Methylenetetrahydrofolate reductase (MTHFR), an enzyme expressed in mammalian testes exerts direct effect on spermatogenesis; however, its protein characteristics in bovine testes remain unknown. Here, we analysed bovine testicular structure, MTHFR bioinformatics profile, mRNA, and protein expression characteristics in yellow-cattle (y-c) and yak testis using histological procedures, bioinformatics analysis, qRT-PCR, and western blot. Testes from 13 bovines, ≤ 2 years juvenile (y-c, n = 3; yak, n=3) and ≥ 3 years adult (y-c, n = 3; yak, n = 4) were collected and analysed. Anatomical characteristics of testis in y-c and yak were similar except the weight or size for which that of y-c was significantly higher or greater than yak. In y-c, an open reading frame (ORF) for 2600 nucleotides sequence, encoding 655 amino acids showed high homology with zebu cattle (99.51%) and wild yak (98.68%). Secondary and 3D protein structures were similar to that of humans with differences in number of nucleotides, amino acids, and some physico-chemical characteristics. MTHFR mRNA expression in y-c and yak were significantly higher in adult testes compared with juvenile ones. However, its protein expression was higher but not statistically significant in adult y-c and yak compared to the juvenile ones. The highlights and inferences of these and other findings are discussed.

Spermatogenesis is a complex, tightly regulated biological process crucial for male fertility in mammals. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) play essential functions in regulating gene expression during spermatogenesis, and their specific roles in Holstein bull testicular development remain unclear. The study analyzed testicular tissues from young and mature Holstein bulls, unveiling the post-sexual maturation transcriptome landscape with a focus on spermatogenesis-associated miRNAs and mRNA profiles. The cDNA libraries from the testicular tissues of three young Holstein bulls (X1, X2, and X3; group YB) and three mature Holstein bulls (D5, D6, and D7; group AB) were established for RNA-sequencing analysis using Illumina high-throughput sequencing technology, followed by bioinformatics analysis. A total of 1114 miRNAs were identified in the X vs. D groups: 683 known miRNAs and 431 novel miRNAs. Notably, 221 known miRNAs exhibit differential expression between the X and D groups, with 118 up-regulated and 103 down-regulated genes. Moreover, a total of 237 previously unidentified novel miRNAs were also observed to exhibit differential expression between X and D, with 77 up-regulated and 160 down-regulated miRNA genes. These miRNA genes were linked to Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to male reproduction and spermatogenesis. The DE analysis of miRNA genes identified the top five up-regulated miRNAs (bta-miR-127, bta-miR-495, bta-miR-493, bta-miR-432, and bta-miR-3956) and down-regulated miRNAs (bta-miR-34c, bta-miR-34b, bta-miR-449a, bta-miR-449b, and 23_28233-5p (hsa-miR-6774-3p)) between the young bull and adult bull groups. The miRNA-mRNA network analysis also identified 17_16057-5p, X_45311-3p (cpo-miR-12072-3p), bta-miR-34c, bta-miR-375, 17_15431-5p (eca-miR-8912), 10_2547-3p (isc-miR-5313), bta-miR-449b, and mRNA genes such as BCL2L11, ADAMTSI1, MED1, and PRDM1; these genes were associated with reproductive system development. The mRNA genes (STRB and BCL2L11) and miRNA genes (bta-miR-134, bta-let-7e, bta-miR-181b, and bta-miR-214) were also found to have a significant correlation with the spermatogenic process. Thus, the comprehensive analysis of miRNA and mRNA enhances our knowledge of the regulatory networks involved in testicular development and identifies potential targets for further investigation and manipulation to improve cattle reproductive efficiency and genetic selection.

The morphology and maturation process of gonads of 70 Burmeister´s porpoises, with body lengths ranging 135–183 cm (n= 34 females) and 64.5-182 cm (n= 36 males) were described. Samples were collected in six ports of central and northern Peru in 1987-1999. In the field, females were classified as immature, mature (resting, lactating, pregnant) and males as immature, pubescent and mature based on, respectively, the presence of ovarian corpora and the relative quantity of semen in cut epididymides. The ovaries of P. spinipinnis are ovoid or bean-shaped and flattened, with corpora modifying surface appearance. In the laboratory, ovaries were examined macro- and microscopically, measured, weighed and sliced in 1-3 mm sections. The number of corpora ovarica (lutea, albicantia, atretica) found in each ovary as well as their macroscopic and microscopic characteristics were documented in some detail. The follicles, their oocytes and nucleus were measured. Follicular development in P. spinipinnis is predominantly left-sided, but occurs in both ovaries in 16.3% of females, mainly in those with numerous corpora. Macroscopically, several corpora atretica with luteinization had characteristics similar to those of corpora albicantia, making microscopic determination essential. Inconclusive evidence of recent ovulation was found in January. Two of three immature females showed good follicular development in March and April. The body length at 50% sexual maturity in females was estimated at 152.7 cm. There was no evidence of reproductive senescence. The testes of P. spinipinnis are elongated and cylindrical. Of the 36 males examined macroscopically, 7 were immature, 5 pubescent and 24 mature. The histological analysis determining the presence and abundance of Sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa, the relative amount of interstitial tissue, the elongation and mean diameter of the seminiferous tubules and the relative size of the lumen allowed to confidently determine sexual maturity status. The field evaluation of maturity based on the presence of sperm in the epididymides is a useful but, in 8.3% of cases, not exact method. For males the body length at 50% sexual maturity was estimated at 157 cm. No histological evidence of male reproductive seasonality was found. Spermatogenesis was perceptible year-round and tubule diameters had non-specific variations for each month.

Theoretic and empirical studies show that social surroundings experienced by male insects during their larval or adult stage can influence their testicular investment in diverse ways. Although insect pupae do not feed and crawl, they can communicate using sex-specific and/or non-sex specific cues. Yet, it is unknown, in any insect, whether and how male pupae can fine-tune their resource allocation to sperm production and testis size in response to socio-sexual environment. We investigated this question using a moth, Ephestia kuehniella, which produces fertile eupyrene sperm and unfertile apyrene sperm. We held male pupae individually or in groups with different sex ratios, and dissected adults upon eclosion, measured their testis size, and counted both types of sperm. We demonstrated that after exposure to conspecific pupal cues regardless of sex, male pupae increased production of eupyrenes and apyrenes at the same rate but kept testis size unchanged. We suggest that testis size is fixed after pupation because most morphological traits are formed during the larval stage, allowing little room for pupae to adjust testis size. Like adults, male pupae with fully grown testes have sufficient resources to produce more sperm of both types according to the perceived increase of sperm competition risk.

Spermatogonial stem cells (SSCs) are the only primitive spermatogonial cells in males that can naturally transmit genetic information to their offspring and replicate throughout their lives. Phospholipase D family member 6 (PLD6) has recently been found to be a surface marker for SSCs in mice and boars; however, it has not been validated in cattle. The results of RT-PCR and qRT-PCR found that the relative expression of PLD6 gene in the testicular tissues of 2-year-old Simmental calves was significantly higher than that of 6-month-old calves. Immunofluorescent staining further verified the expression of PLD6 protein in bovine spermatogenic cells like germ cell marker VASA. Based on multiple bioinformatic databases, PLD6 is a conservative protein which has high homology with mouse Q5SWZ9 protein. It is closely involved in the normal functioning of the reproductive system. Molecular dynamics simulation analyzed the binding of PLD6 as a phospholipase to cardiolipin (CL), and PLD6-CL complex showed high stability. The protein interaction network analysis showed that there is a significant relationship between PLD6 and piRNA binding protein. PLD6 acts as an endonuclease and participates in piRNA production. In addition, PLD6 in bovine and mouse testes has a similar expression pattern with the spermatogonium-related genes VASA and PIWIL2. In conclusion, these analyses imply that PLD6 has a relatively high expression in bovine testes and could be used as a biomarker for spermatogenic cells including SSCs.