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D P Wolf

Publications and source records attributed to D P Wolf.

At least 19 recordsLinked to original sources
PubMed2006

A comparative approach to somatic cell nuclear transfer in the rhesus monkey.

BACKGROUND: Despite the potential utility of primate somatic cell nuclear transfer (SCNT) to biomedical research and to the production of autologous embryonic stem (ES) cells for cell- or tissue-based therapy, a reliable method for SCNT is not yet available. Employing the rhesus monkey as a clinically relevant animal model, we have compared a conventional electrofusion method for SCNT with a one-step micromanipulation (OSM) method. METHODS: A prospective, randomized trial was conducted using only oocytes that were mature [metaphase II (MII)] at collection and a fibroblast-like cell line as nuclear donor cells (fetal fibroblasts). The embryos produced were characterized for in vitro developmental potential, cell number, karyotype and expression of nuclear mitotic apparatus (NuMA) and OCT-4. RESULTS: An in vitro blastocyst development rate of 24.4% was achieved with the OSM method, significantly higher than the 12.2% obtained following electrofusion. SCNT-produced embryos expressed normal karyotypes, cell numbers and NuMA and OCT-4 proteins in most cases. SCNT with male nuclear donor cells resulted in the production of male, SCNT blastocysts, eliminating the possibility of a parthenogenetic origin. Of the four fibroblast cell lines tested as nuclear donor cells, two supported the routine production of blastocysts following SCNT. CONCLUSIONS: The application of a modified SCNT technique (OSM) followed by embryo culture in hamster embryo culture medium-10 (HECM-10) allows, for the first time, the routine production of SCNT blastocysts, most of which appear normal by immunochemical, cytochemical and in vitro developmental criteria. These embryos will provide a resource for isolating ES cells and for studies of nuclear reprogramming by monkey cytoplasts.

Animals↗
PubMed2005

Development of a monkey model for the study of primate genomic imprinting.

An understanding of the role of imprinted genes in primate development requires the identification of suitable genetic markers that allow analysis of allele-specific expression and methylation status. Four genes, NDN (Necdin), H19, SNRPN and IGF2, known to be imprinted in mice and humans, were selected for study in rhesus monkeys along with two imprinting centres (ICs) associated with the regulation of H19/IGF2, NDN and SNRPN. GAPD was employed as a non-imprinted control gene. Primers designed to amplify polymorphic regions in these genes and ICs were based on human sequences. Genomic DNA was isolated from peripheral blood leukocytes of 93 rhesus macaques of Indian or Chinese-origin. Sequence analysis of amplicons resulted in the identification of 32 unique SNPs. Country-of-origin related differences in SNP distributions were evident. Since disruptions in imprinted gene expression and associated developmental abnormalities may result from in vitro embryo manipulation, we also examined imprinting in NDN, H19, SNRPN and IGF2 in rhesus monkey infants produced by natural mating or by ICSI. Muscle biopsies followed by RT-PCR and sequence analysis were performed in four heterozygous animals produced by natural mating and all four genes were expressed monoallelically supporting the conclusion that these genes are normally imprinted in monkeys. In the case of ICSI, five informative infants were selected based on parental analysis. Allele-specific studies indicated that the expected uniparental expression patterns were retained in animals produced from manipulated embryos. Moreover, methylation analysis revealed that CpG islands within H19/IGF2 and SNURF/SNRPN ICs were differentially methylated. The approach described here will allow examination of imprinting in the embryos and embryonic stem cells of the monkey.

Alleles↗
PubMed2004

Use of assisted reproductive technologies in the propagation of rhesus macaque offspring.

The assisted reproductive technologies (ARTs) as tailored to the production of rhesus monkeys at the Oregon National Primate Research Center (ONPRC) are described. Efficient fertilization of mature oocytes recovered by aspiration from females subjected to follicular stimulation was achieved with fresh or frozen sperm by intracytoplasmic sperm injection (ICSI). Embryo development to the early cleavage stage occurred at high frequency. Cryopreserved embryos showed high postthaw survival and were also transferred in efforts to establish pregnancies. Three methods of transfer were evaluated, two involving embryo placement into the oviduct, laparoscopy and minilaparotomy, and a nonsurgical, transcervical approach that resulted in uterine deposition. Early cleaving embryos (Days 1-4) were transferred into the oviducts of synchronized recipients with optimal results and pregnancy rates of up to 36%. Pregnancy rates were similar when two fresh or frozen embryos were transferred (28- 30%), although more than two embryos had to be thawed to compensate for embryo loss during freeze-thawing. Normal gestational lengths, birth weights, and growth curves were seen with ART-produced infants compared with infants produced by natural mating in the timed mated breeding (TMB) colony at the ONPRC. In 72 singleton pregnancies established following the transfer of ART-produced embryos, the live-birth rate, at 87.5%, was statistically identical to that for the TMB colony. Further development of the ARTs should result in increasing use of these techniques to augment conventional approaches to propagating monkeys, especially those of defined genotypes.

Animals↗
PubMed2004

Live birth after ovarian tissue transplant.

Radiation and high-dose chemotherapy may render women with cancer prematurely sterile, a side-effect that would be avoided if ovarian tissue that had been removed before treatment could be made to function afterwards. Live offspring have been produced from transplanted ovarian tissue in mice and sheep but not in monkeys or humans, although sex steroid hormones are still secreted. Here we describe the successful transplantation of fresh ovarian tissue to a different site in a monkey, which has led to the birth of a healthy female after oocyte production, fertilization and transfer to a surrogate mother. The ectopically grafted tissue functions without surgical connection to major blood vessels and sets the stage for the transplantation of cryopreserved ovarian tissue in humans.

Animals↗
PubMed2001

Human spermicidal activity of inorganic and organic oxidants.

OBJECTIVE: To identify prospective oxidants that rapidly immobilize human sperm upon contact with human semen. DESIGN: Inorganic, organic, and enzymatically-generated oxidants were mixed with human semen and spermicidal activity was tracked by a modified Sander-Cramer assay. SETTING: Commercial and university-based laboratories. PATIENT(S): Semen samples obtained through a university-based andrology laboratory. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Quantitation of spermicidal activity of test oxidants. RESULT(S): Sperm lost motility within 20 seconds of exposure to enzymatically generated free iodine (I(2)). Toluidine blue, phenazine methosulfate, or methylene blue exhibited some, albeit much less, spermicidal activity. Oxidants formed by mixing ascorbic acid with Fe(III)-EDTA, xanthine with xanthine oxidase, or by exposing sperm to the nitric oxide generator, SIN-1 (3-morpholinosydnonimine hydrochloride), were far less potent spermicidal agents. CONCLUSION(S): Free I(2) formed in situ and presented to semen is an extremely potent spermicide. Additional studies on methods of generating de novo I(2) may be beneficial in developing a novel new class of nondetergent-based spermicides.

Dose-Response Relationship, Drug↗
PubMed2001

Intrauterine insemination-ready versus conventional semen cryopreservation for donor insemination: a comparison of retrospective results and a prospective, randomized trial.

OBJECTIVE: To compare fecundity rates following intrauterine insemination (IUI) with donor sperm frozen conventionally versus an IUI-ready preparation. DESIGN: Both retrospective results and a prospective, randomized study where recipients were assigned to one of two sperm cryopreservation methods in each cycle of intrauterine insemination are reported. SETTING: University-based infertility practice, affiliated private practices, and andrology laboratory. PATIENT(S): Women desiring therapeutic insemination in an effort to establish pregnancy. INTERVENTION(S): Intrauterine insemination with donor sperm frozen conventionally or by an IUI-ready protocol. MAIN OUTCOME MEASURE(S): Cycle fecundity in donor IUI recipients. RESULT(S): In a retrospective analysis involving 642 inseminations in 209 recipients, 79 pregnancies were recorded for an overall pregnancy rate of 12.3% per insemination (or cycle): 11.3% with IUI-ready sperm and 13.9% with conventionally preserved sperm. In a follow-up prospective, randomized study, the pregnancy rate for IUI-ready sperm preparations was 36% per cycle (14 of 39) whereas that for conventionally preserved sperm was 19.6% per cycle (9 of 46). Thirteen of the 23 pregnancies occurred in the first study cycle of insemination; only two pregnancies were observed in patients undergoing more than four cycles of insemination. CONCLUSION(S): Cycle fecundity for IUI-ready donor sperm is equivalent to conventional cryopreserved sperm based on both prospective and retrospective assessments.

Cryopreservation↗
PubMed2001

Developmental competence of oocytes after ICSI in the rhesus monkey.

Oocyte quantity and quality are critical to assisted reproductive technology (ART), yet few assessments beyond counting metaphase II (MII) oocytes exist. In this study, 30 +/- 2 oocytes per cycle were recovered from rhesus monkeys subjected to follicular stimulation with human gonadotrophins, of which 15 +/- 1 were MII. Oocyte quality was investigated by monitoring the developmental potential of oocytes subjected to intracytoplasmic sperm injection (ICSI). Despite uniform fertilization rates (71 +/- 4%), progression of embryos to blastocysts varied when expressed as a monthly average, from 20 to 85%, with lows from February to April and again in October, which could be attributed to developmental failure of a significant number of oocyte cohorts (14 of 55). Blastocyst rates, after elimination of failed cohorts, were uniform over time (59 +/- 4%). Neither culture conditions, the number of follicular stimulations, nor the individual sperm or oocyte donor were associated specifically with developmental failure, suggesting that intrinsic differences between stimulation cycles account for the observed variation in developmental potential. The in-vivo developmental competence of ICSI-produced embryos grown to blastocysts in vitro was also assessed. Two ongoing pregnancies and the birth of a normal female, 'Blastulina', represent landmarks in efforts to expand the use of ART in the rhesus monkey.

Animals↗
PubMed2001

Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts.

BACKGROUND: Vitrification using the cryoloop procedure was evaluated for preservation of non-human primate blastocysts by comparing survival results from two different cryoprotectant mixtures with prior results from controlled rate cooling. METHODS: Rhesus monkey blastocysts were produced by intracytoplasmic sperm injection of mature oocytes from cycling females stimulated with recombinant human hormones. Morphologically well-formed blastocysts were divided between Procedure A (2.8 mol/l dimethylsulphoxide and 3.6 mol/l ethylene glycol with 0.65 mol/l sucrose and 25 micromol/l Ficoll in TALP-HEPES with 20% fetal bovine serum (TH20)) and Procedure B (3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20). After >48 h in liquid nitrogen, the removal of cryoprotectants was accomplished in the presence of a 3-step series of decreasing sucrose concentrations in TH20. Surviving embryos were co-cultured on buffalo rat liver cells. RESULTS: Of 16 blastocysts vitrified via Procedure A, 38% survived with minimal lysis and only one hatched in culture; in contrast, of 33 blastocysts vitrified by Procedure B, 85% survived and 71% hatched. Of 22 blastocysts cryopreserved by conventional slow cooling, 36% survived and 6% hatched. Transfer into three recipients, each with two embryos vitrified with Procedure B, resulted in a successful twin-term pregnancy. CONCLUSION: Modified cryoloop vitrification with a final solution of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol is a promising procedure for preserving Rhesus monkey blastocysts that is simple, rapid, and inexpensive.

Animals↗
PubMed2001

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.

Animals↗
PubMed2001

Nuclear transfer and cloning.

The use of nuclear transfer in human reproductive and therapeutic cloning is reviewed with attention on the origins of this technology from its evolution to the present. The successes and limitations of mammalian reproductive cloning are itemized. A case is made against the use of human reproductive cloning to reproduce an existing person, based on the unacceptable risks to the embryo, fetus, or newborn. However, support is extended for human therapeutic cloning involving the derivation and use of embryonic stem cells to treat human disease.

Animals↗
PubMed2000

Mammalian cloning: possibilities and threats.

The cloning of mammals originated with the production of limited numbers of genetically identical offspring by blastomere separation or embryo splitting. In the past few years, remarkable progress has been reported in cloning by nuclear transfer (NT) with donor nuclei recovered from embryonic, fetal or adult cells. Factors that contribute to the successful reprogramming of the transferred nucleus and the normal term development of the newly reconstructed embryo include the cell cycle stage of both the donor nucleus and recipient cytoplast, the timing of fusion and cytoplast activation, and the source of donor nuclei. The possibility of producing live offspring by somatic cell NT carries potential applications in animal husbandry, biotechnology, transgenic and pharmaceutical production, biomedical research, and the preservation of endangered species. However, the low efficiencies of cloning by NT coupled with high embryonic, fetal and neonatal losses may restrict immediate commercial applications in agriculture. These limitations notwithstanding, the greatest benefits and practical implications of this new technology could be in transplantation medicine and therapeutic cloning.

Animals↗
PubMed1999

Fecundability trends among sperm donors as a measure of donor performance.

OBJECTIVE: To examine fecundability trends among sperm donors. DESIGN: Retrospective analysis. SETTING: University-based sperm bank and donor insemination program. PATIENT(S): Sperm donors and recipients. INTERVENTION(S): A group of recipients underwent IUI with cryopreserved donor sperm. Fecundability was calculated for 20 sperm donors over 800 insemination cycles. MAIN OUTCOME MEASURE(S): Average fecundability per donor was compared for the first 40 cycles of a donor's use and for those donors within a group of more fertile recipients. Sperm parameters, recipient ages, and number of unique recipients for each donor were analyzed. RESULT(S): Average donor fecundability is constant; however, individual donors demonstrated differences among their fecundabilities (overall mean, 0.09; range, 0.01-0.26). These differences persisted for donors among a group of more fertile recipients (overall mean, 0.12; range, 0.02-0.35). A donor's fecundability at 15 cycles is predictive of his future performance. CONCLUSION(S): Differences in fecundability exist among sperm donors which cannot be discerned through routine semen parameters. Sperm donor fecundability should be analyzed periodically, and directors of sperm banks should consider discontinuing use of a donor whose outcome is substandard.

Fertility↗
PubMed1999

Development and integration of an extended embryo culture program.

OBJECTIVE: To study and evaluate a sequential, extended embryo culture system. DESIGN: Prospective study. SETTING: University-affiliated IVF clinic. PATIENT(S): All couples who were treated between October 1997 and July 1998. INTERVENTION(S): A standard human tubal fluid plus 10% serum substitute supplement (SSS) culture medium was used. The embryos were transferred to extended culture medium (S2 or G2) on day 3. MAIN OUTCOME MEASURE(S): Blastocyst formation and implantation and pregnancy rates. RESULT(S): Forty percent of the 20 donated cryopreserved embryos progressed to the blastocyst stage by day 6. Clinically, 7 (5.6%) of the 125 cycles did not result in a transfer. Blastocyst formation rates ranged from 33%-63% in the five study groups. Implantation rates ranged from 15%-52% and pregnancy rates ranged from 37%-75%. CONCLUSION(S): Extended culture to day 5 or 6 results in acceptable blastocyst formation rates, implantation rates, and pregnancy rates.

Adult↗
PubMed1999

Nuclear transfer in the rhesus monkey: practical and basic implications.

In early 1997, the birth of a lamb after transfer of the nucleus from an adult mammary gland cell into an enucleated oocyte, along with the production of rhesus monkeys by nuclear transfer of embryonic cells, marked a reemergence of the field of mammalian cloning. Clonally derived rhesus monkeys would be invaluable in biomedical research, and the commercial interests in transgenic sheep and cattle propagated by cloning are substantial. Nuclear transfer technology is under consideration in human in vitro fertilization clinics to overcome infertility secondary to advanced maternal age or mitochondrial-based genetic disease. Nuclear transfer involves preparing a cytoplast as a recipient cell, in most cases a mature metaphase II oocyte from which the chromosomes have been removed. A donor nucleus cell is then placed between the zona and the cytoplast, and fusion, as well as cytoplast activation, is initiated by electrical stimulation. Successful reprogramming of the donor cell nucleus by the cytoplast is critical--a step that may be influenced by cell cycle stage. Embryos produced by nuclear transfer are cultured in vitro for several cell divisions before cryopreservation or transfer to the oviduct or uterus of a host mother. The efficiency of producing live young by nuclear transfer in domestic species is low, with a high frequency of developmental abnormalities in both preterm and term animals. However, a number of pregnancies have now been established using fetal cells as the source of donor nuclei. The use of cell lines not only allows large clone sizes but also supports the ability to genetically manipulate cells in vitro before nuclear transfer. Ongoing research focused on the production of clonally derived rhesus monkeys using fetal fibroblasts and embryonic stem cells as the source of donor nuclei will be reviewed.

Animals↗
PubMed1998

Characterization of protein phosphatases in mouse oocytes.

Okadaic acid (OA) enhances the resumption of meiosis in mouse oocytes, indicating that serine/threonine protein phosphatase-1 (PP1) and/or PP2A is involved. However, specific identification of PP1 and/or PP2A in mouse oocytes has not been reported. Here we demonstrate that fully grown germinal vesicle-intact (GVI) mouse oocytes contain mRNA corresponding to two isotypes of PP1, PP1alpha and PP1gamma. In addition, the transcript for PP2A was also present. At the protein level only PP1alpha and PP2A were recognized in fully grown GVI oocytes by Western blot analysis. Neither of the PP1gamma spliced variant proteins, PP1gamma1 and PP1gamma2, was detectable. Immunohistochemical analysis of ovarian tissue from gonadotropin-stimulated adult mice resulted in subcellular localization of both PP1alpha and PP2A, but not PP1gamma, in oocytes from all stages of folliculogenesis. In primordial oocytes, PP1alpha and PP2A were present in the cytoplasm. In more advanced stages of oogenesis, PP1alpha, although still present in the cytoplasm, was highly concentrated in the nucleus, whereas PP2A was predominantly cytoplasmic with a distinct reduction in the nuclear area. Both PP1alpha and PP2A were immunodetectable in oocytes during the prepubertal period. Eleven-day-old mouse oocytes, considered OA-insensitive and germinal vesicle breakdown (GVB)-incompetent, displayed both PP1alpha and PP2A predominantly in the cytoplasm. By 15 days of age mouse oocytes, which are beginning to acquire OA sensitivity and GVB competence, showed a relocation of PP1alpha into the nucleoplasm while PP2A remained predominantly cytoplasmic. This is the first specific identification of PP1alpha and PP2A in mouse oocytes. The differential localization of PP1alpha and PP2A, in addition to the relocation of PP1alpha during the acquisition of meiotic competence, suggests that these PPs have distinct regulatory roles during the resumption of meiosis.

Animals↗
PubMed1998

Oocytes are a source of catecholamines in the primate ovary: evidence for a cell-cell regulatory loop.

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine beta-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes-but not those from which the oocytes had been removed-responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a beta-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced beta-adrenoreceptor activation.

Amino Acid Sequence↗
PubMed1998

Recent progress in mammalian cloning.

PURPOSE: Our purpose was to review recent progress in the use of nuclear transfer technology to produce genetically identical mammals. METHODS: A literature review was conducted. RESULTS: The reasons for cloning nonhuman mammals are manyfold including commercial, biomedical, and basic research applications. Individual steps in the nuclear transfer process are itemized, along with a detailed description of the specific approaches used in the production of Dolly, NETI, and DITTO. The potential application of nuclear transfer in the treatment of human infertility is also considered, along with bioethical concerns. Finally, insights are provided concerning the future application of cloning technology in rhesus macaques. CONCLUSIONS: The cloning of a lamb (Dolly) from an adult, mammary gland cell coupled with the successful production of rhesus monkeys (NETI and DITTO) by nuclear transfer of embryonic cells marks the beginning of a "Golden Age" in the development and application of somatic cell cloning technology in mammals.

Animals↗