Expressions of estrogen receptors in the bovine corpus luteum: cyclic changes and effects of prostaglandin F2alpha and cytokines

Estrogen (E) exerts its function by binding to two intracellular estrogen receptors, ERalpha and ERbeta. Although ERs have been reported to be expressed in the bovine corpus luteum (CL), the mechanisms that control ER expression in the bovine CL are not fully understood. To determine the possible regulatory mechanisms of ERalpha and ERbeta that meditate distinct E functions, we examined 1) the changes in the protein expressions of ERs in the CL throughout the luteal phase and 2) the effects of prostaglandin (PG) F2alpha, tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) on the expressions of ERs in cultured bovine luteal cells. Western blot analyses revealed that ERalpha and ERbeta proteins were expressed throughout the luteal phase. The ERalpha protein level was high at the early luteal (Days 2-3 after ovulation) and mid-luteal stages (Days 8-12) and was extremely low at the regressed luteal stage (Days 19-21). The ERbeta protein level increased from the early to developing luteal stage, remained at the same level at the mid-luteal stage and decreased thereafter. The ratio of ERbeta to ERalpha was higher in the regressed stage than in the other stages. Luteal cells obtained from mid-stage CLs (Days 8-12) were incubated with PGF2alpha (0.01-1 microM), TNFalpha (0.0145-1.45 nM) or IFNgamma (0.0125-1.25 nM) for 24 h. PGF2alpha and TNFalpha inhibited ERa and ERbeta mRNA expressions. IFNgamma suppressed ERbeta mRNA expression but did not affect the expression of ERalpha mRNA. However, the ERalpha and ERbeta protein levels were not affected by any of the above treatments. These data indicate that PGF2alpha, TNFalpha and IFNgamma regulate ERalpha and ERbeta mRNA expressions in bovine luteal cells. Moreover, the changes in the ERbeta/ERalpha ratio throughout the luteal phase suggest that ERalpha is associated with luteal maintenance. Therefore, a dramatic decrease in ERalpha at the regressed luteal stage could result in progression of structural luteolysis in the bovine CL.     

Reproductive performance of dairy cows not detected in oestrus but with a detectable corpus luteum, in response to treatment with progesterone, oestradiol benzoate and prostaglandin F2alpha

AIM: To determine if the reproductive performance of dairy cows not previously detected in oestrus but with a detectable corpus luteum before the planned start of mating (PSM), could be improved by treatment with progesterone, oestradiol benzoate (ODB) and prostaglandin F2alpha (PGF). METHODS: Cows in 18 herds which had not been detected in oestrus, but which had a detectable corpus luteum present at veterinary examination 7 days prior to the PSM (Day -7), were allocated to 1 of 2 groups. Treated cows (n=232) received an injection of 2 mg ODB and an intravaginal progesterone releasing device (CIDR insert) on Day -7, and an injection of PGF on the day of insert removal 7 days later (Treated group). The Control group (n=243) remained untreated. Cows were mated to detected oestrus from Day 0, and conception dates confirmed by manual palpation or transrectal ultrasonography. RESULTS: During the first 7 days of mating, 37.4% of Control cows and 65.9% of Treated cows were inseminated on detection of oestrus (p<0.001). Pregnancy rates for this period were 20.4% and 36.3%, respectively (p=0.001). Conception rates to first insemination, pregnancy rates after 21 days of mating and at the end of the mating period were similar between groups (p>0.1). Median interval from the PSM to conception did not differ between treatment groups (24 and 23 days for Control and Treated, respectively, p>0.1). CONCLUSION: Treating postpartum dairy cows which had not previously been detected in oestrus but which had a detectable corpus luteum, with progesterone, ODB and PGF did not significantly improve their reproductive performance compared with no hormonal intervention. KEY WORDS: dairy cattle, postpartum, anoestrous, reproduction, progesterone treatment.     

Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.     

Maintenance of the corpus luteum of early pregnancy in the ewe. IV. Changes in luteal sensitivity to prostaglandin F2 alpha throughout early pregnancy

Two experiments were conducted to examine the temporal aspects of luteal resistance to the luteolytic effect of prostaglandin (PG) F2 alpha during early pregnancy. In Exp. 1, 14 pregnant and 12 nonpregnant ewes were treated with PGF2 alpha either on d 10 or 13 post-estrus. Jugular venous blood samples were collected at -30 min, 0, 6, 12, 18, 24, 30 and 36 h post-injection for quantification of progesterone. The difference (delta P) between pre-treatment and post-treatment concentrations of progesterone was calculated for each ewe. There was a significant interaction between pregnancy status and day of treatment on delta P (P less than .05). Pregnant and nonpregnant ewes treated on d 10 showed a large delta P. A large delta P also was observed in nonpregnant ewes treated on d 13 post-estrus. However, delta P in pregnant ewes treated on d 13 was smaller than in the other three groups (P less than .05). The temporal patterns of concentrations of progesterone in serum were different among treatment groups (P less than .05). A suppression in the concentration of progesterone was observed by 24 h post-injection in all four treatment groups. Progesterone returned to pre-treatment levels only in pregnant ewes treated on d 13. In Exp. 2, 47 pregnant ewes were treated with PGF2 alpha on d 10, 13, 16, 19, 22, 26 or 30 postestrus. Blood samples were collected and data were analyzed as described for Exp. 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormone secretion and characteristics of estrous cycles after treatment of heifers with human chorionic gonadotropin or prostaglandin F2 alpha during corpus luteum formation

Fertility in cattle is related positively to concentrations of progesterone in blood during the estrous cycle preceding insemination. This study determined whether treatment of heifers with prostaglandin F2 alpha (PGF2 alpha) or human chorionic gonadotropin (hCG) during d 2 to 4 of an estrous cycle affected progesterone during that cycle and whether hormone secretion during the cycle and onset of subsequent estrus were related to progesterone secretion. Nine Holstein heifers were assigned to an experiment designed as a triplicate Latin square, and each heifer received each of three treatments during three consecutive estrous cycles. Treatments were: saline (control, 1 ml) on d 2, 3 and 4 after estrus; hCG, 1000 IU on d 2, 3 and 4; and PGF2 alpha, 25 mg on d 3 with repeated doses 12 and 24 h later. Progesterone throughout the estrous cycle was higher in heifers given hCG than in those given saline. Progesterone during the first week of the cycle was lower in heifers given PGF2 alpha than those given saline, but means for these two groups were similar thereafter. Number of peaks of 15-keto,13,14-dihydro-PGF2 alpha (PGFM) during 24 h after onset of luteolysis was lower in heifers given hCG than in those given saline or PGF2 alpha. Patterns of secretion of luteinizing hormone and estradiol at subsequent estrus were not affected by treatment. Temporal relationships among hormone secretion and onset of estrus were unaffected by treatment.     

Maintenance of the corpus luteum of early pregnancy in the ewe. III. Differences between pregnant and nonpregnant ewes in luteal responsiveness to prostaglandin F2 alpha

The effects of pregnancy and number of corpora lutea on luteal regression induced with prostaglandin F2 alpha (PGF2 alpha) were examined in 93 ewes. Bred and nonpregnant ewes were assigned randomly to receive a single im injection of PGF2 alpha: 0, 2, 4, 6, 8 or 10 mg/58 kg body weight. Injections were given on d 13 postestrus. The concentration of progesterone in serum 24 h after PGF2 alpha injection was affected by dose (P less than .001). The effect of pregnancy and the interaction of pregnancy with number of corpora lutea on levels of progesterone in serum were significant (P less than .05); therefore, data were partitioned according to pregnancy status and analyzed separately. There was an effect of number of corpora lutea on serum concentration of progesterone in pregnant (P less than .01) but not nonpregnant ewes (P greater than .10). Similar relationships among groups were observed for the concentration of progesterone in luteal tissue. In nonpregnant ewes the minimum dose of PGF2 alpha to produce a significant suppression of progesterone in serum (P less than .05) was 4 mg/58 kg body weight. In pregnant ewes with one or two corpora lutea, the minimum effective doses were 6 and 10 mg/58 kg body weight, respectively. The concentration of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) in serum was related to the dose of PGF2 alpha injected. There were no differences in the concentration of PGFM in serum between pregnant and nonpregnant ewes either before or after injection. Corpora lutea of early pregnancy appear to be resistant to the luteolytic effect of PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma levels of prostaglandin F2 alpha metabolite and progesterone in repeat breeder heifers.

A detailed clinical-endocrine investigation was performed in 6 repeat breeder heifers (RBH) with the aim being to ascertain whether endocrine asynchronism exists at luteal regression and during early pregnancy. The heifers were first studied during an open cycle and then after insemination when 3 heifers became pregnant. Circulating plasma levels of PGF2 alpha metabolite were measured every 2nd h, while progesterone (P4) levels were measured every 6th h. The oestrous period and intervals between the onset of oestrus and ovulation were relatively longer, compared with what is normally seen in heifers. Plasma levels of P4 at the onset of oestrus were higher than normal, but it was concluded that the plasma levels of PGF2 alpha metabolite and P4 in RBH at luteal regression and early pregnancy were normal.     

Resynchrony of previously anoestrous cows and treatment of cows not detected in oestrus that had a palpable corpus luteum with prostaglandin F(2 alpha)

AIMS: To determine if resynchrony, using a progesterone (P4) / oestradiol benzoate (ODB) regime, of previously treated, anovulatory anoestrous (AA) cows would increase the probability of oestrus, conception and pregnancy compared to no resynchrony. Additionally, the effect of prostaglandin F2alpha (PG) treatment of cows not detected in oestrus, but in which a corpus luteum (CL) was palpable transrectally (NDO/CL+), was compared with no treatment. METHODS: Cows not detected in oestrus by 1 week before the start of the seasonal breeding programme were categorised as AA or NDO/CL+, based on absence or presence of a palpable CL, respectively. All AA cows were treated with an intravaginal device containing 1.36 g P4 (CIDR-B) for a period of 6 days, and with 1 mg ODB by intramuscular (I/M) injection 1 day after the device was removed (Day 0). Half the AA cows that were detected in oestrus between Days 0 and 3 were randomly assigned to be resynchronised by reinsertion of a clean used CIDR-B device on Day 15, for a period of 6 days, followed by an I/M injection of 0.5 mg ODB, 1 day after the device was removed (resynchrony, RS), while the other half were not resynchronised (no-RS). NDO/CL+ cows were alternately assigned to be either treated with 25 mg of the PG, dinoprost tromethamine, on Day 0 or left as untreated controls (Con). RESULTS: Resynchrony increased the percentage of cows detected in oestrus between Days 14 and 28 (212/282 = 75.2% vs 155/285 = 54.4%, for RS vs no-RS, respectively; p<0.001), but had no effect on conception rate to a service within the first 3 days of the mating period (146/397 = 36.8% vs 148/399= 37.1%, for RS vs no-RS cows, respectively; p>0.9), or to a service between Days 14 and 28 (84/159 = 52.8% vs 114/217 = 52.5%, for RS vs no-RS cows, respectively; p>0.9). Resynchrony increased the Day 28 pregnancy rate (264/432 = 61.1% vs 237/435 = 54.5%, for RS vs no-RS cows, respectively; p=0.03), but had no effect on the Day 56 or final pregnancy rates (p>0.1).Prostaglandin treatment of NDO/CL+ cows did not affect the percentage of cows detected in oestrus by Day 7 (43/106 = 40.6% vs 51/101 = 50.5%, for Con vs PG, respectively; p=0.15), or Day 28 (92/106 = 86.8% vs 91/101 = 90.1%, for Con vs PG, respectively; p>0.4). Treatment did not affect the Day 28 pregnancy rate (55/103 = 53.4% vs 54/98 = 55.1%, for Con vs PG, respectively; p>0.9), the Day 56 pregnancy rate (81/103 = 78.6% vs 74/98 = 75.5%, for Con vs PG, respectively; p>0.6), or the final pregnancy rate (98/103 = 95.1% vs 89/97 = 91.8%, for Con vs PG, respectively; p>0.3). CONCLUSIONS: Resynchrony of AA cows treated using the present protocol increased the proportion of non-pregnant cows detected in oestrus between Days 14 and 28 and increased the Day 28 pregnancy rate. This resynchrony protocol may be useful for increasing the proportion of the herd pregnant in the first month of the breeding programme, especially in herds that have a history of a low proportion of non-pregnant cows detected in oestrus between Days 14 and 28. There was no benefit in treating NDO/CL+ cows with PG compared to no treatment. KEYWORDS: Dairy cows, anoestrus, treatment, resynchrony, prostaglandin, progesterone, oestradiol benzoate.     

Changes in bovine luteal progesterone metabolism in response to exogenous prostaglandin F(2alpha)

An experiment was conducted to determine the effect of prostaglandin F2alpha (PGF2alpha) on luteal synthesis of progesterone (P4) and related progestins. Sixteen beef heifers were assigned in equal numbers to four groups in a 2x2 factorial arrangement of treatments. The experiment consisted of two levels of PGF2alpha analog (0 and 500 microg) and two levels of time (4 and 24 h after injection) of corpus luteum collection. All heifers were injected intravenously with saline (2 ml) or PGF2alpha (cloprostenol) on day 8 of the estrous cycle (estrus=day 0). Jugular blood was collected at 0, 1, 2, 3, 4 and 20, 21, 22, 23, and 24 h after injection. Resulting sera were analyzed for P4 by use of radioimmunoassay. Luteal tissue was analyzed by gas chromatography/mass spectrometry for P4, 20beta-hydroxyprogesterone, pregnenolone, and allopregnanolone (3beta-hydroxy-5alpha-pregnan-20-one). Treatment with PGF2alpha reduced serum concentrations of P4 as early as 1 h after injection (P<0.005) and steroid levels remained low over 24 h. Similarly, administration of PGF2alpha caused a decline in luteal P4 (P<0.005), 20beta-hydroxyprogesterone (P<0.10), and pregnenolone (P<0.05). In contrast, treatment with PGF(2alpha) caused an increase in luteal allopregnanolone over time (time x treatment interaction; P<0.05). These data are interpreted to suggest that PGF2alpha promotes conversion of P4 to the metabolite allopregnanolone.     

Hormonal regulation of oxytocin-induced prostaglandin F(2alpha) secretion by the bovine and ovine uterus in vivo

In long-term ovariectomized ewes and cows, endometrial oxytocin receptors rest at relatively high levels but oxytocin is unable to induce prostaglandin F(2alpha) release. A series of studies were carried out to investigate the roles of physiological levels of progesterone and estradiol in "activating" these receptors in terms of permitting oxytocin-induced prostaglandin F(2alpha) release. In long-term ovariectomized cows, treatment with progesterone, but not estradiol, resulted in the induction of responsiveness to oxytocin. This responsiveness appeared within 2 d of progesterone treatment, reached a maximum by 6 d and was maintained to Day 18. In ovariectomized ewes, while estradiol treatment did induce temporary responsiveness to oxytocin after 3 d of treatment, treatment with progesterone was required to induce sustained responsiveness that appeared by Day 9 of treatment and was maintained to Day 12. Measurement of endometrial receptors for oxytocin revealed a significant decline in oxytocin receptors by Day 6 of progesterone treatment when responsiveness to oxytocin was maximal, demonstrating that receptor concentrations were not a limiting factor. The most likely mechanism by which progesterone treatment induces responsiveness to oxytocin may be through the up regulation of post receptor signaling pathways and/or enzymes involved in prostaglandin synthesis.     

Fish meal supplementation alters uterine prostaglandin F2alpha synthesis in beef heifers with low luteal-phase progesterone

The objective of the current study was to evaluate the effect of omega-3 fatty acids in fish meal on mitigating uterine PGF2alpha synthesis in heifers with low luteal-phase concentrations of progesterone. Animals were individually fed a corn silage-based diet supplemented with fish meal (5% of DMI; n = 12) or corn gluten meal (6% of DMI; n = 13). Estrous cycles were synchronized using PGF2alpha beginning on d 25 of supplementation. Random heifers from each supplement group (n = 6 fish meal, and n = 7 corn gluten meal) were given three additional i.m. injections of PGF2alpha (25 mg) at 12-h intervals beginning at 0600 on d 3 after estrus to induce formation of corpora lutea that secrete lower concentrations of progesterone. Jugular blood samples were collected daily commencing on d 1 and continuing through d 16 of the estrous cycle to determine serum progesterone concentrations. Oxytocin was administered i.v. (100 IU) to heifers on d 16 after estrus to stimulate uterine PGF2alpha synthesis. Before statistical analyses, heifers were sorted to either normal or low luteal-phase progesterone as determined from serum progesterone on d 9 of the estrous cycle. After sorting, treatment groups consisted of 1) normal luteal progesterone + fish meal (n = 6); 2) low luteal progesterone + fish meal (n = 6); 3) normal luteal progesterone + corn gluten meal (n = 6); and 4) low luteal progesterone + corn gluten meal (n = 7). Serum concentrations of the PGF2alpha metabolite following oxytocin stimulation tended (P = 0.09) to be greater in heifers with low luteal-phase progesterone compared with heifers with normal luteal-phase progesterone. Fish meal supplementation mitigated this response in heifers with low luteal-phase progesterone (P < 0.05), but had no effect on heifers with normal luteal-phase progesterone. In conclusion, the omega-3 fatty acids in fish meal seem to decrease uterine PGF2alpha synthesis in heifers with low luteal-phase serum concentrations of progesterone.     

Correlations among body temperature,plasma progesterone,cortisol and prostaglandin F2alpha of the periparturient bitch

The results of this study suggest that, besides the irrelevant role of body temperature measurement to predict the impending parturition in the bitch, progesterone and 15-ketodihydroprostaglandin F2alpha plasma level records could be more suitable to detect the approaching whelping in this species. More interesting was the statistically significant substantial increase in body temperature beginning 12 h after the onset of parturition. Therefore, if any significant increase in body temperature is recorded at the end of pregnancy without the beginning of the expulsion of fetuses, it could indicate problems at parturition. In this study, cortisol levels increased significantly at the time of delivery and remained high 12 h after the beginning of parturition, decreasing within 36 h after the onset of whelping. 15-ketodihydro-prostaglandin F2alpha levels increased significantly 24 h before parturition and again at the onset of whelping. Progesterone levels decreased significantly, starting 24 h before the onset of whelping and remained low after delivery.     

The effect of estrogen, progesterone and prostaglandin F2 alpha on uterine contractions in seasonally anovulatory mares

Uterine contractions were studied in two experiments utilizing ultrasonography and seasonally anovulatory mares. A one-minute ultrasound scan was done to produce longitudinal real-time images of the uterine body and an overall uterine contractile activity score (0 = no or minimal activity to 4 = maximal activity) was assigned to each scan. In experiment 1, a two-hour uterine activity trial (one score every 10 minutes) was done in mares given a single injection of prostaglandin F2 alpha (PGF2 alpha group; n = 4) and in control mares (n = 4). There was no difference between the two groups over the two-hour trial (mean activity score averaged over the two-hour trial: PGF2 alpha group, 0.2; control group, 0.1). In experiment 2, 16 mares were randomly assigned to one of four groups: 1) controls (corn oil vehicle), 2) 1 mg estradiol 17 beta on days 0 to 9 and 100 mg progesterone on days 10 to 20 (E2--greater than P4 group), 3) 100 mg progesterone on days 0 to 20 (P4 group), and 4) 100 mg progesterone on days 0 to 9 and 1 mg estradiol 17 beta + 100 mg progesterone on days 10 to 20 (P4--greater than E2 + P4 group). Uterine activity was assessed for each mare daily. The day by group interaction was significant. Scores for the E2--greater than P4 group were greater on days 4 to 11 (P less than .05) than for the other three groups. From day 14 to 21, scores did not differ among the three steroid-treated groups (except on day 15), but the scores averaged over each steroid-treated group were greater for each day (P less than .1 or .05) than for the controls (except on day 17).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pregnancy and peripheral plasma progesterone levels in cows inseminated after synchronization of estrus with prostaglandin F2 alpha

Fifteen Holstein cows were treated with two doses of 25 mg of a prostaglandin F₂α (PGF₂α as dinoprost tromethamine) administered intramuscularly 11 days apart. The cows were then divided into three groups and inseminated either at 72, 80 or 72 and 96 hours after the second dose of PGF₂α. Thirteen cows ovulated after the second prostaglandin treatment. Eight cows were diagnosed pregnant by rectal palpation 42 days after insemination but only five calved. PGF₂α induced luteolysis in cows with active corpora lutea as evidenced by the dramatic decreases in the plasma progesterone concentrations after treatment. In contrast, following PGF₂α administration to cows in follicular or late luteal phase the concentrations of plasma progesterone either increased gradually or remained low for several days before increasing to maximal levels. The ovulatory rate after the two doses of PGF₂α11 days apart was high. However, the pregnancy rate after this treatment was influenced by other factors including abnormal ovarian function.     

Oxytocin stimulates secretion of prostaglandin F(2alpha) from endometrial cells of swine in the presence of progesterone

Oxytocin (OT) stimulates endometrial secretion of prostaglandin (PG) F(2 alpha) during corpus luteum regression in swine but there is differential responsiveness to OT among endometrial cell types. To determine if progesterone influenced responsiveness of luminal epithelial, glandular epithelial, and stromal cells to 100 nM OT during luteolysis in swine, cells were isolated from endometrium of 15 gilts by differential enzymatic digestion and sieve filtration on day 16 postestrus and cultured continuously in the presence of 0, 10 or 100 nM progesterone. For phospholipase C (PLC) activity and PGF(2 alpha) secretion, stromal cells were most responsive to OT (P<0.01) in the absence of progesterone, whereas luminal epithelial cells were unresponsive and glandular epithelial cells displayed an intermediate response to OT (P<0.09). Progesterone enhanced PLC activity linearly in glandular epithelial cells (P<0.05) and influenced it quadratically in stromal cells (P=0.05). The effect of OT and progesterone on PLC activity in luminal epithelial cells was not significant, and progesterone did not increase PLC activity in response to OT in any cell type. Culture in the presence of progesterone, enhanced PGF(2 alpha) secretion in response to OT in luminal epithelial cells (P<0.05) but not in glandular epithelial or stromal cells. Progesterone also increased overall PGF(2 alpha) release from glandular epithelial (P<0.05) and stromal cells (P<0.06) across both levels of OT treatment. These results indicate that progesterone enhanced PGF(2 alpha) secretion from luminal epithelial cells in response to OT and increased basal PGF(2 alpha) release from glandular epithelial and stromal cells.