SummaryCilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actinbased cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

The female steroid hormone progesterone regulates ovulation and supports pregnancy, but also controls human sperm function within the female reproductive tract. Progesterone causes elevation of sperm intracellular Ca 2+ leading to sperm hyperactivation, acrosome reaction, and perhaps chemotaxis toward the egg. Although it has been suggested that progesterone-dependent Ca 2+ influx into human spermatozoa is primarily mediated by cationic channel of sperm (CatSper), the principal flagellar Ca 2+ channel of sperm, conclusive loss-of-function genetic evidence for activation of CatSper by progesterone has yet to be provided. Moreover, it is not clear whether the responsiveness of CatSper to progesterone is an innate property of human spermatozoa or is acquired as the result of exposure to the seminal plasma. Here, by recording ionic currents from spermatozoa of an infertile CatSper-deficient patient, we demonstrate that CatSper is indeed the principal Ca 2+ channel of human spermatozoa, and that it is strongly potentiated by progesterone. In addition, by recording CatSper currents from human epididymal and testicular spermatozoa, we show that CatSper sensitivity to progesterone arises early in sperm development and increases gradually to a peak when spermatozoa are ejaculated. These results unambiguously establish an important role of CatSper channel in human sperm nongenomic progesterone signaling and demonstrate that the molecular mechanism responsible for activation of CatSper by progesterone arises early in sperm development concurrently with the CatSper channel itself.CatSper ion channel | male fertility | nongenomic steroid action | sperm physiology

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Progesterone induced a rapid influx of calcium in capacitated human sperm, followed by a long-lasting, dose-dependent increase of intracellular free calcium. Thereafter, progesterone increased the fraction of hyperactivated sperm and the acrosome reaction. On the contrary, the progesterone antagonist RU486 (mifepristone) induced an immediate and transient, dose-dependent decrease of intracellular free calcium and a drop in the values of sperm movement parameters related to hyperactivation. Moreover, RU486 counteracted the effects of progesterone on calcium influx, lateral sperm head displacement, and the acrosome reaction. Therefore, RU486 effects were opposite to those of progesterone. The nature of the membrane receptor(s) involved is unknown. Several steroids bearing 11(-phenyl substitutions, with different pharmacological profiles, were also investigated. It was concluded that the steroid structure and chemical groups added to the 11(-phenyl influence effects on calcium influx.Capacitation leads to functional changes ofthe sperm that are characterized by modified movement parameters designated as hyperactivation and by exocytotic events (the acrosome reaction) (1-3). The acrosome reaction is an essential step in the complex series of processes implied in Eutherian fertilization, necessary for sperm penetration through the zona pellucida and fusion with the oocyte plasma membrane. It can be induced by follicular fluid, cumulus cells (4), or zona pellucida (5). Progesterone (PROG) was identified as a major component of the follicular fluid for inducing the acrosome reaction in human sperm (6), preceded by an immediate, transient calcium influx into spermatozoa (7,8). These rapid biological effects suggest a nongenomic mechanism and a cell surface receptor for PROG like the one responsible for the meiotic maturation of Xenopus laevis oocyte (9).The antiprogestin RU486 {RU38486, mifepristone, 17(-hydroxy-11(8-[4-(dimethylamino)phenyl]-17a-propynylestra-4,9-dien-3-one} binds with high affinity to the intracellular PROG receptor in most vertebrate species (10). It has been reported that this potent antiprogestin has either a small or negligible inhibitory effect on PROG-mediated calcium influx into human sperm (11,12). In this study, we evaluated the effect of RU486, alone or in combination with PROG, on intracellular free calcium concentration ([Ca2l]i) and the movement of human sperm and acrosome reaction, two calcium-related events (3, 13).MATERIALS AND METHODS Materials. PROG, human serum albumin (HSA) fraction 5, Fura2-AM, Fluo3-AM, Hoechst 33258, fluorescein-labeled Pisum sativum agglutinin, verapamil, EGTA, Triton X-100, sodium pyruvate, and Hepes were purchased from Sigma.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.RU486, 17p-hydroxy-11p-{4-[2-dimethylamino)ethoxy]phenyl}-17a-propynylestra-4,9-dien-3-one(RU39009), 11-[4-(di-phenyl}estra...

Two groups of men were retrospectively selected according to their observed success in in-vitro fertilization. Seminal and post-migration sperm samples from a low fertilization rate group (less than or equal to 33% cleaved embryos) have been compared to results obtained from a high fertilization rate group (greater than or equal to 66%). It was found that a low mean value of the amplitude of lateral sperm head displacement and an increased percentage of abnormal acrosomes were related to in-vitro fertilization failure. None of the individual sperm factors studied was found to determine in-vitro fertilization success with certainty; only when they were considered in combination was it possible to predict the likelihood of successful in-vitro fertilization of human oocytes.

A microcinematographic (50 f/s) study was performed on motile human spermatozoa. Eighty percent were found to have a linear trajectory and a pseudo‐sinusoidal head displacement pattern. Throughout their progression, the spermatozoa periodically rotated on their longitudinal axis at a frequency equal to that of flagellar wave formation. These waves were found always to begin on the same side of the cell and to propagate in the flattened plane of the head until the moment of rotation. At this time the wave had reached a point near the middle of the flagellum. Beyond this point, the flagellum moves out of the plane of the head. Different variables used to characterize the movement of spermatozoa included the velocity of progression, amplitude and velocity of head displacement, frequency of rotation, wave amplitude, and propagation velocity of the flagellar wave. Among these variables, it was the propagation velocity of the wave that was found to be best correlated with the velocity of spermatozoan progression. This flagellar wave exhibited two stages, one of initiation and one of propagation.

Not applicable.