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Sa R.,University of Porto | Cremades N.,University of Alicante | Malheiro I.,Laboratory of Cytogenetics | Sousa M.,University of Porto | Sousa M.,Center for Reproductive Genetics Alberto Barros
Andrologia | Year: 2012

For patients with threatened fertility, preservation of it is a major concern. Although promising results have been obtained in animal models using testicular germ cell suspensions, in humans, it is crucial to first develop an efficient method of cryopreservation to be able to apply to transplantation. Thus, four reliable and available cryopreservation techniques in any fertility centre were tested to cryopreserve an enriched fraction of diploid germ cells isolated from human testicular biopsies. The protocols were evaluated based on cell viability, and the results showed significant differences between the four methods. The semen and tissue cryopreservation methods appeared to be inadequate for diploid germ cell suspensions, and programmed slow freezing gave significantly lower results than open pulled straw vitrification; the latter was found to be the protocol that best preserved cell viability. The vitrification of isolated human diploid germ cells is innovative and constitutes valuable information for cryopreservation in cases of transplants or in vitro maturation. © 2012 Blackwell Verlag GmbH.


Jesus T.T.,University of Beira Interior | Bernardino R.L.,University of Beira Interior | Martins A.D.,University of Beira Interior | Sa R.,University of Porto | And 4 more authors.
Biochemical and Biophysical Research Communications | Year: 2014

Sertoli cells (SCs) form the blood-testis barrier (BTB) that controls the microenvironment where the germ cells develop the cystic fibrosis transmembrane conductance regulator (CFTR) plays an essential role to male fertility and it was recently suggested that it may promote water transport. Interestingly, Aquaporin-4 (AQP4) is widely expressed in blood barriers, but was never identified in SCs. Herein we hypothesized that SCs express CFTR and AQP4 and that they can physically interact. Primary SCs cultures from 20-day-old rats were maintained and CFTR and AQP4 mRNA and protein expression was assessed by RT-PCR and Western blot, respectively the possible physical interaction between CFTR and AQP4 was studied by co-immunoprecipitation. We were able to confirm the presence of CFTR at mRNA and protein level in cultured rat SCs. AQP4 mRNA analysis showed that cultured rat SCs express the transcript variant c of AQP4, which was followed by immunodetection of the correspondent protein the co-immunoprecipitation experiments showed a direct interaction between AQP4 and CFTR in cultured rat SCs. Our results suggest that CFTR physically interacts with AQP4 in rat SCs evidencing a possible mechanism by which CFTR can control water transport through BTB the full enlightenment of this particular relation between CFTR and AQP4 may point towards possible therapeutic targets to counteract male subfertility/infertility in men with Cystic Fibrosis and mutations in CFTR gene, which are known to impair spermatogenesis due to defective water transport. © 2014 Elsevier Inc. All rights reserved.


Monteiro S.M.,Royal University | Fontainhas-Fernandes A.,Royal University | Sousa M.,University of Porto | Sousa M.,Center for Reproductive Genetics Alberto Barros
Folia Histochemica et Cytobiologica | Year: 2010

This study reports the first complete mapping of the gill epithelium in a tilapia species. Different gill epithelial cell types of the Nile tilapia, Oreochromis niloticus L. have been identified and located using different antisera against mammalian proteins and various histochemical techniques: Periodic Acid Schiff (PAS), Alcian Blue pH 1.0, 2.5, 3.5, Giemsa and Grimelius. The results show that the stratified filament epithelium of O. niloticus gill can be divided into two distinct regions, a superficial layer, where pavement, mucous and mitochondria rich cells can be found, and a deep layer, constituted by undifferentiated, myoepithelial-like, granular and neuroendocrine cells. V-ATPase and Na+/K+-ATPase presence allowed the identification of pavement and mitochondria-rich cells, respectively, suggesting that, in O. niloticus, pavement cells are implicated in Na+ uptake, whereas mitochondria-rich cells have a role in Cl- uptake. The use of PAS and Alcian Blue allowed the recognition of different sub-populations of mucous cells that differentiate from a common deeper precursor. Neuroendocrine markers were detected in different cell populations, stating evidence for a neuroendocrine role of mitochondria-rich cells, and suggesting the existence of distinct neural pathways, a putative O2-chemosensory and an ion regulatory pathway. A defence role was attributed to the deep filament epithelium, suggested by the presence of resident giemsa positive- eosinophil granular cells. The antibody raised against proliferating cell nuclear antigen identified two different cell types, the undifferentiated cells and myoepithelial-like cells. In the superficial layer, it is here stated for the first time the existence of vimentin positive support cells. © Polish Histochemical et Cytochemical Society.


Laurentino S.S.,University of Beira Interior | Correia S.,University of Beira Interior | Cavaco J.E.,University of Beira Interior | Oliveira P.F.,University of Beira Interior | And 6 more authors.
Reproduction | Year: 2011

Regucalcin (RGN) is a calcium (Ca2+)-binding protein which regulates intracellular Ca2+ homeostasis by modulating the activity of enzymes regulating Ca2+ concentration and enhancing Ca2+-pumping activity. Several studies have described the pivotal role of proper Ca2+ homeostasis regulation to spermatogenesis and male fertility. Recently, RGN was identified as a sex steroid-regulated gene in prostate and breast; however, a possible role of RGN in spermatogenesis has not been examined. In this study, the expression and localization of RGN in rat and human testis, and other rat reproductive tissues was analyzed. Moreover, we studied whether RGN protein was present in seminiferous tubule fluid (STF). Finally, we examined the effect of 5α-dihydrotestosterone (DHT) on the expression of Rgn mRNA in rat seminiferous tubules (SeT) cultured ex vivo. The results presented in this study show that RGN is expressed in Leydig and Sertoli cells, as well as in all types of germ cells of both rat and human testis. RGN is also expressed in rat prostate, epididymis, and seminal vesicles. Moreover, RGN protein is present in rat STF. The results also demonstrate that Rgn expression is age dependent in rat testis, and is upregulated by the non-aromatizable androgen DHT in rat SeT cultured ex vivo. Taken together, these findings indicate that Rgn is a novel androgen-target gene in rat testis and that it may have a role in male reproductive function, particularly in the control of spermatogenesis. © 2011 Society for Reproduction and Fertility.


Alves M.G.,University of Beira Interior | Socorro S.,University of Beira Interior | Silva J.,Center for Reproductive Genetics Alberto Barros | Barros A.,Center for Reproductive Genetics Alberto Barros | And 5 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2012

Background: Several important functions for a successful spermatogenesis are dependent on Sertoli cells (SCs). Besides their unique characteristics as support cells, they produce essential cofactors and metabolites, and are responsible for nurturing the developing germ cells. The continuous production of lipids, phospholipids and proteins by germ cells must require high amounts of metabolic precursors. Thus, we hypothesized that hSCs could produce acetate in a hormonally-regulated manner. Methods: hSC-enriched primary cultures were maintained in the absence of insulin or in the presence of 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT). Acetate production was determined by 1H-NMR. mRNA gene expression levels of Acetyl CoA hydrolase (ACoA Hyd) and Acetyl CoA synthase (ACoA Synt) were determined by RT-PCR. Results: hSCs produced high amounts of acetate suggesting that this metabolite should play a key role on the progression of spermatogenesis, namely as a metabolic precursor for the synthesis of cellular constituents. In addition, acetate metabolism proved to be under strict hormonal regulation. In the presence of E2 or DHT, hSCs produced different amounts of acetate. While E2 treatment increased acetate production, increasing ACoA Hyd gene transcript levels, DHT-treated cells showed decreased acetate production, differently modulating the ratio ACoA Hyd/ACoA Synt. Surprisingly, insulin-deprivation completely suppressed acetate production/export and significantly decreased the ACoA Hyd gene transcript levels. General significance: Taken together, these results suggest that, although hSCs are primarily described as lactate producers, the elevated production of acetate deserves special attention, in order to clarify the mechanisms behind its hormonal regulation and its role on a successful spermatogenesis. © 2012 Elsevier B.V.

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