Influence of noradrenaline on progesterone synthesis and post-translational processing of oxytocin synthesis in the bovine corpus luteum

Noradrenaline (NA) influences secretory function of the bovine corpus luteum (CL), stimulating secretion of progesterone and ovarian oxytocin (OT). To study whether NA is able to stimulate progesterone synthesis and to affect post-translational OT processing, different doses of NA alone or in combination with different doses of OT were added to bovine CL slices from 8 to 13 d of the estrous cycle. To determine which receptors NA affects, and if dopamine (DA) also affects CL function, we used NA or DA combined with a beta-antagonist (propranolol). The results indicated that NA stimulates both luteal progesterone and OT content; furthermore, it increased the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA), terminal enzymes in synthesis of these 2 hormones. The stimulating effect of NA was inhibited by propranolol and by pre-treatment of CL slices with high OT doses. Post-translational processing of OT synthesis by PGA activation was also stimulated by DA, but this effect was inhibited by beta-receptor blocker. Thus DA acts in CL as a NA precursor. In conclusion, it can be assumed that the noradrenergic system affects CL secretory function on different levels of regulation. Furthermore, a high concentration of OT in CL prevents NA from activating PGA and thus decreases post-translational OT synthesis.     

Contractility of the ovarian vascular bed during the oestrous cycle and early pregnancy in gilts

The vasoconstrictor activity of the ovarian vascular bed in vitro was investigated during the oestrous cycle and early pregnancy. Gilts were killed during the follicular phase (Days 20 to +1; N = 5) or luteal phase (Days 11 to 13; N = 4) of the oestrous cycle, or on Day 13 of pregnancy (N = 5). Immediately before death, a sample of vena cava blood was obtained for determination of progesterone and oestrogen (oestrone and oestradiol-17 beta) concentrations. One ovary was removed, cannulated, perfused in vitro, and subjected to 10-min infusions of saline (vehicle control) and noradrenaline. Vasoconstriction was provoked by electrical stimulation at the end of each infusion. Ovaries from luteal-phase gilts exhibited greater (P less than 0.01) vasoconstriction than did ovaries from follicular-phase and pregnant gilts at the end of saline and noradrenaline infusions. The oestrogen to progesterone ratio was less (P less than 0.01) for luteal-phase and pregnant than for follicular-phase gilts. Vasoconstriction was negatively correlated (r = -0.99, P less than 0.01) with the oestrogen to progesterone ratio in systemic blood of gilts during the oestrous cycle but not during early pregnancy (r = +0.39, P greater than 0.10), possibly due to an effect of the conceptuses.     

Oxytocin (OT) action in uterine tissues of cyclic and early pregnant gilts: OT receptors concentration, prostaglandin F(2)alpha 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(2)alpha 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 (20IU; i.v.) for three consecutive days starting on days 14 and 15 of the oestrous cycle and plasma PGF(2)alpha metabolite-13,14-dihydro-16-keto PGF(2)alpha (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).     

Peripheral plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2α and progesterone around luteolysis and during early pregnancy in the goat

Peripheral plasma concentrations of 13,14-dihdyro-15-keto-prostaglandin F_2α (PGFM) and progesterone were determined during both luteolysis in the oestrous cycle and early pregnancy in four goats. Luteal regression, characterised by decreasing progesterone concentrations, began on day 12 or 13. PGFM concentrations showed a pulsatile pattern around this time, with peak concentrations increasingly markedly as progesterone levels fell and oestrus approached. During early pregnancy progesterone concentrations did not fall after day 12 and no marked elevation of PGFM above basal values of 50–150 pg/ml was detected.     

Uterine blood flow of cows during the oestrous cycle and early pregnancy: effect of the conceptus on the uterine blood supply.

Blood flow to each uterine horn of cows during the oestrous cycle and early pregnancy was determined daily by use of electromagnetic blood flow probes placed around both middle uterine arteries. The pattern of blood flow to uteri of pregnant and non-pregnant cows was similar until Day 14 after mating or oestrus. Between Days 14 and 18 of pregnancy blood flow to the uterine horn containing the conceptus increased (P less than 0.01) 2- to 3-fold, whereas blood flow to the non-gravid uterine horn in these cows remained constant. No corresponding increase in blood flow to the uterine horn ipsilateral to the ovary bearing the CL was observed in non-pregnant cows during this 4-day period. By Day 19 of pregnancy, blood flow to the gravid uterine horn had returned to a level similar to that observed on Day 13. Blood flow to both uterine horns of pregnant cows remained constant from Days 19 to 25 and then increased to the gravid horn (P less than 0.01) markedly until Day 30 whereas blood flow to the non-gravid horn remained low. Uterine blood flow during the oestrous cycle of non-pregnant cows was positively correlated (P less than 0.01) with systemic concentrations of oestradiol and the ratio of oestradiol (pg/ml) to progesterone (ng/ml). There was no association between oestradiol concentrations and blood flow to the gravid uterine horn. These data indicate local control of uterine blood flow by the bovine conceptus which may function to create optimal conditions for the continuation of pregnancy.     

Faecal steroid metabolites for non-invasive assessment of reproduction in common warthogs (Phacochoerus africanus), red river hogs (Potamochoerus porcus) and babirusa (Babyrousa babyrussa)

The objectives of this study were to analyse faecal steroid metabolites in African and South East Asian pig species kept in European zoos. Species studied were the warthog (Phacochoerus africanus), the red river hog (Potamochoerus porcus) and the babirusa (Babyrousa babyrussa). Faecal samples were collected 1-3 times per week from non-pregnant and pregnant captive female warthogs (n = 9), red river hogs (n = 7) and babirusas (n = 5). Enzyme-immunoassays for faecal progesterone, androgen, and oestrogen metabolites, were tested for their ability to determine follicular and luteal phases. In all three species, oestrous cycles could be monitored with 20alpha-OH- and 20-oxo-pregnane assays. In contrast, oestrogens and androgens were not useful in characterising follicular activity during the oestrous cycle in any species. Faecal 20alpha-OH- and 20-oxo-pregnane values were significantly correlated. Faecal pregnane concentrations revealed species-specific differences. Luteal phase values of 20alpha-OH-pregnanes were considerably higher than 20-oxo-pregnanes; 20alpha-OH-pregnanes were in the range of 3-10 microg/g in warthogs and red river hogs, whereas concentrations were 30-200 microg/g faeces in the babirusa. Regular oestrus cycles had a length of about 35 days in all three species studied. Results indicated a seasonal influence on the occurrence of reproductive cycles in the warthog with anoestrous periods in the European summer. The red river hog was found to be a seasonal and poly oestrous breeder; oestrus cycles started by January and continued until summer. In contrast, the babirusa showed non-seasonal ovarian cyclicity. In pregnant red river hogs, progesterone metabolites were comparable to luteal phase values of the oestrous cycle during the first 3 months of gestation, but did further increase during the last month of pregnancy. Oestrogens and 17-oxo-androstanes were significantly elevated during the second half of gestation. In summary, the reproductive biology of three exotic pig species was studied using non-invasive faecal steroid analysis and these methods were used for comparative investigations of oestrous cycles, pregnancy and seasonality.     

Weight, composition, mitosis, cell death and content of progesterone and DNA in the corpus luteum of pregnancy in the ewe

Changes in luteal weight from about Day 20 to near term, and in quantitative histology as assessed by ultrastructural morphometry and light microscopic counts of mitosis and cell death on Days 30, 60, 100 and 142, were studied in 168 pregnant ewes. Luteal weight (mean +/- s.d.) remained constant at 0.56 +/- 0.11 g until Day 120, and fell thereafter to reach 0.31 +/- 0.11 g after Day 140 (P less than 0.01). Up to Day 100, quantitative aspects of the composition of the luteal tissue showed no significant change, and values for volume density, cytoplasmic:nuclear ratio, cell number/mm3 and cell volume were comparable to values previously obtained for corpora lutea (CL) of the cycle. By Day 142 structural evidence of luteal regression was present, but regressive changes were much more marked in some CL than others. Mitosis was seen in a few cells (0.02-0.04%) on all of the days studied, but never in large luteal cells. Cell death was rarely seen up to Day 100, but had increased in incidence by Day 142 (P less than 0.01). Luteal progesterone content, 55.2 +/- 15.9 nmol/g on Day 30, was not significantly changed on Days 60, 100 or 142. It is concluded that (1) structural regression of the CL of pregnancy does not begin until much later than the time (about Day 50) when pregnancy ceases to depend on the CL; (2) structural luteal regression begins before parturition, but its time of onset and/or rate of progression vary widely between animals; and (3) large and small luteal cells remain as distinctive populations throughout pregnancy, and their numbers at all stages can be accounted for by survival of the cells which differentiate during the genesis of the CL.     

Effects of indomethacin, luteinizing hormone (LH), prostaglandin E2 (PGE2), trilostane, mifepristone, ethamoxytriphetol (MER-25) on secretion of prostaglandin E (PGE), prostaglandin F2alpha (PGF2alpha) and progesterone by ovine corpora lutea of pregnancy or the estrous cycle

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P > or = 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P < or = 0.05) by LH or PGE2. Secretion of progesterone in vitro by CL slices from day-90 pregnant ewes was not affected by LH (P > or = 0.05) while PGE2 increased (P < or = 0.05) secretion of progesterone. Day 8 ovine CL of the estrous cycle did not secrete (P > or = 0.05) detectable quantities of PGF2alpha or PGE while day-90 ovine CL of pregnancy secreted PGE (P < or = 0.05) but not PGF2alpha. Secretion of progesterone and PGE in vitro by day-90 CL of pregnancy was decreased (P < or = 0.05) by indomethacin. The addition of PGE2, but not LH, in combination with indomethacin overcame the decreases in progesterone by indomethacin (P < or = 0.05). In experiment 2, secretion of progesterone in vitro by day-11 CL of the estrous cycle was increased at 4-h (P < or = 0.05) in the absence of treatments. Both day-11 CL of the estrous cycle and day-90 CL of pregnancy secreted detectable quantities of PGE and PGF2alpha (P < or = 0.05). In experiment 1, PGF2alpha secretion by day-8 CL of the estrous cycle and day-90 ovine CL of pregnancy was undetectable, but was detectable in experiment 2 by day-90 CL. Day 90 ovine CL of pregnancy also secreted more PGE than day-11 CL of the estrous cycle (P < or = 0.05), whereas day-8 CL of the estrous cycle did not secrete detectable quantities of PGE (P > or = 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF2alpha by day- 11 CL of the estrous cycle or day-90 CL of pregnancy (P > or = 0.05). It is concluded that PGE2, not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF2alpha.     

Influence of nitric oxide and noradrenaline on prostaglandin F(2)(alpha)-induced oxytocin secretion and intracellular calcium mobilization in cultured bovine luteal cells

Although prostaglandin (PG) F(2alpha) released from the uterus has been shown to cause regression of the bovine corpus luteum (CL), the neuroendocrine, paracrine, and autocrine mechanisms regulating luteolysis and PGF(2alpha) action in the CL are not fully understood. A number of substances produced locally in the CL may be involved in maintaining the equilibrium between luteal development and its regression. The present study was carried out to determine whether noradrenaline (NA) and nitric oxide (NO) regulate the sensitivity of the bovine CL to PGF(2alpha) in vitro and modulate a positive feedback cascade between PGF(2alpha) and luteal oxytocin (OT) in cows. Bovine luteal cells (Days 8-12 of the estrous cycle) cultured in glass tubes were pre-exposed to NA (10(-5) M) or an NO donor (S-nitroso-N:-acetylpenicillamine [S-NAP]; 10(-4) M) before stimulation with PGF(2alpha) (10(-6) M). Noradrenaline significantly stimulated the release of progesterone (P(4)), OT, PGF(2alpha), and PGE(2) (P: < 0.01); however, S-NAP inhibited P(4) and OT secretion (P: < 0.05). Oxytocin secretion and the intracellular level of free Ca(2+) ([Ca(2+)](i)) were measured as indicators of CL sensitivity to PGF(2alpha). Prostaglandin F(2alpha) increased both the amount of OT secretion and [Ca(2+)](i) by approximately two times the amount before (both P: < 0.05). The S-NAP amplified the effect of PGF(2alpha) on [Ca(2+)](i) and OT secretion (both P: < 0.001), whereas NA diminished the stimulatory effects of PGF(2alpha) on [Ca(2+)](i) (P: < 0.05). Moreover, PGF(2alpha) did not exert any additionally effects on OT secretion in NA-pretreated cells. The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL function. While NA stimulates P(4) and OT secretion, NO may inhibit it in bovine CL. Both NA and NO are likely to stimulate the synthesis of luteal PGs and to modulate the action of PGF(2alpha). Noradrenaline may be the factor that is responsible for the limited action of PGF(2alpha) on CL and may be involved in the protection of the CL against premature luteolysis. In contrast, NO augments PGF(2alpha) action on CL and it may be involved in the course of luteolysis.     

Effect of prostaglandins on luteal function during early pregnancy in pigs.

Luteal cells were obtained by digestion of luteal tissue of cyclic (day 12) and early pregnant (days 12, 20 and 30) pigs. Suspensions of the dispersed luteal cells (5 x 10(4) cells ml-1) were incubated for 2 h in minimum essential medium (MEM) alone (control) and MEM with different concentrations of prostaglandin F2 alpha (PGF2 alpha) and PGE2 (0.01, 0.1, 1, 10, 100 and 1000 ng ml-1) and luteinizing hormone (LH) 100 and 1000 ng ml-1, or with combinations of LH + PGF2 alpha and LH + PGE2. Net progesterone production was measured in the incubation media by direct radioimmunoassay. The overall response pattern of the luteal cells to exogenous hormones on day 12 of the oestrous cycle and pregnancy differed (P < 0.05) from treatment on day 20 and 30 of pregnancy. In general progesterone production was higher (P < 0.05) and the response to PGF2 alpha and PGE2 treatment was most obvious on day 12 of the oestrous cycle and pregnancy. Overall, PGF2 alpha stimulated progesterone production in a dose-dependent manner (P < 0.05). The response to PGE2 was of a quadratic nature (P < 0.05) in which the lowest and the highest doses of PGE2 were associated with a greater production of progesterone than were the intermediate doses. Treatment of luteal cells with PGF2 alpha + LH or PGE2 + LH caused overall inhibition (P < 0.05) of progesterone production compared with treatment with each hormone alone. This interaction was not affected by the dose of LH used. These findings indicate that PGF2 alpha and PGE2 are involved in the autocrine control of corpus luteum function.     

An ultrasonographic study of luteal function in breeds of sheep with different ovulation rates

Development and demise of luteal structures were monitored using daily transrectal ultrasonography in 2 breeds of sheep differing in ovulation rates (nonprolific Western white-faced cross-bred, n = 12 and prolific pure-bred Finn sheep, n = 7), during 1 estrous cycle in the mid-breeding season. Jugular blood samples were collected once a day for radioimmunoassay (RIA) of progesterone. The mean diameter of ovulatory follicles was higher in Western white-faced than in Finn ewes (6.4 +/- 0.2 and 5.3 +/- 0.2 mm, respectively; P < 0.001). The mean volume of luteal structures was higher (P < 0.05) in Western white-faced compared with Finn sheep from Days 5 to 15 of the cycle (Day 0 = day of ovulation). This accounted for the higher (P < 0.05) total luteal volumes recorded in Western white-faced ewes on Day 7 and from Days 11 to 15, despite the higher ovulation rate in Finn ewes (2.7 +/- 0.3 and 1.7 +/- 0.2, respectively; P < 0.05). Mean serum progesterone concentrations were higher (P < 0.05) in Western white-faced than in Finn ewes from Days 4 to 14. Daily total luteal volumes were positively correlated with daily serum progesterone concentrations throughout the cycle in Finn sheep (r > or = 0.40, P < 0.02), and during luteal growth and regression (r > 0.60, P < or = 0.00001) but not during mid-cycle in white-faced ewes (r = 0.16; P = 0.22). During the growth of the corpora lutea (CL), luteal tissue volume increased faster (P < 0.05) than serum progesterone concentrations in both breeds of sheep. During luteolysis, the decrease in luteal volumes parallelled that in serum progesterone concentrations in Finn (P = 0.11) but not in Western white-faced ewes, where luteal volumes decreased more slowly (P = 0.02) in relation to progesterone secretion. Increased ovulation rate in prolific Finn ewes resulted in more but smaller CL, and lower serum progesterone levels compared with nonprolific Western white-faced ewes. We conclude that breed-specific mechanisms exist to control the formation of luteal tissue and progesterone secretion in cyclic ewes differing in prolificacy. The mechanisms may involve ovulation of Graafian follicles at different sizes and inhibitory paracrine effects of CL on co-existing CL.     

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

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

Different expression of PGE synthase,PGF receptor,TNF, Fas and oxytocin in the bovine corpus luteum of the estrous cycle and pregnancy

Functional differences between the corpus luteum (CL) of pregnancy and CL of the cycle in cows were examined. Messenger RNA and protein levels of prostaglandin (PG) E synthase (PGES), PGF2α receptor (PGFR), tumor necrosis factor-α (TNF) and Fas were found to be higher in the CL of pregnancy than in CL of the cycle. Oxytocin (OT) mRNA and protein levels were lower in the CL of pregnancy. Messenger RNA levels of progesterone receptor (PR), luteinizing hormone receptor (LHR), PGE2 receptor (PGER), PGF synthase (PGFS), TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) did not differ between the cycle and pregnancy. PGE2 and PGF2α production by cultured bovine endometrial tissues was decreased by a supernatant derived from the homogenized CL of pregnancy but not by that of the CL of the cycle, suggesting that specific substances in the CL of pregnancy affect endometrial PG production in cows. Collectively, PGES, PGFR, TNF, Fas or OT may contribute to differences between the CL of pregnancy and CL of the estrous cycle in cows.     

The relationship between vaginal mucous impedance and serum concentrations of estradiol and progesterone throughout the sheep estrous cycle

The objective of this experiment was to assess the relationship between electrical resistance of the vaginal mucosa and serum concentrations of estradiol (E2) and progesterone (P4) during the estrous cycle in ewes. Vaginal impedance was recorded daily using a 2-electrode impedometer in 10 nonprolific Western white-faced and 7 prolific Finn ewes, during the mid-breeding season (October to December). Transrectal ultrasonography of ovaries was performed once a day to confirm ovulation and monitor follicle growth (follicles > or =3 mm in diameter) and development of corpora lutea (CL). Jugular blood samples were collected daily for radioimmunoassay (RIA) of estradiol and progesterone. In all ewes, a decline in vaginal impedance (to <40 ohms) was closely associated with the onset of behavioral estrus. In both breeds of sheep, there was no significant correlation between daily serum concentrations of estradiol and vaginal impedance throughout the estrous cycle. Daily serum concentrations of progesterone and the E2:P4 ratio were correlated with vaginal impedance during the period of luteolysis and follicular phase in both breeds (Western white-faced ewes: r = 0.62, P = 0.0002 and r = -0.56, P = 0.0002; Finn ewes: r = 0.61, P = 0.001 and r = -0.45, P = 0.03, respectively) and early in the cycle (Days 0 to 2, Day 0 = day of ovulation) in white-faced ewes (r = 0.61, P = 0.0003 and r = -0.36, P = 0.052, respectively) but not during the remaining portion of the luteal phase in either breed. In conclusion, vaginal mucous impedance appears to be primarily controlled by progesterone, but it also changes in response to shifts in the E2:P4 ratio when progesterone concentrations are low. Impedometric characteristics of the vaginal mucosa in cyclic ewes are an indicator of serum concentrations of progesterone and E2:P4 ratios during the terminal stage of the estrous cycle.     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Effect of diethylstilboestrol on the relationship between LH, PMSG and progesterone during pregnancy in the mare.

Two studies were conducted to determine the relationship between LH and progesterone and between PMSG and progesterone during pregnancy in mares. In the first, samples of jugular blood were collected daily from 7 mares from the first day of oestrus until Day 28 of pregnancy, and in the second, samples were collected weekly from 14 mares from Day 35 of gestation until parturition. In an attempt to prolong secretion of progesterone from accessory corpora lutea, 7 of these 14 mares were injected with increasing doses (2--10 mg) of diethylstilboestrol (DES) between Days 84 and 142 of gestation. The remaining 7 mares received injections of vehicle. Concentrations of LH, PMSG and progesterone in serum were determined by radioimmunoassay. From the onset of oestrus until Day 4 of gestation, serum concentrations of LH and progesterone were negatively correlated (r = 0.67, P less than 0.01), but from Days 5 to 28 a positive correlation (r = 0.80, P less than 0.01) was noted. Likewise, serum concentrations of PMSG and progesterone were highly correlated between Days 35 and 196 in mares injected with DES (r = 0.72, P less than 0.01) and the vehicle (r = 0.75, P less than 0.01). Injections of DES did not influence serum concentrations of LH, PMSG or progesterone, or affect the length of gestation. It was concluded that DES does not influence the maintenance of pregnancy in the mare.     

Role of intraluteal prostaglandin F(2alpha), progesterone and oxytocin in basal and pulsatile progesterone release from developing bovine corpus luteum

The present study examined the role of intra-luteal prostaglandin (PG) F(2alpha), progesterone (P4) and oxytocin (OT) on the corpus luteum function by using specific hormone antagonists. Luteal cells from the developing CL (days 5-7 of the estrous cycle) were exposed to P4 antagonist (onapristone, OP, 10(-4)M), OT antagonist (atosiban, AT; 10(-6)M) or indomethacin (INDO; 10(-4)M), for 12h and then stimulated with PGF(2alpha) (10(-8)M) for 4h. Pre-treatment of the cells with OP, AT or INDO resulted in an increase in P4 secretion in response to PGF(2alpha). To examine the temporal effects of P4, OT and PGs on P4 secretion, dispersed luteal cells were pre-exposed to OP, AT or INDO for 1, 2, 4, 6 or 12h. Prostaglandin F(2alpha) stimulated P4 secretion (P<0.05) after 2h of pre-exposition. In the microdyalisis study, the spontaneous release of P4 from developing CL tissue was of pulsatile nature with irregular peaks at 1-2h intervals. Treatment with OP increased the number of P4 peaks (P<0.05), whereas AT and INDO significantly reduced the number of P4 peaks detected (P<0.05). Interestingly, INDO completely blocked the pulsatile nature in the release of P4, but it secretion remained stable throughout the experimental period. These results demonstrate that luteal PGF(2alpha), OT, and P4 are components of an autocrine/paracrine intra-ovarian regulatory system responsible for the episodic (pulsatile) release of P4 from the bovine CL during the early luteal phase.     

Changes in ovarian oxytocin secretion as an indicator of corpus luteum response to prostaglandin F(2alpha) treatment in cattle

Exogenous prostaglandin F(2alpha) (PGF(2alpha)) rapidly increases ovarian oxytocin (OT) release and decreases progesterone (P4) secretion in cattle. Hence, the measurement of OT secretion (the area under the curve and the height of the peak) after different doses of Oestrophan - PGF(2alpha) analogue (aPGF(2alpha)) on Days 12 and 18 of the estrous cycle (estrus = day 0), could be a suitable indicator of corpus luteum (CL) sensitivity to PGF(2alpha) treatment. Mature heifers (n = 36) were used in this study. Blood samples were collected from the jugular vein for the estimation of OT, P4 and 13, 14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM). In Experiment 1, different doses of aPGF(2alpha) (400, 300, 200 and 100 microg) given on Day 12 of the estrous cycle (n = 8) shortened (P < 0.05) the cycle duration (15.2 +/- 0.6 d) compared with that of the control (21.7 +/- 0.4 d). Successive heifers were also treated on Day 12 with 200 (n = 2), 100 (n = 2), 75 (n = 2) or 50 microg aPGF(2alpha) (n = 2). Only the 50 microg aPGF(2alpha) dose did not cause CL regression, although it increased OT concentrations to levels comparable to those observed during spontaneous luteolysis (50 to 70 pg/ml). In Experiment 2, on Day 18 of the cycle heifers (n = 8) were treated with 50, 40, 30 and 20 microg aPGF(2alpha). There was a dose-dependent effect of aPGF(2alpha) on OT secretion on Day 18 of the estrous cycle (r = 0.77; P < 0.05). In Experiment 3, an injection of 500 microg aPGF(2alpha) on Day 12 (n = 4) and 50 microg aPGF(2alpha) on Day 18 (n = 4) caused a similar (P > 0.05) increase in the OT concentration (288.5 +/- 23.0 and 261.5 +/- 34.7 pg/ml, respectively). Thus the effect of the same dose of aPGF(2alpha) (50 microg) on OT secretion was different on Days 12 and 18 of the cycle. To evoke similar OT secretion on Days 12 and 18 the dose of aPGF(2alpha) on Day 18 could be reduced 10-fold, confirming that CL sensitivity to PGF(2alpha) appears to increase in the late luteal phase.     

Adenylate cyclase activity of the corpus luteum during the oestrous cycle of the pig

Basal adenylate cyclase values for corpora lutea (CL) removed from cyclic gilts on Days 3, 8, 13 and 18 were 178 +/- 61, 450 +/- 46, 220 +/- 25 and 208 +/- 18 pmol cAMP formed/min/mg protein, respectively. Basal activity was significantly elevated on Day 8 (P less than 0.001). LH-stimulatable adenylate cyclase values for CL from Days 3, 8, 13 and 18 were 242 +/- 83, 598 +/- 84, 261 +/- 27 and 205 +/- 17 pmol cAMP formed/min/mg protein respectively. Serum progesterone concentrations of 12 gilts bled every 2 days through one complete oestrous cycle ranged from 1.1 to 26.9 ng/ml with highest values between Days 8 and 12. The decline in serum progesterone concentrations was coincident with the decrease in basal adenylate cyclase activity. There was no LH-stimulatable adenylate cyclase activity present in the CL at the specific times of the oestrous cycle examined. We conclude that progesterone secretion by the pig CL is apparently dependent on basal activity of adenylate cyclase.