With the development of science and technology, the molecular mechanism of azoospermia has aroused great interest. The study of azoospermia depends on human studies, animal models, organ culture models and cell lines [9, 15]. The increase in the number of genes / proteins associated with male infertility has been confirmed [9, 16]. However, how these biological processes are regulated at the molecular level remains to be elucidated. Therefore, it is necessary to further study the pathogenesis of azoospermia at the molecular level. In this study, we identified the genes associated with azoospermia and systematically constructed a comprehensive framework of genes and miRNAs.
In the present study, we sought to identify associated genes by reanalyzing
separate studies from GEO datasets (GSE45885, GSE45887, and GSE9210) and validation dataset (GSE4797)[13, 17, 18]. The results showed that several shared genes significantly associated with azoospermia. Finally, we effectively screen out two genes (KIF2C and TEKT2) for validation by GSE4797 in spermatozoa of infertile men with Johnsen score. Among these two genes, KIF2C and TEKT2 significantly down-regulated in spermatozoa of infertile men. The regulatory network of TF-miRNA‐target gene was established, we found KIF2C-miRNAs(has-miR-3154,6075,6760-5p,1251-5p,186-sp)-TFs(EP300,SP1) might work in spermatozoa of infertile men. Interestingly, the relative expression levels of KIF2C and TEKT2 had a negative correlation with Johnsen score, which showed potential role of spermatogenesis.
KIF2C (also known as the mitotic centromere-associated kinesin, MACK) is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins, which is critical in the regulation of microtubule dynamics. During cell division, KIF2C plays an important role in inhibiting the wrong connection between MT and chromosome[19, 20]. The function of kif2c in interphase cells is not obvious, although its main localization in nucleus suggests that kif2c may play a role in nuclear processes. Kif2c promotes the formation of DNA damage foci, which may involve the migration and aggregation of DSBs (DNA Double Strand Break) [21–25]. We found KIF2C might play a significant role on testis cancer and azoospermia.
Tektins (TEKTs), the proteins of the microtubules in Cilia, Flagella, Basal bodies and centrioles [26–28], have been found in various animals, including Filariae, including silk-worms , mice[26, 30] and humans [31, 32]. They were originally isolated from sea urchins and are a group of proteins: TEKT-A, -B and -C [33, 34]. On the other hand, five types of TEKTs have been identified in mammals. TEKT2, which is similar to Tektin-t, locates in the main part of human spermatozoa but no immune signal was detected in the middle or at the end of the human sperm. Tektin2, a membrane protein, is responsible for sperm flagellum movement. Previous studies show that CatSper and tektin are associated with male infertility because they play an important role in sperm motility. Tektin2 is essential for the integrity of motilin arm in sperm flagellum. Lack of tektin2 can lead to impaired sperm motility and male infertility . The low expression of tektin2 mRNA was observed in frozen spermatozoa, suggesting that the decrease of sperm motility after cryopreservation may be due to the transcriptional damage of some sperm motility related genes .
The miRNAs play an important role in infertility. In 2009, for the first time, expression of miRNAs in a testicular sample of NOA patients compared to fertile control samples evaluated by microarray technology, identified 19 upregulated and 154 downregulated miRNAs . Hsa-miR-141, hsa-miR-429, hsa-miR-7-1- 3p, hsa-miR-34b, hsa-miR-34c-5p, hsa-miR-122 expression levels were significantly different in azoospermia [39, 40]. Through luciferase experiments, miR- 525-3p which targets SEMG1 gene and hsa-miR-210 which targets insulin-like growth factor II (IGF2) [41, 42]. The lower expression of hsa-miR-188-3p results in higher expression of MLH1 gene in azoospermia patients and leads to apoptosis in spermatozoa.
Functional classification of the miRNA/mRNA pairs using bioinformatics tools indicated that they play a role in spermatogenesis, cell meiosis, cell cycle. We found KIF2C-miRNAs(has-miR-3154,6075,6760-5p,1251-5p,186-sp)-TFs (EP300,SP1) might work in spermatozoa of infertile men.
Our study also has some limitations. First, more samples could be included in this study and we assessed our results based on published observations. Further in vitro and/or in vivo experiments would need to be carried out to test reliability of our results. This might reduce the error caused by individual differences of patients.