Carolina Almeida

Caspase signalling pathways in human spermatogenesis

PurposeLittle is known about the apoptotic mechanisms involved in abnormal spermatogenesis. In order to describe the significance of apoptosis in azoospermia, testicular tissue from abnormal spermatogenesis was analysed.MethodsTesticular treatment biopsies were obtained from 27 men. Five presented oligozoospermia, 9 obstructive azoospermia (4 congenital bilateral absence of the vas deferens; 5 secondary azoospermia) and 13 non-obstructive azoospermia (5 hypospermatogenis; 3 maturation arrest; 5 Sertoli-cell-only syndrome). Immunohistochemical staining was performed for active caspases-3, −8 and −9. The presence of active caspases in Sertoli cells and germ cells was analyzed using stereological tools.ResultsIncreased active caspase-3 was found in Sertoli-cell-only syndrome. No significant differences were found in maturation arrest. In hypospermatogenesis, primary spermatocytes were the germ cells with higher active caspases. Oligozoospermia and secondary obstruction showed significant differences among germ cells for the presence of all active caspases. In oligozoospermia, spermatogonia presented significant increased active caspase-9 in relation to active caspase-8. In primary obstruction and hypospermatogenesis, germ cells presented significant increased active caspases-3 and −9.ConclusionsResults suggest that increased active caspase-3 might be involved in Sertoli-cell-only syndrome etiology. In cases of hypospermatogenesis, intrinsic lesions at the meiotic stage seem to be related to the pathology. In secondary obstruction apoptosis is suggested to be initiated due to extrinsic and intrinsic lesions, whereas in primary obstruction only the intrinsic apoptotic pathway seems to be present. Finally, in oligozoospermic patients spermatogonia death by mitochondrial damage additionally to meiosis malfunctioning, might be on the origin of the decreased sperm output.

Caspase-3 detection in human testicular spermatozoa from azoospermic and non-azoospermic patients

The apoptotic mechanisms underlying spermatogenesis in testis are poorly understood. In the present study, the rates of testicular spermatozoa with active caspase-3 were quantified in testicular samples with normal and impaired spermatogenesis. Testicular spermatozoa were collected from 18 men after testicular biopsy during assisted reproductive treatments: five presented oligozoospermia, four congenital bilateral absence of the vas deferens (CBAVD), five secondary obstructive azoospermia (sOAZ) and four hypospermatogenesis. Ejaculated samples were derived from six normozoospermic patients. Testicular spermatozoa were analysed using a fluorescence microscope and differences among groups were calculated using regression logistic models. Total rates of spermatozoa with active caspase-3 were significantly higher in sOAZ (78.6±13.9), followed by hypospermatogenesis (70.8±5.8), CBAVD (55.9±25.5), oligozoospermia (31.7±31.0) and normozoospermia (20.4±15.5). Distinct patterns of active caspase-3 were observed in testicular spermatozoa compartments: midpiece, equatorial region, acrosomal vesicle region, nucleus and cytoplasm. Hypospermatogenesis showed active caspase-3 mainly in the midpiece. In CBAVD, sOAZ and oligozoospermia, active caspase-3 was mainly in the nucleus, although no differences were found between oligozoospermia and hypospermatogenesis. In sOAZ, active caspase-3 in the spermatozoa nucleus was 1.89-fold higher than in CBAVD. Results suggest that tubular obstruction may induce nuclear lesions and that disrupted spermatozoa production observed in cases of hypospermatogenesis might be associated with mitochondrial lesions.

Comparative study of gene expression in patients with varicocele by microarray technology

The aim of the present study was to evaluate gene expression profile by microarray technology and validation by real‐time PCR in paired samples of testicular biopsies (pre‐surgery and post‐surgery) in two patients with varicocele. The microarray analysis showed increased expression levels after surgery in 215 and 52 genes in patient 1 and 2, respectively, as well as decreased expression levels in 65 genes in patient 1 and 358 genes in patient 2. Real‐time PCR confirmed the differential expression of the five selected genes: MT1M, PHLDA1 and INSL3 had decreased expression levels and CCIN and PRM2 increased expression levels compared with pre‐surgery biopsies. In conclusion, both techniques showed decreased expression levels of genes involved in stress situations associated with varicocele and increased expression levels of genes involved in normal function of spermatogenesis.

Ultrastructural and cytogenetic analyses of mature human oocyte dysmorphisms with respect to clinical outcomes.

PurposeThe study aimed to describe the ultrastructure of two human mature oocyte intracytoplasmic dysmorphisms, the bull-eye inclusion and the granular vacuole, with evaluation of clinical outcomes after intracytoplasmic sperm injection (ICSI) treatment.MethodsWe retrospectively evaluated 4099 consecutive ICSI cycles during the period 2003–2013. Three groups were compared: controls, those with a bulls-eye inclusion, and those with granular vacuoles. Oocyte dysmorphisms were evaluated by transmission electron microscopy and in situ fluorescence hybridization (FISH). Detailed data on demographic and stimulation characteristics, as well as on embryological, clinical, and newborn outcomes, are fully presented.ResultsThe bull-eye inclusion is a prominent smooth round structure containing trapped vesicles, being surrounded by lipid droplets. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when transferred embryos were exclusively derived from dysmorphism oocytes. The granular vacuole is delimited by a discontinuous double membrane and contains lipid droplets and vesicles. As FISH analysis revealed the presence of chromosomes, they probably represent pyknotic nuclei. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when at least one transferred embryo was derived from dimorphic oocytes.ConclusionsPoor clinical outcomes were observed with transfer of embryos derived from dysmorphism oocytes, although without causing gestation or newborn problems. The bull-eye inclusion and granular vacuoles may thus be new prognostic factors for clinical outcomes.

Integrated Analysis of Biological Samples by Imaging Flow Cytometry.

Instituto de Engenharia Biomédica (INEB). University of Porto, Porto, Portugal b.IMAGE – Bioimaging Center for Biomaterials and Regenerative Therapies, INEB, Porto, Portugal Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar (ICBAS). University of Porto. Porto, Portugal Faculdade de Engenharia da Universidade do Porto (FEUP), Porto, Portugal Faculdade de Medicina da Universidade do Porto (FMUP), Porto, Portugal Centre for Cell Biology and Department of Biology, University of Aveiro, Aveiro, Portugal College of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter, Devon, United Kindgom.

A system level boundary scan controller board for VME applications [to CERN experiments]

This work is the result of a collaboration between INESC and LIP in the CMS experiment being conducted at CERN. The collaboration addresses the application of boundary scan test at system level namely the development of a VME boundary scan controller (BSC) board prototype and the corresponding software. This prototype uses the MTM bus existing in the VME64/spl times/ backplane to apply the 1149.1 test vectors to a system composed of nineteen boards, called here units under test (UUTs). A top-down approach is used to describe our work. The paper begins with some insights about the experiment being conducted at CERN, proceed with system level considerations concerning our work and with some details about the BSC board. The results obtained so far and the proposed work is reviewed in the end of this contribution.

Ultrastructure and molecular cytogenetics of human metaphase II oocytes with granular vacuoles

*Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar of University of Porto, Lg Prof Abel Salazar 2, 4009-003 Porto, Portugal **Centre for Reproductive Genetics Alberto Barros, Av. do Bessa 591 1o Dto. Frente, 4100-009 Porto, Portugal ***Department of Genetics, Faculty of Medicine of University of Porto, Alameda Prof. Hêrnani Monteiro, 4200-319 Porto, Portugal celia.cmsoares@gmail.com