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ABSTRACT: Cystic fibrosis is the most frequent autosomal recessive disease in the Caucasian population, with an incidence of 1:2500 newborn and a frequency of 1:25. The associated gene is Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and it encodes the CFTR protein that functions as a chloride (Cl(-)) channel. It is found in the apical membrane of exocrine epithelial cells, responsible for the regulation of the movement of water and solutes through biological membranes. To our knowledge, there are no studies on protein localization in the different cell types of the seminiferous epithelium with different pathologies. The aim of the present study was to analyze the expression of the CFTR protein in the human seminiferous epithelium of infertile males with different pathologies. CFTR protein expression was studied by immunohistochemistry in paraffin sections of testicular biopsies of six infertile men: Sertoli cell only syndrome, maturation arrest, secondary obstructive azoospermia, primary obstructive azoospermia due to congenital bilateral absence of the vas deferens (CBAVD), severe oligozoospermia, and retrograde ejaculation. All cell types of the seminiferous epithelium were studied: Sertoli cells, spermatogonia, primary spermatocytes at the leptotene/zygotene and at the pachytene stages, secondary spermatocytes, round, elongating and elongated spermatids, and spermatozoa. With the exception of sperm, all cells were labeled in the cytoplasm and in the cytoplasmic membrane. In the patient with CBAVD labeling was light at the cell membrane and absent in the cytoplasm of Sertoli cells and diploid germ cells. Generally, labeling was stronger after the diploid stage, which is probably related to cell volume reduction during spermiogenesis. The results obtained also suggest that the CFTR protein may impact CBAVD spermatogenesis and other pathologies.
Systems biology in reproductive medicine 09/2012; · 0.80 Impact Factor
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ABSTRACT: During spermatogenesis, germ cells undergo a complex process of cell differentiation and morphological restructuring, which depends on the coordinated expression of different genes. Some vital examples are those involved in cell energy metabolism, namely the genes encoding the E1α subunit of pyruvate dehydrogenase complex: the somatic PDHA1 (X-linked) and the testis-specific PDHA2 (autosomal). There are no data related to the study at the RNA and protein levels of PDHA genes during human spermatogenesis. The present study aimed to describe the mRNA and protein expression patterns of the human PDHA genes during spermatogenesis. Expression profiles of the PDHA1 and PDHA2 genes were characterized using different human tissues and cells. Diploid and haploid germ cells fractions were obtained from testis tissues. The mRNA profiles were analyzed by quantitative RT-PCR, whereas the protein profiles were evaluated by immunohistochemistry, western blotting and two-dimensional electrophoresis. Expression of the PDHA1 gene was found in all somatic cells, whereas expression of PDHA2 gene was restricted to germ cells. The switch from X-linked to autosomic gene expression occurred in spermatocytes. Data suggest the activation of PDHA2 gene expression is most probably a mechanism to ensure the continued expression of the protein, thus allowing germ cell viability and functionality.
Gene 06/2012; 506(1):173-8. · 2.34 Impact Factor
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ABSTRACT: The aim of the present work was to quantitate the temporal and stage-specific effects of follicle-stimulating hormone (FSH) and testosterone on the proliferation and differentiation capacities of the human seminiferous epithelium. Seminiferous tubule fragments were kept in culture for 28 days and 5-bromo-2'-deoxyuridine incorporation was used to determine cell proliferation. Data demonstrated a gradual loss of germ cells during the culture period, no decrease in Sertoli cell numbers, and maintenance of the general architecture of the seminiferous tubules. Both FSH and testosterone increased germ cell survival, spermatogonia proliferation, and germ cell differentiation, especially during the first week of culture. At the end of the first week, differentiation of spermatocytes was observed, especially when 50 IU/L FSH and 1 µmol/L testosterone were used. In conclusion, using this methodology, it was possible to quantify germ cell proliferation and differentiation, in a reproducible way, with results compatible with the timing of human spermatogenesis in vivo.
Reproductive sciences (Thousand Oaks, Calif.) 04/2012; 19(10):1063-74. · 2.31 Impact Factor
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ABSTRACT: To compare demographic, embryologic, pregnancy, and newborn outcomes after intracytoplasmic sperm injection (ICSI) cycles with or without mature oocytes (metaphase II [MII]) showing visible aggregates of tubular smooth endoplasmic reticulum (aSERT) and to describe the ultrastructure of this dysmorphism.
Retrospective study.
Private fertility center and university cell biology and genetics departments.
There were 721 ICSI cycles, 520 carrying morphologically normal MII (control group) and 60 containing aSERT-MII (study group).
None.
Embryologic and clinical and live birth outcomes, including malformations and ultrastructural characterization of aSERT-MII.
Compared with the control group there was a significant decrease in the fertilization, embryo cleavage, and blastocyst rates in the study group. The only child born after transfer of embryos derived from aSERT-MII presented a major cardiovascular malformation. Ultrastructurally, large aSERT were surrounded by abnormal-shaped mitochondria and clusters of small dense bodies formed by very small vesicles, and they had curvilinear dense tubules in the interior. The same pathology was observed in small peripheral aSERT.
The presence of large aSERT, showing attainment of the periphery, demonstrated that the cytoplasm is pathologic. The compromised embryo development and implantation was associated with decreased clinical outcomes and newborn malformations. Therefore, oocytes with large aSERT should not be used for embryo transfer.
Fertility and sterility 05/2011; 96(1):143-149.e7. · 3.97 Impact Factor
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ABSTRACT: DNA methylation is an important epigenetic modification that has profound roles in gene expression and, in particular, is thought to be crucial for regulation of tissue-specific genes in animal cells. The pivotal E(1)alpha subunit of human pyruvate dehydrogenase complex, an essential and rate-limiting enzyme system in energy metabolism, is encoded by two distinct genes: PDHA1 gene, located on chromosome X is expressed in somatic tissues, whereas PDHA2 gene, located on chromosome 4, is exclusively expressed in spermatogenic cells. The objective of this study is to elucidate the role of DNA methylation as an epigenetic mechanism controlling the regulation of PDHA2 gene expression in human tissues, namely its repression in somatic tissues and its activation in testicular germ cells. Genomic DNA was isolated from human somatic tissues (circulating lymphocytes and gastric cells) and from testis, including isolated fractions of haploid and diploid germ cells. After primer design with appropriate software, it was performed the sodium bisulfite PCR sequencing of the PDHA2 promoter and coding regions. Total RNA of the same tissues was isolated, reverse transcribed and PDHA1and PDHA2 transcripts were amplified with specific primers and analysed by agarose gel electrophoresis. The analysis of the genomic sequence of the PDHA2 gene revealed the presence of 61 CpG sites whose distribution matches the criteria for the presence of two CpG islands. Sequence analysis of both CpG islands upon bisulfite treatment displayed several differences, either between islands or among tissues. In particular, the methylation pattern of one of the CpG islands revealed a perfect correlation with transcriptional activity of the PDHA2 gene either in testis or in somatic tissues. Surprisingly, it is the full demethylation of the CpG island located in the coding region that seems to play a crucial role upon PDHA2 gene transcription in testis.
Molecular Genetics and Metabolism 11/2009; 99(4):425-30. · 3.19 Impact Factor
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ABSTRACT: In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.
Biology of Reproduction 08/2008; 79(5):962-75. · 4.01 Impact Factor
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ABSTRACT: Approximately 20% of patients with congenital absence of the vas deferens remain without two mutations identified. We applied a strategy of serial screening steps to 45 patients with congenital absence of the vas deferens and characterized cystic fibrosis transmembrane conductance regulator gene mutations in all cases.
DNA samples of 45 patients with congenital absence of the vas deferens were screened by successive different molecular genetics approaches.
Initial screening for the 31 most frequent cystic fibrosis mutations, IVS8 poly(TG)m, poly(T)n, and M470V polymorphisms, identified 8 different mutations in 40 patients (88.9%). Extensive cystic fibrosis transmembrane conductance regulator gene analysis by denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, and DNA sequencing detected 17 further mutations, of which three were novel. Cystic fibrosis transmembrane conductance regulator gene rearrangements were searched by semiquantitative fluorescent multiplex polymerase chain reaction, which detected a CFTRdele2,3 (21 kb) large deletion and confirmed two homozygous mutations. Overall, 42 patients (93.3%) had two mutations and 3 patients (6.7%) had one mutation detected.
The present screening strategy allowed a higher mutation detection rate than previous studies, with at least one cystic fibrosis transmembrane conductance regulator gene mutation found in all patients with congenital absence of the vas deferens.
Genetics in Medicine 04/2007; 9(3):163-72. · 4.76 Impact Factor
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ABSTRACT: The DAZ gene, a contributing factor in infertility, lies on the human Y chromosome's AZFc region, whose deletion is a common cause of spermatogenic failure. Y chromosome binary polymorphisms on the non-recombining Y (NRY) region, believed to be a single occurrence on an evolutionary scale, were typed in a sample of fertile and infertile men with known DAZ backgrounds. The Y single-nucleotide polymorphisms (Y-SNPs) with low mutation rates are currently well characterized and permit the construction of a unique phylogeny of haplogroups. DAZ haplotypes were defined using single-nucleotide variant (SNV)/sequence tagged-site (STS) markers to distinguish between the four copies of the gene. The variation of 10 Y chromosome short tandem repeat (STRs) was used to determine the coalescence age of DAZ haplotypes in a comparable time frame similar to that of SNP haplogroups. An association between DAZ haplotypes and Y chromosome haplogroups was found, and our data show that the DAZ gene is not under selective constraints and its evolution depends only on the mutation rate. The same variants were common to fertile and infertile men, although partial DAZ deletions occurred only in infertile men, suggesting that those should only be used as a tool for infertility diagnosis when analysed in combination with haplogroup determinations.
Molecular Human Reproduction 09/2006; 12(8):519-23. · 3.85 Impact Factor
