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Logo of mcpMolecular & Cellular Proteomics : MCP
 
Mol Cell Proteomics. 2015 December; 14(12): 3185–3195.
Published online 2015 October 2. doi:  10.1074/mcp.M115.050005
PMCID: PMC4762626

Aberrant Expression of Dynein light chain 1 (DYNLT1) is Associated with Human Male Factor Infertility*An external file that holds a picture, illustration, etc.
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Abstract

DYNLT1 is a member of a gene family identified within the t-complex of the mouse, which has been linked with male germ cell development and function in the mouse and the fly. Though defects in the expression of this gene are associated with male sterility in both these models, there has been no study examining its association with spermatogenic defects in human males. In this study, we evaluated the levels of DYNLT1 and its expression product in the germ cells of fertile human males and males suffering from spermatogenic defects. We screened fertile (n = 14), asthenozoospermic (n = 15), oligozoospermic (n = 20) and teratozoospermic (n = 23) males using PCR and Western blot analysis. Semiquantitative PCR indicated either undetectable or significantly lower levels of expression of DYNLT1 in the germ cells from several patients from across the three infertility syndrome groups, when compared with that of fertile controls. DYNLT1 was localized on head, mid-piece, and tail segments of spermatozoa from fertile males. Spermatozoa from infertile males presented either a total absence of DYNLT1 or its absence in the tail region. Majority of the infertile individuals showed negligible levels of localization of DYNLT1 on the spermatozoa. Overexpression of DYNLT1 in GC1-spg cell line resulted in the up-regulation of several cytoskeletal proteins and molecular chaperones involved in cell cycle regulation. Defective expression of DYNLT1 was associated with male factor infertility syndromes in our study population. Proteome level changes in GC1-spg cells overexpressing DYNLT1 were suggestive of its possible function in germ cell development. We have discussed the implications of these observations in the light of the known functions of DYNLT1, which included protein trafficking, membrane vesiculation, cell cycle regulation, and stem cell differentiation.

The t-complex of the mouse occupies the proximal half of chromosome 17 and contains genes which have profound effects on spermatogenesis. Multiple mutations in several loci in the t-complex appear to interact to cause complete male sterility (1, 2). Tctex-1 (t-complex testis expressed-1), lately renamed as dynein light chain 1 (Dynlt1)1, is identified as a candidate gene involved in male sterility in mice (1) and maps to the t-complex in mice (3). Dynlt1 is a member of a multigene family which is virtually germ cell-specific and is eightfold over expressed in t-homozygotes and 200-fold higher in testis than in other adult tissues (1). The human homologue of the mouse Dynlt1 is located on chromosome 6q25.2–25.3. The amino acid sequence shows a high degree of similarity to the predicted product of the Dynlt1 gene of the mouse t complex (4).

DYNLT1 gene encodes a 14 kDa protein constituting the inner arm L1 of cytoplasmic and flagellar dynein complexes (5, 6). DYNLT1 is localized to Golgi complexes as well (7). DYNLT1 protein is present in sperm tails and oocytes (8, 9). A wide range of cellular events are brought about by cytoplasmic dynein and its association with the accessory intermediate, light intermediate, and light chain subunits. These subunits define the interaction of cytoplasmic dynein motor complex with other molecules (10). DYNLT1 is involved in cargo binding (11), lymphocyte division (8), vesicle transport (1214), and human embryo implantation (15). DYNLT1 is known to undergo phosphorylation during apical delivery of rhodopsin (16) and during its interaction with the bone morphogenetic receptor type II (BMPRII) (17). DYNLT1 can function in dynein-independent fashion as a cell fate regulator by its interaction with G-protein β γ subunit regulating initial neurite sprouting (18), axonal specification, and elongation of hippocampal neurons in culture (11, 19). GEF-H1 is bound to microtubules by DYNLT1 and its release without microtubule depolymerization is mediated through the interaction of DYNLT1 with G proteins (20). DYNLT1 is a novel marker for neural progenitors in adult brain (21). DYNLT1 regulatory element was identified which selectively marked nestin+/GFAP+/Sox2+ neural stem-like cells in developing and adult brain (22). The genetic knockdown of DYNLT1 in radial precursors promoted neurogenesis (23). The use of GFP placed under the control of DYNLT1 promoter to mark adult neural stem cells and thus allowing the insertion of any nucleotide sequence selectively into neural progenitors has been patented (24).

DYNLT1 is reported to have functional roles in non-murine germ cells as well. DYNLT1 was found to be essential during spermatid differentiation in Drosophila (10) and a mouse DYNLT1 homolog was identified in the dynein light chain of sea urchin sperm flagella (25, 26). However, the expression of DYNLT1 in human testicular germ cells and its association, if any, with human male factor subfertility are not yet evaluated. This study evaluates the association between DYNLT1expression and spermatogenesis in infertile human males and the possible function of DYNLT1 in spermatogonial cell division and differentiation.

MATERIALS AND METHODS

Reagents

Ready-To-Go T-primed First-Strand Kit andHybondTM-P PVDF membrane (GE Healthcare, NA, UK), antibodies to DYNLT1 and β-ACTIN, goat anti-rabbit IgG-HRP, goat anti-rabbit IgG-FITC (Santa Cruz Biotechnology, Santa Cruz, CA), Reverse Transcriptase PCR primers (Sigma Genosys, Bangalore, India), Big Dye Terminator v3.1 Cycle Sequencing Kit, ChampionTM pET 100 Directional TOPO® Expression kit, Lipofectamine 2000 (Invitrogen, Carlsbad, CA), QIA quick gel extraction kit (Qiagen Gmbh, Hilden, Germany), GC1-spg cell line (CRL:2053) (ATCC, VA, USA), and pEGFPN1 vector (Clonetech, Mountain View, CA) were procured. TRI reagent, agarose, ethidium bromide, Trizma Base, glycine, SDS, CHAPS, Glycerol, PMSF, EGTA, sodium orthovanadate, DAB, Hydrogen peroxide, nickel chloride, poly-l-lysine, paraformaldehyde were purchased from Sigma-Aldrich.

Semen Collection from Fertile and Infertile Individuals

This study was approved by the Institutional Review Board and Human Ethics Committee of Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India. Volunteering semen donors for the study program were recruited after obtaining a written consent from the subjects. Men who have fathered a child in the preceding 2 years and presenting normal seminogram were considered as fertile males (controls). Male partners of sexually active infertile couple visiting Samad Infertility Clinic presenting abnormal seminogram indicative of male factor infertility were recruited as infertile males (cases) in this study. These infertile males did not have psychosomatic disorders affecting normal insemination and their female partners were clinically fertile. The volunteers produced semen samples by masturbation following 2 days of sexual abstinence. The samples were allowed to liquefy for 30 min at 37 °C prior to analysis with a Computer Assisted Semen Analysis system at Samad Hospital and the semen parameters were recorded following WHO guidelines. Our study group consisted of normozoospermic (n = 14), asthenozoospermic (n = 15), oligozoospermic (n = 20) and teratozoospermic (n = 23) subjects (Supplemental Table S1). The spermatozoa were sedimented by centrifugation at 3000 × g at 37 °C for 10 min. The pellet was resuspended in 5 ml PBS and was divided into two aliquots of 2.5 ml each, centrifuged as mentioned above and supernatant discarded. One aliquot was used for RNA preparation, whereas the other aliquot was used for protein extraction.

Reverse Transcriptase PCR Analysis

Each sperm pellet (~107 cells) was homogenized in 1 ml of TRI reagent at 1600 × g for 30 s with 30 s interval using a PT-100 homogenizer probe (Kinematica AG, Luzernerstrasse, Lucerne). Total RNA was extracted following protocol recommended by the reagent manufacturer. Briefly, 200 μl of chloroform was added to the homogenate, shaken vigorously for 15 s, incubated for 15min and centrifuged at 12,000 × g for 15 min at 4 °C. The upper transparent layer was transferred into a new eppendorf tube and RNA precipitated using 0.5 ml isopropanol. The RNA pellet was washed in 70% ethyl alcohol, air dried and suspended in 35 μl sterile DEPC water. 20 μg of the total RNA was heated at 65 °C for 5 min and reverse transcribed to cDNA at 37 °C for 90 min using the Ready To Go T-Prime first strand synthesis kit. The kit utilizes the Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase and an oligo (dt) 18 primer to generate the first strand cDNA. The prepared cDNA was stored at −20 °C.

PCR primers specific for DYNLT1 (DYNLT1_5F, DYNLT1_324R), and ACTB (ACTB -71F, ACTB -490R) were designed using Primer3 software (http://primer3.ut.ee/). The details of the primers used are given in Table II. The polymerase chain reaction was set up as follows: 1 μg of cDNA was mixed with 1X reaction buffer containing 1.5 mm MgCl2, 0.2 mm dNTPs, 0.5 units thermostable Taq Polymerase and 1 μm each of forward and reverse primers, in a 22 μl reaction mixture. An initial denaturation at 94 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30s, extension at 72 °C for 30s and a final extension at 72 °C for 10 min were given to amplify DYNLT1 from the cDNA in the Gene Amp PCR System 9700 (AB Applied Biosystems, Carlsbad, CA). For the amplification of ACTB, an annealing temperature of 58°C was used, and other conditions remained the same. The PCR products were mixed with 2 μl of gel loading buffer and the products were separated by electrophoresis (100 V, constant voltage) on a 1% agarose gel containing 0.5% of ethidium bromide, using Bio-Rad Gel Electrophoresis SubCell (model 192) and 0.5% TBE running gel buffer. The PCR products were visualized on a UV Transilluminator (GeNei, Bangalore, India) and images captured on Gel Doc image analyzer system using the Quantity One software system (Bio-Rad Laboratories, Hercules, CA). Band intensities of DYNLT1 were quantitated and were normalized to the corresponding levels of β-ACTIN using Phoretix 1D Advanced software, Version 4.01 (Phoretix International, Newcastle upon Tyne, UK).

Table II
List of primers used for Polymerase Chain Reaction.

The DYNLT1 bands were excised, eluted using QIA quick gel extraction kit according to manufacturer's instruction, subcloned in pCR4-TOPO vector and recombinant plasmids were isolated. The integration of the gene into the vector was confirmed by DNA sequencing. Automated sequencing reaction was performed using the Big Dye Terminator v3.1 Cycle Sequencing Kit. The sequencing reaction was performed in a Gene Amp PCR System 9700 (Applied Biosystems, Carlsbad, CA), the dye-terminated products were precipitated and were run in an ABI 3730 automated DNA analyzer. The sequences were analyzed by using NCBI nucleotide-nucleotide BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). DNA sequences were aligned using ClustalW (www.ebi.ac.uk/clustalw), the alignments were shaded using GENEDOC, version 2.5.0 and a consensus sequence was generated. The conceptual translation of the sequenced data was generated using the JustBio Translator (http://www.justbio.com/index.php?page=translator).

SDS-PAGE and Western Blotting

Each sperm pellet was resuspended in 200 μl of the solubilization buffer (187 mm Tris-HCl, pH- 6.8, 2% SDS, 0.05% CHAPS, 10% Glycerol, 1 mm PMSF, 1 mm EGTA and 1 mm sodium orthovanadate) and disrupted by sonication (15 MHz, three pulses of 30 s each; Branson Sonifier, Danbury, CT). It was then centrifuged at 7000 × g for 10 min at 4 °C. The supernatant was taken as protein extract and stored at −80 °C.

The protein extracts (20 μg) were mixed with equal volumes of Laemmli buffer (1:1) and were heat denatured at 95 °C for 5 min. Extracts were clarified by centrifugation at 18,404 × g for 10 min at 27°C. Proteins were resolved on a 12% SDS-PAGE at constant voltage (100 V). The proteins were transferred to the PVDF electroblotting membrane at constant current of 30 mA for 8 h at 4 °C, using a Mini Trans Blot cell (Bio-Rad laboratories). The blotting buffer (pH 8.2) composition was 20% methanol, 25 mm Tris (pH 8.2) and 190 mm Glycine. After blotting, the membranes were prewet in methanol and incubated in blocker of 5% skimmed milk powder in PBS-T for two hours. Then the membranes were washed three times with PBS-T at 5 mins interval. The membranes were incubated with DYNLT1 antibody at a dilution of 1:1000 in PBS-T for two hours and were washed three times with PBS-T at 5 mins interval. Goat anti rabbit IgG-HRP was added at a dilution of 1:2000 in blocker for one hour. The blots were washed with PBS-T three times at 5 mins interval. The blots were developed by incubating the membranes in 0.05% DAB, 0.1% Hydrogen peroxide and 0.04% nickel chloride in PBS-T until the desired contrast was obtained. The blots were photographed on Gel Doc image analyzer system using the Quantity One software system (Bio-Rad Laboratories). Band intensities of DYNLT1 and were quantitated and were normalized to the corresponding levels of β-ACTIN using Phoretix 1D Advanced software, Version 4.01 (Phoretix International).

Immunofluorescence assay

Fifty microliters of the sperm suspension was coated onto Poly-l-Lysine-coated cover slips, fixed using 4% paraformaldehyde for 10 min and permeabilized in 0.25% Triton X-100 for 10 min. These cover slips were incubated for 2 h in PBS containing 2 mg/ml BSA and 100 mm glycine to block the nonspecific binding of cellular proteins to the primary antibodies. The cover slips were washed in three changes of PBS and were subsequently incubated with anti-DYNLT1 antibody at a dilution 1:1000 in PBS-T for 2 h at room temperature. The cover slips were again washed three times in PBS and incubated with FITC conjugated goat anti rabbit secondary antibody (dilution 1:2000 in PBS-T) for 1 h in the dark. The cover slips were washed with PBS-T and stored in dark. The cells were imaged under Leica TCS SP2 Confocal Laser Scanning Microscope (Leica TCS SP-II AOBS system, Wetzlar, Germany).

Generation of Dynlt1 Expression Construct

The mouse homolog of DYNLT1 (Dynlt1b; Accession No. CCDS49936.1) was amplified (342 bp) from mature mouse total testicular cDNA using gene specific forward (5′-ATGGAAGACTTCCAGGCCTCCGA) and reverse (5′-TGGATGGACAGTCCGAAGGTACTGAC) primers with HindIII and SalI restriction sites respectively. Both the Dynlt1 amplicon and the pEGFP-N1 vector were double digested with HindIII and SalI restriction enzymes and the products were ligated and transformed into competent E. coli (DH5α) cells. The transformants were screened by colony PCR using pEGFP-N1 vector specific forward (5′-CGCAAATGGGCGGTAGGCGTG and reverse (5′-CGTCGCCGTCCAGCTCGACAG) primers. The proper integration and orientation of the insert in the vector was confirmed by sequencing using vector specific primers. The recombinant plasmid from the positive transformants was isolated for transfection experiments.

Culturing of Cells and Transfection

The GC-1 spg (CRL: 2053) [ATCC, VA] mouse-derived spermatogonial cell line were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% FBS and 1% antibiotics in a CO2 incubator at 37 °C and 5% CO2 (Thermo Scientific). The cells were trypsinized and resuspended in fresh DMEM medium and reseeded in 10 cm culture dishes containing DMEM supplemented with 10% FBS and 1% antibiotics and incubated at above mentioned conditions to reach 70–80% confluent growth. The DMEM medium was replaced with OptiMEM medium 1 h prior to transfection, and the transfection was carried out with 50 μg of expression construct (pEGFPN1-Dynlt1) per plate using lipofectamine 2000 reagent and incubated for 12 h. After 12 h incubation, the OptiMEM medium was replaced with DMEM containing 10% FBS and 1% antibiotics and incubated for 36 h at 37 °C in an atmosphere containing 5% CO2.

Mass Spectrometry

The GC-1 spg cells transfected with pEGFPN1-Dynlt1 construct were harvested and lysed after 48 h post transfection and the total crude protein was extracted using detergent free lysis buffers. GC1-spg cells transfected with empty pEGFPN1 vector served as control. The detergent free hypotonic lysis buffer (10 mm HEPES-pH-7.9, 1.5 mm MgCl2, 10 mm KCl) and extraction buffer (20 mm HEPES, pH-7.9; 1.5 mm MgCl2; 0.42 m NaCl; 0.2 mm EDTA; 25% (V/V) Glycerol) yielded crude cytoplasmic and the nuclear fractions respectively and both these fractions were pooled together as crude protein sample. The concentration of the crude protein samples was measured by Bradford assay and the concentration of all the samples were normalized using 50 mm ammonium bicarbonate (ABC) buffer to yield a final concentration of 1 μg/μl.

One hundred micrograms of proteins from each sample was subjected to in-solution trypsin digestion according to the recommendations of the manufacturer. The digested peptide solutions were centrifuged at 18,404 × g for 12 min and the supernatant was collected. The supernatant was transferred to autosampler vials (Total Recovery Vial, Waters) for peptide analysis via LC-MSE with ion-mobility. The tryptic peptides were separated using a nanoACQUITY UPLC® chromatographic system (Waters, Manchester, UK) employing reversed-phase chromatography technology. Instrument control and data processing were done with MassLynx4.1 SCN781 software. Mass spectral analysis of eluting peptides from the nanoACQUITY UPLC® were carried out on a SYNAPT® G2 High Definition MS™ System [HDMSE System (Waters)]. The acquired ion mobility enhanced MSE spectra was analyzed using ProteinLynx Global SERVER™ v2.5.3 (PLGS, Waters) that uses integrated ProteinLynx™ and MASCOT® database searching (multithreaded) method to maximize number and confidence of protein identifications for protein identification as well as for the label-free relative protein quantification. Data processing included lock mass correction post acquisition. Processing parameters for PLGS were set as follows: noise reduction thresholds for low energy scan ion, 150 counts, high energy scan ion, 50 counts, and peptide intensity, 500 counts (as suggested by manufacturer). The protein identifications were obtained by searching against the mouse database (downloaded from NCBI (ftp://ftp.ncbi.nih.gov/refseq/M_musculus/mRNA_Prot/dated July 22, 2014) containing 77,623 entries. During database search, the protein false positive rate was set to 4%. The parameters for protein identification was made in such a way that a peptide was required to have at least one fragment ion match, a protein was required to have at least three fragment ion matches and a protein was required to have at least 1 peptide match for identification. Mass tolerance was set to 10 ppm for precursor ions and 20 ppm for fragment ions. Oxidation of methionine was selected as variable modification and cysteine carbamidomethylation was selected as a fixed modification. Trypsin was chosen as the enzyme used with a specificity of 1 missed cleavage. Proteins identified with at least two distinct peptides with a probability of 0.95 or above were considered as correct identifications. Data sets were normalized using the “auto-normalization” function of PLGS and label-free quantitative analyses was performed by comparing the normalized peak area/intensity of identified peptides between the samples. Furthermore, only a fold change higher than 50% difference (ratio of either <0.50 or >1.5) was considered to be indicative of significantly altered levels of expression.

Bioinformatics

The lists of differentially displayed proteins were analyzed using PANTHER classification system (http://www.pantherdb.org/) to categorize the proteins based on their functional relevance. Functional networks of differentially displayed proteins in DYNLT1 overexpressed GC1-spg cells were generated using STRING 9.0 (http://string-db.org/newstring_cgi/show_network_section.pl). A confidence view was generated setting the filter to medium confidence (0.400), limiting the interactions below 50 and representing the strength of associations with the thickness of connecting lines.

Statistical Analysis

Normalized band intensities from RT-PCR and Western blot experiments on subjects of four categories, viz., normozoospermia (n = 14), asthenozoospermia (n = 15), oligozoospermia (n = 20) and teratozooserpmia (n = 23) were subjected to one-tailed t test using Mircosoft Excel package. Comparisons were made between normozoospermia and each of the case groups and a p value < 0.05 was considered as statistically significant. The mean ± S.D. of each of the subject groups was computed and plotted as a histogram.

RESULTS

Western Blot Analysis

On Western blots, protein extracts from the spermatozoa of all fertile males showed a prominent band at 14 kDa that was recognized by anti- DYNLT1 antibody (Fig. 1A, lanes N5, N3, N6, N7, N8, and N9). This band was very weak in the protein extracts of spermatozoa from many infertile men (Fig. 1A, lanes A32, A209, A239, O253, O238, T228, T233, and T255), though some of the infertile men showed normal levels of expression of DYNLT1 (Fig. 1A, lanes A34, A254, A257, O75, O250, O243, O1, O19, O20, T 36, T242, T244, T246, T247, and T234). ACTB was used as loading control. The band intensities of DYNLT1 were normalized to the respective ACTB levels following quantitation using the phoretix 1D Advanced software, Version 4.01 (Phoretix International) (Fig. 1B). The expression levels of DYNLT1 in infertile men were significantly lower when compared with that of fertile men.

Fig. 1.
A, Expression of DYNLT1in the spermatozoa of fertile and infertile males as detected by Western blot analysis. ACTB served as a loading control. B, Bar diagrams show the computed band intensities of DYNLT1 normalized to ACTB levels in fertile (n = 6), ...

Immunofluorescence Analysis

The localization of DYNLT1 on spermatozoa was performed using immunocytochemistry. The spermatozoa from fertile males showed DYNLT1 localization on the head, mid-piece and tail regions (Fig. 2, N5). The spermatozoa from infertile males showed a complete absence of DYNLT1 from the tail region, though this protein was present on the head and mid piece of these cells in asthenozoospermic (Fig. 2, A254), oligozoospermic (Fig. 2, O19) and teratozoospermic (Fig. 2, T244) men.

Fig. 2.
Immunocytochemical localization of DYNLT1 in human spermatozoa. Representative images of DYNLT1 expression in spermatozoa of normozoospermic (N5), asthenozoospermic (A254), oligozoospermic (O19) and teratozoospermic (T244) conditions are shown. Propidium ...

RT-PCR Analysis

PCR using DYNLT1 specific primers from the cDNA prepared from the germ cells obtained from men of proven fertility produced a 350 bp amplicon (Fig. 3A, N5, N3, N6, and N7). This amplicon was eluted and its authenticity was confirmed by direct sequencing (supplemental Fig. S1). The sequence was deposited in the Genbank (Accession No. EU862237). A conceptual translation of this gene yielded a protein with 119 amino acids (supplemental Fig. S1).

Fig. 3.
A, A representative RT-PCR indicating the expression of DYNLT1 in the germ cells of fertile and infertile human males. The levels of β-ACTIN in all these conditions are also shown, which served as an internal control. M - 100 bp DNA ladder; N ...

Human males clinically diagnosed to have spermatogenic insufficiency showed absence (Fig. 3A, lanes A209, A231, A122, A202, A30, A32, A38, O41, O12, O28, O29, T229, T121, T213, T242, T246, T247, T26, T27, T31, T33, T39, and T34) or low levels (Fig. 3A, lanes O227 and T228) of expression of DYNLT1 in the germ cells as interpreted from RT-PCR analysis. However, a few infertile men showed normal levels of DYNLT1 expression (Fig. 3A, lanes A204, A34, A37, O75, O124, O207, O35, O244, O232, T5, T233, T234, T187, T244, T245, T36). ACTB served as the endogenous control. The expression levels of DYNLT1 in infertile men which were significantly lower than those of fertile individuals were expressed as relative band intensities, following quantitation using the phoretix 1D software (Fig. 3C).

CD45 was not detected in the cDNA prepared from germ cells of fertile and infertile individuals indicating absence of lymphocyte contamination in the immature germ cells harvested from the semen (Fig. 3B). CD45 amplified from the cDNA prepared from WBC isolated from human blood is shown as positive control.

Proteome Profiling of GC1-spg Cells Over-expressing DYNLT1

Single colonies of the E. coli (DH5α) transformed with pEGFPN1-Dynlt1 were screened by colony PCR (Supplemental Fig. S2, A). The amplicons were sequenced and the authenticity of the insert was confirmed by subjecting the sequence obtained to BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

GC1-spg cells transfected with pEGFPN1-Dynlt1 and the empty pEGFPN1 vector were incubated for 48 h to achieve the over expression of DYNLT1 (Fig. 4 and supplemental Fig. S2B–S2D). The cells were harvested and lysed for protein preparation and subsequent mass spectrometry. The number of distinct peptides and the percentage coverage for each protein assigned for peptide and protein identification are listed in supplemental Tables S2 and S3. The proteomic analysis showed 121 differentially expressed proteins and among them 112 was up-regulated and nine were down-regulated. Among the 121 differentially expressed proteins, only 100 proteins were identified by Uniprot and the respective proteins with Uniprot gene name were listed with their expression level changes (supplemental Table S4). The mass spec predicted proteins which were not identified by Uniprot were tabulated with their fold expression level changes (supplemental Table S5).

Fig. 4.
Confocal microphotographs of GC1 cells transfected with recombinant EGFP-N1 Dynlt1 over expressing vector and EGFP-N1 empty vector constructs. Nuclei were counter stained with Hoechst 33342. Scale bar - 10 μm.

The PANTHER classification system was used to categorize the up and down-regulated proteins with respect to DYNLT1 over expression. Those differentially expressed up-regulated proteins were classified into 17 categories according to protein class. The major proportion of the proteins were categorized under chaperones, followed by nucleic acid binding proteins and cytoskeletal proteins (Fig. 5A). Likewise, the down regulated proteins were classified into three categories (Fig. 5B). The STRING prediction tool was used to identify the interactions among the differentially expressed proteins and it showed four clusters of interacting protein sets like ribosomal protein group, cytoskeletal proteins group, chaperones, and metabolic proteins (Fig. 6A). There were no interacting clusters in case of the down regulated proteins (Fig. 6B).

Fig. 5.
PANTHER classification of proteins up-regulated (A) and down-regulated (B) in GC-1 cells over-expressing DYNLT1.
Fig. 6.
Functional interactome of up-regulated (A) and down-regulated proteins (B) in GC1-spg cells over-expressing DYNLT1, generated using STRING network prediction algorithm.

DISCUSSION

DYNLT1 gene family was identified as a member of the t-complex of the mouse, which has been linked with male germ cell development and function in mouse (1, 27) and the fly (28, 29). Though defects in DYNLT1 expression were linked to defective spermatogenesis in both mouse and Drosophila, there has been no report evaluating the association between the expression of this gene and defective spermatogenesis in human males. Efforts to assess the role of DYNLT1 by creating mice transgenic for a wild-type bacterial artificial chromosome (BAC) derived from the S1-critical region (containing genes Synj2 and Serac1 which are mutated in t haplotypes) bred onto t haplotype mice revealed that introduction of BAC was sufficient to restore fertility in mice (30). However, the fertility status of the heterozygotes generated in this study and their subsequent progeny was not mentioned. Also, the limited number of progeny containing both BAC ends with the intervening genomic DNA (3.9%) indicates the frailty of the above mentioned study in ruling out DYNLT1 candidature as a fertility determining factor. The data presented in this paper documents for the first time that human males with spermatogenic insufficiency present defective expression of DYNLT1in germ cells. The mean values of various semen quality parameters of the subjects who participated in this study are given in Table I.

Table I
Mean sperm quality indicators of the semen samples used in this study. Values indicated are Mean ± S.D. of the observations made. N-Normozoospermia, A-Asthenozoospermia, O-Oligozoospermia, T-Teratozoospermia

DYNLT1 was expressed aberrantly in infertile individuals compared with fertile individuals upon analysis at the genomic (Fig. 3A and and33C) and proteomic (Fig. 1A and and11B) levels. Our lab reported the abnormal expression of TAR DNA-binding protein (TDP-43) leading to spermatogenic dysfunction recently (31). The probability of DYNLT1 being amplified from non germ cells (like WBC) if any present in the semen was nullified by checking the expression of CD45 in these individuals (Fig. 3B). The localization of DYNLT1 in the tail segments of spermatozoa from fertile human males (Fig. 2), and its striking absence in the spermatozoa from infertile males, strongly suggests a role for this molecule in flagellar dynamics. DYNLT1 was identified as a component of the microtubular network (5) of the cells and was implicated in functions involving cytoskeletal network and cellular motility (32, 33). Tctex-2, another t-complex protein was also localized in mouse sperm tails (34) and phosphorylated at activation of sperm motility (35).

The genes in t-complex locus of the mouse have important role in male germ cell development and function. The t-complex locus has several genes essential for normal tail length, embryogenesis and spermatogenesis (36). Dynlt1 is one of the t complex genes and its chromosomal position and germ cell specific expression make it as a candidate gene for male sterility (37). The presence of DYNLT1 in mouse sperm and its involvement in meiotic drive were reported (38). It is also reported that, DYNLT1 expression is limited to cell cycling progenitors (39) and in young neuronal progenitors (40) in the adult brain. This stem cell/progenitor specific expression in the adult organ raises the possibility of role in maintaining stemness in the mature organs. Stem cells (SCs) are unique cells, posses the capacity to self-renew and as well as have ability to generate differentiated cells (41). The expression of transcription factors like Oct3/4, Sox2, Klf4, c-Myc (42), Nanog and Lin28 (43) were necessary for the maintenance of pluripotency or stemness in both embryo and adult tissues (44). However, the list of proteins which showed significant changes in their levels of expression as a result of Dynlt1 over-expression in GC1 cells did not include any protein associated with stemness.

Spermatogenesis is a complex process during which the transformation of spermatogonial stem cell to spermatozoa occurs via the processes like mitosis, meiosis, and morphological differentiation. The differentiation processes includes nuclear condensation, acrosome and flagellum formation and removal of excess cytoplasm (45). In the present study, majority of the up-regulated proteins categorized by panther classification were molecular chaperones, cytoskeletal proteins, and nucleic acid binding proteins which are involved in cell cycle regulation. In our data, we could see the up-regulation of HSP 70 and HSP 90 classes of chaperones proteins and chaperonins like T-complex protein (TCP-1) subunits in abundance. The stress induced expression of HSP70 family of chaperones in all population of mouse spermatogenic cells (46) and the abundant expression of TCP-1 during spermatogenesis and its localized distribution over the developing acrosome were reported (47). Also, the targeted disruption of chaperone proteins results in compromised sperm maturation and spermatogenic arrest (48). Therefore the molecular chaperones identified in the present study might have prominent role in spermatogenesis and sperm maturation. The cytoskeleton changes in the developing male germ cell were reviewed as important feature in spermatogenic process (49). The next major up-regulated proteins were tubulin class of cytoskeletal proteins, which are involved in cell cycle regulation, mitosis and cytokinesis. This suggests that Dynlt1 might have control in the maintenance of germ cell population during spermatogenesis. The next major up-regulated proteins were nucleic acid binding proteins like 40S and 60S ribosomal proteins and various transcriptional and translational regulating factors. The up-regulation of chaperones, cytoskeletal proteins and nucleic acid binding proteins with respect to DYNLT1 over expression suggests that Dynlt1 might have role in maintenance of germ cells by self renewal during spermatogenesis in the testis.

The STRING network prediction analysis showed four distinct clusters of interacting proteins among the up-regulated proteins, which included ribosomal proteins (RPS3, RPS5, RPS7A, RPS10, RPS4X, RPS3A, RPS17, RPS25), tubulin cytoskeletal proteins (TUBB1, TUBB1A, TUBB1B, TUBB2A, TUBB2C, TUBB3, TUBB5, TUBB6), various molecular chaperones and heat shock proteins (CCT3, CCT7, TCP1, HSPA4, HSPA4L, HSPA5, HSPA8, HSP90AA1, HSP90AB1). Incidentally, some of the up-regulated proteins in our list, viz., moesin (MSN), cytoplasmic 1 actin (ACTB), transforming protein RhoA (RHOA), and the molecular chaperones including T-complex protein 1subunits (CCT3 and CCT7) were found to be expressed in mouse stromal mesenchymal stem cells (50). These results suggest that Dynlt1 might have prominent role in contributing to stemness. Chuang et al. reported that Dynlt1 modulated the actin cytoskeleton through a RhoGTPase-dependent pathway during neurite growth (33). Our data also documents the up-regulation of cytoskeletal proteins associated with RhoGTPase in GC1 cells in response to the overexpression of Dynlt1.

Research over the past few decades transformed DYNLT1 from being a sterility locus gene into a gene with multitude of functions. DYNLT1 has recently been used for marking the progenitor cells in a cell population (24). DYNLT1 plays a key role in multiple steps of hippocampal neuron development, including initial neurite sprouting, axon specification, and later dendritic elaboration and these effects are independent from its cargo adaptor role for dynein motor transport (11). DYNLT1 phosphorylated at Thr 94 has a pivotal role in cell cycle progression (26). However, whether male infertility is an outcome of the defective expression of DYNLT1 in the germline stem cells is yet to be evaluated. In conclusion, this study reports a defect in the expression of DYNLT1 in the germ cells of infertile human males and implicates DYNLT1 in spermatogonial cell division and differentiation.

Supplementary Material

Supplemental Data:

Footnotes

Contributed by

Author contributions: S.I., M.L., and P.G.K. designed research; S.I., S.C.S., J.S., A.K., and M.L. performed research; S.I., S.C.S., J.S., M.L., and P.G.K. analyzed data; S.I., J.S., A.K., and P.G.K. wrote the paper; S.M.P. diagnosed and provided clinical samples.

* This work was supported by Grants 2005/37/24/BRNS and 2011/37B/39/BRNS from Board of Research in Nuclear Sciences, Department of Atomic Energy, Mumbai, to Kumar PG. Indu S was supported by Grant No: (I. no. 10–2(5).2005(ii)-E.U.II, May10th 2006) from University Grants Commission, New Delhi, India. Jiji V at the Confocal Imaging Core Facility provided technical assistance in imaging and Arun Surendran at the Mass Spectrometry and Proteomics Core Facility assisted in 2D LC-MS/MS analysis.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpg This article contains supplemental Figs. S1 and S2 and Tables S1 to S5.

1 The abbreviations used are:

Dynlt1
dynein light chain 1
DMEM
Dulbecco's Modified Eagles Medium
BAC
bacterial artificial chromosome.

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