Apoptosis, autophagic cell death, and necroptosis: different types of programmed cell death in bovine corpus luteum regression.

In mammals, the corpus luteum (CL) is a transient organ that secretes progesterone (P4). In the absence of pregnancy, the CL undergoes regression (luteolysis), which is a crucial preparation step for the next estrous cycle. Luteolysis, initiated by uterine prostaglandin F2α (PGF) in cattle, is usually divided into two phases, namely functional luteolysis characterized by a decline in P4 concentration and structural luteolysis characterized by the elimination of luteal tissues from the ovary. Programmed cell death (PCD) of luteal cells, including luteal steroidogenic cells (LSCs) and luteal endothelial cells (LECs), plays a crucial role in structural luteolysis. The main types of PCD are caspase-dependent apoptosis (type 1), autophagic cell death (ACD) via the autophagy-related gene (ATG) family (type 2), and receptor-interacting protein kinase (RIPK)-dependent programmed necrosis (necroptosis, type 3). However, these PCD signaling pathways are not completely independent and interact with each other. Over the past several decades, most studies on luteolysis have focused on apoptosis as the principal mode of bovine luteal cell death. Recently, ATG family members were reported to be expressed in bovine CL, and their levels increased during luteolysis. Furthermore, the expression of RIPKs, which are crucial mediators of necroptosis, is reported to increase in bovine CL during luteolysis and is upregulated by pro-inflammatory cytokines in bovine LSCs and LECs. Therefore, apoptosis, ACD, and necroptosis may contribute to bovine CL regression. In this article, we present the recent findings regarding the mechanisms of the three main types of PCD and the contribution of these mechanisms to luteolysis.

Effects of cortisol on prostaglandin F2α secretion and expression of genes involved in the arachidonic acid metabolic pathway in equine endometrium - In vitro study.

Glucocorticoids (GCs) are known to play an important role in maintaining basal and stress-related homeostasis by interacting with endocrine mediators and prostaglandins (PGs). Although a growing body of evidence shows that GCs exert their regulatory action at a multitude of sites in the reproductive axis through corticosteroid receptors, little is known about the direct role of cortisol, an active form of GCs, in the equine endometrium. Thus, the study aimed to determine the effect of cortisol on PGF2α synthesis in the endometrial tissue and cells in vitro. In Exp.1, the immunolocalization and the expression of the glucocorticoid receptor (GCR) in the endometrium throughout the estrous cycle were established. In Exp. 2 and 3, the effects of cortisol on PGF2α secretion and transcripts associated with the arachidonic acid (AA) cascade in endometrial tissues, and cells were defined. Endometrial tissues obtained from the early, mid, and late luteal phases and the follicular phase of the estrous cycle were exposed to cortisol (100, 200, and 400 nM) for 24 h. Endometrial epithelial and stromal cells (early phase of estrous cycle) were exposed to cortisol (100 nM) for 24 h. Then, PGF2α secretion and transcripts associated with the AA cascade (PLA2G2A, PLA2G4A, PTGS2, and PGFS) were assessed. GCR was expressed in the cytoplasm and the nucleus in the luminal and glandular epithelium as well as in the stroma. Endometrial GCR protein abundance was up-regulated at the late luteal phase compared to the mid-luteal phase of the estrous cycle. Cortisol dose-dependently decreased PGF2α secretion, PLA2G2A and PLA2G4A transcripts in endometrial tissues. Additionally, cortisol treatment decreased PGF2α secretion from endometrial epithelial and stromal cells. Moreover, it affected PLA2G2A, PLA2G4A, and PTGS2 transcripts in endometrial stromal cells. These findings suggest that cortisol suppresses the synthesis of PGF2α by affecting the AA cascade in the equine endometrium during the estrous cycle.

Heat stress influences the attenuation of prostaglandin synthesis by interferon tau in bovine endometrial cells.

Heat stress (HS) reduces reproductive performance of cattle, possibly by disrupting endocrine regulation such as prostaglandin (PG) production from uterus and estradiol 17ß production from the dominant follicle. Prostaglandin F2α (PGF2α) secretion from endometrium surges during the luteal phase due to tumor necrosis factor (TNF) α stimulation and a positive-feedback loop with oxytocin (OT) from the corpus luteum, ultimately triggering luteolysis, while interferon τ (IFNT) inhibits upregulation of PGF2α production by TNFα and OT, thereby preventing luteolysis and triggering recognition of pregnancy. In the present study, we investigated the effect of OT, TNFα, and IFNT on PGF2α production in both types of endometrial cells under HS conditions. Stimulation of PGF2α production in endometrial epithelial cells by OT was unaffected by HS, while stimulation of PGF2α production in endometrial stromal cells by TNFα was enhanced by HS, and this increased PGF2α production was not significantly suppressed by IFNT. These results suggest that HS disrupted the regulation of PGF2α production by TNFα and IFNT in bovine endometrial stromal cells and it might be one of causes for low conception rate of cattle in summer.

Involvement of activin signal pathway in cyclic apoptosis of the oviductal isthmic epithelium in cows.

Activin (ACV) A induces various cellular functions via activin receptor type 2 (ACVR2A/2B)-activin receptor-like kinase (ALK) 4 -Smad 2/3 pathway. Although the production of ACVA is indicated in bovine oviducts, its role on the oviduct is unclear. Oviductal isthmus needs to change its function rapidly at peri-fertilization, however, the mechanism is unknown. This study was aimed to clarify the role of ACVA in the morphological changes of oviductal isthmus in cows. First, mRNA expressions of INHBA (ACVA component) and its receptors (ALK4, ACVR2A and ACVR2B) in the isthmic tissues were examined throughout the estrous cycle. INHBA was the highest, however, ACVR2A was the lowest on the day of ovulation, suggesting reduced ACV signal transduction in the isthmus just after ovulation. Proteins of ACVRs and Smad2/3 were clearly detected in the cultured epithelial cells. It is known that ACVA regulates cellular apoptosis. Our data showed that the number of cleaved caspase-3-positive epithelial cells was largest at 2-3 days after ovulation in the isthmus. Interestingly, our study demonstrated that follistatin (ACV/TGFB/BMP inhibitor) significantly decreased the BCL2/BAX ratio in the cultured isthmic epithelial cells. To clarify which ALK pathway is involved in the regulation of BCL2/BAX ratio, the effects of SB431542 (ACV signaling (ALK4) and TGFB signaling (ALK5) inhibitor), SB525334 (ALK5 inhibitor) and LDN193189 (BMP signaling (ALK2/3) inhibitor) were investigated in the next study. The results showed that only SB431542 significantly decreased BCL2/BAX and the others had no effects. These results suggest that decreased ACVA-ACVR2A-ALK4 signal at the post-ovulation induces cyclic apoptosis of isthmic epithelial cells in bovine oviducts.

Multiple roles of hypoxia in bovine corpus luteum.

There has been increasing interest in the role of hypoxia in the microenvironment of organs, because of the discovery of hypoxia-inducible factor-1 (HIF1), which acts as a transcription factor for many genes activated specifically under hypoxic conditions. The ovary changes day by day during the estrous cycle as it goes through phases of follicular growth, ovulation, and formation and regression of the corpus luteum (CL). These phenomena are regulated by hypothalamic and pituitary hormones, sex steroids, peptides and cytokines, as well as oxygen conditions. Hypoxia strongly induces angiogenesis via transcription of a potent angiogenic factor, vascular endothelial growth factor (VEGF), that is regulated by HIF1. A CL forms with a rapid increase of angiogenesis that is mainly induced by HIF1-VEGF signaling. Hypoxia also contributes to luteolysis by down-regulating progesterone synthesis and by up-regulating apoptosis of luteal cells. This review focuses on recent studies on the roles of hypoxia- and HIF1-regulated genes in the regulation of bovine CL function.

Matrix metallopeptidase expression and modulation by transforming growth factor-ß1 in equine endometrosis.

Equine endometrial fibrosis (endometrosis) is described as a degenerative chronic condition in the uterus. Its characteristic feature is excessive deposition of extracellular matrix (ECM) components around the endometrial glands and stroma. Although matrix metallopeptidases (MMPs) that mediate ECM turnover are important factors in the process of fibrosis, knowledge of their expression and regulation in endometrosis is limited. In other species, one of the important regulators of MMPs and tissue inhibitors of MMPs (TIMPs) is transforming growth factor (TGF)-ß1. The goal of this study was to determine (i) endometrial expression of MMPs and TIMPs during endometrosis and (ii) the effect of TGF-ß1 on expression of MMPs and TIMPs in equine endometrial fibroblasts and epithelial cells. In the follicular phase of the estrous cycle, MMP-1, -2, -9, and TIMP concentrations were higher during endometrosis than in healthy endometrium (P < 0.05). In the midluteal phase, MMP-3 concentration was lower in severe endometrosis compared to healthy endometrium (P < 0.05). In fibroblasts, TGF-ß1 upregulated MMP-1, -9, -13, and TIMP1, but downregulated MMP-3 secretion (P < 0.05). In epithelial cells, TGF-ß1 upregulated MMP-1, -9, -13, and TIMP secretion (P < 0.05). Endometrial expression of MMPs and TIMPs is altered during endometrosis. TGF-ß1 is a regulator of endometrial ECM remodeling via its effect on MMPs and TIMPs in equine endometrial fibroblasts and epithelial cells.

Receptor interacting protein kinases-dependent necroptosis as a new, potent mechanism for elimination of the endothelial cells during luteolysis in cow.

Necroptosis is an alternative form of programmed cell death regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent. In the present study, to clarify if necroptosis in luteal endothelial cells (LECs) participates and contributes for bovine luteolysis, we investigated RIPK1 and RIPK3 localization in luteal tissue and their expression in cultured LECs after treatment with selected immune factors - mediators of luteolytic action of prostaglandin F2α (PGF). In addition, effects of tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM), and/or nitric oxide donor - NONOate (100 µM) on viability and CASP3 activity in the cultured LECs were investigated. Furthermore, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 µM) on RIPKs and CASPs expression, were evaluated. Localization of RIPK1 and RIPK3 protein in the cultured LECs were determined. In cultured LECs, expression of RIPKs mRNA were up-regulated by TNF + IFNG at 12 h, and by PGF (1 µM) or NONOate at 24 h, respectively (P < 0.05). Although NONOate decreased cell viability, it prevented TNF + IFNG-stimulated CASP3 activity in cultured LECs. Nec-1 prevented TNF + IFNG-induced RIPK1 and CASP3 mRNA expression at 12 h and prevented RIPK3 mRNA expression. These findings suggest that RIPKs-dependent necroptosis which are induced by TNF + IFNG, PGF or NO could be potent mechanism responsible for LECs cell death and disappearance of luteal capillaries in regressing bovine CL.

Effect of hypoxia on progesterone production by cultured bovine early and mid luteal cells.

A major role of the corpus luteum (CL) is to produce progesterone (P4). The CL has immature vasculature shortly after ovulation, suggesting it exists under hypoxic conditions. To elucidate the mechanism involved in regulation of luteal cell function during CL development, we compared the effect of hypoxia on P4 production by cultured bovine early and mid luteal cells. Luteal cells obtained from early and mid CL were incubated under different O2 concentrations (20% and 3%) with or without hCG (1 U/ml) for 6 h and 24 h. After 6 h of culture in the presence of hCG, P4 production was not affected by hypoxia whereas decrease in its production by mid luteal cells was observed. After 24 h of culture, P4 production was significantly decreased by hypoxia in both stages of luteal cells regardless of the use of hCG. At 6 h of culture, hypoxia increased mRNA expression of hydroxyl-Δ-5-steroid dehydrogenase, 3ß- and steroid Δ-isomerase 1 (HSD3B1) in early luteal cells, and decreased mRNA expression of cytochrome P450 cholesterol side chain cleavage (CYP11A1) enzyme in mid luteal cells. At 24 h of culture, mRNA expressions of steroidogenic acute regulatory protein (STAR), CYP11A1, and HSD3B1 were not affected by hypoxia in both stages of luteal cells. The overall results suggest that early luteal cells maintain P4 production under hypoxic conditions, and hypoxia-induced HSD3B1 may support this P4 production in the bovine early CL.

Heat stress affects prostaglandin synthesis in bovine endometrial cells.

Heat stress (HS) negatively affects reproduction in cattle; however, its effect on endocrine function in bovine endometrial cells remains unclear. In this study, we examined the effects of HS on the production of prostaglandin (PG) E2 and PGF2α in the cultured bovine endometrial epithelial and stromal cells separately. To evaluate the effect of HS on endocrine function, the cells were cultured at 38.5°C (control) or 40.5°C (HS). After treatment, PGE2 and PGF2α levels were measured via enzyme immunoassay (EIA) and mRNA expressions of enzymes involved in PG synthesis were examined via quantitative reverse transcription polymerase chain reaction (RT-PCR). HS did not influence the production of PGE2 or PGF2α in the epithelial cells; however, HS significantly enhanced the production of both PGE2 and PGF2α in the stromal cells (P < 0.05). In addition, HS significantly increased phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), and carbonyl reductase 1 (CBR1) mRNA expression in the stromal cells (P < 0.05). The overall results suggest that HS induces mRNA expression of enzymes involved in PG synthesis, resulting in the upregulation of PGE2 and PGF2α production in the stromal cells, but not in the epithelial cells. The HS-induced increase of PGE2 and PGF2α secretion in bovine endometrial stromal cells may disrupt the normal estrous cycle and cause infertility in cows during summer.

Evaluating the electrical impedance and mucus-related gene expression of uterine endometrial tissues in mares.

We investigated the electrical impedance of the reproductive tracts (vagina and uterine endometrial tissues) and the expression of mucus-related genes to identify the stage of the estrous cycle in mares. We first examined vaginal impedance in native Hokkaido mares during their estrous cycle and found no significant differences. However, impedance levels tended to decrease towards ovulation. Furthermore, we investigated the estrous cycle by measuring the electrical impedance of the uterine endometrial tissues obtained from carcasses of mares. We found that impedance levels in the endometrial tissues decreased in the regressed phase of the corpus luteum (CL). Expression of mucus-related genes (ATP1A1, CFTR, AQP3, and AQP5) varied at different stages of the estrous cycle. Among them, AQP3 expression was consistent with previous reports. We concluded that electrical impedance in the uterine endometrial tissues of mares could be potentially used to verify the presence of active CL in horses for experimental purposes. However, further studies are needed to determine the reference value and to identify the day of the estrous cycle in mares.