P4 Secretion Research Articles

Porcine theca cells produce immunoreactive beta-endorphin and change steroidogenesis in response to opioid agonist.

In earlier in vitro experiments opioids affected steroidogenesis in porcine luteal and granulosa cells. The present studies were undertaken to examine the effects of FK 33-824 (opioid agonist) alone or in combination with LH, PRL or naloxone (NAL, opioid antagonist) on steroidogenesis in cultured porcine theca cells. Moreover, we have tested beta-endorphin-like immunoreactivity (beta-END-LI) concentrations in culture media under control conditions and following treatments of theca cells with LH, PRL, progesterone (P4), oestradiol (E2) or testosterone (T). FK 33-824 and NAL significantly increased P4 release by theca cells and inhibited stimulatory effect of LH on this steroid output. PRL-induced P4 secretion from the cells was blunted only by FK 33-824. Secretion of androstenedione (A4) and T was essentially elevated in the presence of FK 33-824 and this potentiation of both androgen release was completely abolished by PRL. NAL blocked stimulatory effect of the opioid agonist only in case of T. Secretion of oestradiol and oestrone was completely free from the influence of both the opioid agonist and antagonist. Pig theca cells were able to produce beta-END-LI but none of tested hormones (LH, PRL, P4, E2 and T alone or in combination) significantly affected this production. In conclusion, these data indicate that porcine theca cells may produce beta-END-LI and change their steroidogenesis in response to opioid peptides.

Prolactin binding analysis and immunohistochemical localization of prolactin receptor in porcine ovarian cells.

In the present study we searched for prolactin receptor (PRL-R) in porcine ovarian theca tissue (Tc) of small, medium and large follicles, as well as in early corpus luteum (ECL). The objectives of this investigation were: 1) comparison of the direct effect of PRL action on progesterone (P4) and estradiol (E2) secretion from Tc and ECL cells in culture with adequate effects caused by luteinizing hormone (LH). 2) detection of the presence and distribution of PRL-R in thecal tissue of porcine follicles and in ECL. Tissues were cultured as monolayers either in control M199 medium with calf serum or in medium either with PRL (100 ng/ml) or with LH (100 ng/ml). After 2 days in vitro cultured media were assayed for steroid concentrations by radioimmunoassays. Content and distribution of PRL-R were evaluated by Scatchard analysis and by an immunohistochemical assay. Separated theca layers as well as fragments of ECL were excised on dry ice, homogenized, and incubated with [125I]-PRL. PRL stimulated P4 secretion from Tc 10-fold versus controls. LH stimulated P4 secretion only 2.5-fold. E2 secretion was stimulated by PRL 2.7-fold and by LH 2.4-fold. LH enhanced P4 secretion from ECL cells by 18% while PRL increased P4 secretion by as much as 73%. Femtomol amounts of PRL-R protein were detected in theca tissues of medium and large follicles and also in ECL, which was in accordance with immunohistochemical results. The results showed for the first time the presence of PRL-R in porcine Tc and ECL.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters follicular steroidogenesis in time- and cell-specific manner.

To show the direct effect of TCDD on ovarian steroidogenesis theca (Tc) and granulosa (Gc) cells were cultured separately or as a co-culture (GT). Cells were cultured in M199 medium supplemented every day with 0.1 nM TCDD or only at the beginning of the culture with 10 nM of TCDD. After 48, 96 and 144 h culture media were collected for steroids content analysis. In Tc cultured alone TCDD caused an increase in E2 production after 48 and 96 h of culture, while exposure of Tc on TCDD for 144 h caused the decrease in both T and E2 secretion. In Gc cultured alone and in GT cultures the increase in E2 secretion was observed only after 48 h, while a long term exposure to TCDD (96 and 144 h) caused a decrease of both P4 and E2 secretion. The results of the present study suggest various, time-dependent mechanisms of TCDD action on ovarian cells.

Different actions of noradrenaline and nitric oxide on the output of prostaglandins and progesterone in cultured bovine luteal cells

The effects of noradrenaline (NA) and nitric oxide (NO) on prostaglandins (PGs) and progesterone (P4) secretion during the development of the bovine corpus luteum (CL) were investigated. Bovine luteal cells of early and mid-cycle CL were cultured for 20 to 24 h in medium containing 10% calf serum, washed, and treated with NA or nitrergic agents for an additional 16 h in a serum-free medium. NA (10 −5 M) stimulated P4 from early and mid-cycle CL by 238% and 154% ( P < 0.01), respectively. Moreover, although NA induced a twofold increase in PGE 2 secretion ( P < 0.01) in both examined periods, the effect of NA on PGF 2α secretion was approximately 1.5 times higher ( P < 0.05) in early than in mid-cycle CL. Two NO synthase inhibitors, l-NAME and l-NOARG (both 10 −4 M), stimulated P4 secretion only in mid-luteal cells ( P < 0.01), although they did not affect the cells from early CL. Although a NO donor, S-NAP (10 −4 M) inhibited P4 secretion from mid-cycle luteal cells ( P < 0.05), it strongly stimulated PGE 2 in both examined phases ( P < 0.001). On the other hand, the output of PGF 2α was stimulated by S-NAP only in the cells of the mid-cycle CL ( P < 0.01). The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL functions. Whereas NA may play a supporting role in luteal development, NO may participate in the functional regression of the bovine CL by inhibiting steroidogenesis.

The physiological role of beta-endorphin in porcine ovarian follicles.

Beta-endorphin-like immunoreactivity (beta-END-LI) was measured by radioimmunoassay in porcine ovarian follicular fluid (FF) from small, medium and large follicles throughout the oestrous cycle. The concentration of beta-END-LI in FF from small follicles collected on days 1-5 of the cycle was at least tenfold higher than in the fluid from any other follicles independently from their size and the period of the cycle. The level of beta-END-LI in small follicles on days 6-10 was drastically decreased. Subsequently, on days 11-16 its concentration was enhanced and reduced again in pre-ovulatory period of the cycle. Concentrations of beta-END-LI in FF from medium follicles were relatively equal throughout the cycle (days 6-21). No significant differences in beta-END-LI levels were found between small, medium and large follicles from days 17-21. However, beta-END-LI concentrations in medium follicles on days 11-13 and 14-16 were statistically lower than those in small follicles. Moreover, the effects of FSH, prolactin (PRL), progesterone (P4), testosterone (T) and 17 beta-oestradiol (E2) on beta-END-LI release by granulosa cells (GCs) from large follicles and, on the other hand, the effects of the opioid agonist FK 33-824 alone or in combination with FSH, PRL or naloxone (NAL) on follicular steroidogenesis were studied. FSH drastically increased beta-END-LI output in a dose-dependent fashion. This stimulatory effect of the gonadotrophin was inhibited by the highest dose of P4 (10(-5) M). The effect of PRL and the steroids added to the cultures on beta-END-LI release was negligible. FSH- or PRL-induced P4 secretion by GCs was essentially abolished by both FK 33-824 and NAL. However, androstenedione (A4) and testosterone output by the cells was greatly potentiated by FK 33-824. In the presence of NAL, FSH or PRL, A4 release stimulated by FK 33-824 was suppressed to the basal level. Secretion of E2 was completely free from the influence of FK 33-824 or NAL; only oestrone (E1) output was modulated by them in cultures where FSH or PRL was present. In conclusion, FSH appears to be the key regulator of beta-END-LI secretion by porcine granulosa cells. Moreover, steroidogenesis in pig granulosa cells is modulated by opioid peptides acting both alone and by way of interaction with FSH or PRL.

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro

The main purpose of this study was to investigate the direct effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on ovarian function including ovulation and steroidogenesis. In vivo effects of TCDD were investigated on ovulation and alteration of circulating and ovarian steroid hormones in immature hypophysectomized rats (IHR) primed with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In addition, in vitro effects of TCDD on the steroidogenesis of granulosa cells (GC), theca-interstitial cells (TIC), and whole ovarian dispersates derived from the ovary of IHR were investigated. In the ovulation model, rats were hypophysectomized on Day 23 of age. On Day 26, the IHR were given 20 μg TCDD/kg by gavage. The next day eCG (10 IU) was injected sc to stimulate follicular development. Fifty-two hours after eCG, 10 IU hCG was given to induce ovulation. TCDD (20 μg/kg) blocked ovulation and reduced ovarian weight in IHR. Concentrations of progesterone (P4), androstenedione (A4), and estradiol (E2) in sera and ovaries were not altered by TCDD at 12, 24, 48, and 72 h after eCG, except for a two-fold increase in ovarian concentration of A4 at 48 h after TCDD. However, this higher concentration of A4 at 48 h after TCDD did not reflect that of A4 in sera and did not correlate with E2 in either sera or ovaries. In isolated GC from untreated IHR, TCDD (0.1 to 100 nM) had no significant effect on P4 and E2 after stimulation by LH or FSH. In TIC and whole ovarian dispersates containing GC, TIC, and other ovarian cells, TCDD (0.1 to 800 nM) had no effect on A4 and P4 secretion stimulated by LH. Using RT-PCR, AhR mRNA was shown to be expressed constitutively in the whole ovary of IHR with maximum down-regulation at 6 h after TCDD (20 μg/kg). Ovarian CYP1A1 was induced maximally at 6 h after TCDD, whereas CYP1B1 could not be detected. The induction of AhR related genes by TCDD in the ovary implies the existence of AhR-mediated signal transduction pathways. In summary, these results indicate that TCDD does not affect ovulation in IHR by altering ovarian steroidogenesis. It seems that inhibition of ovulation by TCDD is due to processes related to follicular rupture.

Action of the opioid agonist FK 33-824 on porcine small and large luteal cells from the mid-luteal phase: effect on progesterone, cAMP, cGMP and inositol phosphate release.

The present study was designed to investigate the effect of the opioid agonist FK 33-824 on basal and hCG-induced progesterone (P4), cAMP and cGMP secretion and on the phosphoinositide-specific phospholipase C signalling system in separated porcine small (SLCs) and large luteal cells (LLCs). Unit gravity sedimentation was used to produce cultures of small and large luteal cells from corpora lutea (CL) on days 8-10 of the oestrous cycle. In order to examine the effect of FK 33-824 on P4 and cyclic nucleotide release, SLCs and LLCs were incubated in M199 medium at 37 degrees C in 5% CO2:95% air, for 12 h. Small and large luteal cells were treated with hCG (100 ng/ml) alone, FK 33-824 (10(-9) M) alone or were co-treated with FK 33-824 and hCG and with the opioid antagonist, naloxone (NAL, 10(-5) M). FK 33-824 alone did not influence P4 secretion by LLCs and SLCs. However, FK 33-824 completely abolished the stimulatory effect of hCG on P4 secretion by SLCs. The addition of FK 33-824 was followed by a significant increase in cAMP release (p<0.01) by LLCs and a decrease in cGMP secretion by SLCs (p<0.05). The effect of FK 33-824 was blocked by NAL, which strongly suggests that the observed influence of this opioid agonist was achieved through its binding to opioid receptors in luteal membranes. In the presence of hCG, cAMP secretion by both SLCs and LLCs was many-fold higher than in the control group. As regards cGMP output, only LLCs showed elevated secretion of this cyclic nucleotide under the influence of hCG. With the aim of examining the influence of FK 33-824 on phosphatidylinositol hydrolysis, LLCs, SLCs and mixed small and large cells were labelled with [3H]-myo-inositol (100 microCi/ml) for 3 h at 37 degrees C. The cells were then incubated in M199 medium supplemented with 10 mM LiCl, 1% BSA, and antibiotics in the presence and absence of FK 33-824 (10(-9) M) at 37 degrees C for 30 min. Liberated labelled inositol mono-, bis-, and trisphosphates (IPs) were isolated and quantified by affinity chromatography on columns of AG 1-X8 resin, followed by liquid scintillation spectroscopy. Inositol phosphate accumulation in LLCs, SLCs, and mixed small and large cells was not altered by treatment with FK 33-824 at the dose used. In view of these findings we suggest that opioid peptides affect pig corpus luteum steroid secretion, and the response is probably mediated through cyclic nucleotides, but not IPs.

Sensitivity of bovine corpora lutea to prostaglandin F2alpha is dependent on progesterone, oxytocin, and prostaglandins.

Prostaglandin (PG) F2alpha that is released from the uterus is essential for spontaneous luteolysis in cattle. Although PGF2alpha and its analogues are extensively used to synchronize the estrous cycle by inducing luteolysis, corpora lutea (CL) at the early stage of the estrous cycle are resistant to the luteolytic effect of PGF2alpha. We examined the sensitivity of bovine CL to PGF2alpha treatment in vitro and determined whether the changes in the response of CL to PGF2alpha are dependent on progesterone (P4), oxytocin (OT), and PGs produced locally. Bovine luteal cells from early (Days 4-5 of the estrous cycle) and mid-cycle CL (Days 8-12 of the estrous cycle) were preexposed for 12 h to a P4 antagonist (onapristone: OP; 10(-4) M), an OT antagonist (atosiban: AT; 10(-6) M), or indomethacin (INDO; 10(-4) M) before stimulation with PGF2alpha. Although OP reduced P4 secretion (p < 0.001) only in early CL, it reduced OT secretion in the cells of both phases examined (p < 0.001). OP also reduced PGF2alpha and PGE2 secretion (p < 0.01) from early CL. However, it stimulated PGF2alpha secretion in mid-cycle luteal cells (p < 0.001). AT reduced P4 secretion in early and mid-cycle CL (p < 0.05). Moreover, PGF2alpha secretion was inhibited (p < 0.05) by AT in early CL. The OT secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2alpha. PGF2alpha had no influence on OT secretion, although [Ca2+]i increased (p < 0.05) in the early CL. However, the effect of PGF2alpha was augmented (p < 0.01) in cells after pretreatment with OP, AT, and INDO in comparison with the controls. In mid-cycle luteal cells, PGF2alpha induced 2-fold increases in OT secretion and [Ca2+]i. However, in contrast to results in early CL, these increases were magnified only by preexposure of the cells to AT (p < 0.05). These results indicate that luteal P4, OT, and PGs are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and may be responsible for the resistance of the early bovine CL to the exogenous PGF2alpha action.

Pro- and anti-inflammatory cytokines during human CMV infection

Knowledge on the regulation of corpus luteum (CL) function in the mare is scarce. In this study, the presence of cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), and their receptors (TNFRI, TNFRII and IFNRI), was investigated in equine CL throughout the luteal phase. The effects of TNF and IFNG on secretory function and viability of luteal cells were defined in vitro. Cytokine ligands and receptors were present in steroidogenic and endothelial cells. Protein expression for TNF was greater in mid-phase and regressing CL, while TNFRI was increased in regressing CL and TNFRII did not change. IFNG and IFNRI showed the highest expression in regressing CL. Transcription of mRNA for TNF increased from mid-phase to regressing CL and both TNFRI and TNFRII decreased from early to regressing CL. Transcription of mRNA for IFNG was lower in CL from early phase than in mid or regressing luteal phase, while IFNRI expression was not changed. In the early CL, TNF acted to increase P4 and PGE2 but decrease PGF2α secretion. In the mid luteal phase, TNF increased PGF2α secretion and TNF+IFNG decreased PGE2 secretion. In the regressing luteal phase, TNF, IFNG and TNF+IFNG decreased P4 and PGE2 secretion, but TNF and TNF+IFNG increased PGF2α secretion by luteal cells. Cell viability was reduced by TNF+IFNG in regressing CL. These data show the presence of cytokines TNF and IFNG, and their receptors, in the equine CL and indicate their potential involvement in regulation of luteal function.

Luteinizing hormone inhibits conversion of pregnenolone to progesterone in luteal cells from rats on day 19 of pregnancy.

We have previously reported that intrabursal ovarian administration of LH at the end of pregnancy in rats induces a decrease in luteal progesterone (P4) synthesis and an increase in P4 metabolism. However, whether this local luteolytic effect of LH is exerted directly on luteal cells or on other structures, such as follicular or stromal cells, to modify luteal function is unknown. The aim of the present study was to determine the effect of LH on isolated luteal cells obtained on Day 19 of pregnancy. Incubation of luteal cells with 1, 10, 100, or 1000 ng/ml of ovine LH (oLH) for 6 h did not modify basal P4 production. The addition to the culture medium of 22(R)-hydroxycholesterol (22R-HC, 10 microgram/ml), a membrane-permeable P4 precursor, or pregnenolone (10(-2) microM) induced a significant increase in P4 accumulation in the medium in relation to the control value. When luteal cells were preincubated for 2 h with oLH, a significant (p < 0.01) reduction in the 22R-HC- or pregnenolone-stimulated P4 accumulation was observed. Incubation of luteal cells with dibutyryl cAMP (1 mM, a cAMP analogue) plus isobutylmethylxanthine (1 mM, a phosphodiesterase inhibitor) also inhibited pregnenolone-stimulated P4 accumulation. Incubation with an inositol triphosphate synthesis inhibitor, neomycin (1 mM), or an inhibitor of intracellular Ca2+ mobilization, (8,9-N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (1 mM), did not prevent the decrease in pregnenolone-stimulated P4 secretion induced by oLH. It was concluded that the luteolytic action of LH in late pregnancy is due, at least in part, to a direct action on the luteal cells and that an increase in intracellular cAMP level might mediate this effect.

Effects of induced hypothyroidism on ovarian response to superovulation in Brahman (Bos indicus) cows.

To evaluate the effects of hypothyroidism on ovarian function, multiparous, nonlactating Brahman cows (n = 18) were assigned randomly to dietary treatments containing either 0 (C; n = 9) or 4 mg x kg BW(-1) x d(-1) 6-n-propyl-2-thiouracil (PTU; n = 9), to suppress thyroid function, in the feed concentrate. Weekly changes in BW and body condition score (BCS) were recorded. Dietary treatments began on d 10 of the estrous cycle. Ten days after the first treatment estrus, all cows received daily i.m. injections of 25 IU of porcine FSH over a 3-d period. Seven days after AI, embryos were collected nonsurgically, and the ovaries were removed via midflank laparotomy. Based on thyroxine (T4) concentrations after 49 d of treatment, five cows were hypothyroid (H-PTU) and four were partially suppressed (P-PTU). Cows in the PTU group had greater (P<.01) ADG, (P<.05) ovarian weights, and numbers of large (> or =8 mm) (P<.05) follicles. Cows in the PTU group had lower embryo recovery rate (P<.001), fertilization rate (P<.001), and percentage of blastocysts (P<.1) than C cows. The H-PTU cows had greater numbers of luteinized follicles (P<.06), greater concentrations of progesterone (P4) in the follicular fluid at all size categories (P<.1), and greater numbers of corpora lutea (P<.05) than C cows. The ratio of luteal to serum P4 on d 7 was greater (P<.05) in hypothyroid cows. Induced hypothyroidism improved weight gain and BCS, increased ovarian response to FSH, and affected ovulation, fertility, and P4 secretion in superovulated Brahman cows.

Synergistic effects of prostaglandin F2alpha and tumor necrosis factor to induce luteolysis in the pig.

There is ample evidence that prostaglandin F2alpha (PGF2alpha) is a luteolytic substance in sows, however, there is also some evidence that it may stimulate progesterone (P4) secretion in young corpora lutea (CL). In vitro studies also suggested that tumor necrosis factor alpha (TNF) is inhibitory to luteal cell P4 and estradiol-17beta (E2) release. Since E2 is a strong luteotropic substance in porcine CL, we studied the effects of intraluteal application of PGF2alpha and TNF alone and in combination on the secretion of P4 and E2 in freely moving sows. Furthermore, the effects of intraluteal infusion of E2 and its stereoisomer, estradiol-17alpha, on luteal function, were also determined. Microdialysis systems were implanted into CL at Day 10 of the estrous cycle. After a 24-h recovery period, PGF2alpha (10(-6) M) or E2 (10(-6) M) was applied daily for 6 h into the CL. PGF2alpha caused a stimulation of E2 and P4, and E2 also stimulated P4 secretion at Days 11 and 12, but the stimulatory effect of both substances diminished as the CL approached luteolysis. Intraluteal TNF application resulted in a transient increase of P4 secretion, which was followed by a dramatic reduction of P4 release. When TNF-pretreated CL were exposed to PGF2alpha at Day 11 of the estrous cycle, the prostaglandin was no longer able to stimulate but rather inhibited E2 and P4 secretion. Intraluteal application of estradiol-17alpha had no effect on P4 secretion. These results are suggestive that the PGF2alpha-induced E2 secretion in young and middle-aged CL is stimulatory to P4 secretion. Under the influence of macrophage-derived TNF production, E2 secretion is inhibited, and thereby PGF2alpha and TNF cause functional luteolysis.

Importance of endometrial quality in women with tubal infertility during a natural menstrual cycle for the outcome of IVF treatment.

The importance of endometrial maturation at estimated time of implantation for the outcome of IVF treatment in regularly menstruating women with tubal infertility was evaluated. FSH was measured on cycle day 3, on days 10-15 urine and blood were collected to estimate the day of the LH peak, and E2 and P4 were measured during the luteal phase, on cycle days 19-26. An endometrial biopsy was obtained on days LH + 3 to LH + 6. The number of subjects with delayed endometrial maturation was larger in the group of infertile women who did not become pregnant compared to pregnant women and controls. Those infertile women who did not become pregnant after IVF treatment also presented with a higher basal FSH on cycle day 3 and lower E2 and P4 AUC in the luteal phase. Six infertile women and two controls presented with mid- and late-proliferative endometrium in the luteal phase on cycle days LH + 3 to LH + 6, in the presence of adequate E2 and P4 secretion. Six morphological characteristics were compared in the three groups: (1) 17 infertile women who became pregnant, (2) 18 who did not become pregnant, and (3) 28 controls. The pregnant infertile women did not differ from the controls. The numbers of glandular and stromal mitoses were significantly higher in those women who did not become pregnant (P < 0.01) compared with those who became pregnant. Endometrial biopsies obtained on cycle days LH + 5 and LH + 6 showed significant differences in glandular epithelial height (P < 0.05) and number of vacuolated cells among the nonpregnant women (P < 0.01), the pregnant women (P < 0.05), and controls. A higher frequency of retarded endometrial development in women who did not become pregnant following IVF treatment was found. In some cases, endometrial insensitivity could most likely cause retarded endometrial development and failure of implantation after IVF treatment, which could not be overcome by routine luteal-phase support. However, our results do not allow conclusions concerning its relative importance compared to preembryo quality; this has to be investigated further.

Corticotropin-releasing hormone (CRH) inhibits steroid biosynthesis by cultured human granulosa-lutein cells in a CRH and interleukin-1 receptor-mediated fashion.

The presence of immunoreactive CRH was recently demonstrated in human ovaries. CRH immunoreactivity was localized by immunohistochemistry in the cytoplasm of thecal cells surrounding the ovarian follicles, in luteinized cells of the stroma, and in large granulosa-derived luteinized cells of developing corpora lutea. Also, CRH and its receptors were identified in Leydig cells of the testis where CRH was shown to inhibit testosterone biosynthesis. To examine the role of CRH in the ovary, we studied its effect on estradiol (E2) and progesterone (P4) release by human granulosa cells obtained from women undergoing in vitro fertilization for male factor infertility or uni- or bilateral tubal impatency. In all subjects, superovulation was induced by treatment with gonadotropins. The effects of graded doses of ovine CRH (10[-11]-10[-6] mol/liter) were evaluated in the conditioned medium obtained after 24 h incubation of the cells. All CRH concentrations employed except for the lowest one (10[-11] mol/liter) caused a significant decrease of media E2 and P4 levels. Maximal inhibition for both E2 and P4 production was obtained by 10[-6] mol/liter CRH concentration, which decreased hormone production by 39% and 34%, respectively. The alpha-helical CRH9-41 antagonist at 10(-6) and 10(-7) mol/liter blocked the suppressive effect of 10(-9) mol/liter CRH on both E2 and P4 secretion, while it had no effect when added to the culture media without CRH. Since interleukin (IL-1)-1 mediates certain actions of CRH on leukocytes, we examined whether the CRH effect on ovarian steroidogenesis was IL-1-mediated. Interleukin-1 receptor antagonist at 10(-7) and 10(-6) mol/liter blocked the inhibitory effects of CRH on E2 and P4 secretion, while it had no effect in the absence of CRH. In conclusion, CRH exerts a CRH- and IL-1 receptor-mediated inhibitory effect on ovarian steroidogenesis and might be actively involved in the still enigmatic processes of follicular atresia and luteolysis.

Roles of leukotrienes in bovine corpus luteum regression: an in vivo microdialysis study.

A microdialysis system (MDS) was surgically implanted into the corpora lutea (CL) of 12 normally cycling Holstein heifers. Heifers were either allowed to undergo spontaneous luteolysis (Spontaneous, n = 6) or received an intramuscular injection of prostaglandin F2 alpha (PGF2 alpha) on Day 12 of the estrous cycle (Induced, n = 6). The MDS was implanted on Day 11 in the induced heifers and on Day 17 in Spontaneous heifers. CL were perfused with Ringer's solution at a flow rate of 3 ml/hr beginning immediately after surgery. Dialysate samples were collected hourly for 3-4 days. Samples were assayed for progesterone (P4), oxytocin (OT), PGF, and leukotrienes B (LTB) and C (LTC). Dialysate OT was undetected in all but one Spontaneous and one induced heifer. Lipoxygenase products of arachidonic acid (AA) metabolism (LTB and LTC) in the dialysate were found to be closely associated with luteal regression. In Spontaneous heifers, the mean interval from the first hormone peak to the onset of P4 decline was similar for PGF, LTB, and LTC, with the first peak occurring at 12.8 +/- 8.1, 22.0 +/- 6.1, and 11.0 +/- 8.9 hr before the onset of P4 decline, respectively. The peak LTC value was greater (P < 0.05) than peak LTB or PGF. The 12-hr sampling interval with the highest LTC peak frequency was highly correlated (r = 1.0; P < 0.01) with the onset of P4 decline, but the highest LTB and PGF peak frequencies were not associated with the onset of P4 decline. Indeed, the mean numbers of PGF and LTB hormone peaks were higher (P < 0.05) after the onset of P4 decline than before. Administration of PGF2 alpha on Day 12 of the estrous cycle stimulated a decline in P4 secretion and an increase in the secretion of PGF, LTB, and LTC from the CL. In induced animals, the peak level of PGF was greater (P < 0.05) than peak LTB. These results suggest that the AA metabolites LTB and, especially, LTC play important roles during normal regression of the bovine CL.

Involvement of adrenoceptors in the ovarian vascular pedicle in the regulation of counter current transfer of steroid hormones to the arterial blood supplying the oviduct and uterus of pigs.

1. On Day 10 of the oestrous cycle in pigs, after laparotomy noradrenaline (NA), methoxamine (alpha 1-adrenomimetic, M), Prazosin (alpha 1-adrenolytic, Pr) in total doses of 4 mumol, and saline were infused (10 min) into the superficial layer of mesovarium on both sides of the ovarian pedicle vasculature, close to the ovary. 2. Blood flow in the ovarian artery, heart rate and progesterone (P4) and androstenedione (A4) secretion from the ovary and their concentrations in the ovarian venous effluent, as well as the concentrations of P4 and A4 in the blood supplying the oviduct and the uterus, were determined. 3. A significant increase of P4 and A4 secretion after NA and M infusion and increased concentrations of P4 and A4 in the ovarian venous effluent were found, but these changes did not influence the counter current transfer of hormones from the venous effluent into arterial blood supplying the oviduct and the uterus. 4. Infusion of Pr caused a significant decrease of P4 and A4 secretion and their concentrations in the ovarian venous effluent and significantly increased A4 concentration in the blood supplying the oviduct and uterus. 5. The results indicate that stimulation of alpha 1-adrenoceptors in the area of ovarian vasculature did not influence, whereas block of alpha 1-adrenoceptors affected, the local concentration of steroid hormones in the blood supplying the oviduct and the part of the uterus proximal to the ovary, despite the changes in the concentrations of steroid hormones in the ovarian effluent.

Effects of luteinizing hormone and prostaglandin F2α on gap junctional intercellular communication of ovine luteal cells throughout the estrous cycle

Cellular interactions mediated by contact-dependent pathways may be important to maintain luteal function. The objective of the present experiment was to evaluate the role of LH and prostaglandin F(2α) (PGF) in regulation of contact-dependent, gap junctional intercellular communication (GJIC) of ovine luteal cells from several stages of luteal development. Corpora lutea (CL) obtained from superovulated ewes on days 5 (n=7), 10 (n=8), and 15 (n=9) after estrus were dispersed with collagenase and cell types were separated by elutriation. Cells were plated as a mixed population (nonelutriated), or as small or large luteal cell fractions, and incubated in serum-free media containing no hormone, LH (100 ng/mL), PGF (100 ng/mL), LH+PGF, or dibutyryl cAMP (dbcAMP; 2 mM) for 18-24 h. Media were collected for evaluation of progesterone (P4) concentrations and replaced with media containing fluorescent dye. Then the rate of GJIC was evaluated by using the fluorescence recovery after photobleaching technique and laser cytometry. The rate of GJIC was determined for selected cells: small luteal cells in contact only with small luteal (S-S) cells; large luteal cells in contact only with small luteal (L-S) cells; and large luteal cells in contact only with large luteal (L-L) cells. LH increased (p<0.01) GJIC for S-S on d 5 and 10 and for L-S cells across the estrous cycle, but did not affect GJIC for L-L cells. PGF increased (p<0.05) GJIC for L-L cells on d 10 and 15, and decreased (p<0.05) GJIC for S-S cells from d 5 and 10 of the estrous cycle. LH+PGF increased (p<0.05) GJIC for S-S cells on d 5 and 10, and for L-S and L-L cells on d 10 and 15 of the estrous cycle. In addition, PGF diminished (p<0.05) LH-stimulatory effects on GJIC for S-S cells from d 5 and 10, and for L-S cells from d 5 of the estrous cycle. Dibutyryl cAMP stimulated (p<0.05) GJIC between all evaluated cell types across the estrous cycle. LH and dbcAMP stimulated (p<0.05) P4 secretion by mixed and small luteal cell fractions, PGF alone did not affect basal P4 secretion, but LH+PGF stimulated (p<0.05) P4 production by small luteal cells across the estrous cycle. PGF diminished (p<0.05) LH-stimulatory effects on P4 production in mixed populations of luteal cells across the estrous cycle.These data demonstrate that both luteal cell types communicate with each other, and the rate of communication was affected by LH, PGF, and dbcAMP. Modulation of gap junctional contact-dependent intercellular communication may be an important mechanism by which regulatory signals are transduced during luteal growth, differentiation, and regression in sheep.

Effects of local growth factors on the secretory function of bovine corpus luteum during the oestrous cycle and pregnancy in vitro.

The impact of insulin-like growth factor-I (IGF-I) basic fibroblast growth factor (bFGF), endothelin-1 (ET-1), tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha) and platelet-derived growth factor (PDGF) on the release of progesterone (P4) and oxytocin (OT) from individual bovine corpora lutea at different stages of the oestrous cycle and pregnancy was evaluated with a microdialysis system (MDS) in vitro. IGF-I (1 microgram mL-1) induced significantly the acute effects on P4 release at the late luteal stage (Days 15-18) and early pregnancy (Days 60-120), whereas bFGF (100 ng mL-1) was extremely effective in stimulating P4 release particularly during the mid-luteal stage (Days 8-12). Both peptides stimulated (P < 0.05) the release of OT throughout the three luteal stages and during early and late pregnancy (Days 30-60 and Days 150-210). ET-1 (100 ng mL-1) clearly inhibited P4 release during the early (Days 5-7) and mid-luteal phase and stimulated OT release only during the mid-luteal stage (P < 0.001). TNF-alpha (100 ng mL-1) stimulated the release of P4 exclusively at the early luteal phase (P < 0.05), whereas OT secretion was increased by TNF-alpha during all stages of the oestrous cycle (P < 0.001). TGF-alpha and PDGF (100 ng mL-1) were effective in stimulating P4 release particularly during late pregnancy (P < 0.05). In contrast, stimulation of OT secretion by TGF-alpha was maximal during the late-luteal stage (P < 0.001), whereas PDGF significantly increased OT secretion during the oestrous cycle (except the early luteal stage) and pregnancy (P < 0.001). The data demonstrate distinct and stage-specific effects of growth factors on P4 and OT secretion in vitro. IGF-I, bFGF and TGF-alpha may play an important role in corpus luteum (CL) function during the oestrous cycle and pregnancy since they are locally expressed and synthesized, there are receptors for these growth factors, and they have been demonstrated to exert biological effects on the CL.

Alcohol inhibits epidermal growth factor-stimulated progesterone secretion from human granulosa cells.

In this study, luteinized human granulosa cells (GC) obtained during in vitro fertilization procedures were used as a model system to evaluate the effects of ethanol (EtOH), a well-known reproductive toxin, on epidermal growth factor (EGF) and gonadotropin-stimulated steroidogenesis. Our results demonstrate that the basal progesterone (P4) and estradiol (E2) secretion by human GC in vitro was dependent on the ovarian stimulation protocol. EGF significantly enhanced P4, but not E2, secretion in human GC from clomiphene citrate (CC), human menopausal gonadotropin (hMG), and hMG/gonadotropin-releasing hormone agonist (GnRH-a)-treated patients. The effects of EGF plus luteinizing hormone (LH) were additive in cells from the CC group, but less than additive in hMG and hMG/GnRH-a groups. EtOH at 20 mM or more inhibited EGF stimulated P4 secretion in human GC from all three patient groups. EtOH inhibited P4 secretion stimulated by EGF and LH cotreatment in the CC and hMG/GnRH-a groups, but not in human GC from the hMG-treated patients. These results suggest that basal and EGF or LH-stimulated P4 secretion by human GC, as well as the effects of EtOH, are profoundly influenced by the follicle's hormonal milieu.

Interleukin-1 beta (IL-1 beta) is increased in the follicular fluids of patients with premature luteinization.

Most, but not all, studies indicate that premature luteinization correlates with poor pregnancy outcome in in-vitro fertilization (IVF) programs. It remains unclear whether cytokines (IL-1 beta, TNF alpha), the established immune mediators, play a role in regulation or initiation of an abnormal follicular or embryo development in patients with premature luteinization. Levels of cytokines (IL-1 beta, TNF alpha), estradiol (E2) progesterone (P4), and androstenedione (A'ione) were examined in 18 preovulatory follicular fluid (FF) samples from patients with premature luteinization (group 1) and 3 FF samples from patients without premature luteinization (group 2). The number of oocytes recovered, fertilization rate, and pregnancy outcome were evaluated in these two groups. IL-1 beta (25.4 +/ 11.9 pg/ml, mean +/ SD) and TNF alpha (13.4 +/ 10.7 pg/ml) were present in these FF samples. The mean level of IL-1 beta in group 1 was significantly higher than that in group 2 (37.3 +/ 12.3 vs. 20.0 +/ 7.6 pg/ml; P < 0.00001) and the mean level of E2 was significantly lower in group 1 than that in group 2 (1064 +/ 686 vs. 1570 +/ 641 ng/ml; P = 0.02). The levels of TNF alpha, P4, and A'ione showed no distinction between these two groups. There was no correlation between the levels of either IL-1 beta or TNF alpha and P4, E2 or A'ione. The fertilization rate in group 1 (62/77; 80%) was similar to that in group 2 (124/160; 78%). Five of 7 patients in group 1 and seven of 20 patients in group 2 achieved pregnancy following embryo transfer. One of five pregnancies in group 1 aborted. The exaggerated levels of IL-1 beta in patients with premature luteinization may arise from accumulation of this cytokine owing to sustained high LH stimulation, and this may be a protective response to the abnormal LH surge and function to inhibit prematurely increased secretion of P4. These data indicate the important role of LH in the induction of IL-1 beta secretion and the possible regulatory action of IL-1 beta in luteinization. According to the diminution of E2 in group 1, there may be a subtle atretic process progressing in follicles primed with prematurely elevated LH. However, the detrimental effect of premature luteinization, if it exists, may work at the stage ¿during or after implantation.