Apoptosis in preimplantation mammalian embryo and genetics.

The presence of dead cells in the preimplantation mammalian embryo has been well described. Since Kerr et al. (1972), it has become apparent that these cells die by apoptosis, a form of programmed cell death. This review analyses the recent morphological and biochemical evidence that apoptosis play a role in early mammalian embryo development. Normal and apoptotic (i.e. fragmented) embryos express several apoptosis-related genes during mammalian preimplantation embryo development, with severe changes when apoptosis is activated; these findings support a model in which mammalian preimplantation embryo development is regulated by the ratio of pro- and -anti- apoptotic genes. Apoptosis may be a normal feature in human preimplantation development, even in vivo, and may play an active role in the developing embryo through the removal of genetically abnormal cells. Contrary to these beneficial effects, apoptosis may have detrimental effects if either the number of apoptotic cells or ratio of these cells to the normal cells is elevated. According to this value, embryos could either continue to develop or arrest.

Transmission risk of hepatitis C virus in assisted reproductive techniques.

Medical assistance for procreation in a couple where one or both parents has hepatitis C viral infection (HCV) raises the issue of the transmission of the infection to the baby and/or of possible contamination of both the technicians and the gametes or embryos from virus-free parents in the laboratory. It becomes essential to assess transmission risk in assisted reproductive techniques in order to define clearly the management of couples according to their viral status. To define the HCV transmissibility risk in assisted reproduction related to the presence of virus in semen from infected infertile men, HCV RNA detection was performed in sera, and semen and sperm fractions obtained after Percoll gradient centrifugation. HCV RNA was detected in 5% (2/39) of the semen samples tested: in the raw semen, in the seminal fluid and in the cell pellet but never after Percoll selection. According to these results, we suggest a strategy for HCV-infected infertile men who need assistance for procreation.

[Apoptosis in the pre-implantation embryo]. / Apoptose dans l'embryon pré-implantatoire.

It has been well shown that apoptosis occurs in mammalian embryos as early as the blastocyst stage, in order to regulate the importance of the inner cell mass. We have looked for apoptosis at the cleavage stage, in human embryos that could not be transferred because of a high degree of fragmentation (grade IV) or a blockage in embryo development. Most of these embryos had blastomeres with condensed or fragmented chromatin, evocating apoptosis. Two markers of programmed cell death, detecting either early (Annexin V) or late (TUNEL technique) apoptosis events, were positive in our study: 100% and 30% of embryos were marked by Annexin V and TUNEL, respectively. Therefore, it seems that apoptosis occurs very early in human embryos conceived in vitro; this could represent a response to suboptimal culture conditions.

Laser scanning confocal imaging of abnormal or arrested human preimplantation embryos.

PURPOSE: The improved resolution and optical sectioning of a confocal microscope make it an ideal instrument for extracting three-dimensional information, especially from extended biological specimens such as human embryos. The staining of actin together with chromatin allowed us to specify the architecture of the embryo and the appearance of the nucleus. METHODS: F-Actin and chromatin distributions were visualized using laser scanning confocal microscopy in "fresh" and "cryopreserved" human preimplantation embryos obtained by in vitro fertilization. RESULTS: The current study revealed a high rate of multinucleation in arrested or poor-quality embryos (89%, in grade IV embryos). CONCLUSIONS: Confocal microscopy revealed high levels of multinucleated blastomeres, suggesting that the probable cause of arrested development in these embryos was due to multinucleation of blastomeres.

Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos.

Confocal laser scanning microscopy was used to observe human arrested and fragmented preimplantation embryos obtained by in-vitro fertilization. Observation of the cellular actin cortex and chromatin showed a high frequency of embryos with blastomeres exhibiting two or more nuclei, while others had nuclei displaying chromatin condensation and fragmentation patterns. Many of the abnormal chromatin images could be due to the process of programmed cell death (apoptosis). The possible link between abnormalities of the blastomeres and apoptosis was investigated using two detection methods for cells undergoing apoptosis. Detection of phosphatidylserine exposure was performed using annexin V; the chromosomal breakdown preceding the nuclear collapse of apoptotic nuclei was tested using the terminal transferase-mediated DNA end labelling (TUNEL) assay. Annexin V staining was observed in all arrested and/or fragmented human embryos, but not in cryopreserved embryos which continued to develop normally after thawing. The TUNEL assay was positive in 30% (15/50) of arrested embryos, all of which had cytoplasmic fragments. In contrast, embryos showing regular size blastomeres without fragments were TUNEL negative.

Applications of laser scanning confocal microscopy to the observation of human oocytes and embryos.

Laser scanning confocal microscopy (LSCM) is a powerful and useful tool in developmental biology. The diverse applications of LSCM in biomedical field has led to advances in the microscopes themselves and the synthesis of novel specific probes for the observation of biological structures and the hypothesis of physiological process. LSCM was used to visualize the cellular actin cortex together with the chromatin in human arrested preimplantation embryos and in unfertilized oocytes obtained by in vitro fertilization. In embryos, we observed a majority of multinucleated blastomeres and some with fragmented nuclei or anucleated. LSCM also showed nuclei displaying the chromatin condensation and fragmentation patterns suggesting the phenomenon of apoptosis. Moreover, in arrested fragmented embryos of poor morphology, unequal sized blastomeres often showed cellular fragments without nuclei, compatible with aspects of apoptotic bodies.

Effect of gastrin-releasing peptide on sperm functions.

Male infertility can be related to defects in motility, capacitation, acrosome reaction, binding and penetration of the zona pellucida. While different in-vitro techniques (such as micromanipulation which is complicated and expensive) are available for the treatment of male infertility, several pharmacological agents have been shown to increase fertilizing capacity under accurate experimental conditions. Gastrin-releasing peptide (GRP, the mammalian homologue of the amphibian skin peptide bombesin) is present in the reproductive tract and expressed by the pregnant ovine endometrium prior to attachment and throughout the pregnancy. A bombesin-like peptide resulting from alternate splicing of the GRP gene in testis has been detected in primates. In this study, we have tested the ability of GRP to enhance human sperm functions such as motility, capacitation, zona binding and acrosome reaction. Analysis of sperm motility was performed with the ATS 20 computer-assisted semen analysis (CASA) system. Zona binding was analysed using intact human unfertilized oocytes and selective labelling of spermatozoa with two fluorochromes. Our results did not show any positive effect of GRP on these parameters under our experimental conditions. However, when GRP at the concentration of 100 nM was added after ionophore treatment, the percentage of reacted cells increased. significantly (P < 0.05) compared with situations where each agent was used alone. This led us to suppose that the role of bombesin in the different stages of fertilization might not exclude other unknown factors.

Effects of progesterone on human spermatozoa prepared for in-vitro fertilization.

Progesterone has been tested in vitro with human spermatozoa to verify its physiological effects and its possible therapeutic use in cases of male infertility. Progesterone induced a rapid, dose-dependent influx of calcium in capacitated and non-capacitated spermatozoa with a half-maximally effective dose of 30 nM. The agonist, 19-nortestosterone, was much less potent that progesterone itself. Progesterone-induced calcium influx was not inhibited by a similar concentration (0.1 microgram/ml) of RU 486, a classical progesterone antagonist. The increase in intracytoplasmic calcium levels was unable to induce the acrosome reaction (AR) even after incubation for 5 h, when this was evaluated by double staining, using a monoclonal antibody GB24 raised against the inner acrosome membrane and ethidium homodimer as a vital probe. However, after incubation for 5 h, progesterone was able to enhance the tyrosine phosphorylation of a 95 kD sperm protein, which is phosphorylated progressively during capacitation in well-defined culture media. Incubation of spermatozoa with 1 or 10 micrograms/ml progesterone for 3 or 30 min did not induce major modifications of hyperactivated movement when analysed by computer-assisted semen analysis. Progesterone secreted by cumulus cells may physiologically increase sperm intracytoplasmic free calcium during capacitation. This priming effect may facilitate the acrosome reaction, induced by binding to the zona pellucida, without enhancing spontaneous acrosome reaction prematurely. It therefore seems useful to propose progesterone as a means of accelerating capacitation during in vitro fertilization in cases of male infertility.

Variability of the response to pentoxifylline in vitro in infertile normozoospermic and asthenozoospermic patients.

The in vitro effect on the motility of spermatozoa of the methylxanthine pentoxifylline was studied in 30 patients consulting for infertility (13 normozoospermic and 17 asthenozoospermic). After separation by centrifugation on Percoll gradient, the spermatozoa were incubated for 30 min in pentoxifylline (3.6 mM), then the pentoxifylline was removed by washing and centrifugation. The residual effect of pentoxifylline was studied by computer-assisted videomicrographic analysis of the sperm motion parameters. In both groups of patients a decrease in the percentage of motile spermatozoa after exposure to pentoxifylline was observed, as compared with a control group. The effect on the motion parameters varied according to the time of observation (30, 120, 240 min). In the asthenozoospermic patients, the curvilinear velocity (VCL) was not modified in a statistically significant manner. In the normozoospermics, the VCL was increased after exposure to pentoxifylline at 120 min (p = .03) and 240 min (p = 10(-5)); similarly, the amplitude of the lateral head displacement (ALH) was greater in this group at 120 and 240 min (p = .01 for maximum ALH). However, a heterogeneity was noted in the response between individuals, which required that the statistical analyses take into account these differences.