Vincenza Giuffrida | PubMed Health

PubMed2006

Gene expression in mouse spermatogenesis during ontogenesis.

In this study, we evaluated the expression of genes probably involved in spermatogenesis in the mouse. We examined cytosolic chaperonin theta subunit (CCTtheta), Ngg1 interacting factor 3 like 1 binding protein 1 (NIF3L1 BP1) and apolipoprotein H (ApoH) expression during mouse onto-geny using RT-PCR. Testicular tissue was obtained from mice 3, 6, 8, 10, 12, 14, 18, 20 and 40 (adult) days after birth. For each mouse, one testis was used for histological examination, whereas RNA was extracted from the controlateral testis for expression analysis. RT-PCR analysis showed that CCTtheta gene expression was low until day 10, but increased drastically afterwards. At this age, spermatocytes started to be present in the mouse testis. Therefore, CCT protein could be involved in chromatin packaging and remodeling during spermiogenesis, as also suggested by other studies. NIF3L1 BP1 expression increased steadily during ontogenesis reaching maximum levels in the adult mouse when all germ cell stages are present. This finding suggests that NIF3L1 BP1 is a gene not expressed by a specific germ cell type. ApoH expression was very low or absent during prepuberal stages, whereas it was detectable in the adult testis when spermatogenesis was completed. This suggests that ApoH may be involved in clearing apoptotic bodies during spermatogenesis since apoptotic events increase during spermatogenesis. This study contributes to understanding the role played by genes important for spermatogenesis.

Animals↗

PubMed2006

Quantitative evaluation of partial deletions of the DAZ gene cluster.

Partial deletions of the DAZ gene cluster are thought to cause spermatogenesis impairment. The presence of homologous copies of this gene in the Y chromosome does not allow PCR to be used for the identification of this abnormality. Hence, sequence family variants (SFV), following amplification of sY581, sY587 and sY586 and subsequent enzymatic digestion with Sau3A, DraI and TaqI, respectively, and the dual fiber fluorescence in situ hybridization (FISH) have been used to this aim. However, SFV is not always able to identify single DAZ gene copy deletions. We report a quantitative real-time PCR application to evaluate partial deletions of the DAZ gene cluster. To accomplish this, we designed a probe on exon 6 of the DAZ gene which is repeated 3 times in DAZ1, once in DAZ2 and DAZ3 and twice in DAZ4. Five normozoospermic healthy men (C1-C5) having 4 DAZ gene copies by SFV were selected. Fiber-FISH confirmed this outcome in C1-C4, but not in C5 who had an incomplete DAZ gene cluster. The men underwent then quantitative real-time PCR and C1 was arbitrarily selected as calibrator for the calculation of the DAZ gene signals because of the lowest variation in the threshold cycles. Real-time PCR identified 7.2+/-0.05 signals in C2-C4 and 5.4+/-0.05 signals in C5. The overall coefficient of variation was 1.4+/-0.2%. The loss of two signals in this subject may relate to a deletion of both DAZ2 and DAZ3 or of DAZ4 gene. Since SFV showed clearly the presence of DAZ2, it may be hypothesized that C5 lacks DAZ4. In conclusion, these data suggested that quantitative real-time PCR seems to be an effective and reproducible technique that can be used to study the DAZ gene cluster. In addition, the probe chosen for this approach may give indication on the DAZ gene copy deleted.

Adult↗