Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice

• DOI: 10.1016/j.jbc.2021.101231
• Publication Date: 2021-09-29T13:53:23Z
• Authors: Kaitlyn M. Campbell; Yiding Xu; Chintan Patel; Jeremy M. Rayl; Helena D. Zomer; Hari Prasad Osuru; Michael Pratt; Patcharin Pramoon...; Madeline Timken; Lyndzi M. Miller; Abigail Ralph; Kathryn M. Storey; Yiheng Peng; Jenny Drnevich; Clotilde Lagier-T...; Philip C. Wong; Huanyu Qiao; Prabhakara P. Reddi
• Keywords: Synaptonemal complex; Synapsis; Meiosis II

Meiotic arrest is a common cause of human male infertility, but the causes this are poorly understood. Transactive response DNA-binding protein 43 kDa (TDP-43) highly expressed in spermatocytes preleptotene and pachytene stages meiosis. TDP-43 linked to several neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring functional requirement for spermatogenesis first time, we show here that conditional KO (cKO) Tardbp gene (encoding TDP-43) germ cells mice leads reduced testis size, depletion cells, vacuole formation within seminiferous epithelium, sperm production. Fertility trials also indicated severe subfertility. Spermatocytes cKO showed failure complete prophase I meiosis with at midpachytene stage. Staining synaptonemal complex 3 γH2AX, markers meiotic DNA damage, respectively, super illumination microscopy revealed nonhomologous pairing synapsis defects. Quantitative RT–PCR reduction expression genes critical meiosis, including Spo11 (initiator double-stranded breaks), Rec8 (meiotic recombination protein), Rad21L (RAD21-like, cohesin component), as well those involved retinoic acid pathway entry into RNA-Seq 1036 upregulated 1638 downregulated (false discovery rate <0.05) testis, impacting pathways. Our work reveals crucial role suggests some forms seen infertile men may result from loss function TDP-43. ubiquitously evolutionarily conserved DNA/RNA-binding varied functions, transcription, mRNA splicing, exon skipping, micro-RNA biogenesis (1Ou S.H. Wu F. Harrich D. García-Martínez L.F. Gaynor R.B. Cloning characterization novel cellular protein, TDP-43, binds immunodeficiency virus type 1 TAR sequence motifs.J. Virol. 1995; 69: 3584-3596Crossref PubMed Google Scholar, 2Lagier-Tourenne C. Polymenidou M. Cleveland D.W. FUS/TLS: Emerging roles RNA processing neurodegeneration.Hum. Mol. 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CpG-free vertebrate silences SP-10 tissues: Role function.J. 2007; 282: 36143-36154Abstract (91) Chromatin immunoprecipitation occupancy gene, both spermatids. Interestingly, chromatin pol II pause machinery were loaded spermatids Taken together, our previous studies mainly focused thus far, anticipate plays global regulation post-transcriptional level. Immunolocalization begins intermediate B spermatogonia, peaks (PL) remains high (15Osuru H.P. Pramoonjago P. Abhyankar Swanson Roker L.A. Cathro epithelium.Mol. Reprod. Dev. 2017; 84: 675-685Crossref (3) express gradually tapers off late-stage addition Sertoli location was all aforementioned pattern spatiotemporal epithelium (highest PL spermatocytes) indicates cell differentiation formation, particularly during support this, found aberrantly testicular spermatozoa (16Varghese D.S. Chandran Soumya Pillai Jayakrishnan K. Kumar P.G. Aberrant protein-43 associated spermatogenic men.Reprod. Fertil. 2016; 28: 713-722Crossref (5) On basis data, hypothesized would fertility. To test, have generated lacking adult cells. experimental results absence unable leading maturation arrest. Male bearing produced fewer morphologically abnormal sperm. severely subfertile. Consistent multifunctional resulted changes testis: downregulated. order investigate mice, crossed floxed stimulated (RA) 8 (Stra8)–improved Cre (iCre) deleter delete spermatogonial stage differentiation. (gene symbol TDP-43), codes motifs, flanked loxP (locus X-over P1) sites. Previous using these Cre-mediated excision (10Chiang Stra8–iCre–mediated specific postnatal day 4 (PND4) undifferentiated spermatogonia (17Wu Q. Song R. Ortogero N. Zheng Evanoff Small C.L. Griswold M.D. Namekawa Royo Turner J.M. W. RNase III enzyme DROSHA microRNA production spermatogenesis.J. 2012; 287: 25173-25190Abstract (133) 18Sadate-Ngatchou P.I. Payne Dearth A.T. Braun R.E. recombinase activity postnatal, premeiotic mice.Genesis. 2008; 46: 738-742Crossref (179) appears ((15Osuru Scholar) Fig. S1). Thus, expected lead types. assess phenotypic effect, analyzed genotype TardbpFlox/null, Stra8–Cre+ (referred homozygous cKO). heterozygous Flox/wt served control (het control). We testes PND35 males, time point wave will completed. Testis size compared littermates (Fig. 1, inset). cross section, diameter tubules appeared narrower B). Histological examination extensive presence vacuoles tubules. (heterozygous) luminal interface proper completion 1A). It must noted there no difference terms levels S1B) or phenotype. testes, however, differentiating types 1B). Immunohistochemistry (IHC) performed anti-TDP43 antibody verify status expression. Control 1C), only 1D), confirming At higher magnification, few could 1D). mice. then extended analysis older ranging 2 21 months age (n = 30). Overall, observed 2.9-fold decrease weight (p < 0.0001) 1.6-fold tubule 2, Testes 3, 7, progressively worse C–H). advanced ages, consisted reminiscent infertility condition cell–only syndrome G H). IHC marker Sox confirmed remaining fact S2, variation penetrance phenotype 30; data not shown). majority spermatozoa, males progression portion sections. total caudal averaging 2.2 million per 16 2I). quantified different flow cytometry isolated aged 2J). Compared controls, statistically significant N (round spermatids) an increase (spermatogonia, Sertoli, peritubular, Leydig cells) (primary Accumulation mimicking biopsies obtained azoospermic men. Majority collected cauda epididymides abnormal. Sperm deformed detached head accounted roughly half population. Approximately 55% midpiece bent over principal piece tail S3, A–E). address fertility conducted trials. Three-month-old TardbpFlox/null cohabited age-matched females. four separate used trial. Breeding pairs 3-month-old 5) females controls. end 6-month trial period, gave birth average 6 litters, 0.6 litters same period 2K). 0.0001). Next, wanted systematically examine what halted because lack spermatogonia. Since detected did expect see defects expression, Therefore, potentially affect spermatocytes. explored Stra8 intact expressing C D). RA signaling occurred normally up point. Prophase five stages, leptonema, zygonema, pachynema, diplonema, diakinesis. immunolabeling (SC) (SYCP3) chromosome spreads determine dynamics A–G). until 3C). consistent earlier findings leptotene zygotene started peaked late pachytene, remained diplotene spermatocyte stage, nearly eliminated diakinesis C–G). fluorescent signal normalized element SYCP3 plotted. agreement immunofluorescence images, highest 3H), play processes occurring arrested mostly progressed later 3). staining various 3I), diplotene-like (arrowhead) 3J). Zygotene-like synapsis, short length, chromosomes 17 K, n 25 L numbers chromosomes. Some pachytene-like abnormally long (white arrow 3M). disrupted such homologous occur pachynema versus 4A). accumulation zygotene, stages. decline advance pachynema. further characterize defects, simultaneously stained antibodies SYCP3, 4, B–E). γH2AX restricted sex-body regions 4B, right panel). X Y pair pseudoautosomal unpaired chromosomal regions, transcriptionally silenced, γH2AX. contrast, persistent staining, pseudosex bodies, autosomes C–E, panels). These bodies indicative problems K L). Furthermore, retention foci/flares indicate delay repair double-strand breaks (DSBs). sections nuclei γ-H2AX S4, undergoing apoptosis TUNEL C–E). structure obtain resolution images axes. parallel lines 5A). inset Figure 5A shows end-to-end partner exchange 5B). Examples defective patterns insets 5B. Partner depicted schematics accompanying left sides 5B, respectively. Collectively, suggest normal factor/RNA asked if altered candidate initiation Evaluation pronounced atrophy epithelium. reasoned observe direct effects more appropriate probe differences initial onset testis. begun examined histology prepubertal ages. Histologically, PND8 6, By PND12; noticeable cellularity appearance severity PND15 (data PND12 coincides under signaling. Based (19Gely-Pernot Raverdeau Teletin Vernet Féret B. Klopfenstein Dennefeld Davidson Benoit G. Mark Ghyselinck N.B. Retinoic receptors cell-fate induce SALL4A factor.PLoS 2015; 11e1005501Crossref (43) receptor (RAR) beta, RAR gamma, retinoid (RXR) alpha, RXR retinol-binding (RBP4), Rdln11, (Crbp2), responder cytochrome P450 26A1, spalt-like 4a (Sall4a), 4b (Sall4b), transporter retinol STRA6 PND12. RT (qRT)–PCR RBP4, Crbp2, Sall4b 0.05). beta decreased significantly level 7A). functionally impact execution effect regulating DSB recombination, Mei1 break 1), Prdm9 (PR/SET 9), Dmc1 (DNA (RAD21 component like REC8), Hormad1 (HORMA domain-containing Hormad2 2), Sycp1, (synaptonemal Sycp2 2) Sycp3 3), Msh4 (MutS homolog 4). All (with exception Hormad1, Sycp2, Sycp3) 7B). Finally, qRT–PCR 2.3-fold 0.05) controls had meiosis.Figure 7Dysregulation (A) (B). Average log2 fold change three biological replicates 12 (PND12) (log2 0).