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     

Intérêt du recueil chirurgical de spermatozoïdes déférentiels dans certaines indications masculines de PMA (IAC,FIV)

Two pregnancies have been obtained after assisted procreation using deferential sperm: one by IVF, one by AIC. The indications of this technique are: obstruction of deferens or of ejaculatory ductus, some anejaculations or retrograde ejaculations resistant to classical treatments.     

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.     

Étude en microscopie électronique et en fluorescence des médiateurs chimiques du système nerveux des Nereidae (Annélides Polychètes)

Résumé Le système nerveux central et, accessoirement, le système nerveux périphérique de Nereis pelagica L. et de Nereis diversicolor O. F. Müller ont été étudiés selon la méthode de fluorescence de Falck, Hillarp et al. La majorité des péricaryons fluorescents présentent la couleur jaune caractéristique d'une indole-amine. Les résultats de techniques cytochimiques, spectrofluorométriques et chimiques réalisées antérieurement, permettent de penser que le composé présent n'est pas la sérotonine mais la bufoténine. Celle-ci pourrait être localisée dans de grandes vésicules granulaires (diamètre moyen: 1300 Å) observées au microscope électronique. Les terminaisons axonales des neurones contenant la bufoténine présenteraient néanmoins des figures présynaptiques formées par les accumulations de petites vésicules claires (diamètre moyen: 400 à 500 Å). Il est supposé que la substance est un médiateur chimique associé à une fonction sécrétomotrice dans le neuropile, à une fonction motrice au niveau des jonctions neuro-muculaires.L'action de la bufoténine pourrait, dans certains cas, être liée à celle d'un autre composé découvert également par voie biochimique, l'acide -amino-butyrique. Des comparaisons avec les résultats de travaux réalisés chez d'autres Annélides laissent présager d'une homogénéité dans la répartition des monoamines chez ces animaux. Des études biochimiques et physiologiques sont toutefois nécessaires pour conclure à l'identité des composés découverts par la méthode de fluorescence.

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Electron and fluorescence microscopic study of neurochemical mediators in the central nervous system of nereidae

Summary The central and peripheral nervous systems of Nereis pelagica L. and Nereis diversicolor O.F. Müller were studied with the fluorescence method of Falck, Hillarp et al. Many perikarya show a yellow fluorescence which is characteristic of an indole-amine. Previous results obtained with cytochemical, spectrofluorometric and chemical techniques suggest that the compound in question is not serotonin but bufotenin. This could be located in large granular vesicles (mean diameter: 1300 Å) observed with the electron microscope.The axon terminals of neurons presumed to contain bufotenin show presynaptic clear vesicles (mean diameter: 400 to 500 Å). It is postulated that the material they contain is a chemical mediator with secretomotor function in the neuropile and with motor function at the neuromuscular junction. The action of bufotenin could, in certain cases, be linked with that of another compound also discovered with biochemical methods, -aminobutyric acid.Comparison with results obtained in other annelids show a corresponding distribution of monoamines. Biochemical and physiological studies will be necessary to determine the identity of the compounds discovered with the fluorescence method.

Nous tenons à remercier très vivement Monsieur le Professeur Barry qui nous a permis d'effectuer ces recherches dans son Laboratoire. Nous adressons également nos remerciements à Messieurs les Ingénieurs de la Société Leitz qui ont réalisé l'étude en microspectrofluorométrie, ainsi qu'à Mademoiselle Warembourg et à Monsieur Léonardelli pour l'aide technique qu'ils nous ont apportée.     

La déficience en alpha-1 antitrypsine: corrélation entre épreuves biochimiques et phénotypes dans une famille

Phenotypes (Pi) for serum alpha-1 antitrypsin (AAT) were determined in a family of 19 members spanning three generations and presenting a deficiency in this protein. The standard Fagerhol crossed immunoelectrophoresis procedure was used for this purpose. The individual phenotypes consisted of seven MM, three ZZ, five MZ, one SZ and three MS. AAT levels were obtained by radial immunodiffusion, trypsininhibitory capacity measurements and from cellulose acetate electrophoresis. Good correlation was found between the phenotype and the three biochemical methods for demonstrating the normal phenotype MM and the severe deficiency state ZZ. Some discrepancies were observed for the heterozygotes. It is concluded that these various assays are inaccurate for the interpretation of intermediate AAT concentrations. Since Pi typing is not suitable for use on a routine basis, it is suggested that this analysis should be performed by a specialized laboratory if intermediate deficiency is suspected.     

Apport de l’exploration cytogénétique et ultrastructurale dans le pronostic de fertilité des sujets globozoospermiques

Globozoospermia is a severe form of teratozoospermia characterized by round-headed sperms with absence or presence of a rudimentary acrosome. The objective of this study is to analyze sperm from six patients with globozoospermia syndrome and report the results of 11 intracytoplasmic sperm injection (ICSI) attempts. The investigation of these issues was carried out by studying the sperm aneuploidy rate by fluorescent in situ hybridization (sperm-FISH) for chromosomes X, Yand 18. The rate of DNA fragmentation was studied by using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique and a detailed ultrastructural morphology study of the sperm using transmission electron microscopy. Eleven ICSI attempts were performed in patients with low fertilization rate, (9.37%) and pregnancy did not occur. This study confirmed the variability of sperm phenotypes observed in this syndrome and the low fertilization rates after IVF-ICSI regardless of the phenotype.     

Cinétique et caractérisation du phénomène “apoptosis-like” induit au cours de la congélation des spermatozoïdes bovins

Cryopreservation of ejaculated bovine spermatozoa induces severe cell death. In a preliminary study, we observed that cryopreservation and/or thawing was also associated with early apoptotic features in living spermatozoa: i) decrease of the mitochondrial membrane potential^αψm), ii) caspase activation, iii) increase of membrane permeability, without the appearance of late characteristics: iv) no DNA fragmentation. This process has been called “apoptosis-like”. In this study, we evaluated the consequences of each step of cryopreservation of bovine spermatozoa: dilution in cryopreservation medium, equilibration and cryopreservation in liquid nitrogen/thawing. Apoptosis and acrosomal reaction were analysed by flow cytometry and the presence of AIF (Apoptosis Inducing Factor) was analysed by western blot. We observed that dilution in cryopreservation medium induced a marked and immediate increase of the proportion of living spermatozoa with a low^Δψm. After equilibration, the proportion of living spermatozoa with active caspases then began to increase. After the complete cryopreservation/thawing process, this population reached a maximum, and a significant increase of membrane permeability was observed. These results, showing that some features of the “apoptosis-like” phenomenon are initiated in the early steps of cryopreservation, suggest that ice formation may not be the only factor affecting spermatozoa. The consequence of this^Δψm decrease could be the release and/or activation of various pro-apoptotic factors in the cytoplasm. Presence of the pro-apoptotic AIF factor in bovine spermatozoa suggests a possible role of this protein during the cryopreservation process. We also confirmed that cryopreservation of bovine spermatozoa induced an acrosomal reaction. It would be of interest to investigate the relationship between this acrosomal reaction and membrane permeability. A better understanding of the cellular mechanisms involved in sperm cryopreservation would help to improve the preservation of bovine sperm.     

Composition lipidique membranaire durant la préparation de spermatozoïdes à la fécondation

The final modifications that the spermatozoa undergo correspond with the destabilization of their plasma membrane. This indispensable step facilitates the fusion of membranes and primes the signal transduction during fertilization. This destabilization is composed of a series of changes and modulation of the lipids in membranes such as cholestérol, phospholipids and glycolipids. Several differences exist in the lipid composition of the plasma, acrosome, nuclear and mitochondrial membranes of spermatozoa. The principal membrane phospholipids are phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin. Plasma membrane of sperm is also rich in polyunsaturated fatty acids (PUFA) linked to phospholipids. Such as C₁₈∶2n?6, C₂₀∶4n?6 and large amounts of docosahexaenoic acid (C₂₂∶6n?6). The amount of membrane lipids in human sperm varies considerably between patients. This variation, could influence certain functional properties of the sperm cells such as their ability to undergo capacitation, the acrosome reaction and the fusion between sperm and oocyte membranes. The lipid composition of the human sperm cell can be altered during the process of freezing-thawing. A significant decrease in phospholipids (phosphatidyl choline, phosphatidyl ethanolamine), and PUFA in particular docosahexaenoic acid and arachidonic acid was observed. Human spermatozoa have a molar cholestérol/phopholipid ratio ≤1.0, and reduces during capacitation due to loss of cholestérol. In addition, the decrease in the levels of cholestérol and the methylation of phospholipids is involved in the modification of membrane fluidity and in the maturation of the sperm plasma membrane receptors. Therefore it seems that the methylation is important for the fusion between sperm and oocyte membranes. Intrinsic sperm phospholipase A2 also plays a role in the destabilization of the plasma membrane by producing of lysophospholipid. Therefore this enzyme and free fatty acids are believed to play a role in the acrosome reaction, an indispensable event facilitating the fusion between sperm and oocyte membranes.     

Altérations de la spermiogenèse de Locusta migratoria migratorioïdes après implantation de corps allates et après irradiation

Résumé Une faible dose de rayonnement gamma appliquée à des larves de Locusta migratoria provoque des altérations de la spermiogenèse et entraine la stérilité des mâles. Ces résultats sont comparables à ceux obtenus après implantation des corps allates.Au niveau des organites cellulaires, les altérations morphologiques des spermatides sont semblables dans les deux cas: vacuolisation des mitochondries et transformation du Nebenkern, pycnoses nucléaires, multiplication centriolaire, invagination de la membrane plasmique. Certaines de ces altérations se manifestent tardivement, au moment de la fixation des spermatides sur les cellules pariétales. Celles-ci n'élaborent pas les matériaux de la coiffe des spermatodesmes: leur fonction physiologique est donc également perturbée.Le rapport entre cette altération physiologique et l'évolution anormale des spermatides est envisagé. Le mode d'action de l'hormone juvénile sur la gonade est également discuté.

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Alterations in spermiogenesis of Locusta migratoria migratorioïdes after corpora allata implantation and after irradiation

Summary Low doses of gamma-rays, given to larvae of Locusta migratoria, lead to alteration of spermiogenesis and male sterility. These results are similar to those which follow corpora allata implantation.At cellular level, morphological alterations in spermatids are the same in both treatments: mitochondrial vacuolization, nuclear pycnosis, centriole multiplication, plasmic membrane invagination. Some of these alterations reveal themselves late, at the time the spermatids fix on the parietal cells. These don't elaborate the substances of the spermatodesm's cap: their physiological functions are therefore also disturbed.The relationship beetween this physiological disturbance and abnormal evolution of spermatid are considered and the way in which juvenile hormone acts upon gonads is discussed.

     

Apoptose et ségrégation méiotique dans les spermatozoïdes d'hommes porteurs de translocations

Men with a chromosomal translocation produce a significant percentage of unbalanced spermatozoa. In order to determine a correlation between chromosomal anomalies and apoptosis in human sperm, we analysed DNA fragmentation and meiotic segregation in sperm from men with a (13;14) Robertsonian translocation. We studied sperm from 12 (13;14) translocation carriers and 9 proven fertile men with a normal karyotype. Meiotic segregation of chromosomes 13 and 14 was analysed using dual-colour fluorescencein situ hybridization with locus-specific probes for chromosomes 13 and 14. Apoptosis in spermatozoa was measured byin situ TUNEL assay. The meiotic segregation study showed a significantly increased frequency of unbalanced spermatozoa for chromosomes 13 and 14 in (13;14) carriers (15.9%) compared to the control population (1.3%) (p=0.00016). The study of apoptosis showed an increase of DNA fragmentation in (13;14) carriers (34.9%) compared to the control population (13.8%) (p=0.0036). This increased apoptosis was observed in spermatozoa presenting an increase of unbalanced chromosomal anomalies concerning chromosomes 13 and 14, but with a predominance of balanced spermatozoa compared to the theoretical risk of meiotic segregation. These results suggest that apoptosis could be involved as a regulatory mechanism to eliminate unbalanced chromosomal spermatozoa in men with a (13;14) Robertsonian translocation.     

Étude en microscopie électronique et par cytophotométrie à balayage de la structure et de la distribution de la chromatine dans les noyaux des cellules cartilagineuses deTriturus cristatus âgés au cours de la régénération

Résumé Les cellules cartilagineuses des membres postérieurs deTriturus cristatus en régénération après amputation, ont été étudiées en microscopie électronique et par cytophotométrie à balayage. Nous nous sommes intéressés à la structure et à la distribution de la chromatine mais aussi à différents organites cytoplasmiques. Dans l'étude de cytophotométrie à balayage, la chromatine a été considérée à travers son constituant majeur, l'ADN, coloré par la réaction de Feulgen. Au cours de la régénération du membre, l'hétérochromatine initialement condensée, essentiellement accolée à la membrane nucléaire se décondense. Les vacuoles du cytoplasme, caractéristiques des animaux âgés par rapport aux animaux jeunes, disparaissent, les mitochondries et le reticulum endoplasmique rugueux deviennent plus abondants. Les caractéristiques nucléaires de l'activation cellulaire apparaissent précocement, précédent les modifications cytoplasmiques et conduisent à des cellules en tous points identiques aux cellules d'animaux jeunes en dehors de tout processus régénératif. Cette phase d'euchromatisation et de restructuration cytoplasmique est peut-être nécessaire à l'accroissement d'activité métabolique et à la division cellulaire qui suivent. Son déroulement peut expliquer tout au moins le ralentissement de la régénération observé chez les animaux âgés par rapport aux animaux jeunes.

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Electron microscopic and scanning cytophotometric study of chromatin structure and distribution in nuclei of cartilaginous cells of agedTriturus cristatus during regeneration

Summary Cartilaginous cells of aged newts (Triturus cristatus) were studied during hind limb regeneration. The electron microscope was used to study the structure and distribution of chromatin in the cell nuclei, while the DNA content of the chromatin was measured by means of a scanning cytophotometer.Changes in the ultrastructure of the cytoplasm during regeneration were also studied.It was observed that the structure and distribution of chromatin in the activated cell is greatly modified. In the non-activated cell of the aged newt, the chromatin is found highly condensed and distributed peripherally close to the nuclear membrane. In contrast, in the activated cells, the chromatin is much less condensed and is distributed throughout the nucleus. Moreover, cytoplasmic vacuoles, found only in the non-activated aged cells, disappear and an increase in the mitochondria and rough endoplasmic reticulum is also observed.Changes in the nuclear structure are observed prior to the cytoplasmic modifications.It is interesting to note that the process of activation induces structural changes in the aged cells which make these cells appear to be structurally identical to the young cells. This process of rejuvenation takes 3–5 days in the newt.We suggest that these structural changes of the chromatin and cytoplasm in the aged cells are necessary to increase the metabolic activity which precedes cell division. It may also explain why regeneration takes a longer time in the aged animals than in the young ones.

     

Comparaison de différentes méthodes de segmentation en TEP/TDM pour le ciblage en radiothérapie des carcinomes épidermoïdes des VADS

IntroductionTo assess interobserver variability for biological target volume (BTV) delineation and to compare the reproductibility of different semiautomatic segmentation methods in pretreatment 18-fluorodeoxyglucose positron emission tomography (PET/CT) of head and neck squamous cell carcinoma (HNSCC).Patients and methodsPatients with histologically proved HNSCC referred to the nuclear medicine service in Brest for pretreatment PET/CT were prospectively included from February 2009 to June 2010. Three nuclear medicine physicians (two specialized in oncology) delineated manually and independently BTV on each primary tumor. Four semiautomatic segmentation methods have been studied; three using a fixed threshold and one applying an adaptive threshold based on the signal-to-background ratio (Daisne). The variability between κ observers and/or methods has been assessed. The concordance between the various BTV intersections and unions has been also assessed.ResultsThirty patients (29M; 1F) were included. The primary site location was oropharynx in six patients, oral cavity in 10 patients, hypopharynx in five patients and larynx in nine patients. A statistically significant global interobserver variability (P = 0.01) was showed, but without statistically difference between the two experienced oncologists (P = 0.15). The maximal concordance of the two experienced observers with the semiautomatic methods was found for the Daisne method (CI = 61.5%; κ = 0.68), expressing a good agreement according to the Landis and Koch criteria, better than with the segmentation method using a fixed threshold with 40% of maximal signal intensity (CI = 52.1%; κ = 0.53).ConclusionOur results suggest the feasibility of achieving HNSCC BTV delineation by PET/CT using semiautomatic methods, in particular those which apply an adaptative threshold but under the supervision of an experienced operator.     

Présence en Guadeloupe de deux phénotypes femelles du copépode ascidicole Pachypygus macer Illg, 1958

Resumé Deux formes femelles différentes de Pachypygus macer sont décrites des ascidies Styela partita et Microcosmus exasperatus, récoltées dans un port de Guadeloupe. L'auteur suggère que ces deux formes sont des phénotypes dérivés de parents communs qui auraient été introduits en Guadeloupe par un navire entre 1974 et 1980. Styela partita semble avoir une influence régressive sur le copépode. Ce phénomène est discuté en relation avec l'existence de deux phénotypes mâles chez l'espèce européenne Pachypygus gibber.

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Summary Two distinctive female forms of Pachypygus macer are described from the ascidians Styela partita and Microcosmus exasperatus, collected from a harbour in Guadeloupe. The author suggests that these two forms are phenotypes derived from common parents that could have been introduced into Guadeloupe by a ship between 1974 and 1980. S. partita appears to have a regressive influence upon the copepod. This phenomenon is discussed in relation to the existence of two male phenotypes in a European species, Pachypygus gibber. ac]19850412

     

Estimation du taux d’aneuploïdies des spermatozoïdes épididymaires prélevés en vue d’ICSI chez des hommes à caryotype normal

Over the last ten years, fluorescent in situ hybridization in decondensed sperm nuclei has been used to study the chromosomal constitution of human spermatozoa. Studies have estimated that the disomy rate per chromosomal pairs is between 0.15% and 0.3%. The aim of this study was to evaluate the aneuploidy rate of human epididymal spermatozoa extracted from five men with obstructive azoospermia undergoing IVF. Genetic studies (karyotypes, Y micodeletion syndrome and mutation of the CFTR gene) did not reveal any abnormality. Disomy frequencies were determined by X-Y-8 multicolour fluorescence in situ hybridisation on 18,013 epididymal spermatozoa and 20,000 spermatozoa from healthy donors (control group). No significant difference was found between epididymal and ejaculated samples. However, isolated non-significant differences were observed between one of the patients and the control group. In conclusion, the present findings suggests that there is no increased risk for de novo chromosomal aberrations after IVF therapy with epididymal spermatozoa of men with obstructive azoospermia.     

Innervation de l'hypophyse intermédiaire deRana esculenta,et identification des fibres aminergiques par autoradiographie au microscope électronique

Résumé L'hypophyse intermédiaire deRana esculenta, se compose de deux zones distinctes: une zone externe formée de cellules glandulaires d'aspect normal, une zone interne constituée de cellules à globules.Son innervation est assurée par un faisceau d'axones d'origine hypothalamique qui se subdivise dans l'angle rostro-ventral en deux groupes de fibres distinctes. Le premier groupe se termine sur les capillaires de cette zone, les fibres du deuxième groupe se terminent au contact des cellules glandulaires. Elles sont nombreuses dans la zone interne, moins abondantes dans la zone externe. D'après leurs caractères morphologiques et cytologiques, on distingue 3 types de fibres: des axones neurosécréteurs à grains d'environ 1000 à 1800 Å de diamètre (type 1), des terminaisons à grains de 550 à 950 Å de diamètre (type 2) et des terminaisons pratiquement dépourvues de grains (type 3).Après l'injection de DL noradrénaline H³ et étude autoradiographique au microscope électronique, les terminaisons de type 2 à fines granulations se marquent intensément tandis que les fibres du type 1 et du type 3 ne présentent pas de marquage. Cette méthode met en évidence les fibres aminergiques dont le rôle est discuté.

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Innervation of thePars intermedia ofRana esculenta and identification of aminergic fibres by autoradiography in electron microscopy

Summary In thepars intermedia ofRana esculenta, two zones may be distinguished: an external zone with normal glandular cells and an internal zone with large globular cells.The innervation is assumed by a hypothalamic nervous tract which divides in two distinct bundles: The first group of terminals innervates the capillaries in the rostro-ventral corner of thepars intermedia. The second one penetrates the glandular parenchyma where they are numerous in the internal zone and less numerous in the external zone.On the basis of their morphological and cytological characteristics three kinds of fibres are distinguished: neurosecretory axones with granules of about 1,000 to 1,800 Å in diameter (type 1) terminals with fine granules of 550 to 950 Å, (type 2) and terminals without granules (type 3).After injection of DL noradrenaline H³ and autoradiography on electron microscopy, the type 2 terminals with fine granules are strongly labeled while type 1 and type 3 fibres are devoid of labeling. The fixation of DL noradrenaline H³ demonstrates the aminergic fibres the function of which is discussed.

Avec la collaboration technique de Mme Y. Rohr et de M. C. Chevalier.