The Impact of FBN1-α5ß1 Axis in Fibro/Adipogenic Progenitor Cells (FAPCD9-) on Intramuscular Fat Content in Pigs.

Intramuscular fat (IMF) plays a crucial role in enhancing meat quality, enriching meat flavor, and overall improving palatability. In this study, Single-cell RNA sequencing was employed to analyze the longissimus dorsi (LD) obtained from Guangdong small-ear spotted pigs (GDSS, with high IMF) and Yorkshire pigs (YK, with low IMF). GDSS had significantly more Fibro/Adipogenic Progenitor (FAPs), in which the CD9 negative FAPs (FAPCD9-) having adipogenic potential, as demonstrated by in vitro assays using cells originated from mouse muscle. On the other hand, Yorkshire had more fibro-inflammatory progenitors (FIPs, marked with FAPCD9+), presenting higher expression of the FBN1-Integrin α5ß1. FBN1-Integrin α5ß1 could inhibit insulin signaling in FAPCD9-, suppressing adipogenic differentiation. Our results demonstrated that fat-type pigs possess a greater number of FAPCD9-, which are the exclusive cells in muscle capable of differentiating into adipocytes. Moreover, lean-type pigs exhibit higher expression of FBN1-Integrin α5ß1 axis, which inhibits adipocyte differentiation. These results appropriately explain the observed higher IMF content in fat-type pigs.

Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function.

Bone morphogenetic protein 15 (BMP15) plays a crucial role in the porcine follicular development. However, its exact functions in the in vitro maturation (IVM) of porcine oocytes remain largely unknown. Here, through cytoplasmic injection of a preassembled crRNA-tracrRNA-Cas9 ribonucleoprotein complex, we achieved BMP15 disruption in approximately 54 % of the cultured porcine oocytes. Editing BMP15 impaired the IVM of porcine oocytes, as indicated by the significantly increased abnormal spindle assembly and reduced first polar body (PB1) extrusion. The editing also impaired cytoplasmic maturation of porcine oocytes, as reflected by reduced abundant of Golgi apparatus and impaired functions of mitochondria. The impaired IVM of porcine oocytes by editing BMP15 possibly was associated with the attenuated SMAD1/5 and EGFR-ERK1/2 signaling in the cumulus granulosa cells (CGCs) and the inhibited MOS/ERK1/2 signaling in oocytes. The attenuated MOS/ERK1/2 signaling may contribute to the inactivation of maturation promoting factor (MPF) and the increased abnormal spindle assembly, leading to reduced PB1 extrusion. It also may contribute to reduced Golgi apparatus formation, and impaired functions of mitochondria. These findings expand our understanding of the regulatory role of BMP15 in the IVM of porcine oocytes and provide a basis for manipulation of porcine reproductive performance.

Efficient generation of cloned cats with altered coat colour by editing of the KIT gene.

Coat colour largely determines the market demand for several cat breeds. The KIT proto-oncogene (KIT) gene is a key gene controlling melanoblast differentiation and melanogenesis. KIT mutations usually cause varied changes in coat colour in mammalian species. In this study, we used a pair of single-guide RNAs (sgRNAs) to delete exon 17 of KIT in somatic cells isolated from two different Chinese Li Hua feline foetuses. Edited cells were used as donor nuclei for somatic cell nuclear transfer (SCNT) to generate cloned embryos presenting an average cleavage rate exceeding 85%, and an average blastocyst formation rate exceeding 9.5%. 131 cloned embryos were transplanted into four surrogates, and all surrogates carried their pregnancies to term, and delivered 4.58% (6/131) alive cloned kittens, with 1.53% (2/131) being KIT-edited heterozygotes (KITD17/+). The KITD17/+ cats presented an obvious darkness reduction in the mackerel tabby coat. Immunohistochemical analysis (IHC) of skin tissues indicated impaired proliferation and differentiation of melanoblasts caused by the lack of exon17 in feline KIT. To our knowledge, this is the first report on coat colour modification of cats through gene editing. The findings could facilitate further understanding of the regulatory role of KIT on feline coat colour and provide a basis for the breeding of cats with commercially desired coat colour.

Whole-Genome Sequencing Reveals Rare Off-Target Mutations in MC1R-Edited Pigs Generated by Using CRISPR-Cas9 and Somatic Cell Nuclear Transfer.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used to create animal models for biomedical and agricultural use owing to its low cost and easy handling. However, the occurrence of erroneous cleavage (off-targeting) may raise certain concerns for the practical application of the CRISPR-Cas9 system. In this study, we created a melanocortin 1 receptor (MC1R)-edited pig model through somatic cell nuclear transfer (SCNT) by using porcine kidney cells modified by the CRISPR-Cas9 system. We then carried out whole-genome sequencing of two MC1R-edited pigs and two cloned wild-type siblings, together with the donor cells, to assess the genome-wide presence of single-nucleotide variants and small insertions and deletions (indels) and found only one candidate off-target indel in both MC1R-edited pigs. In summary, our study indicates that the minimal off-targeting effect induced by CRISPR-Cas9 may not be a major concern in gene-edited pigs created by SCNT.

Single-base editing in IGF2 improves meat production and intramuscular fat deposition in Liang Guang Small Spotted pigs.

BACKGROUND: Chinese indigenous pigs are popular with consumers for their juiciness, flavour and meat quality, but they have lower meat production. Insulin-like growth factor 2 (IGF2) is a maternally imprinted growth factor that promotes skeletal muscle growth by regulating cell proliferation and differentiation. A single nucleotide polymorphism (SNP) within intron 3 of porcine IGF2 disrupts a binding site for the repressor, zinc finger BED-type containing 6 (ZBED6), leading to up-regulation of IGF2 and causing major effects on muscle growth, heart size, and backfat thickness. This favorable mutation is common in Western commercial pig populations, but absent in most Chinese indigenous pig breeds. To improve meat production of Chinese indigenous pigs, we used cytosine base editor 3 (CBE3) to introduce IGF2-intron3-C3071T mutation into porcine embryonic fibroblasts (PEFs) isolated from a male Liang Guang Small Spotted pig (LGSS), and single-cell clones harboring the desired mutation were selected for somatic cell nuclear transfer (SCNT) to generate the founder line of IGF2T/T pigs. RESULTS: We found the heterozygous progeny IGF2C/T pigs exhibited enhanced expression of IGF2, increased lean meat by 18%-36%, enlarged loin muscle area by 3%-17%, improved intramuscular fat (IMF) content by 18%-39%, marbling score by 0.75-1, meat color score by 0.53-1.25, and reduced backfat thickness by 5%-16%. The enhanced accumulation of intramuscular fat in IGF2C/T pigs was identified to be regulated by the PI3K-AKT/AMPK pathway, which activated SREBP1 to promote adipogenesis. CONCLUSIONS: We demonstrated the introduction of IGF2-intron3-C3071T in Chinese LGSS can improve both meat production and quality, and first identified the regulation of IMF deposition by IGF2 through SREBP1 via the PI3K-AKT/AMPK signaling pathways. Our study provides a further understanding of the biological functions of IGF2 and an example for improving porcine economic traits through precise base editing.

KLF4 regulates skeletal muscle development and regeneration by directly targeting P57 and Myomixer.

Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell proliferation, apoptosis, and differentiation. Our previous study showed that KLF4 expression is upregulated in skeletal muscle ontogeny during embryonic development in pigs, suggesting its importance for skeletal muscle development and muscle function. We revealed here that KLF4 plays a critical role in skeletal muscle development and regeneration. Specific knockout of KLF4 in skeletal muscle impaired muscle formation further affecting physical activity and also defected skeletal muscle regeneration. In vitro, KLF4 was highly expressed in proliferating myoblasts and early differentiated cells. KLF4 knockdown promoted myoblast proliferation and inhibited myoblast fusion, while its overexpression showed opposite results. Mechanically, in proliferating myoblasts, KLF4 inhibits myoblast proliferation through regulating cell cycle arrest protein P57 by directly targeting its promoter; while in differentiated myoblasts, KLF4 promotes myoblast fusion by transcriptionally activating Myomixer. Our study provides mechanistic information for skeletal muscle development, reduced muscle strength and impaired regeneration after injury and unveiling the mechanism of KLF4 in myogenic regulation.

Molecular characterization of the follicular development of BMP15-edited pigs.

In brief: The regulatory role of BMP15 on porcine ovarian follicular development still remains unclear. This study reveals that biallelic editing of BMP15 impairs SMAD signaling and inhibits granulosa cell proliferation, resulting in porcine follicular development arrest and ovarian hypoplasia. Abstract: Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta (TGF-ß) superfamily, which is critical for facilitating ovarian folliculogenesis in mono-ovulatory mammalian species but is not essential in polyovulatory mice. Our previously established BMP15-edited pigs presented varied female reproductive phenotypes, suggesting the important role of BMP15 in ovarian folliculogenesis in polyovulatory pigs. To understand the regulatory mechanism underlying the effect of BMP15 on porcine ovarian follicular development, we molecularly characterized infertile biallelic-BMP15-edited gilts with ovarian hypoplasia. We found that an absence of BMP15 proteins in biallelic-BMP15-edited gilts can lead to premature activation of primordial follicles, possibly through the upregulation of KITLG-KIT-PI3K-AKT signaling pathways. However, this absence severely impaired SMAD (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling, causing severely reduced granulosa cell proliferation, leading to the arrest of follicular development during the preantral stage and ovarian hypoplasia, resulting in complete infertility. Our study expands the understanding of the molecular functions of BMP15 in nonrodent polyovulatory mammals.

Efficient editing BMP15 in porcine oocytes through microinjection of CRISPR ctRNP.

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/µL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/µL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.

c-Mpl-del, a c-Mpl alternative splicing isoform, promotes AMKL progression and chemoresistance.

Acute megakaryocytic leukemia (AMKL) is a clinically heterogeneous subtype of acute myeloid leukemia characterized by unrestricted megakaryoblast proliferation and poor prognosis. Thrombopoietin receptor c-Mpl is a primary regulator of megakaryopoeisis and a potent mitogenic receptor. Aberrant c-Mpl signaling has been implicated in a myriad of myeloid proliferative disorders, some of which can lead to AMKL, however, the role of c-Mpl in AMKL progression remains largely unexplored. Here, we identified increased expression of a c-Mpl alternative splicing isoform, c-Mpl-del, in AMKL patients. We found that c-Mpl-del expression was associated with enhanced AMKL cell proliferation and chemoresistance, and decreased survival in xenografted mice, while c-Mpl-del knockdown attenuated proliferation and restored apoptosis. Interestingly, we observed that c-Mpl-del exhibits preferential utilization of phosphorylated c-Mpl-del C-terminus Y607 and biased activation of PI3K/AKT pathway, which culminated in upregulation of GATA1 and downregulation of DDIT3-related apoptotic responses conducive to AMKL chemoresistance and proliferation. Thus, this study elucidates the critical roles of c-Mpl alternative splicing in AMKL progression and drug resistance, which may have important diagnostic and therapeutic implications for leukemia accelerated by c-Mpl-del overexpression.

Quercetin protects porcine oocytes from in vitro aging by reducing oxidative stress and maintaining the mitochondrial functions.

Quercetin (QUE) is a component of the flavonoid family that shows various therapeutic properties, such as antioxidant effects. However, whether QUE affects porcine oocyte in vitro aging has not yet been investigated. Therefore, in this study, we applied various doses of QUE to freshly isolated porcine oocytes and found that 10 µM QUE improved the oocyte maturation rate in vitro, as reflected by the increased degree of cumulus cell expansion and first polar body extrusion. More importantly, we found that QUE reduced in vitro aging and improved the maturity level of porcine oocytes after another 24 h of culturing, accompanied by the upregulated expression levels of bone morphogenetic protein 15, growth differentiation factor 9, Moloney sarcoma oncogene, and cyclin-dependent kinase 2. In addition, we found that QUE treatment significantly reduced the intracellular reactive oxygen species levels, apoptosis, and autophagy and upregulated the expression levels of superoxide dismutase 2 and catalase in aged porcine oocytes. In addition, QUE restored impaired mitochondrial membrane potential and spindle assembly in aged porcine oocytes. Our findings demonstrate that QUE can protect porcine oocytes from in vitro aging by reducing oxidative stress and maintaining mitochondrial function.

Impact of KIT Editing on Coat Pigmentation and Fresh Meat Color in Yorkshire Pigs.

The white coat color of Yorkshire pigs is caused by the dominant white I allele, which has been associated with at least one copy of the 450-kb duplication encompassing the entire KIT gene and a splice mutation (G > A) at the first base of intron 17. The splice mutation in KIT has an adverse effect on pigmentation in mice. Therefore, removing the 450 kb duplications harboring the KIT copy with splice mutations is expected to affect Yorkshire pig pigmentation. In this study, we describe the use of a Yorkshire pig kidney cell strain with the I?/IBe-ed genotype, previously created by CRISPR-Cas9, as donor cells for somatic cell nuclear transfer to generate gene-edited Yorkshire pigs. The removal of the 450 kb duplications harboring the KIT copy with splice mutation did not alter the white coat color of Yorkshire pigs, which was confirmed by the absence of fully mature melanocytes and melanin accumulation in the hair follicles. Except for the improved transcription of tyrosinase, and slight increase in microphthalmia transcription factor and tyrosinase-related protein 1 protein expression, there was no significant impact of the removal of splice mutations on genes and signaling pathways (PI3K/AKT) involved in melanogenesis. However, the removal of the 450 kb duplications harboring the KIT copy with splice mutation substantially improved fresh meat color accompanied by significantly increased red blood cell number, which merits further investigation. Our study provides new insights into the role of structural mutations of the KIT gene in the formation of white coat color and erythropoiesis in Yorkshire pigs.

Characterization and Pathogenicity of Two Novel PRRSVs Recombined by NADC30-like and NADC34-like Strains in China.

Porcine reproductive and respiratory syndrome viruses (PRRSVs) pose a serious threat to the swine industry in China, which has caused great difficulties for porcine reproductive and respiratory syndrome (PRRS) immune prevention and control, due to its easily mutable and recombinant nature. In this study, two novel PRRSV strains, which were named GD-H1 and GD-F1, were isolated and fully sequenced from pig farms in Guangdong province, China. The phylogenetic analysis and recombination analysis revealed that the GD-H1 and GD-F1 were generated by the recombination of NADC30-like and NADC34-like strains which were different from the previously prevalent strain. Further pathogenic studies on piglets and sows found that the recombinant strains could cause piglets high fever, loss of appetite and lung lesions, but no piglets died. However, the recombinant strains could cause acute death and abortion in pregnant sow infection models together with average survival rates of 62.5% and 37.5% abortion rates, respectively. These findings indicated that the recombinant strains were extremely pathogenic to sows. Therefore, we report two clinical novel recombinant strains of PRRSV that are different from the traditional epidemic strains in China, which may provide early warning and support for PRRS immune prevention and control.

Editing MC1R in human melanoma cells by CRISPR/Cas9 and functional analysis.

MC1R (melanocortin 1 receptor) encodes the melanocortin-1 receptor, which can activate intracellular cAMP synthesis under the stimulation of the α-melanocyte stimulating hormone (α-MSH) ligand. Increased cAMP then activates the protein kinase A (PKA) pathway, resulting in the up-regulation of the expression of the microphthalmia-associated transcription factor (MITF) which is a critical regulatory factor of melanin synthesis, and tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis tyrosinase (TYR), and ultimately affects production of eumelanin and pheomelanin, and the coat color phenotype of mammalian species. Previous reports have indicated that the mutation A243T in the transmembrane domain 6 (TM6) of MC1R protein might disrupt the function of MC1R, contributing to the red phenotype in Duroc pig. However, functional analysis of the A243T mutation in MC1R has not yet been carried out. In this study, we attempted to used single-stranded oligo-deoxyribonucleotides (ssODN) as donor templates to introduce the c.727G>A (A243T) mutation into MC1R in human melanoma cell line SK-MEL-2 by CRISPR/Cas9 to analyze its effects on MC1R functions. We found the occurrence of ssODN recombination reached to 10%. Unfortunately, Sanger sequencing MC1R in six single-cell clones revealed that none carried the c.727G>A mutation, but all carried undesired mutations surrounding the target site. Cells transfected with CRISPR/Cas9 plasmids and ssODN presented significantly attenuated cAMP activation, and down-regulated MITF and TYR expression, indicating that the editing MC1R could affect the melanin synthesis function in cells. This study provides a basis for further investigation the mechanism of MC1R mutation on animal coat color.

Functional characterization of cAMP signaling of variant porcine MC1R alleles in PK15 cells.

The melanocortin 1 receptor (MC1R), encoded by the classical extension (E) coat color locus, is expressed on the surface of melanocytes and plays a critical role in switching melanin synthesis from pheomelanin (red/yellow) to eumelanin (black/brown). Different MC1R alleles associated with various coat color patterns in pigs have been identified over the past decades. However, functional analysis of variant porcine MC1R alleles has not yet been performed. Therefore, in this study, we examined the subcellular localization and cyclic adenosine monophosphate (cAMP) signaling capability of MC1R variants in porcine kidney epithelial cells (PK15) overexpressing different MC1R alleles. Transcriptional slippage may partially restore the reading frame of the EP allele, possibly accounting for the observed spot phenotype. The A243T substitution in the e allele severely disrupted the membrane localization of the MC1R receptor, resulting in a severely impaired cAMP signaling capability. Both the V95M and L102P substitutions in the ED1 allele may contribute to the constitutively active function of MC1R, thus accounting for the dominant black phenotype. The D124N substitution in the ED2 allele severely attenuated the cAMP signaling capability of MC1R; however, whether this mutation contributes to the distinct phenotype of Hampshire pigs requires further investigation. Thus, our results provide new insights into the functional characteristics of MC1R variants and their roles in porcine coat color formation.

HMGB2 orchestrates mitotic clonal expansion by binding to the promoter of C/EBPß to facilitate adipogenesis.

High-mobility group box 2 (HMGB2) is an abundant, chromatin-associated protein that plays an essential role in the regulation of transcription, cell proliferation, differentiation, and tumorigenesis. However, the underlying mechanism of HMGB2 in adipogenesis remains poorly known. Here, we provide evidence that HMGB2 deficiency in preadipocytes impedes adipogenesis, while overexpression of HMGB2 increases the potential for adipogenic differentiation. Besides, depletion of HMGB2 in vivo caused the decrease in body weight, white adipose tissue (WAT) mass, and adipocyte size. Consistently, the stromal vascular fraction (SVF) of adipose tissue derived from hmgb2-/- mice presented impaired adipogenesis. When hmgb2-/- mice were fed with high-fat diet (HFD), the body size, and WAT mass were increased, but at a lower rate. Mechanistically, HMGB2 mediates adipogenesis via enhancing expression of C/EBPß by binding to its promoter at "GGGTCTCAC" specifically during mitotic clonal expansion (MCE) stage, and exogenous expression of C/EBPß can rescue adipogenic abilities of preadipocytes in response to HMGB2 inhibition. In general, our findings provide a novel mechanism of HMGB2-C/EBPß axis in adipogenesis and a potential therapeutic target for obesity.

KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors.

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.

Editing the cystine knot motif of MSTN enhances muscle development of Liang Guang Small Spotted pigs.

Myostatin (MSTN) is a member of the transforming growth factor-ß (TGF-ß) family, and functions as an inhibitor of muscle growth. Disrupting the inhibitory effect of MSTN on growth can provide an effective way to increase the muscle yield of livestock and poultry. The cysteine knot motif of TGF-ß can stabilize the structure of MSTN protein and plays an important regulatory role in the biological function of MSTN. Accordingly, in this study, we used the CRISRP/Cas9 to edit the exon 3 of MSTN in the kidney cells of Liang Guang Small Spotted pig (LPKCs), in order to disrupt the cysteine knot motif of MSTN and remove the inhibitory effect of MSTN on its target genes.MSTN-edited LPKCs were obtained through fluorescence-activated cell sorting (FACS) and used as donor cells for somatic cell nuclear transfer (SCNT) to generate cloned embryos, which were then transferred to surrogate sows to finally obtain eight MSTN-edited Liang Guang Small Spotted piglets. Among them, two survived to 10 days old. Genotyping revealed that these two piglets were gene edited heterozygotes with base deletion and substitution occurred within the coding sequence of C106 and C108 at the cystine knot motif of MSTN. These changes resulted in frameshift mutations, and conversion of C106 and C108 to other amino acids. More developments of muscles were observed at the shoulders and hips of the heterozygotes of MSTN-edited Liang Guang Small Spotted pigs. H&E analysis showed that the cross-sectional area (CSA) of myofiber inMSTN-edited pigs was significantly decreased, and the number of myofiber were significantly increased. Western blot analysis showed that the disruption of C106 and C108 did not affect the expression of MSTN protein, but significantly up-regulated the expression of its target genes such as Myf5, MyoD, Myogenin and other myogenic regulatory factors. In summary, the gene-edited pig model obtained in this study did not cause complete loss of MSTN expression, and could retain other biological functions of MSTN, thereby promoting muscle growth while minimizing the potential adverse effects on complete loss of MSTN in the Liang Guang Small Spotted pigs.

EZH2 is essential for spindle assembly regulation and chromosomal integrity during porcine oocyte meiotic maturation†.

Enhancer of zeste homolog 2 (EZH2) has been extensively investigated to participate in diverse biological processes, including carcinogenesis, the cell cycle, X-chromosome inactivation, and early embryonic development. However, the functions of this protein during mammalian oocyte meiotic maturation remain largely unexplored. Here, combined with RNA-Seq, we provided evidence that EZH2 is essential for oocyte meiotic maturation in pigs. First, EZH2 protein expression increased with oocyte progression from GV to MII stage. Second, the siRNA-mediated depletion of EZH2 led to accelerated GVBD and early occurrence of the first polar body extrusion. Third, EZH2 knockdown resulted in defective spindle assembly, abnormal SAC activity, and unstable K-MT attachment, which was concomitant with the increased rate of aneuploidy. Finally, EZH2 silencing exacerbated oxidative stress by increasing ROS levels and disrupting the distribution of active mitochondria in porcine oocytes. Furthermore, parthenogenetic embryonic development was impaired following the depletion of EZH2 at GV stage. Taken together, we concluded that EZH2 is necessary for porcine oocyte meiotic progression through regulating spindle organization, maintaining chromosomal integrity, and mitochondrial function.

Novel mutations in the PLCZ1 gene associated with human low or failed fertilization.

BACKGROUND: Fertilization failure (FF) is a complex reproductive disorder characterized by the failure of pronuclei formation during fertilization. In addition to some cases caused by iatrogenic problems and known genetic factors, there are still many unexplained aspects of FF. Here, we aimed to assess the clinical and genetic characteristics of two families experiencing primary infertility with FF. METHODS: We have characterized two families from China. All of the infertile couples presented with similar clinical phenotypes, that is, partial or total fertilization failure in repeated cycles. We performed Sanger sequencing of their WEE2, TLE6, and PLCZ1 genes, and further bioinformatics and functional analyses were performed to identify the pathogenic elements of the variants. RESULTS: We identified novel compound heterozygous mutations c.1259C>T (p.P420L) and c.1733T>C (p.M578T) in the PLCZ1 gene in a male patient of family 1 with total fertilization failure, and another novel homozygous mutation c.1727T>C (p.L576P) in the same gene in a male patient of family 2 with partial fertilization failure. These three novel mutations were absent in the control cohort and in the databases. The amino acids were conserved at their positions among six different species. All mutant amino acids were located in key domains and were predicted to impair hydrolytic activity and lead to PLCZ1 dysfunction. Further functional detection revealed that the three mutations could significantly impair the catalytic activity of PLCZ1. CONCLUSIONS: We identified three novel mutations in PLCZ1 associated with partial and total fertilization failure and have provided new evidence about the genetic basis of FF.

Efficient generation of bone morphogenetic protein 15-edited Yorkshire pigs using CRISPR/Cas9†.

Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed that BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in polyovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in polyovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15-edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at preantral stages, with formation of numerous structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in nonrodent polyovulatory species may be as important as that in mono-ovulatory species.