The positive feedback loop of the NAT10/Mybbp1a/p53 axis promotes cardiomyocyte ferroptosis to exacerbate cardiac I/R injury.

Ferroptosis is a nonapoptotic form of regulated cell death that has been reported to play a central role in cardiac ischemia‒reperfusion (I/R) injury. N-acetyltransferase 10 (NAT10) contributes to cardiomyocyte apoptosis by functioning as an RNA ac4c acetyltransferase, but its role in cardiomyocyte ferroptosis during I/R injury has not been determined. This study aimed to elucidate the role of NAT10 in cardiac ferroptosis as well as the underlying mechanism. The mRNA and protein levels of NAT10 were increased in mouse hearts after I/R and in cardiomyocytes that were exposed to hypoxia/reoxygenation. P53 acted as an endogenous activator of NAT10 during I/R in a transcription-dependent manner. Cardiac overexpression of NAT10 caused cardiomyocyte ferroptosis to exacerbate I/R injury, while cardiomyocyte-specific knockout of NAT10 or pharmacological inhibition of NAT10 with Remodelin had the opposite effects. The inhibition of cardiomyocyte ferroptosis by Fer-1 exerted superior cardioprotective effects against the NAT10-induced exacerbation of post-I/R cardiac damage than the inhibition of apoptosis by emricasan. Mechanistically, NAT10 induced the ac4C modification of Mybbp1a, increasing its stability, which in turn activated p53 and subsequently repressed the transcription of the anti-ferroptotic gene SLC7A11. Moreover, knockdown of Mybbp1a partially abolished the detrimental effects of NAT10 overexpression on cardiomyocyte ferroptosis and cardiac I/R injury. Collectively, our study revealed that p53 and NAT10 interdependently cooperate to form a positive feedback loop that promotes cardiomyocyte ferroptosis to exacerbate cardiac I/R injury, suggesting that targeting the NAT10/Mybbp1a/p53 axis may be a novel approach for treating cardiac I/R.

Extreme precipitation accelerates nitrate leaching in the intensive agricultural region with thick unsaturated zones.

Nitrate accumulation in the soil profile in the intensive agricultural region has been widely concerned in the world. However, the changes in nitrate accumulation characteristics caused by climate change, such as extremely high precipitation, are not well quantified, particularly for the regions with thick unsaturated zones. Here, we resampled the soil profiles taken in normal year (2020) after extreme precipitation year (2021) (>800 cm) in three regions in the southern Loess Plateau (LP) with three different water managements including rainfed orchards (n = 10), well-irrigated orchards (n = 4) and canal-irrigated orchards (n = 8). The accumulation amounts, peak depths, and accumulation depths of nitrate soil profiles of the different regions of two years were compared. The results showed that average nitrate accumulation in normal year at the rainfed region (800-cm depth), well-irrigated region (800-cm depth) and canal-irrigated region (1400-cm depth) were 5995 kg N ha-1, 9765 kg N ha-1, and 19,608 kg N ha-1, respectively. Compared with 2020, extreme precipitation in 2021 led to 56-91% reductions (2060-3702 kg N ha-1) in nitrate accumulation in 0-200 cm soil layer, and average nitrate leaching into the aquifer was >1390 kg N ha-1 in the canal-irrigated region. Average migration depths of nitrate peak in rainfed, well-irrigated and canal-irrigated regions were 92 cm, 115 cm, and 188 cm, respectively; as for nitrate accumulation depths, they were 10 cm, 80 cm and 108 cm, respectively. Vertically, the dried soil layer and paleosol layer (high clay content) in the canal-irrigated region significantly hindered nitrate deep migration caused by the extreme precipitation. The result highlights that extreme precipitation significantly accelerated nitrate leaching in the deep soil profiles, and future vulnerability and risk assessment studies must account for the impacts of extreme precipitation on nitrate leaching.

Transcriptomic analysis of the HPG axis-related tissues reveals potential candidate genes and regulatory pathways associated with testicular size in Hu sheep.

Testicular size is an excellent proxy for selecting high-fertility rams. The hypothalamus-pituitary-gonadal (HPG) axis plays an important role in regulating reproductive capacity in vertebrates, while key genes and regulatory pathways within the HPG axis associated with testicular size remain largely unknown in sheep. This study comprehensively compared the transcriptomic profiles in the hypothalamus, pituitary and testis of rams after sexual maturity between the large-testis group (LTG, testicular weight = 454.29 ± 54.24 g) and the small-testis group (STG, testicular weight = 77.29 ± 10.76 g). In total, 914, 795 and 10518 differentially expressed genes (DEGs) were identified in the hypothalamus, pituitary and testis between LTG and STG, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these DEGs were mainly involved in the biological processes of reproduction, biological regulation, and development process. Notably, the neuroactive ligand-receptor interaction and cAMP signaling pathways, commonly enriched by the DEGs in the hypothalamus and pituitary between two groups, were considered as two key signal pathways regulating testicular development through the HPGs axis. Weighted gene co-expression network analysis (WGCNA) identified two modules that were significantly associated with testicular size, and 97 key genes were selected with high module membership (MM) and gene significance (GS) in these two modules. Finally, a protein-protein interaction (PPI) network was constructed, and ten genes with the highest degree were represented as hub genes, including FOS, NPY, SST, F2, AGT, NTS, OXT, EDN1, VIP and TAC1. Taken together, these results provide new insights into the molecular mechanism underlying the HPG axis regulating testicular size of Hu sheep.

Effect of Codonopsis pilosula polysaccharide on the quality of sheep semen preservation at 4°C.

Objective: This study aimed to investigate the effect of Codonopsis pilosula polysaccharide (CPP) on the motility, mitochondrial integrity, acrosome integrity rate, and antioxidant ability of sheep sperm after preservation at 4 °C. Methods: Semen from healthy adult rams were collected and divided into four groups with separate addition of 0, 200, 400, and 1000 mg/L CPP. Sperm motility was analyzed using the Computer-Assisted Semen Analysis software after preservation at 4 °C for 24, 72, 120, and 168 h. Sperm acrosome integrity rate was analyzed by Giemsa staining at 24, 72, and 120 h, and mitochondrial membrane integrity was analyzed by Mito-Tracker Red CMXRos. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of spermatozoa were measured after 120 h of preservation. Results: The sperm viability and forward-moving sperm under 200 mg/L CPP were significantly higher than that in the control group at 72 h (61.28% ± 3.89% vs. 52.83% ± 0.70%,51.53% ±4.06% vs.42.84% ± 1.14%), and 168 h (47.21% ±0.85% vs.41.43% ± 0.37%, 38.68% ±0.87% vs. 31.68% ± 0.89%). The percentage of fast-moving sperm (15.03% ± 1.10% vs.11.39% ± 1.03%) and slow-moving sperm (23.63% ± 0.76% vs.20.29% ± 1.11%) in the 200 mg/L group was significantly higher than control group at 168 h. The mitochondrial membrane integrity of the sperm in the group with 200 mg/L CPP was significantly higher than those in the control group after storage at 4 °C for 120 h (74.76% ± 2.54% vs. 65.67% ± 4.51%, p < 0.05). The acrosome integrity rate in the group with 200 mg/L (87.66%±1.26%) and 400 mg/L （84.00%±2.95%）was significantly higher than those in the control group (80.65%±0.16%) after storage for 24 h (p < 0.05). CPP also increased T-AOC and decreased the MDA concentration after preservation at 4 °C (p< 0.05). Conclusion: Adding CPP could improve the T-AOC of sperm, inhibit lipid peroxidation, and facilitate semen preservation.

Localization and expression of phospholipase A2 and polyunsaturated fatty acid profile in the testis tissues of Hu sheep.

The fatty acid content and the localization and expression of phospholipase A2 (PLA2) in the testis of Hu sheep were investigated. A total of 18 six-month-old Hu sheep were divided into small group (S, with left testis weight < 50 g), medium group (M, with left testis weight among 90-110 g), and large group (L, with left testis weight >160 g), which had six individuals each. The expression of PLA2 in testicular tissues of different sizes was analyzed by immunohistochemistry, RT-qPCR, and Western blot. The fatty acid profile was detected by gas chromatography. Immunohistochemical labeling determined that PLA2 protein was expressed in the Leydig and Sertoli cells of testis, and the immunohistochemical average optional density in the S group was significantly greater than the L group (P < 0.05). RT-qPCR and Western blot analysis showed that PLA2 in the S group was greater than that in the L group (P < 0.05). Docosahexaenoic acid, ω-3 polyunsaturated fatty acid (PUFA), and total PUFA content in the testis of the L group were significantly less than those of the S and M groups (P < 0.01). This study showed that PLA2 content in the S group was greater than that in the L group.

Molecular characterization of SPATA6 and association of its SNPs with testicular size in sheep.

The testis is an important organ for maintaining fertility in males, and testis size is positively correlated with ejaculate volume, sperm motility, thus fertility. Spermatogenesis-associated 6 (SPATA6) is an evolutionarily conserved testis-specific gene reported in many species. However, the effect of SPATA6 expression levels on testicular development and the effect of single nucleotide polymorphisms (SNPs) on testis and epididymis phenotype in sheep have not been studied. The purpose of the research was to investigate the expression profile of SPATA6 and its effect on testicular development and to confirm the effect of SNPs on the testis and epididymis phenotype. In this study, we detected a 1245bp coding sequence (CDS) of SPATA6 and encoded 414 amino acids. The expression levels of SPATA6 were significantly higher in the testis than in other tissues and gradually increased with testis development. Moreover, the expression level in the large testis was significantly higher than that in the small testis at six months. A total of 11 SNPs were detected in the coding region of SPATA6 by cDNA-pooling sequencing and improved multiplex ligation detection reaction (iMLDR) methods. Correlation analysis showed that SNP2 (c. 3631C > G) significantly affected left epididymis weight (LEW) and right epididymis weight (REW), and SNP10 (c. 937 A > G) significantly affected REW. And the combined genotype of SNP1 (c. 4245 G > A) and SNP2 significantly affected REW. The current study concluded that SPATA6 plays an important role in testicular development and the SNPs significantly associated with the epididymis phenotype can provide molecular markers for the early selection of high-fertility Hu sheep.

Heritability and recursive influence of host genetics on the rumen microbiota drive body weight variance in male Hu sheep lambs.

BACKGROUND: Heritable rumen microbiota is an important modulator of ruminant growth performance. However, no information exists to date on host genetics-rumen microbiota interactions and their association with phenotype in sheep. To solve this, we curated and analyzed whole-genome resequencing genotypes, 16S rumen-microbiota data, and longitudinal body weight (BW) phenotypes from 1150 sheep. RESULTS: A variance component model indicated significant heritability of rumen microbial community diversity. Genome-wide association studies (GWAS) using microbial features as traits identified 411 loci-taxon significant associations (P < 10-8). We found a heritability of 39% for 180-day-old BW, while also the rumen microbiota likely played a significant role, explaining that 20% of the phenotypic variation. Microbiota-wide association studies (MWAS) and GWAS identified four marker genera (Bonferroni corrected P < 0.05) and five novel genetic variants (P < 10-8) that were significantly associated with BW. Integrative analysis identified the mediating role of marker genera in genotype influencing phenotype and unravelled that the same genetic markers have direct and indirect effects on sheep weight. CONCLUSIONS: This study reveals a reciprocal interplay among host genetic variations, the rumen microbiota and the body weight traits of sheep. The information obtained provide insights into the diverse microbiota characteristics of rumen and may help in designing precision microbiota management strategies for controlling and manipulating sheep rumen microbiota to increase productivity. Video Abstract.

Domestication, breeding, omics research, and important genes of Zizania latifolia and Zizania palustris.

Wild rice (Zizania spp.), an aquatic grass belonging to the subfamily Gramineae, has a high economic value. Zizania provides food (such as grains and vegetables), a habitat for wild animals, and paper-making pulps, possesses certain medicinal values, and helps control water eutrophication. Zizania is an ideal resource for expanding and enriching a rice breeding gene bank to naturally preserve valuable characteristics lost during domestication. With the Z. latifolia and Z. palustris genomes completely sequenced, fundamental achievements have been made toward understanding the origin and domestication, as well as the genetic basis of important agronomic traits of this genus, substantially accelerating the domestication of this wild plant. The present review summarizes the research results on the edible history, economic value, domestication, breeding, omics research, and important genes of Z. latifolia and Z. palustris over the past decades. These findings broaden the collective understanding of Zizania domestication and breeding, furthering human domestication, improvement, and long-term sustainability of wild plant cultivation.

iTRAQ-based proteomic analysis provides novel insight into the postnatal testicular development of Hu sheep.

Testicular development is an intricate and coordinated process in which thousands of proteins are involved in the regulation of somatic cells development and spermatogenesis. However, knowledge about the proteomic changes during postnatal testicular development in Hu sheep is still elusive. The study was conducted to characterize the protein profiles at four key stages during postnatal testicular development, including infant (0-month-old, M0), puberty (3-month-old, M3), sexual maturity (6-month-old, M6) and body maturity (12-month-old, M12), and between the large- and small-testis groups at 6 months in Hu sheep. Consequently, 5252 proteins were identified using isobaric tags for relative and absolute quantification (iTRAQ) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, and 465, 1261, 231 and 1080 differentially abundant proteins (DAPs) were found between M0_vs_M3, M3_vs_M6L, M6L_vs_M12, and M6L_vs_M6S, respectively. GO and KEGG analysis revealed that the majority of DAPs were involved in cellular process, metabolic process and immune system-related pathways. Furthermore, a protein-protein interaction network was constructed using 86 fertility-related DAPs, and five proteins with the highest degree were represented as hub proteins, including CTNNB1, ADAM2, ACR, HSPA2 and GRB2. This study provided new insights into the regulation mechanisms of postnatal testicular development and identified several potential biomarkers for selecting the high-fertility rams. SIGNIFICANCE OF THE STUDY: Testicular development is an intricate developmental process in which thousands of proteins are involved in regulating the somatic cells development and spermatogenesis. However, knowledge about the proteome changes during postnatal testicular development in Hu sheep is still elusive. This study provides comprehensive insights into the dynamic changes in the sheep testis proteome during postnatal testicular development. Additionally, testis size is positively correlated with semen quality and ejaculation volume, also for the merits of easy measurement, high heritability and selection efficiency, is an important indicator to select candidate rams with high fertility. The functional analyses of the acquired candidate proteins may help us gain a better understanding of the molecular regulatory mechanisms of testicular development.

Region-Controlled Framework Interface Mediated Anion Exchange Chemical Transformation to Designed Metal Phosphosulfide Heteronanostructures.

Postsynthetic chemical transformation provides a powerful platform for creating heteronanostructures (HNs) with well-defined materials and interfaces that generate synergy or enhancement. However, it remains a synthetic bottleneck for the precise construction of HNs with increased degrees of complexity and more elaborate functions in a predictable manner. Herein, we define a general transformative protocol for metal phosphosulfide HNs based on tunable hexagonal Cu1.81S frameworks with corner-, edge- and face-controlled growth of Co2P domains. The region-controlled Cu1.81S-Co2P framework interfaces can serve as "kinetic barriers" in mediating the direction and rate between P and S anion exchange reactions, thus leading to a family of morphology and phase designed Cu3P1-xSx-Co2P HNs with hollow (branched, dotted and crown), porous and core-shell architectures. This study reveals the internal transformation mechanism between metal sulfide and phosphide nanocrystals, and opens up a new way for the rational synthesis of metastable HNs that are otherwise inaccessible.

PDGF-D-induced immunoproteasome activation and cell-cell interactions.

Platelet-derived growth factor-D (PDGF-D) is abundantly expressed in ocular diseases. Yet, it remains unknown whether and how PDGF-D affects ocular cells or cell-cell interactions in the eye. In this study, using single-cell RNA sequencing (scRNA-seq) and a mouse model of PDGF-D overexpression in retinal pigment epithelial (RPE) cells, we found that PDGF-D overexpression markedly upregulated the key immunoproteasome genes, leading to increased antigen processing/presentation capacity of RPE cells. Also, more than 6.5-fold ligand-receptor pairs were found in the PDGF-D overexpressing RPE-choroid tissues, suggesting markedly increased cell-cell interactions. Moreover, in the PDGF-D-overexpressing tissues, a unique cell population with a transcriptomic profile of both stromal cells and antigen-presenting RPE cells was detected, suggesting PDGF-D-induced epithelial-mesenchymal transition of RPE cells. Importantly, administration of ONX-0914, an immunoproteasome inhibitor, suppressed choroidal neovascularization (CNV) in a mouse CNV model in vivo. Together, we show that overexpression of PDGF-D increased pro-angiogenic immunoproteasome activities, and inhibiting immunoproteasome pathway may have therapeutic value for the treatment of neovascular diseases.

Effect of corn straw or corncobs in total mixed ration during peri-puberty on testis development in Hu lambs.

Corn straw and corncobs contain large amounts of crude fibers and are widely used in mutton sheep husbandry in northwest China. The aim of this study was to determine whether feeding with corn straw or corncobs affects lamb testis development. A total of 50 healthy Hu lamb at two-month-old (average body weight of 22.3 ± 0.1 kg) were randomly and equally divided into two groups, and the lambs were equally allocated to five pens in each group. The corn straw group (CS) received a diet containing 20% corn straw, whereas the corncobs group (CC) received a diet containing 20% corncobs. After a 77-day feeding trial, the lambs, except the heaviest and lightest in each pen, were humanely slaughtered and investigated. Results revealed no differences in body weight (40.38 ± 0.45 kg vs. 39.08 ± 0.52 kg) between the CS and CC groups. Feeding diet containing corn straw significantly (P < 0.05) increased testis weight (243.24 ± 18.78 g vs. 167.00 ± 15.20 g), testis index (0.60 ± 0.05 vs. 0.43 ± 0.04), testis volume (247.08 ± 19.99 mL vs. 162.31 ± 14.15 mL), diameter of seminiferous tubule (213.90 ± 4.91 µm vs. 173.11 ± 5.93 µm), and the number of sperm in the epididymis (49.91 ± 13.53 × 108/g vs. 19.34 ± 6.79 × 108/g) compared with those in the CC group. The RNA sequencing results showed 286 differentially expressed genes, and 116 upregulated and 170 downregulated genes were found in the CS group compared with the CC group. The genes affecting immune functions and fertility were screened out. Corn straw decreased the mtDNA relative copy number in the testis (P < 0.05). These results suggest that compared with corncobs, feeding corn straw in the early reproductive development stage of lambs increased the testis weight, diameter of seminiferous tubule and the number of cauda sperm.

Differences of the anti-oxidative capability, GPX3, and Cu/ZnSOD expression in Hu sheep testis with different size at six-month-old.

This study aimed to investigate the differences in the anti-oxidant capabilities and related gene expressions of six-month-old Hu sheep with different testis sizes. A total of 201 Hu ram lambs were fed up to 6 months in the same environment. Based on their testis weight and sperm count, 18 individuals were selected and divided into large (n = 9) and small (n = 9) groups, with an average testis weight of 158.67 g ± 5.21 g and 44.58 g ± 4.14 g, respectively. The total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and malondialdehyde (MDA) concentration in testis tissue were tested. The localization of antioxidant-related genes, GPX3 and Cu/ZnSOD in testis were detected by immunohistochemistry. The GPX3, Cu/ZnSOD expression, and relative mitochondrial DNA (mtDNA) copy number were detected by quantitative real-time PCR. Compared with the small group, the T-AOC (2.69 ± 0.47 vs. 1.16 ± 0.22 U/mgprot) and T-SOD (22.35 ± 2.59 vs. 9.92 ± 1.62 U/mgprot) in the large group were significantly higher, whereas the MDA (0.72 ± 0.13 vs. 1.34 ± 0.17 nM/mgprot) and relative mtDNA copy number in the large group was significantly lower (p < .05). Immunohistochemistry results indicated that the GPX3 and Cu/ZnSOD were expressed in Leydig cells and seminiferous tubule. The expressions of GPX3 and Cu/ZnSOD mRNA in the large group were significantly higher than those in the small group (p < .05). In conclusion, Cu/ZnSOD and GPX3 widely expressed in the Leydig cells and seminiferous tubule, high expression of Cu/ZnSOD and GPX3 in a large group has a higher potential in addressing oxidative stress and contribute to spermatogenesis.

Characterization of highly polymorphic microsatellite markers for the chinese monal (Lophophorus lhuysii, Galliformes) using Illumina MiSeq sequencing.

BACKGROUND: The Chinese monal (Lophophorus lhuysii, Galliformes) is a vulnerable and endemic bird from southwestern China. To better protect this species and increase its population size, genetic markers are urgently needed for investigation and conservation of both wild and captive populations. METHODS AND RESULTS: By using next-generation sequencing, we developed and characterized markers for seven microsatellite loci of the Chinese monal. PCR examination and statistical analysis indicated that these microsatellites exhibited moderate to high levels of polymorphism, with the expected heterozygosity and polymorphic information content ranging from 0.578 to 0.858 and from 0.540 to 0.841, respectively. Cross-species genome comparison further suggests that these microsatellites are a feature of certain galliform species rather than being specific to the Chinese monal. CONCLUSION: A combination of the seven highly polymorphic loci may provide a fundamental genetic toolkit to assess genetic backgrounds and will contribute to design conservation plan, breeding management and other possible studies of the Chinese monal and other evolutionarily related species in the future.

Variants of ADPGK gene and its effect on the male reproductive organ parameters and sperm count in Hu sheep.

ADP-dependent glucokinase (ADPGK) plays an important role instead of hexokinase in regulating energy metabolism via the Embden-Meyerhof-Parnas Pathway. And energy provided via glycolysis promotes testis development and spermatogenesis. In this study, 466 Hu sheep were screened for mutations in the ADPGK gene to examine the association of the ADPGK gene polymorphisms with the testis traits and spermatogenesis. The NC_056060.1: g.31295 C > T SNP was found in the 3'-UTR region, resulting in two genotypes CC and TC type with genotypic frequencies of 0.66 and 0.34, respectively. This mutation was significantly associated with testis weight, testis long circumference, testis short girth, epididymis weight, and sperm concentration (p < 0.05). Moreover, TC genotype individuals had an increased tendency in the expression of the ADPGK gene and had significant reproductive performance advantages compared with CC genotype individuals in the study. And compared with the small testes (<50 g), the ADPGK gene expression of big testes (>160 g) increased significantly. This indicates an association between the ADPGK gene and reproductive organ parameters and sperm count in selected Hu sheep breed, and this SNP may serve as an effective DNA molecular marker for marker-assisted selection in Hu sheep breeding programs.

Phase Transformation from Amorphous RuSx to Ru-RuS2 Hybrid Nanostructure for Efficient Water Splitting in Alkaline Media.

Ruthenium (Ru)-based materials, as a class of efficient hydrogen evolution reaction (HER) catalysts, play an important role in hydrogen generation by electrolysis of water in an alkaline solution for clean hydrogen energy. Hybrid nanostructure (HN) materials, which include two or more components with distinct functionality, show better performance than their individual materials, since HN materials can potentially integrate their advantages and overcome the weaknesses. However, it remains a challenge to construct Ru-based HN materials with desired crystal phases for enhanced HER performances. Herein, a series of new Ru-based HN materials (t-Ru-RuS2, S-Ru-RuS2, and T-Ru-RuS2) through phase engineering of nanomaterials (PEN) and chemical transformation are designed to achieve highly efficient HER properties. Owing to the plentiful formation of heterojunctions and amorphous/crystalline interfaces, the t-Ru-RuS2 HN delivers the most outstanding overpotential of 16 mV and owns a small Tafel slope of 29 mV dec-1 at a current density of 10 mA cm-2, which exceeds commercial Pt/C catalysts (34 mV, 38 mV dec-1). This work shows a new insight for HN and provides alternative opportunities in designing advanced electrocatalysts with low cost for HER in the hydrogen economy.

Composition of Fatty Acids and Localization of SREBP1 and ELOVL2 Genes in Cauda Epididymides of Hu Sheep with Different Fertility.

The epididymis is an organ that transports, matures and stores sperm, and has functions such as secretion and absorption. Polyunsaturated fatty acid (PUFA) compositions in sperm membrane were changed during the process of epididymis maturation and influence the male fertility. This study aimed to investigate differences in crude fat and fatty acid content in cauda epididymis between high and low fertility of Hu sheep. One hundred and seventy-nine Hu ram lambs were fed from 56 days to 6 months under the same environment. After the feeding trial, all rams were slaughtered, and the body weight, testicular weight, epididymal weight and sperm density were measured. Pearson correlation analysis showed significantly moderate positive correlation between epididymal weight and sperm density and testicular weight. Eighteen rams were selected and divided into the high fertility group (H, n = 9) and low fertility group (L, n = 9) according to the epididymal weight, sperm density and histomorphology. The crude fat content, fatty acid profile and genes related to fatty acid metabolism were detected. The crude fat content, total fatty acid, total n-3 PUFA and docosahexaenoic acid (C22:6n-3, DHA) content of cauda epididymis in high fertility group was significantly higher than those in low fertility group (p < 0.05). However, the ratio of n-6/n-3 PUFA was significantly lower than that in group L (p < 0.05). Immunohistochemistry results showed that SREBP1 and ELOVL2 were expressed in pseudostratified columnar ciliated epithelium and smooth muscle cells. The mRNA expression of SREBP1 (p = 0.09) and ELOVL2 (p < 0.05) in the high fertility group were increased. In conclusion, the high expression of SREBP1 and ELOVL2 may contribute to high n-3 PUFA content in cauda epididymis of high-fertility Hu sheep.

PRAMEY: A Bovid-Specific Y-Chromosome Multicopy Gene Is Highly Related to Postnatal Testicular Growth in Hu Sheep.

PRAMEY (preferentially expressed antigen in melanoma, Y-linked) belongs to the cancer-testis antigens (CTAs) gene family and is predominantly expressed in testis, playing important roles in spermatogenesis and testicular development. This study cloned the full-length cDNA sequence of ovine PRAMEY using the rapid amplification of cDNA ends (RACE) method and analyzed the expression profile and copy number variation (CNV) of PRAMEY using quantitative real-time PCR (qPCR). The results revealed that the PRAMEY cDNA was 2099 bp in length with an open reading frame (ORF) of 1536 bp encoding 511 amino acids. PRAMEY was predominantly expressed in the testis and significantly upregulated during postnatal testicular development. The median copy number (MCN) of PRAMEY was 4, varying from 2 to 25 in 710 rams across eight sheep breeds. There was no significant correlation between the CNV of PRAMEY and testicular size, while a significant positive correlation was observed between the mRNA expression and testicular size in Hu sheep. The current study suggests that the expression levels of PRAMEY were closely associated with testicular size, indicating that PRAMEY may play an important role in testicular growth.

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.

The zinc finger protein 280B-like, Y-linked ZNF280BY is a bovid-specific Y chromosome gene, playing important roles in testicular development and male fertility. In this study, we found that the ovine ZNF280BY was highly expressed in the testis and significantly upregulated during testicular development. The copy number of ZNF280BY was negatively correlated with testicular size in Hu sheep. Our results demonstrated that the CNV of ZNF280BY could be used as an important candidate marker for selecting elite rams at an early stage in Hu sheep.

Antioxidant properties and transcriptome of cauda epididymis with different levels of fertility in Hu lambs.

Oxidative stress damages the structure of sperm and affects its fertility. Further maturation and storage of spermatozoa occur in the epididymis. Thus, a balanced environment between free-radical production and the antioxidant-enzyme system is important. This study aimed to investigate differences in antioxidative activity in epididymis between high and low fertility levels of Hu sheep. A total of 179 Hu ram lambs aged 56 days were fed up to 6 months in the same environment. After feeding trial, all rams were slaughtered. According to the epididymis weight, cauda sperm concentration, and histo-morphological observation, 18 individuals were selected and divided into low fertility (n = 9) and high fertility (n = 9) groups. The RNA sequencing results showed 1404 differentially expressed genes (DEGs), with 735 genes upregulated and 669 genes downregulated in the high fertility group compared with the low fertility group. Sixteen DEGs related to lipid metabolism and oxidative stress, including GPX3, GPX5, LRP1, PLCB1, CRISP2, BSPH1, and ACSBG1, were screened by qPCR, and results were correlated well with the sequencing data. Antioxidative indices such as total antioxidant capacity (21.29 ± 5.97 vs. 30.23 ± 4.56 µM/gprot) and glutathione peroxidase (39.75 ± 7.61 vs. 68.84 ± 12.51 U/mgprot) increased in the high fertility group. However, the relative expression of mtDNA and malondialdehyde concentration (0.75 ± 0.15 vs. 0.54 ± 0.10 nmol/mgprot) and the reactive oxygen species (ROS) content significantly decreased (P < 0.05). Therefore, epididymis from high-fertility group had strong antioxidative activity and protected spermatozoa from ROS.