Chronic alterations in ovine maternal corticosteroid levels influence uterine blood flow and placental and fetal growth

Previous work from this laboratory demonstrated that the elevation of maternal plasma corticosteroid concentrations during pregnancy is important for the support of fetal development. Reducing ovine maternal plasma cortisol concentrations to nonpregnant levels stimulates homeostatic responses that defend fetal blood volume. The present study was designed to test the hypothesis that chronic decreases or increases in maternal plasma cortisol concentration alter uterine and placental blood flow and morphology. Three groups of pregnant ewes and their fetuses were chronically catheterized and studied: ewes infused with cortisol (1 mg.kg(-1).day(-1); high cortisol), ewes adrenalectomized and underreplaced with cortisol (0.5 mg.kg(-1).day(-1); low cortisol), and control ewes. The normal increment in uterine blood flow between 120 and 130 days was eliminated in the low-cortisol ewes; conversely, uterine blood flow was increased in the high-cortisol group compared with the control group. Fetal arterial blood pressure was increased in the high-cortisol group compared with controls, but there was no increase in fetal arterial pressure from 120 to 130 days of gestation in the low-cortisol group. The fetuses of both low-cortisol and high-cortisol groups had altered placental morphology, with increased proportions of type B placentomes, and overall reduced fetal placental blood flow. The rate of fetal somatic growth was impaired in both low-cortisol and high-cortisol groups compared with the fetuses in the intact group. The results of this study demonstrate that maternal plasma cortisol during pregnancy is an important contributor to the maternal environment supporting optimal conditions for fetal homeostasis and somatic growth.     

Uterine blood flow in sows: effects of pregnancy stage and litter size

Female pigs were assigned to three groups at 94 days of age: a control group (CTR), a group undergoing the ligation and severing of the left oviduct (LIG), and a group undergoing right hysteroovariectomy (HHO). They were inseminated at 307 days of age. At 35 days of pregnancy, an ultrasonic transit time flow probe was implanted around the middle artery of one uterine horn in 33 sows and uterine blood flow was measured during thirteen 24-h periods between 44 and 111 days. Despite large differences in ovulation rate per uterine horn (4.8, 8.3 and 16.9 in the LIG, CTR and HHO groups, respectively), variation of litter size was considerably reduced with advancement of pregnancy (3.0, 6.6 and 10.8 foetuses per uterine horn at 35 days, and 3.0, 5.8 and 4.9 at 112 days (slaughter), respectively). Uterine blood flow increased linearly during pregnancy. It was lower in the LIG sows (0.82 to 1.74 L x min(-1) x horn(-1) from 44 to 111 days) than in the CTR and HHO sows (1.22 to 2.84 and 1.09 to 2.63 L x min(-1) x horn(1), respectively). It was more closely related to litter weight than to litter size and amounted to 0.42 L x min(-1) x kg foetus(-1) at 111 days. Uterine blood flow per foetus decreased when litter size increased. It increased from 0.31 to 0.72, 0.26 to 0.60 and 0.20 to 0.43 L x min(-1) x foetus(-1) from 44 to 111 days when there were 2 to 3, 4 to 5, and 6 to 8 foetuses in the uterine horn, respectively. This explains why piglets from large litters are lighter at birth.     

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.     

Unilateral luteotropic effect of uterine venous effluent of a gravid uterine horn in sheep.

The involvement of the main uterine vein in the unilateral maintenance of CL was studied in bilaterally ovulating unilaterally pregnant ewes. Ewes were mated at estrus (Day 0) and bilaterally ovulating ewes were randomized into three groups at surgery on Day 5. In all ewes, the uterine horns were separated through the intercornual area and one was ligated and transected near the internal bifurcation to produce a nongravid horn. One group served as controls (five ewes). In the other two groups the main uterine vein on one side was surgically anastomosed (end to side) to the corresponding vein of the opposite side (gravid side to nongravid side in one group--three ewes, and nongravid side to gravid side in the other--three ewes). Necropsies were done on Day 20. Mean CL weight was less, (P less than .01) on the nongravid side in control ewes than on the gravid side in control ewes or for either side in the other two groups. There were no significant differences among mean weights of CL on the gravid side in control ewes and either side in the other two groups. The CL regressed when the ipsilateral uterine vein contained blood from only the nongravid horn whereas the CL was maintained when the ipsilateral uterine vein contained venous blood from a gravid horn, whether or not it also contained blood from a nongravid horn. Results indicate that the uterine venous effluent from a gravid uterine horn in sheep has a luteotropic effect on the ipsilateral CL.     

Jugular levels of 13,14-dihydro-15-keto-prostaglandin F and progesterone around luteolysis and early pregnancy in the ewe

Six non-pregnant ewes at day 12 of the estrous cycle each had a day-12 embryo transferred into the uterine horn ipsilateral to the corpus luteum, and 4 non-pregnant ewes at day 13 each had a day-13 embryo similarly transferred. Four control ewes, 2 at day 12 and 2 at day 13 received sheep serum into the uterine horn ipsilateral to the corpus luteum. Jugular blood samples were taken at 2-hourly intervals for 3 days post-surgery, then twice-daily for a further 4 days, and the plasma radioimmunoassayed for progesterone and 13,14-dihydro-15-keto-prostaglandin F. All control ewes exhibited estrus within the expected time range and pulsatile peaks of 13,14-dihydro-15-keto-prostaglandin F occurred coincident with declining progesterone levels. With one exception, the recipient ewes had prolonged cycles and those ewes found pregnant at necropsy, 30 days after transfer, showed no progesterone decline and no pulsatile peaks of prostaglandin during days 12 to 16 after estrus. These observations suggest that the presence of the embryo at a critical stage after mating suppresses the release of uterine prostaglandin F_2α.     

Responses of the uterine circulation to sympathetic nerve stimulation

Pregnant and non-pregnant sheep uteri were perfused in situ with arterial blood at a constant flow rate. Unilateral stimulation (1-2 ma, u ms pulse) of the distal end of the severed sympathetic chain (L3-L4) at frequencies between 5 and 25 Hz produced a graded increase in uterine artery pressure in both horns. At 25 Hz, pressure in the horn ipsilateral to the stimulated sympathetic chain increased by 28 +/ 2% in four pregnant animals and 32 +/ 5% in six non-pregnant ewes. The response of the contralateral horn was significantly smaller than that of the ipsilateral horn (P less than or equal to 0.05). The response was alpha-mediated since it was abolished by local injection of dibenzyline into the middle uterine artery. The responses of the pregnant and non-pregnant animals were similar, indicating that pregnancy did not alter the alpha-adrenergic responses of the uterine vasculature.     

Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes

One or two trophoblastic vesicles (0.4-2 mm diam.) from cow (Day 14) or ewe (Day 11-13) embryos without their disc were transferred, after culture for 24 h, into recipients. Each vesicle was transferred into the uterine horn ipsilateral to the CL by the cervical route in heifers and surgically in ewes on Day 12 of the oestrous cycle. In cows, daily measurements of plasma progesterone concentrations and checks for return to oestrus showed that the CL was maintained in 8 out of 12 recipients. These 8 cows had 25- to 37-day cycles while 4 recipient heifers returned to oestrus normally. Three recipients with an extended cycle were slaughtered. The dissected uterus showed that trophoblastic vesicles had developed in the uterine horns. In ewes, the serum progesterone curve, determined in each recipient, showed that the CL was maintained in 7 out of 12 recipients. These 7 ewes had 20- to 54-day cycles and the other 5 ewes had a normal cycle of 15-19 days comparable to that of 17.0 +/- 0.5 days for the 6 control ewes. Whenever the CL was maintained, high blood progesterone levels were followed by rapid luteolysis. In cattle and sheep, therefore, a trophoblastic vesicle transferred into the uterus can develop in vivo, secreting the embryonic signals when there is no embryonic disc control and transforming the cyclic CL into a CL of pregnancy in about 60% of the cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pregnancy rates and gravid uterine parameters in single, twin and triplet pregnancies in naturally bred ewes and ewes after transfer of in vitro produced embryos

The objectives of this study were to: (1) evaluate the pregnancy rates after transfer of embryos produced in the presence or absence of epidermal growth factor (EGF) during in vitro maturation, and (2) compare several variables of the gravid uterus on day 140 after fertilization in single, twin and triplet pregnancies in ewes (n = 12) bred naturally and in ewes (n = 18) after transfer of embryos produced in vitro. Oocytes collected from FSH-treated ewes (n = 18) were collected from all visible follicles and cultured in maturation medium with or without EGF. Oocytes were then fertilized in vitro by frozen-thawed semen. On day 5 after fertilization, embryos with > or = 16 cells were transferred to recipient ewes (n = 39). In addition 12 ewes were bred naturally. Pregnancy was verified by real-time ultrasonography on day 45 or later after embryo transfer (ET) or breeding. On day 140 of pregnancy, the reproductive tract was collected from all ewes and the following parameters were determined: the number, sex, weight and crown to rump length (CRL) of fetuses, weights of gravid uterus and fetal membranes, and weight and number of placentomes. Presence of EGF in maturation medium increased (P < 0.04) cleavage rates (78% versus 59%) and percentage of > or = 16 cell embryos on day 5 after fertilization (62% versus 40%). Pregnancy rates tended to be greater (P < 0.1) after transfer of embryos matured in the presence of EGF (52%) than in the absence of EGF (39%). EGF presence in maturation medium did not affect any variables of gravid uterus or fetal weight. For single pregnancies in naturally bred ewes and ewes after ET all uterine variables were similar. For twin pregnancies, weight of gravid uterus, weight of uterus plus fetal membranes, total weight of placentomes/ewe, mean weight of individual placentome, mean weight of fetus, total fetal weight/ewe and CRL were greater (P < 0.0001-0.04) for ewes after ET than for ewes bred naturally. The weights of gravid uterus, fluid, uterus plus fetal membranes, fetal membranes, total placentomes/ewe, mean weight of individual placentome and total fetal weight/ewe were greater (P < 0.0001-0.08) for triplet pregnancies in ewes after ET than single and twin pregnancies in ewes naturally bred or after ET. The number of placentomes/fetus was greatest (P < 0.0001-0.06) in single pregnancies in ewes bred naturally and after ET fewer in twin pregnancies in ewes bred naturally and after ET and fewest in triplet pregnancies in ewes after ET. The total number of placentomes/ewe was greatest (P < 0.0001-0.06) for twin pregnancies in ewes naturally bred, fewer in single pregnancies in ewes naturally bred and twin and triplet pregnancies after ET, and fewest in single pregnancies in ewes after ET. The mean weight of fetus was greater (P < 0.0001-0.07) in single pregnancies in ewes naturally bred or after ET than in twin or triplet pregnancies in ewes naturally bred or after ET. The CRL was the lowest (P < 0.01) in twin pregnancies in ewes bred naturally. For pregnancies after natural breeding and after ET, the number of fetuses/ewe was negatively correlated (P < 0.03-0.0001) with the weight of placentomes/fetus, the number of placentomes/fetus, the mean weight of the fetus and CRL, and was positively correlated (P < 0.0001-0.05) with weight of gravid uterus, the total number of placentomes/ewe and total fetal weight/ewe. These data demonstrate that the presence of EGF in maturation medium increases the rates of cleavage and early embryonic development, and has a tendency to enhance rates of pregnancy but does not affect variables of the gravid uteri in ewes after transfer of in vitro produced embryos. Transfer of embryos produced in vitro affected some uterine variables in twin but not single pregnancies to compare with pregnancies after natural breeding. In addition, culture conditions in the present experiment did not create large offspring syndrome. The low number of placentomes/fetus seen in triple pregnancies appears to be compensated for by the increase in the weight of each individual placentome.     

Determination of gestational age in sheep and goats using transrectal ultrasonographic measurement of placentomes

Three experiments were conducted to determine gestational age in the ewe and doe by measuring placentomes with a B-mode ultrasonograph and a 5 MHz transducer. Transrectal measurements were obtained by placing the female over a bale of hay. In Experiment 1, ewes (n = 12) and does (n = 15) were examined by transrectal ultrasonography every week from breeding to parturition to determine the growth pattern of placentomes during pregnancy. In Experiment 2, placentomes from 132 ewes and 169 does were measured between 30 and 90 d of gestation. A linear regression relationship between fetal age in days and placentome size in mm was calculated and adjusted for does (gestational age = 28.74 + 1.80PL + e, r(2) = 70.34) and for ewes (age = 47.98 + 0.62PL + e, r(2) = 15.59). In Experiment 3, the placentomes of 63 does were measured to validate this relationship by using linear regression. Gestational age was determined correctly in 66% of the does, with a range of +/- 7 d and in 96% with a margin of +/- 14 d. In conclusion, transrectal ultrasonography allowed for the measurement of placentome size, which increased rapidly during the first 70 to 90 d of gestation in ewes and does. In ewes, however, there was a poor correlation of placentome size with gestational age, while in goats, measurement of placentomes could be used along with pregnancy diagnosis by transrectal ultrasonography as an indication of gestation age.     

Effects of heating the testes and epididymides of rams by scrotal insulation on fertility and embryonic mortality in ewes inseminated with frozen semen.

Fertilization rate and embryonic mortality were assessed in 636 ewes inseminated in each uterine horn with 50 x 10(6) frozen spermatozoa from four control rams and from four rams submitted to a moderate (1.4-2.2 degrees C), but repeated, intermittent (16 h/day for 21 consecutive days) increase in their subcutaneous scrotal temperature by means of scrotal insulation. Pregnancy was assessed twice in each ewe from concentration of progesterone in blood plasma at 17 days and by ultrasound at 65 days after insemination. No differences were observed in the pregnancy rate at 17 days between ewes inseminated with semen collected from control rams (56.0, 65.2, 66.7 and 60.3%) and from heated rams (60.6, 71.8, 63.6 and 48.2%) before or after 4, 15 and 21 days of heating, respectively. In contrast, the rate of embryonic mortality between 17 and 65 days after insemination was significantly higher at days 4, 15 and 21 in the heated rams (78.7, 78.6 and 93%) than in the control rams (55, 59 and 65.7%). These results indicate that an intermittent slight, but repeated, increase in the subcutaneous scrotal temperature could induce a significant increase in the embryonic mortality rate. As these changes were apparent on day 4 of heating, an effect must have occurred on sperm stored in the epididymis.     

Triplex Doppler evaluation of uterine arteries in cyclic and pregnant domestic cats

The aims were to determine resistance index (RI) and pulsatility index (PI) in the uterine arteries of cyclic and pregnant domestic cats comparing the left and right uterine horns, as well as the majority or minority uterine horns, based on fetus number per horn; to determine the presence or absence of an early diastolic notch (EDN) in the uterine artery of pregnant queens. Ten domestic cats were followed during one cycle and one pregnancy until 63rd days after mating. The estrous cycle length was 16 ± 9.57 days. The uterine horn with the highest number of fetuses (majority uterine horn - MUH) presented 2.0 ± 1.0 fetus and the lower (minority uterine horn - miUH) presentes 0.78 ± 0.67 fetus. There were no differences in indexes between uterine arteries during the cycles and pregnancies. The RI and PI of MUH were lower than miUH (P<0.05). Uterine artery of the MUH presented lower indexes than miUH during the acceptance period (P<0.05). On D14 of pregnancy, uterine artery presented reductions in both indexes for the miUH. On D56, the PI was reduced in the miUH. The indexes depended on the week of pregnancy. EDN was present on the uterine arteries of all cats until D35, but disappeared by D49. The blood flow varied according to the category of horn.     

Temperature scrotale et fertilite chez le belier

Numerous animal studies have shown that elevated testicular, scrotal or ambient temperature induces alterations in spermatogenesis that reduce fertility. In this study, fertilization rate and embryonic mortality were assessed in 636 ewes inseminated in each uterine horn with 50 × 10⁶ cryopreserved spermatozoa from one of either four control rams or four rams submitted to a moderate (2°C), but repeated intermittent (16 hours/day for 21 consecutive days), elevation in their subcutaneous scrotal temperature by means of scrotal insulation. Pregnancy was assessed twice in each ewe at 17 days (blood plasma progesterone) and 65 days (ultrasound) after insemination. No differences were observed in the 17-day pregnancy rate between ewes inseminated with semen from control or experimental rams at up to 21 days of scrotal heating. In contrast, the rate of embryonic mortality between 17 and 65 days post-insemination was significantly higher after 4, 15 and 21 days of treatment in the experimental rams (78.7, 78.6 and 92.7 % respectively) compared to the control rams (55.0, 59.1 and 69.4 % respectively). These results indicate that ani intermittent slight increase in scrotal temperature induces a significant increase in embryonic mortality rate. As these changes were already apparent after only 4 days of scrotal heating, the effect must have ocurred either on the epididymis or on the spermatozoa stored in the epididymis     

Effect of prostaglandin F2 alpha (PGF2 alpha) on sources of progesterone and pregnancy in intact, ovariectomized and hysterectomized 90-100 day pregnant ewes.

A single dose of 8 or 16 mg of PGF2 alpha per 58 kg body weight was injected intramuscular into intact, ovariectomized or hysterectomized 90-100 day pregnant sheep in three separate experiments. Both doses of PGF2 alpha decreased the weights of the corpora lutea (P less than or equal to 0.05) and the concentration of progesterone in ovarian venous plasma at 72 hr (P less than or equal to 0.05) compared to the 0 hr sample within treatment groups and to control ewes at 72 hr in intact and hysterectomized pregnant ewes. In hysterectomized pregnant ewes, progesterone in jugular plasma declined (P less than or equal to 0.05) from 0 to 72 hr but never fell below 4 mg/ml and this decrease in progesterone after 8 or 16 mg PGF2 alpha was greater than in control hysterectomized ewes (P less than or equal to 0.05). There was a significant decrease in progesterone over time in jugular or uterine venous plasma in the presence of absence of the ovaries in 90-100 day pregnant ewes (P less than or equal to 0.05) but the profiles of progesterone were not different between vehicle and PGF2 alpha-treated ewes (P greater than or equal to 0.05). Uterine venous progesterone never declined below 30 ng/ml in the presence or absence of the ovaries and there was a significant quadratic increase (P less than or equal to 0.05) in uterine venous progesterone toward the end of the 72 hr sampling period indicating an increase in steroidogenic activity of the placenta. PGF2 alpha did not affect the number of abortions in intact or ovariectomized pregnant ewes (P greater than 0.05). Thus, the corpus luteum of sheep at 90-100 days of pregnancy is functional and responsive to PGF2 alpha, placentomes are functional but do not appear to be responsive to the doses of PGF2 alpha tested and PGF2 alpha was not an abortifacient over the 72 hr treatment period.     

Effect of PGF2alpha, indomethacin, tamoxifen, or estradiol-17beta on incidence of abortion, progesterone, and pregnancy-specific protein B (PSPB) secretion in 88- to 90-day pregnant sheep

One objective of this experiment was to evaluate our hypotheses that estradiol-17beta regulates secretion of pregnancy specific protein B (PSPB) and that secretion of progesterone during pregnancy is regulated by a prostanoid by examining the effects of prostaglandin F2alpha (PGF2alpha), a luteolyic agent; indomethacin, a prostanoid synthesis inhibitor; tamoxifen, an estrogen receptor antagonist; estradiol 17-beta; and interaction of these factors on the incidence of abortion and progesterone and PSPB secretion. Another objective was to determine if there is a luteal source of PSPB. Weights of corpora lutea were decreased (P < or = 0.05) by PGF2alpha, indomethacin, PGF2alpha + tamoxifen, PGF2alpha + indomethacin, and PGF2alpha + estradiol-17beta but not (P > or = 0.05) by tamoxifen or estradiol-17beta alone. No ewe treated with PGF2alpha alone aborted (P > or = 0.05). Forty percent of ewes treated with PGF2alpha + estradiol-17beta aborted (P < or = 0.05), but ewes were not aborted by any other treatment within the 72-h sampling period. Profiles of progesterone in jugular venous blood differed (P < or = 0.05) among control, indomethacin-, tamoxifen-, and PGF2alpha + indomethacin-treated ewes. Progesterone in jugular venous blood of control ewes decreased (P < or = 0.05) by 24 h, followed by a quadratic increase (P < or = 0.05) from 24 to 62 h. Progesterone in jugular venous blood of indomethacin-, PGF2alpha-, PGF2alpha- + tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + estradiol-17beta-, and tamoxifen-treated ewes was reduced (P < or = 0.05) by 18 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Progesterone in vena cava and in uterine venous blood was reduced (P < or = 0.05) at 72 h in PGF2alpha-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + tamoxifen-, and PGF2alpha + estradiol-17beta-treated ewes. Weights of placentomes did not differ among treatment groups (P > or = 0.05). Profiles of PSPB in inferior vena cava blood differed (P < or = 0.05) among control, estradiol-17beta-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, and PGF2alpha + tamoxifen-treated 88- to 90-day pregnant ewes. Concentrations of PSPB in inferior vena cava blood were increased (P < or = 0.05) in indomethacin-, estradiol-17beta-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated 88- to 90-day pregnant ewes within 6 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Concentrations of PSPB in uterine venous blood of indomethacin-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated ewes were greater (P < or = 0.05) at 72 h than at 0 h. PSPB in ovarian venous blood did not differ (P > or = 0.05) adjacent or opposite to the ovary with the corpus luteum. It is concluded from these data that estrogen regulates placental secretion of PSPB and that a prostanoid, presumably prostaglandin E, regulates placental secretion of progesterone during 88-90 days of gestation in sheep and that there is no luteal source of PSPB.     

Effects of neonatal progestin exposure on female reproductive tract structure and function in the adult ewe

Endometrial glands are present in all mammalian uteri and produce secretions that are hypothesized to support conceptus (i.e., embryo/fetus and placental membranes) survival and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by chronic neonatal exposure to a progestin from birth, thereby producing an adult uterine gland knock-out (UGKO) phenotype. This study determined the long-term effects of neonatal progestin exposure on adult ovine reproductive tract structure and function. Neonatal ewes were exposed to norgestomet (Nor) from birth to 32 wk of age. Unexposed ewes served as controls. After puberty, adult Nor-treated (n = 6) and control (n = 6) ewes were repeatedly bred at estrus (Day 0) to intact rams of proven fertility. In contrast to a pregnancy rate of 80% for control ewes, pregnancy was never detected on Day 25 after mating (or thereafter) in bred UGKO ewes. Control and Nor-treated ewes were then bred and necropsied on Day 9. Similar numbers of hatched blastocysts were present in uterine flushings from control and Nor-treated ewes. Weights of the ovaries and cervices were not affected by treatment. No histoarchitectural differences between control and Nor-treated ewes were detected for ovaries, oviducts, cervices, or vaginae. However, uterocervical and uterine weight as well as uterine horn length were less for Nor-treated ewes. The uteri of Nor-treated ewes were devoid of endometrial glands and lacked the stromal delineation characteristic of intercaruncular endometrium in control ewes. Endometrial width, area, and lumenal epithelial length were decreased in uteri from Nor-treated ewes, but myometrial width and morphology were not affected. Expression of a number of mRNAs that are expressed predominantly in the endometrial epithelia was not different between uteri from control and from Nor-treated ewes. Collectively, these results indicate that neonatal exposure of ewes to a progestin from birth appears to only affect development of the uterus and not any extrauterine reproductive tract tissues. The infertility of the UGKO ewes appears to result from a lack of endometrial glands and, by extension, of their secretions that are required to support growth and development of peri-implantation conceptuses.     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uterine blood flow in the pregnant rabbit.

Two methods are described for the measurement of uterine blood flow in the pregnant rabbit. The first involves the use of a Parks ultrasonic Doppler probe placed over the exposed uterine artery. The second method uses a drop counter system connected between the uterine and jugular veins. The Doppler flowmeter was used to measure uterine arterial blood flow in twenty rabbits on Day 28 or 29 of pregnancy. No significant difference was observed between blood flow on these 2 days and the absolute blood flow to one horn (plus or minus S. E.) was found to be 16.8 plus or minus 1.4 ml/min, equivalent to 27.1 plus or minus 1.8 ml/100 g tissue/min. Using the drop recorder technique, the flow to one uterine horn in eleven rabbits on Day 27 or 28 of pregnancy was 12.5 plus or minus 1.9 ml/min, equivalent to 23.6 plus or minus 3.2 ml/100 g tissue/min. The pressure-flow relationship in the uterine vascular bed was studied using the Doppler flowmeter and graded mechanical occlusion of the arterial supply. Within the range of pressures studied, the flow was found to be linearly related to the arterio-venous pressure difference. This suggests that the uterine vascular bed was fully dilated under the conditions of study.     

Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle

Ovine endometrial gland development is a postnatal event that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin from birth to puberty. As adults, these neonatally progestin-treated ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype that is useful as a model for study of endometrial function. Here, objectives were to determine: 1) length of progestin exposure necessary from birth to produce the UGKO phenotype in ewes; 2) if UGKO ewes display normal estrous cycles; and 3) if UGKO ewes could establish and/or maintain pregnancy. Ewe lambs (n = 22) received a Norgestomet (Nor) implant at birth and every two weeks thereafter for 8 (Group I), 16 (Group II), or 32 (Groups III and IV) weeks. Control ewe lambs (n = 13) received no Nor treatment (Groups V and VI). Ewes in Groups I, II, III, and VI were hemihysterectomized (Hhx) at 16 weeks of age. After puberty, the remaining uterine horn in Hhx ewes was removed on either Day 9 or 15 of the estrous cycle (Day 0 = estrus). Histological analyses of uteri indicated that progestin exposure for 8, 16, or 32 weeks prevented endometrial adenogenesis and produced the UGKO phenotype in adult ewes. Three endometrial phenotypes were consistently observed in Nor-treated ewes: 1) no glands, 2) slight glandular invaginations into the stroma, and 3) limited numbers of cyst- or gland-like structures in the stroma. Overall patterns of uterine progesterone, estrogen, and oxytocin receptor expression were not different in uteri from adult cyclic control and UGKO ewes. However, receptor expression was variegated in the ruffled luminal epithelium of uteri from UGKO ewes. Intact UGKO ewes displayed altered estrous cycles with interestrous intervals of 17 to 43 days, and they responded to exogenous prostaglandin F(2 approximately ) (PGF) with luteolysis and behavioral estrus. During the estrous cycle, plasma concentrations of progesterone in intact control and UGKO ewes were not different during metestrus and diestrus, but levels did not decline in many UGKO ewes during late diestrus. Peak peripheral plasma concentrations of PGF metabolite, in response to an oxytocin challenge on Day 15, were threefold lower in UGKO compared to control ewes. Intact UGKO ewes bred repeatedly to intact rams did not display evidence of pregnancy based on results of ultrasound. Collectively, results indicate that 1) transient, progestin-induced disruption of ovine uterine development from birth alters both structural and functional integrity of the adult endometrium; 2) normal adult endometrial integrity, including uterine glands, is required to insure a luteolytic pattern of PGF production; and 3) the UGKO phenotype, characterized by the absence of endometrial glands and a compact, disorganized endometrial stroma, limits or inhibits the capacity of uterine tissues to support the establishment and/or maintenance of pregnancy.     

Transplacental diffusion in a bicornuate uterus: comparison of uterine blood flow and oxygen uptake between horns

The purpose of this study was to determine whether samples from the veins of the pregnant and the nonpregnant horn of the uterus lead to similar estimates of uterine blood flow and oxygen consumption. To accomplish this, a comparison of uterine blood flow, arteriovenous differences of oxygen content, and oxygen consumption measured by sampling the venous drainages of the two uterine horns was performed on eight pregnant sheep during the last 20 days of pregnancy. Each sheep carried a single fetus. Umbilical and uterine blood flows were measured with the test substances ethanol and antipyrine by application of the steady-state diffusion method. Twenty-three measurements of uterine blood flow comparing the two horns were not significantly different (P greater than 0.1), and were highly correlated (r = 0.98). The ratio of the oxygen content arteriovenous difference in the pregnant to that in the nonpregnant horn and the ratio of the uterine blood flow in the nonpregnant to that in the pregnant horn were significantly correlated (r = 0.7). As a consequence, paired calculations of oxygen consumption for the whole pregnant uterus had a small coefficient of variation (+/- 3.7%). These results demonstrate that the use of highly diffusible test substances for the measurement of uterine blood flow in pregnant sheep can provide accurate data for the calculation of uterine oxygen uptake, in part because the oxygen and test substance molecules are similarly affected by local variations in placental perfusion.     

Identification of relaxin in the placenta of the ewe

Sheep placentomes were collected at the abattoir and the stage of gestation was estimated from the crown-rump length and appearance of the fetus. Samples were extracted and either freeze dried (crude extracts) or fractionated on Sephadex G-50 and CM-cellulose. Relaxin immunoreactivity (RXN-IR) was detected in all samples by a pig relaxin RIA and diluted in parallel with the standard curve. Two patterns of RXN-IR were seen after Sephadex G50 purification: (a) a single main peak of RXN-IR eluting at a position similar to pig relaxin; or (b) a 3-peak pattern with additional higher (void volume) and lower (approximately 1000) molecular weight peaks. These peaks were all found with 4 different and specific antisera. The 6000 molecular weight peak eluted at a similar position to pig relaxin on CM cellulose and inhibited electrically stimulated rat uterine contractions in vitro. The amount of relaxin measured in crude extracts of placentomes from different ewes was very variable. Most samples were within the range 0.05-11.2 ng/g wet weight of tissue (3.0 +/- 0.45 (s.e.m.), n = 44) but a few contained much higher concentrations (25.5-61.4 ng/g, n = 3). There was no obvious variation in concentration with stage of pregnancy (20 days to term). Samples of intercotyledonary endometrium, allantochorion and whole ovaries from pregnant ewes were also extracted. All contained low concentrations of RXN-IR (0.6 +/- 0.13 ng/g, n = 4; 0.6 +/- 0.29 ng/g, n = 3; 1.0 +/- 0.66 ng/g, n = 7, respectively). We conclude that relaxin-like peptides are present in the pregnant ewe and that, as the placentomes are the largest component by weight, they represent the major source.