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Ovine uterine cells that represented Day 14 cyclic and pregnant endometrium were fractionated with Percoll and evaluated for suppression of cocultured phytohemagglutinin (PHA)-induced peripheral blood lymphocyte (PBL) proliferation and for the presence of T-lymphocyte markers. Uterine cells were then evaluated for suppressor activity following the depletion of conventional lymphocyte classes (i.e., T-, B-, and NK-like) with complement + antibody treatment. In addition, supernatant (derived from cultured uterine cells) was tested for transforming growth factor beta (TGFbeta) activity using neutralization antibodies to TGFbeta. Fractionated uterine cells (density range of 1.002-1.056 g/ml) from cyclic and pregnant ewes suppressed PHA-induced proliferation of PBL, and the majority (69.5%) of these cells were < or = 5.2 microm in diameter. Percentages of CD5+, CD4+, and CD8+ lymphocytes recovered from endometrial curettage were less for cells in this density range than for cells with greater densities. Uterine cells released suppressor factor(s) into the culture medium (supernatant); however, suppressor activity was unaffected by either anti-TGFbeta or complement + antibody treatment. In conclusion, low-density uterine cells from Day 14 cyclic and pregnant ewes suppressed the proliferation of cocultured PBL and released a suppressor factor(s) into the medium that did not exhibit TGFbeta activity. It is unlikely that the suppressor cells comprise conventional T-, B-, or NK-like lymphocyte lineages.

Bovine uterine luminal protein (ULP) components (> or = 4 x 10(6) and 21,000 M(r)) were obtained from uterine flushings on Day 17 of pregnancy. Experiments were conducted to determine the capability of these components to interfere with the cytolytic activity of interleukin-2 (IL-2)-treated peripheral blood lymphocytes (PBL), designated LAK (lymphokine-activated killer) cells, upon K-562 tumor cells. Proteins (10-100 micrograms) were added at the onset of a 5-day culture period to wells containing PBL (3 x 10(6) + bovine recombinant IL-2 (12,000 IU). After culture, percentage cytotoxicity (Cyt) was assessed by the 51Cr release assay at 4 and 22 h of incubation for ratios (5:1-200:1) of LAK:K-562 cells. Percentage Cyt was affected (p < 0.0001) by time, ratio, protein treatment, and all two-way interactions. Although trends were apparent, neither ULP component affected (p > 0.05) percentage Cyt at 4 h. By 22 h of incubation, mean percentage Cyt values for the > or = 4 x 10(6) and 21,000 M(r) components at effector:target cell ratios of 150:1 (p < 0.002) and 200:1 (p < 0.0001) were less than percentages for LAK cells alone. BSA and serum protein (control preparations) each failed (p > 0.05) to affect percentage Cyt. In conclusion, both ULP components interfered with the cytolytic activity of LAK cells, presumably by the suppression of LAK cell generation.

The secretion of ovine lymphocyte suppressor factor from jugular vein (JV), uterine vein (UV), and curetted hemopoietic uterine luminal (UL) mononuclear cells was evaluated on d 14 of the cycle, following ovariectomy (OVX) and after 14 d of progesterone injections (OVX + P4, 1 mg/kg BW). Mononuclear cells (predominantly lymphocytes) were harvested by Ficoll-Paque and placed into culture (2.5 x 10(6).mL-1.well-1 in RPMI-1640). Cellular supernatants were obtained at 72 h and volumes (5 to 50 microL) were tested for suppression of phytohemagglutinin (PHA [.08 microgram l)-treated peripheral blood lymphocytes (1.0 x 10(5)). In a concurrent experiment, PHA-treated JV, UV, and UL cells (1.0 x 10(5)) were cultured singly and JV cells (1.0 x 10(5)) were also cocultured with each of 1.0 x 10(5), 5.0 x 10(4), and 2.5 x 10(4) UV and UL cells. The incorporation of thymidine into DNA was quantified at 60 h. For the cellular supernatant experiment, thymidine incorporation was affected by reproductive phase (P less than .036), lymphocyte source (P less than .0001), and phase x source (P less than .004). For UL cells, the degree of suppressor activity follows: d 14 greater than OVX greater than OVX+P4 (P less than .05). The UL supernatant from OVX+P4-treated ewes and supernatants of JV and UV cells, irrespective of reproductive phase, lacked suppressor activity. Sephacryl S-200 chromatography revealed that UL supernatant from d-14 ewes contained a greater than or equal to 248,000 molecular weight suppressor macromolecule. For the cellular coculture experiment, thymidine incorporation was affected by reproductive phase (P less than .05) and lymphocyte source (P less than .0001).(ABSTRACT TRUNCATED AT 250 WORDS)

T-lymphocytes were quantitated within luminal, stromal and glandular areas of ovine endometrium. In experiment 1, ovariectomized (OVX), estrus (E) and day 13 (D13) ewes (six/group) received 500 micrograms of phytohemagglutinin (PHA) or vehicle in ligated right and left uterine horns, respectively. At 48 h, uteri were removed for the immunohistochemical evaluation of T-lymphocyte subsets. In experiment 2, T-lymphocytes were quantitated within non-pregnant and pregnant uterine horns on day 19. For experiment 1, mean numbers of T4 and T8 lymphocytes within luminal and stromal areas of PHA-treated horns were greatest (P less than 0.05) for D13 ewes and least (P less than 0.05) for E ewes. Numbers of T6 lymphocytes for these same areas were greatest (P less than 0.05) for PHA-treated horns of OVX ewes. Overall, the T4/T8 ratio (P less than 0.004) and mean number of T19 cells (P less than 0.009) were increased by PHA. Numbers of CD45R lymphocytes were not affected by PHA but were greater (P less than 0.05) in glandular and luminal than stromal areas. For experiment 2, mean numbers of endometrial T4, T6, T8 and T19 lymphocytes were similar (P greater than 0.05) between non-pregnant and pregnant horns; however, the number of CD45R lymphocytes was greater (P less than 0.05) in endometrial tissue of pregnant than non-pregnant horns. The data indicate that the in vivo response of specific ovine T-lymphocytes to PHA was generally dependent upon reproductive stage and the presence of conceptus tissue influenced the infiltration of CD45R lymphocytes.

Uterine luminal protein (ULP) secretions collected on day 17 of bovine pregnancy contain high (greater than or equal to 248 kDa)- and low (7, 21.0, and greater than or equal to 72 kDa)-molecular weight (Mr) components that suppressed incorporation of thymidine into both phytohemagglutinin (PHA)-stimulated and interleukin-2 (IL-2)-stimulated bovine lymphocytes. The pattern of suppressor activity for ULP was similar for both PHA and IL-2 cultures. For IL-2-treated lymphocytes (2 X 10(4)/culture well), mean percentage of control (no test protein) values for 8 and 32 micrograms/ml of high Mr ULP were 94.6% and 4.5%, respectively; whereas, mean values for 8 and 64 micrograms/ml of combined low Mr ULP components were 51.2% and 5.5%, respectively. The data indicate that specific bovine ULP components may locally affect T-lymphocyte function by altering some facet of the IL-2 activation system.

Two experiments were conducted to examine seasonal changes in circulating LH concentrations in ovariectomized heifers. In experiment 1, four Holstein heifers were ovariectomized in April 1977 during middiestrus. Blood samples were collected daily for 30 days surrounding each equinox and solstice for one year to examine changes in plasma LH levels at the time of seasonal photoperiod changes. The LH concentrations were highest during the winter solstice period and lowest during the summer solstice period. In addition, samples taken at two-week intervals indicated a distinct LH profile with maximal LH concentrations during November-April and minimal concentrations during May-October. In experiment 2, eight Holstein heifers were ovariectomized in June-July, 1979 and given an estradiol or a control implant in October. A distinct LH profile for the interval extending from January, 1980 to February, 1981 was found in the heifers that were not treated with estradiol. Concentrations were maximal during December-April and minimal during May-November. The LH profile followed a similar pattern in the estradiol-treated heifers; however, the overall profile was at a higher level. These data indicate that underlying seasonal reproductive mechanisms are present in cattle even though the species ovulates and breeds throughout the year.

Uterine luminal protein (ULP) collected from ovariectomized steroid-treated crossbred heifers was tested for immunosuppressive activity in vitro. Heifers were allotted to treatment groups and for 16 d received daily injections of the following steroids or vehicle: Control (C, corn oil only, n=10); estradiol-17beta (E(2), 1.1 mug/kg body wt, n=10); progesterone (P(4), 2.2 mg/kg body wt, n=10); and E(2)+P(4) (1.1 mug + 2.2 mg/kg body wt, n=9). On Day 17, uterine flushings were collected, concentrated and quantitated for total ULP. ULP was tested for suppression of lymphocyte blastogenesis. For each experiment, 5 x 10(5) bovine lymphocytes were incubated with 0.4 mug of phytohemagglutinin (PHA) and ULP (25 to 400 mug ULP/ml) using standard culture conditions. At 48 h, 0.5 muCi of (3H) thymidine was added to cultures with cells harvested at 60 +/- 1 h by automation. Incorporated thymidine was measured by scintillation chromatography. Mean total ULP values for C-, E(2)-, P(4)- and E(2)+P(4)-treated groups were 4.7, 8.4, 13.6, and 25.5 mg, respectively (E(2)+P(4)>C and E(2), P<0.05). ULP from all treatment groups suppressed (P<0.0001) lymphocyte blastogenesis (thymidine incorporation) to PHA; however, suppression was greater (P<0.0001) for ULP from E(2)- and P(4)-than C-treated heifers at 100 and 200 mug ULP/ml. In conclusion, E(2) and P(4) injections enhanced immunosuppressive activity of ULP secretions.