Different enzymatic antioxidative pathways operate within the sheep caruncular and intercaruncular endometrium throughout the estrous cycle and early pregnancy.

There has been a growing interest in the role played by antioxidant enzymes in the regulation of endometrial function in mammals. However, little is known about enzymatic antioxidative pathways involved in conditioning the cyclic and early pregnant endometrium for conceptus attachment and implantation in domestic ruminants. We aimed to investigate changes in activities of superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT) in sheep caruncles (CAR) and intercaruncles (ICAR) endometrial tissues of cyclic and early pregnant ewes. Irrespective of day of cycle or pregnancy, CAR demonstrated higher activities of SOD1 and SOD2 than in ICAR. On day 12 of the estrous cycle, ICAR demonstrated higher activity of GPX and GR than in CAR tissues. On days 12 and 16 the estrous cycle, ICAR demonstrated higher activity of CAT than in CAR. CAR demonstrated higher activity of GPX on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. CAR demonstrated higher activity of CAT on day 18 than on days 4, 8, 12 and 16 of the estrous cycle. ICAR demonstrated higher activity of CAT on day 18 than on days 4, 8, and 16 of the estrous cycle. The activity of CAT in ICAR increased from days 4 and 8 to day 12 of the estrous cycle. The activity of SOD2 in CAR increased from day 16 to day 18 of pregnancy. On day 12 of pregnancy, CAR demonstrated higher activity of GPX than in ICAR. On day 16 of pregnancy, ICAR demonstrated higher activity of GPX than in CAR. The activity of GPX in ICAR increased from day 12 to day 16 of pregnancy. The activity of GPX in CAR increased from day 16 to day 18 of pregnancy. The activity of GR in CAR and ICAR increased from days 12 and 16 to day 18 of pregnancy. On days 16 and 18 of pregnancy, ICAR demonstrated higher activity of CAT than in CAR. The activity of CAT in CAR decreased from day 12 to days 16 and 18 of pregnancy. The activity of CAT in ICAR decreased from day 12 to day 16 of pregnancy and then increased from day 16 to day 18 of pregnancy. In conclusion, different antioxidant mechanisms operate within CAR and ICAR endometrium throughout the estrous cycle and during early pregnancy. This might be related to the different but important roles of CAR and ICAR endometrial tissues for the establishment of pregnancy.

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy.

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.

Abortion-prone mating influences placental antioxidant status and adversely affects placental and foetal development.

Oxidative stress is associated with decreased female fertility and adversely affects prenatal development. Mammalian cells have developed a network of enzymatic and non-enzymatic antioxidant defence systems to prevent oxidative stress. Little attention has been paid to the antioxidative pathways in placentas of normal and disturbed pregnancies, leaving a gap in our knowledge about the role of antioxidants in the control of foeto-placental development. The challenges in studying early human pregnancy can partly be overcome by designing animal models of abnormal pregnancy. We aimed to determine whether the antioxidant status of placentas from the CBA/J × DBA/2 abortion-prone pregnant mice differed from that of normal pregnant mice. The foetal/placental weight ratio was lower in abortion-prone matings compared with that in non-abortion-prone matings. The increased placental malondialdehyde (MDA) content, the end products of lipid peroxidation, with concomitants alterations in placental antioxidants, namely copper-zinc containing superoxide dismutase (SOD1), manganese containing (SOD2), glutathione peroxidases (GPX), glutathione reductase (GR) and catalase (CAT) activities may be involved in placental and foetal growth restriction. We show that placental oxidative stress is linked with poor prenatal development and pregnancy losses in CBA/J × DBA/2 mice matings. This animal model may be useful in the evaluation of nutritional antioxidant therapies for oxidative stress and associated prenatal developmental disorders.

Proteomic analysis of the sheep caruncular and intercaruncular endometrium reveals changes in functional proteins crucial for the establishment of pregnancy.

The expression and regulation of endometrial proteins are crucial for conceptus implantation and development. However, little is known about site-specific proteome profiles of the mammalian endometrium during the peri-implantation period. We utilised a two-dimensional gel electrophoresis/mass spectrometry-based proteomics approach to compare and identify differentially expressed proteins in sheep endometrium. Caruncular and intercaruncular endometrium were collected on days 12 (C12) and 16 (C16) of the oestrous cycle and at three stages of pregnancy corresponding to conceptus pre-attachment (P12), implantation (P16) and post-implantation (P20). Abundance and localisation changes in differentially expressed proteins were determined by western blot and immunohistochemistry. In caruncular endometrium, 45 protein spots (5% of total spots) altered between day 12 of pregnancy (P12) and P16 while 85 protein spots (10% of total spots) were differentially expressed between P16 and C16. In intercaruncular endometrium, 31 protein spots (2% of total spots) were different between P12 and P16 while 44 protein spots (4% of total spots) showed differential expression between C12 and C16. The pattern of protein changes between caruncle and intercaruncle sites was markedly different. Among the protein spots with implantation-related changes in volume, 11 proteins in the caruncular endometrium and six proteins in the intercaruncular endometrium, with different functions such as protein synthesis and degradation, antioxidant defence, cell structural integrity, adhesion and signal transduction, were identified. Our findings highlight the different but important roles of the caruncular and intercaruncular proteins during early pregnancy.

Reconsideration of the proposed luteotropic and luteoprotective actions of ovine placental lactogen in sheep: in vivo and in vitro studies.

Ovine placental lactogen (oPL) is produced by the conceptus trophectoderm and is secreted into both the maternal and fetal circulations. The present study was designed to examine in vivo the luteotropic effect of recombinant oPL (roPL), as determined by monitoring progesterone concentration and cycle length (experiment 1), and the antioxidative and antiapoptotic effects of roPL, as determined respectively by monitoring antioxidant enzymatic activity and apoptosis in the corpus luteum (CL) of cyclic ewes (experiment 2). We also studied whether roPL is capable of stimulating progesterone secretion in vitro by cultured luteal tissue of functionally active CL obtained from day-10 cyclic ewes (experiment 3) and day-60 pregnant ewes (experiment 4). Circulating concentrations of progesterone and cycle length were not affected by treatment of ewes with 80 microg/kg body weight per day of roPL (n = 4 ewes) for 10 days beginning on day 11 post-estrus, as compared with saline-treated ewes (n = 4 ewes). Luteolysis occurred between days 15 and 16 post-estrus in the four saline-treated ewes and in 3/4 roPL-treated ewes. The activities of the key antioxidant enzymes copper-zinc superoxide dismutase (Cu,Zn-SOD), manganese SOD (Mn-SOD), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) were unaffected by treatment of ewes with 80 microg/kg per day of roPL (n = 4 ewes) for 3 days, between days 11 and 14 post-estrus, as compared with saline-treated ewes (n = 4 ewes). In situ TUNEL method revealed that the number of apoptotic cells was not different between the two groups of ewes. There was no significant change in progesterone secretion by explants from day-10 estrous cycle (n = 3 ewes) or day-60 pregnancy (n = 3 ewes) CL cultured with different concentrations (10, 100 and 1000 ng/ml) of roPL, whereas treatment with oLH at the concentration of 100 or 1000 ng/ml caused a significant increase in progesterone secretion by explants from day-10 estrous cycle CL (P < 0.05) and by explants from day-60 pregnancy CL (P < 0.01). In conclusion, our results demonstrate that oPL has no luteotropic and/or luteoprotective actions in sheep, either in vivo or in vitro.

Demonstration of a non-steroidal, non-inhibin factor in the ovine corpus luteum of pregnancy that reduces pituitary responsiveness to GnRH-induced LH secretion in vitro.

The decline in pulsatile LH secretion and pituitary responsiveness to GnRH as pregnancy advances may be due to non-steroidal factors secreted by the ovine corpus luteum of pregnancy. Corpora lutea from ten ewes on days 70-80 of gestation were homogenized, charcoal-treated and, together with charcoal-treated follicular fluid from superovulated women, were subjected to inhibin immunoaffinity chromatography, reducing dimeric inhibin A and B by >90% and abolishing inhibin bioactivity. These preparations were investigated using cultures of rat pituitary cells. GnRH-induced LH and FSH secretion in vitro was reduced by ovine corpus luteum extract and human follicular fluid by 47+/-5% and 42+/-5% of control LH and by 37+/-5% and 50+/-10% of control FSH, respectively (P<0.001). Extracts prepared from corpora lutea and placentae that were collected on days 50, 90 and 120 of pregnancy (five ewes per stage of pregnancy) showed increased GnRH-induced LH-suppressing bioactivity, particularly in the case of the placental extracts, with a threefold increase in activity. When partially purified by pseudochromatofocusing, GnRH-induced LH-suppressing bioactivity in extracts of ovine corpora lutea was identified at pH 5.40 and 5.77. Although these values are similar to published gonadotrophin surge-attenuating factor (GnSAF) bioactivity pI values, a GnSAF-blocking antiserum had no consistent effect on ovine corpus luteum extract GnRH-induced LH-suppressing bioactivity. It was concluded that the ovine corpus luteum of pregnancy contains a non-steroidal, non-inhibin factor, probably not GnSAF, that has the ability to reduce pituitary responsiveness to GnRH in vitro.

Effects of pregnancy on pulsatile secretion of LH and gonadotrophin-releasing hormone-induced LH release in sheep: a longitudinal study.

Pulsatile LH secretion and its control throughout pregnancy have not been fully determined in sheep. Expt 1 determined the patterns of LH secretion in five ewes on days 10, 20, 60 and 120 of pregnancy and on day 10 postpartum, compared with those on day 10 of the oestrous cycle. Mean (+/- SEM) concentrations of LH declined steadily throughout pregnancy (ANOVA, P < 0.01) and were lower (P < 0.01) on day 60 (0.19 +/- 0.3 ng ml(-1)) and on day 120 (0.18 +/- 0.4 ng ml(-1)) of pregnancy than on day 10 of the oestrous cycle (0.55 +/- 0.04 ng ml(-1)). This decrease was due to a significant reduction in the number and the amplitude of LH pulses. Only on day 120 of pregnancy were progesterone concentrations higher (P < 0.01) than on day 10 of the oestrous cycle. Although concentrations of progesterone on day 10 postpartum were barely detectable, mean LH concentration (0.45 +/- 0.09 ng ml(-1)) was not different from that on day 10 of the oestrous cycle. Expt 2 examined the LH responses in a separate group of four ewes to a physiological dose of GnRH (0.2 microg) on days 10, 20, 60 and 120 of pregnancy and on day 10 postpartum, compared with those on day 10 of the oestrous cycle. The area under the LH response curve and the maximum LH concentrations induced by GnRH declined steadily throughout pregnancy (ANOVA, P < 0.01) and were lower (P < 0.01) on days 60 and 120 of pregnancy than on day 10 of the oestrous cycle, but these parameters were not different between day 10 postpartum and day 10 of the oestrous cycle. Expt 3 examined the LH responses in a separate group of four ewes to a potent GnRH agonist, buserelin (0.5 microg), on days 10, 60 and 120 of pregnancy. The area under the LH response curve and the maximum LH concentrations induced by GnRH were lower (P < 0.01) on days 60 and 120 than on day 10 of pregnancy, but were not different between days 60 and 120. This longitudinal study demonstrates that the pulsatile LH release and pituitary responsiveness to GnRH decreases progressively as pregnancy advances, but does not support the hypothesis that high concentrations of progesterone are solely responsible for the inhibition of pulsatile LH secretion and GnRH-induced LH release during pregnancy in sheep.

Control of ovarian follicular growth and maturation by the corpus luteum and the placenta during pregnancy in sheep.

Ovarian follicular growth and maturation and its control throughout pregnancy have not been described fully in sheep. Experiment 1 characterized the size and maturation (steroid production in vitro and aromatase activity) of ovarian follicles obtained at days 20, 50, 80 and 110 of pregnancy compared with those obtained at day 12 of the oestrous cycle. There was no difference in the number of small follicles (< 3 mm in diameter) between cyclic and pregnant ewes, regardless of the stage of pregnancy. There was a marked reduction (P < 0.01) in the number of medium follicles (3-5 mm) starting at day 80 of pregnancy. Large follicles (> 5 mm) were not detected at day 110 of pregnancy. In vitro testosterone output by follicles was constant throughout pregnancy. Oestradiol output remained steady until day 80, but decreased markedly at day 110 of pregnancy. This decrease was associated with a reduction in aromatase activity in follicles obtained at this stage. Experiment 2 examined the effect of administration of high concentrations of progesterone between day 100 and day 120 after mating on resumption of follicular growth in ewes that underwent Caesarean section at day 99 of pregnancy. In ewes that underwent Caesarean section, progesterone supplementation was successful in mimicking the profile found in pregnant ewes, but did not prevent re-initiation of follicular growth, as demonstrated by the presence of large follicles (> 5 mm) at day 120 after mating. Experiment 3 examined the effects of PGF(2alpha)-induced regression of the corpus luteum of day 100 of pregnancy on resumption of follicular growth. High concentrations of PGF(2alpha) (0.28 mg kg(-1) body weight) administrated at day 100 of pregnancy were required to initiate regression of the corpus luteum. At day 120 after mating, the mean (+/- SEM) diameter of the largest follicle in PGF(2alpha)-treated ewes (3.40 +/- 0.47 mm) was significantly greater (P < 0.05) than that in control pregnant ewes (2.52 +/- 0.34 mm). Experiment 4 examined the effect of removal of the fetus and of the corpus luteum at day 100 of pregnancy on resumption of ovulation. Removal of the corpus luteum by PGF(2alpha) treatment at the time of removal of the fetus resulted in earlier occurrence of short luteal phases (27.8 versus 40.6 days, PGF(2alpha)-treated versus non-treated) but did not alter the timing of the first normal luteal phases (41 days). In conclusion, the results from these experiments indicate that placental compounds play a major role in inhibiting follicular growth and maturation during late pregnancy in sheep.

Absence of regular pulsatile LH secretion during pre- and post-implantation periods in sheep.

Two experiments were conducted to determine the patterns of LH secretion and to evaluate the LH responses to pulsatile administration of GnRH during early pregnancy in ewes. In experiment 1, pregnant ewes (n=16) were used to determine the concentration of LH in plasma of jugular blood samples collected every 15 min for 6h before (day 10 post-mating) and after (days 20 and 30 post-mating) implantation. In experiment 2, the pituitary LH responses to exogenous pulsatile administration of GnRH were examined on day 10 post-mating in 4 pregnant ewes. A small dose of GnRH (200 ng/ml saline) was injected (i.v.) every 3h and jugular blood samples were collected every 15 min for 12h beginning at the onset of GnRH administration and continuing through the 4th GnRH pulse. During the frequent-sample bleeding at any of the stages of pregnancy examined, LH concentrations oscillated in a pulsatile manner. However, pulsatile LH release occurred irregularly and infrequently. Overall mean LH concentrations, frequency and amplitude of LH pulses were not significantly different between any of the stages of pregnancy examined. Pulsatile administration of GnRH on day 10 post-mating induced regular pulses of LH. In conclusion, these data demonstrate that: (i) pulsatile LH secretion occurs irregularly during early pregnancy, and (ii) the absence of regular pulsatile LH release during early pregnancy is not attributed to a lack of pituitary responsiveness to GnRH.

Intracerebroventricular administration of copper-zinc superoxide dismutase inhibits pulsatile luteinizing hormone secretion in ovariectomized ewes.

Intracerebroventricular (i.c.v.) injection of an inhibitor of nitric oxide synthase (NOS) abolishes pulsatile luteinizing hormone (LH) secretion. It has been demonstrated that structural and functional analogs of copper-zinc superoxide dismutase (Cu,Zn-SOD) inhibit neuronal NOS. The present study examined the ability of Cu,Zn-SOD to affect pulsatile LH release in the ewe. Bovine Cu,Zn-SOD was administrated into the third cerebral ventricle of unanesthetized, freely moving, ovariectomized (OVX) ewes. Jugular blood samples were taken every 15 min for 5 h before and 8 h after i.c.v. injections. In a pilot trial using three OVX ewes, i.c.v. injection of Cu,Zn-SOD at a dose of 0.5, 1.0 or 2.0 microg in 100 microl saline decreased plasma LH levels and abolished LH pulses, without affecting FSH secretion. In the main experiment, i.c.v. injection of 100 microl saline had no effect on mean LH levels and LH pulse frequency, whereas i.c.v. injection of Cu,Zn-SOD at a dose of 1 microg/100 microl saline significantly (P < 0.01) decreased mean LH levels and LH pulse frequency. In conclusion, this study provides the first evidence for the role of Cn,Zn-SOD in the control of LH secretion at the level of the brain in female mammals.

The secretion of prolactin in intact and lutectomized pregnant ewes. Effect of the anti-progesterone steroid RU 486.

To study the role, if any, of luteal factors in the control of prolactin secretion during the last two thirds of pregnancy in the ewe, we examined: a) the effect of RU 486 administration on prolactin secretion on days 97, 112 and 131 of pregnancy in five intact ewes and in five ewes from which the corpus luteum (CL) was removed on day 78 of pregnancy; and b) the secretory patterns of prolactin on days 60, 80, 100 and 120 of pregnancy in five intact ewes and in five ewes from which the CL was removed on day 70 of pregnancy. In a pilot experiment, we showed that daily i.v. injections (from day 91 to day 105 of pregnancy) of RU 486 at a dose of 50 mg caused a marked release of prolactin, without any effect on the secretion of progesterone and progression of pregnancy. In experiment 1, a single i.v. injection of 50 mg of RU 486 resulted in a significant (P < 0.01) increase in plasma prolactin concentrations on any day of pregnancy examined in the intact and lutectomized ewes. The prolactin responses (the maximum concentrations, the time to maximum concentrations and the area under the response curves) were not different between the two groups in any stage of pregnancy examined. In the two groups, spontaneous parturition occurred at term with alive lambs. There was no difference between the two groups in gestation length and lamb birth weight. In experiment 2, we showed that plasma concentrations of prolactin fluctuated in a pulsatile manner during the last two-thirds of pregnancy. The mean prolactin concentrations, the frequency and the amplitude of prolactin pulses were not significantly different between the intact and the lutectomized ewes in any stage of pregnancy examined. In conclusion, these experiments demonstrated that the ovine CL of pregnancy is not involved in the control of prolactin secretion in the ewe. The stimulation of prolactin secretion by the RU 486 is probably due to its anti-progesterone action exerted at the level of the receptor. The placental progesterone plays a central role in the control of prolactin secretion during the last two-thirds of pregnancy.

The secretory patterns of growth hormone in pregnant and hysterectomized ewes.

This work was undertaken to determine the secretory patterns of GH during pregnancy, and to evaluate the effect, if any, of hysterectomy during early pregnancy on subsequent secretion of GH in ewes. The concentrations of GH were determined in the plasma of jugular blood samples collected at 15-min intervals during a 6-h period on days 20, 40, 60, 80, 100 and 120 post-mating, and three times per week between days 29 and 120 post-mating from 5 pregnant ewes and from 5 ewes from which the gravid uterus was removed on day 30 post-mating. A pulse analysis program (Pulsar) was used to analyse the secretory patterns of GH in individual profiles of the serial sampling period. In the two groups of ewes, peripheral concentrations of GH fluctuated in an episodic manner during the frequent blood sampling of any stage of the post-mating period examined. The overall GH concentrations, the basal GH concentrations, the frequency and the amplitude of GH pulses remained fairly stable between days 20 and 120 post-mating in the two groups of ewes. The parameters of GH secretion were not different between the two groups of ewes. The secretory patterns of GH, as determined in plasma of blood collected three times per week between days 29 and 120 post-mating were also not different between the two groups of ewes. In conclusion, results of this study show that (i) the pulsatile secretion of GH does not change as pregnancy advances, and (ii) hysterectomy performed during early pregnancy does not subsequently affect the secretory patterns of GH. These findings suggest that the gravid uterus and/or the feto-placental unit secretory products are unlikely to be involved in the control of GH secretion during pregnancy in the ewe.

Effects of luteectomy on the maintenance of pregnancy, circulating progesterone concentrations and lambing performance in sheep.

In sheep, there have been few and conflicting data regarding the necessity of the corpus luteum (CL) for the maintenance of pregnancy. The aims of the present study were to examine the effect of luteectomy on and after Day 50 of pregnancy on maternal plasma progesterone concentrations and the progression of pregnancy, to determine the minimum placental progesterone support required for the maintenance of pregnancy, and to evaluate the effect of luteectomy on lambing performance. In Experiment 1, four ewes luteectomized on Day 50 of pregnancy aborted 2-7 days after surgery, whereas pregnancy progressed and parturition occurred between Days 143 and 149, with live lambs, in three of four ewes and in four ewes luteectomized on Days 60 and 70 of pregnancy respectively. The mean (+/- SEM) progesterone concentrations on the day before and one day after luteectomy decreased from 4.87+/-0.85 to 0.42+/-0.06 ng mL(-1) (P<0.01), from 4.57+/-0.51 to 0.80+/-0.12 ng mL(-1) (P<0.02) and from 6.05+/-0.52 to 1.67+/-0.11 ng mL(-1) (P<0.01), respectively, for the ewes luteectomized on Days 50, 60 and 70 of pregnancy. The fall in progesterone concentrations was 90%, 80% and 71%, respectively, for the ewes luteectomized on Days 50, 60 and 70 of pregnancy. In Experiment 2, pregnancy progressed in four ewes luteectomized on Day 70 and parturition occurred between Days 146 and 149, with live lambs. The mean progesterone concentrations declined (P<0.01) from 6.9+/-0.7 ng mL(-1) on the day before luteectomy to 2.1 = 0.3 ng mL(-1) the day after surgery. The concentrations of progesterone in blood collected every 3 h during a 24-h period were stable on Days 60 and 80 of pregnancy, but they were lower (P<0.03) on Day 80 than on Day 60 of pregnancy, for each time period examined. In Experiment 3, the gestation length and birthweights of single, twin and triplet lambs were not different between the control intact ewes (n = 111) and the ewes luteectomized on Days 70-80 of pregnancy (n = 71). Lamb mortality was not different between the two groups (7.2% v. 8.4%, control v. luteectomized). In conclusion, these results showed that (1) the sheep CL is necessary to maintain pregnancy until at least Day 60, (2) progesterone withdrawal induced by luteectomy on and after Day 50 of pregnancy must be of a critical magnitude to provoke abortion, (3) after Day 60 of pregnancy, the CL and the placenta together secrete more progesterone than required for pregnancy maintenance, (4) there is no apparent 24-hour rhythm in maternal plasma progesterone concentrations before and after luteectomy, and (5) luteectomy at mid pregnancy has no apparent effect on gestation length, lamb birthweight or lamb mortality.

Effects of the presence of rams during pregnancy on lambing performance in ewes.

Ewes of the Préalpes-du-Sud breed (n = 112) were mated with fertile rams and were used to investigate the effect of the presence of vasectomized rams during pregnancy on reproductive outcomes. Ewes in the control group (n = 56) were isolated from rams during the whole period of pregnancy, whereas those in the experimental group (n = 56) were kept with vasectomized rams from day 10 post-mating until lambing. At day 10 post-mating, a series of blood samples was collected every 15 min for 8 h from five control ewes and five experimental ewes to determine their patterns of LH secretion. The introduction of the ram was associated with a rapid increase of pulsatile LH release. The lag between the introduction of the ram and the onset of the first episodic LH release was less than 15 min. The mean(+/- sem) number of LH pulses/4 h after the introduction of the ram (2.8 +/- 0.4) was significantly higher (P < 0.01) than that observed/4 h before the introduction of the ram (1.4 +/- 0.2). Although more ewes were pregnant in the control group (87.5%) than in the ram-exposed group (82.1%), the difference was not significant. The presence of rams did not affect gestation length (145.8 days), overall lamb mortality (3.5%) or birth weights of single (3.96 kg), twin (3.24 kg) or triplet (2.59 kg) lambs. The proportion of ewes with multiple births in the control group (69.4%) was significantly greater (P < 0.05) than that in the ram-exposed ewes (47.8%). The ewes in the control group had significantly more (P < 0.01) twin lambs born alive (72.3%) than the ewes in the ram-exposed group (50.0%). In conclusion, the presence of vasectomized rams during early pregnancy affected the incidence of multiple births but did not affect pregnancy rate or gestation length. The altered fertility of ewes exposed to vasectomized rams may reflect changes in embryonic loss during early pregnancy.

Lack of suppressive action of luteal factors from sheep corpus luteum on LH response to exogenous LHRH in ovariectomized, pregnant and post-partum ewes.

In vivo studies have provided evidence of a non-steroidal factor from ovine corpora lutea (CL) of pregnancy called LH-release inhibiting factor (LH-RIF), which specifically inhibits tonic LH release without affecting FSH secretion. To study the possible pituitary site of action of LH-RIF and other factors from sheep CL of pregnancy, the LH response to LHRH was investigated in ovariectomized (OVX) ewes pretreated with ovine luteal extract (oLE) from CL of mid-pregnancy, and in pregnant and post-partum ewes from which the CL were removed at mid-pregnancy. In view of the fact that human follicular fluid (hFF) contains a non-steroidal factor, called gonadotrophin surge-attenuating factor, which attenuates the LHRH-induced LH release, it was clearly important to examine the LHRH-induced LH release in OVX ewes pretreated with oLE or hFF In experiment 1, the LH and FSH responses (the maximum LH and FSH concentrations, the time to maximum LH and FSH concentrations and the area under the LH and FSH response curves) to a single i.v. injection of 5 microg of the LHRH agonist buserelin was not significantly different between ewes pretreated with oLE and ewes pretreated with BSA. Although the LHRH-induced LH and FSH release was slightly attenuated in OVX ewes pretreated with hFF as compared with OVX ewes pretreated with BSA, the maximum LH and FSH concentrations and the area under the LH and FSH response curves were not significantly different between the two groups of ewes. In experiment 2, the LH responses to a single i.v. injection of 0.5 microg LHRH on days 80 and 100 of pregnancy were not significantly different between intact ewes and ewes from which the CL were removed on day 70 of pregnancy. In both groups of ewes the maximum LH concentration and the area under the LH response curve were significantly lower (P <0.02) on day 100 than on day 80 of pregnancy. In experiment 3, the LH responses to a single i.v. injection of 50 microg LHRH on day 140 of pregnancy and on day 10 post-partum were not significantly different between intact ewes and ewes from which the CL were removed on day 70 of pregnancy. In both groups of ewes the maximum LH concentration and the area under the LH response curve were significantly lower (P <0.01) on day 140 of pregnancy than on day 10 post-partum. These experiments provide evidence for a lack of suppressive action of luteal secretory products from the ovine CL of pregnancy on the pituitary LH response to LHRH in OVX, pregnant and post-partum ewes.

Evidence that the conceptus contributes to the inhibition of follicular growth in the ewe.

The present study was designed to determine the contribution of the gravid uterus and the conceptus in the inhibition of antral follicular growth. All antral follicles were counted and measured on day 48 post mating in ovaries of pregnant ewes (Group 1, n = 5) and in ovaries of ewes from which the uterus (Group 2, n = 5) or the conceptus (Group 3, n = 5) had been surgically removed on days 32-33 post mating. The secretory activity of the corpora lutea was maintained after removal of the uterus or the conceptus until slaughter of ewes. No difference was found between the three groups of ewes in the mean (+/-SEM) total number (non-atretic and atretic) of follicles per ewe, being 59.8 +/- 7.6, 35.0 +/- 7.6 and 50.8 +/- 4.6, respectively for Groups 1, 2 and 3. The mean (+/-SEM) number of follicles 2 mm or more in diameter was greater (P < 0.01) in Group 2 ewes (3.4 +/- 0.5) and Group 3 ewes (4.2 +/- 0.5) than in Group 1 ewes (1.6 +/- 0.4). Of all follicles counted which were 2 mm or larger, 90% in Group 1 were atretic, this value being higher (P < 0.05) than in Group 2 (68%) and Group 3 (55%). The mean (+/-SEM) diameter of the largest (F1) and second largest (F2) follicles was greater (P < 0.01) in Group 2 (F1, 4.0 +/- 0.4; F2, 3.2 +/- 0.4) and in Group 3 (F1, 3.9 +/- 0.2; F2, 3.3 +/- 0.2) than in Group 1 (F1, 2.6 +/- 0.1; F2, 2.2 +/- 0.1). The mean diameter (+/-SEM) of the largest non-atretic follicles was greater (P < 0.01) in Group 2 (4.0 +/- 0.4) and Group 3 (3.9 +/- 0.2) than in Group 1 (1.7 +/- 0.3). In conclusion, the results from this in vivo experiment provide evidence that the conceptus prevents the development of large healthy follicles by inducing atresia during the first trimester of pregnancy. The inhibition of follicular growth would reduce oestradiol production and permit the maintenance of luteal function during early pregnancy.

Evidence that a non-steroidal factor from ovine placenta inhibits aromatase activity of granulosa cells in vitro.

Previous studies in vivo provide evidence that extra-ovarian factors, currently unknown but nevertheless present and associated with pregnancy, directly prevent the growth of follicles beyond a diameter of 2 mm during the last trimester of pregnancy in the ewe. In the present study, the effect of charcoal-treated extract from sheep placenta on total aromatase activity, as determined by the conversion of [3H] androstenedione to estradiol and measurement of [3H] water, was investigated using primary culture of human granulosa cells in serum-free medium as a model. Addition of different doses (50, 150, 300 and 600 micrograms protein) of cotyledons extract of day 110 of pregnancy produced a dose-dependent diminution of granulosa cell aromatase activity in the absence of FSH. The maximal inhibition of aromatase activity occurred at a dose of 600 micrograms. These results showed that the cotyledons of late pregnancy contain a non-steroidal factor that inhibits basal aromatase activity in granulosa cells. Extracts prepared from cotyledons of days 90 and 110 of pregnancy but not extracts of days 50 and 70 significantly reduced basal aromatase activity in a dose-dependent manner. These results suggest that the production of the aromatase inhibiting factor increases with the advance of pregnancy. The aromatase inhibiting activity was lost after heating (80 degrees C, 30 min) or after treatment with trypsin (1 mg/ml) of cotyledons extract of day 110 of pregnancy, demonstrating the protein nature of the aromatase inhibiting factor. In conclusion, these experiments demonstrate that ovine placenta contains a heat- and trypsin-sensitive factor likely to be a protein which inhibits granulosa cell aromatase activity.

Evidence that a non-steroidal factor from corpus luteum of pregnant sheep inhibits aromatase activity of ovarian follicles in vitro.

Porcine and ovine follicular tissues were used to investigate, in vitro, the effect of charcoal-treated aqueous extract from ovine corpora lutea of pregnancy on aromatase activity as determined by the conversion of [3H]testosterone to oestradiol by follicular walls and measurement of 3H2O release. Extract (500 micrograms protein) prepared from corpora lutea of day 112 of pregnancy but not extract (500 micrograms) prepared from ovine fetal cotyledonary tissue obtained at a similar time significantly decreased (P < 0.02) aromatase activity of pig follicles in the absence of FSH. These results demonstrate that a non-steroidal factor in the corpora lutea of late pregnancy directly inhibits aromatase activity. When the effects of different doses (300, 600 or 1200 micrograms) of luteal extract from corpora lutea of day 100 of pregnancy on aromatase activity of pig follicles were studied, the dose by treatment (presence or absence of FSH) interaction was not significant. Luteal extract dose at 300 micrograms did not affect aromatase activity but a significant decrease in activity occurred at 600 micrograms of luteal extract (600 versus 300 micrograms, P < 0.02). There was no further significant increase in the inhibitory effect with 1200 micrograms luteal extract. When the effects of 600 micrograms luteal extract from corpora lutea of days 15, 75 or 100 of pregnancy on aromatase activity of pig follicles were studied, a significant (P < 0.05) stage of pregnancy effect was detected, but the stage of pregnancy by treatment (presence or absence of FSH) interaction was not significant. No effect was noted with day 15 or day 75 luteal extract.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a gonadal nonsteroidal factor that specifically inhibits release of luteinizing hormone in ewes.

Bilaterally ovariectomized ewes were used to investigate the effect of systemic administration (i.v.) of charcoal-treated aqueous luteal extracts from ovine corpora lutea on plasma concentrations of pituitary gonadotrophins. Jugular blood samples were taken every 15 min at least 5 h before (control period) and 5 h after (treatment period) injection. In Expt 1, the administration of luteal extract from corpora lutea of days 70-76 of pregnancy, but not of the extract prepared from muscular tissue, resulted in a significant decrease of mean concentrations of luteinizing hormone (LH) (P < 0.02) and frequency of LH pulses (P < 0.01). Plasma follicle-stimulating hormone (FSH) concentrations were not affected by injections of either extract. These findings provide the first demonstration of the presence of a nonsteroidal factor in the corpus luteum of midpregnancy that selectively suppresses the secretion of LH. In Expt 2, mean concentrations of LH and FSH and frequency of LH pulses were unaffected by injections of luteal extracts from ovine corpora lutea of days 10-12 of the oestrous cycle or day 15 of pregnancy. These data suggest that some factor(s), probably from the fetoplacental endocrine unit, is required to ensure the production of a significant quantity of the luteal LH-inhibiting factor after day 15 of pregnancy. In Expt 3, treatment of luteal extract from corpora lutea of day 70 of pregnancy with proteolytic enzymes destroyed the LH-inhibiting activity, suggesting the proteic nature of the luteal LH-inhibiting factor. In Expt 4, plasma concentrations of LH were not affected by injection of charcoal-treated extract prepared from fetal cotyledonary tissue of days 110-120 of pregnancy suggesting that the LH-inhibiting factor exclusively originates from the corpus luteum during pregnancy. These experiments provide the first direct evidence for the existence of a potent nonsteroidal factor of luteal origin that specifically inhibits pulsatile secretion of LH, without influencing FSH release in female animals. We propose the term LH-release-inhibiting factor (LH-RIF) to describe this activity.

Role of the corpus luteum of pregnancy in controlling pituitary gonadotrophin secretion during the early post-partum period in the ewe.

No difference was found between 5 intact ewes and 5 ewes from which the CL had been excised at Day 70 of pregnancy in the plasma concentration of progesterone at Day 140, and concentrations of progesterone remained below 0.2 ng/ml during the first 20 days post partum. Plasma concentrations of LH, frequency and amplitude of LH pulses were low at Day 140 and increased considerably, particularly in the CL-excised ewes, as early as Day 5 post partum. No significant differences were found between the two groups of ewes in the mean plasma concentrations of FSH for any of the 5 stages examined. Taken together, these results suggest that some factor, other than progesterone, associated with the CL of pregnancy is involved in the inhibition of pulsatile LH secretion during the early post-partum period.