Cows with two waves of follicular growth during the estrous cycle yield follicles that are older and larger at ovulation compared with cows having three waves. The objectives of the current research were 1) to compare fertility in cows with two or three follicular waves and 2) to examine associations between luteal function, follicular development, and fertility after breeding. Follicular waves were monitored by ultrasonography during the estrous cycle before insemination in 106 dairy cows. Fewer cows had three follicular waves before next estrus and ovulation than two waves (P < 0.01; 30% vs 68%, respectively), but pregnancy rate was higher (P = 0.058; 81 vs 63%, respectively). Cows with two waves had shorter estrous cycles (P < 0.01), with the ovulatory follicle being both larger (P < 0.05) and older (P < 0.01). In cows with three waves, luteal function was extended (P < 0.05) and the peak in plasma progesterone occurred later (P < 0.05) in the estrous cycle compared to two wave cows. Considering cows that became pregnant, luteal phase length was shorter (P < 0.05) during the estrous cycle preceding insemination than for nonpregnant cows. In conclusion, fertility was greater in lactating cows inseminated after ovulation of the third-wave follicle that had developed for fewer days of the estrous cycle as compared with two-wave cows.

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The corpus luteum produces progesterone, which is essential for the maintenance of pregnancy. In the absence of a viable embryo, the corpus luteum must regress rapidly to allow for development of new ovulatory follicles. In many species, luteal regression is initiated by uterine release of PGF(2alpha), which inhibits steroidogenesis and may launch a cascade of events leading to the ultimate demise of the tissue. Immune cells, primarily macrophages and T lymphocytes, are present in the corpus luteum, particularly at the time of luteolysis. The macrophages are important for ingestion of cellular remnants that result from the death of luteal cells. However, it has also been hypothesized that immune cells are involved directly in the destruction of luteal cells, as well as in the loss of steroidogenesis; this hypothesis is reviewed in the first part of this article. An alternative hypothesis is also presented, namely that immune cells serve to abate an inflammatory response generated by dead and dying luteal cells, in effect, preventing a response that would otherwise damage surrounding ovarian tissues. Finally, the changes in immune cells that accompany maternal recognition of pregnancy and rescue of the corpus luteum are discussed briefly. Inhibition of immune cells in the corpus luteum during early pregnancy may be due to embryonic or uterine signals, or to maintenance of high progesterone concentrations within the luteal tissue.

Embryonic and fetal mortality reduce lambing rates and litter sizes, thus contributing to economic losses in the sheep industry. In the current study, the timing of late embryonic and fetal loss in ewes and the factors with which these losses were associated were examined. Ewes lambing and lambs born were compared with pregnancy diagnosis and counts of embryos by ultrasonography near d 25, 45, 65, or 85 of gestation. Approximately 19.9% of the ewes experienced late embryonic loss, fetal loss, or both; and 21.2% of the embryos or fetuses were lost from d 25 to term. Potential offspring were lost throughout gestation; 3.7% of embryos from d 25 to 45, 4.3% of fetuses from d 45 to 65, 3.3% from d 65 to 85, and 11.5% from d 85 to parturition; thus, approximately 3 to 4% of the potential offspring were lost for each 20-d period of pregnancy beyond d 25. A greater proportion of ewes lost one (36.7%) rather than all (20.5% single; 3.8% multiple) embryos or fetuses. The patterns of loss were similar in ewes mated during the anestrous season and the transitional period and did not vary with service period within breeding season or method of synchronization of estrus. Late embryonic or fetal losses were not related to the temperature-humidity index. Maternal serum collected near d 25, 45, 65, or 85 of gestation was assayed for concentrations of progesterone, estradiol-17beta , and vascular endothelial growth factor (VEGF). The proportions of embryos or fetuses lost were associated with breed type (P < 0.05), as were concentrations of progesterone (P < 0.01), estradiol (P < 0.05), and VEGF (P < 0.01). The relationships of loss or retention of pregnancy to hormonal variables at the 4 stages studied were limited. Complete and partial losses increased rapidly as maternal progesterone at d 25 decreased below 2 ng/mL (P < 0.05). Survival of fetuses within a litter from d 25 to 65 was greater for ewes with medium concentrations of VEGF near d 25 and from d 65 to parturition was greater for ewes with high concentrations of VEGF near d 45 (P < 0.05). In summary, late embryonic or fetal losses occurred from d 25 throughout gestation and varied with breed type and with concentrations of progesterone in maternal serum on d 25.

ResearchTumour necrosis factor α (TNF-α) and gamma-interferon (IFN-γ) are cytotoxic to bovine luteal cells in vitro and may contribute to cell death during luteolysis in vivo. In this study, the mechanism by which luteal cells are killed by TNF-α and IFN-γ was investigated. Luteal cells were cultured for 7 days in the presence or absence of TNF-α and IFN-γ. Inhibitors of arachidonate metabolism or scavengers of free radicals were included in the culture media. In addition, the effect of IFN-α on the viability of cytokine-treated luteal cells was tested. Lastly, untreated and cytokine-treated cells were subjected to single cell gel electrophoresis for quantification of DNA fragmentation. Neither indomethacin nor nordihydroguaiaretic acid, which are inhibitors of cyclooxygenase and lipoxygenase, respectively, were able to prevent cytokine-induced cell death. Similarly, both the phospholipase A 2 inhibitor arachidonyltrifluoromethyl ketone and the nitric oxide synthase inhibitor N G -monomethyl-L-arginine, were largely without effect. In contrast, while vitamin C did not significantly affect viability, superoxide dismutase plus catalase increased viability of cytokine-treated cells (P < 0.05), and IFN-α prevented cell death (P < 0.05). Finally, while control cells remained free of DNA damage, TNF-α plus IFN-γ induced significant amounts of DNA damage by 48 h after initiation of treatment (P < 0.05). In conclusion, reactive oxygen species, but not arachidonate metabolism or nitric oxide, contribute to cytokine-induced luteal cell death in vitro, and the process of cell death may be via apoptosis. Furthermore, IFN-α may confer protective effects against cytokine-induced cell death in bovine luteal cells.

Tumor necrosis factor-alpha (TNF-alpha) is a macrophage-derived cytokine that is also reportedly produced by granulosal cells and is localized in luteal cells. The present study employed serum-free culture of midcycle bovine luteal cells to investigate the effects of TNF-alpha, alone and with other cytokines, on luteal function. TNF-alpha (1-1000 ng/ml) produced a dose-dependent increase in prostaglandin (PG)F2 alpha and 6-keto-PGF1 alpha synthesis on all days of culture, but had no effect on basal progesterone (P4) production. TNF-alpha, in combination with other known stimulators of luteal PG synthesis, interleukin-1 beta (2.5 ng/ml) or interferon-gamma (IFN-gamma, 100 U/ml), had synergistic effects on PGF2 alpha production (greater than 50-fold above control, P less than 0.05) whereas interferon-alpha (1000 U/ml) significantly suppressed TNF-alpha-stimulated PGF2 alpha production. By day 7 of culture, TNF-alpha inhibited LH-stimulated P4 production (P less than 0.05). Luteal cell numbers were significantly reduced by IFN-gamma but not by TNF-alpha alone. However, the combination of TNF-alpha + IFN-gamma was extremely cytotoxic (only 20% of cells maintained as compared to control). Finally, TNF-alpha (100 ng/ml) enhanced the expression of Class I major histocompatibility complex antigens on cultured bovine luteal cells but did not alter IFN-gamma induction of Class II major histocompatibility complex antigens. In light of these findings, it appears that TNF-alpha, in conjunction with other cytokines, is a modulator of luteal cell function in vitro. The stimulation of PG synthesis, as well as cytotoxic effects of TNF-alpha, may suggest a role in luteolysis.

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In most mammals, prostaglandin F2alpha (PGF2alpha) is believed to be a trigger that induces the regression of the corpus luteum (CL), whereby progesterone synthesis is inhibited, the luteal structure involutes, and the reproductive cycle resumes. Studies have shown that the early growth response 1 (EGR1) protein can induce the expression of proapoptotic proteins, suggesting that EGR1 may play a role in luteal regression. Our hypothesis is that EGR1 mediates the actions of PGF2alpha by inducing the expression of TGF beta1 (TGFB1), a key tissue remodeling protein. The levels of EGR1 mRNA and protein were up-regulated in the bovine CL during PGF2alpha-induced luteolysis in vivo and in PGF2alpha-treated luteal cells in vitro. Using chemical and genetic approaches, the RAF/MAPK kinase (MEK) 1/ERK pathway was identified as a proximal signaling event required for the induction of EGR1 in PGF2alpha-treated cells. Treatment with PGF2alpha increased the expression of TGFB1 mRNA and protein as well as the binding of EGR1 protein to TGFB1 promoter in bovine luteal cells. The effect of PGF2alpha on TGFB1 expression was mimicked by a protein kinase C (PKC)/RAF/MEK1/ERK activator or adenoviral-mediated expression of EGR1. The stimulatory effect of PGF2alpha on TGFB1 mRNA and TGFB1 protein secretion was inhibited by blockade of MEK1/ERK signaling and by adenoviral-mediated expression of NAB2, an EGR1 binding protein that inhibits EGR1 transcriptional activity. Treatment of luteal cells with TGFB1 reduced progesterone secretion, implicating TGFB1 in luteal regression. These studies demonstrate that PGF2alpha stimulates the expression of EGR1 and TGFB1 in the CL. We suggest that EGR1 plays a role in the expression of genes whose cognate proteins coordinate luteal regression.