Maintenance of male fertility via RNA binding proteins

The National Institute of Child Health and Human Development awarded University of Massachusetts Boston $577,974 on August 24, 2026, under the Child Health and Human Development Extramural Research program (CFDA 93.865) to investigate RNA binding proteins in the maintenance of male fertility and spermatogenesis.

The research uses zebrafish as a model organism to elucidate how RNA regulation by ADAD1 proteins controls spermatogonial stem cell maintenance and differentiation during continuous sperm production. The project's three aims test the hypothesis that ADAD1 directly regulates RNA targets: Aim 1A identifies testis RNAs directly bound by ADAD1 and determines whether these RNAs are stabilized, destabilized, or unaffected during spermatogenesis; Aim 1B employs cell-based assays to determine how RNAs are affected upon interaction with ADAD1 protein and which protein domains are critical; and Aim 1C tests conservation of human and mouse ADAD1 function.

Work is performed at the University of Massachusetts Boston location in Dorchester, Massachusetts. The period of performance runs through July 31, 2029. The award is classified as a Project Grant and represents assistance funding to support extramural research training and investigation within NICHD's child health mission.

Abstract

Infertility affects over 17% of couples and in about half of these cases the male partner contributes to the inability to conceive. Continuous sperm production is dependent on the regulation of spermatogenesis, including proliferation and differentiation of spermatogonial stem cells (sscs). The regulation of sscs is controlled by a complex regulatory environment which includes signals from several testicular cell types as well external hormones. How sscs integrate this complex signaling milieu to balance stem cell maintenance and differentiation is poorly understood. Post-transcriptional regulation via RNA binding proteins is essential for correct progression through all stages of spermatogenesis, however the scope of post-transcriptional regulation, particularly in sscs, is limited. This study utilizes the genetic and experimental attributes of the zebrafish model to elucidate processes important for continuous sperm production and maintenance of fertility. Our previous work identified the conserved ADAD1 RNA binding protein as essential for SSC maintenance. How ADAD1 affects rnas during gametogenesis is not known in any organism. Our central hypothesis is that RNA regulation by adad proteins is key for germ cell maintenance and differentiation. AIM1 will test the hypothesis that ADAD1 directly regulates rnas. In AIM1A, testis rnas directly bound by ADAD1 will be identified to understand mechanisms and processes regulated by ADAD1. By comparing transcript abundance and expression patterns of ADAD1-target rnas in ADAD1 mutant and wild-type testes, we will determine if these rnas are stabilized, destabilized, or unaffected by ADAD1 during spermatogenesis. In AIM1B, cell-based assays will be used for functional studies to determine how rnas are affected upon interaction with ADAD1 protein and which protein domains are important. In AIM1C, we will test the hypothesis that human and mouse ADAD1 proteins have shared molecular functions to zebrafish by testing if these orthologous proteins affect transcripts in similar ways. AIM2 will test the hypothesis that ADAD1 acts redundantly with the related ADAD2 gene in germ cells. Our preliminary data shows that, like ADAD1, ADAD2 is expressed specifically in germ cells and is essential for germ cell maintenance. Therefore, ADAD2 may also have an important function in sscs. In AIM2A, we will characterize the ADAD2 mutant phenotype to better understand its function in germ cell development and maintenance. Then, in AIM2B, we will test if ADAD1; ADAD2 double mutants have additional germ cell phenotypes compared to each single mutant, which will point to redundant functions in these cells. Uncovering redundancy will reveal additional functions of these genes that could not be found with single mutant analysis. Knowledge gained from this study can be applied to a better understanding of reproductive health and to improved methods for diagnosis and treatment of infertility, assisted reproductive technologies, and development of male-targeted family planning methods.