Upon entry into the oviduct, the sperm attach to the epithelium to ensure their own survival until fertilization. Recently, we showed that sperm interaction with bovine endometrial epithelial cells in vitro triggers a “pro-inflammatory” response mediated by Toll-like receptor 2 (TLR2) (PLoS ONE 2019). In contrast, sperm interaction with bovine oviduct epithelial cells in vitro generates an “anti-inflammatory” response (PLoS ONE 2017), but its mechanism remains unknown. Thus, we hypothesized that TLR2 may mediate sperm-oviduct epithelium interaction. We developed a bovine oviduct ex vivo explant model to investigate sperm-epithelium interaction and whether TLR2 is involved. Tissue explants were obtained from ampullary folds of bovine oviducts in the pre-ovulatory phase, and co-incubated with heparin-treated cryopreserved-thawed sperm labeled with JC-1 mitochondrial stain. Epifluorescence video microscopy was used to visualize and quantify sperm binding after 5 min and 15 min of co-culture. Video microscopy showed that sperm immediately bound and uniformly attached to the epithelium surface. Addition of TLR2 antagonist into the sperm-explant co-culture significantly suppressed the number of sperm attachment (49.99±3.68%) compared to control. Further, RT-PCR showed that sperm stimulated TLR2 expression in the explant at 3 h. Blocking of TLR2 using specific antagonist suppressed TGFb expression, and prevented stimulation of TLR2 expression. Hyaluronan (HA) fragment, a natural endogenous ligand for TLR2, is secreted by bovine oviducts. Thus, we examined whether HA is also involved in sperm-oviduct epithelium interaction. Whereas addition of HA fragment did not affect sperm binding, it suppressed TGFb expression at 3 h in the explant. Our data suggest that sperm-oviduct epithelium interaction is mediated by TLR2, but further research is needed to investigate whether endogenous hyaluronan shares TLR2 receptor with sperm but with different effect on immune response.

Blood Anti-Müllerian hormone (AMH) level significantly indicates fertility in animals, including women and cows, via unclarified physiological mechanisms. AMH stimulate the LH and FSH secretions from bovine gonadotrophs via its main receptors, AMH receptor type 2 (AMHR2) (Kereilwe et al. 2018). We hypothesized that AMH and AMHR2 are expressed in the most important parts of brain for reproduction: preoptic area (POA), arcuate nucleus (ARC), and median eminence (ME).We collected brain blocks containing either the POA or the ARC and ME (ARC&ME) from Japanese Black heifers (n = 5), following the method of Tanco et al. (2016). We previously developed a new method to quench autofluorescence in bovine brain samples. After our original quenching, we used the free-floating method to stain the POA or ARC&ME coronal sections [50 µm thickness, prepared based on the bovine brain atlas (Okamura 2002))] using the verified anti-human AMH and anti-bovine AMHR2 antibodies with bovine ovaries (Kereilwe et al. 2018; Kereilwe and Kadokawa 2019).We also collected tissues containing either the POA or the ARC&ME from other heifers (n = 5) to perform RT-PCR or western blotting (WB) using previously reported methods.The RT-PCR and WB detected both AMH and AMHR2 expressions in the POA tissues as well as in the ARC&ME tissues.Immunohistochemistry revealed that approximately 65% of GnRH cells express AMHR2, and 50% of GnRH cells express AMH in the POA, ARC, and ME, including the secretory zone to the pituitary portal blood. We also found that GnRH cells were situated around other AMH-positive GnRH and non-GnRH cells.Therefore, both AMH and AMHR2 are expressed in the bovine POA, ARC, and ME.

Embryo transfer technology allows the donor mare to potentially produce multiple foals in a year. In Japan, there has been no report of foal born through embryo transfer after artificial insemination using frozen semen. The aims were to develop a special riding crossbred horse and multiply its number through embryo transfer technology. In both donor and recipient mares, luteolysis was induced by administration of 0.1 mg PGF2α analogue to synchronize the onset of estrus, and ovulation was induced by administering 2000 IU of hCG. Frozen semen from an Irrish Connemara pony was used to breed a Hokkaido native pony by deep-horn artificial insemination (dose, 400 × 10⁶ sperms). A non-surgical transcervical procedure was used to collect embryo from the uterus of the donor mare at day 7 post-ovulation and transferred fresh into the uterus of recipient mare. Weekly blood sampling for monitoring pregnancy associated hormones and transrectal ultrasound for observing fetal development were done throughout the pregnancy. Four embryos were collected from a single donor mare and were transferred to recipients in spring 2018. Three out of 4 recipient mares (75 %) established successful pregnancy and 2 of them have already delivered a healthy foal each in late April 2019, another recipient is expected to give birth in around 1 month. A normal progesterone and equine chorionic gonadotropin hormone profile were observed during the gestation in all recipient mares. This are the first foals produced through embryo transfer in Japan after artificial insemination using frozen semen. We are expecting that this new crossbred (Connemara pony x Hokkaido native pony) would be an ideal riding breed for disable people.

Sperm interaction with the uterus and subsequent immune responses by the endometrium to remove excess, defective and dead sperm are important to prepare the endometrium for embryo implantation. We have recently developed a bovine ex-vivo explant culture model to study the link between sperm interaction with endometrium and immune responses. It was shown that sperm entering bovine pre-ovulatory uterine glands induce an acute inflammatory response by upregulation of pro-inflammatory cytokines mRNA expression (SRD, 2018). We used the developed co-culture model to study the effect of sperm on protein expression of TNFA which is a strong pro-inflammatory marker. Further, sperm interaction and immune responses in the luteal phase were evaluated. Sections of bovine pre-ovulatory endometrial explants stained with hematoxylin and eosin showed normal tissue architecture with intact surface epithelial layer and simple tubular uterine glands that present throughout the extracted endometrium. Pre-ovulatory uterine explants were co-incubated with 10⁶/ml washed fresh sperm for 4 h and TNFA protein expression was detected using immunofluorescence and immunohistochemical staining. Both staining methods showed that sperm upregulate TNFA protein expression in uterine glands. Luteal phase uterine explants were co-incubated with 10⁶/ml sperm. JC-1 labeled sperm were used in fluorescence microscopy. Live, motile sperm did not appear to enter the luteal glands. Also, co-incubation for 2 h did not alter gene expression of TNFA, IL8 or IL1B in the luteal explants. Our observations reveal that sperm entering bovine pre-ovulatory uterine glands, trigger the uterine inflammatory cascade which begins in the glands.

Sperm are antigenic to the female genital tract. Thus, in most of animal species, semen deposition induces rapid inflammatory responses characterized by recruitment of polymorphonuclear neutrophils (PMNs) into the uterine lumen. Little is known about the inflammatory responses induced by cryopreserved sperm in bovine uterus especially after routine artificial insemination (AI). This in vivo study aimed to investigate the time-lapse changes in sperm transport and PMNs infiltrations into bovine uterus after AI. In this study, 12 multiparous Japanese Black cows were synchronized (estrous and ovulation) and divided into 2 groups: intra-uterine saline infused group (control, n=6) and intra-uterine AI group (n=6). In AI groups, vaginal smears were prepared at 1, 6, and 10 h after AI to investigate sperm backflow. Also, uterine body and uterine horn ipsilateral to ovary containing mature follicle in both groups, were flushed by 2 ml of RPM1-140, centrifuged, and sediments were examined under the microscope at different time points (0, 1, 6, 10, 24, 48 h and 7 days after AI). Our results showed that, most of sperm were excluded backward into vagina within 1 h, continued until 6 h, and disappeared 10 h after AI. Similarly, most of sperm were rapidly transported forward to uterine horn within 1 h, and this continued until 6 h after AI. The rapid transport and elimination of sperm in opposite directions may be completely dependent on the acute contractions of uterine smooth muscles. Semen deposition into uterine body did not induce PMN migration into uterine lumen within 1 h, but induced their massive recruitment after 6 hr, gradually decreased from 10 h, and completely disappeared 24 h after AI. Our data provide the first visual in vivo evidence that a number of sperm are transported and eliminated from bovine uterus within 1 h followed by a massive PMN infiltrations into the uterine lumen 6 h after AI which remove the excess or remaining dead sperm and ascending infection that may be impregnated with sperm during AI.

Anti-Müllerian hormone (AMH) is a glycoprotein, and its blood concentration can indicate fertility in various animals, but little is known for physiological mechanisms. Its most well-known secreting organ is ovaries, where AMH has the important roles. The main receptor of AMH is expressed in bovine oviducts and endometria (our unpublished data). Ovaries, oviduct, and endometria exchange various hormones using the systemic circulation or counter-current transfer. Therefore, this study investigated whether AMH is expressed in bovine oviducts and endometria. Reverse transcription-polymerase chain reaction and western blotting detected expressions of AMH mRNA and protein in oviductal and endometrial samples. Immunohistochemistry revealed robust AMH expression in the tunica mucosa of the ampulla and isthmus, and in the grandular and luminal epithelium of endometrium. We compared AMH mRNA (measured by real-time PCR) and protein expression in the ampulla, isthmus, and endometrium among old Holsteins, postpubertal heifer Wagyus, and old Wagyus. The heifer Wagyus had lower AMH expression in ampulla than old Holsteins (P < 0.05), and lower AMH expression in the isthmus than both old Holsteins and Wagyus (P < 0.05), but not in endometrium (P > 0.1). In conclusion, AMH is expressed in bovine oviducts and endometria.

Despite its crucial role in the regulation of the sleep-wake cycle and seasonal-reproduction animals, this study aimed to investigate for the first time the potential role of melatonin on testicular blood flow (TBF) in Shiba goats. Twelve sexually mature (1.5–3 years) male Shiba goats were exposed to a single S/C injection of either melatonin in corn oil suspension (Mel group; 36 mg/goat; n=5) or corn oil only (Cont. group; n=7). Monitoring the changes in TBF was performed 1 week before, at time of injection (W0), and once a week for W8 after injection using color Doppler ultrasonography. Plasma levels of FSH, LH, testosterone (T) and estradiol (E2) were determined by RIA. Plasma levels of melatonin and nitric oxide were measured using enzyme immunoassay kits. Results revealed significant decreases in the Doppler indices (resistive index; RI, pulsatility index; PI) of the testicular artery starting from W2 till W6 in Mel group. Peak systolic velocity and end diastolic velocity of TBF showed non-significant changes among the two groups. Significant increases in FSH, LH, T, and Mel in the Mel group. E2 and nitric oxide were significantly increased coinciding with decreases in the values of Doppler indices. Three explanations may be involved. First is the direct effect of Mel on TBF due to previous evidence of expressions of Mel receptors in the testis. Second is attributable to indirect effects of Mel as a powerful biological antioxidant inside the body. The third may be due to its influence on E2 and nitric oxide that have vasodilatory effects on the testicular artery. In conclusion, Mel may have a stimulatory effect on testicular blood flow in Shiba goats and pursue improve male fertility.