The relationship between sperm and viruses depends on the type of virus present as well as the presence or absence of seminal fluid. Motility is not a good enough criterion on which to prove the presence of viral elements, either in the medium or on the sperm.

Ten framesisec microcinematography ("film"), 1 second timed-exposure photomicrography ("photo"), and laser Doppler velocimetry (LDV) were used to analyse the swimming patterns of human spermatozoa after migration (1 h at 37°C) into an overlying layer of either BWW or Menezo's B2 media. The upper layer of medium was carefully removed and further incubated at 37°C for either 4 h (B2) or 5 h (BWW) and the spcrm motility analysed again. Five experiments were performed using semen from different donors. Film and photo analyses gave the relative incidence of nonprogressive and progressively motile spermatozoa plus, for the progressive spermatozoa, the velocities of progression (Vp) and amplitudes of lateral head displacement (Ah). LDV gave the percentage of motile spermatozoa and the modal instantaneous velocity (Vm). All postmigration sperm populations showed large significant increases in the percentage of motile spermatozoa, with good survival during incubation. The progressive postmigration spermatozoa generally moved with greater Vp and Ah than in the initial seminal plasma-diluted material; Vm was also increased. There were further increases in both Vp and Ah during incubation, but no change in Vm was detected. While the majority of spermatozoa were progressive, some showed a highly active pattern of movement which resulted in no net forward progression. The possible homology between these spermatozoa and the "hyperactivated" motility of capacitated spermatozoa in other mammalian species is discussed. Apparent discrepancies between the three methods used for motility analysis were seen, the possible causes and significances of which are also discussed.

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report, human mesenchymal cells (hMSCs) generated from the human ES cell line H9, were reprogrammed back to induced pluripotent state using Oct-4, Sox2, Nanog, and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes, lipid droplets, glycogen, scarce reticulum) and nuclear levels (features of nuclear plasticity, presence of euchromatin, reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal–epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming.

The location of the phospholipase C β1-isoform (PLC-β1) 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-β1 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-β1a. 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-β1 monoclonal antibody into the germinal vesicle (GV) only when this accumulation was at its maximum. In contrast, PLC-γ1 was absent from the GV from the time of release from the follicle until 1 h later, and microinjection of anti-PLC-γ1 into the GV did not affect GVBD. Our results demonstrate a relationship between the relocation of PLC-β1 and its role in the first step of meiosis.

In this study, we investigated the spatio-temporal distribution of conventional protein kinases C (cPKC) isoforms PKC-α, PKC-βI, PKC-βII and PKC-γ in mouse oocytes. The cPKCs were present in the cytoplasm at the start of the process and migrated to the nucleus (or germinal vesicle) before germinal vesicle breakdown, except for PKC-γ which remained cytoplasmic. In both compartments, the fully phosphorylated form corresponding to the `mature' enzyme was revealed for PKC-α, PKC-βI and PKC-βII. Microinjection of specific antibodies against each isozyme in one or the other cell compartment at different times of the meiotic process, permitted us to observe the following: (1) When located in the cytoplasm at the beginning of the process, PKC-α is not implicated in germinal vesicle breakdown, PKC-βI and PKC-γ are involved in maintaining the meiotic arrest, and PKC-βII plays a role in meiosis reinitiation. Furthermore, just before germinal vesicle breakdown, these cytoplasmic cPKCs were no longer implicated. (2) When located in the germinal vesicle, PKC-α, PKC-βI and PKC-βII are involved in meiosis reinitiation. Our data highlight not only the importance of the nuclear pathways in the cell cycle progression, but also their independence of the cytoplasmic ones. Further investigations are however necessary to discover the molecular targets of these cPKCs to better understand the links with the cell cycle progression.

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.

Our observations suggest that even if MCMV DNA persists from the zygote to the blastocyst stage, its presence has no detrimental effect on pre-implantation or post-implantation development.

As highlighted in this review, the phosphoinositide-phospholipase C pathway is strongly implicated in the control of mouse oocyte meiosis. The pathway becomes progressively functional as oocyte growth advances, and it appears to play a role in the G2/M transition when meiosis resumes, at least in the in vitro spontaneous model. Even if the inositol 1,4,5-trisphosphate receptors are present from the beginning, they function and release Ca2+ when the follicular antrum appears. Phospholipase C beta1 (PLC beta 1) is first exclusively localized to the nucleus and then migrates to the cytoplasm when the oocyte is fully grown. During oocyte maturation PLC beta 1 is active in the cytoplasm before it migrates and becomes active in the nucleus just prior to germinal vesicle breakdown. Because a similar circuit is observed for protein kinase C alpha (PKC alpha), PKC beta 1, PKC beta 2, and active mitogen-activated protein kinase, it is tempting to envisage that a feedback loop occurs between these pathways as demonstrated in other cell types. The chronology of these molecular movements into the oocyte reveals the particular and important role of the nucleus phosphoinositide cycle during oocyte meiosis. It appears also that this chronology is crucial and that defects leading to an inappropriate intracellular localization can have dramatic consequences. Such anomalies can prevent the production of competent oocytes and lead to fertility problems.