Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material

The mouse oocyte can be activated by injection of a single, intact mouse spermatozoon or its isolated head. Isolated tails are unable to activate the oocyte. Active sperm-borne oocyte-activating factor(s) (SOAF) appears during transformation of the round spermatid into the spermatozoon. The action of SOAF is not highly species-specific: mouse oocytes are activated by injection of spermatozoa from foreign species, such as the hamster, rabbit, pig, human, and even fish. Some SOAF can be extracted by simple freeze-thawing of (hamster) spermatozoa; additional SOAF is obtained by sequential treatment of spermatozoa with Triton X-100 and SDS. Electron microscopic examination of sperm heads during SOAF extraction suggests that the relatively insoluble SOAF is associated with perinuclear material. When microsurgically injected into oocytes, Triton X-100-treated sperm heads (with perinuclear material, but without any membranes) can activate the oocytes, leading to normal embryonic development. Whereas perinuclear components have been believed to play a purely structural role, these data suggest an additional function for them in oocyte activation.     

Intracytoplasmic sperm injection in the mouse

Intracytoplasmic sperm injection (ICSI) into mouse oocytes involves a very low survival rate. This study was designed to determine why ICSI frequently fails in mice. Metaphase II oocytes were obtained from superovulated 4-6 week old F1 hybrid mice. Spermatozoa were retrieved from the epididymis of 12-14 week old F1 hybrid mice. The spiked microinjection pipette used to inject a spermatozoon into the ooplasm had outer and inner diameters of 10 and 8 microns respectively. The oocytes used in the first part of the study were not activated (group 1). Some oocytes were incubated with calcium ionophore for 5 min (group 2). The injected oocytes were evaluated 6, 20, 48 and 72 h after injection. A total of 143 eggs in each group underwent ICSI. In group 1, sperm heads escaped into the perivitelline space. In all, 63 (47%) of the remaining oocytes were damaged during the injection or had degenerated by the first evaluation. The survival rate was 53%, but fertilization did not occur. In group 2, 31 oocytes (22%) were damaged during microinjection or soon degenerated. Two oocytes underwent accidental subzonal insemination. Six oocytes were fertilized (4.2%) among the 78% of survivors. After injection, the sperm tail was found in the cytoplasm (27 and 31% in groups 1 and 2 respectively), the perivitelline space (45% in both groups) or protruding through the zona pellucida (28 and 23% respectively). More oocytes degenerated when the tail remained in the cytoplasm, i.e. 78% in group 1 and 36% in group 2.     

Gamete interactions: which sperm ligands for the oocyte membrane?

Fertilization includes sperm-oocyte recognition, adhesion, binding, fusion and egg activation. Integrin receptors which are adhesion molecules are expressed on unfertilized oocytes. By comparing to several cell-cell interactions such as lymphocyte activation, viral fusion, bacterial infection or macrophage phagocytosis, potential sperm ligands have been identified.     

Effect of methoxychlor on implantation and embryo development in the mouse

Methoxychlor (MXC) has been shown to have adverse effects on reproductive functions. However, it has not been fully determined whether the effects of MXC on reproduction are due to its estrogenic or antiestrogenic effects. Therefore, to further elucidate the estrogenic action of this pesticide in the mouse, we studied the effect of MXC on implantation and embryo development. MXC was found to initiate implantation in most delayed implanting mice at 400 microg/g body weight. However, at the higher dose of 800 microg/g body weight, MXC initiated implantation in only 50% of animals and the number of embryos implanting was significantly decreased (P < 0.05). It was determined that MXC inhibited implantation in intact pregnant mice only when given on Day 1 or Day 2 at 800 microg/g but not at lower doses or later in the preimplantation period. Embryonic development and transport were delayed on Days 3 and 4 in these animals. Finally, reciprocal embryo transfers with donor embryos recovered from MXC-treated animals (800 microg/g body weight on Day 1) transferred to untreated recipients resulted in no implantation compared to 79% implantation when donor embryos were treated with vehicle. These data indicate that MXC acts as an estrogen agonist at the level of the uterus and oviduct but as an antiestrogen in the ovary. In addition, MXC appears to alter normal preimplantation embryonic development. These results suggest the need for further studies to assess the mechanism of action of MXC in preimplantation embryos.     

Bilirubin helps to overcome the two-cell block in mouse oocyte cultures

PURPOSE: In vitro fertilization and culture of mouse oocytes, under normal atmospheric oxygen tension, subjects them to severe oxidative stress. Oocytes from some strains of mice lack the natural protective mechanism that guards them against this oxidative stress and fail to develop beyond the two-cell stage. METHODS: We could overcome the toxic effects of oxygen metabolites by adding 0.2-0.4 mg/dl bilirubin in a lactate-pyruvate culture medium defined by Whitten (1971). Six- to 8-week-old ICR (Institute of Cancer Research) female mice were super ovulated by intra peritoneal injection of 5 IU PMSG (pregnant mare serum gonadotropin) followed by 10 IU hCG 48 h later. The oocytes were collected from the distended fallopian tubes and inseminated with 1-2 million sperm from 3-4-month-old ICR male mice. The eggs were scored at 24, 48, and 72 h after the hCG injection. CONCLUSIONS: With 0.4 mg/dl bilirubin supplement, by the end of 72 h, 82% of the eggs progressed from the two-cell stage to the four-cell stage. Routine inclusion of bilirubin can improve embryo development in vitro.     

Sperm-induced oocyte activation in the rhesus monkey: nuclear and cytoplasmic changes following intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has increased the potential of the assisted reproductive technologies to propagate mammalian species and has provided an opportunity for research into cell cycle control and the mechanisms involved in sperm-induced oocyte activation. We have investigated the efficacy of ICSI in the rhesus monkey, the mechanism of fertilization following sperm injection and the cytoskeletal rearrangement that occurs upon oocyte activation. These studies were conducted on mature, and to a lesser extent, immature oocytes. Ejaculated spermatozoa, washed, capacitated and activated before immobilization, were injected into oocytes using conventional ICSI methodology. Sperm injection into mature oocytes induced oocyte activation (19/22; 86%) and pronuclear formation. In contrast, sham-injected oocytes did not activate readily (2/16; 13%). To localize oocyte activation factor(s), spermatozoa were separated mechanically into heads and tails which were then injected individually into mature oocytes. Activation occurred in 87% (20/23) of oocytes receiving heads. After tail injection, a single microtubule aster was nucleated and one pronucleus (PN) was seen in four of 21 oocytes. Intracytoplasmic injection of sperm extract (SE) resulted in oocyte activation at a significantly higher rate than occurred following sham injection (76 versus 13%). Sperm-induced oocyte activation was also evaluated in immature metaphase (MI) oocytes; activation occurred in 46% (12/26) of cases; however, only 8% of the activated oocytes exhibited 2 PN. Finally, beta-tubulin localization in untreated and taxol-treated oocytes was established as a marker for cytoplasmic changes associated with oocyte activation. These results are consistent with the hypothesis that spermatozoa contain an oocyte activating factor(s) which is primarily localized in the sperm head. Moreover, an activation response is limited to mature oocytes and is accompanied by cytoskeletal changes analogous to those seen following conventional fertilization.     

Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection

During the intracytoplasmic sperm injection (ICSI) procedure, the collected oocytes are incubated until just before ICSI. The ideal preincubation time of oocytes was investigated in 544 treatment cycles. Oocyte retrieval was carried out 35 h after human chorionic gonadotrophin administration. Oocytes were cultured for between 1 and 11 h before ICSI. Embryo transfer was performed 48 h after oocyte collection. The survival, fertilization and cleavage rates of injected oocytes indicated no statistically significant differences between oocytes preincubated for different lengths of time. The proportion of good-quality embryos (grades 1 and 2) was lower at 9-11 h of preincubation time than for all the other preincubation times (P < 0.001). No statistically significant differences were detected in the pregnancy rate between each group (mean: 15.9%), although the pregnancy rate at 9-11 h of preincubation time appeared to be low (7.7%). These results suggest that the oocyte retained sufficient potential for fertilization between 1 and 9 h after oocyte collection in ICSI. For the researchers who practise more complex ICSI procedures than IVF, it would be convenient to be able to perform ICSI at any time between 1 and 9 h after oocyte collection.     

Fertilization and embryo development to blastocysts after intracytoplasmic sperm injection in the rhesus monkey

Notwithstanding the thousands of seemingly healthy children born after intracytoplasmic sperm injection (ICSI), it is not yet possible to conclude absolutely that the ICSI procedure might induce some altered development or that the ICSI protocol might not be improved even further. To address this in a clinically relevant system, the developmental potential of rhesus monkey embryos produced by ICSI is reported. Oocytes collected by laparoscopy from gonadotrophin-stimulated fertile females were fertilized by ICSI using spermatozoa obtained from fertile males by electro-ejaculation. Neither sperm immobilization prior to injection nor an additional chemical stimulus were necessary to achieve oocyte activation and pronuclear formation. Survival and activation of the injected oocytes were judged by the extrusion of the second polar body. Successful fertilization was confirmed by the presence of two pronuclei within 12 h post-ICSI. Some oocytes were fixed and processed for the detection of microtubules and chromatin. Fluorescent labelling revealed that by 12 h post-ICSI the male and female pronuclei were closely apposed and eccentrically positioned within a large microtubule aster. ICSI resulted in a 76.6 +/- 14.9% fertilization rate. First cleavage was completed within 24 h post-ICSI. Two-cell ICSI embryos were co-cultured in CMRL medium on a buffalo rat liver cell monolayer until the hatched blastocyst stage. Oocytes collected laparoscopically from stimulated monkeys can be fertilized by ICSI and will complete preimplantation embryo development in vitro demonstrating that the rhesus monkey is an excellent preclinical model for examining and understanding many aspects of human ICSI.     

The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation

The objective of this study was to examine the effects of reactive oxygen species (ROS) on bovine sperm function and on the developmental competence of in vitro-matured bovine oocytes. In a first series of experiments, spermatozoa were exposed to ROS generated through the use of the hypoxanthine-xanthine oxidase system +/- catalase prior to the conduct of in vitro fertilization (IVF). Reactive oxygen species exposure reduced significantly (P < 0.001) the rates of oocyte penetration (control: 56% +/- 4 SEM; ROS: 16 +/- 2-23% +/- 7 SEM), and this effect was reversed by adding catalase (ROS+catalase: 67% +/- 0.3 SEM). During IVF, addition of superoxide dismutase (SOD: 1, 10, or 100 U/ml) had no effect on penetration rates. However, increasing concentrations of catalase (0.1 or 1 mg/ml) reduced these rates significantly (control: 70% +/- 3 SEM; treated: 45% +/- 5 and 1% +/- 1 SEM; P < 0.001). In a second series of experiments, when oocytes were matured in vitro in the presence of exogenous antioxidants (SOD: 10, 100, or 1000 U/ml; beta-mercaptoethanol: 0.01, 0.1, or 0.5 mM; ascorbic acid: 0.05 mg/ml), the developmental competence of the oocytes after IVF was not significantly improved. On the other hand, presumed production of ROS using the hypoxanthine-xanthine system at the beginning of the in vitro maturation period did improve subsequent developmental competence of the oocytes under some conditions and when catalase was present (control: 14% +/- 4 SEM and treated: 23% +/- 9 and 27% +/- 8 SEM; P < 0.05). These observations demonstrate that ROS may be beneficial to gamete function under specific conditions.     

Genetic regulation of preimplantation mouse embryo survival

The preimplantation period of mammalian development is characterized by cleavage of a one-cell embryo to a blastocyst stage embryo. During preimplantation development, 15%-50% of the embryos die as a result of factors that are largely unknown. Two parameters of preimplantation development, a fast rate of development and a low degree of fragmentation, are indicative of good embryo quality. There is mounting evidence that genes control both rate of development and degree of fragmentation. We have discovered a gene, Ped (preimplantation embryo development), which controls the rate of preimplantation embryonic cleavage. The Ped gene is encoded by two similar genes, Q7 and Q9, in the Q region of the mouse major histocompatibility complex (MHC). The Ped gene product is an MHC class Ib protein, the Qa-2 antigen. The mechanisms by which the Ped gene controls rate of embryonic cleavage division are being explored. In order to understand genetic mechanisms underlying the second criterion of embryo quality, degree of fragmentation, we have begun to assess expression of the genes that could potentially regulate apoptosis in preimplantation embryos. We have shown that staurosporine can induce apoptosis in mouse blastocysts. By using RT-PCR, we have shown that genes encoding protein in the two major gene families that regulate apoptosis, the Bcl-2 and caspase gene families, are present in preimplantation embryos. We hypothesize that there is a homeostatic mechanism by which genes that regulate cell survival and those that regulate cell death determine the overall viability of preimplantation embryos.     

Mitosis in the human embryo: the vital role of the sperm centrosome (centriole)

The pattern of sperm centrosomal (centriolar) inheritance, centrosomal replication and perpetuation during mitosis of the human embryo is reviewed with a series of electron micrographs. Embryonic cleavage involves repeated mitoses, a convenient sequence to study centriolar behaviour during cell division. After the paternal inheritance of centrioles in the human was reported (Sathananthan et al., 1991a), there has been an upsurge of centrosomal research in mammals, which largely follow the human pattern. The human egg has an inactive non-functional centrosome. The paternal centrosome contains a prominent centriole (proximal) associated with pericentriolar material which is transmitted to the embryo at fertilization and persists during sperm incorporation. Centriolar duplication occurs at the pronuclear stage (interphase) and the centrosome initially organizes a sperm aster when male and female pronuclei breakdown (prometaphase). The astral centrosome containing diplosomes (two typical centrioles) splits and relocates at opposite poles of a bipolar spindle to establish bipolarization, a prerequisite to normal cell division. Single or double centrioles occupy pivotal positions on spindle poles and paternal and maternal chromosomes organize on the equator of a metaphase spindle, at syngamy. Bipolarization occurs in all monospermic and in most dispermic ova. Dispermic embryos occasionally form two sperm asters initially and produce tripolar spindles (tripolarization). Anaphase and telophase follows producing two or three cells respectively, completing the first cell cycle. Descendants of the sperm centriole were found at every stage of perimplantation embryo development and were traced from fertilization through cleavage (first four cell cycles) to the morula and hatching blastocyst stage. Centrioles were associated with nuclei at interphase, when they were often replicating and occupied pivotal positions on spindle poles during mitosis. Sperm remnants were associated with centrioles and were found at most stages of cleavage. Centrioles were found in trophoblast, embryoblast and endoderm cells in hatching blastocysts. Pericentriolar, centrosomal material nucleated astral and spindle microtubules. Abnormal nuclear configurations observed in embryos reflect mitotic aberrations. The bovine embryo closely resembles the human embryo in centriolar behaviour during mitosis. It is concluded that the sperm centrosome is the functional active centrosome in humans and is likely the ancestor of centrioles within centrosomes in foetal and adult somatic cells. The role of the sperm centrosome in embryogenesis and male infertility is discussed, since it is of clinical importance in assisted reproduction.     

Amino acids and ammonium regulate mouse embryo development in culture

The conformation of the bulge formed between the hairpin ribozyme R derived from (-)sTRSV and noncleavable all-deoxy-substrate analogues dS was studied by photoaffinity labelling. The photolabel deoxy-6-thioinosine was inserted in place of residue G+1 or A-1, located immediately 3' and 5' to the cleavage site, respectively. Upon 335 nm irradiation both substrate analogues were linked to ribozyme at multiple sites. Formation of the R-dS complex is absolutely required for the generation of the crosslinks, since they were detected neither in the absence of Mg2+ nor upon using a ds6I containing 14-mer, unable to interact with the ribozyme. The fraction of ribozyme crosslinked at completion of the reaction increased with increasing analogue concentrations, yielding apparent KD values for the R-dS complex in the range of 5 +/- 2 microM. Multiple crosslinks between ribozyme and each one of the substrate analogues provide clear evidence for a large flexibility of the bulge region.     

Demonstration of lateral asymmetry in G-banded mouse embryo chromosomes

Cells obtained from 10-day mouse embryos were cultured for 3 h in medium containing colcemid. Chromosome preparations were subjected to G-banding in either calcium-magnesium free Hank's solution or the ASG method. From one to several occurrences of lateral asymmetry (unequal banding of sister chromatids) were observed in the majority of karytotypes analyzed.     

Characterization of the zebrafish Orb/CPEB-related RNA binding protein and localization of maternal components in the zebrafish oocyte

The animal/vegetal axis of the zebrafish egg is established during oogenesis, but the molecular factors responsible for its specification are unknown. As a first step towards the identification of such factors, we present here the first demonstration of asymmetrically distributed maternal mRNAs in the zebrafish oocyte. To date, we have distinguished three classes of mRNAs, characterized by the stage of oocyte maturation at which they concentrate to the future animal pole. We have further characterized one of these mRNAs, zorba, which encodes a homologue of the Drosophila Orb and Xenopus CPEB RNA-binding proteins. Zorba belongs to the group of earliest mRNAs to localize at the animal pole, where it becomes restricted to a tight subcortical crescent at stage III of oogenesis. We show that this localization is independent of microtubules and microfilaments, and that the distribution of Zorba protein parallels that of its mRNA.     

BMP4 is essential for lens induction in the mouse embryo

Vertebrate lens development is a classical model system for studying embryonic tissue interactions. Little is known, however, about the molecules mediating such inductive events. Here, we show that Bmp4, which is expressed strongly in the optic vesicle and weakly in the surrounding mesenchyme and surface ectoderm, has crucial roles during lens induction. In Bmp4(tm1) homozygous null mutant embryos, lens induction is absent, but the process can be rescued by exogenous BMP4 protein applied into the optic vesicle in explant cultures. This is associated with rescue of ectodermal expression of Sox2, an early lens placode marker. Substituting the optic vesicle in explant cultures with BMP4-carrying beads, however, does not lead to lens induction, indicating that other factors produced by the optic vesicle are involved. BMP4 appears to regulate expression of a putative downstream gene, Msx2, in the optic vesicle. No change in Pax6 expression is seen in Bmp4(tm1) mutant eyes, and Bmp4 expression appears unaffected in the eyes of homozygous Pax6(Sey-1Neu), suggesting that PAX6 and BMP4 function independently. Based on these results we propose that BMP4 is required for the optic vesicle to manifest its lens-inducing activity, by regulating downstream genes and/or serving as one component of multiple inductive signals.     

Fusion of nasal swellings in the mouse embryo: surface coat and initial contact

The nasal region of 12-day-old mouse embryos was examined with the electron microscope to determine whether a surface coat and membrane specializations are involved in epithelial fusion between the medial and lateral nasal swellings. Ruthenium red was used to examine the distribution of the surface coat. Prior to contact, a surface coat is always present over the epithelial linings of the nasal swellings in the region of presumptive fusion, and it is often heavier in the fusing than in the non-fusing regions. At the point of initial contact, the coat is present as a thin film between touching superficial cells, suggesting that it may mediate epithelial contact. The initial contact between the cells of the medial and lateral nasal swellings is made by short projections from one superficial cell to the surface of an opposing superficial cells meet. The contacting membranes, which are separated by a distance of approximately 6-10 nm and show an increased electron-density, probably provide a firm adhesion between the nasal swellings.