鳥類における就巣行動制御遺伝子の解析

ブンチョウのプロラクチン（PRL）及び小腸血管作用性ペプチド（VIP）cDNAのクローニングを行なった。さらに，PRLの近位プロモーター領域の配列も同定された。ブンチョウPRLのcDNA及び近位プロモーターはニワトリ，シチメンチョウ，アヒルと高い相同性を示し，中でも近位プロモーターはVIP反応領域が晩成性と早成性鳥類ともに保存されている事が明らかになった。晩成性と早成性ともに生理的PRL放出因子と考えられるVIPのcDNAクローニングも行なった。ブンチョウのVIP前駆体基本構造はニワトリやシチメンチョウと類似しているが，5\u27非翻訳領域の上流はニワトリのものと比べ類似性がほとんど認められなかった。晩成性鳥類において，みられる血中PRLの濃度変化は早成性鳥類と異なり抱卵後期に上昇する。晩成性と早成性ともにPRL遺伝子の発現はVIPにより制御されているが，VIP遺伝子の発現時期が異なることにより，結果としてPRL遺伝子の発現増加時期が異なっている可能性が考えられた。Complementary DNA (cDNA) of prolactin (PRL) and vasoactive intestinal polypeptide (VIP) of the Java sparrow (Padda oryzivora) were cloned and sequenced. Java sparrow PRL was found to have 88.3, 88.3, and 89.1 % sequence identity to PRL cDNA of chicken, turkey, and duck, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (91.4 %), turkey (88.9 %) and duck (92.0 %) PRL. Based on the cDNA sequence and genomic DNA sequence of the chicken PRL gene, the proximal promoter was characterized. Sequence analysis of the proximal region of Java sparrow PRL promoter revealed a high degree of similarity to that of chicken, turkey and duck PRL promoter. Moreover, cDNA of prepro-VIP was also cloned and sequenced. Java sparrow prepro-VIP shows high similarity to chicken and turkey prepro-VIP. However, upstream of the 5\u27 untranslated region of Java sparrow prepro-VIP did not show similarity to that of chicken. These results suggest that the mechanisms, which regulate expression of the VIP gene, may be different between precocial and altricial birds, but expression of the PRL gene may be widely conserved in avian species

Nurturing a gender-responsive approach to climate-smart agriculture in Guinayangan, Quezon

Coconut-based farming systems in Guinayangan, Quezon offer special opportunities for achieving multiple objectives, including carbon sequestration, economic empowerment of women and reduction of risks from variable and extreme weather. This info note discusses the gender-based role inequalities within coconut-based farming systems that can be addressed through agroforestry-based, climate-smart agriculture that features small livestock, fruit trees and root and tuber crops as understory crops. Numerous Climate-Smart Villages, spread across the municipality of Guinayangan, now serve as proof of concept, providing evidence that climate-smart agriculture based on agroforestry interventions are gender sensitive

Cloning and Comparison of Prolactin Promoter in Galliformes

To investigate the mechanism regulating transcription of the prolactin (PRL) gene in avian species, the PRL promoter region in Ceylon junglefowl, Japanese quail, ring-necked pheasant, turkey, Indian peafowl and helmeted guineafowl were cloned and sequenced. In each species, approximately 4,800-5,900bp were sequenced. The PRL promoters of 7 galliformes including red junglefowl were found to have, on average, 91.2% sequence identity over the entire region and 97% sequence identity was observed in the proximal promoter (from the initiation codon (+55) to -130). Moreover, average of sequence identities was 91.6% among 11 avian species (7 galliformes, duck, Java sparrow, budgerigar and ostrich). In the PRL proximal promoter, putative Pit-1 binding site and vasoactive intestinal peptide response element were conserved among avian species. These results suggest that that the mechanisms involved in gene expression of PRL may be conserved in Galliformes

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

Prolactin and Growth Hormone in Birds: Protein Structure, Gene Structure and Genetic Variation

Prolactin (PRL) and Growth hormone (GH) are secreted from the anterior pituitary gland and affect a wide variety of physiological functions in birds. A causal relationship between plasma PRL concentration and broody behaviour is well known. Similarly, a relationship between plasma GH concentration and body growth is well documented. Following the cloning of cDNA of PRL and GH in the chicken and turkey, the levels of mRNA were measured. Levels of PRL or GH mRNA in the anterior pituitary gland were generally correlated with the plasma levels of PRL or GH. Using the PRL or GH cDNA clone as a probe, the genomic DNA clone of PRL and GH was obtained in several galliform species. The availability of cDNA and genomic DNA sequence also allowed the determination of sequence variation of PRL and GH genes in chicken and turkey. The genetic variation of the turkey and chicken PRL promoter and allelic variation of PRL have been shown to be associated with the plasma levels of PRL and expression of incubation behaviour. Likewise, allelic variants of the GH have been proposed as a mechanism to explain variation in egg production in the chicken. Recently, the duck PRL and GH gene was cloned and sequenced. Genetic variations were also detected in the duck GH gene. The presence of variation and/or polymorphic sequence may be important since a variety of body types and egg production traits are found among the duck breeds. Thus, genetic variation in the PRL and GH genes of domestic fowl may applicable to marker assisted selection. Cloning of cDNA and genomic DNA of PRL and GH was conducted mainly in the domestic species. The sequence information of PRL and GH in the domestic species makes it possible to clone and characterize the PRL and GH in altricial species. The sequence of PRL and GH in the various avian species will provide useful information for studying the physiological function and general and species specific mechanisms of gene expression in birds

Characterization of Chicken Prolactin Regulatory Element Binding Protein and its Expression in the Anterior Pituitary Gland during Embryogenesis and Different Reproductive Stages

The PRL regulatory element binding protein (PREB) is a transcription factor that specifically binds to a Pit-1 binding element in the PRL promoter to regulate PRL gene expression in mammals. However, it is unknown whether chicken PREB involves the expression of PRL mRNA or not. This study aimed to clone and characterize the chicken PREB gene and to investigate the mRNA expression during embryogenesis and different reproductive stages. Based on the conserved sequence of the human, rats and mouse, primers pairs were designed and applied to amplify the PREB cDNA fragment. After sequencing of PCR products, 5′- and 3′- end of mRNA were amplified and determined. To identify the structure of the PREB gene, PCR amplification was conducted. The chicken PREB gene consisted of 9 exons and 8 introns and encoded for a 411 amino acid protein. The expression of PREB mRNA in the anterior pituitary gland was measured during embryogenesis and at different reproductive stages. PREB mRNA was detectable at embryonic day 12. Significant increase of levels of PREB mRNA was detected at embryonic day 18 and a maximum level was detected at day 1 of chick. In adult Silkie hens, the lowest level of PREB mRNA was detected in non-photostimulated hens whereas the highest level was observed in incubating hens. Since both the expression of PREB mRNA and PRL mRNA show a similar profile, PREB might be involved in expression of PRL mRNA. An alternative-splicing isoform, which lacks exon 7, was also detected. Because the predicted translated protein originating from this splicing isoform lacked most of the region of WD3 repeat region, this isoform may either have no function or have an alternative function. The results of this study support the possibility of involvement of PREB in PRL mRNA expression in the chicken anterior pituitary gland

Characterization and Expression of Turkey Prolactin Regulatory Element Binding in the Anterior Pituitary Gland and Pancreas During Embryogenesis

The PRL regulatory element-binding (PREB) protein is a transcription factor that was originally cloned from the rat anterior pituitary gland and characterized as a regulator of the PRL promoter. It is also strongly expressed in several extrapituitary tissues; however, its functional role is not well understood to date. In this study, we aimed to clone and characterize the turkey PREB gene and investigate its mRNA expression in the anterior pituitary gland and pancreas during embryogenesis. Based on the conserved sequence of chicken and mammalian PREB cDNAs, a turkey PREB cDNA fragment was obtained, and after sequencing of the fragment, the 5′- and 3′-ends of mRNA were amplified and determined. To identify the PREB gene structure, polymerase chain reaction (PCR) amplification was performed. The turkey PREB gene consists of 9 exons and 8 introns, and it encodes a 411-amino-acid protein. The expression of PREB mRNA in the anterior pituitary gland was measured during embryogenesis. Levels of PREB mRNA significantly increased at embryonic day 22, with maximum levels being detected on day 25 of ontogeny, which correlated with similar changes in levels of PRL mRNA. The highest level of PREB mRNA was detected on day 19 in the pancreas. However, the highest level of insulin mRNA was detected at embryonic day 25. These results indicate that PREB may be involved in the expression of PRL mRNA in the anterior pituitary gland, whereas insulin mRNA may be expressed independently of the expression of PREB mRNA in the pancreas during embryogenesis

Inositol-1,4,5-Trisphosphate Receptor-1 and-3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation

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 1,4,5trisphosphate 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 in creases 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