Junping He

Identification and characterization of microRNAs in white and brown alpaca skin

BackgroundMicroRNAs (miRNAs) are small, non-coding 21–25 nt RNA molecules that play an important role in regulating gene expression. Little is known about the expression profiles and functions of miRNAs in skin and their role in pigmentation. Alpacas have more than 22 natural coat colors, more than any other fiber producing species. To better understand the role of miRNAs in control of coat color we performed a comprehensive analysis of miRNA expression profiles in skin of white versus brown alpacas.ResultsTwo small RNA libraries from white alpaca (WA) and brown alpaca (BA) skin were sequenced with the aid of Illumina sequencing technology. 272 and 267 conserved miRNAs were obtained from the WA and BA skin libraries, respectively. Of these conserved miRNAs, 35 and 13 were more abundant in WA and BA skin, respectively. The targets of these miRNAs were predicted and grouped based on Gene Ontology and KEGG pathway analysis. Many predicted target genes for these miRNAs are involved in the melanogenesis pathway controlling pigmentation. In addition to the conserved miRNAs, we also obtained 22 potentially novel miRNAs from the WA and BA skin libraries.ConclusionThis study represents the first comprehensive survey of miRNAs expressed in skin of animals of different coat colors by deep sequencing analysis. We discovered a collection of miRNAs that are differentially expressed in WA and BA skin. The results suggest important potential functions of miRNAs in coat color regulation.

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.

Expression and localization of Smad2 and Smad4 proteins in the porcine ovary.

The objective of the present study was to investigate the temporal and spatial expression of Smad2 and Smad4 proteins, the downstream signaling molecules of the transforming growth factor beta (TGF-β) superfamily, in the porcine ovary. Cellular localization of Smad2 and Smad4 proteins was examined using immunohistochemistry. The specificity of the antibodies was examined using Western blot assay. Western blot analyses demonstrated that 52kDa Smad2 and 60kDa Smad4 proteins were expressed in the porcine ovary. Immunohistochemistry revealed that Smad2 and Smad4 were widely expressed in the porcine ovary, mainly localized in the oocyte, granulosa and thecal cells at different stages of folliculogenesis. Within the primordial and primary follicles, Smad2 and Smad4 showed strong staining in oocytes and follicular cells. In the antral follicle, strong staining was observed in oocytes, granulosa and theca cells. These findings suggest that Smad2 and Smad4 may be a key regulator of follicular development and growth of oocytes in the porcine ovary.

Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions

The aim of this study was to examine whether an elevated ambient temperature (37-40°C) had an effect on the apoptosis of germ cells and the expression of Bcl-2 and Bax in porcine testis. Six boars were used. Three boars were subjected to an elevated ambient temperature (37-40°C, 7days, 3h per day) as a heat stress (HS) group. The other 3 boars were kept in a room temperature house (20-27°C) as a control group. All boars were castrated and the testes were harvested. TUNEL assay was used for the detection of apoptotic cells. Immunohistochemistry, Western blotting and quantitative real-time PCR were used to analyze protein and mRNA levels of Bcl-2 and Bax in response to heat treatment. The results showed that apoptotic signals increased under heat stress conditions compared with the control (P<0.01), and the cell types most affected by heat treatment were spermatocytes and spermatids. In both the control and experimental groups, Bcl-2 was expressed in the cytoplasm and nucleus of spermatogonia, spermatocytes and differentiating spermatids and Bcl-2 preferentially localized close to the seminiferous tubules luminal surface in late spermatocytes and spermatids. Compared with the control group, the expression levels of Bcl-2 protein and mRNA significantly increased in heat treatment group, while the expression levels of Bax protein and mRNA did not show significant changes between the control and experimental group. Low to moderate Bax immunoreactivity staining was observed in all kinds of germ cells in the control group. Strong staining was observed in spermatogonia, and low to moderate Bax staining was observed in spermatocytes and spermatids. A redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed in the spermatogonia, spermatocytes and spermatids obtained in the heat treated group. These results showed that elevated ambient temperatures induced germ cell apoptosis. In response to heat stress, the expression of Bcl-2 increased and a redistribution of Bax from a cytoplasmic to a perinuclear or nuclear localization. This indicates that Bcl-2 and Bax may be involved in regulation of germ cell apoptosis induced by heat stress in boars.

Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors

Hepatocyte growth factor (HGF)/c-Met signaling has been considered as a key pathway in both melanocyte development and melanogenesis. To understand better the expression patterns and tissue distribution characterization of HGF and its receptor c-Met in skin of white versus brown alpaca (Vicugna pacos), we detected the tissue distribution of HGF and c-Met using immunohistochemistry and analyzed the expression patterns by using Western blot and quantitative real time PCR (qPCR). Immunohistochemistry analysis demonstrated that HGF staining robustly increased in the dermal papilla and mesenchymal cells of white alpaca skin compared with that of brown. However, c-Met staining showed strongly positive result, particularly inhair matrix and root sheath in brown alpaca skin. Western blot and qPCR results suggested that HGF and c-Met were expressed at significantly high levels in white and brown alpaca skins, respectively, and protein and transcripts possessed the same expression pattern in white and brown alpaca skins. The results suggested that HGF/c-Met signaling functions in alpaca coat color formation offer essential theoretical basis for further exploration of the role of HGF/c-Met signaling in pigment formation.

Immunolocalization of β-catenin and Lef-1 during postnatal hair follicle development in mice.

It is well recognized that the Wnt pathway, in which β-catenin and Lef-1 are important factors, is associated with many physiological processes, including embryogenesis and postnatal development. The Wnt pathway also plays a critical role in the development of skin. It regulates the formation of the dorsal dermis and epidermal appendages in the skin and the activity of epithelial stem cells. In this study, we investigated the presence and localization of β-catenin and Lef-1 in murine hair follicles through the first postnatal month, which encompasses the first hair cycle in mice, using Western blotting and immunohistochemistry. Our results show that β-catenin and Lef-1 are expressed during all stages in a hair cycle, most strongly in the anagen and weakly in the catagen and telogen phases. The results also suggest that the β-catenin-Lef-1 complex may regulate hair follicle cycling. This process will be of considerable interest to future studies.

Comprehensive circRNA expression profile and construction of circRNA-associated ceRNA network in fur skin

Circular RNA (circRNA), a class of non‐coding RNAs, is a new group of RNAs that are related to tumorigenesis. The role of circRNAs in various diseases has been already highlighted. However, the expression levels and functions of circRNAs related to the melanocytes in the skin are poorly understood. RNA sequence was performed to analyse the expression profiles of circRNAs in black fur skin and white fur skin during different differentiation stages and investigate the relevant metabolism mechanisms. Differentially expressed circRNAs were detected using empirical Bayes sequencing (EBSeq) and then verified through the quantitative real‐time PCR method. The EQSeq analysis of circRNAs identified 11 downregulated and 32 upregulated circRNAs in the embryo of black fur skin and white fur skin, as well as 21 downregulated and 17 upregulated circRNA in the postnatal stage. A circRNA–microRNA (miRNA)–messenger RNA (mRNA) network was established to predict the circRNA targets. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were applied to enrich the mRNA data further. Results showed that the specific mRNAs mainly involved in the transcription‐related GOs, especially GO:0042802, GO:0005080 and GO:0032403, demonstrate their specific actions in transcriptional regulation. In the circRNA–miRNA–mRNA network, the most enriched GO terms of the mRNAs were pigmentation, protein autophosphorylation and protein complex. Therefore, the circRNA–miRNA–mRNA pathway may reveal novel mechanisms for pigmentation, and circRNAs may serve as candidates in pigmentation.

Expression of glucose transporter 8 (GLUT8) in spermatogenesis of adult boar testes

The present study was designed to investigate the glucose transporter 8 (GLUT8) expression and localization in adult boar testis. Localization and expression of GLUT8 were conducted with Western Blotting, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) methods GLUT8 protein and mRNA were expressed in the boar testes. The results of Western Blotting analysis showed specificity of the antibody for protein of boar testes. The immunohistochemistry results showed that GLUT8 protein mainly localized in spermatocytes, round spermatids and elongated spermatids of the seminiferous tubules in the adult boar testes. And the GLUT8 expression persists during eight stages of boar spermatogenesis. GLUT8 may mainly provide glucose for the later stage of germ cell differentiation in adluminal compartment in adult boar testes. These results suggested that GLUT8 is important for the spermatogenesis in the adult boar testes.