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. 2025 Apr 18;71(3):154–160. doi: 10.1262/jrd.2025-012

Mice lacking two testis-specific cytoplasmic poly(A)-binding proteins, PABPC2 and PABPC6, exhibit normal spermatogenesis and fertility

Yuka ISONO 1, Yuko KAKU 1, Yoshinori KANEMORI 1, Shin-ichi KASHIWABARA 1
PMCID: PMC12151635  PMID: 40254460

Abstract

The cytoplasmic poly(A)-binding protein (PABPC) plays a central role in the life of poly(A) mRNAs, including their stability, translation, and decay. In addition to the nearly ubiquitous PABPC1, two testis-specific PABPCs, PABPC2 and PABPC6, are present in rodents, while one specific PABPC, PABPC3, is found in primate testes. These three PABPC proteins are each encoded by intronless genes that may have diverged independently due to the retroposition of prototypical Pabpc1 or PABPC1. PABPC2 and PABPC6 are distinguished from PABPC1 in that they barely associate with translationally active polysomal mRNAs and are enriched in male germ cell-specific nuage, termed chromatoid bodies. Despite these unique characteristics, spermatogenesis and male fertility were not compromised in mutant mice lacking either PABPC2 or PABPC6, suggesting functional redundancy between the two proteins. Here, we produced double-mutant mice lacking both PABPC2 and PABPC6 and found that the simultaneous absence of these two proteins failed to affect testicular protein synthesis, spermatogenesis, or male fertility in vivo. These results suggest that the functions of PABPC2 and PABPC6 are redundant with those of other co-existing PABPC proteins, including PABPC1. We propose that testis-specific PABPC proteins emerged because of transcriptional promiscuity in the testis.

Keywords: PABPC2, PABPC6, Poly(A)-binding protein, Retrogene, Spermatogenesis

Spermatogenesis is a lengthy (approximately 35 days in mice) and complicated process of cellular differentiation, through which male gametes, spermatozoa, are produced in the seminiferous epithelium and comprises three major phases. These include mitotic proliferation of spermatogonia; meiotic prophase accompanying homologous recombination of parental chromosomes, followed by two meiotic divisions to produce round spermatids; and spermiogenesis, which is the haploid phase of spermatogenesis. Drastic morphological alterations in spherical spermatids occur during spermiogenesis, such as the formation of a highly compacted nucleus, acrosome, and flagellum. These differentiation processes are driven by a concerted action of stage-specific gene expression that is regulated at the transcriptional, post-transcriptional, and translational levels [1,2,3,4,5,6,7,8]. At least two types of translational regulation are evident during mammalian spermatogenesis. For instance, a substantial portion of almost all mRNAs, including housekeeping mRNAs, is sequestered into translationally inert messenger ribonucleoprotein particles (mRNPs), resulting in “global/general translational repression” [1, 2, 7]. In contrast, haploid cell-specific mRNAs, such as those encoding flagellar and sperm nuclear proteins, which must be transcribed in round spermatids due to global transcriptional silencing towards the end of spermiogenesis (around step 10 of the 16 steps in mouse spermiogenesis) [9], are also packaged into mRNPs and stored in an inactive state for several days until they are translated in elongating and elongated spermatids [2, 5, 8]. In late haploid cells, silenced transcripts are partially released from mRNPs to form polysomes, where they are translated. This type of uncoupling of transcription and translation is called “translational delay” [2, 5, 8].

The poly(A) tail of eukaryotic mRNAs is responsible for virtually every aspect of mRNA metabolism, including mRNA stability, translation, and decay. These processes are mediated by the cytoplasmic form of poly(A)-binding protein, PABPC, which functions as a multifaceted protein [10,11,12,13,14]. PABPC molecules on the poly(A) tails not only protect mRNAs from exonucleolytic attack by deadenylases but also recruit a wide range of translation-associated factors [e.g., eukaryotic translation initiation factor 4G (eIF4G), translation-activating PABPC-interacting protein 1 (PAIP1), translational repressor PAIP2, translation-terminating GSPT/eRF3, and deadenylation enzymes] [10,11,12,13,14]. Two distinct patterns of poly(A) tail dynamics have been described during mammalian spermatogenesis. The most prominent is the observation of partially deadenylated mRNA species (~A150–A30) in elongating/elongated spermatids generated upon translational activation of stored haploid-specific mRNAs with homogeneous, long poly(A) tracts (~A180) [15, 16]. The other, conversely, is the poly(A) tail extension of a subset of mRNAs, including housekeeping mRNAs, in round spermatids [1, 17,18,19], some of which are catalyzed by the testis-specific cytoplasmic poly(A) polymerase PAPOLB [18, 19]. Mutant mice lacking the ubiquitous PAIP2A or Cnot7/Caf1a subunit of the CCR4-NOT deadenylase complex [4, 5, 20], as well as testis-specific PABPC-interacting proteins (DAZL and BOLL) or PAPOLB [4, 5, 18], exhibit spermatogenesis disorders and male infertility, suggesting the special importance of the poly(A) tail in mammalian spermatogenesis.

Besides the nearly ubiquitous PABPC1, testis-specific PABPC2 and PABPC6 have been identified in rodent species [21,22,23,24], whereas a sole specific PABPC, PABPC3, has been documented in primate testes [25]; either protein is encoded by intronless gene (also called “retrogene” or “retroposon”). PABPC3 expression is decreased in infertile men with non-obstructive azoospermia [26], implying the functional relevance of testis-specific PABPC proteins in male fertility. Our previous studies have shown that PABPC2 and PABPC6 exhibit biochemical properties characteristic of the PABPC family proteins, including non-specific binding to poly(A) mRNAs and interaction with translation-associated factors [22, 24]. However, in contrast to PABPC1, both proteins are barely loaded on translationally active polysomal mRNAs and are concentrated in the male germ cell-specific nuage, the chromatoid body [22, 24], suggesting their involvement in the aforementioned testis-specific translational regulations. Despite these unique properties, genetic ablation of either Pabpc2 or Pabpc6 resulted in no overt abnormalities in spermatogenesis or male fertility, raising the possibility that they are functionally redundant with each other [24, 27]. To evaluate this hypothesis, we generated mutant mice doubly deficient in PABPC2 and PABPC6. Contrary to our expectations, the double mutant mice demonstrated no discernible abnormalities in testicular protein synthesis, spermatogenesis, and male fertility in vivo, indicating that the unique features shared between PABPC2 and PABPC6, including chromatoid body localization and exclusive association with non-polysomal, inactive mRNAs, are not essential for male reproductive processes. The evolutionary implications of the testis-specific PABPC proteins are also discussed.

Materials and Methods

Ethics statement

All animal experiments at the University of Tsukuba were approved and performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (approval numbers: 23-398 and 24-344).

Generation of Pabpc2 and Pabpc6 double knock-out mice

Mice with homozygous mutation in Pabpc2 (Pabpc2-/-) or Pabpc6 (Pabpc6-/-) [24, 27] were used to produce mice lacking both proteins. Briefly, Pabpc6-/- males (ICR) were crossed with Pabpc2-/- females (129S2/SvPas × C57BL/6) to obtain mice heterozygous for each allele (Pabpc2+/-;Pabpc6+/-). Subsequently, mice with a double deficiency of PABPC2 and PABPC6 (Pabpc2-/-;Pabpc6-/-) were generated by Pabpc2+/-;Pabpc6+/- intercrosses.

PCR genotyping

Genotypes were determined by polymerase chain reaction (PCR) analysis of genomic DNA prepared from tail biopsies. The following primers were used for Pabpc2: 5'-ATGGATGACGAGACCCTGAATG-3' (forward primer for Pabpc2), 5'-GGTCTCTGGTCAGTTTAAACAGTTGGG-3' (reverse primer for Pabpc2), and 5'-GCGCTGCGAATCGGGAGCGGCGATACCGT-3' (forward primer for neomycin resistance gene) [27]. A set of primers, 5'-GACCGAGGCGATGCTCTAT-3' (forward primer) and 5'-AAACATTGGTGAACTCCTTGG-3' (reverse primer), was used to genotype Pabpc6 [24].

Protein extract preparation

Testes were homogenized at 4°C in 10 volumes (vol/wt) of the buffer comprising 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol, and proteinase inhibitors (1 μg/ml leupeptin, 1 μg/ml pepstatin, and 0.5 mM phenylmethanesulfonyl fluoride), using a Polytron homogenizer (max speed, 1 min). The homogenates were centrifuged twice at 13,400 ×g for 10 min at 4°C in a microcentrifuge, and the supernatants were used as protein extracts for immunoblot analyses. Protein concentration was determined using a Coomassie protein assay reagent kit (Thermo Fischer Scientific, Waltham, MA, USA) with bovine serum albumin as the standard.

Immunoblot analysis

Protein extracts were mixed with half a volume of 3×Laemmli buffer (30 mM Tris/HCl, pH 6.8, 3% SDS, 3% β-mercaptoethanol, 0.3% bromophenol blue, and 30% glycerol), boiled for 3 min, and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Proteins separated by SDS-PAGE were transferred onto Immobilon-P membranes (Merck Millipore, Billerica, MA, USA), incubated with primary antibodies, and then with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The antibodies used in this study are listed in Supplementary Table 1. The antibodies prepared in our laboratory are available upon request.

Histological analysis

The testicular and epididymal specimens were fixed in Bouin’s solution, dehydrated in an ascending ethanol series, and embedded in paraffin. Paraffin sections (4-μm thick) were prepared by using a MICROM HM340E (Microedge Instruments, White Rock, BC, Canada) and mounted on MAS-coated glass slides (Matsunami glass, Osaka, Japan). After deparaffinization in xylene and hydration in a descending ethanol series, slides were stained with hematoxylin and eosin (FUJIFILM Wako, Osaka, Japan) and photographed using a BioRevo BZ-9000 microscope (KEYENCE, Osaka, Japan).

Sperm counting and staining

Spermatozoa were flushed from a pair of cauda epididymides into 1 ml of phosphate-buffered saline (PBS, pH 7.2), diluted, and counted using a hemocytometer. For sperm staining, the sperm suspension was centrifuged at 800 ×g for 5 min at 4°C and resuspended in PBS containing 4% paraformaldehyde. After fixation on ice for 1 h, followed by three washes with PBS, the spermatozoa were suspended in 1 ml of PBS, smeared onto glass slides, and air-dried. The slides were boiled in 10 mM citrate buffer (pH 6.0) in a microwave oven at 750 W for 1 min, cooled to room temperature, washed three times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature. After washing three times with PBS, followed by blocking with 3% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS containing 0.05% Tween-20, the sperm cells were counterstained with 1 μg/ml Alexa Fluor 568-conjugated peanut agglutinin (PNA; Thermo Fisher Scientific), 1 μM MitoTracker Green FM (Thermo Fisher Scientific), and 5 μg/ml Hoechst 33342 (Thermo Fisher Scientific) in blocking buffer. After washing thrice with PBS, the slides were mounted in 30% glycerol in PBS and observed under an Olympus IX71 fluorescence microscope (EVIDENT, Tokyo, Japan).

Phylogenetic analysis

The coding sequences of putative homologs of murine and human PABPC proteins were obtained from GenBank. Nucleotide sequences were aligned using MAFFT v7 [28]. A phylogenetic tree was constructed using the maximum likelihood (ML) method with the Randomized Axelerated Maximum Likelihood (RAxML) v8 [29]. The coding sequence of transcript variant X6 of the gray short-tailed opossum PABPC1L was used as an outgroup. ML trees were analyzed using the GTR+GAMMA nucleotide model with 1,000 bootstrap replicates. The phylogenetic tree was visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Statistical analysis

Data were analyzed by unpaired Student t-test and expressed as mean ± SD (n ≥ 3). Statistical significance was set at P < 0.05.

Results

Generation of Pabpc2 and Pabpc6 double knock-out mice

As mentioned above, testis-specific PABPC2 and PABPC6 differ from the nearly ubiquitous PABPC1 by their enrichment in chromatoid bodies and inability to associate with polysomal mRNAs undergoing translation [22, 24]. Despite these unique properties, mice lacking either PABPC2 or PABPC6 exhibit normal spermatogenesis and fertility [24, 27]. These findings led us to hypothesize that PABPC2 and PABPC6 are functionally redundant with each other. To clarify whether the loss of function of one protein is compensated for by the other, we generated double knock-out (DKO, Pabpc2-/-;Pabpc6-/-) mice lacking both PABPC2 and PABPC6 by crossing Pabpc2-/- males with Pabpc6-/- females, followed by Pabpc2+/-;Pabpc6+/- intercrosses (Fig. 1A). Genotypic analysis of tail DNA by PCR using specific primer sets revealed the absence of bands amplified from the wild-type (WT) Pabpc2 and Pabpc6 alleles in DKO mice (Fig. 1B). The disappearance of both proteins in the DKO testes was confirmed by immunoblot analysis (Fig. 1C).

Fig. 1.

Fig. 1.

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Generation of Pabpc2 and Pabpc6 double knock-out mice. (A) Schematic of the production of Pabpc2 and Pabpc6 double knock-out (DKO) mice. Mice lacking both PABPC2 and PABPC6 were produced by crossing mice with a null mutation of each allele followed by doubly heterozygous intercrosses. Het, heterozygous mutant; KO, homozygous mutant. (B) Genotypic PCR of Pabpc2 and Pabpc6 alleles. The tail DNA from the wild-type (WT) and DKO mice was analyzed by PCR. (C) Immunoblot analysis of testicular extracts. Testicular protein extracts were subjected to immunoblot analysis using specific antibodies against PABPC2 or PABPC6. Asterisks denote non-specific signals. ACTB served as loading control. Uncropped images of the gels (B) and immunoblots (C) are given in the Supplementary Data Set.

Normal spermatogenesis and male fertility in Pabpc2 and Pabpc6 DKO mice

Pabpc2 and Pabpc6 DKO mice showed no discernible abnormalities in behavior, body weight, or health. Visual inspection of the testicular tissues revealed no apparent anatomical differences between WT and DKO mice (Fig. 2A). In fact, the testicular weight of DKO mice was comparable to that of WT mice (116.2 ± 14.7 and 112.2 ± 10.8 mg in WT and DKO mice, respectively, n = 10, Fig. 2B). Microscopic examination of the testicular and cauda epididymal paraffin sections revealed that spermatogenesis and sperm migration into the cauda epididymis were unaffected by the concomitant absence of PABPC2 and PABPC6 (Fig. 2C). Sperm-specific organelles, including the compacted nucleus, acrosome, and midpiece containing mitochondria, of DKO mice were morphologically indistinguishable from those of the WT controls (Fig. 2D). Furthermore, no significant difference was found in the number of spermatozoa flushed from cauda epididymides (5.4 × 106 ± 5.9 × 105 and 5.5 × 106 ± 3.5 × 105 in WT and DKO mice, respectively, n = 3, Fig. 2E). Finally, when male fertility was assessed by mating DKO males with WT females, DKO males sired offspring with litter sizes (9.6 ± 2.2 pups/litter, n = 9) comparable to the WT controls (10.0 ± 2.2 pups/litter, n = 9). Taken together, these observations indicate normal spermatogenesis and male fertility in vivo in Pabpc2 and Pabpc6 DKO mice.

Fig. 2.

Fig. 2.

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Spermatogenesis and fertility of Pabpc2 and Pabpc6 double knock-out mice. (A) Gross morphology of the WT, and Pabpc2 and Pabpc6 DKO testes. (B) Testicular weight. Testicles from adult mice (3- to 4-month-old) were weighed (n = 10). n.s., not significant. (C) Histological analysis. Testicular and cauda epididymal paraffin sections were stained with hematoxylin and eosin and observed under a microscope. Scale bars, 100 μm. (D) Sperm morphology. Cauda epididymal sperm from the WT and DKO mice were smeared onto glass slides and counterstained with Hoechst 33342 (blue), MitoTracker Green FM (green), and PNA (red). The specimens were photographed with a fluorescence microscope. Scale bars, 10 μm. (E) Sperm count. Sperm flushed from a pair of cauda epididymides were counted using a hemocytometer (n = 3). n.s., not significant. (F) Fertility of male mice. Ten-week-old males were housed with 8-week-old ICR females. The number of pups born was counted (10 litters). n.s., not significant. Uncropped images of the tissue sections (C) and sperm smears (D) are provided in the Supplementary Data Set.

Unaltered testicular protein profiles by the absence of PABPC2 and PABPC6

To clarify the molecular basis of normal spermatogenesis in DKO mice, the abundance of testis-specific proteins was examined using immunoblot analysis. These include PAPOLB, PIWIL1, and ACRBP-W, which are present in pachytene spermatocytes and round spermatids [30, 31]; ACRBP-C, which is processed from ACRBP-W mainly in elongating and elongated spermatids [31]; and TNP2 and PRM2, which are exclusively synthesized in elongating and elongated spermatids [15, 16]. As shown in Fig. 3A, the levels of these testis-specific proteins were not compromised in the DKO mice, indicating the dispensability of PABPC2 and PABPC6 in the synthesis of testicular proteins. We next examined whether the simultaneous loss of PABPC2 and PABPC6 affects the synthesis of other co-existing PABPC proteins, since, as reported for Paip2a KO mice, increased PABPC1 results in impaired spermiogenesis and male infertility [32]. Immunoblot analysis revealed that PABPC1 was not upregulated by the combined absence of PABPC2 and PABPC6 (100:97.9 for WT:DKO, n = 3) (Figs. 3B and C). In addition, the anti-PABPC common antibody (hereafter referred to as anti-PABPCs) [27] gave equivalent signal intensities between the WT and DKO testes (100:104 for WT:DKO, n = 3) (Figs. 3B and C). Two PABPC-interacting proteins (PAIP1 and PAIP2) in the DKO testes were comparable in abundance to those in WT controls (100:102 and 100:110, respectively, for WT:DKO, n = 3) (Figs. 3B and C). These results indicate that the simultaneous absence of PABPC2 and PABPC6 failed to increase PABPC1 and possibly other co-existing PABPC proteins, including PABPC4 and germline-specific PABPC1L/ePAB, and that the amounts of PABPC2 and PABPC6 were negligibly low compared to PABPC1 [24, 27].

Fig. 3.

Fig. 3.

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Testicular protein levels in Pabpc2 and Pabpc6 double knock-out mice. (A) Testis-specific proteins. Protein extracts from the WT and DKO testes were analyzed on immunoblots using antibodies against the indicated testis-specific proteins. ACTB was used as a loading control. (B) PABPC and PABPC-interacting proteins. The blots prepared as described in (A) were probed with the indicated antibodies. “PABPCs” denotes antibodies raised against the RRM domain of PABPC1 that is highly conserved among PABPC family proteins. (C) Quantitative analysis of PABPCs and PABPC-interacting proteins. Protein bands in (B) were quantified using Image J software (n = 3). ACTB was used for normalization. Uncropped images of the immunoblots (A and B) are shown in the Supplementary Data Set.

Evolutionary implications of testis-specific PABPCs

As described above, two intronless Pabpc genes, Pabpc2 and Pabpc6, are specifically expressed in rodent testes [21,22,23,24], whereas only one retrogene, PABPC3, is expressed in primate testes [25]. Based on sequence conservation, we previously proposed that Pabpc2 and Pabpc6 diverged independently from intron-containing Pabpc1 by retroposition [24]. To gain further insight into the molecular evolution of testis-specific PABPCs, pairwise nucleotide sequence comparisons were performed between murine Pabpc1, Pabpc2, Pabpc6, human PABPC1, and PABPC3. The nucleotide sequence identities of the open reading frames between Pabpc2 and PABPC3 and between Pabpc6 and PABPC3 were 79.8% and 79.5%, respectively, which were significantly lower than those between PABPC1 and PABPC3 (94.7%) and between Pabpc1 and PABPC1 (95.2%) (Fig. 4A). These results suggest that human PABPC3 arose by retroposition of PABPC1 rather than from mouse Pabpc2 or Pabpc6. Fig. 4B shows the phylogenetic tree generated using RAxML v8.

Fig. 4.

Fig. 4.

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Phylogenetic analysis of testis-specific PABPCs. (A) Nucleotide sequence identities in the open reading frame among murine Pabpc1, Pabpc2, Pabpc6, human PABPC1, and PABPC3. Pairwise sequence alignments were carried out using the ClustalW algorithm. (B) Phylogenetic tree of testis-specific PABPCs. A phylogenetic tree was generated using the RAxML v8 program following sequence alignment using the MAFFT v7 program. The coding sequence of the transcript variant X6 of the gray short-tailed opossum PABPC1L (oPABPC1LX6) was used as an outgroup. Mouse Pabpc2 and Pabpc6, and human PABPC3 are highlighted in red and blue letters, respectively. The scale bar indicates branch length.

Discussion

As described above, PABPC2 and PABPC6 possess unique properties not shared with PABPC1, including testis-specific expression, retrogene products, enrichment in chromatoid bodies, and nearly exclusive association with translationally dormant mRNP complexes [22, 24]. We previously reported that no obvious abnormalities in spermatogenesis or fertility were found in mice lacking either PABPC2 or PABPC6, suggesting a functional redundancy between the two proteins [24, 27]. In this study, we generated mutant mice doubly deficient in PABPC2 and PABPC6 by crossing mice with a null mutation in each allele (Fig. 1). Intronless genes expressed specifically in the testis, as well as testis-specific chromatoid body components, are often essential for spermatogenesis [33,34,35]. Given that PABPC2 and PABPC6 fulfilled both the criteria, it was surprising that the double deficiency of these proteins did not affect testicular protein synthesis or spermatogenesis (Figs. 2 and 3). These observations provide convincing evidence that testis-specific, retrogene-encoded PABPC2 and PABPC6 are both dispensable for testicular mRNA regulation rather than the loss of function of one protein being compensated for by the other. It is possible that other co-existing PABPCs, including PABPC1, complement the function(s) of these testis-specific PABPC proteins due to the following reasons: (i) the contents of PABPC2 and PABPC6 are extremely low compared to PABPC1 (Figs. 3B and C) [24, 27], (ii) PABPC1 also associate with translationally inactive mRNPs and, albeit not concentrated in chromatoid bodies, is included in the nuages [34, 36], (iii) female and male germline-specific PABPC1L, which is essential for oocyte development, is dispensable for male germ cell differentiation [37]. Although it is currently unclear whether PABPC3, a unique primate testis-specific PABPC [25], possesses the same properties as PABPC2 and PABPC6 in rodents, our present study suggests that PABPC3 is also dispensable for human spermatogenesis. This hypothesis may be further supported by phylogenetic analysis implying that PABPC3 was created by retroposing PABPC1 after the emergence of primates, rather than from Pabpc2 or Pabpc6 (Fig. 4).

Spermatogenesis involves unique cellular events, including meiosis, which is accompanied by homologous recombination between the paternal and maternal genomes, and spermiogenesis, through which round spermatid differentiates into sperm with unique organelles, such as acrosome, condensed nucleus, and flagellum. To accomplish this drastic metamorphosis, spermatogenesis requires the expression of a diverse set of male germ cell-specific genes [3]. To meet this demand, transcriptional activity is significantly upregulated in spermatocytes and round spermatids, which is thought to be facilitated by overall relaxed chromatin resulting from extensive chromatin remodeling [38,39,40] and/or by increased levels of RNA polymerase II and its associated factors in male germ cells [41]. Although this situation, termed “transcriptional promiscuity,” may favor the expression of various spermatogenic genes, it increases the risk of excessive protein synthesis. As one possible means to evade protein overproduction and to maintain proteostasis, significant portions of almost all mRNAs, including housekeeping mRNAs, are sequestered into translationally inactive mRNPs in meiotic and post-meiotic cells, leading to “global/general translational repression” [1, 2, 7]. Another type of translational regulation, called “translational delay”, is essential for spermatid-specific mRNAs, such as those encoding sperm nuclear and flagellar proteins; these mRNAs are transcribed in the early stages of spermiogenesis and stored as totally inert mRNPs for several days until they are partially activated after global silencing of transcription in later stages [2, 5, 8]. Chromatoid bodies have also been considered as mRNA storage sites for post-meiotic mRNAs [34, 42]. Because PABPC2 and PABPC6 are exclusively present in translationally inactive non-polysomal mRNP fractions and are concentrated in chromatoid bodies [22, 24], we hypothesized that PABPC2 and PABPC6 are implicated in the overall repression of translation and/or delayed translation of post-meiotic mRNAs. It has also been proposed that testis-specific PABPCs have been created to meet the high poly(A) mRNA levels in the testes resulting from transcriptional derepression described above [21]. Considering that PABPC2 and PABPC6 contents are extremely low compared to that of PABPC1, the high poly(A) levels in this tissue may be covered by increased PABPC1 [22, 24]. Further studies are necessary to elucidate translational regulation during spermatogenesis.

Intronless genes (also called “retrogenes” or “retroposons”) are generated from intron-containing progenitor genes by making a reverse transcriptase copy of an mRNA and inserting the DNA copy into the genome. Because intronless genes tend to be preferentially expressed in the testes, this tissue has been proposed to promote the creation of new mammalian genes [35, 43, 44]. Retroposed paralogs of X-linked genes are, in many cases, essential for spermatogenesis and/or fertility as a compensatory mechanism for the depletion of somatic counterparts caused by meiotic X chromosome inactivation [35, 43,44,45]. Retrogenes are also created for mammalian reproductive purposes from parental paralogs on autosomes [17, 18, 43]. However, regardless of whether they originate from X-linked or autosomal genes, some testis-specific retrogenes are dispensable for male germ cell development [35]. Pabpc2 (chromosome 18) and Pabpc6 (chromosome 17), which are derived from Pabpc1 (chromosome 15), fall into this category. Given the negligibly low abundance of PABPC2 and PABPC6 compared to that of PABPC1, the emergence of these proteins may be a mere reflection of accidental, leaky expression caused by transcriptional promiscuity in meiotic and early post-meiotic cells.

In conclusion, this study using double knock-out mice provides irrefutable evidence that retrogene-derived, testis-specific PABPC2 and PABPC6 are both dispensable for male germ cell differentiation and that the loss of one protein is not functionally compensated for by the other.

Conflict of interests

We declare that there is no conflict of interest.

Supplementary

Supplementary Materials
jrd-71-154-s001.pdf (1.2MB, pdf)

Acknowledgments

We thank Prof. Akiyoshi Fukamizu, the Gene Research Center, and the Life Science Center for Survival Dynamics at the University of Tsukuba for providing experimental equipment. We also thank Dr. Tadashi Baba (Professor Emeritus at the University of Tsukuba) for various support. This work was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (Grants #21K05498 and #24K08822 to S.K.).

Data availability

All data in this study are available in the main text and supplementary materials.

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Associated Data

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Supplementary Materials

Supplementary Materials
jrd-71-154-s001.pdf (1.2MB, pdf)

Data Availability Statement

All data in this study are available in the main text and supplementary materials.

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