Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.
SUMMARYSperm-head elongation and acrosome formation, which take place during the last stages of spermatogenesis, are essential to produce competent spermatozoa that are able to cross the oocyte zona pellucida and to achieve fertilization. During acrosome biogenesis, acrosome attachment and spreading over the nucleus are still poorly understood and to date no proteins have been described to link the acrosome to the nucleus. We recently demonstrated that a deletion of DPY19L2, a gene coding for an uncharacterized protein, was responsible for a majority of cases of type I globozoospermia, a rare cause of male infertility that is characterized by the exclusive production of round-headed acrosomeless spermatozoa. Here, using Dpy19l2 knockout mice, we describe the cellular function of the Dpy19l2 protein. We demonstrate that the protein is expressed predominantly in spermatids with a very specific localization restricted to the inner nuclear membrane facing the acrosomal vesicle. We show that the absence of Dpy19l2 leads to the destabilization of both the nuclear dense lamina (NDL) and the junction between the acroplaxome and the nuclear envelope. Consequently, the acrosome and the manchette fail to be linked to the nucleus leading to the disruption of vesicular trafficking, failure of sperm nuclear shaping and eventually to the elimination of the unbound acrosomal vesicle. Finally, we show for the first time that Dpy19l3 proteins are also located in the inner nuclear envelope, therefore implying that the Dpy19 proteins constitute a new family of structural transmembrane proteins of the nuclear envelope.KEY WORDS: Dpy19l2, Acrosome biogenesis, Globozoospermia, Nuclear envelope, Nuclear lamina, Spermiogenesis, MouseAbsence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus
An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization.
Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A 2 (sPLA 2 ) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA 2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA 2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.
The acrosome is an organelle that is central to sperm physiology and a defective acrosome biogenesis leads to globozoospermia, a severe male infertility. The identification of the actors involved in acrosome biogenesis is therefore particularly important to decipher the molecular pathogeny of globozoospermia. We recently showed that a defect in the DPY19L2 gene is present in more than 70% of globozoospermic men and demonstrated that Dpy19l2, located in the inner nuclear membrane, is the first protein involved in the attachment of the acrosome to the nuclear envelope (NE). SUN proteins serve to link the nuclear envelope to the cytoskeleton and are therefore good candidates to participate in acrosome-nucleus attachment, potentially by interacting with DPY19L2. In order to characterize new actors of acrosomal attachment, we focused on Sun5 (also called Spag4l), which is highly expressed in male germ cells, and investigated its localization during spermatogenesis. Using immunohistochemistry and Western blot experiments in mice, we showed that Sun5 transits through different cellular compartments during meiosis. In pachytene spermatocytes, it is located in a membranous compartment different to the reticulum. In round spermatids, it progresses to the Golgi and the NE before to be located to the tail/head junction in epididymal sperm. Interestingly, we demonstrate that Sun5 is not, as initially reported, facing the acrosome but is in fact excluded from this zone. Moreover, we show that in Dpy19l2 KO spermatids, upon the detachment of the acrosome, Sun5 relocalizes to the totality of the NE suggesting that the acrosome attachment excludes Sun5 from the NE facing the acrosome. Finally, Western-blot experiments demonstrate that Sun5 is glycosylated. Overall, our work, associated with other publications, strongly suggests that the attachment of the acrosome to the nucleus does not likely depend on the formation of SUN complexes.
Different mammalian secreted phospholipases A(2) (sPLA(2) s) are expressed in male reproductive organs and/or in sperm cells but their cellular functions are still not fully characterized. Because several reports indicate a link between cellular lipids and sperm motility, we have investigated the effect of mouse group IIA, IID, IIE, V, and X sPLA(2) s on sperm motility. Among these enzymes, only mouse group X sPLA(2) (mGX sPLA(2) ) acts as a potent inhibitor of sperm motility that decreases track speed (VCL) and lateral displacement of the head (ALH) of both noncapacitated and capacitated sperm. The inhibitory effect of mGX sPLA(2) is dependent on its enzymatic activity because (i) both the proenzyme form of mGX sPLA(2) (pro-mGX) and the H48Q mutant of mGX sPLA(2) have very weak enzymatic activity and are unable to modulate sperm motility and (ii) LY329722, a specific inhibitor of sPLA(2) s, blocks the inhibitory effect of mGX sPLA(2) . Moreover, mGX sPLA(2) exerts a gradual potency on sperm subpopulations with different velocities, an effect which may be linked to the heterogeneity of lipid composition in these sperm subpopulations. Finally, we found that endogenous mGX sPLA(2) released during spontaneous acrosome reaction modulates sperm motility of capacitated sperm. Together, our results suggest a new role of sPLA(2) in sperm physiology where the sPLA2 selects a sperm subpopulation for fertilization based on its effect on sperm motility.
> Chez les mammifères, la physiologie du spermatozoïde dans les voies génitales femelles est complexe, et plusieurs étapes bien définies doivent être franchies pour que le spermatozoïde puisse féconder l'ovocyte. Les phospholipases, qui permettent de contrôler la composition lipidique du spermatozoïde en hydrolysant les phospholipides de manière très spécifique, sont impliquées dans des mécanismes aussi variés que le contrôle du battement flagellaire, la capacitation, la réaction acrosomique, ainsi que dans le déclenchement des oscillations calciques de l'ovocyte qui entraînent l'activation ovocytaire. Deux phospholipases, la PLC et la PLA 2 sécrétée de groupe X, pourraient représenter des cibles thérapeutiques pour lutter contre l'infertilité masculine. < constituées d'une tête polaire phosphoalcoolique reliée, via une molécule de glycérol, à une queue hydrophobe constituée de deux acides gras [1]. Choline et éthanolamine sont les rési-dus alcool les plus courants au niveau de la partie polaire, mais d'autres, tels que la sérine ou l'inositol biphosphate, composent certaines GPL (Figure 1). Tous ces GPL sont hydrolysés par une superfamille de protéines, les phospholipases (PL). Les PL sont sépa-rées en quatre familles sans homologie structurale, sur la base de la localisation du site enzymatique d'hydrolyse :
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