Identification of voltage-dependent Ca2+ channels in sea urchin sperm

AbstractFunctional evidence indicates that voltage-dependent Ca2+ (Cav) channels participate in sea urchin sperm motility and the acrosome reaction (AR), however, their molecular identity remains unknown. We have identified transcripts for two Ca2+ channel α1 subunits in sea urchin testis similar in sequence to Cav1.2 and Cav2.3. Antibodies against rat Cav1.2 and Cav2.3 channels differentially label proteins in the flagella and acrosome of mature sea urchin sperm. The Cav channel antagonists nifedipine and nimodipine, which inhibit the AR, diminish the intracellular Ca2+ elevation induced by a K+-induced depolarization in valinomycin-treated sperm. These findings reveal that Cav1.2 and Cav2.3 channels could participate in motility and/or the AR in sea urchin sperm

Sirt1 Deficiency Attenuates Spermatogenesis and Germ Cell Function

In mammals, Sirt1, a member of the sirtuin family of proteins, functions as a nicotinamide adenine dinucleotide-dependent protein deactylase, and has important physiological roles, including the regulation of glucose metabolism, cell survival, and mitochondrial respiration. The initial investigations of Sirt1 deficient mice have revealed a phenotype that includes a reduced lifespan, small size, and an increased frequency of abnormal sperm. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (−/−) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Numbers of mature sperm and spermatogenic precursors, as early as d15.5 of development, are significantly reduced (∼2-10-fold less; P≤0.004) in numbers in Sirt1−/− mice, whereas Sirt1 deficiency did not effect the efficiency oocyte production following superovulation of female mice. Furthermore, the proportion of mature sperm with elevated DNA damage (∼7.5% of total epididymal sperm; P = 0.02) was significantly increased in adult Sirt1−/− males. Analysis of global gene expression by microarray analysis in Sirt1 deficient testis revealed dysregulated expression of 85 genes, which were enriched (P<0.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirt1 deficient germ cells, we compared the efficiency of generating embryos and viable offspring in in vitro fertilization (IVF) experiments using gametes from Sirt1−/− and sibling Sirt1+/− mice. While viable animals were derived in both Sirt1−/− X wild type and Sirt1−/− X Sirt1−/− crosses, the efficiency of producing both 2-cell zygotes and viable offspring was diminished when IVF was performed with Sirt1−/− sperm and/or oocytes. Together, these data support an important role for Sirt1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function

Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease

Molekular Charakterisierung der Hook1 und TSEP22 Gene, die in Männlicher Kernzellen exprimiert sind -

Im Rahmen dieser Arbeit wurden die murinen Gene Hook1 und TSEP22 molekulargenetisch charakterisiert. Beide Gene werden in männlichen Keimzellen exprimiert und das Vorkommen der Hook1 und TSEP22Genprodukte in elongierten Spermatiden lässt vermuten, dass beide Gene eine Rolle bei der Reifung bzw. für die Funktion der Spermatozoen spielen.Das Hook1 Gen besteht aus 22 Exons. Sowohl die Hook1-Transkripte als auch das Hook1-Protein kann in runden und elongierten Spermatiden nachgewiesen werden. Vermutlich ist das Hook1-Genprodukt in Mikrotubuli-abhängige Transportprozesse oder in endozytotische Vorgänge involviert. Das Hook1-Gen konnte auf dem murinen Chromosom 4 im Bereich C2-D5 lokalisiert werden. In dieser Region liegt der azh-Locus (abnormal spermatozoon head shape). Durch die Analyse der DNA wie auch der RNA homozygoter azh-Mäuse konnte belegt werden, dass eine Deletion der Exons 10 und 11 des Hook1-Gens vorliegt und dies zu einer Verschiebung des Leseraster führt. Zudem belegen Untersuchungen mit Hook1-spezifischen Antikörpern, dass kein funktionelles Hook1-Genprodukt in testikulären Proteinextrakten von azh-Mäusen nachgewiesen werden kann. Die Analyse der Nachkommen aus Verpaarungen heterozygoter Tiere zeigt, dass die Deletion mit dem Phänotyp segregiert. Diese Ergebnisse belegen, dass der Verlust der Hook1-Funktion die Ursache der azh-Mutation darstellt.Das murine TSEP22-Gen besteht aus drei Exons, die in der Telomerregion des Chromsoms 12 lokalisiert sind. Das Ge kodiert für mehrere Transkripte, die in pachytänen Spermatozyten und in Spermatiden nachweisbar sind. Diese Transkripte variieren in den 5 -nicht-translatierten Bereichen, wobei diese Sequenzen in die Kontrolle der Translation der TSEP22-Transkripte involviert sein könnten. Unterstützung für diese Annahme kommt aus der Beobachtung, dass die TSEP22-Transkripte bereits in Testes 15 Tage alter Mäuse nachgewiesen werden können, während das Protein erst drei Tage später detektierbar ist. Das TSEP22-Protein konnte im Mittelstück der Spermatozoenflagelle nachgewiesen werden. Hier könnte es in die Regulation der Spermienmotilität involviert sein, wobei die Phosphorylierung putativer Erkennungsstellen im TSEP22-Protein eine Rolle spielen könnten. Weitere Untersuchungen werden zeigen, ob die Vermutung hinsichtlich der Funktion von TSEP22 zutrifft

Docking and QSAR studies of aryl-valproic acid derivatives to identify anti-proliferative agents targeting the HDAC8.

Histone deacetylase 8 (HDAC8) is a plausible target for the development of novel anticancer drugs using a metal-chelating group and hydrophobic moieties as pharmacophores. It is known that valproic acid (administered as its salt, sodium valproate; VPANa+) is an HDAC8 inhibitor characterized by its hydrophobic chains. Nevertheless, VPA is hepatotoxic and VPA analogues might be explored for less hepatotoxic antiproliferative compounds. In this work, docking and QSAR studies of 500 aryl-VPA derivatives as possible HDAC8 inhibitors were performed in order to explore and select potential anti-proliferative compounds. Docking results identified π-π hydrogen bonds as the most important noncovalent interactions between HDAC8 (PDB: 3F07) and the ligands tested, whereas Belm4 was the best QSAR descriptor and classified as a 2D-BCUT descriptor. Based on theoretical studies, compound DAVP042 was synthesized and evaluated in vitro for its anti-proliferative activities on several cancer cell lines (A549 - lung, MCF-7 - breast, HCT116 - colon and U937 - lymphoid tissue) in comparison to VPA, as well as for its inhibitory activity on HDAC8 using in vitro models. DAVP042 demonstrated to have antiproliferative activity on all cancer cell lines employed, not only suggesting that this compound should be further studied, but also demonstrating that the methodology herein employed is appropriated to identify new therapeutic candidates