Under endoplasmic reticulum (ER)‐stress conditions, the unfolded protein response (UPR) generates a defense mechanism in mammalian cells. The regulation of UPR signaling is important in oocyte ...maturation, embryo development, and female reproduction of pigs. Recent studies have shown that melatonin plays an important role as an antioxidant to improve pig oocyte maturation. However, there is no report on the role of melatonin in the regulation of UPR signaling and ER‐stress during in vitro maturation (IVM) of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidative effects of melatonin on porcine oocyte maturation through the regulation of ER‐stress and UPR signaling. We investigated the changes in the mRNA/protein expression levels of three UPR signal genes (Bip/Grp78, ATF4, P90/50ATF6, sXbp1, and CHOP) on oocytes, cumulus cells, and cumulus‐oocyte complexes (COCs) during IVM (metaphase I; 22 hours and metaphase II; 44 hours) by Western blot and reverse transcription‐polymerase chain reaction analysis. Treatment with the ER‐stress inducer, tunicamycin (Tm), significantly increased expression of UPR markers. Additionally, cumulus cell expansion and meiotic maturation of oocytes were reduced in COCs of Tm‐treated groups (1, 5, and 10 μg/mL). We confirmed the reducing effects of melatonin (0.1 μmol/L) on ER‐stress after pretreatment with Tm (5 μg/mL; 22 hours) in maturing COCs. Addition of melatonin (0.1 μmol/L) to Tm‐pretreated COCs recovered meiotic maturation rates and expression of most UPR markers. In conclusion, we confirmed a role for melatonin in the modulation of UPR signal pathways and reducing ER‐stress during IVM of porcine oocytes.
The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer ...adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.
Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a ...technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase SpCas9(H840A)-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.
NIMA‐related kinase 2 (NEK2) is a serine/threonine protein kinase that regulates mitosis and plays pivotal roles in cell cycle regulation and DNA damage repair. However, its function in porcine ...embryonic development is unknown. In this study, we used an NEK2‐specific inhibitor, JH295 (JH), to investigate the role of NEK2 in embryonic development and the underlying regulatory mechanisms. Inhibition of NEK2 after parthenogenesis activation or in vitro fertilization significantly reduced the rates of cleavage and blastocyst formation, the numbers of trophectoderm and total cells and the cellular survival rate compared with the control condition. NEK2 inhibition delayed cell cycle progression at all stages from interphase to cytokinesis during the first mitotic division; it caused abnormal nuclear morphology in two‐ and four‐cell stage embryos. Additionally, NEK2 inhibition significantly increased DNA damage and apoptosis, and it altered the expression levels of DNA damage repair‐ and apoptosis‐related genes. Intriguingly, NEK2 inhibition downregulated the expression of β‐catenin and its downstream target genes. To validate the relationship between Wnt/β‐catenin signalling and NEK2 during porcine embryonic development, we cultured porcine embryos in JH‐treated medium with or without CHIR99021, a Wnt activator. CHIR99021 co‐treatment strongly restored the developmental parameters reduced by NEK2 inhibition to control levels. Our findings suggest that NEK2 plays an essential role in porcine embryonic development by regulating DNA damage repair and normal mitotic division via the Wnt/β‐catenin signalling pathway.
Schematic diagram for NEK2 functions during porcine embryonic development. NEK2 plays essential roles in normal mitotic division, DNA damage repair and cell cycle progression via the Wnt/β‐catenin signalling pathway in porcine early embryogenesis.
The glycosylphosphatidylinositol‐anchored sperm hyaluronidases (Hyals), sperm adhesion molecule 1 (SPAM1) and HYAL5, have long been believed to assist in sperm penetration through the cumulus‐oocyte ...complex (COC), but their role in mammalian fertilization remains unclear. Previously, we have shown that mouse sperm devoid of either Spam1 or Hyal5 are still capable of penetrating the COC and that the loss of either Spam1 or Hyal5 alone does not cause male infertility in mice. In the present study, we found that Spam1/Hyal5 double knockout (dKO) mice produced significantly fewer offspring compared with wild‐type (WT) mice, and this was due to defective COC dispersal. A comparative analysis between WT and Spam1/Hyal5 dKO epididymal sperm revealed that the absence of these 2 sperm Hyals resulted in a marked accumulation of sperm on the outside of the COC. This impaired sperm activity is likely due to the deficiency in the sperm Hyals, even though other somatic Hyals are expressed normally in the dKO mice. The fertilization ability of the Spam1/Hyal5 dKO sperm was restored by adding purified human sperm Hyal to the in vitro fertilization medium. Our results suggest that Hyal deficiency in sperm may be a significant risk factor for male sterility.—Park, S., Kim, Y.‐H., Jeong, P.‐S., Park, C., Lee, J.‐W., Kim, J.‐S., Wee, G., Song, B.‐S., Park, B.‐J., Kim, S.‐H., Sim, B.‐W., Kim, S.‐U., Triggs‐Raine, B., Baba, T., Lee, S.‐R., Kim, E. SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus‐oocyte complex penetration defect. FASEB J. 33, 14440‐14449 (2019). www.fasebj.org
Background
Melatonin is a multifunctional hormone synthesized in the pineal gland and peripheral reproductive tissues that regulates many biological processes. There is increasing evidence for a role ...of melatonin in oocyte maturation and embryonic development in various mammals. However, no study has reported evidence for the existence of melatonergic system, such as melatonin synthesis enzymes, melatonin membrane receptors, or melatonin binding sites in non‐human primate cumulus–oocyte complexes (COCs).
Methods
Reverse transcription polymerase chain reaction (RT‐PCR) and immunocytochemistry were performed to detect transcripts and proteins of the rate‐limiting enzyme in melatonin synthesis (arylalkylamine N‐acetyltransferase, AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NRH: quinone oxidoreductase 2, NQO2) in cynomolgus monkey COCs.
Results
RT‐PCR analyses revealed the presence of AANAT, MT1, MT2, and NQO2 transcripts in granulosa cells, germinal vesicle (GV)‐ and metaphase II (MII)‐stage cumulus cells, and oocytes. Immunocytochemistry revealed the presence of AANAT, MT1, MT2, and NQO2 proteins in GV‐ and MII‐stage COCs.
Conclusions
Our results provide the first evidence for the existence of the rate‐limiting enzyme required for melatonin synthesis, melatonin membrane receptors, and a melatonin binding site in non‐human primate COCs.
Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation ...(H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (
and
) and DNA methyltransferases (
,
, and
) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.
X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of ...the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.
This paper is concerned with the dynamic tensile characteristics of transformation-induced plasticity (TRIP)-type and dual phase (DP)-type steel sheets at intermediate strain rates ranging from 0.003 ...to 200
s
−1. The dynamic responses of TRIP600, TRIP800, DP600 and DP800 steel sheets are investigated with the evaluation of stress–strain curves, the strain rate sensitivity, the fracture elongation and the effect of pre-strain. The dynamic responses were acquired from dynamic tensile tests at the intermediate strain rates with a high-speed material testing machine developed. Experiments were carried out with specimens whose dimensions were carefully determined by finite element analyses and experiments to induce uniform deformation in the gauge section at the intermediate strain rates. The tensile tests provide stress–strain curves and the strain rate sensitivity. Experimental results show two important aspects for TRIP-type and DP-type steel sheets quantitatively: The flow stress increases as the strain rate increases and the fracture elongation and the formability of TRIP-type sheets are better than those of DP-type sheets at the intermediate strain rates. The pre-strain effect was also investigated for two types of metals at the intermediate strain rates. TRIP600 and DP600 steel specimens pre-stained by 5% and 10% were elongated at the strain rate of 0.003
s
−1 for quasi-static loading, and then tested at strain rates of 0.003, 1, 10 and 100
s
−1. The results demonstrate that the mechanical properties of TRIP600 and DP600 steels are noticeably influenced by the pre-strain when the strain rate is over 1
s
−1. The ultimate tensile strength as well as the yield stress increases due to the pre-strain.
Heat stress (HS) is an emerging issue that greatly impairs the reproductive performance of animals and humans. In particular, disruption of oocyte maturation due to HS is considered a major cause of ...impaired reproductive performance. HS is known to induce ceramide generation, which causes reactive oxygen species (ROS) production and mitochondrial dysfunction, thereby inducing apoptosis. Therefore, we investigated whether inhibition of ceramide generation ameliorates HS-induced apoptosis in porcine cumulus–oocyte complexes (COCs) using specific inhibitors of the de novo (fumonisin B1, FB1) and hydrolytic pathways (desipramine, Des) of ceramide formation. We investigated the effects of FB1 and Des supplementation under HS conditions (41.5 °C for 44 h) on in vitro maturation (IVM) of porcine COCs. After IVM, HS significantly reduced proportion of COCs exhibiting fully expanded cumulus cells and the rate of metaphase II in oocytes. After parthenogenetic activation (PA), HS significantly reduced the rates of cleavage and blastocyst formation with a lower total cell number and a higher percentage of apoptosis in blastocysts. However, FB1 or Des supplementation under HS avoided detrimental effects of HS on expansion of cumulus cells, nuclear maturation of oocytes, and embryonic development after PA including the rates of cleavage and blastocyst formation, total cell number, and the percentage of apoptosis in blastocysts. Furthermore, FB1 or Des addition under HS, compared with HS alone, significantly decreased ceramide generation, ROS production, cytochrome C expression, and apoptosis and increased mitochondrial membrane potential in COCs, reaching levels comparable with those of the control. Taken together, our results indicate that HS impaired oocyte maturation through ceramide-mediated apoptosis.
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•Heat stress (HS) impairs porcine oocyte maturation.•Ceramide generation inhibitors avoid impaired oocyte maturation exposed to HS.•Ceramide generation inhibitors prevent ceramide-mediated apoptosis induced by HS.•Ceramide generation is involved in HS disruption of oocyte maturation.
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