Abstract
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.