Yongfeng Sun

Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function

The objective was to confirm the presence of nerve growth factor (NGF) and its specific receptor, TrkA, in ejaculated bovine sperm, and to investigate the effects of NGF on specific aspects of bovine sperm function. Both TrkA transcripts and immunoreactivity typical of the translated protein were detected by RT-PCR and western blotting. However, only the NGF protein was detected in bovine sperm using western blotting, and there was no RT-PCR evidence for NGF transcripts in sperm. Using an immunofluorescent technique, NGF-immunoreactivity was localized to the sperm head and tail, whereas that of TrkA was detected in the acrosomal cap, nucleus, and tail regions When sperm were treated with exogenous NGF, both leptin secretion and sperm viability were increased (P < 0.05); moreover, the percentages of late apoptotic and dead sperm were increased (P < 0.05). However, NGF had no effects on insulin secretion, mitochondrial activity, intracellular calcium levels, or the acrosome reaction of sperm (P > 0.05). In conclusion, the presence of TrkA transcript, as well as NGF and TrkA immunoreactivity were confirmed in bovine sperm. Furthermore, exogenous NGF had significant effects on the secretion of leptin, cell viability, and sperm apoptosis. This study provided strong evidence that NGF/TrkA may have roles in regulation of sperm physiology and perhaps male fertility and infertility.

Relationship Between Apoptosis and Proliferation in Granulosa and Theca Cells of Cystic Follicles in Sows

To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.

Comparative Proteomic Analysis of Follicular Fluids from Normal and Cystic Follicles in Sows

Follicular fluid (FF) includes various biologically active proteins which can affect follicular growth and maturation. Certain proteins could reflect the physiological and pathological status of follicles. The aim of the present study was to explore the key proteins associated with pathogenesis of follicular cysts, some of which may be candidate biomarkers for the condition. We analysed the proteomes of FF from small, medium, large and cystic follicles by two-dimensional electrophoresis (2-DE) combined with mass spectrometry (MS). The protein components in FF were found to be significantly different among groups; about 300 proteins spots in each group were examined, and 32 differentially expressed proteins were identified from different groups. To further reveal the source of identified proteins, transcripts encoding two of these, transferrin and RBP-4, were detected in granulosa cells (GCs) by RT-PCR, as well as the proteins were detected in 24 h culture media of GCs by ELISA. High levels of RBP-4 were examined in FF of cystic follicles by 2-DE analysis, which were significantly different to those in large follicles (p < 0.05). In conclusion, the study enriches our understanding of the proteins of FF; RBP-4 and transferrin originate from passive transfer and follicular synthesized secretion, and RBP-4 might be a candidate biomarker for porcine follicular cysts. Combined with histological studies, these results further suggest that changes of the type and quantity of proteins in FF might be attributed to an abnormal metabolism of follicular cells and structure of follicular wall in cystic follicles. Our findings will contribute to further insight into the pathogenesis of follicular cysts.

Expression of brain-derived neurotrophic factor in mature spermatozoa from fertile and infertile men.

BACKGROUND Neurotrophins, a family of growth factor, are not only required for the survival and differentiation of the nervous system but also important for the development of reproductive tissues. We identified the expression of brain-derived neurotrophic factor (BDNF) transcript and protein in human spermatozoa. METHODS The presence of BDNF in human spermatozoa was investigated using RT-PCR, immunofluorescence and Western blotting. Real-time PCR and ELISA were employed to determine expression levels of BDNF. RESULTS BDNF mRNA and protein were detected in human spermatozoa. Immunofluorescence staining showed that BDNF protein was localized in the head, neck, and tail of human spermatozoa. The amount of BDNF mRNA expressed in spermatozoa of oligoasthenozoospermic group was lower than that of fertile group (P < 0.05). The concentration of BDNF in seminal plasma from oligoasthenozoospermic group (2.8 ± 0.7 ng/ml) was both lower than that from fertile group (3.6 ± 0.4 ng/ml) and asthenozoospermic group (3.4 ± 0.5 ng/ml) (P < 0.05). CONCLUSIONS The data showed that the decrease in BDNF transcript and protein in oligoasthenozoospermic group may be associated with pathogenesis in some types of male infertility.

Molecular cloning, polymorphism and tissue distribution of the MHC class IIB gene in the Chinese goose (Anser cygnoides).

1. The goose major histocompatibility complex (MHC) class IIB cDNA (Ancy-MHCII) was cloned by homology cloning and rapid amplification of cDNA ends by polymerase chain reaction (RACE-PCR), and the genomic structure and tissue expression were investigated. 2. Three different 5′-RACE sequences (Ancy-MHC II5′-1, Ancy-MHC II5′-2, Ancy-MHC II5′-3), one 3′-RACE sequence (Ancy-MHC II-3′) and two different full length Ancy-MHC IIB cDNA sequences (Ancy-CD01, Ancy-CD02), which came from different alleles at one locus or different loci, were determined. 3. The genomic organisation is composed of 6 exons and 5 introns, with a longer intron region than that of the chicken. The alleles encode 259 and 260 amino acids in the mature protein. 4. The number of non-synonymous substitutions (dN) in the peptide-binding region of exon 2 from 8 alleles was higher than that of the synonymous substitutions (dS). 5. Tissue-specific expression of Ancy-MHC II mRNA was detected in an adult goose using RT-PCR. These results showed that Ancy-MHC II mRNA was expressed in the lung, spleen, liver, intestine, heart, kidney, pancreas, brain, skin and muscle. This is consistent with the expression of MHC class IIB in various tissues from the chicken. 6. Sequences from goose, snipe and duck clustered together when compared with known MHC class IIB sequences from the other species, significantly differing from mammals and aquatic species, indicating a pattern consistent with accepted evolutionary pathways.

Interaction between neurotrophin 4 and gonadotrophin in bovine oviducts.

The expression and localization of neurotrophin 4 (NT4) and its receptor, tyrosine kinase B (TRKB), in the bovine oviduct, and their interaction with gonadotrophins in bovine oviduct epithelial cells (BOECs), were examined. Transcripts for NT4 and TRKB were detected by reverse transcription polymerase chain reaction (RT-PCR) in bovine oviducts in the follicular and luteal phases, and their proteins were immunolocalized in BOECs. Based on real time PCR, NT4 mRNA did not differ significantly between the two phases of the cycle, although TRKB mRNA expression was higher (P < 0.05) in the luteal phase than that in follicular phase. The BOECs were treated with various concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in vitro; for NT4, mRNA and protein were higher (P < 0.05) than those in the control (based on real time PCR and enzyme-linked immunosorbent assay (ELISA) assays). The effects of NT4 and the TRKB inhibitor (K252a) on the expression of LH receptor (LHR) and FSH receptor (FSHR) in the oviduct epithelial cells were also studied using a monolayer culture model. Expression levels of LHR and FSHR mRNA in BOECs treated with various concentrations of NT4 were higher (P < 0.05) than those in the control. However, these expressions were blocked by treatment with K252α. We concluded that neurotrophin 4 may have a role in regulating the function of bovine oviducts by interacting with gonadotrophins.

Follicle-stimulating hormone (FSH) promotes retinol uptake and metabolism in the mouse ovary

BackgroundRetinoids (retinol and its derivatives) are required for the development and maintenance of normal physiological functions of the ovary. However, the mechanisms underlying the regulation of ovarian retinoid homeostasis during follicular development remain unclear.MethodsThe present study determined retinoid levels and the expression levels of genes involved in the retinol uptake and its metabolic pathway in the ovaries of follicle-stimulating hormone (FSH)-treated mice and in granulosa cells treated with FSH using ultra performance liquid chromatography (UPLC) combined with quadrupole time-of-flight high-sensitivity mass spectrometry (Q-TOF/HSMS) and real-time PCR analysis.ResultsThe levels of total retinoids and retinoic acid (RA) and expressions of retinol-oxidizing enzyme genes alcohol dehydrogenase 1 (Adh1) and aldehyde dehydrogenase (Aldh1a1) are increased in the ovaries of mice treated with FSH; in contrast, the retinyl ester levels and retinol-esterifying enzyme gene lecithin: retinol acyltransferase (Lrat) expression are diminished. In FSH-treated granulosa cells, the levels of retinyl esters, retinaldehyde, and total retinoids are augmented; and this is coupled with an increase in the expressions of stimulated by retinoic acid 6 (Stra6) and cellular retinol-binding protein 1 (Crbp1), genes in the retinol uptake pathway, and Adh1, Adh7, and Aldh1a1 as well as a diminution in Lrat expression.ConclusionsThese data suggest that FSH promotes retinol uptake and its conversion to RA through modulating the pathways of retinol uptake and metabolism in the mouse ovary. The present study provides a possible mechanism for the regulation of endogenous RA signaling in the developing follicles.

De Novo Transcriptome Sequencing Analysis of Goose (Anser anser) Embryonic Skin and the Identification of Genes Related to Feather Follicle Morphogenesis at Three Stages of Development

The objective of this study was to evaluate the changes in the goose embryo transcriptome during feather development. RNA-Sequencing (RNA-Seq) was used to find the transcriptome profiles of feather follicles from three stages of embryonic dorsal skin at embryonic day 13, 18, and 28 (E13, E18, E28). The results showed that 3001, 6634, and 13,780 genes were differently expressed in three stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that differentially expressed genes (DEGs) in E13 vs. E18 were significantly mapped into the GO term of extracellular structure organization and the pathway of extracellular matrix (ECM)-receptor interaction. In E18 vs. E28, the top significantly mapped into GO term was the single-organism developmental process; the pathway was also the ECM-receptor interaction. DEGs in E13 vs. E28 were significantly mapped into the GO term of the multicellular organismal process and the pathway of cell adhesion molecules. Subsequently, the union of DEGs was categorized by succession cluster into eight profiles, which were then grouped into four ideal profiles. Lastly, the seven genes spatio-temporal expression pattern was confirmed by real-time PCR. Our findings advocate that interleukin 20 receptor subunit alpha (IL20RA), interleukin 6 receptor (IL6R), interleukin 1 receptor type 1 (IL-1R1), Wnt family member 3A (WNT3A), insulin-like growth factor binding protein 3 (IGFBP3), bone morphogenetic protein 7 (BMP7), and secreted-frizzled related protein 2 (SFRP2) might possibly play vital roles in skin and feather follicle development and growth processes.

Study on the Changes of IGF-I mRNA Expression Levels in the Developmental Muscles of the Geese

In this study,we detected the expression of IGF-I mRNA at 1(D1),30(D30),60(D60),and 90(D90) days of age in the breast muscle and leg muscle of geese from meat-type Jilin goose,egg-type Jilin goose and hortobagyi goose.We found that the expression of IGF-Ⅰ mRNA in skeletal muscle tissues differed based on developmental age.The expression of IGF-Ⅰ mRNA exhibited a trend that ascended first and then descended after birth.IGF-Ⅰ mRNA was expressed in all breast muscle tissues during each stage with higher levels in meat-type goose and hortobagyi goose than that in egg-type Jilin goose(P0.05).The expression pattern of IGF-Ⅰ mRNA in breast muscle of meat-type goose was similar to that in and hortobagyi goose,and in leg muscle,IGF-Ⅰ mRNA expression patterns were all down-regulated from D30.Similarity in IGF-Ⅰ mRNA expression pattern was found in breast among meat-type jilin goose,egg-type jilin goose and hortobagyi goose,which indicates that IGF-Ⅰ may be related to skeletal muscle differentiation and growth and suggests IGF-Ⅰ mRNA maybe tissue-specific expression and individual difference.