Superior performance of biocomposite nanoparticles PLGA-RES in protecting oocytes against vitrification stimuli.

Irreversible cryogenic damage caused by oocyte vitrification limits its widespread use in female fertility preservation. In recent years, nanoparticles (NPs) have gained great attention as potential alternatives in protecting oocytes against cryoinjuries. In this paper, a novel composite nanoparticle, poly (lactic-co-glycolic acid)-resveratrol (PLGA-RES) was designed to improve the biocompatibility and sustained release properties by encapsulating natural antioxidant RES into PLGA NPs. Firstly, biotoxicity and oxidation resistance of PLGA-RES were determined, and the results showed that PLGA-RES had nontoxic effect on oocyte survival during in vitro maturation (IVM) (97.08% ± 0.24% vs. 98.89% ± 1.11%, p > 0.05). Notably, PLGA-RES even increased maturation (65.10% ± 4.11% vs. 52.85% ± 2.87%, p < 0.05) and blastocyst rate (56.13% ± 1.36% vs. 40.91% ± 5.85%, p < 0.05). Moreover, the reduced reactive oxygen species (ROS) level (13.49 ± 2.30 vs. 34.07 ± 3.30, p < 0.01), increased glutathione (GSH) (44.13 ± 1.57 vs. 37.62 ± 1.79, p < 0.01) and elevated mitochondrial membrane potential (MMP) levels (43.10 ± 1.81 vs. 28.52 ± 1.25, p < 0.01) were observed in oocytes treated with PLGA-RES when compared with that of the control group. Subsequently, the role of PLGA-RES played in oocytes during vitrification was systematically evaluated. The results showed that the addition of PLGA-RES during vitrification and thawing significantly improved the survival rate (80.42% ± 1.97% vs. 75.37% ± 1.3%, p < 0.05). Meanwhile, increased GSH (15.09 ± 0.86 vs. 14.51 ± 0.78, p < 0.01) and mitochondrial membrane potential (22.56 ± 3.15 vs. 6.79 ± 0.60, p < 0.01), decreased reactive oxygen species levels (52.11 ± 2.95 vs. 75.41 ± 7.23, p < 0.05) and reduced mitochondrial abnormality distribution rate (25.00% ± 0.29% vs. 33.33% ± 1.15%, p < 0.01) were assessed in vitrified MII oocytes treated with PLGA-RES. Furthermore, transcriptomic analyses demonstrated that PLGA-RES participated in endocytosis and PI3K/AKT/mTOR pathway regulation, which was verified by the rescued expression of ARRB2 and ULK3 protein after PLGA-RES treatment. In conclusion, PLGA-RES exhibited potent antioxidant activity, and could be used as an efficacious strategy to improve the quality of vitrified oocytes.

Mito-TEMPO Improves the Meiosis Resumption and Mitochondrial Function of Vitrified Sheep Oocytes via the Recovery of Respiratory Chain Activity.

Vitrification is a crucial method for preserving animal germ cells. Considering the increased oxidative stress and organelle damage incurred, it is still necessary to make the process more efficient for oocytes. As the energy source of oocytes, mitochondria are the most abundant organelle in oocytes and play a crucial role in their maturation. Here, we found that Mito-TEMPO, a mitochondria-targeted antioxidant, could efficaciously improve the oxidative stress injury of vitrified oocytes by recovering mitochondrial function via the mitochondrial respiratory chain. It was observed that Mito-TEMPO not only improves oocyte viability and meiosis but also maintains spindle structure. A subsequent study indicated that Mito-TEMPO effectively rescued mitochondrial dysfunction and attenuated vitrification-induced oxidative stress. Further investigation revealed that Mito-TEMPO regulates vitrified oocytes' intracellular Ca2+ homeostasis and ATP content and provides strong antioxidant properties. Additionally, an analysis of the transcriptome at the single-cell level revealed that the respiratory chain mediates the beneficial effect of Mito-TEMPO on vitrified oocytes. Overall, our findings indicate that supplementing oocytes with Mito-TEMPO is an effective method to shield them from the damage caused by vitrification. In addition, the beneficial effects of Mito-TEMPO on vitrified sheep oocytes could inspire further investigations of the principles underlying oocyte cryobiology in other animals.

Vitrification of Mammalian Oocytes: Recent Studies on Mitochondrial Dysfunction.

Vitrification of reproductive cells is definitely essential and integral in animal breeding, as well as in assisted reproduction. However, issues accompanied with this technology such as decreased oocyte competency and relatively low embryo survival rates appear to be a tough conundrum that has long perplexed us. As significant organelles in cell metabolism, mitochondria play pivotal roles in numerous pathways. Nonetheless, extensive evidence has demonstrated that vitrification can seriously impair mitochondrial function in mammalian oocytes. Thus, in this article, we summarize the current progress in oocyte vitrification and particularly outline the common mitochondrial abnormalities alongside subsequent injury cascades seen in mammalian oocytes following vitrification. Based on existing literature, we tentatively come up with the potential mechanisms related to mitochondrial dysfunction and generalize efficacious ways which have been recommended to restore mitochondrial function.

Fetal hypoxia expose caused autophagy in ovary granulosa cells via PI3K/Akt/FoxO1 pathway and mitigated by melatonin subheading.

INTRODUCTION: Fetal hypoxia has long-term effects on postnatal reproductive functions and the mitochondrial impairments of ovarian granulosa cells may be one of the causes. Melatonin applied to mitigate mitochondrial dysfunction and autophagy in mammalian cells has been reported. However, the potential mechanisms by which fetal hypoxia damages reproductive function in neonatal female mice and the melatonin effects on this problem remain unclear. OBJECTIVES: This research aimed to explore the mechanism that fetal hypoxia damages reproductive function in neonatal female mice and attempt to improve the reproductive function by treating with melatonin in vivo and in vitro. METHODS: We established a fetal hypoxia model and confirmed that fetal hypoxia affects ovarian function by inducing GC excessive autophagy. Transcriptomic analysis, gene interference, cell immunofluorescence, immunohistochemistry and western blot were conducted to explore and verify the underlying mechanisms in mice GCs and KGN cells. Finally, melatonin treatment was executed on hypoxia-treated mice GCs and KGN cells and melatonin injection to fetal-hypoxia-treated mice to determine its effect. RESULTS: The results of in vitro experiments found that fetal hypoxia led to mitochondrial dysfunction in ovarian GCs causing autophagic cell death. And the PI3K/Akt/FoxO pathway mediated the occurrence of this process by transcriptome analysis of ovarian GCs from normal and fetal hypoxia mice, which was further verified in mice GCs and KGN cells. Additionally, melatonin administration prevented autophagic injuries and mitochondrial impairments in hypoxia-treated mice GCs and KGN cells. Meanwhile, in vivo experiments by melatonin injection ameliorated oxidative stress of ovary in fetal-hypoxia-treated mice and improved their low fertility. CONCLUSION: Our data found that fetal hypoxia causes ovarian GCs excessive autophagy leading to low fertility in neonatal female mice and mitigated by melatonin. These results provide a potential therapy for hypoxic stress-related reproductive disorders.

Integrated transcriptomics and proteomics assay identifies the role of FCGR1A in maintaining sperm fertilization capacity during semen cryopreservation in sheep.

Semen cryopreservation is a promising technology employed in preserving high-quality varieties in animal husbandry and is also widely applied in the human sperm bank. However, the compromised qualities, such as decreased sperm motility, damaged membrane structure, and reduced fertilization competency, have significantly hampered the efficient application of this technique. Therefore, it is imperative to depict various molecular changes found in cryopreserved sperm and identify the regulatory network in response to the cryopreservation stress. In this study, semen was collected from three Chinese Merino rams and divided into untreated (fresh semen, FS) and programmed freezing (programmed freezing semen, PS) groups. After measuring different quality parameters, the ultra-low RNA-seq and tandem mass tag-based (TMT) proteome were conducted in both the groups. The results indicated that the motility (82.63% ± 3.55% vs. 34.10% ± 2.90%, p < 0.05) and viability (89.46% ± 2.53% vs. 44.78% ± 2.29%, p < 0.05) of the sperm in the FS group were significantly higher compared to those in the PS group. In addition, 45 upregulated and 291 downregulated genes, as well as 30 upregulated and 48 downregulated proteins, were found in transcriptomics and proteomics data separately. Moreover, three integrated methods, namely, functional annotation and enrichment analysis, Pearson's correlation analysis, and two-way orthogonal partial least squares (O2PLS) analysis, were used for further analysis. The results suggested that various differentially expressed genes and proteins (DEGs and DEPs) were mainly enriched in leishmaniasis and hematopoietic cell lineage, and Fc gamma receptor Ia (FCGR1A) was significantly downregulated in cryopreserved sperm both at mRNA and protein levels in comparison with the fresh counterpart. In addition, top five genes (FCGR1A, HCK, SLX4, ITGA3, and BET1) and 22 proteins could form a distinct network in which genes and proteins were significantly correlated (p < 0.05). Interestingly, FCGR1A also appeared in the top 25 correlation list based on O2PLS analysis. Hence, FCGR1A was selected as the most potential differentially expressed candidate for screening by the three integrated multi-omics analysis methods. In addition, Pearson's correlation analysis indicated that the expression level of FCGR1A was positively correlated with sperm motility and viability. A subsequent experiment was conducted to identify the biological role of FCGR1A in sperm function. The results showed that both the sperm viability (fresh group: 87.65% ± 4.17% vs. 75.8% ± 1.15%, cryopreserved group: 48.15% ± 0.63% vs. 42.45% ± 2.61%, p < 0.05) and motility (fresh group: 83.27% ± 4.15% vs. 70.41% ± 1.07%, cryopreserved group: 45.31% ± 3.28% vs. 35.13% ± 2.82%, p < 0.05) were significantly reduced in fresh and frozen sperm when FCGR1A was blocked. Moreover, the cleavage rate of embryos fertilized by FCGR1A-blocked sperm was noted to be significantly lower in both fresh (95.28% ± 1.16% vs. 90.44% ± 1.56%, p < 0.05) and frozen groups (89.8% ± 1.50% vs. 82.53% ± 1.53%, p < 0.05). In conclusion, our results revealed that the downregulated membrane protein FCGR1A can potentially contribute to the reduced sperm fertility competency in the cryopreserved sheep sperm.

Metabolomic and Transcriptomic Changes Underlying the Effects of L-Citrulline Supplementation on Ram Semen Quality.

This study examined the effects of L-Cit supplementation on ram semen quality through metabolomics and transcriptomics. A total of 16 rams were randomly categorized into two groups. The control group was fed a basic diet, whereas the experimental group received feed supplemented with 12 g/d of L-Cit. Semen and blood were collected from the rams on days 0 and 72 to measure sugar, pyruvate, amino acid, and nontargeted metabolite contents. Additionally, hypothalamic and testicular tissues were collected for a transcriptomic analysis. We found 27 differential metabolites between the control and experimental groups, of which 21 were downregulated (p < 0.05) and 6 were upregulated (p < 0.05). Compared with the control group, xylose and pyruvate contents in seminal plasma increased by 43.86% and 162.71%, respectively (p < 0.01). Additionally, the levels of 11 amino acids showed a significant increase in seminal plasma (p < 0.01). Furthermore, 961 and 715 differentially expressed genes were detected in the hypothalamic and testicular tissues, respectively. The pathways of significant enrichment in the hypothalamus and testes were protein digestion, absorption, glycolysis/gluconeogenesis, and amino as well as nucleotide sugar metabolisms. In the present study, L-Cit improved protein synthesis and blood metabolism, consequently increasing the contents of most amino acids in ram seminal plasma. Specifically, the hypothalamus controlled the expression of glycolysis/gluconeogenesis-related genes in the testes through its metabolites released into the serum, thereby providing energy for sperm production, which led to a decrease in the sugar content of seminal plasma.

Leonurine improves bovine oocyte maturation and subsequent embryonic development by reducing oxidative stress and improving mitochondrial function.

It is acknowledged that excessive reactive oxygen species (ROS) level attributes greatly to the compromised developmental potential of oocytes matured in vitro. Although agents were applied to alleviate ROS levels, results were varied because of the distinct antioxidative activity and cell toxicity. Leonurine (LEO), extracted from the natural Chinese herb motherwort, is considered to be a potent free radical scavenger. Yet, it is undetermined whether LEO is benefit for oocyte development during in vitro maturation (IVM). In the present study, the effect of LEO on the quality of bovine oocyte as well as the underlying mechanism was investigated. We found that maturation rate (P < 0.01), subsequent blastocyst formation rate (P < 0.05), and the total blastocyst cell number (P < 0.05) after parthenogenetic activation were significantly increased in the group treated with 20 µM LEO. Moreover, a dramatic decline in ROS (P < 0.01), decreased lipid content (P < 0.01), elevated MMP level (P < 0.05), increased ATP content (P < 0.05), and reduced mitochondrial temperature (P < 0.01) were observed in oocytes treated with LEO. Furthermore, the expression level of anti-apoptotic protein BCL2 was significantly higher in LEO treated oocytes (P < 0.01), and the ratio of BAX/BCL2 was obvious decreased (P < 0.01). Finally, we found that LC3B intensity was significantly reduced (P < 0.05) while the rate of EdU positive nuclei was markedly increased (P < 0.05) in embryos derived from LEO-treated oocytes. Our results demonstrate that LEO exhibits a potent protective role in the acquisition of oocyte development capacity against oxidative stress during IVM, and provides a new solution for optimizing the in vitro culture system of bovine embryos.

Antioxidant procyanidin B2 protects oocytes against cryoinjuries via mitochondria regulated cortical tension.

BACKGROUND: Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation. Antioxidants were always used to antagonist the oxidative stress caused by vitrification. However, the comprehensive mechanism underlying the protective role of antioxidants has not been studied. Procyanidin B2 (PCB2) is a potent natural antioxidant and its functions in response to vitrification are still unknown. In this study, the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored, and the mechanisms underlying the protective role of PCB2 were systematically elucidated. RESULTS: Vitrification induced a marked decline in oocyte quality, while PCB2 could improve oocyte viability and further development after parthenogenetic activation. A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress, rescued mitochondrial dysfunction, and improved cell viability. Moreover, PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties. Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration, ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes. Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation, and PCB2 effectively increased the cortical tension through the electron transfer chain pathway. Additionally, PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality. What's more, targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism. CONCLUSIONS: Antioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.

Mito-Q promotes porcine oocytes maturation by maintaining mitochondrial thermogenesis via UCP2 downregulation.

Mitochondrial thermogenesis is an adaptive response of cells to their surrounding stress. Oxidative stress is one of the common stresses during in vitro maturation (IVM) of oocytes, which leads to mitochondrial dysfunction. This study aimed to probe the effects of the mitochondria-targeted antioxidant Mito-Q on oocyte development and unravel the role of Mito-Q in mitochondrial ATP production and thermogenesis regulation. Our results showed that Mito-Q had a positive effect on porcine oocytes maturation and subsequent embryo development. During oocytes IVM, Mito-Q could reduce ATP levels and ROS, increase lipid droplets accumulation, induce autophagy, and maintain mitochondrial temperature stability. Moreover, in metaphase II (MII) oocytes, Mito-Q would induce mitochondrial uncoupling manifested by decreased ATP, attenuated mitochondrial membrane potential (MMP), and increased mitochondrial thermogenesis. Notably, the expression of mitochondrial uncoupling protein (UCP2) was significantly reduced in oocytes treated with Mito-Q. Further study indicated that specific depletion of UCP2 in oocytes also resulted in increased thermogenesis, decreased ATP and declined MMP, suggesting that UCP2 downregulation may participate in Mito-Q-induced mitochondrial uncoupling. In summary, our data demonstrate that Mito-Q promotes oocyte maturation in vitro and maintains the stability of mitochondrial thermogenesis by inhibiting UCP2 expression.

Mitochondrial Calcium Disorder Affects Early Embryonic Development in Mice through Regulating the ERK/MAPK Pathway.

The homeostasis of mitochondrial calcium ([Ca2+]mt) in oocytes plays a critical role in maintaining normal reproductive cellular progress such as meiosis. However, little is known about the association between [Ca2+]mt homeostasis and early embryonic development. Two in vitro mouse MII oocyte models were established by using a specific agonist or inhibitor targeting mitochondrial calcium uniporters (MCU) to upregulate or downregulate [Ca2+]mt concentrations. The imbalance of [Ca2+]mt in MII oocytes causes mitochondrial dysfunction and morphological abnormity, leading to an abnormal spindle/chromosome structure. Oocytes in drug-treated groups are less likely to develop into blastocyst during in vitro culture. Abnormal [Ca2+]mt concentrations in oocytes hindered epigenetic modification and regulated mitogen-activated protein kinase (MAPK) signaling that is associated with gene expression. We also found that MAPK/ERK signaling is regulating DNA methylation in MII oocytes to modulate epigenetic modification. These data provide a new insight into the protective role of [Ca2+]mt homeostasis in early embryonic development and also demonstrate a new mechanism of MAPK signaling regulated by [Ca2+]mt that influences epigenetic modification.

Proteomic profile of mouse oocytes after vitrification: A quantitative analysis based on 4D label-free technique.

Mature oocyte cryopreservation represents an important trend for future fertility preservation, however, the relatively low efficiency has hampered its clinical application. Proteomic profiling is a method of choice for the exploration of the molecular mechanism underlying cryoinjuries. Here, a systematic comparison of protein expression between fresh and vitrified oocytes was performed based on the 4D label-free technique, an informative method with high sensitivity. Our results indicated that the oocyte survival rate was significantly reduced after vitrification. Proteomic results showed that 32 proteins were up-regulated, while 77 proteins were down-regulated in vitrified oocytes compared with the fresh counterparts. Gene Ontology (GO) functional analysis revealed that differentially expressed proteins (DEPs) were involved in metabolism, mitochondrial function, cytoskeleton and other cell functions. Moreover, proteins that participated in signaling transduction mechanisms were the largest category based on Clusters of Orthologous Groups of protein/EuKaryotic Orthologous Groups (COG/KOG) functional classification. In addition, over-expressed DEPs were enriched for "nucleus", "protein binding", "membrane", "cytoplasm" as well as mitochondrial function. Furthermore, we discovered that the DEPs were clustered in pyruvate metabolism, citric acid (TCA) cycle and glucose metabolism by Protein-Protein Interaction (PPI) network evaluation. In conclusion, our data demonstrate that vitrification induces multi-level damages in oocytes, the dynamic proteomic profiling will provide systematic insights into uncovering the mechanism underlying cryoinjuries.

Procyanidin B2 Protects Aged Oocytes Against Meiotic Defects Through Cortical Tension Modulation.

Defects in meiotic process are the main factors responsible for the decreased developmental competence in aged oocytes. Our recent research indicated that natural antioxidant procyanidin B2 (PCB2) promoted maturation progress in oocytes from diabetic mice. However, the effect of PCB2 on aging-induced chromosome abnormalities and the underlying mechanism have not been explored. Here, we found that PCB2 recovered aging-caused developmental arrest during meiotic maturation, germinal vesicle breakdown (GVBD) rate was significantly higher in aged oocytes treated with PCB2 (P < 0.05). Furthermore, we discovered that cortical mechanics were altered during aging process, cortical tension-related proteins were aberrantly expressed in aged oocytes (P < 0.001). PCB2 supplementation efficaciously antagonized aging-induced decreased cortical tension (P < 0.001). Moreover, PCB2 restored spindle morphology (P < 0.01), maintained proper chromosome alignment (P < 0.05), and dramatically reduced reactive oxygen species (ROS) level (P < 0.05) in aged oocytes. Collectively, our results reveal that PCB2 supplementation is a feasible approach to protect oocytes from reproductive aging, contributing to the improvement of oocytes quality.

Artificially Increasing Cortical Tension Improves Mouse Oocytes Development by Attenuating Meiotic Defects During Vitrification.

Oocyte cryopreservation demonstrates great benefits in the conservation of animal germplasm resources and assisted reproductive technology. However, vitrification causes damages in oocytes, which would lead to the decrease of oocyte quality, and embryonic development post fertilization. Cytoskeleton plays an important role in regulating cell shape, organelle migration, cell division and mechanical signal transduction. Cortical tension is a reflection of the physiological state and contractile ability of cortical cytoskeleton. Appropriate cortical tension is prerequesite for normal oocyte meiosis. In the present study, oocyte cortical tension was examined by evaluating the levels of cortical tension-related protein pERM (Phospho-Ezrin/Radixin/Moesin) and pMRLC (Phospho-Myosin Light Chain 2). We found that the cortical tension of vitrified oocytes was decreased. Increasing cortical tension of vitrified oocytes by adding 10 µg/ml ConA during in vitro culture could significantly improve the polar body extrusion rate and embryo development. Furthermore, increasing the cortical tension could improve spindle positioning, maintain kinetochore-microtubule (KT-MT) attachment, strengthen spindle assembly checkpoint (SAC) activity, and reduce the aneuploidy rate in vitrified oocytes. In conclusion, vitrification induced a remarkable decrease in cortical tension, and increasing the cortical tension could rescue the meiosis defect and improve oocyte quality.

Estrogen Receptor Function: Impact on the Human Endometrium.

The physiological role of estrogen in the female endometrium is well established. On the basis of responses to steroid hormones (progesterone, androgen, and estrogen), the endometrium is considered to have proliferative and secretory phases. Estrogen can act in the endometrium by interacting with estrogen receptors (ERs) to induce mucosal proliferation during the proliferative phase and progesterone receptor (PR) synthesis, which prepare the endometrium for the secretory phase. Mouse knockout studies have shown that ER expression, including ERα, ERß, and G-protein-coupled estrogen receptor (GPER) in the endometrium is critical for normal menstrual cycles and subsequent pregnancy. Incorrect expression of ERs can produce many diseases that can cause endometriosis, endometrial hyperplasia (EH), and endometrial cancer (EC), which affect numerous women of reproductive age. ERα promotes uterine cell proliferation and is strongly associated with an increased risk of EC, while ERß has the opposite effects on ERα function. GPER is highly expressed in abnormal EH, but its expression in EC patients is paradoxical. Effective treatments for endometrium-related diseases depend on understanding the physiological function of ERs; however, much less is known about the signaling pathways through which ERs functions in the normal endometrium or in endometrial diseases. Given the important roles of ERs in the endometrium, we reviewed the published literature to elaborate the regulatory role of estrogen and its nuclear and membrane-associated receptors in maintaining the function of endometrium and to provide references for protecting female reproduction. Additionally, the role of drugs such as tamoxifen, raloxifene, fulvestrant and G-15 in the endometrium are also described. Future studies should focus on evaluating new therapeutic strategies that precisely target specific ERs and their related growth factor signaling pathways.

Estrogen Biosynthesis and Signal Transduction in Ovarian Disease.

Estrogen mainly binds to estrogen receptors (ERs) to regulate menstrual cycles and reproduction. The expression of ERalpha (ERα), ERbeta (ERß), and G-protein-coupled estrogen receptor (GPER) mRNA could be detected in ovary, suggesting that they play an important role in estrogen signal transduction in ovary. And many studies have revealed that abnormal expression of estrogen and its receptors is closely related to ovarian disease or malignant tumors. With the continuous development and research of animal models, tissue-specific roles of both ERα and ERß have been demonstrated in animals, which enable people to have a deeper understanding of the potential role of ER in regulating female reproductive diseases. Nevertheless, our current understanding of ERs expression and function in ovarian disease is, however, incomplete. To elucidate the biological mechanism behind ERs in the ovary, this review will focus on the role of ERα and ERß in polycystic ovary syndrome (PCOS), ovarian cancer and premature ovarian failure (POF) and discuss the major challenges of existing therapies to provide a reference for the treatment of estrogen target tissue ovarian diseases.

Effect of L-citrulline supplementation on sperm characteristics and hormonal and antioxidant levels in blood and seminal plasma of rams.

With the aim of providing a theoretical basis for the application of L-citrulline (L-Cit) in animal husbandry, the effects of L-Cit on reproductive hormone levels, antioxidant capacity and semen quality of rams were studied by feeding them varying doses of L-Cit. A total of 32 rams were randomly divided into four groups with eight rams each. After all rams were trained to donate sperm normally, the control group was fed a basic diet, whereas the experimental groups I, II and III were provided with feed supplemented with 4, 8 and 12 g/d of L-Cit respectively. The experiment was conducted for 70 days, during which blood samples were collected from the jugular vein on days 0, 15, 30, 45 and 60, and semen samples were collected on days 0, 20, 40 and 60. In the same group, 100 µl of semen was used to test for quality, The rest of the semen sample and blood samples were centrifuged at 600 g for 15 min, and the supernatant and serum, respectively, were used to determine the levels reproductive hormones and antioxidant indices. Ram semen samples were also collected on day 70 and used to study sperm plasma membrane, substitution and mitochondrial membrane potential. Compared with the control group, the groups receiving L-Cit showed an increase in sperm concentration and number of linear motile sperm (p < .01); a decrease in the number of dead sperm (p < .01); an increase in sperm viability, particularly in groups II and III (p < .01); and an increase in sperm mitochondrial membrane potential (p < .01). Moreover, groups I, II and III showed significantly higher levels of serum gonadotropin-releasing hormone (GnRH), glutathione peroxidase (GSH-Px) and nitric oxide (NO) (p < .01). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels increased in groups I (p < .05), II (p < .05) and III (p < .01), whereas testosterone (T), catalase (CAT) and superoxide dismutase (SOD) levels increased in groups I and II (p < .01). Serum total antioxidant capacity (T-A) increased (p < .05), whereas both hydroxyl radical (·OH) and peroxy radical ( O 2 · - ) levels decreased (p < .01). Compared with the control, all groups had significantly higher SOD and GSH-Px in their seminal plasma (p < .01), and groups I, II (p < .05 for both) and III (p < .01) had higher levels of GnRH and FSH. LH, CAT and NO levels increased in group I (p < .05), II and III (p < .01 for both); malondialdehyde levels decreased in groups I, II (p < .05 for both) and group III (p < .01); and O 2 · - levels decreased in groups I, II and III (p < .01). Under our experimental conditions, GnRH, FSH, LH, T, CAT, SOD, T-A, GSH-PX and NO levels in the serum and seminal plasma of rams receiving L-Cit increased, whereas Oestradiol (E2 ), O 2 · - and ·OH levels in the seminal plasma decreased; this improved the semen quality of rams supplemented with L-Cit. Moreover, supplementation with 12 g/d gave the best results.

Nampt affects mitochondrial function in aged oocytes by mediating the downstream effector FoxO3a.

Maternal aging can impair the quality and decrease the developmental competence of ovulated oocytes. In this study, compromised germinal vesicle breakdown (GVBD) was found in aged mice oocytes. Furthermore, we observed increased reactive oxygen species (ROS) and mitochondrial Ca2+ levels, along with reduced mitochondrial temperature in aged oocytes. Maternal aging also changed the crotonylation level in oocytes. Forkhead box O3 (FoxO3a), a member of the forkhead protein family involved in the regulation of cell survival and life span reached a peak level in the metaphase II stage. Compared with a younger group, FoxO3a expression increased in aged oocytes. Intracellular localization of FoxO3a changed from the cytoplasm to chromatin in response to aging. The expression of the upstream regulator nicotinamide-phosphoribosyltransferase (Nampt) peaked in the GVBD stage. Moreover, Nampt expression was increased in aged oocytes, and more intense staining of Nampt was found in aged mice ovary. To further study the role of Nampt in mitochondrial function, specific agonist P7C3 and inhibitor FK866 were applied to aged oocytes, and FK866 significantly decreased adenosine triphosphate and mitochondrial membrane potential. In conclusion, mitochondrial dysfunction in aged oocytes was associated with elevated FoxO3a, and suppression of Nampt could further impair mitochondrial function.

Polystyrene microplastics induced female reproductive toxicity in mice.

Plastics have caused serious environmental pollution. In recent years, microplastics (MPs) have caused widespread concern about their potential toxicity on animals and humans, especially on organ and tissue deposition. However, there is little known about the reproductive toxic effects of MPs in female mammals. In this study, the reproductive toxicity of polystyrene MPs (PS-MPs) in female mice was evaluated after continued exposure for 35 days. Results showed that PS-MPs could accumulate in heart, liver, spleen, lung, kidney, brain, large intestine, small intestine, uterus, ovary and blood of exposed mice. Moreover, PS-MPs exposure increased the IL-6 level and decreased malondialdehyde (MDA) level in mouse ovaries. The results also showed that PS-MPs exposure decreased the first polar body extrusion rate and the survival rate of superovulated oocytes. Meanwhile, PS-MPs reduced the level of glutathione (GSH), mitochondrial membrane potential (MMP), endoplasmic reticulum calcium ([Ca2+]ER) and increased reactive oxygen species (ROS) in oocytes. In conclusion, our study illustrated that PS-MPs exposure induced the inflammation of ovaries and reduced the quality of oocytes in mice, which provided a basis for studying the reproductive toxic mechanism of PS-MPs in female mammals.

Identifying the heat resistant genes by multi-tissue transcriptome sequencing analysis in Turpan Black sheep.

Heat stress not only affects the physical condition but also affects reproductive performance in sheep. A thorough understanding of the molecular and physiological mechanisms underlying heat stress would certainly improve livestock productivity and provide genetic evaluation ways for heat resistant breeds selection. In this study, 85 Turpan Black sheep, a breed exhibited excellent heat resistance from long-term artificial selection, and 85 heat sensitive Kazakh sheep in Turpan basin were tested for physiological and reproductive performance from July to August in summer. The results showed that the estrus rate was significantly higher in Turpan Black sheep (P < 0.05), while the heart rate and respiratory rate of Turpan Black sheep are significantly lower than that of Kazakh sheep (P < 0.05). Furthermore, to clarify genes participated in heat stress response, the pituitary, ovarian and hepatic tissues from three Turpan Black sheep and three Kazakh sheep were subjected to RNA-seq. The results indicated that 32, 49 and 69 genes were up-regulated, and 39, 60 and 145 genes were down-regulated in pituitary, ovarian and hepatic tissues in Turpan Black sheep compared with that of the Kazakh sheep, respectively. KEGG and gene set enrichment analysis showed that the differentially expressed genes were mainly involved in signal transduction pathways. In particular, the differentially expressed genes in hepar were enriched in the energy metabolism pathway, while the differentially expressed genes in ovarian tissue were enriched in the ovarium steroidogenesis pathway. In conclusion, our results implied that the pituitary-ovary axis might include hepar as downstream targeted organism in heat resistant regulation. Under heat stress, the signals released from pituitary would impact steroidogenesis in ovary, and further alter energy metabolism in hepar. As we know, this is the first comparative study to investigate the gene expression in multi-tissue in sheep under heat stress.