Characterization of spermatogonial cells and niche in the scorpion mud turtle (Kinosternon scorpioides).

Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male's life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.

Duration of spermatogenesis and identification of spermatogonial stem cell markers in a Neotropical catfish, Jundiá (Rhamdia quelen).

Spermatogenesis is a process driven by stem cell, where germ cell cycle is under the control of a specific genotype species. Considering that Jundiá (Rhamdia quelen) is a Neotropical catfish with great economical importance and useful experimental model, little information is available on basic aspects of its reproductive biology, especially on spermatogenesis. As a result, this study aimed to characterize the male germ cells, estimate the duration of spermatogenesis and evaluate the expression of selected stem cell genes in Jundiá testis. Similar to other fish species, our results showed a remarkable decrease of germ cell nuclear volume during Jundiá spermatogenesis, particularly from type A undifferentiated to late type B spermatogonia and from diplotene to late spermatids. Using a S-phase marker, bromodeoxyuridine (BrdU), the combined duration of meiotic and spermiogenic phases in this species was estimated in approximately 7 days. This is considered very short when compared to mammals, where spermatogenesis last from 30 to 74 days. Selected stem cell genes were partially sequenced and characterized in Jundiá testis. Expression analysis showed higher plzf and pou5f3 mRNA levels in the cell fractions enriched by type A undifferentiated spermatogonia. These results were further confirmed by in situ hybridization that showed strong signal of plzf and pou5f3 mRNA in type A undifferentiated spermatogonia. Altogether, these information will expand our knowledge of the reproductive biology of this species, contributing to improve its production and management, and also for biotechnological applications, such as germ cell transplantation.

Successful xenogeneic germ cell transplantation from Jundia catfish (Rhamdia quelen) into adult Nile tilapia (Oreochromis niloticus) testes.

Fish germ cell transplantation presents several important potential applications for aquaculture, including the preservation of germplasm from endangered fish species with high genetic and commercial values. Using this technique in studies developed in our laboratory with adult male Nile tilapias (Oreochromis niloticus), all the necessary procedures were successfully established, allowing the production of functional sperm and healthy progeny approximately 2months after allogeneic transplantation. In the present study, we evaluated the viability of the adult Nile tilapia testis to generate sperm after xenogeneic transplant of germ cells from sexually mature Jundia catfish (Rhamdia quelen) that belong to a different taxonomic order. Therefore, in order to investigate at different time-periods post-transplantation, the presence and development of donor PKH26 labeled catfish germ cells were followed in the tilapia seminiferous tubules. From 7 to 20days post-transplantation, only PKH26 labeled spermatogonia were observed, whereas spermatocytes at different stages of development were found at 70days. Germ cell transplantation success and progression of spermatogenesis were indicated by the presence of labeled PKH26 spermatids and sperm on days 90 and 120 post-transplantation, respectively. Confirming the presence of the catfish genetic material in the tilapia testis, all recipient tilapias evaluated (n=8) showed the genetic markers evaluated. Therefore, we demonstrated for the first time that the adult Nile tilapia testis offers the functional conditions for development of spermatogenesis with sperm production from a fish species belonging to a different order, which provides an important new venue for aquaculture advancement.

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.

Functionalized nanomaterials: are they effective to perform gene delivery to difficult-to-transfect cells with no cytotoxicity?

Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death.