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The common marmoset (Callithrix jacchus) is commonly used as a subject model animal in experimental research. The species has several advantages compared with other laboratory primates and we succeeded in creating a transgenic (Tg) marmoset with germline transmission of the transgene, the first time in a nonhuman primate. We have been attempting to further improve marmoset reproductive technology, which is more similar to that of humans than rodent experimental animals, such as mice. We have produced many genetically modified marmosets as human disease models and have also improved marmoset reproductive techniques to obtain many fertilized embryos and neonates. For ethical reasons, it is difficult to perform human reproductive studies; thus, we must rely on nonhuman primate models in basic research. For this reason, reproductive studies of marmosets may help the development of assisted reproduction technologies (ART) for humans and may also be useful in human preclinical studies. In this minisymposium, we describe practical marmoset reproductive technologies performed at the Central Institute for Experimental Animals (CIEA) and discuss our planned future research using marmosets in reproductive studies.
Assisted reproductive technologies (ARTs) in mice were recently advanced when two long-existing technical barriers were overcome. The first barrier was the limited number of mature oocytes after conventional superovulation, especially in inbred strains of mice. A combination of estrous cycle synchronization and antiinhibin serum treatments increased the number of collected oocytes from female mice by approximately 3–4 times in many strains. The second barrier was the low fertilization rate after in vitro fertilization (IVF) using frozen-thawed spermatozoa. The addition of reduced glutathione in the fertilization medium dramatically increased the IVF yields, even in cryopreserved/warmed spermatozoa from the C57BL/6J strain, which is one of the strains most sensitive to cryoinjury. This result encouraged the use of cryopreserved spermatozoa in mouse strains worldwide for the preservation and transportation of their genetic characteristics. The final yield to produce offspring from one female was increased from 9 to 30. In IVF with cryopreserved spermatozoa from the C57BL/6J strain, the final yield using these technological innovations was estimated to be ninefold higher than previously. Following this improvement, the efficiency of ARTs in mice was increased dramatically and the decrease in the number of euthanized animals contributes to animal welfare and reduces labor and expense.
The regulation of mammalian oogenesis in vivo is complicated because of numerous constantly changing events caused by ovarian cells interacting with or influencing each other. One of the most intractable questions for nearly the last 80 years has been the mechanism controlling the maintenance of meiotic arrest and the resumption of oocyte meiosis in a pre-ovulatory follicle. The question is now mostly resolved, as the regulatory mechanisms of cGMP, cAMP, and the NPPC/NPR2 system in the follicle, have recently been uncovered. Oocyte growth in vitro has also been the subject of extensive research utilizing growing oocytes at various stages in several species, including mice, cattle, pig, sheep, goat, and horse. Remarkably, the first reconstitution of the entire process of mammalian oogenesis in vitro from primordial germ cells (PGCs) was recently achieved in mice. Furthermore, even PGC-like cells, originally produced from mouse embryonic stem cells and induced pluripotent stem cells, can develop into functional oocytes in vitro with the help of gonadal somatic cells of female mouse fetuses. These updated findings and newly developed culture systems will assist in gaining a better understanding of the mechanisms of oogenesis and will also lead to the creation of new gamete resources for mammals.
Human oocytes have the aggregated chromosome phase (AC phase) during the first and second meiosis. This needs to be better understood, as the timing of ICSI significantly influences ART outcomes. In fact, performing ICSI after the completion of MII spindle formation is known to improve successful fertilization and embryo development. This human AC phase should also be taken into consideration in the application of nuclear transfer/mitochondrial replacement for patients suffering from severe mitochondrial diseases, to prevent the transmission of these diseases to their offspring, with the aim of limiting the risk of mitochondrial carryover. The possible risks and benefits of AC transfer and other procedures for mitochondrial replacement are reviewed and discussed in this paper.
Age-associated telomere shortening in oocytes and granulosa cells is considered a sign of ageassociated decline in oocyte quality. The present study examined the effect of aging on telomere lengths (TLs) in bovine oocytes, embryos, and granulosa cells, as well as the relationship between the TLs in oocytes and granulosa cells. TL was directly assessed by real-time PCR, using a telomeric standard of 84 bp length TTAGGG, repeated 14 time). TLs in immature oocytes derived from early antral follicles (EAFs) and antral follicles (AFs) as well as for in vitro matured oocytes derived from aged cows (>120 months) were shorter than their respective counterparts in younger cows (20–70 months, 0.45-, 0.82-, and 0.84- fold, respectively, P < 0.05). Telomeres elongate extensively during embryo development until the blastocyst stage (4.2-fold, P < 0.05); however, TLs in the blastocysts did not differ between the two age groups. TLs in the granulosa cells of both AFs and EAFs were shorter in aged cows than in younger cows, and showed a positive correlation with TLs in oocytes (r=0.66, P < 0.05). In conclusion, aging affects TL in oocytes, and the TLs in granulosa cells and oocytes are correlated.