Post-partum oestrus in the sow in relation to the concentration of plasma oestrogens.

Ten sows, three entire and seven which had been ovariectomized at different stages of late gestation, were observed for post-partum oestrus. Serial blood samples were collected from six sows during the pre-and post-partum periods, and plasma oestrogen concentrations determined by radioimmunoassay. Morphological aspects of ovarian activity in the entire sows were examined at laparotomy and at autopsy. Although peak values of oestrogen concentration in the plasma varied from 3-9 to 8-0 ng/ml between individuals, the pattern of oestrogen levels was similar for control and ovariectomized sows. Peak concentrations occurred just before parturition, and the timing of ovariectomy did not affect the incidence of the oestrogen peak. Oestrus was detected in one control and one ovariectomized sow at 46 and 40 hr post partum respectively, there being no evidence of ovarian activity in the entire sows. The occurrence of post-partum oestrus in a sow ovariectomized at Day 108 of gestation indicates that this phenomenon is not directly connected with ovarian secretion of oestrogens. The post-partum oestrus is apparently a result of the peak of feto-placental oestrogens that occurs at parturition.     

Effect of an inhibitor of prostaglandin synthesis on uterine motility in the ovariectomized ewe during induced oestrus. A preliminary report

The myometrial electromyographic activity (EMG) of three ovariectomized ewes was studied after two consecutive treatments used for inducing oestrus: oestrogen administration and oestrogen plus an inhibitor of prostaglandin synthesis (indomethacin). The myometrial activity preceding treatments was used as a reference. The results of this study demonstrated that indomethacin modified the EMG of the myometrium recorded 24 hours after oestrogen injection. The rhythmic activity normally induced by oestrogens was suppressed, so that there was little or no difference between uterine activity recorded before and 24 hours after oestrogen injection.     

Increase in uterine peroxidase activity in the rat uterus during oestrogen hyperaemia.

The administration of oestrogen results in increased arterial blood flow in all mammalian species studied to date, but its mechanism of action has not been elucidated. Because an interval of 30-60 min is observed between oestrogen injection and uterine hyperaemia, it has been suggested that a vasoactive intermediate is involved and recent evidence suggests that catechol oestrogens are the vasoactive oestrogen intermediates. Uterine peroxidase catalyses the conversion of oestrogens to their catechol forms and thus may play an important role in oestrogen-induced uterine hyperaemia. The present studies evaluated the time course and dose-response effects of oestrogen on uterine peroxidase activity and related these to changes in uterine blood volume, an index of uterine hyperaemia in immature rats. These data demonstrated that the minimal effective hyperaemic dose of oestradiol also increased (P less than 0.05) uterine peroxidase activity. The oestradiol-induced increase in uterine peroxidase activity preceded significant increases in uterine blood volume (1 h versus 2 h, respectively). These data are consistent with a role for peroxidase-mediated conversion of oestradiol to catechol oestradiol in facilitating uterine hyperaemia in rats.     

Total unconjugated oestrogen and progesterone concentrations in peripheral blood during the oestrous cycle of the dog.

From the beginning of pro-oestrus to the end of metoestrus, daily peripheral blood samples were withdrawn from six bitches. Further samples were obtained during anoestrus. Oestrogen values rose from the onset of pro-oestrus to attain a peak value of 25-3 +/- 4-8 pg/ml on Day 1 of oestrus. Progesterone concentrations began to rise 2 days before the oestrogen peak, and reached their highest levels of 18-9 +/- 1-0 ng/ml 16 days after the end of oestrus. After oestrus, an oestrogen rise was detected which reached a peak at Day 18 of metoestrus. The duration of oestrus was unrelated to the length of time that oestrogen levels were raised, or the maximum values were attained. No significant oestrogen levels were detected during anoestrus.     

Testicular endocrine function, seasonality and semen quality of the stallion

To gain further information on gonadal function of the stallion, concentrations of testicular steroids in blood plasma (bpl) and seminal plasma (spl) and their distribution in the ejaculate were determined. Blood and semen samples from a total of 11 stallions were collected from November to July. Estrone (E1), estrone sulfate (E1S), estradiol-17beta (E2beta) and testosterone (T) were determined in bpl and spl, and in addition androstenedione (A), dehydroepiandrosterone (DHEA) and 5alpha-dihydrotestosterone (5alpha-DHT) were measured in spl. At certain points of time, aliquots of an ejaculate were centrifuged, washed and the distribution of E1, E1S, E2beta and T into seminal plasma and the sperm fraction was assessed. Hormone assay was by RIA, partly after prior separation by HPLC. Mean concentrations (X(g) x DF) were as follows: E2beta (bpl) 31.1 (1.16), (spl) 24.2 (1.42) pg ml(-1); E1 (bpl) 143.3 (1.21), (spl) 117.7 (1.53) pg ml(-1); E1S (bpl) 157.3 (1.44), (spl) 2.92 (1.42) ng ml(-1); T (bpl) 570.6 (1.43), (spl) 23.1 (1.68) pg ml(-1); A (spl) 17.9 (1.39) pg ml(-1); DHEH (spl) 12.4 (1.51) pg ml(-1); 5alpha-DHT (spl) 9.7 (1.29) pg ml(-1). Except for E2beta and A in seminal plasma, a seasonal pattern was established for all other steroids with lowest mean values occurring from November to April. From the semen parameters determined, only motility was correlated to season. There was a higher correlation among oestrogen in blp than in spl and the only correlation identified between oestrogenic and androgenic steroids was between T and E2beta in blp. In spl, T was correlated with A and 5alpha-DHT. T was the dominant free steroid in bpl while it was E1 in spl; T and E1S concentrations were about 23- and 54-fold lower in spl compared to bpl with E1S, however, showing the highest absolute values in both fluids. In the fractionated ejaculate an association of free oestrogens, particularly E2beta, with spermatozoa was observed.     

Influence of sperm number and seminal plasma on fertility of progestagen-treated sheep in confinement

Progestagen-impregnated vaginal sponges + PMSG were used to synchronize oestrus in crossbred adult ewes which were inseminated 56 h after sponge removal with 0.5 ml diluted semen containing 400, 200, 100, 50 or 25 x 10(6) spermatozoa per insemination. The diluent was skim milk-citrate or pooled seminal plasma. There was no difference in reproductive performance due to the insemination medium. Fertility (no. of ewes lambing) after insemination of 400 or 200 x 10(6) spermatozoa was 68% and was similar to that observed after natural service at progestagen-induced oestrus. When less than or equal to 100 x 10(6) spermatozoa were inseminated, fertility fell markedly and the number of lambs per ewe inseminated decreased. A decrease in litter size also occurred. The data indicate that insemination of 200 x 10(6) spermatozoa, i.e. less than 10% of the number in a single ram ejaculate, allows normal conception rates in progestagen-treated ewes.     

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.     

Spontaneous electromyographic activity throughout the cycle in the sow and its change by intrauterine oestrogen infusion during oestrus

Two experiments were carried out to monitor influences on the uterine electromyographic activity (EMG) in cyclic gilts with chronic uterine EMG electrodes. In Exp. 1 the EMG was recorded continuously from Day -1 for 24 days and was evaluated for frequency, duration and amplitude. Progesterone and oestradiol in peripheral plasma were measured daily. As high amounts of oestrogens are characteristic for boar semen, in Exp. 2 the influence of seminal oestrogens on uterine contractions at Day 0 (first day of standing reflex) was investigated in gilts with chronic intrauterine catheters. They were infused with 10 ml saline (N = 4) or saline with physiological amounts of oestrogens (5 micrograms oestradiol + 2 micrograms oestrone + 4.5 micrograms oestrone sulphate; N = 4). Sham-treated gilts (infusion catheters, no infusion; N = 5) served as controls. The EMG was recorded for 2 h before and 9 h after infusion. In Exp. 1 the maximal amplitude (2040 +/- 98 microV) and duration (32 +/- 1.7 sec) but the lowest frequency (15.8 +/- 2.1 contractions/h) were found on Day 0. With decreasing oestrogen and increasing progesterone concentrations the frequency increased continuously until Day 5 (63.5 +/- 1.0 contractions/h) while the amplitude (183 +/- 13 microV) and duration (3.3 +/- 0.7 sec) decreased. During Days 6-13 the EMG activity was not detectable. The reverse pattern was found from the onset of luteolysis until the following Day 0. On Day 0 a significant correlation between oestradiol and the duration (r = 0.81; P less than 0.01; n = 10) but not the frequency was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous uterine activity and plasma progesterone and oestrogens in the ewe with live and stillborn lambs

Peripheral plasma progesterone and oestrogen concentrations were measured during late pregnancy and the parturient period in 12 ewes producing live lambs and three others producing stillborn lambs. Progesterone values started declining by 10 days before lambing but at minus 24 hours were still 6.1 +/- 3 ng/ml in the ewes bearing live lambs; during the last 24 hours progesterone was significantly lower in the ewes producing stillborn lambs. Oestrogens reached a maximum level of 550 +/- 280 pg/ml at the time of delivery and declined rapidly to basal values shortly after lambing. Oestrogens did not rise at lambing in the ewes producing stillborn lambs. In seven of the 12 ewes bearing live lambs, uterine activity, as determined by intrauterine pressure changes, was recorded throughout parturition and compared with the plasma values for progesterone and oestrogens. It was found that there was a highly significant positive correlation between uterine activity and plasma oestrogen concentrations, and a highly significant negative correlation between uterine activty and both plasma progesterone concentrations and the progesterone: oestrogen ratio.     

The relationship of the oestrogen and progestin receptors in the abnormal uterus of the adult anovulatory rat. Effects of neonatal treatment with testosterone propionate or clomiphene citrate.

The neonatal administration of testosterone propionate to Wistar rats resulted in anovulatory adults in persistent vaginal oestrus. Clomiphene citrate had a similar effect. In both groups of adults, hyperplasia of the uterine epithelium and occasional metaplasia was observed. The uterine nuclear and cytosol oestrogen and progestin receptors of these anovulatory rats were found to have affinities for their respective ligands similar to those of normal females. The nuclear oestrogen receptor comprised occupied and unoccupied components, as in normal females. The content of the nuclear oestrogen receptor was comparable with that of females in the late dioestrous or pro-oestrous phase. This content was higher in the clomiphene-treated group. Despite the relatively high nuclear oestrogen receptor content the content of progestin receptors, a putative index of the oestrogenic response, was lower in the treated rats than in normal adult females throughout the cycle. Administration of oestradiol to both treatment groups resulted in depletion of cytosol oestrogen receptor content 1 h later, which, however, was not reflected by an increase in the content of nuclear oestrogen receptors. There was no measurable increase in progesterone receptor content in treated rats after daily administration of oestrogen (5 microgram/rat) for 3 days. These changes in sex-hormone-receptor interactions involving an impairment of the normal oestrogenic response may be associated with the abnormal differentiation of the uterus in these sterile, anovulatory animals.     

Dynamics of Eosinophils in the Uterus after Oestrogen Administration

To investigase the role of the eosinophil leukocytes in the early oestrogenic responses in the uterus, the kinetics of oestrogen-induced uterine eosinophilia and other parameters of oestrogen stimulation were studied at very early times. Uterine eosinophils increase as early as 5 min after an intravenous injection of oestradiol to immature rats, much earlier than several other changes in the early parameters of oestrogen stimulation. Large number of uterine eosinophils are found attached to the wall of small uterine blood vessels at early times. To elucidate the mechanisms involved in the specific attraction of eosinophils to the uterus in the presence of oestrogens, the in vivo localisation of oestrogens in the rat uterus at early times was studied using a radioautographic technique. Oestrogen receptors were found in the surface of eosinophils and in the wall of small uterine blood vessels. This simultaneous presence of both oestrogen receptors is proposed to explain the specific attachment of eosinophils to uterine blood vessels in the presence of oestrogens, which is the initial step toward eosinophil penetration into the uterus.     

In-vivo myometrial electrical activity in the cyclic mare

Uterine electromyography was performed by means of chronically implanted surface electrodes in 3 Pony mares during spontaneous oestrous cycles and following luteolysis induced by a prostaglandin analogue (fluprostenol). Three distinct patterns were recognized during the oestrous cycle. (1) During oestrus well defined phases of activity with closely grouped high-amplitude spikes were separated by long periods (10-45 min) of complete inactivity. (2) During dioestrus more diffuse phases of activity with low-amplitude spikes were separated by variable periods of relative inactivity. (3) During luteolysis, short and frequently occurring phases of activity were propagated between the two electrodes on one uterine horn; a similar pattern also occurred between 1 and 3 h after injection of fluprostenol. Peripheral plasma progesterone, but not total inconjugated oestrogen, concentrations were closely related to characteristics of the myographic activity during the cycle. Insemination during oestrus and injection of fluprostenol during dioestrus caused a marked and prolonged increase in myometrial electrical activity. Almost any non-specific environmental stimulus, including entry by palpation of the genital tract per rectum and vaginoscopic examination, but these were of brief duration and the normal resting pattern of activity was quickly re-established after completion of the manipulations.     

Plasma progesterone concentrations, ovarian and endocrinological response and sperm transport in ewes with synchronized oestrus

Two experiments involving 24 and 54 Australian Merino ewes were conducted in which the establishment of a cervical population of spermatozoa and several endocrinological events were studied after several regimens for the synchronization of oestrus. Intravaginal sponges impregnated with 500 mg (Exp. 1) or 200, 400 or 600 mg (Exp. 2) progesterone resulted in the maintenance of plasma progesterone concentrations of 1.5-4.9 ng/ml over a 12-day insertion period compared with 1.9-6.9 ng/ml during dioestrus in control ewes. In Exp. 1 basal concentrations of less than or equal to 0.25 ng/ml plasma were attained by 4 h after sponge withdrawal and this decline was much more rapid than in normal luteolysis. This was associated with fewer spermatozoa recovered from the cervix 2 h after insemination, and PMSG had no significant effect. In Exp. 2 injection of a supplementary dose of progesterone at sponge withdrawal resulted in a rapid increase in plasma progesterone concentrations followed by an equally rapid decrease and an attenuation of the rise in plasma oestradiol-17 beta, the LH surge, and the onset of oestrus. The numbers of spermatozoa recovered 4 h after insemination were not increased, and PMSG had no significant effect. Two factors were significant, namely the dose of progesterone in the sponge (600 mg greater than 400 or 200 mg, P less than 0.05) and stage of oestrus when inseminated (mid- or late oestrus greater than early). The data demonstrated that an adequate dose of progesterone/progestagen incorporated into intravaginal sponges and accurate timing of insemination relative to the LH surge are the most important factors involved in penetration of the cervix by spermatozoa.     

Prolactin as a factor in the uterine response to progesterone in rabbits

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.     

Porcine spermatozoa inhibit post-breeding cytokine induction in uterine epithelial cells in vivo

Early endometrial cytokine responses after exposure to various inseminate components were investigated for a better understanding of the immunological reactions occurring in the porcine uterus after insemination. Baseline values were established for the mRNA concentrations of GM-CSF, IL-6, IL-10, CXCL8 (interleukin-8), Tumour Necrosis Factor α (TNF-α), TGF-β, cyclooxygenase-2 (COX-2) and arachidonate 5-lipooxygenase (ALOX-5) in periovulatory uterine endometrial tissue using quantitative RT-PCR. Synchronized gilts were inseminated with spermatozoa diluted either in the semen extender Androhep™ or seminal plasma. Uterine infusions of media without spermatozoa were used as controls. Three hours after insemination sows were slaughtered and the expression of the above mentioned cytokines was measured in uterine epithelial cells. Simultaneously, the influx of polymorphonuclear neutrophilic (PMN) granulocytes into the uterus was quantified. Compared to baseline values seminal plasma (SP) and Androhep™ (AH) respectively, if used alone, caused a significant increase in mRNA concentrations of IL-10 (SP: 1.5-fold), TGF-β (AH: 1.5-fold), CXCL8 (AH: 7.1-fold), TNF-α (AH: 1.9-fold) and COX-2 (AH: 7-fold). Surprisingly, in the presence of spermatozoa, none of the tested cytokines revealed mRNA concentrations higher than baseline values. The number of immigrated, intra-luminal PMN correlated only with mRNA concentrations of CXCL8 in presence of Androhep™ (r = 0.51). None of the other cytokines tested seemed to be involved in the regulation of neutrophil recruitment. However, the most interesting result was the sperm-induced down-regulation in the expression of TNF-α, TGF-β, IL-10, CXCL8 and COX-2 to mRNA concentration levels similar to or even below baseline values. In conclusion the results show that CXCL8 contributes significantly to uterine PMN recruitment and indicate a so far underestimated role of porcine spermatozoa in the general regulation of the uterine post-mating inflammatory response.     

Monitoring ovarian function and pregnancy by evaluating excretion of urinary oestrogen conjugates in semi-free-ranging Przewalski's horses (Equus przewalskii).

Immunoreactive urinary oestrogen conjugates were assessed in daily urine samples (approximately 5 samples/week) collected from 8 Przewalski's mares maintained under semi-free-ranging pasture conditions. The relative percentage contributions of immunoreactive urinary oestrogens during different reproductive stages (oestrus, luteal phase, early, mid- and late gestation) were determined using high-pressure liquid chromatography. In general, conjugated forms of oestrone (oestrone sulphate and oestrone glucuronide) were the major excreted immunoreactive oestrogens in nonpregnant and pregnant Przewalski's mares. Variations in urinary oestrogen conjugates indicated that the onset of oestrous cyclicity coincided with increasing daylengths, and the non-conception oestrous cycle was 24.1 +/- 0.7 days (n = 17) in duration. Most copulations (29/35, 82.9%) were observed between Day -4 and Day +1 from the preovulatory oestrogen conjugates peak (Day 0). Based on known copulation dates, the mean gestation length was 48.6 +/- 0.4 weeks (range 47.3-50.3 weeks). During pregnancy, urinary excretion of oestrogen conjugates increased approximately 300-fold over levels in non-pregnant mares, reaching peak concentrations by Week +24 (51% of gestation). These results demonstrate that longitudinal reproductive events, including oestrous cyclicity and pregnancy, can be monitored precisely by evaluating urinary oestrogen conjugates in samples from Przewalski's mares maintained under semi-free-ranging conditions.     

The effect of oestrogen administered during the progestational phase of the cycle on transport of spermatozoa in ewes.

Two split-plot factorial experiments are described, the first with 72 entire cyclic ewes and the second with 80. The pattern of transport of spermatozoa through the reproductive tract was studied, following treatments with progestagen and oestrogen or with oestrogen alone during 2 weeks preceding insemination. A daily dose of 25 mug oestradiol-17 beta administered to ewes for 14 days preceeding oestrus had a deleterious effect on the passage of spermatozoa through the cervix into the uterus within the first 2 hr after insemination. The numbers of spermatozoa recoverable from the cranial region of the cervix 2 hr after insemination appeared to be related to the numbers in the oviducts at 24 hr. These numbers were related to fertility data from an earlier experiment using similar treatments. The data for log numbers of spermatozoa recoverable from the cervix formed a near-normal distribution and so were suitable for formal statistical analysis. There was an interaction between progestagen and oestrogen influence before mating on the pattern of sperm transport through the cervix.     

Metabolism of [4-14C]oestradiol by oestrogen-induced uterine peroxidase

1. An enzyme that catalyses the metabolism and binding of [4-(14)C]oestradiol to protein and to other high-molecular-weight substances in the presence of H(2)O(2) was shown to be absent from the uteri of immature rats and to be induced by physiological doses of oestrogen or pregnant-mare-serum gonadotrophin. 2. The pH optimum, stability to heat and other characteristics of the uterine enzyme system as well as its subcellular distribution were determined. 3. The increase in the ability of uterine preparations to convert [4-(14)C]oestradiol into water-soluble products as a result of oestrogen treatment was accompanied by an increase in peroxidase and NADH oxidase activities and was inhibited by actinomycin D and cycloheximide. 4. The results support the proposal that the increase in peroxidase activity after oestrogen treatment might be part of an adaptive response of the uterus permitting it to bind and inactivate oestrogens and thus limit the duration of their effect upon this target tissue.     

New Concepts on the Action of Oestrogens in the Uterus and the Role of the Eosinophil Receptor System

Two receptor systems for oestrogens have been demonstrated in the uterus: the cytosol-nuclear receptor system and the eosinophil receptor system. It has been proposed that the cytosol-nuclear receptor system mediates the genomic response to oestrogens in the uterus, while the eosinophil receptor system is thought to mediate the uterine edema and other early oestrogenic responses in the uterus. Cortisol is known to decrease drastically the number of eosinophils in the blood and therefore to limit their availability for migration to the uterus. The present results show that cortisol also drastically reduces both the oestrogen-induced uterine eosinophilia and the uterine wet weight responses, but does not interfere with the oestrogen-induced uterine RNA and protein increases. Oestradiol-17 beta has a higher affinity than oestriol for the cytosol-nuclear receptors and is now found to be the more potent oestrogen in inducing the genomic activation in the uterus. Estriol has a higher affinity than oestradiol-17 beta for the eosinophil receptors, and therefore, oestriol is the stronger oestrogen in inducing those oestrogenic effects which are mediated by the eosinophil receptor system. We conclude that the eosinophil receptor system for oestrogens is a new system, independent of Jensen's cytosol-nuclear receptor system, and this eosinophil receptor system is involved in the mechanism of oestrogen action in the uterus.     

Oestrogen pattern during early pregnancy in the mare.

Plasma total (conjugated + unconjugated) oestrogens were measured from Day 0 to 100 of pregnancy and compared with the levels found during the oestrous cycle. From Day 0 to 35 of gestation, the concentrations were similar to those during dioestrus. An increase in total oestrogens between Days 35 and 40 was followed by a plateau of 3 ng/ml between Days 40 and 60 which was slightly higher than preovulatory concentrations. This first increase in total oestrogen level was produced by the ovaries since values were suppressed after ovariectomy; stimulation may be due indirectly to PMSG causing follicular growth. After Day 60, a second increase was detected and considered to be of feto-placental origin as the levels at this time were not suppressed after ovariectomy. By Day 85, oestrogen concentrations exceeded thos detected in non-pregnant mares so that a direct measurement of total oestrogens in plasma by a simplified radioimmunoassay after Day 85 of gestation offers a reliable method of pregnancy diagnosis.