Uterine contractile activity in mares during the estrous cycle and early pregnancy

Transrectal ultrasonography was used to quantitate uterine contractile activity during the estrous cycle and early pregnancy in pony mares (nonbred, n = 9; pregnant, n = 16). Continuous 1-min scans of longitudinal sections of the uterine body were videotaped, and uterine activity scores (1=minimal activity, 5=maximal activity) were assigned to each tape segment. There was a tendency (P<0.06) for a main effect of reproductive status (nonbred versus pregnant), a main effect of day (P<0.0001), and a reproductive status by day interaction (P<0.006). Uterine activity scores were higher (P<0.05) in pregnant mares on Days 1, 11, 12, and 17 (Day 0=day of ovulation) than in nonbred mares. Maximal activity in pregnant mares occurred on Days 11 to 14 during the reported period of maximal embryo mobility. Activity scores decreased (P<0.05) between the day prior to and the day of fixation (mean = Day 15) of the embryonic vesicle. Activity scores were maintained at an intermediate level for several days following fixation before declining to minimal levels by 7 d postfixation. A postovulatory decrease (P<0.04) in activity scores was observed in nonbred mares, but not in pregnant mares, between Days 0 and 1 followed by a progressive increase (P<0.03) between Days 2 and 4. Maximal activity in nonbred mares occurred during the late luteal phase (Days 13 to 14), corresponding temporally to the reported onset of luteolysis.     

Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-alpha-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12-18). However, bikunin unbound to the inter-alpha-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.     

Blood constituents during the estrous cycle and early pregnancy in dairy cows

The objective of this study was to determine if maternal platelet count, white blood cell count or other blood constituents undergo sustained alterations in concentration following fertilization. Blood samples from 17 Holstein females were collected over an 18-d period starting at estrus. Blood was analyzed for levels of platelets, white blood cells, red blood cells, hemoglobin and hematocrit. Results were analyzed for differences between nonpregnant and pregnant groups. Analysis of variance revealed a day-by-group interaction in the platelet count (P<0.01). White blood cell count showed both a day-by-group interaction and a difference between days (P<0.01). Red blood cell count, hematocrit and hemoglobin levels resulted in no significant difference between the two groups (P>0.05). While statistically significant differences were observed in platelet and white blood cell count, neither of these were sustained over a period longer than 2 d.     

Follicular development during early pregnancy and the estrous cycle of the sow

The objective of this study was to monitor and compare follicle populations and follicular development in pregnant and nonpregnant sows from Day 3 to Day 20 after breeding. Twenty-four sows were paired within parity on the day of artificial insemination and were randomly allocated within pair for insemination with either killed (n=12) or live spermatozoa (n=12). All the sows were artificially inseminated with the pooled ejaculate of the same boar. From Day 3 through Day 20 post estrus, ovarian follicles were scanned daily by ultrasonography. Ultrasound images were recorded on videotape and were retrospectively analyzed. Follicles were mapped to indentify the existence of follicular waves. The follicles were then classified as small (< 3 mm), medium (3-5 mm), or large (>/=5 mm). Pregnancy diagnosis was performed on Day 21 by ultrasonography. Pregnant sows maintained a constant proportion of the follicle population in the small, medium and large follicle categories. However, in the nonpregnant sows, the proportion of follicles in the various size categories remained constant until Day 15. Thereafter, the proportion of small follicles decreased (P < 0.05) from Day 15 to 20, and the proportions of medium and large follicles increased (P < 0.05). The predictability of pregnancy status on Day 20 based on follicle populations in any of the 3 follicle categories was low. Moreover, there was no evidence of follicular waves during the estrous cycle or early pregnancy. In conclusion, the proportion of small follicles decreased while medium and large follicle increased from Day 15 through Day 20 of the estrous cycle, but not during a similar stage of pregnancy. This latter finding concurs with follicle recruitment from the pool of small follicles for ovulation following PGF2alpha secretion to induce luteolysis, which reduces progesterone concentrations and thereby allows for the stimulation of the pool of small follicles by gonadotropins.     

Oxytocin receptors in the porcine endometrium during the estrous cycle and early pregnancy

Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using ¹²⁵I-labeled OT antagonist [d(CH₂)₅,Tyr(Me)²,Thr⁴,Tyr-NH⁹₂]-vasotocin. Binding specificity was demonstrated by displacement with four peptides related to oxytocin ([Arg⁷]-vasopressin, [Thr⁴,Gly⁷]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile³]-pressinoic acid (tocinoic acid)). The dissociation constant (K_d) of endometrial OT receptors on D0 (0.59 ± 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 ± 0.21; D15, 0.60 ± 0.14 nM; mean ± SEM). In the early luteal stage (D5), K_d (2.41 ± 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, K_d values were 3.25 ± 0.29 nM on D5 and 2.44 ± 0.44 nM on D15. Binding site concentration (B_max) on D0 (910.0 ± 25.1 fmol mg⁻¹ protein) was significantly higher than on D5 and D10 (D5, 322.5 ± 71.7; D10, 147.5 ± 25.8 fmol mg⁻¹ protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 ± 82.6; D15, 315.0 ± 20.1 fmol mg⁻¹ protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, B_max values were constant and similar to that on D5 of the early luteal stage.Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.     

Quantification of receptors for estradiol-17 beta and progesterone in the pituitary and hypothalamus of gilts during the estrous cycle and early pregnancy

Nuclear and cytoplasmic exchange assays were utilized to quantify receptors for estradiol-17 beta (E2) and progesterone (P4) in hypothalamic and pituitary tissues from 4-6 gilts each on Days 1, 5, 10, 15 and 18 of the estrous cycle and from 4-5 gilts each on Days 5, 10, 15, 21 and 30 of pregnancy. No differences in the number of cytoplasmic E2 or P4 receptors in the pituitary were found from Days 1 to 15 of the estrous cycle (P greater than 0.05). However, on Day 18, the quantities of E2 and P4 receptors were 64-fold and 25-fold lower (P less than 0.01) than those found during Days 1 to 15 of the estrous cycle. No differences in the number of nuclear receptors for E2 in the pituitary were observed from Days 1 to 18 of the estrous cycle, but nuclear receptors for P4 were 2-fold higher (P less than 0.01) on Day 1 than Days 5 to 18. In hypothalamic tissue, the numbers of cytoplasmic and nuclear receptors for E2 and P4 were lower (P less than 0.05) on Day 18 than Day 10 of the cycle. The quantity of most steroid receptors decreased between Days 15 and 18 in nonpregnant gilts as luteolysis occurred and a new follicular phase was initiated. Pregnant pigs on Days 5, 10 and 15 had decreased pituitary receptors for E2 and P4 when compared with cycling animals on these days. In general, numbers of receptors in hypothalamic tissue did not differ between pregnant and nonpregnant pigs except for decreased (P less than 0.01) nuclear P4 receptors on Day 15.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circulating concentrations of oxytocin during the estrous cycle and early pregnancy in sheep

Plasma concentrations of oxytocin and progesterone have been measured by radioimmunoassay in jugular venous blood obtained daily from 5 sheep during 2 estrous cycles and in early pregnancy.Concentrations of oxytocin were relatively high (15–30 pg/ml) during the luteal phase of the cycle, but fell at estrus (to 1–17 pg/ml). A fall in oxytocin was also observed on day 15 of pregnancy, when, as expected, progesterone levels remained high. It is suggested that raised basal levels of oxytocin are unlikely to cause the increasing uterine release of prostaglandin F_2α which occurs at the end of the estrous cycle.     

Endometrial immunoreactive beta-endorphin increases during mid-estrous cycle and early pregnancy in gilts.

Immunoreactive (ir) beta-endorphin (BEND) was recently identified in porcine uterine fluids. In the study reported here, we examined the hypothesis that porcine endometrium serves as a source of uterine fluid ir-BEND during the estrous cycle and early pregnancy. Endometrial ir-BEND was chromatographically characterized, sites of ir-BEND synthesis were immunocytochemically localized, and concentrations of endometrial ir-BEND during the estrous cycle and early pregnancy were measured. Sephadex G-50 chromatographic profiles of endometrial extracts from Day 15 of the estrous cycle revealed three distinct peaks of ir-BEND, with the first peak occurring near void volume and the second and third peaks coinciding with standard porcine beta-lipotropin and standard porcine BEND, respectively. Reverse-phase HPLC C18 chromatographic profiles indicated that endometrial ir-BEND contained both standard BEND and alpha-N-acetylated BEND. Immunocytochemical studies demonstrated ir-BEND in the surface and glandular epithelial cells of the endometrium, with immunostaining most prominent in the apical portion of epithelial cells. Concentrations of ir-BEND in endometrial tissues were higher on Days 14-15 than on Days 8-12 during the estrous cycle and pregnancy (p less than 0.05); however, values were not different in pregnant and cyclic gilts. Biochemical and immunocytochemical evidence supports our hypothesis that ir-BEND present in uterine fluids is derived from the endometrium. The increase in endometrial ir-BEND concentration during Days 14-15 in cyclic and pregnant gilts indicates that ovarian steroids may influence the synthesis of endometrial ir-BEND.     

Conjugated estrogens in the endometrium during the estrous cycle in pigs

Estrogen metabolism results in the formation of inactive estrogen sulphates and glucuronides. Despite the lack of receptor binding, circulating conjugated estrogens might serve as a reservoir for the active form through the involvement of specific cleaving enzymes. In order to elucidate the potential role that estrogen conjugates play in the regulation of the estrous cycle, we determined the concentration of progesterone, estrogen and estrogen conjugates in serum and endometrial homogenates of cycling gilts. In addition, we determined the mRNA expression changes of enzymes (UDP glucuronosyltransferase (UGT), β-glucuronidase (GUSB), sulphotransferases (SULT) and steroid sulphatase (STS)) and transporters (multidrug resistance-associated protein (MRP), organic anion-transporting polypeptide (OATPs)) involved in the estrogen metabolism in the endometrium across the estrous cycle. GUSB displayed highest expression at estrous (day 0), decreasing expression during metestrus (day 3 and 6), minimal expression on day 10 and 12, and increasing expression towards proestrus (day 18), suggesting either a stimulation by estrogens or a negative impact of progesterone. The mRNA expression of the influx-transporter OATP1A2 significantly increased from day 0 to 6 and decreased again by day 10, while the efflux-transporters (MRP1, MRP2, and MDR1) displayed minimal expression at day 3 and 6. The mRNA expression of the UDP-glucuronsyltransferases followed a similar pattern, with minimal expression found at day 6. The analyses of the concentration of local and circulating steroid hormones points towards an interaction of the analyzed transporters and enzymes with steroid hormones, thereby possibly regulating the reservoir of active steroids contributing to the endometrial function.     

Physical characteristics of the uterus during the bovine estrous cycle and early pregnancy

Uterine tone, uterine contractility and endometrial echotexture were monitored daily in heifers during the estrous cycle (n = 6; Days 0 to 21; ovulation = Day 0) and during early pregnancy (n = 7; Days 0 to 26). Uterine tone was assessed by transrectal palpation and scored from 1 (flaccid) to 5 (turgid) by an operator who had no knowledge of reproductive status, day, or group. The main effect of day was significant, but the group effect and the group-by-day interaction were not. Uterine tone scores were high during the periovulatory period (Days--1, 0, 1), decreased (P < 0.05) to low levels on Days 3 and 4, and then increased (P < 0.05) from Days 4 to 10. The increase in tone during early diestrus was confirmed (P < 0.05) in a second experiment. Uterine contractility was assessed by transrectal ultrasonography during a five-minute scan of the caudal portions of the uterine horns and scored from 1 (minimal contractility) to 4 (maximal contractility). The main effects of day and the group-by-day interaction were significant. Contractility scores in both groups were highest just before or on the day of ovulation (Days--1,0) and then decreased (P < 0.05) until Day 11. After Day 16, the scores increased (P < 0.05) in the nonbred heifers and remained low in the pregnant heifers. Endometrial echotexture scores were different among days (P < 0.0001), between the 2 groups (P < 0.02), and for the group-by-day interaction (P < 0.0001). Echotexture scores in both groups peaked just before ovulation (Day--1) and then decreased (P < 0.05) until Day 4. After Day 16, the scores increased in the nonbred group but remained low in pregnant heifers. In summary, uterine contractility and endometrial scores had similar profiles, being high during the periovulatory period and low thereafter; the levels rose in association with the end of the interovulatory interval in nonbred heifers, but remained at low levels in pregnant heifers. Uterine tone scores were also high during the periovulatory period and decreased to low levels several days postovulation, but then, in contrast with the other end points, began to increase in both the nonbred and pregnant heifers.     

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.     

Uterine luminal proteins during the preimplantation period in the ferret

The pattern of synthesis of uterine luminal proteins during the 6-day preimplantation period of the ferret has been analyzed by polyacrylamide gel electrophoresis and fluorography. The results demonstrate that several classes of protein, in particular those of molecular weights of 75,000, 58,000, 50,000, 18,000, and 8,000, are actively synthesized and appear in the uterine lumen during this period. Quantitative changes in the amount of radioactivity incorporated by specific proteins as viewed on autoradiograms are also suggested in association with the observed qualitative changes and both become more prevalent as implantation approaches.     

Uterine and ovarian blood flow during the estrous cycle in mares

Uterine and ovarian blood flow was investigated in four mares during two consecutive estrous cycles using transrectal color Doppler sonography. The uterine and ovarian arteries of both sides were scanned to obtain waves of blood flow velocity. The pulsatility index (PI) reflected blood flow. There were significant time trends in PI values of all uterine and ovarian blood vessels during the estrous cycle (P < 0.05). PI values did not differ between the uterine arteries ipsi- and contralateral to the corpus luteum or the ovulatory follicle. PI values of the uterine arteries showed a wave shaped profile throughout the estrous cycle. The highest PI values occurred on Days 0 and 1 (Day 0 = ovulation) and around Day 11, and the lowest PI values were measured around Days 5 and -2 of the estrous cycle. During diestrus (Days 0-15) PI values of the ovarian artery ipsilateral to the corpus luteum were significantly lower than PI values of the contralateral ovarian artery (P < 0.0001). No differences (P > 0.05) in resistance to ovarian blood flow occurred between sides during estrus (Days -6 to -1). In this cycle stage PI values decreased in both ovarian vessels (P < 0.05). During diestrus, high PI values of the ovarian artery ipsilateral to the corpus luteum were measured between Days 0 and 2, followed by a decline until Day 6 (P < 0.05). From this time on, the resistance to blood flow increased continuously until Day 15 (P < 0.05). The cyclic blood flow pattern in the contralateral ovarian artery was similar to that in the uterine arteries (r = 0.68; P < 0.0001). No correlations occurred between the diameter of the corpus luteum and the PI values of the ipsilateral ovarian artery (P > 0.05) during diestrus. During estrus, there was a negative relationship between growth of the diameter of the ovulatory follicle and changes in PI values of the dominant ovarian artery (r = -0.41; P < 0.05). PI values of the uterine arteries and of the ovarian artery ipsilateral to the ovulatory follicle were negatively related to estrogen (E) levels in plasma during estrus (uterine arteries: r = -0.21; P < 0.05; dominant ovarian artery: r = -0.35; P < 0.05). In diestrus, PI values of the dominant ovarian artery were negatively related to plasma progesterone levels (r = -0.38; P < 0.0001), but not the PI values of the uterine arteries (P > 0.05). The findings of this study show that there are characteristic changes in blood supply of the uterus and the ovaries throughout the equine estrous cycle. There are negative correlations between resistance to blood flow in the uterine and ovarian arteries and the plasma estrogen levels during estrus. In diestrus, there is a negative relationship between the resistance to ovarian blood flow and the progesterone levels.     

Interaction of 4-hydroxylated estradiol and potential-sensitive Ca2+ channels in altering uterine blood flow during the estrous cycle and early pregnancy in gilts

This study was conducted to define further the role of catechol estrogens (CE) as intermediates in estrogen-stimulated uterine hyperemia. Previous studies from our laboratory strongly suggest that changes in uterine blood flow (UBF) result from alterations in uterine arterial tone (distensibility) and/or contractility (reactivity to alpha 1-adrenergic agonists). Tone changes appear to set the baseline rate of flow, whereas contractility changes result in short-term reductions in luminal diameter. Changes in uterine arterial tone and contractility result from alterations in Ca2+ uptake through potential-sensitive channels (PSCs) and receptor-operated channels (ROCs), respectively. Uterine and mesenteric arteries were removed from 6 gilts at estrus (Day 0), 9 gilts on Day 13 of gestation (high estrogen, high UBF), and 8 gilts on Day 13 of the estrous cycle (low estrogen, low UBF). Arterial measurements included initial tone (baseline perfusion pressure [BPP] to a constant intraluminal flow) and increased tone after exposure to KCl, the contractility in response to the alpha 1-agonist phenylephrine, and specific uptake of 45Ca before and after exposure to the CE 4-hydroxylated estradiol (4OH-E2). Contractility of uterine arteries from Day 13 nonpregnant (NP) and Day 13 pregnant (P) gilts to phenylephrine were similar and significantly greater (p less than 0.01) than contractility of vessels from estrous gilts. The BPP and responses of uterine arteries from Day 13 NP gilts to KCl were greater (p less than 0.05) than the BPP and responses of arteries from Day 13 P and estrous gilts, which were similar to each other.(ABSTRACT TRUNCATED AT 250 WORDS)