Microbial and metabolomic mechanisms mediating the effects of dietary inulin and cellulose supplementation on porcine oocyte and uterine development

Background Dietary fiber (DF) is often eschewed in swine diet due to its anti-nutritional effects, but DF is attracting growing attention for its reproductive benefits. The objective of this study was to investigate the effects of DF intake level on oocyte maturation and uterine development, to determine the optimal DF intake for gilts, and gain microbial and metabolomic insight into the underlying mechanisms involved. Methods Seventy-six Landrace × Yorkshire (LY) crossbred replacement gilts of similar age (92.6 ± 0.6 d; mean ± standard deviation [SD]) and body weight (BW, 33.8 ± 3.9 kg; mean ± SD) were randomly allocated to 4 dietary treatment groups (n = 19); a basal diet without extra DF intake (DF 1.0), and 3 dietary groups ingesting an extra 50% (DF 1.5), 75% (DF 1.75), and 100% (DF 2.0) dietary fiber mixture consisting of inulin and cellulose (1:4). Oocyte maturation and uterine development were assessed on 19 d of the 2nd oestrous cycle. Microbial diversity of faecal samples was analysed by high-throughput pyrosequencing (16S rRNA) and blood samples were subjected to untargeted metabolomics. Results The rates of oocytes showing first polar bodies after in vitro maturation for 44 h and uterine development increased linearly with increasing DF intake; DF 1.75 gilts had a 19.8% faster oocyte maturation rate and a 48.9 cm longer uterus than DF 1.0 gilts (P < 0.05). Among the top 10 microbiota components at the phylum level, 8 increased linearly with increasing DF level, and the relative abundance of 30 of 53 microbiota components at the genus level (> 0.1%) increased linearly or quadratically with increasing DF intake. Untargeted metabolic analysis revealed significant changes in serum metabolites that were closely associated with microbiota, including serotonin, a gut-derived signal that stimulates oocyte maturation. Conclusions The findings provide evidence of the benefits of increased DF intake by supplementing inulin and cellulose on oocyte maturation and uterine development in gilts, and new microbial and metabolomic insight into the mechanisms mediating the effects of DF on reproductive performance of replacement gilts.

Pterostilbene Alleviates Chlorpyrifos-Induced Damage During Porcine Oocyte Maturation

Chlorpyrifos (CPF), a widely used organophosphate pesticide, is reported to severely impair mammalian reproductive system. Pterostilbene (PTS), an effective free radical scavenger, is considered as beneficial for mammalian reproduction. However, the toxicity of CPF on oocyte maturation and whether PTS can eliminate the detrimental effect of CPF on oocytes remain unclear. Here, porcine oocytes were applied to investigate the potential effect and possible mechanism of CPF and PTS during oocyte maturation. This work demonstrated that CPF significantly delayed the meiotic progression and decreased the polar body extrusion by disturbing spindle assembly and chromosome alignment and causing DNA damage in oocytes (p &lt; 0.05). And, CPF significantly impaired oocyte cytoplasmic maturation by inducing the high level of reactive oxygen species and decreasing glutathione content (p &lt; 0.05). Moreover, CPF significantly triggered embryo apoptosis and reduced the blastocyst rate and cell number following parthenogenetic activation (p &lt; 0.05). Whereas CPF-exposed oocytes were treated with PTS, these defects caused by CPF were obviously rescued, and oocyte maturation and subsequent embryonic development were also significantly ameliorated (p &lt; 0.05). In conclusion, these results revealed that CPF exerted the toxic effect on porcine oocytes, while PTS effectively alleviated CPF-induced damage on oocytes. This work provides a potential strategy to protect oocyte maturation in mammalian species.

Tannin Supplementation Improves Oocyte Cytoplasmic Maturation and Subsequent Embryo Development in Pigs

To investigate the effects of tannins (TA) on porcine oocyte in vitro maturation (IVM), different concentrations of TA (0, 1, 10 and 100 μg/mL) were supplemented with a maturation medium and the COCs and subsequent embryonic development were examined. The results showed that 10 µg/mL TA significantly improved the cumulus expansion index (CEI), cumulus-expansion-related genes (PTGS1, PTGS2, PTX-3, TNFAIP6 and HAS2) expression and blastocyst formation rates after parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) compared to the control groups, but not oocyte nuclear maturation. Nevertheless, 10 µg/mL TA dramatically enhanced the mRNA expression of oocyte-development-related genes (BMP15, GDF9, CDC2 and CYCLIN B1), GSH, ATP, SOD1, PGC1α, BMP15, GDF9 and CDC2 levels and reduced intracellular ROS level in porcine oocytes. These results indicated that porcine oocyte cytoplasmic maturation was improved by 10 µg/mL TA treatment during IVM. In contrast, a high concentration of TA (100 μg/mL) significantly decreased the CEI and PTGS1, PTGS2, PTX-3 and HAS2 mRNA expressions in cumulus cells, and reduced oocyte nuclear maturation and the total cell numbers/blastocyst. In general, these data showed that 10 μg/mL TA supplementation has beneficial effects on oocyte cytoplasmic maturation and subsequent embryonic development in pigs.

Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant.

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal-to-zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here we report that porcine ssc-miR-205 and bovine bta-miR-10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc-miR-205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to porcine oocyte-to-embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.

Melatonin Protects Against Mdivi-1-Induced Abnormal Spindle Assembly and Mitochondrial Superoxide Production During Porcine Oocyte Maturation

Mitochondrial division inhibitor 1 (Mdivi-1) reportedly provides a close connection between oocyte maturation and mitochondrial function in pigs. N-acetyl-5-methoxy-tryptamine (melatonin) is known to be a representative antioxidant with the ability to rehabilitate meiotic maturation of porcine oocytes. However, the ability of melatonin to recover Mdivi-1-mediated disruption of spindle formation during meiotic maturation of porcine oocytes during in vitro maturation (IVM) has not been studied. Here, we first investigated changes in mitochondrial length, such as fragmentation and elongation form, in mature porcine oocytes during IVM. Mature oocytes require appropriate mitochondrial fission for porcine oocyte maturation. We identified a dose-dependent reduction in meiotic maturation in porcine oocytes following Mdivi-1 treatment (50, 75, and 100 μM). We also confirmed changes in mitochondrial fission protein levels [dynamin-related protein 1 phosphorylation at serine 616 (pDRP1-Ser616) and dynamin-related protein 1 (DRP1)], mitochondrial membrane potential, and ATP production in 75 μM Mdivi-1-treated oocytes. As expected, Mdivi-1 significantly reduced mitochondrial function and DRP1 protein levels and increased spindle abnormalities in porcine oocytes. In addition, we confirmed that melatonin restores abnormal spindle assembly and reduces meiotic maturation rates by Mdivi-1 during porcine oocyte maturation. Interestingly, the expression levels of genes that reduce DNA damage and improve tubulin formation were enhanced during porcine meiotic maturation. Taken together, these results suggest that melatonin has direct beneficial effects on meiotic maturation through tubulin formation factors during porcine oocyte maturation.