Roles of MAPS and SPDYA in XY chromosomal dynamics during male pachynema progression

Abstract

During mammalian meiotic prophase I, chromosome remodeling and nuclear compartmentalization process vigorously, particularly at pachynema, where events concentrate on the sex chromosomal regions. During this period, active DNA damage response (DDR) in these regions initiates meiotic sex chromosome inactivation (MSCI). Epigenetic modifications of histones and variant depositions, along with the relocation of sex chromosomes, contribute to distinct compartments. These culminate in a membrane-less organelle, the XY body, which sustains MSCI. Additionally, dynamic synapsis and remodeling of DNA loops in the pseudoautosomal regions (PARs) of sex chromosomes lead to recombination within PARs. This process ensures the end-to-end connection of X and Y chromosomes through the recombination nodule. Here, we report that the male pachynema-specific protein (MAPS) regulates these processes to drive pachynema progression through liquid-liquid phase separation (LLPS). MAPS was detected during the pachytene stage, initially dispersing throughout the nucleus, and subsequently accumulating in sex chromosome regions by mid-pachytene. Structural analysis revealed that MAPS comprises a simple three-dimensional structure with several α-helices and two intrinsically disordered regions (IDR1: residues 1–153 and IDR2: residues 208–252). Recombinant MAPS formed condensates independently or with nucleosome arrays in vitro. Expression of MAPS-GFP in cells led to the formation of GFP condensates that coalesced into larger, brighter spherical aggregates. Mouse spermatocytes lacking MAPS exhibited multiple defects, including failed XY body formation, disrupted MSCI, altered histone composition and variant deposition, impaired chromatin accessibility, and abnormal XY bivalent morphology. Ectopic reintroduction of MAPS expression completely rescued these defects. Furthermore, a frameshift mutation in C3orf62, the human homolog of Maps, was associated with nonobstructive azoospermia due to pachynema arrest. C3ORF62 also possesses a simple three-dimensional structure with a lengthy IDR spanning residues 37 to 267. This frameshift mutation might truncate C3ORF62, disrupting the IDR and impairing condensate formation in cellulo. Therefore, the LLPS of MAPS is fundamental for pachynema progression in both mice and humans. To study the abnormal XY chromosomal morphology in pachytene spermatocytes, we employed RNA sequencing and mass spectrometry, revealing reduced expression of telomere-associated proteins in Maps–/– pachytene cells, including SPDYA, CDK2, and SUN1. Given the localization of SPDYA and CDK2 on the telomeres and unsynapsed XY chromosomal axes during pachynema, our initial focus was on these two genes. Employing the Ddx4-CreERT2/Loxp system, we created conditional knockout mice through a tamoxifen-induced strategy to bypass the defects caused by Spdya and Cdk2 global knockout, achieving targeted gene deletion in pachytene spermatocytes. Pachytene spermatocytes lacking SPDYA exhibited a high ratio of Y-X full alignment configuration. Mechanistically, the absence of SPDYA disrupted the connection between centromere-proximal telomeres of XY chromosomes and the LINC complex, impairing cytoskeletal forces perception, which led to Y-X full alignment. The deletion of Cdk2 during pachynema did not rapidly remove SPDYA from telomeres, thereby preserving typical pachytene XY chromosomal morphology. However, CDK2 was shown to play a synergistic role with SPDYA in regulating XY chromosomal morphology. Therefore, we propose that SPDYA primarily governs the dynamic association of XY chromosomes at pachynema, and maintaining telomeric-nuclear envelope attachment is crucial for pachynema progression.