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ContentsSpermatogenesis is a process in which differentiated cells are produced and the adult stem cell population-known as spermatogonial stem cells (SSCs)-is continuously replenished. However, the molecular mechanisms underlying these processes are not fully understood in the canine species. We addressed this in this study by analysing the expression of specific markers in spermatogonia of seminiferous tubules of canine testes. SSCs at different stages of reproductive development (prepubertal and adult) were examined by immunohistochemistry and flow cytometry. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRA1), deleted in azoospermia-like (DAZL) and promyelocytic leukaemia zinc finger (PLZF) were expressed in SSCs, while stimulated by retinoic acid gene 8 (STRA8) was detected only in undifferentiated spermatogonia in prepubertal testis and differentiated spermatogonia and spermatocytes in adult canine.Octamer-binding transcription factor 4 (OCT4) showed an expression pattern, and the levels did not differ between the groups examined. However, C-kit expression varied as a function of reproductive developmental stage. Our results demonstrate that these proteins play critical roles in the self-renewal and differentiation of SSCs and can serve as markers to identify canine spermatogonia at specific stages of development.

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

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 o...

ResumoO Procyon cancrivorus é um carnívoro silvestre amplamente distribuído e relativamente comum, mas ainda é pouco estudado, existindo poucos trabalhos relatando sobre a biologia dessa espécie. Este trabalho tem como objetivo, caracterizar morfologicamente a glândula pineal de Procyon cancrivorus, através de estudos macro, microscópicos, radiográficos e compará-los com outros animais já estudados. Foram utilizados quatro animais adultos de ambos os sexos, provenientes do Criatório Científico CECRIMPAS, IBAMA (Processo nº 02027.003731/04-76). Macroscopicamente, a glândula pineal de Procyon cancrivorus foi localizada entre os lobos occipitais dos hemisférios cerebrais e cranialmente ao vermis cerebelar, posicionava-se rostralmente aos colículos rostrais e caudal à comissura das habênulas. Microscopicamente, a glândula é revestida externamente por uma cápsula derivado da meninge pia-máter. Foi notada a presença de três tipos de células no parênquima glandular: pinealócitos, células gliais e mastócitos. Não foram encontradas concreções calcáreas na glândula pineal nos estudos radiográficos e microscópicos. Unitermos: glândula pineal, morfologia, Procyon cancrivorus, radiografia AbstractMorphological study of the pineal gland of (crab eater raccoon) Procyon cancrivorus (Cuvier, 1798). The Procyon cancrivorus is a wild carnivore that is widely distributed and relatively common, but it remains little studied, and few works report on the biology of this species. The aim of this work was to characterize morphologically the pineal gland of Procyon cancrivorus through macro, microscopic and radiographic studies, and to compare them with those from other animals. In this work, four adult animals of both sexes were used, originating from the Scientific Herd of CECRIMPAS IBAMA (Process nº 02027.003731/04-76). Macroscopically, the pineal gland of Procyon cancrivorus was located between the occipital lobes of the cerebral hemispheres, cranially to the vermis cerebelar. It was positioned rostrally to the rostral colliculus and caudally to the habenular comissure. Microscopically, the gland was covered externally by a capsule deriving from the meningeal pia mater.