Expression of Cnnm1 and Its Association with Stemness, Cell Cycle, and Differentiation in Spermatogenic Cells in Mouse Testis.

Cyclin M1 (CNNM1) functions as a copper storage protein in neuronal cells. We report that Cnnm1 is expressed in mouse testis and brain and has a coding sequence of 1761 bp that encodes a 586 amino acid protein with a molecular weight of 66 kDa. Cnnm1 is expressed in the testes of mice from neonatal to adult stages with relatively higher levels in neonates. CNNM1 expression appeared to be restricted to c-KIT- and OCT3/4-positive cells in the testis, indicating that they are early spermatogonial cells. Spermatogonial stem cells in primary culture expressed Cnnm1, and their differentiation into embryoid body-like clusters in vitro resulted in the loss of Cnnm1 expression. Silencing of Cnnm1 in GC1-spg cells resulted in a significant reduction in the number of cells in G1 phase with concomitant increase in the numbers of cells in both S and G2/M phases. Further, retinoic acid downregulated the expression of Cnnm1 in GC1-spg cells. We conclude that CNNM1 is associated with stemness and self-renewal, and its downregulation triggers differentiation in spermatogonial cells in mouse.

Expression profiles of NPHP1 in the germ cells in the semen of men with male factor infertility.

NPHP1, the gene that encodes the protein nephrocystin-1 has been identified to be mutated in Juvenile Nephronophthisis, an autosomal recessive cystic kidney disorder which is the most frequent genetic cause of end-stage renal disease (ESRD) in children and young adults. Nphp1-targeted mutant mice studies have shown that it did not express renal manifestations of nephronophthisis; instead male mice were infertile with oligoteratozoospermia signifying the crucial role of Nphp1 in relation to murine spermatogenesis. Whether an aberrant expression of NPHP1 in testis might lead to spermatogenic defects in human and ultimately male factor infertility is a possibility that have not been investigated so far. In this regard, characterization of NPHP1 in spermatozoa from fertile and infertile males was carried out by employing RT-PCR, western blotting, and immunofluorescence analysis. In all the 23 normozoospermic samples we screened, NPHP1 was significantly expressed at the target message and protein level and also prominent localization pattern of NPHP1 was observed at the head, midpiece, and tail segments of spermatozoa. Conversely, in majority of the 103 infertile samples we screened, aberrant pattern of NPHP1 expression was detected at the transcript and protein level and abnormal localization pattern of expression was observed in spermatozoa. Anomalies detected in infertile cases when compared with the normozoospermic controls points to the indispensable role of NPHP1 in relation to spermatogenesis. Thus, besides the decisive association with juvenile nephronophthisis, our study provides the first direct evidence that NPHP1 is associated with male factor infertility and also could be a possible biomarker for the assessment of male fertility status. GENBANK NM_000272.3

AIRE1A might be involved in cyclin B2 degradation in testicular lysates.

The autoimmune regulator gene Aire shows predominant expression in thymus and other immunologically relevant tissues, and is assigned the major function of programming autoreactive T-cell deletion. However, the expression of this gene in tissues outside the immune system raises a question about its possible function beyond the T-cell deletion dogma. We detected Aire in mouse testis, and the expression of AIRE protein was remarkably high in postmeiotic germ cells. Sequencing results indicate that testis expressed Aire variant 1a. AIRE could be detected in spermatozoa, with heavy localization on the principal acrosomal domains. Mouse oocytes stained negatively for AIRE before fertilization, but stained positively for AIRE 30 min after fertilization. In the zygote, the levels of AIRE correlated negatively with cyclin B2 levels. Goat testicular lysates spiked with recombinant human AIRE exhibited augmented cyclin B2 degradation in the presence of protease inhibitors, which was inhibited by MG-132, indicating the operation of proteasomal pathways. Thus, this study identifies a correlation between the presence of AIRE and proteasomal breakdown of cyclin B2, which leads us to speculate that cyclin B2 could be a target of AIRE's E3-ubiquitin ligase activity.