Bovine Testis Research Articles

Expression of brain-derived neurotrophic factor and neurotrophic tyrosine receptor kinase-2 in bovine testes

Expression of brain-derived neurotrophic factor and neurotrophic tyrosine receptor kinase-2 in bovine testes

P-097 Studying epididymal sperm using cryo-electron microscopy

Abstract Study question What are the structural changes in epididymal sperm leading to gain of motility? Summary answer We resolved the contents of the cytoplasmic droplet in immature sperm. We further found that the outer dynein arms are in a pre-power stroke conformation. What is known already Cells exiting the testis are morphologically complete but incapable of fertilization. They first need to mature in the epididymis, where sperm gain progressive motility. Intriguingly, during this epididymal maturation the cytoplasmic droplet, a cytoplasmic remnant from spermiogenesis, migrates distally from the connecting piece to the principal piece. At the heart of the sperm’s flagellum is the axoneme, the molecular motor of the cell. It is a microtubule-based structure that houses protein complexes with different functions. For instance, it has radial spokes for signaling and rigidity. It also houses dyneins, motor proteins attached to neighboring doublets that ultimately generate flagellar beating. Study design, size, duration Bovine testes were acquired from a slaughterhouse, and sperm were isolated from different regions of the epididymis. After a washing step, these cells were directly used for imaging. Participants/materials, setting, methods Cells were prepared for imaging by rapid freezing on cryo-Electron Microscopy (cryo-EM) grids. Frozen cells were either imaged directly or first thinned by Focused Ion Beam (FIB) – milling. We applied the image analysis approaches subtomogram averaging and single particle analysis. Main results and the role of chance We resolved the structure of the 96-nm repeat of the axonemal doublet from immature sperm and showed a complete axoneme, containing radial spokes and different sets of dynein arms. Focusing our refinement on a specific set of dyneins, the outer dynein arms, revealed that they are in the pre-power stroke state. In this primed state the dyneins have ATP bound and are active. Interestingly, these cells display no movement. This could indicate that a different blocking mechanism is present in the axoneme. Furthermore, we used 3D cryo-EM to elucidate the contents of the cytoplasmic droplet. We showed that it contains many vesicles, some display a shape that is reminiscent of an autophagosome. We also find vesicles displaying a double membrane with a highly repetitive density connecting in between. This suggests that sperm cells bring along auxiliary proteins and molecules in the cytoplasmic droplet that may be required for proper maturation. Altogether we show that cryo-EM can be used to study immature sperm cells in detail, with a focus on structures important in the gain of movement. Limitations, reasons for caution Cryo-EM imaging requires frozen cells and uses electrons as imaging source, therefore we lack information on cell state (e.g. viability/motility) or protein identity. By complementing our visual data with other techniques such as CASA and mass spectrometry, we aim to get a full picture. Wider implications of the findings Elucidating protein conformation or the structural mechanism behind sperm activation could open doors into developing new therapeutics for diseases on one hand. On the other hand, understanding how sperm motility arises (and how to potentially put it to a halt) could also lay ground for developing male contraception. Trial registration number not applicable

Ultrastructure of the Bovine Testis in Cattle (Bos taurus): New View.

The purpose of this study was to analyze the ultrastructure of the testes of sexually immature calves and reproductive bulls of the Polish Holstein-Friesian Black-and-White breed. Utilizing TEM, this study identified three distinct stages of seminiferous tubule development in calves, characterized by varying shapes, distributions, and arrangements of individual cells. In immature animals, early developing spermatocytes, prespermatogonia, and pre-Sertoli cells were observed within the seminiferous tubules. In sexually mature bulls, all cells of the spermatogenic series were observed, situated on a thin, multilayered basal lamina, which forms characteristic undulations. An abundant smooth endoplasmic reticulum was observed in the cytoplasm of spermatogonia in both groups of animals, forming characteristic membranous swirls. In adult bulls, spermatogonia maintain contact with each other through numerous cytoplasmic bridges and cell connections, forming small spaces with visible microvilli between them. The ultrastructural analysis facilitated the identification of morphological changes occurring during the maturation of pre-Sertoli cells, transitioning from a large euchromatic nucleus to a nucleus in which the formation of characteristic vesicles and tubules could be observed. It should also be emphasized that two types of Sertoli cells, namely dark and light electron-dense cells, can be found in cattle. These cells differ from each other, indicating that they may perform different functions. The widespread recognition of the presence of two types of Sertoli cells in cattle will undoubtedly contribute to a better understanding of the processes occurring within the testes and provide a basis for further research in this area.

A Comparative Full-Length Transcriptome Analysis Using Oxford Nanopore Technologies (ONT) in Four Tissues of Bovine Origin.

The transcriptome complexity and splicing patterns in male and female cattle are ambiguous, presenting a substantial obstacle to genomic selection programs that seek to improve productivity, disease resistance, and reproduction in cattle. A comparative transcriptomic analysis using Oxford Nanopore Technologies (ONT) was conducted in bovine testes (TESTs), ovaries (OVAs), muscles (MUSCs), and livers (LIVs). An average of 5,144,769 full-length reads were obtained from each sample. The TESTs were found to have the greatest number of alternative polyadenylation (APA) events involved in processes such as sperm flagellum development and fertilization in male reproduction. In total, 438 differentially expressed transcripts (DETs) were identified in the LIVs in a comparison of females vs. males, and 214 DETs were identified in the MUSCs between females and males. Additionally, 14,735, 36,347, and 33,885 DETs were detected in MUSC vs. LIV, MUSC vs. TEST, and OVA vs. TEST comparisons, respectively, revealing the complexity of the TEST. Gene Set Enrichment Analysis (GSEA) showed that these DETs were mainly involved in the "spermatogenesis", "flagellated sperm motility", "spermatid development", "reproduction", "reproductive process", and "microtubule-based movement" KEGG pathways. Additional studies are necessary to further characterize the transcriptome in different cell types, developmental stages, and physiological conditions in bovines and ascertain the functions of the novel transcripts.

A clinical study of the local injection of a freshly manufactured 35 kDa hyaluronan fragment for treating chronic wounds.

This study manufactured a 35 kDa hyaluronan fragment (HA35) by enzymatically degrading high-molecular-weight HA using hyaluronidase PH20 derived from bovine testis. The research then examined the therapeutic efficacy of locally administered, tissue-permeable HA35 in alleviating chronic wounds and their associated neuropathic pain. For 20 patients with nonhealing wounds and associated pain lasting over three months, 100 mg of HA35 was injected daily into the healthy skin surrounding the chronic wound for 10 days. Self-assessmentsbefore and after treatment indicated that HA35 significantly enhanced wound healing. This was evidenced by the formation of fresh granulation tissue on the wounds (p < 0.0001); reduceddarkness, redness, dryness, and damage in the skin surrounding the wounds (p < 0.0001), and a decrease in wound size (p < 0.001). Remarkably, HA35 injections alleviated pain associated with chronic wounds within 24 hours (p < 0.0001). It can be concluded that the low-molecular-weight hyaluronan fragment HA35 potentially enhancesthe immune response and angiogenesis during wound healing.

Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids

Mesenchymal stem cells (MSC) display self-renewal and mesodermal differentiation potentials. These characteristics make them potentially useful for in vitro derivation of gametes, which may constitute experimental therapies for human and animal reproduction. Organoids provide a spatial support and may simulate a cellular niche for in vitro studies. In this study, we aimed at evaluating the potential integration of fetal bovine MSCs derived from adipose tissue (AT-MSCs) in testicular organoids (TOs), their spatial distribution with testicular cells during TO formation and their potential for germ cell differentiation. TOs were developed using Leydig, Sertoli, and peritubular myoid cells that were previously isolated from bovine testes (n = 6). Thereafter, TOs were characterized using immunofluorescence and Q-PCR to detect testicular cell-specific markers. AT-MSCs were labeled with PKH26 and then cultured with testicular cells at a concentration of 1 × 106 cells per well in Ultra Low Attachment U-shape bottom (ULA) plates. TOs formed by testicular cells and AT-MSCs (TOs + AT-MSCs) maintained a rounded structure throughout the 28-day culture period and did not show significant differences in their diameters. Conversely, control TOs exhibited a compact structure until day 7 of culture, while on day 28 they displayed cellular extensions around their structure. Control TOs had greater (P < 0.05) diameters compared to TOs + AT-MSCs. AT-MSCs induced an increase in proportion of Leydig and peritubular myoid cells in TOs + AT-MSCs; however, did not induce changes in the overall gene expression of testicular cell-specific markers. STAR immunolabelling detected Leydig cells that migrated from the central area to the periphery and formed brunches in control TOs. However, in TOs + AT-MSCs, Leydig cells formed a compact peripheral layer. Sertoli cells immunodetected using WT1 marker were observed within the central area forming clusters of cells in TOs + AT-MSCs. The expression of COL1A associated to peritubular myoids cells was restricted to the central region in TOs + AT-MSCs. Thus, during a 28-day culture period, fetal bovine AT-MSCs integrated and modified the structure of the TOs, by restricting formation of branches, limiting the overall increase in diameters and increasing the proportions of Leydig and peritubular myoid cells. AT-MSCs also induced a reorganization of testicular cells, changing their distribution and particularly the location of Leydig cells.

Structural variants and short tandem repeats impact gene expression and splicing in bovine testis tissue.

Structural variants (SVs) and short tandem repeats (STRs) are significant sources of genetic variation. However, the impacts of these variants on gene regulation have not been investigated in cattle. Here, we genotyped and characterized 19,408 SVs and 374,821 STRs in 183 bovine genomes and investigated their impact on molecular phenotypes derived from testis transcriptomes. We found that 71% STRs were multiallelic. The vast majority (95%) of STRs and SVs were in intergenic and intronic regions. Only 37% SVs and 40% STRs were in high LD (R2 > 0.8) with surrounding SNPs/Indels, indicating that SNP-based association testing and genomic prediction are blind to a non-negligible portion of genetic variation. We showed that both SVs and STRs were more than two-fold enriched among expression and splicing QTL (e/sQTL) relative to SNPs/Indels and were often associated with differential expression and splicing of multiple genes. Deletions and duplications had larger impacts on splicing and expression than any other type of structural variant. Exonic duplications predominantly increased gene expression either through alternative splicing or other mechanisms, whereas expression- and splicing-associated STRs primarily resided in intronic regions and exhibited bimodal effects on the molecular phenotypes investigated. Most e/sQTL resided within 100 kb of the affected genes or splicing junctions. We pinpoint candidate causal STRs and SVs associated with the expression of SLC13A4 and TTC7B, and alternative splicing of a lncRNA and CAPP1. We provide a catalogue of STRs and SVs for taurine cattle and show that these variants contribute substantially to gene expression and splicing variation.

Expression of Phospholipase D Family Member 6 in Bovine Testes and Its Molecular Characteristics.

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 reversed transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) found that the relative expression of the PLD6 gene in the testicular tissues of two-year-old Simmental calves was significantly higher than that of six-month-old calves. Immunofluorescent staining further verified the expression of PLD6 protein in bovine spermatogenic cells like germ cell marker DEAD box helicase 4 (DDX4, also known as 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 the PLD6-CL complex showed high stability. The protein interaction network analysis showed that there is a significant relationship between PLD6 and piwi-interacting RNA (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 piwi like RNA-mediated gene silencing 2 (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.

Dynamic transcriptome profiles and novel markers in bovine spermatogenesis revealed by single-cell sequencing

Testicular development is an important biological process in male and requires interaction between the male germ cells and somatic cells. However, the mechanisms of testicular development in livestock, particularly in cattle, are poorly understood. Furthermore, cellular heterogeneity hinders the profiling of different cell types at different developmental stages. In this study, we first performed a single-cell transcriptomic study of the bovine testis development during puberty by using 10× genomics single-cell RNA sequencing (scRNA-seq). By collecting the scRNA-seq data from 11,083 cells from prepubertal and pubertal bovine testes, a high-resolution scRNA-seq atlas was described, identifying 9 somatic and 13 spermatogenic clusters. We also distinguished several stage-specific marker genes for bovine germ cells and somatic cells, such as GRAF2 and MORC1 for SSC (spermatogonial stem cells), HJURP and TCF19 for differentiating spermatogonia, ARSE for immature Sertoli, CLEC12B for mature Sertoli, LOC112441470 for Leydig. In conclusion, we have examined the transcription levels and constructed the single-cell developmental maps of germ cells and somatic cells during testicular development in Angus cattle. The datasets provided new insights into spermatogenesis and testicular somatic cell development in cattle.

РЕЗУЛЬТАТЫ ЭКСПЕРИМЕНТОВ ПО РАЗРАБОТКЕ ВЫСОКООЧИЩЕННОЙ ГИАЛУРОНАТ-ЭНДО-β-N-АЦЕТИЛГЕКСОЗАМИНИДАЗЫ В КАЧЕСТВЕ ФАРМАЦЕВТИЧЕСКОЙ СУБСТАНЦИИ ДЛЯ ЛЕКАРСТВЕННОГО ПРЕПАРАТА

Enzyme preparations, including hyaluronidase, are widely used in medical practice. Most drugs with this active substance on the pharmaceutical market are produced mainly from testicular hyaluronidase, the raw material for which are bovine testes. The technologies of enzyme preparation production are quite well known and diverse. However, the creation of a different method will allow the highly purified hyaluronate-endo-β-N-acetylhexosaminidase with high specific enzymatic activity. Objective: develop a technology and obtain a laboratory batch of the highly purified pharmaceutical substance hyaluronate-endo-β-N-acetylhexosaminidase. Materials and Methods. In the experimental study, preparations of bovine testes extracts were taken to obtain highly purified hyaluronate-endo-β-N-acetylhexosaminidase. In the first stage the protein spectrum of the testes extracts was studied, proteins were fractionally precipitated, and substances of different chemical nature were fractionated by chromatography methods. At the second stage, the development of a method to obtain the pharmaceutical substance hyaluronate-endo-β-N-acetylhexosaminidase was carried out using the methods described further in the work. Results. Based on the results obtained during the experimental study, P-II, a preparation of bovine testes extract produced by Samson-Med LLC (St. Petersburg), was chosen as the initial substance of crude hyaluronidase for further studies. The most optimal is the purification scheme, which includes as the main method affinity chromatography on heparin-sepharose using affinity elution with heparin solution in buffer with a concentration of 3 mg/ml. Conclusions As a result of the experimental study, the optimal scheme was presented to obtain the pharmaceutical substance HEBNA with high enzymatic total activity of 289 (unit of action) was presented, and a laboratory batch of highly purified hyaluronate-endo-β-N-acetylhexosaminidase was obtained.

A Comprehensive Sequencing Analysis of Testis-Born miRNAs in Immature and Mature Indigenous Wandong Cattle (Bos taurus).

Micro RNAs (miRNAs) have been recognized as important regulators that are indispensable for testicular development and spermatogenesis. miRNAs are endogenous transcriptomic elements and mainly regulate the gene expression at post-transcriptional levels; however, the key role of miRNA in bovine testicular growth is not clearly understood. Thus, supposing to unveil the transcriptomics expression changes in the developmental processes of bovine testes, we selected three immature calves and three sexually mature bulls of the local Wandong breed for testicular-tissue sample collection. The cDNA libraries of experimental animals were established for RNA-sequencing analysis. We detected the miRNA expression in testes by using high-throughput sequencing technology, and bioinformatics analysis followed. The differentially expressed (DE) data showed that 151 miRNAs linked genes were significantly DE between immature and mature bull testes. Further, in detail, 64 were significantly up-regulated and 87 were down-regulated in the immature vs. mature testes (p-value &lt; 0.05). Pathway analyses for miRNA-linked genes were performed and identified JAG2, BCL6, CFAP157, PHC2, TYRO3, SEPTIN6, and BSP3; these genes were involved in biological pathways such as TNF signaling, T cell receptor, PI3KAkt signaling, and functions affecting testes development and spermatogenesis. The DE miRNAs including MIR425, MIR98, MIR34C, MIR184, MIR18A, MIR136, MIR15A, MIR1388 and MIR210 were associated with cattle-bull sexual maturation and sperm production. RT-qPCR validation analysis showed a consistent correlation to the sequencing data findings. The current study provides a good framework for understanding the mechanism of miRNAs in the development of testes and spermatogenesis.

A SNP of the COX4I2 gene associated with environmental adaptation in Chinese cattle

COX4I2 is an isoform of cytochrome C oxidase subunit IV (COX4), which plays an important role in mitochondrial oxidative phosphorylation. This gene affects heat production and thus affects thermoregulatory capacity in mammals. A splice region variant (rs109072064, NC_037340.1:g.61202988C > T) was identified in COX4I2 by using Ensembl, which transforms the amino acid arginine into cysteine in XP_005214921.1. In this study, we sought to determine the relationship between the mutant locus and the environment in which the cattle are located. We verified that mRNA (XM_005214864.4), which translated XP_005214921.1, is expressed in bovine muscle, fat, heart, liver, kidney, lung and testis tissues. The g.61202988C > T variant was then genotyped in 569 individuals of 34 cattle breeds. Compared with the CC genotype, southern cattle carried more the CT and TT genotypes. Furthermore, the association results carried out that the frequencies of genotypes (CC, CT, TT) and the value of climate parameters (mean annual temperature (T), relative humidity (RH) and temperature humidity index (THI)) were significantly correlated (P < 0.01). Hence, we speculated that the g.61202988C > T variant of COX4I2 gene was associated with the environmental adaptation trait in Chinese cattle and the locus may be considered as a molecular marker for Chinese cattle breeding.

Localization and characterization of SSCs from pre-pubertal bovine testes

Self renewal and proliferation ability of spermatogonial stem cells (SSCs) support spermatogenesis during adult life. Theoretically, these stem cells can be utilized for transmission of genetic information to descendants via testicular transplantation. However, lack of knowledge in methodologies for identification of SSCs limits the application of SSCs transplantation in domestic animals. Accumulated studies have shown that SSCs specific markers (DBA, UCHL1) and stem cell marker (Sox2, Oct4) are useful to screen SSCs that able to be used for transplantation. However, in cattle, less information is available on the expression status of these markers till date. Therefore, a study was carried out in 2019 at Tsukuba University, Japan where testes from 3, 5 and 7 months old calves were utilized to examine testicular localization and in vitro propogation of stem cell markers. SSCs were isolated by enzymatic digestion combined with centrifugal separation on discontinuous Percoll density gradient. Cell propagation and SSCs marker expression were determined at 5, 10 and 15 days post-culture. Immunostaining in conjunction with Western Blot analysis of cultured cells showed that stem cell markers (UCHL1, Oct4 and Sox2) were expressed in SSCs suggesting that differentiation of gonocyte started by 3 months and SSCs differentiation begins after 5 months of age. Taken together, these results demonstrated marker expression and localization of bull SSCs and showed that in vitro culturing of bull SSCs is implementable.

Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations.

Simple SummaryOrganoids are 3D-cell culture systems composed of tissue-specific cells that create structures similar to those of their tissue of origin. Organoids have generated great interest in recent years, since they are considered a useful tool to perform laboratory studies in different scientific areas including reproduction. Testicular organoids (TOs) may provide an innovative model for the study of testicular physiology in various species; however, no previous studies have reported on the production of TOs in the bovine species. Thus, in the present study, we sought to generate and characterize bovine TOs, derived from testicular cell populations that include Leydig, Sertoli and peritubular myoid cells. After isolation from testes and characterization, testicular cells were cultured in ultra-low attachment dishes. Testicular cells formed TOs after 3 days of culture. Leydig, Sertoli and peritubular myoid cells displayed specific locations and changed in number in TOs. Moreover, bovine TOs were able to produce and increase the concentration of testosterone after 27 days of culture. The present study represents the first report on the generation and characterization of bovine TOs. These TOs could be useful tools to evaluate the impact of exogenous factors on the physiology of sperm production and testis development in domestic and wild cattle.Organoids are 3D-culture systems composed of tissue-specific primary cells that self-organize and self-renew, creating structures similar to those of their tissue of origin. Testicular organoids (TOs) may recreate conditions of the testicular niche in domestic and wild cattle; however, no previous TO studies have been reported in the bovine species. Thus, in the present study, we sought to generate and characterize bovine TOs derived from primary testicular cell populations including Leydig, Sertoli and peritubular myoid cells. Testicular cells were isolated from bovine testes and cultured in ultra-low attachment (ULA) plates and Matrigel. TOs were cultured in media supplemented from day 3 with 100 ng/mL of BMP4 and 10 ng/mL of FGF2 and from day 7 with 15 ng/mL of GDNF. Testicular cells were able to generate TOs after 3 days of culture. The cells positive for STAR (Leydig) and COL1A (peritubular myoid) decreased (p < 0.05), whereas cells positive for WT1 (Sertoli) increased (p < 0.05) in TOs during a 28-day culture period. The levels of testosterone in media increased (p < 0.05) at day 28 of culture. Thus, testicular cells isolated from bovine testes were able to generate TOs under in vitro conditions. These bovine TOs have steroidogenic activity characterized by the production of testosterone.

Copy number variation of ZNF280BY across eight sheep breeds and its association with testicular size of Hu sheep.

ZNF280BY, a bovid-specific Y chromosome gene, was firstly found to be highly expressed in bovine testis, indicating that it may play important roles in testicular development and male fertility. In this study, we firstly cloned the full-length cDNA of ovine ZNF280BY containing 1,993 bp, and with a 1,632 bp open reading frame. ZNF280BY was predominantly expressed in the testis, and its expression level was significantly higher in large testis than in small testis in Hu sheep at 6 months of age. In addition, the expression level of ZNF280BY significantly increased during testicular development, showing the highest expression level at 12 mo of age. ZNF280BY showed copy number variation (CNV) in 723 rams from eight sheep breeds, ranging from 17 to 514 copies, with a median copy number of 188. Pearson correlation analysis showed that the CNV of ZNF280BY was negatively correlated with testis size in Hu sheep. Furthermore, its mRNA expression level in testis had no significant correlation with the CNV but was significantly correlated with testis size. This study concluded that the expression of ZNF280BY was closely related to testicular development, and the CNV of ZNF280BY could be used as an important genetic marker to evaluate the ram reproductive capacity at an early stage in Hu sheep.

Characteristics of alpha smooth muscle actin-positive peritubular cells in prepubertal bovine testes

Characteristics of peritubular myoid cells (PMCs) in sexually immature cattle remain largely unknown. Here, we report the character and behavior of peritubular cells expressing alpha smooth muscle actin (αSMA), a marker of PMCs in adult testes, in prepubertal testes procured from 5-months-old bulls. Elastin distribution around αSMA + PMCs was dim and discontinuous in prepubertal testes, but strong and continuous in adult testes. Fibroblast-specific protein 1 (FSP1) was rarely expressed in αSMA + PMCs of prepubertal testes, while in adult testes, majority of αSMA + PMCs were FSP1+. Moreover, αSMA + PMCs in prepubertal testes proliferated more actively than those in adult testes. In vitro culture of isolated seminiferous tubules from prepubertal testes revealed that αSMA + PMCs migrate from peritubular to interstitial area. Hence, in prepubertal bovine testes, (1) PMCs rarely portray fibroblast-like properties, (2) PMCs exhibit heterogeneity in FSP1 expression, (3) PMCs proliferate more actively than those in adult testes, and (4) PMCs have a potential to migrate to the interstitium. Our observations help to understand the maturation of PMCs and their involvement in bovine testicular function.

The Dynamic of PRAMEY Isoforms in Testis and Epididymis Suggests Their Involvement in Spermatozoa Maturation.

The preferentially expressed antigen in melanoma, Y-linked (PRAMEY) is a cancer/testis antigen expressed predominantly in bovine spermatogenic cells, playing an important role in germ cell formation. To better understand PRAMEY’s function during spermatogenesis, we studied the dynamics of PRAMEY isoforms by Western blotting (WB) with PRAMEY-specific antibodies. The PRAMEY protein was assessed in the bovine testicular and epididymal spermatozoa, fluid and tissues, and as well as in ejaculated semen. The protein was further examined, at a subcellular level in sperm head and tail, as well as in the subcellular components, including the cytosol, nucleus, membrane, and mitochondria. RNA expression of PRAMEY was also evaluated in testis and epididymal tissues. Our WB results confirmed the previously reported four isoforms of PRAMEY (58, 30, 26, and 13 kDa) in the bovine testis and spermatozoa. We found that testicular spermatozoa expressed the 58 and 30 kDa isoforms. As spermatozoa migrated to the epididymis, they expressed two additional isoforms, 26 and 13 kDa. Similarly, the 58 and 30 kDa isoforms were detected only in the testis fluid, while all four isoforms were detected in fluid from the cauda epididymis. Tissue evaluation indicated a significantly higher expression of the 58 and 13 kDa isoforms in the cauda tissue when compared to both the testis and caput tissue (p < 0.05). These results indicated that testis samples (spermatozoa, fluid, and tissue) expressed predominantly the 58 and 30 kDa PRAMEY isoforms, suggesting their involvement in spermatogenesis. In contrast, the 26 kDa isoform was specific to epididymal sperm and the 13 kDa isoform was marked in samples derived from the cauda epididymis, suggesting their involvement in sperm maturation. Results from the sperm head and tail experiments indicated that the 13 kDa isoform increased 4-fold in sperm tails from caput to cauda, suggesting this isoform may have a significant role in tail function. Additionally, the 13 kDa isoform increased significantly (p < 0.05) in the cytosol during epididymal passage and tended to increase in other subcellular components. The expression of PRAMEY in the sperm subcellular components during epididymal maturation suggests the involvement of PRAMEY, especially the 13 kDa isoform, in sperm motility.

Transcriptome-Wide m6A Analysis Provides Novel Insights Into Testicular Development and Spermatogenesis in Xia-Nan Cattle.

Testis is the primary organ of the male reproductive tract in mammals that plays a substantial role in spermatogenesis. Improvement of our knowledge regarding the molecular mechanisms in testicular development and spermatogenesis will be reflected in producing spermatozoa of superior fertility. Evidence showed that N6-Methyladenosine (m6A) plays a dynamic role in post-transcription gene expression regulation and is strongly associated with production traits. However, the role of m6A in bovine testis has not been investigated yet. In this study, we conducted MeRIP-Seq analysis to explore the expression profiles of the m6A and its potential mechanism underlying spermatogenesis in nine bovine testes at three developmental stages (prepuberty, puberty and postpuberty). The experimental animals with triplicate in each stage were chosen based on their semen volume and sperm motility except for the prepuberty bulls and used for testes collection. By applying MeRIP-Seq analysis, a total of 8,774 m6A peaks and 6,206 m6A genes among the studied groups were identified. All the detected peaks were found to be mainly enriched in the coding region and 3′- untranslated regions. The cross-analysis of m6A and mRNA expression exhibited 502 genes with concomitant changes in the mRNA expression and m6A modification. Notably, 30 candidate genes were located in the largest network of protein-protein interactions. Interestingly, four key node genes (PLK4, PTEN, EGR1, and PSME4) were associated with the regulation of mammal testis development and spermatogenesis. This study is the first to present a map of RNA m6A modification in bovine testes at distinct ages, and provides new insights into m6A topology and related molecular mechanisms underlying bovine spermatogenesis, and establishes a basis for further studies on spermatogenesis in mammals.

Circular RNA Expression and Regulation Profiling in Testicular Tissues of Immature and Mature Wandong Cattle (Bos taurus).

Wandong cattle are an autochthonous Chinese breed used extensively for beef production. The breed tolerates extreme weather conditions and raw feed and is resistant to tick-borne diseases. However, the genetic basis of testis development and sperm production as well as breeding management is not well established in local cattle. Therefore, improving the reproductive efficiency of bulls via genetic selection is crucial as a single bull can breed thousands of cows through artificial insemination (AI). Testis development and spermatogenesis are regulated by hundreds of genes and transcriptomes. However, circular RNAs (circRNAs) are the key players in many biological developmental processes that have not been methodically described and compared between immature and mature stages in Bovine testes. In this study, we performed total RNA-seq and comprehensively analyzed the circRNA expression profiling of the testis samples of six bulls at 3 years and 3 months of developmental age. In total, 17,013 circRNAs were identified, of which 681 circRNAs (p-adjust < 0.05) were differentially expressed (DE). Among these DE circRNAs, 579 were upregulated and 103 were downregulated in calf and bull testes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the identified target genes were classified into three broad functional categories, including biological process, cellular component, and molecular function, and were enriched in the lysine degradation, cell cycle, and cell adhesion molecule pathways. The binding interactions between DE circRNAs and microRNAs (miRNAs) were subsequently constructed using bioinformatics approaches. The source genes ATM, CCNA1, GSK3B, KMT2C, KMT2E, NSD2, SUCLG2, QKI, HOMER1, and SNAP91 were found to be actively associated with bull sexual maturity and spermatogenesis. In addition, a real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed a strong correlation with the sequencing data. Moreover, the developed model of Bovine testes in the current study provides a suitable framework for understanding the mechanism of circRNAs in the development of testes and spermatogenesis.

Comprehensive Analysis of miRNAs and Target mRNAs between Immature and Mature Testis Tissue in Chinese Red Steppes Cattle.

Simple SummaryMicroRNAs are small molecules that can regulate the relative abundance of their target genes by binding to the 3′ untranslated region of the target genes at the post-transcriptional level to affect various biological processes, such as biosynthesis, fat metabolism and proliferation, apoptosis, and cell differentiation. Fertility is one of the most important economic traits in livestock production. Bulls require the continuous production of high-quality spermatozoa in abundance. The quality of semen is an exceptionally important factor affecting the fertilization rate of the dairy cow and is also associated with the increasing conception rate in the process of artificial insemination. Therefore, accurately predicting fertility potential for a semen sample from donor bull for artificial insemination is crucial for consistently high reproductive efficiency. The present study performed a genome-wide sequencing analysis of microRNAs and mRNAs between immature and mature testes of Chinese Red Steppes. These results provide novel candidate microRNAs and functional genes related to bull reproduction traits and the networks between microRNAs and target genes, which will provide a useful genetic mechanism and epigenetic information for marker-assisted selection of bulls with excellent sperm quality in the future.This study aims to screen potential regulators and regulate fecundity networks between microRNAs (miRNAs) and target genes. The bovine testes of immature and mature Chinese Red Steppes were performed by genome-wide analysis of mRNAs and miRNAs. Compared with testicular tissues of newborns, 6051 upregulated genes and 7104 downregulated genes in adult cattle were identified as differentially expressed genes (DEGs). The DEGs were significantly enriched in 808 GO terms (p < 0.05) including male gonad development, male genitalia development, spermatogenesis, and sperm motility. Moreover, DEGs were also significantly enriched in 105 KEGG pathways (p < 0.05), including cGMP-PKG signaling pathway and calcium signaling pathway. To explore the expression of miRNA-regulated gene expression, 896 differentially expressed target genes negatively regulated with the expression levels of 31 differentially expressed miRNAs (DERs) were predicted and analyzed, and a network-integrated analysis was constructed. Furthermore, real-time PCR was performed to verify the expression levels of DEGs and DERs. Our results identified novel candidate DEGs and DERs correlated with male reproduction and intricate regulating networks between miRNAs and genes, which will be valuable for future genetic and epigenetic studies of sperm development and maturity, as well as providing valuable insights into the molecular mechanisms of male fertility and spermatogenesis in cattle.