山羊卵泡发育相关基因的筛选及分析

【背景】山羊第一卵泡波中的优势卵泡（dominant follicles, DF）和从属卵泡（subordinate follicles, SF）是整个卵泡发育过程中最为关键的两个阶段。随着卵泡的进一步发育,最终DF可能发育成为成熟卵泡,直到排卵;SF将走向闭锁,其中颗粒细胞的凋亡是导致卵泡发生闭锁的关键因素。然而目前对促进卵泡的优势化或导致其闭锁的分子机理尚不清楚。【目的】通过对山羊第一卵泡波中DF和SF颗粒细胞进行高通量测序,旨在筛选影响卵泡发育的关键基因,为深入探究卵泡发育的调控机制提供理论依据。【方法】选取10只1岁龄健康的贵州白山羊分别注射前列腺素F_2α,使其同期发情,此后每天用B超检测并记录卵泡的生长情况,发情3 d后,统一屠宰并采集第一卵泡波中DF (直径4.5—6 mm)与SF (直径3 —4.5 mm),分别分离其中的颗粒细胞,提取总RNA、构建文库后通过Illumina Hiseq 2500平台进行测序。利用FastQC对测序产出raw reads进行质量评估并经过过滤后,获得品质较高的clean reads;使用Trinity对得到的clean reads进行重新组装,从而获得unigenes;使用CLC Genomics Workbench将unigenes与山羊RefSeq数据库进行比对获得mRNA;使用DESeq2 软件对获得的mRNA进行差异表达分析;分别采用goseq和kobas软件对得到的差异表达基因进行GO分析及KEGG信号通路分析;最终通过qRT-PCR对筛选出的可能影响卵泡发育的关键基因进行验证。【结果】分别对测序得到的raw reads进行过滤后,在DF颗粒细胞中获得43 217 934条clean reads,占raw reads的比例为95.19%;SF颗粒细胞中获得40 766 348条clean reads,占raw reads的比例为95.35%。将得到的unigenes与山羊的RefSeq 数据库进行比对后,共得到33 896条带有注释的转录本,再通过设定FPKM>1, q value<0.05,共在两种卵泡颗粒细胞中获得13 644个基因。设定参数：FPKM≥1,SF-FPKM/DF-FPKM>1,P<0.05,获得695个差异表达mRNA,其中233个在SF颗粒细胞中表达显著上调,462个表达显著下调;对所获得695个差异表达mRNA进行GO功能富集分析,共分为三大类42组：其中生物学过程占47.6%,细胞组分占47.6%,分子功能占4.8%;KEGG信号通路分析,发现20条通路,其中与核糖体通路相关的基因富集最为显著。通过在Genecard中进行功能分析后,筛选6个可能与山羊卵泡发育密切相关的基因,其中PRLR、PTX3、RGN在SF颗粒细胞中表现为上调;DKK3、ALDH1A2、RARRES1则表现为下调。qRT-PCR显示PRLR、RGN、DKK3、ALDH1A2、RARRES1的表达趋势与高通量测序结果一致,且RGN在从属卵泡颗粒细胞中的表达量极显著地高于优势卵泡（P<0.01）;DKK3、ALDH1A2、RARRES1在优势卵泡颗粒细胞中的表达量极显著地高于从属卵泡（P<0.01）。【结论】DKK3、ALDH1A2、RARRES1和RGN在优势卵泡和从属卵泡中表达量存在极显著差异,推测在山羊卵泡发育过程中可能促进卵泡的优势化或导致闭锁,对深入探究卵泡发育的调控机制具有重要意义。     

Bilateral apex pubis distance：a novel index for follicular development and egg laying status in domestic hens (Gallus gallus domesticus)

ABSTRACT

1. Measures of the bilateral apex pubis (BAP) in White Leghorn (WLH) hens aged from 16 to 64 weeks (from the time ovaries were completely undeveloped to senility) were ranked into various groups so the relationship between external traits (BAP distance, comb length, comb height, wattle length, wattle height, body weight) and internal traits (largest ovarian follicle diameter (LFD) and oviduct weight and length) could be determined.

2. Using correlation and regression analysis with data from 16 weeks of age to the onset of lay, measurements of the apex pubis showed a strong correlation with LFD (r = 0.80, P < 0.01) and oviduct weight (r = 0.80, P < 0.01).

3. Multiple comparisons among WLH hens from 16 to 43 weeks of age showed that the BAP distance increased in all groups ranked by LFD or by laying period.

4. In the late laying period (>60 weeks of age), BAP distance showed a significant decrease (P < 0.01) in long-term non-laying hens compared to laying hens.

5. These findings suggest BAP distance can serve as an index for evaluating the onset and probable stages of follicular development during sexual maturation and/or aid in the identification and selection of non-laying hens during reproduction.     

Wnt10b基因的生理功能及其在绒毛周期性生长中的作用研究进展

绒山羊的绒毛生长具有周期性的特点,受多种因素的影响。Wnt10b基因作为Wnt家族的重要成员之一,在动物体脂肪的生成、骨骼的生成和某些疾病（如肿瘤）的产生、毛囊的生成,以及毛发基板的生长与保持活性状态等方面发挥着重要作用。对Wnt10b的来源、结构、生理功能、调控机制及其对毛囊周期性生长的影响进行综述,以期为Wnt10b促进绒毛周期性生长的作用机理研究提供参考。     

Comparison of bacterial endotoxin lipopolysaccharide concentrations in the blood,ovarian follicular fluid and uterine fluid: a clinical case of bovine metritis

We investigated the concentration of the bacterial endotoxin lipopolysaccharide (LPS) in the blood, ovarian follicular fluid and uterine fluid of a clinical case of bovine metritis. A 2-year-old lactating Holstein cow exhibited continuous fever >39.5°C for more than 2 weeks after normal calving. The cow produced a fetid, watery, red-brown uterine discharge from the vagina and was diagnosed with metritis. The LPS concentrations in plasma and uterine fluid were 0.94 and 6.34 endotoxin units (EU)/ml, respectively. One of seven follicles showed an extremely high level of LPS (12.40 EU/ml) compared to the other follicles (0.62–0.97 EU/ml). These results might suggest the presence of high concentration of LPS in follicles in cows with postpartum metritis.     

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.     

绵羊毛囊及其角蛋白研究进展

毛囊是毛纤维的生长基础,决定毛纤维的性状,角蛋白是毛囊组成的重要成分之一。近年来,对毛囊生长发育和角蛋白基因的挖掘等取得了一定的进展。文章回顾了绵羊角蛋白基因的发现,已经与人类角蛋白基因相当,但在数量和分类上存在差异;笔者介绍了绵羊毛囊的形态学结构,简述了毛囊角蛋白基因的表达定位和影响毛纤维性状的角蛋白,并分析了毛囊中的蛋白质组学技术的应用对毛囊生长发育和毛纤维性状的重要作用,以期为绵羊毛囊生长发育和羊毛性状的分子选育提供新的研究思路。     

人绒毛膜促性腺激素对母驴促排卵效果的研究

[目的]研究人绒毛膜促性腺激素（human chorionic gonadotropin,hCG）对母驴卵泡发育、排卵率、受胎率以及血清生殖激素水平的影响。[方法]选择优势卵泡直径在30~35 mm以及大于35 mm的母驴各30头,不同优势卵泡直径的母驴群体分别设置1个500 IU/头hCG处理组（n=10）、1个1 000 IU/头hCG处理组（n=10）、1个不接受hCG处理的对照组（n=10）。采用肌肉注射方法对各组母驴进行hCG处理。每隔24 h进行1次B超检查,观察各组母驴卵泡发育情况,测量卵泡直径;记录各组发生排卵的母驴数量,计算排卵率。对各组母驴进行人工输精,输精后第18天进行孕检,记录各组受胎母驴头数,计算各组受胎率。于hCG处理后0、24、48、72 h分别测定各组母驴血清中雌二醇（estradiol,E₂）和孕酮（progesterone,PROG）水平。[结果]2个群体母驴的卵泡直径随hCG注射剂量的增加而增大;优势卵泡直径大于35 mm的母驴群体中,肌肉注射hCG的2个组在处理后24 h内均出现排卵,而对照组母驴没有排卵;优势卵泡直径不同的2个母驴群体,在hCG处理48 h后排卵母驴数和排卵率与对照组相比均有所提高,其中,hCG处理后72 h,优势卵泡直径大于35 mm的母驴群体中,1 000 IU/头 hCG处理组的排卵率达到100%。2个母驴群体中,接受hCG处理的母驴,受胎率均高于对照,并且随hCG剂量的增加,受胎率有所提高;优势卵泡直径大于35 mm的母驴群体中,1 000 IU/头 hCG处理组的受胎率达到50%。2个母驴群体中,1 000 IU/头处理组在hCG处理后24 h的血清E₂浓度均较0 h时有较大幅度的提升,在0~72 h内血清PROG浓度的总体提升幅度较大。[结论]hCG处理可提高母驴的排卵率、受胎率以及血清中E₂和PROG水平,1 000 IU/头剂量的效果更好。     

不同日龄滩羊皮肤组织化学特征比较

为比较2、35日龄滩羊皮肤毛囊的发育特点与血管内皮生长因子（vascular endothelial growth factor，VEGF）和血管内皮生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）的分布特征，探究出生后滩羊被毛生长发育的变化特点，试验应用常规HE染色及改良Masson胶原纤维染色、Gomori银氨法染色、磷钨酸染色等特殊染色观察2与35日龄滩羊皮肤组织结构特点；应用免疫组织化学法结合免疫荧光染色法观察VEGF及VEGFR2在2与35日龄滩羊皮肤组织中的分布定位，并用IPP图像分析软件进行定量分析。结果显示：与2日龄滩羊皮肤组织比较，35日龄滩羊表皮与真皮间界限更加清晰，毛囊结构发育完整；毛囊密度显著降低（P<0.05）；胶原纤维与弹性纤维含量增加，形成网格状分布。免疫组化及免疫荧光结果显示，VEGF及VEGFR2在滩羊皮肤表皮及毛囊外根鞘、皮脂腺上均有表达。统计表明，VEGF及VEGFR2在2日龄滩羊皮肤组织中的表达量均显著高于35日龄（P<0.05）。综合上述结果，滩羊毛囊发育过程中，胶原纤维和弹性纤维增加明显；VEGF与VEGFR2通路在毛囊角质形成中起直接调节作用。     

Badger hair in shaving brushes comes from protected Eurasian badgers

The Eurasian badger (Meles meles) is included in Appendix III of the Bern Convention and protected by national laws in many European countries. Badger hair is used to manufacture luxury shaving brushes, although it is frequently argued that the hog badger (Arctonyx collaris), which in Europe is an introduced and unprotected species, is the origin of the hair used. We applied an extraction protocol to recover DNA from the unrooted hair of shaving brushes obtained from commercial companies in The Netherlands and Spain. The tested brushes originated from The Netherlands, Spain, France, Italy and the United Kingdom where the Eurasian badger is a protected species. We sequenced 191 bp of the mitochondrial DNA control region and 170 bp of the cytochrome b gene and compared the sequences obtained with Eurasian badger and hog badger reference sequences of the same mitochondrial DNA regions obtained in our laboratory and from GenBank. Sequences obtained from four shaving brushes were clearly hog badger sequences, whereas four sequences clustered with Eurasian badger sequences of both European and Asian origins. One of the shaving brushes made of Eurasian badger was produced in France, where it is legal to capture or trade the species under certain conditions. The remaining three brushes originated from The Netherlands, where it is illegal to possess, sell, transport or use for commercial purposes dead Eurasian badgers or products derived from them.