Évolution de la composante lipidique de la membrane plasmique des spermatozoïdes durant la maturation épididymaire

One aspect of mammalian post-testicular sperm maturation is the progressive change in their plasma membrane lipid composition. These modifications in lipids allow sperm cells to fuse with oocytes during fertilization. A significant share of these sperm lipid changes occurs during their descent through the epididymal tubule. It then continues within the female genital tract during the capacitation process, an essential prerequisite for acrosomic reaction and hence fertilization. This review presents what is known concerning the sperm plasma membrane lipid changes during epididymal maturation in various mammalian models. In the first section, after a brief presentation of the classic eukaryotic cell plasma membrane lipid organization, the emphasis is on the particularities of sperm plasma membrane lipids. The second section presents the different changes occurring in the three major classes of lipids (i.e. phospholipids, sterols and fatty acids) during the sperm’s epididymal descent. The final section briefly describes the mechanisms by which these lipid changes might happen in the epididymal lumen environment. The role played by lipid-rich vesicles secreted by the epididymal epithelium via apocrine secretory processes is highlighted.     

Principes de préparation et de congélation des spermatozoïdes testiculaires humains

The advantages and feasibility of human testicular spermatozoa cryoconservation for intracytoplasmic sperm injection (ICSI) have now been clearly demonstrated. However, the freezing protocol is based on empirical knowledge obtained from freezing of ejaculated spermatozoa. Testicular spermatozoa may not be fully mature gametes and may also be retrieved in only limited quantities. Little research has been conducted to determine whether they have the same cryobiological requirements as ejaculated spermatozoa. A better understanding of their cryobiological features and assessment of possible subcellular changes after thawing would help to optimize testicular preparations for cryopreservation (whole biopsies, seminiferous tubules, shredded suspension, single spermatozoa, etc.), freezing-thawing procedure, freezing media, and storage. Finally, there is a growing need for welldefined criteria (nuclear quality, etc.) to evaluate the tolerance of testicular spermatozoa to freezing-thawing procedure for ICSI     

Le chimiotactisme des spermatozoïdes a-t-il un rôle dans la fécondation?

Sperm chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime between 50 min and 4 h) and they occur only once in the sperm’s lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in man) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the duration of time over which capacitated spermatozoa would be available in the female reproductive tract. The sperm chemoattractants have not been identified but they appear to be heat-stable peptides. Thein vivo location of sperm chemotaxis is not known; a number of possible locations are discussed.     

Les spermatozoïdes immobiles frais ou décongelés en fécondation assistée

Contrasting with sperm count or morphology, complete lack of mobile sperm may seriously impair ICSI fertilization and pergnancy rate. In three cases with flagellar skeleton abnormalities [dynein arm absence] only immobile sperm were found in the ejaculate. Following repeated ejaculations, higher rates of viable spermatozoa and even some motile spermatozoa could be found. Some times, in nonobstructive azoospermia, extensive sperm search didn't allow us to find but immobile sperm mostly, with very few motile sperm cells, not enough for the microinjection of all oocytes. The third group of immobile sperm is iatrogenic, following freezing and thawing surgically retrieved, testicular or epididymal spermatozoa in order to avoid repeated surgical retrieval. Following thawing, one find frequently very few motile spermatozoa that may be not enough for all retrieved oocytes and it might be necessary to inject some eggs with immobile spermatozoa. The outcome of ICSI using mobile and immobile sperm was compared in the three above mentioned groups: 1-immobile ejaculated sperm with flagellar defects, 2-immobile sperm discovered in the ejaculate after extensive sperm search and 3- immobile frozen-thawed testicular or epididymal spermatozoa. The results of ICSI in these groups show that fertilizing ability of fresh or frozenthawed immobile spermatozoa is not significantly different from ICSI with mobile sperm from the same origin. More over, in the first group with flagellar abnormalities, repeated ejaculations allowed us significantly increase sperm viability and fertilization ability. Finding only immobile fresh or frozen-thawed sperm the day of egg retrieval should not lead us to ICSI cancellation. Pregnancies may occur with such immobile sperm.     

La sélection des spermatozoïdes à fort grossissement permet-elle une diminution de la fréquence des aneuploïdies spermatiques?

Severe male infertility concerns two categories of men. Men with abnormal karyotype, who represent 2 to 14% of infertile men and who can produce sperm cells carrying unbalanced chromosomes related to the patients initial chromosomal reorganization inducing a variable risk of transmission of the abnormality to their conceptus. The second category is men with a normal karyotype but an increased rate of spermatic aneuploidy in a context of severe oligo- and/or asthenozoospermia and men from couples in implantation failure. ICSI is the standard Assisted Medical Reproductive technique for most of these 2 categories despite the obvious increased chromosomal risk. This raises the question of how to morphologically identify sperm cells with abnormal chromosome content during ICSI ? Unfortunately, no relationship has yet been found between sperm morphology in the ICSI sperm fraction (×200) and their chromosome content. Nevertheless, since the end of the 1990s, Bartoov’s team has developed MSOME (Motile Sperm Organelle Morphology Examination) consisting of high-power examination of sperm cells up to × 12,250. This technique was indicated for cases of repeated ICSI failures and appeared to increase pregnancy rates. But was this improvement due to better selection of the chromosomal content of sperm cells to be injected? The present study addressed this question by estimating the value of MSOME in the selection of euploid sperm cells in 2 groups of patients known to have an increased rate of sperm aneuploidy. Group 1 was composed of 2 patients with normal karyotype who presented a macrocephalic sperm syndrome with more than 99% of aneuploid sperm. Group 2 was composed of 11 patients with abnormal karyotype: 6 patients with reciprocal translocation and 5 patients with Robertsonian translocation. The purpose of this study was to compare spermatozoa aneuploidy rates in fresh semen, to those obtained after ICSI selection (×200) and MSOME selection (×6000). Three specific steps of the protocol were (1) all sperm cells selected in MSOME were “top sperm cells“ (2) fixation of selected sperm cell (average loss of 15% during FISH washes) (3) FISH results were validated by two different examiners. FISH analysis of X, Y and 18 chromosomes showed that MSOME eliminates polyploid and diploid sperm cells in patients with macrocephalic sperm syndrome, but the 6 sperm cells selected were all haploid and aneuploid. FISH analysis of X, Y and 18 chromosomes of all other patients did not show any influence of the selection method on the aneuploidy rate. For the 5 subjects with a Robertsonian translocation, the global results of FISH analysis paradoxically showed a significant decrease of the euploidy rate in MSOME selection. The global results of FISH analysis for the 6 patients with mutual reciprocal translocations, showed that the various mutual translocations were not modified between whole sperm and the 2 selection methods. On the other hand, a significant decrease of adjacent 1 and 2 segregation frequency was observed between whole sperm and MSOME selection, associated with a significant increase of 3:1 segregation frequency suggesting that the segregations which modify the structure of chromosomes, for example adjacent 1 and 2 segregations, would induce visible morphological modifications selected by MSOME. We hypothesized that the efficacy of spermatic apoptosis could be modulated by morphology but also by the chromosome contents of the sperm cell. In conclusion, MSOME does not provide any guarantee of the normal chromosome contents of the TOP selected sperm cell. However, these results obtained in a small series of patients suggest that MSOME can eliminate some chromosome abnormalities (adj1 and 2) which would alter sperm nuclear structures.     

Sera-T-Il possible de mécaniser la préparation des tableux phytosociologiques?

Sans résuméReçu par la rédaction le 30.X.1953.     

Perspectives de l’analyse de la mobilité des spermatozoïdes

Several studies have shown a good correlation between sperm motility and fertility though the microscopic evaluation of the percentage of motile sperm is highly subjective by nature. Therefore in the last decade, various objectives methods have been proposed to overcome this problem. Two types of methods were developed: The methods based on the analysis of images obtained by microphotography, microcinematography and microvideography and the global, undirect methods based on physical principles. Several systems based on video and image analysis (Computer Aided Sperm Analysis, CASA) have been developed and are used in numerous laboratories of reproductive biology. CASA technology offers the possibility to analyse some characteristics of sperm motion which are related to the fertilization potential and to develop new parameters related to some important aspects of sperm behavior such as hyperactivation. However, there is a large amount of interactions between the operator and the CASA machine. CASA instruments are not “ready-to-use” robots: the reliability of CASA depends largely on the expertise and training of the user and the application of standardized procedures and quality control schemes. By contrast, there is only minimal interaction between the operator and the Sperm Quality Anlyser which is a new device measuring and index of sperm motility highly correlated to the concentration of progressively motile sperm. The device uses light passed through a small sample of semen introduced in a capillary tube to detect variations in optical density that result from moving particles. The reproducibility of the measurements is excellent, the device is easy to use and this is a potentially useful tool for field-work studies. Further investigations of this device in the managment of male infertility is warranted. Finally, both types of objectives approaches are complementary to the conventional analysis of sperm motility and they will not replace it. Standardized procedures have been proposed by the World Health Organization for the subjective evaluation of sperm motility. Such procedures are very useful to reduce significantly the intra- and interlaboratory variations but internal and external quality controls schemes indicate that they are not sufficient to achieve acceptable levels of variation and regular quality controls followed by the definition and the application of corrective procedures are required.     

Evaluation de l’apport de la méthode d’observation des spermatozoïdes à fort grossissement en ICSI

Due to the growing interest in the method of high-magnification sperm observation and selection proposed for the specific indication of ICSI failure, the authors evaluated the technique in unselected ICSI. The aim of this study was to evaluate the relevance of Motile Sperm Organelle Morphology Examination (MSOME) compared with usual selection performed in ICSI. In a series of conventionally selected sperm for ICSI, the number with an abnormal appearance on high magnification was determined and the predictive value of this parameter on ICSI outcome was assessed. The study included 25 successive unselected ICSI attempts in the IVF Laboratory of Poissy Hospital (France). ICSI were performed according to usual protocols used in the laboratory. Twenty five motile spermatozoa of the migrated fraction, still available after ICSI, and “injectable— according to conventional morphology assessment in ICSI (“normal” or “as normal as possible” with magnification of ×200–400) were assessed by MSOME (higher than ×4500) and classified according to criteria adapted from Bartoov’s work and taking into account David’s sperm morphology classification. We compared the results of MSOME and ICSI results. In this small series of ICSI with diverse indications, we found very high frequencies of abnormalities (more than 70%), particularly nuclear vacuoles. No predictive value of the morphology of sperm assessed with high magnification (including vacuoles) was found for fertilization rate, embryo quality and ICSI outcome. In contrast with previous reports, pregnancies were obtained with very abnormal sperms. In this series of unselected ICSI, nuclear vacuoles do not seem to have a pejorative impact on pregnancy outcome. This study raises several perspectives. It would be interesting to understand the “anatomical” basis for vacuoles observed with MSOME and their meaning. The question of the phenotype-genotype relation, i.e. the possible correlation between sperm morphology and genetic content could be investigated. Finally, a prospective analysis should be performed in clearly defined indications to validate the potential applications of the method for high-magnification sperm observation and selection.     

Formation de la membrane de fécondation de l'oeuf d'Artemia salina

Résumé L'oeuf vierge d'Artemia salina n'est pas entouré de membranes exocellulaires. Le plasme sous-cortical ne contient pas d'organites spéciaux. Dès la fécondation, une membrane est secrétée par l'oeuf. La substance membranogène, contenue dans le reticulum endoplasmique lisse, passe par les éléments golgiens, où elle semble modifiée, et est expulsée dans des vésicules qui se détachent du Golgi. Retenue par un enduit granuleux, qui couvre le plasmolemme, et qui peut être un glycocoat ou du suc du tractus génital, elle s'étale en une membrane de fécondation, qui se soulève pour constituer l'espace périvitellin. Le processus est progressif et dure environ une heure et demi.

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Formation of the fertilization membrane of the egg inArtemia salina

Summary The unfertilized egg ofArtemia salina is not covered with any extracellular structure. No special organelles are found in the sub-cortical plasma. From the moment of fertilization, a membrane is progressively secreted by the egg. The membranogenous substance is first seen as large granules in the smooth endoplasmic reticulum, presumably transformed within Golgi elements and extruded in vesicles liberated from the Golgi apparatus. Retained by a glycocoat or by contact with the fluid of the genital tract, it spreads out into a fertilization membrane, soon surrounding a perivitelline space. The process lasts till 1 1/2 h after fertilization.

     

Capacité des spermatozoïdes à se fixer sur la zone pellucide

After liberation from the seminiferous epithelium, the spermatozoa (SPZ), undergo in the epididymis a serie of functional and metabolic modifications resulting the capacity to ensure fertilization. Fertilization is the fundamental process in sexual reproduction as it permits the initiation and the formation of a new being by the fusion of two germinal cells: the male gamete (spermatozoa) and the female gamete (oocyte). For fertilization to occur the SPZ must recognize the zona pellucida (ZP), bind to it, penetrate it and fuse with the oocyte plasma membrane. Sperm binding to the ZP is an early, crucial event leading to fertilization and pre-embryo development. In mammals, sperm-ZP binding follows a serie of steps that occur in a well-defined chronological order: a) A loose association between SPZ and ZP referred to as «attachment». This shortlived interaction is heterospecific. b) Attachment is followed by a more distinct and persistent association of SPZ with ZP, thus called «binding». This sperm-zona interaction is species-specific, irreversible and mediated by complementary receptors present on the SPZ head and the ZP. c) The bound SPZ then undergoes the acrosome reaction (AR). Which involves fusion and vesiculation of the SPZ outer acrosomal membrane and plasma membrane leading to the release of acrosomal contents and the exposure of the inner acrosomal membrane. This AR is essential for SPZ passage through the ZP and to access to the oocyte plasma membrane where gamete fusion occurs.     

Techniques de préparation des microhyménoptères

Summary The author proposes to standardize the preparation technique for microhymenoptera. The insects have to be presented for identification in a dry state, mounted on a minute pin or glued on a heavy paper point (double-mounting). Preference is given to the mounting on minute pin, as this method has the advantage that a specimen pinned with a minuten may be removed from its support and each morphological detail easily observed. The minuten is fixed to a short strip of soft material such as polyporus (bracket fungus); if this is not available, stiff paper may be used. The support with the pinned specimen is then attached to a pin no. 3 (fig. 1–3). The absence of glue is advantageous, especially in subtropical countries, where the glue is generally destroyed by bacteria and fungi. When the pins are unavailable for this double-mounting technique, the author proposes to glue the thoracic pleurae of the insect to the previously folded tip of a heavy paper point (fig. 6). The material for identification may be preserved in a liquid medium (as alcohol with some drops of glycerine) after a series of individuals have been prepared according to the double-mounting technique. Also, specimens in excess may be sent dry in a plastic or glass tube between two cellucotton masses.      

Utilisation de la thyreostimuline humaine recombinante dans la préparation au traitement par iode-131 des pathologies thyroïdiennes

The introduction of recombinant human TSH (rhTSH) as a method of preparation for radioiodine therapy of follicular-derivated thyroid tumors (benign and malignant) is a significant medical advance. RhTSH has been approved for use in remnants ablation after total thyroidectomy for carcinoma. There are other potential uses for rhTSH that have not yet been licensed. The use of rhTSH allows to reduce administrated doses in goiters through an increase of iodine uptake and a more homogeneous distribution of radioiodine in the gland. RhTSH also improves thyroid cancer patients’quality of life by avoiding hypothyroidism.     

Syndrome de Klinefelter et microinjections intraovocytaires de spermatozoïdes (revue de la littérature)

Historically, Klinefelter’s is the typical clinical form of secretory azoospermia with no hope of achieving biological paternity. However, ICSI, either from ejaculated sperm or following testicular sperm extraction, have been recently applied to patients with Klinefelter’s syndrome. Papers published until June 2002 have reported microinjections with ejaculated sperm in 9 cases of Klinefelter’s syndrome with extreme oligospermia with the following overall results: 79 mature oocytes, 52 fertilized oocytes, 31 embryos, 18 transferred embryos, 6 pregnancies, 5 births, or from testicular spermatozoa, in 93 cases, with the following results: 347 oocytes, 193 fertilized oocytes, 149 embryos, 78 embryos transferred out of 37 cycles, 24 clinical pregnancies and 2 positive pregnancy tests, and 32 births. Although a publication bias was very likely (successful attempts were published, failed attempts were probably not published), these results were unexpected based on the traditional view on Klinefelter’s syndrome. The rate of aneuploid spermatozoa also appeared to be lower than expected. The real proportion of Klinefelter patients in whom spermatozoa with a good potential for embryonic development can be retrieved and the means of identifying these patients remain to be established.     

L’analyse automatisée de la morphologie du spermatozoïde

Examination of sperm morphology is one factor of evaluation of sperm function, but it can also be considered as a biomarker of testicular function. All publications showed a high variability in observed results, in relation with different methods of staining slides and classifying sperm morphology, and a large subjectivity in the visual assessment. Automated sperm morphology analysis (ASMA) have the potential to provide more objective, accurate, and precise morphometric measurements of spermatozoa. Standardisation of the methods of slides preparation is first essential. Analysis of the sperm head morphometry appears the more accessible for the ASMA and could give selective parameters in the evaluation of fertility, in complement with motion sperm analysis. In the other hand automated analysis of all sperm abnormalities appears illusory with actual instruments, because the midpiece or the flagellum is a little structure weakly stained, and thus difficult to be identified by the computer. Until more rigorous and consistent definitions of sperm features can be developped, in relation with testicular function, the pronostic value of existing sperm abnormalities classifications is limited.