Skin proteomic screening and functional analysis of differential proteins associated with coat color in sheep (Ovis aries).

Objective: Coat color is an important characteristic and economic trait in domestic sheep. In this study, we explored the potential mechanisms and the signaling pathways involved in coat color regulation for sheep. Methods: Isobaric tags for relative and absolute quantification (iTRAQ) technology was used to catalog global protein expression profiles in skin of sheep with black versus white coat color. Immunofluorescence was used to observe the expression localization of differential protein. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used to evaluate their role in the coat color formation of sheep. Results: A total of 136 differential proteins were obtained in different coat colors, including 101 up-regulated and 35 down-regulated. Pigmentation function entries were enriched through GO annotation. Tyrosine metabolism and platelet activation signaling pathway were extracted by KEGG analysis. APOA1 (Apolipoprotein A-1) and FGA (Fibrinogen alpha chain) were found to be critical differential proteins by the interaction of differential proteins in the direct-interaction network diagram. Strikingly, twenty candidate differential proteins were screened, from which ACTB (Beta-actin) protein showed higher expression in white sheep skin, while ALB (albumin), APOA1 MAOA (Amine oxidase) and FGA proteins showed higher expression in black sheep skin, which validated by immunofluorescence, western blot and qRT-PCR. Conclusion: Our studies identified several novel proteins that may involved in the coat color formation of sheep. The white and black sheep skin proteome profiles obtained provide a valuable resource for future research to understand the network of protein expression controlling skin physiology and melanogenesis in sheep.

Platycodin D ameliorates ammonia-induced pulmonary fibrosis by repressing TGF-ß1-mediated extracellular matrix remodeling.

Ammonia can induce pulmonary fibrosis in humans and animals. Platycodin D (PLD) possesses various bioactive activities including anti-fibrotic properties. In this study, we aimed to explore the activity and mechanism of PLD in pulmonary fibrosis induced by ammonia. The mouse model of ammonia-induced lung fibrosis was established, and the role of PLD was assessed by H&E and Masson's trichrome staining. The differentially expressed genes (DEGs) were identified by RNA-seq and subjected to GO and KEGG pathway analyses. BEAS-2B cells were treated with NH4 Cl alone or along with PLD. Results showed that PLD attenuated ammonia-induced pulmonary inflammation and fibrosis in vivo. The extracellular matrix (ECM)-receptor interaction pathway was predicted as a prominent pathway underlying the anti-fibrotic function of PLD. In ammonia-induced mouse models and NH4 Cl-treated BEAS-2B cells, PLD could repress the activation of the TGF-ß1 pathway. By incubating lung fibroblast HFL1 cells with the conditioned medium of BEAS-2B cells treated with NH4Cl alone or along with PLD, PLD was confirmed to attenuate NH4 Cl-induced ECM deposition in HFL1 cells. Our findings demonstrate that PLD exerts a protective function in ammonia-induced pulmonary fibrosis by repressing TGF-ß1-mediated ECM remodeling, suggesting the potential therapeutic value of PLD in this disease.

Protective effects of puerarin on liver tissue in Salmonella-infected chicks: a proteomic analysis.

Salmonella enterica is a zoonotic bacterium that not only causes serious economic losses to the livestock and poultry industries but also seriously endangers human health. Long-term indiscriminate use of antibiotics has led to drug resistance in Salmonella, and thus the identification of alternatives to antibiotics is crucial. In this study, the effects of puerarin on the S. enterica-infected chickens were investigated. A total of 360 chicks were randomly assigned as the control group (CON), the S. enterica group (S), and puerarin-treatment group (P). Chicks in the P group were fed the basal diet supplemented with 50 (P50), 100 (P100), 200 (P200), and 400 (P400) mg/kg puerarin, respectively. It was found that puerarin treatment markedly altered the serum activities of aspartate aminotransferase (AST), alanine transaminase (ALT), and superoxide dismutase (SOD), together with the malondialdehyde (MDA) and total antioxidant capacity (T-AOC) contents in the serum. The mRNA expression of IL-6, IL-1ß, TNF-α, Bcl-2, and caspase-8 in the livers of S. enterica-infected chicks was increased after infection but significantly reduced after treatment with puerarin. Histologic analysis showed that puerarin effectively mitigated morphological damage in the liver caused by S. enterica. Proteomic analysis revealed that S. enterica infection led to metabolic disorders in the liver, resulting in oxidative stress, increased inflammation, and significantly elevated levels of hepatocellular carcinoma biomarkers. The findings of the filtered sequencing were verified by using quantitative PCR (qPCR). Treatment with 100 mg/mL puerarin thus effectively alleviated disordered liver metabolism, reduced inflammation and oxidative damage and significantly reduced the levels of hepatocellular carcinoma biomarkers in the liver. The results suggest that puerarin has the potential to replace antibiotics to control Salmonella infection in poultry and thus improve food safety.

Metabolomic and microbiome analysis of the protective effects of Puerarin against Salmonella Enteritidis Infection in chicks.

BACKGROUND: Salmonella Enteritidis is a zoonotic pathogen and poses a substantial risk to human health, as well as significant financial losses to the livestock and poultry industries. It is currently urgent to identify alternatives to antibiotic treatment. RESULTS: In this study, we explored the influence of Puerarin on the immunological response, intestinal flora, serum metabolome, and growth performance of chicks infected with Salmonella Enteritidis. Chicks were weighed at specific time points and the average daily gain (ADG) was calculated. Serum, intestinal, and cecal content samples were collected on days 10 and 17. The results showed that 100 mg/kg of Puerarin significantly suppressed inflammation and enhanced immune function. Metabolomic analysis showed significant differences in serum metabolites after Puerarin treatment and suggested that Puerarin may regulate abnormal amino acid and lipid metabolism after Salmonella Enteritidis infection through the autophagic and ABC transporter pathways. In addition, Puerarin suppressed Salmonella Enteritidis-induced intestinal flora dysbiosis through modulation of the microbial community structures (increased Lactobacillus, Faecalibacterium, and Subdoligranulum), as demonstrated by 16S rRNA analysis. CONCLUSIONS: In conclusion, Puerarin can improve growth performance in chicks, suppress the inflammatory response in vivo, enhance immunity, and regulate lipid and amino acid metabolism and the intestinal flora.

TMT-based quantitative proteomics reveals the targets of andrographolide on LPS-induced liver injury.

BACKGROUND: Andrographolide (Andro) is a diterpenoid derived from Andrographis paniculate, which has anti-inflammatory, antibacterial, antiviral and hepatoprotective activities. Gram-negative bacterial infections can cause varying degrees of liver injury in chickens, although Andro has been shown to have a protective effect on the liver, its underlying mechanism of action and effects on liver proteins are not known. METHODS: The toxicity of Andro on the viability of leghorn male hepatoma (LMH) cells at different concentrations and times was analyzed by CCK-8 assays. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the culture supernatants were measured using an automatic biochemical analyzer to evaluate the protective effect of androscopolide on LPS-induced injury of LMH cells. Subsequently, TMT proteomics analysis were performed on the negative control group (NC group), LPS, and LPS-Andro groups, and bioinformatics analysis was performed on the differentially expressed proteins (DEPs). RESULTS: It was found that Andro reduced ALT and AST levels in the cell supernatant and alleviated LPS-induced injury in LMH cells. Proteomic analysis identified 50 and 166 differentially expressed proteins in the LPS vs. NC group and LPS-Andro vs. LPS group, respectively. Andro may be involved in steroid metabolic processes, negative regulation of MAPK cascade, oxidative stress, and other processes to protect against LPS-induced liver injury. CONCLUSIONS: Andro protects against LPS-induced liver injury, HMGCS1, HMGCR, FDPS, PBK, CAV1, PRDX1, PRDX4, and PRDX6, which were identified by differential proteomics, may be the targets of Andro. Our study may provide new theoretical support for Andro protection against liver injury.

Effect of overexpression of KLF4 on the growth and development of hair follicles in mice.

Hair follicle growth is cyclical, and hair cycle dysfunction can lead to hair follicle-related disorders, including alopecia and hirsutism. The objective was to investigate the influence and underlying mechanism of Krüppel-like factor 4 (KLF4) overexpression on hair follicle growth and development in C57BL/6 mice. To provide a theoretical basis for the biological functions of KLF4 gene in hair follicle development and hair follicle cycle, mice were assigned to three groups: experimental, overexpressing KLF4 (Ad-KLF4); control, expressing green fluorescent protein (Ad-NC); and blank, no treatment. Fur was removed from the dorsal surface, and the mice were intradermally injected with 25 µL 1 × 1010 PFU/mL adenovirus vector (Ad-KLF4 or Ad-NC) at three points. Samples were collected for molecular biological and histological analysis. It was found that mRNA and protein levels of Wnt pathway-associated factors ß-catenin, LEF1, hair follicle cell proliferation-related factor Ki67, and hair follicle inner caledrin marker AE15 were all significantly greater in the Ad-NC and blank groups than in Ad-KLF4 mice (P < 0.01). These findings were confirmed by immunohistochemical analysis. Hair growth was monitored photographically for 14 days, showing an absence of growth in the injected region of the KLF4-overexpressing mice in contrast to non-overexpressing areas where hair growth was normal. HE staining showed that hair follicles in the blank and Ad-NC mice were normal, while those in the KLF4-overexpressing areas remained in telogen or early anagen with spherical dermal papillae situated at the edge of the dermis and subcutaneous tissue without an inner heel sheath. In conclusion, it was found that KLF4 downregulated key Wnt/ß-catenin-associated factors during follicular regeneration in mice, reducing both follicular development and growth.

Expression and tissue distribution analysis of vimentin and transthyretin proteins associated with coat colors in sheep (Ovis aries).

OBJECTIVE: Pigment production and distribution are controlled through multiple proteins, resulting in different coat color phenotypes of sheep. METHODS: The expression distribution of vimentin (VIM) and transthyretin (TTR) in white and black sheep skins was detected by liquid chromatography-electrospray ionization tandem MS (LC-ESI-MS/MS), gene ontology (GO) statistics, immunohistochemistry, Western blot, and quantitative real time polymerase chain reaction (qRT-PCR) to evaluate their role in the coat color formation of sheep. RESULTS: LC-ESI-MS/MS results showed VIM and TTR proteins in white and black skin tissues of sheep. Meanwhile, GO functional annotation analysis suggested that VIM and TTR proteins were mainly concentrated in cellular components and biological process, respectively. Further research confirmed that VIM and TTR proteins were expressed at significantly higher levels in black sheep skins than in white sheep skins by Western blot, respectively. Immunohistochemistry notably detected VIM and TTR in hair follicle, dermal papilla, and outer root sheath of white and black sheep skins. qRT-PCR results also revealed that the expression of VIM and TTR mRNAs was higher in black sheep skins than in white sheep skins. CONCLUSION: The expression of VIM and TTR were higher in black sheep skins than in white sheep skins and the transcription and translation were unanimous in this study. VIM and TTR proteins were expressed in hair follicles of white and black sheep skins. These results suggested that VIM and TTR were involved in the coat color formation of sheep.

Effects of MITF on marker protein expression of multivesicular bodies and miRNA omics of extracellular vesicles of mice melanocyte cell line.

Extracellular vesicles (EVs) are heterogeneous membrane-bound complexes of cell-derived and nanosized structures originating from the endosomal system and subsequently released from the plasma membrane. EVs contribute significantly to intercellular communication and are involved in pigmentation processes that rely on tight communication between keratinocytes and melanocytes in the epidermis. Microphthalmia-associated transcription factor (MITF) induces melanogenesis and modulates the expression factors involved in melanosome biogenesis, maturation and dispersal in melanocytes. Here, we evaluated the effects of MITF on the fate of multivesicular bodies and the biogenesis of extracellular vesicles of melanocytes. It was found that MITF increased the expression of subunits of the endosomal sorting complex, required for transport (ESCRT), including VPS37, VPS36B, and tetraspanin CD81, which are key mediators of multivesicular body biogenesis. Over 110 miRNAs, including miR-211-5p, miR-335-5p, let-7g-5p and miR-28a-3p, were differentially expressed in melanocyte-derived EVs after overexpression of MITF in melanocytes. These miRNAs have been reported to be key regulators of plasma protein binding, changes in the cell membrane system and transferase activity. These results suggest that while enhancing melanogenesis, melanocytes may mediate intercellular communication with surrounding cells by serving as EV delivery vehicles.

Host Cell Receptors Implicated in the Cellular Tropism of BVDV.

Bovine viral diarrhea virus (BVDV) is one of the most hazardous viruses, which causes huge economic losses in the cattle industry around the world. In recent years, there has been a continuous increase in the diversity of pestivirus worldwide. As a member of the genus Pestivirus in the Flaviviridae family, BVDV has a wide range of host animals including cattle, goat, sheep, pig, camel and other cloven-hoofed animals, and it has multi-tissue tropism as well. The recognition of their permissive cells by viruses via interaction with the cellular receptors is a prerequisite for successful infection. So far, little is known about the cellular receptors essential for BVDV entry and their detailed functions during BVDV infection. Thus, discovery of the cellular receptors involved in the entry of BVDV and other pestiviruses is significant for development of the novel intervention. The viral envelope glycoprotein Erns and E2 are crucial determinants of the cellular tropism of BVDV. The cellular proteins bound with Erns and E2 potentially participate in BVDV entry, and their abundance might determine the cellular tropism of BVDV. Here, we summarize current knowledge regarding the cellular molecules have been described for BVDV entry, such as, complement regulatory protein 46 (CD46), heparan sulfate (HS), the low-density lipoprotein (LDL) receptor, and a disintegrin and metalloproteinase 17 (ADAM17). Furthermore, we focus on their implications of the recently identified cellular receptors for pestiviruses in BVDV life cycle. This knowledge provides a theoretical basis for BVDV prevention and treatment by targeting the cellular receptors essential for BVDV infection.

The role of KLF4 in melanogenesis and homeostasis in sheep melanocytes.

KLF4 expression has been associated with hair color in mammals and has also been found to regulate melanoma cell growth. Here, we assessed the influence of KLF4 on coat color formation and melanocytes. We found that KLF4 was highly expressed in the black skin of sheep both at the mRNA and protein levels compared with white skin. KLF4 immunostaining further showed that KLF4 protein was mainly expressed in epidermal, outer root, and hair bulb regions. In sheep melanocytes, the proliferation of melanocytes was inhibited by KLF4 overexpression and this decrease in cell proliferation was coupled with induction of the S phase, cell cycle arrest, and apoptosis. In vitro cell migration assays showed that KLF4 suppressed cell migration. In addition, KLF4 overexpression significantly increased melanin production and pigment-related gene expression. Collectively, our findings show that KLF4 is important for coat color formation and melanocyte homeostasis.

Expression and distribution of bone morphogenetic protein 4 and its antagonist Noggin in the skin of Kazakh sheep (Ovis aries) with a white and brown coat color.

The natural coat color is an important trait of vertebrate animals. For example, the coat color can help avoid harm to human beings caused by chemical dyeing, and it has economic significance for domestic animals. The bone morphogenetic protein 4 (BMP4) and its antagonist Noggin can regulate pigmentation and the generation of coat color in mice; thus, they may also regulate the coat color of Kazakh sheep. To gain mechanistic insight into this possibility, we determined the relative expression levels of BMP4 and Noggin in the skin of white and brown Kazakh sheep by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis. The localization of BMP4 and Noggin were detected by immunohistochemistry (IHC). The results of qPCR and western blot analysis demonstrated that the relative expression levels of BMP4 and Noggin in the skin of brown Kazakh sheep were significantly higher than those in white Kazakh sheep. Our IHC results showed that the BMP4 protein was expressed in the epidermis and root sheath of the Kazakh sheep skin. The Noggin protein was expressed in the epidermis, root sheath, hair shaft, and dermal papilla of the Kazakh sheep skin. These results provide a theoretical basis for additional studies regarding the association and mechanism of BMP4 and Noggin in coat-color formation in Kazakh sheep. These results may provide new methods for developing treatment strategies for pigmentation disorders and diseases.

Bacillus amyloliquefaciens BLCC1-0238 Can Effectively Improve Laying Performance and Egg Quality Via Enhancing Immunity and Regulating Reproductive Hormones of Laying Hens.

In this study, we sought to evaluate the effects of dietary Bacillus amyloliquefaciens (B. amyloliquefaciens) BLCC1-0238 supplementation on laying performance, egg quality, antioxidant enzyme activities, reproductive hormone, and immunity of laying hens. A total of 240 Hy-Line Brown laying hens (28 weeks old) were randomly divided into four groups, and three replicates per group (n = 20 per replicate). The control group was fed a standard basal diet, and the three treatment groups were provided the basal diet supplemented with either 0.01%, 0.03%, or 0.06% B. amyloliquefaciens BLCC1-0238 (2 × 1010 CFU/g), respectively. Hens were allowed 2 weeks to acclimate prior to initiation of the 8-week experiment. It was observed that dietary supplementation with 0.01% or 0.03% B. amyloliquefaciens BLCC1-0238 significantly increased egg production and egg mass. However, no significant differences in feed intake, egg weight, and feed conversion ratio among the four groups were observed. Different levels of B. amyloliquefaciens BLCC1-0238 supplementation also significantly increased egg shell strength and thickness. With respect to the levels of reproductive hormones in the hens, B. amyloliquefaciens BLCC1-0238 supplementation significantly reduced serum adrenal cortical hormone (ACTH) levels, while increasing estradiol (E2) and follicle-stimulating hormone (FSH) secretion in the treatment groups compared to the control group. Relative to the control group, supplementation with 0.03% and 0.06% B. amyloliquefaciens BLCC1-0238 was observed to significantly increase serum glutathione S-transferase (GST) concentration, and supplementation significantly reduced serum IL-1 and IL-6 levels, whereas IL-4 levels increased for all concentrations tested. In conclusion, supplementation of a basal chicken diet with B. amyloliquefaciens BLCC1-0238 can improve laying performance and egg quality through the reduction of stress responses, up-regulation of growth hormones, and supporting immunity in laying hens.

Expression and tissue distribution analysis of Angiotensin II in sheep (Ovis aries) skins associated with white and black coat colors.

Angiotensin II (AngII) regulates pigment synthesis by tyrosinase in melanocytes. To evaluate the association between AngII and coat color formation, we detected the expression distribution of AngII in white and black sheep skins by LC-ESI-MS/MS, western blot, quantitative real-time-PCR (qPCR) and distribution of AngII by immunohistochemistry.Liquid chromatography-electrospray ionization tandem MS (LC-ESI-MS/MS) results showed that AngII was found in white and black skin tissues of sheep. Western blot results verified the LC-ESI-MS/MS results and suggested that AngII was expressed at significantly higher levels in black sheep skins compared with the white sheep skins. Quantitative real time PCR (qRT-PCR) results also revealed that the expression level of AngII mRNA was higher in black sheep skins than that in white sheep skins. Immunohistochemical analysis further demonstrated that AngII protein was localized in the hair bulb and outer root sheath of hair follicle in sheep. In summary, protein and transcripts exhibited the same expression pattern in white and black sheep skins. Furthermore, the expressions of AngII in the hair bulb and outer root sheath of black sheep were stronger than those in white sheep. These results suggested that AngII functions in sheep coat color regulation and offer a novel insight for further investigation on the role of AngII in the coat color formation in sheep.

A small-scale study on airborne transmission of H9N2 avian influenza virus under field conditions.

INTRODUCTION: H9N2 avian influenza viruses (AIV) can transmit in chicken flocks through direct contact and aerosols. Nevertheless, data on airborne transmission of AIV is very limited, especially under field conditions. To fill this literature gap, this study was designed to investigate airborne transmission of H9N2 AIV originating from infected chicken flocks under field conditions, with the aim to further characterize the airborne transmission of H9N2 AIV. METHODOLOGY: Oropharyngeal swabs were collected from different diseased chickens to confirm H9N2 AIV infection. All glass impingers 30 (AGI-30) were used to collect indoor, upwind and downwind air samples for three chicken houses with H9N2 AIV infected chickens. Swabs and air samples were tested for H9N2 AIV using a real-time reverse transcription polymerase chain reaction (RRT-PCR). H9N2 AIV was isolated in embryonated chicken eggs and hemagglutinin (HA) gene sequence similarity of the isolated AIV was compared. RESULTS: The results showed that indoor air samples were all RRT-PCR positive for H9N2 AIV. Downwind air samples collected between 10 m and 1.5 km away from the chicken houses were also found positive with an average load 2.62-5.21×103 RNA copies/m3. However, upwind air samples were all negative for H9N2 AIV. In addition, H9N2 AIV was isolated from swabs and indoor air samples. CONCLUSION: In summary, this study provides insights into the airborne transmission of H9N2 AIV under field conditions.

Conjugation Reaction with 8-Arm PEG Markedly Improves the Immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 Fusion Protein.

Mycobacterium tuberculosis (Mtb) is a serious fatal pathogen responsible for tuberculosis (TB). Effective vaccination is highly desired for immunoprotection against Mtb infection. CFP10 and TB10.4 are two important immunodominant Mtb-secreted protein antigens, which suffer from poor immunogenicity. Thus, an antigen delivery system and adjuvants are needed to improve the immunogenicity of the two proteins. A CFP10-TB10.4 fusion protein (CT) was used as the antigen in the present study. Conjugation of 4-6 CT molecules in one entity with 8-arm polyethylene glycol (PEG) acted as an antigen delivery system. Aluminum-loxoribine mixture (A-L) and poly(I:C) functioned as the adjuvants. As compared with CT, the polymerized CT (CT-PEG) elicited significantly higher CT-specific IgG titers, higher Th1- and Th2-type cytokines and higher percentages of CD4+ IFN-γ+ and CD4+ IL-4+ cells in BALB/c mice. The presence of A-L and poly(I:C) could both increase the immune response to CT-PEG. Conjugation reaction with 8-arm PEG showed a predominant driving force to improve the immunogenicity of CT. Pharmacokinetic study in SD rats revealed that conjugation reaction with 8-arm PEG prolonged the systemic circulation of CT and exposure to the immune system. CT-PEG with A-L showed no apparent toxicity to organs, whereas CT-PEG with poly(I:C) displayed some toxicity to organs. Thus, an effective and safe vaccine against Mtb infection could be rationally designed by conjugation reaction of Mtb-secreted protein antigen with 8-arm PEG and subsequent addition of A-L.

Toxicity Studies of Ethyl Maltol and Iron Complexes in Mice.

Ethyl maltol and iron complexes are products of ethyl maltol and the iron found in the cooking pots used to prepare the Chinese dish, hot-pot. Because their safety is undocumented, the toxicity study of ethyl maltol and iron complexes was conducted in male and female Kunming (KM) mice. The animal study was designed based on the preliminary study conducted to determine the median lethal dose (LD50). The doses used in the study were 0, 1/81, 1/27, 1/9, and 1/3 of the LD50 (mg kg body weight (BW)-1 day-1) dissolved in the water. The oral LD50 of the ethyl maltol and iron complexes was determined to be 743.88 mg kg BW-1 in mice. The ethyl maltol and iron complexes targeted the endocrine organs including the liver and kidneys following the 90 D oral exposure. Based on the haematological data, the lowest-observed-adverse-effect level (LOAEL) of the ethyl maltol and iron complexes was determined to be 1/81 LD50 (9.18 mg kg BW-1 day-1) in both male and female mice. Therefore, we suggest that alternative strategies for preparing the hot-pot, including the use of non-Fe-based cookware, need to be developed and encouraged to avoid the formation of the potentially toxic complexes.

Expression and localization of guanine nucleotide-binding protein alpha S in the testis and epididymis of rams at different developmental stages.

The guanine nucleotide-binding alpha S subunit (Gαs) is an important element of key signaling pathways, which is widely expressed in mammalian tissues; however, its role in the reproductive system is still unclear. In this study, we investigated the expression and localization of Gαs in the testes and epididymis of rams at different developmental stages using quantitative RT-PCR, immunohistochemistry, and western blotting. In 1-, 6-, and 12-month-old rams, the transcription of Gαs-encoding gene (Gnαs) was significantly upregulated in the corpus and cauda epididymis compared to the testes and caput epididymis (P<0.05). At 12 months, the level of Gnαs mRNA was higher than that at 1 and 6 months for all tested tissues (P<0.05). The Gαs protein was detected in the principal cells and interstitial epididymal cells, including Sertoli and Leydig cells, as well as in testicular cells, spermatogonial stem cells, and spermatocytes. Gαs expression was the highest in the cauda epididymis (P<0.05), followed by the corpus epididymis, caput epididymis, and testes. The results indicate that in the reproductive organs of rams, Gαs is expressed in a tissue-specific and age-dependent manner. The high levels of Gαs observed in the epididymis suggest that Gαs may influence the composition of the epididymal lumen fluid and, consequently, the microenvironment for spermatozoa maturation. Thus, Gαs could play an important role in spermatogenesis and the development of the testes and epididymis in the reproductive system of rams.

Expression and Distribution of the Guanine Nucleotide-binding Protein Subunit Alpha-s in Mice Skin Tissues and Its Association with White and Black Coat Colors.

Guanine nucleotide-binding protein subunit alpha-s (Gnαs) is a small subunit of the G protein-couple signaling pathway, which is involved in the formation of coat color. The expression level and distribution of Gnαs were detected by quantitative real-time-polymerase chain reaction (qPCR), western blot, and immunohistochemistry to investigate the underlying mechanisms of coat color in white and black skin tissues of mice. qPCR and western blot results suggested that Gnαs was expressed at significantly higher levels in black mice compared with that of white mice, and transcripts and protein possessed the same expression in both colors. Immunohistochemistry demonstrated Gnαs staining in the root sheath and dermal papilla in hair follicle of mice skins. The results indicated that the Gnαs gene was expressed in both white and black skin tissues, and the expression level of Gnαs in the two types of color was different. Therefore, Gnαs may be involved in the coat color formation in mice.

Molecular Cloning, mRNA Expression, and Localization of the G-protein Subunit Galphaq in Sheep Testis and Epididymis.

The reproductive function of G-protein subunit Galphaq (GNAQ), a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR). The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01) and testis (p<0.05). Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation.