Isolation, in vitro study, and stem cell markers for type A spermatogonia in a Characiformes species.

The objective of this study was to establish a protocol for the characterization, isolation, and culture of type A spermatogonia using specific molecular markers for these cells in fish. To this end, adult Prochilodus lineatus testes were collected and digested enzymatically and the resulting testicular suspension was separated using a discontinuous Percoll gradient, followed by differential plating. The cell cultures obtained were monitored for 15 days and analyzed using the immunofluorescence method with anti-Vasa, anti-GFRα1, and anti-OCT4 antibodies. Spermatogonial enrichment was also performed using flow cytometry. Although discontinuous Percoll gradient centrifugation followed by differential plating enabled the removal of differentiated germ cells and somatic cells, enriching the pool of type A spermatogonia, the enrichment of type A spermatogonia through flow cytometry of samples without Percoll proved to be more efficient. Prominent cell agglomerates that were characterized according to different stem cell markers as type A spermatogonia were observed during the 15 days of the cell culture. The use of immunoperoxidase and western blot analysis methods confirmed the specificity of the markers for type A spermatogonia of P. lineatus. When combined with specific cell culture conditions, the positive characterization of these molecular markers clarified certain aspects of spermatogonial regulation, such as survival and proliferation. Finally, understanding the regulation of the in vitro germ cell maintenance process may contribute to the enhancement of in vivo and in vitro reproduction techniques of endangered or aquaculture fish species.

Testicular structure and development of germ cells of Hypophthalmus marginatus Valenciennes 1840 (Siluriformes: Pimelodidae).

The present study was conducted to assess the testicular structure and germ cell ultrastructure of Hypophthalmus marginatus during spermatogenesis. Semen and sections of the mid-region of the testis were collected, processed, and analyzed using optical and electron microscopy. Macroscopically, the testes of H. marginatus were filiform, and the testicular parenchyma was composed of spermatogenic cells that proliferated, organized within spermatic cysts. During spermiogenesis, spermatids had no nuclear rotation. The proximal centriole was perpendicular to the distal centriole, characteristic of type III spermiogenesis. Spermatozoa were released into the lumen of the seminiferous tubules and had an ovoid head without an acrosome, condensed nucleus, and shallow nuclear fossa. The midpiece was short, with a single long flagellum. The flagellum had the typical axoneme structure, with nine pairs of peripheral and a central pair of microtubules. The thin end piece comprised only peripheral microtubules. Spermatogenesis in H. marginatus features filiform testes, cystic spermatogenesis, and type III spermiogenesis.

Microsatellites Associated with Growth Performance and Analysis of Resistance to Aeromonas hydrophila in Tambaqui Colossoma macropomum.

Tambaqui, Colossoma macropomum, is the main native fish species produced in Brazil, and is an important species for genetic improvement in aquaculture. In addition, breeding studies on this species can be optimized with the use of molecular markers associated with productive phenotypes. The objective of the present study was to test the performance of growth traits and resistance to the bacteria, Aeromonas hydrophila, in association with microsatellite markers in C. macropomum. In this study, three full-sib families were subjected to bacterial challenge and morphometric growth assessments. Tambaqui families subjected to the bacterial challenge differed significantly in death time and mortality rate. There was, however, no association between resistance to bacteria and microsatellite markers. In relation to growth traits, we observed a marker/phenotype association in two microsatellites. The marker in the 6b isoform x5 gene (TNCRC6b) was associated with length, whereas an anonymous marker was associated with height. The present study highlighted the evaluation of molecular markers associated with growth traits, and can serve as the basis for future marker-assisted selection (MAS) of tambaqui.

Effects of re-stripping on the seminal characteristics of pacu (Piaractus mesopotamicus) during the breeding season.

Seminal characteristics in teleost fish with an annual reproductive period, such as pacu (Piaractus mesopotamicus), may vary during the breeding season. The sperm formed before the beginning of the spawning period may be stored for a long time, causing damage to the cells. Therefore, re-stripping may be an important way to eliminate the "old" and allow for the collection of "new" spermatozoids. In this study, we analyzed the seminal characteristics of hormonally induced pacu at the beginning, middle and end of the breeding season, and we analyzed samples from re-stripped males (stripped first at the beginning, re-stripped in the middle, and re-stripped again at the end of the season) during two breeding seasons. The sperm density, ionic composition, pH, and osmolality were similar among the groups. The semen volume, seminal plasma protein concentration and incidence of morphologically anomalous sperm increased over time. In addition, some parameters that are associated with good-quality semen decreased, such as sperm motility, viability and DNA integrity. Moreover, we observed a positive association among motility, viability and DNA integrity for sperm with elevated 11-ketotestosterone, but there was no such association for fshb or lhb mRNA levels in the pituitary. The semen that was obtained earlier (at the beginning) or from re-stripped males exhibited better characteristics than the other samples collected. In conclusion, collecting semen from pacu at the end of breeding season should be avoided; it is preferable to strip early and then re-strip later in the season, and this approach may be used for diverse aquaculture purposes.

Inappropriate management conditions, especially for the regressed class, are related to sperm quality in Prochilodus lineatus.

The aims of this study were to evaluate the characteristics of the reproductive classes and semen quality in curimbatá (Prochilodus lineatus) breeders maintained in two different rearing systems. To achieve this goal, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively. The fish were maintained under the experimental conditions for 18 months. During this period, males were randomly sampled every 2 months for biometric analysis (n = 30 per sample) and for an evaluation of selected characteristics of the testes (n = 5 per sample). After this period, males maintained in EPs and males maintained in Cs (CMs) were evaluated in induced breeding experiments. We observed that rearing P. lineatus in a C at a high stocking density for the long 18-month period of study produced reductions in growth, testis development, gonadosomatic index values, and sperm quality in the fish. We found differences between the groups in all the reproductive classes examined, especially in the regression class, which showed a pronounced accumulation of immature germ cells in the CMs. In this group, we also noted a less intense transition from a continuous to discontinuous germinal epithelium, with an extended and abnormal but less intense spermatogenic period resulting in decreases in semen volume and sperm concentration in the breeding season. Together, such dysfunctions resulted in the production of low-quality sperm in the CMs, as demonstrated by lower-quality DNA (as evaluated by the comet assay), low fertilization success, and low hatching success. In conclusion, to ensure high-quality semen in P. lineatus, appropriate management conditions must be provided throughout the reproductive cycle, especially for the regressed class, even in winter, two seasons before the breeding season.

Germ cell transplantation in felids: a potential approach to preserving endangered species.

With the exception of the domestic cat, all members of the family Felidae are considered either endangered or threatened. Although not yet used for this purpose, spermatogonial stem cell (SSC) transplantation has a high potential to preserve the genetic stock of endangered species. However, this technique has not previously been established in felids. Therefore, we developed the necessary procedures to perform syngeneic and xenogeneic SSC transplants (eg, germ cell [GC] depletion in the recipient domestic cats, enrichment and labeling of donor cell suspension, and the transplantation method) in order to investigate the feasibility of the domestic cat as a recipient for the preservation and propagation of male germ plasm from wild felids. In comparison with busulfan treatment, local x-ray fractionated radiation was a more effective approach to depleting endogenous spermatogenesis. The results of both syngeneic and xenogeneic transplants revealed that SSCs were able to successfully colonize and differentiate in the recipient testis, generating elongated spermatids several weeks posttransplantation. Specifically, ocelot spermatozoa were observed in the cat epididymis 13 weeks following transplantation. As donor GCs from domestic cats and ocelots were able to develop and form mature GCs in the recipient environment seminiferous tubules, these findings indicate that the domestic cat is a suitable recipient for SSC transplantation. Moreover, as modern cats descended from a medium-size cat that existed approximately 10 to 11 million years ago, these results strongly suggest that the domestic cat could be potentially used as a recipient for generating and propagating the genome of wild felids.

Selective ablation of the androgen receptor in mouse sertoli cells affects sertoli cell maturation, barrier formation and cytoskeletal development.

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

A new and fast technique to generate offspring after germ cells transplantation in adult fish: the Nile tilapia (Oreochromis niloticus) model.

BACKGROUND: Germ cell transplantation results in fertile recipients and is the only available approach to functionally investigate the spermatogonial stem cell biology in mammals and probably in other vertebrates. In the current study, we describe a novel non-surgical methodology for efficient spermatogonial transplantation into the testes of adult tilapia (O. niloticus), in which endogenous spermatogenesis had been depleted with the cytostatic drug busulfan. METHODOLOGY/PRINCIPAL FINDINGS: Using two different tilapia strains, the production of fertile spermatozoa with donor characteristics was demonstrated in adult recipient, which also sired progeny with the donor genotype. Also, after cryopreservation tilapia spermatogonial cells were able to differentiate to spermatozoa in the testes of recipient fishes. These findings indicate that injecting germ cells directly into adult testis facilitates and enable fast generation of donor spermatogenesis and offspring compared to previously described methods. CONCLUSION: Therefore, a new suitable methodology for biotechnological investigations in aquaculture was established, with a high potential to improve the production of commercially valuable fish, generate transgenic animals and preserve endangered fish species.

Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations.

The zebrafish has become an important vertebrate model for basic and biomedical research, including the research field of the biology of reproduction. However, very few morphological and stereological data are available regarding zebrafish testis structure and spermatogenesis. In this careful histomorphometric evaluation of the testis, we studied spermatogonial cells using molecular markers, determined the combined duration of meiotic and spermiogenic phases, and examined the formation of the Sertoli cell barrier (tight junctions). We found at least nine spermatogonial generations and propose a morphology-based nomenclature for spermatogonial generations that is compatible with the one used in higher vertebrates. The number of germ cells per cyst increased dramatically (1 to approximately 1360 cells) from undifferentiated spermatogonia type A to early spermatids. The combined duration of meiotic and spermiogenic phases is approximately 6 days, one of the shorter periods among the teleost fish investigated to date. The number of Sertoli cells per cyst increased 9-fold during the maturational cycle of spermatogenic cysts and stabilized in the meiotic phase at a ratio of approximately 100 early spermatids per Sertoli cell (Sertoli cell efficiency). Similarly to mammals, Sertoli cell proliferation ceased in the meiotic phase, coinciding with the formation of tight junctions between Sertoli cells. Hence, the events taking place during puberty in the germinal epithelium of mammals seem to recapitulate the "life history" of each individual spermatogenic cyst in zebrafish.