Exogenous Expression of an Alternative Splicing Variant of Myostatin Prompts Leg Muscle Fiber Hyperplasia in Japanese Quail.

Myostatin (MSTN) negatively regulates muscle growth and development through inhibiting myoblast proliferation and differentiation. Five alternative splicing isoforms of MSTN (MSTN-A to MSTN-E) have been discovered in domestic avian species. MSTN-A has high expression in skeletal muscle and encodes the full-length peptide with anti-myogenic activity. Another isoform, MSTN-B, is also highly expressed in skeletal muscle and encodes a truncated peptide that has pro-myogenic capabilities in vitro, which include promoting the proliferation and differentiation of quail muscle precursor cells. The objective of this study was to investigate overexpression of MSTN-B in vivo by using two independent lines of transgenic Japanese quail with expression directed in the skeletal muscle. Unexpectedly, the chicken skeletal muscle alpha actin 1 (cACTA1) promoter resulted in restricted exogenous MSTN-B protein expression to certain skeletal muscles, such as the gastrocnemius and tibialis anterior, but not the pectoralis major muscle. Gastrocnemius weight as a percentage of body weight in transgenic quail was increased compared to non-transgenic quail at posthatch day 21 (D21) and posthatch D42. An increase in the size of the gastrocnemius in transgenic quail was attributed to an increase in fiber number but not fiber cross-sectional area (CSA). During embryonic development, paired box 7 (PAX7) expression was prolonged in the transgenic embryos, but other myogenic regulatory factors (MRFs) were unchanged after MSTN-B overexpression. Taken together, these data provide novel insights into the regulation of skeletal muscle development by alternative splicing mechanisms in avians.

Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells.

Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species.

Myostatin (MSTN) is a key negative regulator of muscle growth and development, and an increase of muscle mass is achieved by inhibiting MSTN signaling. In the current study, five alternative splicing isoforms of MSTN mRNAs in avian species were identified in various tissues. Among these five, three truncated forms of myostatin, MSTN-B, -C, and -E created premature stop codons and produced partial MSTN prodomains encoded from exon 1. MSTN-B is the second dominant isoform following full-length MSTN-A, and their expression was dynamically regulated during muscle development of chicken, turkey, and quail in vivo and in vitro. To clarify the function of MSTN-B, two stable cell lines of quail myoblasts (QM7) were generated to overexpress MSTN-A or MSTN-B. Interestingly, MSTN-B promoted both cell proliferation and differentiation similar to the function of the MSTN prodomain to counteract the negative role of MSTN on myogenesis. The coimmunoprecipitation assay revealed that MSTN-B binds to MSTN-A and reduces the generation of mature MSTN. Furthermore, the current study demonstrated that the partial prodomain encoded from exon 1 is critical for binding of MSTN-B to MSTN-A. Altogether, these data imply that alternative splicing isoforms of MSTN could negatively regulate pro-myostatin processing in muscle cells and prevent MSTN-mediated inhibition of myogenesis in avian species.

Developmental regulation of adipose tissue growth through hyperplasia and hypertrophy in the embryonic Leghorn and broiler.

The United States is a world leader in poultry production, which is the reason why achieving better performance and muscle growth each year is a necessity. Reducing accretion of adipose tissue is another important factor for poultry producers because this allows more nutrients to be directed toward muscle growth, but the effect of embryonic adipose growth on posthatch development has not been fully understood. The purpose of this study was to investigate the total DNA mass, morphological characteristics, differentiation markers, and triglyceride breakdown factors of embryonic adipose tissue, and their relation to hyperplastic and hypertrophic growth within layers (Leghorn) and meat-type chickens (broilers). After embryonic day (E) 12, broiler weight was significantly higher than Leghorn, and this trend continued throughout the rest of incubation and posthatch (P < 0.05). Neck and leg fat pad weights between the 2 breeds did not differ at most of the time points. A remarkable increase in total DNA mass was observed between E12 and E14 in both Leghorn and broilers (P < 0.05), indicating a high potential for hyperplastic growth during this time. Histological analysis revealed clusters of preadipocytes at E12; however, the majority of these cells differentiated by E14 and continued to grow until the time of hatch. The adipocyte sizes between both breeds did not generally differ, even though broilers are known to have larger adipocytes posthatch. Fatty acid-binding protein 4 expression levels in Leghorn and broilers continued to rise with each time point, which paralleled the expansion of mature adipocytes. Adipose triglyceride lipase was highly expressed at E20 and d 1 posthatch to mobilize triglyceride degradation for energy during hatching. Thus, embryonic chicken adipose tissue was found to develop by hyperplastic mechanisms followed by hypertrophy. At embryonic stages and early posthatch, layer- and meat-type chicken adipose growth does not differ, which suggests breed differences occur posthatch.

Heart-specific expression of the green fluorescent protein gene in avian embryos by administration of recombinant adenovirus type-5 vector into the embryonic blood vessel.

Genetic modification in vivo could provide direct functions of genes that could potentially contribute to diverse areas of research including genetics, developmental biology, and physiology. It has been reported that genes of interest could be introduced via recombinant adenovirus type 5 (Ad5) in poultry. Successful gene delivery to mammal fetuses in utero promises substantial progress in clinical and developmental biology, but it is limited because of difficulties in injecting specific sites and invasiveness. On the other hand, developing avian embryos are easily accessible by making a window on the eggshell. Therefore, the objective of this study is to determine permissive embryonic stages for gene transfer into specific avian tissue/organs by injection of Ad5 containing the green fluorescent protein (GFP) gene into blood vessels. At 2 d of post-injection, a strong GFP signal was predominantly identified in the heart of chicken embryos injected at Hamilton-Hamburger (HH) 14, 15, 16 and17 stages with the percentages (44%, 53%, 25%, and 14%, respectively) of GFP positive embryos. In quail embryos, the injection at the HH 15 resulted in heart-specific expression of GFP. Western blot analysis revealed that GFP was exclusively expressed in the avian hearts. These results suggest that the GFP gene is specifically delivered to the avian embryonic hearts when Ad5 is injected through the blood vessel at HH 14-17. This adenoviral transduction of genes of interest in avian embryonic hearts can provide new models for understanding functions of genetic factors on embryonic heart development and unravel genetic etiology of congenital heart diseases.

Chordin-like 1, a Novel Adipokine, Markedly Promotes Adipogenesis and Lipid Accumulation.

White adipose tissue serves as a metabolically dynamic organ that can synthesize and secrete biologically active compounds such as adipokines as well as a caloric reservoir for maintaining energy homeostasis. Adipokines are involved in diverse biological and physiological processes and there have been extensive attempts to characterize the effects of over two dozen adipokines. However, many of these adipokines are produced by not only adipose tissue, but also other tissues. Therefore, investigations into the effects of adipokines on physiological functions have been challenged. In this regard, we aimed to identify a new secreted protein that is encoded by genes specifically expressed in white adipose tissue through analysis of multi-tissue transcriptome and protein expression. As a result, we report a novel adipokine that is encoded by the adipose-specific gene, chordin-like 1 (Chrdl1), which is specifically expressed in white adipose tissue in mice; this expression pattern was conserved in the human orthologous CHRDL1 gene. The expression of Chrdl1 was enriched in fat cells and developmentally regulated in vitro and in vivo, and moreover, its retrovirus-mediated overexpression and recombinant protein treatment led to markedly increased adipogenesis. Further pathway enrichment analysis revealed enriched pathways related to lipogenesis and adipogenic signaling. Our findings support a pro-adipogenic role of CHRDL1 as a new adipokine and pave the way toward animal studies and future research on its clinical implications and development of anti-obesity therapy.

Transdifferentiation of Myoblasts Into Adipocytes by All-Trans-Retinoic Acid in Avian.

Increased adipogenesis in muscle tissues is related to metabolic syndromes and muscle weakness in humans and improvement of meat quality in animal production. With growing evidence for pro-adipogenic functions of all-trans-retinoic acid (atRA), the current study investigated whether atRA can transdifferentiate myoblasts into adipocytes using a quail myogenic cell line (QM7) and avian primary myoblasts. atRA increased cytoplasmic lipid droplet accumulation and mRNA expression for adipogenic genes in these cells. An acute induction of Pparγ expression by atRA under cycloheximide treatment indicated a direct regulation of Pparγ by atRA. In addition, the induction of Pparγ expression was mediated by retinoic acid receptors . At high levels of Pparγ by atRA, BADGE, an antagonist of Pparγ, inhibited, and rosiglitazone, an agonist of Pparγ, further enhanced atRA-induced transdifferentiation. However, at very low levels of Pparγ in the absence of atRA treatment, rosiglitazone could not induce transdifferentiation of avian myoblasts. These data suggest that the induction of Pparγ expression by atRA is an essential molecular event in myoblasts for atRA-induced transdifferentiation into adipocytes. Based on our findings, atRA can be a new transdifferentiation factor of myoblasts to adipocytes, providing a potential nutrient to enhance marbling in poultry.

Differential Expression of MSTN Isoforms in Muscle between Broiler and Layer Chickens.

Myostatin (Mstn)-A, the main isoform among Mstn splicing variants, functions as a negative regulator, whereas Mstn-B functions as a positive regulator in muscle development. Because broiler chickens are a fast-growing breed raised for meat production and layer chickens are a slow-growing breed raised for egg production, differences in the expression of Mstn isoforms between the two distinct breeds were analyzed in this study. There was no difference in the expression levels of total Mstn (Mstn-A and -B forms) during embryonic development and at D33 between the two breeds. Interestingly, the ratios of Mstn-B to -A were significantly higher in the broiler compared to the layer at most ages. In pectoralis major muscle (PM) tissue, the cross-sectional area (CSA) of muscle fiber was significantly greater in the broiler. The broiler also showed greater bundle CSA and a similar fiber number per bundle compared to the layer at D5 and D33. These data suggest that the greater bundle CSA with myofiber hypertrophy in the broilers is associated with greater muscle growth. The relationship between the expression of Mstn isoforms and growth rate can be used as a potential genetic marker for the selection of higher muscle growth in chickens.

Retinol Binding Protein 7 Promotes Adipogenesis in vitro and Regulates Expression of Genes Involved in Retinol Metabolism.

Retinol is an essential nutrient in animals. Its metabolites, specifically retinoic acid (RA), are crucial for cell differentiation, including adipogenesis. Retinol binding protein 7 (Rbp7) is under the control of PPARγ, the master regulator of adipogenesis. However, the role of RBP7 in adipogenesis is unclear. Our study showed that Rbp7 was abundantly expressed in white and brown mouse adipose tissues and had a higher expression in adipocytes than in stromal vascular fraction. Rbp7 overexpression promoted 3T3-L1 preadipocyte differentiation with increased triglyceride accumulation and up-regulation of Pparγ, Fabp4, C/ebpα, and AdipoQ. Rbp7 deficient adipocytes had opposite effects of the overexpression, which were rescued by RA supplementation. Indirect assessment of relative nuclear RA levels using RAR response element (RARE)-Luc reporter assay demonstrated that Rbp7 overexpression significantly increased RARE-Luc reporter activity. Rbp7 overexpression significantly increased expression of Raldh1, responsible for RA production, and up-regulation of Lrat and Cyp26a1, involved in retinol storage and RA catabolism, respectively, in 3T3-L1 adipocytes. Rbp7 deficient adipocytes had opposite effects of the overexpression of those genes involved in retinol metabolism. These data suggest that RBP7 increases transcriptional activity of RARE that may induce negative feedback responses via regulation of the gene expression for retinol homeostasis. Our data indicate critical RBP7 functions in adipocytes: regulation of transcriptional activity of RARE and adipocytes differentiation, potentially providing a new target for obesity therapy.

A novel testis-enriched gene, Samd4a, regulates spermatogenesis as a spermatid-specific factor.

Spermatogenesis is the highly orchestrated process involving expression of a series of testicular genes. Testis-enriched genes are critical for cellular processes during spermatogenesis whose disruption leads to impaired spermatogenesis and male infertility. Nevertheless, among poorly investigated testicular genes are the mouse Samd4a and human SAMD4A which were identified in the current study as novel testis-enriched genes through transcriptomic analyses. In particular, as orthologous alternative splicing isoforms, mouse Samd4a E-form and human SAMD4AC-form containing the SAM domain were specific to testes. Western blot analyses revealed that the murine SAMD4AE-form was predominantly found in the testis. Analyses on GEO2R and single-cell RNA-seq datasets revealed that the Samd4a/SAMD4A expression was enriched in spermatids among various types of cells in adult testes. To investigate in vivo functions of Samd4a, Samd4a knockout mice were generated using the CRISPR/Cas9 system. The Samd4a deficiency resulted in lower testis weight, absence of elongated spermatids, and an increased number of apoptotic cells. Profiling of gene expression in human testis samples revealed that the SAMD4A expression was comparable between obstructive azoospermia patients and normal controls, but significantly lowered in nonobstructive azoospermia (NOA) patients. Among three subgroups of NOA, pre-meiotic arrest (NOA-pre), meiotic arrest (NOA-mei), and post-meiotic arrest (NOA-post), expression level of SAMD4A was higher in the NOA-post than the NOA-mei, but there was no difference between the NOA-pre and NOA-mei. The current studies demonstrated spermatid stage-specific expression of Samd4a/SAMD4A, and impairment of the late stages of spermatogenesis by disruption of the mouse Samd4a gene. These data suggest that Samd4a/SAMD4A plays an essential role in normal spermatogenesis, and SAMD4A, as a spermatid specific marker, can be used for subcategorizing NOA patients. Further understanding the molecular role of SAMD4A will advance our knowledge on genetic regulations in male infertility.

Research Note: Potential usage of DF-1 cell line as a new cell model for avian adipogenesis.

Current research of avian adipogenesis has been dependent on primary preadipocytes culture due to the lack of commercially available immortal preadipocyte cell lines in avian species. In addition to primary stromal vascular cells, primary chicken embryonic fibroblasts (CEF) were suggested as new in vitro models for adipogenesis study, because CEF can be differentiated into adipocytes by a combination of fatty acids and insulin (FI), or all-trans retinoic acid (atRA) alone in the media containing chicken serum (CS). However, there are decreases in differentiation of primary cells due to diverse population of cell types and low adipogenic potential of cells after passages. In the present study, adipogenic differentiation of DF-1 cells, immortal fibroblasts derived from an embryonic chicken, was tested with 4 different medium; 10% fetal bovine serum (FBS), 10% CS, 10% CS with FI, and 10% CS with FI and atRA. Lipid droplets stained with Oil Red O were not shown in DF-1 cells under 10% FBS, appeared with very small sizes under 10% CS, significantly increased under 10% CS with FI, and most significantly accumulated under 10% CS with FI and atRA. In addition, expressions of markers for adipogenesis (Znf423, C/ebpß, Pparγ, and Fabp4), fatty acid uptake (CD36), triglyceride synthesis (Gpd1, Dgat2), and lipid droplet stabilization (Plin1) were significantly upregulated by supplementation of 10% CS with FI and atRA. Morphological evidence for formation of lipid droplets and dramatic induction of adipogenic marker genes support the adipogenic potential of DF-1 cells, offering DF-1 cells as a new cell model to investigate various research studies involving avian adipogenesis.

Research Note: Adipogenic differentiation of embryonic fibroblasts of chicken, turkey, duck, and quail in vitro by medium containing chicken serum alone.

The study of adipogenesis is one of the most important areas for not only regulating meat quality, but production efficiency associated with fat accretion in the poultry species. Current in vitro models for avian adipogenesis require adipogenic inducers including dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), fatty acids, or insulin. However, problems still remain in these models for testing/screening potential nutritional, hormonal, and pharmaceutical factors because of interfering/overriding effects of the inducing factors. Therefore, the purpose of this study was to develop a simple in vitro method for avian adipogenesis. In this study, chicken serum (CS) and fetal bovine serum (FBS) were compared for adipogenic potential using chicken embryonic fibroblasts (CEF). Oil-red O staining at 4 d in culture of CEF under CS revealed that lipid droplet formation was increased by CS in a dose-dependent manner (0 to 10%). On the contrary, all concentrations of FBS (0 to 10%) alone did not show lipid droplet formation. In accordance with the morphological data of CEF, mRNA expression of genes involved in adipocyte differentiation/determination, fatty acid uptake, and triacylglycerol (TAG) synthesis, were most significantly up-regulated by 10% CS at d 4 compared to 1 or 5% CS. In addition, embryonic cells isolated from quail (QEF) at E5, duck (DEF) at E6, and turkey (TEF) at E6, were tested for adipogenic differentiation by media containing the same concentrations of CS. Similar to the morphological data from CEF, quantitative data of the Oil-red O staining showed that lipid droplet formation in QEF, DEF, and TEF was increased by CS in a dose-dependent manner (0 to 10%). The current study demonstrates that CS alone can induce adipogenesis on embryonic fibroblasts of various poultry species. By providing a new simple in vitro method of avian adipogenesis, diverse nutritional, hormonal, and pharmaceutical factors can be broadly and easily tested for scientific and industrial purposes.

Necessity for Validation of Effectiveness of Selected Guide RNA In Silico for Application of CRISPR/Cas9.

Selection of guide RNA (gRNA) is important to increase the efficiency of gene editing in the CRISPR/Cas9 system. Due to the variation in actual efficiency of insertion/deletion (indel) mutation among selected gRNAs in silico, reliable methods for validation of efficiency of gRNA need to be developed. Three gRNAs with high on-target scores (72.0 for target 1, 65.4 for target 2, and 62.9 for target 3) were designed to target the quail retinol binding protein 7 (qRbp7) gene, and indel efficiencies were predicted by the Sanger sequencing and Inference of CRISPR Edits (ICE) analysis of sorted cell populations receiving the CRISPR/Cas9 vector. Unlike the order of on-target scores among 3 gRNAs, predicted rates of indel mutations were highest in gRNA2, intermediate in gRNA1, and lowest in gRNA3. This was confirmed by actual indel mutation rates, 51.8% in gRNA2, 31% in gRNA1, and 12.9% in gRNA3, which were calculated by sequencing individual allele cloned into a vector. These data showed a rapid and reliable method for estimation of the efficiency of selected gRNAs, providing a critical necessary step for successful gene editing for further applications.

Research Note: Increased myostatin expression and decreased expression of myogenic regulatory factors in embryonic ages in a quail line with muscle hypoplasia.

Genetic selection of quail for a low body weight for more than 80 generations established a low-weight (LW) Japanese quail line that has been previously characterized to have a muscle hypoplasia phenotype. The aim of this study is to investigate the relationship of temporal expression levels of myostatin (Mstn) and myogenic regulatory factors (MRFs) with hypoplastic muscle growth in the LW line. During embryonic day (E) 13 to 15, gain of embryo weight was 2-fold lower (P < 0.001) in the LW line than that in the random bred control (CON). Gains in body weight and pectoralis muscle weight from hatch to posthatch day (P) 28 were also significantly lower (P < 0.01) in the LW line but increased by 4-fold (P < 0.05) during P42 to P75. PCR analysis showed that expression levels of Mstn were greater in the LW at embryonic stage (E12 to E14, P < 0.05), but there was no difference after hatch. In addition, expression levels of Pax7 and myogenin (MyoG) at E12 were 23-fold (P < 0.05) and 3.4-fold (P < 0.05) lesser in the LW line, respectively. At E14, expression of Pax3, Pax7, and MyoG gene was 3.5-fold (P < 0.05), 6.5-fold (P = 0.065), and 4.4-fold (P < 0.01) less than that in the CON. Taken together, high expression levels of Mstn and low expression of MRFs during embryonic stages can be associated with development of muscle hypoplasia and delayed muscle growth in the LW quail line. These data provide evidence that genetic selection for a low body weight resulting in an avian model with muscle hypoplasia has altered the expression profiles of myogenic factors.

Research Note: All-trans retinoic acids induce adipogenic differentiation of chicken embryonic fibroblasts and preadipocytes.

Adipocytes store excess energy in the form of lipids, whereas fat accretion contributes to feed efficiency, meat quality, and female reproduction in poultry. As a metabolite of vitamin A, all-trans retinoic acid (atRA) has been shown to have influence over metabolic functions such as lipid and energy homeostasis, as well as adipogenesis. Although atRA has been known to function as a regulating factor in mammalian adipogenesis, the effects of atRA on adipogenesis has not been studied in chickens. In this study, chicken preadipocytes isolated from leg fat tissues at embryonic day (E) 14 and chicken embryonic fibroblasts (CEF) harvested at E5 were cultured. The preadipocytes and CEF in culture with 10% chicken serum were treated with various concentrations (0 µmol, 100 µmol, or 150 µmol) of supplemented atRA for 48 h. In these cells, cytoplasmic lipid droplet accumulation and mRNA expression for adipogenic genes were analyzed by Oil-Red-O staining and quantitative real-time PCR, respectively. Analysis of the relative amount of Oil-Red-O staining (lipid accumulation) revealed that all 3 variables increased in a dose-dependent manner, in response to increasing atRA supplementation. Genes involved in adipocyte differentiation, fatty acid transport, and triacylglycerol synthesis in both E14 preadipocytes and E5 CEF were upregulated by supplementation of atRA. These data demonstrated that atRA alone promoted adipogenesis of embryonic preadipocytes and fibroblasts in vitro, suggesting that atRA has an influential role in multiple stages of adipogenesis in chicken embryos.

Research Note: Association of temporal expression of myostatin with hypertrophic muscle growth in different Japanese quail lines.

Myostatin (MSTN) negatively regulates in muscle growth and development. Among alternative splicing isoforms of avian MSTN, MSTN-A has antimyogenic activities and MSTN-B functions as a promyogenic factor. In this study, different lines of Japanese quail were used: a random bred control (RBC) and a heavy weight (HW) quail line with muscle hypertrophy. The objectives of the current study are to compare temporal expression of the MSTN isoforms in pectoralis major muscle (PM) between 2 quail lines and to relate MSTN expression with temporal changes in muscle growth and total amounts of DNA in PM. Gains of body weight (BW) and PM weight were greater until posthatch day (D) 28 (P < 0.001), and the fold increases in total DNA contents of PM were greater in the HW line compared with the RBC line during D7 to D28 (P < 0.05). PCR analysis showed that MSTN-A expression was greater at 14 D (E14) of embryonic age (P < 0.01), D7 (P = 0.052), and D14 (P < 0.01) in the RBC line compared with the HW line. At D28 and D75, expression of MSTN-A was greater in the HW line compared with the RBC line (P < 0.05). MSTN-B expression was barely detectable from E14 to D14 and measurable from D28 to D75 in the muscle of both lines. Ratios of the MSTN-B/-A form ranging from 0.15 to 0.29 indicate a minor expression of the B form. Taken together, the lesser expression levels of MSTN-A at E14, D7, and D14 are associated with the fast growth of PM, and greater MSTN-A expression at D28 and D75 are associated with a slowdown of PM growth in the HW line. These data indicate a negative association of MSTN expression with PM growth and provide a scientific basis for potential usage of MSTN expression as a selection marker for greater muscle growth in poultry.

Adipose-Specific Expression, Developmental and Nutritional Regulation of the Gene-Encoding Retinol-Binding Protein 7 in Pigs.

Modulation of expression of adipose tissue-specific transcripts has been known to regulate adipogenesis and lipid metabolism. Recently, adipose-specific expression patterns and developmental regulation of the gene-encoding retinol-binding protein 7 (RBP7) was identified. However, its expression in adipose tissue of the porcine species has yet to be explored. In this study, adipose tissue-specific expression of porcine RBP7 was identified and conservation of the fatty acid-binding domains and evolutionary relationship of the RBP7 gene were verified comparatively across mammalian species. Our in vitro and in vivo analysis of gene expression revealed that RBP7 expression was significantly high in fat cell fraction compared to stromal vascular cells (p < 0.05) and increased during development (p < 0.05). The level of RBP7 expression was upregulated during a 24-h short-term fasting intervention and restored 6 h after refeeding (p < 0.05). Taken together, these studies provide insights into the role of RBP7 in adipose tissue of pigs during development and nutritional intervention and pave the way for future studies on the regulation of retinol homeostasis in porcine adipose tissue.

Adipose-specific expression of mouse Rbp7 gene and its developmental and metabolic changes.

Alternative splicing and alternative promoter usage have been shown to have an integral role in creating flexibility in the regulation of gene expression. Previous studies collectively showed that expression of the retinol binding protein 7 (Rbp7) gene was adipose tissue-specific across species. Nevertheless, alternative splicing and alternative promoter usage of the Rbp7 gene in adipose tissue and other tissues have not been investigated. The objectives of this study were to investigate protein isoforms of RBP7 produced from alternative splicing, alternative promoter usage and pre-mRNA trans-splicing, and to examine expression patterns of RBP7 isoforms during adipogenesis, cold exposure, and retinol or retinoic acid treatment. Our RT-PCR analysis revealed that mouse Rbp7 isoforms were present, but only one protein isoform was detected which was specific to adipose tissue. In addition, a fusion transcript of the Nmnat1 gene and the Rbp7 gene was produced by pre-mRNA trans-splicing in several tissues; however, its protein expression was not detectable. During adipogenesis, RBP7 expression was prominent in both neonatal and after-weaning stages and its expression was significantly higher in fat cells than in preadipocytes. Exposure to cold led to an increased expression of RBP7 in brown adipose tissue (BAT). Furthermore, Rbp7 mRNA expression in 3T3-L1 cells was significantly up- and down-regulated by retinol and retinoic acid, respectively. Our data showed that the mouse Rbp7 gene produces a predominant isoform in adipose tissue during adipocyte development, cold exposure, and nutritional treatments, which can be a potential target for future investigation on reduced adiposity.

Comparative expression profiling of testis-enriched genes regulated during the development of spermatogonial cells.

The testis has been identified as the organ in which a large number of tissue-enriched genes are present. However, a large portion of transcripts related to each stage or cell type in the testis still remains unknown. In this study, databases combined with confirmatory measurements were used to investigate testis-enriched genes, localization in the testis, developmental regulation, gene expression profiles of testicular disease, and signaling pathways. Our comparative analysis of GEO DataSets showed that 24 genes are predominantly expressed in testis. Cellular locations of 15 testis-enriched proteins in human testis have been identified and most of them were located in spermatocytes and round spermatids. Real-time PCR revealed that expressions of these 15 genes are significantly increased during testis development. Also, an analysis of GEO DataSets indicated that expressions of these 15 genes were significantly decreased in teratozoospermic patients and polyubiquitin knockout mice, suggesting their involvement in normal testis development. Pathway analysis revealed that most of those 15 genes are implicated in various sperm-related cell processes and disease conditions. This approach provides effective strategies for discovering novel testis-enriched genes and their expression patterns, paving the way for future characterization of their functions regarding infertility and providing new biomarkers for specific stages of spematogenesis.

Role of neuronal energy status in the regulation of adenosine 5'-monophosphate-activated protein kinase, orexigenic neuropeptides expression, and feeding behavior.

Nutrient sensing in the hypothalamus is tightly related to food intake regulation. However, the mechanisms by which the nutrient-sensing cells of the brain translate this signal of energy need into feeding behavior via regulation of neuropeptide expression are not known. To address this issue, we investigated two neuronal cell lines expressing agouti-related protein (AgRP), ex vivo hypothalamic tissues, and in vivo whole animals. Maintaining cells in a low cellular ATP concentration generated by low glucose, 2-deoxyglucose (2-DG), ATP synthesis inhibitor, and 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside increased phosphorylation of AMP-activated protein kinase (AMPK) and increased AgRP expression, whereas maintaining cells in high ATP status by high glucose and pyruvate supplementation in 2-DG-treated cells decreased phosphorylation of AMPK and decreased AgRP expression. Overexpression of a dominant-inhibitory mutant of AMPK significantly decreased low-glucose- or 2-DG-induced AgRP expression. Furthermore, ex vivo hypothalamus culture in high glucose concentrations decreased both expression and phosphorylation of AMPK and expression of both AgRP and neuropeptide Y, whereas pyruvate supplementation suppressed a 2-DG-induced AgRP expression. Finally, our in vivo studies clearly show that central administration of pyruvate dramatically delayed 2-DG-induced food intake. These data indicate that modulation of ATP levels in neuronal cells triggers a cascade of events via AMPK that modulate feeding behavior to restore energy status of cells.