Impact of adrenocorticotropin hormone administration on the endocrinology, estrus onset, and ovarian function of weaned sows.

Chronic stress affects the reproductive health of mammals; however, the impact of adrenocorticotropin hormone (ACTH) level elevation during chronic stress on the reproduction of weaned sows remains unclear. In this study, nine weaned sows with the same parturition date were randomly divided into control group (n = 4) and ACTH group (n = 5). Each group received intravenous administration of ACTH three times daily for 7 days. Blood samples were collected every 3 h after injection. A radioimmunoassay was used to measure the concentrations of cortisol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4) and estradiol-17ß (E2) in the blood. Estrus was determined according to changes in the vulva and the boar contact test. The mRNA expressions of glucocorticoid receptor, FSH receptor, LH receptor (LHR) in the corpus luteum (CL) were detected by qRT-PCR. The results showed that ACTH administration substantially delayed the initiation of estrus and the pre-ovulatory LH peak. The sows of control group ovulated within 10 days and the ovulation rate was 100%, while it was 60% in the ACTH group. Two sows of ACTH group showed pseudo-estrus. The E2 concentrations significantly decreased in the ACTH group at 36 h, 42 h and 66 h of the experimental period. The P4 concentrations in the ACTH group significantly decreased at 132, 138, and 147 h of the experimental period. ACTH significantly reduced the LHR mRNA expression in CLs. In conclusion, long-term repeated ACTH administration affects the endocrinology, estrus onset, and ovarian function of weaned sows.

Isolation, Culture and Identification of Porcine Skeletal Muscle Satellite Cells.

The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse) have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells.

[Association of porcine ATF4 gene polymorphism and production traits and analysis of gene expression].

In order to understand the function of gene ATF4 and identify new DNA markers involved in pig production traits, the cDNA fragment of porcine ATF4 was cloned and sequenced. Sequence comparison revealed an A159G substitution downstream of the initiation codon (ATG). We then carried out PCR-AluⅠ-RFLP analysis in Large white, Landrace, Tongcheng and Meishan pigs, followed by association analysis in F2 "Large white ×Meishan" resource family. In all the individuals tested, Large White and Landrace pigs possessed the AA genotype, while Meishan and Tongcheng pigs pos-sessed the GG genotype. Association analysis in F2 resource family showed that this site was highly associated with buttock fat thickness (BFT) (Pamp;0.01) and had significant effect on thorax-waist fat thickness (TFT), average backfat thickness (ABT), loin eye height (LEH), and loin eye area (LEA)(Pamp;0.05). Real-time PCR was used to analyze the expression patterns of porcine ATF4 gene in longissimus dorsi at different development stages of Large White and Meishan pigs. The results showed that the gene expression levels of ATF4 were low 65 days after conception and 3 days after birth, but no signifi-cant differences were observed in both breeds. Meanwhile, the expression levels of porcine ATF4 gene were up-regulated 60 days and 120 days after birth in both breeds and the expression level in Meishan pigs was obviously higher than that in Large White pigs. These data could lay the foundation for further study on the molecular mechanism of porcine ATF4 gene in lipid metabolism.

[Polymorphism in coding region of pig PRDX6 gene and its genetic effects analysis].

PRDX6, a member of antioxidant protein superfamily, plays an important role in oxidative stress, catabolism of lipids and phospholipid lipisomes. Therefore, we used PRDX6 as an important candidate gene for meat quality according to its physiological and biochemical function. Partial coding sequence of porcine PRDX6 was isolated and two potenial SNPs, one at 417 bp (C/T) and the other at 423 bp (A/G), were found in the fourth exon by comparison of the obtained sequence from different pig breeds. In order to explore the relationship between PRDX6 polymorphism and meat quality, genetic variation and trait association of these two SNPs were separately performed in 6 purebred pig population and 247 F2 "Large White x Meishan" resource population by pyrosequencing. The results showed that allele C was predominant in western pig breeds, while allele T was predominant in Chinese indigenous breeds at 417 bp (C/T). This SNP was significantly associated with the intramuscular fat and water moisture (P < 0.05). The A/G mutation at 423 bp was significantly associated with drip water rate, water holding capacity, intramuscular fat, and water moisture (P < 0.05). Allele A was predominant in western pig breeds, while allele G was predominant in Chinese indigenous breeds. These two SNPs were likely to be important markers affecting meat quality traits (especially the muscle tenderness).

Expression and preliminary functional profiling of the let-7 family during porcine ovary follicle atresia.

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.

Expression of PUMA in Follicular Granulosa Cells Regulated by FoxO1 Activation During Oxidative Stress.

Many studies have demonstrated that oxidative stress-induced apoptosis is a main cause of follicular atresia. Reactive oxygen species (ROS)-induced granulosa cell (GC) apoptosis is regulated by a variety of signaling pathways involving numerous genes and transcription factors. In this study, we found expression of the p53-upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 subfamily protein, in ovarian GCs during oxidative stress. By overexpression and knockdown of Forkhead box O1 (FoxO1), we found that FoxO1 regulates PUMA at the protein level. Moreover, as c-Jun N-terminal kinase (JNK) has been shown to activate FoxO1 by promoting its nuclear import, we used a JNK inhibitor to reduce FoxO1 activation and detected decreased PUMA messenger RNA expression and protein levels during oxidative stress. In addition, in vivo oxidative stress-induced upregulation of PUMA was found following injection of 3 nitropropionic acid in mice. In conclusion, oxidative stress increases PUMA expression regulated by FoxO1 in follicular GCs.