Simple Culture System for Bobwhite Quail and Japanese Quail Embryos from the Blastoderm Stage to Hatching using a Single Surrogate Eggshell

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.ArticleJOURNAL OF POULTRY SCIENCE. 51(2):202-205 (2014)journal articl

Culture System for Bobwhite Quail Embryos from the Blastoderm Stage to Hatching

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 egg (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.ArticleJOURNAL OF POULTRY SCIENCE. 50(2):155-158 (2013)journal articl

Fertilization 2: Polyspermic Fertilization

During fertilization in animals, a haploid egg nucleus fuses with a haploid sperm nucleus to restore the diploid genome. In most animals including mammals, echinoderms, and teleostei, the penetration of only one sperm into an egg is ensured at fertilization because the entry of two or more sperm is prevented by polyspermy block systems in these eggs. On the other hand, several animals such as birds, reptiles, and most urodele amphibians exhibit physiological polyspermy, in which the entry of several sperm into one egg is permitted. However, in these polyspermic eggs, only one sperm nucleus is involved in zygotic formation with a female nucleus, thereby avoiding syngamy with multiple sperm nuclei. In the chicken, 20-60 sperm are generally found within the egg cytoplasm at fertilization and this number is markedly higher than that of other polyspermic species; however, avian-specific events such as the degeneration and mitosis of supernumerary sperm nuclei during early embryo development allow a polyspermic egg to develop normally. This chapter describes current knowledge on polyspermy-related events in avian eggs during fertilization, and is characterized by a comparison to the fertilization modes of other vertebrates. The close relationship between sperm numbers and egg sizes, and the movement of supernumerary sperm nuclei towards the periphery of the egg cytoplasm and their degeneration are summarized. The molecular mechanisms by which polyspermy initiates egg activation to start embryo development are also discussed

Research Note: Diethylstilbestrol reduces primordial germ cells in male Japanese quail

ABSTRACT: This study investigated the detrimental effects of diethylstilbestrol (DES), an estrogenic endocrine-disrupting chemical, on the viability of primordial germ cells (PGCs), embryonic precursors of germ cells, in Japanese quail. We injected 50 or 100 nmol DES solubilized in sesame oil into the yolk of stage X embryos and assessed changes in the population and cell cycle properties of circulating PGCs in blood vessels and gonadal PGCs after 2.5- and 7-day incubations, respectively. Liquid chromatography tandem mass spectrometer and Western blotting analyses identified DEAD-box polypeptide 4 (DDX4) and proliferating cell nuclear antigen (PCNA) as a stem cell marker and proliferation marker of quail PGCs, respectively. Immunochemical analyses revealed significant decreases in the number of DDX4- and PCNA-positive blood-circulating PGCs in males treated with 50 and 100 nmol DES than in the oil-treated control group. These reductions were not observed in females. Furthermore, the number of DDX4-positive gonadal PGCs was smaller in males treated with 50 and 100 nmol DES than in the control group, and these reductions were not observed in females. The protein expression of the Sertoli cell marker showed normal testis development in DES-treated embryos on d 7. These results demonstrate the potentially cytotoxic effects of DES on male germ cells, namely, the inhibition of cell cycle progression and induction of apoptosis in Japanese quail

Knockdown of DEAD-box helicase 4 (DDX4) decreases the number of germ cells in male and female chicken embryonic gonads

DEAD-box helicase 4 (DDX4; also known as vasa) is essential for the proper formation and maintenance of germ cells. Although DDX4 is conserved in a variety of vertebrates and invertebrates, its roles differ between species. This study investigated the function of DDX4 in chicken embryos by knocking down its expression using retroviral vectors that encoded DDX4-targeting microRNAs. DDX4 was effectively depleted invitro and invivo via this approach. Male and female gonads of DDX4-knockdown embryos contained a decreased number of primordial germ cells, indicating that DDX4 is essential to maintain a normal level of these cells in chicken embryos of both sexes. Expression of doublesex and mab-3 related transcription factor 1 (DMRT1) and sex determining region Y-box 9 (SOX9), which are involved in testis determination and differentiation, was normal in male gonads of DDX4-knockdown embryos. In contrast, expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which encodes aromatase and is essential for ovary development, was significantly decreased in female gonads of DDX4-knockdown embryos. Expression of forkhead box L2 (FOXL2), which plays an important role in ovary differentiation, was also slightly reduced in DDX4-knockdown embryos, but not significantly. Based on several pieces of evidence FOXL2 was hypothesised to regulate aromatase expression. The results of this study indicate that aromatase expression is also regulated by several additional pathways

cDNA Cloning and mRNA Expression of Androgen Receptor in Male Japanese Quail (Coturnix coturnix japonica)

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

Cyclin D1 gene expression is essential for cell cycle progression from the maternal-to-zygotic transition during blastoderm development in Japanese quail

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

Expression of Transferrin and Albumin in the Sperm-Storage Tubules of Japanese Quail and their Possible Involvement in Long-Term Sperm Storage

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

Egg Envelope Glycoproteins ZP1 and ZP3 Mediate Sperm-Egg Interaction in the 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