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Sperm drastically change their flagellar movement in response to the surrounding physical and chemical environment. Testicular sperm are immotile; however, they gain the competence to initiate motility during passage through the male reproductive tract. Once ejaculated, the sperm are activated and promptly initiate motility. Unlike mammals, ejaculated sperm in birds are stored in specialized tubular invaginations referred to as sperm storage tubules (SSTs), located between the vagina and uterus, before fertilization. The resident sperm in the SSTs are in a quiescent state and then re-activated after release from the SSTs. It is thought that avian sperm can undergo motility change from quiescent to active state twice; however, the molecular mechanism underlying sperm motility regulation is poorly understood. In this short review, we summarize the current understanding of sperm motility regulation in male and female bird reproductive tracts. We also describe signal transduction, which regulates sperm motility, mainly derived from in vitro studies.

Objective: This study investigated the effects of 5-aminolevulinic acid (5-ALA) on the motility parameters, mitochondrial membrane depolarization, and ATP levels in chicken sperm.Methods: The pooled semen from Barred Plymouth Rock males was used. In the first experiment, the semen was diluted 4-times with phosphate-buffered saline (PBS (-)) containing various concentrations (0, 0.01, 0.05, and 0.1 mM) of 5-ALA, and then the sperm motility parameters after incubation were evaluated by computer-assisted sperm analysis (CASA). In the second experiment, the semen was diluted 4-times with PBS (-) containing 0.05 mM 5-ALA, and then sperm mitochondrial membrane depolarization and ATP levels after 1.5 h of incubation were analyzed with the MitoPT® JC-1 Assay and ATP Assay kits, respectively. In the third experiment, the semen was removed from the seminal plasma and resuspended with the mediums of PBS (-), PBS (-) supplemented with CaCl2 and MgCl2 (PBS (+)) + 5-ALA, PBS (+) + caffeine, and PBS (+) + caffeine + 5-ALA. Then, the sperm motility parameters after incubation were evaluated by CASA. In the last experiment, the semen was treated with the mediums of PBS (-), PBS (-) + 5-ALA, 5.7% glucose, 5.7% glucose + 5-ALA after removing the seminal plasma, and then the sperm motility parameters were evaluated by CASA.Results: The addition of 0.05 mM 5-ALA significantly increased the chicken sperm motility, progressive motility, linearity, average path velocity, curvilinear velocity, straight-line velocity, and the wobble. The sperm mitochondrial membrane depolarization was also increased by the 5-ALA treatment. The 5-ALA treatment decreased the sperm ATP levels. Both the caffeine treatment and glucose treatment decreased the sperm motility during incubation period.Conclusion: 5-ALA might increase sperm mitochondrial membrane depolarization to utilize the ATP for enhancing sperm movement.

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°C. 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.

CpG-oligodeoxynucleotides (CpG-ODN) has been shown to possess immunostimulatory features in both mammals and birds. However, compared to its pro-inflammatory effects, little is known about the anti-inflammatory responses triggered by CpG-ODN in avian cells. Hence, in this study, we aimed to characterize the anti-inflammatory response in the chick macrophage cell line HD11 under the stimulation of five kinds of CpG-ODN: CpG-A1585, CpG-AD35, CpG-B1555, CpG-BK3, and CpG-C2395. Single-stimulus CpG-B1555, CpG-BK3, and CpG-C2395 induced the interleukin (IL)-10 expression without causing cellular injury. The effects of pretreatment with each CpG-ODN before subsequent lipopolysaccharide stimulation were also evaluated. Interestingly, only CpG-C2395 maintained a high expression level in this situation. Finally, expression analysis of inflammation-related genes, such as the tumor necrosis factor-α, IL-1β, IL-6, and Toll-like receptor 4, was conducted, and pretreatment with CpG-C2395 significantly reduced their expression. Overall, our results shed light on the anti-inflammatory responses triggered by CpG-C2395 stimulation using a comparative analysis of three major classes of CpG-ODN in chick macrophages.

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.

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.

In birds, the ejaculated spermatozoa do not directly pass to the site of fertilization but rather are stored initially in specialized structures, referred to as sperm storage tubules (SSTs), located in the utero-vaginal junction (UVJ) of the oviduct. The fertilizing capacity of spermatozoa in the SSTs is maintained for an extended period (i.e., several days to months). Although many studies have been conducted to ascertain the mechanisms involved in sperm storage, the understanding of the phenomenon is limited. In this study, there was investigation of the effects of sperm surface oligosaccharides in sperm passage into SSTs in Japanese quail. Results from lectin staining of ejaculated spermatozoa indicated galactose/N-Acetylgalactosamine (Gal/GalNAc), N-Acetylglucosamine (GlcNAc) or mannose/glucose (Man/Glc) moieties were present on the sperm surface, indicating the presence of glycoproteins/glycolipids containing these oligosaccharides. When ejaculated spermatozoa were co-incubated with UVJ explants, the lectins derived from Agaricus bisporus and Canavalia ensiformis had marked inhibitory effects on sperm passage into SSTs. Preincubation of UVJ explants with these lectins, however, had no effect indicating there were no effects of UVJ oligosaccharides in this process. Furthermore, none of these lectin had effects on values of sperm motility variables. These results indicate that O-glycans with terminal β-Gal or GalNAc and N-glycans with terminal α-D-Man or α-D-Glc may have functions in the process of sperm passage into SSTs.

Fertilization in animals that employ sexual reproduction is an indispensable event for the production of the next generation. A significant advancement in our understanding of the molecular mechanisms of sperm-egg interaction in mammalian species was achieved in the last few decades. However, the same level of knowledge has not been accumulated for birds because of egg size and the difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. In this review, we summarize the current understanding of sperm-egg interaction mechanism during fertilization in birds, especially focusing on sperm-egg binding, sperm acrosome reaction and the authentic sperm protease required for the hole formation on the perivitelline membrane. We explain that the zona pellucida proteins (ZP1 and ZP3) in the perivitelline membrane play important roles in sperm-egg binding, induction of the acrosome reaction as well as sperm penetration by digestion of sperm protease. We anticipate that a deeper understanding of avian fertilization will open up new avenues to create powerful tools for a myriad of applications in the poultry industries including the production of transgenic and cloned birds.

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 (

Prolactin receptor (PRLR) is expressed in a wide variety of tissues and mediates diverse biological actions of prolactin (PRL). In mammals, PRL signaling is thought to be involved not only in the process of spermatogenesis and steroidogenesis in the testis, but also in the survival of ejaculated sperm. In avian species, although the expression of PRLR with several variants in the testis was reported, the role of PRL in testicular function is still unclear. The aim of this study was to examine the expression of PRLR in the testis and mature sperm in quail. It is revealed that PRLR was mainly localized in the round- and elongated-spermatid by immunohistochemical analysis on the testis suggesting that PRL signaling may participate in the spermatogenesis. Western blot analysis confirmed the presence of PRLR in the plasma membrane of the ejaculated sperm (SPML), whereas the size of PRLR in the sperm was smaller than that in the hypothalamus. Moreover, PRLR was detected on the surface of the midpiece and flagellum of sperm by immunostaining. To evaluate the functionality of the sperm PRLR, the dot blot assay was performed to test the binding of pituitary PRL to PRLR in the SPML, and resulted in the detection of specific binding of PRL to the component of SPML, most likely to sperm PRLR. Furthermore, when the ejaculates were incubated with pituitary PRL to investigate the role of PRL on the sperm, the occurrence of spontaneous acrosome reaction was significantly decreased. In addition, the expression of PRL on the surface of utero-vaginal junction of oviduct was detected by immunohistochemistry. These results may suggest a novel system that the interaction between oviductal PRL and sperm PRLR is involved in the maintenance of the fertilizability of the spermatozoa through the prevention of the spontaneous acrosome reaction in Japanese quail.

Biased mating due to female preferences towards certain traits in males is a major mechanism driving sexual selection, and may constitute an important evolutionary force in organisms with sexual reproduction. In birds, although the role of male ornamentation, plumage coloration, genetic dissimilarity, and body size have on mate selection by females have been examined extensively, few studies have clarified exactly how these characteristics affect female mate preferences. Here, we show that testosterone (T)-dependent male attractiveness enhances female preference for males of a polygamous species, the Japanese quail. A significant positive correlation between female mating preference and circulating T in the male was observed. The cheek feathers of attractive males contained higher levels of melanin and were more brightly colored. The ability of females to distinguish attractive males from other males was negated when the light source was covered with a sharp cut filter (cutoff

To reproduce sexually, males and females produce very different gametes (sperm and eggs) in many animals. This difference gives rise to very different strategies in the two sexes and in gamete cells from the two sexes. Sperm meet eggs in harmony; however, the male and female do not always have common interests in reproduction. The battle of the sexes continues even after copulation. Female promiscuity is key to the understanding of reproductive behaviors not only in male individuals but also in sperm cells, because sexual selection continues after mating through sperm competition. Here, we highlight multiple sperm traits—the sperm acrosome reaction in sea urchins, sperm storage in birds, and sperm dimorphism in squid—that are tightly associated with postcopulatory reproductive strategies.

In internal fertilizers including mammals, fertilization success depends on the timely arrival of sperm and egg at the site of fertilization. Males should transfer their spermatozoa to the female reproductive tract by copulation during or prior to ovulation in order to achieve this aim. However, such a collaborative mating behavior is often disconnected from the efficiency of the sperm-egg encounter, i.e., ovulation by females occurs independently from insemination by males in many species. To compensate for this time lag, females are capable of storing spermatozoa in their reproductive tracts until the eggs are ready to be fertilized. In avian species, simple tubular invaginations referred to as sperm storage tubules (SSTs) are located between the vagina and uterus as sperm storage sites. Spermatozoa, once ejaculated, migrate to and are thereafter stored in the lumen of the SSTs without loss of fertilizing capacity for up to 15 weeks at a body temperature of 41 °C. This is astonishing, because terminally differentiated cells that lack new protein synthesis are still capable of being functional for a long period at a high temperature; however, the actual mechanism has been an enigma for more than half a century. In this chapter, we will first describe the physiological importance and adoptive benefits of sperm storage in the female genital tract for successful fertilization in animals, and next, we will describe our recent findings in birds with regard to the specific mechanism of sperm uptake into the SST, sperm maintenance within it, and controlled release from it.

A characteristic biological property of avian gamete (e.g., extremely large egg and polyspermic fertilization) does not allow the direct observation of sperm-egg interactions in vitro, but recent research advances make it possible to manipulate the gamete in vitro. Here, we describe the techniques for the handling of gametes required for in vitro fertilization assay. In addition, we also introduce the procedures for sperm-perivitelline membrane assay, intracytoplasmic sperm injection, and ex ovo culture.

A characteristic biological property of avian gamete (e.g., extremely large egg and polyspermic fertilization) does not allow the direct observation of sperm-egg interactions in vitro, but recent research advances make it possible to manipulate the gamete in vitro. Here, we describe the techniques for the handling of gametes required for in vitro fertilization assay. In addition, we also introduce the procedures for sperm-perivitelline membrane assay, intracytoplasmic sperm injection, and ex ovo culture.

In internal fertilizers including mammals, fertilization success depends on the timely arrival of sperm and egg at the site of fertilization. Males should transfer their spermatozoa to the female reproductive tract by copulation during or prior to ovulation in order to achieve this aim. However, such a collaborative mating behavior is often disconnected from the efficiency of the sperm–egg encounter, i.e., ovulation by females occurs independently from insemination by males in many species. To compensate for this time lag, females are capable of storing spermatozoa in their reproductive tracts until the eggs are ready to be fertilized. In avian species, simple tubular invaginations referred to as sperm storage tubules (SSTs) are located between the vagina and uterus as sperm storage sites. Spermatozoa, once ejaculated, migrate to and are thereafter stored in the lumen of the SSTs without loss of fertilizing capacity for up to 15 weeks at a body temperature of 41 °C. This is astonishing, because terminally differentiated cells that lack new protein synthesis are still capable of being functional for a long period at a high temperature; however, the actual mechanism has been an enigma for more than half a century. In this chapter, we will first describe the physiological importance and adoptive benefits of sperm storage in the female genital tract for successful fertilization in animals, and next, we will describe our recent findings in birds with regard to the specific mechanism of sperm uptake into the SST, sperm maintenance within it, and controlled release from it.

Systems for maintaining the viability of ejaculated sperm in the female reproductive tract are widespread among vertebrates and invertebrates. In birds, this sperm storage function is performed by specialized simple tubular invaginations called sperm storage tubules (SSTs) in the uterovaginal junction (UVJ) of the oviduct. Although the incidence and physiological reasons for sperm storage in birds have been reported extensively, the mechanisms of sperm uptake by the SSTs, sperm maintenance within the SSTs, and control of sperm release from the SSTs are poorly understood. In this study, we demonstrated that the highly conserved heat shock protein 70 (HSP70) stimulates sperm motilityin vitroand also that HSP70 expressed in the UVJ may facilitate the migration of sperm released from the SSTs. Quantitative RT-PCR analysis demonstrated that the expression ofHSP70mRNA in the UVJ increases before ovulation/oviposition. Gene-specificin situhybridization and immunohistochemical analysis with a specific antibody to HSP70 demonstrated that HSP70 is localized in the surface epithelium of the UVJ. Furthermore, injection of anti-HSP70 antibody into the vagina significantly inhibited fertilizationin vivo. In addition, we found that recombinant HSP70 activates flagellar movement in the sperm and that the binding of recombinant HSP70 to the sperm surface is mediated through an interaction with voltage-dependent anion channel protein 2 (VDAC2). Our results suggest that HSP70 binds to the sperm surface by interacting with VDAC2 and activating sperm motility. This binding appears to play an important role in sperm migration within the oviduct.

The ability to store sperm in the female genital tract is frequently observed in vertebrates as well as in invertebrates. Because of the presence of a system that maintains the ejaculated sperm alive in the female reproductive tract in a variety of animals, this strategy appears to be advantageous for animal reproduction. Although the occurrence and physiological reasons for sperm storage have been reported extensively in many species, the mechanism of sperm storage in the female reproductive tract has been poorly understood until recently. In avian species, the specialized simple tubular invaginations referred to as sperm storage tubules (SSTs) are found in the oviduct as a sperm storage organ. In this review, we summarize the current understanding of the mechanism of sperm uptake into the SSTs, maintenance within it, and controlled release of the sperm from the SSTs. Since sperm storage in avian species occurs at high body temperatures (i.e., 41 C), elucidation of the mechanism for sperm storage may lead to the development of new strategies for sperm preservation at ambient temperatures, and these could be used in a myriad of applications in the field of reproduction.

The capability for sperm storage in the female genital tract is frequently observed in vertebrates as well as in invertebrates. Because of the presence of a system that maintains the ejaculated sperm alive in the female reproductive tract in a variety of animals, including insects, fish, amphibians, reptiles, birds, and in mammals, this strategy appears to be advantageous for animal reproduction. Although the occurrence and physiological reasons for sperm storage have been reported extensively in many species, the mechanism for sperm storage in the female reproductive tract has been poorly understood until recently. In this chapter, we report our recent findings on the mechanism of sperm storage in avian oviducts, especially data obtained from the Japanese quail (Coturnix japonica), as an experimental model. Because sperm storage in birds occurs at body temperature (i.e. 41 °C), elucidation of the mechanism of sperm maintenance in the avian oviduct may open up new avenues for the development of novel strategies for sperm storage in vitro without cryopreservation.

A wide diversity of domesticated chicken breeds exist due to artificial selection on the basis of human interests. Miniature variants (bantams) are eminently illustrative of the large changes from ancestral junglefowls. In this report, the genetic characterization of seven Japanese miniature chicken breeds and varieties, together with institute-kept Red Junglefowl, was conducted by means of typing 40 microsatellites located on 21 autosomes. We drew focus to genetic differentiation between the miniature chicken breeds and Red Junglefowl in particular. A total of 305 alleles were identified: 27 of these alleles (8.9%) were unique to the Red Junglefowl with high frequencies (>20%). Significantly high genetic differences (F(ST)) were obtained between Red Junglefowl and all other breeds with a range of 0.3901-0.5128. Individual clustering (constructed from combinations of the proportion of shared alleles and the neighbour-joining method) indicated high genetic divergence among breeds including Red Junglefowl. There were also individual assignments on the basis of the Bayesian and distance-based approaches. The microsatellite differences in the miniature chicken breeds compared to the presumed wild ancestor reflected the phenotypic diversity among them, indicating that each of these miniature chicken breeds is a unique gene pool.

Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41 °C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (

Fertilization is an indispensable step for formation of a zygote in sexual reproduction, leading to species survival. When mating occurs, sperm is transported to the female reproductive tracts via the seminal plasma (SP). SP is derived from male accessory sex glands and it plays pivotal roles for fertilization in animals. However, molecular mechanisms of SP or a fluid derived from male accessory sex glands for successful fertilization remain unclear. Here, we report that in male quail the cloacal gland (CG) produces prostaglandin F2α (PGF2α) that contributes to successful fertilization. PGF2α, as well as the secretion of CG (CGS), induced vaginal contractions and caused the opening of the entrance of the sperm storage tubules, the structures responsible for the long-term sperm storage and fertilization. The removal of CGS from the male before mating reduced the fertility, but the supplementation of CGS or PGF2α rescued the subfertility. We further showed that male CG contains glucose that is utilized as energy source for the intrinsic sperm mobility after transportation to female vagina. This mechanism, in concert with the excitatory effects of PGF2α enables successful fertilization in the domestic bird.