The PDC addresses various aspects of reproduction and fertility including Assisted Reproductive Technologies (ART), the cell biology of fertilization, and clinical problems of pregnancy loss due to genetic, epigenetic and environmental influences.

Variations between ART and natural fertilization are studied both in-vitro, comparing embryo development and viability, as well as in-vivo studying the behavior and development of nonhuman primate offspring produced through ART techniques. This information may lead to improvements in ART safety, as well as inform and reassure infertile patients and their physicians about the safety and efficacy of novel therapeutic approaches to infertility.

PDC investigators are also developing new fertility preserving options for male cancer survivors. Basic cell biology studies in fertilization are performed using quantitative computer-based and laser-scanning confocal microscopy for the discovery of critical, but subtle, events occurring during fertilization. The fundamental research implications include understanding the variations among male meiosis, female meiosis, embryonic mitosis and somatic mitosis; the unique features of the fertilization cell cycle that permit the restoration of totipotency; extranuclear inheritance of the centrosome, mitochondria and endomembranes; cytoplasmic complementation with pronuclei and aberrations after cloning.

Molecular Basis of Infertility

A detailed understanding of the cellular and molecular events during fertilization in humans is essential for diagnosing and treating infertility, as well as for the development of new approaches for managing reproduction and avoiding or treating birth defects. Some of our recent findings include chromosomal anomalies in intracytoplasmic sperm injection (ICSI) produced rhesus embryos (Luetjens, M., et al, 1999), centriole degeneration during spermatogenesis (Manandhar, G., et al 1999; 2000), and biparental inheritance of g-tubulin during fertilization (Simerly, C.R., 1999). We continue to study proteins involved in the sperm centrosome, the joining of the male and female pronuclei, and the biogenesis of the acrosome using embryos produced through a variety of artificial reproductive techniques.

Flawless sperm functioning is essential for the successful completion of fertilization in humans. Some of the questions we will answer in order to understand the causes of male infertility include: What are the constituents, roles and fates of centrosome molecules and microtubule-based molecular motors that form fertilization competent sperm during human spermiogenesis?; What membrane trafficking events are crucial to human spermiogenesis and is the SNARE hypothesis valid for membrane-mediated events in human sperm? (in collaboration with Dr. Joao Ramalho-Santos at the University of Coimbra, Portugal); and What are the behaviors and fates of the centrosome, molecular motors, and membrane trafficking molecules during infertility therapies, including ICSI, ELSI, ROSI, and can innovative assays with predictive valves be derived? Collectively, these approaches characterize the molecules present in human sperm membranes and centrosomes, the molecules bound to the sperm centrosome after exposure to egg cytoplasm, and the membrane dynamics and microtubule-organizing abilities of the sperm. This investigation, which explores defects in interactions of the sperm and oocyte as well as subtle aspects of sperm function not currently detected with existing methods, can translate into clinical applications for the diagnosis and possible treatment of idiopathic male infertility.

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Mitochondrial Inheritance during Primate Reproduction

Using the hypervariable regions of the D-loop found in the mitochondrial genome, we can follow inheritance patterns of mitochondria after nonhuman primate embryos are produced through various ART techniques. An infrequently used ART technique, cytoplasmic transfer (CT), invigorates an older egg by injection of part of a ‘younger’ egg’s cytoplasm prior to fertilization. Concerns have been raised as to the pattern of mitochondrial inheritance in these offspring. Similar concerns have been raised for offspring produced through nuclear transfer. In addition, male infertility has been linked to mutations within the mitochondrial genome. This work is being performed in collaboration with Dr. Justin St. John at the University of Birmingham, UK.

Fertility after Cancer Treatment

Prospects for surviving cancer are increasing with improved therapies. As the population of cancer survivors burgeons, quality of life after cancer is a growing concern. Infertility is a common side effect of chemotherapy and radiation treatment. In men this condition potentially can be treated by cryopreservation of germline stem cells prior to treatment and reintroduction of these cells into the testis upon its completion. Thus, through autologous transplantation of germ cells, it may be feasible for male cancer patients to recover natural fertility and father their own genetic offspring. The proof-in-principle for this approach is already established in mice, rats and goats. Development of a non-human primate model for fertility after cancer is an essential step that will enable translation of this cell-based therapy from the laboratory to the bedside. The principal investigator of these studies, Kyle Orwig, PhD has established expertise in germline development and stem cell transplantation, focusing primarily on rodent models. Working with Stefan Schlatt, PhD, who was the first person to perform germline stem cell transplantation in non-human primates, their studies are developing a chemo- and radiotherapy models in non-human primates that recapitulates the male reproductive deficits (oligospermia and azoospermia) observed in human cancer patients.

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Cloning by Somatic Cell Nuclear Transfer (SCNT) and Embryo Splitting

Researchers at the PDC are perfecting several methods for cloning in a variety of species to propagate valuable research models, provide identical twins or triplets to use in environmental, stem cell or vaccine studies, and determine the safety and efficacy of cloning capabilities to develop new animal resources. We have been successful in embryo splitting, with the birth of the first nonhuman primate from this technique, Tetra (Chan, A.W.S., et al, 2000), and have gone on to produce several additional offspring successfully. We are currently optimizing strategies for nuclear transfer (Dominko, T., et al., 2000) including comparing embryos prepared with somatic nuclei from fetal and adult rhesus tissue as well as embryonic nuclei. In addition, we are studying the organization of the microtubule cytoskeleton, the centrosome and the DNA during bovine and primate nuclear transfer of embryonic and somatic cells, in vitro.

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Genetic and Environmental Causes of Adverse Pregnancy Outcomes

Life begins in utero typically. Prenatal environmental exposures, coupled with each zygote’s genetics and epigenetic imprints, trace a life history path of health outcomes. The central theme of the Pittsburgh Specialized Center of Research on Sex and Gender Factors Affecting Women’s Health is the “Genetic and Environmental Causes of Adverse Pregnancy Outcomes”. This major, but still under-investigated, priority for women’s health urgently requires multidisciplinary research both for the health of adult women and also for the health of developing fetuses and infants. For women, recurrent spontaneous abortions (RSA) are devastating. We have identified transgenerational transmission of a ‘miscarriage gene’ that may be an extreme example of deviant genomic imprinting. Fetal outcomes are also of great importance, since in utero development of the fetus, both female and male, has implications for infant, adolescent, and adult health.

To address these issues, three research projects along with two research cores and an administrative core are funded under the directorship of Gerald Schatten, PhD and Kristine Lain, MD. Project I, “Pregnancy Loss: Genomic Imprinting and Skewed X-Inactivation” (J. Richard Chaillet, MD, PhD, P.I), investigates DNA methylation defects in mice responsible for genomic imprinting as well as skewed X-chromosome inactivation, responsible for RSA in women. Project II, “Epigenetic, Genetic and Environmental Regulation of Pregnancy in Primates” (Gerald Schatten, PhD, P.I. and Steve Caritis, MD, Co-P.I.), imaging primate pregnancies and inflammatory responses, addresses sex-specific genomic imprints in genetically controlled and experimentally-manipulated pregnancies. Project III (Julie DeLoia, PhD, P.I.), “Maternal and Fetal Consequences of Tobacco Smoke Exposure”, analyzes the consequences of smoke exposure in pregnant women and in murine models to understand the interaction of genetic variants that jeopardize fetal development and pregnancy. The Imaging Core A performs non-invasive micro-PET and MRI imaging with specific probes, including transgenic MRI/PET reporters. The Pregnancy Core B establishes and maintains pregnancies through conventional and artificial reproductive technologies (ART) in nonhuman primates and mice. The Administrative Core fosters intra- and inter-SCOR cooperation to facilitate and accelerate basic and clinical research. The multi-disciplinary, interactive, and collegial environment of the new Pittsburgh Development Center at Magee-Womens Research Institute and Foundation, on the contiguous campuses of the University of Pittsburgh and Carnegie-Mellon University, brings together accomplished teams of clinical and basic investigators inspiring innovations in non-invasive imaging of pregnancy outcomes. Taken together, this comprehensive investigation will answer major women’s health problems regarding the dynamic interplay among fetal and maternal genetics, sex-specific genomic imprints and consequences of our first environmental exposures.

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