SPOC domain-containing protein 1 regulates the proliferation and apoptosis of human spermatogonial stem cells through adenylate kinase 4

Abstract

Spermatogonial stem cells (SSCs) are the origin of male spermatogenesis, which can reconstruct germ cell lineage in mice. However, the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans. To investigate the role and mechanism of SPOC domain-containing protein 1 (SPOCD1) in human SSC proliferation. We analyzed publicly available human testis single-cell RNA sequencing (RNA-seq) data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages. Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis. Subsequently, we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes. In addition, we examined SPOCD1 expression in some non-obstructive azoospermia (NOA) patients to explore the correlation between SPOCD1 and NOA. The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC, and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs. SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis. RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4 (AK4). Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes, indicating that AK4 is a functional target gene of SPOCD1. In addition, we found a significant downregulation of SPOCD1 expression in some NOA patients, suggesting that the downregulation of SPOCD1 may be relevant for NOA. Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.