Chromatin remodelers HELLS, WDHD1 and BAZ1A are dynamically expressed during mouse spermatogenesis.

In brief: Proper regulation of heterochromatin is critical for spermatogenesis. This study reveals the dynamic localization patterns of distinct chromatin regulators during spermatogenesis and disrupted sex chromatin status in spermatocytes in the absence of DICER. Abstract: Heterochromatin is dynamically formed and organized in differentiating male germ cells, and its proper regulation is a prerequisite for normal spermatogenesis. While heterochromatin is generally transcriptionally silent, we have previously shown that major satellite repeat (MSR) DNA in the pericentric heterochromatin (PCH) is transcribed during spermatogenesis. We have also shown that DICER associates with PCH and is involved in the regulation of MSR-derived transcripts. To shed light on the heterochromatin regulation in the male germline, we studied the expression, localization and heterochromatin association of selected testis-enriched chromatin regulators in the mouse testis. Our results show that HELLS, WDHD1 and BAZ1A are dynamically expressed during spermatogenesis. They display limited overlap in expression, suggesting involvement in distinct heterochromatin-associated processes at different steps of differentiation. We also show that HELLS and BAZ1A interact with DICER and MSR chromatin. Interestingly, deletion of Dicer1 affects the sex chromosome heterochromatin status in late pachytene spermatocytes, as demonstrated by mislocalization of Polycomb protein family member SCML1 to the sex body. These data substantiate the importance of dynamic heterochromatin regulation during spermatogenesis and emphasize the key role of DICER in the maintenance of chromatin status in meiotic male germ cells.

STAGETOOL, a Novel Automated Approach for Mouse Testis Histological Analysis.

Spermatogenesis is a complex differentiation process that takes place in the seminiferous tubules. A specific organization of spermatogenic cells within the seminiferous epithelium enables a synchronous progress of germ cells at certain steps of differentiation on the spermatogenic pathway. This can be observed in testis cross-sections where seminiferous tubules can be classified into distinct stages of constant cellular composition (12 stages in the mouse). For a detailed analysis of spermatogenesis, these stages have to be individually observed from testis cross-sections. However, the recognition of stages requires special training and expertise. Furthermore, the manual scoring is laborious considering the high number of tubule cross-sections that have to be analyzed. To facilitate the analysis of spermatogenesis, we have developed a convolutional deep neural network-based approach named "STAGETOOL." STAGETOOL analyses histological images of 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI)-stained mouse testis cross-sections at ×400 magnification, and very accurately classifies tubule cross-sections into 5 stage classes and cells into 9 categories. STAGETOOL classification accuracy for stage classes of seminiferous tubules of a whole-testis cross-section is 99.1%. For cellular level analysis the F1 score for 9 seminiferous epithelial cell types ranges from 0.80 to 0.98. Furthermore, we show that STAGETOOL can be applied for the analysis of knockout mouse models with spermatogenic defects, as well as for automated profiling of protein expression patterns. STAGETOOL is the first fluorescent labeling-based automated method for mouse testis histological analysis that enables both stage and cell-type recognition. While STAGETOOL qualitatively parallels an experienced human histologist, it outperforms humans time-wise, therefore representing a major advancement in male reproductive biology research.

Novel expression of zona pellucida 3 protein in normal testis; potential functional implications.

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.