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.

BNIP3 expression in bovine follicle and corpus luteum.

BNIP3 (BCL2/adenovirus E1B nineteen kilodalton interacting protein-3), a member of the BCL2 family, is activated under hypoxic conditions and induces apoptosis or mitochondrial autophagy for adapting cells to hypoxia. The physiological roles of BNIP3 in the mammalian ovary are still unclear. In order to understand the role of BNIP3 in the bovine ovary, we examined its mRNA and protein expressions of BNIP3 in follicular granulosa cells and corpus luteum (CL). BNIP3 mRNA and protein expressions in granulosa cells from large follicles (>10 mm) at the follicular stage were much higher than those in small follicles (2-8 mm). BNIP3 mRNA and protein expressions in the CL peaked at the early luteal stage. In bovine granulosa cells cultured for 6 hr under hypoxia (3% O2) and normoxia (20% O2), BNIP3 mRNA expression was higher under hypoxia. These results of the present study suggest that BNIP3 has some roles in luteal formation in the bovine ovary, and that the highly expressed BNIP3 in the granulosa cells from large follicles at the follicular stage is related to the roles of BNIP3 in the luteal formation.

Hypoxia increases glucose transporter 1 expression in bovine corpus luteum at the early luteal stage.

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. Hypoxia-inducible factor-1 (HIF1) induces the expression of glucose transporter 1 (GLUT1). To clarify the physiological roles of GLUT1 in bovine CL, we examined GLUT1 mRNA expression in the CL under hypoxic conditions by quantitative RT-PCR. We also measured the effects of glucose (0-25 mM) and GLUT1 inhibitors (cytochalasin B, STF-31) on P4 production in bovine luteal cells. GLUT1 mRNA expression in bovine CL was higher at the early luteal stage compared to the other later stages. Hypoxia (3% O2) increased GLUT1 mRNA expression in early luteal cells, but not in mid luteal cells. Glucose (0-25 mM) increased P4 production in early luteal cells, but not in mid luteal cells. Both GLUT1 inhibitors decreased P4 production in early and mid luteal cells. Overall, the results suggest that GLUT1 (possibly induced by hypoxic conditions in the early CL) plays a role in the establishment and development of bovine CL, especially in supporting luteal P4 synthesis at the early luteal stage.

Adrenomedullin regulates the speed of oviductal fluid flow in cattle.

Unidirectional flow of oviductal fluid from the ovarian to uterine side of the ampulla plays a significant role in successful pregnancy, and is produced by ciliary beating. Various systems regulate ciliary beating, such as paracrine, autocrine, and endocrine. We hypothesized that Adrenomedullin (ADM)-a peptide hormone that acts via its receptors, which are complexes of Calcitonin receptor-like receptor (CRLR) and Receptor activity-modifying protein (RAMP) 2 or 3 - promotes oviductal fluid flow in the ampulla of bovine oviducts. First, we examined the expression of ADM, CRLR, RAMP2, and RAMP3 mRNAs in isolated epithelial cells throughout the estrous cycle, and the localization of ADM receptor protein constituents in the ampulla. RAMP2 expression was significantly higher in the follicular phase. Furthermore, RAMP2 protein was detected only in ciliated cells, whereas CRLR and RAMP3 were detected in all epithelial cells. The effects of ADM and an ADM antagonist on fluid-flow speed were examined using microbeads in ampullary tissue. ADM antagonist decreased bead transport speed, and this decrease was reversed by ADM. In addition, ADM recovered the bead transport speed that decreased in the absence of calcium. Overall, our results suggest that ADM contributes to the regulation of oviductal fluid flow in ampulla.

Bovine embryo induces an anti-inflammatory response in uterine epithelial cells and immune cells in vitro: possible involvement of interferon tau as an intermediator.

Recent observations suggest that the bovine uterus starts to react to the early embryo immediately after its arrival from the oviduct. The present study aimed to investigate the effect of the early developing embryo on the immune-related gene profile in bovine uterine epithelial cells (BUECs) in vitro, and to further examine the impact of conditioned media (CM), either from embryo-BUEC co-culture or embryo culture alone, on gene expression in peripheral blood mononuclear cells (PBMCs). First, BUECs were co-cultured with morulae (n = 10) for D5-D9 (D0 = IVF), and gene expression in BUECs was analyzed. Subsequently, PBMCs were cultured in CM from embryo-BUEC co-culture or D5-D9 embryo culture, and gene expression was evaluated. In BUECs, the embryo induced interferon (IFN)-stimulated genes (ISGs: ISG15, OAS1, and MX2), a key factor for IFN-signaling (STAT1), and type-1 IFN receptors (IFNAR1 and IFNAR2), with suppression of NFkB2, NFkBIA and pro-inflammatory cytokines (TNFA and IL1B). The embryo also stimulated PTGES and PGE2 secretion in BUECs. In PBMCs, both CM from embryo-BUEC co-culture and embryo culture alone induced ISGs, STAT1 and TGFB1, while suppressing TNFA and IL17. Similarly, interferon tau (IFNT) at 100 pg/ml suppressed NFkB2, TNFA and IL1B in BUECs, and also stimulated TGFB1 and suppressed TNFA in PBMCs. Our findings suggest that the bovine embryo, in the first four days in the uterus (D5-D9), starts to induce an anti-inflammatory response in epithelial cells and in immune cells. IFNT is likely to act as one of the intermediators for induction of the anti-inflammatory response in the bovine uterus.

Expressions of lipoprotein receptors and cholesterol efflux regulatory proteins during luteolysis in bovine corpus luteum.

The corpus luteum (CL) synthesises and secretes progesterone (P4), which is essential for the establishment and maintenance of pregnancy in mammals. P4 is synthesised from cholesterol. Cholesterol is internalised by low-density lipoprotein receptor (LDLR) and/or scavenger receptor B1 (SR-BI), and is effluxed by ATP-binding cassette (ABC) transporter A1 (ABCA1) and G1 (ABCG1). To test the hypothesis that lipoprotein receptors and ABC transporters are involved in functional luteolysis, we examined the expression of LDLR, SR-BI, ABCA1 and ABCG1 in bovine CL during the luteal stages and after injection of prostaglandin (PG) F2α on Day 10 after ovulation. Expression of LDLR and SR-BI mRNA and protein was lower in the regressed luteal than late luteal stage. Injection of cows with a PGF2α did not affect LDLR mRNA and protein levels in the CL. Although expression of SR-BI mRNA did not change, SR-BI protein expression decreased 12 and 24h after PGF2α injection. The overall findings of the present study suggest that the decreased expression of SR-BI induced by PGF2α is one of the factors responsible for the continuous decrease in P4 production during functional luteolysis.

Hypoxia-inducible factor 1 mediates hypoxia-enhanced synthesis of progesterone during luteinization of granulosa cells.

Hypoxia has been suggested to enhance progesterone (P4) synthesis in luteinizing granulosa cells (GCs), but the mechanism is unclear. The present study was designed to test the hypothesis that the hypoxia-induced increase in P4 synthesis during luteinization in bovine GCs is mediated by hypoxia-inducible factor 1 (HIF-1). GCs obtained from small antral follicles were cultured with 2 µg/ml insulin in combination with 10 µM forskolin for 24 h as a model of luteinizing GCs. To examine the influence of HIF-1 on P4 synthesis, we determined the effect of changes in protein expression of the α-subunit of HIF-1 (HIF1A) on P4 production and on the expression levels of StAR, P450scc, and 3ß-HSD. CoCl2 (100 µM), a hypoxia-mimicking chemical, increased HIF-1α protein expression in luteinizing GCs. After the upregulation of HIF-1α, we observed an increase in P4 production and in the gene and protein expression levels of StAR in CoCl2-treated luteinizing GCs. In contrast, CoCl2 did not affect the expression of either P450scc or 3ß-HSD. Echinomycin, a small-molecule inhibitor of HIF-1's DNA-binding activity, attenuated the effects of CoCl2 and of low oxygen tension (10% O2) on P4 production and StAR expression in luteinizing GCs. Overall, these findings suggest that HIF-1 is one of the factors that upregulate P4 in GCs during luteinization.

Programmed necrosis - a new mechanism of steroidogenic luteal cell death and elimination during luteolysis in cows.

Programmed necrosis (necroptosis) is an alternative form of programmed cell death that is regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent, but is a caspase (CASP)-independent pathway. In the present study, to determine if necroptosis participates in bovine structural luteolysis, we investigated RIPK1 and RIPK3 expression throughout the estrous cycle, during prostaglandin F2α (PGF)-induced luteolysis in the bovine corpus luteum (CL), and in cultured luteal steroidogenic cells (LSCs) after treatment with selected luteolytic factors. In addition, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 µM) on cell viability, progesterone secretion, apoptosis related factors and RIPKs expression, were evaluated. Expression of RIPK1 and RIPK3 increased in the CL tissue during both spontaneous and PGF-induced luteolysis (P < 0.05). In cultured LSCs, tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM) up-regulated RIPK1 mRNA and protein expression (P < 0.05). TNF + IFNG also up-regulated RIPK3 mRNA expression (P < 0.05), but not RIPK3 protein. Although Nec-1 prevented TNF + IFNG-induced cell death (P < 0.05), it did not affect CASP3 and CASP8 expression. Nec-1 decreased both RIPK1 and RIPK3 protein expression (P < 0.05). These findings suggest that RIPKs-dependent necroptosis is a potent mechanism responsible for bovine structural luteolysis induced by pro-inflammatory cytokines.

Histological analysis of arteriovenous anastomosis-like vessels established in the corpus luteum of cows during luteolysis.

BACKGROUND: The mechanisms regulating the function and regression of the corpus luteum (CL) have not yet been elucidated in detail. The regressed CL of cows was previously reported to be filled with unusual vessels like arteriovenous anastomosis (AVA); however how these vessels are being established during luteolysis remains unknown. METHODS: The bovine CL at different luteal stages and regressing bovine CL induced by prostaglandin F2α (PGF) were histologically analyzed using light and electron microscopic levels. The changes in mRNA expression of genes encoding α-smooth muscle actin (SMA; Acta2) and transforming growth factor ß1 (Tgfb1) in luteal tissues were analyzed by quantitative RT-PCR. RESULTS: AVA-like vessels appeared in the regressed CL with a diameter less than 1.5 cm in which no functional luteal cells and macrophages were observed. Epithelioid cells in the AVA-like vessel wall were immunoreactive for SMA, and the lumen of the vessels were narrow. Immunoreaction for SMA was found in the tunica media of typical arteries and arterioles, and pericytes around capillary vessel. Cells with elongated cytoplasmic processes -resident fibroblasts expressing vimentin- distributed in the CL parenchyma without any association with blood vessels are also immunoreactive for SMA, and accumulated around arteries and arterioles during the late-luteal stage. In the regressed CL, walls of arteries and arterioles consisted of more than two layers of epithelioid cells positive for both SMA and desmin, suggesting that they are myofibroblasts transformed from fibroblasts. The percentage of the area positive for SMA and the mRNA expression of Acta2 were significantly increased in the regressed CL; however, they did not alter when a luteolytic dose of PGF was injected in vivo and collected within 24 h after the injection. On the other hand, Tgfb1, a known regulator for myofibroblast transformation, was significantly increased in PGF-induced regressing CL as well as in the CL during the late-luteal stage. CONCLUSIONS: SMA-positive myofibroblasts accumulates around the arteries and arterioles to form AVA-like vessels during luteolysis in cows. PGF indirectly regulates myofibroblast transformation through enhancing the expression of TGFß1. These peculiar AVA-like vessels may be involved in the regulation of blood flow in the bovine CL during luteolysis.

Possible Contribution of Alpha2,6-Sialylation to Luteolysis in Cows by Inhibiting the Luteotropic Effects of Galectin-1.

The corpus luteum (CL) is essential for establishing pregnancy. If pregnancy does not occur during the estrous cycle, luteolysis is induced by prostaglandin (PG) F2alpha secreted from the uterus. Galectin-1, a beta-galactose-binding protein, is expressed in the functional CL of cows and increases the viability of bovine luteal steroidogenic cells (LSCs) by modifying the functions of membrane glycoproteins. The binding of galectin-1 to glycoproteins is blocked by alpha2,6-sialylation of the terminal galactose residues of glycoconjugates, which is catalyzed by a sialyltransferase (ST6Gal-I). However, the physiological role of alpha2,6-sialic acid in bovine CL is unclear. The level of alpha2,6-sialylation of the bovine CL was higher during the regressed-luteal stage than in other luteal stages. Lectin histochemistry revealed that alpha2,6-sialylated glycoconjugates were localized to luteal endothelial cells throughout the estrous cycle. In addition, alpha2,6-sialylated glycoconjugates concentrated to the membrane of LSCs during the regressed-luteal stage. PGF2alpha treatment for 72 h enhanced the expression of ST6Gal-I mRNA and the level of alpha2,6-sialylated glycoproteins in mid-LSCs. The level of alpha2,6-sialylated glycoproteins of late-stage LSCs (Days 15-17 after ovulation) was higher than that of mid-stage LSCs (Days 8-12 after ovulation), and galectin-1 increased the viability of mid-LSCs but not that of late-stage LSCs. Furthermore, galectin-1 increased the viability of late-LSCs when alpha2,6-sialic acid residues were removed by neuraminidase. The overall findings suggest that alpha2,6-sialylation stimulated by PGF2alpha contributes to luteolysis by inhibiting the luteotropic effects of galectin-1 in bovine CL.

Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.