Porcine Primordial Germ Cell-Like Cells Generated from Induced Pluripotent Stem Cells Under Different Culture Conditions.

Culture conditions regulate the process of pluripotency acquisition and self-renewal. This study aimed to analyse the influence of the in vitro environment on the induction of porcine induced pluripotent stem cell (piPSCs) differentiation into primordial germ cell-like cells (pPGCLCs). piPSC culture with different supplementation strategies (LIF, bFGF, or LIF plus bFGF) promoted heterogeneous phenotypic profiles. Continuous bFGF supplementation during piPSCs culture was beneficial to support a pluripotent state and the differentiation of piPSCs into pPGCLCs. The pPGCLCs were positive for the gene and protein expression of pluripotent and germinative markers. This study can provide a suitable in vitro model for use in translational studies and to help answer numerous remaining questions about germ cells.

Central nervous system development in rabbits (Oryctolagus cuniculus L. 1758).

The present study describes the embryonic and fetal development of the central nervous system in rabbits from the seventh day after conception until the end of the full-term fetal period. A total of 19 embryonic and fetal samples were carefully dissected and microscopically analyzed. Neural tube closure was observed between 7.5 and 8 days of gestation. Primordial encephalic vesicle differentiation and spinal canal delimitation were observed on the 12th day of gestation. Histologically, on the 15th day of gestation, the brain, cerebellum, and brain stem were delimited. On the 18th day of gestation, the cervical and lumbar intumescences of the spinal cord were visible. On the 28th day of gestation, four-cell layers could be distinguished in the cerebral cortex, while the cerebellar cortex was still differentiating. Overall, the morphological aspects of the embryonic and fetal developmental phases in rabbits were highly similar to those in humans. Thus, the present study provides relevant information highlighting rabbits as an excellent candidate animal model for preclinical research on human neurological diseases given the high adaptability of rabbits to bioterium conditions and the similarity of morphological events between rabbits and humans.

Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice.

BACKGROUND: Xenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis. METHODS: SSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L-1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis. RESULTS: Supplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice. CONCLUSIONS: In conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

Stem cells on regenerative and reproductive science in domestic animals.

Stem cells are undifferentiated and self-renewable cells that present new possibilities for both regenerative medicine and the understanding of early mammalian development. Adult multipotent stem cells are already widely used worldwide in human and veterinary medicine, and their therapeutic signalling, particularly with respect to immunomodulation, and their trophic properties have been intensively studied. The derivation of embryonic stem cells (ESCs) from domestic species, however, has been challenging, and the poor results do not reflect the successes obtained in mouse and human experiments. More recently, the generation of induced pluripotent stem cells (iPSCs) via the forced expression of specific transcription factors has been demonstrated in domestic species and has introduced new potentials in regenerative medicine and reproductive science based upon the ability of these cells to differentiate into a variety of cells types in vitro. For example, iPSCs have been differentiated into primordial germ-like cells (PGC-like cells, PGCLs) and functional gametes in mice. The possibility of using iPSCs from domestic species for this purpose would contribute significantly to reproductive technologies, offering unprecedented opportunities to restore fertility, to preserve endangered species and to generate transgenic animals for biomedical applications. Therefore, this review aims to provide an updated overview of adult multipotent stem cells and to discuss new possibilities introduced by the generation of iPSCs in domestic animals, highlighting the possibility of generating gametes in vitro via PGCL induction.

Seasonal variations cause morphological changes and altered spermatogenesis in the testes of viscacha (Lagostomus maximus).

This study complements the previous investigations of the reproductive biology of male viscachas, a rodent of a seasonal Hystricognathi that exhibits photoperiod-induced morphological variations in the reproductive system. In the present study, a quantitative analysis of spermatogenesis was performed during the summer and the spring. Spermatogonial cells were analyzed to determine by immunolabelling for STRA8 and DAZL, which are essential for spermatogenesis. Six free-living male viscachas were captured, three animals in the summer during the period of reproductive activity and three animals in the spring during the period of testicular regression. The testes of the viscachas were collected and processed for light microscopy, macroscopic and immunochemical analyses. The germ and Sertoli cells present in the seminiferous tubules were quantitatively analyzed in each animal. The efficiency coefficient for spermatogonial mitosis, meiotic yield, overall spermatogenesis yield and Sertoli cell index, revealed that the Sertoli cells in male viscachas captured during the summer had a reduced capacity to structurally and nutritionally support the developing round spermatids compared with the male viscachas captured during the spring. The animals produced less sperm during the spring than the summer, suggesting a seasonal impact on spermatogenesis. Immunolabelling for STRA8 and DAZL was detected during summer and spring seasons. These results suggest that in seasonal rodents, such as the male viscachas, the photoperiod promotes significant changes in the testis and in the germ cell yield.

The carnivore pregnancy: the development of the embryo and fetal membranes.

The aim of this research was to compare the morphological aspects during the development of pregnancy in dogs and cats, distinguishing features of the fetal membranes, such as yolk sac evolution and differentiation of hemangioblasts, and the degree of elaboration of the amnion and allantois. Canine and feline placentae from 20, 24, 35, 45 and 55 d of pregnancy were perfusion-fixed for histological investigation and vascular corrosion casts were produced. The casts were prepared for scanning electron microscopy (SEM) and the embryo and fetal membrane development was analyzed. The growth patterns of the conceptuses were compared with the organization of the placentation process, and changes of the morphology during pregnancy were recorded. In feline placentae, an incomplete zonary shape was present in 62.5% out of 60 studied cases. This was located distal to the insertion of the umbilical cord. In the lamellar zone, the interhemal membrane or placental barrier resembled endotheliochorial conditions, and the maternal-fetal microvascular blood flow interrelationship was of simple crosscurrent type. Dogs have a zonary placenta, completely surrounding the fetus, and complex lamellar organization of maternal and fetal tissues. At the border, two marginal hematomes with green colouration delimited the central placental girdle. The yolk sac consisted of one large sacculation with an inverted "T" shape and an enormous number of blood vessels; it had hemangioblast cells in contact with the epithelium. The amnion was avascular in early stages, but became vascularized by blood vessels of the internal allantoic membrane in later stages of pregnancy by intrinsic relation.