Effects of testosterone on skin structure and factors related to androgen conversion and binding in Hetian sheep.

The effects of androgens on human skin mainly include the regulation of growth and differentiation of hair follicles and sebaceous glands. Androgens may have some physiological roles in sheep skin that are similar to those of humans, but further confirmation is needed. Therefore, Hetian sheep were chosen in this study as an animal model to explore the effects of testosterone on skin structure and factors related to androgen conversion and binding in Hetian sheep. The sheep were treated with different concentrations of testosterone for 42 days. Skin tissue sections were prepared and then subjected to hematoxylin-eosin, Sacpic, Masson's trichrome, and Oil Red O staining to observe changes in skin morphology. Changes in the content of blood-related factors were also detected using ELISA kits. The skin tissue distribution of androgen receptor was explored by immunohistochemistry and immunofluorescence assays. The results showed that testosterone significantly increases the sebaceous gland area and stimulates the formation of new sebaceous glands. Further exploration revealed that testosterone promotes the proliferation of sebaceous gland juvenile cells. However, testosterone was found to have no significant effect on hair follicle density and hair follicle structure. Testosterone increased dihydrotestosterone levels but decreased 5α-reductase 1 and 5α-reductase 2 levels. The androgen receptors were distributed in the hair follicles, sebaceous glands, and some major skin appendages of Hetian sheep. This study suggests that androgens can be effective in regulating sebum production in sheep. This study will help advance research efforts to further explore the molecular and cellular mechanisms by which androgens modify sheep follicles and sebaceous glands.

Effects of different concentrations of androgens on KAP24.1 gene expression in Hetian sheep and Karakul sheep.

The purpose of the experiment was to clone and eukaryotic expression of hair follicle keratin associated protein 24.1 (KAP24.1), study the effect of different concentrations of androgen on protein expression, and compare KAP24.1 gene in skin and hair follicles of different breeds of sheep expression, explore KAP24.1 Expression difference of gene among local sheep breeds in southern Xinjiang and its effect on wool quality. The body-side hair follicles of Plain-type Hetian sheep, Mountain-type Hetian sheep and Karakul sheep were used as experimental materials, and the KAP24.1 gene sequence of sheep in GenBank (accession number: JX112014.1) was used as the reference to design primers. The KAP24.1 gene was amplified by PCR, and the pMD19-T-KAP24.1 cloning plasmid was constructed. After double digestion and identification, the pEGFP-N1-KAP24.1 eukaryotic recombinant expression plasmid was constructed. After PCR and double digestion and identification, sequencing and sequence analysis were performed, and the expression was transfected into Hela cells. SDS-PAGE and Western blotting were used to detect the expression levels of androgen at different concentrations. The expression of KAP24.1 gene in different sheep skin follicles was detected by real-time fluorescent quantitative PCR. Three sheep KAP24.1 were cloned The CDS region sequence of gene is 759 bp, encoding 252 amino acids, all of which are unstable hydrophobic proteins.The results of similarity comparison showed that compared with the reference gene, the gene sequence similarity of Mountain-type Hetian sheep and Karakul Sheep was 99.47%, and that of Plain-type Hetian sheep was 99.34%. Phylogenetic tree analysis showed that the three sheep had the closest genetic relationship with Capra hircus and the furthest genetic relationship with Cervus canadensis.The secondary structure of KAP24.1 was mainly composed of random coil.PEGFP-N1-KAP24.1 was successfully constructed eukaryotic recombinant expression plasmid was successfully transfected into HeLa cells to obtain 58 kDa KAP24.1 recombinant protein. When the concentration of androgen is 10-8 mol / L, the protein expression is the highest. The expression of KAP24.1 gene in skin and hair follicles of Mountain-type Hetian sheep was significantly different from that of plain-type Hetian sheep (P < 0.05), and there was significant difference between Mountain-type Hetian sheep and Karakul Sheep (P < 0.05). The expression of Karakul Sheep was significantly higher than that of Plain-type Hetian sheep (P < 0.05). The 759 bp CDS sequence of KAP24.1 gene in sheep was cloned, and PEGFP-N1-KAP24.1 was constructed eukaryotic recombinant expression plasmid to obtain 58 kDa KAP24.1 recombinant protein. When the concentration of androgen was 10-8mol / L, the protein expression was the highest, and KAP24.1 gene was expressed in the skin and hair follicles of three sheep breeds, and the expression of Mountain-type Hetian sheep was the highest.

Identification of Signatures of Selection for Litter Size and Pubertal Initiation in Two Sheep Populations.

Fecundity is an important economic trait in sheep that directly affects their economic and productive efficiency. Our study aimed to identify SNP loci associated with sheep puberty or litter size which could be used in future breeding programs to improve fertility. Genomic DNA was obtained from Hetian and Cele Black sheep breeds and used for reduced-representation genome sequencing to identify SNP loci associated with pubertal initiation and litter size. Selective signatures analysis was performed based on the fixation index and nucleotide diversity, followed by pathway analysis of the genes contained in the selected regions. The selected SNP loci in the genes associated with pubertal initiation and litter size were validated using both sheep breeds. In total, 384,718 high quality SNPs were obtained and 376 genes were selected. Functional annotation of genes and enrichment analysis identified 12 genes associated with pubertal initiation and 11 genes associated with litter size. SNP locus validation showed that two SNP on PAK1 and four on ADCY1 may be associated with pubertal initiation, and one SNP on GNAQ gene (NC_040253.1: g.62677376G > A) was associated with litter size in Cele Black sheep. Our results provide new theoretical support for sheep breeding.

The effect of androgen on wool follicles and keratin production in Hetian sheep / Efeito do androgênio na produção de folículos e queratina em ovinos Hetianos

Abstract To investigate the optimal androgen concentration for culturing Hetian sheep wool follicle and to detect effects of androgen concentration on wool follicle cell proliferation and apoptosis using immunofluorescence labeling and real-time quantitative fluorescence determinations of wool keratin-associated protein gene expression levels. Wool follicles were isolated by microdissection and wool follicles and skin pieces were cultured in various concentrations of dihydrotestosterone (DHT) in culture medium. Next, daily lengthwise growth measurements of wool follicles were obtained using a microscopic micrometer. Cultured Hetian wool follicles were stained using the SACPIC method to reveal wool follicle structure, while sheep skin slices were used to observe cell proliferation by immunostaining and cell apoptosis using the TUNEL method. At the molecular biological level, keratin-associated protein (Kap) gene expression was studied using wool follicles cultured for various numbers of days in vitro. Effects of androgen concentrations on Hetian wool follicle growth and development were experimentally studied. EdU proliferation assays revealed that androgen promoted cell proliferation within wool follicle dermal papillae. TUNEL apoptosis detection demonstrated that androgen treatment could delay cell apoptosis. Quantitative reverse transcription polymerase chain reaction (qPCR) results demonstrated that gene expression level patterns of Hetian mountain sheep super-high sulfur protein. Kap1.1, KIF1.2, Kap2.12 and Kap4.2 gene expression level of the mountainous experimental group was significantly higher than plains Hetian sheep. An androgen concentration of 100 nM can promote the growth of Hetian wool follicle cells in vitro, resulting in overexpression of some genes of the Kap family.

Resumo Investigar a concentração ideal de andrógenos em cultura de folículos pilosos de carneiro Hetiano e detectar os efeitos da concentração de andrógenos na proliferação e apoptose de células foliculares, por meio de imunofluorescência e de determinação quantitativa, em tempo real, da fluorescência dos níveis de expressão gênica de proteína associada à queratina. Folículos pilosos foram isolados por microdissecção, e folículos de lã e pedaços de pele foram cultivados em várias concentrações de di-hidrotestosterona (DHT) em meio de cultura. Em seguida, medições diárias de crescimento longitudinal dos folículos capilares foram obtidas usando um micrômetro microscópico. Folículos de lã cultivados de Hetianos foram corados pelo método SACPIC para revelar a estrutura do folículo piloso, enquanto fatias de pele de carneiro foram usadas para observar a proliferação celular por imunocoloração e apoptose celular por meio do método TUNEL. Em âmbito da biologia molecular, a expressão gênica da proteína associada à queratina (Kap) foi estudada usando folículos capilares cultivados por vários dias, in vitro. Os efeitos das concentrações de andrógenos no crescimento e desenvolvimento dos folículos de lã de Hetianos foram estudados experimentalmente. Ensaios de proliferação de EdU revelaram que o andrógeno promoveu a proliferação celular dentro das papilas dérmicas do folículo piloso. A detecção de apoptose por TUNEL demonstrou que o tratamento com andrógeno poderia atrasar a apoptose celular. Os resultados da reação em cadeia da polimerase transcrição reversa quantitativa (qPCR) demonstraram que os padrões de expressão gênica da proteína de enxofre Kap1.1, KIF1.2, Kap2.12 e Kap4.2 foram significativamente maiores no grupo de ovinos Hetianos de montanha. Uma concentração de androgênio de 100 nM pode promover o crescimento de células foliculares de lã de Hetianos in vitro, resultando na superexpressão de alguns genes da família Kap.