Regional differences of pheromone production in the sebaceous glands of castrated goats treated with testosterone

The primer pheromone is responsible for the "male effects" in goats and produced in the sebaceous glands testosterone-dependently. In the present study, the responses of sebaceous glands obtained from the head and rump regions of castrated goats were examined by our bioassay system after testosterone treatment to demonstrate the presence of regional differences in the pheromone production in male goats. The testosterone treatment resulted in the marked development of sebaceous glands and the induction of pheromone bioactivity in the head region of the goats. On the contrary, this treatment brought neither development of the sebaceous glands nor induction of pheromone bioactivity in the rump region. The treatment increased immunoreactivities to androgen receptors (AR) and 5alpha-reductase in the sebaceous glands of both regions, although the activities were more apparent in the head region than the rump region. These findings suggest that the primer pheromone of male goats is produced specifically in the sebaceous glands of the head region due partly to regional differences in the expression of AR and 5alpha-reductase mediating testosterone bioactivities.     

Androgen induces production of male effect pheromone in female goats

Previously we showed that the primer pheromone responsible for the "male effect" was produced in specific skin regions of castrated male goats by androgen treatments. In the present study, we examined whether androgen can also induce production of the male effect pheromone in female goats. Capsules containing dihydrotestosterone (DHT) or testosterone (T) were subcutaneously implanted into six ovariectomized (OVX) goats for 28 days. Small skin samples were collected from the head and rump regions, and the pheromone activity of their ether extracts was examined using a bioassay that monitors the electrophysiological manifestation of the hypothalamic gonadotropin-releasing hormone pulse generator as multiple-unit activity. Behaviors of OVX goats towards ovary-intact estrous goats were also examined before and at the end of DHT or T treatment. Before androgen treatment, neither the head nor rump skin samples in OVX goats showed pheromone activity. DHT treatment induced pheromone activity in the head skin sample of six OVX goats and in the rump skin sample of two OVX goats. Similar results were obtained by T treatment. In addition, OVX goats treated with T showed masculine-type sexual behaviors such as courtship and mounting behaviors towards the estrous goats. These results demonstrate that androgen is capable of inducing primer pheromone activity in the female and suggest that the synthesis pathway of the male effect pheromone exists in both sexes in the goat.     

Induction of primer pheromone production by dihydrotestosterone in the male goat

Castrated goats were treated with dihydrotestosterone (DHT) for four weeks. Skin samples were collected from the head and the rump regions before and after the DHT treatment. The primer pheromone activities of these samples were assessed neurophysiologically by recording electrophysiological manifestations of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator activity. Pheromone activity was detected in both the head and rump skin samples following the DHT treatment, although the development of sebaceous glands was limited to the head region. Taken together with our previous finding that testosterone treatment results in the appearance of primer pheromone activity in the skin sample of the head region but not of the rump region. these observations suggests that the regional difference of pheromone production would be ascribed to intrinsic expression levels of 5alpha-reductase, an enzyme converting testosterone to DHT.     

简州大耳羊肌内脂肪细胞成脂分化差异表达基因的筛选与鉴定

旨在通过转录组测序技术获得简州大耳羊肌内前体脂肪细胞成脂分化前后的差异表达基因,并经生物信息学分析获得相关信号通路及可能发挥作用的关键功能候选基因。本研究以7日龄的健康简州大耳羊公羊为试验动物（n=3）,采用胶原酶消化法分离获得其肌内前体脂肪细胞;利用Illumina平台对肌内前体脂肪细胞和诱导分化5 d的肌内脂肪细胞cDNA样品（n=3）进行高通量测序;以|log₂fold change|>0和P<0.05为阈值筛选获得差异表达基因,并利用clusterProfiler R包对其进行GO功能和KEGG通路富集;最后利用实时荧光定量PCR（quantitative real-time PCR,qRT-PCR）技术检测功能候选基因在细胞分化前后的表达量变化。结果显示,共获得差异表达基因7 916个,其中4 143个为表达上调基因,主要富集到氧化磷酸化、核糖体合成和三羧酸循环通路等305条通路;3 773个为表达下调基因,且主要富集到甲状腺激素信号通路等303条通路;差异基因GO功能注释中52.8%为生物过程、13.4%是细胞组成和33.8%是分子功能。qRT-PCR结果表明,UCP3、ACACB、ACOT11、ACOX3、APOA1和WISP2在分化前后的山羊肌内脂肪细胞中的表达趋势与RNA-seq结果一致,提示这些基因适合作为下一步研究的功能候选基因。本研究筛选得到简州大耳羊肌内脂肪细胞成脂分化的差异基因,并确认了6个基因可作为功能候选基因。研究结果为阐明肉用山羊肌内脂肪细胞成脂分化的分子调控网络提供基础数据和系统资料。     

Genome-wide Selection Signals Reveal Candidate Genes Associated with the Cashmere Traits of Cashmere Goats

The purpose of this study was to screen the genome-wide selection signals of Liaoning cashmere goat and Inner Mongolia cashmere goat. Based on the Illumina Caprine 50K SNP chip, Liaoning cashmere goat, Inner Mongolia cashmere goat and Huanghuai goat were genotyped, and the common 50 010 SNPs were obtained after quality control. Using population differentiation coefficients Fst and XP-EHH, Huanghuai goats were used as reference populations to detect selection signals for Inner Mongolia cashmere goats and Liaoning cashmere goats. The top 5% of Fst and XP-EHH values was used as the threshold. The result showed that 501 candidate genes were selected by Fst and 145 candidate genes were selected by XP-EHH in cashmere goats. Based on these SNPs, we detected 12 SNPs under strong selection by Fst and XP-EHH. After gene annotation, 21 candidate genes were identified, such as EXOC4, ASIC2, PCDH9, RHBDD1, IRS1 and PDE10A. These genes were found to be mainly enriched in PI3K-Akt signaling pathway, protein digestion and absorption and relaxin signaling pathways. The study indicated that genes associated with cashmere traits were subjected to strong artificial selection pressure during domestication of the cashmere goats. These findings also help us better understand the selection progress of cashmere goats and provide a new theoretical basis for the protection and utilization of germplasm resources of Chinese cashmere goat breeds.     

全基因组选择信号揭示绒山羊绒毛性状相关的候选基因

旨在对辽宁绒山羊和内蒙古绒山羊的基因组选择信号进行筛选。本研究利用Illumina 50K的山羊芯片，对内蒙古绒山羊、辽宁绒山羊和黄淮山羊进行基因型检测，质控后获得50 010个共同SNPs位点。通过群体分化系数Fst和XP-EHH，以黄淮山羊为参考群体分别对内蒙古绒山羊和辽宁绒山羊进行选择信号检测，Fst和XP-EHH值的top 5%作为阈值。结果，利用Fst在绒山羊基因组中筛选到501个候选基因，利用XP-EHH在绒山羊基因组中筛选到145个候选基因。其中有12个SNPs在绒山羊基因组中受到较强选择。通过基因注释筛选到21个候选基因，包括EXOC4、ASIC2、PCDH9、RHBDD1、IRS1和PDE10A等。这些基因主要富集在PI3K-Akt信号通路、蛋白质消化吸收和松弛素信号通路等。研究结果发现，绒山羊在驯化过程中其基因组很多与毛囊相关的基因受到了强烈的选择。这些发现也有助于更好地了解绒山羊的选择进展，为中国绒山羊品种的种质资源保护和利用提供重要参考。     

Isolation and Identification of Goat Dermal Papilla Cells and Expression of TGF/β-smad Pathway Related Genes

The purpose of this study was to isolate goat hair follicle cells,and to explore the expression of TGF-β/smad pathway-related genes,providing a good model for the research on the mechanism of goat hair follicle development in vitro.In this study,two steps of neutral protease and collagenase digestion were used to treat the skin on the back of goats,the hair follicle cells were isolated and purified under stereomicroscope.Cellular immunofluorescence and Western blotting method were used to verify the expression of α-SMA (α-smooth muscle actin,α-SMA) and VIM (vimentin,VIM),and the expression of related genes of the TGF-β/smad pathway in goat hair papilla cells were examined.The results showed that the isolated goat hair follicle cells in adherent culture after separation grew slowly and had mature morphology after 15 days of separation and could be subcultured.The results of immunofluorescence and Western blotting showed that both α-SMA and VIM were positive in vitro cultured hair papilla cells.The expression of smad4 and smad5 genes in the TGF-β/smad pathway were significantly higher than those in control group (P<0.05),and the expression of smad2,smad6,and smad7 were extremely significantly higher than those in control group (P<0.01).These key genes related to hair follicle development were highly expressed.This study successfully isolated goat dermal papilla cells,which would give a good theoretical basis and cell model for studying the hair follicle development mechanism in vitro.     

山羊毛乳头细胞分离鉴定及TGF-β/smad通路相关基因的表达

试验旨在分离山羊毛乳头细胞,并探索其中TGF-β/smad通路相关基因的表达,为体外开展山羊毛囊发育的相关机制研究提供良好模型。采用中性蛋白酶与胶原酶两步消化法对山羊背部皮肤做处理,在体视显微镜下分离毛乳头细胞并进行纯化。细胞免疫荧光和Western blotting验证平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）和波形蛋白（vimentin,VIM）在体外培养的毛乳头细胞中的表达,并检测TGF-β/smad通路中相关基因在山羊毛乳头细胞中的表达情况。结果显示,分离后进行贴壁培养的毛乳头细胞生长较慢,在分离15 d后具有成熟形态,可进行传代培养。细胞免疫荧光和Western blotting结果表明,α-SMA和VIM在体外培养的毛乳头细胞中均表达,TGF-β/smad通路中smad4、smad5基因的表达水平显著高于对照组（P<0.05）,smad2、smad6、smad7基因的表达量极显著高于对照组（P<0.01）,这些与毛囊发育相关的关键基因均呈现高表达,表明毛乳头细胞可能通过TGF-β/smad通路的调控从而影响毛囊的发育。本研究成功分离出山羊毛乳头细胞,为体外研究山羊毛囊发育机制奠定良好的理论基础和细胞模型。     

内蒙古绒山羊Hox基因家族成员在毛囊中表达的研究

Hox基因作为一类重要的转录因子,在胚胎发育组织分化过程中有重要作用。为了探索Hox基因在绒山羊毛囊发育中的作用机制,本研究采用原位杂交技术,检测Hox基因家族成员Hoxa4、Hoxa5、Hoxa6基因在绒山羊毛囊的表达模式。结果显示,Hoxa4、Hoxa5、Hoxa6基因分别在绒山羊胚胎期和兴盛期毛囊的不同部位表达,说明Hox基因在绒山羊毛囊生长发育过程中扮演着重要的角色。     

催乳素及其受体在青年公羊驼皮肤中分布的研究

羊驼是一种经济价值很高的毛用动物，研究包括激素在内的各种因素对羊驼毛生长的调控极为重要。作者采用基因芯片杂交技术发现青年公羊驼皮肤中不仅存在PRL及其受体基因，而且还存在PRL相关蛋白基因，提示羊驼皮肤是PRL的一个垂体外合成部位，同时羊驼皮肤还可以合成PRL相关蛋白及PRL受体；通过荧光定量分析比较，认为羊驼皮肤合成的PRL是极少量的，垂体是PRL合成的主要部位；通过免疫组织化学技术确定PRL及其受体蛋白主要分布在羊驼毛囊上皮根鞘、表皮、皮脂腺等，提示PRL对羊驼皮肤及其衍生物可能以内分泌、自分泌和旁分泌的方式发挥作用。     

内蒙古绒山羊Hox基因家族成员在毛囊中表达的研究

Hox基因作为一类重要的转录因子,在胚胎发育组织分化过程中有重要作用。为了探索Hox基因在绒山羊毛囊发育中的作用机制,本研究采用原位杂交技术,检测Hox基因家族成员Hoxa4、Hoxa5、Hoxa6基因在绒山羊毛囊的表达模式。结果显示,Hoxa4、Hoxa5、Hoxa6基因分别在绒山羊胚胎期和兴盛期毛囊的不同部位表达,说明Hox基因在绒山羊毛囊生长发育过程中扮演着重要的角色。     

山羊经济性状候选基因研究进展

通过对山羊经济性状候选基因和山羊经济性状的常用分子标记方法的研究，提出了小羊经济性状候选基因的研究意义，以亟利用这些信息选育产肉香、产毛好、产奶多的优良山羊新品种。     

内蒙古绒山羊105 d胎儿皮肤的基因表达

选择105d绒山羊胎儿体侧部皮肤组织构建cDNA文库，随机挑取克隆进行表达序列标签（ESTs）测序，共测序1152个质粒，合格的大于100bp的高质量ESTs846个，平均长度为443．2bp。对525个绒山羊胎儿皮肤组织已知功能基因的ESTs进行分类，可分为细胞分裂类18个，细胞信号类65个；细胞结构蛋白基因73个；细胞防御类21个；基因／蛋白表达126个；代谢类的69个；未分类的153个。发现了Wnt-4、Bmpr-Ib、ASIP gene、Ectodysplasin等重要的与毛囊分化相关的基因。     

Immunolocalization of c-Fos,ELOVL5 and oestradiol in the ewe vulva in relation to oestrus behaviour after treatment with lipopolysaccharide

Sudden activation of the stress axis by a lipopolysaccharide endotoxin (LPS) significantly reduces ewes' sexual attractivity to rams by delaying all signs of oestrous behaviour. To understand mechanisms involved in attracting male interest, we examined c-Fos (nuclear activation), ELOVL5 (enzyme involved in pheromone synthesis) and oestradiol receptors (ER) using immunohistochemistry on ewe vulval tissue at 0, 31 and 40 hr in the ovarian follicular phase with or without exposure to LPS at 28 hr (5 groups of 4 ewes per group). While there was intense staining for immunoreactive (IR)-c-Fos and IR-ELOVL5 in the vulval epithelium and sebaceous glands, there were no differences in intensity between groups of ewes. The absence of IR-ER staining in vulval epithelium and sebaceous/sweat glands was unexpected. Differences in ram behaviour towards ewes in the ovarian follicular phase and after LPS treatment do not appear to involve quantitative changes in vulval c-Fos, ELOVL5 or ER, but subtle qualitative differences in individual-specific compounds (attraction pheromones) remain an option.     

Nuclear survivin expression as a potentially useful tool for the diagnosis of canine cutaneous sebaceous lesions

Background – Sebaceous glands are specialized cutaneous adnexal glands, which work under constant hormonal control to produce sebum. They can give rise to several proliferative lesions, such as hamartoma, hyperplasia and neoplasms (adenoma, epithelioma and carcinoma). Their nomenclature is currently confusing, both in veterinary and in human medicine, owing to the difficulty of differentiating between some of these lesions. Methods – The present study used immunohistochemistry to determine the expression levels and patterns of survivin and Ki67 in five samples of normal canine skin and 44 cases of canine cutaneous lesions with sebaceous differentiation (10 hamartomas, nine hyperplasia, eight adenomas, eight epitheliomas and nine carcinomas). Results – In normal glands, survivin, as well as Ki67, was expressed in scattered reserve cells. In hamartomas, survivin was more highly expressed than in normal skin, indicating a possible role of this molecule in the pathogenesis of these congenital lesions. In tumours, a moderate or high level of survivin and Ki67 expression (more than two and four and more than two positive cells, respectively) were significantly correlated with a malignant histotype, infiltrative growth and a moderate or high number of mitoses (more than two). Conclusions and clinical importance – The level of survivin expression increased with increasing malignancy, designating survivin as a new diagnostic marker in the assessment of malignancy of sebaceous tumours.     

非编码RNA调节羊绒细度候选基因的研究进展

羊绒细度是评价羊绒品质和衡量羊绒价格的关键数量性状,近年来,随着羊绒细度功能标记基因的深入研究,通过高通量数据解析及全基因组关联分析,逐步筛选出羊绒细度性状的候选基因,但是羊绒细度关键调控基因、表达量及分子调节机制尚未明了。基因表达受多种调节因子调控,其中非编码RNA的调节作用比较活跃,主要包括microRNA、lncRNA和circRNA等。经高通量测序和生物信息分析发现,非编码RNA在绒山羊品种间和品种内的不同羊绒细度个体皮肤、次级毛囊及次级毛囊周期性生长发育中差异表达并调节羊绒细度相关靶基因,但多种非编码RNA共同调节同一靶基因的信号通路甚少。目前通过对羊绒细度差异基因进行SNP标记辅助选择、转录组筛选、高通量解析非编码RNA调节及全基因组关联分析等相关研究发现,两两通路聚焦相同的羊绒细度靶基因较多,两条通路以上共同聚焦到某个或某几个羊绒细度的关键靶基因较少。作者主要讨论了羊绒细度候选基因及转录水平上microRNA、lncRNA和circRNA对羊绒细度相关靶基因分子调控机制的研究进展。     

CTSD对黔北麻羊卵泡颗粒细胞的调控机制及功能分析

旨在分析组织蛋白酶D(cathepsin D,CTSD)对黔北麻羊卵泡颗粒细胞的调控机制并初步探究其对产羔性状的影响机理.本研究以36周龄、健康、多胎黔北麻羊母羊(n=5)为研究对象,屠宰后采集卵巢组织分离培养卵泡颗粒细胞,构建真核表达载体pEGFP-N3-CTSD,将其导入细胞后在转录与翻译水平验证真核表达效率;通过...     

全基因组选择信号揭示绵羊毛囊发育及脱毛性状相关的候选基因

利用全基因组选择信号研究不同羊毛类型绵羊的群体结构及遗传分化程度,以挖掘与毛囊发育及脱毛性状相关的候选基因。本研究以3种羊毛类型的21个品种共290只绵羊群体为研究对象,利用Illumina Ovine SNP 50K芯片基因分型数据,基于群体分化指数Fst和核苷酸多样性比值θπ Ratio方法对无绒毛型、细毛型和中毛型绵羊进行选择信号检测。将Fst和θπ Ratio的top 5%作为阈值检测受到强烈选择的SNPs位点并进行注释。结果表明,SOX18、ALX4、FGF1和LRP4等与毛囊发育及脱毛性状相关的基因受到强烈选择。本研究通过Fst和θπ Ratio两种方法检测出与毛囊发育周期、羊毛形成以及毛囊和皮脂腺部分细胞相关的重要基因,这将为进一步研究绵羊重要经济性状形成的机制提供有意义的参考。     

基于UniGene寻找山羊新的miRNA及其试验验证

根据miRNA进化的保守性,以已知动物的miRNA为搜索序列与山羊的UniGene进行BLAST比对,预测山羊新的miRNA,并通过RT-PCR和克隆测序进行验证。对新发现的山羊miRNA进行实时荧光定量,检测其在12个月皮肤及肌肉中的表达量;用基于UniGene的方法获得了5条山羊miRNA候选分子,通过RT-PCR和克隆测序验证,发现这5条miRNA分子在山羊的皮肤和肌肉中均有表达,其中由chi-miR-374b、chi-miR-421和chi-miR-421*构成了山羊miRNA基因簇,chi-miR-2284n是牛和山羊特异的。实时荧光定量结果显示,chi-miR-1839、chi-miR-374b和chi-miR-2284n在2月份皮肤中的表达量明显高于其他月份,chi-miR-421和chi-miR-421*在10月份皮肤中的表达量最高;发现了5条山羊新的miRNA(chi-miR-2284n、chi-miR-421*、chi-miR-421、chi-miR-1839和chi-miR-374b,登录号:JQ002550-JQ002554),补充了山羊miRNA数据库的不足。     

内蒙古绒山羊角蛋白辅助蛋白1.1基因真核表达载体构建及基因转染

In this study, Cashmere goat skin by extracting total RNA, cloning keratin-associated protein 1.1 (KAP1.1) gene eukaryotic expression vector pEGFP-N3-KAP1.1, using liposome-mediated recombinant plasmids were transformed into Inner Mongolia Cashmere goats cultured fibroblasts, temporal and spatial expression of the genes in eukaryotic cells, subcellular localization and expression pattern. The results showed that 2.0 μg plasmid and 6.0 μL ratio liposomes suitable for the optimization of the experimental protocol, KAP1.1 gene could be successfully expressed in fibroblasts in Inner Mongolia Cashmere goat, and 48 h after transfection, the transfection efficiency reaches a peak, the gene in eukaryotic cells in vitro on the nucleus, cytoplasm and cell membrane are expressed, but the strongest expression of the nucleus.