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Expression of ciliated bronchial epithelium 1 during human spermatogenesis
ORIGINAL ARTICLE: ANDROLOGY
Expression of ciliated bronchial epithelium 1 during human spermatogenesis Christiane Pleuger, M.Sc.,a Daniela Fietz, Dr.Med.Vet.,a Katja Hartmann, Dr.Med.Vet.,a Hans-Christian Schuppe, Dr.Med.,b Wolfgang Weidner, Dr.Med.,b Sabine Kliesch, Dr.Med.,c Mark Baker, Ph.D.,d Moira K. O'Bryan, Ph.D.,e and Martin Bergmann, Dr.Rer.Medic.a a Institute for Veterinary Anatomy, Histology und Embryology, and b Clinic for Urology, Pediatric Urology and Andrology, Justus Liebig University, Giessen, Germany; c Department of Clinical Andrology, Centre for Reproductive Medicine € nster, Mu € nster, Germany; d School of Environmental and Life Science, University and Andrology, University Hospital Mu of Newcastle, Callaghan, New South Wales, Australia; and e Development and Stems Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia
Objective: To define the precise cellular localization of ciliated bronchial epithelium 1 (CBE1) in the human testis and test its relationship to impaired spermatogenesis. Design: Gene expression analysis, and histologic and immunohistochemical evaluation. Setting: University research laboratories and andrologic outpatient clinic. Patient(s): Forty-three human testicular biopsies: 12 biopsies showing normal spermatogenesis (NSP), 8 with maturation arrest at level of spermatocytes (STA), 8 with maturation arrest at level of spermatids (SDA), 4 with scattered elongating spermatids, and 12 with Sertoli cell-only syndrome, with an additional 5 semen samples from healthy donors. Intervention(s): None. Main Outcome Measure(s): Evaluation of CBE1 expression in normal as well as impaired spermatogenesis on mRNA (quantitative reverse-transcription polymerase chain reaction and in situ hybridization) and protein level (immunohistochemistry, Western blot analysis). Result(s): In normal spermatogenesis, CBE1 mRNA was expressed in late pachytene spermatocytes, and the protein was localized within the flagellum of elongating spermatids from stage V up to the spermiation in stage II. Immunoelectron microscopy showed CBE1 clearly associated with microtubules at the manchette, the head-tail coupling apparatus, and the flagellum, but the protein was absent in spermatozoa. Compared with normal spermatogenesis, CBE1 mRNA was statistically significantly reduced in samples with a maturation arrest at the level of round spermatids and primary spermatocytes, and was absent in samples showing Sertoli cell-only syndrome. CBE1 protein was completely missing in SDA samples showing few elongating spermatids. Conclusion(s): Our data strongly suggest an influence of CBE1 in ciliogenesis in spermatids due to the localization at the microtubules of the elongating spermatids, indicating a role in the intramanchette and/or intraflagellar transport mechanism. The absence of CBE1 in spermatozoa suggests that CBE1 is important for the spermatid development but not for the maintenance of mature spermatozoa as a component of the flagellum. (Fertil SterilÒ 2017;-:-–-. Ó2017 by American Society for Reproductive Medicine.) Key Words: Intraflagellar transport, intramanchette transport, male fertility, microtubules, spermiogenesis Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/ 16110-fertility-and-sterility/posts/16846-24101
Received March 29, 2017; revised May 5, 2017; accepted May 14, 2017. C.P. has nothing to disclose. D.F. has received grants from the German Research Foundation (DFG). K.H. has nothing to disclose. H.-C.S. has received grants from the International Research Training Group ‘‘Molecular Pathogenesis of Male Reproductive Disorders’’ (German Research Foundation; DFG) and honoraria from Ferring, Germany, and Merck-Serono, Germany. W.W. has nothing to disclose. S.K. has nothing to disclose. M.B. has nothing to disclose. M.K.O. has received grants from the Monash IVF Research and Education Foundation. M.B. has nothing to disclose. Supported by the DFG (German Research Foundation)-IRTG 1871; and a fellowship from the National Health and Medical Research Council of Australia (APP1058356) (to M.K.O.). € ttingen, Germany, Presented as posters at 111th Annual Meeting of Anatomische Gesellschaft, Go September 21–24, 2016; and the 50th Annual Meeting of Physiologie und Pathologie der Fortpflanzung, Munich, Germany, February 15–17, 2017. Reprint requests: Christiane Pleuger, M.Sc., Institute for Veterinary Anatomy, Histology and Embryology, Justus Liebig University, Frankfurter Straße 98, 35392 Gießen, Germany (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2017 0015-0282/$36.00 Copyright ©2017 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2017.05.019 VOL. - NO. - / - 2017
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icrotubules play a crucial role during cell-division processes, such as mitosis and meiosis, and the development of microtubule-based structures during spermiogenesis. After meiotic reductive divisions, haploid round spermatids undergo many modifications to achieve the required characteristics for fertilization. This process includes the polarization of the nucleus with the acrosome to one side of the cell, the concomitant elongation of the 1
ORIGINAL ARTICLE: ANDROLOGY spermatid, the nuclear condensation and sperm head shaping by the manchette, as well as the assembly of the axoneme (1). The inappropriate formation of these microtubule-based structures and defects in the associated transport mechanisms along the manchette and flagellum of spermatids can lead to asthenozoospermia (reduced motility) and/or teratozoospermia (abnormal morphology), thus causing male infertility (2). The formation of the axoneme of the sperm flagellum shows parallels to the development of motile cilia in other tissues, such as the brain and the lung (3). The axoneme, the core of the flagellum, consists of a centrally located microtubule pair surrounded by nine outer microtubule doublets that are connected by the dynein heavy chains of the inner and outer dynein arms. Axoneme formation in many cilia and likely in the sperm tail involves a microtubulebased protein system known as intraflagellar transport (IFT). The mammalian sperm tail has additional characteristic peri-axonemal structures, such as nine outer dense fibers, the mitochondrial sheath, and fibrous sheath and is connected to the nucleus via the connecting piece (4). The structural features enable motility of the mature sperm and maintain structural integrity (5). Concordant with the onset of spermatid head elongation, the transient microtubule-based manchette comes into existence and persists until the elongation and condensation of the spermatid nucleus is completed (6). The manchette is thought to serve essential roles in nuclear shaping and to act as a protein transport platform for proteins involved in tail formation, known as intramanchette transport (IMT) (7). While not completely characterized and temporally distinct, the IFT likely shares similar molecular components to IMT. It is proposed that in both transport mechanisms, a multicomplex protein raft linked to cargo proteins and vesicles moves along a microtubule track (6, 8, 9). During IFT, essential proteins for the assembly and maintenance of the flagellum are transported in an anterograde direction by kinesins to the distal part of the flagellum (10) and in a retrograde direction by dyneins back toward the basal body (11). It is proposed that the IMT, which is transient just like the manchette itself, additionally enables an exchange of cargo between the Golgi, nucleus, and cytoplasm as well as the head-tail coupling apparatus (HTCA) (2, 8, 9, 12). Ciliated bronchial epithelium 1 (CBE1 alias SMRP1, 1110017D15Rik, C9orf24, NYD-SP22) is a protein that has been described in association with ciliated cells in bronchial tissue (13) and in the manchette of murine elongating spermatids (14). During pulmonary development, CBE1 shows a biphasic expression. CBE1 expression starts during the formation of the lung buds and shows a further expression in later stages (pseudoglandular stage) concurrent with the expression of the transcription factor forkhead box factor FOXJ1 (hepatocyte nuclear factor-3/forkhead homologue 4) (15), which is closely linked to ciliogenesis (16). Although these data indicate a role for CBE1 in establishing differentiation of the mucociliary epithelium (15), CBE1 is localized intracellularly in ciliated epithelial cells but not in the cilia structure (13). Previous investigations of the murine orthologue (called Smrp1 spermatid-manchette-related protein 1 in the original 2
study) detected the protein at the spermatid manchette around the nucleus where it colocalized with a-tubulin. Although three mRNA variants have been described, only one transcript variant was translated into the protein (14). To date, no precise molecular function has been described for CBE1. Although the murine orthologue shows a clear localization at the spermatid manchette (14), it is unknown whether CBE1 is either an integral part of this structure or it plays a role in the cargo transport. To clarify its potential relevance to human spermatogenesis, we investigated the mRNA expression and protein localization of CBE1 in human testes with normal and impaired spermatogenesis to gain information regarding the molecular function and a possible association with the phenotype of maturation arrests.
MATERIAL AND METHODS Testicular Tissue and Semen Samples Human testicular biopsy samples were obtained (with consent) from patients of the Centre of Reproductive Medicine and Andrology of the University Hospital in M€ unster (Germany) and the Department of Urology and Andrology of the University Hospital in Giessen (Germany). The surgical procedure was indicated because of obstructive (refertilization after vasectomy) or nonobstructive azoospermia as defined using the criteria according to Bergmann and Kliesch (17). In total, 75 testicular biopsy samples were used (44 samples to analyze mRNA expression and 31 to evaluate the protein localization in normal and impaired spermatogenesis). Immediately after surgical removal, the testicular tissues were immersed in Bouin's solution and ultimately embedded in paraffin. We stained 5-mm-thick sections with hematoxylin and eosin (H&E) to evaluate the quality of the spermatogenesis according to the score count protocol of Bergmann and Kliesch (17). The stages of spermatogenesis were differentiated in accordance with Clermont (18). To analyze the mRNA expression, we selected 12 biopsy samples showing normal spermatogenesis (NSP); 8 biopsy samples with a maturation arrest at the level of early round spermatids (SDA), whereby samples showed scattered elongating spermatids; 12 biopsy samples with a maturation arrest at the level of primary spermatocytes (STA); and 12 biopsy samples showing Sertoli cell-only syndrome (SCO). Human ejaculates (n ¼ 5) were received from five different healthy volunteers. A semen analysis was performed according to the World Health Organization's 2010 guidelines to measure sperm concentration, motility, morphology, and vitality (19). After the evaluation, the ejaculates were centrifuged at 500 g for 15 minutes, and the pellet was washed three times with sperm washing medium containing 5 mg/ mL of human serum albumin (Irvine Scientific) and stored at 20 C. The study was performed following the declaration of Helsinki. Written informed consent was obtained from all men involved. The study was approved by the ethics committee of the medical faculty of the Justus-Liebig-University Giessen (AZ 100/07; AZ 32/11). VOL. - NO. - / - 2017
Fertility and Sterility® CBE1 mRNA Expression We obtained messenger RNA (mRNA) from Bouin's fixed and paraffin embedded tissue by use of the RNeasy FFPE Kit (Qiagen) including proteinase K treatment as recommended by the manufacturer. To remove any contaminating genomic DNA, the extracted mRNA was incubated with RNase-free DNase (Peqlab Biotechnology), RNase-free incubation buffer 10x (Roche), RNase Inhibitor (2,000 U), and MgCl2 solution (Applied Biosystems by Thermo Fisher Scientific) for 25 minutes at 37 C. To synthesize the complementary DNA (cDNA), 8 mL of mRNA were mixed with 60 mL of RT-Mix (components from Applied Biosystems by Thermo Fisher Scientific) consisting of 10 mL of GeneAmp 10x PCR Gold Buffer, 10 mL of nucleotide mix (10 mM each), 10 mL of MgCl2 (25 mM), 5 mL of random hexamer primers (50 mM), 5 mL of RNase inhibitor (20 U/mL), 5 mL of MultiScribe Reverse Transcriptase (50 U/mL), and 15 mL of sterile distilled water. The negative control samples contained no reverse transcriptase. The synthesized cDNA was subsequently mixed with 2.5 mL of GeneAmp 10x PCR Gold Buffer, 2 mL of MgCl2 (25 mM), 1 mL of each forward and reverse primer (10 pmol/mL), 1 mL of nucleotide mix (10 mM each), 0.15 mL of AmpliTaq Gold polymerase, and 12.35 mL of sterile distilled water. We selected specific primers for human CBE1, b-actin, KASH5, and GAPDH (Table 1) using OligoExplorer 1.1.0 (T. Kuulasma, University of Kuopio, Kuopio, Finland); and purchased them from biomers.net (Ulm, Germany). After reversetranscription polymerase chain reaction (RT-PCR) (cycling conditions: 1 9 minutes at 95 C, 35 [45 seconds at 94 C, 45 seconds at 60 C, 45 seconds 72 C] and 7 minutes at 72 C), the PCR products were separated and visualized using gel electrophoresis (1.5 % agarose gel) with GelGreen Nucleic Acid Stain (Biotium). For quantitative RT-PCR (qRT-PCR), the same primers as previously were used with the 8 NSP and 8 SDA samples. We added 1 mL of cDNA of each sample with 19 mL of mastermix containing 4 mL of the intercalating fluorescent dye EvaGreen (Bio&Sell) as detection mode, 0.6 mL of forward and reverse primer, and 13.8 mL of sterile distilled water. We performed qRT-PCR using CFX96 RealTime cycler system (BioRad Laboratories) under the following conditions: 15 minutes at
95 C, 40 (15 seconds at 95 C, 30 seconds at 60 C, 20 seconds at 72 C). The melt curve analysis consisted of 10 seconds at 95 C, then 65 C to 95 C in increments 0.5 C for 5 seconds. The relative expression levels were calculated using the 2DDCt method whereby b-actin and KASH5 (for the primer sequence, Table 1), a specific spermatocyte marker (Klarsicht/ANC-1/ Syne/homology 5), were used as internal reference genes. We used GraphPad Prism 5.01 (GraphPad Software) for statistical analyses and diagram generation, and statistical significance was tested by one-way analysis of variance.
In Situ Hybridization To detect CBE1 mRNA in the cellular context, we generated a 467-bp amplicon (primer sequences in Table 1) as described earlier. To generate DIG-labeled cRNA probes, the RT-PCR product was ligated into pCR II TOPO vector (Invitrogen) as recommended by the manufacturer and subsequently transformed in One Shot TOP 10 Chemically Competent E. coli (Invitrogen) followed by plasmid extraction using QIAprep Spin Miniprep (Qiagen). After validating all plasmids by sequencing (performed by Scientific Research Development GmbH), the correct insertion of the PCR products was controlled by double digest using BamHI and NotI (New England Biolabs). We applied the same restriction enzymes for linearization of the plasmid DNA. For the subsequent in vitro transcription, we used RNA-DIG Labeling Mix (Boehringer Mannheim) and RNA polymerase T7 and SP6 (Promega). In situ hybridization was performed as described by Fietz et al. (20) with the following modifications. Deparaffinized and rehydrated tissue sections (5 mm) were incubated with proteinase K (10 mg/mL) in sterile diethylpyrocarbonate (DEPC)-treated water for 20 minutes at 37 C. Digestion was stopped by incubation in 0.2% glycine solution in phosphate-buffered saline supplemented with 1 mM MgCl2 (PBS-M) for 5 minutes. Subsequently, the sections were postfixed with 4% paraformaldehyde for 10 minutes, incubated in 0.25% acetic acid in 10 nM triethanolamine solution for 10 minutes, and prehybridized in 20% glycerol solution in DEPC-treated water for 60 minutes.
TABLE 1 Sequences of used primers for RT-PCR, RT-qPCR, and in situ hybridization. GenBank accession number CBE1 PCR primer (NM_032596.3) CBE1 ISH primer (NM_032596.3) b-actin (NM_001101.3) KASH5 (NM_144688.4) GAPDH (NM_002046.3)
Sequence Forward 50 -GAGGAATGCCCTTGGAATG-30 reverse 50 -TCCGCGACAGTGAGTTCAG-30 Forward 50 -AGGCTGTTGGGGAAGGAAG-30 reverse 50 -GTTTGGGCTGCATTTGTCTG-30 Forward 50 -TTCCTTCCTGGGCATGGAGT-30 reverse 50 -TACAGGTCTTTGCGGATGTC-30 Forward 50 -TGGACTTGGACACTTTCCTG-30 reverse 50 -CCTCTTCCAGCTCTAATCCC-30 Forward 50 -CCAGGTGGTCTCCTCTGACTTC-30 reverse 50 -GTGGTCGTTGAGGGCAATG-30
PCR product length 93 bp 467 bp 89 bp 81 bp 81 bp
Note: bp ¼ base pair; ISH ¼ in situ hybridization; PCR ¼ polymerase chain reaction. Pleuger. CBE1 in human spermatogenesis. Fertil Steril 2017.
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ORIGINAL ARTICLE: ANDROLOGY For hybridization, the probe was used at a dilution of 1:50. Therefore, 4 mL of sense or antisense cRNA was mixed with 4 mL of yeast RNA and 2 mL of salmon sperm (both Sigma-Aldrich). We added 10 mL of probe mix to 190 mL of hybridization buffer consisting of 20 mL of 20 standard saline citrate (SSC), 40 mL of dextran sulfate, 4 mL of Denhardt's reagent, 100 mL of deionized formamide, and 26 mL of DEPC water. Hybridization was performed at 48 C overnight in a humidified chamber containing 50% formamide in 2 SSC. For posthybridization, the sections were washed stringently as follows: 2 15 minutes in 2 SSC at room temperature, 4 15 minutes in 2 SSC at 58 C, 2 30 minutes in 0.2 SSC at 58 C, 5 minutes in 2 SSC at room temperature, and 10 minutes in 1 TNMT (Tris, NaCl, MgCl, and Tween 20) at room temperature. To prepare for immunohistochemical detection, the sections were incubated for 1 hour in 3% bovine serum albumin (BSA; Roth) in 1 TNMT. The digoxigenin (DIG)-labeled probes were detected using a DIG-Fab antibody (Roche) at a dilution of 1:1,000 in 1 TNMT and 3% BSA at 4 C overnight in a humidified chamber. Immunohistochemical staining was visualized using a ready-to-use 5-bromo-4-chloro-3-indolyl phosphate/nitro-blue tetrazolium chloride (BCIP/NBT) solution (Amresco).
Immunohistochemistry For antigen retrieval, deparaffinized and rehydrated 5-mmthick sections of testicular biopsy samples were boiled in 1 Tris-EDTA solution (pH 9.0) for 20 minutes. Endogenous peroxidases were blocked in 3% hydrogen peroxide for 15 minutes. To minimize the nonspecific binding sites, the slides were incubated in 1.5% BSA for 45 minutes. We applied the CBE1 antibody (rabbit polyclonal, NBP2-14425; Novus Biological) in a dilution of 1:50 in 1 Tris buffer overnight at 4 C. As the secondary antibody we used a biotinylated polyclonal goat anti-rabbit immunoglobulin 1.6 g/L (Dako A/S) in a dilution of 1:200 in 1 Tris-buffer for 1 hour at room temperature; subsequently we incubated the sections with Vectastain Elite ABC Standard Kit (Vector Laboratories) for 1 hour. Immunohistochemical binding was visualized with Peroxidase Substratkit AEC (BioLogo Dr. Hartmut Schultheiss e.K.) for 10 minutes as recommended by the manufacturer. Nuclei were counterstained with hematoxylin. The samples were observed with the Leica DM750 microscope. Before continuing with the same protocol as for testicular biopsy sections, ejaculate sperm smears were postfixed with 4 % paraformaldehyde for 30 minutes after air drying. They were treated for 30 minutes with 1% sodium dodecyl sulfate (SDS) for antigen retrieval.
Immunoelectron Microscopy Immunoelectron microscopy was performed on testicular biopsy samples showing normal spermatogenesis that had been fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.35). The samples were rinsed in PBS, dehydrated, and embedded in LRWhite Resin (TAAB Laboratories) as described previously elsewhere (21). Sections were cut into ultrathin sections with the ultramicro4
tome Ultracut (Reichert). For immunodetection, the sections were placed on collodion-coated nickel grids and incubated for 45 minutes in blocking buffer (PBS þ 0.2% BSA and 0.1% Tween 20). The sections were then incubated with rabbit polyclonal CBE1-antibody (1:1) for 2 hours at room temperature and subsequently rinsed five times for 3 minutes with blocking buffer. Secondary 10 nm gold-coupled goat F(ab)2 anti-rabbit IgG (British Biocell) was diluted 1:5 in PBS with 0.1% BSA. In the last step, the sections were rinsed threetimes for 5 minutes in PBS and two times for 5 minutes in double-distilled water. Contrasting was performed with uranyl acetate for 7 minutes and lead citrate for 4 minutes.
Western Blot Analysis Proteins were extracted from frozen testicular biopsy samples using TRIzol reagent as recommended by the manufacturer (Life Technologies). Proteins from formalin-fixed and paraffin-embedded (FFPE) material were extracted using QProtreome FFPE tissue Kit (Qiagen) as per the manufacturer's recommendation. Proteins from purified sperm pellets that had been washed three times with sperm washing medium containing 5 mg/mL of HAS (Irvine Scientific) were extracted using T-Per Tissue Protein Extraction Reagent (Thermo Fisher Scientific) as recommended by manufacturer. The same amount of protein homogenate (defined by BCA assay) was loaded per lane. We mixed 9.1 mL of protein sample with 3.5 mL of NuPAGE LDS sample buffer (4; Invitrogen) and 1.4 mL of NuPAGE Reducing Agent (10, Invitrogen) and heated it for 10 minutes at 75 C. The samples were fractionated on NuPAGENovex 4%–12% Bis-Tris Protein Gels with 1 NuPAGE SDS MOPS Running Buffer (180 V, 60 minutes, with the inner chamber containing 500 mL of NuPAGE Antioxidant) and the protein marker PageRuler Prestained Protein Ladder (10–180 kDa; Thermo Fisher Scientific). Subsequently, the proteins were electrophoretically transferred to Invitrolon polyvinylidene fluoride membranes (Invitrogen) at 30 V for 60 minutes. The transfer buffer contained 50 mL of NuPAGE Transfer buffer (Invitrogen), 1 mL of NuPAGE Antioxidant, 200 mL of methanol, and 749 mL of distilled water. After blocking with 1.5% skimmed milk powder and 1.5% BSA in PBS (pH 7.4) for 30 minutes, the membrane was probed with the primary antibody against SMRP1 (CBE1, ab136688; Abcam) in a dilution of 1:5,000; and as loading control we used a-tubulin (ab4074; Abcam) in a dilution of 1:20,000 (4 C overnight). Membranes were washed three times for 5 minutes in PBS-T (PBS þ 1% Tween; Sigma-Aldrich) containing 1% BSA and were blocked with normal serum from the same host species as the labeled secondary antibody (1 mL of goat serum in 20 mL of PBS). Membranes were then incubated with appropriate secondary antibody coupled to horseradish peroxidase (goat anti-rabbit-antibody; Dianova) in a dilution of 1:100,000 for 1 hour at room temperature. After washing three times for 10 minutes, the signals were visualized using Amersham ECL Select Western Blotting Detection Reagent and Amersham Hyperfilm ECL high performance chemiluminescence film (GE Healthcare Life Science) according to the manufacturer's recommendations. VOL. - NO. - / - 2017
Fertility and Sterility®
RESULTS CBE1 Expression in Normal Spermatogenesis In normal human spermatogenesis, CBE1 mRNA was localized only in late primary pachytene spermatocytes. The postmeiotic germ cells showed no specific staining. The incubation with the sense probe (insets) showed no staining (Fig. 1A and 1B). By Western blot analysis, we detected the CBE1 protein in human testicular biopsy samples but not in ejaculated spermatozoa (although both samples contained similar amounts of a-tubulin, which was used as loading control) (Fig. 1C). The individual donors of the semen showed no differences. In the testis, CBE1 was clearly localized in the flagellum of elongating spermatids from step 5 onward (Fig. 1D). In contrast to the developing spermatids, CBE1 was completely absent in ejaculated spermatozoa (Fig. 1E). Using immunoelectron microscopy, we were able to show that the protein was clearly associated with the microtubules of the manchette (Fig. 1F), the head-tail coupling apparatus, and the flagellum (Fig. 1G). The highest signal was seen at the microtubules of the flagellum (Fig. 1H) of elongating spermatids.
CBE1 Expression is Altered in Impaired Spermatogenesis The CBE1 mRNA was completely missing in samples showing Sertoli cell-only syndrome (SCO, n ¼ 12) compared with samples representing normal spermatogenesis, indicating that CBE1 is only expressed in germ cells and not in somatic Sertoli cells (Fig. 2A). Furthermore, CBE1 was statistically significantly reduced in all specimens showing maturation arrest at the level of spermatids (Fig. 2B, SDA, P¼ .0002) and at the level of primary spermatocytes (Fig. 2B, STA; P¼ .0001), although the mRNA was localized in primary pachytene spermatocytes during normal spermatogenesis (Fig. 1A). To avoid bias introduced by changes in cell content between samples, we normalized the CBE1 expression with b-actin and KASH5 (a specific spermatocyte marker). In the next step, we selected four SDA samples that contained very low numbers of elongating spermatids and examined the CBE1 protein localization. Western blot analysis revealed that the protein was not present in these samples in contrast to the specimens showing normal spermatogenesis (Fig. 2C). In the ensuing procedure, we evaluated the SDA samples with few elongating spermatids using H&E staining (Fig. 2D). Immunohistochemical analysis of these samples showed the absence of CBE1 in the flagellum (Fig. 2E).
DISCUSSION In this study, we showed for the first time the expression pattern of CBE1 during human spermatogenesis and were able to show a clear association of CBE1 with the microtubules of the manchette, the HTCA, and the flagellum, which could hint at a possible involvement in the development of elongating spermatids. VOL. - NO. - / - 2017
In ciliated epithelial cells of the lung, CBE1 has been immunohistochemically observed intracellularly but not in the ciliary structures per se. This fact suggests that CBE1 is involved in ciliogenesis but is not a component of the cilia. The nuclear and perinuclear localization of CBE1 in the bronchial epithelium implies that CBE1 might be a nucleocytoplasmic shuttle protein (13). However, we did not observe any nuclear staining of CBE1. Rather, CBE1 showed a transient localization in association with the manchette and the flagellum of elongating spermatids in normal spermatogenesis. The localization at the microtubules of the manchette could hint at a transport between the nucleus and the cytoplasm and distal parts of the spermatids whereby CBE1 plays a role in ciliogenesis in spermatids. Like ciliated bronchial epithelium, we were not able to detect CBE1 in human spermatozoa. Immunoelectron microscopy confirmed a close association between CBE1 and the microtubule-based structures in elongating spermatids. The localization at the manchette in humans corresponds with the described manchette-related localization of the murine orthologue SMRP1 (14). We have additionally shown that CBE1 is localized at the microtubules of the flagellum during spermiogenesis but not in mature sperm. Spermatids show a high polarity which in part likely originates via the action of two cargo transport pathways, IMT and IFT. The IMT is a part of the transfer of cargos between the nucleus and the cytoplasm (nucleocytoplasmic exchange) and the HTCA (22). Additionally, the IMT is thought to be required for the delivery of structural sperm tail proteins to basal body regions (23, 24). The IFT is involved in the development of the sperm tail. Both transport mechanisms have a similar cytoskeletal track, provided by microtubules. In combination, both transport pathways are involved in the development of the HTCA (25, 26). Our data strongly support an involvement of CBE1 in both pathways not only due to the localization at the microtubules of manchette and flagellum but also due to the localization at the HTCA. Hence, it could also have an influence in the formation of this structure. Furthermore, we have shown that CBE1 is localized in developing spermatids and is missing in spermatozoa, indicating that it could be a cargo protein. Previous investigations of proteins of the IFT complex A (IFT140) and of the IFT complex B (IFT88, IFT57, IFT20) in mice show that they are highly testis-enriched but are absent in spermatozoa, which indicates an important role of the IFT in the sperm development but not in the sperm maintenance. For example, male IFt88/ mice are sterile as a consequence of a reduced sperm count, sperm immotility, and abnormal morphology, especially of the flagellum (27). Both the localization of CBE1 and the absence in spermatozoa indicate that it could be related to the IFT either as a component of the transport mechanism or as cargo that is transported by IMT and IFT. After we characterized CBE1 expression in normal spermatogenesis, we investigated its significance for male fertility. As published most recently by Miyata et al. (28), Cbe1 knockout using CRISR/Cas9 technology did not lead to infertility in the mouse line. But even if a gene is not 5
ORIGINAL ARTICLE: ANDROLOGY
FIGURE 1
mRNA expression and protein localization of CBE1 in normal spermatogenesis. (A) CBE1 mRNA is clearly localized in cytoplasm of primary pachytene spermatocytes, showing by in situ hybridization. Insets show the lower magnification and the matching negative control using the sense probe. (B) Staging according to Bergmann and Kliesch (17). (C) Western blot analysis revealed that CBE1 protein (29.9 kDa) was only localized in testicular biopsy samples but not in ejaculate samples (a-tubulin, 50 kDa, was used as loading control). (D) Immunohistochemical detection showed a clear stage-specific localization of CBE1 within the flagellum of spermatids from stage V (black arrow) to the sperm release in stage II. In the flagellum of spermatids in stage IV, CBE1 was missing (white arrow). (E) CBE1 was not detected in the flagellum of mature sperm, in contrast to the control a-tubulin. (F–H) Immunoelectron microscopy showed CBE1 (black arrows) clearly associated with the microtubules of the manchette (F), the head-tail coupling area (G), and the flagellum (H) of elongating spermatids, indicating strongly an influence not only in the intramanchette but also in the intraflagellar transport. Pleuger. CBE1 in human spermatogenesis. Fertil Steril 2017.
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FIGURE 2
CBE1 expression is altered in impaired spermatogenesis. (A) In contrast to the constant mRNA expression in normal spermatogenesis (NSP), CBE1 was not expressed in specimens showing a Sertoli cell only syndrome (SCO), indicating that CBE1 is only expressed in germ cells and not in somatic Sertoli cells. (B) Although the mRNA was localized in primary pachytene spermatocytes in normal spermatogenesis (n ¼ 8), CBE1 was significantly reduced in maturation arrests at the level of early round spermatids (SDA, n ¼ 8) and primary spermatocytes (STA, n ¼ 8). The CBE1 expression was normalized with b-actin and KASH5 (specific spermatocyte marker) to account for variations in the amount of spermatocytes in the samples. (C) Western blot shows that the protein was missing in SDA samples in comparison to normal spermatogenesis (a-tubulin was used as loading control). (D and E) Immunohistochemical analysis of selected SDA samples, which contain (D) a rare amount of elongating spermatids, showed (E) an absence of CBE1 in the flagellum. Pleuger. CBE1 in human spermatogenesis. Fertil Steril 2017.
responsible for infertility in mice, its significance in human spermatogenesis and spermatogenic impairment has to be elucidated, as was shown for Kr€ uppel-like factor 4 (KLF4/ Klf4). In a Sertoli cell–specific knockout, the significance of Kfl4 in proper Sertoli cell differentiation was shown, but adult knockout mice were nevertheless fertile (29). Moreover, a germ cell–specific knockout of Klf4 did not lead to infertility as spermatogenesis was unaltered (30). In contrast, human KLF4 showed altered expression during spermiogenesis in spermatogenic arrest samples (31). Accordingly, we were able to show an altered expression of CBE1 mRNA in impaired spermatogenesis in the human. Although CBE1 mRNA was localized in primary pachytene spermatocytes in normal spermatogenesis, the expression was significantly reduced in maturation arrests at the level of primary spermatocytes and early round spermatids. Furthermore, the protein was completely missing in the selected SDA samples with rare elongating spermatids. VOL. - NO. - / - 2017
Many factors are necessary for the correct nuclear and cytoplasmic modification of round spermatids to spermatozoa (7, 32). However, the factors that lead to SDA are largely unknown. Male infertility can have various causes, including a disturbed assembly of microtubules, a disorder of important processes during spermatogenesis (3, 7). The absence of CBE1 in the SDA sample group with few elongating spermatids reinforced that CBE1 could be a part of the intraflagellar transport system and thus important for the correct differentiation and elongation of spermatids. To sum up, our data strongly suggest an influence of CBE1 on the development of the sperm tail in spermatids due to the localization at the microtubules of the manchette and the flagellum via a role in the IMT and/or IFT mechanism. Given that the highest level of CBE1 was detected in spermatid development, it likely plays a role in spermatogenesis but is not important for the maintenance of mature sperm as a component of the flagellum. 7
ORIGINAL ARTICLE: ANDROLOGY Acknowledgments: The authors thank the staff for the collaboration and skillful technical assistance, especially Susanne Schubert-Porth, Alexandra Hax, and Jutta DernWieloch.
17.
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Expression of ciliated bronchial epithelium 1 during human spermatogenesis Christiane Pleuger, M.Sc.,a Daniela Fietz, Dr.Med.Vet.,a Katja Hartmann, Dr.Med.Vet.,a Hans-Christian Schuppe, Dr.Med.,b Wolfgang Weidner, Dr.Med.,b Sabine Kliesch, Dr.Med.,c Mark Baker, Ph.D.,d Moira K. O'Bryan, Ph.D.,e and Martin Bergmann, Dr.Rer.Medic.a a Institute for Veterinary Anatomy, Histology und Embryology, and b Clinic for Urology, Pediatric Urology and Andrology, Justus Liebig University, Giessen, Germany; c Department of Clinical Andrology, Centre for Reproductive Medicine € nster, Mu € nster, Germany; d School of Environmental and Life Science, University and Andrology, University Hospital Mu of Newcastle, Callaghan, New South Wales, Australia; and e Development and Stems Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia
Objective: To define the precise cellular localization of ciliated bronchial epithelium 1 (CBE1) in the human testis and test its relationship to impaired spermatogenesis. Design: Gene expression analysis, and histologic and immunohistochemical evaluation. Setting: University research laboratories and andrologic outpatient clinic. Patient(s): Forty-three human testicular biopsies: 12 biopsies showing normal spermatogenesis (NSP), 8 with maturation arrest at level of spermatocytes (STA), 8 with maturation arrest at level of spermatids (SDA), 4 with scattered elongating spermatids, and 12 with Sertoli cell-only syndrome, with an additional 5 semen samples from healthy donors. Intervention(s): None. Main Outcome Measure(s): Evaluation of CBE1 expression in normal as well as impaired spermatogenesis on mRNA (quantitative reverse-transcription polymerase chain reaction and in situ hybridization) and protein level (immunohistochemistry, Western blot analysis). Result(s): In normal spermatogenesis, CBE1 mRNA was expressed in late pachytene spermatocytes, and the protein was localized within the flagellum of elongating spermatids from stage V up to the spermiation in stage II. Immunoelectron microscopy showed CBE1 clearly associated with microtubules at the manchette, the head-tail coupling apparatus, and the flagellum, but the protein was absent in spermatozoa. Compared with normal spermatogenesis, CBE1 mRNA was statistically significantly reduced in samples with a maturation arrest at the level of round spermatids and primary spermatocytes, and was absent in samples showing Sertoli cell-only syndrome. CBE1 protein was completely missing in SDA samples showing few elongating spermatids. Conclusion(s): Our data strongly suggest an influence of CBE1 in ciliogenesis in spermatids due to the localization at the microtubules of the elongating spermatids, indicating a role in the intramanchette and/or intraflagellar transport mechanism. The absence of CBE1 in spermatozoa suggests that CBE1 is important for the spermatid development but not for the maintenance of mature spermatozoa as a component of the flagellum. (Fertil SterilÒ 2017;-:-–-. Ó2017 by American Society for Reproductive Medicine.) Key Words: Intraflagellar transport, intramanchette transport, male fertility, microtubules, spermiogenesis Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/ 16110-fertility-and-sterility/posts/16846-24101
Received March 29, 2017; revised May 5, 2017; accepted May 14, 2017. C.P. has nothing to disclose. D.F. has received grants from the German Research Foundation (DFG). K.H. has nothing to disclose. H.-C.S. has received grants from the International Research Training Group ‘‘Molecular Pathogenesis of Male Reproductive Disorders’’ (German Research Foundation; DFG) and honoraria from Ferring, Germany, and Merck-Serono, Germany. W.W. has nothing to disclose. S.K. has nothing to disclose. M.B. has nothing to disclose. M.K.O. has received grants from the Monash IVF Research and Education Foundation. M.B. has nothing to disclose. Supported by the DFG (German Research Foundation)-IRTG 1871; and a fellowship from the National Health and Medical Research Council of Australia (APP1058356) (to M.K.O.). € ttingen, Germany, Presented as posters at 111th Annual Meeting of Anatomische Gesellschaft, Go September 21–24, 2016; and the 50th Annual Meeting of Physiologie und Pathologie der Fortpflanzung, Munich, Germany, February 15–17, 2017. Reprint requests: Christiane Pleuger, M.Sc., Institute for Veterinary Anatomy, Histology and Embryology, Justus Liebig University, Frankfurter Straße 98, 35392 Gießen, Germany (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2017 0015-0282/$36.00 Copyright ©2017 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2017.05.019 VOL. - NO. - / - 2017
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icrotubules play a crucial role during cell-division processes, such as mitosis and meiosis, and the development of microtubule-based structures during spermiogenesis. After meiotic reductive divisions, haploid round spermatids undergo many modifications to achieve the required characteristics for fertilization. This process includes the polarization of the nucleus with the acrosome to one side of the cell, the concomitant elongation of the 1
ORIGINAL ARTICLE: ANDROLOGY spermatid, the nuclear condensation and sperm head shaping by the manchette, as well as the assembly of the axoneme (1). The inappropriate formation of these microtubule-based structures and defects in the associated transport mechanisms along the manchette and flagellum of spermatids can lead to asthenozoospermia (reduced motility) and/or teratozoospermia (abnormal morphology), thus causing male infertility (2). The formation of the axoneme of the sperm flagellum shows parallels to the development of motile cilia in other tissues, such as the brain and the lung (3). The axoneme, the core of the flagellum, consists of a centrally located microtubule pair surrounded by nine outer microtubule doublets that are connected by the dynein heavy chains of the inner and outer dynein arms. Axoneme formation in many cilia and likely in the sperm tail involves a microtubulebased protein system known as intraflagellar transport (IFT). The mammalian sperm tail has additional characteristic peri-axonemal structures, such as nine outer dense fibers, the mitochondrial sheath, and fibrous sheath and is connected to the nucleus via the connecting piece (4). The structural features enable motility of the mature sperm and maintain structural integrity (5). Concordant with the onset of spermatid head elongation, the transient microtubule-based manchette comes into existence and persists until the elongation and condensation of the spermatid nucleus is completed (6). The manchette is thought to serve essential roles in nuclear shaping and to act as a protein transport platform for proteins involved in tail formation, known as intramanchette transport (IMT) (7). While not completely characterized and temporally distinct, the IFT likely shares similar molecular components to IMT. It is proposed that in both transport mechanisms, a multicomplex protein raft linked to cargo proteins and vesicles moves along a microtubule track (6, 8, 9). During IFT, essential proteins for the assembly and maintenance of the flagellum are transported in an anterograde direction by kinesins to the distal part of the flagellum (10) and in a retrograde direction by dyneins back toward the basal body (11). It is proposed that the IMT, which is transient just like the manchette itself, additionally enables an exchange of cargo between the Golgi, nucleus, and cytoplasm as well as the head-tail coupling apparatus (HTCA) (2, 8, 9, 12). Ciliated bronchial epithelium 1 (CBE1 alias SMRP1, 1110017D15Rik, C9orf24, NYD-SP22) is a protein that has been described in association with ciliated cells in bronchial tissue (13) and in the manchette of murine elongating spermatids (14). During pulmonary development, CBE1 shows a biphasic expression. CBE1 expression starts during the formation of the lung buds and shows a further expression in later stages (pseudoglandular stage) concurrent with the expression of the transcription factor forkhead box factor FOXJ1 (hepatocyte nuclear factor-3/forkhead homologue 4) (15), which is closely linked to ciliogenesis (16). Although these data indicate a role for CBE1 in establishing differentiation of the mucociliary epithelium (15), CBE1 is localized intracellularly in ciliated epithelial cells but not in the cilia structure (13). Previous investigations of the murine orthologue (called Smrp1 spermatid-manchette-related protein 1 in the original 2
study) detected the protein at the spermatid manchette around the nucleus where it colocalized with a-tubulin. Although three mRNA variants have been described, only one transcript variant was translated into the protein (14). To date, no precise molecular function has been described for CBE1. Although the murine orthologue shows a clear localization at the spermatid manchette (14), it is unknown whether CBE1 is either an integral part of this structure or it plays a role in the cargo transport. To clarify its potential relevance to human spermatogenesis, we investigated the mRNA expression and protein localization of CBE1 in human testes with normal and impaired spermatogenesis to gain information regarding the molecular function and a possible association with the phenotype of maturation arrests.
MATERIAL AND METHODS Testicular Tissue and Semen Samples Human testicular biopsy samples were obtained (with consent) from patients of the Centre of Reproductive Medicine and Andrology of the University Hospital in M€ unster (Germany) and the Department of Urology and Andrology of the University Hospital in Giessen (Germany). The surgical procedure was indicated because of obstructive (refertilization after vasectomy) or nonobstructive azoospermia as defined using the criteria according to Bergmann and Kliesch (17). In total, 75 testicular biopsy samples were used (44 samples to analyze mRNA expression and 31 to evaluate the protein localization in normal and impaired spermatogenesis). Immediately after surgical removal, the testicular tissues were immersed in Bouin's solution and ultimately embedded in paraffin. We stained 5-mm-thick sections with hematoxylin and eosin (H&E) to evaluate the quality of the spermatogenesis according to the score count protocol of Bergmann and Kliesch (17). The stages of spermatogenesis were differentiated in accordance with Clermont (18). To analyze the mRNA expression, we selected 12 biopsy samples showing normal spermatogenesis (NSP); 8 biopsy samples with a maturation arrest at the level of early round spermatids (SDA), whereby samples showed scattered elongating spermatids; 12 biopsy samples with a maturation arrest at the level of primary spermatocytes (STA); and 12 biopsy samples showing Sertoli cell-only syndrome (SCO). Human ejaculates (n ¼ 5) were received from five different healthy volunteers. A semen analysis was performed according to the World Health Organization's 2010 guidelines to measure sperm concentration, motility, morphology, and vitality (19). After the evaluation, the ejaculates were centrifuged at 500 g for 15 minutes, and the pellet was washed three times with sperm washing medium containing 5 mg/ mL of human serum albumin (Irvine Scientific) and stored at 20 C. The study was performed following the declaration of Helsinki. Written informed consent was obtained from all men involved. The study was approved by the ethics committee of the medical faculty of the Justus-Liebig-University Giessen (AZ 100/07; AZ 32/11). VOL. - NO. - / - 2017
Fertility and Sterility® CBE1 mRNA Expression We obtained messenger RNA (mRNA) from Bouin's fixed and paraffin embedded tissue by use of the RNeasy FFPE Kit (Qiagen) including proteinase K treatment as recommended by the manufacturer. To remove any contaminating genomic DNA, the extracted mRNA was incubated with RNase-free DNase (Peqlab Biotechnology), RNase-free incubation buffer 10x (Roche), RNase Inhibitor (2,000 U), and MgCl2 solution (Applied Biosystems by Thermo Fisher Scientific) for 25 minutes at 37 C. To synthesize the complementary DNA (cDNA), 8 mL of mRNA were mixed with 60 mL of RT-Mix (components from Applied Biosystems by Thermo Fisher Scientific) consisting of 10 mL of GeneAmp 10x PCR Gold Buffer, 10 mL of nucleotide mix (10 mM each), 10 mL of MgCl2 (25 mM), 5 mL of random hexamer primers (50 mM), 5 mL of RNase inhibitor (20 U/mL), 5 mL of MultiScribe Reverse Transcriptase (50 U/mL), and 15 mL of sterile distilled water. The negative control samples contained no reverse transcriptase. The synthesized cDNA was subsequently mixed with 2.5 mL of GeneAmp 10x PCR Gold Buffer, 2 mL of MgCl2 (25 mM), 1 mL of each forward and reverse primer (10 pmol/mL), 1 mL of nucleotide mix (10 mM each), 0.15 mL of AmpliTaq Gold polymerase, and 12.35 mL of sterile distilled water. We selected specific primers for human CBE1, b-actin, KASH5, and GAPDH (Table 1) using OligoExplorer 1.1.0 (T. Kuulasma, University of Kuopio, Kuopio, Finland); and purchased them from biomers.net (Ulm, Germany). After reversetranscription polymerase chain reaction (RT-PCR) (cycling conditions: 1 9 minutes at 95 C, 35 [45 seconds at 94 C, 45 seconds at 60 C, 45 seconds 72 C] and 7 minutes at 72 C), the PCR products were separated and visualized using gel electrophoresis (1.5 % agarose gel) with GelGreen Nucleic Acid Stain (Biotium). For quantitative RT-PCR (qRT-PCR), the same primers as previously were used with the 8 NSP and 8 SDA samples. We added 1 mL of cDNA of each sample with 19 mL of mastermix containing 4 mL of the intercalating fluorescent dye EvaGreen (Bio&Sell) as detection mode, 0.6 mL of forward and reverse primer, and 13.8 mL of sterile distilled water. We performed qRT-PCR using CFX96 RealTime cycler system (BioRad Laboratories) under the following conditions: 15 minutes at
95 C, 40 (15 seconds at 95 C, 30 seconds at 60 C, 20 seconds at 72 C). The melt curve analysis consisted of 10 seconds at 95 C, then 65 C to 95 C in increments 0.5 C for 5 seconds. The relative expression levels were calculated using the 2DDCt method whereby b-actin and KASH5 (for the primer sequence, Table 1), a specific spermatocyte marker (Klarsicht/ANC-1/ Syne/homology 5), were used as internal reference genes. We used GraphPad Prism 5.01 (GraphPad Software) for statistical analyses and diagram generation, and statistical significance was tested by one-way analysis of variance.
In Situ Hybridization To detect CBE1 mRNA in the cellular context, we generated a 467-bp amplicon (primer sequences in Table 1) as described earlier. To generate DIG-labeled cRNA probes, the RT-PCR product was ligated into pCR II TOPO vector (Invitrogen) as recommended by the manufacturer and subsequently transformed in One Shot TOP 10 Chemically Competent E. coli (Invitrogen) followed by plasmid extraction using QIAprep Spin Miniprep (Qiagen). After validating all plasmids by sequencing (performed by Scientific Research Development GmbH), the correct insertion of the PCR products was controlled by double digest using BamHI and NotI (New England Biolabs). We applied the same restriction enzymes for linearization of the plasmid DNA. For the subsequent in vitro transcription, we used RNA-DIG Labeling Mix (Boehringer Mannheim) and RNA polymerase T7 and SP6 (Promega). In situ hybridization was performed as described by Fietz et al. (20) with the following modifications. Deparaffinized and rehydrated tissue sections (5 mm) were incubated with proteinase K (10 mg/mL) in sterile diethylpyrocarbonate (DEPC)-treated water for 20 minutes at 37 C. Digestion was stopped by incubation in 0.2% glycine solution in phosphate-buffered saline supplemented with 1 mM MgCl2 (PBS-M) for 5 minutes. Subsequently, the sections were postfixed with 4% paraformaldehyde for 10 minutes, incubated in 0.25% acetic acid in 10 nM triethanolamine solution for 10 minutes, and prehybridized in 20% glycerol solution in DEPC-treated water for 60 minutes.
TABLE 1 Sequences of used primers for RT-PCR, RT-qPCR, and in situ hybridization. GenBank accession number CBE1 PCR primer (NM_032596.3) CBE1 ISH primer (NM_032596.3) b-actin (NM_001101.3) KASH5 (NM_144688.4) GAPDH (NM_002046.3)
Sequence Forward 50 -GAGGAATGCCCTTGGAATG-30 reverse 50 -TCCGCGACAGTGAGTTCAG-30 Forward 50 -AGGCTGTTGGGGAAGGAAG-30 reverse 50 -GTTTGGGCTGCATTTGTCTG-30 Forward 50 -TTCCTTCCTGGGCATGGAGT-30 reverse 50 -TACAGGTCTTTGCGGATGTC-30 Forward 50 -TGGACTTGGACACTTTCCTG-30 reverse 50 -CCTCTTCCAGCTCTAATCCC-30 Forward 50 -CCAGGTGGTCTCCTCTGACTTC-30 reverse 50 -GTGGTCGTTGAGGGCAATG-30
PCR product length 93 bp 467 bp 89 bp 81 bp 81 bp
Note: bp ¼ base pair; ISH ¼ in situ hybridization; PCR ¼ polymerase chain reaction. Pleuger. CBE1 in human spermatogenesis. Fertil Steril 2017.
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ORIGINAL ARTICLE: ANDROLOGY For hybridization, the probe was used at a dilution of 1:50. Therefore, 4 mL of sense or antisense cRNA was mixed with 4 mL of yeast RNA and 2 mL of salmon sperm (both Sigma-Aldrich). We added 10 mL of probe mix to 190 mL of hybridization buffer consisting of 20 mL of 20 standard saline citrate (SSC), 40 mL of dextran sulfate, 4 mL of Denhardt's reagent, 100 mL of deionized formamide, and 26 mL of DEPC water. Hybridization was performed at 48 C overnight in a humidified chamber containing 50% formamide in 2 SSC. For posthybridization, the sections were washed stringently as follows: 2 15 minutes in 2 SSC at room temperature, 4 15 minutes in 2 SSC at 58 C, 2 30 minutes in 0.2 SSC at 58 C, 5 minutes in 2 SSC at room temperature, and 10 minutes in 1 TNMT (Tris, NaCl, MgCl, and Tween 20) at room temperature. To prepare for immunohistochemical detection, the sections were incubated for 1 hour in 3% bovine serum albumin (BSA; Roth) in 1 TNMT. The digoxigenin (DIG)-labeled probes were detected using a DIG-Fab antibody (Roche) at a dilution of 1:1,000 in 1 TNMT and 3% BSA at 4 C overnight in a humidified chamber. Immunohistochemical staining was visualized using a ready-to-use 5-bromo-4-chloro-3-indolyl phosphate/nitro-blue tetrazolium chloride (BCIP/NBT) solution (Amresco).
Immunohistochemistry For antigen retrieval, deparaffinized and rehydrated 5-mmthick sections of testicular biopsy samples were boiled in 1 Tris-EDTA solution (pH 9.0) for 20 minutes. Endogenous peroxidases were blocked in 3% hydrogen peroxide for 15 minutes. To minimize the nonspecific binding sites, the slides were incubated in 1.5% BSA for 45 minutes. We applied the CBE1 antibody (rabbit polyclonal, NBP2-14425; Novus Biological) in a dilution of 1:50 in 1 Tris buffer overnight at 4 C. As the secondary antibody we used a biotinylated polyclonal goat anti-rabbit immunoglobulin 1.6 g/L (Dako A/S) in a dilution of 1:200 in 1 Tris-buffer for 1 hour at room temperature; subsequently we incubated the sections with Vectastain Elite ABC Standard Kit (Vector Laboratories) for 1 hour. Immunohistochemical binding was visualized with Peroxidase Substratkit AEC (BioLogo Dr. Hartmut Schultheiss e.K.) for 10 minutes as recommended by the manufacturer. Nuclei were counterstained with hematoxylin. The samples were observed with the Leica DM750 microscope. Before continuing with the same protocol as for testicular biopsy sections, ejaculate sperm smears were postfixed with 4 % paraformaldehyde for 30 minutes after air drying. They were treated for 30 minutes with 1% sodium dodecyl sulfate (SDS) for antigen retrieval.
Immunoelectron Microscopy Immunoelectron microscopy was performed on testicular biopsy samples showing normal spermatogenesis that had been fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.35). The samples were rinsed in PBS, dehydrated, and embedded in LRWhite Resin (TAAB Laboratories) as described previously elsewhere (21). Sections were cut into ultrathin sections with the ultramicro4
tome Ultracut (Reichert). For immunodetection, the sections were placed on collodion-coated nickel grids and incubated for 45 minutes in blocking buffer (PBS þ 0.2% BSA and 0.1% Tween 20). The sections were then incubated with rabbit polyclonal CBE1-antibody (1:1) for 2 hours at room temperature and subsequently rinsed five times for 3 minutes with blocking buffer. Secondary 10 nm gold-coupled goat F(ab)2 anti-rabbit IgG (British Biocell) was diluted 1:5 in PBS with 0.1% BSA. In the last step, the sections were rinsed threetimes for 5 minutes in PBS and two times for 5 minutes in double-distilled water. Contrasting was performed with uranyl acetate for 7 minutes and lead citrate for 4 minutes.
Western Blot Analysis Proteins were extracted from frozen testicular biopsy samples using TRIzol reagent as recommended by the manufacturer (Life Technologies). Proteins from formalin-fixed and paraffin-embedded (FFPE) material were extracted using QProtreome FFPE tissue Kit (Qiagen) as per the manufacturer's recommendation. Proteins from purified sperm pellets that had been washed three times with sperm washing medium containing 5 mg/mL of HAS (Irvine Scientific) were extracted using T-Per Tissue Protein Extraction Reagent (Thermo Fisher Scientific) as recommended by manufacturer. The same amount of protein homogenate (defined by BCA assay) was loaded per lane. We mixed 9.1 mL of protein sample with 3.5 mL of NuPAGE LDS sample buffer (4; Invitrogen) and 1.4 mL of NuPAGE Reducing Agent (10, Invitrogen) and heated it for 10 minutes at 75 C. The samples were fractionated on NuPAGENovex 4%–12% Bis-Tris Protein Gels with 1 NuPAGE SDS MOPS Running Buffer (180 V, 60 minutes, with the inner chamber containing 500 mL of NuPAGE Antioxidant) and the protein marker PageRuler Prestained Protein Ladder (10–180 kDa; Thermo Fisher Scientific). Subsequently, the proteins were electrophoretically transferred to Invitrolon polyvinylidene fluoride membranes (Invitrogen) at 30 V for 60 minutes. The transfer buffer contained 50 mL of NuPAGE Transfer buffer (Invitrogen), 1 mL of NuPAGE Antioxidant, 200 mL of methanol, and 749 mL of distilled water. After blocking with 1.5% skimmed milk powder and 1.5% BSA in PBS (pH 7.4) for 30 minutes, the membrane was probed with the primary antibody against SMRP1 (CBE1, ab136688; Abcam) in a dilution of 1:5,000; and as loading control we used a-tubulin (ab4074; Abcam) in a dilution of 1:20,000 (4 C overnight). Membranes were washed three times for 5 minutes in PBS-T (PBS þ 1% Tween; Sigma-Aldrich) containing 1% BSA and were blocked with normal serum from the same host species as the labeled secondary antibody (1 mL of goat serum in 20 mL of PBS). Membranes were then incubated with appropriate secondary antibody coupled to horseradish peroxidase (goat anti-rabbit-antibody; Dianova) in a dilution of 1:100,000 for 1 hour at room temperature. After washing three times for 10 minutes, the signals were visualized using Amersham ECL Select Western Blotting Detection Reagent and Amersham Hyperfilm ECL high performance chemiluminescence film (GE Healthcare Life Science) according to the manufacturer's recommendations. VOL. - NO. - / - 2017
Fertility and Sterility®
RESULTS CBE1 Expression in Normal Spermatogenesis In normal human spermatogenesis, CBE1 mRNA was localized only in late primary pachytene spermatocytes. The postmeiotic germ cells showed no specific staining. The incubation with the sense probe (insets) showed no staining (Fig. 1A and 1B). By Western blot analysis, we detected the CBE1 protein in human testicular biopsy samples but not in ejaculated spermatozoa (although both samples contained similar amounts of a-tubulin, which was used as loading control) (Fig. 1C). The individual donors of the semen showed no differences. In the testis, CBE1 was clearly localized in the flagellum of elongating spermatids from step 5 onward (Fig. 1D). In contrast to the developing spermatids, CBE1 was completely absent in ejaculated spermatozoa (Fig. 1E). Using immunoelectron microscopy, we were able to show that the protein was clearly associated with the microtubules of the manchette (Fig. 1F), the head-tail coupling apparatus, and the flagellum (Fig. 1G). The highest signal was seen at the microtubules of the flagellum (Fig. 1H) of elongating spermatids.
CBE1 Expression is Altered in Impaired Spermatogenesis The CBE1 mRNA was completely missing in samples showing Sertoli cell-only syndrome (SCO, n ¼ 12) compared with samples representing normal spermatogenesis, indicating that CBE1 is only expressed in germ cells and not in somatic Sertoli cells (Fig. 2A). Furthermore, CBE1 was statistically significantly reduced in all specimens showing maturation arrest at the level of spermatids (Fig. 2B, SDA, P¼ .0002) and at the level of primary spermatocytes (Fig. 2B, STA; P¼ .0001), although the mRNA was localized in primary pachytene spermatocytes during normal spermatogenesis (Fig. 1A). To avoid bias introduced by changes in cell content between samples, we normalized the CBE1 expression with b-actin and KASH5 (a specific spermatocyte marker). In the next step, we selected four SDA samples that contained very low numbers of elongating spermatids and examined the CBE1 protein localization. Western blot analysis revealed that the protein was not present in these samples in contrast to the specimens showing normal spermatogenesis (Fig. 2C). In the ensuing procedure, we evaluated the SDA samples with few elongating spermatids using H&E staining (Fig. 2D). Immunohistochemical analysis of these samples showed the absence of CBE1 in the flagellum (Fig. 2E).
DISCUSSION In this study, we showed for the first time the expression pattern of CBE1 during human spermatogenesis and were able to show a clear association of CBE1 with the microtubules of the manchette, the HTCA, and the flagellum, which could hint at a possible involvement in the development of elongating spermatids. VOL. - NO. - / - 2017
In ciliated epithelial cells of the lung, CBE1 has been immunohistochemically observed intracellularly but not in the ciliary structures per se. This fact suggests that CBE1 is involved in ciliogenesis but is not a component of the cilia. The nuclear and perinuclear localization of CBE1 in the bronchial epithelium implies that CBE1 might be a nucleocytoplasmic shuttle protein (13). However, we did not observe any nuclear staining of CBE1. Rather, CBE1 showed a transient localization in association with the manchette and the flagellum of elongating spermatids in normal spermatogenesis. The localization at the microtubules of the manchette could hint at a transport between the nucleus and the cytoplasm and distal parts of the spermatids whereby CBE1 plays a role in ciliogenesis in spermatids. Like ciliated bronchial epithelium, we were not able to detect CBE1 in human spermatozoa. Immunoelectron microscopy confirmed a close association between CBE1 and the microtubule-based structures in elongating spermatids. The localization at the manchette in humans corresponds with the described manchette-related localization of the murine orthologue SMRP1 (14). We have additionally shown that CBE1 is localized at the microtubules of the flagellum during spermiogenesis but not in mature sperm. Spermatids show a high polarity which in part likely originates via the action of two cargo transport pathways, IMT and IFT. The IMT is a part of the transfer of cargos between the nucleus and the cytoplasm (nucleocytoplasmic exchange) and the HTCA (22). Additionally, the IMT is thought to be required for the delivery of structural sperm tail proteins to basal body regions (23, 24). The IFT is involved in the development of the sperm tail. Both transport mechanisms have a similar cytoskeletal track, provided by microtubules. In combination, both transport pathways are involved in the development of the HTCA (25, 26). Our data strongly support an involvement of CBE1 in both pathways not only due to the localization at the microtubules of manchette and flagellum but also due to the localization at the HTCA. Hence, it could also have an influence in the formation of this structure. Furthermore, we have shown that CBE1 is localized in developing spermatids and is missing in spermatozoa, indicating that it could be a cargo protein. Previous investigations of proteins of the IFT complex A (IFT140) and of the IFT complex B (IFT88, IFT57, IFT20) in mice show that they are highly testis-enriched but are absent in spermatozoa, which indicates an important role of the IFT in the sperm development but not in the sperm maintenance. For example, male IFt88/ mice are sterile as a consequence of a reduced sperm count, sperm immotility, and abnormal morphology, especially of the flagellum (27). Both the localization of CBE1 and the absence in spermatozoa indicate that it could be related to the IFT either as a component of the transport mechanism or as cargo that is transported by IMT and IFT. After we characterized CBE1 expression in normal spermatogenesis, we investigated its significance for male fertility. As published most recently by Miyata et al. (28), Cbe1 knockout using CRISR/Cas9 technology did not lead to infertility in the mouse line. But even if a gene is not 5
ORIGINAL ARTICLE: ANDROLOGY
FIGURE 1
mRNA expression and protein localization of CBE1 in normal spermatogenesis. (A) CBE1 mRNA is clearly localized in cytoplasm of primary pachytene spermatocytes, showing by in situ hybridization. Insets show the lower magnification and the matching negative control using the sense probe. (B) Staging according to Bergmann and Kliesch (17). (C) Western blot analysis revealed that CBE1 protein (29.9 kDa) was only localized in testicular biopsy samples but not in ejaculate samples (a-tubulin, 50 kDa, was used as loading control). (D) Immunohistochemical detection showed a clear stage-specific localization of CBE1 within the flagellum of spermatids from stage V (black arrow) to the sperm release in stage II. In the flagellum of spermatids in stage IV, CBE1 was missing (white arrow). (E) CBE1 was not detected in the flagellum of mature sperm, in contrast to the control a-tubulin. (F–H) Immunoelectron microscopy showed CBE1 (black arrows) clearly associated with the microtubules of the manchette (F), the head-tail coupling area (G), and the flagellum (H) of elongating spermatids, indicating strongly an influence not only in the intramanchette but also in the intraflagellar transport. Pleuger. CBE1 in human spermatogenesis. Fertil Steril 2017.
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FIGURE 2
CBE1 expression is altered in impaired spermatogenesis. (A) In contrast to the constant mRNA expression in normal spermatogenesis (NSP), CBE1 was not expressed in specimens showing a Sertoli cell only syndrome (SCO), indicating that CBE1 is only expressed in germ cells and not in somatic Sertoli cells. (B) Although the mRNA was localized in primary pachytene spermatocytes in normal spermatogenesis (n ¼ 8), CBE1 was significantly reduced in maturation arrests at the level of early round spermatids (SDA, n ¼ 8) and primary spermatocytes (STA, n ¼ 8). The CBE1 expression was normalized with b-actin and KASH5 (specific spermatocyte marker) to account for variations in the amount of spermatocytes in the samples. (C) Western blot shows that the protein was missing in SDA samples in comparison to normal spermatogenesis (a-tubulin was used as loading control). (D and E) Immunohistochemical analysis of selected SDA samples, which contain (D) a rare amount of elongating spermatids, showed (E) an absence of CBE1 in the flagellum. Pleuger. CBE1 in human spermatogenesis. Fertil Steril 2017.
responsible for infertility in mice, its significance in human spermatogenesis and spermatogenic impairment has to be elucidated, as was shown for Kr€ uppel-like factor 4 (KLF4/ Klf4). In a Sertoli cell–specific knockout, the significance of Kfl4 in proper Sertoli cell differentiation was shown, but adult knockout mice were nevertheless fertile (29). Moreover, a germ cell–specific knockout of Klf4 did not lead to infertility as spermatogenesis was unaltered (30). In contrast, human KLF4 showed altered expression during spermiogenesis in spermatogenic arrest samples (31). Accordingly, we were able to show an altered expression of CBE1 mRNA in impaired spermatogenesis in the human. Although CBE1 mRNA was localized in primary pachytene spermatocytes in normal spermatogenesis, the expression was significantly reduced in maturation arrests at the level of primary spermatocytes and early round spermatids. Furthermore, the protein was completely missing in the selected SDA samples with rare elongating spermatids. VOL. - NO. - / - 2017
Many factors are necessary for the correct nuclear and cytoplasmic modification of round spermatids to spermatozoa (7, 32). However, the factors that lead to SDA are largely unknown. Male infertility can have various causes, including a disturbed assembly of microtubules, a disorder of important processes during spermatogenesis (3, 7). The absence of CBE1 in the SDA sample group with few elongating spermatids reinforced that CBE1 could be a part of the intraflagellar transport system and thus important for the correct differentiation and elongation of spermatids. To sum up, our data strongly suggest an influence of CBE1 on the development of the sperm tail in spermatids due to the localization at the microtubules of the manchette and the flagellum via a role in the IMT and/or IFT mechanism. Given that the highest level of CBE1 was detected in spermatid development, it likely plays a role in spermatogenesis but is not important for the maintenance of mature sperm as a component of the flagellum. 7
ORIGINAL ARTICLE: ANDROLOGY Acknowledgments: The authors thank the staff for the collaboration and skillful technical assistance, especially Susanne Schubert-Porth, Alexandra Hax, and Jutta DernWieloch.
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