Effect of melatonin on ATG2B-mediated autophagy regulation in sheep granulosa cells with different FecB genotypes.

Melatonin (MLT) protects cells by reducing reactive oxygen species (ROS) levels, which are key for inducing cellular autophagy. The aim of this study was to investigate the molecular mechanisms underlying MLT regulation of autophagy in granulosa cells (GCs) with BMPR-1B homozygous (FecB BB) and wild type (FecB ++) mutations. GCs collected from small-tailed Han sheep with different FecB genotypes were typed using a TaqMan probe assay, and autophagy levels were found to be significantly higher in GCs with FecB BB than the levels in those with FecB ++. Autophagy-related 2 homolog B (ATG2B) was associated with cell autophagy and was highly expressed in GCs with the FecB BB genotype in small-tailed Han sheep. Overexpression of ATG2B in the GCs of sheep with both FecB genotypes promoted GC autophagy, and the contrary was observed after the inhibition of ATG2B expression. Subsequently, treatment of GCs with different genotypes of FecB and MLT revealed a significant decrease in cellular autophagy and an increase in ATG2B expression. Addition of MLT to GCs with inhibited ATG2B expression revealed that MLT could protect GCs by decreasing ROS levels, especially in GCs with FecB ++ genotype. In conclusion, this study determined that autophagy levels were significantly higher in sheep GCs with FecB BB genotype than the levels in those with FecB ++ genotype, which may have contributed to the difference in lambing numbers between the two FecB genotypes. Autophagy was regulated by ATG2B and was able to protect GCs by reducing the high levels of ROS produced following inhibition of ATG2B through the addition of MLT in vitro.

The mechanism of circadian clock and its influence on animal circannual rhythm.

The circadian clock exists in almost all life forms, and is an internal activity generated by organisms adapting to the daily periodic changes of the external environment. The circadian clock is regulated by the transcription-translation-negative feedback loop in the body, which can regulate the activities of tissues and organs. Its normal maintenance is important for the health, growth, and reproduction of organisms. In contrast, due to the season changes of the environment, organisms have also formed annual cycle physiological changes in their bodies, such as seasonal estrus, etc. The annual rhythm of living things is mainly affected by environmental factors such as photoperiod, and is related to gene expression, hormone content, morphological changes of cell and tissues in vivo. Melatonin is an important signal to recognize the changes of photoperiod, and the circadian clock plays an important role in the pituitary to interpret the signal of melatonin and regulate the changes of downstream signals, which plays an important guiding role in the recognition of annual changes in the environment and the generation of the body's annual rhythm. In this review, we summarize the progress of research on the mechanism of action of circadian clocks in influencing annual rhythms, by introducing the mechanisms of circadian and annual rhythms generation in insects and mammals, and in the context of annual rhythms in birds, with the aim of providing a broader range of ideas for future research on the mechanism of annual rhythms influence.

Progress on the effect of FecB mutation on BMPR1B activity and BMP/SMAD pathway in sheep.

BMPR1B is the first major gene of litter size identified in sheep. However, the molecular mechanism of the FecB mutation that increases the ovulation rate in sheep is still unclear. In recent years, it has been demonstrated that BMPR1B activity is regulated by the small molecule repressor protein FKBP1A, which acts as a key activity switch of the BMPR1B in the BMP/SMAD pathway. The FecB mutation is located close to the binding site of FKBP1A and BMPR1B. In this review, we summarize the structure of BMPR1B and FKBP1A proteins, and clarify the spatial interactive domains of the two proteins with respect to the location of the FecB mutation. Then the relationship between the FecB mutation and the degree of affinity of the two proteins are predicted. Finally, the hypothesis that FecB mutation causes change of activity in BMP/SMAD pathway by affecting the intensity of the interactions between BMPR1B and FKBP1A is proposed. This hypothesis provides a new clue to investigate the molecular mechanism of FecB mutation affecting ovulation rate and litter size in sheep.

Expression characteristics of pineal miRNAs at ovine different reproductive stages and the identification of miRNAs targeting the AANAT gene.

BACKGROUND: Many recent studies have shown that miRNAs play important roles in the regulation of animal reproduction, including seasonal reproduction. The pineal gland is a crucial hub in the regulation of seasonal reproduction. However, little is known about the expression characteristics of pineal miRNAs in different reproductive seasons (anestrus and breeding season). Therefore, the expression profiles and regulatory roles of ovine pineal miRNAs were investigated during different reproductive stages using Solexa sequencing technology and dual luciferase reporter assays. RESULTS: A total of 427 miRNAs were identified in the sheep pineal gland. Significant differences in miRNA expression were demonstrated between anestrus and the breeding season in terms of the frequency distributions of miRNA lengths, number of expressed miRNAs, and specifically and highly expressed miRNAs in each reproductive stage. KEGG analysis of the differentially expressed (DE) miRNAs between anestrus and the breeding season indicated that they are significantly enriched in pathways related to protein synthesis, secretion and uptake. Furthermore, transcriptome analysis revealed that many target genes of DE miRNAs in the ribosome pathway showed relatively low expression in the breeding season. On the other hand, analyses combining miRNA-gene expression data with target relationship validation in vitro implied that miR-89 may participate in the negative regulation of aralkylamine N-acetyltransferase (AANAT) mRNA expression by targeting its 3'UTR at a unique binding site. CONCLUSIONS: Our results provide new insights into the expression characteristics of sheep pineal miRNAs at different reproductive stages and into the negative regulatory effects of pineal miRNAs on AANAT mRNA expression.

Current genetic diversity in eight local Chinese sheep populations.

China has numerous local domestic sheep breeds. In this study, the genetic diversity of eight sheep populations was estimated using 17 microsatellites. Knowledge of such diversity provides novel insight into the degree of breed protection needed and the prediction of hybrid advantage. In total, 17 microsatellites were genotyped in 186 individuals from eight populations. The mean number of alleles (± SD) ranged from 3.71 ± 1.36 in Zhaotong sheep to 11.94 ± 3.58 in small-tailed Han sheep. The observed heterozygote frequency (± SD) within a population ranged from 0.482 ± 0.025 in Zhaotong sheep to 0.664 ± 0.023 in Tibetan sheep. In addition, using pairwise difference (FST) analysis, the highest within-population diversity was observed in Tibetan sheep (πX = 12.8098) and small-tailed Han (πX = 12.67873), and the lowest diversity was observed in Zhaotong sheep (πX = 7.90337). The results for genetic divergence between populations indicated that the populations were significantly different (P < 0.05) based on the average number of pairwise differences between populations (πXY) and the corrected average pairwise differences. Both phylogenetic networks and structure analysis showed that these eight populations were separated into three clusters in accordance with their geographical habitat, except Tibetan and Hu sheep. In short, we genotyped eight local Chinese sheep populations using 17 microsatellites, and the results indicated that their current genetic diversity is decreasing and that new conservation strategies are needed. In addition, significant genetic differences between populations could be used in cross breeding.

Cashmere growth control in Liaoning cashmere goat by ovarian carcinoma immunoreactive antigen-like protein 2 and decorin genes.

OBJECTIVE: The study investigated the biological functions and mechanisms for controlling cashmere growth of Liaoning cashmere goat by ovarian carcinoma immunoreactive antigen-like protein 2 (OCIAD2) and decorin (DCN) genes. METHODS: cDNA library of Liaoning cashmere goat was constructed in early stages. OCIAD2 and DCN genes related to cashmere growth were identified by homology analysis comparison. The expression location of OCIAD2 and DCN genes in primary and secondary hair follicles (SF) was performed using in situ hybridization. The expression of OCIAD2 and DCN genes in primary and SF was performed using real-time polymerase chain reaction (PCR). RESULTS: In situ hybridization revealed that OCIAD2 and DCN were expressed in the inner root sheath of Liaoning cashmere goat hair follicles. Real-time quantitative PCR showed that these genes were highly expressed in SF during anagen, while these genes were highly expressed in primary hair follicle in catagen phase. Melatonin (MT) inhibited the expression of OCIAD2 and promoted the expression of DCN. Insulin-like growth factors-1 (IGF-1) inhibited the expression of OCIAD2 and DCN, while fibroblast growth factors 5 (FGF5) promoted the expression of these genes. MT and IGF-1 promoted OCIAD2 synergistically, while MT and FGF5 inhibited the genes simultaneously. MT+IGF-1/MT+FGF5 inhibited DCN gene. RNAi technology showed that OCIAD2 expression was promoted, while that of DCN was inhibited. CONCLUSION: Activation of bone morphogenetic protein (BMP) signaling pathway up-regulated OCIAD2 expression and stimulated SF to control cell proliferation. DCN gene affected hair follicle morphogenesis and periodic changes by promoting transforming growth factor-ß (TGF-ß) and BMP signaling pathways. OCIAD2 and DCN genes have opposite effects on TGF-ß signaling pathway and inhibit each other to affect the hair growth.

[Comparison of different single cell whole genome amplification methods and MALBAC applications in assisted reproduction].

Single-cell whole genome amplification (WGA) is a new technology, which can amplify small amounts of DNA from single cell and obtain the high coverage whole genome DNA template for revealing cell heterogeneity. Single cell WGA methods mainly include primer extension preamplification PCR (PEP-PCR), degenerate oligonucleotide primed PCR (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). In this review, we describe the principles and applications of different single cell genome wide amplification, and we evaluate and compare their amplification efficiency, including the coverage of genome, homogeneity, reproducibility, and detection power of single-nucleotide variants (SNV) and copy number variants (CNV). The results show that MALBAC have the highest amplification homogeneity, the lowest allelic gene knockdown rate, the best reproducibility, and the best detection effect on CNV and SNV. We also describe the applications of MALBAC in human single sperm meiosis, aneuploidy analysis, and human oocyte genome research.

[The molecular mechanism of sheep seasonal breeding and artificial regulatory techniques for estrus and mating in anestrus].

Seasonal breeding is an important factor limiting sheep production efficiency. Detailed analysis on the molecular mechanisms of seasonal breeding is the premise for improving estrus and mating rate of sheep during anestrus. Recent research showed that under long-photoperiod and short-photoperiod conditions, a series of changes in signaling molecules and cell morphology could be observed in ovine seasonal reproduction pathway. Based on the molecular mechanisms of seasonal reproduction, several technologies or methods for inducing estrus and mating of ewes in anestrus have been developed. In this review, photoperiod-induced changes in signaling molecules and cell morphology in pituitary and hypothalamic tissue are first summarized in terms of the molecular mechanisms and characteristics of seasonal reproduction. The application effect, advantages and disadvantages for applying these technologies for inducing estrus and mating of ewes in anestrus are then discussed, thereby providing the critical insights in identifying a new technology, which is environmentally friendly and efficient, to improve breeding rate in anestrus.

Selection signature in domesticated animals.

Domesticated animals play an important role in the life of humanity. All these domesticated animals undergo same process, first domesticated from wild animals, then after long time natural and artificial selection, formed various breeds that adapted to the local environment and human needs. In this process, domestication, natural and artificial selection will leave the selection signal in the genome. The research on these selection signals can find functional genes directly, is one of the most important strategies in screening functional genes. The current studies of selection signal have been performed in pigs, chickens, cattle, sheep, goats, dogs and other domestic animals, and found a great deal of functional genes. This paper provided an overview of the types and the detected methods of selection signal, and outlined researches of selection signal in domestic animals, and discussed the key issues in selection signal analysis and its prospects.

[A mechanistic review of how hypoxic mircroenvironment regulates mammalian ovulation].

Mammalian ovulation is a complicated process that includes development of follicles, ovulation, formation of corpus luteum and luteolysis. The three different stages of the ovulation activity are affected by hypoxic microenvironment and hypoxia-induced factors (HIF), which play a crucial role in physiologyical processes, such as angiogenesis and inflammation. Although the process of ovulation has been well elucidated, the molecular mechanism regulated by hypoxia needs an in depth study. In this review, we summarize how hypoxic and HIF regulate gene expression during mammalian ovulation in order to provide a better understanding of ovulation mechanism, which may lay a theoretical basis for prevention and therapy of various ovarian diseases.

[The mechanism of miRNA-mediated PGR signaling pathway in regulating female reproduction].

MicroRNAs (miRNAs) are involved in several physiological processes as important post-transcriptional regulators. Progesterone (P4), an important steroid hormone, produces physiological effect through binding specific receptor progesterone receptors (PGR) which regulates functions of both reproductive and non-reproductive tissues as a member of the nuclear receptor superfamily. P4/PGR and miRNAs could regulate female reproduction independently, however, it is still unclear how miRNAs and P4/PGR interaction regulates female reproductive activities such as ovulation in female reproduction. In this review, we summarize the possible ways in which miRNAs regulate P4 production and PGR gene expression as well as P4/PGR regulate miRNAs expression, which provide a theoretical basis for further studying the role of miRNAs and P4/PGR in female reproduction.

Application of epigenetic markers in molecular breeding of the swine.

Livestock phenotypes are determined by the interaction of a variety of factors, including the genome, the epigenome and the environment. Epigenetics refers to gene expression changes without DNA sequence alterations. Epigenetic markers mainly include DNA methylation, histone modifications, non-coding RNAs, and imprinting genes. More and more researches show that epigenetic markers play an important role in the traits of pigs by modulating phenotype changes via gene expression. However, the role of epigenetic markers has caught little attention in swine breeding. The mechanism that influences important traits of swine has not been analyzed in detail, and it still lacks adequate scientific basis for practical applications. From the aspects of nutrition, diseases, important economic traits and trans-generational inheritance, we summarize the research, application prospects and challenges in the field of utilizing epigenetic markers in molecular breeding of pigs, thus providing a more comprehensive theoretical basis to promote more rapid research development in this field.

Identification and verification of differentially expressed genes in the caprine hypothalamic-pituitary-gonadal axis that are associated with litter size.

Litter size is a favorable economic trait for the goat industry, but remains a complex trait controlled by multiple genes in multiple organs. Several genes have been identified that may affect embryo survival, follicular development, and the health of fetuses during pregnancy. Jining Grey goats demonstrate the largest litter size among goat breeds indigenous to China. In order to better understand the genetic basis of this trait, six suppression subtractive hybridization (SSH) cDNA libraries were constructed using pooled mRNAs from hypothalamuses, pituitaries, and ovaries of sexually mature and adult polytocous Jining Grey goats, as testers, versus the pooled corresponding mRNAs of monotocous Liaoning Cashmere goats, as drivers. A total of 1,458 true-positive clones--including 955 known genes and 481 known and 22 unknown expressed sequence tags--were obtained from the SSH libraries by sequencing and alignment. The known genes were categorized into cellular processes and signaling information storage and processing, and metabolism. Three genes (FTH1, GH, and SAA) were selected to validate the SSH results by quantitative real-time PCR; all three were up-regulated in the corresponding tissues in the tester group indicating that these are candidate genes associated with the large litter size of Jining Grey goats. Several other identified genes may affect embryo survival, follicular development, and health during pregnancy. This study provides insights into the mechanistic basis by which the caprine hypothalamic-pituitary-gonadal axis affects reproductive traits and provides a theoretical basis for goat production and breeding.

A 1.1 Mb duplication CNV on chromosome 17 contributes to skeletal muscle development in Boer goats.

The Boer goat is one of the top meat breeds in modern animal husbandry and has attracted widespread attention for its unique growth performance. However, the genetic basis of muscle development in the Boer goat remains obscure. In this study, we identified specific structural variants in the Boer goat based on genome-wide selection signals and analyzed the basis of the molecular heredity of related candidate genes in muscle development. A total of 9 959 autosomal copy number variations (CNVs) were identified through selection signal analysis in 127 goat genomes. Specifically, we confirmed that the highest signal CNV (HSV) was a chromosomal arrangement containing an approximately 1.11 Mb (CHIR17: 60062304-61171840 bp) duplicated fragment inserted in reverse orientation and a 5 362 bp deleted region (CHIR17:60145940-60151302 bp) with overlapping genes (e.g., ARHGAP10, NR3C2, EDNRA, PRMT9, and TMEM184C). The homozygous duplicated HSV genotype (+/+) was found in 96% of Boer goats but was not detected in Eurasian goats and was only detected in 4% of indigenous African goats. The expression network of three candidate genes ( ARHGAP10, NR3C2, and EDNRA) regulating dose transcription was constructed by RNA sequencing. Results indicated that these genes were involved in the proliferation and differentiation of skeletal muscle satellite cells (SMSCs) and their overexpression significantly increased the expression of SAA3. The HSV of the Boer goat contributed to superior skeletal muscle growth via the dose effects of overlapping genes.

[The neuroendocrine regulatory mechanisms of mammalian seasonal reproduction].

The seasonal reproduction of mammal means the reproduction experiences an annual period from quiescence to renaissance. Studies have shown that kisspeptin and RFRP play an important role in the reproductive seasonality. The non-breeding season is characterized by an increase in the negative feedback effect of estrogen on GnRH, and this effect is transmitted by kisspeptin neurons, which may be an important factor affecting the reproduction activities. The expression of RFRP depends on melatonin secretion, and shows an apparent inhibition on reproduction in non-breeding season. In addition, thyroid hormones influence termination of the breeding season. Dopaminergic neuron A14/A15 also contributes to the seasonal changes in estrogen negative feedback. These neural systems may synergistically modulate the seasonal changes of reproductive function with the photoperiod. This review makes a systematic expatiation on the relationship between seasonal reproduction and these neural systems.

[Progesterone receptor-mediated molecular mechanisms on mammalian female reproduction].

The steroid hormone, progesterone, plays a critical role in regulation of mammalian female reproductive activities. Besides the non-genomic activity of progesterone on target cells, its main physiological effect is caused through genomic action by the ligand-dependent nuclear progesterone receptor. The genomic and non-genomic effects of progesterone collectively mediate various female reproductive functions, including ovulation, embryo implantation, maintenance of pregnancy, initiation of parturition, and development of mammary gland. Although a large number of candidate genes regulated by progesterone have been identified by gene chip technology, the traditional progesterone response elements located in the promoter region of downstream target genes havenot been detected. Accordingly, it was suggested thatthe mechanism of nuclear progesterone receptors regulating transcription may be different from other nuclear steroid receptors. In this review, we summarized the mechanisms of progesterone receptors mediating the physiological effects in various female re-productive activities.

[Effects of RFRP-3 on reproductive function and energy balance in mammals].

The hypothalamo-pituitary-gonadal (HPG) axis integrates internal and external cues via a balance of stimulatory and inhibitory neurochemical systems to regulate reproductive function in mammals. However, RFRP-3 is a unique inhibitor of HPG axis at the hypothalamuic level in mammals to date. A large number of previous studies have confirmed that RFamide-related peptide (RFRP-3) suppresses gonadotropin-releasing hormone (GnRH) system and luteinizing hormone (LH) secretion, thereby affecting the reproduction. However, whether the inhibition of LH secretion by RFRP-3 occurs at the pituitary level or the hypothalamus level is not clear. It is interesting that RFRP-3 is also related to signal pathway of melatonin modulating mammal seasonal reproduction, but little is known about the effects of melatonin on the RFRP-3 neuron up to now. In addition, RFRP-3 also plays an important role in the regulation of energy balance and behavior. The regulatory mechanism of RFRP-3 in HPG axis and role of RFRP-3 in modulating mammalian energy balance, as well as behavior, are systematically elaborated and the remaining unsolved problems are also discussed in this paper.

[Research progress in molecular mechanism of animal seasonal reproduction].

Animal seasonal reproduction involves complicated neuroendocrine processes of the hypothalamo-pituitary-gonadal axis. It is dominantly regulated by photoperiod, a crucial environmental cue. Melatonin, as internal photoperiod signal, regulates seasonal reproduction of animals. In recent years, it has been found that Kiss1/GPR54 system, which may influence GnRH secretion evidently, is regulated by both melatonin and feedback action of gonadal steroid hormones. Consequently, Kiss1/GPR54 system may play a key role in seasonal reproduction. Additionally, there exists another potential retrograde control pathway of seasonal breeding, which involves TSH-DIO2/DIO3 system. TSH-DIO2/ DIO3 system affects synthesis and secretion of GnRH and is regulated by melatonin, as well as Kiss1/GPR54 system. In this article, melatonin signal, especially the research advances of Kissl/GPR54 system and TSH-DIO2/DIO3 system were reviewed.

Expression analysis and single-nucleotide polymorphisms of SYNDIG1L and UNC13C genes associated with thoracic vertebral numbers in sheep (Ovis aries).

The objective of the current study was to analyze expression levels of synapse differentiation inducing 1-like (SYNDIG1L) and unc-13 homolog C (UNC13C) genes in different tissues, while single-nucleotide polymorphisms (SNPs) of two genes were associated with multiple thoracic vertebrae traits in both Small-tailed Han sheep (STH) and Sunite sheep (SNT). The expression levels of SYNDIG1L and UNC13C were analyzed in the brain, cerebellum, heart, liver, spleen, lung, kidney, adrenal gland, uterine horn, longissimus muscle, and abdominal adipose tissues of two sheep breeds with different thoracic vertebral number (TVN) sheep (T13 groups and T14 groups) by real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the polymorphisms of UNC13C gene g.52919279C > T and SYNDIG1L gene g.82573325C > A in T14 and T13 were genotyped by the Sequenom MassARRAY® SNP assay, and association analysis was performed with the TVN. The results demonstrated that UNC13C gene was extensively expressed in 11 tissues. The expression of UNC13C gene in longissimus muscle of T14 groups of STH was significantly higher than that of T13 groups ( P < 0.05 ). SYNDIG1L gene was overexpressed in brain and cerebellum tissues, and the expression level of UNC13C gene in the brain and cerebellum of T13 groups in SNT was significantly higher than that of T14 groups ( P < 0.01 ). Association analysis showed that SNPs found in the UNC13C gene had no significant effects on TVN for both two genes. The polymorphism of SYNDIG1L g.82573325C > A was significantly correlated with the TVN in both STH ( P < 0.05 ) and SNT ( P < 0.01 ). Taken together, the SYNDIG1L gene was related to thoracic vertebral development, and this variation may be potentially used as a molecular marker to select the multiple thoracic vertebrae in sheep.

The expression and mutation of BMPR1B and its association with litter size in small-tail Han sheep (Ovis aries).

Previous studies have shown that BMPR1B promotes follicular development and ovarian granulosa cell proliferation, thereby affecting ovulation in mammals. In this study, the expression and polymorphism of the BMPR1B gene associated with litter size in small-tail Han (STH) sheep were determined. The expression of BMPR1B was detected in 14 tissues of STH sheep during the follicular phase as well as in the hypothalamic-pituitary-gonadal (HPG) axis of monotocous and polytocous STH sheep during the follicular and luteal phases using quantitative polymerase chain reaction (qPCR). Sequenom MassARRAY® single nucleotide polymorphism (SNP) technology was also used to detect the polymorphism of SNPs in seven sheep breeds. Here, BMPR1B was highly expressed in hypothalamus, ovary, uterus, and oviduct tissue during the follicular phase, and BMPR1B was expressed significantly more in the hypothalamus of polytocous ewes than in monotocous ewes during both the follicular and luteal phases ( P < 0.05 ). For genotyping, we found that genotype and allele frequencies of three loci of the BMPR1B gene were extremely significantly different ( P < 0.01 ) between the monotocous and polytocous groups. Association analysis results showed that the g.29380965A > G locus had significant negative effects on the litter size of STH sheep, and the combination of g.29380965A > G and FecB (Fec - fecundity and B - Booroola; A746G) at the BMPR1B gene showed that the litter size of AG-GG, AA-GG, and GG-GG genotypes was significantly higher compared with other genotypes ( P < 0.05 ). This is the first study to find a new molecular marker affecting litter size and to systematically analyze the expression of BMPR1B in different fecundity and physiological periods of STH sheep.