localization of the enzyme in the ovary was performed by the avidin-biotin-complex (ABC) method for paraffin sections. Positive immunoreactivity with OCR antibody was observed for the theca cells and interstitial gland cells at 72 hr after pregnant mare serum gonadotropin (PMSG) treatment when ovulation was confirmed, and the granulosa cells were consistently negatively stained. The OCR activity was significantly increased by PMSG, human chorionic gonadotropin (hCG) and PMSG-hCG treatments, but estradiol and tamoxifen overcame the effect of PMSG on the enzyme activity. Moreover, estradiol enhanced the effect of hCG, but tamoxifen did not. Changes in the OCR activity well-correlated with those in the OCR content. These findings indicate that the OCR is regulated by gonadotropin and estrogen and that metabolites formed by the enzyme could be closely involved in ovarian cell function.

Effect of hCG on the 13,14-dihydro-prostaglandin F2-alpha forming capacity of the ovary after ovulation in PMSG-primed immature female rats.

We have examined the change in the ovarian 13,14-dihydro-prostaglandin F2 alpha (13,14H2-PGF2 alpha) forming capacity after the first ovulation induced by injection of pregnant mare serum gonadotropin (PMSG 5 IU, sc) at 26 days of age. After ovulation, the 13,14H2-PGF2 alpha forming capacity in the whole ovary (WO) and in non-luteal ovarian tissues (WO-CL) gradually decreased, whereas a rapid decrease of the synthesizing capacity was observed in corpus luteum (CL). The capacity in WO 4 days after ovulation (33 days of age) was markedly stimulated by human chorionic gonadotropin (hCG 10 IU, ip) administration, whereas CL at 33 days of age did not respond to the stimulatory effect of hCG. A single injection of hCG on day 7 after hypophysectomy resulted 12 hrs later in a significant increase in the forming capacity of 13,14H2-PGF2 alpha in WO-CL. These results indicate that the 13,14H2-PGF2 alpha forming capacity in CL rapidly decreases after the first ovulation and the WO-CL, but not CL, retain the ability to form 13,14H2-PGF2 alpha in response to exogenous gonadotropin for a long time.

A study of prostaglandin F2 alpha as the luteolysin in swine: V. Comparison of prostaglandin F, progestins, estrone and estradiol in uterine flushings from pregnant and nonpregnant gilts.

Uterine flushings were collected from 38 gilts representing Days 6,8,10,12,14,15,16 and 18 of the estrous cycle and pregnancy. The same group of gilts were represented within each of the respective days of the estrous cycle and pregnancy, i.e., three to six gilts per day per status. Uterine flushings (about 40ml) were assayed for prostaglandin F (PGF), estrone (E1), estradiol (E2), progestins (P) and protein. Nonpregnant gilts had higher (P < .01) concentrations of P in uterine flushings than pregnant gilts, but pregnant gilts had higher (P < .01) E1 and E2 concentrations. Significant day by status interactions were detected for E1 (P < .05), but not for E2 concentrations in uterine flushings. Total recoverable PGF and PGF concentrations in uterine flushings were greater (P < .01) in pregnant than nonpregnant gilts and significant (P < .01) day by status interactions were detected. In nonpregnant gilts, PGF increased between Days 12 and 16, i.e., during the period of corpora lutea (CL) regression. In pregnant gilts, PGF in uterine flushings increased markedly between Days 10 and 18. Total recoverable PGF on Day 18 of the estrous cycle was only 464.5 +/- 37.6 ng as compared to 22,688.1 +/- 1772.4 ng on Day 18 of pregnancy. Total recoverable protein was also higher (P < .01) in pregnant gilts. These data indicate that PGF synthesis and secretion by the uterine endometrium and/or conceptuses is not inhibited during pregnancy and suggest that PGF is sequestered within the uterine lumen of pregnant gilts, as is the total protein component of endometrial secretions referred to as histotroph.

The disposition and metabolism of the synthetic prostaglandin fluprostenol (ICI 81,008) in the horse.

1. Following single intramuscular doses of [14C]fluprostenol (0.5--2.4 micrograms/kg) to three female horses and to three gelded male horses, radioactivity was present in the plasma within 5 min; peak concn. (0.32--1.30 ng/ml fluprostenol equiv.) occurred 5 to 90 min after injection. Radioactivity was still present in the plasma of the females after three days. About 88% of fluprostenol is bound to plasma proteins. 2. Radioactivity was present in the parotid saliva of the gelded male horses within 10 min. Peak concn. (45--91 pg/ml fluprostenol equiv.) occurred from 5 min to 1 h after injection. Saliva : plasma concn. ratios varied inversely with saliva flow rate and limiting ratios were 0.33 and 0.41 for the combined results of two experiments on each of two male horses; the calculated value is 0.46 Chromatography indicated that the majority of plasma and saliva radioactivity was [14C]fluprostenol. 3. Excretion of radioactivity in the urine was rapid and virtually complete 12 h after dosing. The total radioactivity excreted in urine by the female horses was 45% of the dose (96 h) and by the gelded male horses 53% (30 h). About 30% of the radioactivity present in the urines was unchanged fluprostenol. 4. Faecal excretion, which was substantially complete after 2 days, accounted for 32% of the radioactivity administered to the female horses. 5. Tissue conc. of radioactivity in the female horses at four days were below the limits of detection (90 pg/g), but 0.2--0.9% of the dose was detected at the site of injection.

Prostaglandin E1 and F2alpha specific binding in bovine corpora lutea: comparison with luteolytic effects.

Preliminary characterization indicated the presence of separate prostaglandin (PG)E1 and (PG)F2alpha binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 x 10(-9)M and 1.1 x 10(-8)M for PGE1 and PGF2alpha, respectively. Competition of several natural prostaglandins for the PGE1 and PGF2alpha bovine luteal specific binding sites indicates specificity for the 9-keto or 9alpha-hydroxyl moiety, respectively. Differences in relative ability to inhibit 3H-PG binding were found due to sensitivity to the absence or presence of the 5, 6-cis-double bond as well. Bovine luteal function was affected following treatment of heifers with 25 mg PGF2alpha as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contract, treatment with 25 mg PGE1 resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE1 treatment compared to pretreatment values. In so far as data obtained in vitro on PGF2alpha relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF2alpha induced luteolysis in the bovine, PGF2alpha relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE1 relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.