Determination of annual reproductive cycle in male sterlet, Acipenser ruthenus using histology and ultrasound imaging.

The aim of this study was to evaluate seasonal testicular development in the cultured sterlet, Acipenser ruthenus. During annual sexual cycle of male sterlet, stages of gonad maturity were examined using histology and ultrasonography approaches. The histology identified males at different stages of maturity among fish sampled monthly. According to the seasonal changes in the testes, reproductive cycle was divided into four stages including resting, pre-spawning, spawning, and post-spawning. The histology examination revealed considerable variation in testicular developmental stages. These changes were identified based on persistent spermatogenesis and asynchronous gonad development in testes, showing that regulation of annual gonadal cycle is influenced by season. Also, the results obtained using ultrasound suggested that reproductive stages can be identified based on morphology and tissue echogenicity. At each phase of testicular development, gonadosomatic index (GSI) and number of spermatogenic cysts were variable. The present study focused on determination of annual reproductive development in cultured male sterlet which clearly identifies reproductive stage in each season as valuable guide for future researches on reproductive biology of sterlet. This study presents basic knowledge about reproductive biology in sterlet contributing to optimal broodstocks management that allows comparison of reproductive development among sturgeon species.

Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae).

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml -1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

Triploid or hybrid tetra: Which is the ideal sterile host for surrogate technology?

This work was aimed at developing an effective procedure to obtain sterile ideal host fish in mass scale with no endogenous germ cells in the germinal epithelium, owning permanent stem-cell niches able to be colonized by transplanted germ cells in surrogate technology experiments. Thus, triploids, diploid hybrids, and triploid hybrids were produced. To obtain hybrid offspring, oocytes from a single Astyanax altiparanae female were inseminated by sperm from five males (A. altiparanae, A. fasciatus, A. schubarti, Hyphessobrycon anisitsi, and Oligosarcus pintoi). Triploidization was conducted by inhibition of the second polar body release using heat shock treatment at 40 °C for 2 min. At 9-months of age, the offspring from each crossing was histologically evaluated to access the gonadal status of the fish. Variable morphological characteristics of the gonads were found in the different hybrids offspring: normal gametogenesis, gametogenesis without production of gametes, sterile specimens holding germ cells, and sterile specimens without germ cells, which were considered "ideal hosts". However, only in the hybrid derived from crossing between A. altiparanae and A. fasciatus, 100% of the individuals were completely sterile. Among them 83.3% of the male did not present germ cells inside germinal epithelium, having only somatic cells in the gonad. The other 16.7% also presented spermatogonia inside the niches. Such a methodology allows the production of sterile host in mass scale, opening new insights for application of surrogate technologies.

Early depletion of primordial germ cells in zebrafish promotes testis formation.

As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.

Germ cells are not the primary factor for sexual fate determination in goldfish.

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.

Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae.

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 ± 108 DsRed-positive germ cells was recovered from each female. For transplantations, a total of approximately 620 ± 242 EGFP-positive cells of which 12 ± 4.7 were DsRed-positive germ cells were introduced into the abdominal cavity under the swim bladder of 2-wk-old sterile hybrid larvae. Six weeks after transplantation, a total of 10 recipients, obtained from 2 different transplantations, were examined, and 2 individuals (20%) were identified that possessed a large number of DsRed- and EGFP-positive cells in the gonadal region. The transplanted ovarian germ cells successfully colonized the gonads and differentiated into sperm in the male hybrid recipients. Of 67 adult recipients, 12 (18%) male chimeric fish reproduced and generated normal offspring when paired with wild-type zebrafish females. The fertilization efficiency ranged from 23% to 56%. Although the fertile male chimeras were generated by transplantation of ovarian germ cells, the F1 generation produced by the male chimeras contained both male and female progeny, indicating that male sex determination in zebrafish is not controlled by sex chromosome heterogamy. Our findings indicate that a population of ovarian germ cells that are present in the ovary of adult zebrafish can function as germline stem cells, able to proliferate and differentiate into testicular germ cells and functional sperm in male recipients. The high frequency of germline chimera formation achieved with the ovarian germ cells and the convenience of identifying the chimeras in the sterile host background should make this transplantation system useful for performing genetic manipulations in zebrafish.

Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation.

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.

Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor-beta, activin A and microphthalmia-associated transcription factor.

Plasminogen activator inhibitor-1 (PAI-1) is a key molecule that regulates turnover of the extracellular matrix. In the present study, we characterized PAI-1 gene expression in mast cells and melanocytes. In bone marrow-derived cultured mast cells, up-regulation of the PAI-1 gene was observed upon treatment with TGF-beta1, and was regulated at the transcriptional level. Microphthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix leucine zipper family of tissue-specific transcription factors predominantly expressed in mast cells, melanocytes and osteoclasts, also stimulated PAI-1 gene transcription, and TGF-beta1 did not increase PAI-1 mRNA levels in mast cells from mi/mi mice expressing dominant-negative MITF. MITF isoforms regulated TGF-beta1-induced transcription of PAI-1 differently; MITF-E-mediated transcription was further increased by TGF-beta1, whereas transcriptional activation by TGF-beta1 was blocked by MITF-M or MITF-mc expression. In contrast, activin A, another member of the TGF-beta family, enhanced transcription induced by MITF-M, as well as by MITF-E, although MITF-mc blocked activin A-induced transcription of PAI-1. Different regulation of PAI-1 gene expression upon TGF-beta1 and activin A treatment was also detected in B16 melanocytes; TGF-beta1 transiently increased the PAI-1 mRNA level, whereas activin A had prolonged effects on up-regulation of PAI-1. Our results on the control of PAI-1 gene expression by MITF isoforms, TGF-beta1 and activin A suggest that discrete regulation of the plasminogen activator system occurs in a cell type-specific manner.