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Biomedical subjects

Gerald Schatten

Publications and source records attributed to Gerald Schatten.

30 records · Page 2Linked to original sources
PubMed2003

LIS1 association with dynactin is required for nuclear motility and genomic union in the fertilized mammalian oocyte.

Mutations in the human LIS1 gene cause the devastating brain disorder lissencephaly. LIS1 also regulates microtubule dynamics; it interacts with the molecular motor cytoplasmic dynein and its cofactor dynactin, and is necessary for neuronal migration. Recently, LIS1 has been suggested to mediate pronuclear migration during fertilization. Here we use rhesus monkey and bovine oocytes, as well as pronucleate-stage bovine zygotes, to determine: Lis1 RNA expression using reverse transcription-polymerase chain reaction; LIS1 protein association with dynactin using immunoprecipitation, Western blot analysis, and immunocytochemistry; and LIS1 function in mediating genomic union using antibody transfection. We find that Lis1 RNA expression increases during fertilization, that LIS1 and dynactin subunit p150/(Glued) co-immunoprecipitate and co-localize to pronuclear surfaces, and that anti-LIS1 antibodies transfected into zygotes dramatically inhibit pronuclear migration and apposition. LIS1 is, therefore, essential to mediate genomic union in a process that involves the dynein-dynactin complex. These results shed light on an additional role for LIS1 and raise implications for human reproduction.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2002

Ubiquitin-dependent sperm quality control mechanism recognizes spermatozoa with DNA defects as revealed by dual ubiquitin-TUNEL assay.

Defective mammalian spermatozoa become ubiquitinated during epididymal passage, a mechanism that may mark the abnormal spermatozoa for proteolytic destruction (Sutovsky et al., 2001a: J Cell Sci 114:1665-1675). It is not known how such spermatozoa are recognized by the epididymal ubiquitination pathway and whether there is a selection against certain types of sperm defects. We examined the relationship between sperm ubiqutination, lifelong sperm morphology and sperm DNA defects using a single chanel, ubiquitin-activated flow cytometric assay, and a dual, ubiquitin-TUNEL assay. Semen samples from nine service sires of good-to-average fertility were screened. A positive correlation was found between sperm ubiquitination and the average frequency of morphological semen abnormalities from field evaluations performed throughout the reproductive life of individual sires. Sample correlation coefficients were r=0.65 for primary (head and tail) and r=0.60 for total semen abnormalities in the single channel assay. In a dual assay, we found a high, positive correlation (r=0.93) between the ubiquitin-positive sperm and the TUNEL positive sperm. Substantial correlations (r=0.47-0.64) were observed when the measurements from these two respective assays were compared for individual sires. While anti-ubiquitin antibodies recognized most of the TUNEL-positive sperm cells, the TUNEL-positive spermatozoa represented only a subset (approximately 20-40%) of all ubiquitin-positive cells. It appears that the ubiquitin-dependent sperm quality control, residing in the epididymal epithelium, has the ability to detect spermatozoa with apoptotic or necrotic DNA, while spermatozoa with defects other than DNA fragmentation are also recognized and ubiquitinated.

Animals↗
PubMed2002

VAMP/synaptobrevin as an acrosomal marker for human sperm.

OBJECTIVE: To determine the possible use of the mammalian acrosomal marker vehicle-associated membrane protein (VAMP)/synaptobrevin to detect acrosome abnormalities in human sperm. DESIGN: Analysis of human sperm after fixation and staining with an anti-VAMP antibody. SETTING: An academic research institution. PATIENT(S): Semen samples from consenting patients who were participating in an infertility treatment program. INTERVENTION(S): Human sperm samples were fixed, permeabilized with detergent, and examined by immunocytochemistry. MAIN OUTCOME MEASURE: Immunostaining. RESULT(S): Observation of sperm from patients with no obvious sperm morphological defects revealed normal looking acrosomes, as assessed by VAMP immunostaining. However, severe acrosome malformations were detected in other cases. The observations registered varied from the absence of a fully formed organelle in samples of patients with globozoospermia to abnormal VAMP staining in samples from patients with acrosomal defects. CONCLUSION: VAMP/synaptobrevin may be a useful marker for the functional assessment of acrosomal status in human sperm.

Acrosome↗
PubMed2002

Rhesus offspring produced by intracytoplasmic injection of testicular sperm and elongated spermatids.

OBJECTIVE: To establish pregnancies in rhesus monkeys using testicular sperm and elongated spermatids injected into oocytes. DESIGN: Comparative animal study. SETTING: Regional Primate Research Center. ANIMAL(S): Prime, fertile rhesus monkeys. INTERVENTION(S): Oocytes collected by laparoscopy from gonadotropin-stimulated female rhesus monkeys were injected with testicular sperm or elongated spermatids obtained from the testis of males. Cleavage stage embryos were transferred to surrogate females. MAIN OUTCOME MEASURE(S): Fertilization, embryo cleavage, and the establishment of pregnancies. Fertilization failures were fixed and processed for the detection of microtubules and chromatin configurations. RESULT(S): Fertilization, assessed by the presence of two pronuclei within 15 hours after injection, was 60% for intracytoplasmic sperm injection with testicular sperm and 47% for elongated spermatid injection. Fertilized zygotes co-cultured in Connaughts Medical Research Labs (CMRL) medium on a Buffalo Rat Liver cell monolayer resulted in hatched blastocysts after testicular sperm extraction-intracytoplasmic sperm injection and elongated spermatids. Embryos transferred at the 4- to 8-cell stage gave rise to three pregnancies: 2/3 from testicular sperm and 1/1 from an elongated spermatid. Three healthy infants were delivered by cesarean. Oocytes that failed to fertilize typically remained arrested in metaphase of meiosis. CONCLUSION(S): Testicular sperm and elongated spermatids can be used for fertilization in the rhesus monkey resulting in live births.

Animals↗
PubMed2002

Fate of sperm components during assisted reproduction: implications for infertility.

Studies in non-human primates highlight their suitability as preclinical models for investigating assisted reproduction techniques. The cytoskeletal events of fertilization in non-human primates are similar to those in humans in that they require a paternally derived centrosome. The centrosome, introduced by the sperm at fertilization, organizes a microtubule array that is responsible for bringing the parental genomes together at first mitosis. Incomplete functioning of the sperm centrosome during fertilization has been identified as a novel form of infertility that would not necessarily benefit from intracytoplasmic sperm injection (ICSI). The global use of ICSI to overcome male infertility has been very successful, although concerns remain regarding the long-term effects on children born after ICSI. The cytoskeletal events that occur during ICSI are quite different from the events of in vitro fertilization: a sperm selected for ICSI does not undergo typical oocyte interactions, and abnormal remodelling of the male pronucleus may result. The implications of these findings are discussed in relation to the safety of the ICSI technique.

Animals↗
PubMed2002

Golgi apparatus dynamics during mouse oocyte in vitro maturation: effect of the membrane trafficking inhibitor brefeldin A.

We have studied Golgi apparatus dynamics during mouse oocyte in vitro maturation, employing both live imaging with the fluorescent lipid BODIPY-ceramide and immunocytochemistry using several specific markers (beta-COP, giantin, and TGN38). In germinal vesicle oocytes the Golgi consisted of a series of structures, possibly cisternal stacks, dispersed in the ooplasm, but slightly more concentrated in the interior than at the cortex. A similar pattern was detected in rhesus monkey germinal vesicle oocytes. These "mini-Golgis" were functionally active because they were reversibly disrupted by the membrane trafficking inhibitor brefeldin A. However, the drug had no visible effect if the oocytes had been previously microinjected with GTP-gamma-S. During in vitro maturation the large Golgi apparatus structures fragmented at germinal vesicle breakdown, and dispersed homogenously throughout the ooplasm, remaining in a fragmented state in metaphase-II oocytes. Similarly to what has been reported using protein synthesis inhibitors, the presence of brefeldin A blocked maturation at the germinal vesicle breakdown stage before the assembly of the metaphase-I spindle. These results suggest that progression of murine oocyte maturation may require functional membrane trafficking.

Animals↗
PubMed2002

Control of membrane fusion during spermiogenesis and the acrosome reaction.

Membrane fusion is important to reproduction because it occurs in several steps during the process of fertilization. Many events of intracellular trafficking occur during both spermiogenesis and oogenesis. The acrosome reaction, a key feature during mammalian fertilization, is a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome. Once the sperm has crossed the zona pellucida, the gametes fuse, but in the case of the sperm this process takes place through a specific membrane domain in the head, the equatorial segment. The cortical reaction, a process that prevents polyspermy, involves the exocytosis of the cortical granules to the extracellular milieu. In lower vertebrates, the formation of the zygotic nucleus involves the fusion (syngamia) of the male pronucleus with the female pronucleus. Other undiscovered membrane trafficking processes may also be relevant for the formation of the zygotic centrosome or other zygotic structures. In this review, we focus on the recent discovery of molecular machinery components involved in intracellular trafficking during mammalian spermiogenesis, notably related to acrosome biogenesis. We also extend our discussion to the molecular mechanism of membrane fusion during the acrosome reaction. The data available so far suggest that proteins participating in the intracellular trafficking events leading to the formation of the acrosome during mammalian spermiogenesis are also involved in controlling the acrosome reaction during fertilization.

Acrosome↗
PubMedDate not supplied

The use of primates as models for assisted reproduction.

Evidence from donated human oocytes and embryos demonstrates that the spermatozoon contributes the 'centrosome', which is critical to fertilization, and that some cases of infertility in couples are related to defects in the pathways that reconstitute the zygotic centrosome. A greater understanding of these microtubule-mediated motility events that ensure normal sperm-oocyte interactions has been made easier by the use of non-human primate gametes. Our studies using rhesus monkey gametes have shown that the cytoskeletal events during fertilization by IVF and intracytoplasmic sperm injection (ICSI) are very similar to those of human fertilization, and that manipulations of non-human primate gametes may help to test the safety and improve current strategies for reproduction, as well as develop new techniques. ICSI results in abnormal nuclear remodelling, in part due to the persistence of VAMP (vesicle-associated membrane protein), the acrosome and the perinuclear theca on the sperm head, all of which are normally removed at, or close to, the oocyte cortex during natural and in-vitro fertilization. Progression through the first cell cycle in ICSI oocytes cannot be completed until these structures have been removed from the forming male pronucleus, demonstrating unique differences between ICSI and IVF. While ICSI is of enormous therapeutic value for the treatment of male infertility, fundamental research using clinically relevant animal models is only now unravelling the cellular and molecular events that permit fertilization by sperm microinjection.

Animals↗