Molecular and developmental mechanisms of phosducin-like protein 3 function in spermatogenesis

The National Institute of Child Health and Human Development awarded Loyola University of Chicago $545,625 on July 1, 2026, to investigate the molecular and developmental mechanisms of phosducin-like protein 3 function in spermatogenesis under the Child Health and Human Development Extramural Research program (CFDA 93.865).

The project addresses male infertility, which accounts for approximately one-third of infertility cases in couples experiencing reproductive difficulty in the United States. The research focuses on phosducin-like protein 3 (PHLP3), a conserved thioredoxin-domain-containing protein identified as essential for male fertility. PHLP3 mutant males are sterile and cannot produce mature sperm due to defects in spermatid nuclear clustering, elongation, and individualization. The project employs a multi-faceted approach to elucidate PHLP3's biochemical function, identify key cysteine residues involved in redox activity, and determine how PHLP3 regulates cytoskeletal changes necessary for nuclear elongation and sperm individualization. The research leverages the conservation of PHLP3 redox activity across species, from protozoans to humans and Drosophila, to establish mechanistic understanding applicable across eukaryotes.

Performance occurs at Loyola University of Chicago's Lake Shore Campus in Chicago, Illinois. The period of performance runs from July 1, 2026, through June 30, 2029. This is a Project Grant, the standard assistance type for extramural research awards under NICHD.

Abstract

Approximately 10-15% of couples in the united states experience infertility. In one-third of these cases, infertility is linked to male factors, primarily due to abnormal sperm production and function. Addressing this issue requires identifying and characterizing the genetic mechanisms that control sperm production. Recently, we identified the drosophila phosducin-like protein 3 (PHLP3) as essential for male fertility. PHLP3 mutant males are sterile and cannot produce mature sperm. Spermatid nuclei fail to cluster, complete nuclear elongation, and individualize, resulting in seminal vesicles without sperm and sterility. To understand how PHLP3 functions in vivo, IT's important to consider its molecular role. PHLP3 belongs to a family of small thioredoxin- domain-containing proteins that are highly conserved among all eukaryotes. Members of the PHLP3 family exhibit cysteine-dependent redox activity, and their functions are linked to the regulation of microtubules and actin filaments. Recent studies have shown conservation of this redox activity across species, from the protozoan parasite plasmodium berghei to humans and drosophila, although the cysteine dependence of drosophila PHLP3's redox activity has not yet been confirmed. Given the proposed role for PHLP3 as a regulator of cytoskeletal proteins and the critical importance of cytoskeletal remodeling during spermatid maturation, we hypothesize that PHLP3 is necessary for the cytoskeletal changes that promote nuclear elongation and sperm individualization. We also hypothesize that the conserved redox activity of PHLP3 is essential for its biological function. This project aims to elucidate the molecular and developmental mechanisms of PHLP3 function during spermatogenesis through a multi-faceted approach: (1) understanding the biochemical function of PHLP3 by identifying the key cysteine(s) involved in redox activity and its in vivo binding partners, and (2) characterizing the tissue and subcellular localization of PHLP3 and examining how PHLP3 mutations affect critical stages of late spermatogenesis. These studies will shed light on the biochemical and genetic mechanisms by which PHLP3 regulates spermatogenesis. Considering the high level of sequence conservation across species, we propose that PHLP3 performs similar functions related to spermatogenesis across different organisms.