Initiation Of Luteolysis Research Articles

Luteal haemodynamics during spontaneous and induced luteolysis in crossbred cattle

The haemodynamics of CL during natural and induced luteolysis was studied in crossbred cattle using colour Doppler ultrasonography. In natural cycle, penetration of Doppler signals deep into the CL was observed during Day 15 – 17 coinciding with the initiation of luteolysis. When PG was administered in the early luteal phase (Day 4), there was an increase in blood flow intensity in the periphery of CL, but when PG was administered in the mid luteal phase (Day 10), the intensified Doppler signals penetrated deep into the luteal parenchyma within two hours. There were no significant differences in velocity parameters and RI values between the treatment groups. Thus it was concluded that the characteristic difference in the intensity and nature of vascular perfusion was the determining factor for the initiation of luteolytic process in cattle.

Dihydrotestosterone regulation of cyclooxygenase-2 expression in bovine endometrial epithelium cells by androgen receptor mediated EGFR/PI3K/Akt pathway

Uterine prostaglandins F2α (PGF2α) is essential for implantation, initiation of luteolysis and delivery. Previous studies have demonstrated that the expression of Cyclooxygenase-2 (COX-2), an enzyme limiting PGF2α rate, is regulated by steroid hormones, and also dihydrotestosterone (DHT) may be involved in regulating COX-2 expression both positively and negatively. However, it remains unclear how whether DHT regulates COX-2 expression and consequent PGF2α release in bovine endometrial epithelial cells (EECs). In this study, we evaluated the localization of the two isoforms of DHT synthetase 5α-reductase (5α-red1 and 5α-red2) and androgen receptor (AR) in bovine endometria by immunohistochemistry, and investigated 5α-red1, 5α-red2, AR, and DHT levels at the different stages of endometria (follicle, early-, mid-, and late-pregnancy phases). The results showed that 5α-red1, 5α-red2 and AR all were expressed in endometria, and their expressions and the level of DHT significantly increased in the late-pregnancy phase compared with the mid-pregnancy phase. Moreover, we cultured EECs from the mid-pregnancy phase and the in vitro study showed that DHT dose-dependently increased COX-2 expression and PGF2a release, but AR antagonist (flutamide) inhibited the stimulating effect via DHT. In addition, the DHT-induced COX-2 expression and PGF2α release were subjected to the regulation of both EGFR/PI3K/Akt/NFkB signaling as the inhibitors of EGFR (AG1478) and PI3K/Akt (LY294002) and NFkB (QNZ) attenuated the DHT mediated effect. Taken together, the results demonstrated that DHT-induced COX-2 expression and consequent PGF2α release in bovine EECs were mediated through AR-derived EGFR transactivation and PI3K/Akt cascade leading to NFkB activation.

Up-regulation of endometrial oxytocin receptor is associated with the timing of luteolysis in heifers with two and three follicular waves†.

Initiation of luteolysis in ruminants is variable due to ill-defined mechanisms. Cycles of two follicular waves are shorter and have earlier luteolysis than three-wave cycles. This study validated a cytobrush technique for evaluating dynamics of endometrial gene expression and associated changes in mRNA with timing of luteolysis, based on circulating progesterone and ultrasound-determined changes in blood flow and volume of corpus luteum (CL). On day 8 (ovulation=day 0), Holstein heifers were randomized into two groups: cytobrush group (n=9) had an endometrial sample collected every 48h from day 8 until end of luteolysis (CL blood flow ≤ 20%) and control group was sampled only once either before (day 12; n=4) or at the end of luteolysis (n=5). Concentrations of progesterone, CL blood flow, CL volume, and the frequency of two and three-wave cycles were similar between groups. Endometrial mRNA for progesterone receptors and estradiol receptors 1 and 2 was greater on day 8 and decreased thereafter similarly in two and three-wave cycles. Oxytocin receptor mRNA increased earlier in two vs three-wave cycles (day 14 vs 18), and the increase was associated with the onset of luteolysis. In conclusion, the cytobrush technique allowed in vivo collection of multiple endometrial samples during the estrous cycle. Endometrial mRNA expression of steroid receptors did not explain the variability in timing of onset of luteolysis in heifers while the later onset of luteolysis in three-wave cycles was associated with later up-regulation of oxytocin receptor mRNA.

Prostaglandins as Mediators of the Conceptus-Maternal Interactions During Peri-implantation Period.

Ovarian progesterone induces changes leading to a temporary state of uterine receptivity for conceptus implantation. Estrogen produced by the porcine conceptus on days 11-12 of pregnancy, provide the primary signal for the maternal recognition of pregnancy and prolongs luteal progesterone production. In pigs, as in other species, implantation and establishment of pregnancy is associated with up-regulation of expression of proinflammatory factors, including cytokines, growth factors and prostaglandins (PGs), that probably activate inflammatory pathways in endometrium. Moreover, PGF2alpha and PGE2 exert an opposite action on corpus luteum. Therefore, a control over their synthesis and secretion is involved either in the initiation of luteolysis, or pregnancy establishment. One of the supportive mechanisms by which the porcine conceptus inhibits luteolysis is changing PG synthesis in favour of luteoprotective PGE2. We have found that selective changes in PG synthesis enzymes are regulated by estrogen. Using gene silencing PGE2 synthase (mPGES-1) in endometrial cells, we indicated an interdependence between the expression of this enzyme and PGE2 receptor, PTGER2. Moreover, we have established that PGE2 through endometrial PTGER2 elevates expression of enzymes involved in PGE2 synthesis, as well as endometrial and trophoblast PTGER2. These findings lead to the suggestion that conceptus PGE2 could be amplified by PGE2 feedback loop in endometrium. After reaching ovaries, PGE2 may act through luteal PTGER2 and PTGER4. On the other hand, elevated endometrial and trophoblast synthesis of PGE2 could have a direct effect to promote uterine function and conceptus development. We found that expression of PTGER2 is up-regulated in the endometrium on days 11-12 of pregnancy and in the trophoblast during implantation period. Now, we have used in vivo model to elucidate local and systemic effects of conceptus on endometrial gene expression. Understanding mechanisms of pregnancy establishment requires establishment of a hierarchy and timing of molecular relationships in the maternal-conceptus cross-talk. Supported by the State Committee for Scientific Research in Poland grants (DWM/N106/COST/2008 and NN311319135) and the basic grant of the Institute (IRZBZ PAN-2008-ZMDH). (platform)

Transcriptional Profiling of Equine Endometrium During the Time of Maternal Recognition of Pregnancy1

Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. To gain new insights into this complex process in the horse, transcriptional profiling of Day 13.5 pregnant and cyclic endometrial tissue samples was carried out using custom-designed microarrays. Selected array data were validated using quantitative RT-PCR, and proteins of interest were localized using immunohistochemistry. One hundred and six transcripts were up-regulated, whereas 47 transcripts showed lower expression levels in pregnant mares, that is, were down-regulated in pregnant mares. Half of the genes with known or inferred function are classically regulated by estrogens. Elevated transcript levels were found for genes involved in cell-cell signaling, heat shock response, and secretory proteins, among others. Solute carrier family 36 (proton/amino acid symporter), member 2, SLC36A2, was one of the most highly up-regulated genes, potentially reflecting the nutritional needs of the rapidly developing embryo. Among the genes showing lower expression in pregnant mares, estrogen receptor 1 was of particular interest because of its potential involvement in the initiation of luteolysis in cyclic mares. We hypothesize that either conceptus' estrogens or luteinizing hormone of uterine origin is involved in the observed down-regulation of estrogen receptor 1. Several of the genes identified in the current study are known to play a role in early pregnancy in species other than the horse. Thus, products of these commonly expressed genes likely contain universal activities for controlling endometrial receptivity to the conceptus, whereas other factors play unique roles within specific species in ensuring ongoing corpus luteum function. This is the first systematic study of endometrial transcriptome changes in response to the presence of an embryo during maternal recognition of pregnancy and an important step toward deciphering the embryo-maternal dialogue in equids.

141 TRANSCRIPTIONAL PROFILING OF EQUINE ENDOMETRIUM DURING THE TIME OF MATERNAL RECOGNITION OF PREGNANCY

Early embryonic development, implantation, and maintenance of a pregnancy are critically dependent upon a precisely orchestrated embryo-maternal interaction leading to a receptive uterine environment. The embryo has to signal its presence to the dam in order for estrous cyclicity to subside; however, the horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into molecular mechanisms underlying this complex process in the horse, transcriptional profiling of Day 13.5 pregnant and cyclic endometrial tissue samples was carried using custom-designed 2-color microarrays. Endometrial tissue samples collected from 4 pregnant and cyclic mares each were labeled with either Cy3 or Cy5, paired, and analyzed on 4 individual arrays. Transcripts showing a difference in signal intensity of at least 1.3 and a P-value of 0.01 or less in a mixed model analysis were considered differentially expressed. Array data were validated performing quantitative RT-PCR and changes in relative gene expression between pregnant and non-pregnant mares were determined using the delta delta cycle threshold method. Proteins of interest were localized using immunohistochemistry. One hundred six transcripts were up-regulated, whereas 47 transcripts showed lower expression levels in pregnant mares. Quantitative RT-PCR for five up- and down-regulated transcripts each confirmed expression data obtained by microarray with a validation rate of 100%. Half of the genes with known or inferred function have previously been described as regulated by estrogens. Given the large quantities of estrogen synthesized by the equine conceptus, this finding confirms validity of the data. Elevated transcript levels were found for genes involved in cell-cell signaling, heat shock response, and secretory proteins among others. Solute carrier 36 member 2, SLC36A2, was the most highly up-regulated gene, reflecting the nutritional needs of the rapidly developing embryo. The beta subunit of luteinizing hormone (LHB) showed higher expression levels in pregnant mares. It is likely that the observed up-regulation of LHB leads to increased release of bioactive LH/CG into the uterine environment during early pregnancy where we hypothesize it to exert paracrine effects preparing the uterus for conceptus implantation. Among the down-regulated genes, estrogen receptor 1 was of particular interest because of its potential involvement in the initiation of luteolysis in cyclic mares. We hypothesize that either embryonic estrogen or uterine-derived choriogonadotropin is involved in the observed down-regulation of estrogen receptor 1, which may be the key to maternal recognition of pregnancy. Several of the genes identified in the current study are known to play a role in early pregnancy in species other than the horse. We thus hypothesize that a subset of genes crucial to endometrial receptivity does not differ between species, representing a common pattern during early pregnancy, whereas each species has a distinct mechanism ensuring ongoing corpus luteum function.

Oxytocin (OT) action in uterine tissues of cyclic and early pregnant gilts: OT receptors concentration, prostaglandin F 2α secretion, and phosphoinositide hydrolysis

Oxytocin (OT) is involved in the regulation of luteolysis in pigs. However, it is still not clear if OT is responsible for initiation of luteal regression in this species. The objectives of the study were: (1) to compare OT receptors (OTr) concentrations in endometrium and myometrium of cyclic and early pregnant pigs, (2) to examine the effect of OT on plasma PGF 2α secretion during the progressive luteal regression, (3) to ascertain the effect of OT on inositol phosphates (IPs) accumulation in endometrial and myometrial cells of cyclic and early pregnant pigs. Concentrations of OTr on the endometrium and myometrium of cyclic ( n = 33) (days 2–4; 11–13; 14–16; 18–20; day 21) and early pregnant ( n = 4) (days 14–16) gilts were determined and they ranged from 7 ± 3 (days 11–13) to 377 ± 113 fmol/mg protein (day 21) in the endometrium and from 33 ± 11 (days 2–4) to 167 ± 28 fmol/mg protein (days 18–20) in the myometrium. In both tissues, concentrations of OTr were low during the luteal phase and increased ( P < 0.01) during the follicular phase. In contrast to myometrial OTr, endometrial OTr during pregnancy were undetectable. In next experiment, mature gilts ( n = 12) were injected with OT (20 IU; i.v.) for three consecutive days starting on days 14 and 15 of the oestrous cycle and plasma PGF 2α metabolite—13,14-dihydro-16-keto PGF 2α (PGFM) concentration was determined. On days 15–16 and 16–17, OT increased plasma PGFM level. This effect was not observed on days 14–15 of the estrous cycle. A negative correlation was noticed between plasma concentrations of PGFM and progesterone ( r = −0.3; P < 0.05). In last experiment, OT (100 nM) augmented ( P < 0.01) an accumulation of inositol phosphates (IPs) in isolated myometrial cells on days 14–16 ( n = 4) and 18–20 ( n = 3) of the estrous cycle and on days 14–16 ( n = 4) of pregnancy. Oxytocin-stimulated accumulation of IPs was not observed in endometrial cells. In summary: (1) concentrations of OTr on both the endometrium and myometrium were the highest during perioestrus-period in pigs, (2) myometrium of early pregnant sows possessed functional OTr, (3) oxytocin increased plasma PGFM concentration after initiation of luteolysis; and (4) OT-stimulated accumulation of IPs in myometrial, but not in endometrial cells. In conclusion, OT appears to not be involved in the initiation of luteal regression in sows and functional OTr are still present in the myometrium during early pregnancy (days 14–16).

The oestrous cycle and early pregnancy – a new concept of local endocrine regulation

Facts discovered in recent decades have compelled us to revise long-established views on the physiological regulation of cyclic adjustments to the reproductive system in preparation for pregnancy in females. Evidence has been presented to show that changes in the uterine blood supply induced by the oestrogen/progesterone ratio in the blood and cytokines are important in the regulation of the secretory function of the endometrium. Progressive reduction in uterine blood flow during the luteal phase of the oestrous cycle causes regressive changes in endometrial cells and release of prostaglandin (PG) F 2α, resulting in initiation of luteolysis. Retrograde transfer of PGF 2α in the area of the mesometrium vasculature is an important element in the mechanism protecting the corpora lutea against luteolysis before day 12 of the porcine oestrous cycle and during early pregnancy and pseudopregnancy. Results of many studies presented in this review indicate that PGF 2α pulses in uterine venous blood during the follicular phase of the oestrous cycle may not be due to PGF 2α secretion by endometrial cells, but occur due to remodeling of the endometrium and pulsatile exretion of PGF 2α in accordance with rhythmic uterine contractions caused by oxytocin.

Roles of tumor necrosis factor-alpha of the estrous cycle in cattle: an in vivo study.

We have suggested in a previous in vitro study that tumor necrosis factor-alpha (TNFalpha) plays a role in the initiation of luteolysis in cattle. The aim of the present study was to examine the influence of different doses of TNFalpha on the estrous cycle in cattle by observing the standing behavior and measuring peripheral concentrations of progesterone (P4) during the estrous cycle. Moreover, we evaluated the secretion of P4, oxytocin (OT), nitric oxide (NO), and luteolytic (prostaglandin F2alpha [PGF2alpha] and leukotriene C4 [LTC4]) and luteotropic (PGE2) metabolites of arachidonic acid in peripheral blood plasma as parameters of TNFalpha actions. Mature Holstein/Polish black and white heifers (n = 36) were treated on Day 14 of the estrous cycle (Day 0 = estrus) by infusion into the aorta abdominalis of saline (n = 8), an analogue of PGF2alpha (cloprostenol, 100 microg; n = 3) or saline with TNFalpha at doses of 0.1 (n = 3), 1 (n = 8), 10 (n = 8), 25 (n = 3), or 50 microg (n = 3) per animal. Peripheral blood samples were collected frequently before, during, and up to 4 h after TNFalpha treatment. After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. Lower doses of TNFalpha (0.1 and 1 microg) decreased the P4 level during the estrous cycle and consequently resulted in shortening of the estrous cycle (18.8 +/- 0.9 and 18.0 +/- 0.7 days, respectively) compared with the control (22.3 +/- 0.3 days, P < 0.05). One microgram of TNFalpha increased the PGF2alpha (P < 0.001) and NO (P < 0.001) concentrations and decreased OT secretion (P < 0.01). Higher doses of TNFalpha (10, 25, 50 microg) stimulated synthesis of P4 (P < 0.001) and PGE2 (P < 0.001), inhibited LTC4 secreton (P < 0.05), and consequently resulted in prolongation of the estrous cycle (throughout 30 days, P < 0.05). Altogether, the results suggest that low concentrations of TNFalpha cause luteolysis, whereas high concentrations of TNFalpha activate corpus luteum function and prolong the estrous cycle in cattle.

Expression and regulation of plasminogen activators, plasminogen activator inhibitor type-1, and steroidogenic acute regulatory protein in the rhesus monkey corpus luteum.

The corpus luteum (CL) is a transient endocrine organ that secretes progesterone to support early pregnancy. Using primate materials obtained from rhesus monkeys, we have in this study investigated the expression and regulation of the plasminogen activators (PAs) and PA inhibitor type 1 (PAI-1) during CL development and regression. Adult (5-7 yr old) female rhesus monkeys were treated with pregnant mare serum gonadotropin/human chorionic gonadotropin to induce ovulation and follicular luteinization. At various luteal developmental stages, CL or whole ovaries were obtained for preparing luteal cells, Northern blot, in situ hybridization, and immunohistochemistry. We demonstrated that luteal cells from the rhesus monkey were able to produce both tissue type PA (tPA) and urokinase type PA, as well as the physiological PAI-1. During luteal development in the monkey, urokinase type PA was the major PA species taking part in the active angiogenesis and tissue remodeling processes in the forming CL. However, the mRNA as well as the enzymatic activity levels of tPA increased dramatically in monkey CL with the advent of luteolysis. This change of tPA levels was in a temporal coordination with the regulation of PAI-1 expression, resulting in an increased tPA activity at the initiation of luteolysis. Therefore, we suggest that tPA might be a luteolytic factor to the monkey CL. A PAI-1 modulated tPA activity might be important for the initiation of luteolysis in the monkey. In addition, we have also demonstrated that the expression of steroidogenic acute regulatory protein in the monkey CL was in accordance with the changes of progesterone production, suggesting that steroidogenic acute regulatory protein expression may be considered as a reliable marker for CL function in primates.

A prostaglandin f(2alpha) analog induces suppressors of cytokine signaling-3 expression in the corpus luteum of the pregnant rat: a potential new mechanism in luteolysis.

PRL and placental lactogen (PL) play key roles in maintaining the rodent corpus luteum through pregnancy. Suppressors of cytokine signaling (SOCS) have been shown to decrease cell sensitivity to cytokines, including PRL, and so here we have addressed the issue of whether luteolysis induced by prostaglandin F(2alpha) (PGF(2alpha)) might up-regulate SOCS proteins to inhibit PRL signaling. In d 19 pregnant rats, cloprostenol, a PGF(2alpha) analog, rapidly induced transcripts for SOCS-3 and, to a lesser extent, SOCS-1. We also found increased SOCS-3 protein in the ovary by immunoblot and in the corpus luteum by immunohistochemistry. Increased SOCS-3 expression was preceded by an increase in STAT3 tyrosine phosphorylation 10 min after cloprostenol injection and was maintained for 4 h, as determined by gel shift and immunohistochemistry. Induction of SOCS-3 was accompanied by a sharp decrease in active STAT5, as determined by gel-shift assay and by loss of nuclear localized STAT5. Four hours after cloprostenol administration, the corpus luteum was refractory to stimulation of STAT5 by PRL administration, and this was not due to down-regulation of PRL receptor. Therefore, induction of SOCS-3 by PGF(2alpha) may be an important element in the initiation of luteolysis via rapid suppression of luteotropic support from PL.

Regulation of endometrial prostaglandin F 2α synthesis during luteolysis and early pregnancy in cattle

Luteal regression is caused by a pulsatile release of prostaglandin (PG) F 2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF 2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF 2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF 2α secretion. It has been recently demonstrated that tumor necrosis factor-α (TNF-α) stimulates PGF 2α output from bovine endometrial tissue not only during the follicular phase but also during the late luteal phase, suggesting that TNF-α is a factor in the initiation of luteolysis in cattle. Furthermore, our recent study has shown that IFN-τ suppresses the action of TNF-α on PGF 2α synthesis by the bovine endometrium in vitro, suggesting that IFN-τ plays a luteoprotective role by inhibiting TNF-α-induced PGF 2α production in early pregnancy. On the other hand, factors other than oxytocin or TNF-α have also been suggested to be involved in the regulation of PGF 2α synthesis by bovine endometrium. The purpose of this review is to summarize our current understanding of the endocrine mechanisms that regulate the timing and pattern of uterine PGF 2α secretion during the estrous cycle and early pregnancy.

Old, new and the newest concepts of inhibition of luteolysis during early pregnancy in pig

In 1977 Bazer and Thatcher proposed that maternal recognition of pregnancy in the pig involves the secretion of PGF 2α towards the uterine lumen (exocrine) rather than towards the uterine venous drainage (endocrine) as occurs in the non-pregnant pig during the mid to late stages of the estrous cycle. The retrograde transfer of PGF 2α from the venous blood and uterine lymph into the uterus and the ability of the uterine vein and artery wall to accumulate PGF 2α could constitute a part of putative mechanism of corpus luteum protection during early pregnancy. A luteotropic/anti-luteolytic effect of PGE 2 in the pig also has been frequently demonstrated and it seems that the most effective agent in changing PGE 2:PGF 2α secretion is estradiol. The role for oxytocin during luteolysis and early pregnancy is controversial. It appears, however, that the main function of this hormone is autocrine and/or paracrine stimulation of PGF 2α secretion. Pig trophoblastic interferons, unlike those of ruminants, do not themselves exert an anti-luteolytic effect in pigs. It is likely, that cytokines and angiogenic growth factors are involved in the initiation of luteolysis and/or maintenance of corpora lutea (CL). A discovery of functional LH receptors in porcine endometrium opened a new possibility for this hormone in luteolysis and perhaps in recognition of pregnancy in pigs. The endogenous LH pulses can provoke prostaglandin secretion from endometrium in pigs. On the other hand prolongation of up-regulation of LH receptors in endometrium of early pregnant gilts can additionally increase angiogenic factor production before the process of implantation is completed. Finally new integrated concepts of luteolysis and inhibition of luteolysis in pigs based on selectively reviewed information are presented.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10 ,100 and 1000 ng/ml). Following 48 hours of incubation ,the levels of progesterone ,20α-hydroxy-4-pregnene-3-one (20α-OH-P) and adenosine 3′ ,5′-monophosphate (cAMP) were measured in the culture media. PACAP alone significantly stimulated the production of progesterone and 20α-OH-P in a dose-dependent manner (p < 0.01 and 0.05 ,respectively ,ANOVA). LH-induced production of progesterone and accumulation of cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each ,ANOVA). Conversely ,LH-stimulated 20α-OH-P production was enhanced by PACAP in a dose-dependent manner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20α-OH-P production in LH-stimulated cells ,PACAP-mediated inhibition of the stimulatory action of LH on progesterone production may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects of LH on luteal cell function and that PACAP might be closely linked to reproduction.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnantmare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10 ,100 and 1000 ng/ml). Following48 hours of incubation ,the levels of progesterone ,20α-hydroxy-4-pregnene-3-one (20α-OH-P) and adenosine 3′ ,5′-monophosphate (cAMP) were measured in the culture media. PACAP alonesignificantly stimulated the production of progesterone and 20α-OH-P in a dose-dependent manner (p < 0.01 and 0.05 ,respectively ,ANOVA). LH-induced production of progesterone and accumulationof cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each ,ANOVA). Conversely ,LH-stimulated 20α-OH-P production was enhanced by PACAP in a dose-dependentmanner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20α-OH-P production in LH-stimulated cells ,PACAP-mediated inhibition of the stimulatory action of LH on progesteroneproduction may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects ofLH on luteal cell function and that PACAP might be closely linked to reproduction.

Is tumor necrosis factor alpha a trigger for the initiation of endometrial prostaglandin F(2alpha) release at luteolysis in cattle?

To determine the physiological significance of tumor necrosis factor alpha (TNFalpha) in the regulation of luteolytic prostaglandin (PG) F(2alpha) release by the bovine endometrium, the effect of TNF-alpha on PGF(2alpha) output by the endometrial tissues in vitro was investigated and compared with the effect of oxytocin (OT). Furthermore, the presence of specific receptors for TNFalpha in the bovine endometrium during the estrous cycle was determined. Endometrial slices (20-30 mg) taken from six stages of the estrous cycle (estrus: Day 0; early I: Days 2-3; early II: Days 5-6; mid-: Days 8-12; late: Days 15-17; and follicular: Days 19-21), as determined by macroscopic examination of the ovaries and uterus, were exposed to TNFalpha (0.06-6 nM) and/or OT (100 nM). OT stimulated PGF(2alpha) output at the follicular stage and at estrus (P < 0.001), but not at the late luteal stage. On the other hand, the stimulatory effects of TNFalpha on PGF(2alpha) output were observed not only at the follicular stage but also at the late luteal stage (P < 0.001). When the endometrial tissues at late luteal stage were simultaneously exposed to TNFalpha (0.6 nM) and OT (100 nM), the stimulatory effect on PGF(2alpha) output was higher than the effect of TNFalpha or OT alone (P < 0.05). Specific binding of TNFalpha to the bovine endometrial membranes was observed throughout the estrous cycle. The concentration of TNF-alpha receptor at the early I luteal stage was less than the concentrations at other luteal stages (P < 0.01). The dissociation constant (K(d)) values of the endometrial membranes were constant during the estrous cycle. The overall results lead us to hypothesize that TNFalpha may be a trigger for the output of PGF(2alpha) by the endometrium at the initiation of luteolysis in cattle.

The effect of pregnancy on the expression of uterine oxytocin, oestrogen and progesterone receptors during early pregnancy in the cow.

The expression of oxytocin receptor (OTR) in the uterine endometrium plays an important role in the initiation of luteolysis. During early pregnancy, the conceptus secretes interferon tau (IFN|gt) which inhibits OTR up-regulation and luteolysis. In this study, uterine horn cross sections were collected on day 16 from 15 pregnant cows (PREG), 9 uninseminated controls and 5 inseminated cows with no embryo present. The latter two groups had similar results and were combined to form a single non-pregnant (NP) group. The animals were given an oxytocin challenge shortly before tissue collection to assess prostaglandin F2alpha (PGF2alpha) release through the measurement of the metabolite 13,14-dihydro-15-keto PGF2alpha (PGFM). The mRNAs for OTR, oestrogen receptor (ER) and progesterone receptor (PR) were localised by in situ hybridisation. The results were quantified by optical density (OD) measurements from autoradiographs using image analysis. OTR protein was measured by autoradiography with iodinated oxytocin antagonist and ER and PR protein was detected by immunocytochemistry. The release of PGFM after the oxytocin challenge was significantly higher in the 14 NP cows (187%+/-15%) compared with the PREG group (131%+/-11%) (P<0.01). Low concentrations of OTR mRNA were localised to the luminal epithelium (LE) in 6 out of the 14 NP cows, of which 2 also expressed OTR protein, while OTR mRNA and protein were undetectable in all the pregnant animals. These results indicated that the sampling time coincided with the onset of the luteolytic mechanism in the NP cows. On day 16 ER mRNA was detectable in both the LE and glands of both PREG and NP animals. There were no differences in either ER mRNA or protein between NP and PREG samples. PR mRNA was moderately expressed in the caruncular stroma, with lower levels in the dense caruncular-like stroma and glands. There were no differences between PREG and NP animals. The expression of PR mRNA and protein in the deep glands was variable between animals. These results suggested that, in cows, the presence of an embryo suppressed the expression of OTR, but had no effect on the expression of the transcriptionally regulated ER on day 16.

Differentiation of bovine preovulatory follicles during the follicular phase is associated with increases in messenger ribonucleic acid for cytochrome P450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, and P450 17 alpha-hydroxylase, but not P450 aromatase.

In cattle, a dramatic increase in plasma estradiol occurs during the short 2- to 3-day follicular phase. The objective of this study was to investigate the molecular mechanisms that mediate this critical change, specifically whether increases in the steroidogenic ability of granulosa and thecal cells of the preovulatory follicle are associated with increases in the levels of messenger RNA (mRNA) for steroidogenic enzymes. Luteolysis and a follicular phase were induced cycling Holstein heifers (n=15) by injection of a luteolytic dose of prostaglandin F2 alpha (PGF 2 alpha) on day 6 or 7 of the estrous cycle (day 0 = estrus), and preovulatory follicles were obtained at three stages of differentiation (0, 12, or 24 h post-PGF2 alpha treatment). To assess developmental changes in steroidogenesis in vivo, estradiol and androstenedione were measured in follicular fluid and in culture medium after a 3-h incubation of granulosa and thecal cells in defined medium with or without gonadotropins. To determine whether changes in mRNA for steroidogenic enzymes are associated with changes in follicular steroidogenesis, levels of mRNA for cytochrome P450 side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome P450 17 alpha-hydroxylase, and cytochrome P450 aromatase (P450arom) were measured in thecal and granulosa cells using ribonuclease protection assays. Concentrations of estradiol in follicular fluid were relatively high at time zero, increased significantly by 12 h, and increased further by 24 h post-PGF2 alpha treatment. However, the aromatizing activity of granulosa cells was high at the time of PGF2 alpha injection and did not increase significantly during the first 24 h after the initiation of luteolysis. The aromatizing activity of granulosa cells was reflected in levels of mRNA for P450arom, which was relatively abundant in granulosa cells obtained before luteolysis and did not increase further during the first 24 h of the follicular phase. Concentrations of androstenedione were virtually undetectable in follicular fluid at time zero and had increased dramatically by 12 and 24 h post-PGF2 alpha treatment. Similarly, thecal cells isolated at 24 h secreted 3-fold more androstenedione than cells isolated at the time of PGF2 alpha injection. Androstenedione production by thecal cells in response to LH was also markedly higher at 12 and 24 h than at the time of PGF2 alpha injection. Likewise; levels of mRNA for P450 17 alpha-hydroxylase increased significantly by 12 h post-PGF2 alpha treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxytocin stimulates progesterone production by bovine granulosa cells isolated before, but not after, the luteinizing hormone surge

Oxytocin and its mRNA have been detected in bovine granulosa cells, but the function of follicular oxytocin is not well understood. We have shown previously that oxytocin exerts a specific, dose-dependent, stimulatory effect on progesterone secretion by granulosa, but not theca cells isolated from bovine preovulatory follicles obtained 48 h after the initiation of luteolysis. The objective of the present study was to characterize the development of granulosa cell responsiveness to oxytocin during the follicular phase. Granulosa cells and theca interna were isolated from preovulatory follicles early in the follicular phase (24 h after the initiation of luteolysis) or after the luteinizing hormone (LH) surge and cultured in defined medium for 5 days with or without oxytocin and in the presence or absence of gonadotropins. Granulosa, but not theca cells obtained before the LH surge increased progesterone production 3.3-fold in response to oxytocin. However, late in the follicular phase, after the LH surge, granulosa cells did not respond to oxytocin (or to follicle-stimulating hormone (FSH) or LH). These findings suggest that the LH surge (1) stimulates granulosa cells to maximal progesterone secretion, so that they cannot be further stimulated, (2) abolishes the responsiveness of granulosa cells to oxytocin, or (3) stimulates granulosa cells to increase oxytocin production, so that exogenous oxytocin has no additional effect.

Effects of Oxytocin on Steroidogenesis by Bovine Theca and Granulosa Cells*

Oxytocin (OT) is secreted during the final stages of bovine follicular development. To test OT's potential role as a regulator of follicular steroidogenesis, theca and granulosa cells were isolated from bovine preovulatory follicles 48 h after initiation of luteolysis with prostaglandin F2 alpha, and cultured with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml). Granulosa cells were cultured with testosterone (0.5 microM) in either defined medium or medium containing 10% fetal bovine serum in the presence or absence of FSH (300 ng/ml); medium was collected and replaced daily for 5 days. In defined medium, oxytocin alone significantly increased progesterone production by granulosa cells (P less than 0.001) in a dose-dependent manner; over 5 days, doses of 0.5, 5, 50, and 500 mIU/ml OT caused 1.7-, 2.0-, 2.2-, and 2.6-fold increases. FSH enhanced progesterone 5-fold, but no dose of OT increased progesterone in the presence of FSH. OT also elevated progesterone in serum-containing medium (P less than 0.005), but the magnitude of its effects was lower (1.07-, 1.1-, 1.2-, and 1.4-fold increases with 0.5, 5, 50, and 500 mIU/ml OT). OT had little effect on estradiol secretion by granulosa cells cultured with or without FSH. To test the specificity of OT's effects on progesterone production by granulosa cells, granulosa cells were treated with graded doses of an OT antagonist (0, 1, 10, 100, and 1000 ng/ml) in the presence or absence of OT (5 and 50 mIU/ml). Progesterone production by granulosa cells in the presence of the antagonist alone was similar to production in control cultures. The stimulatory effects of 5 and 50 mIU OT were completely abolished in the presence of 100 or 1000 ng antagonist, respectively (P less than 0.01). Preparations of theca interna were cultured in defined medium with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml) in the presence or absence of LH (300 ng/ml), with collection and replacement of medium at 3, 6, 12, 24, 48, and 72 h. LH alone increased both progesterone (12-fold) and androstenedione (4-fold) production over controls. However, no dose of OT significantly affected either progesterone or androstenedione production. These results show that OT stimulates progesterone production by granulosa cells, and thus, suggest that OT regulates steroidogenesis in bovine granulosa cells in vivo.