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When pregnancy is established, interferon tau (IFNT), a well-known pregnancy recognition signal in ruminants, is secreted by embryonic trophoblast cells and acts within the uterus to prepare for pregnancy. IFNT acts as an endocrine factor on the corpus luteum (CL) to induce refractory ability against the luteolytic action of PGF_{2 α}. Hypothesising that IFNT may influence not only the uterine environment but also the CL in cows via local or peripheral circulation, we investigated qualitative changes in the CL of pregnant cows during the maternal recognition period (day 16) and the CL of non-pregnant cows. The CL of pregnant animals had a higher number of neutrophils, and the expression of interleukin 8 (IL8) mRNA and its protein was higher as well as compared with the CL of non-pregnant animals. Although IFNT did not affect progesterone (P₄) secretion and neutrophil migration directly, it stimulated IL8 mRNA expression on luteal cells (LCs), influencing the neutrophils, resulting in the increased migration of IFNT-activated neutrophils. Moreover, both IFNT-activated neutrophils and IL8 increased P₄ secretion from LCs in vitro. Our novel finding was the increase in neutrophils and IL8 within the CL of pregnant cows, suggesting the involvement of IFNT function within the CL toward establishment of pregnancy in cows. The present results suggest that IFNT upregulates neutrophil numbers and function via IL8 on LCs in the CL of early pregnant cows and that both neutrophils and IL8, stimulated by IFNT, are associated with an increase in P₄ concentrations during the maternal recognition period in cows.

Abstract

Endothelins (EDNs) participate in various physiological events including smooth muscle contraction, nitric oxide (NO) synthesis, and embryonic development. In this study, we investigated the regional roles of EDNs produced by bovine oviductal epithelial cells in NO synthesis and smooth muscle motility. Quantification of mRNA expressions indicated that expression of EDN receptor B (EDNRB) in the ampullary region was higher after ovulation than before ovulation, whereas expression of EDNRA in the isthmic region was higher after ovulation than before ovulation. Immunohistochemistry revealed that the EDN receptors (EDNRA and EDNRB) were expressed in the epithelium, whereas smooth muscle showed positive staining only for EDNRA. The expressionsPlease suggest whether 'NOS2' can be treated as the updated symbol for 'iNOS' as per gene nomenclature. of inducible NO synthase (iNOS) protein and its mRNA (NOS2) in cultured epithelial cells isolated from the ampulla were stimulated by EDN1, but not by EDN2 or EDN3, after 1h of incubation. In isthmic epithelial cells, none of the EDNs affected the expression of NOS2. Isometric contraction tests indicated that spontaneous waves were strong in the isthmic region but weak in the ampullary region. EDN1 modulated smooth muscle motility in both the regions. The overall findings suggest that EDN1 plays region-specific roles in smooth muscle motility and epithelial NO synthesis, providing an optimal oviductal microenvironment for transport of gametes, fertilization, and development/transport of early embryo.

The concentrations of electrolytes (Na, K, Cl, Mg and Ca) and glucose in small follicle (SF) follicular fluid (SFF) and large follicle (LF) follicular fluid (LFF) from slaughterhouse-derived ovaries were studied. Oocytes were matured in medium based on synthetic oviductal fluid. The effects of various concentrations of electrolytes (Na, K, Ca and Mg) and glucose in the maturation medium on the progression of nuclear maturation and subsequent development were also studied. K in SFF was significantly greater than that in LFF. The Mg concentration in follicular fluid (FF) is 2.0–2.3 mM, which is greater than the concentration present in medium generally used for culture. The glucose concentration in FF is about 3.5–3.9 mM and rapidly decreases during the preservation of ovaries. LF oocytes resumed nuclear maturation and progressed to the M2 stage significantly faster than those collected from SF oocytes. In addition, more LF oocytes developed to blastocysts than did SF oocytes. Changing the Na/K ratio in the maturation medium from 16 to 24 did not affect either the progression of nuclear maturation or the rate of development. A low concentration of Mg (0.5 mM) combined with a low Ca concentration (0.5 mM) inhibited the rate of development, but did not affect the progression of nuclear maturation. On the other hand, increasing the Mg concentration to 2.0 mM from 0.5 mM hastened the progression of nuclear maturation and improved the rate of blastulation, irrespective of the Ca concentration. The progression of nuclear maturation was faster and the rate of development was greater with 5.56 mM glucose than with 1.5 mM glucose. The difference in time needed to progress to M2 among the experiment was about 2–3 h. Therefore, prolonging the maturation periods from 21 to 24 h did not change the rate of development. Our results show that the concentrations of Mg and glucose in the maturation medium and the follicle size enveloping the oocyte affect the progression of nuclear maturation and subsequent development. The time requirement for oocytes to reach M2 is strongly related to the developmental competence of the oocytes.

The corpus luteum (CL), which secretes large amounts of progesterone and is thus essential for establishing pregnancy, contains various types of immune cells that may play essential roles in CL function by generating immune responses. The lymphatic system is the second circulation system and is necessary for immune function, but the lymphatic system of the bovine CL has not been characterized in detail. We collected bovine CLs on days 12 and 16 of the estrous cycle (C12 and C16) and days 16 and 40 of early pregnancy (P16 and P40). Lymphatic endothelial hyaluronan receptor 1 (LYVE1) protein was detected in the CL by immunohistochemistry and western blotting and increased at P40 compared with C16. The mRNA expression levels of lymphangiogenic factors, such as vascular endothelial growth factor-C (VEGFC), VEGFD, and their common receptor VEGFR3, as well as the lymphatic endothelial cell (LyEC) marker podoplanin, increased in P16 and P40 CLs. Thus, it is suggested that the lymphatic system of the bovine CL reconstitutes during early pregnancy. Interferon tau (IFNT) from the conceptus in the uterus is a candidate for activating luteal lymphangiogenesis during the maternal recognition period (MRP). We found that treatment of LyECs isolated from internal iliac lymphatic vessels with IFNT stimulated LyEC proliferation and significantly increased mRNA expression of VEGFC and IFN-stimulated gene 15. Moreover, both IFNT and VEGFC induced LyECs to form capillary-like tubes in vitro. In conclusion, it is suggested that new lymphangiogenesis in the bovine CL begins during the MRP and that IFNT may mediate this novel phenomenon.

Abstract

Nitric oxide (NO) is a regulator of sperm motility, oocyte/embryo survival, and waves of contraction/relaxation in mammalian oviducts. As follicles control oviductal functions by two routes at least, (1) a systemic way via blood vessels before ovulation, (2) a direct way by entering of follicular fluid through fimbria at ovulation, we hypothesized that NO synthesis in the bovine oviduct is regulated by follicular steroids and prostaglandins (PGs). Quantification of mRNA expressions in the ampullary tissues showed that inducible NO synthase (NOS2) mRNA expression was highest on the day of ovulation (day 0). By contrast, NOS2 mRNA expression in the isthmus was highest on days 5–6 and lowest on days 19–21. Endothelial NOS (NOS3) mRNA expressions in either the ampulla or the isthmus did not change during the estrous cycle. PGE2 and PGF2α increased NOS2 mRNA expressions in cultured ampullary oviductal epithelial cells after 1-h incubation. These increases were suppressed by an antagonist of E-prostanoid receptor type 2, one of the PGE2 receptor. Estradiol-17β decreased the expression of NOS2 mRNA expression in cultured isthmic epithelial cells 24h after treatment. This effect was suppressed by an antagonist of estrogen receptorα(ESR1). Expression of ESR1 was highest on days 19–21 in the isthmic tissues. The overall findings indicate region-specific difference of NO synthesis in the oviduct. PGs flowed from ruptured follicle may up-regulate NO synthesis in the oviductal epithelium, whereas circulating E2 seems to inhibit NO synthesis via ESR1 in the isthmus at the follicular stage.

In ruminants, interferon-tau (IFNT)-mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy.

Abstract

In mares, prostaglandin F_2α (PGF_2α) secreted from the endometrium is a major luteolysin. Some domestic animals have an auto-amplification system in which PGF_2α can stimulate its own production. Here, we investigated whether this is also the case in mares. In an in vivo study, mares at the mid-luteal phase (days 6–8 of estrous cycle) were injected i.m. with cloprostenol (250 µg) and blood samples were collected at fixed intervals until 72 h after treatment. Progesterone (P₄) concentrations started decreasing 45 min after the injection and continued to decrease up to 24 h (P < 0.05). In turn, 13,14-dihydro-15-keto-PGF_2α (PGFM) metabolite started to increase 4h after an injection and continued to increase up to 72 h (P < 0.05). PGF receptor (PTGFR) mRNA expression in the endometrium was significantly higher in the late luteal phase than in the early and regressed luteal phases (P < 0.05). In vitro, PGF_2α significantly stimulated (P < 0.05) PGF_2α production by endometrial tissues and endometrial epithelial and stromal cells and significantly increased (P < 0.05) the mRNA expression of prostaglandin-endoperoxide synthase-2 (PTGS2), an enzyme involved in PGF_2α synthesis in endometrial cell. These findings strongly suggest the existence of an endometrial PGF_2α auto-amplification system in mares.

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.