Sperm morphology and motility in men: Investigations of defective male germ cell differentiation

Abstract

Abnormalities in transcriptional expression and translational modifications during spermatogenesis can lead to defective sperm morphology and motility that contribute to approximately 80% of male infertility cases. Both RNA sequencing (RNAseq) and mass spectrometry (MS) are powerful tools to identify differentially expressed genes (DEG) and proteins (DEP) that are altered in these pathological conditions. This thesis aimed to identify DEG in normal and impaired spermatogenesis, and DEP in normal and phenotypically abnormal human spermatozoa, to elucidate new mechanisms contributing to human sperm development and function. Testis biopsies from men were classified as normal spermatogenesis (NSP), spermatid arrest (SDA, lack of elongated spermatids and spermatozoa), or Sertoli cell only (SCO, lack of all germ cells) (n=2-3 per group) and subjected to RNAseq. DEGs were filtered based on predominant expression in spermatids and functional gene annotations relevant to sperm morphology and motility. Of these, 10 DEGs were validated by qRT-PCR and the localisation of two proteins was determined in testis biopsies by immunohistochemistry (IHC). Secondly, human ejaculates classified as normozoospermia (NORM, n=3) or asthenoteratozoospermia (defective morphology and motility, AT, n=3) were subjected to MS. Proteins selected through in silico analysis were examined by IHC in testicular biopsies and protein changes between AT vs NORM verified by quantitative Western blotting (qWB). Finally, importin 4 (IPO4) was selected for further investigation and identification of its potential cargos (ASF1B and TNP1) using dual-label immunofluorescence (IF). Out of 10 DEG with potential roles in sperm morphology and/or motility, six (SPATA31E1, TEKT3, SLC9C1, PDE4A, CFAP47, and TNC) were identified as novel, human spermatid-enriched genes. IHC revealed localisation of ORAI1 and SPATA31E1 in developing human germ cells, with SPATA31E1 enriched in late spermatocytes and spermatids. 35 DEP were identified between AT and NORM sperm by MS. Through in silico analysis, five candidates were selected for further investigation. MS data showed IPO4, ELSPBP1, and IFT57 were higher abundance 6 in AT whereas CCDC105 and ACTRT2 were lower. IHC revealed their localisation in human germ cells (ACTRT2, IPO4, IFT57, CCDC105) with a predominant localisation of ACTRT2, IPO4 and IFT57 in round/elongated spermatids. ELSPBP1 was identified as an epididymal-specific protein suggesting its sperm binding ability could occur in a phenotype-dependent manner during post-testicular maturation. qWB on a different set of ejaculates did not reveal statistically significant differences for ACTRT2, ELSPBP1, and IFT57 between AT and NORM. IHC/IF analysis of IPO4 showed its localisation in the Golgi apparatus, acrosome of round spermatids, and elongating spermatid nuclei. IPO4-ASF1B co-localisation was observed in the developing human acrosome in round spermatids and IPO4-TNP1 in step 3-5 spermatid nuclei, suggesting IPO4’s potential role in cargo transportation, acrosome biogenesis and spermatid nucleus condensation. In conclusion, both transcriptomic and proteomic strategies discovered novel candidate genes and proteins likely involved in human sperm morphology and motility. Further investigation will reveal their functional properties that maintain normal sperm function. Furthermore, these genes/proteins are candidates for future studies that aim to define new diagnostic markers for the differentiation of obstructive and non obstructive azoospermia and identify new targets for male contraception.