Germ cells have exceptionally diverse transcriptomes. Furthermore, the progress of spermatogenesis is accompanied by dramatic changes in gene expression patterns, the most drastic of them being near-to-complete transcriptional silencing during the final steps of differentiation. Therefore, accurate RNA regulatory mechanisms are critical for normal spermatogenesis. Cytoplasmic germ cell-specific ribonucleoprotein (RNP) granules, known as germ granules, participate in posttranscriptional regulation in developing male germ cells. Particularly, germ granules provide platforms for the PIWI-interacting RNA (piRNA) pathway and appear to be involved both in piRNA biogenesis and piRNA-targeted RNA degradation. Recently, other RNA regulatory mechanisms, such as the nonsense-mediated mRNA decay pathway have also been associated to germ granules providing new exciting insights into the function of germ granules. In this review article, we will summarize our current knowledge on the role of germ granules in the control of mammalian male germ cell's transcriptome and in the maintenance of fertility.Reproduction (2018) 155 R77-R91

Ribonucleoprotein (RNP) granules play a major role in compartmentalizing cytoplasmic RNA regulation. Haploid round spermatids that have exceptionally diverse transcriptomes are characterized by a unique germ cell-specific RNP granule, the chromatoid body (CB). The CB shares many characteristics with somatic RNP granules but also has germline-specific features. The CB appears to be a central structure in PIWI-interacting RNA (piRNA)-targeted RNA regulation. Here, we identified a novel CB component, FYCO1, which is involved in the intracellular transport of autophagic vesicles in somatic cells. We demonstrated that the CB is associated with autophagic activity. Induction of autophagy leads to the recruitment of lysosomal vesicles onto the CB in a FYCO1-dependent manner as demonstrated by the analysis of a germ cell-specific Fyco1 conditional knockout mouse model. Furthermore, in the absence of FYCO1, the integrity of the CB was affected and the CB was fragmented. Our results suggest that RNP granule homeostasis is regulated by FYCO1-mediated autophagy.

Androgen receptor (AR) is regulated by SUMOylation at its transactivation domain. In vitro, the SUMOylation is linked to transcriptional repression and/or target gene-selective regulation. Here, we generated a mouse model (ArKI) in which the conserved SUMO acceptor lysines of AR are permanently abolished (ArK381R, K500R). ArKI males develop normally, without apparent defects in their systemic androgen action in reproductive tissues. However, the ArKI males are infertile. Their spermatogenesis appears unaffected, but their epididymal sperm maturation is defective, shown by severely compromised motility and fertilization capacity of the sperm. Fittingly, their epididymal AR chromatin-binding and gene expression associated with sperm maturation and function are misregulated. AR is SUMOylated in the wild-type epididymis but not in the testis, which could explain the tissue-specific response to the lack of AR SUMOylation. Our studies thus indicate that epididymal AR SUMOylation is essential for the post-testicular sperm maturation and normal reproductive capability of male mice.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I-XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.