TEX15 Regulates Spermiogenesis via the HUSH2 Complex to Ensure Proper Chromatin Packaging

Fang Yang¹, Ankit Jaiswal¹, Zhenlong Kang¹, N. Adrian Leu¹, Kevin Monahan¹, Lan Ye¹, and P. Jeremy Wang^1,*

¹Department of Biomedical Sciences, University of Pennsylvania School of Veterinary

^{Medicine, Philadelphia, PA, USA.}

Abstract Text: Infertility is a global reproductive health issue affecting both men and women equally. Genetic studies in model organisms have identified pathways that regulate fertility, with over 400 genes implicated in reproductive function. Male infertility, particularly nonobstructive azoospermia (NOA), is characterized by maturation arrest or Sertoli cell-only syndrome, often caused by genetic mutations. We previously identified Tex15 as a testis-specific gene. We found that TEX15 is required for male meiosis. Furthermore TEX15 is associated with PIWI proteins and is essential for retrotransposon silencing in male germ cells in mice. Notably, mutations in TEX15 cause NOA in humans. Since TEX15 global knockout cause early meiotic arrest, we generated a TEX15 conditional knockout (cKO), to study its functional aspect in meiotic and post-meiotic germ cells. For this, we generated a floxed TEX15 allele in which exons 6,7 and 8- i.e 80% of the coding sequence were flanked by loxP sites. By crossing these mice with Ngn3-Cre mice, which express Cre recombinase in spermatogonia from postnatal day 7, TEX15 was specifically inactivated in meiotic and postnatal germ cells. TEX15^cko male mice were sub-fertile. Additionally, transmission electron microscopy shows that loss of TEX15 disrupts sperm head morphology, highlighting its role in sperm chromatin packaging. This study identifies a novel role for TEX15 as a critical regulator of spermiogenesis. IP/mass spec screen identified TASOR2 and MPP8 as TEX15-associated proteins in testis, which are components of the HUSH2 complex (TASOR2-MPP8-PPHLN1). TEX15 interacts specifically with TASOR2 but not with TASOR in mouse testis, a key component of the canonical HUSH complex. In round spermatids, TEX15, TASOR2, and MPP8 colocalize at XY chromatin, with TASOR2 essential for TEX15 recruitment. Interestingly, Tasor2-deficient mice exhibit teratozoospermia and male subfertility, phenocopying Tex15 mutants. RNA-seq analysis of Tex15^cKO or Tasor2-deficient testes reveals dysregulation of X-linked histone-like (H2al) genes, implicating HUSH2 in chromatin silencing during spermiogenesis. Additionally, transmission electron microscopy shows that loss of TEX15 or TASOR2 disrupts sperm head morphology, highlighting their role in sperm chromatin packaging. To further elucidate this pathway, we will perform RNA-seq on Tex15-deficient round spermatids and TASOR2 ChIP-seq to determine genomic targets. Additionally, we aim to investigate the role of histone variants in spermiogenesis. Understanding chromatin regulation in the testis may provide novel insights into NOA pathogenesis and potential clinical applications, including ICSI. Our findings establish TEX15 as a key regulator of sperm chromatin organization and male fertility, bridging fundamental biology with translational research.