Development of an enzyme-linked immunosorbent assay based on viral antigen capture by anti-spike glycoprotein monoclonal antibody for detecting immunoglobulin A antibodies against porcine epidemic diarrhea virus in milk.

BACKGROUND: Porcine epidemic diarrhea (PED), caused by PED virus (PEDV), is a severe enteric disease burdening the global swine industry in recent years. Especially, the mortality of PED in neonatal piglets approaches 100%. Maternal antibodies in milk, particularly immunoglobulin A (IgA) antibodies, are of great importance for protection neonatal suckling piglets against PEDV infection as passive lactogenic immunity. Therefore, appropriate detection methods are required for detecting PEDV IgA antibodies in milk. In the current study, we prepared monoclonal antibodies (mAbs) against PEDV spike (S) glycoprotein. An enzyme-linked immunosorbent assay (ELISA) was subsequently developed based on PEDV antigen capture by a specific anti-S mAb. RESULTS: The developed ELISA showed high sensitivity (the maximum dilution of milk samples up to 1:1280) and repeatability (coefficient of variation values < 10%) in detecting PEDV IgA antibody positive and negative milk samples. More importantly, the developed ELISA showed a high coincidence rate with a commercial ELISA kit for PEDV IgA antibody detection in clinical milk samples. CONCLUSIONS: The developed ELISA in the current study is applicable for PEDV IgA antibody detection in milk samples, which is beneficial for evaluating vaccination efficacies and neonate immune status against the virus.

Identification and characterization of circRNAs related to meat quality during embryonic development of the longissimus dorsi muscle in two pig breeds.

Meat quality, an important economic trait, is regulated by many factors, especially by genetic factors, including coding genes, miRNAs, and lncRNAs. Recent studies have elucidated that circRNAs also play a key role in muscle development and lipid deposition. However, the functions and regulatory mechanisms of circRNAs in meat quality remain mostly unknown. The circRNA expression profiles between Huainan pigs (Chinese indigenous pigs, fat-type, Huainan HN) and Large White pigs (Western commercial pigs, lean-type, LW) in the longissimus dorsi (LD) muscle at 38, 58, and 78 days post conception (dpc) were compared by sequencing. In total, 39,887 circRNAs were identified in 18 samples, and 60, 78, and 86 differentially expressed circRNAs (DECs) were found at the three stages mentioned above between these two breeds. The parent genes of DECs were enriched in myogenesis, proliferation, adipogenesis and muscle fiber-type transition. The circRNA-miRNA interaction networks included 38 DECs and 47 miRNAs, and these miRNAs were involved in muscle development and lipid metabolism. Two shared DECs (circ_0030593 and circ_0032760) of these three stages were selected, their head-to-tail junction sites were validated by Sanger sequencing, and RT‒qPCR results suggested that these two DECs might be involved in intramuscular fat deposition. These findings provide a basis for understanding the role of circRNAs in meat quality.

Downregulation of miR-192 Alleviates Oxidative Stress-Induced Porcine Granulosa Cell Injury by Directly Targeting Acvr2a.

Follicular atresia is primarily caused by breakdown to granulosa cells (GCs) due to oxidative stress (OS). MicroRNAs (miRNAs) elicit a defense response against environmental stresses, such as OS, by acting as gene-expression regulators. However, the association between miRNA expression and OS in porcine GCs (PGCs) is unclear. Here, we examined the impact of H2O2-mediated OS in PGCs through miRNA-Seq. We identified 22 (14 upregulated and 8 downregulated) and 33 (19 upregulated and 14 downregulated) differentially expressed miRNAs (DEmiRNAs) at 100 µM and 300 µM H2O2, respectively, compared with the control group. Among the DEmiRNAs, mi-192 was most induced by H2O2-mediated OS, and the downregulation of miR-192 alleviated PGC oxidative injury. The dual-luciferase reporter assay results revealed that miR-192 directly targeted Acvr2a. The Acvr2a level was found to be remarkably decreased after OS. Furthermore, grape seed procyanidin B2 (GSPB2) treatment significantly reduced the H2O2-induced upregulation of miR-192, and decreased PGC apoptosis and oxidative damage. Meanwhile, GSPB2 prevented an H2O2-induced increase in caspase-3 activity, which was enhanced by the application of the miR-192 inhibitor. These results indicate that GSPB2 protects against PGC oxidative injury via the downregulation of miR-192, the upregulation of Acvr2a expression, and the suppression of the caspase-3 apoptotic signaling pathway.

Notch signaling leads to a slower progression of embryonic myogenic differentiation in Landrace than in Langtang pigs.

Delving into porcine embryonic myogenesis is the key to elucidate the complex regulation of breed-specific differences in growth performance and meat production. Increasing evidence proves that pigs with less meat production show earlier embryonic myogenesis, but little is known about the underlying mechanisms. In this study, we examine the longissimus dorsi muscle (LDM) by immunohistochemistry and confirm that the differentiation of myogenic progenitors is increased ( P<0.05) in Lantang (LT, fatty) pigs compared with that in Landrace (LR, lean) pigs, which results in more ( P<0.001) differentiated myoblasts (Pax7 -/MyoD +) and less ( P<0.001) myogenic progenitors (Pax7 +/MyoD -) in LT pigs at 35 days post-conception (35dpc). Additionally, embryonic myogenic progenitors isolated from LT pigs show greater ( P<0.001) differentiation capacity with earlier expression of MyoD compared with those from LR pigs. Moreover, Notch signaling is more active ( P<0.05) in LR pig myogenic progenitors than in LT pig myogenic progenitors. Inhibition of Notch signaling in LR myogenic progenitors suppresses Pax7 expression and increases MyoD expression, thus promoting myogenic differentiation. Consistently, the process of myogenic progenitors differentiating into myoblasts in ex vivo embryo limbs is accelerated when Notch signaling is inhibited. These results indicate that Notch signaling facilitates the maintenance of myogenic progenitors and antagonizes myogenic differentiation by promoting Pax7 expression and preventing MyoD expression in LR pigs.

Analysis of differentially expressed circRNAs in longissimus muscle between castrated and intact male pigs.

Castration can reduce odor and fights in boars, but the carcass yield is reduced, and the intramuscular fat content is increased. Understanding its molecular mechanism is of great significance for production. Recent studies have shown that circular RNAs (circRNAs) play an important role(s) in the regulation of muscle development. To explore the effects of circRNAs on the development of longissimus dorsi (LD) muscle after castration, six Huainan male pigs were selected and three of which were randomly castrated. Six pigs were slaughtered when their body weight reached around 130 kg, and the LD muscle samples were collected. The differentially expressed circRNAs (DECs) were screened by high-throughput sequencing and functionally analyzed using the KEGG databases. DECs-miRNAs network was constructed, and the expression profiles of candidate circRNAs and their interactions with miRNAs were verified in porcine skeletal muscle satellite cells. The results showed that a total of 5866 circRNAs were obtained, and 370 DECs were identified in LD muscle between the castrated and intact groups (| log2Foldchange | > 1, padj <0.8). KEGG enrichment indicated that the parental genes for the DECs were mainly enriched in the pathways associated with muscle development, muscle fiber type transformation, and energy metabolism. There were 8 miRNAs and 69 circRNAs enriched in the DECs-miRNA network. circRNA_2241 and circRNA_4237 were selected for verification, which showed that these two circRNAs really existed and their expression profiles were consistent with the sequencing results. Further, preliminary analysis showed that circRNA_2241 interacted with miR-1, and testosterone promoted circRNA_2241 but inhibited miR-1 expression. These results confirmed that circRNAs might participate in the regulation of LD muscle development after castration by interacting with miRNAs, thereby providing new materials and references for analyses on the molecular mechanisms of castration on the regulation of muscle development.

Castration induced circRNA expressional changes in subcutaneous adipose tissue of male pigs.

Circular RNAs (circRNAs) participated in regulation of lipid metabolism; however, its functional role on castration-induced lipid deposition has not been deeply researched. So in this research, we firstly compared circRNAs expressional differences in subcutaneous adipose tissue between intact and castrated male Huainan pigs. A total of 6116 differentially expressed circRNAs (DECs) were detected between these two groups (|log2 foldchange| ≥ 1 and padj ≤ 0.05); GO and KEGG analysis showed that their parent genes were mainly enriched in metabolism-related pathway. And TGF-beta, insulin, AMPK, and MAPK pathways might play vital role in castration-induced lipid deposition. The miRNAs enriched in the constructed circRNA-miRNA network were mainly participated in adipogenesis and lipid metabolism, such as miR-143a-3p, miR-378, and miR-195. And it was verified that testosterone upregulated miR-181a but downregulated circ_0005912 expression in a dose-dependent manner in porcine intramuscular adipocytes, and overexpression of miR-181a inhibited circ_0005912. Taken together, these DECs may participate in the regulation of lipid metabolism after castration by reaction with miRNAs, which indicated the novel role of circRNAs in castration-induced lipid deposition.

Autophagy Contributes to Oxidative Stress-Induced Apoptosis in Porcine Granulosa Cells.

Oxidative stress-induced granulosa cell (GC) death is a major cause of follicular atresia. As the major types of programmed cell death, autophagy and apoptosis have been observed in response to H2O2-mediated oxidative stress and have been demonstrated to be responsible for porcine GC death. To date, however, the cellular reactions linking autophagy to the apoptosis of porcine GC under oxidative stress are still poorly understood. Porcine GC were treated with H2O2, and autophagic flux was examined by western blotting. Cell viability and cell death assays were performed after cotreatment of porcine GC with autophagy activator (rapamycin) or inhibitor (3-methyladenine, 3-MA) together with H2O2. We revealed that short exposure (1-3 h) of porcine GC to H2O2 dramatically increased autophagic flux (1.8- to 2.5-fold over that in the control), whereas 6-12 h prolonged treatment decreased autophagy but elevated the caspase-3 activity and GC apoptotic rate. Furthermore, we showed that pretreatment with rapamycin exacerbated H2O2-mediated cytotoxicity and caspase-3 activation but that 3-MA or siRNAs specific for Beclin 1 and Atg7 genes ameliorated H2O2-mediated GC apoptosis. Together, our results indicate that autophagy plays a pivotal role in H2O2-mediated porcine GC apoptosis. Importantly, we show that the early induction of autophagic flux contributes to oxidative stress-induced apoptosis in porcine GC. The results also suggest that regulating the autophagy response in porcine GC under oxidative stress might be a new strategy for abnormal follicular atresia.

LncRNA IMFlnc1 promotes porcine intramuscular adipocyte adipogenesis by sponging miR-199a-5p to up-regulate CAV-1.

BACKGROUND: Local Chinese local pig breeds have thinner muscle fiber and higher intramuscular-fat (IMF) content. But its regulation mechanism has not been discussed in-depth. Studies indicated that long non coding RNAs (lncRNAs) play important role in muscle and fat development. RESULTS: The lncRNAs expressional differences in the longissimus dorsi (LD) muscle were identified between Huainan pigs (local Chinese pigs, fat-type, HN) and Large White pigs (lean-type, LW) at 38, 58, and 78 days post conception (dpc). In total, 2131 novel lncRNAs were identified in 18 samples, and 291, 305, and 683 differentially expressed lncRNAs (DELs) were found between these two breeds at three stages, respectively. The mRNAs that co-expressed with these DELs were used for GO and KEGG analysis, and the results showed that muscle development and energy metabolism were more active at 58 dpc in HN, but at 78 dpc in LW pigs. Muscle cell differentiation and myofibril assembly might associated with earlier myogenesis and primary-muscle-fiber assembly in HN, and cell proliferation, insulin, and the MAPK pathway might be contribute to longer proliferation and elevated energy metabolism in LW pigs at 78 dpc. The PI3K/Akt and cAMP pathways were associated with higher IMF deposition in HN. Intramuscular fat deposition-associated long noncoding RNA 1 (IMFlnc1) was selected for functional verification, and results indicated that it regulated the expressional level of caveolin-1 (CAV-1) by acting as competing endogenous RNA (ceRNA) to sponge miR-199a-5p. CONCLUSIONS: Our data contributed to understanding the role of lncRNAs in porcine-muscle development and IMF deposition, and provided valuable information for improving pig-meat quality.

miR-181a promotes porcine granulosa cell apoptosis by targeting TGFBR1 via the activin signaling pathway.

Activin/Smad3 signaling plays a pivotal role in follicle development and atresia. However, the precise mechanisms underlying this process are not yet fully understood. Herein, we identified miR-181a as a central component of activin/Smad3-mediated follicle atresia. miR-181a was strikingly upregulated in porcine atretic follicles, which induced the apoptosis of porcine granulosa cells (GCs) in vitro. Furthermore, the transforming growth factor-ß type 1 receptor (TGFBR1) was confirmed as a direct target of miR-181a by bioinformatics analysis and luciferase assays. Transfection with an miR-181a agomir repressed the TGFBR1 mRNA and protein levels. In addition, TGFBR1 overexpression repressed GC apoptosis, whereas TGFBR1 inhibition promoted GC apoptosis. miR-181a overexpression downregulated the phosphorylation of Smad3 and blocked the activation of TGF-ß signaling. Moreover, activin A downregulated miR-181a expression and upregulated the TGFBR1 and p-Smad3 protein levels. Collectively, these data suggest that miR-181a regulates porcine GC apoptosis by targeting TGFBR1 via the activin signaling pathway.

Grape Seed Procyanidin B2 Protects Porcine Ovarian Granulosa Cells against Oxidative Stress-Induced Apoptosis by Upregulating let-7a Expression.

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.

Pig SOX9: Expression profiles of Sertoli cell (SCs) and a functional 18 bp indel affecting testis weight.

Sex determining region Y-box 9 (SOX9), an important member of the SRY- type HMGbox (SOX) gene family, plays an important role in the regulation of mammalian reproduction, including sex differentiation during the embryonic development stage and spermatogenesis after birth. To explore the roles of polymorphism and expression of the SOX9 gene in the development of testes, we analyzed the indel of SOX9 in pigs and the corresponding expression level of the SOX9 gene in 7-day and 5-month-old porcine Sertoli cells. Results revealed that the DD haplotype of SOX9 gene as well as the ID genotype were significantly associated with larger testicular weight, while the II haplotype was closely related to the smaller testicular weight. More importantly, the SOX9 gene expression of ID genotyped group was significantly higher than that in II genotyped group. Our results first revealed that the indel polymorphism and expression of SOX9 were significantly associated with pig reproduction traits indicating the critical roles of SOX9 gene in testes development. The study provides a new clue for understanding the regulation of animal reproductive activities.

Comprehensive Analysis of Differentially Expressed mRNA, lncRNA and circRNA and Their ceRNA Networks in the Longissimus Dorsi Muscle of Two Different Pig Breeds.

Circular RNA (circRNA) and long non-coding RNA (lncRNA) are known to participate in adipogenesis and myogenic differentiation, but their impact on porcine muscle traits is not well understood. We compared their expressional profiles in the longissimus dorsi muscle of Chinese Huainan pigs (HN, the fat type) and Western commercial Duroc×(Landrace×Yorkshire) (DLY, the thin type) pigs, and 854 mRNAs, 233 lncRNAs, and 66 circRNAs (p < 0.05 and ｜log2FoldChange｜>1) were found to be differentially expressed. The differentially expressed mRNA and circRNA parental genes were enriched in the Wnt signaling pathway (adipogenesis), the transition between fast and slow fibers (myogenic differentiation), and alanine, aspartate and glutamate metabolism (pork flavor). The potential lncRNAs/circRNAs-miRNAs-mRNAs regulatory networks shared MYOD1, PPARD, miR-423-5p and miR-874, which were associated with skeletal muscle muscular proliferation, differentiation/regeneration and adipogenesis. Taken together, these differentially expressed non-coding RNAs may be involved in the molecular basis of muscle traits, acting as the competing endogenous RNA (ceRNA) for miRNAs.

Identification and characterization of long non-coding RNAs in subcutaneous adipose tissue from castrated and intact full-sib pair Huainan male pigs.

BACKGROUND: Long non-coding RNAs (lncRNAs) regulate adipose tissue metabolism, however, their function on testosterone deficiency related obesity in humans is less understood. For this research, intact and castrated male pigs are the best model animal because of their similar proportional organ sizes, cardiovascular systems and metabolic features. RESULTS: We identified lncRNAs in subcutaneous adipose tissue by deep RNA-sequencing using the intact and castrated Huainan male pigs. The results showed that castration reduced serum testosterone but increased body fatness-related traits (serum triglyceride levels, backfat thickness, intramuscular fat content, and adipocyte size). Meanwhile, 343 lncRNAs from subcutaneous adipose tissue were identified, including 223 intergenic lncRNAs (lincRNAs), 68 anti-sense lncRNAs, and 52 intronic lncRNAs. It was predicted that there were 416 recognition sites for C/EBPα in the 303 lncRNA promoter region, and 13 adipogenesis-promoting miRNAs and five adipogenesis-depressing miRNAs target these lncRNAs. Eighteen lncRNAs, including nine up- and nine down-regulated had more than 2-fold differential expression between the castrated and intact male pigs (q-value < 0.05). Functional analysis indicated that these 18 lncRNAs and their target genes were involved in fatty acid, insulin, and the adipocytokine signaling pathway. We further analyzed the features of a conserved mouse lncRNA gene ENSMUST00000189966 and found it mainly expressed in the cell nucleus and target the Nuclear Receptor Subfamily 2 Group F Member 2 (NR2F2) gene. In 3 T3-L1 cells, differentiation down-regulated their expression, but dihydrotestosterone (DHT) significantly up-regulated their expression in a concentration-dependent manner (P < 0.05). CONCLUSIONS: These results suggested that lncRNAs and their target genes might participated in the castration-induced fat deposition and provide a new therapeutic target for combatting testosterone deficiency-related obesity.

Long non-coding RNA analysis of muscular responses to testosterone deficiency in Huainan male pigs.

Long non-coding RNAs (lncRNAs) participated in growth and development of skeletal muscle; however, little is known about their response to testosterone deficiency in porcine skeletal muscle. We compared lean mass related carcass traits and lncRNAs expression files in Longissimus dorsi (LD) muscle between intact and castrated Huainan male pigs. The results showed that castration significantly reduced eye muscle area and lean meat percentage (P < 0.05), but increased the fat mass weight (P < 0.05). Meanwhile, 8946 lncRNAs, including 6743 intergenic lncRNAs (lincRNAs), 498 anti-sense lncRNAs, and 1705 intronic lncRNAs, were identified in porcine LD, among which, 385 lncRNAs were considered as the differentially expressed candidates between intact groups and castrated groups (q-value < 0.05). Functional analysis indicated that these differently expressed lncRNAs and their target genes were involved in the estrogen receptor signaling pathway and skeletal and muscular system development and function. We first detect porcine muscular lncRNA response to castration, and the results suggested that lncRNAs and their target genes participated in the regulation of testosterone deficiency-related skeletal muscle growth.

Castration-induced changes in the expression profiles and promoter methylation of the GHR gene in Huainan male pigs.

Castration plays a regulatory role in growth and carcass traits, particularly in fat deposition, but its molecular mechanisms are still not clear. The present study showed that castration significantly reduced the serum growth hormone and the responses of the growth hormone receptor (GHR), insulin-like growth factor 1 (IGF-I), IGF-IR and peroxisome proliferator-activated receptor gamma (PPARγ) to castration were similar in different adipose tissues. However, the GHR expression trends were opposite between the liver and the adipose tissues; bisulfite sequencing PCR (BSP) showed that its methylation in these two tissues was different. In particular, the GHR methylation rate in the liver of castrated and intact pigs were 93.33% and 0, respectively, which was consistent with its higher expression level in the intact group. It was predicted that there were potential binding sites for 11 transcription factors in the ninth CpG site (which was methylated and demethylated in subcutaneous adipose tissue of the intact and castrated groups, respectively), including androgen receptor (AR), CCAAT/enhancer binding protein-α (C/EBPα) and C/EBPß, all of which are important factors in lipid metabolism. These results indicate that DNA methylation may participate in castration-induced fat deposition.

Critical Role of FoxO1 in Granulosa Cell Apoptosis Caused by Oxidative Stress and Protective Effects of Grape Seed Procyanidin B2.

Reactive oxygen species (ROS) are closely related to the follicular granulosa cell apoptosis. Grape seed procyanidin B2 (GSPB2) has been reported to possess potent antioxidant activity. However, the GSPB2-mediated protective effects and the underlying molecular mechanisms in granulosa cell apoptosis process remain unknown. In this study, we showed for the first time that GSPB2 treatment decreased FoxO1 protein level, improved granulosa cell viability, upregulated LC3-II protein level, and reduced granulosa cell apoptosis rate. Under a condition of oxidative stress, GSPB2 reversed FoxO1 nuclear localization and increased its level in cytoplasm. In addition, FoxO1 knockdown inhibited the protective effects of GSPB2 induced. Our findings suggest that FoxO1 plays a pivotal role in regulating autophagy in granulosa cells, GSPB2 exerts a potent and beneficial role in reducing granulosa cell apoptosis and inducing autophagy process, and targeting FoxO1 could be significant in fighting against oxidative stress-reduced female reproductive system diseases.

Chronic Exposure to Diquat Causes Reproductive Toxicity in Female Mice.

Diquat is a bipyridyl herbicide that has been widely used as a model chemical for in vivo studies of oxidative stress due to its generation of superoxide anions, and cytotoxic effects. There is little information regarding the toxic effects of diquat on the female reproductive system, particularly ovarian function. Thus, we investigated the reproductive toxic effects of diquat on female mice. Chronic exposure to diquat reduced ovary weights, induced ovarian oxidative stress, resulted in granulosa cell apoptosis, and disrupted oocyte developmental competence, as shown by reactive oxygen species (ROS) accumulation, decreased polar body extrusion rates and increased apoptosis-related genes expression. Additionally, after diquat treatment, the numbers of fetal mice and litter sizes were significantly reduced compared to those of control mice. Thus, our results indicated that chronic exposure to diquat induced reproductive toxicity in female mice by promoting the ROS production of gruanousa cells and ooctyes, impairing follicle development, inducing apoptosis, and reducing oocyte quality. In conclusion, our findings indicate that diquat can be used as a potent and efficient chemical for in vivo studies of female reproductive toxicity induced by oxidative stress. Moreover, the findings from this study will further enlarge imitative research investigating the effect of ovarian damage induced by oxidative stress on reproductive performance and possible mechanisms of action in large domestic animals.

cDNA cloning, bioinformatic and tissue-specific expression analysis of porcine JARID1C gene.

Jumonji, AT-rich interactive domain 1C (JARID1C) protein belongs to the highly conserved ARID protein family, which is involved in chromatin remodeling and transcriptional regulation during cell growth, differentiation, and development. In humans, this gene plays a vital role in normal brain development and function. Using an in silico approach in combination with 5' rapid amplification of cDNA ends (5' RACE), the full-length cDNA of JARID1C (GenBank accession No. EF139241) from porcine ovary, which contains 5,908 bp nucleotides, with an open reading frame (ORF) of 4,548 bp, has been cloned. The putative porcine JARID1C protein, which is located in the nucleus, encodes 1,516 amino acids with a molecular weight of 170 kDa and a pI of 5.44. Bioinformatic prediction indicates that the protein contains several conserved domains: a JmjN domain, an ARID domain, a JmjC domain, a C5HC2 zinc finger domain, and a PHD zinc finger domain. Similarity comparisons for nucleic and amino acid sequences reveal that the porcine JARID1C protein shares a high identity with its dog, mouse, rat, and human counterparts. The phylogenetic tree of the JARID1 subfamily proteins has been constructed to reveal the evolutionary relationship of various species. Real-time PCR analysis shows that the JARID1C gene is expressed in various tissues, but at different levels. The expression levels of this gene are higher in the brain and gonad than in other tissues, suggesting that the JARID1C protein plays a role in porcine brain and gonad functions.

Overexpression of IGF2R and IGF1R mRNA in SCNT-produced goats survived to adulthood.

The procedure of somatic cell nuclear transfer (SCNT) is likely to affect the expression level of growth-related genes especially imprinting genes. In this study, expressions of growth-related genes including three imprinting genes (H19, IGF2, and IGF2R) and four non-imprinting genes (IGF1, IGF1R, GHR, and GHSR) in adult nuclear transferred (NT) goats were investigated by real-time PCR. The expressions of these genes in adult clones were found largely normal, but IGF2R and IGF1R were more highly expressed in cloned goats than in non-NT goats (P < 0.01). Analysis on mono-allelic expression pattern of imprinting genes indicated that mono-allelic expression patterns of H19 and IGF2 in cloned goats were similar to that in non-NT goats. In addition, the sequence of goat IGF2 gene and the putative amino acid sequence were obtained. The 986 nucleotide cDNA of goat IGF2 gene contained an open-reading frame of 540 nucleotides coding for 179 amino acids. Both cDNA sequence and amino acid sequence of IGF2 in goat showed their higher homology with that in sheep than in cattle; the partial cDNA fragments of H19, IGF2R, GHSR, IGF1R, and GHR in goat were also cloned and sequenced, which shared higher sequence identities with those in sheep than in cattle.