Search PubMed⌕ Search

Biomedical subjects

G Schatten

Publications and source records attributed to G Schatten.

At least 73 records · Page 4Linked to original sources
PubMed1997

Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization.

Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.

Animals↗
PubMed1997

Microtubule organization and chromatin configurations in hamster oocytes during fertilization and parthenogenetic activation, and after insemination with human sperm.

The cytoskeletal components of hamster oocytes, zygotes, and spontaneously activated parthogenotes were examined after immunocytochemical labeling. Microtubules were found only in the anastral, tangentially arranged second meiotic spindle of unfertilized oocytes. Taxol treatment of unfertilized oocytes greatly augmented astral microtubules in both the metaphase II spindle and the cortex. Disruption of the meiotic spindle microtubules with nocodazole resulted in cortical chromosomal scattering. During hamster sperm incorporation and pronuclear formation, no sperm aster was detected in association with the male DNA. Instead, a large overlapping array of microtubules assembled in the cortex. By mitosis, this interphase array disassembled and an anastral metaphase spindle formed. Microtubule and chromatin configurations were also imaged in hamster oocytes injected with human sperm. Astral microtubules were absent from the sperm centrosome. The implications of these results are discussed in relation to the hamster oocyte penetration assay, a test commonly used by in vitro fertilization clinics to demonstrate the fertilizing ability of human sperm. We conclude that since hamsters and humans follow different methods of centrosome inheritance, maternal and paternal, respectively, the hamster may be an inappropriate model for exploring microtubule and centrosomal defects in humans or for assaying postinsemination forms of human male fertility defects.

Animals↗
PubMed1997

The implications of a paternally derived centrosome during human fertilization: consequences for reproduction and the treatment of male factor infertility.

PROBLEM: Successful fertilization in humans follows a complex series of events, including the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei, the restoration of the sperm centrosome, and the nucleation of microtubule-mediated motility necessary to bring the male and female pronuclei into close apposition. These events occur after both fertilization in vitro and after intracytoplasmic sperm injection (ICSI), a new technique which is currently being applied in many clinics to overcome severe male infertility. Defects in any of the events leading to fertilization can be lethal to the zygote and may prove to be causes of infertility. METHODS: Imaging of inseminated human and rhesus oocytes using immunohistochemical techniques reveals several phases at which fertilization arrests. RESULTS: Oocytes from some infertile patients failed to complete fertilization due to failure of the sperm aster microtubules in uniting the sperm and egg nuclei. The rate of sperm aster formation, size, and organization during fertilization has been used as a measurement of bovine sperm quality. The development of an assay using Xenopus laevis oocyte extract can also be used to test sperm from various species for their ability to form esters and perform other centrosomal functions in vitro, as well as another indicator of sperm quality. Semen from men with questionable fertility was found to contain sperm which are generally incapable of producing sperm asters. In addition, the activity of centrosomal proteins such as gamma-tubulin and centrin have been detected in mammalian eggs and sperm. The levels of gamma-tubulin increase markedly after exposure to X. laevis egg extract. CONCLUSION: Defects in either male or female nucleus decondensation also resulted in the arrest of fertilization and was found to occur in both inseminated human oocytes and in rhesus oocytes fertilized by ICSI. These discoveries on the molecular basis of infertility in humans have important implications for infertility diagnosis and managing reproduction.

Animals↗
PubMed1997

A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte.

Casein kinase I is a family of serine/threonine protein kinases common to all eukaryotes. In yeast, casein kinase I homologues have been linked to the regulation of growth, DNA repair and cell division. In addition, their subcellular localization to membraneous structures and the nucleus is essential for function. In higher eukaryotes, there exist seven genetically distinct isoforms: (alpha), ss, (gamma)1, (gamma)2, (gamma)3, (delta) and (epsilon). Casein kinase I(alpha) exhibits a cell cycle-dependent subcellular localization including an association with cytosolic vesicular structures and the nucleus during interphase, and the spindle during mitosis. casein kinase I has also been shown to modulate critical regulators of growth and DNA synthesis/repair in mammalian cells such as SV40 large T antigen and p53. These results suggest that casein kinase I may be involved in processes similar to those ascribed to the yeast casein kinase I homologues. To define a role for casein kinase I(alpha) in cell cycle regulation, the mouse oocyte was utilized because of its well-defined cell cycle and ease of micromanipulation. Immunofluorescence studies from meiosis I of maturation to the first zygotic cleavage demonstrated that the kinase was associated with structures similar to those previously reported. Microinjection of casein kinase I(alpha) antibodies at metaphase II-arrest and G2 phase, had no effect on the completion of second meiosis or first division. However, microinjection of these antibodies during the early pronucleate phase prior to S-phase onset blocked uptake of the kinase into pronuclei and interfered with proper and timely cell cycle progression to first cleavage. These results suggest that the kinase regulates the progression from interphase to mitosis during the first cell cycle.

Animals↗
PubMed1997

Inheritance defects of the sperm centrosome in humans and its possible role in male infertility.

During fertilization in humans, the sperm introduces the centrosome, the oocyte's microtubule organizing centre (MTOC), restoring centrosome function. The newly activated oocyte initiates extrusion of the second polar body and begins pronuclear formation. Apposition of the male and female pronuclei requires microtubule-mediated motility in the form of an aster of microtubules emanating from the sperm centrosome. The centrosome then duplicates and separates, forming the two poles of the mitotic spindle, upon which the parental genomes intermix, completing fertilization. The restoration and function of the centrosome is critical for successful fertilization suggesting that a defective sperm centrosome will lead to fertilization arrest and may be a new cause of male infertility. Several types of fertilization failure, associated with the sperm centrosome, are documented. These include: i) failure of the sperm to nucleate microtubules after sperm incorporation; ii) detachment of the sperm centrosome from the sperm head; and iii) failure in microtubule elongation after successful sperm aster formation. Although some types of severe male infertility can be overcome with the use of intracytoplasmic sperm injection (ICSI), men with defective sperm centrosomes are unlikely to benefit. The rate of sperm aster formation, size and organization during bovine fertilization has been used as a measurement of bovine sperm quality. Sperm from bulls which developed large highly organized sperm asters resulted in a higher rate of fertilization in vitro. The development of an assay using Xenopus laevis oocyte extracts has also been used to test centrosome function and sperm aster formation using sperm from men with questionable fertility. In general, these sperm were incapable of forming sperm asters and when used for in vitro fertilization, resulted typically in fertilization failure. These discoveries on the inheritance and function of the sperm centrosome have revealed a new cause of fertilization failure linked to male infertility which may not be circumvented using ICSI.

Animals↗
PubMed1996

Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and developmental success.

This study describes a paternal effect on sperm aster size and microtubule organization during bovine fertilization. Immunocytochemistry using tubulin antibodies quantitated with confocal microscopy was used to measure the diameter of the sperm aster and assign a score (0-3) based on the degree of radial organization (0, least organized; 3, most organized). Three bulls (A-C) were chosen based on varying fertility (A, lowest fertility; C, highest fertility) as assessed by nonreturn to estrus after artificial insemination and in vitro embryonic development to the blastocyst stage. The results indicate a statistically significant bull-dependent difference in diameter of the sperm aster and in the organization of the sperm astral microtubules. Insemination from bull A resulted in an average sperm aster diameter of 101.4 microm (76.3% of oocyte diameter). This significantly differs (P < or = 0.0001) from the average sperm aster diameters produced after inseminations from bull B (78.2 microm; 60.8%) or bull C (77.9 microm; 57.8%), which themselves displayed no significant differences. The degree of radial organization of the sperm aster was also bull-dependent. Sperm asters organized by bull A-derived sperm had an average quality score of 1.8, which was higher than that of bull B (1.4; P < or = 0.0005) or bull C (1.2; P < or = 0.0001). Results with bulls B and C were also significantly different (P < or = 0.025). These results indicate that the paternally derived portion of the centrosome varies among males and that this variation affects male fertility, the outcome of early development, and, therefore, reproductive success.

Analysis of Variance↗
PubMed1996

Cold-treated centrosome: isolation of centrosomes from mitotic sea urchin eggs, production of an anticentrosomal antibody, and novel ultrastructural imaging.

A novel isolation of centrosomes is described and it was used to both generate a centrosome-specific monoclonal antibody and to image with high-resolution low-voltage scanning electron microscopy the surface details of the isolated centrosome. At first mitotic prometaphase, sea urchin zygotes are chilled on ice overnight. While most of the microtubules disassemble, the mitotic centrosomes collapse into aggregated masses. These centrosomes have been isolated, and used to generate a monoclonal antibody, designated 4D2, which is reactive with interphase and mitotic centrosomes. 4D2 staining of centrosomes is similar, but not identical, to that of other centrosomal antibodies like Ah6 and 5051. Centrosomal material is detected as a compact sphere after cold treatment; upon recovery the sphere expands and undergoes the shape changes previously described [Mazia et al., 1987: J. Cell Biol. 105:206a] to eventually reorganize a normal mitotic apparatus.

Animals↗
PubMed1996

Excision and disassembly of sperm tail microtubules during sea urchin fertilization: requirements for microtubule dynamics.

To determine the fate of the sperm tail during fertilization, the microtubules of the incorporated axoneme are measured using a monoclonal antibody against acetylated alpha-tubulin in zygotes from the sea urchin Strongylocentrotus purpuratus. This antibody recognizes axonemal microtubules, but does not recognize egg cytoplasmic tubulin or microtubules. The detachment of the axoneme from the male pronucleus occurs as early as 15 min post-insemination. Following excision, the axoneme is often found in close association with the female pronucleus during its migration to the male pronucleus. Fragmentation of the sperm tail, detected at 25 min, continues with only a few micrometers remaining at 85 min post-insemination. The fluorescence intensity of the axonemal fragments diminishes over time as compared to intact axonemes. At 100 min post-insemination, the sperm axoneme is no longer detected. Alternative imaging approaches using brief cold or elevated calcium extraction to disrupt the labile cytoplasmic, but not axonemal, microtubules, indicate that these observations are not due to changes in the post-translational modifications of alpha-tubulin. In the presence of nocodazole, a microtubule assembly inhibitor, a large portion of the tail remains visible at 100 min post-insemination; this suggests that microtubule dynamics are required for the disassembly of the sperm tail. Furthermore, the detachment of the axoneme from the male pronucleus requires the formation of the sperm aster. This suggests that the sperm aster microtubules both detach the axoneme from the male pronucleus, and also cause the translocation of the tail towards the female pronucleus after pronuclear union. In summary, the sperm tail is excised from the male pronucleus and the tail microtubules disassembled during the first cell cycle of sea urchin fertilization, and these events require new microtubule assembly within the zygote.

Animals↗
PubMed1996

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation.

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both microtubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes.

Actin Cytoskeleton↗
PubMed1996

Propranolol induces polyspermy during sea urchin fertilization.

Propranolol, a beta-adrenergic receptor blocker, is found to induce polyspermy in sea urchin eggs. Unfertilized sea urchin eggs treated for 10 min with 50 microM of propranolol, and then inseminated, become polyspermic and show a fertilization envelope which is barely visible to the light microscope. Examination of treated eggs by transmission and scanning electron microscopy shows that the drug does not alter the cortex of the unfertilized egg. However, after insemination an incomplete cortical reaction occurs. This might well account for both polyspermy and the defective elevation of the fertilization envelope. Since the effects of the drug are reversed by simultaneous treatment with adrenalin, perhaps propranolol interferes with the monoaminergic system that has been proposed to be active. The involvement of the monoaminergic system in the fertilization process is present in the sea urchin egg.

Animals↗
PubMed1996

Intracytoplasmic sperm injection for Rhesus monkey fertilization results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly.

The disassembly and reorganization of sperm-derived structures are landmarks for the onset of embryonic development. Since complete information on these events is not yet available, we examined the disassembly of the sperm axoneme, the formation of the sperm aster, and the decondensation and development of the male and female pronuclei in inseminated Rhesus monkey oocytes conceived by in-vitro fertilization (IVF) or by intracytoplasmic sperm injection. During IVF, the spermatozoa lose their acrosomes after contacting the zona pellucida, and the plasma membrane and nuclear envelope disappear after fusion with the oolemma. Subsequently, a sperm aster of microtubules forms around the proximal centriole, which is bound to the sperm connecting piece. This process is then followed by the formation of both pronuclei, which single sperm centriole later duplicates and the bipolar mitotic apparatus is observed. Following sperm injection, the spermatozoa have both an intact plasma membrane and acrosome. Although the microtubules form the sperm aster in a fashion identical to that seen during IVF, the presence of an intact acrosome appears to be associated with a heterogeneity in the decondensation of sperm chromatin. While this may indicate an abnormal pattern of chromatin decondensation during the formation of the male pronucleus following sperm injection, the male pronucleus eventually fully decondenses, as during IVF. Sperm mitochondria are displaced as the sperm centriole is exposed. Annulate lamellae and a previously undescribed organelle which seems to contain annulate lamellae precursors, as well as maternal mitochondria, are found in association with the developing pronuclear envelopes. This information increases understanding of fertilization in primates, and may also be of significance for use in assisted human reproduction as well as in the preservation of endangered mammalian species. In addition, these results demonstrates the similarities between fertilization in Rhesus monkeys and humans, providing additional evidence for the use of this non-human primate as a model system in which to investigate the cellular and molecular biological basis of human reproduction.

Animals↗
PubMed1996

Microtubule organization in porcine oocytes during fertilization and parthenogenesis.

Microtubule configurations in porcine oocytes after sperm penetration or after artificial activation by electrical stimulation were imaged by immunocytochemistry and laser scanning confocal microscopy. Soon after sperm penetration, an aster was seen adjacent to the incorporated sperm head. Polyspermic penetrations led to the presence of multiple sperm asters in association with each sperm. The sperm aster enlarged and, at the time of pronuclear apposition, filled the cytoplasm. After male and female gamete union, the microtubule matrix was reduced. At the mitotic metaphase stage, microtubules were detected in the spindle, which was anastral and fusiform. At anaphase, asters assembled at each spindle pole, and at telophase, large asters filled the cytoplasm. Artificial activation by electrical stimulation induced in the cytoplasm a dense network of microtubules, which seem to be involved in proper positioning of the female pronucleus. At mitotic metaphase, microtubules were concentrated around the chromatin. The results of experiments using taxol, a microtubule stabilizing agent, suggest that maternal centrosomal material is present in the mature porcine oocyte as dispersed undetectable material that can form a microtubule network after parthenogenetic activation. However, at fertilization, the paternal centrosome collects centrosomal material to form a sperm aster. These results suggest that the functional centrosome that forms during fertilization is a result of the blending of paternal and maternal centrosomal components.

Animals↗
PubMed1996

Microtubule and chromatin dynamics during fertilization and early development in rhesus monkeys, and regulation by intracellular calcium ions.

To explore primate fertilization, oocytes and zygotes from fertile rhesus monkeys were imaged throughout fertilization, polyspermy, and artificial activation using confocal microscopy for microtubules and DNA, as well as ratiometric computer-enhanced video microscopy for intracellular calcium. Unfertilized oocytes displayed microtubules only in the radially oriented meiotic spindles. At insemination, a large calcium transient was followed by a series of smaller oscillations, and sperm astral microtubules had assembled from the sperm centrosome by 2.5 h after transient onset. This aster enlarged, and later duplicated, as the pronuclei converged near the cortex. Pronuclear apposition was prevented by microtubule inhibitors. At mitotic prophase, microtubules ensheathed both sets of condensing chromosomes. At metaphase, the spindle was barrel-shaped and eccentrically positioned with two small asters at the pole with the sperm tail. Microtubules emanating from the telophase spindle interacted with the adjacent cortex and displaced the spindle toward the cell center as first cytokinesis ensued. During polyspermy, each sperm nucleated an aster, and the frequency of calcium oscillations increased. Activation resulted initially in disarrayed microtubules that eventually organized into functional mitotic spindles. These kinetic results demonstrate that rhesus monkeys accomplish fertilization in a fashion nearly identical to that of humans and are, therefore, ideal models in which to investigate cytoskeletal events during human reproduction.

Adenine↗
PubMed1996

Microtubule and chromatin configurations during rhesus intracytoplasmic sperm injection: successes and failures.

Intracytoplasmic sperm injection (ICSI) was performed on rhesus monkey oocytes, and the resultant microtubule and DNA configurations were imaged by laser-scanning confocal microscopy. In addition, polyspermic oocytes fertilized by ICSI were examined by transmission electron microscopy (TEM). Successful rhesus fertilization by ICSI revealed microtubule and DNA configurations similar to those observed during in vitro fertilization of human and rhesus monkey oocytes, including sperm aster formation, pronuclei decondensation, spindle formation, and cell division. Several abnormalities, however, were also observed: 1) inability to complete meiosis; 2) inability to undergo male or female pronucleus formation; 3) separation of the sperm tail from the sperm nucleus; 4) premature chromosome condensation with the formation of a paternal meiotic spindle; and 5) formation of multiple female pronuclei (karyomeres) during chromosome decondensation. TEM analysis revealed that sperm can undergo decondensation in the presence of an intact acrosome at least 18 h after sperm injection. These results demonstrate the utility of rhesus ICSI in pre-clinical applications as well as with endangered species. However, the different types of fertilization failures observed here indicate that although ICSI may be a readily accepted means of fertilization of human oocytes in many clinics, we should further characterize the cellular and genetic abnormalities associated with ICSI in both human and nonhuman primates.

Animals↗
PubMed1996

Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization.

Sperm incorporation and the conversion of the sperm-derived components into zygotic structures during in vitro fertilization of bovine oocytes was explored by combining ultrastructural studies with observations of the fertilizing sperm tagged with a mitochondrion-specific vital dye MitoTracker green FM. The zygotes fertilized by the MitoTracker-labeled sperm were fixed at various times after fertilization and then processed for immunocytochemistry to examine the distribution of DNA, microtubules, and sperm tail components, including the fibrous sheath and axonemal microtubules. We show here that the complete incorporation of the sperm, but not sperm-oocyte binding and oocyte activation, depends upon the integrity of oocyte microfilaments and is inhibited by the microfilament disrupter cytochalasin B. After sperm incorporation, the mitochondria are displaced from the sperm's connecting piece, and the sperm centriole is exposed to the egg cytoplasm. This event is followed by the formation of the microtubule-based sperm aster, which is responsible for the union of male and female pronuclei. Concomitantly, the major structure of the sperm principal piece, the fibrous sheath, disappears. After the first mitosis, the compact mitochondrial sheath can be seen in one of the blastomeres. An aggregate of the sperm mitochondria is observed at the entry of the second mitosis, although they remain in the vicinity of the nucleus and can later be seen at one pole of the metaphase spindle. The mitochondrial cluster is occasionally found in one of the blastomeres in the early-stage four-cell embryos, but it is no longer detected by the beginning of the third mitotic cycle. These data suggest that the disassembly of the sperm tail during bovine fertilization occurs as a series of precisely orchestrated events involving the destruction (fibrous sheath and mitochondrial sheath) and transformation (DNA, sperm centriole) of particular sperm structures into zygotic and embryonic components.

Acrosome↗
PubMed1996

Molecular medical approaches for alleviating infertility and understanding assisted reproductive technologies.

Fertilization is a precisely orchestrated cascade of events that results in the union of paternal and maternal genomes and in the establishment of mitotic potential of the zygote. To initiate embryonic development, the structures of the fertilizing sperm have to be disassembled and transformed into zygotic components by interactions with the cytoplasm of the egg. These interactions include the decondensation of the sperm nucleus into male pronucleus, the assembly of the zygotic centrosome, and the gathering of centrosomal proteins and sperm aster microtubules around the sperm centriole. Both the formation of the male pronucleus and the assembly of the zygotic centrosome are crucial steps required for pronuclear apposition and genomic union. The discovery of previously undetected fertilization failures that are due to defects in the assembly of the zygotic centrosome, abnormal pronuclear development, and compromised cytoskeletal dynamics enforces the development of new diagnostic strategies. Moreover, the introduction of new methods of infertility treatments, such as intracytoplasmic sperm injection and round spermatid nucleus injection into assisted human reproductive technology programs, emphasizes our lack of understanding of the cellular and molecular basis of human fertilization and evokes the need for additional experimentation. These efforts, however, are compromised by the sensitive nature of human embryo research and thus are severely restricted. Animal models that are reliable and cost-effective and that feature the characteristics of human fertilization have therefore been sought. Rodents such as the rat, mouse, and hamster are poor models owing to their maternal inheritance of the zygotic centrosome that is in strong contrast with the biparentally contributed assembly of the human zygotic centrosome during fertilization. Although rabbits are similar to humans from the standpoint of mitotic potential inheritance, information on postfertilization events in rabbits are lacking. Nonhuman primates represented by the rhesus monkey proved to be a reliable model for human in vitro fertilization and intracytoplasmic sperm injection, an advantage that is further emphasized by phyllogenetic similarity. In situations in which the high cost of primate research does not allow for large-scale experimentation (i.e., when large numbers of oocytes and embryos are needed), ruminants would be an ideal solution. Represented by the cow and sheep, domestic ruminants feature a fertilization strategy similar to that of the human. In addition, large numbers of gametes can be obtained wherever farms and slaughterhouses are accessible. Moreover, the detailed information on ruminant fertilization is strengthened by years of research and well-defined reproductive technology aimed at increasing the productivity of farm animals. Ruminants and rhesus monkeys have been extensively studied, and the data from these studies have been extrapolated in order to propose new strategies for the diagnosis and treatment of human infertility.

Animals↗
PubMed1995

Protein tyrosine phosphorylation during sea urchin fertilization: microtubule dynamics require tyrosine kinase activity.

Protein tyrosine phosphorylation plays an important role in cell growth, mitosis, and tumorigenesis. It has also been implicated in meiotic maturation and fertilization. We have used anti-phosphotyrosine immunofluorescence and immunoblotting to identify sperm and egg proteins which are phosphorylated on tyrosine residues prior to and during sea urchin fertilization. On immunoblots of sperm proteins, the monoclonal anti-phosphotyrosine antibody detected three major proteins with molecular weights of 44, 82, and 100 kD, and six minor bands at 46, 48, 70, 76, 95, and 150 kD. These phosphotyrosyl proteins were localized to the sperm acrosomal and centriolar fossae. In contrast, staining was found globally in unfertilized eggs, and the antibody recognized two major egg phosphotyrosyl proteins of molecular weights 42 and 50 kD, and five minor bands at 40, 90, 116, 130, and 150 kD. While immunofluorescent staining remained throughout the fertilized egg cytoplasm, there were dynamic changes in the staining intensity of single bands. The 90 kD immunoreactive band increased in intensity, and the 40 and 42 kD bands disappeared by 15 min after fertilization. Loss of the 40 and 42 kD bands was due to dephosphorylation by okadaic acid-sensitive phosphatase(s). The 50 kD immunoreactive protein was unchanged up to the 8-cell stage and was still present in blastulae, indicating its importance throughout fertilization and early development. Alterations in the pattern of phosphotyrosine-containing proteins during fertilization did not depend on nascent proteins and could not be completely mimicked by increasing intracellular calcium, pH, and protein kinase C activity alone. Since changes in the fertilization pattern of phosphotyrosyl proteins occurred during formation of the sperm aster and mitotic spindle, we analyzed the role of protein tyrosine kinase activity in these processes using the tyrosine kinase specific inhibitor, erbstatin. Both the sperm aster and mitotic spindle were disrupted, indicating an involvement of tyrosine phosphorylation in these processes during interphase and mitosis. We conclude that the changes in phosphotyrosyl proteins play an important role in fertilization and early development of sea urchin eggs. Control of microtubule assembly into the sperm aster and mitotic spindle of the first cell cycle are examples of such roles.

Animals↗