The male pachynema-specific protein MAPS drives phase separation in vitro and regulates sex body formation and chromatin behaviors in vivo.

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.

A pathogenic DMC1 frameshift mutation causes nonobstructive azoospermia but not primary ovarian insufficiency in humans.

Nonobstructive azoospermia (NOA) and diminished ovarian reserve (DOR) are two disorders that can lead to infertility in males and females. Genetic factors have been identified to contribute to NOA and DOR. However, the same genetic factor that can cause both NOA and DOR remains largely unknown. To explore the candidate pathogenic gene that causes both NOA and DOR, we conducted whole-exome sequencing (WES) in a non-consanguineous family with two daughters with DOR and a son with NOA. We detected one pathogenic frameshift variant (NM_007068:c.28delG, p. Glu10Asnfs*31) following a recessive inheritance mode in a meiosis gene DMC1 (DNA meiotic recombinase 1). Clinical analysis showed reduced antral follicle number in both daughters with DOR, but metaphase II oocytes could be retrieved from one of them. For the son with NOA, no spermatozoa were found after microsurgical testicular sperm extraction. A further homozygous Dmc1 knockout mice study demonstrated total failure of follicle development and spermatogenesis. These results revealed a discrepancy of DMC1 action between mice and humans. In humans, DMC1 is required for spermatogenesis but is dispensable for oogenesis, although the loss of function of this gene may lead to DOR. To our knowledge, this is the first report on the homozygous frameshift mutation as causative for both NOA and DOR and demonstrating that DMC1 is dispensable in human oogenesis.

The male germline-specific protein MAPS is indispensable for pachynema progression and fertility.

Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a number of mouse genes that were dramatically elevated in spermatocytes, relative to their very low expression in spermatogonia and somatic organs. Here, we investigated in detail 1700102P08Rik, one of these genes, and independently conclude that it encodes a male germline-specific protein, in agreement with a recent report. We demonstrated that it is essential for pachynema progression in spermatocytes and named it male pachynema-specific (MAPS) protein. Mice lacking Maps (Maps-/- ) suffered from pachytene arrest and spermatocyte death, leading to male infertility, whereas female fertility was not affected. Interestingly, pubertal Maps-/- spermatocytes were arrested at early pachytene stage, accompanied by defects in DNA double-strand break (DSB) repair, crossover formation, and XY body formation. In contrast, adult Maps-/- spermatocytes only exhibited partially defective crossover but nonetheless were delayed or failed in progression from early to mid- and late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult Maps-/- pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in Maps-/- pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general.

The novel male meiosis recombination regulator coordinates the progression of meiosis prophase I.

Meiosis is a specialized cell division for producing haploid gametes in sexually reproducing organisms. In this study, we have independently identified a novel meiosis protein male meiosis recombination regulator (MAMERR)/4930432K21Rik and showed that it is indispensable for meiosis prophase I progression in male mice. Using super-resolution structured illumination microscopy, we found that MAMERR functions at the same double-strand breaks as the replication protein A and meiosis-specific with OB domains/spermatogenesis associated 22 complex. We generated a Mamerr-deficient mouse model by deleting exons 3-6 and found that most of Mamerr-/- spermatocytes were arrested at pachynema and failed to progress to diplonema, although they exhibited almost intact synapsis and progression to the pachytene stage along with XY body formation. Further mechanistic studies revealed that the recruitment of DMC1/RAD51 and heat shock factor 2-binding protein in Mamerr-/- spermatocytes was only mildly impaired with a partial reduction in double-strand break repair, whereas a substantial reduction in ubiquitination on the autosomal axes and on the XY body appeared as a major phenotype in Mamerr-/- spermatocytes. We propose that MAMERR may participate in meiotic prophase I progression by regulating the ubiquitination of key meiotic proteins on autosomes and XY chromosomes, and in the absence of MAMERR, the repressed ubiquitination of key meiotic proteins leads to pachytene arrest and cell death.

Anti-CXCR4 Single-Chain Variable Fragment Antibodies Have Anti-Tumor Activity.

Monoclonal antibodies (mAbs) are large and have limitations as cancer therapeutics. Human single-chain variable fragment (scFv) is a small antibody as a good alternative. It can easily enter cancer tissues, has no immunogenicity and can be produced in bacteria to decrease the cost. The chemokine receptor CXCR4 is overexpressed in different cancer cells. It plays an important role in tumor growth and metastasis. Its overexpression is associated with poor prognosis in cancer patients and is regarded as an attractive target for cancer treatment. In this study, a peptide on the CXCR4 extracellular loop 2 (ECL2) was used as an antigen for screening a human scFv antibody library by yeast two-hybrid method. Three anti-CXCR4 scFv antibodies were isolated. They could bind to CXCR4 protein and three cancer cell lines (DU145, PC3, and MDA-MB-231) and not to 293T and 3T3 cells as negative controls. These three scFvs could decrease the proliferation, migration, and invasion of these cancer cells and promote their apoptosis. The two scFvs were further examined in a mouse xenograft model, and they inhibited the tumor growth. Tumor immunohistochemistry also demonstrated that the two scFvs decreased cancer cell proliferation and tumor angiogenesis and increased their apoptosis. These results show that these anti-CXCR4 scFvs can decrease cancer cell proliferation and inhibit tumor growth in mice, and may provide therapy for various cancers.