Sertoli Cells Require NELF-B for Spermatogenesis in Mice

Shivi Khurana¹, Helena D. Zomer¹, Kavyashree Basavaraju¹, Prabhakara P. Reddi¹

1. Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, USA

Abstract Text:

The binding of RNA polymerase II (Pol II) to gene promoters is crucial for transcription initiation. Negative elongation factor (NELF) associates with Pol II at the promoter region, pauses transcription and regulates the expression of genes. Sertoli cells (SC), the only somatic cells in the testicular seminiferous tubules (ST), support germ cells and maintain spatiotemporal gene regulation as germ cell cohorts transition through different stages of spermatogenesis.

We hypothesize that NELF in SC controls the expression of genes essential for germ cell development during spermatogenesis and male fertility.

The NELF-B gene was conditionally knocked out (cKO) in mouse SC using Amh-Cre. Mice were analyzed during the first wave of spermatogenesis (postnatal days 12 and 21; PND-12 and 21) and adults (3 months old; 3MO) (n=3/control and cKO/age). Testicular weight was recorded. Hematoxylin Eosin and TUNEL staining were performed to histologically analyze ST abnormalities and apoptosis, respectively. Further, immunostaining was performed to quantify the impacted spermatogenic stages using γH2AX (pachytene spermatocytes) and SP-10 (round spermatids). For each staining analysis, fifty ST/testis cross-sections were counted and analyzed using t-test. Additionally, to evaluate the reproductive outcomes, measured the sperm parameters (concentration, motility, and morphology) and conducted fertility trial (n=8) of four months. Furthermore, for mechanistic insights into SC-mediated infertility in cKO mice, bulk RNA sequencing was performed on SC isolated from PND-12 (n=3/control and cKO). RNA-seq data were analyzed and validated by real-time PCR (qPCR).

cKO mice across all age groups showed a reduction (p < .001) in testicular weight. Histological analysis revealed that testicular atrophy started at PND 12 and persisted in adults. There was a decrease (p < .001) in ST diameter and an increase (p < .05) in apoptotic cell numbers within the ST. Immunostaining showed a decrease in pachytene spermatocytes (p < .01) and round spermatids (p < .05) at PND-21 and 3MO. Consistent with the spermatogenic defects, there was a decrease (p < .001) in sperm concentration, motility, and morphologically normal sperm. Also, adult cKO mice were infertile, with no pups born in the fertility trial. These findings suggest severe spermatogenic defects and male infertility, likely due to impaired support from SC. RNA-seq analysis identified 178 upregulated and 81 downregulated SC-enriched differentially expressed genes (DEGs) in cKO. qPCR validation confirmed upregulation of Cldn23, Inhbb, Amh and downregulation of Corin, Serpina5, and Rhox5. Notably, dysregulated transcripts, such as Inhbb, Amh, Corin and Serpina5, are known key regulators of hormonal regulation by SC and the hypothalamic-pituitary-testicular (HPT) axis. These results suggest that NELF-B in SC could be playing a crucial role in endocrine feedback signalling to the hypothalamus and pituitary.

Our data supports the hypothesis that NELF in SC regulates gene expression essential for maintaining the endocrine niche crucial for germ cell development and male fertility. Interestingly, the cKO mice resemble the male infertility of the type oligoasthenoteratozoospermia (OAT, the most complex form of male infertility, characterized by a decreased sperm count, motility, and normal morphology).

Future studies will explore hormonal changes, especially in young males. Also, this mouse model and siRNA-based in-vitro models will be used to study pathways by which SC influences the gene expression of the germ cells.

Together, this study highlights NELF as an essential regulator of spermatogenesis and could provide potential leads to therapeutic approaches for OAT (male fertility).

Financial support by NIH, R01HD094546 (PPR)