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In vitro fertilization has been widely used to produce offspring in several mammalian species. We previously successfully produced Japanese quail chicks using intracytoplasmic sperm injection (ICSI), whereas in vitro insemination was not successful. This may be due to the difficulties associated with mimicking the sperm-egg fusion process and subsequent events in physiological polyspermic fertilization in vitro. In the present study, we observed egg development after in vitro insemination and investigated the inactivation of metaphase-promoting factor (MPF) and cytostatic factor (CSF), which are downstream of the Ca2+ signaling pathway in the egg, due to fertilizing sperm. We found a sperm number-dependent increase in hole formation caused by sperm penetration of the perivitelline membrane, the extracellular coat surrounding the egg. Egg development was observed following in vitro insemination; however, the developmental rate and stages after 24-h culture were inferior to those of ICSI eggs, even when insemination was performed with a high number of sperm (2 × 104). We also noted the downregulation of inositol 1,4,5-trisphosphate receptor-1, ryanodine receptor-3, cyclin B1, and c-MOS, which are important regulatory components of MPF and CSF in the egg, which was dependent on the number of sperm used for insemination. However, the decreases observed in these components did not reach the levels observed in the ICSI eggs. Collectively, the present results suggest that a sperm number higher than 2 × 104 is required for the progression of the Ca2+ signaling pathway, which initiates subsequent egg development in Japanese quail.

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca2+ ([Ca2+]i): transient elevations in [Ca2+]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca2+ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca2+]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca2+ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca2+]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca2+ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate increases in [Ca2+]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca2+ wave and spiral-like Ca2+ oscillations, respectively.

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.

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 (

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.

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.

The ex vivo culture of avian embryos is a technique for the long-term culturing of embryos outside of their own shell and shell membrane. It allows easy access to the developing embryos and embryo manipulation. The two-step system is widely applied when the culture is performed after oviposition. Japanese quail as well as bobwhite quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been more limited than those on the Japanese quail. We have developed a more simplified ex vivo culture protocol for the two species of quail embryos from the blastoderm stage through to hatching using a single surrogate eggshell. Hatchabilities of 31% and 27% were obtained in bobwhite quail and Japanese quail embryos, respectively. The simple system described in the present study is an easy and acceptable procedure.

Quail are divided phylogenetically into two groups, Old World quail and New World quail. Old World quail, such as the Japanese quail (Coturnix japonica), belong to the Phasianidae and distributed in the Palaearctic region (Europe, North Africa, and Asia), whereas New World quail, such as the bobwhite quail (Colinus virginianus), belong to the Odontophoridae and are restricted to North and South America. Both the bobwhite quail and the Japanese quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been limited compared with those on the Japanese quail. We have therefore now developed an ex vivo culture protocol for bobwhite quail embryos from the blastoderm stage through hatching. Of the various culture conditions examined in the present study, a good hatching rate (39%) was obtained when the embryos were cultured ex vivo in a two-step procedure. Unincubated embryos (with egg yolk) were first cultured inside the shell of a Japanese quail eggs (11.5 to 13.0 g whole egg weight) together with chicken thin albumen for 63 to 65 h and were then transferred to the shell of a small-sized chicken egg (38 g whole egg weight) until hatching. This ex vivo culture system should provide to be widely applicable to the maintenance and generation of manipulated birds for basic and applied studies on the bobwhite quail.

In mammals, a normal offspring can be obtained even from infertile male by intracytoplasmic sperm injection (ICSI). Although ICSI technique has permitted significant progress in clinical practice in humans and mouse to date, it has been established recently in birds. In addition, efficiency of fertility and developmental rates has been low and no chick has been produced by in vitro fertilization and culture. Furthermore, polyspermic fertilization and subsequent normal developmental processes remains unknown. The enhancement of fertility and developmental rates is the first step in the avian ICSI system to be applied for protection of endangered species and production of transgenic and clone birds. This review paper describes (1) the establishment of ICSI technique in Japanese quail, (2) molecular mechanisms whereby polyspermy activates development of quail oocyte, (3) improvement of ICSI efficiency by phospholipase Cζ cRNA. Also, possible application of ICSI for avian sex manipulation and transgenic birds was summarized.

Although many studies have shown that P450aromatase (P450arom), anti-Müllerian hormone (AMH) and estrogen receptor α (ERα) play pivotal roles in sexual differentiation of the gonads during early embryonic development in chickens and quail, few studies have been reported for other domestic birds. Furthermore, little information is available in relation to the mRNA expression in gonads after sexual differentiation in posthatching birds. The present study was conducted to assay mRNA expression of P450arom, AMH and ERα in gonads at day of hatch in turkey and duck and 2 days after hatching in goose using the real-time PCR. The mRNA expression was also determined at one week of age in gonads of these birds. At the time of collection of the left gonads for total RNA extraction, the external appearance of the left and right gonads of male and female was documented by digital camera. Clear asymmetry was observed in the female ovary in which the right ovary was regressed completely by 1-2 days in posthatching turkey, duck and goose. Although gonadal asymmetry was as remarkable as in females, the left testis was larger than the right one in males. Remarkable expression of P450arom mRNA was observed only in females in all the 3 species but substantially no expression was detected in males. Significantly higher expression of AMH mRNA was detected in males than females only in goose but there was no sex difference in turkey and duck at 1-2 days posthatching and one week of age. Weak ERα mRNA expression without a sex difference was detected in the 3 birds. These results suggest that estrogen plays a key role for ovarian development via P450arom mRNA expression after hatching, whereas absence of its expression in males leads to testis development in turkey, duck and goose.

The present study was conducted to investigate (1) the effective day of treatment with Fadrozole, nonsteroidal aromatase inhibitor (AI), during incubation for sex reversal, (2) the mRNA expression of aromatase (P450arom), anti-Müllerian hormone (AMH) and estrogen receptor α (ERα) in quail gonad and gonadal structures after the AI treatment before hatching and (3) the effects of AI on gonad growth, left-right asymmetry of the gonad and body weight gain between 1 to 7 weeks posthatch. (1) Females AI-treated at 0, 2 and 4 days of incubation had symmetrical gonads at hatch similar to normal males, whereas those treated at 6 and 8 days of incubation had asymmetrical gonads. (2) In females AI-treated at day 0 of incubation, the left gonad became an ovotestis at day 15 of incubation, displaying seminiferous tubules with testicular-like cords and ovarian follicle with ovum. In the control group, P450arom mRNA expression was significantly higher in females than males. However, AI-treated females showed 2 response-patterns of either higher or lower expression. The latter was similar to that of control males. No significant differences in levels of AMH and ERα mRNA between sexes either in control or AI-treated groups was observed. (3) A gradual increase in body weight of the control males and females without sex difference was observed up to 5 weeks of age. However, at 6-7 weeks the average weight of females was heavier than males each in control and treated group. These results indicate that shortly before day 6 of incubation P450arom mRNA expression, at least in a part, is important for asymmetrical formation of gonads in female quail. In AI-treated females, the high responder show similar level of P450arom mRNA in control males and the low responder do not respond to the AI. Body weight is controlled non-gonadally in quail.

The present study was conducted to show a profile of mRNA expression of sex-differentiation related genes in the duck gonad. Fertile duck eggs (Cherry-valley) were incubated (hatching at day 28 of incubation) and the gonads were collected at days 7, 8, 10, 12 and 15 of incubation after taking photos of the gonads. Left gonad was used for identification of genetic sex and for total RNA extraction. Reverse transcription-polymerase chain reaction (PCR) was performed using appropriate forward and reverse primers for each of FOXL2, P450arom, DMRT1, AMH, P450c17, SF1, ER α and AR genes of the chicken and the corresponding cDNA fragments were sequenced for duck. Subsequently, real-time PCR was performed to detect mRNA expression of those genes in the duck embryo. Male bilateral gonads assumed symmetry in appearance throughout the examined period, whereas the female gonads were noticeably asymmetric at 10 days of incubation with a smaller size of the right. Female asymmetry was distinct by day 15 of incubation. In females, FOXL2 and P450arom mRNA expression began at day 8 of incubation and remained high thereafter, but in males only negligible expression was detected. In males, mRNA expression of DMRT1 and AMH was detected at day 7 of incubation and tended to increase gradually thereafter, whereas in females the expression remained low throughout the examined period. Though the slight expression of P450c17 mRNA started at day 7 only in males, the expression was low throughout the examined period, whereas in female the expression was distinct at day 8 of incubation and remained high thereafter. Expressions of the other genes were variable with no differences in each expression between the sexes. The results indicate that FOXL2 and P450arom play an important role for the ovarian formation.

The present study was conducted to improve the rate and stage of blastoderm and embryo development of quail oocytes by intracytoplasmic sperm injection (ICSI) with quail phospholipase Cζ (PLCζ) cRNA. Blastoderm development of quail oocytes cultured for 24 or 72h were staged under a stereomicroscope. The blastoderms were stained with 4,6-diamidino-2-phenylindole (DAPI) for cell division progress. A quail PLCζ cDNA clone was isolated from sperm by RT-PCR. The 1978bp nucleotide sequence contained an ORF encoding 637 amino acids of protein. The deduced quail PLCζ protein consists of EF-hand domain, X and Y catalytic domain and C2 domain, but lacked a plextrin-homology (PH) domain at the N-terminus. RT-PCR analysis revealed that PLCζ mRNA is expressed only in the testis. ICSI into a quail oocyte without PLCζ cRNA induced blastodermal development in 16% (4/25) of the oocytes which reached stages III-VI 24h after culture. In contrast, ICSI together with PLCζ cRNA (in 3nl at 60μg/ml) into a quail oocyte induced blastoderm development more than 2 fold (36.8%, 7/19) with embryos reaching stages VI-VII. Furthermore, 72h after culture ICSI into a quail oocyte without PLCζ cRNA induced embryo development in 15.4% (2/13) of the oocytes and reached stages V-VII. In contrast, ICSI together with PLCζ cRNA (in 3nl at 60μg/ml) into a quail oocyte induced 3 fold more embryo development (50%, 9/18) with embryos between stages VI and over X. The microinjection of PLCζ cRNA significantly improved the conventional ICSI method by enhancing the proportion and the stages of embryo development. Accordingly, the present PLCζ cRNA microinjection method in birds may contribute to assist in the production of transgenic birds and protection of endangered species of birds.

A polymerase chain reaction (PCR) primer set for a chicken pkci gene on the Z-chromosome, chPKCI, amplified a 329 bp fragment of genomic DNA in chickens but not quail. Likewise, a PCR primer set for the quail pkci gene on the Z-chromosome, quPKCI, amplified a 95 bp fragment of genomic DNA in quail but not chickens. These chicken and quail primers validly identified chicken-quail hybrids derived from Z-chromosome carrying sperm of chicken and the hybrid derived from Z-chromosome carrying oocyte of quail, respectively.

The biological activities of androgen are largely mediated via androgen receptor (AR). Although a partial sequence AR cDNA was revealed in canaries and zebra finch, AR cDNA has not been cloned in quail and chickens. To understand the physiological function of androgen and to apply it for other purposes, AR cDNA is necessary. Hence, this study was aimed to isolate the cDNA of AR and to reveal changes in mRNA expression using RT-PCR analysis in male reproductive organs of quail under different day-lengths and castration treatment. We found that a partial length of quail AR cDNA contains a 1385bp sequence encoding 343 amino acid residues and had high homology to other vertebrates. Quail were raised under continuous light regimen from 4 to 6 weeks old (6LL) and they were divided into 3 groups under the following conditions up to 9 weeks old : (1) continuous light regimen group (24h light ; 9LL), (2) short days regimen group (8h light and 16h darkness ; 9SD) and (3) castration group in which testis were surgically removed at 6 weeks old and raised under continuous light regimen (9CAS). Although weekly changes in the cloacal gland protrusion area showed significantly progressive increase at 7-9 weeks old in 9LL group and a progressive decrease in 9SD group, there were no changes in AR mRNA levels in the gland. In contrast, in 9CAS group, although the cloacal gland protrusion area decreased after castration treatment, AR mRNA levels increased in the gland. On the other hand, AR mRNA levels in epididymis and vas deferens increased in 9LL group, but there were no differences in 9SD and 9CAS groups from 6LL. The testicular AR mRNA levels did not change after long or short days treatment. These results indicate there is tissue-specific regulation in AR mRNA expression in quail.