The present study was conducted to explore the source of acetylcholine (ACH) in the corpus luteum (CL) and to test our hypothesis of an antiapoptotic role of ACH in the bovine CL and, further, to investigate whether nerve growth factor (NGF), insulin-like growth factor 1 (IGF1), and transforming growth factor beta1 (TGFB1) influence the expression of choline acetyltransferase (CHAT), the biosynthetic enzyme of ACH, in cultured bovine luteal cells. Protein expression and immunolocalization of CHAT were carried out at different stages throughout the luteal phase and in cultured luteal and endothelial cells. ACH was measured in luteal tissue at the different luteal stages and in luteal cells cultured for 8 and 24 h. Cell viability and TUNEL assays were performed on cultured midluteal cells treated with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG) in the presence of ACH and its muscarinic (atropine) and nicotinic (mecamylamine) receptor antagonists. The CL was devoid of cholinergic nerve fibers. CHAT immunostaining was evident in luteal, endothelial, and stromal cells in luteal tissue sections and in cultured luteal and endothelial cells. CHAT protein was expressed throughout the cycle without any significant changes. ACH concentration in luteal tissue was not changed during the luteal stages but increased over time and with increased cell numbers in luteal cell cultures. ACH increased cell viability and prevented cell death induced by TNF/IFNG. Atropine significantly attenuated ACH action, whereas mecamylamine had no effect. TNF/IFNG treatment downregulated CHAT expression, whereas NGF, IGF1, and TGFB1 upregulated CHAT expression, in cultured luteal cells. The overall findings strongly suggest a nonneural source and antiapoptotic role of ACH in the bovine CL. Locally produced ACH appears to be regulated by NGF, IGF1, and TGFB1.

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In mammals, the corpus luteum (CL) is an essential endocrine gland for the establishment and maintenance of pregnancy. If pregnancy is not established, the CL regresses and disappears rapidly from the ovary. A possible explanation for the rapid disappearance of the CL is that luteal cells are transported from the ovary via lymphatic vessels. Here, we report the presence of cells positive for 3β-hydroxysteroid dehydrogenase (3β-HSD), an enzyme involved in progesterone synthesis, in the lumen of lymphatic vessels at the regressing luteal stage and in the lymphatic fluid collected from the ovarian pedicle ipsilateral to the regressing CL. The 3β-HSD positive cells were alive and contained lipid droplets. The 3β-HSD positive cells in the lymphatic fluid were most abundant at days 22–24 after ovulation. These findings show that live steroidogenic cells are in the lymphatic vessels drained from the CL. The outflow of steroidogenic cells starts at the regressing luteal stage and continues after next ovulation. The overall findings suggest that the complete disappearance of the CL during luteolysis is involved in the outflow of luteal cells from the CL via ovarian lymphatic vessels.

Abstract. The objective of the present study was to investigate the potential mechanisms regulating cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic factor, in the bovine corpus luteum (CL). Expression of cFLIP mRNA was highest at the developing stage and then decreased significantly during the mid, late and regressed stages (P<0.05). Western blot analysis revealed that expression of the long isoform of cFLIP (cFLIPL) protein was high during the early and developing luteal stages, remained steady during the mid and late luteal stages and then decreased significantly (P<0.05) by the regressed stage. However, the expression levels of the short isoform of cFLIP (cFLIPS) remained low during the early, developing and mid luteal stages. Immunostaining of cFLIP was strongest in the cytoplasm of luteal and non-luteal cells, including endothelial and immune cells, remained high during the early, developing and mid luteal stages and then decreased significantly (P<0.05) in the late and regressed luteal stages. Immunostaining of cFLIP was observed only in macrophage-like cells in the regressing CL. However, cultured mid luteal cells had a higher percentage of cFLIP-positive cells and a lower percentage of TUNEL-positive cells than luteal cells treated with tumor necrosis factor α (TNF)/interferon γ (IFNG; P<0.01). These results indicate downregulation of cFLIP during structural luteal regression, suggesting that cFLIP plays a survival role in the bovine CL. Key words: Anti-apoptotic factor, Apoptosis, Bovine, Cellular FLICE-like inhibitory protein (cFLIP), Corpus luteum (J. Reprod. Dev. 56: [230][231][232][233][234][235] 2010) poptosis is a crucial mechanism in ovarian function through its significant contribution to cell deletion during follicular atresia [1][2][3][4][5] and luteal regression [6]. It is generally accepted that apoptosis is the main type of cell death during structural luteolysis in many species [7]. Apoptosis is a highly regulated process, and various factors are known to be involved in apoptosis in the corpus luteum (CL), such as hormones (progesterone), cytokines [tumor necrosis factor (TNF), interferons, interleukins] and others [BCL2 family, FAS and Fas ligand (FASLG) system, caspase family members, reactive oxygen species (nitrogen oxide, etc.)] [8]. A better understanding of pro-and anti-apoptotic factors may improve manipulation techniques of luteolysis, which is a key element in estrous cycle control in clinical bovine reproduction. Although many proteins (e.g., BCL2 family proteins, FASLG and caspases) have been demonstrated to be involved in the apoptotic cascade process during luteolysis [8], little information about one anti-apoptotic factor, cellular FLICE-like inhibitory protein, cFLIP, is available in the bovine CL.cFLIP is an important regulator of death receptor-mediated apoptosis [2,[9][10][11]. At the mRNA level, cFLIP exists as multiple splice variants, but at the protein level, it occurs in two endogenous forms, cFLIP long (cFLIPL) and cFLIP short (cFLIPS) [12]. ...

Abstract.To investigate the possible role of the vasculature in the local regulation of corpus luteum (CL) function, we determined the densities of capillaries and large blood vessels in the center of the bovine CL during the estrous cycle and following prostaglandin (PG) F2α-induced luteolysis. The CLs at the early (Days 2-3 post-ovulation), developing (Days 5-7), mid , late and regressed (Days 19-21) stages were collected. In addition, the CLs were collected by transvaginal ovariectomy from 12 cows (Day 10 after ovulation), i.e., non-treated (n=3, 0 h, control), at 0.5 (n=3), 2 (n=3) and 12 h (n=3) after injection of a luteolytic dose of PGF2α. Immunohistochemical staining with von Willebrand Factor (specific for endothelial cells that are found in both types of blood vessels) revealed that the density of the luteal blood vessels was significantly higher at the developing and late luteal stages (P<0.05) than at the other stages, whereas the number of larger blood vessels (those stained with α-smooth muscle actin) was higher at the late and regressed luteal stages (P<0.05) than at the other stages. Furthermore, both the density of blood vessels and the number of blood vessels with smooth muscle were significantly higher in the CLs obtained at 2 h and 12 h after PGF2α administration (P<0.05) than in those without PGF2α treatment. These results suggest that the number of blood vessels with smooth muscle per unit area in the regressing CL increased as a result of losing steroidogenic cells and capillaries. The overall results demonstrate that the capillaries disappeared earlier than the large blood vessels during structural luteolysis and suggest that the loss of capillaries in the CL results in a reduced supply of nutrients and oxygen to luteal cells followed by cell death. . It reaches structural and functional maturity by the mid-luteal phase and then begins to regress after Day 17 post-ovulation of the nonfertile cycle. Luteal regression in cows is known to be induced by uterine prostaglandin (PG) F2α until the CL is completely degraded. In cows, luteal regression is characterized by a reduction in P4 production (functional luteolysis) and tissue degeneration by apoptosis (structural luteolysis) [2,3].The development of the bovine CL is known to be associated with an intensive angiogenic process. The intensity of this angiogenic process in the developing CL is similar to the intensities of angiogenesis in the fastest growing tumors [4], and this results in the mature CL becoming one of the most highly vascularized organs in the body [5][6][7] and vascular endothelial cells accounting for up to 50% of the total cells of the mid CL [8,9]. Furthermore, luteal endothelial cells have been known to secrete some vasoactive substances that directly regulate P4 secretion within the CL [10][11][12]. Therefore, in order to know the mechanisms regulating the function of the bovine CL, it is important to investigate the changes in the luteal vasculature.Recently, capillaries and a few blood vessels with smooth muscle were...