 MTHFR proteins. Interestingly, for every other 18 amino acid residue along the entire sequence, these parameters changed, and the change was consistent with human MTHFR protein suggesting a high level of similarity in character and function. Predicted secondary structure of Bos taurus MTHFR are shown in Figure S4.
Y-c MTHFR mRNA in testicular tissues of y-c and yaks were investigated by real-time polymerase chain reaction (RT-PCR). As shown in Figure 7a,b relative mRNA expression levels in both adult y-c and yak testes were highly significant compared to their juvenile counterparts and this expression level was 5.586 folds higher in the adult y-c testes than the juvenile ones (1.000) (p < 0.05, p < 0.01). In a like-wise manner, the expression level in the adult yak testes was 5.686 folds higher than that in the juvenile ones (1.500) (p < 0.05, p < 0.01). This shows that the highest expression level of MTHFR mRNA was observable during adulthood in both species. On the other hand, MTHFR protein expression was higher but not statistically significant in adult yellow-cattle and yak testes compared to their juvenile ones. MTHFR protein expression tendencies were consistent at the different ages between y-c and yak. According to our data, levels of MTHFR were high in almost all adult testes studied compared to the juvenile ones (Figure 8a–d).
For the first time, this study evaluated testicular histopathology, and analysed DNA sequence, amino acids, relative mRNA expression, and predicted protein characteristics of MTHFR in the testes of juvenile and adult y-c or yak. It highlighted the unique characteristics of this enzyme in bovine testes and examined roles MTHFR could play in the physiology of bovine spermatogenesis. MTHFR is pivotal in folate metabolism and controls the relative distribution of one-carbon moieties between two important biochemical pathways that support general housekeeping methylation reactions and purine/pyrimidine biosynthesis. Precise control of MTHFR expression may therefore be essential to ensure that the optimum balance between the above pathways is tailored to tissue-specific requirements. Characterization of the structure of its mRNA in both human and mouse has also provided a foundation for studies on the regulation of MTHFR [33].
In bovines, testicular growth increases markedly between 7 and 10 months of age [34]. Anatomical characteristics of testis in y-c and yak were generally similar except for the weight or size for which that of y-c was significantly higher or greater than yak. Analysis of testicular structure revealed that healthy juvenile and adult y-c or yak testes had a continuous arrangement of seminiferous tubule epithelium and germ cells also had normal morphology and regular arrangement. Primary spermatocytes and early spermatids were also observed in all tissues. The lumina (with spermatocytes) in the seminiferous tubules were more developed in the adult testes than in the juvenile ones. This could probably be due to age-related physiological changes. In bovines, lumen appears around the age of 20 weeks and increase from 2% to 98 µm by the age of 32 weeks [35].
Bioinformatics analysis showed that y-c MTHFR nucleotide sequence had several ORFs including a 1968 bp, with 5′ and 3′—terminal UTR (untranslated regions) corresponding to the 192 bp (initiation codon) and 2159 bp (termination codon) respectively. This was different in the case of human MTHFR as reported by Homberger et al. [36]. In this study, we found that y-c MTHFR had 10,402 atoms with a molecular weight of 74.49 kDa whereas in human studies, 70 to 77 kDa was reported. Six hundred and fifty-five (655) amino acids constituted MTHFR protein, and a sequence length of 2600 bp covered the full coding region with 13 exons whereas in humans, MTHFR had sequence length of 2196 bp with 11 exons encoding 679 amino acids in one instance. The human MTHFR encoded three putative polypeptides of 657, 679, and 698 amino acids [36]. Y-c MTHFR coding sequence was highly similar with that of other organisms and of all species considered, the highest level of homology was observed in wild yak (Bos mutus) and the lowest in horse (Equus caballus); this suggests a common evolutionary origin.
The MTHFR protein N-terminal was composed of methionine (Met, M) with a half-life of 30 h in mammals. It is reported that more than 80% of proteins of any given proteome has methionine at its N-terminal [37]. Protein N-terminal methionine (NM) is an essential cotranslational factor that occurs in the cytoplasm of all organisms and in two organelles (i.e., mitochondria and plastids) capable of protein synthesis [38,39]. Amino acids have their unique and specific chemical characteristics enabling them to perform exclusive roles in protein structure and function. Depending on the inclination and proximity of the side chains to water, amino acids can be classified as hydrophobic (lowly inclined to be in contact with water), polar and charged (energetically favoured to be in contact with water) [40]. In our study, we found that the predicted MTHFR protein contained 322 hydrophobic and 154 hydrophilic residues and the total number of negatively (Asp + Glu) and positively (Arg + Lys) charged residues were 93 and 72 respectively. Instability index (II) was found to be 52.27 with an aliphatic index and grand average of hydropathicity (GRAVY) of 79.62 and −0.430 respectively. These data suggest that y-c MTHFR gene codes for an unstable and very reactive protein.
In this study, it was also found that y-c MTHFR had no transmembrane region, thus belonging to the extra-membrane category of proteins. Proteins within a cell are often localized to specific cellular compartments, such as the cytoplasm, nucleus, mitochondria, plasma membrane, or vesicles, and their specific localization can provide crucial information about the function of the protein [41]. The distribution of MTHFR proteins was subcellularly predicted to be more in the cytoplasm (78.3%). Subcellular localization determines the access of proteins to interacting partners and the post-translational modification machinery, which enables the integration of proteins into functional biological network [42]. It has also been demonstrated that the abnormalities of subcellular locations of protein are potentially involved in the pathogenesis of many human diseases [42,43,44,45].
Protein phosphorylation is the most abundant form of cellular regulation, essentially organizing all cell functions, including metabolism, proliferation, differentiation, motility, survival, and death. Protein phosphorylation is a post-translational modification of proteins whereby a phosphate group is covalently attached to either a serine, threonine or tyrosine residue [46,47]. Phosphorylation sites in y-c MTHFR predicted protein were analysed and we found that several phosphorylation sites existed in the protein. Scores greater than the threshold (0.500) indicated potential presents of phosphorylation sites, however, scores just above the threshold implied that the confidence for categorizing the site as phosphorylated was low.
To a large extent, secondary (α-helices and β-sheet) and tertiary structures of proteins are defined by amino acids, although the effect of local environment on structure stabilization is also crucial. The α-helix is considered to be the most abundant form of secondary structure, accounting for about 31% of amino acid secondary structure states [48]. In our study, we found that the secondary structure of y-c MTHFR protein was made of α-helix 255 (Hh: 38.93%), extended strand 86 (Ee: 13.13%), beta-turn 37 (Tt: 5.65%), and random coil 277 (Cc: 42.29%). The predicted 3D structure of y-c MTHFR protein had 91.61% sequence identity, modeled with 100.0% confidence and 90% coverage. The result showed general folding pattern and the 3D structure was highly similar to that of Homo sapiens MTHFR.
A comprehensive understanding of the cell biology, protein structure, and genetics of spermatogenesis in most species is arduous because it occurs within a complex testicular environment characterized by the intimate association of developing sperms with accessory cells [49]. Nevertheless, in recent years, advances in genomics have greatly improved our knowledge of spermatogenesis by identifying numerous genes/proteins essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened insights into normal and pathological spermatogenesis [50].
Studying MTHFR mRNA expression in testicular tissues of juvenile and adult y-c or yak, we found a relatively high expression level in the adult testes compared to the juvenile ones. This suggests that with the passage of time or maturity, there are increased/pronounced activities of MTHFR enzyme in the testes to promote its core functions, which is the production of spermatozoa through spermatogenesis. This could partly explain why the highest MTHFR mRNA expression levels were observed during adulthood in healthy bovines. Liu et al. [51], working on bovines, reported that mRNA and protein expression were mostly consistent at different developmental stages. They added that mRNA expression levels were significantly higher for some testicular genes in almost all developmental stages of y-c and yak [51]. Chen et al. in 2001 also reported that animal model studies suggested that MTHFR plays a critical role in spermatogenesis due to exceptionally higher activity in adult mouse testes [26]. Meanwhile, Kelly et al. [20] observed that inactivation of MTHFR results in infertility with abnormal testicular histology characterized by absence of germ