Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice
2021; Elsevier BV; Volume: 297; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2021.101231
ISSN1083-351X
AutoresKaitlyn M. Campbell, Yiding Xu, Chintan Patel, Jeremy M. Rayl, Helena D. Zomer, Hari Prasad Osuru, Michael F. Pratt, Patcharin Pramoonjago, Madeline Timken, Lyndzi M. Miller, Abigail Ralph, Kathryn M. Storey, Yiheng Peng, Jenny Drnevich, Clotilde Lagier‐Tourenne, Philip C. Wong, Huanyu Qiao, Prabhakara P. Reddi,
Tópico(s)Amyotrophic Lateral Sclerosis Research
ResumoMeiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43. Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43. Transactive response DNA-binding protein of 43 kDa (TDP-43) is a ubiquitously expressed and evolutionarily conserved DNA/RNA-binding protein with varied functions, including gene transcription, mRNA splicing, exon skipping, as well as micro-RNA biogenesis (1Ou S.H. Wu F. Harrich D. García-Martínez L.F. Gaynor R.B. Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs.J. Virol. 1995; 69: 3584-3596Crossref PubMed Google Scholar, 2Lagier-Tourenne C. Polymenidou M. Cleveland D.W. TDP-43 and FUS/TLS: Emerging roles in RNA processing and neurodegeneration.Hum. Mol. Genet. 2010; 19: R46-R64Crossref PubMed Scopus (672) Google Scholar). TDP-43 contains two RNA recognition motifs with which it binds to DNA and RNA (3Buratti E. Baralle F.E. Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9.J. Biol. Chem. 2001; 276: 36337-36343Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). The C-terminal region contains a glycine-rich domain. The very first report on TDP-43 showed that it functions as a sequence-specific transcriptional repressor in human cells (1Ou S.H. Wu F. Harrich D. García-Martínez L.F. Gaynor R.B. Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs.J. Virol. 1995; 69: 3584-3596Crossref PubMed Google Scholar). A subsequent study demonstrated a role for TDP-43 in exon skipping in mammalian cells (4Buratti E. Dörk T. Zuccato E. Pagani F. Romano M. Baralle F.E. Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping.EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (477) Google Scholar). TDP-43 has been shown to interact with heterogeneous nuclear ribonucleoprotein proteins to mediate alternative splicing (5Buratti E. Brindisi A. Giombi M. Tisminetzky S. Ayala Y.M. Baralle F.E. TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: An important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing.J. Biol. Chem. 2005; 280: 37572-37584Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 6Wang H.Y. Wang I.F. Bose J. Shen C.K. Structural diversity and functional implications of the eukaryotic TDP gene family.Genomics. 2004; 83: 130-139Crossref PubMed Scopus (225) Google Scholar). The C-terminal part of TDP-43 also consists of a prion-like intrinsically disordered domain involved in forming protein aggregates (7Cushman M. Johnson B.S. King O.D. Gitler A.D. Shorter J. Prion-like disorders: Blurring the divide between transmissibility and infectivity.J. Cell Sci. 2010; 123: 1191-1201Crossref PubMed Scopus (224) Google Scholar, 8Fuentealba R.A. Udan M. Bell S. Wegorzewska I. Shao J. Diamond M.I. Weihl C.C. Baloh R.H. Interaction with polyglutamine aggregates reveals a Q/N-rich domain in TDP-43.J. Biol. Chem. 2010; 285: 26304-26314Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Germline deletion of TDP-43 in mice proved to be embryonic lethal indicating the essential nature of the protein (9Wu L.S. Cheng W.C. Hou S.C. Yan Y.T. Jiang S.T. Shen C.K. TDP-43, a neuro-pathosignature factor, is essential for early mouse embryogenesis.Genesis. 2010; 48: 56-62PubMed Google Scholar, 10Chiang P.M. Ling J. Jeong Y.H. Price D.L. Aja S.M. Wong P.C. Deletion of TDP-43 down-regulates Tbc1d1, a gene linked to obesity, and alters body fat metabolism.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 16320-16324Crossref PubMed Scopus (202) Google Scholar). Although TDP-43 became widely known as a protein involved in the pathology of a number of neurodegenerative diseases following the publication by Neumann et al., in 2006 (11Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. et al.Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4084) Google Scholar), prior to that, we reported cloning of TDP-43 from a mouse testis complementary DNA (cDNA) library as a transcription factor binding to the promoter of the testis-specific Acrv1 gene (12Acharya K.K. Govind C.K. Shore A.N. Stoler M.H. Reddi P.P. Cis-requirement for the maintenance of round spermatid-specific transcription.Dev. Biol. 2006; 295: 781-790Crossref PubMed Scopus (55) Google Scholar). The mouse Acrv1 gene is expressed exclusively in round spermatids, and its promoter contains two TGTGTG motifs—canonical TDP-43-binding sites to which TDP-43 binds in vitro. Using transgenic mice as a reporter system, we showed that mutation of the two TGTGTG motifs within the Acrv1 promoter caused premature transcription of a reporter gene in spermatocytes, whereas the WT promoter maintained round spermatid-specific expression in vivo (12Acharya K.K. Govind C.K. Shore A.N. Stoler M.H. Reddi P.P. Cis-requirement for the maintenance of round spermatid-specific transcription.Dev. Biol. 2006; 295: 781-790Crossref PubMed Scopus (55) Google Scholar). This suggested that TDP-43 might repress the Acrv1 gene expression in spermatocytes in vivo. Using the Gal4 reporter assay, we showed that TDP-43 represses gene transcription (13Lalmansingh A.S. Urekar C.J. Reddi P.P. TDP-43 is a transcriptional repressor: The testis-specific mouse acrv1 gene is a TDP-43 target in vivo.J. Biol. Chem. 2011; 286: 10970-10982Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In addition, we also demonstrated a role for TDP-43 in the insulator function required to keep the Acrv1 gene silent in the somatic tissues (14Abhyankar M.M. Urekar C. Reddi P.P. A novel CpG-free vertebrate insulator silences the testis-specific SP-10 gene in somatic tissues: Role for TDP-43 in insulator function.J. Biol. Chem. 2007; 282: 36143-36154Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Chromatin immunoprecipitation showed occupancy of TDP-43 at the promoter of the Acrv1 gene, both in spermatocytes as well as round spermatids. Interestingly, chromatin immunoprecipitation also showed that RNA pol II and the pol II pause machinery were loaded on the Acrv1 promoter in spermatocytes prior to the expression of Acrv1 mRNA in round spermatids (13Lalmansingh A.S. Urekar C.J. Reddi P.P. TDP-43 is a transcriptional repressor: The testis-specific mouse acrv1 gene is a TDP-43 target in vivo.J. Biol. Chem. 2011; 286: 10970-10982Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Taken together, our previous work showed that TDP-43 functions as a transcriptional repressor and that Acrv1 is a TDP-43 target gene in vivo. Although our studies mainly focused on its role as a transcriptional repressor of the Acrv1 gene thus far, we anticipate that TDP-43 plays a global role in the regulation of gene expression in the testis, both at the transcriptional as well as post-transcriptional level. Immunolocalization studies of the mouse testis showed that TDP-43 expression begins in the intermediate and type B spermatogonia, peaks in preleptotene (PL) spermatocytes, and remains high in pachytene spermatocytes (15Osuru H.P. Pramoonjago P. Abhyankar M.M. Swanson E. Roker L.A. Cathro H. Reddi P.P. Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium.Mol. Reprod. Dev. 2017; 84: 675-685Crossref PubMed Scopus (3) Google Scholar). The round spermatids express TDP-43, but the expression gradually tapers off in late-stage spermatids. In addition to germ cells, Sertoli cells also express TDP-43. The location of TDP-43 was nuclear in all the aforementioned cells (15Osuru H.P. Pramoonjago P. Abhyankar M.M. Swanson E. Roker L.A. Cathro H. Reddi P.P. Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium.Mol. Reprod. Dev. 2017; 84: 675-685Crossref PubMed Scopus (3) Google Scholar). The pattern of spatiotemporal expression of TDP-43 within the seminiferous epithelium (highest expression seen in PL and pachytene spermatocytes) indicates a functional role for the protein in male germ cell differentiation and sperm formation, particularly during meiosis. In support of this, TDP-43 was found to be aberrantly expressed in testicular germ cells and spermatozoa of some infertile men (16Varghese D.S. Chandran U. Soumya A. Pillai S.M. Jayakrishnan K. Reddi P.P. Kumar P.G. Aberrant expression of TAR DNA binding protein-43 is associated with spermatogenic disorders in men.Reprod. Fertil. Dev. 2016; 28: 713-722Crossref PubMed Scopus (5) Google Scholar). On the basis of the aforementioned data, we hypothesized that TDP-43 would be essential for spermatogenesis and male fertility. To test, we have generated conditional KO (cKO) mice lacking Tardbp in adult male germ cells. Our experimental results show that in the absence of TDP-43, spermatocytes were unable to complete prophase I of meiosis leading to maturation arrest. Male mice bearing germ cell KO of Tardbp produced fewer and morphologically abnormal sperm. Tardbp KO male mice were severely subfertile. Consistent with its role as a multifunctional protein, loss of TDP-43 resulted in global changes in the expression of genes in the testis: 1036 genes were upregulated and 1638 were downregulated. In order to investigate the functional requirement of TDP-43 for spermatogenesis in mice, we crossed floxed TDP-43 mice with stimulated by retinoic acid (RA) gene 8 (Stra8)–improved Cre (iCre) deleter mice to delete the TDP-43 gene in the spermatogonial stage of male germ cell differentiation. In floxed TDP-43 mice, exon 3 of Tardbp (gene symbol for TDP-43), which codes for two critical RNA recognition motifs, is flanked by the loxP (locus of X-over P1) sites. Previous studies using these mice showed that Cre-mediated excision leads to loss of TDP-43 protein in target tissues (10Chiang P.M. Ling J. Jeong Y.H. Price D.L. Aja S.M. Wong P.C. Deletion of TDP-43 down-regulates Tbc1d1, a gene linked to obesity, and alters body fat metabolism.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 16320-16324Crossref PubMed Scopus (202) Google Scholar). Stra8–iCre–mediated excision of floxed genes is specific for the male germ cells and begins by postnatal day 4 (PND4) within the undifferentiated spermatogonia of the testis (17Wu Q. Song R. Ortogero N. Zheng H. Evanoff R. Small C.L. Griswold M.D. Namekawa S.H. Royo H. Turner J.M. Yan W. The RNase III enzyme DROSHA is essential for microRNA production and spermatogenesis.J. Biol. Chem. 2012; 287: 25173-25190Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 18Sadate-Ngatchou P.I. Payne C.J. Dearth A.T. Braun R.E. Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice.Genesis. 2008; 46: 738-742Crossref PubMed Scopus (179) Google Scholar). TDP-43 protein first appears in intermediate and type B spermatogonia ((15Osuru H.P. Pramoonjago P. Abhyankar M.M. Swanson E. Roker L.A. Cathro H. Reddi P.P. Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium.Mol. Reprod. Dev. 2017; 84: 675-685Crossref PubMed Scopus (3) Google Scholar) and Fig. S1). Thus, Stra8–iCre–mediated excision is expected to lead to loss of TDP-43 in intermediate spermatogonia and all subsequent male germ cell types. To assess the phenotypic effect, we analyzed male mice with the genotype TardbpFlox/null, Stra8–Cre+ (referred to as homozygous cKO). The heterozygous Tardbp Flox/wt Stra8–Cre+ mice served as control (het control). We first analyzed the testes of PND35 males, the time point at which the first wave of spermatogenesis will have completed. Testis size was severely reduced in cKO mice compared with control littermates (Fig. 1, inset). In cross section, the diameter of the seminiferous tubules appeared narrower compared with control (Fig. 1, A and B). Histological examination showed extensive germ cell depletion and the presence of vacuoles within the tubules. The control (heterozygous) testes showed germ cells at all stages of differentiation including spermatozoa at the luminal interface indicating proper completion of the first round of spermatogenesis (Fig. 1A). It must be noted here that there was no difference between the heterozygous and WT mice in terms of TDP-43 protein levels (Fig. S1B) or testis phenotype. The cKO testes, however, showed fewer differentiating cell types in the epithelium and the absence of spermatozoa (Fig. 1B). Immunohistochemistry (IHC) was performed using anti-TDP43 antibody to verify the status of TDP-43 expression. Control mice expressed TDP-43 in germ cells as well as Sertoli cells (Fig. 1C), whereas the cKO testis showed TDP-43 only in Sertoli cells (Fig. 1D), thus confirming KO of Tardbp within the spermatogonia and all subsequent germ cell types. At higher magnification, spermatogonia and a few meiotic cells could be seen in cKO testis but no round spermatids (Fig. 1D). Thus, absence of round spermatids at PND35 suggested failure to complete meiosis in Tardbp cKO mice. We then extended the analysis to older mice ranging from 2 to 21 months of age (n = 30). Overall, we observed a 2.9-fold decrease in testis weight (p < 0.0001) and 1.6-fold reduction in the diameter of the seminiferous tubule (p < 0.0001) (Fig. 2, A and B). Testes of cKO mice at 3, 7, and 21 months of age showed progressively worse depletion of germ cells and vacuole formation within the seminiferous tubules (Fig. 2, C–H). At advanced ages, the seminiferous epithelium only consisted of Sertoli cells, reminiscent of the human infertility condition known as Sertoli cell–only syndrome (Fig. 2, G and H). IHC using antibody to the Sertoli marker Sox 9 confirmed that the cells remaining within the seminiferous epithelium of the cKO mice are in fact Sertoli cells (Fig. S2, A and B). We observed a variation in the penetrance of the phenotype in terms of germ cell loss in cKO mice (n = 30; data not shown). A majority of the cKO mice showed severe germ cell depletion with few spermatozoa, whereas some cKO males showed progression of spermatogenesis in a portion of seminiferous tubule cross sections. Consistent with this, the total number of caudal sperm varied averaging at 2.2 million per cKO mouse compared with 16 million per control mouse (Fig. 2I). We quantified the different types of germ cells in cKO mice testes by flow cytometry using testicular cells isolated from mice aged 3 to 4 months (Fig. 2J). Compared with WT controls, cKO mice testes showed a statistically significant decrease in 1 N cells (round spermatids) and an increase in 2 N (spermatogonia, Sertoli, peritubular, and Leydig cells) and 4 N (primary spermatocytes) cells. Accumulation of 2 N and 4 N cells in Tardbp cKO testes indicated arrest in meiosis, mimicking the meiotic arrest phenotype seen in biopsies obtained from some azoospermic infertile men. Majority of spermatozoa collected from cauda epididymides of Tardbp cKO mice were morphologically abnormal. Sperm with deformed or detached head accounted for roughly half of the population. Approximately 55% of the cKO sperm showed head and midpiece bent over the principal piece of the tail (Fig. S3, A–E). To address the requirement of TDP-43 for fertility in male mice, we conducted fertility trials. Three-month-old TardbpFlox/null Stra8–Cre+ males were cohabited with age-matched WT females. A total of four separate cKO males were used in the fertility trial. Breeding pairs of 3-month-old WT males (n = 5) and females served as controls. At the end of the 6-month fertility trial period, control mice gave birth to an average of 6 litters, whereas the cKO mice produced an average of 0.6 litters during the same period (Fig. 2K). Thus, TDP-43 deletion in male germ cells caused a significant reduction in male fertility in mice (p < 0.0001). Next, we wanted to systematically examine at what point spermatogenesis was halted because of the lack of TDP-43 in spermatogonia. Since TDP-43 expression was not detected in undifferentiated spermatogonia (15Osuru H.P. Pramoonjago P. Abhyankar M.M. Swanson E. Roker L.A. Cathro H. Reddi P.P. Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium.Mol. Reprod. Dev. 2017; 84: 675-685Crossref PubMed Scopus (3) Google Scholar), we did not expect to see defects in early stage spermatogonia. TDP-43 expression, however, begins in the intermediate spermatogonia of WT mice. Therefore, depletion of TDP-43 could potentially affect the progression of type B spermatogonia to PL spermatocytes. To address this, we explored the expression of Stra8 as a marker of PL spermatocytes. IHC showed intact PL spermatocytes expressing Stra8 in cKO mice (Fig. S2, C and D). Stra8 expression in cKO mouse PL spermatocytes also indicated that the RA signaling pathway for entry into meiosis occurred normally up to that point. Prophase I of meiosis consists of five stages, including leptonema, zygonema, pachynema, diplonema, and diakinesis. We performed immunolabeling with TDP-43 and synaptonemal complex (SC) protein 3 (SYCP3) on WT mouse testis meiotic chromosome spreads to determine the dynamics of expression of TDP-43 during meiotic prophase I (Fig. 3, A–G). TDP-43 was not found until the midpachytene stage (Fig. 3C). This was consistent with earlier findings in which IHC showed lack of TDP-43 expression in leptotene and zygotene spermatocytes ((15Osuru H.P. Pramoonjago P. Abhyankar M.M. Swanson E. Roker L.A. Cathro H. Reddi P.P. Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium.Mol. Reprod. Dev. 2017; 84: 675-685Crossref PubMed Scopus (3) Google Scholar) and Fig. S1). TDP-43 expression started in midpachytene spermatocytes, peaked in late pachytene, and remained high until the diplotene spermatocyte stage, and nearly eliminated in diakinesis (Fig. 3, C–G). Next, we quantified TDP-43 expression during meiotic prophase I. TDP-43 fluorescent signal was normalized with that of the lateral element protein SYCP3 and plotted. In agreement with the immunofluorescence images, the highest expression of TDP-43 was seen from late pachytene to late diplotene stages (Fig. 3H), indicating that TDP-43 might play an important role in the meiotic processes occurring in pachytene and diplotene spermatocytes. Consistent with this, immunofluorescence of meiotic chromosome spreads from the cKO testis showed cells arrested mostly at midpachytene stage, whereas some progressed to later stages of prophase I (Fig. 3). SYCP3 staining showed various synapsis defects in the spermatocytes of cKO mice. Compared with the WT spermatocytes (Fig. 3I), diplotene-like spermatocytes from Tardbp cKO showed nonhomologous chromosome synapsis (arrowhead) (Fig. 3J). Zygotene-like spermatocytes from Tardbp cKO showed nonhomologous chromosome synapsis, short chromosome length, and abnormal number of chromosomes (n = 17 in K, n = 25 in L of Fig. 3). The abnormal numbers of chromosomes in cKO testes may have resulted from abnormal synapsis between nonhomologous chromosomes. Some pachytene-like spermatocytes showed abnormally long chromosomes (white arrow in Fig. 3M). The data indicated that loss of TDP-43 in male germ cells disrupted critical processes such as homologous chromosome pairing and recombination that occur at pachynema during prophase I of meiosis. Next, we compared the number of cells at various stages of prophase I in the WT versus cKO testis to determine the stage at which meiotic arrest occurred in cKO mice (Fig. 4A). Tardbp cKO mice showed accumulation of cells in zygotene, early pachytene, and mostly at midpachytene stages. Tardbp cKO mice showed a decline in the number of late pachytene and diplotene spermatocytes, indicating failure to advance to these stages in the absence of TDP-43. This suggested that TDP-43 is required for meiotic processes that occur during pachynema. To further characterize the meiotic defects, meiotic chromosome spreads were simultaneously stained with antibodies to γH2AX, SYCP3, and TDP-43 (Fig. 4, B–E). In WT pachytene spermatocytes, γH2AX staining is restricted to the sex-body regions (Fig. 4B, right panel). This is because the X and Y chromosomes pair at the pseudoautosomal region and the remaining unpaired X and Y chromosomal regions, which are transcriptionally silenced, are stained with γH2AX. In contrast, extensive and persistent γH2AX staining, pseudosex bodies, was seen over the autosomes in Tardbp cKO spermatocytes (Fig. 4, C–E, right panels). These pseudosex bodies are indicative of synapsis problems including nonhomologous chromosome pairing consistent with the abnormal numbers of chromosomes in meiotic spreads (Fig. 3, K and L). Furthermore, retention of γH2AX foci/flares on autosomes in cKO testis could also indicate a delay in the repair of DNA double-strand breaks (DSBs). IHC of testis cross sections further confirmed that the nuclei of cKO spermatocytes show extensive γ-H2AX staining (Fig. S4, A and B). Consistent with failure to complete meiosis, several spermatocytes are seen undergoing apoptosis as revealed by TUNEL staining (Fig. S4, C–E). We performed structure illumination microscopy microscopy to obtain higher resolution images of homologous chromosome pairing and synapsis in WT and cKO spermatocytes. SYCP3 is a marker for the meiotic chromosome axes. WT spermatocytes at pachynema showed two parallel SYCP3 lines indicating proper synapsis between homologous chromosomes (Fig. 5A). The inset in Figure 5A shows the end-to-end pairing of homologous chromosomes in WT mice. In contrast, Tardbp cKO spermatocytes showed synapsis partner exchange between nonhomologous chromosomes (Fig. 5B). Examples of defective synapsis patterns are shown in insets of Figure 5B. Partner exchange between five and four different nonhomologous chromosomes is depicted in the schematics accompanying the insets on the left and right sides of Figure 5B, respectively. This showed that loss of TDP-43 causes nonhomologous chromosome synapsis in pachytene spermatocytes. Collectively, these data suggest that TDP-43 is required for normal synapsis of chromosomes in mouse spermatocytes. Since TDP-43 is a transcription factor/RNA binding protein, we asked if the loss of TDP-43 altered the expression of candidate genes known to be involved in the initiation and progression of meiosis. Evaluation of the cKO testis in adult mice showed that over time there was pronounced atrophy of the seminiferous epithelium. Therefore, we reasoned that in order to observe direct effects of loss of TDP-43, it would be more appropriate to probe for differences in gene expression at the time of the initial onset of pathology in cKO testis. To determine the time point at which phenotypic changes have begun in the testis of the KO mice, we examined the histology of the testis at prepubertal ages. Histologically, there was no difference between the control and cKO testis at PND8 (Fig. 6, A and B). By PND12; however, there were noticeable changes in the cellularity of the seminiferous epithelium and the appearance of spermatocytes in the cKO testis (Fig. 6, C and D). The severity of pathology became progressively worse at PND15 (data not shown). The period between PND8 and PND12 coincides with the onset of prophase I of meiosis in the testis under the control of RA signaling. Based on a previous publication of RA pathway genes in the testis (19Gely-Pernot A. Raverdeau M. Teletin M. Vernet N. Féret B. Klopfenstein M. Dennefeld C. Davidson I. Benoit G. Mark M. Ghyselinck N.B. Retinoic acid receptors control spermatogonia cell-fate and induce expression of the SALL4A transcription factor.PLoS Genet. 2015; 11e1005501Crossref PubMed Scopus (43) Google Scholar), we analyzed the expression of retinoic acid receptor (RAR) beta, RAR gamma, retinoid X receptor (RXR) alpha, RXR beta, RXR gamma, retinol-binding protein 4 (RBP4), Rdln11, cellular retinol-binding protein 1, cellular retinol-binding protein 2 (Crbp2), retinoic acid receptor responder 1, cytochrome P450 26A1, spalt-like transcription factor 4a (Sall4a), spalt-like transcription factor 4b (Sall4b), and signaling receptor and transporter of retinol STRA6 at PND12. Quantitative RT (qRT)–PCR showed that RBP4, Crbp2, RAR beta, and Sall4b showed significant differences (p < 0.05). RBP4, Crbp2, and RAR beta showed decreased gene expression in Tardbp cKO testes, whereas Sall4b showed a significantly high level of expression (Fig. 7A). This suggested that TDP-43 may functionally impact the execution of RA signaling for the initiation and maintenance of meiosis in spermatocytes. Next, to investigate the effect of TDP-43 on the expression of genes regulating prophase I of meiosis, we analyzed the expression of genes involved in DSB formation, synapsis, and recombination, including Mei1 (meiotic double-stranded break formation protein 1), Prdm9 (PR/SET domain 9), Spo11 (initiator of meiotic double-stranded breaks), Dmc1 (DNA meiotic recombinase 1), Rad21L (RAD21 cohesin complex component like 1), Rec8 (meiotic recombination protein REC8), Hormad1 (HORMA domain-containing protein 1), Hormad2 (HORMA domain-containing protein 2), Sycp1, (synaptonemal complex protein 1), Sycp2 (synaptonemal complex protein 2) Sycp3 (synaptonemal complex protein 3), and Msh4 (MutS homolog 4). All these (with the exception of Hormad1, Sycp2, and Sycp3) showed a statistically significant decrease in gene expression in PND12 cKO testes (Fig. 7B). Finally, qRT–PCR confirmed conditional deletion of the Tardbp gene and showed 2.3-fold reduction in the expression of Tardbp mRNA (p < 0.05) in the cKO testes compared with controls (Fig. 7B). The data suggest that loss of TDP-43 in testicular germ cells had an impact on the mRNA expression of candidate genes critical for prophase I of meiosis.Figure 7Dysregulation of gene expression in Tardbp cKO testis. Quantitative RT–PCR of genes involved in the retinoic acid pathway (A) and meiosis (B). Average log2 fold change in mRNA transcription of genes for three biological replicates of postnatal day 12 (PND12) cKO mice compared with WT as no change (log2 fold change = 0). In
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