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Departments of Pharmacology, University of Texas Southwestern Medical Center in Dallas, TexasCecil H & Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center in Dallas, Texas
Departments of Pharmacology, University of Texas Southwestern Medical Center in Dallas, TexasCecil H & Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center in Dallas, Texas
Departments of Pharmacology, University of Texas Southwestern Medical Center in Dallas, TexasCecil H & Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center in Dallas, Texas
Departments of Pathology, University of Texas Southwestern Medical Center in Dallas, TexasDepartments of Molecular Biology, University of Texas Southwestern Medical Center in Dallas, Texas
Departments of Pharmacology, University of Texas Southwestern Medical Center in Dallas, TexasCecil H & Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center in Dallas, Texas
To whom correspondence should be addressed: Department of Pharmacology and Cecil H and Ida Green Center for Reproductive Biology Sciences, UT Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390. Tel.: (214) 645-6279; Fax: (214) 645-6276;
Departments of Pharmacology, University of Texas Southwestern Medical Center in Dallas, TexasCecil H & Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center in Dallas, Texas
* This work was supported by NIH grants RO1HD036022 and RO1HD053889 from the National Institute of Child Health and Human Development; R24RR03232601 from the National Center for Research Resources; R24OD011108 from the Office of the Director. This article contains supplemental Figs S1 to S3, Tables S1 and S2, Data S1 to S4, and Discussion.
Spermiogenesis is a postmeiotic process that drives development of round spermatids into fully elongated spermatozoa. Spermatid elongation is largely controlled post-transcriptionally after global silencing of mRNA synthesis from the haploid genome. Here, rats that differentially express EGFP from a lentiviral transgene during early and late steps of spermiogenesis were used to flow sort fractions of round and elongating spermatids. Mass-spectral analysis of 2D gel protein spots enriched >3-fold in each fraction revealed a heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, TDRD7) abundantly expressed in round spermatids prior to their elongation. Notably, each protein within this ontology cluster regulates alternative splicing, sub-cellular transport, degradation and/or translational repression of mRNAs. In contrast, elongating spermatid fractions were enriched with glycolytic enzymes, redox enzymes and protein synthesis factors. Retrogene-encoded proteins were over-represented among the most abundant elongating spermatid factors identified. Consistent with these biochemical activities, plus corresponding histological profiles, the identified RNA processing factors are predicted to collectively drive post-transcriptional expression of an alternative exome that fuels finishing steps of sperm maturation and fitness.
Post-transcriptional regulation of gene expression is essential for cells to transition into and out of distinct developmental, physiological, and pathological states (
). Accordingly, post-transcriptional control of gene expression plays essential roles during gametogenesis and embryo development by helping cells undergo dynamic changes in fate and function (
). Then, in response to meiotic maturation, polyadenylation of the stored mRNAs signals their translation into maternal proteins required for early embryogenesis (
). However, in contrast to many oocyte mRNAs, translational activation in developing spermatozoa is commonly associated with poly-A tail shortening, rather than polyadenylation (
). This equates to >25,000 spermatozoa generated/male/minute throughout a 65 year reproductive life span to help parent an average family of ∼3 children (
). In adult males, haploid gametes that form spermatozoa are continuously being produced from spermatogonial stem cells in the testes through the developmental process of spermatogenesis (
). During spermatogenesis, a subset of spermatogonial stem cells within the basal compartment of seminiferous tubules give rise to differentiating spermatogonia that amplify in number through a series of mitotic divisions. Differentiating spermatogonia then initiate meiosis and traverse the Sertoli cell blood-testis barrier to enter the seminiferous tubules as newly formed spermatocytes (
). Once inside the adluminal compartment of the seminiferous epithelium, spermatocytes complete meiosis to generate nascent haploid germ cells, termed round spermatids (
). As requisite for round spermatids to mature into spermatozoa, they must dramatically transform anatomically in size, shape, and organelle composition through the post-meiotic process of spermiogenesis (
In rodents, newly formed round spermatids undergo up to 19 well-defined steps of spermiogenesis before being shed into the lumen of the seminiferous epithelium as fully elongated, yet functionally immature spermatozoa (reviewed by Yves Clermont) (
). Acrosome biogenesis is a sperm-specific process adapted from the Golgi complex, and is commonly used to classify spermatids at distinct steps of spermiogenesis as they mature during progressive stages of the seminiferous epithelium cycle (
). The periodic acid-Schiff's (PAS) staining method differentially highlights step-specific morphological changes to the acrosome and nucleus of developing spermatids (
). This is because the epithelial stages are defined by physical associations formed between different testis cell types during an epithelial cycle (see Supplemental Fig. 1 for review). Each unique cellular association defining a respective stage is organized vertically in space within a tubular segment by successive generations of spermatogenic cell types (
). Developmental gaps between each generation of germ cells comprising a given stage is defined by the time taken to complete one cycle of the seminiferous epithelium (i.e. ∼12.9 days/cycle in rats) (
). As such, each epithelial stage merely represents subsequent snap-shots in cycle time within the same seminiferous tubule segment (Supplemental Fig. 1).
In rodents, it is estimated that >5% of mRNAs are specifically expressed to support the meiotic and post-meiotic processes of sperm development and fertilization (
). Spermatogenic cells also express an unusually high percentage of retrogenes, a subset of which encode glycolytic enzymes hypothesized to have been selected by enhancing sperm fitness (
). Still, global silencing of transcription occurs during spermatid elongation as haploid nuclei remodel their chromatin into a hyper-compacted state within the spermatozoan head piece (
). As a result, major fractions of “silenced” transcripts are stored for up to a week in messenger ribonucleic acid particles (mRNPs) until factors in the elongation phase of spermiogenesis trigger their translation to support final steps in spermatozoan development and fertilization (
). Thus, sperm development provides an extraordinary system to study molecular events that regulate post-transcriptional gene expression.
Here, we present mass spectral analysis of proteins distinctly detected in fractions of round and elongating spermatids collected by flow cytometry using Green Elongating Spermatid (GESptd1)
transgenic rats. This cell sorting approach was made possible because GESptd1 rats differentially express EGFP post-transcriptionally from viral elements by mechanisms that mimic many spermatid genes. Protein profiles revealed by these studies demonstrate global down-regulation of an abundant RNA binding proteome expressed prior to up-regulation of metabolic proteins during spermatid elongation. Biochemical activities reported for these RNA-processing factors shed light on how the multiplicity of germline specific mRNA variants are uniquely generated, stored and then expressed to drive spermatozoan biology following transcriptional silencing of the male haploid genome.
MATERIALS AND METHODS
Complete methodologies used for spermatid isolation, Western blot, Northern blot, immunofluorescence, histological staining, in situ hybridization, and sucrose gradient fractionation are detailed in Supplementary Information.
Animal Protocols
Protocols for use of rats in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at UT-Southwestern Medical Center in Dallas, as certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AALAC).
Transgenic Rats
GESptd1–12 transgenic rat strains [i.e. S.D.-Tg(ESptd-EGFP)Fkh1−12] were produced in Sprague-Dawley (Harlan, Inc.) backgrounds using freshly isolated laminin-binding spermatogonia that were treated overnight in culture with lentiviral particles (produced using plasmids pHR'-CMV-EGFP-SIN18, pBH10ψ−E−, and pLVSV-G) prior to transplantation into recipient testes, as described (
). Lentiviral integration sites for GESptd1 and GESptd2 rat strains were identified by splinkerette PCR using genomic DNA digested with HaeIII and ligated to a splinker adapter. Two rounds of PCR were performed using lentiviral vector-specific primers to either the 5′ LTR or the 3′ LTR in combination with linker-specific primers. 5′-LTR-specific primers: “outer” 5′-ctctcgcacccatctctctc-3′; “inner” 5′-tttttggcgtactcaccagtc-3′; 3′-LTR-specific primers: “outer” 5′-gaattcgagctcggtaccttt-3′; “inner” 5′-caatgacttacaaggcagctgta-3′.
Mass Spectrometry and Analyses
Round and elongating spermatid fractions were collected from adult rats between 165–240 days of age by flow cytometry, as described. A total ∼2 × 107 cells for each respective, EGFP Dim and Bright fraction were collected from five cumulative sorts. Post-sort, each fraction was washed twice with PBS and centrifugation at ∼200 × g and pellets were flash frozen in liquid nitrogen and then stored at −80 °C. Frozen pellets were shipped on dry ice to Applied Biomics, Hayward, CA, for 2D DIGE expression profiling. Protein from pooled round spermatid fractions were labeled with Cy3, whereas proteins from pooled elongating spermatid fractions were labeled with Cy5 prior to fractionation. 2D gels were scanned with a Typhoon image scanner (G.E. Healthcare Life Sciences, Inc.) and images were generated with ImageQuant software (G.E. Healthcare Life Sciences, Inc.). Differential protein expression profiles were analyzed with Decyder software (G.E. Healthcare Life Sciences, Inc.). Spots of interest were picked with the Ettan Spot Picker (G.E. Healthcare Life Sciences, Inc.), treated with trypsin and identified by MALDI TOF/TOF mass spectrometry (Applied Biosystems, Foster City, CA) using the non-redundant NCBI database to search 111,467 entries (October, 2010) allowing 1 missing cleavage/entry including the following variable modifications: carbamidomethylation of cysteine and oxidation of methionine. Mass tolerance for precursor ions and fragment ions were set at 100ppm and 0.5 Da, respectively; Cutoff score/expectation value for accepting individual MS/MS spectra = 20; noise threshold <5%. Accession numbers of identified proteins were imported into the Database for Annotation, Visualization, and Integrated Discovery (DAVID) for pathway analysis (http://david.abcc.ncifcrf.gov/). Peaklist-generating software and release version was GPS Explorer™ v3.6; search engine and release version MASCOT 2.0 (Matrix Science).
RESULTS
Spermiogenic Expression of EGFP in Rats
Novel strains of transgenic rats were generated by transducing donor spermatogonial stem cells with a self-inactivating, lentiviral vector (
), and evaluated as animal models for studying sperm development. The lentiviral vector used was designed to express EGFP from an internal cytomegalovirus (CMV) promoter (Fig. 1A) (
), testicular expression of EGFP was visualized by fluorescence microscopy in 7 of 12 (∼58%) rat strains generated (Fig. 1B). Genomic sites where the lentiviral vector integrated were defined for two of the single-copy strains expressing highest relative levels of EGFP in testes (i.e. GESptd1 and GESptd2 rats) (Fig. 1C). In all 7 strains where transgene expression could be visualized microscopically, a consistent pattern of fluorescence was observed in spermatogenic cells nearest to the luminal centers of seminiferous tubules (Figs. 2A, 2B). Northern and Western blot analyses of GESptd1 rats demonstrated selective expression of the reporter transgene in testes (Fig. 2C, 2D). The same Western blot profile was detected for EGFP in GESptd2 rat tissues (not shown). This localization pattern indicated that EGFP was being expressed in the germline during late steps of spermiogenesis, thus, coining the name “Green Elongating Spermatid rats.”
Fig. 1Transgenic rats produced with lentiviral reporter vector.A, Diagram of self-inactivating lentiviral vector, pHR'CMV-EGFP-SIN18 (
Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector.
), used to generate transgenic rat lines by in vitro transduction of donor spermatogonia. Depicted are the U3, R, and U5 regions of the 5′ and 3′ long terminal repeat regions (LTRs); surface glycoprotein element (G); rev response element (RRE); splice acceptor sequence (SA); splice donor sequence (S.D.); cytomegalovirus promoter (CMV); enhanced green fluorescent protein (EGFP). Also shown is the region deleted from the 3′ LTR (ΔU3), which helps to disrupt viral replication (
). B, Fluorescence microscopy of EGFP expression (green) in seminiferous tubules of transgenic rat strains GESptd1, GESptd2, and GESptd3 hemizygous for a single copy of the lentiviral construct. Wild-type rat seminiferous tubules illustrate background fluorescence. Right panels show respective bright field images of seminiferous tubules from each rat. Scale bar, 1 mm. C, Lentiviral transgenes integrated between the Trmp6 and Rorb genes in 1q43 of chromosomes 1 in GESptd1 rats, and between the Sema5a and LOC100360282 genes in 2q23 on Chromosome 2 in GESptd2 rats.
Fig. 2GESptd rats express EGFP specifically in spermatids.A, Phase contrast and green fluorescence microscopy images identify EGFP expression (green) near the lumen of seminiferous tubules in transgenic rat strains GESptd1 and GESptd2. Scale bars, 200 μm. B, Histological cross-section of seminiferous tubules form GESptd1 rats illustrates EGFP expression in elongating spermatids (green). Image overlay shows nuclear with Hoechst 33342 dye in all testis cells (blue). Scale Bar, 200 μm. C, Top: Northern analysis of EGFP expression in tissues of transgenic rat strain, GESptd1. T, testis; M, skeletal muscle; I, small intestine; K, kidney; Lv, liver; Lg, lung; H, heart. Germ Cell Specific EGFP transgenic rat testis (GCS) (
); Wildtype rat testis (WT). Total RNA isolated from day 45 homozygous transgenic and wildtype littermates. Bottom: Same blot after stripping and re-probing for Rn18S (◀ ∼1.9 kb). D, Western analysis of EGFP (◀ ∼27 kDa) and GAPDH (◀ ∼55 kDa) expression in tissues of the transgenic (GESptd1 and GCS) and wildtype rats used in panel “A”.
To more precisely define steps of spermiogenesis expressing EGFP in GESptd1 rats, PAS-Feulgen staining and immunofluorescence were performed on parallel cross-sections prepared from their testes. Expression of EGFP was most abundant in the cytoplasm of step 12–19 spermatids during stages XI to VIII of the seminiferous epithelium cycle (Fig. 3). By comparison, the spermatocyte and round spermatid transcription factor, CREM-Τ (
), was most abundant in late pachytene spermatocytes during stages XI to XIV, and in step 1 to 11 spermatids during stages I to XI of the epithelial cycle (Fig. 3). However, an antisense probe to EGFP specifically hybridized to RNA in both meiotic and post-meiotic spermatogenic cells of GESptd1 rat testes (Fig 4A). Silver grains produced from the EGFP antisense probe became apparent over pachytene spermatocytes after stage IV of the epithelial cycle and then most densely labeled subsequent steps in spermatocyte and spermatid development (Fig. 4B). Thus, in adult rats, EGFP expression appeared to be up-regulated at the transcriptional level during meiotic prophase-I (Fig. 4C; blue gradient bar) and at the translational level during elongation steps of spermiogenesis (Fig. 4C; green gradient bar).
Fig. 3GESptd rats robustly express EGFP in elongating spermatids. Testis cross sections from GESptd1 rats show EGFP expressed during elongating steps 11–19 of spermiogenesis (green fluorescence); by comparison, immunolabeling detects the transcription factor, Crem-tau, in round spermatids during Steps 1–11 of spermiogenesis (red fluorescence). Right panel shows respective images of PAS-Feulgen staining in adjacent histological sections to define stages of the seminiferous epithelium cycle (Roman numerals). Scale Bar, 100 μm.
Fig. 4GESptd1 rats initiate transcription of EGFP in spermatocytes.A, In Situ hybridization of a radio-labeled, EGFP antisense probe to testis cells from GESptd1 rats, but not wildtype rats. Scale Bar, 200 μm. B, Autoradiographic detection of silver grains developing from the EGFP antisense probe most densely formed over late prophase-I spermatocytes (pachytene-secondary) and step 1–17 spermatids. Sertoli Cell (SC); zygotene spermatocyte (Z); pachytene spermatocyte (P); secondary spermatocyte (2nd); round spermatids (RS); elongating spermatids (ES). Scale Bar, 30 μm. C, Illustration of GESptd1 rat transgene expression in spermatozoan progenitor cell types based on in situ hybridization studies in adult rats (top blue and green gradient bars). Note: EGFP transcripts are depicted expressed in GESptd1 rats several days prior to EGFP. As a comparison, arrows at the bottom show relative postnatal days (D) when first generation spermatogenic cell types are initially formed in rat testes. D, Northern analysis of EGFP (◀ ∼1.2 kb), DAZL (◀ ∼3 kb), and Rn18S (◀ ∼1.9 kb) transcripts in GESptd1 rat testes on postnatal days 10, 21, 25, 30, 35, 40, 45; and in D45 testes of wild-type rats (WT). E, Western analysis of EGFP (◀ ∼27 kDa), DAZL (◀ ∼36 kDa) and TUBA1a (◀ ∼55 kDa) in GESptd1 rat testes on postnatal days 21, 25, 30, 35, 40, 45; and in D45 testes of wild-type rats (WT).
The relative timing of EGFP expression in GESptd1 rats was analyzed during the onset of spermiogenesis, which initiates in rats after ∼25 days of age when round spermatids are initially produced through meiotic divisions of first-generation spermatocytes (Fig. 4C; arrows). During postnatal development, EGFP transcripts were first detectable by Northern analysis in testes of 30-day-old rats, which then steadily increased in relative abundance as a function of age (Fig. 4D). Subsequent to detection of EGFP mRNA, EGFP was initially detected by Western blot on day 40, after development of the first elongating spermatids, and proceeded to increase in abundance by day 45 (Fig. 4E). By comparison, detection of testicular transcripts and protein encoded by the deleted in azoospermia-like (dazl) gene each peaked by postnatal day 21, consistent with expression of DAZL in spermatogonia and spermatocytes (Figs. 4D, 4E) (
). Accordingly, transcription and translation of the GESptd1 transgene appeared uncoupled postnatally during sperm development.
However, because mature pachytene spermatocytes are present in 21–25-day-old GESptd1 rats, signals for EGFP detection by Northern blot in total testis lysates at these ages did not correlate precisely with in situ hybridization silver grains formed over pachytene spermatocytes in adult rat testes (Fig. 4B). This could reflect differences in detection thresholds for Northern versus in situ hybridization assays at these respective ages because 1st generation spermatocyte and spermatid populations take time to accumulate in abundance relative to other testis cell types (seesupplemental Materials and Methods), and/or, actual differences in the onset of EGFP expression by pachytene spermatocytes in adolescent versus adult rats.
Packaging EGFP into mRNPs
To help explain the presumptive delay between expression of EGFP transcripts and EGFP during spermiogenesis, lysates prepared from GESptd1 rat testes were fractionated over sucrose density gradients (Fig. 5). Most EGFP transcripts ranging from 0.8–1.5 kb were detected in fractions near the top of the gradient (Fractions 1–3), whereas, lower molecular weight EGFP transcripts of ∼1 kb migrated into EDTA-sensitive polysome-like fractions at the bottom of the gradient (Fractions 9–11) (Fig. 5A). Similarly, the mRNP marker protein, MSY2 (
), comigrated predominantly with a majority of EGFP transcripts in fraction 2 and 3, but was also detected in fractions throughout the sucrose gradient (Fig. 5B). By contrast, a majority of transcripts encoding DAZL migrated in Fractions 9–11 of the sucrose gradient, and thus, were enriched in the polysome-like fractions (Fig. 5C). Hybridization of total RNA from testes of 45-day-old rats with oligo-dt18−24 and treatment with RNase-H increased the relative mobility of EGFP transcripts, shifting a larger portion into bands migrating closer in size to, or less than the ∼1 kb transcript within the polysome-like fractions (Fig. 5A). RNase-H treatment also increased the relative mobility of DAZL transcripts (Fig. 5C). Thus, larger EGFP transcripts potentially represent longer poly-adenylated species, which like many endogenous spermatid genes, could be translationally repressed in mRNPs of spermatocytes and/or round spermatids prior to de-adenylation and incorporation into polysomes for translation in elongating spermatids (
). However, such modifications to GESptd1 EGFP poly-A tails in nonpolysome and polysome fractions will require verification by sequence analysis.
Fig. 5EGFP transcripts localize to mRNP-like particles in GESptd1 rats.A, Northern blot of EGFP transcripts in whole testis lysates from an adult GESptd1 rat after centrifugation into a sucrose density gradient. RNase-H (RH) treated samples after hybridizing with Oligo(dt). Note: a majority of EGFP transcripts are detected in low density mRNP-like particles. Controls (Ct) = testis lysates prepared from GCS-EGFP transgenic (+) and wildtype (−) rats. B, Western blot of MSY2 protein in whole testis lysates (T) from an adult GESptd1 rat after centrifugation into a sucrose density gradient. C, Northern blot of DAZL transcripts in whole testis lysates from an adult GESptd1 rat after centrifugation into a sucrose density gradient. Note: a majority of DAZL transcripts are detected in polysome-like particles. Controls (Ct) = testis lysates prepared from tgGCS-EGFP transgenic (+) and wildtype (−) rats.
Based on robust expression of EGFP in GESptd1 rat spermatids, we postulated that enriched fractions of elongating spermatids could be isolated from their testes by flow cytometry to facilitate biochemical analyses on spermiogenesis. Testis cells from GESptd1 rats were enzymatically disaggregated, and then two major peaks of EGFP-positive cells were identified by flow cytometry and collected by cell sorting (Figs. 6A, 6B). A dimmer peak of EGFP+ cells expressed ∼eightfold lower levels of EGFP when compared with the second, brighter peak of EGFP+ cells (Figs. 6A, 6B). However, only 34.7 ± 3.3% (S.E.; n = 3) of collected EGFP-Bright cells were nucleated; whereas, 92 ± 3.6% (S.E.; n = 3) of collected EGFP-Dim cells were nucleated when scored post-sorting using Hoechst 33342 dye. Based on transgene expression (Fig. 2, Fig. 3, Fig. 4), nuclear morphologies (Fig. 6C;supplemental Fig. S2) and testis cell markers (supplemental Figs. S3A, S3B; supplemental Tables S1, S2), the EGFP-Dim fraction was enriched with round spermatids; whereas, the EGFP-Bright fraction was enriched in both elongating spermatid cytoplasts (also termed residual bodies), in addition to intact elongating spermatids. Both spermatid fractions were depleted of somatic testis cells (supplemental Fig. S3A, S3B).
Fig. 6Flow sorting round and elongating spermatids from GESptd1 rats.A, Flow cytometry analysis of EGFP+ testis cells sorted from GESptd1 rats. Left: Total EGFP+ testis cell population gated within region 1 (R1) of forward and side scatter plot (R1 = 31.9 ± 5.7% total) Center: Background green fluorescence gates G1 and G2 set using a wild-type rat littermate; Right: Dominant peaks of EGFP+GESptd1 rat testis cells sorted from gates G1 and G2 (G1 = 27.8 ± 3.4% R1; G2 = 31.6 ± 0.9% R1); ±S.D. n = 5 sorts. B, Relative purities of “EGFP-Dim” and “EGFP-Bright” GESptd1 rat cells post-sorted from gates G1 and G2, respectively. C, Top: Images of testis cells isolated from wildtype and GESptd1 rats before and after sorting into EGFP-Bright and EGFP-Dim fractions. Bottom: Hoechst 33342 nuclear labeling (blue) of cells in top panels. Scale bars, 8 μm.
Due to the relative purity and abundance of each sorted population, we conducted mass-spectral analysis to identify proteins differentially sorted into the EGFP-Dim and EGFP-Bright spermatid fractions after purification by 2D-electrophoresis (Fig. 7). Respective EGFP-Dim and EGFP-Bright spermatid fractions from five total sorts were pooled for the analysis (∼20 million cells/fraction). A total of 88 differentially expressed (≥twofold) protein-like factors were submitted for analysis (Fig. 7A) (supplemental Data Files S1–S3). Consistent with histological and cytometric fluorescence signals in round and elongating spermatids, EGFP was found by mass spectrometry to be ∼10-fold more abundant in the EGFP-Bright fraction versus the EGFP-Dim fraction (Fig. 7B). Similarly, the EGFP signal was 9.4-fold higher in the EGFP-Bright fraction than the EGFP-Dim fraction by Western blot (supplemental Fig. S3A; supplemental Table S2); in contrast, EGFP transcript levels were similar, but slightly more abundant in the EGFP-Dim fraction than in the EGFP-Bright fraction based on qtPCR (supplemental Table S1). Thus, the GESptd1 rat transgene provided an internal standard for analyzing the relative expression of round and elongating spermatid proteins (Fig. 7B).
Fig. 7Analysis of differentially expressed rat spermatid proteins.A, Differential display of molecules in round and elongating spermatids from GESptd1 rats after separation by 2D gel electrophoresis. Round spermatids in EGFP Dim fractions were prelabeled with a green fluorescent dye; Elongating spermatids in the EGFP Bright fractions were prelabeled with a red dye. The circled molecules were submitted for sequence analysis by mass spectrometry. B, Relative abundance of fluorescently labeled molecules identified by mass spectrometry in fractions of round spermatids (EGFP Dim Spermatids; green), and elongating spermatids (EGFP Bright Spermatids; red) from GESptd1 rats. Ratios of elongating spermatid/round spermatid molecular fluorescence signal intensities (ES/RS) were plotted for positively identified rat proteins (symbols) changing ≥threefold.
Sequences for 20 proteins in spots enriched ≥threefold in the EGFP-Dim fraction (Table I), and 28 proteins in spots enriched ≥3-fold in the EGFP-Bright fraction were positively identified (Table II). Histological localization studies for 26 of the 50 non-EGFP proteins listed in Table I, Table II were previously reported in round and elongating spermatids of rats or mice (
Isolation and characterization of a haploid germ cell-specific novel complementary deoxyribonucleic acid; testis-specific homologue of succinyl CoA:3-Oxo acid CoA transferase.
Molecular complex of three testis-specific isozymes associated with the mouse sperm fibrous sheath: hexokinase 1, phosphofructokinase M, and glutathione S-transferase mu class 5.
Mice lacking Raf kinase inhibitor protein-1 (RKIP-1) have altered sperm capacitation and reduced reproduction rates with a normal response to testicular injury.
). In all reported cases (i.e. 26 of 26 reports), immunolocalization of the identified proteins during spermiogenesis within testicular cross-sections and isolated spermatids were in agreement with results of this study (Table I, II). As additional standards, immunolabeling rat testis sections with antibodies to three of these proteins confirmed the mass-spectral results in this study by matching the reported localization of hnRNPK and GLUL in mice; and STMN in rats (Fig. 8) (
). To our knowledge, up to ∼56% (13 of 23) and ∼29% (8 of 27; excluding EGFP) of proteins in spots that differed by ≥threefold in relative abundance between respective round and elongating spermatid fractions represent newly reported spermiogenic factors (Table I, II). Moreover, in rat testis sections, PCBP1 (Fig. 9A) and EE2F (Fig. 9B) were selectively expressed in round and elongating spermatids, respectively. This was also consistent with mass-spectrometry results (Tables I, II). Interestingly, PCBP1 antibody labeling demonstrated a diffuse granular patter throughout the cytoplasm and in the nucleus, but was also concentrated in distinct germinal granule-like foci in spermatocytes and round spermatids; PCBP1 was also highly localized to a relatively large type of cytoplasmic granule in elongating spermatids (Fig. 9A). EE2F immunolabeling was most prominent in differentiating spermatogonia, preleptotene spermatocytes, step 14–17 elongating spermatids and small nuclear granules in Sertoli cells (Fig. 9B).
Table IProteins enriched in round spermatid fraction. (Common Acronym); ES, Elongating Spermatid; RS, Round Spermatid; rg, retrogene-like containing 0–1 introns; p-rg, Proteins encoded by parent gene and retrogene identified in same protein spot. Supplemental Data File S1 contains corresponding protein accession numbers and mass spec data. Supplemental Data File S2 contains additional gene information
Table IIProteins enriched in elongating spermatid fraction. ES, Elongating Spermatid; RS, Round Spermatid; rg, retrogene-like containing 0–1 introns; p-rg, Proteins encoded by parent gene and retrogene identified in same protein spot. Supplemental Data File S1 contains corresponding protein accession numbers and mass spec data. Supplemental Data File S3 contains additional gene information
Isolation and characterization of a haploid germ cell-specific novel complementary deoxyribonucleic acid; testis-specific homologue of succinyl CoA:3-Oxo acid CoA transferase.
Molecular complex of three testis-specific isozymes associated with the mouse sperm fibrous sheath: hexokinase 1, phosphofructokinase M, and glutathione S-transferase mu class 5.
Mice lacking Raf kinase inhibitor protein-1 (RKIP-1) have altered sperm capacitation and reduced reproduction rates with a normal response to testicular injury.
Fig. 8Mass spectrometry predicts protein localization in rat spermatids.A, Localization of anti-hnRNPK IgG binding (red) in wild-type rat testis. Note: labeling in pachytene spermatocytes (PS) and round spermatids (RS), but not in elongating spermatids (ES). Hoechst 33342 dye labels nuclei (blue) of all testis cells. Scale bar, 30 μm. B, Localization of anti-STMN1 IgG binding (red) in GESptd1 rat testis. Note: labeling in pachytene spermatocytes (PS) and round spermatids (RS), but not in elongating spermatids (ES). Hoechst 33342 dye labels nuclei (blue) of all testis cells. Asterisks marks stage IX seminiferous tubule containing step 9 spermatids starting to elongate. Scale bar, 100 μm. C, Localization of anti-GLUL IgG binding (red) in rat GESptd1 testis. Note: labeling in elongating spermatids (ES), but not in pachytene spermatocytes (PS) or round spermatids (RS). Hoechst 33342 dye labels nuclei (blue) of all testis cells. Scale bar, 100 μm.
Fig. 9Testicular expression of PCBP1 and EEF2 correlate with mass spectrometry signals.A, Localization of anti-PCBP1 IgG binding (red) in wild-type rat testis. Note: widespread labeling in pachytene spermatocytes (PS) and round spermatids (RS), but predominantly focal localization to granules in elongating spermatids (ES). Hoechst 33342 dye labels nuclei (blue) of all testis cells. Roman numbers estimate spermatogenic stage. Scale bars, 30 μm. B, Localization of Anit-EEF2 IgG Binding in wild-type rat Testis. Round Spermatid (RS); Elongating Spermatid (ES); Pachytene Spermatocyte (Py); Preleptotene Spermatocyte (PL); Intermediate Spermatogonia (Int Spg); Sertoli Cell (SC). Roman numbers estimate spermatogenic stage. Scale bar, 30 μm.
Ontology analysis of all sequenced proteins revealed the round spermatid fraction to be dominated by proteins that regulate RNA binding, processing and transport (supplemental Data File S4). This cluster included hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, and TDRD7 (Table I). Interestingly, with respect to post-transcriptional phases of spermatozoan biology, fractions containing elongating spermatids were dominated by alternative forms of glycolytic/metabolic enzymes, redox enzymes, protein synthesis enzymes, and factors that mediate protein stability (Table II).
Retrogene products were also over-represented within the elongating spermatid proteins sequenced; five of the top seven proteins in Table II are predicted to be encoded by retrogenes.
DISCUSSION
Here, a proteomics approach using flow sorted fractions of round and elongating spermatids revealed global down-regulation of an abundant RNA binding proteome expressed prior to up-regulation of metabolic factors during spermatid elongation. Based on biochemical activities in other biological processes, the identified factors are predicted to drive post-transcriptional RNA processing during meiotic and post-meiotic steps in spermatozoan development. Genome-wide studies find testes to express an extraordinarily diverse repertoire of tissue-specific mRNAs (
). Testes not only express an unusually wide array of total exons compared with most tissues, but also express exons encoding tissue-specific, cis-acting splicing elements; a majority of these elements bind hnRNP-family proteins that mediate exon skipping (
). This conduit of post-transcriptional genetic diversity unleashed by the testis is directly related to its fundamental role in supporting sex-specific processes required for spermatozoan development and fertility (
). Thus, it seems logical that we identified RNA regulatory factors as a major, differentially expressed class of proteins in fractions of round spermatids.
Heterogeneous RNA-binding proteins that mediate pre-mRNA splicing, including: hnRNPA2b1 (
Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands.
Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6A from chicken beta-tropomyosin pre-mRNA.
Heterogeneous nuclear ribonucleoprotein E3 modestly activates splicing of tau exon 10 via its proximal downstream intron, a hotspot for frontotemporal dementia mutations.
) comprised the most notable protein cluster we indentified in round spermatids. Several of these same proteins (i.e. hnRNPA2b/1, hnRNPK, hnRNPL, PCBP1, PTBP2) (
The hnRNA-binding proteins hnRNP L and PTB are required for efficient translation of the Cat-1 arginine/lysine transporter mRNA during amino acid starvation.
) are established mediators of translational regulation. Similarly, hnRNPA2b1, hnRNPI, hnRNPL, PTBP2, TDRD7, and RUVBL2 prevent premature translation, but do so by regulating mRNA stability (
Inflammation modulates the interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) I/polypyrimidine tract binding protein and hnRNP L with the 3′untranslated region of the murine inducible nitric-oxide synthase mRNA.
A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells.
) mediate RNA transport and packaging between subcellular compartments, particularly export from the nucleus. Thus, collective expression and then loss of this protein cluster in spermatids prior to up-regulating glycolytic and protein biosynthetic machinery during spermatid elongation sheds light on how the haploid genome ultimately produces, and then translates such a diverse transcriptome.
With respect to nuclear localization of pre-mRNA splicing factors, a strong positive correlation exists between several nuclear RNA binding proteins identified in the flow-sorted round spermatid fraction and their histological localization in testis sections (
A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells.
). Experimentally, we obtained 100% agreement between our mass-spectrometry results and actual localization of spermatid proteins identified in 26 independent studies previously conducted in rats and mice (Table I, Table II). Moreover, 17 of the 26 validating reports were conducted using mice. This demonstrates clear conservation of processes that control protein expression during spermiogenesis in these rodents.
However, it is interesting that only ∼35% of spermatids in the elongating spermatid fraction retained their nucleus following the isolation procedure. Though biochemically suitable for comparing proteins sorted as different cellular and sub-cellular fractions; theoretically, this should bias our results by ∼threefold when measuring the relative abundance of nuclear proteins down-regulated during spermiogenesis. Thus, the clear absence of false-positive discoveries following histological validation of >50% of the total identified proteins in testis sections was intriguing. This fact potentially underscores dramatic remodeling of nuclear protein composition that normally occurs during late steps in spermiogenesis (
). Consequently, normal depletion of representative round spermatid nuclear proteins from fully compacted and elongated spermatids mirrors the relative abundance of these proteins detected in our collected round and elongating spermatid fractions. As such, this current approach would also be less sensitive for identifying up-regulated elongating spermatid proteins tightly association with more mature flagella and nuclei (
). Optimization of methods for testis cell disaggregation and sorting could result in higher yields of intact elongating spermatids. Additional purification steps could also better resolve intact sorted elongating spermatids from cytoplasts/residual bodies (
). At any rate, additional studies are required to validate localization of ∼48% remaining spermiogenic factors in Table I, Table II.
We also demonstrated that post-transcriptional regulation of EGFP expression in GESptd1 transgenic rats mimics genes up-regulated during later steps of spermiogenesis. This is because, like many spermiogenic genes, transcription and translation of the EGFP reporter appears to be largely uncoupled in GESptd1 rats. This developmental paradigm is highly conserved in sexually reproducing organisms (
), and is well established for a large fraction of total transcripts (∼75%) that are stored in the nonpolysomal fractions of rodent spermatocytes and round spermatids prior to elongation (
). Requirement for this protracted translational delay for many sperm-specific genes is highlighted by early ectopic expression of protamine 1 or transition protein 2 in round spermatids (
). Forced premature expression of these sperm histone proteins in round spermatids stimulates precocious chromatin condensation prior to spermatid elongation, and results in infertility due to a block in spermatid development or function (
), RNA binding proteins identified in this study, such as hnRNPA2b/1, hnRNPK, hnRNPL, PCBP1, and/or PTBP2 potentially represent factors that mediate translational repression of mRNAs in spermatocytes and round spermatids (Fig. 10). This theory further implicates that some unknown signal generated in the seminiferous epithelium inactivates and/or stimulates degradation of such RNA binding factors to facilitate spermatid elongation (Fig. 10).
Fig. 10Post-transcriptional regulation of rat tgGESptd1 expression. Open questions are illustrated on how expression of the GESptd1 rat transgene (tgGESptd1) is regulated post-transcriptionally during spermatid maturation. Here, translation of a tgGESptd1 encoded mRNA upon spermatid elongation is modeled hypothetically in association with hnRNP-family proteins (Fig. 5, Table I), reported small RNA processing pathways (
). Mechanisms controlling relative abundance of hnRNPs upon spermatid elongation remain to be determined. Also illustrated, based on information from other studies, are hypothetical GW182/AGO-like protein components in RNA silencing complex (RISC) (
). Yanagiya and colleagues demonstrated that a double knockout of the poly-A-binding protein-interacting proteins (i.e. PAIP2a & PAIP2b) resulted in pre-mature translation of stored mRNAs and a lack of spermatid elongation (
). It was demonstrated that up-regulation of these proteins in round spermatids just prior to elongation under normal conditions proved instrumental in titrating out repressive effects of PABPC1 on translation (
). Genetically, this study further predicts the existence of multiple Paibp2-like factors that orchestrate translational activation during spermiogenesis (Fig. 10; also see Supplementary Discussion).
It is quite interesting that a relatively high percentage of retrogenes are abundantly expressed specifically in spermatids, many of which encode glycolytic enzymes (
). Five of the top seven most-enriched 2D gel spots in elongating spermatid fractions contained proteins predicted to be expressed from retrogene-like elements (Table II). Again, this is similar to the GESptd1 rat lentiviral transgene, which was identified in the 11th most-enriched protein spot in elongating spermatid fractions (Table II). A recent study by Vemuganti and colleagues used bioinformatics to study this relationship, and found an over-representation of LINE and LTR elements flanking glycolytic retrogenes in rodent and primate genomes (
). Likewise, EGFP is expressed from a LTR-based lentiviral retroelement we experimentally integrated directly into the germline of GESptd1 rats (Fig. 1A). Thus, in addition to chromatin factors regulating transcript levels, the striking abundance of proteins expressed from such retrogenes invokes questions regarding the existence of proteinacious and/or RNA translational enhancers that operate with their mRNA sequences during spermiogenesis (Fig. 10). Additionally, chromosomal DNA generated by reverse transcription, including retrogenes and other LTR-expressed factors, may lack elements that negatively regulate protein synthesis from their encoded mRNAs during spermatid elongation.
The unusually robust expression, and sperm-specific nature of retroelements seems remarkably well tailored for driving sperm fitness by selecting activities that promote ATP production while generating relatively low levels of oxidative stress (
), and three of the six core glycolytic enzymes listed Table II represent retrogenes. Thus, it is fitting that glycolytic and redox enzymes were also identified as enriched in the elongating spermatid fraction isolated from GESptd1 rats. Theoretically, this glycolytic power endows spermatozoa in many nonruminant mammalian species greater potential for driving motility (
). Similarly, it can now be asked how increased heterogeneity and abundance of the round spermatid RNA binding proteome would select for sperm fitness.
Along with regulating the time, location and abundance of alternative mRNAs expressed in spermatids, additional clues into this later question may emerge from studies demonstrating that splicing elements in spermatogenic cells largely incorporate RNA binding proteins that select against exon inclusion (
). Consequently, variants produced by exon skipping uniquely in spermatogenic cells could select for gamete fitness by eliminating RNA/protein coding domains that constrain spermatozoan development and function, but which are beneficial to reproductive success when expressed in somatic tissue. Another intriguing hypothesis alluded to above reflects additional roles of the germline RNA binding proteome in restricting replication and coordinating expression from integrated reverse-transcribed sequences that escape transcriptional repression specifically during gametogenesis (as modeled in GESptd1 rats) (
). As such, deleterious retroelement-derived copy number variation could further drive purifying selection so to enrich genomes with replication-restricted or deficient integrants expressed in nonmitotic cells. Thus, evolutionarily, spermiogenetic down-regulation of the RNA binding proteome, as observed here in GESptd1 rats, would further capture benefits gained from retroelements post-transcriptionally, and selectively during spermatid elongation.
Acknowledgments
We thank Gerardo Medrano, Bray S. Denard and Dalia Saidley-Alsaadi for their help with these experiments.
Isolation and characterization of a haploid germ cell-specific novel complementary deoxyribonucleic acid; testis-specific homologue of succinyl CoA:3-Oxo acid CoA transferase.
Molecular complex of three testis-specific isozymes associated with the mouse sperm fibrous sheath: hexokinase 1, phosphofructokinase M, and glutathione S-transferase mu class 5.
Mice lacking Raf kinase inhibitor protein-1 (RKIP-1) have altered sperm capacitation and reduced reproduction rates with a normal response to testicular injury.
Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands.
Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6A from chicken beta-tropomyosin pre-mRNA.
Heterogeneous nuclear ribonucleoprotein E3 modestly activates splicing of tau exon 10 via its proximal downstream intron, a hotspot for frontotemporal dementia mutations.
The hnRNA-binding proteins hnRNP L and PTB are required for efficient translation of the Cat-1 arginine/lysine transporter mRNA during amino acid starvation.
Inflammation modulates the interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) I/polypyrimidine tract binding protein and hnRNP L with the 3′untranslated region of the murine inducible nitric-oxide synthase mRNA.
A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells.
Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector.
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