Search

Previous studies have shown that melatonin (MT) can ameliorate vitrification-inflicted damage in mouse germinal vesicle (GV) oocytes, however, the key mechanistic basis of this improvement still remains poorly understood. This study was conducted to investigate whether MT can improve in vitro developmental potential of vitrified-warmed GV oocytes through its receptors. The fresh oocytes were randomly divided into four groups: untreated (control group, F), vitrified by open-pulled straw method (vitrification group, V), vitrification group with 100 nmol/L MT supplementation (vitrification + MT group, VM), and with 100 nmol/L MT plus 100 nmol/L luzindole administration (vitrification + MT + luzindole group, VML) or with 50 nmol/L ramelteon addition (vitrification + ramelteon group; VR). After warming, oocytes were cultured in vitro, and MT receptors (MTRs), MAD2 (mitotic arrest deficient 2), Securin and CyclinB1 protein levels and spindle morphology were evaluated. The ratio of oocytes developed to the metaphase I (MI) and metaphase II (MII) stages was also assessed. The results showed that after vitrification-warming, the in vitro maturation rate of GV oocytes was significantly lower compared to the control (F) group. Vitrification also significantly impaired the spindle morphology, decreased the protein level of MTRs and Securin, and decreased MAD2 levels in MI oocytes. However, when MT or ramelteon (MTRs agonist) were added (group wise) to warming and maturation media, the maturation rate of GV oocytes was significantly increased, the normal proportion of the spindle morphology increased, and the expression level of MAD2 increased in their resulting MI oocytes compared to the vitrification group. However, following addition of both MT and ramelteon, the maturation rate of GV oocyte showed no significant difference between VML and vitrification groups. The spindle morphology and MAD2 levels in MI oocytes were comparable to the vitrification group but differed significantly from the VM group. Taken together, finding of the present study shows that MT (100 nmol/L) can ameliorate the in vitro maturation of vitrified-warmed mouse GV oocytes, potentially by improving the spindle morphology, modulating MAD2 protein level and promoting the development of MI stage oocytes through MTRs.

Background This study investigated the effect of melatonin (MT) on cell cycle (G1/S/G2/M) of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II (MII) oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos. Results After vitrification and warming, oocytes were parthenogenetically activated (PA) and in vitro cultured (IVC). Then the spindle morphology and chromosome segregation in oocytes, the maternal mRNA levels of genes including Miss, Doc1r, Setd2 and Ythdf2 in activated oocytes, pronuclear formation, the S phase duration in zygotes, mitochondrial function at G1 phase, reactive oxygen species (ROS) level at S phase, DNA damage at G2 phase, early apoptosis in 2-cell embryos, cleavage and blastocyst formation rates were evaluated. The results indicated that the vitrification/warming procedures led to following perturbations 1) spindle abnormalities and chromosome misalignment, alteration of maternal mRNAs and delay in pronucleus formation, 2) decreased mitochondrial membrane potential (MMP) and lower adenosine triphosphate (ATP) levels, increased ROS production and DNA damage, G1/S and S/G2 phase transition delay, and delayed first cleavage, and 3) increased early apoptosis and lower levels of cleavage and blastocyst formation. Our results further revealed that such negative impacts of oocyte cryopreservation could be alleviated by supplementation of warming, recovery, PA and IVC media with 10− 9 mol/L MT before the embryos moved into the 2-cell stage of development. Conclusions MT might promote cell cycle progression via regulation of MMP, ATP, ROS and maternal mRNA levels, potentially increasing the first cleavage of parthenogenetic zygotes developed from vitrified–warmed mouse oocytes and their subsequent development.

Female reproductive (ovarian) aging is characterized by a marked decline in quantity and quality of follicles and oocytes, as well as alterations in the surrounding ovarian stroma. In our previous report, we have shown that dietary selenium (Se) insufficiency and supplementation differentially impacted the reproductive efficiency in aging mice; however, the precise understanding of such modulation is still incomplete. In the present study, we sought to determine the impact of low (mildly low level) and moderately high (medium level) Se diets on expression profile of non-selenoprotein genes in the ovaries of aging mice. For this purpose, the aged mice were divided in two groups and fed either a low Se (Se-L; 0.08 mg Se/kg) diet or a moderately high Se (Se-M; 0.33 mg Se/kg) diet. RNA-seq analysis revealed that a total of 168 genes were differentially expressed between the two groups. From these, 72 and 96 differentially expressed genes (DEGs) were found to be upregulated and downregulated, respectively. Gene Ontology (GO) and pathways enrichment (KEGG) analyses revealed that these DEGs were enriched in several key GO terms and biological pathways including PI3K-Akt signaling pathway, steroid hormone biosynthesis, signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, ovarian steroidogenesis, and Wnt signaling pathway. Further filtering of RNA-seq data revealed that several DEGs such as Star, Hsd3b6, Scd1, Bmp7, Aqp8, Gas1, Fzd1, and Wwc1 were implicated in key ovarian- and fertility-related functions. In addition, some of the DEGs were related to ER homeostasis and/or proteostasis. These results highlight that dietary low and moderately high (medium level) Se diets, in addition to modulation of selenoproteins, can also have an impact on expression of several non-selenoprotein genes in the ovaries of aging mice. To sum up, these findings add more value to our understanding of Se modulation of ovarian functions and female fertility and will pave a way for the focused mechanistic and functional studies in this domain.

Previous studies have shown that melatonin can mitigate cryopreservation-induced mitochondrial dysfunction in oocytes; however, the underlying molecular mechanism remains unclear. The objective of the present study was to investigate whether melatonin can improve the mitochondrial function during in vitro maturation of vitrified-warmed mouse germinal vesicle (GV) oocytes by modulating phosphorylation of dynamin related protein 1 (Drp1). Vitrification/warming procedures resulted in the following: (1) After cryopreservation of mouse GV oocytes, the phosphorylation level of Drp1 at Ser616 (p-Drp1 Ser616) in metaphase II (MII) oocytes was increased (P < 0.05). Furthermore, the rates of in vitro maturation, cleavage and blastocyst formation after parthenogenetic activation were decreased (P < 0.05). (2) In MII oocytes, the expression levels of translocase of the mitochondrial outer membrane 20 (TOMM20), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, and mRNA levels of mitochondrial biogenesis-related genes (Sirt1, Pgc-1α, Tfam) were all decreased (P < 0.05), and (3) Reactive oxygen species (ROS) level, early apoptosis level, Cytochrome C release and mRNA levels of pro-apoptotic related genes (Bax, Caspase9, Caspase3) in MII oocytes were all increased (P < 0.05). The results of this study further revealed that negative impacts of GV oocyte cryopreservation were mitigated by supplementation of warming and in vitro maturation media with 10−7mol /L melatonin or 2 x 10−5mol/L Mdivi-1 (Drp1 inhibitor). Therefore, we concluded that 10−7mol/L melatonin improved mitochondrial function, reduced oxidative stress and inhibited apoptosis by regulating phosphorylation of Drp1, thereby enhancing in vitro development of vitrified-warmed mouse GV oocytes.

Previously it was reported that melatonin could mitigate oxidative stress caused by oocyte cryopreservation, however, the underlying molecular mechanisms which cause this remain unclear. The objective was to explore whether melatonin could reduce oxidative stress during in vitro maturation of vitrified-warmed mouse germinal vesicle (GV) oocytes through the Nrf2 signaling pathway or its receptors. During in vitro maturation of vitrified-warmed mouse GV oocytes, there were decreases (p &lt, 0.05) in the development rates of metaphase I (MI) oocytes and metaphase II (MII) and spindle morphology grades, increases (p &lt, 0.05) in the reactive oxygen species (ROS) levels, and decreases (p &lt, 0.05) in expressions of Nrf2 signaling pathway-related genes (Nrf2, SOD1) and proteins (Nrf2, HO-1). However, adding 10−7 mol/L melatonin to both the warming solution and maturation solutions improved (p &lt, 0.05) these indicators. When the Nrf2 protein was specifically inhibited by Brusatol, melatonin did not increase development rates, spindle morphology grades, genes, or protein expressions, nor did it reduce vitrification-induced intracellular oxidative stress in GV oocytes during in vitro maturation. In addition, when melatonin receptors were inhibited by luzindole, the ability of melatonin to scavenge intracellular ROS was decreased, and the expressions of genes (Nrf2, SOD1) and proteins (Nrf2, HO-1) were not restored to control levels. Therefore, we concluded that 10−7 mol/L melatonin acted on the Nrf2 signaling pathway through its receptors to regulate the expression of genes (Nrf2, SOD1) and proteins (Nrf2, HO-1), and mitigate intracellular oxidative stress, thereby enhancing in vitro development of vitrified-warmed mouse GV oocytes.

Melatonin (MT) is a potent antioxidant with useful applications in several fields. Due to the capacity to scavenge free radicals and enhance cellular endogenous antioxidant defenses, MT is widely used in sperm cryopreservation to protect against oxidative stress-induced damage in frozen-thawed sperm. In this article, there is a review of positive effects of MT supplementation in cryopreservation of sperm from domestic ruminants and swine. There is direct or indirect scavenging of free radicals, preventing lipid peroxidation (LPO), and reducing oxidative stress, therefore, protecting membrane and DNA integrity, enhancing post-thaw antioxidant and enzymatic functions to maintain mitochondrial functions and activity, and regulating ATP production and utilization leading to maintenance of sperm quality, motility, and viability. In addition, MT reportedly inhibits sperm apoptosis, potentially by enhancing sperm viability and modulating abundances of mRNA transcripts.

In the present report, we determined the impact of dietary selenium (Se) deficiency and supplementation on the expression of two ER-resident selenoproteins i.e., Selenok and Selenom in the ovaries of aging mice. The mRNA expression of Selenok and Selenom (RT-qPCR) was significantly higher in the ovaries of mice fed diets supplemented with inorganic (ISe-S: 0.33 mg Se/kg) and organic (OSe-S: 0.33 mg Se/kg) Se compared to those fed a Se-deficient (Se-D: 0.08 mg Se/kg) diet and both Se-adequate (ISe-A: 0.15 mg Se/kg and OSe-A: 0.15 mg Se/kg) diets. Similarly, the protein signals of SELENOK (immunofluorescence assay) were also significantly higher in the Se-supplemented groups compared to those fed Se-D and Se-adequate (ISe-A and OSe-A) diets. Meanwhile, the rate of in vitro-produced blastocysts developing from MII oocytes was also evaluated and it was revealed that this rate was significantly higher in the Se-supplemented mice compared to those fed a Se-D diet. Altogether, the dietary Se supplementation increased the expression of Selenok (also its protein expression) and Selenom in the ovaries of aging mice, potentially contributing to an improved developmental potential of in vitro-matured M II oocytes.

miR-26a is associated with sperm metabolism and can affect sperm motility and apoptosis. However, how miR-26a affects sperm motility remains largely unknown. Our previous study indicated that the PDHX gene is predicted to be a potential target of miR-26a, which is responsible for pyruvate oxidative decarboxylation which is considered as a key step for connecting glycolysis with oxidative phosphorylation. In this study, we first reported a potential relationship between miR-26a and PDHX and their expressions in fresh, frozen-thawed, and epididymal boar sperm. Then, sperm viability and survival were determined after transfection of miR-26a. mRNA and protein expression level of PDHX in the liquid-preserved boar sperm after transfection were also determined by RT-qPCR and Western Blot (WB). Our results showed that expression level of PDHX was significantly increased during sperm transit from epididymal caput to corpus and cauda. Similarly, expression of PDHX was significantly higher (P &lt, 0.05) in fresh sperm as compared to epididymal cauda and frozen-thawed sperm. However, the expression of miR-26a in epididymal corpus sperm was significantly higher (P &lt, 0.05) than that of caput and cauda sperm. Furthermore, after transfection of boar sperm with miR-26a mimic and inhibitor under liquid storage, the lowest and highest sperm viability was observed in miR-26a mimic and inhibitor treatment (P &lt, 0.05), respectively. The protein levels of PDHX, after 24 and 48 h of transfection of miR-26a mimics and inhibitor, were notably decreased and increased (P &lt, 0.05), respectively, as compared to negative control (NC) group. In conclusion, the novel and enticing findings of our study provide a reasonable evidence that miR-26a via PDHX, a link between glycolysis and oxidative phosphorylation, could regulate the glycometabolic pathway which eventually affect boar sperm viability and survival.

Lipopolysaccharide (LPS) released from Gram-negative bacteria binds to toll-like receptor 4 (TLR4) and induces boar sperm apoptosis. Similarly, polyhistidine (pHis), a TLR4 agonist, can also bind to TLR4. We hypothesized that pHis could inhibit LPS-induced sperm apoptosis by competitively binding to TLR4 to then improve sperm quality. Therefore, the objective of this study was to examine whether pHis can inhibit LPS-induced sperm apoptosis and affect sperm quality. The results showed that the concentrations of bacterial colonies were significantly increased from 36 to 120 h under liquid storage conditions (p &lt, 0.05), however, concentrations of LPS in boar semen showed a relatively constant trend (4.98 &plusmn, 1.55 EU/mL) following 120 h storage. The addition of 100 &mu, g/mL pHis in the BTS extender significantly improved boar sperm motility and viability at 37 &deg, C, and it significantly (p &lt, 0.05) inhibited boar sperm apoptosis under liquid storage (17 &deg, C) and at 37 &deg, C incubation conditions. The co-treatment of LPS and pHis further confirmed that pHis played its role in inhibiting LPS-induced sperm apoptosis. In conclusion, our preliminary findings provide reasonable evidence that pHis could act as an inhibitor of LPS-induced apoptosis in boar sperm stored for longer periods of time. pHis might inhibit LPS-induced sperm apoptosis by competitively binding to TLR4. Nevertheless, further mechanistic studies are awaited to fully elucidate its potential implication in inhibiting LSP-induced apoptosis.

In this study, we evaluated the effects of Cyclosporine A (CsA) on Lipopolysaccharide (LPS)-induced cytokine production in the genital tract of female rabbits. Twelve sexually mature and healthy female rabbits were randomly divided into four groups (n = 3 each). The rabbits in the LPS group were given an intrauterine infusion of Escherichia coli LPS (4 mg/kg body weight (BW)). Rabbits in the CsA group were given CsA (20 mg/kg BW). Rabbits in the LPS + CsA group were given LPS (4 mg/kg BW) and CsA (20 mg/kg BW). The control group received only LPS and CsA carrier. The gene expression and protein levels of pro- and anti-inflammatory cytokines were observed using qRT-PCR and immuno-histochemical (IHC) assay, respectively. Our study showed that IL-1β, IL-6, IL-8, TNF-α, IFN-γ, IL-4, IL-10, IL-13, and TGF-β were expressed in female genital organs. The LPS challenge increased the mRNA expression of IL-6 and TNF-α in the uterine body and IL-1β in the uterotubal junction compared to the control group. CsA increased the basal mRNA expression of anti-inflammatory cytokines (i.e., IL-4 in the uterine body, uterotubal junction, and oviductal ampulla; IL-10 in the cervix, oviductal isthmus, and ampulla; and TGF-β in the uterotubal junction and oviductal ampulla) and pro-inflammatory cytokines (i.e., IL-6 and IL-8 in the cervix; IL-1β in the oviductal isthmus; TNF-α in the oviductal ampulla; and IFN-γ in the uterine body compared to the control group). In addition, CsA inhibited the mRNA expression of pro-inflammatory cytokines, such as IL-6 in the uterine body, uterotubal junction, and oviductal isthmus; TNF-α in the uterine body; and IFN-γ in the uterotubal junction and oviductal isthmus induced by the LPS challenge. The IHC assay showed the LPS-induced increase in protein production of IL-6 in the uterine body and oviductal isthmus. CsA increased the protein production of IL-10 in the cervix, uterine body, oviductal ampulla, and isthmus. Moreover, CsA decreased the protein production of IL-6 in the uterine body and oviductal isthmus induced by LPS.

This study aimed to investigate the effect of melatonin on the cell cycle of parthenogenetic embryos derived from vitrified mouse metaphase II (MII) oocytes. Fresh oocytes were randomly allocated into three groups: untreated (control), or vitrified by the open-pulled straw method without (Vitrification group) or with melatonin (MT) supplementation (Vitrification + MT group). After warming, oocytes were parthenogenetically activated and cultured in vitro, then the percentage of embryos in the G1/S phase, the levels of reactive oxygen species (ROS) and glutathione (GSH), and the mRNA expression of cell cycle-related genes (P53, P21 and E2F1) in zygotes and their subsequent developmental potential in vitro were evaluated. The results showed that the vitrification/warming procedures significantly decreased the frequency of the S phase, markedly increased ROS and GSH levels and the expression of P53 and P21 genes, and decreased E2F1 expression in zygotes at the G1 stage and their subsequent development into 2-cell and blastocyst stage embryos. However, when 10&minus, 9 mol/L MT was administered for the whole duration of the experiment, the frequency of the S phase in zygotes was significantly increased, while the other indicators were also significantly improved and almost recovered to the normal levels shown in the control. Thus, MT might promote G1-to-S progression via regulation of ROS, GSH and cell cycle-related genes, potentially increasing the parthenogenetic development ability of vitrified&ndash, warmed mouse oocytes.

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.

As an important micronutrient, selenium (Se) plays many essential roles in immune response and protection against pathogens in humans and animals, but underlying mechanisms of Se-based control of salmonella growth within macrophages remain poorly elucidated. In this study, using RNA-seq analyses, we demonstrate that Se treatment (at an appropriate concentration) can modulate the global transcriptome of chicken macrophages HD11. The bioinformatic analyses (KEGG pathway analysis) revealed that the differentially expressed genes (DEGs) were mainly enriched in retinol and glutathione metabolism, revealing that Se may be associated with retinol and glutathione metabolism. Meanwhile, Se treatment increased the number of salmonella invading the HD11 cells, but reduced the number of salmonella within HD11 cells, suggesting that enhanced clearance of salmonella within HD11 cells was potentially modulated by Se treatment. Furthermore, RNA-seq analyses also revealed that nine genes including SIVA1, FAS, and HMOX1 were differentially expressed in HD11 cells infected with salmonella following Se treatment, and GO enrichment analysis showed that these DEGs were mainly enriched in an extrinsic apoptotic signaling pathway. In summary, these results indicate that Se treatment may not only affect retinol and glutathione metabolism in macrophages, but could also inhibit salmonella-induced macrophage apoptosis via an extrinsic apoptotic signaling pathway involving SIVA1.

Selenium (Se) is an essential micronutrient that has several important functions in animal and human health. The biological functions of Se are carried out by selenoproteins (encoded by twenty-five genes in human and twenty-four in mice), which are reportedly present in all three domains of life. As a component of selenoproteins, Se has structural and enzymatic functions; in the latter context it is best recognized for its catalytic and antioxidant activities. In this review, we highlight the biological functions of Se and selenoproteins followed by an elaborated review of the relationship between Se and female reproductive function. Data pertaining to Se status and female fertility and reproduction are sparse, with most such studies focusing on the role of Se in pregnancy. Only recently has some light been shed on its potential role in ovarian physiology. The exact underlying molecular and biochemical mechanisms through which Se or selenoproteins modulate female reproduction are largely unknown; their role in human pregnancy and related complications is not yet sufficiently understood. Properly powered, randomized, controlled trials (intervention vs. control) in populations of relatively low Se status will be essential to clarify their role. In the meantime, studies elucidating the potential effect of Se supplementation and selenoproteins (i.e., GPX1, SELENOP, and SELENOS) in ovarian function and overall female reproductive efficiency would be of great value.