Photoperiod Induces the Epigenetic Change of the GNAQ Gene in OVX+E2 Ewes.

GNAQ, a member of the alpha subunit encoding the q-like G protein, is a critical gene in cell signaling, and multiple studies have shown that upregulation of GNAQ gene expression ultimately inhibits the proliferation of gonadotropin-releasing hormone (GnRH) neurons and GnRH secretion, and ultimately affects mammalian reproduction. Photoperiod is a key inducer which plays an important role in gene expression regulation by affecting epigenetic modification. However, fewer studies have confirmed how photoperiod induces epigenetic modifications of the GNAQ gene. In this study, we examined the expression and epigenetic changes of GNAQ in the hypothalamus in ovariectomized and estradiol-treated (OVX+E2) sheep under three photoperiod treatments (short photoperiod treatment for 42 days, SP42; long photoperiod treatment for 42 days, LP42; 42 days of short photoperiod followed by 42 days of long photoperiod, SP-LP42). The results showed that the expression of GNAQ was significantly higher in SP-LP42 than in SP42 and LP42 (p < 0.05). Whole genome methylation sequencing (WGBS) results showed that there are multiple differentially methylated regions (DMRs) and loci between different groups of GNAQ. Among them, the DNA methylation level of DMRs at the CpG1 locus in SP42 was significantly higher than that of SP-LP42 (p < 0.01). Subsequently, we confirmed that the core promoter region of the GNAQ gene was located with 1100 to 1500 bp upstream, and the DNA methylation level of all eight CpG sites in SP42 was significantly higher than those in LP42 (p < 0.01), and significantly higher than those in SP-LP42 (p < 0.01), except site 2 and site 4 in the first sequencing fragment (p < 0.05) in the core promoter region. The expression of acetylated GNAQ histone H3 was significantly higher than that of the control group under three different photoperiods (p < 0.01); the acetylation level of sheep hypothalamic GNAQ genomic protein H3 was significantly lower under SP42 than under SP-LP42 (p < 0.05). This suggests that acetylated histone H3 binds to the core promoter region of the GNAQ gene, implying that GNAQ is epigenetically regulated by photoperiod through histone acetylation. In summary, the results suggest that photoperiod can induce DNA methylation in the core promoter region and histone acetylation in the promoter region of the GNAQ gene, and hypothesize that the two may be key factors in regulating the differential expression of GNAQ under different photoperiods, thus regulating the hypothalamus-pituitary-gonadal axis (HPGA) through the seasonal estrus in sheep. The results of this study will provide some new information to understand the function of epigenetic modifications in reproduction in sheep.

Screening of Differentially Expressed Genes and miRNAs in Hypothalamus and Pituitary Gland of Sheep under Different Photoperiods.

The reproduction of sheep is affected by many factors such as light, nutrition and genetics. The Hypothalamic-pituitary-gonadal (HPG) axis is an important pathway for sheep reproduction, and changes in HPG axis-related gene expression can affect sheep reproduction. In this study, a model of bilateral ovarian removal and estrogen supplementation (OVX + E2) was applied to screen differentially expressed genes and miRNAs under different photoperiods using whole transcriptome sequencing and reveal the regulatory effects of the photoperiod on the upstream tissues of the HPG axis in sheep. Whole transcriptome sequencing was performed in ewe hypothalamus (HYP) and distal pituitary (PD) tissues under short photoperiod 21st day (SP21) and long photoperiod 21st day (LP21). Compared to the short photoperiod, a total of 1813 differential genes (up-regulation 966 and down-regulation 847) and 145 differential miRNAs (up-regulation 73 and down-regulation 72) were identified in the hypothalamus of long photoperiod group. Similarly, 2492 differential genes (up-regulation 1829 and down-regulation 663) and 59 differential miRNAs (up-regulation 49 and down-regulation 10) were identified in the pituitary of long photoperiod group. Subsequently, GO and KEGG enrichment analysis revealed that the differential genes and target genes of differential miRNA were enriched in GnRH, Wnt, ErbB and circadian rhythm pathways associated with reproduction. Combined with sequence complementation and gene expression correlation analysis, several miRNA-mRNA target combinations (e.g., LHB regulated by novel-414) were obtained. Taken together, these results will help to understand the regulatory effect of the photoperiod on the upstream tissues of HPG in sheep.

Transcriptome Analysis of Neuroendocrine Regulation of Ovine Hypothalamus-Pituitary-Ovary Axis during Ovine Anestrus and the Breeding Season.

Most sheep are seasonal estrus, and they breed in autumn when the days get shorter. Seasonal estrus is an important factor that affects the productivity and fertility of sheep. The key point to solve this problem is to explore the regulation mechanism of estrus in sheep. Therefore, in this study, transcriptomic sequencing technology was used to identify differentially expressed mRNAs in the hypothalamus, pituitary and ovary of Small Tail Han sheep (year-round estrus) and tan sheep (seasonal estrus) among luteal, proestrus and estrus stages. There were 256,923,304,156 mRNAs being identified in the hypothalamus, pituitary and ovary, respectively. Functional analysis showed that the photosensor, leucine and isoleucine biosynthesis pathways were enriched significantly. It is speculated that photoperiod may initiate estrus by stimulating the corresponding pathways in hypothalamus. ODC1, PRLH, CRYBB2, SMAD5, OPN1SW, TPH1 are believed to be key genes involved in the estrogen process. In conclusion, this study expanded the database of indigenous sheep breeds, and also provided new candidate genes for future genetic and molecular studies on the seasonal estrus trait in sheep.

Identification of Prolificacy-Related Differentially Expressed Proteins from Sheep (Ovis aries) Hypothalamus by Comparative Proteomics.

Reproduction, as a physiologically complex process, can significantly affect the development of the sheep industry. However, a lack of overall understanding to sheep fecundity has long blocked the progress in sheep breeding and husbandry. In the present study, the aim is to identify differentially expressed proteins (DEPs) from hypothalamus in sheep without FecB mutation in two comparison groups: polytocous (PF) versus monotocous (MF) sheep at follicular phase and polytocous (PL) versus monotocous (ML) sheep at luteal phase. Totally 5058 proteins are identified in sheep hypothalamus, where 22 in PF versus MF, and 39 proteins in PL versus ML are differentially expressed, respectively. A functional analysis is then conducted including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to reveal the potential roles of these DEPs. The proteins ENSOARP00000020097, ENSOARP00000006714, growth hormone (GH), histone deacetylase 4 (HDAC4), and 5'-3' exoribonuclease 2 (XRN2) in PF versus MF, and bcl-2-associated athanogene 4 (BAG4), insulin-like growth factor-1 receptor (IGF1R), hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), and transthyretin (TTR) in PL versus ML appear to modulate reproduction, presumably by influencing the activities of gonadotropin-releasing hormone (GnRH). This study provides an alternative method to identify DEPs associated with sheep prolificacy from the hypothalamus. The mass spectrometry data are available via ProteomeXchange with identifier PXD013822.

Reproductive role of miRNA in the hypothalamic-pituitary axis.

The hypothalamic-pituitary-gonadal (HPG) axis plays a critical role in regulating reproductive function. Gonadotropin-releasing hormone (GnRH), which is secreted by the hypothalamus, acts on pituitary gonadotrophs to stimulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and secretion, ultimately affecting the animal's fertility. MicroRNAs are small, non-coding RNAs that are widely expressed throughout the brain and can fine-tune gene expression post-transcriptionally. Recently, growing evidence has unveiled the central position of miRNAs within a key regulatory process involving GnRH secretion and subsequent activation in the pituitary. Although transcriptional regulation of reproduction has been well studied, the post-transcriptional processes are less well understood. In this review, we elaborate comprehensively on the critical role of miRNAs in the reproductive process, including both temporal and spatial aspects. A better understanding of how miRNAs impact the neuroendocrine system may improve our knowledge of reproduction and provide novel targets for therapeutic development.

Molecular cloning and epigenetic change detection of Kiss1 during seasonal reproduction in Chinese indigenous sheep.

Like most seasonal domesticated species, sheep are short-day breeders, which means that the reproduction axis is activated by short days. The annual photoperiodic cycle affects the amount of daylength information that is transmitted to the hypothalamic-pituitary-gonadal (HPG) axis by regulating pulsatile secretion of gonadotrophin-releasing hormone from the hypothalamus. Kisspeptin, which is encoded by Kiss1, plays a major role in reproductive seasonality. Based on results from our previous Solexa sequencing data obtained from Tan (T) and Small Tail Han (STH) sheep during anoestrus and the breeding season, full-length mRNA information for ovine Kiss1 was obtained; 894bp in T sheep and 1145bp in STH sheep. Both encode 135 amino acids. Additionally, T and STH sheep have different transcription start sites of Kiss1. Kiss1 expression during oestrus was significantly higher than that during dioestrus, both in T and STH sheep (P<0.01). We also found a strong relationship between Kiss1 mRNA levels and histone H3 acetylation status in the 5' promoter region of ovine Kiss1. These data indicated that epigenetic modification occurs during reproduction in sheep, and this is the first report that histone H3 deacetylation occurs in the hypothalamus of seasonal sheep breeders during the transition from dioestrus to oestrus.

Effects of carriers on nutrient removal and membrane fouling in combined process of inclined-plates hydrolytic tank and membrane bioreactor.

A novel process, inclined-plates hydrolytic tank (IHT) and membrane bioreactor (MBR), was used to treat domestic sewage continuously. In this study, the effects of carriers' addition on operational performances of IHT-MBR were studied at the hydraulic retention time of 5.4 h and the recycling rate of 200%. Experimental results indicated the removal efficiencies of chemical oxygen demand, total nitrogen and total phosphorus reached 86.8%, 82.9% and 89.6%, respectively, corresponding trans-membrane pressure decreased to 1.50 kPa/d at a packing ratio of 20%. Simultaneously, the scanning electron microscope and soluble microbial products analysis demonstrated that high nutrient removal and low membrane fouling were attributed to the attached growth of microorganisms on carriers. The bioattachment and adsorption of carriers not only decreased the soluble proteins and polysaccharide in MBR, but also provided good living environments for denitrifying bacteria and phosphorus-accumulating bacteria.

Physiological interactions between the hypothalamic-pituitary-gonadal axis and spleen in rams actively immunized against GnRH.

Hypothalamic-pituitary-gonadal (HPG) axis is strongly implicated in the regulation of immune system. The objective was to determine the effects of immunocastration on splenic reproduction- and immunity-related gene expressions, and serum cytokine profiles in rams. Forty rams were randomly allocated into three groups: control (n=14); surgically castrated (n=13); or immunized (n=13) against 100µg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 6months of age (with a booster 2months later). Blood samples (for hormone and immune cytokine profiles) were collected at 1-month intervals until rams were slaughtered (10months). Compared to intact controls, anti-GnRH immunization reduced (P<0.05) serum concentrations of LH, FSH, and testosterone. Reduced testosterone abrogated its inhibitor feedback effect on the synthesis of GnRH in spleen, as evidenced by increased (P<0.05) protein content and mRNA expressions of GnRH, and simultaneously decreased (P<0.05) mRNA expressions of androgen receptor in spleen. In parallel with the increased GnRH production in spleen, the mRNA expressions of interleukin (IL)-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α) as well as lymphocyte marker CD4, CD8 and CD19 molecules were increased (P<0.05) in spleen. Consistently, serum concentrations of IL-2, IL-4, IL-6, TNF-α were increased (P<0.05) in rams following immunization. Similarly, deprivation of testosterone by surgical castration also increased (P<0.05) GnRH and thus immune cytokine expressions in spleen. Collectively, our data suggested that immunocastration increased GnRH production in spleen by abrogating the inhibitory feedback effects from testosterone, consequently improving the immune markers of spleen and serum immune cytokines in rams.

The Inhibitory Effects of RFamide-Related Peptide 3 on Luteinizing Hormone Release Involves an Estradiol-Dependent Manner in Prepubertal but Not in Adult Female Mice.

The mammalian gonadotropin-inhibitory hormone (GnIH) ortholog, RFamide-related peptide (RFRP), is considered to act on gonadotropin-releasing hormone (GnRH) neurons and the pituitary to inhibit gonadotropin synthesis and release. However, there is little evidence documenting whether RFamide-related peptide 3 (RFRP-3) plays a primary role in inhibition of the hypothalamo-pituitary-gonadal (HPG) axis prior to the onset of puberty. The present study aimed to understand the functional significance of the neuropeptide on pubertal development. The developmental changes in reproductive-related gene expression at the mRNA level were investigated in the hypothalamus of female mice. The results indicated that RFRP-3 may be an endogenous inhibitory factor for the activation of the HPG axis prior to the onset of puberty. In addition, centrally administered RFRP-3 significantly suppressed plasma luteinizing hormone (LH) levels in prepubertal female mice. Surprisingly, centrally administered RFRP-3 had no effects on plasma LH levels in ovariectomized (OVX) prepubescent female mice. In contrast, RFRP-3 also inhibited plasma LH levels in OVX prepubescent female mice that were treated with 17beta-estradiol replacement. Our study also examined the effects of RFRP-3 on plasma LH release in adult female mice that were ovariectomized at dioestrus, with or without estradiol (E2). Our results showed that the inhibitory effects of RFRP-3 were independent of E2 status. Quantitative real-time PCR and immunohistochemistry analyses showed that RFRP-3 inhibited GnRH expression at both the mRNA and protein levels in the hypothalamus. These data demonstrated that RFRP-3 could effectively suppress pituitary LH release, via the inhibition of GnRH transcription and translation in prepubescent female mice, which is associated with estrogen signaling pathway and developmental stages.

Impacts of human lysozyme transgene on the microflora of pig feces and the surrounding soil.

The rapid development of genetic engineering and extensive applications of genetically engineered (GE) animals have provided many research benefits, but concerns have been raised over the potential environmental impact of transgenic animals. We investigated the effects of human lysozyme (hLZ) transgenic pigs which can express hLZ in their mammary glands on the surrounding environment from the angle of the changes of pig feces and the surrounding soil, including the probability of horizontal gene transfer (HGT), the impact on microbial communities in pig gastrointestinal (GI) tracts and soil, and the influence on the total nitrogen (TN) and total phosphorus (TP) content of pig excrement and surrounding soil. Results showed that hLZ gene was not detected by polymerase chain reaction (PCR) or quantitative real-time PCR (Q-PCR) in gut microbial DNA extracts of manure or microbial DNA extracts of topsoil. PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analysis and 16S rDNA sequence analysis showed that hLZ gene had no impact on the microflora structure of pig guts or soil. Finally, TN and TP contents were not significantly different in pig manure or soils taken at different distances from the pig site (P>0.25).