DOCSLIB.ORG

Calmegin Is Required for Fertilin <Alpha>/<Beta> Heterodimerization

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Calmegin Is Required for Fertilin <Alpha>/<Beta> Heterodimerization Developmental Biology 240, 254–261 (2001) doi:10.1006/dbio.2001.0462, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE Calmegin Is Required for Fertilin ␣/␤ provided by Elsevier - Publisher Connector Heterodimerization and Sperm Fertility Masahito Ikawa,* Tomoko Nakanishi,* Shuichi Yamada,*,† Ikuo Wada,‡ Katsuya Kominami,§ Hiromitsu Tanaka,¶ Masami Nozaki,¶ Yoshitake Nishimune,¶ and Masaru Okabe*,‡,1 *Genome Information Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565- 0871, Japan; †Program for Promotion of Basic Research Activities for Innovative Biosciences; ‡Department of Biochemistry, Sapporo Medical School of Medicine, Sapporo 060-8556, Japan; §Research and Development Center, Fuso Pharmaceutical Industries, Osaka 536-8523, Japan; and ¶Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607–611). In the present study, we demonstrate that calmegin ؊/؊ sperm were defective in migrating into the oviducts and in binding to the egg plasma membrane. Despite the impaired adhesive function, calmegin ؊/؊ sperm could fertilize eggs when zonae pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term. Since these sperm characteristics were similar to those found in ,fertilin ␤ ؊/؊ sperm, we investigated the interaction of calmegin with fertilin ␤. Using immunoprecipitation techniques calmegin was found to bind to sperm membrane proteins, fertilin ␣ and ␤, during spermatogenesis. The binding was specific to calmegin: another endoplasmic reticulum chaperone calnexin, a calmegin homologue, was not able to bind to fertilin ␣ and ␤. In the calmegin ؊/؊ mice, a loss of heterodimerization of fertilin ␣ and ␤ was observed and fertilin ␤ was not detectable in mature sperm. The data not only explain why the calmegin and fertilin ␤ knockout mouse lines share a common infertile phenotype, but also reveal the importance of the maturation of sperm membrane proteins in the endoplasmic reticulum. © 2001 Academic Press Key Words: ADAM; chaperone; calmegin; calnexin; cyritestin; egg plasma membrane; fertilin; fertilization; sperm; zona pellucida. INTRODUCTION when the calnexin interaction is disrupted, some nascent membrane proteins are incorrectly folded or assembled and Many complex membrane proteins undergo subunit fold- are retrotranslocated to cytosolic proteasomes (Chevet et ing and assembly in the endoplasmic reticulum (ER) before al., 1999; Fayadat et al., 2000). transport to the cell surface. Among the ER resident pro- Calmegin was first identified as a spermatogenic cell- teins, calnexin, a molecular chaperone for N-linked glyco- specific antigen which is present from pachytene spermato- proteins, functions as a component of ER quality control cytes to round spermatids (Watanabe et al., 1992). Later it machinery (Ellgaard et al., 1999; Trombetta and Helenius, was proved to be a Ca2ϩ binding protein and to be a calnexin 1998). Mutations in membrane proteins are known to cause homologue in the testis (Watanabe et al., 1994). In our some diseases such as cystic fibrosis, where improperly previous paper, we showed that calmegin is essential for folded mutant protein (cystic fibrosis transmembrane con- sperm fertility since the sperm from calmegin Ϫ/Ϫ mice ductance regulator, CFTR) is retained by calnexin inside the lack the ability to bind the zonae pellucidae of unfertilized ER and fails to be transported to the plasma membrane eggs (Ikawa et al., 1997). Our data suggested that the (Chevet et al., 1999; Pind et al., 1994). On the other hand, calmegin chaperones a sperm membrane protein required for adhesion to zona pellucida, but it remained unclear 1 To whom correspondence should be addressed. Fax: ϩ81-6- which membrane proteins are missing and how the loss 6879-8376. E-mail: [email protected]. occurs. 0012-1606/01 $35.00 Copyright © 2001 by Academic Press 254 All rights of reproduction in any form reserved. Calmegin Is Required for Fertilin Dimerization 255 Apart from our work, defective zona binding has also microscope. Following the partial zona dissection, eggs were de- been observed in sperm from mice lacking fertilin ␤, also tached from the dish by introducing BSA-containing medium into known as an ADAM2 (a member of the ADAM family, the drop. The eggs were then washed with TYH medium and transmembrane proteins containing adisintegrin and met- subjected to in vitro fertilization. Fertilization events were judged alloprotease domain) (Cho et al., 1998; Primakoff and by pronuclei formation 8 h after sperm insemination. Fertilized eggs were cultivated until the blastocyst stage and then transferred Myles, 2000). Fertilin consists of an ␣ and a ␤ subunit and to pseudopregnant females. has been proposed to be a sperm/egg fusion protein (Blobel et al., 1992; Cho et al., 2000). The defect of fertilin ␤ Ϫ/Ϫ sperm was reported to include impaired migration of sperm Transgenic Mice Expressing Calmegin into the oviduct and disabled binding of sperm to the egg A rescue construct was prepared in the pBluescript SK IIϩ plasma membrane. Here, we show that calmegin Ϫ/Ϫ plasmid. The SacI–BamHI fragment (330 bp) of the calmegin sperm share phenotypes in common with fertilin ␤ Ϫ/Ϫ promoter region (Watanabe et al., 1995) was placed in front of the sperm. Further analysis revealed that mature type fertilin BamHI–EcoRI fragment (2146 bp) containing calmegin cDNA with was lacking on calmegin Ϫ/Ϫ sperm since calmegin was a polyadenylation signal. TgN(CM-CM)Osb transgenic mouse lines important in fertilin ␣/␤ heterodimerization during sper- were produced by injecting the SacI–EcoRI fragment of the rescue construct into pronuclei of B6C3F1 ϫ B6C3F1 fertilized eggs. matogenesis. Offspring carrying the transgene were identified by PCR using Primer C (5Ј-CCTTCCTgCg gCTTgTTCTC T-3Ј) and Primer D (5Ј-TATCATCCTT CTTTgCTTTT g-3Ј) for calmegin cDNA. The MATERIALS AND METHODS endogenous calmegin mutation was detected by PCR using Primer A(5Ј-TCTTACCACA AAgCACCTCC-3Ј) and Primer-B (5Ј- Sperm Migration Analysis gCACAACAgg ATggATgATT-3Ј). B6C3F1 females were superovulated by intraperitoneal injection of 5 units of pregnant mare’s serum gonadotropin followed 48 h Immunoprecipitation and Western Blotting later by 5 units of human chorionic gonadotropin (HCG). Super- Analysis ovulated females were caged together with calmegin mutant males 12 h after HCG injection, and the formation of vaginal plug was Rabbit polyclonal antisera were raised against synthesized pep- observed every 30 min. About 2 h after copulation, oviducts were tides, murine calnexin (C-terminal, EDEILNRSPRNRKPRRE), excised together with a connective part of the uterus. The sperm calmegin (C-terminal, DESPGSGDAPLKSLRKRRVRKD), and fer- ␤ were flushed out from the remainder of the uterus with 1.0 ml of tilin (C-terminal, MKWRMDDFSSEEQFESESESKD). Rabbit anti- ␣ TYH medium and an aliquot was used for counting sperm num- fertilin “metalloprotease and prodomain” antiserum was a gift bers. To detect sperm in the utero-tubal junction, the oviduct with from Dr. Paul Primakoff. These antibodies were used for immuno- attached uterus was fixed in 4% paraformaldehyde–PBS for 6 h, precipitation and Western blotting for testicular lysates. To detect ␤ followed by washing with PBS, and was then prepared for frozen fertilin on sperm, mAb 9D was purchased from CHEMICON sections. International, Inc. Cell lysates or immunoprecipitates, prepared as before (Ikawa et al., 1997), were subjected to SDS–PAGE under nonreducing (0.5% SDS at room temperature) or reducing condi- Sperm Binding Analysis tions (2% SDS, 5% ␤-mercaptoethanol and boiled) and then ana- lyzed by Western blotting. The eggs were collected from oviducts of superovulated mice at 16 h after HCG injection and placed into a drop of TYH medium under mineral oil. The eggs were freed from cumulus cells by Ϫ1 RESULTS incubating with hyaluronidase (500 ␮gml ; Sigma) for 5 min followed by washing. Half of the cumulus-free eggs were treated Defective Sperm Migration into Oviduct in with acidic Tyrode’s solution (Sigma) for about 30 s to remove the ؊ ؊ zonae pellucidae and were then washed with TYH medium. The Calmegin / Sperm zona-intact and zona-free eggs were transferred into 200-␮l drops of To observe the effect of calmegin disruption on sperm TYH medium covered with mineral oil. Sperm prepared from migration from the uterus into the oviduct, cross sections caudae epididymides of 3-month-old male mice were dispersed into of the utero-tubal junction were prepared and observed 2 h TYH medium and, following 120 min of preincubation, were mixed after coitus. We found few sperm from calmegin Ϫ/Ϫ mice with eggs to a final density of 1–2 ϫ 105 sperm mlϪ1. After 15 min into the colliculus, the initial part of the oviduct, while of coincubation, the eggs were washed gently with TYH medium and sperm bound to zona pellucida or egg plasma membrane were numerous sperm were observed in the nearby uterine lu- observed. men (Figs. 1A–1D). There was no difference in the numbers of wild-type and calmegin Ϫ/Ϫ sperm recovered from the uterus (5.9 Ϯ 2.0 ϫ 106 and 5.7 Ϯ 1.9 ϫ 106, respectively). Partial Zona Dissection and in Vitro Fertilization The partial zona dissection was performed following the method Impaired Adhesion to Zona-Free Eggs in Calmegin described by Nakagata et al. (1997). Briefly, the cumulus-free eggs ؊/؊ Sperm were transferred into 0.3 M sucrose/PB1 without BSA on bacterial- grade culture dish. Zonae pellucidae were then dissected manually In our previous report, sperm adhesion to zona pellucida with the tip of a 30-gauge injection needle under a dissecting was observed to be defective (Ikawa et al., 1997). In the Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 256 Ikawa et al. FIG. 1. Defective migration of calmegin Ϫ/Ϫ sperm into oviduct found.
Load more

Recommended publications
  • SPATA33 Localizes Calcineurin to the Mitochondria and Regulates Sperm Motility in Mice
    SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility in mice Haruhiko Miyataa, Seiya Ouraa,b, Akane Morohoshia,c, Keisuke Shimadaa, Daisuke Mashikoa,1, Yuki Oyamaa,b, Yuki Kanedaa,b, Takafumi Matsumuraa,2, Ferheen Abbasia,3, and Masahito Ikawaa,b,c,d,4 aResearch Institute for Microbial Diseases, Osaka University, Osaka 5650871, Japan; bGraduate School of Pharmaceutical Sciences, Osaka University, Osaka 5650871, Japan; cGraduate School of Medicine, Osaka University, Osaka 5650871, Japan; and dThe Institute of Medical Science, The University of Tokyo, Tokyo 1088639, Japan Edited by Mariana F. Wolfner, Cornell University, Ithaca, NY, and approved July 27, 2021 (received for review April 8, 2021) Calcineurin is a calcium-dependent phosphatase that plays roles in calcineurin can be a target for reversible and rapidly acting male a variety of biological processes including immune responses. In sper- contraceptives (5). However, it is challenging to develop molecules matozoa, there is a testis-enriched calcineurin composed of PPP3CC and that specifically inhibit sperm calcineurin and not somatic calci- PPP3R2 (sperm calcineurin) that is essential for sperm motility and male neurin because of sequence similarities (82% amino acid identity fertility. Because sperm calcineurin has been proposed as a target for between human PPP3CA and PPP3CC and 85% amino acid reversible male contraceptives, identifying proteins that interact with identity between human PPP3R1 and PPP3R2). Therefore, identi- sperm calcineurin widens the choice for developing specific inhibitors. fying proteins that interact with sperm calcineurin widens the choice Here, by screening the calcineurin-interacting PxIxIT consensus motif of inhibitors that target the sperm calcineurin pathway. in silico and analyzing the function of candidate proteins through the The PxIxIT motif is a conserved sequence found in generation of gene-modified mice, we discovered that SPATA33 inter- calcineurin-binding proteins (8, 9).
    [Show full text]
  • Mammalian Male Germ Cells Are Fertile Ground for Expression Profiling Of
    REPRODUCTIONREVIEW Mammalian male germ cells are fertile ground for expression profiling of sexual reproduction Gunnar Wrobel and Michael Primig Biozentrum and Swiss Institute of Bioinformatics, Klingelbergstrasse 50-70, 4056 Basel, Switzerland Correspondence should be addressed to Michael Primig; Email: [email protected] Abstract Recent large-scale transcriptional profiling experiments of mammalian spermatogenesis using rodent model systems and different types of microarrays have yielded insight into the expression program of male germ cells. These studies revealed that an astonishingly large number of loci are differentially expressed during spermatogenesis. Among them are several hundred transcripts that appear to be specific for meiotic and post-meiotic germ cells. This group includes many genes that were pre- viously implicated in spermatogenesis and/or fertility and others that are as yet poorly characterized. Profiling experiments thus reveal candidates for regulation of spermatogenesis and fertility as well as targets for innovative contraceptives that act on gene products absent in somatic tissues. In this review, consolidated high density oligonucleotide microarray data from rodent total testis and purified germ cell samples are analyzed and their impact on our understanding of the transcriptional program governing male germ cell differentiation is discussed. Reproduction (2005) 129 1–7 Introduction 2002, Sadate-Ngatchou et al. 2003) and the absence of cAMP responsive-element modulator (Crem) and deleted During mammalian male
    [Show full text]
  • Experimental Validation of the Regulated Expression of Large Numbers of Non-Coding Rnas from the Mouse Genome
    Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Article Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome Timothy Ravasi,1,4,5 Harukazu Suzuki,2,4 Ken C. Pang,1,3,4 Shintaro Katayama,2,4 Masaaki Furuno,2,4,6 Rie Okunishi,2 Shiro Fukuda,2 Kelin Ru,1 Martin C. Frith,1,2 M. Milena Gongora,1 Sean M. Grimmond,1 David A. Hume,1 Yoshihide Hayashizaki,2 and John S. Mattick1,7 1ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia; 2Laboratory for Genome Exploration Research Group, RIKEN Genomic Science Center, RIKEN Yokohama Institute, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan; 3T Cell Laboratory, Ludwig Institute for Cancer Research, Austin & Repatriation Medical Centre, Heidelberg VIC 3084, Australia Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did not contain an apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-mRNA contamination during library construction, or alternatively represent nonspecific “transcriptional noise.” Here we show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts of unknown function whose expression appears to be regulated.
    [Show full text]
  • Association of Gene Ontology Categories with Decay Rate for Hepg2 Experiments These Tables Show Details for All Gene Ontology Categories
    Supplementary Table 1: Association of Gene Ontology Categories with Decay Rate for HepG2 Experiments These tables show details for all Gene Ontology categories. Inferences for manual classification scheme shown at the bottom. Those categories used in Figure 1A are highlighted in bold. Standard Deviations are shown in parentheses. P-values less than 1E-20 are indicated with a "0". Rate r (hour^-1) Half-life < 2hr. Decay % GO Number Category Name Probe Sets Group Non-Group Distribution p-value In-Group Non-Group Representation p-value GO:0006350 transcription 1523 0.221 (0.009) 0.127 (0.002) FASTER 0 13.1 (0.4) 4.5 (0.1) OVER 0 GO:0006351 transcription, DNA-dependent 1498 0.220 (0.009) 0.127 (0.002) FASTER 0 13.0 (0.4) 4.5 (0.1) OVER 0 GO:0006355 regulation of transcription, DNA-dependent 1163 0.230 (0.011) 0.128 (0.002) FASTER 5.00E-21 14.2 (0.5) 4.6 (0.1) OVER 0 GO:0006366 transcription from Pol II promoter 845 0.225 (0.012) 0.130 (0.002) FASTER 1.88E-14 13.0 (0.5) 4.8 (0.1) OVER 0 GO:0006139 nucleobase, nucleoside, nucleotide and nucleic acid metabolism3004 0.173 (0.006) 0.127 (0.002) FASTER 1.28E-12 8.4 (0.2) 4.5 (0.1) OVER 0 GO:0006357 regulation of transcription from Pol II promoter 487 0.231 (0.016) 0.132 (0.002) FASTER 6.05E-10 13.5 (0.6) 4.9 (0.1) OVER 0 GO:0008283 cell proliferation 625 0.189 (0.014) 0.132 (0.002) FASTER 1.95E-05 10.1 (0.6) 5.0 (0.1) OVER 1.50E-20 GO:0006513 monoubiquitination 36 0.305 (0.049) 0.134 (0.002) FASTER 2.69E-04 25.4 (4.4) 5.1 (0.1) OVER 2.04E-06 GO:0007050 cell cycle arrest 57 0.311 (0.054) 0.133 (0.002)
    [Show full text]
  • Genomic and Expression Profiling of Human Spermatocytic Seminomas: Primary Spermatocyte As Tumorigenic Precursor and DMRT1 As Candidate Chromosome 9 Gene
    Research Article Genomic and Expression Profiling of Human Spermatocytic Seminomas: Primary Spermatocyte as Tumorigenic Precursor and DMRT1 as Candidate Chromosome 9 Gene Leendert H.J. Looijenga,1 Remko Hersmus,1 Ad J.M. Gillis,1 Rolph Pfundt,4 Hans J. Stoop,1 Ruud J.H.L.M. van Gurp,1 Joris Veltman,1 H. Berna Beverloo,2 Ellen van Drunen,2 Ad Geurts van Kessel,4 Renee Reijo Pera,5 Dominik T. Schneider,6 Brenda Summersgill,7 Janet Shipley,7 Alan McIntyre,7 Peter van der Spek,3 Eric Schoenmakers,4 and J. Wolter Oosterhuis1 1Department of Pathology, Josephine Nefkens Institute; Departments of 2Clinical Genetics and 3Bioinformatics, Erasmus Medical Center/ University Medical Center, Rotterdam, the Netherlands; 4Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands; 5Howard Hughes Medical Institute, Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts; 6Clinic of Paediatric Oncology, Haematology and Immunology, Heinrich-Heine University, Du¨sseldorf, Germany; 7Molecular Cytogenetics, Section of Molecular Carcinogenesis, The Institute of Cancer Research, Sutton, Surrey, United Kingdom Abstract histochemistry, DMRT1 (a male-specific transcriptional regulator) was identified as a likely candidate gene for Spermatocytic seminomas are solid tumors found solely in the involvement in the development of spermatocytic seminomas. testis of predominantly elderly individuals. We investigated these tumors using a genome-wide analysis for structural and (Cancer Res 2006; 66(1): 290-302) numerical chromosomal changes through conventional kar- yotyping, spectral karyotyping, and array comparative Introduction genomic hybridization using a 32 K genomic tiling-path Spermatocytic seminomas are benign testicular tumors that resolution BAC platform (confirmed by in situ hybridization).
    [Show full text]
  • Calmegin (CLGN) Rabbit Polyclonal Antibody – TA590180 | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA590180 Calmegin (CLGN) Rabbit Polyclonal Antibody Product data: Product Type: Primary Antibodies Applications: ELISA, IHC, WB Recommended Dilution: WB 1:5000~20000, IHC 1:150,ELISA 1:100-1:2000 Reactivity: Human, Monkey, Rat Host: Rabbit Isotype: IgG Clonality: Polyclonal Immunogen: DNA immunization. This antibody is specific for the C Terminus Region of the target protein. Formulation: 20 mM Potassium Phosphate, 150 mM Sodium Chloride, pH 7.0 Concentration: 1.09635 mg/ml Purification: Purified from mouse ascites fluids or tissue culture supernatant by affinity chromatography (protein A/G) Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Gene Name: calmegin Database Link: NP_004353 Entrez Gene 685504 RatEntrez Gene 698361 MonkeyEntrez Gene 1047 Human O14967 Background: Calmegin is a testis-specific endoplasmic reticulum chaperone protein. CLGN may play a role in spermatogeneisis and infertility. [provided by RefSeq] Synonyms: calmegin Note: This antibody was generated by SDIX's Genomic Antibody Technology ® (GAT). Learn about GAT Protein Families: Transmembrane This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 3 Calmegin (CLGN) Rabbit Polyclonal Antibody – TA590180 Product images: HEK293T cells were transfected with the pCMV6- ENTRY control (Cat# [PS100001], Left lane) or pCMV6-ENTRY CLGN (Cat# [RC205301], Right lane) cDNA for 48 hrs and lysed.
    [Show full text]
  • Novel Targets of Apparently Idiopathic Male Infertility
    International Journal of Molecular Sciences Review Molecular Biology of Spermatogenesis: Novel Targets of Apparently Idiopathic Male Infertility Rossella Cannarella * , Rosita A. Condorelli , Laura M. Mongioì, Sandro La Vignera * and Aldo E. Calogero Department of Clinical and Experimental Medicine, University of Catania, 95123 Catania, Italy; [email protected] (R.A.C.); [email protected] (L.M.M.); [email protected] (A.E.C.) * Correspondence: [email protected] (R.C.); [email protected] (S.L.V.) Received: 8 February 2020; Accepted: 2 March 2020; Published: 3 March 2020 Abstract: Male infertility affects half of infertile couples and, currently, a relevant percentage of cases of male infertility is considered as idiopathic. Although the male contribution to human fertilization has traditionally been restricted to sperm DNA, current evidence suggest that a relevant number of sperm transcripts and proteins are involved in acrosome reactions, sperm-oocyte fusion and, once released into the oocyte, embryo growth and development. The aim of this review is to provide updated and comprehensive insight into the molecular biology of spermatogenesis, including evidence on spermatogenetic failure and underlining the role of the sperm-carried molecular factors involved in oocyte fertilization and embryo growth. This represents the first step in the identification of new possible diagnostic and, possibly, therapeutic markers in the field of apparently idiopathic male infertility. Keywords: spermatogenetic failure; embryo growth; male infertility; spermatogenesis; recurrent pregnancy loss; sperm proteome; DNA fragmentation; sperm transcriptome 1. Introduction Infertility is a widespread condition in industrialized countries, affecting up to 15% of couples of childbearing age [1]. It is defined as the inability to achieve conception after 1–2 years of unprotected sexual intercourse [2].
    [Show full text]
  • V-Set and Immunoglobulin Domain-Containing 1 (VSIG1), Predominantly Expressed in Testicular Germ Cells, Is Dispensable for Spermatogenesis and Male Fertility in Mice
    animals Article V-Set and Immunoglobulin Domain-Containing 1 (VSIG1), Predominantly Expressed in Testicular Germ Cells, Is Dispensable for Spermatogenesis and Male Fertility in Mice Yena Jung 1,2,†, Hyewon Bang 1,†, Young-Hyun Kim 3,†, Na-Eun Park 1, Young-Ho Park 2, Chaeli Park 1, Sang-Rae Lee 4, Jeong-Woong Lee 5 , Bong-Seok Song 2 , Ji-Su Kim 2, Bo-Woong Sim 2 , Dong-Won Seol 6, Gabbine Wee 6, Sunhyung Kim 7, Sun-Uk Kim 2 and Ekyune Kim 1,* 1 College of Pharmacy, Catholic University of Daegu, Gyeongsan-si 38430, Korea; [email protected] (Y.J.); [email protected] (H.B.); [email protected] (N.-E.P.); qkrcofl[email protected] (C.P.) 2 Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 28116, Korea; [email protected] (Y.-H.P.); [email protected] (B.-S.S.); [email protected] (J.-S.K.); [email protected] (B.-W.S.); [email protected] (S.-U.K.) 3 National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 28116, Korea; [email protected] 4 Laboratory Animal Research Center, School of Medicine, Ajou University, Suwon 16499, Korea; [email protected] 5 Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Deajeon 34141, Korea; [email protected] Citation: Jung, Y.; Bang, H.; Kim, 6 Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), Y.-H.; Park, N.-E.; Park, Y.-H.; Park, Daegu 41061, Korea; [email protected] (D.-W.S.); [email protected] (G.W.) C.; Lee, S.-R.; Lee, J.-W.; Song, B.-S.; 7 Department of Environmental Horticulture, University of Seoul, Seoul 02504, Korea; [email protected] Kim, J.-S.; et al.
    [Show full text]
  • BMC Bioinformatics
    BMC Bioinformatics This Provisional PDF corresponds to the article as it appeared upon acceptance. The fully-formatted PDF version will become available shortly after the date of publication, from the URL listed below. Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation BMC Bioinformatics 2006, 7:237 doi:10.1186/1471-2105-7-237 Jamie A Lee ([email protected]) Robert S Sinkovits ([email protected]) Dennis Mock ([email protected]) Eva L Rab ([email protected]) Jennifer Cai ([email protected]) Peng Yang ([email protected]) Brian Saunders ([email protected]) Robert C Hsueh ([email protected]) Sangdun Choi ([email protected]) Shankar Subramaniam ([email protected]) Richard H Scheuermann ([email protected]) In collaboration with the Alliance for Cellular Signaling ([email protected]) ISSN 1471-2105 Article type Research article Submission date 21 September 2005 Acceptance date 2 May 2006 Publication date 2 May 2006 Article URL http://www.biomedcentral.com/1471-2105/7/237 Like all articles in BMC journals, this peer-reviewed article was published immediately upon acceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright notice below). Articles in BMC journals are listed in PubMed and archived at PubMed Central. For information about publishing your research in BMC journals or any BioMed Central journal, go to © 2006 Lee et al., licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
    [Show full text]
  • Update on the Proteomics of Male Infertility: a Systematic Review
    Arab Journal of Urology (2018) 16, 103–112 Arab Journal of Urology (Official Journal of the Arab Association of Urology) www.sciencedirect.com DIAGNOSIS ORIGINAL ARTICLE Update on the proteomics of male infertility: A systematic review Manesh Kumar Panner Selvam, Ashok Agarwal * American Centre for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA Received 28 October 2017, Received in revised form 17 November 2017, Accepted 19 November 2017 Available online 29 December 2017 KEYWORDS Abstract Objective: To assess the role of differentially expressed proteins as a resource for potential biomarker identification of infertility, as male infertility is Sperm proteomics; of rising concern in reproductive medicine and evidence pertaining to its aetiology Male infertility; at a molecular level particularly proteomic as spermatozoa lack transcription and Spermatozoa; translation. Proteomics is considered as a major field in molecular biology to vali- Varicocele; date the target proteins in a pathophysiological state. Differential expression analy- Biomarkers sis of sperm proteins in infertile men and bioinformatics analysis offer information about their involvement in biological pathways. Materials and methods: Literature search was performed on PubMed, Medline, and Science Direct databases using the keywords ‘sperm proteomics’ and ‘male infer- tility’. We also reviewed the relevant cross references of retrieved articles and included them in the review process. Articles written in any language other than Eng- lish were excluded. Results: Of 575 articles identified, preliminary screening for relevant studies elim- inated 293 articles. At the next level of selection, from 282 studies only 80 articles related to male infertility condition met the selection criteria and were included in this review.
    [Show full text]
  • Agricultural University of Athens
    ΓΕΩΠΟΝΙΚΟ ΠΑΝΕΠΙΣΤΗΜΙΟ ΑΘΗΝΩΝ ΣΧΟΛΗ ΕΠΙΣΤΗΜΩΝ ΤΩΝ ΖΩΩΝ ΤΜΗΜΑ ΕΠΙΣΤΗΜΗΣ ΖΩΙΚΗΣ ΠΑΡΑΓΩΓΗΣ ΕΡΓΑΣΤΗΡΙΟ ΓΕΝΙΚΗΣ ΚΑΙ ΕΙΔΙΚΗΣ ΖΩΟΤΕΧΝΙΑΣ ΔΙΔΑΚΤΟΡΙΚΗ ΔΙΑΤΡΙΒΗ Εντοπισμός γονιδιωματικών περιοχών και δικτύων γονιδίων που επηρεάζουν παραγωγικές και αναπαραγωγικές ιδιότητες σε πληθυσμούς κρεοπαραγωγικών ορνιθίων ΕΙΡΗΝΗ Κ. ΤΑΡΣΑΝΗ ΕΠΙΒΛΕΠΩΝ ΚΑΘΗΓΗΤΗΣ: ΑΝΤΩΝΙΟΣ ΚΟΜΙΝΑΚΗΣ ΑΘΗΝΑ 2020 ΔΙΔΑΚΤΟΡΙΚΗ ΔΙΑΤΡΙΒΗ Εντοπισμός γονιδιωματικών περιοχών και δικτύων γονιδίων που επηρεάζουν παραγωγικές και αναπαραγωγικές ιδιότητες σε πληθυσμούς κρεοπαραγωγικών ορνιθίων Genome-wide association analysis and gene network analysis for (re)production traits in commercial broilers ΕΙΡΗΝΗ Κ. ΤΑΡΣΑΝΗ ΕΠΙΒΛΕΠΩΝ ΚΑΘΗΓΗΤΗΣ: ΑΝΤΩΝΙΟΣ ΚΟΜΙΝΑΚΗΣ Τριμελής Επιτροπή: Aντώνιος Κομινάκης (Αν. Καθ. ΓΠΑ) Ανδρέας Κράνης (Eρευν. B, Παν. Εδιμβούργου) Αριάδνη Χάγερ (Επ. Καθ. ΓΠΑ) Επταμελής εξεταστική επιτροπή: Aντώνιος Κομινάκης (Αν. Καθ. ΓΠΑ) Ανδρέας Κράνης (Eρευν. B, Παν. Εδιμβούργου) Αριάδνη Χάγερ (Επ. Καθ. ΓΠΑ) Πηνελόπη Μπεμπέλη (Καθ. ΓΠΑ) Δημήτριος Βλαχάκης (Επ. Καθ. ΓΠΑ) Ευάγγελος Ζωίδης (Επ.Καθ. ΓΠΑ) Γεώργιος Θεοδώρου (Επ.Καθ. ΓΠΑ) 2 Εντοπισμός γονιδιωματικών περιοχών και δικτύων γονιδίων που επηρεάζουν παραγωγικές και αναπαραγωγικές ιδιότητες σε πληθυσμούς κρεοπαραγωγικών ορνιθίων Περίληψη Σκοπός της παρούσας διδακτορικής διατριβής ήταν ο εντοπισμός γενετικών δεικτών και υποψηφίων γονιδίων που εμπλέκονται στο γενετικό έλεγχο δύο τυπικών πολυγονιδιακών ιδιοτήτων σε κρεοπαραγωγικά ορνίθια. Μία ιδιότητα σχετίζεται με την ανάπτυξη (σωματικό βάρος στις 35 ημέρες, ΣΒ) και η άλλη με την αναπαραγωγική
    [Show full text]
  • Calmegin (B-11): Sc-515251
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . Calmegin (B-11): sc-515251 BACKGROUND APPLICATIONS Calmegin, belonging to the calreticulin family, is expressed in testis as an Calmegin (B-11) is recommended for detection of Calmegin of human origin endoplasmic reticulum membrane protein, where it acts as a chaperone pro - by Western Blotting (starting dilution 1:100, dilution range 1:100-1:1000), tein and plays a role in spermatogenesis. Calmegin is a single-pass type I immunoprecipitation [1-2 µg per 100-500 µg of total protein (1 ml of cell membrane protein that is transcriptionally regulated in coordination by CpG lysate)], immunofluorescence (starting dilution 1:50, dilution range 1:50- methyltransferase and histone deacetylase (HDAC). First expressed in meiot - 1:500) and solid phase ELISA (starting dilution 1:30, dilution range 1:30- ic prophase of spermatocytes, Calmegin facilitates sperm-egg zona pellicuda 1:3000). binding through association with sperm membrane proteins (fertilin a and b). Suitable for use as control antibody for Calmegin siRNA (h): sc-60316, A loss in Calmegin results in male sterility. However, if the zona pellicuda is Calmegin shRNA Plasmid (h): sc-60316-SH and Calmegin shRNA (h) partially dissected and fertilized in vitro, the egg will develop normally. Lentiviral Particles: sc-60316-V. REFERENCES Molecular Weight (predicted) of Calmegin: 70 kDa. 1. Siep, M., et al. 2004. Basic helix-loop-helix transcription factor Tcfl5 inter - Molecular Weight (observed) of Calmegin: 93 kDa. acts with the Calmegin gene promoter in mouse spermatogenesis. Nucleic Positive Controls: human testis extract: sc-363781, human heart extract: Acids Res.
    [Show full text]