Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9.

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Loss of One X and the Y Chromosome Changes the Configuration of the X Inactivation Center in the Genus Tokudaia.

INTRODUCTION: X chromosome inactivation (XCI) is an essential mechanism for dosage compensation between females and males in mammals. In females, XCI is controlled by a complex, conserved locus termed the X inactivation center (Xic), in which the lncRNA Xist is the key regulator. However, little is known about the Xic in species with unusual sex chromosomes. The genus Tokudaia includes three rodent species endemic to Japan. Tokudaia osimensis and Tokudaia tokunoshimensis lost the Y chromosome (XO/XO), while Tokudaia muenninki (TMU) acquired a neo-X region by fusion of the X chromosome and an autosome (XX/XY). We compared the gene location and structure in the Xic among Tokudaia species. METHODS: Gene structure of nine genes in Xic was predicted, and the gene location and genome sequences of Xic were compared between mouse and Tokudaia species. The expression level of the gene was confirmed by transcripts per million calculation using RNA-seq data. RESULTS: Compared to mouse, the Xic gene order and location were conserved in Tokudaia species. However, remarkable structure changes were observed in lncRNA genes, Xist and Tsix, in the XO/XO species. In Xist, important functional repeats, B-, C-, D-, and E-repeats, were partially or completely lost due to deletions in these species. RNA-seq data showed that female-specific expression patterns of Xist and Tsix were confirmed in TMU, however, not in the XO/XO species. Additionally, three deletions and one inversion were confirmed in the intergenic region between Jpx and Ftx in the XO/XO species. CONCLUSION: Our findings indicate that even if the Xist and Tsix lncRNAs are expressed, they are incapable of producing a successful and lasting XCI in the XO/XO species. We hypothesized that the significant structure change in the intergenic region of Jpx-Ftx resulted in the inability to perform the XCI, and, as a result, a lack of Xist expression. Our results collectively suggest that structural changes in the Xic occurred in the ancestral lineage of XO/XO species, likely due to the loss of one X chromosome and the Y chromosome as a consequence of the degradation of the XCI system.

Identification of a New Enhancer That Promotes Sox9 Expression by a Comparative Analysis of Mouse and Sry-Deficient Amami Spiny Rat.

INTRODUCTION: Testis differentiation is initiated by the SRY gene on the Y chromosome in mammalian species. However, the Amami spiny rat, Tokudaia osimensis, lacks both the Y chromosome and the Sry gene and acquired a unique Sox9 regulatory mechanism via a male-specific duplication upstream of Sox9, without Sry. In general mammalian species, the SRY protein binds to a testis-specific enhancer to promote SOX9 gene expression. Several enhancers located upstream of Sox9/SOX9 have been reported in mice and humans. In particular, the binding of SRY to the highly conserved enhancer Enh13 is thought to be a common mechanism underlying testis differentiation and sex determination in mammals. METHODS: Sequences of T. osimensis homologues of three Sox9 enhancers that were previously reported in mice, Enh8, Enh14, and Enh13, were determined. We performed in vitro assays to confirm enhancer activity involved in Sox9 regulation in T. osimensis. RESULTS: T. osimensis Enh13 showed enhancer activity when co-transfected with NR5A1 and SOX9. Mouse Enh13 was activated by NR5A1 and SRY; however, T. osimensis Enh13 did not respond to SRY, even though the binding sites of SRY and NR5A1 were conserved. To identify the key sequence that is present in mouse but absent from T. osimensis, we performed reporter gene assays using vectors in which partial sequences of T. osimensis Enh13 were replaced with mouse sequences. For T. osimensis Enh13 in which the second half (approximately 430 bp) was replaced with the corresponding mouse sequence, activity in response to NR5A1 and SRY was recovered. Further, reporter assays revealed that multiple regions in the second half of the mouse Enh13 sequence are required for the response to NR5A1 and SRY. The latter 49 bp was particularly important and contained four binding sites for three transcription factors, POU2F1, HOXA3, and GATA1. CONCLUSION: We showed that there are unknown sequences responsible for the interaction between NR5A1 and SRY and mEnh13 based on comparative analyses of Sry-dependent and Sry-independent species. Our comparative analyses revealed new molecular mechanisms underlying mammalian sex determination.

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.

The Neo-X Does Not Form a Barr Body but Shows a Slightly Condensed Structure in the Okinawa Spiny Rat (Tokudaia muenninki).

X chromosome inactivation (XCI) is an essential mechanism for gene dosage compensation between male and female cells in mammals. The Okinawa spiny rat (Tokudaia muenninki) is a native rodent in Japan with XX/XY sex chromosomes, like most mammals; however, the X chromosome has acquired a neo-X region (Xp) by fusion with an autosome. We previously reported that dosage compensation has not yet evolved in the neo-X region; however, X-inactive-specific transcript (Xist) RNA (long non-coding RNA required for the initiation of XCI) is partially localized in the region. Here, we show that the neo-X region represents an early chromosomal state in the acquisition of XCI by analyses of heterochromatin and Barr body formation. We found no evidence for heterochromatin formation in the neo-X region by R-banding by acridine orange (RBA) assays and immunostaining of H3K27me3. Double-immunostaining of H3K27me3 and HP1, a component of the Barr body, revealed that the entire ancestral X chromosome region (Xq) showed a bipartite folded structure. By contrast, HP1 was not localized to the neo-X region. However, BAC-FISH revealed that the signals of genes on the neo-X region of the inactive X chromosome were concentrated in a narrow region. These findings indicated that although the neo-X region of the inactive X chromosome does not form a complete Barr body structure (e.g., it lacks HP1), it forms a slightly condensed structure. These findings combined with the previously reported partial binding of Xist RNA suggest that the neo-X region exhibits incomplete inactivation. This may represent an early chromosomal state in the acquisition of the XCI mechanism.

Spiny rat SRY lacks a long Q-rich domain and is not stable in transgenic mice.

BACKGROUND: Although Tokudaia muenninki has multiple extra copies of the Sry gene on the Y chromosome, loss of function of these sequences is indicated. To examine the Sry gene function for sex determining in T. muenninki, we screened a BAC library and identified a clone (SRY26) containing complete SRY coding and promoter sequences. RESULTS: SRY26 showed high identity to mouse and rat SRY. In an in vitro reporter gene assay, SRY26 was unable to activate testis-specific enhancer of Sox9. Four lines of BAC transgenic mice carrying SRY26 were generated. Although the embryonic gonads of XX transgenic mice displayed sufficient expression levels of SRY26 mRNA, these mice exhibited normal female phenotypes in the external and internal genitalia, and up-regulation of Sox9 was not observed. Expression of the SRY26 protein was confirmed in primate-derived COS7 cells transfected with a SRY26 expression vector. However, the SRY26 protein was not expressed in the gonads of BAC transgenic mice. CONCLUSIONS: Overall, these results support a previous study demonstrated a long Q-rich domain plays essential roles in protein stabilization in mice. Therefore, the original aim of this study, to examine the function of the Sry gene of this species, was not achieved by creating TG mice.

Regulation of the Sox3 Gene in an X0/X0 Mammal without Sry, the Amami Spiny Rat, Tokudaia osimensis.

Two species of spiny rats, Tokudaia osimensis and Tokudaia tokunoshimensis, show an X0/X0 sex chromosome constitution due to the lack of a Y chromosome. The Sry gene has been completely lost from the genome of these species. We hypothesized that Sox3, which is thought to be originally a homologue of Sry, could function in sex determination in these animals in the absence of Sry. Sox3 was localized in a region of the X chromosome in T. osimensis homologous to mouse. A similar testis- and ovary-specific pattern of expression was observed in mouse and T. osimensis. Although the sequence of the Sox3 gene and its promoter are highly conserved, a 13-bp deletion was specifically found in the promoter region of the 2 spiny rat species. Reporter gene assays were performed to examine the effect of the 13-bp deletion in the promoter region on Sox3 regulation. Although an approximately 60% decrease in activity was observed using the Tokudaia promoters with the 13-bp deletion, the activity was recovered using a mutated promoter in which the deletion was filled with mouse sequence. To evaluate whether SOX3 could regulate Sox9 expression, a reporter gene assay was carried out using testis-specific enhancer of Sox9 core (TESCO). Co-transfection with a combination of mouse SF1 and mouse SOX3 or T. osimensis SOX3 resulted in a greater than 2-fold increase in activity of mouse and T. osimensis TESCO. These results support the idea that the function of SOX3 as a transcription factor, as has been reported in mice and humans, is conserved in T. osimensis. Therefore, we conclude that the Sox3 gene has no function in sex determination in Sry-lacking Tokudaia species.

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.

Unique XCI evolution in Tokudaia: initial XCI of the neo-X chromosome in Tokudaia muenninki and function loss of XIST in Tokudaia osimensis.

X chromosome inactivation (XCI) is an essential mechanism to compensate gene dosage in mammals. Here, we show that XCI has evolved differently in two species of the genus Tokudaia. The Amami spiny rat, Tokudaia osimensis, has a single X chromosome in males and females (XO/XO). By contrast, the Okinawa spiny rat, Tokudaia muenninki, has XX/XY sex chromosomes like most mammals, although the X chromosome has acquired a neo-X region by fusion with an autosome. BAC clones containing the XIST gene, which produces the long non-coding RNA XIST required for XCI, were obtained by screening of T. osimensis and T. muenninki BAC libraries. Each clone was mapped to the homologous region of the X inactivation center in the X chromosome of the two species by BAC-FISH. XIST RNAs were expressed in T. muenninki females, whereas no expression was observed in T. osimensis. The sequence of the XIST RNA was compared with that of mouse, showing that the XIST gene is highly conserved in T. muenninki. XIST RNAs were localized to the ancestral X region (Xq), to the heterochromatic region (pericentromeric region), and partially to the neo-X region (Xp). The hybridization pattern correlated with LINE-1 accumulation in Xq but not in Xp. Dosage of genes located on the neo-X chromosome was not compensated, suggesting that the neo-X region is in an early state of XCI. By contrast, many mutations were observed in the XIST gene of T. osimensis, indicating its loss of function in the XO/XO species.

The birth of quail chicks after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors - phospholipase Cζ (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) - all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca(2+) rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca(2+) oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.

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.

Fertilisation of cryopreserved sperm and unfertilised quail ovum by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an important technique in animal biotechnology for animal cloning and conservation of genetic resources, but has been a challenge for avian species. In the present study, we investigated the ability of cryopreserved quail spermatozoa to achieve fertilisation and embryo development. Female quail were killed 70-120min after previous oviposition to collect unfertilised oocytes from the oviduct. Fresh or cryopreserved-thawed spermatozoa were injected into the cytoplasm of unfertilised oocytes, and the manipulated oocytes were incubated in quail surrogate eggshells. Injection of fresh spermatozoa supplemented with inositol 1,4,5-trisphosphate (IP3) resulted in a significantly increased rate of embryo development compared with injection of fresh spermatozoa alone (90% vs 13%, respectively). Although >80% of embryos stopped cell division and development before Hamburger and Hamilton (HH) Stage 3, approximately 15% of embryos from the fresh sperm injection developed to past HH Stage 4, and one embryo survived up to HH Stage 39 (11 days of incubation). In the case of cryopreserved spermatozoa, the embryo development rate was 30% after ICSI, and this increased significantly to 74% with IP3 supplementation. In conclusion, cryopreserved spermatozoa combined with ICSI followed by surrogate eggshell culture can develop quail embryos.

Sperm activation by heat shock protein 70 supports the migration of sperm released from sperm storage tubules in Japanese quail (Coturnix japonica).

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 motility in vitro and 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 of HSP70 mRNA in the UVJ increases before ovulation/oviposition. Gene-specific in situ hybridization 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 fertilization in 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.

Application of intracytoplasmic sperm injection (ICSI) for fertilization and development in birds.

Intracytoplasmic sperm injection (ICSI) technology in birds has been hampered due to opacity of oocyte. We developed ICSI-assisted fertilization and gene transfer in quail. This paper reviews recent advances of our ICSI experiments. The oocyte retrieved from the oviduct and a quail sperm was injected into the oocyte under a stereomicroscope. The oocyte was cultured for 24h at 41°C under 5% CO2 in air. The fertilization and development was assessed by microscopic observation. The fertility rate ranged 12-18% and development varied from stage II to V in trials. To improve the fertility rate, phospholipase C (PLC) zeta was injected with a sperm. It was increased to 37-50%. Furthermore, injection of inositol trisphosphate increased to over 85%. Quail oocyte can be fertilized with chicken sperm and so can testicular elongated spermatid. To extend embryonic development, chicken eggshell was used as a surrogate culture at 37°C after the 24h incubation at 41°C under 5% CO2 in air. It survived up to 2days thereafter. Finally, gene transfer was attempted in quail egg. The sperm membrane was disrupted with Triton X-100 (TX-100) and was injected with PLCzeta cRNA and enhanced green fluorescent protein (EGFP) gene in oocyte. The GFP expression was evaluated at 24h incubation at 41°C under 5% CO2 in air in the embryos. While the expression was not detected in the control oocytes, the experimental treatment induced blastoderm development (44%) of the oocytes and 86% of blastoderm showed fluorescent emission. In addition, PCR analysis detected EGFP fragments in 50% of GFP-expressing blastoderm. Our ICSI method may be the first step toward the production of transgenic birds.

Initial formation of and sex differences in primordial germ cells in Japanese quail.

DEAD-box RNA helicase 4 (DDX4) is posited to be a key maternal germ cell factor regulating avian germ cell formation. We herein showed that the DDX4 gene product of zygotic genome activation associated with the nuclear localization of the cyclin D1 protein in presumptive primordial germ cells (PGCs) plays an essential role in the proliferation of PGCs using a CRISPR/Cas9 system approach combined with in vitro fertilization techniques in Japanese quail. A proteome analysis also revealed molecular-based differences in the features of early male and female PGCs.

FcRY is a key molecule controlling maternal blood IgY transfer to yolks during egg development in avian species.

Maternal immunoglobulin transfer plays a key role in conferring passive immunity to neonates. Maternal blood immunoglobulin Y (IgY) in avian species is transported to newly-hatched chicks in two steps: 1) IgY is transported from the maternal circulation to the yolk of maturing oocytes, 2) the IgY deposited in yolk is transported to the circulation of the embryo via the yolk sac membrane. An IgY-Fc receptor, FcRY, is involved in the second step, but the mechanism of the first step is still unclear. We determined whether FcRY was also the basis for maternal blood IgY transfer to the yolk in the first step during egg development. Immunohistochemistry revealed that FcRY was expressed in the capillary endothelial cells in the internal theca layer of the ovarian follicle. Substitution of the amino acid residue in Fc region of IgY substantially changed the transport efficiency of IgY into egg yolks when intravenously-injected into laying quail; the G365A mutant had a high transport efficiency, but the Y363A mutant lacked transport ability. Binding analyses of IgY mutants to FcRY indicated that the mutant with a high transport efficiency (G365A) had a strong binding activity to FcRY; the mutants with a low transport efficiency (G365D, N408A) had a weak binding activity to FcRY. One exception, the Y363A mutant had a remarkably strong binding affinity to FcRY, with a small dissociation rate. The injection of neutralizing FcRY antibodies in laying quail markedly reduced IgY uptake into egg yolks. The neutralization also showed that FcRY was engaged in prolongation of half-life of IgY in the blood; FcRY is therefore a multifunctional receptor that controls avian immunity. The pattern of the transport of the IgY mutants from the maternal blood to the egg yolk was found to be identical to that from the fertilized egg yolk to the newly-hatched chick blood circulation, via the yolk sac membrane. FcRY is therefore a critical IgY receptor that regulates the IgY uptake from the maternal blood circulation into the yolk of avian species, further indicating that the two steps of maternal-newly-hatched IgY transfer are controlled by a single receptor.

PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens.

Primordial germ cells (PGCs) are vital for producing sperm and eggs and are crucial for conserving chicken germplasm and creating genetically modified chickens. However, efforts to use PGCs for preserving native chicken germplasm and genetic modification via CRISPR/Cas9 are limited. Here we show that we established 289 PGC lines from eight Chinese chicken populations with an 81.6% success rate. We regenerated Piao chickens by repropagating cryopreserved PGCs and transplanting them into recipient chickens, achieving a 12.7% efficiency rate. These regenerated chickens carried mitochondrial DNA from female donor PGC and the rumplessness mutation from both male and female donors. Additionally, we created the TYRP1 (tyrosinase-related protein 1) knockout (KO) PGC lines via CRISPR/Cas9. Transplanting KO cells into male recipients and mating them with wild-type hens produced four TYRP1 KO chickens with brown plumage due to reduced eumelanin production. Our work demonstrates efficient PGC culture, cryopreservation, regeneration, and gene editing in chickens.

Sperm storage in the female reproductive tract in birds.

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.

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

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.

Egg Development After In Vitro Insemination in Japanese Quail (Coturnix japonica).

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.