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21 December 2011 PABP Interacting Protein 2A (PAIP2A) Regulates Specific Key Proteins During Spermiogenesis in the Mouse
Geraldine Delbes, Akiko Yanagiya, Nahum Sonenberg, Bernard Robaire
Author Affiliations +
Abstract

During spermiogenesis, expression of the specific proteins needed for proper differentiation of male germ cells is under translational control. We have shown that PAIP2A is a major translational regulator involved in the maturation of male germ cells and male fertility. To identify the proteins controlled by PAIP2A during spermiogenesis, we characterized the proteomic profiles of elongated spermatids from wild-type (WT) mice and mice that were Paip2a/Paip2b double-null mutants (DKO). Elongated spermatid populations were obtained and proteins were extracted and separated on gradient polyacrylamide gels. The gels were digested with trypsin and peptides were identified by mass spectrometry. We identified 632 proteins with at least two unique peptides and a confidence level of 95%. Only 209 proteins were consistently detected in WT or DKO replicates with more than five spectra. Twenty-nine proteins were differentially expressed with at least a 1.5-fold change; 10 and 19 proteins were down- and up-regulated, respectively, in DKO compared to WT mice. We confirmed the significantly different expression levels of three proteins, EIF4G1, AKAP4, and HK1, by Western blot analysis. We have characterized novel proteins that have their expression controlled by PAIP2A; of these, 50% are involved in flagellar structure and sperm motility. Although several proteins affected by abrogation of Paip2a have established roles in reproduction, the roles of many others remain to be determined.

INTRODUCTION

Spermatogenesis is the process by which male germ cells become highly differentiated haploid spermatozoa. This complex process requires expression of specific proteins at very specific steps of cell differentiation, and so displays tightly controlled transcriptional and translational regulations. Some major factors of transcriptional regulation in spermatogenesis have been identified as DNA binding proteins, transcription factors, and chromatin remodeling factors (see review in [1]), but translational control during spermatogenesis remains largely elusive. RNA-binding proteins and the shortening of the mRNA poly (A) tail are believed to be involved in this process [29]. RNA binding proteins such as SAM68 (official symbol KHDRBS1) [10], GRTH (official symbol DDX25) [11], and YBX2 (also known as MSY2) [7] have been shown to be essential for translational control during spermatogenesis. Mice carrying a null mutation for Sam68 show an early arrest in spermatogenesis around the pachytene spermatocyte stage, whereas knockouts for Grth and Ybx2 result in spermatogenic arrest in late round spermatids, suggesting that these proteins play different roles at different steps of spermatogenesis.

Other factors, such as PTBP2 and PABP, play important roles in RNA storage and stabilization of mRNA during spermatogenesis [2, 5]. We have recently shown that a PABP partner, PAIP2A, is also an essential player in translational control during late spermiogenesis [12]. PAIP2A has been described as a translational inhibitor in vitro; it competes with EIF4G for PABP binding and decreases the PABP affinity for the mRNA poly (A) tails [13, 14]. There are two isoforms of PAIP2: PAIP2A and PAIP2B. PAIP2A is highly expressed in the testis and pancreas, while PAIP2B is highly expressed in the pancreas. PAIP2A is specifically expressed in the cytoplasm of late spermatids during spermiogenesis [12]. We have previously shown by immunohistochemistry [12] that PAIP2A expression becomes increased in late elongated spermatids as PABP expression becomes decreased, while PAIP2B is barely expressed in these cells.

The regulation of poly (A) tail length by polyadenylation or deadenylation is one of the key mechanisms of gene expression in spermatogenesis. For example, PAPOLB (also known as TPAP) is involved in poly (A) tail extension in round spermatids, and Papolb-KO mice exhibit spermatogenetic arrest [15]. Kleene and colleagues [4, 16] have shown that during spermiogenesis, the shortening of the poly (A) tail in elongated spermatids is correlated with active translation, suggesting an important role for PABP. We have demonstrated that efficient mRNA translation in late spermiogenesis occurs at an optimal concentration of PABP, and that it is regulated by PAIP2A [12].

Male Paip2a/Paip2b-DKO mice, as well as Paip2a-KO mice, are infertile due to specific defects in spermiation and the differentiation of elongated spermatids [12]. In the Paip2a-KO and Paip2a/Paip2b-DKO, elongated spermatids at Stage 7 of spermatogenesis show multiple structural abnormalities, including impairment of flagellum formation, absence of the mitochondrial sheath in the middle piece, as well as abnormal chromatin condensation and acrosomal development [12]. The large numbers of defects observed in the Paip2a/Paip2b-DKO elongated spermatids strongly suggest that key proteins needed for proper cell differentiation are not generated in the absence of PAIPA2.

We hypothesized that PAIPA2 plays an important role in late spermiogenesis by regulating the expression of key proteins for male germ cell differentiation. To investigate which proteins are regulated by PAIP2A during spermiogenesis, a wide-ranging proteomic analysis was undertaken. We have identified a group of proteins that is differentially expressed in elongated spermatids between WT and Paip2a/Paip2b-DKO mice.

MATERIALS AND METHODS

Animals

Male Paip2a/Paip2b-DKO mice [12] were housed on a 12L:12D cycle with food and water provided ad libitum. All animal studies were conducted in accordance with the guidelines outlined in A Guide to the Care and Use of Experimental Animals, prepared by the Canadian Council on Animal Care (McGill Animal Resources Centre protocol 5505). Mice (10 to 12 weeks old) were euthanized by CO2 asphyxiation; their testes were removed, decapsulated, and flash frozen in liquid nitrogen for whole testis extract or used for further isolation of spermatogenic cells by unit gravity cell separation.

Cell Separation

Spermatogenic cells were obtained through cell separation by velocity sedimentation using the STA-PUT method as previously described by Bellvé et al. [17]. Briefly, the testes of five mice were decapsulated and digested by enzymatic treatment at 34°C with 0.5 mg/ml collagenase (C9891; Sigma, Oakville, ON, Canada) for 12 min, followed by 0.5 mg/ml trypsin (T8003; Sigma) for 16 min. Cells were then suspended by gentle pipetting in the presence of 1 μg/ml DNase I (DN-25; Sigma), and filtered through a nylon mesh and washed with RPMI medium containing 0.5% BSA fraction V. Of these, 3×108 cells were loaded in the velocity sedimentation apparatus (STA-PUT; Proscience, Don Mills, ON, Canada), followed by 2%–4% BSA gradient in RPMI. Fractions containing pachytene spermatocytes, round spermatids from spermiogenesis Stages 1 to 8, and a mixture of elongating and elongated spermatids from spermiogenesis Stages 8 to 16 were identified by phase-contrast microscopy using cell morphology criteria as previously described (Supplemental Fig. S1; all Supplemental Data are available online at  www.biolreprod.org) [18]. Fractions with higher than 85% purity were pooled and stored at −80°C for RNA and protein extraction.

As assessed by counting nucleated cells, elongated spermatids constituted 94.1 ± 1.5% and 93.7 ± 1.3% of the cells in the elongated spermatid fraction in the WT and the Paip2a/Paip2b-DKO mice, respectively. Despite the defects in spermiogenesis in Paip2a/Paip2b-DKO mice, no apparent difference could be observed in the different fractions obtained when compared to WT (Supplemental Fig. S1). It is important to note that using the STA-PUT method, the elongated spermatid fraction contains a mixture of nuclear and anucelate cytoplasmic fragments of elongated spermatids, residual bodies, and anucleate cytoplasmic fragments of other cell types that cannot be identified by phase contrast microscopy [22]. We calculated that the residual bodies represented about 70% of the sample (72 ± 7% in WT mice [n = 6], and 73 ± 8% in Paip2a/Paip2b-DKO mice [n = 4]). Therefore, we were analyzing the proteins from each cellular compartment of the spermatids, including the cytoplasm that is shed in later spermiation.

Proteomics

Pellets obtained from 2 × 106 elongated spermatids (n = 3, WT and Paip2a/Paip2b-DKO) were thawed on ice and placed in 175 μl of Laemmli buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.002% bromphenol blue, and 62.5 mM Tris-HCl). The samples were homogenized with an ultrasonicator (Sonics & Materials, Inc., Newtown, CT) and centrifuged at 10 000 × g for 10 min at 4°C. The remaining supernatant from each sample was aliquoted and stored at −20°C for protein assay using the 2D Quant Protein Assay (GE Healthcare, Baie d'Urfe, QC, Canada). In collaboration with Genome Quebec, proteins from each sample (30 μg) were separated on a 2.4-cm, 7%-to-15% SDS-acrylamide gel electrophoresis (Supplemental Fig. S2). Each lane was then robotically excised with the Pro-XCISION Proteomics Gel Cutting Robot (PerkinElmer) in 15 bands that were further subjected to in-gel digestion for 4.5 h with 6 ng/μl of trypsin (Promega Gold) in 100 mM ammonium bicarbonate. Sample injection and HPLC separation was done using an Agilent 1100 series system. Mass spectrometric analysis was conducted using the Micro-Qtof from Waters (Milford, MA). The scan range was 350 m/z to 1600, which means a minimum size of 750 Da for a doubly charged peptide (on average, six amino acids) to an upper limit of 4800 Da for a triply charged peptide. Some small-sized peptides may also have been too hydrophilic to be retained by the c18 column. Peptides and proteins were first identified using Mascot 2.1 ( www.matrixscience.com) against a uniprot_database from November 2008 filtered for Mus musculus proteins (60 636 entries) using trypsin as digestion enzyme with one miscleavage allowed, carboxyamidomethylation of cysteines as fixed modification, methionine oxydation as variable modification, and 0.5 Da precursor and 0.5 fragment search tolerances. An additional search using X! Tandem ( http://www.thegpm.org/TANDEM/) was carried out on the subset database of identified proteins with additional modifications considered for D/E and c-terminal methylation, proprionamide modification of cysteines, deamidation of N/Q, and pyroglutamic acid formation of n-terminal Q. Results were subsequently analyzed using Scaffold 2.0 (Proteome Software, Inc., Portland, OR). Only peptides for which we had a confidence level of 95% were considered; proteins were identified by at least two unique peptides and a false discovery rate lower than 0.1%. For quantification and further statistical analysis, we used the spectral counting method [19]. Data are expressed as unweighted spectrum count for each protein; this represents the number of times a spectrum was assigned to a peptide belonging to a specific protein.

Real-Time PCR

Total RNA was extracted from the elongated spermatid fractions (2–6 × 106 cells) using TRI-ZOL Reagent (Invitrogen, Burlington, ON, Canada) and reverse-transcribed by SuperScript III RT (Invitrogen) following the manufacturer's instructions. The resultant cDNA was used for real-time PCR using the gene-specific primers for mouse Eif4g1, Wdr62, Akap4, Hk1-s (Supplemental Table S1), and β-actin (Actb) (mHKG-110; MCLAB, San Francisco, CA). The SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) was used for real-time PCR using the first-strand cDNA as a template, and real-time PCR was performed in triplicate. The Actb mRNA level in each sample was determined to normalize the differences of total RNA amount. Values indicate the relative mRNA levels in Paip2a/Paip2b-DKO mice as compared with those in WT mice, which were set as one.

Western Blot Analysis

Pachytene spermatocyte, round spermatid, and elongated spermatid pellets (3–8 × 106 cells) were thawed on ice and lysed in RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (10 μl/ml; P8340; Sigma). Cell lysates were clarified by centrifugation at 13 000 × g for 10 min at 4°C. Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad Laboratories, ON, Canada). Samples (20 μg) were resolved by 8% SDS-PAGE and transferred onto a nitrocellulose membrane (PerkinElmer). Membranes were blocked in 5% nonfat milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 and probed with primary antibodies against EIF4GI (1:1000 dilution; #2498; Cell Signaling Technologies, Danvers, MA), rabbit anti-mouse AKAP4 (1:5000 dilution) [20], the SSR antiserum (1:1000 dilution) to detect spermatogenic cell-specific type 1 hexokinase [21], and monoclonal anti-β-actin antibody (1:2000 dilution; A5441; Sigma-Aldrich) overnight at 4°C, and subsequently with secondary antibody (ECL anti-rabbit IgG horseradish peroxidase-linked whole antibody; GE Healthcare). Immunoreactive proteins were visualized using enhanced chemiluminescence (Perkin-Elmer). The intensities of the bands were measured using ImageJ software (National Institutes of Health, Bethesda, MD). The value of each band was normalized by that of β-actin.

Statistical Analysis

Experiments were run in triplicate. Two-tailed, unpaired Student t-test was used for statistical analysis. The level of significance was taken as P < 0.05, unless stated otherwise.

RESULTS

Characterization of Proteins in the Elongated Spermatids

Using 1D gel electrophoresis coupled to mass spectrometry analysis, we were able to characterize the proteins in elongated spermatids and obtained a list of 632 proteins. Of these, 526 proteins were detected in at least one of the three WT replicates, and 530 proteins were detected in one of the three Paip2a/Paip2b-DKO replicates (Fig. 1). Interestingly, only 53.7% (286 proteins) and 54.5% (289 proteins) were consistently detected in the three replicates of the WT mice and Paip2a/Paip2b-DKO mice, respectively (Fig. 1), suggesting a degree of variability in the detection of peptides by the mass spectrometer. For this reason, in further analysis, we only considered proteins that were detected in at least two replicates of each group; these represent 391 (61.9%) and 401 (63.5%) proteins detected in the WT mice and Paip2a/Paip2b-DKO mice, respectively (Fig. 1). In total, 469 proteins were expressed in at least two replicates of one genotype, and 323 were considered expressed in both genotypes (Fig. 2A).

FIG. 1.

Numbers of proteins detected in each biological replicate of elongated spermatids. The proteins were extracted from three different cell separations performed on WT (left) or Paip2a/Paip2b-DKO (right) mice. Further analysis was performed on the proteins that were detected in at least two replicates (red triangles).

i0006-3363-86-3-95-f01.tif

FIG. 2.

A) Numbers of proteins expressed in the elongated spermatids in WT and Paip2a/Paip2b-DKO mice. B) Numbers and names of proteins significantly affected in elongated spermatids from Paip2a/Paip2b-DKO mice when compared to the WT mice.

i0006-3363-86-3-95-f02.tif

To undertake further quantitative analysis and eliminate false positives, we selected the proteins having average spectrum counts of five or more; these represent 209 proteins (Supplemental Table S2). The most abundant protein found in the WT elongated spermatids was the l-lactate dehydrogenase C chain, representing 3.4% of all spectrum count of the mass spectrometry analysis. It is noteworthy that the basic proteins, known to be essential in chromatin remodeling during spermiogenesis, were not detected in this analysis. Protamines were not detected by the proteomics analysis we used, since they do not resolve in SDS gels; in addition, the peptides generated after trypsin digestion of the transition proteins (i.e., TNP1 and TNP2) were too small to be detected in our analysis. Interestingly, many heat-shock proteins were present in great abundance in the elongated spermatids (i.e., HSP90AA1, HSP90B1, HSPA4L, HSPA8, HSP1L, and HSP90B1), representing a total of 14.4% of the total proteins. Moreover, consistent with our previous result obtained using immunohistochemistry [12], PABP in Paip2a/Paip2b-DKO mice was expressed at a higher level than in WT mice (Supplemental Table S2), but the difference was not statistically significant.

Effect of the Lack of PAIP2A and PAIP2B on Protein Expression in Spermiogenesis

In order to identify the proteins whose expression was affected by the lack of PAIP2A and PAIP2B, we undertook a statistical analysis with the proteins having an average spectrum count of five or more (209 proteins; Supplemental Table S2). Interestingly, only 29 (13.8%) of the proteins were significantly affected, with at least a 1.5-fold change in Paip2a/Paip2b-DKO mice when compared to WT mice. Two proteins were only detected in Paip2a/Paip2b-DKO mice, while three proteins detected in WT mice were never detected in Paip2a/Paip2b-DKO mice (Fig. 2B and Table 1). Twenty-four proteins were expressed in both samples, with a significant difference of at least a 1.5-fold change in Paip2a/Paip2b-DKO mice as compared with WT mice; seven were down-regulated and 17 were up-regulated in Paip2a/Paip2b-DKO mice (Fig. 2B and Table 1). It is interesting to note that 27.6% of these 29 proteins are involved in metabolic processes and almost 21% are related to reproduction, while several represent a variety of other cellular functions (Table 2).

TABLE 1.

Proteins that are significantly affected with at least 1.5-fold change in Paip2a/Paip2b-DKO as compared to WT elongated spermatids.*

i0006-3363-86-3-95-t01.tif

TABLE 2.

Biological function, number, and names of proteins significantly up- or down-regulated in the absence of PAIP2A and PAIP2B.

i0006-3363-86-3-95-t02.tif

To confirm the results obtained by mass spectrometry analysis, some of the most affected proteins were analyzed by Western blotting (Fig. 3). We were able to assess the amount of these proteins in the different fractions of male germ cells obtained by the STA-PUT method: the pachytene spermatocytes, the round spermatids, and the elongated spermatids (Fig. 3A). By quantifying the intensities of the bands in the elongated spermatids fraction, we confirmed that EIF4GI protein level was increased 3.5-fold in Paip2a/Paip2b-DKO mice when compared to WT (Fig. 3A and 3B). Interestingly, EIF4GI was increased 2.4-fold in round spermatids, as well (see Fig. 3A: 1.0 ± 0.12 in WT [n = 3], and 2.4 ± 0.3 in Paip2a/Paip2b-DKO mice [n = 3], P < 0.05). The increased levels of EIF4GI in DKO spermatids could be due to increased protein synthesis or decreased degradation. We also confirmed that AKAP4 was decreased to 20% and HK1-s was decreased to 25% in Paip2a/Paip2b-DKO elongated spermatids when compared to WT, suggesting the impaired translational control of these proteins in the absence of PAIP2A and PAIP2B during late spermiogenesis.

FIG. 3.

A) Representative Western blot of three experiments for the EIF4G1, AKAP4, and HK1 proteins in the pachyteme spermatocytes (PS), round spermatids (RS), and elongated spermatids (EL) of WT and Paip2a/Paip2b-DKO mice. Quantitative analysis of the bands in the EL for EIF4GI (B), AKAP4 (C), and HK1-s (D). The intensities of the bands were measured using ImageJ (National Institutes of Health). The value of each band was normalized by that of β-actin. The band intensity in WT mice was set at one. *P < 0.05 using an unpaired t-test (n = 3). Black circles indicate WT mice; gray squares indicate Paip2a/Paip2b-DKO mice.

i0006-3363-86-3-95-f03.tif

Effect of the Lack of PAIP2A and PAIP2B on Transcription in Spermiogenesis

In order to determine whether the results obtained using proteomic analysis were due to an impaired transcription regulation in the absence of PAIP2A and PAIP2B, we quantified the mRNA levels in elongated spermatids by real-time PCR (Fig. 4). We selected the three proteins for which changes had been confirmed by Western blot analysis. The relative mRNA level of the up-regulated protein, EIF4GI, in Paip2a/Paip2b-DKO mice was similar to that in WT mice (1.04-fold; Fig. 4A). The mRNA levels of the down-regulated protein, AKAP4, were unchanged in Paip2a/Paip2b-DKO mice as compared to WT mice (Fig. 4B). There are three isoforms of hexokinase 1 (Hk1) mRNA: the general Hk1 and two spermatogenic cell-specific mHk1-s (mHk1-sa and mHk1-sb) containing a spermatogenic cell-specific sequence region (SSR). The peptide sequences obtained by proteomics assay were consensus sequences of both Hk1 and testis-specific Hk1-s (Supplemental Fig. S3). Primers were designed to detect both the ubiquitous and the testis-specific forms of HK1. No amplification could be obtained using the ubiquitous Hk1. The mRNA corresponding to the testis-specific HK1-s was amplified from elongated spermatids, and we did not observe any change in the amount of this mRNA in Paip2a/Paip2b-DKO mice as compared to WT mice (Fig. 4C). Overall, these results suggest that the changes observed by proteomics were most likely due to regulation at the protein level, such as translation and protein stability, rather than changes in transcripts.

FIG. 4.

Relative mRNA levels of Eif4g1 (A), Akap4 (B), and Hk1-s (C) in the elongated spermatids of WT mice (black bars) and Paip2a/Paip2b-DKO mice (gray bars). The Actb mRNA level in each sample was determined and used to normalize the differences of total RNA amount. Values indicate the relative mRNA levels in Paip2a/Paip2b-DKO mice as compared with those in WT mice, which were set as one. Statistical analysis was performed using an unpaired t-test (n = 3). No significant differences were observed.

i0006-3363-86-3-95-f04.tif

Selective Translation Control by PAIP2A

Since PAIP2A is a key player of translation initiation during spermiogenesis [12], we speculated that proteins whose mRNAs are under translational control by PAIP2A would be down-regulated when compared to WT. For this reason, we further investigated the 10 down-regulated proteins in Paip2a/Paip2b-DKO mice and compared them with those of WT mice. Interestingly, four of these (i.e., AKAP4, ATP1A4, FSCB, and TCP11) are directly related to reproductive function according to the GO term database ( http://www.geneontology.org/; Table 2). This suggests that PAIP2A is involved in the expression of specific proteins for male germ cell differentiation in late spermiogenesis. Moreover, all of these proteins are expressed in male germ cells (Table 3), and 50% of them (i.e., ATP1A4, AKAP4, TCP11, FSCB, and CLMN [CALMIN]) have been suggested to be under translational control, as they are spermatid specific (Table 3). These results strongly indicate that PAIP2A is a major factor controlling translation of mRNAs encoding these proteins.

TABLE 3.

Tissue distribution of the down-regulated proteins in the absence of PAIP2A and PAIP2B, according to the literature.

i0006-3363-86-3-95-t03.tif

DISCUSSION

In this study, we have characterized the proteins in the elongated spermatids from Stages 8 to 16 of spermiogenesis using a 1D gel coupled to MS/MS analysis. We identified 209 proteins that are consistently expressed with a level of expression higher than five unweighted spectrum counts. There is growing interest in the characterization of the proteome of male germ cells [23]. A recent study identified 2116 proteins from ICR mouse haploid germ cells using a similar strategy [24]. The discrepancy between this number and that obtained in our present study can be explained, at least in part, by the differences in cell sorting processes; in the present study, we excluded the early stage of spermiogenesis (i.e., the round spermatids) and characterized the proteins expressing in late spermiogenesis.

We have further demonstrated that the expression of several proteins was affected by the absence of PAIP2A and PAIP2B. Considering that PAIP2A is much more abundant than PAIP2B in the testis [12], and that only the Paip2a- but not the Paip2b-KO male mice are infertile [12], it is very likely that PAIP2A is responsible for translational control in late spermiogenesis.

PAIP2A has been characterized as a general translational regulator [14]. Surprisingly, only some proteins were up- or down-regulated in Paip2a/Paip2b-DKO elongated spermatids, suggesting that PAIP2 might regulate translation of specific mRNAs. Interestingly, other studies have shown that knockouts of genes encoding factors that are expected to affect general translation appear to affect translation of specific mRNAs in male germ cells [15, 25]. We have previously demonstrated that PAIP2A controls, in part, translation of the transition proteins and protamine 1 during late spermiogenesis in Paip2a/Paip2b-DKO mice [12]. Therefore, in this study, we hypothesized that the down-regulated proteins in the Paip2a/Paip2b-DKO mice might be controlled by PAIP2A. Surprisingly, we only observed 10 significantly down-regulated proteins, including two that were totally missing in Paip2a/Paip2b-DKO elongated spermatids: TUBB5 and GSTM1. To our knowledge, translational control of TUBB5 and GSTM1 during spermiogenesis had not been described previously. Imunohistochemical studies showed that GSTs are mainly expressed in testicular somatic cells [2628], especially in the Sertoli cells. However, studies assessing GST activity in purified testicular cell populations have shown activity in all cell types, including the germ cells [29, 30]. Various isoforms of GSTs are present on sperm plasma membranes and in the cytosolic compartment [31]. Their main role is considered to be to help protect sperm from chemical insult, but some isoforms have been localized also to the fibrous sheath [32], anchoring an enzymatic complex to a specific cellular compartment.

Our results suggest that the 10 significantly down-regulated proteins might be targets of PAIP2A in translation control. Interestingly, five of these proteins are exclusively expressed in spermatids. Moreover, protein expression of AKAP4, ATP1A4, and FSCB is regulated under translational control in late spermiogenesis (see Table 3 for references); these observations fit well with our proposed model, in which PAIP2A regulates translation of the proteins needed for late spermiogenesis. It is interesting to note that seven of the 10 down-regulated proteins are expressed in the flagellum of spermatozoa (Table 3). Furthermore, it has been shown that AKAP4, HK1, and FSCB interact and are involved in the late steps of fibrous sheath development [33, 34]. These results suggest that PAIP2A is specifically involved in regulating translation of mRNAs encoding proteins necessary for biogenesis of the flagellum. This clearly correlates with the phenotype observed in the Paip2a/Paip2b-DKO and the Paip2a-KO, where we observed an impairment of flagellum formation associated with the absence of the mitochondrial sheath in the middle piece [12]. Interestingly, Akap4-KO mice [20] have abnormalities in the fibrous sheath and the outer dense fibers similar to those found in the Paip2a/Paip2b-DKO testis [12].

The mechanism by which PAIP2A selectively regulates the expression of specific RNAs is unknown. Transcriptional and post-transcriptional mechanisms during spermatogenesis are unique and have been reviewed [1, 35, 36], but little is known about RNA stabilization and translation during spermiogenesis. The male infertility found in Paip2a/Paip2b-DKO and Paip2a-KO mice suggests an important role for PAIP2A in spermatogenesis [12]. We previously demonstrated that efficient translation occurs at an optimal concentration of PABP, which is regulated by PAIP2A [12]. Indeed, excess amounts of PABP compete with EIF4G1, resulting in translational inhibition [12]. We confirmed in the present study that PABP expression was increased in the elongated spermatids of Paip2a/Paip2b-DKO when compared to WT; nevertheless, such a mechanism alone would not be sufficient to explain the regulation of specific mRNAs in our model. It is possible that there are regulatory cis-elements in these RNAs, such as a specific sequence in the 3′ untranslated region (UTR). For example, Zhong et al. [37] have characterized a highly conserved sequence in the 3′ UTR region of the protamine 1 mRNA that is necessary for translation repression. It is possible that these regulatory cis-elements in specific mRNAs might modulate the affinity of PABP to poly (A) tail together with PAIP2A. Further studies comparing the sequence of the RNAs of up- and down-regulated proteins in the Paip2a/Paip2b-DKO are needed to investigate the possibility of such regulatory sequences.

In this study, we have also shown that 19 proteins were significantly up-regulated in the Paip2a/Paip2b-DKO elongated spermatids. These proteins are involved in various biological functions, but mostly in metabolism. It is important to consider the aberrant spermatid differentiation in the Paip2a/Paip2b-DKO and Paip2a-KO mice [12] that is likely to involve variation in cellular components, indirectly resulting in changes of protein levels. One possible explanation for up-regulated proteins is that the translation of mRNAs encoding proteins involved in degradation processes is inhibited. To our knowledge, no obvious candidate for involvement in protein degradation processes can be found in the significantly up-regulated proteins in the Paip2a/Paip2b-DKO. It has been suggested that the ubiquitin-proteasome pathway plays a major role during spermatogenesis [38, 39], and, interestingly, members of the proteasome complex, such as the proteasome activator complex subunit 4, have been detected by mass spectrometry. Further study of proteins involved in the degradation pathway found by our proteomics analysis using elongated spermatids should be informative.

In summary, these studies have revealed a number of proteins that could be targets of PAIP2A-dependent translational control. Moreover, we identified the up-regulated proteins present in elongated spermatids, and several uncharacterized proteins were also revealed. The function of some of these proteins in late spermiogenesis is known, while that of many others remains to be identified.

ACKNOWLEDGMENT

We would like to thank Dr. E.M. Eddy for the gift of the AKAP4 and HK1 antibodies. We also wish to thank Drs. Eddy Rijntjes and Marcos Di Falco for their help in data analysis under Scaffold 2.0 and discussions of statistical analysis.

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Notes

[1] Financial disclosure Supported by grants from the Canadian Institutes of Health Research.

[2] Equal contributor These authors contributed equally to this work.

Geraldine Delbes, Akiko Yanagiya, Nahum Sonenberg, and Bernard Robaire "PABP Interacting Protein 2A (PAIP2A) Regulates Specific Key Proteins During Spermiogenesis in the Mouse," Biology of Reproduction 86(3), 95-, (21 December 2011). https://doi.org/10.1095/biolreprod.111.092619
Received: 30 March 2011; Accepted: 1 November 2011; Published: 21 December 2011
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