Transplanted bone marrow cells do not provide new oocytes but rescue fertility in female mice following treatment with chemotherapeutic agents.

It is generally accepted that mammalian females are born with a finite pool of oocytes and that this is the sole source of ovules throughout the reproductive life of the adult. This dogma was shaken in 2003 when researchers showed that the oocyte stock might be renewable in adult mammals. It has been proposed that hematopoietic stem cells might be a source of new oocytes. These discoveries have puzzled many researchers and remain controversial. In our study, we attempted to determine if transplanted bone marrow cells could provide new oocytes in PU.1 mice and in severe combined immunodeficiency (SCID) mice after treatment with chemotherapeutic agents. We also examined the possibility that grafted bovine embryonic ovarian cortex might provide an environment favoring such a response. We found no evidence that transplanted bone marrow cells provide new fertilizable oocytes in PU.1 mice, in SCID mice treated with chemotherapeutic agents, or with bovine embryonic ovarian tissue grafts. However, transplanted bone marrow cells have improved the fertility of SCID mice previously treated with chemotherapeutic agents. These data suggest that bone marrow cells cannot provide new oocytes but can positively influence ovarian physiology to improve the fertility of mice previously treated with chemotherapeutic agents.

Comparison of bulk enucleation methods for porcine oocytes.

Cloning of mammalian oocytes requires that the recipient oocyte is enucleated to remove all genetic material associated with the chromosomes. The procedure currently used in most species requires careful micromanipulation of oocytes treated with cytochalasin B to prevent structural damage. Although functional, this procedure requires time and limits the number of oocytes available for cloning, and our ability to understand the mechanisms of nuclear reprogramming. Therefore, this study aimed at evaluating different procedures to enucleate large pools of oocytes in a time-efficient manner. Two different approaches were tested. The first approach involved centrifugation of zona-free oocytes through a percoll gradient to separate the portion containing the chromatin from the cytoplasmic portion. The second used etoposide to prevent chromatin segregation at first metaphase and resulting in the expulsion of all chromosomes in the polar body. Using the chemical approach an average enucleation rate of 39.4 +/- 7.5% was obtained, while the centrifugation approach resulted in an average enucleation rate of 66.9 +/- 6. In terms of time efficiency, the control manipulation method takes 0.11 min and the centrifugation took an average of 0.52 min per oocyte. The MPF activity at the end of procedure was estimated through the measurement of H1 activity and as expected, the etoposide-cycloheximide treated oocytes had lower H1 activity which was restored by further incubation in the maturation medium for 5 hr while the centrifugation gave a nonsignificant intermediary result. In conclusion, the results presented suggest that both the chemical and the mechanical methods are usable alternatives to micromanipulation of oocytes to generate a large number of chromosome free cytoplasm for biochemical analysis. Mol. Reprod. Dev. 67: 70-76, 2004.

Making recombinant proteins in animals--different systems, different applications.

Transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. The ability of transgenic animals to produce complex, biologically active recombinant proteins in an efficient and economic manner has stimulated a great deal of interest in this area. As a result, genetically modified animals of several species, expressing foreign proteins in various tissues, are currently being developed. However, the generation of transgenic animals is a cumbersome process and remains problematic in the application of this technology. The advantages and disadvantages of different transgenic systems in relation to other bioreactor systems are discussed.