Kiyoshi Shimada

Sex differentiation and mRNA expression of P450c17, P450arom and AMH in gonads of the chicken.

The present study was conducted to reveal effects of in ovo injection of nonsteroidal aromatase inhibitor (Fadrozole) or estradiol at day 3 of incubation on mRNA levels of P45017αhydroxylase (P450c17), P450 aromatase (P450arom) and anti‐Müllerian hormone (AMH) in the chicken gonads. The mRNA levels in the gonads at days 4–8 of incubation were assessed by in situ hybridization analysis using digoxigenin labeling method. The in situ hybridization data were analyzed by relative expression of specific hybridizable signals of each mRNA corrected by the non‐specific background by employing an image analyzer. P450c17 mRNA expression increased rapidly at day 6 of incubation in the male but decreased thereafter. In contrast to the transient expression in the male, the expression was gradually increased in the female. P450arom mRNA was not expressed in the male but was detectable in the female as early as day 6 and increased subsequently with days of incubation. AMH mRNA was expressed as early as day 5 of incubation followed by a sharp increase on day 6, which was maintained in the male thereafter. In contrast, the female showed very little expression. The injection of Fadrozole caused no effect on P450c17 mRNA expression, while it suppressed P450arom mRNA expression but increased AMH mRNA expression in the female. In contrast, the injection of estradiol induced P450arom mRNA expression significantly but suppressed AMH mRNA expression in the male. These results indicate that expression of P450arom and AMH is sexually dimorphic and is reciprocally regulated during early ontogenic life in chicken gonads. Mol. Reprod. Dev. 55:20–30, 2000.

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.

Cytogenetic mapping of 31 functional genes on chicken chromosomes by direct R-banding FISH

Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q).

Gonadotropin-stimulated estradiol production in small ovarian follicles of the hen is suppressed by physiological concentrations of prolactin in vitro.

The in vitro effects of physiological levels of prolactin (PRL) on luteinizing hormone (LH)-stimulated estradiol production by four classes of small follicles (super small (SS) less than 1 mm; small white (SW) 1-2 mm; large white (LW) 3-5 mm; and small yellow (SY) 5-10 mm) were compared in laying and out-of-lay (OL) Gifujidori hens. All classes of follicles responded to increasing doses of LH in a dose-dependent manner and the estradiol content of the media increased two to five-fold when 8 ng/ml of ovine LH was included in the incubation media. Ovine PRL (up to 1 micrograms/ml) had no effect on the LH-stimulated rise in media content of estradiol from SW or LW follicles from either the laying or OL hens, whereas the rise in the estradiol content of the media from SS follicles was blocked or reduced by 500 ng/ml PRL in the laying and OL hens, respectively. This suggests that the SS follicles are a target organ for PRL and that PRL may act, in part, at the level of the ovary to reduce estradiol production at the onset of incubation behavior and thus cause ovarian regression.

Gene expression of steroidogenic enzymes in chicken embryonic gonads

This paper reviews the molecular aspects of sex differentiation in birds and describes the sex-reversal effects of aromatase inhibitor on mRNA expression of steroidogenic enzymes. Although it is unknown whether the sex-determining gene resides in the W chromosome or whether a gene-dosage mechanism is involved in sex determination, it is clear that anti-Müllerian hormone (AMH) and androgen play a key role for testicular development, whereas estrogen is important for ovarian formation. Our in situ hybridization results demonstrated that mRNA expression of P450aromatase first takes place on day 6.5 of incubation exclusively in the female gonad. Treatment with aromatase inhibitor in ovo suppressed mRNA expression in genetic females. This suggests that ovarian development is promoted by aromatase expression via estrogen production. On the other hand, previous reports demonstrated that formation of the testis is accompanied by Sox9 and AMH mRNA expression, which start on days 6 and 8, respectively. This suggests that Sox9 plays an important role for the male determining pathway which leads to AMH expression and formation of the testis.

Phospholipase Cζ 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 Cζ (PLCζ) 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 PLCζ 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 Ca2+ chelator) before SE injection. On the other hand, when the oocytes were injected with PLCζ cRNA at 60 µg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCζ 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 PLCζ cRNA can induce development. In addition, RT‐PCR revealed that PLCζ mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCζ 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. Mol. Reprod. Dev. 76: 1200–1207, 2009.

Molecular Cloning of Chicken Vasoactive Intestinal Polypeptide Receptor Complementary DNA, Tissue Distribution and Chromosomal Localization

Abstract Chicken vasoactive intestinal polypeptide receptor (VIPR) cDNA was cloned by the reverse transcription-polymerase chain reaction method using primers designed on the basis of other species of VIPR cDNA. The cDNA obtained was sequenced by the dideoxy-mediated chain-termination method. Of the 2227 nucleotides that were sequenced, 84, 855, and 1338 bases represent the 5′-untranslated region (UTR), the 3′-UTR, and the open reading frame that predicts a peptide of 446 amino acids. The cDNA of the chicken VIPR shows 65% and 60% homologies to human cDNA of VIP1 and VIP2 receptors, respectively. The clone had the expected similarity to highly conserved features of the other G protein-coupled receptors (GPCRs) such as six cysteine residues that are functionally important in the VIPR subfamily. In addition, the seven potential membrane-spanning domains characteristic of the family B group III GPCR superfamily and highly conserved motif within the third cellular loop between transmembrane regions 5 and 6. Northern blot hybridization analysis in this study indicated mRNA expression of VIPRs in the various tissues of the chicken. Strong signal was detected in the brain and anterior pituitary gland. High levels of VIPR mRNA in the brain was consistent with VIP-binding experiments and with the function of VIP in the brain as a neuroendocrine factor or neurotransmitter. Expression of VIPR was detected in the anterior pituitary gland of chick embryos. The expression of VIPR mRNA in the chick anterior pituitary gland may indicate a regulatory function of VIP on prolactin (PRL) production or PRL cell proliferation during embryogenesis. Chicken VIPR shows high homology with mammalian type I VIPR but, in some part, possesses similarity of amino acid sequence. Expression of VIPR in various tissues supports diverse functions for VIP in the chicken.

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 γ is localized in the cytoplasm. Functional studies with C3H10T1/2 cells showed that Vinexin γ acted as a scaffold protein to activate MEK and ERK through interaction with c‐Raf and ERK. Ultimately, Sox9 transcription was induced by Vinexin γ. This up‐regulation of Sox9 expression disappeared when the cells were treated with a specific MEK inhibitor, U0126. To determine the role of Vinexin γ 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 γ−/– XY gonads, and Sox9 expression was down‐regulated. Thus, Vinexin γ seems to be implicated in regulation of Sox9 gene expression by modulating MAPK cascade in mouse fetal gonads.

Identification of sperm-bearing female-specific chromosome in the sex-reversed chicken

Sexual differentiation in the female chick embryo was phenotypically reversed to the male sex by injection of an aromatase inhibitor (Fadrozole, 0.1 mg/egg) into the embryo at day 5 of incubation. The birds were raised to 10 months of age, and the morphology of the gonads of sex-reversed hens were examined by the light-microscopic morphology, and the presence of the W chromosome gene was determined by fluorescence in situ hybridization (FISH) technique and used for PCR analysis of a single isolated sperm. The sex-reversed hens possessed two testes with a fully developed oviduct on the left side. The testes contained essentially the same cellular components as those of normal testes, although sperm counts were low. FISH analysis revealed numerous spermatids and several sperm bearing W-chromosomes, indicating that the second meiosis occurred normally but that the transformation from the spermatid to the spermatozoon is partially impaired. PCR analysis using the DNA of a single sperm also indicates that sperm-carrying the W chromosome were produced.

Changes in plasma levels of prolactin and estradiol, nutrient intake, and time spent nesting during the incubation phase of broodiness in the Chabo hen (japanese bantam)

The plasma concentrations of prolactin (PRL) and estradiol, time spent nesting, consumption of feed and water, body weight, and hematocrit were measured during egg laying, incubating, and brooding of the chicks in a Japanese strain of dwarf bantam hen, Chabo. Plasma levels of PRL increased before incubation, were maintained at high levels during incubation, and decreased rapidly at the onset of hatching the young. The concentration of estradiol decreased before incubation and was maintained at low levels when circulating levels of PRL were high. During incubation, hens spent greater than 95% of the day on the nest, reduced their daily intake of feed and water by 63 and 78%, respectively, and decreased their body weight by 19% by the end of incubation. Thus, Chabo hens show a mode of incubation behavior similar to that reported in larger chickens. The temporal changes between nutrient intake and plasma levels of PRL at the start and end of incubation behavior suggested that changes in nutrient intake may not cause changes in the concentration of PRL, whereas the association between increased levels of PRL and decreased levels of estradiol suggested that they may be causally associated. Plasma levels of PRL also appeared to be associated with time spent on the nest.