Alcaloïdes des feuilles de Holarrhena febrifuga

Leaves of Holarrhena febrifuga from East Africa contain 1·5% of alkaloids, mostly of the amino-3α ceto-20 pregnane-5α type. Six alkaloids have been isolated: funtumine (I), holamine (II), dimethyl- and monomethyl-funtumine (Ia) and (Ib), dimethyl- and monomethylholamine (IIa) and (IIb); these methyl derivatives have been isolated for the first time from natural source. Pregnane-3,20-dione has been isolated from the same leaves.     

Indications de la recherche des anticorps anti-spermatozoïdes

The clinical significance of antisperm antibodies (ASA) is highly controversial. A significant percentage of infertile men and women present immunity to spermatozoa, suggesting that ASA may interfere with the fertilizing capacity. ASA can act negatively on sperm parameters, sperm-cervical mucus interaction, gamete fusion and possibly also on the first step of embryonic development. ASA are present in approximately 2.8% to 26% of the male population and 0.2% to 1.6% of women. The pathogenesis of immunity to spermatozoa had not been fully elucidated: breakdown of normal protective mechanisms, i.e. blood-testis barrier, or epithelial barrier in women, and other mechanisms of immunological sperm tolerance, such as regulation of suppressor T lymphocytes. The indication for antisperm antibody testing is based on clinical and laboratory findings of infertile patients. In men, indictions for ASA testing include a history of genital disease, surgery for genital abnormalities, vasectomy, obstruction or injuries of the male genital tract, infection of accessory glands, long-standing infertility, alteration of semen parameters (agglutination, motility), mucus penetration, and reduced fertilizing capacity in IVF. In many cases, no etiological cause of autoimmunity is found and a genetic predisposition has been suggested. A majority of women do not develop antisperm antibodies, despite repeated contact with spermatozoa during their sexual life. Upper genital tract infection is the main cause of isoimmunization in females, although sexual practices, endometriosis, surgery for cervical neoplasia, recurrent spontaneous abortion and long-term infertility may also be involved. Sperm-cervical mucus impairment is the most obvious effect of immunization in women associated with IVF failure. Autoantibodies are frequently associated with antisperm antibodies. One of the consequences of the success of ICSI has been a decreased research effort to further the understanding of the origin and relevance of antisperm antibodies and specific antibody-antigen interactions. A better understanding of the natural history of immunological infertility would be useful for patient conseiling and to develop the most effective, efficient and safest management strategies. Such data could also be useful for the development of new tests and immunological methods of male contraception.     

Les flavonoïdes de Doronicum grandiflorum

After isolation and identification of the flavonoid compounds of D. grandiflorum Lam., a comparison of the flavonoid patterns of four species of Doronicum was undertaken. The differences observed are discussed from the evolutionary point of view.     

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.     

Alcaloïdes de Zanthoxylum tsihanimposa

From the bark of Zanthoxylum tsihanimposa (Rutaceae) 5 furoquinoline and benzophenanthridine alkaloids were isolated. Three are known compounds: γ-fagarine, skimmianine and dihydrochelerythrinylacetone. The other two, dihydrochelerythrinylacetaldehyde and O-methyldihydrochelerythrinyl-acetone, are probably artefacts.     

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     

Morphologie des spermatozoïdes

Spermatozoa morphology is one of the qualitative characteristics of spermatogenesis. However, because of both the variations in the definition of normal morphology and the existence of different kinds of sperm abnormalities as well as the use of various techniques of morphology assessment, such a parameter is poorly used in usual laboratory work. Morphological sperm anomalies can be from testicular or post-testicular origines, while the latter is still unproved. The causes of such anomalies are either from genetic origines, but in these cases any spermatozoa demonstrate this anomaly, or due to an endogenous factor with varicocele the most usually quoted but unproved pathology, But exogenous factors, either chemical such as drugs and pesticides or physical such as heat, are also responsible for morphological sperm anomalies. Analysis of sperm morphology is indicative of both the testicular health status (in cases of occupational exposure to chemical or physical toxics) and the fertility potential since morphology is correlated to sperm motility and involved in fertilization through the acrosome reaction.     

Les constituants flavonoïdes de Cephalanthus spathelliferus

We have isolated from Cephalanthus spathelliferus (Rubiaceae) five compounds: umbelliferone, skimmin, 7,4′-dimethylkaempferol,7,4′-dimethylaromadendrin and its 5-glucoside. The latter two are new compounds.     

Evaluation morphologique des spermatozoïdes

Semen analysis, as defined by World Health Organisation (WHO), is a fundamental step in the work-up of infertile couples. Spermatozoa morphology has been recognised as the best predictive factor in natural fertility, and in intrauterine insemination and classicalin vitro fertilisation. Ultrastructural spermatozoa abnormalities are the only sperm alterations likely to influence the outcome of ICSI. However, these abnormalities cannot be detected by conventional microscopy (¥100 or ¥200–400). Bartoov et al. (2002) developed a real-time spermatozoa observation system using a ¥6600 magnification called MSOME (Magnification Motile Sperm Morphology Examination). Spermatozoa abnormalities detected with this technique are vacuoles localised on spermatozoa heads with variable number, size and site (nucleus or acrosome). Spermatozoa evaluation using MSOME could be performed to predict the probability of fertilisation by these spermatozoa either spontaneously or after assisted reproductive technology.     

Controle de la multiplication et de la polyploïdie des hepatocytes du rat

Résumé L'injection de substances irritantes de nature chimique variée perturbe les mécanismes qui contrÔlent la multiplication des hépatocytes du rat.Les très jeunes rats réagissent de faÇon intense et reproductible. 15 heures après l'injection il se produit une augmentation brève et intense du nombre des cellules entrant en phase S de sorte que les divisions cellulaires, qui normalement auraient été réparties sur les 30 ou 40 prochaines heures, débutent en l'espace de 2 heures seulement. La croissance hépatique a pris une importante avance qui est suivie par une période de diminution du nombre des mitoses due à une rétroaction inhibitrice.La réaction des rats plus âgés met en évidence un mécanisme inhibiteur permanent qui entraÎne une réponse moins intense et moins régulière.La production d'hépatocytes binucléés et polyploÏdes parait liée à ce mécanisme inhibiteur.La régularité et la sensibilité des réactions des très jeunes rats en fait un matériel de choix pour l'étude des mécanismes régulateurs de la croissance hépatique.

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Control of multiplication and polyploidy of rat hepatocytesA study of the perturbations of physiological regulation induced by injection of irritants

Summary The injection of irritating substances of various chemical nature disturbs the mechanisms regulating the multiplication of rat hepatocytes.The reaction of baby rats is intense and reproducible. It results, 15 hours after the injection, in an increase of the number of cells entering the S phase, so that practically all the divisions which would normally have been scattered over 36 hours are initiated within 2 hours. This burst of synchronized mitosis corresponding to a 36 hours advance in the hepatic growth is followed by a decrease of mitoses due to a feed-back inhibition.The reaction of older rats brings to evidence a permanent inhibitory mechanism that results in a less regular and intensive response.The production of binucleated and polyploÏd hepatocytes seems to be correlated to this inhibitory mechanism.The regularity and sensivity of the baby rat reactions make them a choice material for the study of hepatic growth.

     

En France, la majorité des donneurs de spermatozoïdes souhaite le maintien de leur anonymat

So far in France, sperm donor anonymity, which was a fundamental principle and has been twice confirmed in the law in 1994 and 2004, is debated nowadays. In this context, the Cecos wanted to know the donors opinion on anonymity. In 2006, 193 semen donors recruited in 14 Cecos answered anonymously a questionnaire: 73% were in agreement with the principle of anonymity and less than 30% agreed that the future law should change to allow the children to know the donor identity. In case of anonymity disclosure, 60% would give up their sperm donation. The same proportion of donors would accept that non identifying information on them could be given on request to the parents and the child.     

Anticorps anti-spermatozoïdes: Revue de la littérature

Auto immune infertility represents about 15% of all sterilities. The most commun mechanisms of autoimmunity in males are genitals infections (epidymitis) and traumatisms (vasectomies, testiculat biopsies). Mixt agglutination reaction test and immunobead test are the most commun tests used for detection of antibodies. Since 1981, researchs tried to characterize spermatozoa antigens by using polyclonal and monoclonal antibodies. More than 30 antigens had been found during this last decade. Naz and coll had identified an antigen called FA 1 antigen. FA 1 Ag is a 23 Kd glycoprotein. Anti FA1 antibodies inhibit binding and penetration of sperm to zona intact mice oocytes and also zona free hamster oocytes. They do so without causing agglutination or immobilization of sperm and by the way they cannot be detected by usual tests.     

Les corps myeloïdes de l'epithelium pigmentaire rétinien

Résumé Les corps myéloïdes de l'épithélium pigmentaire rétinien ont été observés après différentes techniques de fixation, déshydratation, inclusion et coloration. Après fixation classique par le glutaraldéhyde et l'osmium, l'espace intersacculaire apparaît réduit (jonctions) et une ligne dense est parfois observable dans l'espace intrasacculaire. La fixation par le permanganate met en évidence de façon permanente cette ligne dense qui correspondrait à la fusion des membranes. La coloration négative caractérise un constituant protéique entre les saccules. La déshydratation par le Durcupan hydrosoluble met en évidence un matériel lipidique intrasacculaire qui est extrait par le méthanol-chloroforme et partiellement par la déshydratation alcoolique classique.Les corps myéloïdes apparaissent plusieurs jours avant l'éclosion chez l'oiseau. Ils se forment à partir du réticulum granulaire. Ils ne peuvent être confondus avec les dictyosomes Golgiens : Ils ne contiennent pas de glycoprotéines.

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Summary Myeloid bodies of the retinal pigment epithelium of different vertebrates were investigated by various techniques of fixation, dehydration, embedding and staining. After classical fixation with buffered glutaraldehyde and osmium, the intersaccular space is narrow (junction complexes) and a dense line is sometimes visible in the middle of the intrasaccular space. With permanganate fixative this dense line is always visible; it represents probably the fusion of the unit-membranes. The negative staining demonstrates the presence of proteins between the saccules. Dehydration by water soluble Durcupan shows a lipidic component in the intrasaccular space, which is removed by methanol-chloroform and partly by the classical ethanol dehydration.The existence of myeloid bodies is demonstrated in the bird-embryo a few days before birth. They originate from granular reticulum cisternae. They are not of Golgi origin: glycoprotein cannot been demonstrated at their level.

Chargé de Recherche à l'I.N.S.E.R.M. (contrat n° 711.152). Avec la collaboration technique de L. Salin et D. Joseph.     

La vitalité des spermatozoïdes

Most of the numerous techniques used to assess sperm viability only have research applications, while only two classical tests, i.e. eosin-Y and hypo-osmotic swelling test (HOST), are currently used in routine sperm analysis to determine the percentage of viable sperm. A viability rate below 50% of living sperm defines necrozoospermia, a condition whose clinical significance is fairly difficult to assess as the mechanisms of sperm cell death are still poorly understood. However, even when a precise cause for necrozoospermia cannot be identified, abnormal viability requires further andrological investigations with particular emphasis on clinical and laboratory signs of chronic infection of the male reproductive tract. Intracytoplasmic sperm injection (ICSI) can yield very good pregnancy rates, even in couples with the most severe forms of male infertility. However, when no motile sperm are available after sperm preparation, the outcome of ICSI is seriously impaired, probably because of a high risk of injecting dead sperm. In these patients, sperm viability could therefore be assessed by the hypo-osmotic swelling test in order to select only viable sperm for ICSI. However, the long incubation time of sperm in the hypo-osmotic solution, as recommended in the classical HOST procedure, has been shown to be detrimental to the spermatozoa. A single sperm test able to assess the viability of each individual spermatozoon within microdroplets covered by mineral oil therefore seems to be preferable. This selection procedure is less suitable in the case of immotile frozen-thawed sperm, as viability does not appear to be reliably predicted by HOST in cryopreserved sperm. Examination of sperm viability now also evaluates programmed cell death or apoptosis, as apoptotic alterations can be detected in spermatozoa by several techniques. The percentage of apoptotic sperm is correlated with deficient sperm parameters and poor outcome of assisted reproductive techniques. More effective selection procedures are therefore needed in order to identify spermatozoa not only with intact membranes but also with an intact genome to be used for ICSI.     

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.     

Étude complémentaire de la structure de trois galactoxyloglucanes (amyloïdes) de graines

D-Galacto-D-xylo-D-glucans (amyloids) from Balsamina, Tropaeolum, and Tamarindus seeds behave in a similar manner in the presence of various glycosidase preparations: slow depolymerization by enzymes from several germinated or non-germinated seeds, and hydrolysis into monosaccharides and oligosaccharides by commercial cellulase and hemicellulase preparations from fungi. A purified cellulase from Penicillium notatum gave a dialyzable fraction almost exclusively composed of α-D-xylopyranosyl-(1→6)-D-glucose residues and a nondialyzable fraction composed of chains of β-D-(1→4)[withsome (1→3)]-glucopyranosyl residues; β-D-galacto-pyranosyl-(1→2)-α-D-xylosyl groups are linked to some of the β-D-glucosyl residues at 0-6. The presence of (1→3)-linkages in the D-glucan chain of the Balsamina was verified by methylation and sequential periodate oxidation-borohydride reduction; the distribution of the substituents on the D-glucan chain is not regular. The main D-glucan backbone, where the β-D-glucosyl residues are partly linked at 0-6 to β-D-galactosyl-(1→2)-D-xylosyl groups, is linked to D-glucan chains where almost all the D-glucose units are linked at 0-6 by one α-D-xylosyl group. The presence of 3,6-di-O-methyl-D-glucose after permethylation and hydrolysis suggests that the xyloglucan chains are linked to 0-2 of the D-glucosyl units of the galactoxyloglucan backbone.     

Chimiotaxonomie des lycopodiales: distribution des alcaloïdes

The distribution of lysine-derived alkaloids in the genus Lycopodium (s.l.) supports the separation of the following taxa, Huperzia (= Urostachys), Lycopodiella s.l. (= Lepidotis, excl. L. volubile and L. deuterodensum) from Lycopodium s. str. Within the latter, the Fastigiatum group and the Complanata section (= Diphasiastrum) can be distinguished. The results obtained are in good agreement with the classification proposed by Wilce.