Preimplantation Development of Somatic Cell Cloned Embryos in the Common Marmoset (Callithrix jacchus)

Abstract

The somatic cell nuclear transfer technique has been applied to various mammals to produce cloned animals; however, a standardized method is not applicable to all species. We aimed here to develop optimum procedures for somatic cell cloning in nonhuman primates, using common marmosets. First, we confirmed that parthenogenetic activation of in vitro matured oocytes was successfully induced by electrical stimulation (three cycles of 150 V/mm, 50 microsec x 2, 20 min intervals), and this condition was applied to the egg activation procedure in the subsequent experiments. Next, nuclear transfer to recipient enucleated oocytes was performed 1 h before, immediately after, or 1 h after egg activation treatment. The highest developmental rate was observed when nuclear transfer was performed 1 h before activation, but none of the cloned embryos developed beyond the eight-cell stage. To investigate the causes of the low developmental potential of cloned embryos, a study was performed to determine whether the presence of metaphase II (MII) chromosome in recipient ooplasm has an effect on developmental potential. As a result, only tetraploid cloned embryos produced by transferring a donor cell into a recipient bearing the MII chromosome developed into blastocysts (66.7%). In contrast, neither parthenogenetic embryos nor cloned embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage. These results suggest that MII chromosome, or cytoplasm proximal to the MII chromosome, plays a major role in the development of cloned embryos in common marmosets.

Oocyte‐activating capacity of fresh and frozen–thawed spermatids in the common marmoset (Callithrix jacchus)

“…Such stimuli also enhanced the development of ICSI-derived embryos in these species (Probst & Rath, 2003;Suttner et al, 2000). For marmosets, information on artificial oocyte activation is limited, although it was reported that diploid parthenogenetic embryos produced by electric stimulation developed to the 8-cell stage (Sotomaru et al, 2009).…”

“…The common marmoset (Callithrix jacchus) has been used extensively for biomedical research because of its advantages of small size, ease of handling, and a relatively short gestation period (143-148 days). In 2009, it was reported that transgenic marmosets expressing enhanced green fluorescent protein were born and the transgene could be transmitted to offspring by in vitro fertilization (Sasaki et al, 2009). This was the first report on the establishment of gene-modified lines of primates.…”

Quantitative molecular assessment of chimerism across tissues in marmosets and tamarins

“…This, coupled with the development of somatic cell cloned embryos [17], the ability to produce induced pluripotent stem cells [18], and the aforementioned husbandry benefits, has ensconced the marmoset as a major new player in non-human primate research going forward. Several years prior, Callithrix jacchus was selected as the first New World monkey genome to be sequenced.…”

Cloning of non-human primates: the road "less traveled by"

“…Controlled ovarian stimulation in non-human primates is routinely induced by administration of exogenous gonadotropins (Sotomaru et al , 2009; Zelinski-Wooten et al , 1995). In rhesus macaques, starting at menses (day 1–4 of their menstrual cycle), females receive twice-daily injections of recombinant human follicle stimulating hormone (FSH; Organon; 30 IU, im) for 8 days and recombinant human luteinizing hormone (LH; Ares Serono; 30 IU, im) on days 7–8 of the stimulation protocol.…”

“…In our current protocols, we recover oocytes 35–36 hours post-hCG to allow oocyte maturation and spindle migration away from the polar body, thus making it easier to visualize and enucleate the meiotic spindle. In the common marmoset, ovarian stimulation commonly entails a single, daily injection of 50 IU FSH for 11 days, followed by the administration of 75 IU hCG on the 12 th day of the stimulation protocol (Sotomaru et al , 2009). Germinal vesicle-stage oocytes, enclosed in cumulus cell-oocyte complexes, are recovered 16 hours post-hCG injection and placed in maturation medium for approximately 22–24 hours (Sotomaru et al , 2009).…”