Proteome profiling of the sperm maturation milieu in the rhesus monkey (Macaca mulatta) epididymis.

The mammalian spermatozoon acquires its fertilising potential during transit through the epididymis, where it interacts with epididymal luminal fluid proteins (the sperm maturation milieu). In order to highlight the epididymal-specific function of the rhesus monkey (Macaca mulatta) in sperm maturation, two-dimensional gel electrophoresis of epididymal luminal fluid proteins was followed by identification by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) or MALDI-TOF/TOF and revealed over five hundred spots, comprising 198 non-redundant proteins. Some mass spectrometric data were confirmed by western blotting identification. Some common epididymal fluid proteins were identified, such as clusterin, α-1-antitrypsin, malate dehydrogenase, L-lactate dehydrogenase B, α-1-acid glycoprotein 1 and α-mannosidase. More than 7% of all proteins were anti-oxidative, which might control oxidative stress within the male tract. When compared with bull and human epididymal luminal fluid proteins, those in the rhesus monkey had more overlap with the human, which provides evidence of a close evolutionary relationship between the rhesus monkey and man. This study provides new proteomic information on possible rhesus monkey epididymal functions and novel potential biomarkers for the noninvasive assessment of male fertility.

WITHDRAWN: Comparative proteome analysis of human testis from newborn, young adult, and aged men identified spermatogenesis-associated proteins.

Ahead of Print article withdrawn by publisher.

Aged men share the sperm protein PATE1 defect with young asthenozoospermia patients.

STUDY QUESTION: Does a defect in the human sperm-located protein prostate and testis expressed 1 (PATE1) exist in both aged men and young asthenozoospermia patients? SUMMARY ANSWER: A defect in sperm PATE1 exists in both aged men and young asthenozoospermia patients, and an antibody against PATE1 can decrease human sperm motility and zona-free hamster oocyte penetration. WHAT IS KNOWN ALREADY: Both aged men and young asthenozoospermia patients have poor sperm quality. The PATE1 protein seems to mediate sperm-egg interactions; however, the mechanisms are still unknown. STUDY DESIGN, SIZE, DURATION: This was a case-control study including 60 young fathers (aged 28-32 years) and 60 aged fathers (68-72 years old) who donated semen by masturbation after 7 days of sexual abstinence. Comparative sperm proteome analysis from the young fathers and aged fathers was performed to discover key proteins. The target protein PATE1 was chosen and validated by western blotting and immunohistochemistry. Quantitative assessment of sperm PATE1 protein was performed on sperm from 60 young fathers, 60 aged fathers and 110 young asthenozoospermia patients. Furthermore, an antibody against PATE1 assay was used to test whether PATE1 participated in sperm motility and penetration of zona-free hamster egg. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were pooled and separated by two-dimensional gel electrophoresis followed by identification by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Western blotting and immunohistochemistry were used to validate the confidence of proteomic data. Sperm immunofluorescence quantification experiments disclosed whether the aged men indeed shared the same PATE1 defect with 110 young asthenozoospermia patients. The sperm motility test and penetration of zona-free hamster egg assay were performed for PATE1. MAIN RESULTS AND THE ROLE OF CHANCE: Twenty-two sperm proteins with significant differential expression between young adults and aged men were identified (P < 0.05, mean ratio >1.5), including 13 proteins with decreased expressions with aging. Based on bioinformatics, PATE1 was chosen for further study, and exhibited similar changes in expression level and localization on sperm from aged men and young asthenozoospermia patients. Antibody blocking revealed that PATE1 was involved in sperm-egg penetration and sperm motility. LIMITATIONS, REASONS FOR CAUTION: Before any clinical application of PATE1 as a biomarker for the diagnosis of male infertility, more cases should be used to evaluate confidence in this approach. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed a common molecular basis underlying the decline in sperm quality in the natural aging process and in young men with asthenozoospermia. The data should greatly contribute to the development of molecular evaluation of sperm quality, and the diagnosis and treatment of asthenozoospermia. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from the National Natural Science Foundation of China (NO. 81300533, 81370013 and 81000277) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002, ZR2014HQ068). The authors declare no competing financial interests.

In-depth Proteomic mapping of mouse (Mus musculus) epididymal constructive basis for sperm maturation.

BACKGROUND: The mouse epididymis performs an essential role in sperm maturation, but global protein expression data in mouse epididymis are still lacking. Here, we reported the first in-depth gel-based profiling of mouse epididymis proteome and established a 2-DE map. RESULTS: A total of 832 protein spots were detected in the reproducible gels, and 625 spots corresponding to 355 unique protein entries have been successfully identified by MALDI-TOF-MS. The confidence of proteome data was validated by Western blot. Functional annotations showed that these proteins were mainly related to general metabolism, antioxidant and structural molecule activity. Immunohistochemistry disclosed two structural proteins (myosin regulatory light polypeptide 9 and alpha-2 type I collagen) continuously expressed in the myoid cell since postpartum. CONCLUSION: This study provides a first-draft reference map of the mouse epididymis proteome, which will greatly expand the knowledge of the epididymal structural basis and contribute to the better understanding of those proteins in the process of mouse epididymal sperm maturation.

Transcriptome analysis of a cDNA library from adult human epididymis.

Mammalian Gene Collection (MGC) verified over 9,000 human full-ORF genes and FLJ Program reported 21,243 cDNAs of which 14,409 were unique ones and 5,416 seemed to be protein-coding. The pity is that epididymis cDNA library was missing in their sequencing target list. Epididymis is a very important male accessory sex organ for sperm maturation and storage. Fully differentiated spermatozoa left from testis acquire their motility and capacity for fertilization via interactions with the epididymal epithelium duct lumen during passage through this convoluted duct. Here, we report that 20,000 clones from a healthy male epididymis cDNA library have been sequenced. The sequencing data provided 8,234 known sequences and 650 unknown cDNA fragments. Hundred and six of 650 unknown cDNA clone inserts were randomly selected for fully sequencing. There were 25 unknown unique sequences and 19 released but unreported sequences came out. By northern blot analysis, four sequences randomly selected from the 19 released sequences with no known function showed positive mRNA signals in epididymis and testis. The signals for three of six from those unknown group showed as epididymis abundant in a region-specific manner but not in the testis and other tissues tested. All the sequencing data will be available on the website www.sdscli.com.

Expression and function of HSP110 family in mouse testis after vasectomy.

HSP110 functions to protect cells, tissues, and organs from noxious conditions. Vasectomy induces apoptosis in the testis; however, little is known about the reason leading to this outcome. The aim of the present study was to evaluate the expression and function of HSP110 in mouse testis after vasectomy. Following bilateral vasectomy, we used fluorescent Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to detect apoptosis, Western blotting and immunohistochemistry to examine HSP110 expression and localization. Serum antisperm antibody (AsAb) and testosterone were measured by Enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Expression of endoplasmic reticulum stress (ERS) sensors and downstream signaling components was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and the phosphorylation of eIF2α and JNK was detected by Western blotting. Vasectomy induced morphologic changes, increased apoptosis in the testis, increased serum AsAb, and decreased testosterone levels. After vasectomy, ORP150 mRNA level was increased first and then decreased, Bcl-2 was decreased, and the expression of HSPA4l, GRP78, GADD153, PERK, ATF6, IRE-1, XBP-1s, Bax, Bak, and caspases and the phosphorylation of eIF2α and JNK were increased. We present that an ER stress-mediated pathway is activated and involved in apoptosis in the testis after vasectomy. HSPA4l and ORP150 may play important roles in maintaining the normal structure and function of testis.

Pancreatic insulin-producing cells differentiated from human embryonic stem cells correct hyperglycemia in SCID/NOD mice, an animal model of diabetes.

BACKGROUND: Human pancreatic islet transplantation is a prospective curative treatment for diabetes. However, the lack of donor pancreases greatly limits this approach. One approach to overcome the limited supply of donor pancreases is to generate functional islets from human embryonic stem cells (hESCs), a cell line with unlimited proliferative capacity, through rapid directed differentiation. This study investigated whether pancreatic insulin-producing cells (IPCs) differentiated from hESCs could correct hyperglycemia in severe combined immunodeficient (SCID)/non-obese diabetic (NOD) mice, an animal model of diabetes. METHODS: We generated pancreatic IPCs from two hESC lines, YT1 and YT2, using an optimized four-stage differentiation protocol in a chemically defined culture system. Then, about 5-7 × 10(6) differentiated cells were transplanted into the epididymal fat pad of SCID/NOD mice (n = 20). The control group were transplanted with undifferentiated hESCs (n = 6). Graft survival and function were assessed using immunohistochemistry, and measuring serum human C-peptide and blood glucose levels. RESULTS: The pancreatic IPCs were generated by the four-stage differentiation protocol using hESCs. About 17.1% of differentiated cells expressed insulin, as determined by flow cytometry. These cells secreted insulin/C-peptide following glucose stimulation, similarly to adult human islets. Most of these IPCs co-expressed mature ß cell-specific markers, including human C-peptide, GLUT2, PDX1, insulin, and glucagon. After implantation into the epididymal fat pad of SCID/NOD mice, the hESC-derived pancreatic IPCs corrected hyperglycemia for ≥ 8 weeks. None of the animals transplanted with pancreatic IPCs developed tumors during the time. The mean survival of recipients was increased by implanted IPCs as compared to implanted undifferentiated hESCs (P<0.0001). CONCLUSIONS: The results of this study confirmed that human terminally differentiated pancreatic IPCs derived from hESCs can correct hyperglycemia in SCID/NOD mice for ≥8 weeks.

Characterisation of Lyzls in mice and antibacterial properties of human LYZL6.

C-type lysozyme genes (Lyzls) belong to the class of lysozymes and are highly expressed in the testis and epididymis. The members Lyzl4 and Spaca3 have been reported to play a role in sperm-egg binding and fertilisation in mice. However, the function of the remaining two mouse c-type lysozyme genes, Lyzl1 and Lyzl6, is still not clear. In the present study, we analysed the tissue expression and androgen-dependent expression of mouse c-type lysozyme genes and the possible contribution of human recombinant LYZL6 (rLYZL6) to immunity. The expression of Lyzls was detected by RT-PCR, Western blots, immunohistochemistry and immunofluorescence. The bacteriolytic activity of rLYZL6 was analysed by a colony-forming assay. In mice, the expression of Lyzl genes was mainly in the testis and epididymis in a developmentally regulated manner and androgen- or testicular factor-regulated manner. Immunodetection revealed the presence of LYZL6 protein in primary spermatocytes and round spermatids of the testis and on the post-acrosomal area and midpiece of mature epididymal spermatozoa. The rLYZL6 protein exhibited antibacterial activity. From the results, Lyzls may play a role in mitochondrial function of spermatozoa and LYZL6 may contribute to the innate immunity of the male genital tract.

Deep sequencing analysis of small non-coding RNAs reveals the diversity of microRNAs and piRNAs in the human epididymis.

The epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. Small non-coding RNAs (sncRNAs), especially microRNAs (miRNAs), can participate in the regulation of various physiological pathways. However, their abundance and whether they are involved in the regulation of gene expression in the human epididymis are unknown. By adopting the Solexa deep sequencing approach, we systematically investigated the sncRNAs in the adult human epididymis. A total of 4903 unique sequences representing 527 known miRNA were discovered. Eighteen novel miRNA genes encoding 23 mature miRNAs were also identified and the expression of some of them was confirmed by qRT-PCR. The presence of Piwi-interacting RNAs (piRNAs) in the library also adds to the diversity of the sncRNA population in the human epididymis. This research will contribute to a preliminary database for their functional study in male reproductive system.

Topical administration of peroxiredoxin-6 on the cornea suppresses inflammation and neovascularization induced by ultraviolet radiation.

PURPOSE: To investigate the effect of topical administration of peroxiredoxin-6 (PRDX6) on ultraviolet-induced corneal injury. METHODS: Corneal transparency and neovascularization were observed with a slit-lamp microscope and hematoxylin and eosin staining. The oxidative damage was determined with a commercial malondialdehyde (MDA) kit. The expressions of PRDX6, polymorphonuclear neutrophil (PMN), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF) were determined by immunohistochemistry and Western blot. The expressions of genes related with antioxidant defense systems and cell apoptosis were detected by RT-PCR. RESULTS: The irradiated corneas appeared opaque and had high levels of MDA. Peripheral neovascularization and neutrophils appeared in the control and buffer-treated groups (with no treatment or PRDX6 diluent, respectively), whereas they were significantly suppressed in the PRDX6-treated group. The MDA content of the corneas in the PRDX6-treated group was significantly lower than that of the control and buffer-treated groups (P < 0.05). In the PRDX6-treated group the immunoreactivity of VEGF was lower, and that of PEDF was higher, than that in the control and buffer-treated groups. In addition, there were expression correlations between PRDX6 and PMN, VEGF, PEDF. The expressions of genes related with antioxidant defense systems and cell apoptosis were significant different between buffer- and PRDX6-treated groups (P < 0.05). CONCLUSIONS: The topically administered PRDX6 maintained the homeostasis of corneal cells, reduced inflammation, and suppressed neovascularization and apoptosis under ultraviolet irradiation.

[Repair of unilateral complete cleft lip with the method of rotation descent step by step].

OBJECTIVE: To explore the clinical effect of the method of rotation descent step by step for unilateral complete cleft lip with. METHODS: The method of rotation descent step by step was used to repair unilateral complete cleft lip. Point X was located on the angular bisector of 123, rotation descent step by step and orbicularis oris degloved dissection were used. Nasal deformities were corrected at the same stage. RESULTS: From Oct. 2006 to Oct. 2008, 68 cases were treated with primary healing. 42 cases were followed up for less than one year. Among them, 3 cases showed asymmetric lip height, and 6 cases showed asymmetric lip width. 26 cases were followed up more than one year with only two cases of asymmetric lip width. CONCLUSIONS: The method of rotation descent step by step is very suitable for repairing unilateral complete cleft lip. The restoration of the malpositioned tissue is especially emphasized in this method.

[Risk factors on anemia among rural elderly women aged 50-75 y in Xinning county, Anhui province, China].

OBJECTIVE: To study the risk factors on anemia among elderly women in rural areas of Xiuning county, Anhui province, China. METHODS: Xiuning county was selected as working field and elderly women aged 50-75 y were selected as subjects. Finger hemoglobin (Hb) was measured and basic health survey was face-to-face interviewed. 220 elderly women with anemia entered into the case group; and matched by age, another 220 women with normal Hb concentration entered the control group. Survey on diet, questionnaire regarding health and lifestyle and related blood indexes were studied and tested. RESULTS: When comparing the data from both case and control groups, weight was (49.4 +/- 7.3) kg vs. (52.5 +/- 8.4) kg (t = 3.97, P < 0.01), waist circumference was (75.8 +/- 7.8) cm vs. (79.1 +/- 9.3) cm (t = 3.85, P < 0.01), BMI was (21.8 +/- 2.6) kg/m2 vs. (22.9 +/- 3.2) kg/m2 (t = 3.775, P < 0.01), respectively. The total protein was (76.4 +/- 5.0) g/L vs. (78.4 +/- 5.6)g/L (t = 3.83, P < 0.01), albumin was (45.7 +/- 3.1) g/L vs. (47.3 +/- 2.9)g/L (t = 5.24, P < 0.01), serum iron was ( 10.3 +/- 4.1) micromol/L vs. (12.7 +/- 4.6) micromol/L (t = 5.48, P < 0.01), and saturation of transferrin was (19.0 +/- 7.6)% vs. (23.1 +/- 9.1) % (t = 4.90, P < 0.01), respectively. Results from multifactor conditioned logistic regression analysis showed that the odd ratios (OR) for anemia with staple food, BMI and vitamin A were 1.54, 1.89, 1.69, and the OR for anemia with BMI, staple food, animal food, carbohydrate and vitamin A were 2.0, 1.6, 1.6, 1.4, 1.6, with their confidence intervals (CI) as 1.3-2.9, 1.1-2.3, 1.0-2.3, 1.0-2.1, 1.1-2.4, respectively. CONCLUSION: The quality of diet, health status and related blood indexes on anemia among elderly women were lower than that in control group. Lower BMI, less staple food and animal food, less carbohydrate and vitamin A intake appeared to be risk factors of anemia.

[The expression of basic fibroblast growth factor 1 during human embryonic yolk sac hematopoiesis].

OBJECTIVE: To explore the effect of basic fibroblast growth factor 1 (bFGF1) during embryonic development on hematopoiesis, to study the expression of FGF1, vascular endothelial growth factor receptor (KDR), CD133, CD34 and transcription factors Ihh, SCL, GATA-1, GATA-2 and PU. 1 in the yolk sac, and to learn about the role and relationships of FGF1, hematopoietic cells and transcription factors during embryonic hematopoiesis. METHODS: 10 microm sections and total RNA were prepared from 107 human embryos aged 3-12 weeks. Immunohischemical SP staining and RT-PCR were performed. RESULTS: The yolk sac blood islands of human 3 approximately 12 weeks embryos consisted of peripheral vascular endothelial cells and central hematopoietic cells. The expression of FGF1 was firstly found in visceral mesoderm around periphery of yolk sac blood island at day 16, while was little inside it. KDR was not or lowly expressed and CD34 and CD133 were not expressed then. The expression increased, gray value decreased and staining enhanced at day 21. Strong staining of CD34+, CD133+ and KDR+ cells were found in blood island and mesoderm at day 30, their gray values changed from 156 +/- 16, 173 +/- 18 and 160 +/- 14 to 53 +/- 7, 52 +/- 6 and 69 +/- 8 respectively. FGF1 expression was strong positive, the gray value declined dramatically from 161 +/- 13 to 40 +/- 5. Some positive cells formed vessel-like structure along the periphery of blood island. Moderate expression of CD34+, CD133+, KDR+ cells increased at day 45, the cells aggregated into mass in blood island and FGF1+ cells did the same in blood island, while little in mesoderm. Its gray valve was increased. After 7 weeks, CD133+, KDR+, CD34+ cells significantly decreased their gray values increased, the staining became week. FGF1 was weakly expressed in yolk sac and its gray value increased to 179 +/- 22. RT-PCR showed Ihh, SCL, GATA-1 and GATA-2 were expressed at different time in yolk sac. PU. 1 were not expressed at day 16, and then expressed. CONCLUSIONS: The hematopoietic properties of yolk sac may be dependent on signaling through FGF receptors and FGF1 plays an important role in hematopoietic stem cell homeostasis. The FGF pathway regulates primitive hematopoiesis by modulating transcription factors such as Gata1 expression level and activity.