No abstract

Progesterone is suggested to be a suppressor of apoptosis in bovine luteal cells. Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells. Furthermore, apoptosis is known to be controlled by the bcl-2 gene/protein family and caspases. This study was undertaken to determine whether intraluteal progesterone (P4) is involved in Fas L-mediated luteal cell death in the bovine corpus luteum (CL) in vitro. Moreover, we studied whether an antagonist of P4 influences gene expression of the bcl-2 family and caspase-3 and the activity of caspase-3 in the bovine CL. Luteal cells obtained from the cows in the midluteal phase of the estrous cycle (Days 8-12 of the cycle) were exposed to a specific P4 antagonist (onapristone [OP], 10(-4) M) with or without 100 ng/ml Fas L. Although Fas L alone did not show a cytotoxic effect, treatment of the cells with OP alone or in combination with Fas L resulted in killing of 30% and 45% of the cells, respectively (P <0.05). DNA fragmentation was observed in the cells treated with Fas L in the presence of OP. The inhibition of P4 action by OP increased the expression of Fas mRNA (P <0.01); however, it did not affect bax or bcl-2 mRNA expression (P >0.05). Moreover, OP stimulated expression of caspase-3 mRNA (P <0.01). The overall results indirectly show that intraluteal P4 suppresses apoptosis in bovine luteal cells through the inhibition of Fas and caspase-3 mRNA expression and inhibition of caspase-3 activation.

Abstract. Hypoxia-inducible factor 1 (HIF1) has been demonstrated to have critical roles in angiogenesis via transcriptional regulation of angiogenic factors, such as vascular endothelial growth factor (VEGF). In the ovary, angiogenesis is known to occur after ovulation in the developing corpus luteum (CL) in mammals. To determine whether HIF1 participates in angiogenesis in bovine CL, the present study investigated the mRNA and protein expressions of the HIF1 alpha subunit (HIF1A) and VEGF in bovine CL during the estrous cycle. The effects of hypoxia on the expressions of HIF1A protein, VEGF mRNA and VEGF protein in bovine luteal cells were also examined by using a cell culture system. HIF1A mRNA expression was less at the regressed stage than at the other stages, whereas protein expression of HIF1A was highest at the early luteal stage and decreased thereafter. VEGF mRNA expression was highest at the developing luteal stage and decreased thereafter. VEGF protein expression was highest at the early luteal stage and decreased significantly at the regressed luteal stage. Hypoxia increased the amounts of HIF1A protein, VEGF mRNA and VEGF protein in cultured bovine luteal cells. Furthermore, we found that hypoxia inhibited progesterone production in the mid luteal cells, but not in the early luteal cells. The overall findings indicate that HIF1 is one of the factors promoting VEGF-induced angiogenesis during luteal development, and suggest that the hypoxic conditions formed after follicle rupture contribute to establishing luteal vascularization in cattle. Key words: Cattle, Corpus luteum, Hypoxia, Hypoxia-inducible factor 1, Vascular endothelial growth factor (J. Reprod. Dev. 56: [110][111][112][113][114][115][116] 2010) he corpus luteum (CL) is an organ that is temporarily formed and regressed during the female reproductive cycle. It is formed from a ruptured follicle after ovulation with rapid angiogenesis [1][2][3]. Angiogenesis is known to be stimulated by a variety of growth factors [1,2,4], one of the strongest which is vascular endothelial growth factor (VEGF) [5]. VEGF also has a role in the angiogenesis of newly formed CL in cattle [6,7]. Another factor, hypoxia-inducible factor 1 (HIF1), is a strong inducer of the transcription of erythropoietin [8] and VEGF [9]. A ruptured follicle just after ovulation is thought to be under hypoxic conditions because of bleeding and because it has an immature vasculature [10]. Therefore, we hypothesized that hypoxic conditions are important for establishing the vascular system during luteal development.HIF1 is an obligatory heterodimeric protein composed of two members of the basic-helix-loop-helix (bHLH)-containing PER-ARNT-SIM (PAS) domain family, HIF1A and the aryl hydrocarbon receptor nuclear translocator (ARNT) [11]. ARNT expression is not affected by oxygen concentration, whereas HIF1A is rapidly ubiquitinated under hypoxia, which targets the protein for degradation by the proteasome [12][13][14]. In bovine CL, HIF1 expression has been reported only in culture...

Oxygen deficiency caused by a decrease in the blood supply is known to induce various responses of cells. Because luteal blood flow has been shown to decrease during luteolysis, a low-oxygen condition seems to be an integral part of the environment during luteolysis. To determine whether a low-oxygen condition is associated with functional luteolysis, we examined the influence of reduced oxygen tension on the luteal progesterone (P4) generating system in cultured bovine midluteal cells. Luteal cells obtained from midcycle corpus luteum (d 8-12) were incubated under different O2 concentrations (20, 10, 5, 3% O2) with or without LH for 24 h. P4 production decreased with decreasing O2 concentration but was significantly stimulated by LH regardless of O2 concentration. After 8 h of culture, both basal and LH-stimulated P4 production was significantly lower under 3% O2 than under 20% O2. Low-oxygen condition also inhibited pregnenolone production. Cytochrome P450 side-chain cleavage enzyme (P450scc) mRNA expression, measured by quantitative PCR, decreased under low-oxygen condition in both non-LH-treated and LH-treated cells. Low-oxygen condition did not affect the expressions of steroidogenic acute regulatory protein mRNA or protein, whereas steroidogenic acute regulatory protein mRNA expression was stimulated by LH during 4 h of culture. Low-oxygen condition also did not affect 3 beta-hydroxysteroid dehydrogenase/Delta 5-Delta 4 isomerase mRNA expression or the activity of the enzyme in the cells, regardless of the incubation period. The overall results indicate that a low-oxygen condition decreases P4 synthesis by attenuating P450scc production and P450scc activity in bovine luteal cells and suggest that oxygen deficiency is an essential condition for the progression of luteolysis in cattle.

Low oxygen caused by a decreasing blood supply is known to induce various responses of cells, including apoptosis. The present study was conducted to examine whether low-oxygen conditions (hypoxia) induce luteal cell apoptosis in cattle. Bovine midluteal cells incubated under hypoxia (3% O(2)) showed significantly more cell death than did those incubated under normoxia (20% O(2)) at 24 and 48 h of culture, and had significantly lower progesterone (P4) levels starting at 8 h. Characteristic features of apoptosis, such as shrunken nuclei and DNA fragmentation, were observed in cells cultured under hypoxia for 48 h. Hypoxia increased the mRNA expressions of BNIP3 and caspase 3 at 24 and 48 h of culture. Hypoxia had no significant effect on the expressions of BCL2 and BAX mRNA. Hypoxia also increased BNIP3 protein, and activated caspase-3. Treatment of P4 attenuated cell death, caspase-3 mRNA expression, and caspase-3 activity under hypoxia. Overall results of the present study indicate that hypoxia induces luteal cell apoptosis by enhancing the expression of proapoptotic protein, BNIP3, and by activating caspase-3, and that the induction of apoptosis by hypoxia is partially caused by a decrease in P4 production. Because hypoxia suppresses P4 synthesis in bovine luteal cells, we suggest that oxygen deficiency caused by a decreasing blood supply in bovine corpus luteum is one of the major factors contributing to both functional and structural luteolysis.