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 , as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes  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  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 , 2. TDP-et al., in 2006  spermatocytes, and remains high in pachytene spermatocytes . The rouTardbp 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.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 (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 . The heterozygous TardbpFlox/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 (inset). In cross section, the diameter of the seminiferous tubules appeared narrower compared with control (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 (A). It must be noted here that there was no difference between the heterozygous and WT mice in terms of TDP-43 protein levels (B) or testis phenotype. The cKO testes, however, showed fewer differentiating cell types in the epithelium and the absence of spermatozoa (B). 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 (C), whereas the cKO testis showed TDP-43 only in Sertoli cells (D), 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 (D). Thus, absence of round spermatids at PND35 suggested failure to complete meiosis in Tardbp cKO mice.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  tissues . Stra8–i tissues , 18. TDP tissues  and Fig. tissues . Thus, Stermates , inset.  control , A and Bogenesis A. It musn levels B or testrmatozoa B. Immunoli cells C, whereali cells D, thus cermatids D. Thus, p < 0.0001) and 1.6-fold reduction in the diameter of the seminiferous tubule (p < 0.0001) (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 (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 (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 (A and B).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 ( 0.0001) , A and B tubules , C–H. Atsyndrome , G and Hli cells , A and BI). 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 (J). Compared with WT controls, cKO mice testes showed a statistically significant decrease in 1 N cells (round spermatids) and an increase in 2 N  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.We observed a variation in the penetrance of the phenotype in terms of germ cell loss in cKO mice . 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 I. We qua4 months J. ComparTardbp 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 (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 (K). Thus, TDP-43 deletion in male germ cells caused a significant reduction in male fertility in mice (p < 0.0001).Majority of spermatozoa collected from cauda epididymides of the tail , A–E. Toe period K. Thus, 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.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 , we did A–G). TDP-43 was not found until the midpachytene stage (C). This was consistent with earlier findings in which IHC showed lack of TDP-43 expression in leptotene and zygotene spermatocytes ((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 (H), 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 (I), diplotene-like spermatocytes from Tardbp cKO showed nonhomologous chromosome synapsis (arrowhead) (J). Zygotene-like spermatocytes from Tardbp cKO showed nonhomologous chromosome synapsis, short chromosome length, and abnormal number of chromosomes . 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.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 , A–G. TDne stage C. This wtocytes  J. Zygoteversus cKO testis to determine the stage at which meiotic arrest occurred in cKO mice (A). 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.Next, we compared the number of cells at various stages of prophase I in the WT cKO mice A. TardbpB–E). In WT pachytene spermatocytes, γH2AX staining is restricted to the sex-body regions . 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 . These pseudosex bodies are indicative of synapsis problems including nonhomologous chromosome pairing consistent with the abnormal numbers of chromosomes in meiotic spreads (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 (A and B). Consistent with failure to complete meiosis, several spermatocytes are seen undergoing apoptosis as revealed by TUNEL staining (C–E).To further characterize the meiotic defects, meiotic chromosome spreads were simultaneously stained with antibodies to γH2AX, SYCP3, and TDP-43 , B–E. In spreads , K and Lstaining , A and Bstaining , C–E.A). 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 (B). 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.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 A. The inomosomes B. ExamplA 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 (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 (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 (A). 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, , Sycp2  Sycp3 , and Msh4 (MutS homolog 4). All these  showed a statistically significant decrease in gene expression in PND12 cKO testes (B). 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 (B). 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.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 , A and BO testis , C and De testis , we analpression A. This sO testes B. Finallcontrols B. The daTardbp cKO  mice (n = 3 each). PND12 was chosen as a time point because at this age overt phenotypical changes of the seminiferous epithelium have not yet set in, thus making it possible to identify direct target genes of TDP-43. A total of 2674 genes were differentially expressed  in the cKO testis compared with controls. Of these, 1036 genes were upregulated and 1638 genes were downregulated (A). We generated a heat map of the top 80 protein-coding differentially expressed genes (DEGs), which showed 21 upregulated and 59 downregulated genes (A). Thus, overall, there were more downregulated than upregulated genes in this early phase of TDP-43 pathology in mouse testis. To gain clues on the underlying causes of testis pathology upon loss of TDP-43, we performed functional classification of the DEGs using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. RNA transport, cell cycle, DNA replication, and basal transcription factor pathways had more DEG genes than expected , and they primarily were downregulated (B). The top pathways with primarily upregulated genes were axon guidance and other signaling pathways although these did not quite meet the threshold for more genes than expected . Next, we performed Gene Ontology (GO) enrichment analysis. Genes that were differentially expressed in the Tardbp cKO testis were associated with a number of biological processes (BPs) and molecular functions (MFs). The top two BPs for downregulated genes were cell cycle and cellular metabolic process (B). Genes known to be involved in meiosis were among these including Hormad2, Sycp2, Syep3, Msh4, Msh5, Rad21L, Rpa2, and Meiob. Upregulated genes including, Tead1, Nrbp2, Erbb4, Nav2, and Mpzl2, were associated with several BPs related to development (C). In terms of MF, nucleic acid–binding function  was among the top four for the downregulated genes (A). Protein binding and DNA binding were enriched for upregulated genes . Thus, the unbiased approach showed that loss of DNA-/RNA-binding protein TDP-43 leads to dysregulation of gene expression early in the disease process (PND12), which might be the precursor for the subsequent phenotypic alterations to male germ cells and disruption of spermatogenesis. Next, we asked if the differentially regulated genes affected the BPs associated with spermatogenesis. Consistent with the observed phenotype of the cKO testis, we found that multiple processes central to the execution of meiosis, including meiotic cell cycle, synapsis, homologous chromosome segregation, and homologous recombination, were significantly enriched for downregulated genes (C).Next, in order to identify global gene expression changes caused by the loss of TDP-43 in the testis, we performed RNA-Seq using testes from PND12 WT (control) and egulated A. We gened genes A. Thus, egulated B. The to process B. Genes elopment C. In tered genes A. Proteied genes C.Figure D), which is many more than expected because of chance . TDP-43 CLIP-Seq study in mouse brain showed that most downregulated genes after TDP-43 knockdown harbored more TDP-43 CLIP clusters and had exceptionally long introns compared with unaffected or upregulated genes. We compared our testis DEG list with the brain TDP-43 CLIP dataset and found that most upregulated as well as most downregulated genes in cKO testis contain longer introns than nonregulated genes   although upregulated genes have slightly longer introns than downregulated genes (p = 0.037). For the mean cluster count in introns, upregulated genes have many more clusters than nonregulated genes (p = 7.9e−05) and a few more clusters than downregulated genes (p = 0.024), whereas there was no significant difference in the number of clusters between downregulated and nonregulated genes (p = 0.443) . Next, using the brain TDP-43 CLIP-Seq data, we asked what proportion of testis DEGs contains TDP-43 binding sites within the UTRs of target mRNAs. Of the 1036 upregulated genes in the testis, 39 and 144 genes showed overlap with brain RNAs containing TDP-43 binding sites within the 5′ and 3′ UTR, respectively (E and F), which is also more than expected because of chance . Of the 1638 downregulated genes in the testis, only 34 and 153 showed overlap with brain target RNAs within the 5′ and 3′ UTR, respectively, which was not more than expected because of chance .TDP-43 contains two canonical RNA recognition motifs with which it binds target RNAs. Therefore, question arises as to how many of the PND12 cKO testis DEGs resulted because of TDP-43 binding to their RNAs. To this end, we explored the published database of TDP-43 binding RNAs in mouse brain , 21. SinFig. 8ectively , E and FAcrv1 gene is a TDP-43 target gene in vivo (Acrv1 gene resulted in premature expression of a reporter gene in spermatocytes of transgenic mice (Acrv1 gene product (SP-10 protein) in spermatocytes in vivo. IHC of testis cross sections showed the typical round spermatid-specific immunoreactivity of the acrosomal protein SP-10 in control mice testis (A and C). In contrast, in addition to round spermatid expression, the cKO testes also showed antibody staining within the cytoplasm of spermatocytes (B and D). There was a statistically significant increase in the expression of SP-10 within the spermatocytes of the cKO mice (E). Thus, lack of endogenous TDP-43 led to premature expression of the Acrv1 gene in spermatocytes. Taken together, the data indicate that one function of TDP-43 is to repress the transcription of its target genes such as the Acrv1 gene in spermatocytes.Our previous work showed that TDP-43 functions as a transcriptional repressor and that the  in vivo , 13. We nic mice . The pree testis , A and Catocytes , B and DcKO mice E. Thus, Tardbp cKO mice showed pathology reminiscent of germ cell aplasia seen in human male infertility including meiotic arrest and sertoli cell–only syndrome.Infertility affects one in six couples in the reproductive age group in the United States; the male factor accounts for approximately half the number of these cases . CompareC and D). Subsequent germ cell types, however, were severely affected. Accumulation of midpachytene stage spermatocytes in cKO mice and relatively low representation of diplotene and diakinesis stages indicate failure to complete prophase I (A). Consequently, the cKO mice produced only a low number of sperm, a majority of which were morphologically abnormal. Tardbp cKO males were severely subfertile (I–L). This shows that TDP-43 plays a critical role in male germ cell differentiation and formation of functional sperm.Although TDP-43 is expressed in the intermediate and type B spermatogonia, loss of TDP-43 in these cells did not appear to affect the formation of PL spermatocytes as judged by the expression of the Stra8 protein , C and Dophase I A. Conseqbfertile , I–L. ThTardbp cKO testes suggesting that TDP-43 might play a role independent of Stra8 during meiosis. Among the genes important for the RA pathway, RBP4, Crbp2, and RAR beta showed decreased gene expression in Tardbp cKO testes, whereas Sall4b showed a significantly high level of expression (A). Previous studies showed the requirement of RARs and RBP4 for male fertility  in cKO mice. Structure illumination microscopy showed synapsis defects and nonhomologous chromosome pairing and synapsis. Given that DSB formation is critical for homologous searching at leptotene stage and that nonhomologous pairing occurs in Spo11 mutant mice  and DMC1 (DSB repair protein) was downregulated in the cKO testes. Extensive γH2AX on meiotic chromosomes could have also resulted from nonhomologous synapsis, resembling the pseudosex body seen in 1 mutant . SYCP3 s1 mutant , J–M in ant mice , it is lInterestingly, a role for TDP-43 in nonhomologous end joining (NHEJ)–mediated DSB repair has been previously reported. In induced pluripotent stem cell–derived motor neurons, TDP-43 was shown to be recruited at DSB sites, where it interacted with factors of the NHEJ-mediated DSB repair pathway . In partTardbp cKO spermatocytes might have also been caused by abnormalities in the loading of the SC/cohesin proteins. Similarly, loading of cohesions, such as REC8 and RAD21L, may have been affected by the lack of TDP-43. Consistent with this, mRNAs coding for the SC (SYCP 1–3) as well as cohesion proteins (Rec8 and Rad21L) were downregulated in Tardbp cKO testes. Previous studies showed that loss of REC8 leads to synapsis between sister chromatids (Rec8/RAD21L mutant mice (B). KO of all the aforementioned genes disrupted the formation of SC leading to failure of meiosis . Sall4a is the regular spliced product, whereas Sall4b is a product of alternative splicing in which the latter portion of exon 2 gets spliced out  within the cytoplasm of the spermatocytes in testis cross sections  promoter in spermatocytes in vivo . Data fratocytes . The presections . Thus, e pausing . PrematuTardbp cKO testis. Loss of TDP-43 caused transcript levels to change in both directions although the trend was that there were more downregulated than upregulated DEGs (A and S5A). TDP-43 is known to play multiple roles in gene regulation including repression of transcription, alternative splicing, and mRNA stability. Improper alternative splicing and loss of mRNA stability could have led to transcript decay and downregulation of gene expression, whereas loss of transcriptional repressor function might have elevated the transcript levels of target DEGs. KEGG pathway analysis showed RNA transport, cell cycle, DNA replication, and basal transcription factor pathways among the top affected pathways. This explains, at least in part, the phenotype seen in Tardbp cKO testis and sperm. The differentiating germ cells have intercellular bridges through which RNAs are transported, and KOs affecting this transport have been shown to be detrimental to spermatogenesis , genes regulated by the transcriptional function (promoter binding but not RNA binding), or those that are indirectly affected by the loss of TDP-43. Second, many of the testis DEGs contained exceptionally long introns and higher mean TDP-43 binding cluster count as reported for the TDP-43 regulated genes in mouse brain .Bouin's fixed testes were processed for paraffin embedding using a Tissue-Tek VIP 1000 processor (Sakura Finetek). About 4-micron thick sections were cut using the Leica RM2125 RTS rotary microtome (Leica Biosystems) and mounted on glass slides. Slides were deparaffinized with xylene and hydrated through a series of graded ethyl alcohols. Antigen retrieval was performed using citrate buffer (pH 6.0) in a vegetable steamer for 60 min. Endogenous peroxidase was blocked using 3.0% hydrogen peroxide for 10 min. Background Punisher  was used for 20 min to block nonspecific background (BG). The sections were incubated for 1 h at room temperature in the primary antibody. Following rinsing, the sections were incubated with secondary antibodies. 3,3′Diamenobenzidine  was used as the chromogen with an incubation time of 5 min. Slides were counterstained with hematoxylin, dehydrated, cleared, and mounted.In Situ Apoptosis Detection Kit  per manufacturer's instructions. Three cKO and three B6 WT control males were used. About 50 cross sections per animal were counted; cross sections containing three or more apoptotic cells were counted as positive.Mouse testis cross sections were stained for the detection of apoptosis using ApopTag Peroxidase For sperm count and morphology analysis, caudal epididymidis of adult cKO and C57bl/6 controls was collected, minced, and incubated in 1 ml of PBS at 37 °C for 20 min to release spermatozoa. Sperms were counted using a hemocytometer under an optical microscope (Laxco). For morphology analysis, sperms were diluted in PBS, and 30 μl drops were placed on slides. The samples were air dried, incubated with 4% paraformaldehyde in PBS for 10 min at RT, washed three times with PBS, and then incubated in hematoxylin (Ricca) for 2 min. Slides were washed with water and mounted in Entellan (Millipore). The morphology of at least 160 random spermatozoa per sample was analyzed under 100× magnification and classified as normal , detached head, deformed head, or bent/curved tail.t test using Prism software  when appropriate. All experiments were performed with at least three biological replicates. Data are described as mean ± SD, with differences considered significant when p < 0.05.Statistical significance was evaluated by Student's g for 5 min, and suspended in PBS. Finally, cells were fixed in 4% paraformaldehyde (PFA), washed with PBS, stained with Hoechst , and analyzed using a flow cytometer (Cytek Aurora).Testicular germ cells were stained with a nuclear stain and subjected to flow cytometry for classification based on DNA content, as described previously . BrieflyFour adult F/-, iCre+ males (cKO) and five adult C57B6 males (control) were used in fertility trials. In each case, the male was paired with a C57B6 female, and the breeding cages were maintained for 6 months. The females were routinely checked for plugs. The number of litters produced and pups born to the cKO and control group was recorded for the 6 months' duration.Chromosome “spreads” were prepared from mouse testes according to previous publication , 66. Aftblue) and antibodies to TDP-43  TDP-43 cKO and WT mice were stained with 4′,6-diamidino-2-phenylindole , an individual region of interest (ROI) was drawn around each nucleus. Each ROI was duplicated in a neighbor region where there is no DNA staining. The software calculated the sum intensity of TDP-43, SYCP3, and 4′,6-diamidino-2-phenylindole. The duplicated ROI was used to account for BG intensity and subtracted out of the sample's sum intensity. The following calculation was used to determine the ratio between TDP-43 and SYCP3:Testis samples were prepared using a handheld homogenizer for 1 min. The homogenizer was cleaned between each sample using four alternating washes of 100% ethanol and sterile deionized water for at least 1 min in each. RNA was extracted from the lysate using spin columns in the PureLink RNA Mini Kit . Samples were stored at −80 °C until the RNA (1 μg) was reverse transcribed using the Qiagen Quantitect RT Kit . The cDNA samples were stored at −20 °C until further analysis.Oligonucleotide primers  and S2 w−ΔΔCt method (qPCR was performed on a QuantStudio 3 Thermocycler (Thermo Fisher Scientific) using SybrGreen PowerUp Master Mix (Thermo Fisher Scientific). The 20 μl reaction included 1 μl target cDNA template, 10 μl SybrGreen Master Mix, 8 μl nuclease-free water, 0.5 μl forward primer, and 0.5 μl reverse primer. The reactions were incubated in a 96-well plate at 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. All qPCR plates included a negative control, actin as the reference gene for each sample, and all reactions were performed in triplicate. The cycle threshold (Ct) is the number of cycles required for the fluorescent signal to exceed BG levels. Relative gene transcription changes were calculated using the 2t method :ΔCt and WT control (n = 3) mice and sent to the Roy J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign for preparation, sequencing, and analysis. The RNA-Seq libraries were prepared with the TruSeq Stranded mRNAseq Sample Prep kit (Illumina), then pooled, quantitated by qPCR, and sequenced on one S4 lane for 101 cycles from one end of the fragment on a NovaSeq 6000. Fastq files were generated and demultiplexed with the bcl2fastq, v2.20, Conversion Software (Illumina), and adaptors were trimmed. Salmon (version 1.2.0)   was usedhttps://www.R-project.org/) using packages as indicated later. Gene-level counts were estimated from transcript-level counts using the “bias corrected counts without an offset” method from the tximport package (version 1.18.0). This method provides more accurate gene-level count estimates and keeps multimapped reads in the analysis compared with traditional alignment-based method  was done using a one-tailed hypergeometric test separately for upregulated and downregulated gene sets using the EGSEA  with the criteria of p value less than 10−5. For comparing germ cell development–related GO, GO terms were selected from Mouse Genome Informatics GO Browser and categorized into three types manually: spermatogenesis, spermatogonia development, and spermiogenesis.GO enrichment analysis was done using the Bioconductor package EdgeR . GO datahttps://CRAN.R-project.org/package=VennDiagram). We also performed the same test of over-representation of genes with CLIP sites specifically in their 3′ or 5′ UTR. To see if intron length or number of CLIP sites in or near a gene is related to a gene's expression after cKO of Tardbp, we followed the method of Lagier–Tourenne et al.  . Of the e et al.  and rankhttps://www.ncbi.nlm.nih.gov/geo/info/linking.html) reported in this publication have been deposited in the Gene Expression Omnibus of NCBI and are accessible through GEO Series accession number GSE17573 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17573). All the RNA-Seq data contained within the article are located as aforementioned (GSE17573). Histopathology of testes of 30 separate cKO mice as well as histopathology of the PND15 cKO mice were reported as “data not shown” in the article. These data will be shared upon reasonable request by the corresponding author (preddi@illinois.edu).The RNA-Seq data (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


A novel approach to preparing the material for intermediate layer by modifying the graphene flakes by gold nanoparticles is presented. With this approach, we observed a large surface area of material and in consequence high electrical capacitance of electrodes. We have obtained satisfactory results demonstrating that the modification of graphene with gold allows for enhancing electrical and wetting properties of carbon nanomaterial. Electrical capacitance of designed nanocomposite-contacted electrode equals to approximately 280 µF, which in consequence ensures great long-term potential stability defined by the potential drift of 36 μV/h. The modification of graphene with nanoparticles completely changed its wetting properties, as the designed material turned out to be hydrophobic with a water contact angle of 115°. Graphene/gold nanoparticles–contacted electrodes are insensitive to the changing light conditions, exhibiting near-Nernstian response in the potassium concentration range between 10 Nanomaterials, commonly known as materials with a single unit sized up to 100 nm, have been widely applied for the construction of ion-selective electrodes (ISEs) , using tAll-solid-state electrodes have their origin in early 1970s when Catrall and Freiser  made a sNanomaterials belong to a group with large surface area, which ensures that high double-layer capacitance is formed in the interface between the solid-contact layer and membrane. The wide implementation of nanomaterials results from their unique physical and chemical properties, such as large surface/volume ratio and large pore volume, ensuring unique surface chemistry and good conductivity . These fThis work describes the design of a nanomaterial composed by modification of graphene flakes (GR) by gold nanoparticles (AuNPs) and its application in ion-selective electrodes’ construction as solid-contact layer. Graphene (GR) belongs to the group of carbon nanomaterials that are among the most widely used materials for solid-contact layer. It is a two-dimensional carbon material with only one atomic layer that shows unique properties such as a fast electron transportation, a high surface area, a high thermal conductivity, and excellent mechanical properties ,12. The Although both materials used in this work were already applied as solid-contact layers individually, combining them into one material allowed us to obtain a hydrophobic layer, of much higher contact angle and greater electrical parameters in comparison with other, as mentioned above, graphene–based composites. To the best of our knowledge, our group is the first one to apply graphene/gold nanoparticles material in the construction of ion-selective electrodes and obtain satisfactory results demonstrating that the modification of graphene with gold allows for enhancing electrical and wetting properties of carbon nanomaterial. Obtained results are promising in the context of designing solid-contact ion-selective electrodes and were presented in the next sections. This paper has been divided into the following parts: material characteristic and electrical and analytical characterization of ion-selective electrodes with nanomaterial layer.4 acting as a reductor. Graphene flakes decorated with gold nanoparticles were dispersed in dimethylformamide (DMF) (POCH).The solid-contact layer consisted of graphene obtained from ACS Material, gold nanoparticles, and was provided by 3D-nano, Poland, with Sodium borohydride NaBHThe membrane components: potassium ionophore I , lipophilic salt–potassium tetrakis(4-chlorophenyl)borate (KTpClPB), 2-nitrophenyl octyl ether (o-NPOE), and poly(vinyl chloride) (PVC) were purchased from Sigma-Aldrich and dissolved in Tetrahydrofuran .−7 to 10−1 M were used for potentiometry, chronopotentiometry, and EIS measurements. Hydrochloric acid and sodium hydroxide used for adjusting the pH value of solutions during the pH sensitivity test were purchased from POCH, Gliwice, Poland.Potassium chloride (KCl) was purchased from POCH  and solutions of K+ ions concentration from 10Graphene modified with gold nanoparticles was implemented onto the glassy carbon disc electrode’s surface and the material was examined for its suitability as intermediate layers in ion-selective electrodes.4  was mixed thoroughly with DMF/graphene dispersion  for 20 min with the assistance of sonification. Afterwards, NaBH4 , acting as a reducing agent, was added rapidly to the solution being mixed under vigorous stirring. Such a prepared solution was washed by centrifugation  in order to remove the excess of the reducing agent, and subsequently redispersed in DMF. The washing procedure was repeated three times.Solid-contact layer was casted onto glassy carbon disc electrode surface using the drop casting method, which among other techniques is considered to be the fastest and the simplest. In order to obtain the solid-contact layer, the DMF-based solution of graphene modified with nanogold was prepared by a simple one-pot method. For this purpose, HAuClAt the beginning of the preparation process, glassy carbon disc electrodes were polished with alumina slurries and rinsed with water and methanol, alternately. Clean and dried electrodes were casted with 20 µL of solid-contact layer solution. DMF was removed from casted layers by evaporation process until only the solid particles were left at the electrode’s surface. At this stage, the layers were tested without membrane in order to examine their electrochemical properties.w/w), KTpClPB 0.25% (w/w), o-NPOE 65.65% (w/w), and PVC 33.00% (w/w). All membrane components of total weight 0.125 g were dissolved in 1 mL of THF. The solvent was evaporated in the room temperature and after 24 h, the conditioning process begun as electrodes were placed into 0.01 M KCl solution in order to saturate the membrane with K+ ions.The experimental part was followed by casting the obtained layers with 60 µL of ion-selective membrane solution of the following composition: potassium ionophore I 1.10% , contact angle microscope, Electrochemical Impedance Spectroscopy (EIS), and chronopotentiometry method were used for material characterization and the potentiometry method was implemented to evaluate the influence of the layers’ presence on the ion-selective electrodes’ analytical performance.The microstructure of studied graphene/gold nanoparticles material was investigated with Transmission Electron Microscope Tecnai 20 X-TWIN  fitted with Energy Dispersive X-Ray Analysis (EDAX) and High Angle Annular Dark Field (HAADF) detectors.In order to examine the wetting properties of the obtained material, contact angle microscope Theta Lite microscope with One Attension software by Biolin Scientific  was implemented into studies.For chronopotentiometry and electrochemical impedance spectroscopy method, glassy carbon disc electrodes were covered with the studied graphene/gold nanoparticles material and placed into a measuring cell in sequence, together with reference Ag/AgCl electrode with 3 M KCl solution  and auxiliary-glassy carbon electrode. All electrodes were connected to the Autolab analyzer . The cell was filled with 0.01 M KCl solution acting as an electrolyte. Both chronopotentiograms—the potentiometric response of electrode recorded with time in the forced current conditions and Nyquist plots, on which the imaginary part of impedance (Z″) is plotted on the y axis, and the real part of impedance (Z′) on the x axis, were collected using NOVA 2.1 software.−1 to 10−7 M concentration were used as K+ ions standard solutions.For potentiometry method all prepared ion-selective electrodes with gold nanoparticles layer and potassium-selective membrane and the coated-disc electrode were connected to the 16-channel mV-meter  and measurements were conducted against Ag/AgCl electrode  reference electrode in the presence of platinum auxiliary electrode. For this measurement, KCl solutions of 10The examined material was graphene modified with gold nanoparticles, which was tested using TEM microscope to explore the microstructure. Further, a contact angle microscope was used to evaluate the wetting properties and Autolab analyzer was used to evaluate the electrochemical behavior.Pictures obtained from the TEM microscope were presented in w/w) and carbon 80.55% (w/w).In the BF scan nanoparticles are visible as dark points on the graphene flakes (of lighter color) while in the HAADF (High-angle Annular Dark-field) scan, gold nanoparticles can be spotted as light points. This was also confirmed by the EDS analysis, as for the lightest point . The wettability of graphene/gold nanoparticles material was tested using the contact angle microscope; the water droplet of 5 µL was discharged onto the materials’ surface, while software measured the angle between the surface and the tangent to the water drop, as shown in The software gave the contact angle a value of 115°, which indicates that the designed material is hydrophobic. The obtained results were compared with those presented in our previous work concerning the graphene/ruthenium dioxide material. The contact angles of 49.5° and 61.9° were reported for graphene individually and graphene/ruthenium dioxide composite, respectively ,19.Implementing hydrophobic intermediate layer into ion-selective electrodes’ construction was repeatedly shown to prevent the formation of water film. This is not only crucial in the context of analytical performance of electrodes but also when it comes to the lifetime of electrodes, as a water layer under an ion-selective membrane causes the delamination of membrane and, in consequence, mechanical damage of the electrode.Designed electrodes were characterized with the use of two electrochemical techniques: chronopotentiometry and electrochemical impedance spectroscopy.Based on the chronopotentiograms recorded with the use of electrodes covered with tested graphene-gold nanoparticles layer, the electrical parameters of the layer were evaluated including electrical capacitance and resistance. With solid-contact layers for ion-selective electrodes, the higher the electrical capacity, the more stable the potentiometric response of electrodes is. Therefore, high values of electrical capacitance and low values of resistance are considered as desirable features. Another method used for the intermediate layer characterization is electrochemical impedance spectroscopy. The Nyquist plot, being the visualization of the imaginary part of the impedance (−Z″) (axis y) and the real part of impedance (Z′) (axis x), is presented in the After being examined, GR-AuNPs–based layers were covered with ion-selective membrane solution in order to obtain ready-to-use solid-contact ion-selective electrodes. The electrical properties of the studied electrodes were also evaluated using chronopotentiometry and electrochemical impedance spectroscopy. The dependencies obtained in the subsequent measurements are shown in Based on the chronopotentiogram, presented in the The EIS measurement results are shown again in the Nyquist plot in For the potentiometric tests, the studied graphene/gold nanoparticles layer was covered with ion-selective membrane, and the ready-to-use electrode was connected to the potentiometer. The characteristics of solid-contact and coated-disc electrodes were recorded in standard KCl solutions in order to determine such parameters of electrodes as standard potential and sensitivity .Calibration curves were recorded after 24, 48, and 72 h of conditioning in 0.01 M KCl solution for each group of electrodes (solid-contact and coated-disc) and presented in All tested electrodes exhibit the slope of calibration curve consistent with the theoretical Nernstian value.−6 to 10−1 M) (arrow pointing up) and subsequently solutions of decreasing concentration (arrow down), as shown in To compare the repeatability of the response of the electrodes with solid-contact layer and coated disc electrode, calibration curves were recorded firstly by measuring standard solutions of increasing concentration  was prepared and the potentiometric response of the GC/GR-AuNP/K+-ISM electrode was recorded in each solution. Results of the test are presented in With ion-selective electrodes it is important to examine the pH range in which the potential response is stable and repeatable, therefore, the range in which electrodes may be applied. For the purpose of this test 0.01 M K−5.9 M to 10−1 M.This work explores the association of two nanomaterials by combining them into one nanocomposite material by modifying the structure of graphene with nanometric particles of gold. Throughout the paper we have repeatedly proven that the properties of both materials translate into the electrodes’ performance. The modification of graphene with gold nanoparticles (size approximately 5 nm) allowed us to create a layer of rough microstructure observed with the TEM microscope a high water contact angle of 115°, ensuring the hydrophobic properties of designed solid-contact layer. This feature of solid-contact layer prevents the water layer formation, therefore protecting the electrodes from mechanical damage caused by the membrane delamination. The addition of nanometric particles to carbon material caused the increase of surface area of material and in consequence the increase of electrical capacitance value. High electrical capacitance of solid-contact layer (of 1.8 mF) translated into high electrical capacity of graphene/gold nanoparticles-contacted electrodes . The designed electrodes are insensitive to the light conditions, exhibit stable potentiometric response  and may be applied in the pH range between 2 and 10.5 and potassium ions concentration range between 10The paper has proved that the modification of carbon nanomaterial allows for obtaining more satisfactory results in the context of solid-contact ion-selective electrodes and presents a universal approach to designing robust potentiometric sensors for future research.