Prostaglandin E-2 as a potential luteotrophic agent during early pregnancy in cattle

Heifers slaughtered on Day 18/19 of pregnancy had significantly higher (P less than 0.001) concentrations of PGE-2 (measured as its methyl oxime) in uterine flushings than did animals slaughtered on Days 6 or 12 of pregnancy, or on Days 6 or 12 of the oestrous cycle. In addition, concentrations were higher in the uterine horn ipsilateral to the corpus lueum on Days 12 (P less than 0.05) and 18/19 (P less than 0.01) than in the contralateral horn. Incubation of dispersed luteal cells for 3 h with LH (0.1 or 100 ng/ml) and/or PGE-2 (0.01-1000 ng/ml) in vitro showed no differences in basal progesterone production or in the responses to exogenous hormones between pregnant and non-pregnant cattle. However, low doses of PGE-2 (0.01-10 ng/ml) inhibited the stimulation of progesterone secretion by the lower dose of LH. These findings indicate that although PGE-2 can stimulate progesterone synthesis by luteal cells it may also have inhibitory effects, and therefore its role in pregnancy requires further definition.     

Plasma progesterone profiles and factors affecting embryo-fetal mortality following embryo transfer in dairy cattle

The relationship between plasma progesterone (P4) levels and embryo survival, and the value of P4 profiles for the selection of cattle embryo transfer recipients is still a matter of controversy. This study reports a comparison between lactating cows and heifers (n = 407) from a single dairy herd, after transfer of either fresh or frozen-thawed good quality embryos, of their ability to sustain embryo-fetal development to term. Plasma P4 concentrations on the day of estrus (Day 0 = D0), Day 4, Day 7 and on Day 21 were measured and related to embryo survival. Plasma P4 levels on Days 0, 4 and 7 were similar in recipients later found pregnant or open. Plasma P4 levels on Day 7 were significantly higher (P < 0.01) in heifers than in cows, but they were similar in pregnant and nonpregnant heifers and in pregnant and nonpregnant cows. Pregnancy rates for fresh and frozen-thawed embryos were higher in heifers than in cows, but the differences did not reach significance. However, the overall late embryonic mortality was significantly higher (P < 0.01) and the calving rate for frozen-thawed embryos was significantly lower (P < 0.05) in cows than in heifers. As expected, plasma P4 on Day 21 was significantly higher (P < 0.001) in pregnant than in nonpregnant recipients, but there was no difference between pregnant cows and pregnant heifers. Plasma P4 levels on Day 7 of recipients presumed pregnant on Day 21 and later found pregnant or nonpregnant were similar, but plasma P4 levels on Day 21 were significantly higher (P < 0.001) in pregnant than in nonpregnant recipients. The results of this study suggest that plasma P4 levels until the day of transfer, except for the rejection of recipients with abnormal luteal function, are of limited practical use for embryo transfer recipient selection. However, in lactating cows low plasma P4 values on Day 7 might negatively affect embryo survival, while in heifers this effect is not noticeable. Lactating cows are more prone to embryo loss than heifers, especially in the case of frozen-thawed embryos; this is associated with a lower competence of the corpus luteum at Day 7.     

Interrelationships between progesterone, 13,14-dihydro-15-keto PGF-2 alpha (PGFM) and LH in cyclic and early pregnant cows

Plasma progesterone and LH secretion patterns were examined in 18 mature dairy cows during the oestrous cycle and after insemination. Blood samples were collected every 15 min for 8 h per day on Days 3, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20 and 21 of the oestrous cycle, then, in the same cows, at the same times during early pregnancy. PGF-2 alpha secretion rates (as determined by plasma PGFM concentrations) were also monitored on Days 14, 16 and the day of, or equivalent to, luteal regression. Mean daily plasma progesterone concentrations were similar until Day 16 in cyclic and pregnant cows, after which values in non-pregnant animals declined. Regression analysis indicated that progesterone concentrations were best described by a quadratic expression with fitted maximum values on Day 13 in non-pregnant animals but values increased linearly over the whole period to Day 21 in pregnant cows. The frequency, amplitude and area under the curve of LH episodes showed no significant differences between cyclic and pregnant animals. In pregnant cows, the amplitude and area under the curve of progesterone episodes increased linearly between Days 8 and 21, although no such increase occurred in cyclic cows. Low-level PGFM episodes were present in cyclic and pregnant cows on Days 14 and 16 after oestrus, and high amplitude episodes occurred in non-pregnant cows during luteal regression. Pregnant cows showed a significant depression of the amplitude, but not the frequency of episodes at the expected time of luteal regression. These results confirm that the corpus luteum of pregnancy secretes an increasing amount of progesterone per se and per unit of LH until at least Day 21 after mating. They further suggest that the corpus luteum of the cyclic cow may experience small episodes of PGF-2 alpha and be subjected to initial degenerative changes by Day 14 after oestrus, some time before the onset of definitive luteolysis.     

Luteotrophic influence of early bovine embryos and the relationship between plasma progesterone concentrations and embryo survival

This study was carried out to evaluate the luteotrophic influence of early (before Day 7 as well as after Day 7; Day 0=estrus) bovine embryos and the relationship between plasma progesterone (P4) concentrations and embryo survival. Virgin Holstein dairy heifers (n=325) from a single herd were randomly allocated to be nonbred, bred by artificial insemination (AI) or by embryo transfer (ET). Bred heifers were either treated with 1500 IU human chorionic gonadotrophin (hCG) on Day 7 of the estrous cycle or received no hCG treatment. Plasma P4 concentrations on Days 0, 5, 7, 10, 13, 15, 17, 19 and 21 were similar in pregnant AI- and ET-bred heifers and, this was observed in both hCG-treated and untreated females. Nonbred, AI- and ET-bred nonpregnant heifers (both hCG-treated and untreated) presented similar plasma P4 concentrations. Plasma P4 concentrations of pregnant heifers significantly deviated from those of nonpregnant and nonbred heifers on Day 17. In hCG-treated heifers, plasma P4 concentrations and Day 28 pregnancy rate were significantly higher in females with an induced accessory corpus luteum (CL) than in those females without an induced accessory CL. Treatment with hCG, although inducing the formation of accessory CL and significantly increasing plasma P4 concentrations had no significant effect on Day 28 pregnancy rate. In conclusion, this study does not support the existence of any peripherally detectable luteotrophic influence from early embryos (Days 5-7). Plasma P4 was only significantly related to embryo survival on Day 17, the time of expected onset of luteolysis.     

Luteal luteinizing hormone receptors during the postovulatory period in the mare

Changes in serum luteinizing hormone (LH) and progesterone concentrations, number of luteal unoccupied LH receptors, receptor affinity constants, luteal weights and luteal progesterone concentrations were determined during the postovulatory period in the mare. The number of unoccupied LH receptors and receptor affinity was less during the early (Days 1-4) and late [Day 15 through 3rd day after start of corpus luteum (CL) regression] luteal phases than during the mid-luteal (Days 9-14) phase of the postovulatory period (P less than 0.01). The number of LH receptors per CL increased 21-fold (P less than 0.001) from Day 1 to Day 14. Receptor affinity increased 5-fold (P less than 0.001) from Day 1 to Day 13. Receptor number was highly correlated with receptor affinity (P less than 0.01) and both were highly correlated with serum and luteal progesterone (P less than 0.01). During regression of the CL, the number of LH receptors and receptor affinity decreased concomitantly with serum and luteal progesterone. Morphologically, luteal cell development and degeneration correlated with the change in receptor numbers, affinity constants and luteal and serum progesterone concentrations. Receptor number and affinity, luteal weight and serum and luteal progesterone concentrations did not differ between the CL from multiple ovulations. Random variations in the data observed between CL from multiple and single ovulations suggested that CL from the two groups were not different in structure and function. In summary, the above results suggest that major factors in regulation of progesterone secretion and maintenance of the equine CL are changes in the number of LH receptors and the affinity constants throughout the postovulatory period.     

Luteotrophic effect, growth and survival of whole versus half embryos and, their relationship with plasma progesterone concentrations of recipient dairy heifers

This prospective and randomised experiment was designed to compare the luteotrophic effect of whole versus half embryos and, to evaluate the relationship between the plasma progesterone (P4) profiles and the rates of early embryonic (from Days 7 to 25), late embryonic (Days 25-42) and foetal (Days 42-63) mortalities of whole and half embryo recipients. Within a single herd, 188 virgin, healthy, cyclic, reproductively sound, with adequate body condition score, Holstein dairy heifers were randomly allocated to receive one whole or one half embryo on Day 7 of the oestrous cycle (Day 0=estrus). In each embryo-transfer (ET) group, half of the recipients were treated with a CIDR (controlled internal drug releasing device) between Days 7 and 19. Pregnancy was evaluated by ultrasound on Days 25, 42 and 63 and plasma P4 profiles were obtained until Day 63 of pregnancy. CIDR-treated and untreated heifers had similar pregnancy rates on Days 25, 42 and 63 and, embryo size on Day 42 was also similar in treated and untreated recipients. Therefore, CIDR treatment failed to promote growth and survival of half and whole embryos. Half embryos presented a significantly higher rate of early and late embryonic mortality than whole embryos. In contrast, foetal mortality was similar in whole and half embryos and, this was coincidental to a similar embryo size on Day 42. Therefore, half embryos exhibited a compensatory growth until Day 42, irrespective of CIDR treatment, after which they presented a similar survival rate to that of whole embryos. Half embryo-derived pregnancies presented significantly lower plasma P4 concentrations on Day 25 than whole embryo-derived pregnancies, suggesting that this lower luteotrophic effect of half embryos could be related to their higher rate of late embryonic mortality. No significant relationship between the early luteal P4 concentrations and embryo survival was observed in whole and half embryo recipients. The first detectable luteotrophic effect of embryonic origin was observed on Day 14 and no detectable second luteotrophic effect was observed until Day 63 of pregnancy. Treatment with CIDR significantly increased plasma P4 concentrations during treatment but induced a significant decrease after removal of the device, suggesting that secretion of luteotropins was downregulated in the course of treatment.     

Effects of antibiotics and medium supplements on steroidogenesis in cultured cow luteal cells

Corpora lutea were removed from regularly cycling dairy cows, dissociated with collagenase and cultured for 8 or 10 days in Ham's F-12 medium. In Exp. 1 treatment with insulin, or an insulin-transferrin-selenium combination (ITS), increased progesterone production from basal levels on Day 4 of culture to 234% (P less than 0.01) above controls on Day 10. LH alone increased progesterone production 45% above controls on Day 10 (P greater than 0.05). When LH was combined with insulin or ITS, progesterone production was stimulated to an average of 1802% (P less than 0.01) above controls on Day 10 of culture. Transferrin or selenium without insulin did not allow LH to stimulate progesterone synthesis. In Exp. II, LH alone or LH plus gentamicin or penicillin-streptomycin increased progesterone production from basal levels on Day 2 steadily to an average of 468% (P less than 0.01) above controls (no antibiotics) by Day 8 of culture. The addition of amphotericin-B, alone or in combination with the other antibiotics, inhibited all LH-stimulated progesterone synthesis, but did not affect basal progesterone levels. We conclude that insulin is essential for maximal steroidogenesis in a bovine luteal cell culture system, and that LH-stimulated progesterone production is inhibited in the presence of amphotericin-B, but is not inhibited by gentamicin or penicillin-streptomycin. The elimination of amphotericin-B, coupled with the addition of insulin to the cell culture system increased the responsiveness of the cells to LH. These culture conditions represent the first report in which LH increased total progesterone production for 10 days, maintaining luteal function in a chemically-defined culture system.     

Termination of pregnancy and induction of premature luteolysis by the antiprogestagen, mifepristone, in dogs

Five pregnant beagle bitches were treated with 2.5 mg mifepristone/kg body weight, twice a day, for 4.5 days starting at Day 32 of gestation. Results of fetal ultrasonography and assay of serum progesterone concentrations every 2-4 days were compared to those in 5 control bitches. Mifepristone resulted in a premature (P less than 0.01) termination of pregnancy (36 +/- 1 vs 65 +/- 1 days), without side effects. The antiprogestagen also caused progesterone to decline to less than 1 ng/ml by Day 40-45 after the preovulatory LH peak (vs 64-67 days in controls) and reduced (P less than 0.05) mean concentrations on Days 34-50 (2.2 +/- 0.5 vs 6.3 +/- 0.3 ng/ml). The results suggest that antiprogestagen therapy is a safe means to terminate unwanted pregnancy in dogs, and that luteal function in pregnant bitches is dependent on luteotrophic support that is blocked by antiprogestagen treatment, directly or indirectly, due to termination of pregnancy.     

Changing responsiveness of luteal cells of the marmoset monkey (Callithrix jacchus) to luteotrophic and luteolytic agents during normal and conception cycles

Dispersed marmoset luteal cells were incubated for 2 h and progesterone production measured after exposure to hCG, cloprostenol, dibutyryl cAMP, PGF-2 alpha, PGF-2, adrenaline or melatonin. The cells were studied on Days 6, 14 and 20 after ovulation in conception and non-conception cycles. Luteal cells from Day 14 non-pregnant marmosets were compared with human luteal cells taken in the mid-luteal phase. All the treatments stimulated progesterone production including cloprostenol, which is luteolytic when administered to the marmoset in vivo, but the degree of response varied with the stage of the cycle or pregnancy and between marmoset and human luteal cells. In the marmoset, overall analysis of the effect of the treatments showed that, on Day 6 after ovulation, there was no significant effect of any of the treatments in cells from pregnant or non-pregnant animals. In contrast, luteal cells from non-pregnant animals on Day 14 showed a significant response to the treatments (F (8,41) = 2.79, P less than 0.0145) whereas cells from pregnant Day-14 animals were responsive; in cells from pregnant animals, the control production of progesterone was high and already equivalent to the levels stimulated by the treatments. By Day 20, cells from pregnant animals produced lower control concentrations of progesterone than did those on Day 14 and there was a significant overall effect of the treatments (F (8,33) = 3.78, P less than 0.003). These results show that the marmoset CL gains responsiveness to treatment between Days 6 and 14 after ovulation in the non-pregnant cycle. In pregnancy, on Day 14, 2 days after attachment of the embryo, the high control concentrations of progesterone and absence of response to treatment suggest that an embryo message may have affected the CL, providing an endogenous stimulus.     

Low peripheral progesterone and late embryonic/early fetal loss in suckled beef and lactating dairy cows

Pregnancy failure during placentation in lactating dairy cows was associated with low concentrations of serum progesterone. Beef cows have greater serum progesterone and less pregnancy failure. Experiment 1 determined that reduction of serum progesterone affected late embryonic/early fetal loss in suckled beef cows. Cows (n = 40) received progesterone from two new or used controlled internal drug releasing devices, replaced every 5 d, beginning on Day 28 of gestation (mating = Day 0); CL were enucleated on Day 29. Retention of pregnancy was 77% in treated cows and 97% in 78 control cows (P < 0.05). Experiment 2 determined how pregnant, lactating dairy cows with high or low progesterone concentrations during Days 28-34 differed in luteal function or in serum progesterone during replacement therapy. Luteal tissue from such cows was assayed for progesterone and expression of mRNA for genes of endothelin and prostaglandin (PG) systems. Secretion of progesterone and prostaglandins by dispersed luteal cells was determined during incubation with LH, endothelin-1, or arachidonic acid. Neither luteal progesterone nor mRNAs for endothelin or prostaglandin systems differed. Endothelin-1 inhibited secretion of progesterone more (P < 0.05) in luteal cells from cows with low versus high serum progesterone, when incubated with arachidonic acid. Secretion of prostaglandin F₂α was increased and that of 6-keto-PGF₁α decreased by endothelin-1 in vitro. Serum progesterone during replacement was lower (P < 0.05) for cows with low than high serum progesterone at lutectomy. Thus, clearance, more than luteal production, determined peripheral progesterone in pregnant, lactating dairy cows.     

A morphometric analysis of the corpus luteum of the cow during the estrous cycle and early pregnancy

Corpora lutea were collected from cows on Days 6, 8, 10, 12, 14, 16, 18 and 19 of the estrous cycle and early pregnancy (n=2/d) and were examined by light microscopy. Mean lutein cell diameter was significantly (P<0.05) greater in pregnant than in cyclic cows on Days 6, 8, 10, 12, 16, 18 and 19 (cyclic versus pregnant: Day 6: 13.9 +/- 0.22 vs 14.9 +/- 0.24; Day 8: 13.8 +/- 0.20 vs 15.4 +/- 0.2; Day 10: 14.8 +/- 0.24 vs 17.4 +/- 0.24; Day 12: 13.2 +/-0.25 vs 17.9 +/- 0.31; Day 16: 13.9 +/- 0.28 vs 16.5 +/- 0.31; Day 18: 13.0 +/- 0.22 vs 16.5 +/- 09.36, and Day 19: 15.0 +/- 0.23 vs 17.6 +/- 0.33 mum, respectively). The distribution of cell sizes was leptokurtotic throughout the estrous cycle and the first 10 d of pregnancy, but tended towards bimodality after Day 14 of pregnancy. The proportion of lutein cell cytoplasm occupied by vacuoles was lower in pregnant than in cyclic cows from the 12th day post estrus, but there was a marked (P<0.05) increase in vacuolation of cells from cows undergoing luteolysis. Stainable intercellular collagen was also less abundant in pregnant than cyclic cows from the 12th day post estrus. The higher rate of progesterone secretion of pregnant, compared with cyclic cows may be attributed to the greater numbers and greater contribution to luteal mass of large lutein cells in the corpus luteum of pregnancy.     

The postpartum induced corpus luteum: functional differences from that of cycling cows and the effects of progesterone pretreatment

Anestrous postpartum (PP) Hereford cows (n = 41) were used to compare corpora lutea (CL) from gonadotropin-releasing hormone (GnRH)-induced ovulation with CL from cycling cows. Postpartum cows were injected i.m. daily with 100 mg progesterone (P4) or oil on Days 25 through 28 PP and then given 200 micrograms GnRH i.m. on Day 30 PP. Corpora lutea were removed from one-half of the PP cows in the oil- and P4-treated groups 6.5 days after GnRH injection, and from the cycling cows 7 days after estrus. Intact PP cows were used to evaluate cycle length. Blood was collected daily from all PP cows from Day 25 PP through luteectomy and on Days 9, 11, and 13 post-GnRH from the oil- and P4-intact cows to determine short (SHORT) versus normal (NORM) luteal phases. Cycling cows were bled daily from estrus until CL removal NORM PP cows had higher (P less than 0.001) P4 levels than did SHORT PP cows from Day 7 through Day 13 post-GnRH, and more (P less than 0.05) P4-intact cows were NORM compared with oil-intact cows (45.5% vs. 14.3%, respectively). Corpora lutea from cycling cows were heavier (P less than 0.05) and had a higher luteinizing hormone (LH) receptor concentration (P less than 0.05), but CL P4 concentration did not differ from PP cows. Corpora lutea weight, LH receptor and P4 concentration, and in vitro P4 production were similar in the oil-and P4-treated PP cows. NORM cows had heavier CL (P less than 0.05) than SHORT cows, although P4 content and LH receptor concentration did not differ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma and luteal concentrations of oxytocin in cyclic and early-pregnant cattle.

Concentrations of oxytocin were measured in corpora lutea obtained from heifers throughout the oestrous cycle and first 30 days of pregnancy. Values were low during the first 3 days of the cycle (less than 250 ng/g tissue), increasing to 1312 ng/g by Day 4. Values then further increased up to a maximum of 2344 ng/g on Day 12. Concentrations were similar in cyclic and pregnant animals throughout the midluteal phase and were maintained at approximately 1500 ng/g until the 18th (cyclic cows) or 19th (pregnant cows) day after oestrus, when they were again low. Values subsequently remained less than 250 ng/g in pregnant cattle. Concentrations of oxytocin in jugular venous plasma of cyclic (n = 5) and pregnant (n = 4) cows were measured in samples collected every 15 min for 8 h on Days 14, 16, 18 and 19 after oestrus. There were no significant differences in mean concentrations (range: 2.5-4.7 pg/ml) or in the number, frequency or area under the curve of episodes between either cyclic and pregnant animals, or between days. Mean basal concentrations were higher on Day 16 than on Day 14 (P less than 0.05), values on Days 18 and 19 being intermediate. These findings suggest that the corpus luteum contains a finite amount of releasable oxytocin, which is exhausted by Day 18-19 after oestrus, whether or not pregnancy occurs, and that there is no further accumulation of oxytocin in the animal during early pregnancy. The contribution of luteal oxytocin to jugular venous concentrations appears to be less than in sheep, in which values in the jugular vein closely parallel those within the corpus luteum.     

Relationships between insulin and glucose metabolism and pituitary-ovarian functions in fasted heifers

The effects of fasting between Days 8 and 16 of the estrous cycle on plasma concentrations of luteinizing hormone (LH), progesterone, cortisol, glucose and insulin were determined in 4 fasted and 4 control heifers during an estrous cycle of fasting and in the subsequent cycle after fasting. Cortisol levels were unaffected by fasting. Concentrations of insulin and glucose, however, were decreased (p less than 0.05) by 12 and 36 h, respectively, after fasting was begun and did not return to control values until 12 h (insulin) and 4 to 7 days (glucose) after fasting ended. Concentrations of progesterone were greater (p less than 0.05) in fasted than in control heifers from Day 10 to 15 of the estrous cycle during fasting, while LH levels were lower (p less than 0.01) in fasted than in control heifers during the last 24 h of fasting. Concentrations of LH increased (p less than 0.01) abruptly in fasted heifers in the first 4 h after they were refed on Day 16 of the fasted cycle. Concentrations (means +/- SEM) of LH also were greater (p less than 0.05) in fasted (11.2 +/- 2.6 ng/ml) than in control (4.7 +/- 1.2 ng/ml) heifers during estrus of the cycle after fasting; this elevated LH was preceded by a rebound response in insulin levels in the fasted-refed heifers, with insulin increasing from 176 +/- 35 pg/ml to 1302 +/- 280 pg/ml between refeeding and estrus of the cycle after fasting. Concentrations of LH, glucose and insulin were similar in both groups after Day 2 of the postfasting cycle. Concentrations of progesterone in two fasted heifers and controls were similar during the cycle after fasting, whereas concentrations in the other fasted heifers were less than 1 ng/ml until Day 10, indicating delayed ovulation and (or) reduced luteal function. Thus, aberrant pituitary and luteal functions in fasted heifers were associated with concurrent fasting-induced changes in insulin and glucose metabolism.     

Difference in luteinizing hormone response to an opioid antagonist in beef heifers and cows

In three experiments, we examined endogenous opioid inhibition of luteinizing hormone (LH) secretion during the bovine estrous cycle. An increase in serum LH in response to the opioid antagonist naloxone (Na; 1 mg/kg i.v.) was the criterion for opioid inhibition. Estrous cycles were synchronized via prostaglandin administration. In Experiment 1, mean serum LH was not different during the luteal phase in yearling heifers (n = 6/group) at Hour 1 after Nal (2.1 ng/ml) compared to controls (1.8 ng/ml). However, LH peak amplitude was increased (p less than 0.05) in the Nal compared to the control group. Serum LH was increased (p less than 0.01) during the follicular phase in heifers at Hour 1 post-Nal compared to controls (4.7 and 3.5 ng/ml, respectively). Again, Nal administration was followed by increased (p less than 0.05) LH pulse amplitude compared to control. In Experiment 2, no effect of Nal upon serum LH was detected in cows (n = 9) during proestrus, metestrus, midluteal and late luteal portions of the estrous cycle. In Experiment 3, the LH response to Nal was examined simultaneously in yearling heifers and cows (n = 5/group) during the luteal and follicular phases. Serum LH increased (p less than 0.001) during Hour 1 post-Nal in heifers compared to cows during the follicular (3.4 vs. 1.7 ng/ml) but not during the luteal phase. LH pulse amplitude also increased (p less than 0.05) during Hour 1 post-Nal in heifers compared to cows during the luteal (2.5 vs. 1.1 ng/nl and follicular (2.5 vs. 1.3 ng/ml) phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of subpopulations of luteal cells obtained from cyclic and superovulated ewes

Peripheral blood samples were collected daily (Days 1-10 after ovulation) and analysed for progesterone content. Luteal tissue was collected on Day 10 after the LH surge, or Day 10 after hCG injection from cyclic and superovulated ewes, respectively. The tissue was enzymically dispersed and an aliquant was utilized for measurement of cell diameters, and staining for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase activity (3 beta-HSD). The remaining cell preparation was separated into small (10-22 micron) and large (greater than 22 micron) cell fractions by elutriation. Small and large cell suspensions were incubated (37 degrees C, 2 h) in the presence or absence or ovine LH (100 ng/ml) or dbcAMP (2 mM) and progesterone content of the medium was measured. Superovulation did not affect circulating progesterone concentrations, when expressed per mg luteal tissue recorded; basal progesterone production by small or large luteal cells; the unresponsiveness of large luteal cells to ovine LH or dbcAMP; the ratio of small:large cells recovered by dissociation the mean diameter of total cells; or the mean diameter of large cells. However, the mean cell diameter and LH stimulation of progesterone production by small cells were greater (P less than 0.05) in luteal tissue collected from superovulated than in that from cyclic ewes. These differences appear to be an amplification of basic function. Therefore, we conclude that corpora lutea obtained from superovulated ewes can be used to study functional aspects of small and large cells.     

Evidence for dopaminergic regulation of prolactin and a luteotropic complex in the ferret

The role of dopaminergic agents in prolactin (Prl) release and the luteotrophic role of Prl and luteinizing hormone (LH) were investigated in pseudopregnant female ferrets. A single injection of the dopamine antagonist pimozide (0.63 mg/kg) resulted in a tenfold elevation of plasma Prl in anestrous females. Subcutaneous injection of pimozide on alternate days from Day 2 through Day 16 of pseudopregnancy elevated both Prl and progesterone levels. Daily treatment with the dopamine agonist 2 alpha-bromoergocryptine (bromocriptine, 4 mg/kg), from Day 2 through Day 16 of pseudopregnancy lowered levels of both plasma Prl and progesterone. Neither pimozide nor bromocriptine had a direct effect on progesterone secretion by luteal cells in vitro. Daily intraperitoneal administration of a monoclonal antibody against gonadotropin-releasing hormone from Day 2 through Day 10 of pseudopregnancy lowered both plasma LH and progesterone, but had no effect on plasma Prl concentrations. Daily administration of equine antisera against bovine LH or 100 IU of human chorionic gonadotrophin to pseudopregnant ferrets lowered progesterone levels. It is concluded that Prl release is influenced by dopaminergic compounds, and both Prl and LH are required for luteal maintenance in the ferret.     

Effect of rate of change of luteinizing hormone concentration on in-vitro progesterone secretion within rat corpora lutea during differentiation.

Immature rats were injected with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Ovaries were removed 0, 2, 5 or 8 days after hCG and either prepared for morphometric analysis or perifused with 0, 5 or 30 ng luteinizing hormone (LH)/min. In a second study, ovaries were removed on Day 2 or 8 and perifused with 0.1 mg 8-br-cyclic adenosine 5'-phosphate/ml (8-br-cAMP). On Day 0, the granulosa cells of the preovulatory follicles were small (53 +/- 0.5 microns2) with a cytoplasmic to nuclear (Cy:Nu) ratio less than or equal to 1.5. By Day 2, corpora lutea (CL) were present and composed of 95% small luteal cells (diameter less than 125 microns2, Cy:Nu greater than or equal to 3.0) and 5% large luteal cells (diameter greater than 125 microns2, Cy:Nu ratio greater than or equal to 3.0). The percentage of large luteal cells increased to 36 +/- 7% by Day 5, suggesting that they are derived from a select population of small luteal cells. Basal progesterone secretion increased from 38 +/- 5 on Day 0 to 1010 +/- 48 pg/mg/ml on Day 8. The rate of 5 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8; 30 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8, but not on Day 5; 8-br-cAMP stimulated progesterone secretion on both Days 2 and 8. These data demonstrate that once granulosa cells are induced to luteinize they lose their capacity to secrete progesterone in response to 5 ng LH/min and do not regain their responsiveness to LH rate until they completely differentiate. The loss of this LH responsiveness appears to be due to an inability to stimulate sufficient intracellular cAMP concentrations, since cAMP stimulates progesterone secretion on both Days 2 and 8.     

Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers.

This study was conducted to compare the superovulatory (SOV) response of dairy cows (n=172) and heifers (n=172), with two SOV treatments started at the mid-luteal-phase of the estrus cycle. Donors were randomly treated either with equine chorionic gonadotrophin (eCG) plus neutra-eCG serum (eCG+N group, n=167) or follicle stimulating gonadotrophin (FSH-P group, n=177).No significant differences were observed among groups in the percentage of superovulatory responsive donors (SR donors; corpora lutea (CL) >/=2), the mean number of total ova, fertilized ova and viable embryos recovered. Cows yielded significantly less total ova and less fertilized ova (P<0.05) and tended to yield less viable embryos (P<0.06) than heifers.Plasma progesterone (P4) concentrations (n=135 donors) on the day of PGF(2alpha) (PGF) injection and on the day of SOV estrus were significantly higher (P<0.01) in eCG+N than in FSH-P donors and, the increase between those 2 days was also significantly higher (P<0.05) in group eCG+N than in group FSH-P, suggesting a higher luteotrophic effect of eCG than FSH-P. SR donors had P4 levels significantly higher (P<0.001) than non-SR donors only on day 5 after the SOV estrus and on the day of embryo recovery. Plasma P4 concentrations at 5 days after the SOV estrus and at embryo recovery correlated significantly (r=0.76, P<0.001).Heifers had significantly higher P4 levels than cows at gonadotrophin injection (P<0.01), PGF injection (P<0.001), 5 days (P<0.01) and 7 days (P<0.001) after the SOV estrus. At day 7 after the SOV estrus, P4 concentrations per ova recovered were significantly higher in heifers than in cows (P<0.01). The increase of plasma P4 per ova recovered, between days 5 and 7 after the SOV estrus, was significantly (P<0.01) higher in heifers than in cows. Also, the increase of plasma P4 between injections of gonadotrophin and PGF was significantly higher (P<0.05) in heifers than in cows.These results suggest that heifers have higher plasma P4 concentrations at diestrus (either before or after the SOV treatment) and this is associated with a higher embryo yield and quality, as compared to lactating cows. These higher plasma P4 concentrations reflect not only differences in ovulation rate as well as the competence of the corpus luteum, which is potentialized by gonadotrophin stimulation.     

Uterine oxytocin receptors in cyclic and pregnant cows.

Binding of [3H]oxytocin to uterine subcellular preparations ('oxytocin receptor concentrations') was measured in uterine tissue of heifers and multiparous dairy cows at various stages of the oestrous cycle and during early pregnancy. A method for the assay of ovine uterine oxytocin receptors was optimized for use on bovine tissue. Oxytocin receptor concentrations were increased in cyclic animals around the period of luteolysis and oestrus, rising on Day 15 in endometrium and on Day 17 in myometrium while pregnant animals showed no comparable rise. Receptor concentrations then declined on Day 3 after oestrus in myometrium and on Day 5 in endometrium. Some cyclic animals did not show the expected rise in receptors in the late luteal phase; these animals had abnormally high progesterone concentrations for this stage of the cycle. In animals slaughtered on Day 18 after oestrus and/or insemination which had low oxytocin receptor levels, plasma progesterone concentrations were consistently high; while all animals showing the late luteal phase elevation in receptor values had low progesterone concentrations. Oxytocin receptor and progesterone concentrations were negatively correlated (P less than 0.05). These data support the hypothesis that oxytocin receptor level is a key factor in the process of luteolysis in cattle and that in pregnancy there is suppression of uterine oxytocin receptor at the expected time of luteolysis. We suggest that uterine oxytocin receptor levels are partly controlled by circulating steroid hormones and are suppressed during early pregnancy.