Mitochondria Associated Germinal Structures in Spermatogenesis: piRNA Pathway Regulation and Beyond

Multiple specific granular structures are present in the cytoplasm of germ cells, termed nuage, which are electron-dense, non-membranous, close to mitochondria and/or nuclei, variant size yielding to different compartments harboring different components, including intermitochondrial cement (IMC), piP-body, and chromatoid body (CB). Since mitochondria exhibit different morphology and topographical arrangements to accommodate specific needs during spermatogenesis, the distribution of mitochondria-associated nuage is also dynamic. The most relevant nuage structure with mitochondria is IMC, also called pi-body, present in prospermatogonia, spermatogonia, and spermatocytes. IMC is primarily enriched with various Piwi-interacting RNA (piRNA) proteins and mainly functions as piRNA biogenesis, transposon silencing, mRNA translation, and mitochondria fusion. Importantly, our previous work reported that mitochondria-associated ER membranes (MAMs) are abundant in spermatogenic cells and contain many crucial proteins associated with the piRNA pathway. Provocatively, IMC functionally communicates with other nuage structures, such as piP-body, to perform its complex functions in spermatogenesis. Although little is known about the formation of both IMC and MAMs, its distinctive characters have attracted considerable attention. Here, we review the insights gained from studying the structural components of mitochondria-associated germinal structures, including IMC, CB, and MAMs, which are pivotal structures to ensure genome integrity and male fertility. We discuss the roles of the structural components in spermatogenesis and piRNA biogenesis, which provide new insights into mitochondria-associated germinal structures in germ cell development and male reproduction.

Aging and chromatoid body assembly: Are these two physiological events linked?

The chromatoid body is a cytoplasmic male germ cell structure that plays a role in the regulation of mRNA transcription during spermatogenesis. A proteomic analysis of this structure has identified the presence of its classic molecular markers (MVH and MIWI), as well as a significant number of transient proteins. Circadian locomotor output cycles protein kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), which are molecular components of the circadian clock, are likely located in the chromatoid body in a transient fashion. This study sought to determine whether aging produces morphological changes in the chromatoid bodies of round spermatids similar to those previously observed in BMAL1 knockout mice. A sample of 30 male mice was divided into three groups: juvenile mice (45 days old), adult mice (120 days old), and old mice (+180 days old). Aging was confirmed by viability and sperm count analyses and testosterone dosage. Squash slides prepared with fragments of seminiferous tubules were immunostained for MVH, MIWI, BMAL1, and CLOCK detection. In juvenile and adult specimens, single round chromatoid bodies were observed using MVH/BMAL1 and MIWI/CLOCK immunostaining. In old specimens, many chromatoid bodies displayed changes in number and morphology, as well as an increase in the interactions between MVH and BMAL1; MIWI and CLOCK. Changes in chromatoid body morphology increased interactions between the proteins analyzed herein, and decreased amounts of these proteins in seminiferous tubules of older mice may indicate that aging influences the assembly and physiology of chromatoid bodies, which may, in turn, affect fertility. Impact statement The results discussed in this paper indicate that aging compromises the structure and physiology of chromatoid bodies (CBs) in post-meiotic male cells. Since CB is a fundamental structure for the differentiation of the mature male germ cell it is possible that this imbalance in CB physiology may play a role in the reduction of fertility in older men. It is important to note that not only the classic CB markers (such as the MIWI and MVH proteins) were used to showcase the structural changes in the CBs but also the main components of circadian cycle control (the CLOCK and BMAL1 proteins), indicating that the reduction of circadian control in aged males may contribute to these changes in CBs as well. Therefore, it is intriguing to evaluate the hypothesis that controlling these physiological/structural changes in CBs may be a way of delaying the effects of aging in males.

KH-type splicing regulatory protein is a new component of chromatoid body

The chromatoid body (CB) is a specific cloud-like structure in the cytoplasm of haploid spermatids. Recent findings indicate that CB is identified as a male germ cell-specific RNA storage and processing center, but its function has remained elusive for decades. In somatic cells, KH-type splicing regulatory protein (KSRP) is involved in regulating gene expression and maturation of select microRNAs (miRNAs). However, the function of KSRP in spermatogenesis remains unclear. In this study, we showed that KSRP partly localizes in CB, as a component of CB. KSRP interacts with proteins (mouse VASA homolog (MVH), polyadenylate-binding protein 1 (PABP1) and polyadenylate-binding protein 2 (PABP2)), mRNAs (Tnp2 and Odf1) and microRNAs (microRNA-182) in mouse CB. Moreover, KSRP may regulate the integrity of CB via DDX5-miRNA-182 pathway. In addition, we found abnormal expressions of CB component in testes of Ksrp-knockout mice and of patients with hypospermatogenesis. Thus, our results provide mechanistic insight into the role of KSRP in spermatogenesis.

KIF1-binding protein interacts with KIF3A in haploid male germ cells

Male fertility relies on the production of functional spermatozoa. Spermatogenesis is a complex differentiation process that is characterized by meiosis and dramatic morphogenesis of haploid cells. Spermatogenesis involves active changes in the microtubular network to support meiotic divisions, cell polarization, the reshaping of the nucleus, and the formation of a flagellum. Previously, we have demonstrated that a microtubule-based anterograde transport motor protein KIF3A is required for the sperm tail formation and nuclear shaping during spermatogenesis. In this study, we show that KIF3A interacts with a KIF1-binding protein (KBP) in the mouse testis. We have characterized the expression and localization pattern of KBP during spermatogenesis and localized both KIF3A and KBP in the cytoplasm of round spermatids and manchette of elongating spermatids. Interestingly, KBP localized also in the late chromatoid body (CB) of elongating spermatids, whose function involves intracellular movement and association with the microtubular network. Altogether our results suggest a role for KBP in spermatid elongation and in the function of the late CB.