Sirtuin 3 regulation: a target to alleviate ß-hydroxybutyric acid-induced mitochondrial dysfunction in bovine granulosa cells.

BACKGROUND: During the transition period, the insufficient dry matter intake and a sharply increased in energy consumption to produce large quantities of milk, high yielding cows would enter a negative energy balance (NEB) that causes an increase in ketone bodies (KBs) and decrease in reproduction efficiency. The excess concentrations of circulating KBs, represented by ß-hydroxybutyric acid (BHBA), could lead to oxidative damage, which potentially cause injury to follicular granulosa cells (fGCs) and delayed follicular development. Sirtuin 3 (Sirt3) regulates mitochondria reactive oxygen species (mitoROS) homeostasis in a beneficial manner; however, the molecular mechanisms underlying its involvement in the BHBA-induced injury of fGCs is poorly understood. The aim of this study was to explore the protection effects and underlying mechanisms of Sirt3 against BHBA overload-induced damage of fGCs. RESULTS: Our findings demonstrated that 2.4 mmol/L of BHBA stress increased the levels of mitoROS in bovine fGCs. Further investigations identified the subsequent mitochondrial dysfunction, including an increased abnormal rate of mitochondrial architecture, mitochondrial permeability transition pore (MPTP) opening, reductions in mitochondrial membrane potential (MMP) and Ca2+ release; these dysfunctions then triggered the caspase cascade reaction of apoptosis in fGCs. Notably, the overexpression of Sirt3 prior to treatment enhanced mitochondrial autophagy by increasing the expression levels of Beclin-1, thus preventing BHBA-induced mitochondrial oxidative stress and mitochondrial dysfunction in fGCs. Furthermore, our data suggested that the AMPK-mTOR-Beclin-1 pathway may be involved in the protective mechanism of Sirt3 against cellular injury triggered by BHBA stimulation. CONCLUSIONS: These findings indicate that Sirt3 protects fGCs from BHBA-triggered injury by enhancing autophagy, attenuating oxidative stress and mitochondrial damage. This study provides new strategies to mitigate the fGCs injury caused by excessive BHBA stress in dairy cows with ketosis.

Identification of spermatogenesis-related lncRNA in Holstein bull testis after sexual maturity based on transcriptome analysis.

Under the large-scale breeding model, the performance of the Holstein bull is directly related to the economic efficiency of the whole dairy farm and is the key factor affecting the genetic quality of the herd. Although the number of reported studies on the association of long noncoding RNAs (lncRNAs) with male reproduction is increasing, there is a lack of research on how lncRNAs regulate Holstein bull testicular development and spermatogenesis. To explore the molecular mechanisms between lncRNAs and spermatogenesis, three 8-week-old Holstein bull (young bull, YB) testes and three 80-week-old Holstein bull (adult bull, AB) testes from the same herd were randomly chosen, and transcriptome analysis was performed to find associations between spermatogenesis and transcriptome profiles. About 16,956 differentially expressed lncRNAs (DELs) and 7970 differentially expressed mRNAs (DEMs) were identified, and 3642 significantly relationship pairs were screened out based on co-expression analysis. Further Hub analysis obtained 6 lncRNAs, and 71 mRNAs regulated by them were enriched subsequently. Functional analysis results of differentially expressed Hub LncRNA-mRNA pairs showed that more positive cell cycle checkpoints, spindle assembly and the sister chromatids separation in AB testes indicated higher production rates of sperm in AB as compared to YB. This study reveals physiological changes and further broaden our understanding of lncRNA regulation of spermatogenesis in the fully mature testis according to the transcriptome profiles.

Uncovering Novel Features of the Pc Locus in Horn Development from Gene-Edited Holstein Cattle by RNA-Sequencing Analysis.

The Polled Celtic (Pc) mutation locus is a genetically simple single mutation that is the best choice for breeding polled cattle using gene editing. However, the mechanism of the Pc locus for regulating horn development is unclear, so we used gene editing, somatic cell nuclear transfer and embryo transfer to obtain polled Holstein fetal bovine (gestation time 90 days) with a homozygous Pc insertion (gene-edited Holstein fetal bovine, EH) and the wild-type 90 days Holstein fetal bovine (WH) as controls. The hematoxylin-eosin (HE) staining results showed that, compared to the WH, the EH horn buds had no white keratinized projections or vacuolated keratinocytes and no thick nerve bundles under the dermal tissue. Furthermore, DNA sequencing results showed that the Pc locus was homozygously inserted into the fetal bovine genome. A total of 791 differentially expressed genes were identified by transcriptome sequencing analysis. Enrichment analysis and protein interaction analysis results of differentially expressed genes showed that abundant gene changes after Pc insertion were associated with the adhesion molecule regulation, actin expression, cytoskeletal deformation and keratin expression and keratinization. It was also noted that the results contained several genes that had been reported to be associated with the development of horn traits, such as RXFP2 and TWIST1. This study identified these changes for the first time and summarized them. The results suggested that the Pc mutant locus may inhibit neural crest cell EMT generation and keratin expression, leading to failures in neural crest cell migration and keratinization of the horn bud tissue, regulating the production of the polled phenotype.

Natural Astaxanthin Improves Testosterone Synthesis and Sperm Mitochondrial Function in Aging Roosters.

Spermatogenesis, sperm motility, and apoptosis are dependent on the regulation of glandular hormones and mitochondria. Natural astaxanthin (ASTA) has antioxidant, anti-inflammatory, and anti-apoptotic properties. The present study evaluates the effects of ASTA on testosterone synthesis and mitochondrial function in aging roosters. Jinghong No. 1 layer breeder roosters (n = 96, 53-week old) were fed a corn−soybean meal basal diet containing 0, 25, 50, or 100 mg/kg ASTA for 6 weeks. The levels of plasma reproductive hormones and the mRNA and protein levels of molecules related to testosterone synthesis were significantly improved (p < 0.05) in the testes of the ASTA group roosters. In addition, antioxidant activities and free radical scavenging abilities in roosters of the ASTA groups were higher than those of the control group (p < 0.05). Mitochondrial electron transport chain complexes activities and mitochondrial membrane potential in sperm increased linearly with dietary ASTA supplementation (p < 0.05). The levels of reactive oxygen species and apoptosis factors decreased in roosters of the ASTA groups (p < 0.05). Collectively, these results suggest that dietary ASTA may improve testosterone levels and reduce sperm apoptosis, which may be related to the upregulation of the testosterone synthesis pathway and the enhancement of mitochondrial function in aging roosters.

The Dynamic Transcription Profiles of Proliferating Bovine Ovarian Granulosa When Exposed to Increased Levels of ß-Hydroxybutyric Acid.

Ketosis is common in high-yield dairy cows. It is a condition that is characterized by the accumulation of serum ß-hydroxybutyric acid (BHBA). Both subclinical ketosis and clinical ketosis can compromise the reproductive performance and cause long-lasting negative effects on reproductive efficiency by affecting the proliferation of follicular and granulosa cells. However, the regulatory mechanisms involved in the development of follicular cells and granulosa cells in cows experiencing subclinical ketosis and clinical ketosis remain largely unknown. To investigate the effect of a ketosis-triggered increase in BHBA on bovine follicular granulosa cell development, we detected a significant reduction in the proliferation of granulosa cells (P < 0.05) in the BHBA-1.2 mM and BHBA-2.4 mM groups and a significant increase in the number of granulosa cells in the G1 phase of the cell cycle (P < 0.05). RNA-seq and trend analysis were used to identify differentially expressed genes by comparing three clusters: low-concentration response to 1.2 mM BHBA, high-concentration response to 2.4 mM BHBA, and the similar trend (up or down) response following BHBA concentration increased. GO and KEGG enrichment analyses were performed separately for each cluster. Analysis showed that two novel down-regulated genes (G0S2 and S100A6), which are associated with cell proliferation and cycle progression, were enriched in the low-concentration response to 1.2 mM BHBA. Another differentially expressed gene (PARP), which plays a role in the apoptotic pathway, was enriched in the high-concentration response to 2.4 mM BHBA. We also found that CYP27B1 and CYP17A1, which are associated with Ca2+ homeostasis and estrogen synthesis, were enriched in a similar trend response. In conclusion, we describe the dynamic transcription profiles of granulosa cells under different levels of ß-hydroxybutyric stress and report key regulators that may underlie the detrimental effects on the development of follicles and granulosa cells, thus representing potential therapeutic targets to improve fertility in dairy cows with subclinical ketosis or clinical ketosis.

RhoA improves cryopreservation of rooster sperm through the Rho/RhoA-associated kinase/cofilin pathway.

Cryopreservation of rooster sperm leads to relatively low semen quality due to cytoskeletal damage during the freeze-thawing process. This study aimed to explore how the addition of RhoA recombinant protein affected the viability and subcellular structure of rooster sperm after freeze-thawing and elucidated the molecular mechanisms of sperm cryopreservation. Semen quality and acrosome integrity testing revealed that the addition of 0.5 µg/mL RhoA recombinant protein to the cryoprotectant fluid significantly increased sperm motility, survival rate, linearity, straight-line velocity, and acrosome integrity after freeze-thawing (P < 0.05). Ultrastructure analysis of cryopreserved sperm showed structural damage to the sperm plasma membrane, nuclear membrane, and tail. However, compared to the control, these structural changes were reduced upon the addition of RhoA recombinant protein to the cryoprotective fluid (P < 0.05). Western blotting revealed that the expression of Rho/RhoA-associated kinase and p-cofilin was increased, and cofilin expression was decreased after sperm cryopreservation with recombinant RhoA protein. Treatment with Y-27632, a ROCK antagonist, suppressed ROCK and p-cofilin expression and decreased semen quality, acrosome integrity, and ultrastructure integrity. In summary, we have demonstrated a cryoprotective effect in spermatozoa involving the Rho/ROCK pathway during freeze-thawing. Furthermore, the addition of 0.5 µg/mL RhoA recombinant protein to the cryoprotective fluid improved rooster semen quality and subcellular structural homeostasis after freeze-thawing via the Rho/ROCK pathway. This pathway may regulate the dynamic reorganization of the actin cytoskeleton by regulating the cofilin phosphorylation.

Mitofusins: from mitochondria to fertility.

Germ cell formation and embryonic development require ATP synthesized by mitochondria. The dynamic system of the mitochondria, and in particular, the fusion of mitochondria, are essential for the generation of energy. Mitofusin1 and mitofusin2, the homologues of Fuzzy onions in yeast and Drosophila, are critical regulators of mitochondrial fusion in mammalian cells. Since their discovery mitofusins (Mfns) have been the source of significant interest as key influencers of mitochondrial dynamics, including membrane fusion, mitochondrial distribution, and the interaction with other organelles. Emerging evidence has revealed significant insight into the role of Mfns in germ cell formation and embryonic development, as well as the high incidence of reproductive diseases such as asthenospermia, polycystic ovary syndrome, and gestational diabetes mellitus. Here, we describe the key mechanisms of Mfns in mitochondrial dynamics, focusing particularly on the role of Mfns in the regulation of mammalian fertility, including spermatogenesis, oocyte maturation, and embryonic development. We also highlight the role of Mfns in certain diseases associated with the reproductive system and their potential as therapeutic targets.

Long Noncoding RNAs: Recent Insights into Their Role in Male Infertility and Their Potential as Biomarkers and Therapeutic Targets.

Long noncoding RNAs (lncRNAs) are composed of nucleotides located in the nucleus and cytoplasm; these are transcribed by RNA polymerase II and are greater than 200 nt in length. LncRNAs fulfill important functions in a variety of biological processes, including genome imprinting, cell differentiation, apoptosis, stem cell pluripotency, X chromosome inactivation and nuclear transport. As high throughput sequencing technology develops, a substantial number of lncRNAs have been found to be related to a variety of biological processes, such as development of the testes, maintaining the self-renewal and differentiation of spermatogonial stem cells, and regulating spermatocyte meiosis. These indicate that lncRNAs can be used as biomarkers and potential therapeutic targets for male infertility. However, only a few comprehensive reviews have described the role of lncRNAs in male reproduction. In this paper, we summarize recent findings relating to the role of lncRNAs in spermatogenesis, their potential as biomarkers for male infertility and the relationship between reproductive arrest and transgenerational effects. Finally, we suggest specific targets for the treatment of male infertility from the perspective of lncRNAs.

Natural astaxanthin enhanced antioxidant capacity and improved semen quality through the MAPK/Nrf2 pathway in aging layer breeder roosters.

BACKGROUND: Natural astaxanthin (ASTA) has strong antioxidant properties and has been widely used as a health product to improve human health. However, the effects of ASTA on the reproductive performance of aging roosters have been poorly studied. We aimed to investigate the effects of dietary ASTA on semen quality and antioxidant capacity in aging roosters and to explore the potential mechanism of semen quality change via anti-oxidation defense system. METHODS: In the present study, 96 53-week-old Jinghong No. 1 layer breeder roosters were fed a corn-soybean meal basal diet containing 0, 25, 50, or 100 mg/kg ASTA for 6 weeks. RESULTS: Semen quality in the ASTA groups remarkably improved than that in the control group, and antioxidant activities, the abilities to scavenge hydroxyl radicals and superoxide anions, increased gradually with ASTA addition (P < 0.05). In addition, the mRNA levels of antioxidant enzymes as well as the mRNA and protein levels of the mitogen-activated protein kinase (MAPK) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were markedly increased in the 50-100 mg/kg ASTA group (P < 0.05). CONCLUSIONS: Collectively, these results demonstrate that dietary ASTA may improve semen quality by increasing antioxidant enzyme activities and the ability to scavenge hydroxyl radicals, which may be related to upregulation of the MAPK/Nrf2 pathway.

Dietary supplementation with natural astaxanthin from Haematococcus pluvialis improves antioxidant enzyme activity, free radical scavenging ability, and gene expression of antioxidant enzymes in laying hens.

The objective of this study was to evaluate the effects of natural astaxanthin (ASTA) from Haematococcus pluvialis on production performance, egg quality, antioxidant enzyme activity, free radical scavenging ability, and gene expression of antioxidant enzymes in laying hens. Nongda No. 3 laying hens (n = 450) were randomly allocated to 1 of 5 dietary treatments. Each treatment had 6 replicates of 15 hens each. All birds were assigned to a corn-soybean meal-based diet containing 0, 20, 40, 80, or 160 mg/kg ASTA for 4 wk. With increasing dietary ASTA, no significant effects were observed on egg weight, feed consumption, feed efficiency, laying rate, Haugh unit, or eggshell strength. Yolk color darkened linearly with increasing dose of ASTA (P < 0.05). Glutathione peroxidase activity was improved in the kidney with dietary ASTA at levels of 40 mg/kg. Total superoxide dismutase (SOD) was significantly increased in the liver, kidney, and plasma with dietary ASTA supplementation at 40 mg/kg. With increasing dietary ASTA, the scavenging abilities of hydroxyl radicals and superoxide anions were linearly increased (P < 0.05), and the malondialdehyde content decreased linearly (P < 0.05). Compared with the control group, mRNA expression of Cu-Zn SOD (SOD1), Mn SOD (SOD2), and nuclear factor E2-related factor 2 (NRF2) in the liver and kidney was significantly increased in the 40 mg/kg ASTA group (P < 0.05). The level of GPX4 mRNA in the liver and kidney was significantly increased with ASTA supplementation at 40 and 80 mg/kg (P < 0.05). The results demonstrate that dietary ASTA improves free radical scavenging ability and antioxidant enzyme activity, which may be related in part to the upregulated mRNA expression of genes encoding antioxidant enzymes and NRF2.

Effects of supplementing natural astaxanthin from Haematococcus pluvialis to laying hens on egg quality during storage at 4°C and 25°C.

The objective of this study was to evaluate the effects of different levels of dietary natural astaxanthin (ASTA) (from the microalga Haematococcus pluvialis) and storage at 4°C and 25°C on the quality of eggs from laying hens. Nongda No. 3 laying hens (n = 450) were randomly allocated to 1 of 5 dietary treatments. Each treatment had 6 replicates of 15 hens each. All birds were assigned to a corn-soybean meal-based diet containing 0, 20, 40, 80, or 160 mg/kg natural ASTA for 4 wk. A total of 540 eggs were collected at the end of the 4-week feeding trial. Sixty fresh eggs were collected and measured for egg quality within 24 h after collection. The other 480 eggs were used in a factorial arrangement with 5 dietary ASTA levels, 4 storage times, and 2 storage temperatures. During the 8-week storage period at 4°C and 25°C, egg quality measurements were performed every 2 wk on 12 eggs per treatment. No significant effects (P > 0.05) on yolk index, yolk pH, Haugh units, weight loss, or eggshell strength were observed with increasing concentrations of dietary ASTA. Yolk color darkened linearly with increasing dose of ASTA (P < 0.05). During storage of eggs, yolk index and Haugh units decreased significantly (P < 0.05), whereas yolk pH and weight loss increased (P < 0.05). An interaction was observed between dietary ASTA level and storage time on yolk index, yolk color, and Haugh units (P < 0.05). These results demonstrated that dietary ASTA from H. pluvialis delayed the decrease in yolk index and yolk color during storage at 4°C and 25°C. Therefore, we speculate that there may be a combined effect of dietary ASTA level and storage time on egg internal quality; this information may provide additional options by which to extend the storage time of eggs.

Effects of dietary supplementation of natural astaxanthin from Haematococcus pluvialis on antioxidant capacity, lipid metabolism, and accumulation in the egg yolk of laying hens.

The present study evaluated the effects of natural astaxanthin (ASTA) from Haematococcus pluvialis on the antioxidant capacity, lipid metabolism, and ASTA accumulation in the egg yolk of laying hens. Hy-Line Brown layers (n = 288, 50 wk old) were randomly assigned to 1 of 4 dietary treatment groups. Each group had 6 replicates of 12 hens each. All birds were given a corn-soybean meal-based diet containing 0, 25, 50, or 100 mg/kg ASTA for 6 wk. The results showed that the total antioxidant capacity, superoxide dismutase level, and glutathione peroxidase level in the plasma, livers, and egg yolks were significantly increased in the ASTA groups compared with those of the control group (P < 0.05), whereas the content of malondialdehyde linearly decreased (P < 0.05). The plasma levels of high-density and very-low-density lipoprotein cholesterol in the ASTA groups were significantly higher than those in the control group (P < 0.05). In addition, ASTA supplementation decreased low-density lipoprotein cholesterol and triglyceride plasma levels (P < 0.05). However, there were no significant differences in the other lipid metabolism parameters among the ASTA-supplemented groups relative to the control group except for an increase in high-density lipoprotein cholesterol in the liver. Compared with the control, dietary ASTA supplementation significantly increased the enrichment of ASTA in egg yolks at the end of week 2, 4, and 6 (P < 0.05). The mRNA expression of scavenger receptor class B type 1 (SCARB1) and very-low-density lipoprotein receptor (VLDLR) in the ASTA groups was markedly higher (P < 0.05) than that in the control group in the liver and ovaries, respectively. In conclusion, these results suggest that dietary ASTA enhances the antioxidant capacity and regulates lipid metabolism in laying hens. ASTA enrichment in egg yolks may be closely related to the upregulation of SCARB1 and VLDLR gene expression.