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.

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.

Novel method of gene transfer in birds: intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms.

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

TonEBP regulates hyperosmolality-induced arginine vasotocin gene expression in the chick (Gallus domesticus).

Arginine vasotocin (AVT) is expressed mainly in the paraventircular and supraoptic nuclei of the hypothalamus in chicken. This peptide is known to act as an antidiuretic hormone and its gene expression is stimulated by hyperosmolality. However, the transcription factors that regulate the AVT gene expression induced by hyperosmolality are still unknown. In this study, we examined the role of hyper-tonicity enhancer binding protein (TonEBP) in the transcriptional regulation of AVT gene in chicken. TonEBP mRNA expression levels increased at 1h after salt-loading treatment in the hypothalamus. This increase preceded that in AVT and c-fos mRNA expression. Intracerebroventricular injections of TonEBP antisense oligonucleotides, before the salt-loading treatment, prevented the increase in AVT gene expression. These results, all together, suggest that the transcription factor TonEBP may be involved in the regulation of AVT genes expression in response to a hyperosmotic environment in chicken.

Differential development of sex-related characters of chickens from the GSP and PNP/DO inbred lines after left ovariectomy.

To elucidate strain differences in the sex reversal of genetic females to phenotypic males, GSP and PNP/DO females were left ovariectomized (ovx) between one to three days after hatching, and the degree of masculinization based on sex-related characters, histological analysis of the right gonad and hormone assay were assessed at one year of age. The GSP and PNP/DO inbred lines were both derived from the Fayoumi breed and are only differentiated based on the red blood cell antigen type carried by each inbred line. Combs and wattles were found to be significantly bigger (P<0.05) in the GSP ovx compared with the PNP/DO ovx chickens, although male plumage patterns were more pronounced in the PNP/DO ovx. Spurs were observed both in the GSP and PNP/DO ovx chickens with no significant difference (P>0.05) in length compared with the respective male controls, and body weight was not significantly different (P>0.05) compared with the female controls. The weight of the right gonad was significantly heavier (P<0.05) in the GSP ovx than in the PNP/DO ovx. Positive correlations were found in the sex-related characters as well as the plasma testosterone level and right gonad weight in both the GSP and PNP/DO ovx chickens, but not in the spur length, which showed a negative correlation in the PNP/DO ovx chickens. Histological analysis revealed that the right gonads of the PNP/DO ovx chickens were morphologically developed compared with the GSP ovx chickens, which showed more advance stages of spermatogenesis. It could be inferred that PNP/DO females that exhibit a hereditary persistent right oviduct are more responsive to the masculinizing effect of ovariectomy compared with GSP females, suggesting that genetic background may have a possible contribution to the degree of masculinization and subsequent development of sex related characters.

Phospholipase Czeta mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor.

This study was conducted to investigate the role of a sperm-borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Czeta (PLCzeta) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCzeta cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca(2+) chelator) before SE injection. On the other hand, when the oocytes were injected with PLCzeta cRNA at 60 microg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCzeta cRNA-induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCzeta cRNA can induce development. In addition, RT-PCR revealed that PLCzeta mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCzeta is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis.

Changes in post-translational modifications of prolactin during development and reproductive cycles in the chicken.

Changes in proportion of glycosylated prolactin in the anterior pituitary glands of chickens were assessed using one- and two-dimensional western blotting analysis during the perihatch stage of embryos and reproductive cycles. Multiple isoforms of prolactin were detected by one-dimensional analysis and glycosylated (G) and non-glycosylated (NG) isoforms were identified by N-glycosidase and neuraminidase treatment. Increases of ratio of G to NG isoforms were observed in both embryonic stages and reproductive cycles by the one-dimensional analysis. Although a similar tendency of increase of proportion of G prolactin was obtained, different values of proportion were observed between one-dimensional and two-dimensional analysis. Since two-dimensional analysis may better resolve isoforms differing slightly in molecular size of G prolactin, the results from two-dimensional analysis may reflect the actual proportion of prolactin isoforms. Furthermore, isoforms differing in isoelectric points were detected after N-glycosidase and neuraminidase treatment. These results indicate that prolactin may also be additionally post-translationally modified such as by phosphorylation. Thus function and biological activity of prolactin were, at least in part, regulated by post-translational modification in the various physiological stages.

Histocompatibility assessment in the chicken colonies of the RIR-Y8/NU, YL, WL-G, and BL-E closed for 28-48 years.

Histocompatibility was assessed in the RIR-Y8/NU, BL-E, YL, and WL-G chicken closed colonies by hemagglutination test using anti-red blood cell (RBC) antibodies (HT), skin transplantation test (STT), and formation of isohemagglutinins (FIHs) during STT. The YL individuals all showed the survival of skingrafts for more than 17 days with no FIHs in STT and no RBC antigenic variations in HT, indicating a histocompatible nature together with high homogeneity at serological loci. The BL-E as well as WL-G closed colonies were also found to be histocompatible in the STT with no FIHs, although the HT showed heterogeneities at serological locus/loci other than the B and C blood group loci which have significant effects on histocompatibility or FIHs in chicken. In the RIR-Y8/NU closed colonies, one individual in 6 reciprocal combinations of the STT showed early skingraft rejection with positive FIHs caused by different B locus alleles, and the HT suggested relatively high heterogeneities at the other serological loci too. The closed colonies of YL, BL-E, and WL-G will be useful avian materials for transplantation or related experiments, but RIR-Y8/NU needs further pedigree selection for serological homogeneity.

Identification of differentially expressed genes involved in the regression and development of the chicken Müllerian duct.

Müllerian ducts of male chickens undergo regression around day 12 of incubation, but the underlining mechanisms remain unclear. The purpose of this study was to identify factors that contribute to regression of the Müllerian duct in the chicken. We first employed annealing control primer-based RT-PCR to screen candidate genes differentially expressed in the Müllerian ducts between male and female. Four differentially expressed genes (MSX2, GAL10, VCP and PLCH1) were partially sequenced. The expression of mRNA of the latter genes and MSX1 in the male and female Müllerian ducts were compared at 7.5, 8 and 9 days of incubation using semi-quantitative RT-PCR. The results indicated that both MSX1 and MSX2 mRNA was highly expressed in the male Müllerian duct at day 9 of incubation, whereas, PLCH1 mRNA was lower in the male duct at day 9 of incubation compared to that of the female duct. Although VCP mRNA was expressed in both left and right female Müllerian ducts, no expression was detected in the male duct. Whole mount in situ hybridyzation analysis showed that the expression of MSX1 and MSX2 mRNA were localized specifically in the mesenchymal cells of the male Müllerian duct at day 9 of incubation. In contrast, VCP mRNA expression was observed in both mesenchymal and epithelial cells of the female Müllerian duct but not detected in the male duct. These results suggest that both up-regulation of MSX1 and MSX2 mRNA expression is involved in the regression of the Müllerian duct in male chicken embryo, whereas VCP expression is involved in development of the female duct.

Sex- and age-related variation in neurosteroidogenic enzyme mRNA levels during quail embryonic development.

Brain can synthesize steroids de novo from cholesterol and this biochemical feature is a conserved property of vertebrates. There is growing evidence indicating that neurosteroids might participate in sexual differentiation of the brain. Therefore, in this study we investigated the presence, the sex differences, and the development-dependent variation of mRNAs coding for key neurosteroidogenic enzymes, namely cytochrome P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid-dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/c17, 20-lyase (P450c17), and aromatase in embryonic prosencephali. Our results indicated that 3beta-HSD mRNA levels were sexually dimorphic and developmental age-dependent. In particular, 3beta-HSD mRNA levels were higher in females than in males at E7, whereas, this dimorphism was reversed at E9 and E15. In females, the relative levels of 3beta-HSD mRNA were highest at E7, whereas, in males they were significantly higher at E9 and E15 than at E7 and at E11. This sexual dimorphism was a peculiar feature of the prosencephalon, it could not be observed before gonadal sexual differentiation and it was not paralleled by a dimorphism in the brain content of progesterone. The level of mRNA coding for P450scc and for P450c17 did not show obvious developmental- or sex-related variation. Aromatase mRNA varied as a function of the embryonic age but not of the sex. These results, taken together, are suggestive of a potential role of some neurosteroidogenic enzymes in the development of quail brain and suggest that sexual differences in the hormonal environment may occur during brain development.

Histocompatible chicken inbred lines: homogeneities in the major histocompatibility complex antigens of the GSP, GSN/1, PNP/DO and BM-C inbred lines assessed by hemagglutination, mixed lymphocyte reaction and skin transplantation.

Chicken inbred lines of the GSP, GSN/1, PNP/DO and BM-C have been established by selection of a specific allele at the B blood group locus (MHC B-G region) and other polymorphic loci through pedigree mating. To extend the potential of these inbred lines as experimental animals in Aves, we assessed the antigenic homogeneities of the MHC antigens by three immunological methods. Antigenic variations of red blood cells (RBCs) were surveyed in the inbred lines and a random-bred line (NG) derived from the Nagoya breed by using ten kinds of intact antisera produced in the inbred line of chickens against RBCs of a red junglefowl and hybrids. In the hemagglutination test, no individual variations were found within the inbred line at all, while all the ten antisera detected highly heterogeneous reactions in individuals of the NG. The reciprocal one-way mixed lymphocyte reactions gave constantly higher stimulation responses (P<0.01) between individual pairs from the inbred lines having different B alleles compared to pairs within the inbred line, while lower stimulation was observed between pairs of the GSP and GSN/1 inbred lines both having the B(21) allele. In reciprocal skin transplantation, the transplanted skingrafts within the inbred line and between individuals from the GSP and GSN/1 inbred lines survived more than 100 days, while all the skingrafts showed signs of rejection within 7 days among the inbred lines having different B alleles. The results obtained by the three practical methods coincidentally indicated that the individuals in the respective four inbred lines were histocompatible, and further, that the GSP and GSN/1 individuals were histocompatible.

Possible role of calcium on oocyte development after intracytoplasmic sperm injection in quail (Coturnix japonica).

Although a rise in intracellular calcium concentration of vertebrate oocytes plays a pivotal role for the initiation of fertilization or oocyte activation, no study on this subject has been reported in birds. This study was conducted to study the role of intracellular calcium in relation to fertilization in avian oocytes. First, immediately after a quail oocyte was injected with a sperm, it was treated with strontium chloride as an inducer for intracellular calcium rise at doses of 0, 2.5, 5, 7.5, 10 mM for 4 hr in the culture medium and was followed by 20-hr culture. Treatment with 5 mM of strontium chloride induced blastodermal development in 24.2% of injected eggs, although no oocytes developed without strontium treatment. Second, quail oocytes were injected with a sperm and 0.1 M calcium chloride or a sperm and saline solution, cultured without calcium for 4 hr and was followed by 20-hr culture without strontium. The calcium solution induced blastodermal development in 20.5% of the oocytes, although no oocytes developed without calcium treatment. Third, quail oocytes were injected with 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) as a calcium chelator, cultured with strontium (5 mM) for 4 hr followed by 20-hr culture without strontium. Only one oocyte developed after BAPTA and strontium treatment of 36 oocytes examined. Developmental stages of all the oocytes ranged from IV to VII. These results suggest that intracellular calcium rise may participate in quail oocyte activation and allow fertilization and blastodermal development.

Effect of water deprivation on aquaporin 4 (AQP4) mRNA expression in chickens (Gallus domesticus).

Aquaporin (AQP) 4 is a member of the AQP gene family of water-selective transport proteins. We studied the effect of water deprivation on AQP4 gene expression in chickens. The nucleotide sequence of a chicken aquaporin 4 (AQP4) cDNA that encodes a protein of 335 amino acids showed high homology to mammalian AQP4. Using Northern blotting analysis, AQP4 mRNA in chickens was observed as a band of approximately 5.5 kb in several tissues in addition to the hypothalamus, proventriculus, kidney, and breast muscle. Quantitative analysis by real-time RT-PCR analysis showed that the mRNA expression of AQP4 in the hypothalamus significantly increased after dehydration. On the other hand, the mRNA expression of AQP4 in the kidney significantly decreased after dehydration. This suggests that AQP4 may play a pivotal role in osmoregulation in the chicken brain.

A novel isoform of Vinexin, Vinexin gamma, regulates Sox9 gene expression through activation of MAPK cascade in mouse fetal gonad.

Recent loss-of-function and gain-of-function studies have revealed that transcription factor Sox9 is required for testis formation by governing Sertoli cell differentiation, and thereafter regulating transcription of Sertoli marker genes. In the present study, we identified a novel isoform of Vinexin, which is expressed in somatic cells but not germ cells of sexually indifferent stages of fetal gonads. After the sex is determined, the expression continues in testicular Sertoli cells. Immunohistochemical analyses with a specific antibody to Vinexin indicated that Vinexin gamma is localized in the cytoplasm. Functional studies with C3H10T1/2 cells showed that Vinexin gamma acted as a scaffold protein to activate MEK and ERK through interaction with c-Raf and ERK. Ultimately, Sox9 transcription was induced by Vinexin gamma. This up-regulation of Sox9 expression disappeared when the cells were treated with a specific MEK inhibitor, U0126. To determine the role of Vinexin gamma during gonad formation, the gene was disrupted by targeted mutagenesis. The phenotype displayed by the mice indicated that ERK activation was decreased in the Vinexin gamma(-/-) XY gonads, and Sox9 expression was down-regulated. Thus, Vinexin gamma seems to be implicated in regulation of Sox9 gene expression by modulating MAPK cascade in mouse fetal gonads.

Cloning of duck PRL cDNA and genomic DNA.

Complementary DNA (cDNA) and genomic DNA, including flanking regions of the prolactin (PRL) gene of domesticated duck, were cloned and sequenced. Duck PRL was found to have 92.0, 91.7, and 91.4% sequence identity at the cDNA level to PRL of chicken, turkey, and quail, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (93.4%), turkey (91.3%), and quail (91.3%) PRL. Mature duck PRL contains the consensus sequence for N-linked glycoslylation at position 6 which is not present in either chickens or turkeys. Thus, duck PRL is likely to be post-translationally modified differently from other avian species. Based on the cDNA sequence, the genomic structure of the gene was characterized. The duck PRL gene consists of 5 exons and 4 introns. Moreover, sequence analysis of the proximal region of duck PRL promoter revealed a high degree of similarity to that of chicken and turkey PRL promoter. These results suggest that the mechanisms, which regulate expression of the PRL gene, may be widely conserved in avian species.

Changes in mRNA expression of MMP-2 in the Müllerian duct of chicken embryo.

Although asymmetric development of the ovary and the oviduct is a unique characteristic in birds, the mechanism of asymmetric development still remains unclear. Recently, degradation of extracellular matrix has been suggested as an important factor related to the regression of the Müllerian duct in mammals. The present study was conducted to examine a possible role of metalloproteinase-2 (MMP-2) in the regression of the right Müllerian duct in the developing chicken embryo. Morphological changes in the Müllerian ducts were studied on day 15 of incubation and mRNA expresseion of MMP-2 was studied on days 12, 15, and 18 of incubation. Morphological observation demonstrated the disappearance of basement membrane in the right Müllerian duct which undergoes the regression. RT-PCR analysis showed that MMP-2 mRNA expression of the right Müllerian duct increased on days 15 and 18 of incubation coincidently with the time of regression. In the right Müllerian duct, regression was prevented by diethylstilbestrol treatment on day 4 of incubation and a coincident decrease in MMP-2 expression was observed when compared to the control group. These results suggest that MMP-2 may be involved in the regression of the right Müllerian duct in the female embryos of the chicken.

Photoinducible phase-specific light induction of Cry1 gene in the pars tuberalis of Japanese quail.

Prolactin (PRL) secretion is regulated by photoperiod in mammals and birds. In mammals, the pars tuberalis (PT) in the pituitary is involved in the regulation of photoperiodic regulation of PRL secretion. In birds, however, hypothalamic vasoactive intestinal peptide is implicated in PRL secretion, and physiological roles of the avian PT remain unknown. In the present study, we show that PRL secretion increases under long days and short days with a night interruptive schedule, both of which also cause gonadal growth in Japanese quail. We have also found Cry1 gene expression in the PT of Japanese quail. Cry1 expression was rhythmic under long and short photoperiods in the PT, and the peak was phase delayed under a lengthened photoperiod. Moreover, expression of Cry1 gene was induced by a light pulse but only when given during the photoinducible phase. In our previous study, we have shown rhythmic Per2 gene expression with a peak in the PT during the early day under various photoperiods. When taken together with the results from the present study, different phase relationships between Per2 and Cry1 in the Japanese quail PT under different photoperiods may decode photoperiodic information and regulate photoperiodic PRL secretion in a manner similar to that of mammals.

Expression of estrogen receptor alpha mRNA in theca and granulosa layers of the ovary in relation to follicular growth in quail.

Changes in expression of estrogen receptor alpha (ERa) mRNA were studied in special reference to follicular growth of the ovarian follicles in laying quail. Levels of mRNA were determined by RT-PCR in the ovarian stroma, each class of the ovarian follicles, oviductal parts and in the liver. Low levels of ERalpha mRNA were detected in stroma, the small white follicles, large white follicles and small yellow follicles and in the theca layer of the three largest preovulatoy follicles. Although the level in the granulosa layer of the F3 was also low, the level significantly increased in F2 and F1. Relatively higher levels were found in the liver and oviduct, and that in the magnum was the highest among all tissues examined. The level in the F1 granulosa layer was comparable to that in the liver which actively synthesises egg yolk proteins for the sake of estrogen and ER. The results of the present study demonstrate that (1) ERalpha mRNA is present in each compartment of the reproductive tissue in quail, (2) the marked expression of ERalpha mRNA in the granulosa layer of the largest follicle may indicate the involvement of estrogens in the biosynthesis of inhibin/activin, progesterone and yolk perivitelline layer protein, (3) very high expression of ERalpha in the oviductal tissues may be related to the role of estrogens in cell proliferation and protein synthesis in the oviduct.

Molecular characterization of chicken growth hormone secretagogue receptor gene.

Synthetic growth hormone secretagogues stimulate growth hormone secretion by binding to a specific receptor, growth hormone secretagogue receptor (GHS-R). In this study, we investigated the cDNA and the genomic structure of chicken GHS-R. Chicken GHS-R gene is composed of two exons separated by an intron. Two GHS-R mRNA species, cGHS-R1a and cGHS-R1a-variant (cGHS-R1aV) are generated by alternative splicing of a primary transcript. cGHS-R1a protein is predicted to have seven transmembrane domains by a high degree of amino acid sequence identity with mammalian and teleost homologs. cGHS-R1aV lacks the transmembrane-6 domain due to a 48 bp deletion. RT-PCR analysis showed widespread tissue distributions of cGHS-R1a and cGHS-R1aV mRNAs with much higher amounts of cGHS-R1a in all the tissues.