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In our previous studies, we demonstrated that the primary source of relaxin 3 (RLN3) in Japanese quail is ovarian granulosa cells. Although several relaxin family peptide (RXFP) receptors have been sequenced, the intricacies of these receptors in avian species remain insufficiently clarified. Therefore, we assessed the expression of RXFP receptors, RXFP1 and 3, in Japanese quail. Using RT-PCR, we found that both RXFP1 and 3 were ubiquitously expressed. The expression level of RXFP1 is significantly higher in the ovarian theca layer, indicating that it is the primary receptor for RLN3 in the ovary. During follicular development, there was an elevation in thecal RXFP1 expression, but it declined after the luteinizing hormone (LH) surge. We found that the protease activity of the 60 kDa band increased after the LH surge, suggesting the involvement of RLN3 signaling in ovulation. These results suggest a paracrine role of RLN3, involving its binding with RXFP1 in ovarian theca cells. This interaction may elicit biological actions, potentially initiating ovulation after the LH surge.

The relaxin (RLN) gene is expressed in the reproductive tracts, such as the ovary and uterus, of mammalian species. Although RLN expression is detected in the chicken ovary, detailed clarification of the physiological role of RLN has not yet been reported. To address this issue, in the present study we aimed to examine the spatiotemporal expression and hormonal control of RLN in Japanese quail. By performing semi-quantitative and quantitative reverse transcription-polymerase chain reaction analysis, we found that RLN mRNA was mainly expressed in the granulosa and theca layers of the ovary. The expression level in the granulosa layer increased with the stage of follicular development. Results from granulosa layer culture experiments revealed that RLN mRNA expression increased with the addition of estradiol-17β, whereas the addition of progesterone suppressed RLN transcription. More detailed analysis indicated that RLN expression was highest in the stigma region of the follicle but significantly decreased as the time of the expected luteinizing hormone (LH) surge approached. Together, our findings demonstrated that the granulosa cells in the mature preovulatory follicles constitute the main source of RLN in the Japanese quail. Because RLN expression was highest in the stigma region and the expression dramatically decreased following the LH surge, the results further suggest that RLN may be related to tissue remodeling for the ovulation process in birds.

Because of the presence of sperm storage tubules (SSTs) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm enter the female reproductive tract, they can survive for a prolonged period in domestic birds; however, the specific mechanisms involved in sperm maintenance within the SST remain to be elucidated. In this study, we showed that transferrin (TF) and albumin (ALB) are expressed in SSTs. When UVJ extracts were subjected to size-exclusion column chromatography, we obtained fractions that extend sperm longevity in vitro. LC-MS/MS analysis of the two major proteins in the fractions identified these proteins as TF and ALB. Immunohistochemical analysis using specific antisera against TF and ALB indicated that both proteins were localized not only in the SSTs, but also in the surface epithelium of the UVJ. When the ejaculated sperm were incubated with either purified TF or ALB, sperm viability increased after 24 h. These results indicated that oviductal TF and ALB are involved in the process of sperm storage in SSTs and may open a new approach for technological improvement to prolong sperm longevity in vitro.

In birds, sperm storage tubules (SST) located in the utero-vaginal junction are thought to be a site of sperm selection; however, the exact mechanism of sperm selection is poorly understood. Here, we investigated sperm entry into the SST and subsequent fertilization success under a competitive situation created by artificial insemination of a sperm mixture obtained from 2 males. We employed 2 quail strains, a wild-type and a dominant black (DB) type, as this allows easy assessment of paternity by feather coloration. We found paternity of embryos was biased toward DB males when a sperm mix with similar sperm numbers from the 2 males strains was artificially inseminated into females. Our novel sperm staining method with 2 different fluorescent dyes showed that the DB-biased fertilization was because of the better ability of DB sperm to enter the SST. Moreover, we found that DB sperm had a longer flagellum and midpiece. These characteristics probably allow sperm to swim faster in a high viscosity medium, which may be a similar environment to the lumen of the female reproductive tract. Our results indicated that sperm competition occurs to win a place in the SST and that filling the SST with their own spermatozoa is a critical step to achieve better fertilization success for the male Japanese quail.

Fertilization is indispensable for zygotic formation leading to the birth of animals and the species-specific sperm-egg binding thought to be the initial step in this important process. In birds, the oocyte, which encounters the spermatozoa at the time of fertilization, is enclosed in a perivitelline membrane (pvm) constructed of several zona pellucida glycoproteins (ZP proteins: ZP1, ZP2, ZP3, ZP4 and ZPD). The aim of this study was to determine the ZP protein in the pvm responsible for sperm-pvm binding in Japanese quail. We tested the effects of anti-ZP protein antibodies on in vitro sperm perforation in the pvm. The results showed that the anti-ZP1 and ZP3 antibody significantly blocked hole formation by sperm, whereas anti-ZP2, ZP4 and ZPD as well as normal rabbit serum had no such effect. When the sperm acrosome reaction was inhibited in the presence of pertussis toxin, sperm-pvm binding was observed. This sperm-pvm binding was significantly prevented when the purified ZP1 or ZP3 was included in the reaction mixture. Moreover, both digoxigenin-labeled ZP1 and ZP3 were found to interact with the sperm head by immunocytochemical observation. Our results indicate that sperm binding to the pvm is, at least in part, mediated by the interaction of ZP1 and ZP3 with the sperm head during fertilization in Japanese quail.

Sperm motility is an essential trait for successful fertilization in animals. In birds, ejaculated sperm migrate into sperm storage tubules before fertilization and are stored in a quiescent state. We previously reported that this type of sperm’s flagellar quiescence was induced by lactic acid through flagellar dynein ATPase inactivation following cytoplasmic acidification (

Sperm motility is considered as one of the most important traits for successful fertilization, but the motility of an ejaculated sperm decreases with time when stored as liquid. It is reported that seminal plasma serves as a nutrient rich medium for sperm and plays an important role in sperm motility and its fertilization ability. Several studies have reported that imidazole dipeptides such as anserine and carnosine affect sperm motility and its fertilization ability in mammals. In this study, we report the presence of anserine and carnosine in the male reproductive tract of the Japanese quail. Abundant levels of anserine (44.46 μM) and carnosine (41.75 μM) were detected in the testicular fluid and seminal plasma respectively using the amino acid analyzer; however, seminal plasma solely contained carnosine. When the ejaculates were incubated with anserine or carnosine, we found that both the dipeptides improve sperm motility parameters such as straight line velocity, curvilinear velocity, average path velocity and amplitude of lateral head displacement after in vitro sperm storage at 15℃. These results indicate that imidazole dipeptides are present in the male reproductive tract and may improve sperm quality during in vitro sperm storage in the liquid states. [ABSTRACT FROM AUTHOR]