We investigated the androgenic effects of 11-ketotestosterone (11-KT) on gonadal sex reversal and spermatogenesis in honeycomb grouper Epinephelus merra by method of gonadal biopsy. 11-KT was injected intramuscularly at a concentration of 1 and $10{\mu}g$ body weight. The proportion of cross sectional area of the gonad occupied by each germ cell type was measured and compared pre- and post-injection group. During the sex change phase, the distribution ratio of oocytes was decreased in all fish of 11-KT treatment group while the distribution ratio of spermatocytes was increased than pre-injection group. In male phase, all fish of 11-KT treatment group shown the increased distribution ratio of spermatocytes, but the distribution ratio of spermatozoa was decreased than pre-injection group. The present results suggest that 11-KT can stimulate degeneration of oocytes, proliferation of spermatocytes and spermiation in honeycomb grouper.

This study attempted to verify the possibility of using germ cell aspiration (GCA) method as a non-fatal technique in studying the life-history of equilateral venus, Gomphina veneriformis (Veneridae) and granular ark, Tegillarca granosa (Arcidae). Using twenty-six gauge 1/2" (12.7mm) needle, GCA was carried out in equilateral venus through external ligament. In granular ark, GCA was performed by preventing closure of the shells by inserting a tongue depressor between the shells while still open. The success rate of sex identification using the GCA method was 95.6% for the equilateral venus (n=650/680) and 94.3% for the granular ark (n=707/750). Mortality of equilateral venus, which spent 33 days under wild conditions, was 13.8% (n=90/650) while the mortality of granular ark, which spent 390 days under wild conditions, was 2.4% (n=17/707). Although we believe that GCA does not appear to cause death in equilateral venus or granular ark, the success rate in employing of this methodology may differ depending on the level of proficiency of the researcher and reproductive stage of the bivalve. This study concludes that GCA is a convenient non-fatal methodology, which can be employed to identify sex and investigate gonadal maturity in Gomphina veneriformis and Tegillarca granosa.

We developed a novel dicistronic system for the expression of target cDNA sequences in the milk of transgenic animals using goat beta-casein/hGH fusion construct, pGbc5.5hGH (Lee, 2006) and internal ribosome entry site (IRES) sequences of encephalomyocarditis virus (EMCV). Granulocyte colony-stimulating factor (hG-CSF) cDNA was linked to 3' untranslated region of hGH gene in the pGbc5.5hGH via EMCV IRES sequences. Transgenic mice were generated by microinjection and transgene expression was examined in the milk and mammary gland of transgenic mice at 10 days of lactation. Northern blot analysis showed that hGH gene and hG-CSF cDNA were transcribed as a single dicistronic mRNA. The hG-CSF and hGH proteins were independently translated from the dicistronic mRNA and secreted into the milk of transgenic mice. The highest concentration of hG-CSF and hGH in the milk of transgenic mice were $237{\mu}g/m{\ell}$ and $8,990{\mu}g/m{\ell}$, respectively. In contrast, another hG-CSF expression cassette, in which hG-CSF genomic sequences were inserted into a commercial milk-specific expression vector (pBC1), generated a lower level ($91{\mu}g/m{\ell}$) of hG-CSF expression in the milk of transgenic mice. These results demonstrated that the novel pGbc5.5hGH-based dicistronic construct could be useful for an efficient cDNA expression in the milk of transgenic animals.

Nile tilapia (Oreochromis niloticus) are mouth-brooders so that the females holding eggs in their mouth sacrifice their somatic growth for reproduction. For this reason, artificial control of reproduction for the culture of this species has been of interest. Manipulation of photoperiod is an emerging technique for such purpose, but little information is available to establish appropriate photoperiod regime. To obtain necessary basic information, sexually mature females were individually accommodated to glass aquarium, and the spawning activity of these females were monitored for two years under natural photoperiod regime. Female tilapia spawned most frequently on March, April and May when the day length gradually increased from 11 hours to 14 hours and least frequently on September, October, November and December when the day length gradually decreased from 13 hours to less than 10 hours in the first year. The decrease of spawning frequency as day length decreased was also observed in the second year, although the increase of spawning frequency as day length increased was less clear. Spawning of female tilapia was less active when the night was dark due to the disappearance of moonlight (Dark Phase), compared to the Phase of Getting Lighter, Light Phase and Phase of Getting Darker. Results from this study suggest that long day length, particularly increasing phase, is favoured for active spawning of Nile tilapia, and that this species, as a tropical fish species, may utilize changing lunar phases as a secondary environmental cue for reproduction.

A fertilized oocyte can get the competence for implantation through cleavage and stage-specific gene expression. These are under the control of autonomous and exogenous regulators including physiological culture condition. Endogenous and exogenous growth factors are considered as critical regulators of cleaving embryos during travel the oviduct and uterus. In this study, an effort was made to evaluate comprehensively the quality of embryos for implantation, grown in media enriched with EGF and PAF. The study evaluated developmental rates on given time, blastulation and hatching rates, and adhesion rates. Developmental rates of blastocyst to the hatching stage were significantly high in PAF treated group compared to the control in a dose-dependent manner but not in EGF group. Implantation rates were significantly high both PAF and EGF in a dose-dependent manner. H7, a PKC inhibitor, blocked the process of hatching of the blastocysts but combined treatment of EGF and PAF enhanced the hatching and implantation of blastocsyts. Based on these results it is suggested that EGF and PAF support acquirement of implantation competence at blastocyst stage through a PKC pathway.

Mammalian reproduction is regulated by a feedback circuit of the key reproductive hormones such as GnRH, gonadotropin and sex steroids on the hypothalamic-pituitary-gonadal axis. In particular, the onset of female puberty is triggered by gain of a pulsatile pattern and increment of GnRH secretion from hypothalamus. Previous studies including our own clearly demonstrated that genistein (GS), a phytoestrogenic isoflavone, altered the timing of puberty onset in female rats. However, the brain-specific actions of GS in female rats has not been explored yet. The present study was performed to examine the changes in the activities of GnRH neurons and their neural circuits by GS in female rats. Concerning the drug delivery route, intracerebroventricular (ICV) injection technique was employed to eliminate the unwanted actions on the extrabrain tissues which can be occurred if the testing drug is systemically administered. Adult female rats (PND 100, 210-230 g BW) were anaesthetized, treated with single dose of GS ($3.4{\mu}g$/animal), and sacrificed at 3 hrs post-injection. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). ICV infusion of GS significantly raised the transcriptional activities of enhanced at puberty1 (EAP-1, p<0.05), glutamic acid decarboxylase (GAD67, p<0.01) which are known to modulate GnRH secretion in the hypothalamus. However, GS infusion could not change the mRNA level of nitric oxide synthase 2 (NOS-2). GS administration significantly increased the mRNA levels of KiSS-1 (p<0.001), GPR54 (p<0.001), and GnRH (p<0.01) in the hypothalami, but decreased the mRNA levels of LH-$\beta$ (p<0.01) and FSH-$\beta$ (p<0.05) in the pituitaries. Taken together, the present study indicated that the acute exposure to GS could directly activate the hypothalamic GnRH modulating system, suggesting the GS's disrupting effects such as the early onset of puberty in immature female rats might be derived from premature activation of key reproduction related genes in hypothalamus-pituitary neuroendocrine circuit.