The Nile tilapia, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying gonadal sex differentiation because genetic all-females and all-males are available. In this study, we used quantitative real-time RT-PCR to determine the precise timing of the gonadal expression of 17 genes thought to be associated with gonadal sex differentiation in vertebrates. Gonads were isolated from allfemale and all-male tilapia before (5–15 days after hatching [dah]) and after (25–70 dah) morphological sex differentiation. The transcript of aromatase (cyp19a1a), an enzyme responsible for producing estradiol-17beta, was expressed only in XX gonads at 5 dah, with a marked elevation in expression thereafter. In contrast, mRNA expression of steroid 11beta-hydroxylase (cyp11b2), an enzyme responsible for the synthesis of 11ketotestosterone (11-KT, a potent androgen in fish), was found in XY gonads from 35 dah only. These results, combined with the presence of transcripts for other steroidogenic enzymes and estrogen receptors in XX gonads at 5–7 dah, are consistent with our earlier suggestion that estradiol-17beta plays a critical role in ovarian differentiation in tilapia, whereas a role for 11-KT in testicular differentiation is questionable. A close relationship between the expression of foxl2, but not nr5a1 (Ad4BP/SF-1), and that of cyp19a1a in XX gonads suggests an important role for Foxl2 in the transcriptional regulation of cyp19a1a. Dmrt1 exhibited a male-specific expression in XY gonads from 6 dah onward, suggesting an important role for Dmrt1 in testicular differentiation. Sox9 and amh (anti-Mullerian hormone) showed a testis-specific expression, being evident only in the later stages of testicular differentiation. It is concluded that the sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads during early gonadal differentiation (5–6 dah) is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia. aromatase, DMRT1, fish, Foxl2, sex differentiation

Sexual Dimorphic Expression of Genes in Gonads During Early Differentiation of a Teleost Fish, the Nile Tilapia Oreochromis niloticus

In this study, we report the purification, cDNA cloning, and characterization of the novel growth hormone-releasing peptide, ghrelin, in the chicken (Gallus gallus). Chicken ghrelin is composed of 26 amino acids (GSSFLSPTYKNIQQQKDTRKPTARLH) and possesses 54% sequence identity with human ghrelin. The serine residue at position 3 (Ser(3)) is conserved between the chicken and mammalian species, as its acylation by either n-octanoic or n-decanoic acid. Chicken ghrelin mRNA is predominantly expressed in the stomach, where it is present in the proventriculus but absent in the gizzard. Using RT-PCR analysis, low levels of expression were also detectable in brain, lung, and intestine. Administration of chicken ghrelin increases plasma GH levels in both rats and chicks, with a potency similar to that of rat or human ghrelin. In addition, chicken ghrelin also increases plasma corticosterone levels in growing chicks at a lower dose than in mammals. The present results indicate that the stimulatory effect of ghrelin on GH secretion is evolutionarily conserved, whereas its effect on adrenal function seems to be unique in the chicken.

CHICKEN GHRELIN : PURIFICATION, cDNA CLONING AND BIOLOGICAL ACTIVITY

Insights into Egg Coat Assembly and Egg-Sperm Interaction from the X-Ray Structure of Full-Length ZP3

The extent to which an animal is affected by the pain or distress of a conspecific will depend on its capacity for empathy. Empathy most probably evolved to facilitate parental care, so the current study assessed whether birds responded to an aversive stimulus directed at their chicks. Domestic hens were exposed to two replicates of the following conditions in a counterbalanced order: control (C; hen and chicks undisturbed), air puff to chicks (APC; air puff directed at chicks at 30 s intervals), air puff to hen (APH; air puff directed at hen at 30 s intervals) and control with noise (CN; noise of air puff at 30 s intervals). During each test, the hens' behaviour and physiology were measured throughout a 10 min pre-treatment and a 10 min treatment period. Hens responded to APH and APC treatments with increased alertness, decreased preening behaviour and a reduction in eye temperature. No such changes occurred during any control period. Increased heart rate and maternal vocalization occurred exclusively during the APC treatment, even though chicks produced few distress vocalizations. The pronounced and specific reaction observed indicates that adult female birds possess at least one of the essential underpinning attributes of empathy.

Avian maternal response to chick distress

Human exposure to endocrine disrupting chemicals and fertility: A case-control study in male subfertility patients

Because of the presence of sperm-storage tubules (SST) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm have entered the female reproductive tract, they can survive for a prolonged time in domestic birds, although the specific mechanisms involved in the sperm uptake into, maintenance within, and controlled release from the SST remain to be elucidated. In this report, we provide evidence that progesterone triggers the release of the resident sperm from the SST in the UVJ. The ultrastructural observation of the SST indicated that the resident sperm are released from the SST around 20 h after oviposition. When laying birds were injected with progesterone, most of the sperm were released from the SST within 1 h of injection. In situ hybridization analyses demonstrated the presence of the transcripts of membrane progestin receptor α in the UVJ, and the translated proteins were detected in the UVJ extracts by Western blotting. Moreover, the number of secretory granules in the SST epithelial cells fluctuates during the ovulatory cycle, and the progesterone administration mimics this phenomena. A binding assay using [(3)H]-progesterone indicated the presence of a high affinity, limited capacity, saturable and single binding site for [(3)H]-progesterone in the membrane fraction of the UVJ, and this receptor did not interact with the synthetic antiprogestin RU486. These results demonstrated for the first time that the progesterone stimulates the release of the resident sperm from the SST and that the release of the sperm might occur via membrane progestin receptor α-mediating signal transduction.

Progesterone is a sperm-releasing factor from the sperm-storage tubules in birds.

Cloning of duck PRL cDNA and genomic DNA.

Effect of broody hens on behaviour of chicks

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.

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

The extracellular matrix surrounding avian oocytes, called the perivitelline membrane (PL), consists of at least two major glycoproteins,ZP3andZP1.OurpreviousstudyusingJapanesequailhaddemonstratedthatthePLobtainedfromthepreovulatory follicleswasincubatedinvitrowithspermatozoa,andperforationswereobserved.ThisresultindicatedthatthePLmightcontaina constituent that possesses activity to initiate the acrosome reaction (AR) in quail. In order to elaborate upon our previous findings, we evaluated the effects of ZP3 and ZP1 on the induction of sperm AR in Japanese quail. Ejaculated sperm were incubated with or withoutthepurifiedPLglycoprotein,andtheiracrosomestatuswasdeterminedbasedonthepresenceorabsenceoftheacrosome. Treatment of spermatozoa with increasing doses of the purified monomeric ZP1 led to a concentration-dependent stimulation of AR. The purified dimeric ZP1 had similar effect. Moreover, we found that the ZP1-induced AR was significantly blocked by the digestion of the PL protein with PNGaseF. In contrast, the addition of purified ZP3 failed to induce AR at any doses tested. These results indicate that N-linked glycans on ZP1 play an important role in triggering the AR in Japanese quail.

/pdf/induction-of-sperm-acrosome-reaction-by-perivitelline-1qvbka7tlg.pdf

Norio Kansaku

Papers

Progesterone is a sperm-releasing factor from the sperm-storage tubules in birds.

Cloning of duck PRL cDNA and genomic DNA.

Effect of broody hens on behaviour of chicks

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

Induction of sperm acrosome reaction by perivitelline membrane glycoprotein ZP1 in Japanese quail (Coturnix japonica)