Integrated proteomics and transcriptomics analysis reveals insights into differences in premature mortality associated with disparate pathogenic RBM20 variants.

Variants in RNA binding motif protein 20 (RBM20) are causative in a severe form of dilated cardiomyopathy referred to as RBM20 cardiomyopathy, yet the mechanisms are unclear. Moreover, the reason(s) for phenotypic heterogeneity in carriers with different pathogenic variants are similarly opaque. To gain insight, we carried out multi-omics analysis, including the first analysis of gene expression changes at the protein level, of mice carrying two different pathogenic variants in the RBM20 nuclear localization signal (NLS). Direct comparison of the phenotypes confirmed greater premature morality in S639G variant carrying mice compared to mice with the S637A variant despite similar cardiac remodeling and dysfunction. Analysis of differentially spliced genes uncovered alterations in the splicing of both RBM20 target genes and non-target genes, including several genes previously implicated in arrhythmia. Global proteomics analysis found that a greater number of proteins were differentially expressed in the hearts of Rbm20S639G mice relative to WT than in Rbm20S637A versus WT. Gene ontology analysis suggested greater mitochondrial dysfunction in Rbm20S639G mice, although direct comparison of protein expression in the hearts of Rbm20S639G versus Rbm20S637A mice failed to identify any significant differences. Similarly, few differences were found by direct comparison of gene expression at the transcript level in Rbm20S639G and Rbm20S637A despite greater coverage. Our data provide a comprehensive overview of gene splicing and expression differences associated with pathogenic variants in RBM20, as well as insights into the molecular underpinnings of phenotypic heterogeneity associated with different dilated cardiomyopathy-associated variants.

RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis.

Arginine-serine (RS) domain(s) in splicing factors are critical for protein-protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm20S637A ) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.

Three novel simple sequence repeats (SSRs) identified by MALDI-TOF-MS method were associated with backfat in pig.

Backfat trait is an important economic trait and highly heritable, but difficult to evaluate. Thus, it is of great significance to explore optimal backfat thickness of pigs by using marker-assisted selection (MAS) to speed up its breeding process and improve economic efficiency. This study aimed to investigate the relationship between genetic variations (e.g., SSRs) and backfat of Qinghai Bamei pigs using MALDI-TOF Mass Spectrometry (MALDI-TOF-MS). Herein, five alternative SSR loci (namely V1, V2, V3, V4 and V5) were selected for subsequent detection. The results suggested that 3 (141-, 143- and 145-), 3 (128-, 130- and 132-), 2 (160- and 162-), 2 (136- and 139-) and 3 (170-, 184- and 192-) alleles of V1, V2, V3, V4 and V5 were found, respectively. Subsequent analysis showed that there was linkage equilibrium among five SSRs and Hap19 (13.1%) (141-/132-/160-/139-/192-) had the highest haplotype frequency. Among these five SSR loci, V1, V2 and V3 loci were significantly associated to the backfat of Qinghai Bamei sows. These findings enriched the study of SSRs in Qinghai Bamei pigs, and (AC)n (Chr15:85485851-85485995), (AC)n (Chr10:52724583-52724713) and (TG)n (Chr4:90732644-90732802) could be utilized as the candidate locus for MAS in pig industry.HIGHLIGHTSFive novel SSR loci was identified in pigs through MALDI-TOF MS.V1, V2 and V3 loci was were significantly associated to the backfat of pigs.

RBM20S639G mutation is a high genetic risk factor for premature death through RNA-protein condensates.

Dilated cardiomyopathy (DCM) is a heritable and genetically heterogenous disease often idiopathic and a leading cause of heart failure with high morbidity and mortality. DCM caused by RNA binding motif protein 20 (RBM20) mutations is diverse and needs a more complete mechanistic understanding. RBM20 mutation S637G (S639G in mice) is linked to severe DCM and early death in human patients. In this study, we generated a RBM20 S639G mutation knock-in (KI) mouse model to validate the function of S639G mutation and examine the underlying mechanisms. KI mice exhibited severe DCM and premature death with a ~ 50% mortality in two months old homozygous (HM) mice. KI mice had enlarged atria and increased ANP and BNP biomarkers. The S639G mutation promoted RBM20 trafficking and ribonucleoprotein (RNP) granules in the sarcoplasm. RNA Seq data revealed differentially expressed and spliced genes were associated with arrhythmia, cardiomyopathy, and sudden death. KI mice also showed a reduction of diastolic stiffness and impaired contractility at both the left ventricular (LV) chamber and cardiomyocyte levels. Our results indicate that the RBM20 S639G mutation leads to RNP granules causing severe heart failure and early death and this finding strengthens the novel concept that RBM20 cardiomyopathy is a RNP granule disease.

Identification of three new microsatellites and their effects on body measurement traits in pigs using time of flight-mass spectrometry (TOF-MS).

The body status of livestock affects their physiological function and productive performances. Microsatellites, one of the most used DNA markers, have been found to be associated with pig productive traits. However, their identifications and effects on body measurement traits of the Chinese Qinghai Bamei pig still uncovered. According to our previous sequencing data, in this study, three novel microsatellites were found in this breed. Using time of flight-mass spectrometry (TOF-MS) method, these microsatellites were further identified in a large Bamei pig population. TOF-MS spectra showed that there are three microsatellites loci, named P1, P2 and P3. These microsatellites were linkage equilibrium based on the values of D' and r2 tests. Association results demonstrated that P1 locus was associated with the body length, body height and chest width and the beneficial genotype was 150-/150-bp (p < 0.05); and P2 locus was associated with the body height (p < 0.05), and the 145-/145-bp, 145-/147-bp and 145-/149-bp were claimed as favorable genotypes and 145-bp allele was considered as the favorable allele. These findings suggested that P1 and P2 microsatellites might be considered as the candidate genetic markers to select pigs with superior body sizes, especially in local breed.

RBM20-Mediated Pre-mRNA Splicing Has Muscle-Specificity and Differential Hormonal Responses between Muscles and in Muscle Cell Cultures.

Pre-mRNA splicing plays an important role in muscle function and diseases. The RNA binding motif 20 (RBM20) is a splicing factor that is predominantly expressed in muscle tissues and primarily regulates pre-mRNA splicing of Ttn, encoding a giant muscle protein titin that is responsible for muscle function and diseases. RBM20-mediated Ttn splicing has been mostly studied in heart muscle, but not in skeletal muscle. In this study, we investigated splicing specificity in different muscle types in Rbm20 knockout rats and hormonal effects on RBM20-mediated splicing both in cellulo and in vivo studies. The results revealed that RBM20 is differentially expressed across muscles and RBM20-mediated splicing is muscle-type specific. In the presence of RBM20, Ttn splicing responds to hormones in a muscle-type dependent manner, while in the absence of RBM20, Ttn splicing is not affected by hormones. In differentiated and undifferentiated C2C12 cells, RBM20-mediated splicing in response to hormonal effects is mainly through genomic signaling pathway. The knowledge gained from this study may help further understand muscle-specific gene splicing in response to hormone stimuli in different muscle types.

Ssc-miR-429 expression proliles and functions on inducing Leydig cells apoptosis.

Leydig cells (LCs) play an indispensable role in testosterone synthesis, and their dysfunction can result in male reproductive disorders. Previous transcriptome sequencing revealed differential expression of MicroRNA-429 (miR-429) in both Leydig stem cells (SLCs) and LCs, indicating its potential regulatory function in LCs. In this study, we examined the expression of miR-429 in seven pig tissues (heart, liver, spleen, lung, kidney, testis, epididymis, brain) and investigated its impact on the proliferation and apoptosis of testicular interstitial cells using various techniques such as CCK-8, EdU, TUNEL, Western blot, among others. The results demonstrated that miR-429 exhibited lower expression levels in the testis, particularly in the LCs of testicular tissue. Upon upregulation of miR-429, TM3 cell density significantly increased, while downregulation led to a slight elevation in cell density. Further research indicated that the observed phenotype was due to miR-429-induced cell apoptosis, independent of cell proliferation. Additionally, a dual-luciferase reporter system revealed no targeting relationship between miR-429 and the predicted target genes (BMI1 and SOX5). Previous reports confirm Bcl2 as a known target of miR-429, leading us to hypothesize that miR-429 diminishes LCs' anti-apoptotic capability by inhibiting Bcl2. In summary, our findings suggest that miR-429 may induce LC apoptosis, supporting its potential as a biomarker for male reproductive disorders linked to Leydig cell dysfunction.

Mechanisms of RBM20 Cardiomyopathy: Insights From Model Systems.

RBM20 (RNA-binding motif protein 20) is a vertebrate- and muscle-specific RNA-binding protein that belongs to the serine-arginine-rich family of splicing factors. The RBM20 gene was first identified as a dilated cardiomyopathy-linked gene over a decade ago. Early studies in Rbm20 knockout rodents implicated disrupted splicing of RBM20 target genes as a causative mechanism. Clinical studies show that pathogenic variants in RBM20 are linked to aggressive dilated cardiomyopathy with early onset heart failure and high mortality. Subsequent studies employing pathogenic variant knock-in animal models revealed that variants in a specific portion of the arginine-serine-rich domain in RBM20 not only disrupt splicing but also hinder nucleocytoplasmic transport and lead to the formation of RBM20 biomolecular condensates in the sarcoplasm. Conversely, mice harboring a disease-associated variant in the RRM (RNA recognition motif) do not show evidence of adverse remodeling or exhibit sudden death despite disrupted splicing of RBM20 target genes. Thus, whether disrupted splicing, biomolecular condensates, or both contribute to dilated cardiomyopathy is under debate. Beyond this, additional questions remain, such as whether there is sexual dimorphism in the presentation of RBM20 cardiomyopathy. What are the clinical features of RBM20 cardiomyopathy and why do some individuals develop more severe disease than others? In this review, we summarize the reported observations and discuss potential mechanisms of RBM20 cardiomyopathy derived from studies employing in vivo animal models and in vitro human-induced pluripotent stem cell-derived cardiomyocytes. Potential therapeutic strategies to treat RBM20 cardiomyopathy are also discussed.

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy.

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knockin (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We showed that Rbm20ΔRS mice manifested DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 was mis-localized to the sarcoplasm in Rbm20ΔRS mouse hearts and formed RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif showed similar mis-splicing of major RBM20 target genes but did not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrated that only DCM-associated mutations in the RS domain facilitated RBM20 nucleocytoplasmic transport and promoted granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain of RBM20. Mutation analysis of phosphorylation sites in the RS domain suggested that this modification may be dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

Ssc-MiR-21-5p and Ssc-MiR-615 Regulates the Proliferation and Apoptosis of Leydig Cells by Targeting SOX5.

Leydig cells (LCs) are the predominant cells of androgen production, which plays key roles in spermatogenesis and maintaining male secondary sexual characteristics. Abnormal development of LCs affects androgen levels in vivo, affects fertility and may even lead to infertility. Little is known about the regulation mechanism on LCs' development and maturation in domestic animals, especially the regulation of non-coding RNAs. In this study, we continued to dig deeper in the previous RNA-seq data of porcine LCs from our group, combined with detecting the expression profiles in different tissues and different types of cells in the testis, to screen out candidate microRNAs (miRNAs) that may affect the regulation of LCs. A total of two miRNAs, ssc-miR-21-5p and ssc-miR-615 ("ssc" is omitted below), were finally determined. After overexpression and interference of miRNAs in vitro, the effects of candidate miRNAs on the proliferation and apoptosis of TM3 (mouse Leydig cell line) were explored. The results showed that miR-21-5p led to a decrease in TM3 cell density and p53 (apoptosis related protein) expression. Meanwhile, miR-21-5p decreased EdU positive cell numbers, but increased TUNEL positive cell numbers, suggesting miR-21-5p could inhibit proliferation and promote apoptosis. Conversely, miR-615 could increase TM3 cell density. Western blot and TUNEL assay indicated miR-615 inhibited apoptosis, but had no effect on proliferation. In addition, Sox5 was identified a potential target gene of these two miRNAs by Dual-Luciferase reporter system assay. Our findings about functions of miRNAs in TM3 and the mapping of miRNAs-target gene regulatory network would provide an important basis for the further elucidation of miRNAs in regulating pig LCs.

SR Protein Kinases Regulate the Splicing of Cardiomyopathy-Relevant Genes via Phosphorylation of the RSRSP Stretch in RBM20.

(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy.

Rbm20 ablation is associated with changes in the expression of titin-interacting and metabolic proteins.

Dilated cardiomyopathy (DCM) is a major risk factor for developing heart failure and is often associated with an increased risk for life-threatening arrhythmia. Although numerous causal genes for DCM have been identified, RNA binding motif protein 20 (Rbm20) remains one of the few splicing factors that, when mutated or genetically ablated, leads to the development of DCM. In this study we sought to identify changes in the cardiac proteome in Rbm20 knockout (KO) rat hearts using global quantitative proteomics to gain insight into the molecular mechanisms precipitating the development of DCM in these rats. Our analysis identified changes in titin-interacting proteins involved in mechanical stretch-based signaling, as well as mitochondrial enzymes, which suggests that activation of pathological hypertrophy and altered mitochondrial metabolism and/or dysfunction, among other changes, contribute to the development of DCM in Rbm20 KO rats. Collectively, our findings provide the first report on changes in the cardiac proteome associated with genetic ablation of Rbm20.

The ryanodine receptor stabilizer S107 ameliorates contractility of adult Rbm20 knockout rat cardiomyocytes.

RNA binding motif 20 (RBM20) cardiomyopathy has been detected in approximately 3% of populations afflicted with dilated cardiomyopathy (DCM). It is well conceived that RBM20 cardiomyopathy is provoked by titin isoform switching in combination with resting Ca2+ leaking. In this study, we characterized the cardiac function in Rbm20 knockout (KO) rats at 3-, 6-, 9-, and 12-months of age and examined the effect of the ryanodine receptor stabilizer S107 on resting intracellular levels and cardiomyocyte contractile properties. Our results revealed that even though Rbm20 depletion promoted expression of larger titin isoform and reduced myocardial stiffness in young rats (3 months of age), the established DCM phenotype required more time to embellish. S107 restored elevated intracellular Ca2+ to normal levels and ameliorated cardiomyocyte contractile properties in isolated cardiomyocytes from 6-month-old Rbm20 KO rats. However, S107 failed to preserve cardiac homeostasis in Rbm20 KO rats at 12 months of age, unexpectedly, likely due to the existence of multiple pathogenic mechanisms. Taken together, our data suggest the therapeutic promises of S107 in the management of RBM20 cardiomyopathy.

Goat DNMT3B: An indel mutation detection, association analysis with litter size and mRNA expression in gonads.

DNA methyltransferase 3ß (DNMT3B) is a gene encoding a de novo methylation enzyme that is required for DNA methylation during mammalian embryo development. Previous genome-wide association analysis suggested DNMT3B is a candidate gene for goat fertility, but there is no study on the effect of DNMT3B on litter size in goat. The aim of this study was to identify possible insertion/deletion (indel) mutations associated with litter size. Seven putative indels were designed to study their association with litter size, but just one 11-bp insertion variant of intron 22 (the last intron) was found in healthy female Shaanbei white cashmere goats (SBWC goats) (n = 1534). Statistical analysis showed that the 11-bp insertion was related to the first-born litter size (P < 0.01) and the goats with the deletion/deletion genotype had a higher average first-born litter size (P < 0.01). In addition, the expression profile of the DNMT3B mRNA in goat was detected, which revealed significant differences in DNMT3B mRNA expression in the gonads. Additionally, the results of western blotting revealed that the ovaries of mothers of multi-lamb (MML) had a higher level of DNMT3B protein than the ovaries of mothers of single-lamb (MSL). Furthermore, the mRNA of DNMT3B was widely expressed in male goats. Differences in mRNA expression levels were observed in the ovaries of MSL and MML. These findings indicated that the 11-bp indel in DNMT3B was significantly associated with first-born litter size, which can be used for marker-assisted selection (MAS) of goats for breeding.

miR-205 Expression Elevated With EDS Treatment and Induced Leydig Cell Apoptosis by Targeting RAP2B via the PI3K/AKT Signaling Pathway.

The adult Leydig cells (ALCs), originated from stem Leydig cells (SLCs), can secrete testosterone which is essential for germ cell development and sexual behavior maintenance. As a synthetic compound, ethane dimethane sulfonate (EDS), a well-known alkylating agent, has been reported to specifically ablate ALCs. In this study, EDS was verified to ablate differentiated pig LCs by experiments. Subsequently, the primary isolated pig LCs (containing SLCs and differentiated LCs) and EDS-treated LCs (almost exclusively SLCs) were collected for RNA-seq 4,904 genes and 15 miRNAs were differently expressed between the two groups. Down-regulated genes in the EDS-treated group were mainly related to steroid hormone biosynthesis. The highest up-regulation miRNAs was miR-205 after EDS treatment. Additionally, miR-205 was expressed more highly in pig SLCs clones compared with differentiated LCs. Through qRT-PCR, western blot (WB), TUNEL, EDU and flow cytometry, miR-205 was found to induce cell apoptosis, but did not affect proliferation or differentiation in both TM3 and GC-1spg mouse cell lines. Through luciferase reporter assays and WB, RAP2B was identified as a target gene of miR-205. Besides, overexpression of miR-205 inhibited the expressions of PI3K, Akt and p-AKT. All these findings were helpful for elucidating the regulation mechanism in pig LCs.

An 11-bp Indel Polymorphism within the CSN1S1 Gene Is Associated with Milk Performance and Body Measurement Traits in Chinese Goats.

The casein alpha s1 (CSN1S1) gene encodes α-s1 casein, one of the proteins constituting milk, which affects milk performance, as well as improving the absorption of calcium and bone development in mammals. A previous study found that an 11-bp insertion/deletion (indel) of this gene strongly affected litter size in goats. However, to our knowledge, the relationships between this polymorphism and the milk performance and body measurement traits of goats have not been reported. In this paper, the previously identified indel has been recognized in three Chinese goat breeds, namely the Guanzhong dairy goat (GZDG; n = 235), Shaanbei white cashmere goat (SBWC; n = 1092), and Hainan black goat (HNBG; n = 278), and the following three genotypes have been studied for all of the breeds: insertion/insertion (II), deletion/deletion (DD), and insertion/deletion (ID). The allele frequencies analyzed signified that the frequencies of the "D" allele were higher (47.8%-65.5%), similar to the previous report, which indicates that this polymorphism is genetically stable in different goat breeds. Further analysis showed that this indel was markedly associated with milk fat content, total solids content, solids-not-fat content, freezing point depression, and acidity in GZDG (p < 0.05), and also affected different body measurement traits in all three breeds (p < 0.05). The goats with II genotypes had superior milk performance, compared with the others; however, goats with DD genotypes had better body measurement sizes. Hence, it may be necessary to select goats with an II or DD genotype, based on the desired traits, while breeding. Our study provides information on the potential impact of the 11-bp indel polymorphism of the CSN1S1 gene for improving the milk performance and body measurement traits in goats.

A 20-bp insertion/deletion (indel) polymorphism within the CDC25A gene and its associations with growth traits in goat.

Cell division cycle 25A (CDC25A), a member of the CDC25 family of phosphatases, is required for progression from G1 to the S phase of the cell cycle. CDC25A provides an essential function during early embryonic development in mice, suggesting that it plays an important role in growth and development. In this study, we used mathematical expectation (ME) methods to identify a 20-bp insertion/deletion (indel) polymorphism of CDC25A gene in Shaanbei White Cashmere (SBWC) goats. We also investigated the association between this 20-bp indel and growth-related traits in SBWC goats. Association results showed that the indel was related to growth traits (height at hip cross, cannon circumference, and cannon circumference index) in SBWC goats. The height at hip cross of individuals with insertion/insertion (II) genotype was higher than those with insertion/deletion (ID) genotype ( P = 0.02 ); on the contrary, the cannon circumference and cannon circumference index of individuals with ID genotype were superior when compared with those with II genotype ( P = 0.017 and P = 0.009 ). These findings suggest that the 20-bp indel in the CDC25A gene significantly affects growth-related traits, and could be utilized as a candidate marker for marker-assisted selection (MAS) in the cashmere goat industry.

A novel missense mutation (L280V) within POU1F1 gene strongly affects litter size and growth traits in goat.

In mammals, POU1F1 is a key transcription factor that directly affects the secretion of pituitary hormones (GH, PRL, and TSH) and maintains growth and development. The aim of this study was to identify novel single nucleotide polymorphisms (SNPs) of the POU1F1 gene and evaluate the relations to litter size and growth performance in Shaanbei white cashmere (SBWC) goats. The ear tissues of female goats (n = 653) were collected, and the corresponding data were recorded. From direct DNA sequencing, a missense mutation (NC_030808.1:g.34236169A > C) was first detected in exon 6 of POU1F1 in SBWC goats that transformed the amino acid leucine into valine (L280V). Further analyses showed that the number of lambs of female goats with the TT genotype was significantly greater than that of female goats with the TG genotype (P < 0.01). In addition, a Chi-square test showed that the distributions of the two genotypes were remarkably different in the litters of two different sizes (P = 2.06E-06). Simultaneously, the frequency of the TT genotype for female goats with multiple lambs (≥2) was much greater than that of the TG genotype. When the relationships between growth traits and L280V were evaluated, the individuals with the TT genotype had superior growth traits compared with those of the TG genotype (P < 0.05), including for body height, body length, chest circumference, chest depth, chest width, height at hip cross, and cannon circumference. Notably, a positive relationship indicated that TT genotype female goats with improved growth traits tended to give birth to more lambs. We hypothesized that the mutation was in linkage disequilibrium with other responsible SNPs or affected the post-transcriptional regulation levels and then affected growth and litter size. This novel SNP may provide a functional genetic marker for improving economically valuable traits in goat breeding.

Relationship between SNPs of POU1F1 Gene and Litter Size and Growth Traits in Shaanbei White Cashmere Goats.

POU (Pit-Oct-Unc) class 1 homeobox 1 (POU1F1, or Pit-1) is a transcription factor that directly regulates pituitary hormone-related genes, as well as affects the reproduction and growth in mammals. Thus, POU1F1 gene was investigated as a candidate gene for litter size and growth performance in goats. In the current study, using direct DNA sequencing, c.682G > T, c.723T > G and c.837T > C loci were genotyped in Shaanbei white cashmere (SBWC) goats (n = 609), but c.876 + 110T > C was monomorphic. Besides, the c.682G > T locus was first identified by HinfI (Haemophilus influenzae Rf) restriction endonuclease. Association analysis results showed that the c.682G > T, c.837T > C loci and diplotypes were significantly associated with goat litter size (p < 0.05). The positive genotypes were GT and TT for the two SNPs, respectively, and the optimal diplotype was H3H7 (GTTT-TTTT). On the other hand, the c.682G > T, c.723T > G and c.837T > C strongly affected growth traits and body measurement indexes in SBWC goats (p < 0.05). The positive genotypes or allele of these SNPs were GT, G and TT, respectively. Additionally, the goats with H3H7 diplotype also had a greater growth status than others (p < 0.05). Here, individuals with same genotype had both a better litter size and growth traits, showing a positive correlation between these economic traits. Meanwhile, the positive genotypes of four SNPs were combined to obtain the optimal diplotype, which was also H3H7. These SNPs, especially the diplotype, could be used for the genomic selection of excellent individuals with a greater litter size and better growth status in goat breeding.

Chlorpyrifos Induction of Testicular-Cell Apoptosis through Generation of Reactive Oxygen Species and Phosphorylation of AMPK.

Chlorpyrifos (CPF) is the most frequently applied insecticide. Aside from effects on the neuronal cholinergic system, previous studies suggested a potential relationship between CPF exposure and male infertility; however, the molecular mechanism remains elusive. The aim of this study was to investigate the toxic effect of CPF on testicular cells and the potential mechanism via in vitro and in vivo experiments. The cytotoxic effects of CPF on mouse-derived spermatogonial cell lines (GC-1), Sertoli cell lines (TM4) and Leydig cell lines (TM3) were assessed by a CCK-8 assay, flow cytometry, a TUNEL assay, quantitative RT-PCR, and Western blotting. Exposure to CPF (10-50 µM) for 12 or 24 h resulted in significant death in all three testicular cell lines. The number of TUNEL-positive apoptotic cells were dose-dependent and increased with raised CPF concentrations. Further investigation indicated that CPF induced cell-cycle arrest and then promoted cell apoptosis. Additionally, CPF increased reactive-oxygen-species (ROS) production and lipid peroxidation (MDA) and reduced mitochondrial-membrane potential. The mechanism of cell apoptosis induced by CPF involved an increase in phosphorylated-AMP-activated-protein-kinase (p-AMPK) levels in the tested cells. In vivo, the expression of steroid-hormone-biosynthesis-related genes in testis, spleen, and lung in F0 and F1 mice were downregulated when there was intraperitoneal injection or dietary supplementation of CPF. This study provides a potential molecular mechanism of CPF-induced toxicity in testicular cells and a theoretical basis for future treatment of male infertility.