[Development and regulations of testicular functions in the human foetus]. / Ontogenèse et régulations des fonctions testiculaires chez le foetus humain.

Two major functions are assumed by the testis: the production of male gametes (that is, spermatozoa) and the production of steroid hormones. Both two functions are established during fetal life and are essential to the adult fertility and the masculinization of the internal tract and genitalia. For many years, our laboratory has been interested in the ontogeny of those two functions in rodents and, since 2003, in collaboration with gynecology and obstetrics service of professor R. Frydman in Antoine-Béclère hospital, we have studied them in human. The first aim of this work was to improve the global knowledge of the human fetal testis development by using both our experimental data and the literature. Then, we focused on the different defects that can occur during the fetal testis development. Indeed, male reproductive abnormalities have been steadily increasing since the last decades and are thought to be related to the concomitant increase of the concentration of contaminants and particularly of endocrine disruptors in the environment. Thus, we decided to study the effect of endocrine disruptors on human fetal testis and, more particularly, the effect of phthalates, by using an organ culture system developed for human. In contrast to the data obtained in rat, mono (ethylhexyl)-phthalate (MEHP), an active metabolite of the most widespread phthalate in the environment, does not disturb the steroidogenic function. On the other hand, it has a negative effect on the male germ cells number. This study is the first experimental demonstration of a negative effect of phthalates directly on human fetal testis.

Spatio-developmental distribution of the prion-like protein doppel in Mammalian testis: a comparative analysis focusing on its presence in the acrosome of spermatids.

The first prion-like protein doppel, officially designed as prion protein dublet, does not seem to be needed for prion disease progression, whereas its physiological function seems to be related to male fertility. Its expression is primarily detected in the male genital tract, and Prnd-inactivated male mice are sterile. We investigated the location of Doppel in the testis of various species of mammal to determine its physiological function. Doppel is expressed early during ontogenesis, and is found in both germ cells and Sertoli cells in mice, rats, boars, and humans. Doppel is permanently expressed in the Sertoli cells but at different levels according to species. Its expression in testicular germ cells was primarily detected in spermatids, with a transient presence in the acrosome. These data suggest that Doppel may play a physiological role in acrosome biogenesis and may be of use in studies of patients suffering from idiopathic infertility.

[Viral transmission and medically assisted procreation: the Herpesviridae case]. / Transmission virale et assistance médicale à la procréation: le cas des Herpesviridae.

Procreation with sperm donation is at present achieved by insemination either in the uterus or in vitro, always from ejaculated and washed spermatozoa. Then, the infectious risk only exists if the donor sperm is capable of transporting the virus or its DNA, either by adhesion or by integration. With CMV, HSV1 and HSV2, medically assisted procreation in couples (AI or IVF-ET) does not increase the risk of viral contamination as compared with natural procreation, except possibly the cases of surgical procedure to pick up testicular sperm to be used in ICSI. Animal experiments show that, even if viral material is introduced in the oocyte, it may be eliminated from the embryo, at least for CMV.

Experimental inoculation of male mice with murine cytomegalovirus and effect on offspring.

BACKGROUND: Murine cytomegalovirus (MCMV) was used to examine aspects of viral infection in male mice, and its possible transmission to their offspring. METHODS AND RESULTS: FVB/N mice inoculated intratesticularly with 5x10(5) plaque forming units (PFU) of MCMV, developed peritoneal haemorrhagic exudates, spleen hypertrophy and acute local infection. Infectiousness was detected until 15 days post-inoculation (D15 PI) in the genital organs, and virus DNA up to D35 PI. Testicular endothelial and Leydig cells were infected, and peritubular cells severely damaged. Spermatogenesis was affected, but neither germ cells nor Sertoli cells were infected. No virus was found in the epididymal epithelial cells. Viral DNA was detected in cells extracted from vas deferens samples until D15 PI. Neither infectious virus nor viral DNA were found in spermatozoa recovered from uterine fluid, fertilized oocytes, blastocysts, fetal tissues or newborn animals following the mating of infected males with uninfected females. CONCLUSIONS: MCMV harboured in the male genital organs was not transmitted to their offspring, even when mating occurred during the acute phase of CMV disease. Although the infection may have had an impact on spermatogenesis, fertility was not affected. These results do not support the hypothesis of conceptus MCMV infection by the fertilizing spermatozoon in natural conception.

Cytoplasmic and nuclear phospholipase C-beta 1 relocation: role in resumption of meiosis in the mouse oocyte.

The location of the phospholipase C beta 1-isoform (PLC-beta 1) in the mouse oocyte and its role in the resumption of meiosis were examined. We used specific monoclonal antibodies to monitor the in vitro dynamics of the subcellular distribution of the enzyme from the release of the oocyte from the follicle until breakdown of the germinal vesicle (GVBD) by Western blotting, electron microscope immunohistochemistry, and confocal microscope immunofluorescence. PLC-beta 1 became relocated to the oocyte cortex and the nucleoplasm during the G2/M transition, mainly in the hour preceding GVBD. The enzyme was a 150-kDa protein, corresponding to PLC-beta 1a. Its synthesis in the cytoplasm increased during this period, and it accumulated in the nucleoplasm. GVBD was dramatically inhibited by the microinjection of anti-PLC-beta1 monoclonal antibody into the germinal vesicle (GV) only when this accumulation was at its maximum. In contrast, PLC-gamma 1 was absent from the GV from the time of release from the follicle until 1 h later, and microinjection of anti-PLC-gamma 1 into the GV did not affect GVBD. Our results demonstrate a relationship between the relocation of PLC-beta 1 and its role in the first step of meiosis.

Calmodulin, gametes and fertilisation.

The role of calmodulin in fertilisation events was examined in a zona-free mouse system by using a selective calmodulin inhibitor, calmidazolium (1 microM). The effects of this antagonist were studied either on the ooplasmic calcium oscillations induced by fertilisation by using the Ca2+ indicator, fluo-3/AM, or on pronucleus formation 4 h later by using the nucleic acid stain, Syto-15. When the calmidazolium treatment was applied to one or the other gamete before insemination, the fertilisation process was affected only when spermatozoa were treated: most of the oocytes were partially fertilised as demonstrated by the profile of Ca2+ oscillations and the presence of polar bodies with no typical male and female pronuclei. When the treatment was applied during insemination, more than half the oocytes were unfertilised and only a few were partially fertilised. These results demonstrate that: (1) the calmodulin-dependent events taking place in spermatozoa before insemination appear essential at least for regular Ca2+ oscillations and for pronucleus formation; (2) the inhibition of calmodulin by calmidazolium applied to metaphase II oocytes before insemination has no major impact on their fertilising ability; and (3) at the time of gamete fusion calmodulin, either from the oocyte or from the spermatozoon, is essential for fertilisation to occur.

Effects of cholic acid and 'Protectaid' formulations on human sperm motility and ultrastructure.

Cholic acid (sodium cholate) and the other active ingredients of F-5 gel preparations in use for the impregnation of a new vaginal sponge (Protectaid) with contraceptive and anti-sexually transmitted disease properties, were assessed for their effects on human sperm motility and ultrastructure. Cholic acid (CA) produced an inhibition of motility which was both dose- and time-dependent. A complete suppression of motility was obtained at 30 s by a CA concentration of 1.25%. Nonoxynol-9 (NX9) compared with benzalkonium chloride (BZC) showed no significant difference at the concentration required (0.025%) to give a total inhibition of sperm motility after exposure for 30 s. The addition of F-5A gel containing 0.5% of each one of the spermicide ingredients (CA, NX9 and BZC) produced the total suppression of sperm motility within 30 s at a dilution of 1/50. Another preparation, F-5B gel, containing the spermicide ingredients at different concentrations (1.25% CA, 0.125% NX9 and 0.05% BZC) produced this same effect with a 1/10 dilution. Exposure of semen to a CA concentration of 1.25% or to 1/10 dilutions of F-5A gel for 30 s led to profound changes of sperm ultrastructure studied by scanning (SEM) and transmission (TEM) electron microscopy. SEM and TEM findings indicate that CA acts as a spermicide through its 'natural detergent' properties, damaging the outer plasma membrane of sperm cells. Protectaid formulations affect sperm motility and viability in a similar way.

Calmodulin during human sperm incorporation into hamster oocyte: an immunogold electron microscope study.

In the present study, immunogold labeling of ultrathin sections of human sperm, before and after incorporation into hamster oocyte, was used to obtain insight into the ultrastructural localization and possible function of calmodulin during fertilization. In heads of ejaculated, capacitated, and acrosome-reacted fixed human sperm, calmodulin was mainly found in two compartments, the subacrosomal layer and the postacrosome. After sperm-egg fusion, the subacrosomal calmodulin was unaltered and surrounded by the fertilization cone in which actin was abundant. There was no co-localization of calmodulin and actin. In contrast, postacrosomal calmodulin disappeared as soon as the sperm head was incorporated into egg cytoplasm. These unique localizations and redistributions are in agreement with the concept of a calmodulin targeting from acrosome toward postacrosome through the subacrosomal layer during spermatogenesis (Weinman et al., 1986b: J Histochem Cytochem 34:118). Moreover, they strongly suggest a role for calmodulin both in sperm-egg fusion and in the initial pulse of Ca2+ occurring during fertilization.

Presence and localization of the 60 KD calicin in human spermatozoa presenting postacrosomal sheath defects: preliminary results.

Using an indirect immunofluorescence technique, we studied the anticalicin antibody distribution in four teratozoospermic human sperm samples with a high rate of spermatozoa with incomplete postacrosomal sheath structure (PAS) detected at the ultrastructural level. The calicin distribution obtained in these samples is compared with the distribution obtained in two normal human sperm samples with a high proportion of normal PAS and in 3 human sperm samples without PAS ("round-headed syndrome"). In contrast to the homogeneous calicin distribution found in the majority of the normal group, similar to that reported by Longo et al. (J Cell Biol 105:1105-1120, 1987) and Paranko et al. (Differentiation 38:21-27, 1988), and the absence of calicin in the three non-PAS samples, we observed new patterns of calicin distribution in the four sperm samples studied. In the light of these preliminary results, the use of anticalicin antibody as a tool for human postacrosomal region evaluation is discussed.

Initial stages of sperm--egg interaction in a heterospecific system: fate of the post-acrosomal sheath and appearance of a particular material within the oocyte.

We describe the initial stages of the sperm--egg interaction in the human--hamster heterospecific system. The ultrastructural study reveals that fusion is initiated on both sides of the spermatozoon in the region extending from the anterior part of the post-acrosomal sheath (PAS) to the acrosome equatorial segment (AES). The splitting of the PAS and the appearance of a particular material within the oocyte subsisting until the pronuclear formation are described. The observation of the available electron micrographs suggests that this material may originate from the PAS. The role, the origin and the nature of this material are discussed.

Impaired ability of human spermatozoa to penetrate zona-free hamster oocytes: is a postacrosomal sheath anomaly involved?

We selected 17 infertile men whose sperm ultrastructural study revealed at least 70% of spermatozoa with postacrosomal sheath (PAS) anomalies. Among the other sperm head defects, those affecting the nuclear shape were most frequently encountered and were highly correlated with PAS anomalies (r = +0.71; P less than .01). PAS anomalies were also correlated with chromatin condensation defects (r = +0.67; P less than .01) and acrosome anomalies (r = +0.53; P less than .05). Those spermatozoa were tested for their ability to penetrate zona-free hamster oocytes and were compared to a control sperm population. It was shown that sperm head morphological anomalies impaired the ability of spermatozoa to attach to and penetrate the oocyte. The highest significant and negative correlations were found between the penetration rate and 1) the percentage of spermatozoa with PAS anomalies (r = -0.81; P less than .01) and 2) the percentage of spermatozoa with nuclear shape anomalies (r = -0.66; P less than .01). The effect of PAS anomalies on human fertilization process are discussed.

In vitro fertilization of hamster and human oocytes by microinjection of human sperm.

In this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influence on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 x 10(6) sperm/ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.

Zona-free hamster ovum penetration by human spermatozoa: influence of various sperm factors.

The present paper evaluates the influence of various technical factors when preparing human spermatozoa to be tested for their ability to penetrate zona-free hamster ova. Higher in vitro fertilization rates were obtained after sperm selection by "swim-up" migration, 4-h incubation, sperm dilution to a concentration of 2.5 X 10(6) spermatozoa/ml, and observation of the oocytes 8 h after insemination. The use of control samples was necessary because of wide intraindividual and interindividual variations in the results. Unless the result has been confirmed several times, it is impossible to conclude, from a negative test, that a human spermatozoon cannot penetrate a hamster oocyte.

Effect of migration and capacitation on the nuclear stability of human sperm.

A possible change in the nuclear stability of the human spermatozoa further than ejaculation was investigated. A nuclear chromatin decondensation ability test using 1% SDS + 6 mM EDTA was used on spermatozoa migrated for 1 h in a swim-up migration (in BWW + human serum albumin 0.8%) and capacitated for 5 h in the same medium. The results, analyzed as paired series, showed that (1) capacitated and migrated spermatozoa have a greater nuclear stability than that of the control population (total sperm), (2) there was no significant difference of the nuclear stability between migrated and capacitated spermatozoa, and (3) there was no effect of the media used (BWW + HSA) on the nuclear stability. Thus, it seemed that migrating spermatozoa definitely selects a specific resistant population to decondensing reagents.

[Stages of organogenesis and cytodifferentiation of the albumen gland in the snail Helix aspersa Muller]. / Les stades de l'organogenèse et la cytodifférenciation de la glande à albumen de l'escargot Helix aspersa Müller.

The differentiation of the albumen gland of the pulmonate stylommatophora Helix aspersa has been divided into five stages. An ultrastructural study showed, the differentiation of undifferentiated epithelial cells into two cell types: ciliated cells and secretory cells. The glandular differentiation of epithelial cells was characterized by the development of ergastoplasma and the Golgi apparatus which were both involved in protein and galactogen synthesis.

Intraindividual variability of concanavalin A binding to the plasmalemma of human spermatozoa.

Certain characteristics of the sperm plasmalemma were studied using 10 ejaculates from the same subject. Measurements of membrane glycoconjugates binding by FTC Con A were made on samples with equal numbers of motile spermatozoa which had been selected by migration in phosphate buffered saline (pH 7.4). Scatchard plots showed that the association constant for Con A binding to membrane glycoconjugate varied between ejaculates, but in 9 of 10 cases the number of receptor sites for this lectin was constant with only one class of receptor.