Suppressor cell activity of ovine caruncular and intercaruncular tissues during the placentation period

We assessed the suppression of lymphocyte proliferation ovine endometrial cells recovered during each trimester (Days 45, 90, and 135) of pregnancy. We evaluated fractionated and unfractionated caruncular (C) and intercaruncular (IC) cells for suppression of cocultured peripheral blood lymphocytes (PBL) in phytohemagglutinin (PHA)-treated cultures. We also evaluated cells for the release of the suppressor factor into the culture medium and tested the factor for transforming growth factor-beta (TGF-beta) activity. Suppression of PHA-induced proliferation of PBL was evident for C and IC cells recovered from each day of pregnancy, and the activity was predominantly attributed to the population(s) of low-density (1.006-1.054 g/ml) cells. The activity was greater for unfractionated C than for IC cells on Day 45, whereas the pattern was reversed by Day 135 of pregnancy. For the C cells, the activity was greatest on Day 45 and least by Day 135. Although suppressor factor was released into the medium from cultured C and IC cells, its activity was not apparently mediated by TGF-beta. In conclusion, we observed a temporal pattern in suppressor activity for unfractionated endometrial cells during pregnancy. Suppression was predominately mediated by a population(s) of low-density cells. In addition, the cells released a soluble suppressor factor that seems to be unrelated to TGF-beta. The suppressor cells may provide immunological protection to the fetoplacental unit by suppressing specific lymphocyte responses directed toward conceptus tissues.     

A morphometric analysis of the corpus luteum of the cow during the estrous cycle and early pregnancy

Corpora lutea were collected from cows on Days 6, 8, 10, 12, 14, 16, 18 and 19 of the estrous cycle and early pregnancy (n=2/d) and were examined by light microscopy. Mean lutein cell diameter was significantly (P<0.05) greater in pregnant than in cyclic cows on Days 6, 8, 10, 12, 16, 18 and 19 (cyclic versus pregnant: Day 6: 13.9 +/- 0.22 vs 14.9 +/- 0.24; Day 8: 13.8 +/- 0.20 vs 15.4 +/- 0.2; Day 10: 14.8 +/- 0.24 vs 17.4 +/- 0.24; Day 12: 13.2 +/-0.25 vs 17.9 +/- 0.31; Day 16: 13.9 +/- 0.28 vs 16.5 +/- 0.31; Day 18: 13.0 +/- 0.22 vs 16.5 +/- 09.36, and Day 19: 15.0 +/- 0.23 vs 17.6 +/- 0.33 mum, respectively). The distribution of cell sizes was leptokurtotic throughout the estrous cycle and the first 10 d of pregnancy, but tended towards bimodality after Day 14 of pregnancy. The proportion of lutein cell cytoplasm occupied by vacuoles was lower in pregnant than in cyclic cows from the 12th day post estrus, but there was a marked (P<0.05) increase in vacuolation of cells from cows undergoing luteolysis. Stainable intercellular collagen was also less abundant in pregnant than cyclic cows from the 12th day post estrus. The higher rate of progesterone secretion of pregnant, compared with cyclic cows may be attributed to the greater numbers and greater contribution to luteal mass of large lutein cells in the corpus luteum of pregnancy.     

Characterization of the uterine environment during early conceptus expansion in the bovine

Fifty-three Angus and Hereford beef cows were utilized to investigate the effect of the conceptus on uterine environment during the period of pregnancy recognition. Blood samples were collected on Days 10, 12, 14, 16 and 18. Cows were randomly assigned to be either mated on the subsequent oestrus or serve as cyclic controls. Blood samples were then collected daily from Day 10 until slaughter on Day 15, 16 or 17 from the initiation of oestrus. Uteri were flushed with physiological saline and flushings analyzed for quantitative and qualitative protein changes, calcium, oestradiol-17β and prostaglandin F content. Endometrial explants of caruncular and intercaruncular tissue, and conceptus tissue recovered from pregnant cows were cultured with [³H]-leucine to determine quantitative and qualitative polypeptide synthesis and release. Plasma progesterone concentrations were similar between pregnant and cyclic cows from Day 10 through 17. Only the uterine content of prostaglandin F significantly increased in the ipsilateral horn of pregnant cows on Days 16 and 17. This increase in prostaglandin content was related to the increase in conceptus length from 25 to 40–80 mm. Conceptus production of bovine trophoblastic protein-1 was also first clearly detectable in fluorographs of medium from conceptuses measuring 25 mm. The complexity of the polypeptides present in the medium increased with conceptus development. Polypeptide synthesis by the endometrium was similar between tissues and days; however, production of two groups of low molecular weight basic polypeptides continued to be intensified on fluorographs from the pregnant horn on Day 17 compared to cyclic cows.     

High-density ovine endometrial cells exhibit natural killer activity during early pregnancy

Natural killer (NK)-like activity was assessed for peripheral blood lymphocytes (PBL) and unfractionated and fractionated endometrial cells recovered from ewes during the estrus cycle (Days 12 to 14) and early pregnancy (Days 16 to 18). The PBL and endometrial cells (each designated as effector cells) were cocultured with chromium-51 (51Cr) labeled NK-sensitive K-562 target cells in effector:target cell ratios ranging from 25:1 to 200:1, respectively. Lytic activity (i.e., release of 51Cr into the medium) was assessed at 22 h of culture. A high-density (> or = 1.088 g/mL) population of endometrial cells from the pregnant ewes exhibited NK-like activity, whereas endometrial cells from the cyclic ewes failed to exhibit activity. Lytic activity of these cells was greater (P < 0.05) for pregnant than for cyclic ewes (12.0 and 2.1%, respectively) at the effector:target cell ratio of 100:1, respectively. For both groups of ewes, PBL exhibited NK-like activity. These data indicate that the ovine endometrium contains NK-like cells with lytic activity between Days 16 and 18 of pregnancy.     

Plasma and luteal concentrations of oxytocin in cyclic and early-pregnant cattle.

Concentrations of oxytocin were measured in corpora lutea obtained from heifers throughout the oestrous cycle and first 30 days of pregnancy. Values were low during the first 3 days of the cycle (less than 250 ng/g tissue), increasing to 1312 ng/g by Day 4. Values then further increased up to a maximum of 2344 ng/g on Day 12. Concentrations were similar in cyclic and pregnant animals throughout the midluteal phase and were maintained at approximately 1500 ng/g until the 18th (cyclic cows) or 19th (pregnant cows) day after oestrus, when they were again low. Values subsequently remained less than 250 ng/g in pregnant cattle. Concentrations of oxytocin in jugular venous plasma of cyclic (n = 5) and pregnant (n = 4) cows were measured in samples collected every 15 min for 8 h on Days 14, 16, 18 and 19 after oestrus. There were no significant differences in mean concentrations (range: 2.5-4.7 pg/ml) or in the number, frequency or area under the curve of episodes between either cyclic and pregnant animals, or between days. Mean basal concentrations were higher on Day 16 than on Day 14 (P less than 0.05), values on Days 18 and 19 being intermediate. These findings suggest that the corpus luteum contains a finite amount of releasable oxytocin, which is exhausted by Day 18-19 after oestrus, whether or not pregnancy occurs, and that there is no further accumulation of oxytocin in the animal during early pregnancy. The contribution of luteal oxytocin to jugular venous concentrations appears to be less than in sheep, in which values in the jugular vein closely parallel those within the corpus luteum.     

Characterization and immunolocalization of bovine uterine retinol-binding protein.

Endometrial explants obtained from cows between Days 13 and 29 of pregnancy were cultured for 24 h in modified minimum essential medium in the presence of [35S]methionine or [3H]leucine. Proteins synthesized and released into medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Uterine luminal flushings were obtained from cyclic cows (Days 2-20 of estrous cycle) and early pregnant cows (Days 17-22). Endometrial tissues from cows on Days 17 and 29 of pregnancy were prepared for immunocytochemistry. A uterine secretory protein, which consisted of five isoelectric variants (pI 5.3-6.1) of identical molecular mass (23,000 Da), was shown to react immunologically with antiserum raised against bovine placental retinol-binding protein (bpRBP). Limited N-terminal sequence analysis of two major isoforms showed that the protein had nearly complete homology with bovine placental and plasma retinol-binding protein (RBP) over the first 25 amino acids. Through use of bpRBP antiserum, immunoreactive RBP was detected in uterine flushings collected from cows in the late luteal phase of the estrous cycle and early pregnancy by Western blotting, and in medium conditioned by uterine explants prepared at Days 13-29 of pregnancy by immunoprecipitation. Immunoreactive RBP was localized in endometrial surface and glandular epithelium on Days 17 and 29 of pregnancy by immunocytochemistry. These results demonstrate that RBP is a product of bovine uterine tissues. The uterine RBP may play an important role in vitamin A transport between maternal tissues and developing embryos.     

Platelet-activating factor alters the dynamics of prostaglandin and protein synthesis by endometrial explants from pregnant and cyclic cows at day 17 following estrus

Factors produced by bovine conceptuses alter prostaglandin (PG) and protein secretion by endometrial explants from cyclic cows and induce an intracellular inhibitor of PG synthesis. Endometrial explants from cyclic (n = 4) and pregnant (n = 3) cows at Day 17 following estrus were incubated for 24 h with 0, 0.1, 0.5, 1 and 5 mug platelet-activating factor (PAF)/ml. Cotyledonary microsomes from parturient cows were utilized to determine levels of an intracellular/cytosolic inhibitor of PG synthesis. Endometrial explants from additional cyclic cows (n = 4) were incubated for 24 h with 0 or 5 mug PAF/ml with and without 50 muCi [(3)H]leucine. Endometrial explants (cyclic cows, n = 3) were also incubated for 12 h with each of the following treatments: 1) Control; 2) PAF (1 mug/ml); 3) lyso-PAF (2 to 10 mug/ml); 4) PAF-receptor antagonist (2 to 10 mug/ml); 5) PAF (1 mug/ml) + antagonist (2 to 10 mug/ml); 6) bovine conceptus secretory proteins (bCSP; 25 mug/ml); and 7) bCSP (25 mug/ml) + antagonist (5 mug/ml). Platelet-activating factor had distinct negative and positive dose effects on PGF and PGE-2 secretion, respectively, by explants from cyclic cows, whereas PG secretion was not altered by PAF in the endometrium of pregnant cows. Platelet-activating factor did not alter the level of an intracellular inhibitor of prostaglandin synthesis, whereas, bCSP increased the level of this inhibitor. Platelet-activating factor decreased the incorporation of [(3)H]leucine into tissue and secreted proteins for explants from cyclic cows. Lyso-PAF did not alter endometrial prostaglandin secretion. The effects of PAF but not of bCSP were blocked by the PAF-receptor antagonist. Platelet-activating factor altered PG and protein secretion by the endometrium from cyclic cows, and it may be a potential regulatory factor during early pregnancy if secreted by the bovine conceptus.     

Characteristics of prostaglandin F measurements in the ovarian circulation during the oestrous cycle and early pregnancy in the cow

Holstein or crossbred beef cows were anaesthetized on Days 15 to 17 after oestrus; the ovarian artery (OA), ovarian (utero-ovarian) vein (OV) and a peripheral artery (PA), were catheterized for chronic blood sampling. Beginning on the day after surgery, 6 sequential blood samples were collected every 30-40 min twice daily from 8 cyclic and 6 pregnant cows during Days 16-20: 818 blood samples (including 216 OA and PA concurrent arterial pairs) were collected. Overall least squares means for PGF concentrations (pg/ml) in the OV, OA and PA of cyclic cows were 562, 228 and 106, respectively. A significant (P less than 0.01) OA-PA difference (122 pg/ml) suggests that a local transfer system, between uterine venous effluent and ovarian arterial affluent, is functional in the cow. A transfer efficiency of about 1% was estimated. In cyclic cows differences in OA-PA concentrations of PGF were minimal on Days 16-18 and increased to about 160 pg/ml during luteal regression (Days 19-20). In pregnant cows a biphasic OA-PA pattern of difference in PGF between days was detected, with a peak on Day 18 (136 pg/ml) which was not apparent on Days 19-20. Amplitude of PGF spikes in the OA was significantly higher in cyclic (725 pg/ml) than in pregnant cows (397 pg/ml). We suggest that pregnancy suppresses PGF delivery to the ovarian circulation, resulting in maintenance of the corpus luteum in pregnant cows.     

Interrelationships between progesterone, 13,14-dihydro-15-keto PGF-2 alpha (PGFM) and LH in cyclic and early pregnant cows

Plasma progesterone and LH secretion patterns were examined in 18 mature dairy cows during the oestrous cycle and after insemination. Blood samples were collected every 15 min for 8 h per day on Days 3, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20 and 21 of the oestrous cycle, then, in the same cows, at the same times during early pregnancy. PGF-2 alpha secretion rates (as determined by plasma PGFM concentrations) were also monitored on Days 14, 16 and the day of, or equivalent to, luteal regression. Mean daily plasma progesterone concentrations were similar until Day 16 in cyclic and pregnant cows, after which values in non-pregnant animals declined. Regression analysis indicated that progesterone concentrations were best described by a quadratic expression with fitted maximum values on Day 13 in non-pregnant animals but values increased linearly over the whole period to Day 21 in pregnant cows. The frequency, amplitude and area under the curve of LH episodes showed no significant differences between cyclic and pregnant animals. In pregnant cows, the amplitude and area under the curve of progesterone episodes increased linearly between Days 8 and 21, although no such increase occurred in cyclic cows. Low-level PGFM episodes were present in cyclic and pregnant cows on Days 14 and 16 after oestrus, and high amplitude episodes occurred in non-pregnant cows during luteal regression. Pregnant cows showed a significant depression of the amplitude, but not the frequency of episodes at the expected time of luteal regression. These results confirm that the corpus luteum of pregnancy secretes an increasing amount of progesterone per se and per unit of LH until at least Day 21 after mating. They further suggest that the corpus luteum of the cyclic cow may experience small episodes of PGF-2 alpha and be subjected to initial degenerative changes by Day 14 after oestrus, some time before the onset of definitive luteolysis.     

Concentrations of insulin-like growth factor-I, estradiol and progesterone in follicular fluid of ovarian follicles during early pregnancy in cattle

To determine if the presence of the developing conceptus is associated with changes in intrafollicular concentrations of insulin-like growth factor-I (IGF-I), estradiol (E2) and/or progesterone during early pregnancy in cattle, either pregnant (n=16) or nonpregnant (n=15) cows were slaughtered on Day 10, 15 or 18 postestrus. Ovaries and follicular fluid were collected. Follicles were grouped by diameter: 1.0 to 3.9 mm (small; n=63), 4.0 to 7.9 mm (medium; n=128), and >/= 8.0 mm (large; n=38). The average diameter of large follicles was greater (P<0.05) in pregnant than in nonpregnant cows on Day 10, but on Day 18 it was greater (P<0.05) in nonpregnant than in pregnant cows (11.3 vs 9.7 mm). Status (pregnant vs nonpregnant) did not affect (P>0.10) follicular fluid progesterone nor IGF-I concentrations. In contrast, the status and days postestrus affected (P<0.05) follicular fluid E2 concentrations. Follicular fluid E2 levels in the three follicle size-categories on Day 10 did not differ (P>0.10) between pregnant and nonpregnant cows. However, on Days 15 and 18 postestrus, follicular fluid E2 concentrations in pregnant cows was lower (P<0.05) in large follicles than in nonpregnant cows. We conclude that the presence of a developing conceptus early in pregnancy may alter follicular growth and inhibit follicular E2 production in cattle. These effects appear to be mediated by factors other than IGF-I.     

Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts

This study was undertaken to characterize uterine immune factors involved in the establishment of pregnancy in gilts. Thirty crossbred Yorkshire-Landrace gilts of similar age and weight were observed twice a day for oestrous behaviour with intact boars. On the day of first standing oestrus (Day 0) and 12h later, 15 gilts were inseminated with pooled semen from Duroc boars of proven fertility. Pregnant gilts were slaughtered either on Days 10, 15 or 25 of gestation (n=5 per day). The other 15 gilts were not inseminated and were slaughtered on either Days 0, 10 or 15 of the oestrous cycle (n=5 per day). Immediately after slaughter, endometrial tissue samples from the mesometrial side were removed for gene expression using RNase protection assay and in situ hybridization methodologies. The other uterine horn was flushed with 20 ml of PBS to collect the uterine fluid. In pregnant gilts, endometrial interleukin (IL)-6 mRNA expression was higher on Day 15 than on Days 10 and 25 (P<0.01 and P<0.1, respectively). On Day 15, IL-6 expression was also significantly higher (P<0.01) in pregnant gilts than in cyclic gilts. In both pregnant and cyclic gilts, transforming growth factor (TGF)-beta2 in uterine fluid was significantly higher (P<0.0001) on Day 15 than on Day 10. At the gene expression level, TGF-beta2 also increased between Days 10 and 15 in both cyclic and pregnant gilts but differences were not significant. On Day 15, concentrations of interferon-gamma and prostaglandin E(2) (PGE(2)) in uterine fluid were markedly higher (P<0.001) in pregnant gilts than in cyclic gilts, whereas the total amount of TGF-beta2 in uterine fluid and its endometrial expression were approximately 70% higher although this increase was not significant. Finally, tumour-necrosis factor-alpha and granulocyte-macrophage/colony-stimulating factor mRNA expressions were undetectable in all endometrial samples. In conclusion, production and/or expression of uterine TGF-beta2, IL-6 and PGE(2) increased during the embryonic attachment period and are coincidental with embryonic interferon-gamma production.     

Relationship of uterine infections and folliculogenesis in dairy cows during early puerperium

Postpartum uterine infections have been associated with reduced fertility in dairy cows; however, the mechanism by which uterine infections limit reproductive function has not been clearly determined. The purpose of this study was to elucidate the relationship between uterine infections in the early puerperium and the onset of folliculogenesis in dairy cows. The pattern and intensity of uterine infections and follicular dynamics were studied in cows that shed fetal membranes (n = 18), and in those that retained fetal membranes after parturition (n = 18). Endometrial swabs collected aseptically from each animal on Days 4, 8 and 12 after parturition were cultured. Ultrasound scanning of the ovaries was carried out on Days 4, B and 12 using a B mode, real-time, linear array ultrasound scanner. The total number of follicles was recorded, and the follicles were classified according to size as small (2 to 4 mm) or medium (5 to 7 mm).

The severity of infection was higher (P<0.05) in retained placenta cows on Days 4, 8 and 12 compared to nonretained placenta cows. The total number of follicles was larger (P<0.05) in nonretained placenta cows on Days 4, 8 and 12 than in retained placenta cows. The distribution of different sizes of follicles on all days of observations was similar in both groups of cows (P>0.05). These data support the hypothesis that uterine infection may delay the initiation of folliculogenesis and suppress the rate of follicular growth in dairy cows in the immediate postpartum period.     

Luteal blood flow increases during the first three weeks of pregnancy in lactating dairy cows

The objectives of this experiment were to characterize luteal blood flow in pregnant and non-pregnant cows and to determine its value for early pregnancy diagnosis. Lactating dairy cows (n = 54), 5.2 ± 0.2 y old (mean ± SEM), average parity 2.4 ± 0.2, and ≥ 6 wk postpartum at the start of the study, were used. The corpus luteum (CL) was examined with transrectal color Doppler ultrasonography (10.0-MHz linear-array transducer) on Days 3, 6, 9, 11, 13, 15, 18, and 21 of the estrus cycle (estrus = Day 0). Artificially inseminated cows (n = 40) were retrospectively classified as pregnant (embryonic heartbeat on Day 25; n = 18), nonpregnant (interestrus interval 15 to 21 d, n = 18), or having an apparent early embryonic loss (interestrus interval >25 d, n = 4). There was a group by time interaction (P < 0.001) for luteal blood flow from Days 3 to 18; it was approximately 1.10 ± 0.08 cm² (mean ± SEM) on Day 3, and increased to approximately 2.00 ± 0.08 cm² on Day 13 (similar among groups). Thereafter, luteal blood flow was numerically (albeit not significantly) greater in pregnant cows, remained constant in those with apparent embryonic loss, and declined (not significantly) between Days 15 and 18 in nonpregnant cows. Luteal blood flow was greater in pregnant than in nonpregnant (P < 0.05) and nonbred cows (P < 0.05, n = 14) on Day 15 (2.50 ± 0.16, 2.01 ± 0.16, and 2.00 ± 0.18 cm², respectively) and on Day 18 (2.40 ± 0.19, 1.45 ± 0.19, and 0.95 ± 0.21 cm²). In cows with apparent early embryonic loss, luteal blood flow was 2.00 ± 0.34 and 2.05 ± 0.39 cm² on Days 15 and 18, which was less (not significantly) than in pregnant cows, but greater (P < 0.05) than in nonbred cows on Day 18. Although mean luteal blood flow was significantly greater in pregnant than nonpregnant (and nonbred) cows on Days 15 and 18, due to substantial variation among cows, it was not an appropriate diagnostic tool for pregnancy status.     

Changes in vascular perfusion of the endometrium in association with changes in location of the embryonic vesicle in mares

The equine embryonic vesicle is mobile on Days 12-14 (Day 0 = ovulation), when it is approximately 9-15 mm in diameter. Movement from one uterine horn to another occurs, on average, approximately 0.5 times per hour. Mobility ceases (fixation) on Days 15-17. Transrectal color Doppler ultrasonography was used to study the relationship of embryo mobility (experiment 1) and fixation (experiment 2) to endometrial vascular perfusion. In experiment 1, mares were bred and examined daily from Day 1 to Day 16 and were assigned, retrospectively, to a group in which an embryo was detected (pregnant mares; n = 16) or not detected (n = 8) by Day 12. Endometrial vascularity (scored 1-4, for none to maximal, respectively) did not differ on Days 1-8 between groups or between the sides with and without the corpus luteum. Endometrial vascularity scores were higher (P < 0.05) on Days 12-16 in both horns of pregnant mares compared to mares with no embryo. In pregnant mares, the scores increased (P < 0.05) between Day 10 and Day 12 in the horn with the embryo and were higher (P < 0.05) than scores in the opposite horn on Days 12-15. In experiment 2, 14 pregnant mares were examined from Day 13 to 6 days after fixation. Endometrial vascularity scores and number of colored pixels per cross-section of endometrium were greater (P < 0.05) in the endometrium surrounding the fixed vesicle than in the middle portion of the horn of fixation. Results supported the hypothesis that transient changes in endometrial vascular perfusion accompany the embryonic vesicle as the vesicle changes location during embryo mobility.     

Characterization of proteins produced in vitro by bovine endometrial explants

Endometrial tissues were obtained from 17 pregnant (P, estrus/mating = Day 0; Day 16, n = 4; Day 19, n = 6; Day 22, n = 3; Day 24, n = 4) and six nonpregnant (NP; Day 16, n = 4; Day 19, n = 2) cattle, as well as from one cyclic (nonbred) cow (Day 4), one later-pregnant cow (Day 69), and both ligated and pregnant uterine horns of three unilaterally pregnant cattle (UP; Day 270). Tissues (approximately equal to 500 mg wet tissue/explant) were cultured for 24 or 48 h in modified minimal essential medium (MEM), in the presence of radioactive amino acid and/or amino sugar substrate(s) (L-[3H] leucine, L-[14C] leucine, and D-[3H] glucosamine), in order to characterize substrate uptake and de novo synthesis and release of proteins and polypeptides in vitro. Endometrial explants from all cattle produced proteins de novo from radiolabeled substrates. Chromatographic (gel filtration, cation, and anion exchange) and two-dimensional polyacrylamide gel electrophoretic analyses revealed complex patterns of primarily acidic radiolabeled polypeptides in dialyzed MEM, which were absent from endometrial tissue homogenates. No qualitative differences were noted in the array of proteins released into MEM associated with either pregnancy status (P vs. NP, UP) or stage of gestation (Days 16, 19, 22, 24, 69, and 270). Medium from all endometrial explants was enriched in polypeptides in four Mr (X 10(3)/pH classes (I, approximately equal to 14/greater than 7.2; II, 19-24/5.4-6.3; III, 28-31/6.9-7.3; and IV, greater than or equal to 150/less than or equal to 5.1).     

Ovine endometrial cells fail to lyse K-562 target cells: a preliminary investigation

The ability of unfractionated and fractionated endometrial cells to lyse K-562 target cells was investigated within ewes during the late luteal phase of the estrous cycle (Days 12 to 14) and pregnancy (Days 16 and 19). In separate experiments, lymphokine activated killer (LAK) cells and endometrial cells, both designated as effector cells, were co-cultured with chromium-51 (51Cr)-labeled K-562 target cells in varying effector: target cell ratios. At 22 h, lytic activity was assessed by the release of 51Cr into the culture medium. The LAK cells exhibited lytic activity in a ratio-dependent manner, whereas the unfractionated and fractionated endometrial cells failed to lyse the target cells. For ratios combined, the rate of cytotoxicity for unfractionated endometrial cells recovered from ewes in the cyclic and pregnant (Days 16 and 19 combined) groups was 13.9 and 5.4%, respectively. Although the findings are preliminary, they indicate that ovine endometrial cells recovered during the late luteal phase and early pregnancy failed to exhibit natural killer activity upon K-562 target cells.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.     

Immunosuppressive activity associated with early pregnancy in the bovine

Immunosuppressive activity was assessed in uterine flushings (UF) and uterine vein serum and plasma from nonpregnant and early-pregnant cows, and in media from the short-term culture of Day 18 bovine embryos. The preparations were tested for their ability to inhibit [3H] thymidine (3H-TdR) incorporation into phytohemagglutinin-stimulated bovine lymphocytes. On Days 2-3 (called Day 3), Days 9-10 (called Day 10), and Days 17-19 (called Day 18) of the estrous cycle (estrus = Day 0) and pregnancy, untreated and superovulated cows were anesthesized and jugular vein and uterine vein blood was collected. The uteri were removed and flushed to obtain UF and embryos. Uterine flushings were concentrated and tested for immunosuppressive activity at 400 micrograms uterine protein/ml culture fluid. Uterine flushings from both Day 18 pregnant and Day 18 nonpregnant cows were immunosuppressive (8/8), whereas Day 10 UF were usually not immunosuppressive (7/10). Day 3 UF were usually stimulatory or only marginally suppressive (8/8). Uterine vein serum and plasma from Day 18 cows were not suppressive when compared to jugular vein serum or plasma from the same cow; neither were Day 18 uterine vein serum or plasma suppressive when compared to those same samples taken from Day 3 cows. Embryo culture media obtained from the 48-h culture of Day 18 embryos was consistently suppressive. The activity was lost after dialysis in 1000-Mr cut-off tubing, removed by charcoal, and reduced by protease digestion. These results suggest two mechanisms whereby the embryo could escape immune rejection: 1) the progesterone-induced secretion of a uterine immunosuppressive substance(s) and 2) the production by the embryo of a low molecular weight immunosuppressive substance(s).     

Regulation of prostacyclin synthase expression and prostacyclin content in the pig endometrium

Prostaglandins (PGs) are critical regulators of a number of reproductive processes, including embryo development and implantation. In the present study, prostacyclin (PGI₂) synthase (PGIS) mRNA and protein expression, as well as 6-keto PGF_1α (a PGI₂ metabolite) concentration, were investigated in the pig uterus. Endometrial tissue and uterine luminal flushings were obtained on Days 4 to 18 of the estrous cycle and pregnancy. Additionally, conceptuses were collected and examined for PGIS mRNA expression and 6-keto PGF_1α concentration. Regulation of PGI₂ synthesis in the porcine endometrium by steroids, conceptus products, and cytokines was studied in vitro and/or in vivo. Endometrial PGIS protein level increased on Days 12 and 16 in pregnant but not in cyclic gilts. Moreover, higher PGIS protein expression on Day 12 of pregnancy was accompanied by a greater content of 6-keto PGF_1α in the endometrium. The concentration of 6-keto PGF_1α in uterine luminal flushings increased substantially on Days 16 and 18 in pregnant gilts and was higher than in cyclic animals. Greater PGIS mRNA expression and PGI₂ metabolite concentration were detected in Day 12 and 14 conceptuses, respectively. Incubation of endometrial explants with conceptus-conditioned medium resulted in upregulation of PGIS protein expression and increased PGI₂ secretion. Moreover, PGIS mRNA and protein expression were upregulated in the endometrium collected from gravid uterine horn on Day 14 of pregnancy. In summary, PGIS is differentially expressed in the endometrium of cyclic and pregnant gilts resulting in higher PGI₂ synthesis in pregnant animals. Porcine conceptuses are important regulators of endometrial PGIS expression and PGI₂ release during the implantation period.     

Suppression of lymphocyte proliferation by a greater than 30,000 molecular weight factor in horse conceptus-conditioned medium

In this experiment we have identified and partially characterized the immunosuppressive activity of preimplantation horse conceptus-conditioned medium (HCCM). Horse conceptuses were nonsurgically flushed from mares at Days 9-10 (n = 6), 15-16 (n = 3), and 25-26 (n = 3). After incubating the conceptuses for 24 h in RPMI-1640 supplemented with 15% fetal calf serum (FCS) and 1% penicillin/streptomycin, HCCM was obtained from cultures and tested for immunosuppressive activity in lymphocyte proliferation assays. Peripheral blood lymphocytes obtained from randomly selected mares were stimulated with mitogens (pokeweed mitogen [PWM], concanavalin A [Con A], and phytohemagglutinin [PHA]) in cultures supplemented with 0%, 25%, or 50% HCCM. HCCM from all cultures suppressed lymphocyte proliferation induced by all three mitogens (p less than 0.001). After being subjected to various treatments (heating, freeze-thawing, and nitrocellulose filtration), HCCM maintained its full biological suppressor activity. Amicon microconcentrators with 10,000 and 30,000 molecular weight (MW) exclusion filter membranes were used to fractionate HCCM by molecular weight. The suppressor factor was found to be in the greater than 30,000 MW fraction. HCCM was further tested interspecifically on donkey and goat lymphocytes stimulated with PWM. HCCM did suppress proliferation of interspecific lymphocytes (p less than 0.01); however, the suppressive capacity of HCCM in caprine lymphocyte cultures was less (p less than 0.05) than that observed in equine cultures. These data support the hypothesis that the horse conceptus produces an immunoregulatory factor. This factor is extremely stabile and appears to exhibit some degree of species-specificity. The production and immunosuppressive effectiveness of such a factor may play an important role in maintaining the fetal allograft throughout gestation.