Science Collected Works - Research Publications
Now showing items 1-12 of 158
An Essential Role for Katanin p80 and Microtubule Severing in Male Gamete Production
(PUBLIC LIBRARY SCIENCE, 2012-05-01)
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.
Flagellar energetics from high-resolution imaging of beating patterns in tethered mouse sperm
(ELIFE SCIENCES PUBLICATIONS LTD, 2021-04-30)
We demonstrate a technique for investigating the energetics of flagella or cilia. We record the planar beating of tethered mouse sperm at high resolution. Beating waveforms are reconstructed using proper orthogonal decomposition of the centerline tangent-angle profiles. Energy conservation is employed to obtain the mechanical power exerted by the dynein motors from the observed kinematics. A large proportion of the mechanical power exerted by the dynein motors is dissipated internally by the motors themselves. There could also be significant dissipation within the passive structures of the flagellum. The total internal dissipation is considerably greater than the hydrodynamic dissipation in the aqueous medium outside. The net power input from the dynein motors in sperm from Crisp2-knockout mice is significantly smaller than in wildtype samples, indicating that ion-channel regulation by cysteine-rich secretory proteins controls energy flows powering the axoneme.
New Insights Into Sperm Ultrastructure Through Enhanced Scanning Electron Microscopy
(FRONTIERS MEDIA SA, 2021-04-22)
The analysis of spermatozoa morphology is fundamental to understand male fertility and the etiology of infertility. Traditionally scanning electron microscopy (SEM) has been used to define surface topology. Recently, however, it has become a critical tool for three-dimensional analysis of internal cellular ultrastructure. Modern SEM provides nanometer-scale resolution, but the meaningfulness of such information is proportional to the quality of the sample preservation. In this study, we demonstrate that sperm quickly and robustly adhere to gold-coated surfaces. Leveraging this property, we developed three step-by-step protocols fulfilling different needs for sperm imaging: chemically fixed monolayers for SEM examination of the external morphology, and two high-pressure freezing-based protocols for fast SEM examination of full cell internal morphology and focused ion-beam SEM tomography. These analyses allow previously unappreciated insights into mouse sperm ultrastructure, including the identification of novel structures within the fibrous sheath and domain-specific interactions between the plasma membrane and exosome-like structures.
A global approach to addressing the policy, research and social challenges of male reproductive health
(OXFORD UNIV PRESS, 2021-01-01)
Male infertility is a global health issue; yet to a large extent, our knowledge of its causes, impact and consequence is largely unknown. Recent data indicate that infertile men have an increased risk of somatic disorders such as cancer and die younger compared to fertile men. Moreover, several studies point to a significant adverse effect on the health of the offspring. From the startling lack of progress in male contraception combined with the paucity of improvements in the diagnosis of male infertility, we conclude there is a crisis in male reproductive health. The Male Reproductive Health Initiative has been organized to directly address these issues (www.eshre.eu/Specialty-groups/Special-Interest-Groups/Andrology/MRHI). The Working Group will formulate an evidence-based strategic road map outlining the ways forward. This is an open consortium desiring to engage with all stakeholders and governments.
Deficiency of theTbc1d21gene causes male infertility with morphological abnormalities of the sperm mitochondria and flagellum in mice
(PUBLIC LIBRARY SCIENCE, 2020-09-01)
Approximately 2-15% of couples experience infertility, and around half of these cases are attributed to male infertility. We previously identified TBC1D21 as a sterility-related RabGAP gene derived from infertile men. However, the in vivo function of TBC1D21 in male fertility remains unclear. Here, we show that loss of Tbc1d21 in mice resulted in male infertility, characterized by defects in sperm tail structure and diminished sperm motility. The mitochondria of the sperm-tail had an abnormal irregular arrangement, abnormal diameter, and structural defects. Moreover, the axoneme structure of sperm tails was severely disturbed. Several TBC1D21 interactors were selected via proteomic analysis and functional grouping. Two of the candidate interactors, a subunit protein of translocase in the outer membrane of mitochondria (TOMM20) and an inner arm component of the sperm tail axoneme (Dynein Heavy chain 7, DNAH7), confirmed in vivo physical co-localization with TBC1D21. In addition, TOMM20 and DNAH7 detached and dispersed outside the axoneme in Tbc1d21-deficient sperm, instead of aligning with the axoneme. From a clinical perspective, the transcript levels of TBC1D21 in sperm from teratozoospermia cases were significantly reduced when compared with those in normozoospermia. We concluded that TBC1D21 is critical for mitochondrial and axoneme development of mammalian sperm.
Rare mutations in the complement regulatory gene CSMD1 are associated with male and female infertility
(NATURE PUBLISHING GROUP, 2019-10-11)
Infertility in men and women is a complex genetic trait with shared biological bases between the sexes. Here, we perform a series of rare variant analyses across 73,185 women and men to identify genes that contribute to primary gonadal dysfunction. We report CSMD1, a complement regulatory protein on chromosome 8p23, as a strong candidate locus in both sexes. We show that CSMD1 is enriched at the germ-cell/somatic-cell interface in both male and female gonads. Csmd1-knockout males show increased rates of infertility with significantly increased complement C3 protein deposition in the testes, accompanied by severe histological degeneration. Knockout females show significant reduction in ovarian quality and breeding success, as well as mammary branching impairment. Double knockout of Csmd1 and C3 causes non-additive reduction in breeding success, suggesting that CSMD1 and the complement pathway play an important role in the normal postnatal development of the gonads in both sexes.
SOX30 is required for male fertility in mice
(NATURE PUBLISHING GROUP, 2017-12-15)
Male infertility is a major and growing problem and, in most cases, the specific root cause is unknown. Here we show that the transcription factor SOX30 plays a critical role in mouse spermatogenesis. Sox30-null mice are healthy and females are fertile, but males are sterile. In the absence of Sox30 meiosis initiates normally in both sexes but, in males, germ cell development arrests during the post-meiotic round spermatid period. In the mutant testis, acrosome and axoneme development are aberrant, multinucleated germ cells (symplasts) form and round spermatids unable to process beyond step 3 of spermiogenesis. No elongated spermatids nor spermatozoa are produced. Thus, Sox30 represents a rare example of a gene for which loss of function results in a complete arrest of spermatogenesis at the onset of spermiogenesis. Our results suggest that SOX30 mutations may underlie some instances of unexplained non-obstructive azoospermia in humans.
A mutation in the viral sensor 2 '-5 '-oligoadenylate synthetase 2 causes failure of lactation
(PUBLIC LIBRARY SCIENCE, 2017-11-01)
We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.
PLAG1 deficiency impairs spermatogenesis and sperm motility in mice
(NATURE PUBLISHING GROUP, 2017-07-13)
Deficiency in pleomorphic adenoma gene 1 (PLAG1) leads to reduced fertility in male mice, but the mechanism by which PLAG1 contributes to reproduction is unknown. To investigate the involvement of PLAG1 in testicular function, we determined (i) the spatial distribution of PLAG1 in the testis using X-gal staining; (ii) transcriptomic consequences of PLAG1 deficiency in knock-out and heterozygous mice compared to wild-type mice using RNA-seq; and (iii) morphological and functional consequences of PLAG1 deficiency by determining testicular histology, daily sperm production and sperm motility in knock-out and wild-type mice. PLAG1 was sparsely expressed in germ cells and in Sertoli cells. Genes known to be involved in spermatogenesis were downregulated in the testes of knock-out mice, as well as Hsd17b3, which encodes a key enzyme in androgen biosynthesis. In the absence of Plag1, a number of genes involved in immune processes and epididymis-specific genes were upregulated in the testes. Finally, loss of PLAG1 resulted in significantly lowered daily sperm production, in reduced sperm motility, and in several animals, in sloughing of the germinal epithelium. Our results demonstrate that the subfertility seen in male PLAG1-deficient mice is, at least in part, the result of significantly reduced sperm output and sperm motility.
HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse
(PUBLIC LIBRARY SCIENCE, 2015-10-01)
piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2' O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program.
LRGUK-1 Is Required for Basal Body and Manchette Function during Spermatogenesis and Male Fertility
(PUBLIC LIBRARY SCIENCE, 2015-03-01)
Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.
A Missense Mutation in the Transcription Factor ETV5 Leads to Sterility, Increased Embryonic and Perinatal Death, Postnatal Growth Restriction, Renal Asymmetry and Polydactyly in the Mouse
(PUBLIC LIBRARY SCIENCE, 2013-10-21)
ETV5 (Ets variant gene 5) is a transcription factor that is required for fertility. In this study, we demonstrate that ETV5 plays additional roles in embryonic and postnatal developmental processes in the mouse. Through a genome-wide mouse mutagenesis approach, we generated a sterile mouse line that carried a nonsense mutation in exon 12 of the Etv5 gene. The mutation led to the conversion of lysine at position 412 into a premature termination codon (PTC) within the ETS DNA binding domain of the protein. We showed that the PTC-containing allele produced a highly unstable mRNA, which in turn resulted in an undetectable level of ETV5 protein. The Etv5 mutation resulted in male and female sterility as determined by breeding experiments. Mutant males were sterile due to a progressive loss of spermatogonia, which ultimately resulted in a Sertoli cell only phenotype by 8 week-of-age. Further, the ETV5 target genes Cxcr4 and Ccl9 were significantly down-regulated in mutant neonate testes. CXCR4 and CCL9 have been implicated in the maintenance and migration of spermatogonia, respectively. Moreover, the Etv5 mutation resulted in several developmental abnormalities including an increased incidence of embryonic and perinatal lethality, postnatal growth restriction, polydactyly and renal asymmetry. Thus, our data define a physiological role for ETV5 in many aspects of development including embryonic and perinatal survival, postnatal growth, limb patterning, kidney development and fertility.