Effect of globin peptide on female fertility in aging granulosa cell-specific Nrg1 knockout mice.

Ovarian fibrosis contributes to age-related ovarian dysfunction. In our previous study, we observed ovarian fibrosis in both obese and aging mice with intracellular lipid droplets in the fibrotic ovaries. Although the importance of mitochondria in ovarian fibrosis has been recognized in pharmacological studies, their role in lipid metabolism remains unclear. Globin peptide (GP), derived from hemoglobin, enhances lipid metabolism in obese mice. This study aimed to elucidate the importance of lipid metabolism in ovarian fibrosis by using GP. Treatment of ovarian stromal cells with GP increased mitochondrial oxygen consumption during ß-oxidation. Lipid accumulation was also observed in the ovaries of granulosa cell-specific Nrg1 knockout mice (gcNrg1KO), and the administration of GP to gcNrg1KO mice for two months reduced ovarian lipid accumulation and fibrosis in addition to restoring the estrous cycle. GP holds promise for mitigating lipid-related ovarian issues and provides a novel approach to safeguarding ovarian health by regulating fibrosis via lipid pathways.

Intrauterine infusion of low levels of interferon-tau on day-8 post-estrus stimulates the bovine endometrium to secrete apolipoprotein-A1: A possible implication for early embryo tolerance.

We previously reported that interferon-tau (IFNT), derived from day-7 blastocyst, generates anti-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. However, the real in vivo impact of early embryo-derived IFNT on the uterine proteomic profile is mostly unknown. This study aimed to investigate proteomic changes of uterine flush (UF) when infused with a low physiological level of IFNT without embryo on day-8 post-estrus and its possible impact on the uterine immunological microenvironment. First, a fresh medium was infused into the uterine lumen on day-6, from which UF was obtained 24 h later, and this procedure was repeated on day-7 (control UF). On day-8, this procedure was done with a medium containing recombinant bovine IFNT (100 pg/ml) (IFNT-supplemented UF). Control and IFNT-supplemented UF were tested for immune responses in peripheral blood mononuclear cells (PBMCs). Real-time PCR results revealed that IFNT-supplemented UF downregulated pro-inflammatory cytokines (TNFA, IL1B) and upregulated anti-inflammatory cytokine (TGFB1) and PTGES in PBMCs. Through 2-D PAGE, followed by TOF/TOF mass spectrometer, apolipoprotein-A1 (Apo-A1) protein was identified in the IFNT-supplemented UF, which was confirmed by ELISA analysis. Proteomic analysis revealed again that the in vitro stimulation of BEECs by IFNT upregulated Apo-A1 expression. Further, stimulation of PBMCs with recombinant bovine Apo-A1 downregulated TNFA and NFKB and upregulated TGFB1 and PTGES in PBMCs. Altogether, our results suggest that minute amounts of IFNT alone, normally secreted from bovine blastocyst, stimulate Apo-A1 secretion from the endometrial epithelium in the absence of embryo that initiates an anti-inflammatory environment, which could pave the way for the acceptance of early embryo in the uterus.

Cortisol induces follicle regression, while FSH prevents cortisol-induced follicle regression in pigs.

During follicular development, a few dominant follicles develop to large antral dominant follicles, whereas the remaining follicles undergo atretic degeneration. Because vascularization on the follicular surface is a morphological feature of dominant follicles, we previously classified these follicles as vascularized follicles (VFs) and non-VFs (NVFs). In NVFs, progesterone producing genes were expressed similarly to that in VFs; however, the progesterone concentration in follicular fluid was low in large NVFs. Therefore, we estimated that progesterone is converted to cortisol, which induces the loss of follicular functions. In this study, we comparative analyzed the expression of genes for progesterone converting enzymes (Cytochrome (CYP)11B1, CYP21A2, Hydroxysteroid (HSD)11B2) and cortisol receptor (NR3C1) in VF and NVF granulosa cells. In NVFs, expression of cortisol producing genes (CYP11B1 and CYP21A2) was higher than in VFs. Expression of the gene for the cortisol metabolizing enzyme HSD11B2 in NVFs was significantly lower than in VFs. In NVFs, accompanied by increasing cortisol concentration in follicular fluid, apoptosis of granulosa and cumulus cells was observed. Cultivation with FSH and metyrapone (a CYP11B1 inhibitor) of NVF cumulus-oocyte complexes inhibited apoptosis of cumulus cells and induced cumulus cell proliferation and oocyte maturation. Cortisol-induced CYP11B1 and CYP21A2 expression, whereas FSH-induced HSD11B2 mRNA expression in VF granulosa cells in the presence of cortisol. Furthermore, an addition of 18ß-glycyrrhetinic acid (18-GA; a HSD17B2 inhibitor) to cortisol and FSH-containing medium increased apoptosis of VF granulosa cells. These results suggested that cortisol is a stimulatory factor that induces follicular atresia; furthermore, inhibition of cortisol production by FSH might increase the number of healthy preovulatory follicles in pigs.

Sperm interaction with the uterine innate immune system: toll-like receptor 2 (TLR2) is a main sensor in cattle.

During the passage through the female reproductive tract, sperm interact with various compartments and their immune systems. The immune system that protects the female against pathogens also could destroy sperm or prevent them from reaching the site of fertilisation. In particular, the uterine innate immune response is crucial from the perspectives of both the sperm and the uterus. Following insemination, sperm immediately start to trigger inflammation in the uterus by entering uterine glands and activating an innate immune response. In cattle, the activation occurs mainly via TLR2 signalling, if not the only one, between sperm and the uterine epithelium lining the glands. This acute immune response is manifested as the upregulation of mRNA expression of IL8, TNFA, IL1B , and PGES . As a consequence, many sperm are trapped by polymorphonuclear neutrophils, the first and major component of innate immunity. The sperm-induced uterine innate immune responses apparently serve to clear the uterus of excess sperm and, importantly, prepare the endometrium for implantation. Pathophysiological conditions in the uterus seriously disrupt this phenomenon, and thus could directly decrease fertility.

[Propofol Infusion Syndrome Treated Successfully by Early Diagnosis].

Propofol infusion syndrome (PRIS) is one of the severe complications which occur during continuous venous infusion of propofol, and has a high mortality rate. It is featured by high fever, oliguria, myogloblin urine, acute renal failure, hepatomegaly, fatty liver, and so on. We have experienced a case of PRIS who was saved by prompt changing of sedatives from propofol to midazolam and dexmedetomidine. The patient was an 82-year-old man, who underwent off-pump coronary bypass grafting due to effort angina pectoris. After the operation, he suffered from continuous high fever over 38 ℃, acute renal impairment, and high level of creatine kinase (CK) without CK-MB increment, suggesting PRIS. We promptly changed sedatives from propofol to midazolam and dexmedetomidine, then the patient recuperated from these abnormalities. It is strongly suggested that meticulous observation is necessary during propofol infusion.

Cutting the ovarian surface improves the responsiveness to exogenous hormonal treatment in aged mice.

PURPOSE: Ovarian vascular abnormality and ovarian fibrosis are observed in the low responder patients and aging mice. Vascularization and fibrosis are regulated by injury-repair system, such as wound. Thus, in this study, the authors tried to investigate the effect of the surgical treatment to ovarian surface with cutting on the functions of ovary in aging mouse model, gcNrg1KO. METHOD: The ovarian surface of gcNrg1KO was surgically cut, and then the ovary was returned inside of bursa ovarica. To assess the effect of cutting on fertility, mating test, smear analysis, and exogenous hormonal treatment were done. Additionally, the histological analysis was used for observing the remodeling of ovarian stroma after the surgical approach. RESULT: Ovarian fibrosis disappeared at 7 days after surgery. With the abrogation of fibrosis, the blood vessels were fluently observed around the follicles, and the follicular development was re-started. The responses against exogenous hormone were recovered at 21 days after the surgery, and estrous cycle and delivery were also recovered by the surgery and the fertility was maintained for 3 months. CONCLUSION: This cutting method of ovarian surface becomes a good option against low responder patients.

Impact of lipopolysaccharide administration on luteinizing hormone/choriogonadotropin receptor (Lhcgr) expression in mouse ovaries.

Lipopolysaccharide (LPS) is isolated from the genital tract of animals suffering from uterine damage and ovarian dysfunction. This study provides direct molecular evidence about the mechanism through which endotoxins cause reproductive disorders. Granulosa cells and ovaries were collected from immature mice treated with eCG or with eCG and LPS injection intraperitoneally. Normal large antral follicles were observed in ovaries obtained from eCG and LPS coinjected mice, and the morphology of the ovaries was similar to that observed in the control group. These antral follicles were not deemed atretic because few TUNEL-positive cells were observed. However, the granulosa cells of large antral follicles did not acquire the ability to respond to hCG stimulation. The number of ovulated oocytes was significantly lower in LPS-injected mice after superovulation compared to mice that were not exposed to LPS. The low reactivity was caused by the limited expression of the Lhcgr gene, which encodes the LH receptor in granulosa cells as well as an LPS-induced increase in the level of Dnmt1 expression. The methylation rate of the Lhcgr promoter region was significantly higher in granulosa cells obtained from the LPS treatment group compared with the control group. Together, these findings demonstrated that the decrease in the expression of Lhcgr due to LPS was a result of the epigenetic regulatory action of LPS. Our studies suggest that ovarian follicular cysts that is characterized by bacterial infection in humans and animals, is closely connected to the level of methylation of the Lhcgr promoter region.

Peptidoglycan disrupts early embryo-maternal crosstalk via suppression of ISGs expression induced by interferon-tau in the bovine endometrium.

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure.

Iron deficiency induces female infertile in order to failure of follicular development in mice.

Iron is important for many cellular functions, including ATP synthesis and cell proliferation. Insufficient of iron in the diet causes iron deficiency anemia (IDA), which often occurs in people living in the world. Since 50% of women with IDA show amenorrhea, the relationship of between iron deficiency and reproductive function was assessed using mice fed a low Fe diet (LFD). The estrous cycle in the LFD mice was blocked at diestrus, which impair follicle development, and fertility. Further, even LFD mice were injected with exogenous pregnant mare serum gonadotropin (PMSG), follicular development was ceased at the secondary follicle stage, and preovulatory follicles were not observed. Amount of ATP decreased in the ovary of the LFD mice, and expression of follicle development markers (Fshr, Cyp19a1, Ccnd2) and estradiol-17ß (E2) was low level compared to levels mice fed a normal diet. Feeding a normal diet with sufficient iron to the LFD mice for an additional 3 weeks completely reversed absence the effects of iron insufficient on the estrous cycle and infertility. Thus, iron restriction depresses ovary functions, especially follicular development from secondary follicle to antral follicles and infertility. These effects are fully reversible by supplementation of a normal diet containing iron.

Roadmap to pregnancy in the first 7 days post-insemination in the cow: Immune crosstalk in the corpus luteum, oviduct, and uterus.

The first 7 days post-insemination are critical for establishment of pregnancy. The pre-ovulatory luteinizing hormone (LH) surge induces ovulation through disruption of the follicle structure that elucidates pro-inflammatory (Th1) responses. Various types of immune cells are recruited into the corpus luteum (CL) to regulate luteal angiogenesis and progesterone (P4) secretion into the circulation to establish pregnancy. The active sperm-uterine crosstalk also induces Th1 responses, mainly via Toll-like receptor (TLR) 2/4 signaling pathway in vitro. The endometrial glands serve as sensors for sperm signals, which trigger Th1 responses. Conversely, the sperm-oviduct binding generates anti-inflammatory (Th2) responses to support sperm survival until fertilization. It is well-established that embryo-maternal crosstalk starts after the embryo hatches out from the zona pellucida (ZP). However most recently, it was shown that the 16-cell stage bovine embryo starts to secrete interferon-tau (IFNT) that induces Th2 immune responses in the oviduct. Once developing embryos descend into the uterine horn, they induce Th2 responses with interferon-stimulated genes (ISGs) expression in the uterine epithelium and local immune cells mainly via IFNT release. Likewise, multiple embryos in the uterus of superovulated donor cows on D7 post-insemination induce Th2 immune responses with ISGs expressions in circulating immune cells. These findings strongly suggest that the maternal immune system reacts to the embryo during the first 7 days post-insemination to induce fetal tolerance. It became evident that the innate immunity of the developing CL, oviduct, and uterus works together to provide optimal conditions for fertilization and early embryonic development during the first 7 days post-insemination.

Peptidoglycan Switches Off the TLR2-Mediated Sperm Recognition and Triggers Sperm Localization in the Bovine Endometrium.

In mammals, the uterine mucosal immune system simultaneously recognizes and reacts to most bacteria as well as allogenic sperm mainly through the Toll-like receptors (TLR)2/4 signaling pathway. Here, we characterized the impact of pathogen-derived TLR2/4 ligands (peptidoglycan (PGN)/lipopolysaccharide (LPS)) on the immune crosstalk of sperm with the bovine endometrial epithelium. The real-time PCR analysis showed that the presence of low levels of PGN, but not LPS, blocked the sperm-induced inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. Immunoblotting analysis revealed that PGN prevented the sperm-induced phosphorylation of JNK in BEECs. Activation or blockade of the TLR2 system in the endometrial epithelium verified that TLR2 signaling acts as a commonly-shared pathway for PGN and sperm recognition. The impairment of endometrial sperm recognition, induced by PGN, subsequently inhibited sperm phagocytosis by polymorphonuclear neutrophils (PMNs). Moreover, using an ex vivo endometrial explant that more closely resembles those in vivo conditions, showed that sperm provoked a mild and reversible endometrial tissue injury and triggered PMN recruitment into uterine glands, while PGN inhibited these events. Of note, PGN markedly increased the sperm attachment to uterine glands, and relatively so in the surface epithelium. However, addition of the anti-CD44 antibody into a PGN-sperm-explant co-culture completely blocked sperm attachment into glands and surface epithelia, indicating that the CD44 adhesion molecule is involved in the PGN-triggered sperm attachment to the endometrial epithelium. Together, these findings demonstrate that, the presence of PGN residues disrupts sperm immune recognition and prevents the physiological inflammation induced by sperm in the endometrial epithelium via the MyD88-dependent pathway of TLR2 signaling, possibly leading to impairment of uterine clearance and subsequent embryo receptivity.

Negative effects of ROS generated during linear sperm motility on gene expression and ATP generation in boar sperm mitochondria.

Mitochondrial oxidative phosphorylation (OXPHOS) is essential for ATP production to maintain sperm linear motility during migration from the uterus to the oviduct. However, ROS are generated as by-products of OXPHOS, causing stress and damaging the sperm quality. This study aimed to clarify the ROS targets in sperm mitochondria that decrease linear motility and to investigate whether mitochondria-target antioxidants (PQQ and CoQ10) affect mitochondrial activity and sperm motility. Sperm linear motility pattern, ATP production, and mitochondrial activity were decreased with increasing ROS levels during incubation in the low-glucose medium. However, sperm motility patterns and ROS levels were not significantly changed in the high-glucose medium. Moreover, the gene expression system (mt-DNA, mitochondrial transcription factor-A (TFAM) and RNA polymerase (POLRMT)) in sperm mitochondria was damaged during incubation in the low-glucose medium. Interestingly, PQQ treatment increased the mt-DNA stability and decreased the damage to TFAM and POLRMT, which resulted in high expression of mitochondrial genes. Furthermore, the antioxidants increased mitochondrial activity and maintained sperm linear motility under the low glucose condition. These results revealed that both ATP production and the mitochondrial transcription system are damaged with increasing ROS levels in sperm that show a linear motility pattern. Treatment with antioxidants, such as PQQ and CoQ10, is beneficial tool to maintain sperm linear motility.

Cyclooxygenase-2 is acutely induced by CCAAT/enhancer-binding protein ß to produce prostaglandin E 2 and F 2α following gonadotropin stimulation in Leydig cells.

Cyclooxygenase 2 (COX-2) is an inducible rate-limiting enzyme for prostanoid production. Because COX-2 represents one of the inducible genes in mouse mesenchymal stem cells upon differentiation into Leydig cells, we investigated COX-2 expression and production of prostaglandin (PG) in Leydig cells. Although COX-2 was undetectable in mouse testis, it was transiently induced in Leydig cells by human chorionic gonadotropin (hCG) administration. Consistent with the finding that Leydig cells expressed aldo-keto reductase 1B7 (PGF synthase) and PGE synthase 2, induction of COX-2 by hCG caused a marked increase in testicular PGF 2α and PGE 2 levels. Using mouse Leydig cell tumor-derived MA-10 cells as a model, it was indicated by reporter assays and electron mobility shift assays that transcription of the COX-2 gene was activated by CCAAT/enhancer-binding protein ß (C/EBPß) with cAMP-stimulation. C/EBPß expression was induced by cAMP-stimulation, whereas expression of C/EBP homolog protein (CHOP) was robustly downregulated. Transfection of CHOP expression plasmid inhibited cAMP-induced COX-2 promoter activity. In addition, CHOP reduced constitutive COX-2 expression in other mouse Leydig cell tumor-derived TM3 cells. These results indicate that COX-2 is induced in Leydig cells by activation of C/EBPß via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF 2α and PGE 2 .

TLR2/4 signaling pathway mediates sperm-induced inflammation in bovine endometrial epithelial cells in vitro.

We have recently shown that sperm attachment to bovine endometrial epithelial cells (BEECs) triggers uterine local innate immunity with induction of a pro-inflammatory response in vitro, however details of the mechanism remain unknown. Here, we investigated the involvement of Toll-like receptor 2/4 (TLR2/4) pathway in mediating sperm-BEECs inflammatory process. Immunohistochemistry of the uterine tissue revealed that TLR2 and TLR4 proteins were present in the luminal and glandular epithelia of bovine endometrium. Moreover, BEECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h, followed by evaluating mRNA expression of the pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs using a real-time PCR. Both Pam and LPS treatments showed a dose-dependent stimulation of mRNA expression of the pro-inflammatory genes. To elucidate the functional role of TLR2/4 in sperm-BEECs interaction, BEECs monolayers were incubated with either TLR2 antagonist or TLR4 antibody for 2 h prior to the co-culture with sperm for 3 h. Importantly, pre-incubation of BEECs with TLR2 antagonist or TLR4 antibody prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs. Furthermore, sperm increased the phosphorylation levels of TLR2/4 downstream targets (p38MAPK and JNK) in BEECs within 1 h of the co-culture. Treatment of BEECs with TLR2 antagonist prior to sperm addition inhibited JNK phosphorylation, while TLR4 antibody inhibited the phosphorylation of both p38MAPK and JNK. In conclusion, the present in vitro findings strongly suggest that bovine endometrial epithelial cells respond to sperm via TLR2/4 signal transduction.

Oviduct epithelium induces interferon-tau in bovine Day-4 embryos, which generates an anti-inflammatory response in immune cells.

Recent studies indicate that communication between the bovine embryo and the mother begins in the oviduct. Here, we aimed to investigate the effect of embryos on bovine oviducts for their immune responses using an in vitro model. First, zygotes were cultured with or without bovine oviduct epithelial cells (BOECs) for 4 days, when embryos had reached the 16-cell stage. At that time, we detected interferon-tau (IFNT) in embryos co-cultured with BOECs, but not in embryos cultured alone. Next, peripheral blood mononuclear cells (PBMCs) were incubated either in media from embryo alone cultures or from co-cultures of embryos with BOECs. The medium from embryo alone cultures did not modulate PBMCs gene expression; whereas the embryo-BOEC co-culture medium increased interferon-stimulated genes (ISGs: ISG15, OAS1, MX2), STAT1, PTGES and TGFB1 but suppressed IL17 expression in PBMCs. Both IFNT-treated BOEC culture medium and IFNT-supplemented fresh medium alone without BOEC, modulated PBMCs gene expressions similar to those by the embryo-BOEC co-culture medium. Further, specific antibody to IFNT neutralized the effect of embryo-BOEC co-culture medium on PBMCs gene expression. Our results indicate that BOECs stimulate embryos to produce IFNT, which then acts on immune cells to promote an anti-inflammatory response in the oviduct.

Evidence that interferon-tau secreted from Day-7 embryo in vivo generates anti-inflammatory immune response in the bovine uterus.

Recent studies suggest that Day-7 bovine embryo starts to communicate with the uterine epithelium through interferon-tau (IFNT) signaling. However, immune modulatory role of IFNT in the uterus just after the embryo moves from the oviduct is unclear. We aimed to examine the hypothesis that Day-7 bovine embryo secretes IFNT in the uterus, which induces anti-inflammatory response in immune cells. The uterine flush (UF) with multiple embryos was collected from Day-7 donor pregnant cows and peripheral blood mononuclear cells (PBMCs) were then cultured in UF. Transcripts detected in PBMCs revealed that UF from pregnant cows down-regulated pro-inflammatory cytokines (TNFA, IL1B) and up-regulated anti-inflammatory cytokine (IL10) expression, with activation of interferon-stimulated genes (ISGs; ISG15, OAS1) as compared with UF from non-pregnant cows. An addition of specific anti-IFNT antibody to the UF inhibited the effect on PBMCs, indicating that IFNT is a major factor for such immune modulation. The observation that conditioned media from bovine uterine epithelial cells both stimulated with IFNT in vitro and supplemented with fresh IFNT induced similar PBMCs gene expression, confirming that IFNT directly acts on this immune crosstalk. This study shows that IFNT secreted from Day-7 embryo in vivo generates anti-inflammatory response in immune cells, which may provide immunological tolerance to accept the embryo.

The Cell Type-Specific Expression of Lhcgr in Mouse Ovarian Cells: Evidence for a DNA-Demethylation-Dependent Mechanism.

The luteinizing hormone receptor (LHCGR) is expressed at low levels in mural granulosa cells and cumulus cells of antral follicles and is induced dramatically in granulosa cells but not in cumulus cells by follicle-stimulating hormone (FSH). Therefore, we hypothesized that FSH not only activates transcription factors controlling Lhcgr expression but also alters other events to permit and enhance Lhcgr expression in granulosa cells but not in cumulus cells. In granulosa cells, the level of DNA methylation in the Lhcgr promoter region was significantly decreased by equine chorionic gonadotropin (eCG) in vivo. However, in cumulus cells, hypermethylation of the Lhcgr promoter remained after eCG stimulation. eCG induced estrogen production from testosterone (T) and retinoic acid (RA) synthesis in granulosa cells. When either T or RA in the presence or absence of FSH was added to granulosa cell cultures, the combined treatment with FSH and RA induced demethylation of Lhcgr-promoter region and Lhcgr expression. FSH-dependent RA synthesis was negatively regulated by coculture of granulosa cells with denuded oocytes, suggesting that oocyte-secreted factors downregulate RA production in cumulus cells where Lhcgr expression was not induced. Strikingly, treatment of cultured cumulus-oocyte complexes with a SMAD inhibitor, SB431542, significantly induced RA production, demethylation of Lhcgr-promoter region, and Lhcgr expression in cumulus cells. These results indicate the demethylation of the Lhcgr-promoter region is mediated, at least in part, by RA synthesis and is a key mechanism regulating the cell type-specific differentiation during follicular development.

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice.

It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both in vivo and in vitro were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of Hsd3b. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.

A proinflammatory response of bovine endometrial epithelial cells to active sperm in vitro.

In the cow, cryopreserved semen is inseminated into the uterus, and most of sperm are removed by backflow and phagocytes. Nevertheless, the mechanism responsible for sperm phagocytosis is unclear. Here, we used cultured bovine uterine epithelial cells (BUECs) to investigate the uterine response to sperm and the mechanism that activates polymorphonuclear neutrophils (PMNs). BUEC monolayers were co-cultured with different numbers of washed sperm obtained from cryopreserved semen (104 , 105 , and 106 sperm/ml) for 3 hr. Sperm dose-dependently up-regulated IL8 (Interleukin 8). Sperm at 106 /ml increased mRNA expression of TNFA (Tumor necrosis factor alpha), IL1B (Interleukin 1B), NFKB2 (Nuclear factor kappa B2), and C3 (Complement factor 3), as well as PGES (Prostaglandin E synthase) expression and PGE2 release. Live sperm, but not dead sperm, attached to BUECs, and dead sperm did not induce an acute inflammatory response. Time-dependent effects were evaluated by co-culture of 106 /ml washed sperm with BUECs for 0, 1, 3, and 6 hr. The number of detached sperm increased gradually toward 6 hr. Maximum mRNA expression of IL8, TNFA, IL1B, and NFKB2 was induced at 3 hr, while C3 continued to increase toward 6 hr. Sperm did not stimulate mRNA expression of anti-inflammatory cytokines TGFB1 (Transforming growth factor beta 1) or IL10 (Interleukin 10). Medium conditioned by sperm co-incubated with BUECs stimulated PMNs phagocytosis of sperm in vitro. Fresh media supplemented with low levels of IL1B, TNFA, and PGE2 up-regulated sperm phagocytosis by PMNs as well. In conclusion, our findings strongly suggest that the active sperm attach to BUECs and trigger uterine local innate immunity with induction of a pro-inflammatory response that enhances sperm phagocytosis by PMNs.