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 preserv

Spermatogonial stem cell markers and niche in equids.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called "niche" that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate the isolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers (GFRA1, PLZF and CSF1R) in three different domestic equid species (stallions, donkeys, and mules). Aund were first characterized according to their morphology and expression of the GFRA1 receptor. Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid species evaluated in this study. These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology are conserved among mammals. We hope that our findings will help future studies needing isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies that aim at preserving the germplasm of valuable animals, and involve germ cell transplantation or xenografts of equids testis fragments/germ cells suspensions

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.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Testis morphometry in horses, during the breeding and non-breeding season, and seminiferous tubules cross-sections subdivisions.

<p>A) Whereas the seminiferous tubules (ST) volume density was not changed during the two periods evaluated, Leydig cells (LC) and connective tissue (CT) were the most prevalent components of the intertubular compartment during the breeding and non-breeding season. Seminiferous tubules cross-sections were subdivided into 4 different regions according to the prevalence of these aforementioned components (B). BV = blood vessels. Figure B, bar = 100 µm.</p

Immunostaining evaluation of the presence of CSF1R in equids.

<p>A) As it can be noted, the expression of this marker was limited to the cytoplasm of Aund (arrowheads) and this pattern was similar for horse (A2–4), donkey (A6–8) and mule (A10–12). A1, A5 and A9 are the negative controls. B) Immunoblotting confirmed the expression of CSF1R in the testis of horse [during the breeding (BS) and non-breeding (NBS) season], donkey and mule. C) Percentage of CSF1R(+) Aund cells showing that approximately 35% of these cells express this membrane receptor (*p<0.05). Figure A, bar = 10 µm.</p

Immunostaining evaluation of the presence of GFRA1 in equids.

<p>A) As it can be noted, the expression of this marker was limited to the cytoplasm of Aund (arrowheads) and this pattern was similar for horse (A2–4), donkey (A6–8) and mule (A10–12). A1, A5 and A9 are the negative controls. B) Immunoblotting confirmed the expression of GFRA1 in the testis of horse [during the breeding (BS) and non-breeding (NBS) season], donkey and mule. C) Percentage of GFRA1(+) Aund cells showing that approximately 90% of these cells express this membrane receptor (*p<0.05). Figure A, bar = 10 µm.</p

Stages of the seminiferous epithelium cycle (A) and their frequencies (B) in stallions.

<p>A) The following symbols were used to designate specific germ cell types: A1, type A1 spermatogonia; A2, type A2 spermatogonia; B1, type B1 spermatogonia; B2, type B2 spermatogonia; P, pachytene spermatocyte; D, diplotene spermatocyte; M, meiotic figure; R, round spermatids; E, elongating/elongated spermatids; SC, Sertoli cell. Arabic numerals (1–12) indicate each step of the spermatid acrosome development. B) Note that stages I, VII and XII presented the highest frequencies, whereas the opposite was observed for stages II, III, IV and XI. White and black bars = 5 µm.</p

Aund distribution in horses according to morphological (A–H) and immunostaining (I–P) criteria.

<p>As indicated by red arrowheads, using both criteria, Aund cells were present in all four regions considered. However, independently of the breeding season, these cells were more frequently observed in the areas facing the interstitium, particularly nearby the blood vessels. TT = Tubule-Tubule contact; TI−BV = Tubule-Interstitium without blood vessels; TI+BV = Tubule-Interstitium with blood vessels; TIC = Tubule-Interstitium containing connective tissue. Figure A–D and H–K, bar = 10 µm.</p

Qualitative evaluation of the co-localization of the three different spermatogonial markers used for horses.

<p>Considering the co-expression of GFRA1 and PLZF the following pattern was observed: A) GFRA1(+) cells (A1; red arrowhead) presenting co-localization with PLZF (A2; yellow arrowhead), as evidenced in the merged figure (A3; white arrowhead); B) this panel illustrates GFRA1(+) cells (B1; red arrowhead) that do not present PLZF expression (B2), as shown in the merged figure (B3; white arrowhead). In relation to the co-expression of GFRA1 and CSF1R the following labeling pattern was observed: C) GFRA1(+) cells (C1; red arrowhead) also expressing CSF1R (C2; green arrowhead), shown in the merged figure (C3; white arrowhead); D) differently, some GFRA1(+) cells (D1; red arrowhead) do not present CSF1R (D2; white arrowhead in D3 merged figure). E) Summarization of the quantitative data obtained for Aund GFRA1, PLZF and CSF1R positive cells in horses, suggesting that these three proteins are differently expressed in this cell population. Yellow bar = 20 µm; White bar = 30 µm.</p