The Role of the Nhlh2 Protein in Regulating Male Fertility and Sexual Development

Abstract

Infertility is a major problem worldwide-about one in six people will experience reproductive issues at some point in their lives. These issues can have many causes, ranging from lifestyle factors to genetic influence. There are a variety of treatments that exist, including lifestyle intervention, medication, and surgery. However, work needs to be done to find more effective solutions to congenital infertility in men. The nescient helix-loop-helix 2 (NHLH2) transcription factor is expressed in the hypothalamic-pituitary-gonadal axis (HPG), which controls the development of primary and secondary sex characteristics. Signaling from the pituitary reaches the Leydig cells of the testis, which in response produce testosterone, leading to proper sexual development. Nhlh2 knockout mice (N2KO) show obesity, infertility, and hypogonadism. Likewise, humans with variants in the NHLH2 protein are infertile and hypogonadal with adult-onset obesity. Furthermore, the newly published Dog10K sequencing database uncovered a Nhlh2 variant in the obesity-prone breed Lhasa Apso. To test the hypothesis that this variant leads to obesity and infertility in this breed, we analyzed the effects of this D37N variant on protein function. Additionally, we worked to generate a Nhlh2-AAV testicular gene therapy construct for treatment of infertility, first in the Nhlh2 knockout mice, then with dogs as an intermediate trial. Results using in silico tools showed that the D37N variant alters the protein's structural and secondary modification characteristics; the mutant protein showed reduced overall stability through delta-delta G, changed helixing structure along the basic domain of the protein, added a PKA phosphorylation site at position 34, and removed a non-specific phosphorylation site at position 36. However, construction of the gene therapy plasmid was unsuccessful, as the plasmid construct had deletions in the Nhlh2 coding region, making it unusable for future projects. These results call for further analysis of phosphorylation effects on Nhlh2 structure and transactivation capabilities, and provide data on a Nhlh2 mutation in a dog breed that commonly has phenotypes associated with Nhlh2 dysfunction in mice and humans, supporting future pre-clinical testicular gene therapy trials in mice and canines.