Guilherme Mattos Jardim Costa

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.

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.

SUB-ACUTE INTRAVENOUS ADMINISTRATION OF SILVER NANOPARTICLES IN MALE MICE ALTERS LEYDIG CELL FUNCTION AND TESTOSTERONE LEVELS

The aim of this study was to determine whether short-term, in vivo exposure to silver nanoparticles (AgNPs) could be toxic to male reproduction. Low dose (1mg/kg/dose) AgNPs were intravenously injected into male CD1 mice over 12 days. Treatment resulted in no changes in body and testis weights, sperm concentration and motility, fertility indices, or follicle-stimulating hormone and luteinizing hormone serum concentrations; however, serum and intratesticular testosterone concentrations were significantly increased 15 days after initial treatment. Histologic evaluation revealed significant changes in epithelium morphology, germ cell apoptosis, and Leydig cell size. Additionally, gene expression analysis revealed Cyp11a1 and Hsd3b1 mRNA significantly upregulated in treated animals. These data suggest that AgNPs do not impair spermatogonial stem cells in vivo since treatment did not result in significant decreases in testis weight and sperm concentrations. However, AgNPs appear to affect Leydig cell function, yielding increasing testicular and serum testosterone levels.

The Spermatogonial Stem Cell Niche in the Collared Peccary (Tayassu tajacu)

ABSTRACT In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the “niche” that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (Aund) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of Aund were GDNF receptor alpha 1 positive (GFRA1+), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1+ Aund. Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of Aund toward type A1 spermatogonia.

Biology and identity of fish spermatogonial stem cell

Although present at relatively low number in the testis, spermatogonial stem cells (SSCs) are crucial for the establishment and maintenance of spermatogenesis in eukaryotes and, until recently, those cells were investigated in fish using morphological criteria. The isolation and characterization of these cells in fish have been so far limited by the lack of specific molecular markers, hampering the high SSCs biotechnological potential for aquaculture. However, some highly conserved vertebrate molecular markers, such as Gfra1 and Pou5f1/Oct4, are now available representing important candidates for studies evaluating the regulation of SSCs in fish and even functional investigations using germ cells transplantation. A technique already used to demonstrate that, different from mammals, fish germ stem cells (spermatogonia and oogonia) present high sexual plasticity that is determined by the somatic microenvironment. As relatively well established in mammals, and demonstrated in zebrafish and dogfish, this somatic environment is very important for the preferential location and regulation of SSCs. Importantly, a long-term in vitro culture system for SSCs has been now established for some fish species. Therefore, besides the aforementioned possibilities, such culture system would allow the development of strategies to in vitro investigate key regulatory and functional aspects of germline stem cells (ex: self-renewal and/or differentiation) or to amplify SSCs of rare, endangered, or commercially valuable fish species, representing an important tool for transgenesis and the development of new biotechnologies in fish production.

Germ cell transplantation as a potential biotechnological approach to fish reproduction

Although the use of germ cell transplantation has been relatively well established in mammals, the technique has only been adapted for use in fish after entering the 2000s. During the last decade, several different approaches have been developed for germ cell transplantation in fish using recipients of various ages and life stages, such as blastula-stage embryos, newly hatched larvae and sexually mature specimens. As germ cells can develop into live organisms through maturation and fertilization processes, germ cell transplantation in fish has opened up new avenues of research in reproductive biotechnology and aquaculture. For instance, the use of xenotransplantation in fish has lead to advances in the conservation of endangered species and the production of commercially valuable fish using surrogated recipients. Further, this could also facilitate the engineering of transgenic fish. However, as is the case with mammals, knowledge regarding the basic biology and physiology of germline stem cells in fish remains incomplete, imposing a considerable limitation on the application of germ cell transplantation in fish. Furthering our understanding of germline stem cells would contribute significantly to advances regarding germ cell transplantation in fish.

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.

Spermatogenic Cycle Length and Sperm Production in a Feral Pig Species (Collared Peccary, Tayassu tajacu)

Although the collared peccary (Tayassu tajacu) is found throughout the Americas, with a high potential for domestication and commercial exploitation, there are few data on the reproductive biology of this mammalian species. The aim of the present study was to investigate testis structure, spermatogenic cycle length, Sertoli cell efficiency, and spermatogenic efficiency. Twelve adult peccaries were used for biometrical, histological, and stereological analyses; 3 of these peccaries received intratesticular injections of (3)H-thymidine for the determination of the duration of spermatogenesis. Testis weight and gonadosomatic index were 23.7 +/- 1.8 g and 0.2% +/- 0.1%, respectively. Seminiferous tubule volume density was 77.4% +/- 1.7%. Leydig cells occupied 12.8% +/- 1.8% of the testis parenchyma and presented a peculiar cytoarchitecture in the periphery of the seminiferous tubule lobes. The premeiotic, meiotic, and postmeiotic stage frequencies were very similar to those found for wild and domestic boars. The spermatogenic cycle and entire spermatogenic process (based on 4.5 cycles) lasted approximately 12.3 +/- 0.2 and 55.1 +/- 0.7 days, respectively. Daily sperm production per gram of testis in the collared peccary was approximately 23.4 +/- 2 x 10(6), which is similar to that of domestic and wild boars. The knowledge generated in the present study could be used in reproduction and animal improvement programs and provides important information that may be used for comparative reproductive biology with previously investigated mammalian species.

Phenotypic characterization and in vitro propagation and transplantation of the Nile tilapia (Oreochromis niloticus) spermatogonial stem cells

In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.

Duration of Spermatogenesis and Spermatogenic Efficiency in 2 Large Neotropical Rodent Species: The Agouti (Dasyprocta leporina) and Paca (Agouti paca)

The agouti (Dasyprocta leporina) and paca (Agouti paca) are among the largest extant rodent species. Although these species have considerable economic potential, there are few reports in the literature concerning male reproductive biology in sexually mature agoutis and pacas. The objectives of the present study were to perform detailed stereologic and histologic analyses of the testis and estimate spermatogenic cycle length in these 2 Neotropical rodent species as well as to compare the data with those from other well-studied rodent members of the Hystricomorpha and Myomorpha suborders. Eight adult agoutis and 6 adult pacas were used. The spermatogenic cycle in agoutis and pacas lasted 9.5 ± 0.03 and 11.5 ± 0.16 days, respectively, whereas the total duration of spermatogenesis for these 2 species was 42.8 ± 0.16 and 51.6 ± 0.7 days, respectively. Most of the parameters investigated were similar to those obtained for the other members of the Hystricomorpha suborder. As a result of the combination of high values of seminiferous tubule volume density, number of Sertoli cells per gram of testis, Sertoli cell efficiency, and a relatively short duration of spermatogenesis, the spermatogenic efficiency found in agoutis (52 ± 2 × 10(6)) and pacas (39 ± 2 × 10(6)) was particularly high when compared with that of previously investigated mammalian species. The data presented in this investigation would be useful for studies related to animal production as well as improvement and conservation programs involving these 2 large Neotropical rodent species.