A number of morphological and biochemical changes occur as the cells of the recently ovulated follicle luteinize and develop into a functional CL. There are two distinct steroidogenic luteal cell types that appear to differentiate from thecal and granulosal cells in the follicle. The control of progesterone secretion is quite different in the two cell types. Prostaglandin F2 alpha (PGF2 alpha) is the primary luteolytic hormone in most mammals. PGF2 alpha appears to exert its antisteroidogenic actions via activation of the protein kinase C system, while its cytotoxic effects appear to be mediated via a dramatic increase in intracellular levels of free calcium. The mechanisms involved in maternal recognition of pregnancy are very diverse between species and may involve direct luteotropic stimulation of the CL, reduced uterine secretion of PGF2 alpha, and/or inhibition of actions of PGF2 alpha at the level of the CL.

The effect of interleukin-3 (IL-3) on conceptus production of ovine interferon tau (olFNτ) was examined using two dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE), western blot, northern/slot blot,in situ hybridization and immunohistochemical methodologies. Ovine conceptuses (day 17,n=14) were culturedin vitro for 24 h in Eagle's Minimum Essential Medium (MEM) containing various doses of human recombinant interleukin-3 (hIL-3). At 75 and 150 colony forming units (units)/ml hIL-3, amounts of oIFNτ released into the culture media increased 3-8-fold over controls while at 300 units/ml (60 pM), hIL-3 did not enhance oIFNτ production. These variations in production of oIFNτ parallel amounts of oIFNτ mRNA detected by northern and slot blot analyses. Ovine IL-3 mRNA was primarily localized at luminal and glandular epithelia with minor staining at stromal regions of uterine endometria collected from day 17 pregnant ewes but its polypeptide was localized in the luminal epithelial region. However, such signals were not detected in day 17 conceptuses. Ovine IL-3 mRNA and its polypeptide were also localized at the same regions of the endometrium obtained from cyclic ewes on days 8 and 12. The effect of hIL-3 on the enhancement of oIFNτ productionin vitro could be mimicked by the addition of phorbol 12-myristate 13-acetate (PMA). The present observations demonstrate that the hemopoietic cytokine, IL-3, stimulates conceptus production of oIFNτin vitro and that ovine IL-3 and its mRNA are localized in the maternal endometrium. These results suggest that a cytokine, IL-3, produced by the maternal endometrium, is involved in conceptus production of oIFNτ which may result from the activation of a second messenger system, protein kinase C.

The association between uteroferrin (UF, measured as acid phosphatase [AP] activity), retinol-binding protein (RBP), and transferrin (TF), within the intrauterine environment during the estrous cycle and pregnancy was examined. Pregnant gilts were killed on Days 7, 10, 13, 16, 19, 25, 30, 40, 50, 60, 70, 80, and 90. Cyclic gilts were killed on Days 7, 10, 13, and 16. On Days 7 through 19, each uterine horn was flushed with 20 ml Minimum Essential Medium. On Days 25 through 90, serum and allantoic fluid samples were collected from each fetus within each gilt and pooled for each gilt. Uterine flushings, allantoic fluid, and fetal serum samples were assayed for AP, RBP, and TF. Endometrium was collected from all gilts and cultured with [3H]-leucine; conditioned medium was measured for nondialyzable radioactive macromolecules, AP, and RBP. In uterine flushings, AP, RBP, and TF were low on Day 7, and then increased markedly (p < 0.01) by Day 13 in both cyclic and pregnant gilts. After log transformation of the data, AP was highly correlated with RBP (r = 0.99) and TF (r = 0.91, p < 0.01). Secretion of RBP and AP by endometrium in culture also increased (p < 0.01) during this period and was similar in cyclic and pregnant gilts, and RBP and AP secretion were highly correlated (r = 0.65, p < 0.01). Endometrial tissue did not secrete detectable amounts of TF in culture. Concentrations of all three proteins were low in allantoic fluid on Day 25, after which time they increased to Day 40, and then either stabilized to Day 70 followed by a decrease to Day 90 (AP and TF) or decreased to Day 90 (RBP). Endometrial secretion of both AP and RBP increased between Day 25 and 30. Then, AP increased further to Day 40 and did not change to Day 60, while RBP did not change from Day 30 to Day 60. Both decreased from Day 60 to 70, did not change to Day 80, and then increased again to Day 90. The association of RBP and TF concentrations with AP in various maternal and conceptus compartments during pregnancy is consistent with the hypothesis that one function of these two proteins may be to protect maternal and fetal tissues from lipid peroxidation that is a possible consequence of iron transport via endometrial secretion of UF.

Experiment I was designed to determine the optimal dose of phorbol 12-myristate 13-acetate (PMA) that inhibited progesterone production when infused into the ovarian artery. The most efficacious dose of PMA was 2 mumol. Experiment II was designed to determine whether activation of protein kinase C (PKC) inhibited progesterone production without initiating luteolysis. Ewes received ovarian arterial infusions of 4 alpha-phorbol (2 mumol, n = 4), PMA (2 mumol, n = 8), or prostaglandin F2 alpha (PGF2 alpha; 1 mumol, n = 5). Concentrations of progesterone in serum decreased by 3 h in PMA-treated ewes and by 5 h in PGF2 alpha treated ewes (p < 0.05). By 48 h, serum levels of progesterone in PMA-treated ewes had returned to control values; but in PGF2 alpha-treated ewes they remained low for the duration of the experiment. Luteal weights and progesterone contents at 48 h were similar in 4 alpha-phorbol- and PMA-treated ewes but were decreased in PGF2 alpha-treated ewes (p < 0.05). Experiment III was designed to determine whether PGF2 alpha or PKC activation induced oligonucleosome formation or influenced mRNA levels for cytochrome P450sec or 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD). Ewes received treatments as in experiment II, and CL were collected at 3, 12, or 24 h (n = 3-4 per group). Luteal weights were decreased (p < 0.05) and oligonucleosome formation was increased (p < 0.05) in PGF2 alpha-treated ewes compared to controls or to PMA-treated ewes by 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of these experiments was to determine the pattern of mRNA expression for cytochrome P450 side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5,delta 4 isomerase (3 beta-HSD) during luteinization of the follicle and in ovine luteal tissue on Days 3, 6, 9, 12, and 15 of the estrous cycle. Mean concentration of mRNA for P450scc was not different in follicles collected 4 or 24 h after the onset of estrus but increased (p < 0.05) 3-fold by 48 h (corpus hemorrhagicum). With the methods used, mRNA for 3 beta-HSD was not detected until after ovulation and formation of the corpus hemorrhagicum (48 h after onset of estrus). In luteal tissue, mean concentration of mRNA for P450scc increased from Days 3 to 9 (p < 0.05) and had decreased (p < 0.05) by Day 15. Mean concentration of mRNA for P450scc was higher (p < 0.05) in small luteal cells on Day 9 than on Day 15, with values on Days 6 and 12 being intermediate. In large luteal cells, mean concentrations of P450scc mRNA increased (p < 0.05) between Days 6 and 12 and then decreased (p < 0.05) on Day 15. Mean concentration of mRNA for 3 beta-HSD was not different (p = 0.33) in luteal tissue on any day examined. In small luteal cells, mean concentrations of mRNA for 3 beta-HSD decreased between Days 6 and 15 (p < 0.05) while in large luteal cells, mean concentrations decreased (p < 0.05) between Days 12 and 15.(ABSTRACT TRUNCATED AT 250 WORDS)