An Essential Role for Katanin p80 and Microtubule Severing in Male Gamete Production

Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development

In vivo evidence that RBM5 is a tumour suppressor in the lung

Abstract Cigarette smoking is undoubtedly a risk factor for lung cancer. Moreover, smokers with genetic mutations on chromosome 3p21.3, a region frequently deleted in cancer and notably in lung cancer, have a dramatically higher risk of aggressive lung cancer. The RNA binding motif 5 (RBM5) is one of the component genes in the 3p21.3 tumour suppressor region. Studies using human cancer specimens and cell lines suggest a role for RBM5 as a tumour suppressor. Here we demonstrate, for the first time, an in vivo role for RBM5 as a tumour suppressor in the mouse lung. We generated Rbm5 loss-of-function mice and exposed them to a tobacco carcinogen NNK. Upon exposure to NNK, Rbm5 loss-of-function mice developed lung cancer at similar rates to wild type mice. As tumourigenesis progressed, however, reduced Rbm5 expression lead to significantly more aggressive lung cancer i.e. increased adenocarcinoma nodule numbers and tumour size. Our data provide in vivo evidence that reduced RBM5 function, as occurs in a large number of patients, coupled with exposure to tobacco carcinogens is a risk factor for an aggressive lung cancer phenotype. These data suggest that RBM5 loss-of-function likely underpins at least part of the pro-tumourigenic consequences of 3p21.3 deletion in humans

Katanin-like 2 (KATNAL2) functions in multiple aspects of haploid male germ cell development in the mouse

<div><p>The katanin microtubule-severing proteins are essential regulators of microtubule dynamics in a diverse range of species. Here we have defined critical roles for the poorly characterised katanin protein KATNAL2 in multiple aspects of spermatogenesis: the initiation of sperm tail growth from the basal body, sperm head shaping via the manchette, acrosome attachment, and ultimately sperm release. We present data suggesting that depending on context, KATNAL2 can partner with the regulatory protein KATNB1 or act autonomously. Moreover, our data indicate KATNAL2 may regulate δ- and ε-tubulin rather than classical α-β-tubulin microtubule polymers, suggesting the katanin family has a greater diversity of function than previously realised. Together with our previous research, showing the essential requirement of katanin proteins KATNAL1 and KATNB1 during spermatogenesis, our data supports the concept that in higher order species the presence of multiple katanins has allowed for subspecialisation of function within complex cellular settings such as the seminiferous epithelium.</p></div

CRISP2 is a regulator of multiple aspects of sperm function and male fertility

The cysteine-rich secretory proteins (CRISPs) are a group of proteins that show a pronounced expression biased to the male reproductive tract. Although sperm encounter CRISPs at virtually all phases of sperm development and maturation, CRISP2 is the sole CRISP produced during spermatogenesis, wherein it is incorporated into the developing sperm head and tail. In this study we tested the necessity for CRISP2 in male fertility using Crisp2 loss-of-function mouse models. In doing so, we revealed a role for CRISP2 in establishing the ability of sperm to undergo the acrosome reaction and in establishing a normal flagellum waveform. Crisp2-deficient sperm possess a stiff midpiece and are thus unable to manifest the rapid form of progressive motility seen in wild type sperm. As a consequence, Crisp2-deficient males are subfertile. Furthermore, a yeast two-hybrid screen and immunoprecipitation studies reveal that CRISP2 can bind to the CATSPER1 subunit of the Catsper ion channel, which is necessary for normal sperm motility. Collectively, these data define CRISP2 as a determinant of male fertility and explain previous clinical associations between human CRISP2 expression and fertility.</p

Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling

Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein 55 kDa (CEP55) is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 (TEX14) protein, has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic ( Cep55Tg/Tg) mice aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55Tg/Tg male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1 ( Sohlh1). Thus, Cep55 amplification leads to a shift toward the initial maintenance of undifferentiated spermatogonia and ultimately results in progressive germ cell loss. Collectively, our findings demonstrate that Cep55 overexpression causes change in germ cell proportions and manifests as a Sertoli cell only tubule phenotype, similar to that seen in many azoospermic men

KATNAL2 interacts with δ- and ε-tubulin.

<p>KATNAL2 interaction with δ-tubulin (TUBD1) (<b>a</b>) and ε-tubulin (TUBE1) (<b>b</b>) was indicated by co-immunoprecipitation of pEGFP-KATNAL2-pmCherry-TUBD1 and pEGFP-KATNAL2-pmCherry-TUBE1 complexes respectively, using anti-GFP beads. (<b>c</b>) Interactions were confirmed by co-immunoprecipitation assays from mouse testis lysate and (<b>d</b>) <i>in situ</i> proximity ligation assays. (<b>a-b</b>) Input: whole cell lysate from transfected cells; GFP IP: immunoprecipitation with GFP conjugated beads. (<b>a</b>) The left upper panel shows mCherry-TUBD1 was successfully transfected into both cell populations and the left lower panel shows EGFP and EGFP-KATNAL2 were successfully transfected into the desired cell population. The right upper panel confirmed mCherry-TUBD1 can bind to EGFP-KATNAL2, but not to EGFP. Right lower panel confirmed EGFP and EGFP-KATNAL2 proteins were successfully precipitated with GFP beads. (<b>b</b>) The left upper panel shows mCherry-TUBE1 was successfully transfected into both cell populations and the left lower panel shows EGFP and EGFP-KATNAL2 were successfully transfected into the desired cell population. The right upper panel confirmed mCherry-TUBE1 can bind to EGFP-KATNAL2, but not to EGFP. Right lower panel confirmed EGFP and EGFP-KATNAL2 proteins were successfully precipitated with GFP beads. (<b>c</b>) Input: whole testis lysate from adult mice; KATNAL2 IP: immunoprecipitation (<b>+</b>) and negative control immunoprecipitation (<b>-</b>) using an antibody directed against KATNAL2. Blots were probed with antibodies directed against KATNAL2, TUBD1 and TUBE1. (<b>d</b>) Representative images of <i>in situ</i> proximity ligation assays using antibodies directed against KATNAL2 and TUBD1 and against KATNAL2 and TUBE1 in <i>Katnal2</i>^WT/WT isolated elongating spermatids. The specificity of labelling was assessed via the parallel labelling of <i>Katnal2</i>^KO/KO cells. Green represents the acrosome as labelled by PNA and blue represents DNA as labelled by DAPI. Manufacturer recommended negative controls are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007078#pgen.1007078.s010" target="_blank">S8 Fig</a>. Scale bars in <b>d</b> = 2 μm.</p

KATNAL2 has essential roles in tail formation and centriole number regulation.

<p>Acetylated tubulin immunolabelling as a marker for sperm tails in <i>Katnal2</i>^WT/WT (<b>a</b>) and <i>Katnal2</i>^Y86C/Y86C (<b>b</b>) testis sections. Sperm tail content was markedly reduced in <i>Katnal2</i>^Y86C/Y86C versus <i>Katnal2</i>^WT/WT. Scale bars in <b>a</b>–<b>b</b> = 10 μm. In both <i>Katnal2</i>^WT/WT (<b>c</b>) and <i>Katnal2</i>^Y86C/Y86C (<b>d</b>) spermatids, normal coupling of the centriole to the nuclear membrane was observed (black arrowheads). Docking of the centriole to the plasma membrane (white arrowhead) was frequently observed in <i>Katnal2</i>^WT/WT spermatids (<b>c</b>), but was never seen in <i>Katnal2</i>^Y86C/Y86C spermatids (<b>d</b>). Supernumerary centrioles (red arrowhead) were frequently observed in the cytoplasm of <i>Katnal2</i>^Y86C/Y86C (<b>d</b>) but not <i>Katnal2</i>^WT/WT (<b>c</b>) spermatids. Immunostaining of centrioles in isolated elongating spermatids of <i>Katnal2</i>^WT/WT (<b>e</b>) and <i>Katnal2</i>^Y86C/Y86C (<b>f</b>) mice confirmed abnormal duplication of centrioles (red arrowhead) in <i>Katnal2</i>^Y86C/Y86C mice. In (<b>e-f</b>) red represents centrioles as labelled by centrin immunostaining and blue represents DNA as labelled by DAPI. Scale bars in <b>c</b>–<b>f</b> = 2 μm. Primary antibody negative controls for (<b>a</b>–<b>b</b>) and (<b>e</b>–<b>f</b>) are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007078#pgen.1007078.s009" target="_blank">S7 Fig</a>.</p