THE ROLE OF TASTE RECEPTORS IN MALE AND FEMALE FERTILITY

Fertility is decreasing worldwide and male infertility factors contribute to approximately 30% of all infertility cases. The causes of this condition are often unknown, and about 40% of infertile men are oligozoospermic or azoospermic despite normal reproductive hormonal profile, presenting a condition of idiopathic infertility. Several studies have demonstrated the expression of taste receptors and their signaling transduction cascade in the male reproductive system, highlighting the potential role of response to chemical stimuli of taste receptors and their possible involvement in sperm maturation as well as in sperm behavior and fertilization. Considering the strong genetic component in male infertility, and the evidence that polymorphic variant in the gene are often functional, this thesis investigates the possible role of taste receptors in spermatogenesis, as well as the functional effect of selected taste receptor polymorphisms in male fertility. We hypothesized that polymorphisms in taste receptors genes might influence sperm functionality, in term of sperm parameters, as well as sperm behavior and fertilization. To this end we conducted a study to identify possible novel markers of susceptibility in human infertility within the taste receptor clusters. We selected 19 SNPs in 12 taste related genes that have been reported to be expressed in human and or mice testis or sperm and analyzed a possible correlation with different types and degrees of male infertility. For this purpose we enrolled 494 male patients, undergoing spermiogram at the Centre of Couple Sterility, Siena University Hospital. All patients were characterized for main sperm parameters, according to WHO (2010) guidelines, and were genotyped for 19 SNPs in taste receptors genes, using the KASPar SNP genotyping system. To assess the possible functional associations between SNPs and sperm parameters, different bioinformatic tools were used. Among the 19 SNPs investigated in this study for their potential association with male infertility, we found that TAS2R14-rs3741843, TAS1R2-rs4920566 and TAS2R3-rs11763979 showed the strongest significant association with specific sperm parameters. In particular, the homozygous carriers of the minor (G) allele of the TAS2R14-rs3741843 SNP showed a decreased sperm progressive motility compared to common (A) homozygous (coefficient= -0.46; P=0.003). Moreover, GTEx we showed that TAS2R14-rs3741843 has a plethora of expression quantitative trait loci (eQTL) in various tissues, and in the testicular tissue the SNP has one of the biggest effect size, suggesting its importance in this tissue. In addition, we analyzed, in human ejaculated sperm, the gene expression profile of the selected taste receptors, as well as of several genes involved in the signal transduction cascade elicited by these receptors. We partially confirmed the obtained data from the gene expression analysis at the protein level both in human sperm as well as in mouse sperm and testis (this study has been carried out during my training period at the Walther Straub Institute, Ludwig-Maximilians-University Munich). In particular, by using the Western blot and immunohistochemical techniques, we focused on the bitter receptor TAS2R4, TAS2R14, and the umami receptor TAS1R1 and the G proteins α-gustducin and α-transducin. Since we observed the expression of the detected proteins in the male reproductive system, we carried out the same analysis in the female somatic follicular cells, namely cumulus and granulosa cells, that play an important role in the oocyte competence acquiring and fertilization process . Immunofluorescence assay revealed a cytoplasmic localization in cumulus cells and granulose cells, in particular around the nucleus, probably in the transition region between rough endoplasmic reticulum and Golgi apparatus, where the protein concentration inside vesicles prelude to an active transport/release from these cells. In conclusion, our results suggest a role of genetic variability of taste receptors in human male infertility, as indicated by the significant correlation with the main sperm, and the expression of several components of taste receptor transduction cascade in male and female gamete

Single nucleotide polymorphisms of USP26 in azoospermic men

Some studies have focused on the association between male infertility and single nucleotide polymorphisms (SNPs) in the ubiquitin-specific protease 26 (USP26) gene, but the results are controversial. In this case-control study including both normozoospermic men and patients with nonobstructive azoospermia, we analyzed both the entire coding region and 5’ and 3’ untranslated regions of USP26 in order to identify genetic variants in this gene to investigate the role of USP26 on spermatogenesis. We reported variations in the USP26 gene sequence in 82% of azoospermic and in 50% normospermic men. The synonymous variation c.576G>A has a frequency significantly different in the azoospermic (60.2%) and normozoospermic (23.6%) groups, while the frequencies in the two groups of both c.1090C>T and c.1737G>A missense mutations did not reach statistical significance. A cluster mutation (c.371insACA, c.494T>C) was detected in 2 normozoospermic men (2.7%). In the 5’UTR we identified the -33C>T variation both in azoospermic (3.8%) and in normozoospermic (2.7%) men. In a normozoospermic man we detected the nonsense mutation c.882C>A, never reported to date. According to our results, we suggest that only the variation c.576G>A has a frequency significantly different in azoospermic compared to normozoospermic men. Moreover, the identification in a normozoospermic man of a nonsense mutation (c.882C>A) which causes the production of a truncated protein, suggests a marginal role of USP26 in male spermatogenesis. Additional studies may be useful as we cannot exclude that the other SNPs may represent risk factors for male fertility acting by an oligogenic/polygenic mechanism

Suppression of galactocerebrosidase premature termination codon and rescue of galactocerebrosidase activity in twitcher cells

Krabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of β-galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in β-galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of β-galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well-characterized compound known to induce ribosomal read-through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense-mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose-dependent manner. The low but sustained expression of β-galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read-through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc

Impaired spermatogenesis in the twitcher mouse: A morphological evaluation from the seminiferous tubules to epididymal transit

Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa. In this developmental process the interactions between different cell types are finely regulated, hence any disruption in these relationships leads to male infertility. The twitcher mouse, the murine model of Krabbe disease, is characterized by deficiency of galactosylceramidase, an enzyme also involved in the metabolism of the galactosyl-alkyl-acyl-glycerol, the precursor of sulfogalactosyl-alkyl-acyl-glycerol, the most abundant glycolipid in spermatozoa. Twitcher mice are sterile due to alterations of spermatogenesis resulting in the production of spermatozoa with abnormally swollen acrosomes and bent flagella, mainly at the midpiece–principal piece junction. The current study employs light, fluorescence, and electron microscopy to examine the defective spermiogenesis leading to the morphological abnormalities of mature sperm. This study reveals that alterations in germ cell development can be initially detected at the stage VIII and IX of spermatogenesis. The disrupted spermatogenetic process leads to a reduced number of elongating spermatids and spermatozoa in these mutant animals. Electron microscopy analysis demonstrates major acrosomal and chromatin condensation defects in the mutants. In addition, in twitcher mice, the epididymal architecture is impaired, with stereocilia of caput and corpus broken, detached and completely spread out into the lumen. These findings indicate that seminolipid expression is crucial for proper development of spermatocytes and spermatids and for their normal differentiation into mature spermatozoa. Abbreviations: GALC: galactosylceramidase; GalAAG: galactosyl-alkyl-acyl-glycerol; SGalAAG: sulfogalactosylalkylacylglycerol; PND: postnatal day; PAS: periodic acid-Schiff stain; TEM: transmission electron microscopy; SEM: scanning electron microscopy; PFA: paraformaldheyd