Testicular germ line cell identification, isolation, and transplantation in two North American catfish species.

Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 µm, diameter with distinct nucleus 7-8 µm diameter) and spermatogonia (12-15 µm, with distinct nucleus 6-7.5 µm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 µm) and type B spermatogonia (diameter 10-11 µm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.

Spermatogonial stem cells specific marker identification in channel catfish, Ictalurus punctatus and blue catfish, I. furcatus.

Testicular germ cells of channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus were separated into four layers with Percoll density gradient centrifugation, containing different cell types (40% in the first layer were spermatogonial stem cells, SSCs). Expression of seventeen genes was analyzed for cells from different layers by real-time quantitative PCR. Pfkfb4, Urod, Plzf, Integrin6, IntegrinV, Thy1 and Cdh1 genes showed the same expression change pattern in both channel and blue catfish as these genes were down-regulated in the spermatocytes and even more so in spermatids. Plzf and Integrin6 had especially high expression in SSCs and can be used as SSCs specific markers. Sox2 gene was up-regulated in spermatocytes and even more highly up-regulated in spermatids, which indicated it could be a spermatid marker. In contrast to channel catfish, Id4, Smad5 and Prdm14 gene expressions were strongly down-regulated in spermatocyte cells, but up-regulated in spermatid cells in blue catfish. Smad5 gene was down-regulated in spermatocytes, but up-regulated in both spermatogonia and spermatids, allowing identification as a marker for spermatocytes in blue catfish. Oct4, Id4, Gfrα2, Pum2 and Prdm14 genes showed different expression patterns in the testicular germ cells of channel and blue catfish. This may be a partial explanation to the differing responses of channel catfish and blue catfish to induced spawning technologies. The SSCs specific markers can be used for further SSCs labeling, which can increase the SSCs sorting efficiency and be applied in various studies involving SSCs and other germ cells.

The gametogenic cycle of Leguminaia whaetleyi (Lea, 1862) in lake Gölbasi, Turkey (Bivalvia: Unionidae).

The gonadal structure and cycle of the Leguminaia whaetleyi is described for the first time indicating that 5% of this species is simultaneous hermaphrodite. A total of 420 individuals were collected in monthly samples from October 2008 to September 2009 in Gölbasi Lake, Turkey. Calculation of the condition index and histological examination of the gonads showed that gametogenesis began in January. Spawning occurred between May and August with one maximum peak in July. Annual maximum oocytes size peaked in July. The population consisted of simultaneous hermaphrodite individuals. Sex ratio of L. whaetleyi was significantly different from the expected 1:1 ratio (P< 0.05), and also female biased sex ratio was recorded. In hermaphrodite specimens, male and female follicles were mixed in the visceral mass but can be clearly distinguished by light microscopy study. L. whaetleyi appears to be a dioecious species in which 25 specimens were simultaneous hermaphrodite and under certain environmental conditions may be capable of self-fertilization. This suggests that detailed studies on sex ratio of L. whaetleyi may be helpful in understanding unionid phylogeny.

The effects of Gokshura, Tribulus terrestris on sex reversal of guppy, Poecilia reticulata.

This study examined the effects of Tribulus terrestris (TT) on sex reversal in guppy, Poecilia reticulata. The objective of this study was to introduce a new environmentally friendly method for masculinization in P. reticulata. Since male guppy has higher commercial value than female. TT is a natural, non-toxic herb which helps enhance testosterone levels in human and animals. It was prepared in a laboratory in France. Different concentration (0.0, 0.05, 0.1 and 0.15 g L(-1)) of TT was investigated for sex reversal in the Poecilia reticulata. TT extract was administered by immersion of newly born offspring once weekly for two months. Among the dosages used in the present study 0.15 g L(-1) TT was the most effective dosage that ensured maximum male ratio (80%, p < 0.01). Although, sex ratios of 0.05 and 0.1 g L(-1) TT were not significantly different from the expected 1:1 ratio, in these two groups treatment with TT also result in higher number of males (58.25 and 59.77%, respectively), than control (p > 0.05). Total survival rates in all treatments and control were uniformly high ranging from 83 to 87% (p > 0.05). It is concluded that TT has no negative effect on survival rate of P. reticulata. All groups of TT-treated fish exhibited successful growth acceleration comparing to the control group, but only TT treatment at the concentration of 0.15 and 0.1 g L(-1) TT significantly improved growth rate of P. reticulata (p < 0.01). Histological examinations revealed that testes of fish treated with TT-extract contained all stages of spermatogenesis. Sex reversal in P. reticulata demonstrated that TT treated new-born progenies showed successful sex reversal, spermatogenesis and better growth rate than untreated progenies.