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Biomedical subjects

Gerald Schatten

Publications and source records attributed to Gerald Schatten.

At least 19 recordsLinked to original sources
PubMed2005

Dogs cloned from adult somatic cells.

Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

Aging↗
PubMed2005

Patient-specific embryonic stem cells derived from human SCNT blastocysts.

Patient-specific, immune-matched human embryonic stem cells (hESCs) are anticipated to be of great biomedical importance for studies of disease and development and to advance clinical deliberations regarding stem cell transplantation. Eleven hESC lines were established by somatic cell nuclear transfer (SCNT) of skin cells from patients with disease or injury into donated oocytes. These lines, nuclear transfer (NT)-hESCs, grown on human feeders from the same NT donor or from genetically unrelated individuals, were established at high rates, regardless of NT donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and matched the NT patient's DNA. The major histocompatibility complex identity of each NT-hESC when compared to the patient's own showed immunological compatibility, which is important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made. Additional work remains to be done regarding the development of reliable directed differentiation and the elimination of remaining animal components. Before clinical use of these cells can occur, preclinical evidence is required to prove that transplantation of differentiated NT-hESCs can be safe, effective, and tolerated.

Adult↗
PubMed2004

WAVE1 intranuclear trafficking is essential for genomic and cytoskeletal dynamics during fertilization: cell-cycle-dependent shuttling between M-phase and interphase nuclei.

A-kinase-anchoring proteins (AKAP) help regulate the intracellular organization of cyclic AMP-dependent kinase (PKA) and actin within somatic cells. Elevated levels of cAMP also help maintain meiotic arrest in immature oocytes, with AKAPs implicated as critical mediators but poorly understood during this process. Here we test the hypothesis that the AKAP WAVE1 is required during mammalian fertilization, and identify a nuclear localization of WAVE1 that is independent of actin and actin-related proteins (Arp). Immunofluorescence and immunoprecipitation experiments show a redistribution of WAVE1 from the cortex in germinal vesicle (GV) oocytes to cytoplasmic foci in oocytes arrested in second meiosis (Met II). Following sperm entry, WAVE1 relocalizes to the developing male and female pronuclei. Association of WAVE1 with a regulatory subunit of PKA is detected in both Met II oocytes and pronucleate zygotes, but interaction with Arp 2/3 is observed only in Met II oocytes. WAVE1 redistributes to the cytoplasm upon nuclear envelope breakdown at mitosis, and concentrates at the cleavage furrow during embryonic cell division. Blocking nuclear pore formation with microinjected wheat germ agglutinin does not inhibit the nuclear localization of WAVE1, suggesting that this event precedes nuclear envelope formation. Neither depolymerization nor stabilization of actin affects WAVE1 distribution. Microtubule stabilization with Taxol, however, redistributes WAVE1 to the centrosome, and anti-WAVE1 antibodies prevent both the nuclear distribution of WAVE1 and the migration and apposition of pronuclei. These findings show that WAVE1 sequestration to the nucleus is required during fertilization, and is an actin-independent event that relies on dynamic microtubules but not nuclear pores.

Active Transport, Cell Nucleus↗
PubMed2004

Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction.

Therapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for "therapeutic cloning," though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or "fertilization failures" might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming.

Animals↗
PubMed2004

WAVE1, an A-kinase anchoring protein, during mammalian spermatogenesis.

BACKGROUND: Proper compartmentalization of signalling cascades is paramount to many intracellular activities during spermatogenesis and sperm function. In the present study we focus on the A-kinase-anchoring protein (AKAP) WAVE1, a member of the Wiskott-Aldrich syndrome (WASP) family of adaptor proteins, to study its localization throughout mammalian spermatogenesis. METHODS: Using transmission electron microscopy, immunocytochemistry and western blotting, we examined the distribution of WAVE1 and putative partners during mammalian spermatogenesis. The localization and association of PKA RII, the regulatory subunit II of protein kinase A, tyrosine kinase Abl, and small GTPase RAC1 were also explored. RESULTS: WAVE1 localization in spermatocytes and round spermatids coincided with Golgi apparatus distribution, whereas in elongated spermatids and testicular sperm WAVE1 localized to the mitochondrial sheath. Following epididymal passage, WAVE1 was found exclusively on the mitochondrial sheath, suggesting that the protein may function in this region. WAVE1 and PKA RII co-localized along the mitochondrial sheath, PKA RII concentrates in the mid-piece, and RAC1 associated with the post-acrosomal region and the connecting piece. The distribution of WAVE1, PKA RII and RAC1 is conserved in mature mouse, bull, baboon and human sperm. CONCLUSIONS: The data support the possibility of a functional signalling unit established by WAVE1 and its associated proteins in the mid-piece of maturing sperm.

Animals↗
PubMed2004

Centrosomal function assessment in human sperm using heterologous ICSI with rabbit eggs: a new male factor infertility assay.

Sperm centrosomal function was assessed by immunocytochemical analysis after the injection of human sperm into mature rabbit eggs. Three hours after intracytoplasmic sperm injection (ICSI), an astral microtubule array from the base of the human sperm was observed in the rabbit eggs. This sperm aster expanded in the egg cytoplasm, concomitant with pronuclear formation, and a dense microtubule array was organized at the time of pronuclear centration. Using fertile donor sperm, the sperm aster formation rate at 3 hr after ICSI was 35.0 +/- 1.5%. Using sperm from infertile patients, the average aster formation rate was lower (25.4 +/- 14.8%, P<0.05). Among infertile cases, there was no correlation between sperm aster formation rates and conventional parameters of semen analysis. However, the sperm aster formation rate correlated with the embryonic cleavage rate following human in vitro fertilization (IVF). These data suggest that this assay reflects sperm function during embryonic development after sperm entry and that reproductive success during the first cell cycle requires a functional sperm centrosome. Furthermore, sperm centrosomal function cannot be predicted from conventional parameters of semen analysis. We propose that insufficient centrosomal function could be the cause of certain cases of idiopathic infertility. These assays may lead to the discovery of new types of infertility, which have previously been treated as "unexplained infertility," and may also lead to the treatment of infertility incurable even by ICSI. Consequently, an accurate and relevant assay to help assure couples of the success of fertilization is warranted, perhaps prior to ICSI therapy.

Animals↗
PubMed2004

Can diabetes be cured by therapeutic cloning?

With the increasing incidence of diabetes mellitus (DM), it is imperative to develop novel treatments. Stem cells offer the potential for use as renewable sources of glucose-responsive, insulin-secreting cells. However, developing a consistent protocol to enrich beta-cells is not a trivial issue. The question whether embryonic, fetal, or adult stem cells offer particular advantages as the starting material remains to be resolved experimentally. While somatic cell nuclear transfer avoids many of the problems associated with heterologous transplantation, the problem of autoimmune destruction of the beta-cells in type 1 DM might still remain. This review summarizes the innovative treatment strategies for DM and considers the possible advantages and problems.

Cloning, Organism↗
PubMed2004

Paternal mitochondrial DNA transmission during nonhuman primate nuclear transfer.

Offspring produced by nuclear transfer (NT) have identical nuclear DNA (nDNA). However, mitochondrial DNA (mtDNA) inheritance could vary considerably. In sheep, homoplasmy is maintained since mtDNA is transmitted from the oocyte (recipient) only. In contrast, cattle are heteroplasmic, harboring a predominance of recipient mtDNA along with varying levels of donor mtDNA. We show that the two nonhuman primate Macaca mulatta offspring born by NT have mtDNA from three sources: (1) maternal mtDNA from the recipient egg, (2) maternal mtDNA from the egg contributing to the donor blastomere, and (3) paternal mtDNA from the sperm that fertilized the egg from which the donor blastomere was isolated. The introduction of foreign mtDNA into reconstructed recipient eggs has also been demonstrated in mice through pronuclear injection and in humans through cytoplasmic transfer. The mitochondrial triplasmy following M. mulatta NT reported here forces concerns regarding the parental origins of mtDNA in clinically reconstructed eggs. In addition, mtDNA heteroplasmy might result in the embryonic stem cell lines generated for experimental and therapeutic purposes ("therapeutic cloning").

Animals↗
PubMed2003

Golgi dynamics during meiosis are distinct from mitosis and are coupled to endoplasmic reticulum dynamics until fertilization.

One current theory of the Golgi apparatus views its organization as containing both a matrix fraction of structural proteins and a reservoir of cycling enzymes. During mitosis, the putative matrix protein GM130 is phosphorylated and relocalized to spindle poles. When the secretory pathway is inhibited during interphase, GM130 redistributes to regions adjacent to vesicle export sites on the endoplasmic reticulum (ER). Strikingly, meiotic maturation and fertilization in nonrodent mammalian eggs presents a unique experimental environment for the Golgi apparatus, because secretion is inhibited until after fertilization, and because the centrosome is absent until introduced by the sperm. Here, we test the hypothesis that phosphorylated GM130 associates not with meiotic spindle poles, but with ER clusters in the mature bovine oocyte. At the germinal vesicle stage, phosphorylated GM130 is observed as fragments dispersed throughout the cytoplasm. During meiotic maturation, GM130 reorganizes into punctate foci that associate near the ER-resident protein calreticulin and is notably absent from the meiotic spindle. GM130 colocalizes with Sec23, a marker for ER vesicle export sites, but not with Lens culinaris agglutinin, a marker for cortical granules. Because disruption of vesicle transport has been shown to block meiotic maturation and embryonic cleavage in some species, we also test the hypothesis that fertilization and cytokinesis are inhibited with membrane trafficking disruptor brefeldin A (BFA). Despite Golgi fragmentation after BFA treatment, pronuclei form and unite, and embryos cleave and develop through the eight-cell stage. We conclude that, while the meiotic phosphorylation cycle of GM130 mirrors that of mitosis, absence of a maternal centrosome precludes Golgi association with the meiotic spindle. Fertilization introduces the sperm centrosome that can reorganize Golgi proteins, but neither fertilization nor cytokinesis prior to compaction requires a functional Golgi apparatus.

Animals↗
PubMed2003

Preferentially localized dynein and perinuclear dynactin associate with nuclear pore complex proteins to mediate genomic union during mammalian fertilization.

Fertilization is complete once the parental genomes unite, and requires the migration of the egg nucleus to the sperm nucleus (female and male pronuclei, respectively) on microtubules within the inseminated egg. Neither the molecular mechanism of pronucleus binding to microtubules nor the role of motor proteins in regulating pronuclear motility has been fully characterized, and the failure of zygotic development in some patients suggests that they contribute to human infertility. Based on the minus-end direction of female pronuclear migration, we propose a role for cytoplasmic dynein and dynactin in associating with the pronuclear envelope and mediating genomic union. Our results show that dynein intermediate and heavy chains preferentially concentrate around the female pronucleus, whereas dynactin subunits p150Glued, p50 and p62 localize to the surfaces of both pronuclei. Transfection of antibodies against dynein and dynactin block female pronuclear migration in zygotes. Both parthenogenetic activation in oocytes and microtubule depolymerization in zygotes significantly reduce the localization of dynein to the female pronucleus but do not inhibit the pronuclear association of dynactin. When immunoprecipitated from zygotes, p150Glued associates with nuclear pore complex proteins, as well as the intermediate filament vimentin and dynein. Antibodies against nucleoporins and vimentin inhibit pronuclear apposition when transfected into zygotes. We conclude that preferentially localized dynein and perinuclear dynactin associate with the nuclear pore complex and vimentin and are required to mediate genomic union. These data suggest a model in which dynein accumulates and binds to the female pronucleus on sperm aster microtubules, where it interacts with dynactin, nucleoporins and vimentin.

Animals↗
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↗