Heat stress causes infertility in male rabbits in summer. This study was conducted to determine the effects of heat stress on semen quality and seminal plasma metabolites of male rabbits. To achieve these objectives, the temperature and humidity index (THI) was used to determine the stress state of male rabbits during different months, thereby the rabbits were divided into heat stress and no heat stress groups. The quality of the semen and the biochemical indices of seminal plasma were then analyzed. Next the plasma metabolites of rabbits in both groups were evaluated using the ultra-high performance liquid chromatography-mass spectroscopy (UPLC-MS)/MS technique. Our results showed that the THI value of the rabbit housing in May was 20.94 (no heat stress). The THI value of the housing in August was 29.10 (heat stress group, n = 10). Compared with the non-heat stress group, the sperm motility, density, and pH in the heat stress group (n = 10) were significantly decreased (P < 0.01); the semen volume decreased significantly (P < 0.05); and the sperm malformation rate increased significantly (P < 0.01). The number of grade A sperm significantly decreased, while the numbers of B and C grade sperm significantly increased (P < 0.01). The total sperm output (TSO), total motile sperm (TMS), and total functional sperm fraction (TFSF) decreased significantly (P < 0.01). Heat stress protein 70 (HSP70) and acid phosphatase (ACP) in the seminal plasma of rabbits in the heat stress group (n = 20) were significantly increased (P < 0.01). Seminal plasma testosterone (T), α-glucosidase (α-Glu), and fructose decreased significantly (P < 0.01). The concentrations of Mg2+ (P < 0.05), Na+ (P < 0.01), and K+ (P < 0.01) in metal ions were significantly decreased. These findings indicated that heat stress severely affected the quality of the male rabbit semen. Furthermore, UPLC-MS/MS technology was used to analyze the seminal plasma samples of rabbits in the heat stress group and non-heat stress group (n = 9 for each group). In total, 346 metabolites were identified, with variable importance in project (VIP) > 1.0, fold change (FC) > 1.5 or < 0.667, and P < 0.05 as the threshold. A total of 71 differential metabolites were matched, including stearic acid, betaine, arachidonic acid, L-malic acid, and indole. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differential metabolites revealed 51 metabolic pathways, including synthesis and degradation of ketones, serine and threonine metabolism, tryptophan metabolism, and the citric acid cycle. Our study has shown that the sperm motility, sperm pH value, and sperm density of male rabbits decreased significantly under heat stress, and the sperm malformation rate increased significantly. Furthermore, the quality of semen was shown to deteriorate and the energy metabolism pathway was disturbed. These findings provide a theoretical reference for alleviating the adaptive heat stress in male rabbits.

Melanocytes play a major role in the formation of mammalian fur color and are regulated by several genes. Despite playing the pivotal role in the study of melanoma, the mechanistic role of NRAS (neuroblastoma RAS viral oncogene homolog) in the formation of mammalian epidermal color is still elusive. First of all, the expression levels of NRAS mRNA and protein in the dorsal skin of different colored Rex rabbits were detected by qRT-PCR and Western blot. Then, the subcellular localization of NRAS was identified in melanocytes by indirect immunofluorescence. Next, the expression of NRAS was overexpressed and knocked down in melanocytes, and its efficiency was verified by qRT-PCR and Western blot. Subsequently, NaOH, CCK-8, and Annexin V-FITC were used to verify the changes in melanin content, proliferation, and apoptosis in melanocytes. Finally, we analyzed the regulation of NRAS on other genes (MITF, TYR, DCT, PMEL, and CREB) that affect melanin production. In silico studies showed NRAS as a stable and hydrophilic protein, and it is localized in the cytoplasm and nucleus of melanocytes. The mRNA and protein expression levels of NRAS were significantly different in skin of different colored Rex rabbits, and the highest level was found in black skin (P < 0.01). Moreover, the NRAS demonstrated impact on the proliferation, apoptosis, and melanin production of melanocytes (P < 0.05), and the strong correlation of NRAS with melanin-related genes was evidently observed (P < 0.05). Our results suggested that NRAS can be used as a gene that regulates melanin production and controls melanocyte proliferation and apoptosis, providing a new theoretical basis for studying the mechanism of mammalian fur color formation.

Dermal papilla cells (DPCs) are key regulators of hair follicle (HF) development and growth, which not only regulate HF growth and cycling but play a role in the pathogenesis of hair loss. The transcription factor Homeobox C13 (HOXC13) can modulate the growth and development of HFs. Nevertheless, the specific genes and pathways regulated by HOXC13 in DPCs have yet to be determined. Thus, to gain a better understanding of genomic binding sites involved in HOXC13-regulated HF development, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) was performed on rabbit DPCs with pcDNA3.1-3 × Flag-HOXC13 overexpression. A complete set of 9670 enrichment peaks was acquired by applying HOXC13-Flag ChIP. Subsequently, the peak sequence was annotated to the rabbit genome, revealing that 6.1 % of the peaks were identified within in the promoter region. Thereafter, five annotated genes were verified using RT-qPCR. The peak-associated genes were mainly enriched in signaling pathways related to HF development, such as MAPK and PI3K-Akt. Furthermore, by using a dual-luciferase reporter assay, we found that HOXC13 can target the protein kinase cAMP‑dependent catalytic β (PRKACB) promoter region (-1596 ∼ -1107 bp) and inhibit its transcription, which was consistent with data obtained from ChIP-seq analysis. Overexpression of PRKACB gene significantly modulated the expression of BCL2, WNT2, LEF1, and SFRP2 genes related to HF development as determined by RT-qPCR (P < 0.01, P < 0.05). The CCK-8 and flow cytometry assays showed that PRKACB significantly inhibited the proliferation of DPCs and promoted apoptosis (P < 0.01). In conclusion, our research revealed that PRKACB has the potential to serve as a novel target gene of HOXC13, contributing to the regulation of the proliferation and apoptosis of DPCs. The process of identifying global target genes can contribute to the understanding of the intricate pathways that HOXC13 regulates in the growth of HFs.

Mammalian hair formation is critically determined by the growth of hair follicles (HF). MiRNAs are crucial in the periodic development of hair follicles; they maintain epidermal homeostasis by targeting genes and influencing the activity of signaling pathways and related regulators. Our study discovered miR-129-5p to be overexpressed in the skin of Angora rabbits during catagen, and was negatively correlated with HOXC13 expression (Pearson’s R = −0.313, p < 0.05). The dual-Luciferase reporter gene detection system and Western blotting confirmed that miR-129-5p targeted HOXC13. In addition, miR-129-5p overexpression was found to significantly inhibit the expression of hair follicle development-related genes (HFDRGs), such as BCL2, WNT2, CCND1, and LEF1 (p < 0.01), and promoted the expression of SFRP2, TGF-β1, and FGF2 (p < 0.01), which was the same as the knockdown of HOXC13. In contrast, the knockout of miR-129-5p was the opposite, and it demonstrated similar results to the overexpression of HOXC13. CCK8 and flow cytometry demonstrated that miR-129-5p mimics significantly promoted the apoptosis of dermal papilla cells (DPCs) and inhibited proliferation (p < 0.01), while the inhibitor was found to reduce the apoptosis of DPCs and promote proliferation (p < 0.01). These results showed that miR-129-5p can participate in the periodic development of HF by targeting HOXC13, and it can induce apoptosis and inhibit proliferation of DPCs. These results will help to understand the role and mechanism of miR-129-5p in the periodic development of HF, and will provide support for subsequent studies, not only providing a theoretical basis for genetically improving the quality of hair in animals in the future, but also a new theory and method for diagnosing and treating hair loss in humans.

[目的]为了研究骨形态发生蛋白4(BMP4)对兔毛囊发育相关基因的影响.[方法]利用显微切割和二步酶消化相结合的方法分离了兔毛乳头细胞,并通过免疫荧光染色检测毛乳头细胞的特异性标记物α-SMA和VIM的表达情况.构建pcDNA3.1-BMP4过表达载体,转染至毛乳头细胞中,利用荧光定量PCR技术分析BMP4基因对毛囊发育相关基因(ID1、ID2、SAMD7、FGF1、TGF-β)mRNA表达的影响,通过CCK-8细胞增殖实验验证BMP4基因对兔毛乳头细胞生长的作用.[结果]免疫荧光结果显示,分离培养的细胞为毛囊真皮源性的毛乳头细胞.进一步成功克隆了家兔BMP4基因,发现其CDS序列全长为1230 bp,编码409个氨基酸,预测为亲水性外分泌蛋白,含有1个信号肽序列,无跨膜结构域.荧光定量PCR结果显示,过表达BMP4能显著升高ID1、ID2、SAMD7的mRNA表达水平(P＜0.01),但显著抑制FGF1的mRNA表达水平(P＜0.05).通过CCK-8细胞增殖实验发现,过表达BMP4基因极显著抑制毛乳头细胞增殖(P＜0.01).[结论]BMP4基因可能通过影响毛乳头细胞中毛囊发育相关基因的表达,抑制毛乳头细胞增殖,参与家兔毛囊发育过程.

Hair follicles (HFs) achieve hair growth and renewal by periodic regeneration. Therefore, exploring the key factors affecting hair growth in rabbits is of great significance for precisely breeding Angora rabbits and improving the competitiveness of the rabbit industry. Based on the results of our previous studies, lncRNA2690 was differentially expressed in the HF cycle using lncRNA-Seq, and the full-length sequence was annotated by bioinformatics analysis. The lncRNA2690 is 363 nt long and is found on chromosome 14 from 163 321 514 to 163 321 872. The lncRNA2690 was predicted to not have the coding ability through open reading frame and CPC2, and the nuclear-cytoplasmic separation experiment showed the lncRNA2690 to be highly expressed in the nucleus (p < 0.01). The expression pattern of lncRNA2690 was further analyzed in the different HF development stages of Angora rabbits using quantitative real-time PCR. The results showed that lncRNA2690 was periodically expressed in HF development, and the expression level was found to be high in the HF resting phases. The overexpression and knockdown of lncRNA2690 were found to significantly upregulate and downregulate the expression of the genes WNT2, CCND1, BMP2, LEF1, and SIAH1 in the rabbit dermal papilla cells (p < 0.01), promoting cell apoptosis and inhibiting cell proliferation (p < 0.01). This indicated that lncRNA2690 negatively regulates the periodic regeneration of the HFs in rabbits. These results provide a basis for the further study of lncRNA2690 in the HF growth cycle of Angora rabbits.

为建立新西兰白兔毛囊体外培养模型,探究其最适的培养环境,本实验选用2月龄新西兰白兔,采集触须毛囊,分离组织和脂肪,保留完整毛囊.将完整无损的毛囊放入24孔板,每孔1根,加入William's E、DMEM、MEM3种不同培养基,每组6个重复,每2 d在倒置显微镜下观察毛囊毛干的生长,并利用HE和PCNA免疫荧光染色检测毛囊的形态和增殖能力.结果显示:3种培养基中毛囊毛干在体外培养4 d后生长减缓,在2 d与4 d时的生长速度均大于6 d(P<0.05).其中,William's E组生长速度高于MEM组(P<0.05),且William's E组生长最快.4 d时,HE染色发现William's E组毛囊结构完整,毛乳头清晰,而此时DMEM和MEM组毛乳头已经消失.进一步用PCNA免疫荧光染色,发现Williams'E组PCNA在毛乳头阳性表达,DMEM和MEM组毛囊表达仅表达在外根鞘.因此,William's E培养基最适宜新西兰白兔触须毛囊生长,毛乳头增殖能力最强.

同源盒C13(Hoxc13)基因与毛囊发育和毛发性状的形成密切相关.在哺乳动物毛囊的周期性发育过程中,Hoxc13通过调控角蛋白关联蛋白(KAP)基因等来控制毛囊在生长期、退行期和休止期之间转换,进而直接控制毛发性状的形成和改变,对皮毛动物产业发展具有重要的经济意义.本文围绕Hoxc13的结构特点、生物学功能以及作用机制进行阐述,对该基因的研究前景进行展望,以期为阐明哺乳动物毛囊发育调控机制提供依据.

胱氨酸/谷氨酸转运蛋白(Solute carrier family7 member11,Slc7a11)基因是新发现决定黑色素沉积的关键基因之一,该基因可调控黑色素在动物体内的转换来决定色素沉积的比例,对动物毛色形成有重要影响.目前,已知Slc7a11基因的编码序列较为保守,主要对棕色毛发的形成具有调控作用.本文综述了Slc7a11基因的来源、功能和结构,以及其在哺乳动物毛色形成中的作用机制,为哺乳动物毛色生成的研究提供理论参考.

本研究通过比较不同培养条件下兔卵巢颗粒细胞的生长状态、增殖情况,了解兔卵巢颗粒细胞体外生长增殖的特性,优化兔卵巢颗粒细胞体外培养体系.选用2只4～6月龄新西兰白兔母兔肌肉注射孕马血清促性腺激素并于6 h后处死,采集卵巢颗粒细胞,在体外培养至贴壁后利用卵泡刺激素受体免疫荧光染色鉴定兔颗粒细胞.进一步分别在含5％、10％、15％血清的DMEM培养基和DMEM/F-12培养基中培养颗粒细胞,观察细胞生长状况,并使用CCK-8法测定细胞在不同浓度血清及培养基条件下的增殖状况.结果显示:随着血清浓度的提高,测得的OD值不断增加并在15％浓度下趋于平稳,表明在一定程度上增加血清浓度有助于颗粒细胞增殖;兔卵巢颗粒细胞在DMEM/F-12培养基中的增殖效果优于DMEM培养基.因此,兔卵巢颗粒细胞在含有15％血清的DMEM/F-12培养基中增殖状况最好.