Early Events Of Fertilization Research Articles

Use of a mitochondrial-specific live sperm stain to confirm penetration of equine oocytes after in vitro fertilization

Intracytoplasmic sperm injection (ICSI) is an important research and clinical tool. However, work with standard in vitro fertilization (IVF) is needed to explore sperm capacitation and the early events of fertilization. Repeatable methods for equine IVF have not yet been reported. Definitive evidence of fertilization, such as presence of the sperm tail within the cytoplasm, is critical when assessing potential methods for IVF. We have previously stained live sperm with the mitochondrial-specific stain, MitoTracker, and successfully visualized the sperm tail in the oocyte cytoplasm after ICSI. In the present study, we evaluated whether staining sperm with MitoTracker would A) be compatible with maintenance of sperm motility in culture and B) allow visualization of sperm tails within the COC or oocyte after co-culture with sperm. Semen was collected from one fertile stallion, and 200 µl of extended semen was layered under 1 mL of MOPS-buffered medium containing 10 or 100 nM Mitotracker Red CMXRos, in a 5-mL tube. The tube was incubated at a 45 o angle at 38 °C for 20 min. The uppermost 750 µL of the medium was collected, washed, centrifuged, and the pellet resuspended in modified TALP. Sperm were added to 50-µL droplets of modified TALP to a concentration of 1 million sperm/mL and these were incubated in 5% CO 2 in air. Sperm motility was assessed visually after 1, 6 and 22 h in culture. At 22 h, sperm were removed, counterstained with DAPI, and evaluated under fluorescence microscopy. In some replicates, in vitro-maturedequine cumulus-oocyte complexes (COCs) were added to the sperm droplets and co-incubated for 1 to 9 h. To assess sperm progress toward oocyte penetration, oocytes were denuded, fixed, and counterstained with DAPI. Some COCs were stained intact for confocal assessment of chromatin, sperm mitochondria, and acrosome status. These were fixed, permeabilized with 0.1% Triton-X 100, labeled with fluorescein-conjugated peanut agglutinin, then stained with Hoechst 33342. A minimum of three replicates were performed with each MitoTracker concentration. MitoTracker-stained sperm showed progressive motility after 1, 6 and 22 h incubation (∼80%, 50%, and 15% respectively) but at 22 h appeared to have a higher proportion of immotile sperm, and less vigorous motility, than did unstained sperm (∼25% progressive at 22 h). Staining with 10 nM MitoTracker supported more vigorous motility than did 100 nM. Both concentrations enabled clear visualization of the sperm tail on fluorescence microscopy. In penetrated oocytes, the sperm tail was clearly visualized in the oocyte cytoplasm, associated with sperm chromatin. On confocal microscopy, acrosomal ghosts were visualized at the periphery of the zona pellucida, indicating that sperm underwent the acrosome reaction in response to the zona. We conclude that staining of live sperm with 10 nM MitoTracker during swim-up provides a simple method to allow confirmation of sperm penetration of oocytes after sperm-oocyte coincubation.

Blastocyst development after fertilization with in vitro spermatids derived from nonhuman primate embryonic stem cells

Objective:To demonstrate that functional spermatids can be derived in vitro from nonhuman primate pluripotent stem cells.Design:Green fluorescent protein-labeled, rhesus macaque nonhuman primate embryonic stem cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatid-like cells (rSLCs) from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection.Setting:Multiple academic laboratory settings.Patient(s):Not applicable.Intervention(s):Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes.Main Outcome Measure(s):Differentiation into spermatogenic cell lineages was measured through multiple assessments including ribonucleic acid sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by intracytoplasmic sperm(atid) injection by assessing early fertilization events such as spermatid deoxyribonucleic acid decondensation and pronucleus formation/apposition. Preimplantation embryo development from the one-cell zygote stage to the blastocyst stage was also assessed.Result(s):Nonhuman primate embryonic stem cells can be differentiated into advanced germ cell lineages, including haploid rSLCs. These rSLCs undergo deoxyribonucleic acid decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and coinjection of ten-eleven translocation 3 protein, undergo embryonic divisions with approximately 12% developing successfully into expanded blastocysts.Conclusion(s):This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. To our knowledge, this work represents, for the first time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells.

O-003 The puzzling unknowns of abnormal fertilization and first cleavage

Abstract text Fertilization is a critical event in development in that it provides the connection between the gametes and the earliest stages of embryogenesis. Yet, despite the central importance of this process in contributing to embryo developmental fate, clinical embryologists have historically assessed fertilization merely by the number of pronuclei and, if two are present, perhaps, by the presence of two polar bodies. Even though over 20 years ago, time lapse imaging was applied for defining early events of fertilization (Payne et al., 1997), it is only with contemporary time-lapse imaging systems in the last few years that detailed evaluation of spatial and temporal events of fertilization have been described (Iwata & Yasuyuki, 2016; Cottichio et al., 2018). These careful analyses allow us to describe typical and atypical events of fertilization and how they are each associated with timing of the first cleavage division and subsequent embryo development. In this lecture, we will first describe the fundamental underpinnings of fertilization and highlight the normal events associated with this process. We will then discuss gross morphological abnormalities as visualized by light microscopy and highlight the unknowns associated with these events. Finally, we will focus on time-lapse imaging studies, which have revealed the remarkable spatial and temporal coordination of meiotic resumption, pronuclear dynamics, chromatin organization and cytoplasmic/cortical modifications that occur during fertilization and the implications of aberrations for the first cleavage division. At the conclusion of this presentation, attendees should be able to: Review the normal events associated with fertilization and the first cleavage division. 1 Describe gross morphological aberrations of these two fundamental processes. 2 Discuss temporal and spatial abnormalities in the coordinated sequence of events that underly these processes. 3 State the potential application of these abnormalities as predictors of abnormal embryo development. 4 Summarize the puzzling unknowns that underly these abnormalities.

Sex hormones alter the response of Toll-like receptor 3 to its specific ligand in fallopian tube epithelial cells.

ObjectiveThe fallopian tubes play a critical role in the early events of fertilization. The rapid innate immune defense is an important part of the fallopian tubes. Toll-like receptor 3 (TLR3), as a part of the innate immune system, plays an important role in detecting viral infections. In this basic and experimental study, the effect of sex hormones on the function of TLR3 in the OE-E6/E7 cell line was investigated.MethodsThe functionality of TLR3 in this cell line was evaluated by cytokine measurements (interleukin [IL]-6 and IL-1b) and the effects of sex hormones on TLR3 were tested by an enzyme-linked immunosorbent assay kit. Additionally, TLR3 small interfering RNA (siRNA) and a TLR3 function-blocking antibody were used to confirm our findings.ResultsThe production of IL-6 significantly increased in the presence of polyinosinic-polycytidylic acid (poly(I:C)) as the TLR3 ligand. Using a TLR3-siRNA-ransfected OE-E6/E7 cell line and function-blocking antibody confirmed that cytokine production was due to TLR3. In addition, 17-β estradiol and progesterone suppressed the production of IL-6 in the presence and absence of poly(I:C).ConclusionThese results imply that sex hormones exerted a suppressive effect on the function of TLR3 in the fallopian tube cell line when different concentrations of sex hormones were present. The current results also suggest that estrogen receptor beta and nuclear progesterone receptor B are likely to mediate the hormonal regulation of TLR3, as these two receptors are the main estrogen and progesterone receptors in OE-E6/E7 cell line.

Ion currents involved in gamete physiology.

Gametes are electrogenic cells that modify their electrical properties in response to different stimuli. This behavior is due to the occurrence of ion currents flowing through ion channels located on the plasma membranes. The modulation of ion channels has been described during the processes of gamete maturation, activation and fertilization in most of the animal models studied. In particular, predominant ions involved in physiological events in oocyte and sperm have been recognized to be sodium, potassium and calcium. In this review, we give an overview on the occurrence, modulation and function of ion fluxes, from gametogenesis to early fertilization events, from marine animals to human. The implications for a dynamic role of ion currents in gamete physiology and their possible clinical and technological applications are discussed.

6 EQUINE SPERM INDUCES PRONUCLEAR FORMATION BY INTRACYTOPLASMIC SPERM INJECTION IN BOVINE, SWINE, AND FELINE OOCYTES INDEPENDENTLY OF CHEMICAL ACTIVATION ASSISTANCE

Interspecific intracytoplasmic sperm injection (ICSI) is a valuable tool to study early events of fertilization in species for which oocyte availability is reduced. Equine in vitro fertilization remains unsuccessful and ICSI is the technique of choice for the in vitro production of high-value embryos. Therefore, the objective of this study was to evaluate the rate of pronuclear (PN) formation after ICSI with stallion sperm in bovine, swine and feline oocytes with or without chemical activation assistance. Ovaries from cows and pigs were collected at abattoirs whereas gonads from female domestic cats were obtained from ovariectomized animals at veterinary sterilization centers. Cumulus-oocyte complexes were matured in TCM-199 supplemented following standard protocols for each species. ICSI was performed in 100-μL drops of TALP-HEPES, using frozen-thawed semen from one stallion. Spermatozoa were held separate in 3-μL droplets of 7% (vol/vol) polyvinylpyrrolidone, where one of them was immobilized by swiping the injection pipette across its tail, and then injected into the matured oocyte. After ICSI, some oocytes were chemically activated with 5 μM ionomycin for 4 min (cow and cat) or with an electric pulse (sow) followed by 3 h in culture medium to allow extrusion of the second polar body and then exposure to 1.9 mM 6-DMAP solution for 3 h. Embryos were cultured in SOF medium. After 17 h of culture, embryos were stained with propidium iodide to identify the percentage of oocytes activated and with PN. Haploid and diploid parthenogenetic controls were included. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of the partenogenetic control groups were assessed. There were no statistical differences (chi-squared analysis) in PN formation between the activated and nonactivated groups within species. When the activated group was compared between the different species, no differences were observed. However, for the nonactivated group, significant differences were observed between species. The feline oocyte showed the higher percentage of PN and activation, whereas the bovine oocyte exhibited the lower rate of PN formation (cat: 22/27, 81.48%; swine: 19/39, 71.64%; cow:18/63, 43.07%). Our results suggest that the feline oocyte can be used as model to study fertilization events associated with the stallion sperm due to the higher efficiency in supporting PN formation. Our results indicate that the equine sperm is capable of inducing PN formation in these 3 species without further chemical activation assistance.

Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules

We previously demonstrated that many aspects of the intracellular Ca2+ increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca2+ wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca2+ signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca2+ spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca2+ decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca2+ waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca2+ waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.

Multiscale Modeling of Exocytosis in the Fertilization Process

We discuss the implementation of a multiscale biophysico-chemical model able to cope with the main mechanisms underlying cumulative exocytosis in cells. The model is based on a diffusion equation in the presence of external forces that links calcium signaling and the biochemistry associated to the activity of cytoskeletal-based protein motors. This multiscale model offers an excellent quantitative spatio-temporal description of the cumulative exocytosis measured by means of fluorescence experiments. We also review pre-existing models reported in the literature on calcium waves, protein motor activation and dynamics, and intracellular directed transport of vesicles. As an example of the proposed model, we analyze the formation of the shield against polyspermy in the early events of fertilization in sea urchin eggs.

Use of sex-sorted and unsorted frozen/thawed sperm and in vitro fertilization events in bovine oocytes derived from ultrasound-guided aspiration

The objective was to investigate the effects of sex-sorting on early fertilization events in ovum pick up (OPU) derived oocytes fertilized in vitro with frozen-thawed sperm at different co-incubation lengths. Eighty-four OPU sessions were carried out in 18 cyclic, dry and non-stimulated Holstein Friesian and German black pied cows. Ovum pick up oocytes were matured in vitro for 24 hours and fertilized with frozen-thawed sex sorted or unsorted sperm from the same ejaculate. Fertilization was achieved by two experimental protocols: 1) short gamete co-incubation length: 4, 8 and 12 hours; and 2) long gamete co-incubation length: 18 and 24 hours. After in vitro fertilization, ova were fixed and stained to identify early fertilization events. Sperm penetration, monospermy, pronuclear formation and syngamy did not differ, whether sexed or unsexed sperm was used. Overall, the findings demonstrate similar fertilizing potential between sex-sorted and unsorted sperm.

On Fertilization in Chaetopleura apiculata and Selected Chitonida

Early events of fertilization are described in Chaetopleura apiculata and other selected Chitonida. C. apiculata egg hulls are elaborated into multi-branched spines with interlocking polygonal bases. Around the perimeter of each base are a series of open pores, ranging in size from 0.1-0.5 microm, which permit sperm direct access to the vitelline layer. In Callochitonidae (Chitonida) even larger pores occur in egg jelly coats, but this is considered to be the plesiomorphic condition, found also in Lepidopleurida such as Deshayesiella curvata. Other Chitonina, such as Rhyssoplax tulipa and Acanthopleura granulata, have a continuous outer dense layer that lacks pores and must be digested by penetrating sperm. Fertilization in Chitonida is unique and involves injection of chromatin into the egg via a narrow tubular nuclear extension that appears to exclude other sperm organelles, including mitochondria, centrioles, and flagellum. New evidence from studies of fertilization in Mopalia muscosa (Chitonida: Acanthochitonina) supports this hypothesis. This type of fertilization implies maternal inheritance of both mitochondria and centrioles, which is highly unusual, because in most animals one sperm centriole assists movements of pronuclei and regulates organization of the mitotic spindle. This mechanism of fertilization is defined by a series of apomorphic characters that unify the order Chitonida.

A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the self‐incompatible species Solanum chacoense Bitt.

We have used 2-DE for a time-course study of the changes in protein and phosphoprotein expression that occur immediately after fertilization in Solanum chacoense. The phosphorylation status of the detected proteins was determined with three methods: in vivo labeling, immunodetection, and phosphoprotein-specific staining. Using a pI range of 4-7, 262 phosphorylated proteins could be mapped to the 619 proteins detected by Sypro Ruby staining, representing 42% of the total proteins. Among these phosphoproteins, antibodies detected 184 proteins from which 78 were also detected with either of the other two methods (42%). Pro-Q Diamond phosphoprotein stain detected 111 proteins, of which 76 were also detected with either of the other two methods (68%). The 32P in vivo labeling method detected 90 spots from which 78 were also detected with either of other two methods (87%). On comparing before and after fertilization profiles, 38 proteins and phosphoproteins presented a reproducible change in their accumulation profiles. Among these, 24 spots were selected and analyzed by LC-MS/MS using a hybrid quadrupole-TOF (Q-TOF) instrument. Peptide data were searched against publicly available protein and EST databases, and the putative roles of the identified proteins in early fertilization events are discussed.

Cracking the egg: increased complexity in the zona pellucida

A functional zona pellucida is critical for both fertilization and the early stages of embryo development. Recent data from genomic and proteomic studies have questioned our simplistic view of the zona as being composed of three proteins whose functions are clearly defined. In the human, for example, the zona pellucida is composed of four proteins, not three. The increased complexity of the zona pellucida in humans and other species across the evolutionary tree now demands that we reconsider our reliance on the mouse model for understanding early fertilization events. Additionally, we are now well placed to examine, for the first time, potential defects in zona genes and their proteins associated with defined pathology.

Nature of telomere dimers and chromosome looping in human spermatozoa

Specific and well-organized chromosome architecture in human sperm cells is supported by the prominent interactions between centromeres and between telomeres. The telomere-telomere interactions result in telomere dimers that are positioned at the nuclear periphery. It is unknown whether composition of sperm telomere dimers is random or specific. We now report that telomere dimers result from specific interactions between the two ends of each chromosome. FISH using pairs of subtelomeric DNA probes that correspond to the small and long arms of seven human chromosomes demonstrates that subtelomeres of one chromosome are brought together. Statistical analysis confirmed that telomere associations could not result from the random proximity of DNA sequences. Therefore, chromosomes in human sperm nuclei adopt a looped conformation. This higher-order chromosome structure is most likely required for chromosome withdrawal/decondensation during the early fertilization events leading to zygote formation.

Characterization of boar sperm cytoskeletal cylicin II as an actin-binding protein

The presence of actin-binding proteins in the perinuclear theca of boar spermatozoa has been investigated, using stepwise extractions of proteins from sperm heads. Proteins extracted with the alkaline buffer 1 M Na 2CO 3, pH 11, were found to contain a 66 kDa protein that binds F-actin in actin pelleting assays. Sequence studies and immunological characterization with antibodies specific for human cylicin II identified the 66 kDa protein as the homologue of bovine and human cylicin II. Immunocytochemical studies showed the presence of porcine cylicin II in the acrosomal region of round spermatids and in the postacrosomal region of late spermatids and spermatozoa, in agreement with the previously described localization of cylicins. Taken together, the results suggest that cylicin II, a protein of the sperm perinuclear cytoskeleton, is a novel actin-binding protein, which probably plays a role in the actin-related events that occur during spermiogenesis and the early events of fertilization.

Cytochalasin-D retards sperm incorporation deep into the egg cytoplasm but not membrane fusion with the egg plasma membrane.

The fertilization process is impaired when spermatozoa are previously incubated with Cytochalasin-D (Cyt-D). Although this fact reveals the participation of polymerized actin in fertilization, the specific event obstructed by Cyt-D treatment has not been determined. To identify this event, we capacitated guinea pig spermatozoa in minimal capacitating medium with pyruvate and lactate (MCM-PL) with Cyt-D, to inseminate hamster zona pellucida (ZP)-free eggs. Cyt-D (70 microM) decreased F-actin relative concentration in capacitated spermatozoa to a larger extent than in spermatozoa incubated under control conditions. Cyt-D also cancelled the F-actin increase normally observed in acrosome-reacted cells, and decreased the number of these cells with normal F-actin localization at the equatorial zone. Insemination of eggs with Cyt-D treated spermatozoa did not change early fertilization events such as the egg cortical reaction (CR), membranes fusion, and egg F-actin new localization, but clearly retarded, by 16 hr, spermatozoa incorporation deep into the egg cytoplasm, and decondensation of egg metaphase II chromosomes. These results show that actin polymerization is necessary for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane fusion nor egg activation early steps.

Spermiogenesis and Sperm Structure in Relation to Early Events of Fertilization in the Limpet Tectura testudinalis (Muller, 1776).

Spermiogenesis and fertilization in the limpet Tectura testudinalis (Mollusca: Archaeogastropoda: Patelloidea) were examined using scanning and transmission electron microscopy, as well as light microscopy. Spermiogenesis was similar to that described for other Lottiidae (Patelloidea), but some key differences were noted. We observed that the acrosomal vesicle forms on the plasma membrane from several Golgi-derived proacrosomal vesicles during the early spermatid. Evidence from anti-actin antibody staining of a related species, T. scutum, indicates that the acrosome probably contains G-actin in three different locations, but there are no preformed actin filaments. The flagellum of mature sperm tapers to a thin, filamentous end-piece. The mature egg of T. testudinalis is enclosed by a thin vitelline layer and a thick jelly coat bound by follicle cells. Where a mature sperm encounters the intact jelly coat, its acrosome tip may elongate, up to twice its original length. The acrosome reaction is not induced, but by this mechanism the acrosome bridges the jelly coat and extends down far enough to contact the vitelline layer. During the elongation of the acrosome, microfilaments are formed inside the tip. As well, new membrane required for this process is supplied by elimination of any slack in the plasma membrane and also perhaps by spontaneous formation of vesicles inside the acrosome. Once the tip of the sperm contacts and binds with the vitelline layer it undergoes the acrosome reaction, in which the plasma and acrosomal membranes fuse and roll back in a manner typical of scaphopods and some polychaetes. Sperm also may bind directly with the vitelline layer when the jelly coat is absent. Furthermore, the acrosome tip may undergo spontaneous fusion with a naked egg microvillus that formerly connected with a follicle cell process. Thus, the sperm either individually or collectively are equipped to deal with any one of several potential routes to successful fertilization of the egg. Current theories of gastropod phylogeny place the limpets as basal to the Gastropoda, but new evidence presented here supports the idea that they are a divergent group, with some unique innovations in sperm design.

Early fertilization events in the sexual and aposporous egg apparatus of Pennisetum ciliare (L.) Link

While detailed descriptions exist on the fine structural characteristics of double fertilization in sexual angiosperms, few studies have described fertilization in apomictic species. The results of this study with buffelgrass (Pennisetum ciliare (L.) Link syn=Cenchrus ciliaris L.) show that differences exist between the early fertilization events in sexual and apomictic (aposporous) female gametophytes. In unpollinated sexual female gametophytes, the egg cell did not show signs of parthenogenetic activity. In contrast, autonomous egg embryony was observed in unpollinated aposporous female gametophytes. Compared with the sexual genotype, degeneration of the synergids in the aposporous female gametophyte was precocious and rapid. During the first 4 h after pollination, the chalazal end of the sexually functional egg cell remained covered by the plasma membrane. In the aposporous embryo sac, a cell wall covered the plasma membrane of the egg cell several hours before penetration of the pollen tube into the female gametophyte.

Synchronous sperm penetration of zona-free mouse eggs in vitro.

To synchronize sperm penetration of zona-free eggs immediately after insemination, zona-free eggs preloaded with Hoechst-33342 were inseminated under various conditions. Insemination was mostly conducted at 10 sperm/microliter. In preliminary experiments, fatty acid-free BSA (FAF) was more satisfactory for sperm penetration than fraction V BSA, and FAF was used in the following experiments. Only 26% of zona-free eggs were fertilized at 10 min after insemination when the eggs were inseminated immediately after zona removal and preloading. However, egg preincubation significantly improved the penetration rate (1 h preincubation: 63%, 2 h preincubation: 82% penetrated 10 min after insemination). Some eggs preincubated for 2 h were already penetrated at 3 min (7%), and the rate gradually increased in a time-dependent manner (3 min: 7%, 5 min: 30%, 10 min: 80%). The rate further improved as the sperm concentration was increased; the maximal level was obtained at 160 sperm/microliters. At 160 sperm/microliters, 54% of the eggs were penetrated at 3 min and 78% were at 5 min. These results indicate that it is possible to synchronize the sperm entry and that not only sperm but zona-free eggs should be preincubated before insemination. These data are considered valuable for investigating early events in fertilization.

Spatiotemporal relationships among early events of fertilization in sea urchin eggs revealed by multiview microscopy

Four early events of egg fertilization, changes in intracellular calcium concentration and intracellular pH, reorientation of the surface membrane, and the elevation of the fertilization envelope, were imaged in real time and in pairs in single sea urchin eggs. The paired imaging allowed the correlation of the four events spatially and temporally. Three of them propagated as waves starting at the sperm entry site. The earliest was the calcium wave, visualized with fluorescent indicator dyes. After a delay of 10 s there followed a large decrease in the fluorescence polarization of membrane-bound dyes, which we interpret as arising from membrane reorientation as a result of cortical granule exocytosis and microvillar elongation. With a further delay of 15 s the fertilization envelope was seen to rise in transmitted light. All three waves propagated with similar velocities of approximately 10 microns/s, supporting the view that calcium triggers the latter two events. The fluorescence polarization changed in two steps with a clear pause of 10-20 s in between. The second step, which also propagated as wave, reflects either further elongation of microvilli or straightening of irregular microvilli. This second step was abolished by cytochalasin B and was coincident with an increase in cytoplasmic pH, suggesting that pH-induced actin reorganization may play a role. The cytoplasmic alkalinization, imaged with a fluorescent probe, was quite different from the other events in that it took place homogeneously throughout the egg and slowly (over 100 s). Apparently, the alkalinization is not on a direct downstream pathway of calcium origin. An opposing possibility, that the alkalinization may in fact be triggered by the traveling calcium wave, is also discussed.

History and efficiency of microassisted fertilization in mammals

The main purpose of early investigations into sperm injection conducted between 1965 and 1980 was to investigate the early events of fertilization, such as membrane fusion between homologous and heterologous gametes, activation of the oocyte cytoplasm and formation of male and female pronuclei. Very few papers were published in the field of gamete manipulation in rodents or mammalian species. In accordance with the rapid advancement of studies on in vitro fertilization (IVF) in mammals and of the development of many human IVF-ET programmes, attention focused upon difficult cases in which the binding of gametes was not achieved by conventional IVF for various reasons, especially infertility stemming from male factors. Since 1980, fundamental studies on the micromanipulation of gametes in non-human mammalian species have increased dramatically, resulting in basic knowledge and techniques that may be useful in overcoming gamete binding problems, including those encountered in human infertility. Recently, studies on the stability of the nucleus of mammalian spermatozoa exposed to severe environmental conditions such as heat shock, dehydration and long-term preservation at 4-25°C have been also carried out using micromanipulation techniques. This chapter reviews the historical background of this technique and the efficiency of microassisted fertilization in mammals. Particular emphasis is placed on physiological studies of gamete interaction and the application of this technique to the production of embryos and offspring in mammals, including humans.