Male Infertility is a Women\u27s Health Issue-Research and Clinical Evaluation of Male Infertility Is Needed

Infertility is a devastating experience for both partners as they try to conceive. Historically, when a couple could not conceive, the woman has carried the stigma of infertility; however, men and women are just as likely to contribute to the couple\u27s infertility. With the development of assisted reproductive technology (ART), the treatment burden for male and unexplained infertility has fallen mainly on women. Equalizing this burden requires reviving research on male infertility to both improve treatment options and enable natural conception. Despite many scientific efforts, infertility in men due to sperm dysfunction is mainly diagnosed by a semen analysis. The semen analysis is limited as it only examines general sperm properties such as concentration, motility, and morphology. A diagnosis of male infertility rarely includes an assessment of internal sperm components such as DNA, which is well documented to have an impact on infertility, or other components such as RNA and centrioles, which are beginning to be adopted. Assessment of these components is not typically included in current diagnostic testing because available treatments are limited. Recent research has expanded our understanding of sperm biology and suggests that these components may also contribute to the failure to achieve pregnancy. Understanding the sperm\u27s internal components, and how they contribute to male infertility, would provide avenues for new therapies that are based on treating men directly for male infertility, which may enable less invasive treatments and even natural conception

Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment

Regulated centrosome biogenesis is required for accurate cell division and for maintaining genome integrity1. Centrosomes consist of a centriole pair surrounded by a protein network known as pericentriolar material (PCM)1. PCM assembly is a tightly regulated, critical step that determines a centrosome’s size and capability2–4. Here, we report a role for tubulin in regulating PCM recruitment via the conserved centrosomal protein Sas-4. Tubulin directly binds to Sas-4; together they are components of cytoplasmic complexes of centrosomal proteins5,6. A Sas-4 mutant, which cannot bind tubulin, enhances centrosomal protein complex formation and has abnormally large centrosomes with excessive activity. These suggest that tubulin negatively regulates PCM recruitment. Whereas tubulin-GTP prevents Sas-4 from forming protein complexes, tubulin-GDP promotes it. Thus, tubulin’s regulation of PCM recruitment depends on its GTP/GDP-bound state. These results identify a role for tubulin in regulating PCM recruitment independent of its well-known role as a building block of microtubules7. Based on its guanine bound state, tubulin can act as a molecular switch in PCM recruitment

Length-dependent disassembly maintains four different flagellar lengths in Giardia.

With eight flagella of four different lengths, the parasitic protist Giardia is an ideal model to evaluate flagellar assembly and length regulation. To determine how four different flagellar lengths are maintained, we used live-cell quantitative imaging and mathematical modeling of conserved components of intraflagellar transport (IFT)-mediated assembly and kinesin-13-mediated disassembly in different flagellar pairs. Each axoneme has a long cytoplasmic region extending from the basal body, and transitions to a canonical membrane-bound flagellum at the 'flagellar pore'. We determined that each flagellar pore is the site of IFT accumulation and injection, defining a diffusion barrier functionally analogous to the transition zone. IFT-mediated assembly is length-independent, as train size, speed, and injection frequencies are similar for all flagella. We demonstrate that kinesin-13 localization to the flagellar tips is inversely correlated to flagellar length. Therefore, we propose a model where a length-dependent disassembly mechanism controls multiple flagellar lengths within the same cell

Plk1/Polo Phosphorylates Sas-4 at the Onset of Mitosis for an Efficient Recruitment of Pericentriolar Material to Centrosomes

Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4's localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and gamma-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and gamma-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion

Sperm centriole assessment identifies male factor infertility in couples with unexplained infertility - a pilot study

Unexplained infertility affects about one-third of infertile couples and is defined as the failure to identify the cause of infertility despite extensive evaluation of the male and female partners. Therefore, there is a need for a multiparametric approach to study sperm function. Recently, we developed a Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC) assay to determine sperm centriole quality. Here, we perform a pilot study of sperm from 10 fertile men and 10 men in couples with unexplained infertility, using three centriolar biomarkers measured at three sperm locations from two sperm fractions, representing high and low sperm quality. We found that FRAC can identify men from couples with unexplained infertility as the likely source of infertility. Higher quality fractions from 10 fertile individuals were the reference population. All 180 studied FRAC values in the 10 fertile individuals fell within the reference population range. Eleven of the 180 studied FRAC values in the 10 infertile patients were outliers beyond the 95% confidence intervals (P = 0.0008). Three men with unexplained infertility had outlier FRAC values in their higher quality sperm fraction, while four had outlier FRAC values in their lower quality sperm fraction (3/10 and 4/10, P = 0.060 and P = 0.025, respectively), suggesting that these four individuals are infertile due, in part, to centriolar defects. We propose that a larger scale study should be performed to determine the ability of FRAC to identify male factor infertility and its potential contribution to sperm multiparametric analysis

A dynamic basal complex modulates mammalian sperm movement

Centrioles are ancient organelles with a conserved architecture and their rigidity is thought to restrict microtubule sliding. Here authors show that, in mammalian sperm, the atypical distal centriole and its surrounding atypical pericentriolar matrix form a dynamic basal complex that facilitates a cascade of internal sliding deformations, coupling tail beating with asymmetric head kinking

Mediation of adenylyl cyclase sensitization by PTX-insensitive Gα oA , Gα i1 , Gα i2 or Gα i3

Chronic activation of mu-opioid receptors, which couple to pertussis toxin-sensitive Gα i/o proteins to inhibit adenylyl cyclase (AC), leads to a compensatory sensitization of AC. Pertussis toxin-insensitive mutations of Gα i/o subtypes, in which the pertussis toxin-sensitive cysteine is mutated to isoleucine (G ), were used to determine whether each of the Gα i/o subtypes is able to mediate sensitization of AC. G , G , G or G were individually transiently transfected into C6 glioma cells stably expressing the mu-opioid receptor, or transiently co-expressed with the mu-opioid receptor into human embryonic kidney (HEK)293T cells. Cells were treated with pertussis toxin to uncouple endogenous Gα i/o proteins, followed by acute or chronic treatment with the mu-opioid agonist, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO). Each Gα i/o subtype mediated acute DAMGO inhibition of AC and DAMGO-induced sensitization of AC. The potency for DAMGO to stimulate sensitization was independent of the Gα i/o subtype, but the level of sensitization was increased in clones expressing higher levels of Gα i/o subunits. Sensitization of AC mediated by a component of fetal bovine serum, which was also dependent on the level of functional Gα i/o subunits in the cell, was observed. This serum-mediated sensitization partially masked mu-opioid-mediated sensitization when expressed as percentage overshoot due to an apparent increase in AC activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65707/1/j.1471-4159.2006.04176.x.pd

Basal body stability and ciliogenesis requires the conserved component Poc1

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis

RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively-spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of photoreceptor connecting cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, Structural Maintenance of Chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain (RLD) of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal photoreceptors and MDCK cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150(Glued) and p50-dynamitin. Inhibition of dynein function by over-expressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia

Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly

Cep120, a protein involved in maintenance of neural progenitor cells, is required for centriole duplication in cycling cells and for centriole amplification in tracheal epithelial cells