ABSTRACT
N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.
Subject(s)
Arginine , Argonaute Proteins , Pachytene Stage , RNA, Small Interfering , Spermatogenesis , Animals , Male , Mice , Arginine/metabolism , Arginine/genetics , Argonaute Proteins/metabolism , Argonaute Proteins/genetics , DNA Transposable Elements , Piwi-Interacting RNA , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins , Tudor DomainABSTRACT
BACKGROUND: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells. METHODS: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs. RESULTS: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts. CONCLUSIONS: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.
Subject(s)
Heat-Shock Response , RNA, Small Interfering , Spermatids , Spermatocytes , Testis , Animals , Male , Spermatids/metabolism , Spermatocytes/metabolism , RNA, Small Interfering/genetics , Rats , Heat-Shock Response/genetics , Heat-Shock Response/physiology , Testis/metabolism , Spermatogenesis/genetics , Spermatogenesis/physiology , Pachytene Stage/genetics , Rats, Sprague-Dawley , Piwi-Interacting RNAABSTRACT
Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation in vitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps-/- pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. MapsΔ2-9/Δ2-9 male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps-/- mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.
Subject(s)
Chromatin , Meiosis , Humans , Male , Mice , Animals , Chromatin/metabolism , Pachytene Stage , Phase Separation , Meiotic Prophase I , Spermatocytes/metabolism , Mammals/geneticsABSTRACT
BACKGROUND: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition. RESULTS: In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents. CONCLUSIONS: The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.
Subject(s)
Animals , Male , Mice , Spermatocytes/ultrastructure , Cell Nucleus/genetics , Chromosomes, Mammalian/ultrastructure , Meiotic Prophase I , Subcellular Fractions , Heterochromatin , Molecular Probes , Cell Nucleus , Ultrasonography , In Situ Hybridization, Fluorescence , Pachytene Stage , Heterozygote , HomozygoteABSTRACT
An analysis of the meiotic chromosomes of Cebus apella (Cebidae, Platyrrhini) has allowed for the visualization of constitutive heterochromatic regions throughout the meiotic cycle and has permitted the identification of four chromosomes in the first and the second divisions. In pachytene nuclei, and in pachytene spreads where synaptonemal complexes were visible, one heterochromatic region was frequently unpaired. No chiasmata were found in heterochromatic regions, and there was no evidence of chiasma terminalization. The role of heterochromatin in homologous pairing and its relationship to chiasma position and number in meiotic configurations were discussed. A comparison was made between bivalent 9 in C. apella and bivalent 21 in man, based on the interspecific homologies between these chromosomes. A similar comparison was made between the Algerian and Asian hedgehogs, where marked differences in the content of constitutive heterochromatin occur in two chromosome pairs.