Ovarian stimulation of marmoset monkeys (Callithrix jacchus) using recombinant human follicle stimulating hormone.

To reduce the number of animals required for controlled studies of marmoset oocytes and early embryos, a superovulation protocol was developed for the common marmoset. Females were given up to 50 i.u./day recombinant human follicle stimulating hormone (FSH)--(r-hFSH) for 6 days. Ovaries were visualized by a modified laparoscopic technique and follicular aspiration was performed using a needle and suction apparatus inserted directly through an otoscope speculum. The number of follicles + ovulation points (+/- S.E.) was 2.9 (+/- 0.2) in controls and 14.1 (+/- 1.6; P < or = 0.001) in the 50 i.u. r-hFSH per day animals. Oocytes, typically at the germinal vesicle stage at collection, extruded a first polar body within 26 hours. In vitro fertilization was performed and embryos developed to the hatched blastocyst stage (34%). With many high quality oocytes and the ability to synchronize cycles, the marmoset is a valuable primate model for examining nuclear reprograming and early embryonic events.

Parthenogenetic activation of marmoset (Callithrix jacchus) oocytes and the development of marmoset parthenogenones in vitro and in vivo.

Mammalian oocytes can be induced to resume meiosis without fertilization, and the resulting parthenogenetic embryos carry only maternal chromosomes. Human oocytes can be activated by many chemical and physical stimuli, but postimplantation studies of human parthenogenetic embryos are not ethically acceptable. The common marmoset monkey (Callithrix jacchus) is a good model for studying primate parthenogenetic development postimplantation, since follicular aspiration, embryo transfer, and early postimplantation development of biparental embryos have already been described. Marmoset oocytes were either subjected to two series of six electrical pulses (DC; 2 kV/cm and 70 microsec) or were incubated in 7% ethanol in PBS. Ninety-two percent (68 of 74) and 20% (8 of 40) of marmoset oocytes were activated by electrical stimulus or ethanol, respectively. Parthenogenetic (n = 3) or in vitro-fertilized (n = 2) embryos were transferred at the 4-cell stage to synchronized recipient female marmosets (n = 5). Progesterone, chorionic gonadotropin, and inhibin in the peripheral plasma of recipient animals were measured. After 33 days of gestation, recipient animals were perfused and the uteri were collected. The 2 females that had received biparental embryos and 2 of the 3 females that had received parthenogenetic embryos displayed biochemical and histological evidence of implantation. This is the first report that a primate embryo comprising only parthenogenetic cells is capable of implantation. This highlights the need to scrutinize levels of parthenogenesis associated with human assisted reproductive technologies. Marmoset parthenogenones also provide a unique model for elucidating the roles of parental genomes in primate development.

Embryonic stem cell lines derived from human blastocysts.

Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

Neural differentiation of rhesus embryonic stem cells.

Primate embryonic stem (ES) cells are capable of indefinite, undifferentiated proliferation and maintain the potential to differentiate to trophoblast and derivatives of embryonic endoderm, mesoderm, and ectoderm. We previously reported that neural differentiation by rhesus ES cells in teratomas includes tissue with a remarkable resemblance to neural tube (Thomson et al. 1995). Here we examine a series of markers including a cell proliferation marker, neurofilament proteins, and glial fibrillary acidic protein (GFAP) in teratomas at 5, 6, 7, 9, and 12 weeks after rhesus ES cell transplantation into muscles of immunodeficient mice. All teratomas examined contained derivatives of all three embryonic germ layers. Neural differentiation included tissues resembling neural tube and embryonic ganglia, as well as individual dispersed neurons, and brain-like gray matter. Tumours of all ages contained neurons and proliferating cells, indicated by staining for neurofilament subunits and Ki67 antigens. Younger tumours contained no or few astrocytes indicated by the absence of GFAP staining, but as these tumours developed, there was an increase in astrocyte differentiation. The results indicate that normal neural differentiation is recapitulated, in part, by the differentiation of rhesus ES cells in teratomas. The differentiation of rhesus ES cells provides an important new model for understanding human neural differentiation.

Primate embryonic stem cells.

Primate embryonic stem (ES) cells are derived from preimplantation embryos, have a normal karyotype, and are capable of indefinite, undifferentiated proliferation. Even after culture for more than a year, primate ES cells maintain the potential to differentiate to trophoblast and derivatives of embryonic endoderm, mesoderm, and ectoderm. In this review, we compare the characteristics of ES cell lines from two primate species, the rhesus monkey (Macaca mulatta) and the common marmoset (Callithrix jacchus), with the characteristics of mouse ES cells and human embryonal carcinoma cells. We also discuss the implications of using primate ES cells to understand early human development and discuss the practical and ethical implications for the understanding and treatment of human disease.

Nonsurgical embryo transfer in the common marmoset monkey.

A technique for nonsurgical embryo transfer in common marmosets was developed. Transfers were either synchronous (ST) or asynchronous (AT). Synchronous transfers (embryo donor and the embryo recipient ovulated on the same day) were performed 5 to 8 days post-ovulation. Asynchronous transfers (embryo donor had ovulated at least 2 days before the embryo recipient) were performed when the recipient was 2 to 4 days post-ovulation (donor was 6 to 8 days post-ovulation). Four pregnancies from nine transfers (44%) were established by AT, and three pregnancies were carried to term. Only 1 of 11 transfers (9%) from ST resulted in a pregnancy, which was lost by Day 40 of gestation. Significantly more infants were born from AT (6 infants from 17 embryos; 35%) than from ST (0 infants from 22 embryos; 0%; p < 0.005). This technique allows experimental analysis of primate postimplantation development and provides a tool for conservation of endangered Callithrichid species.

Successful embryo transfer following artificial insemination of superovulated fallow deer (Dama dama).

Thirty-four European fallow deer (Dama dama dama) were randomly allocated into embryo donor (n = 12) or embryo recipient (n = 22) groups. All does were treated with controlled internal drug release (CIDR) devices for 14 days. Animals in the embryo donor group were further treated with 200 I.U. pregnant mare serum gonadotrophin (PMSG) and 0.5 units ovine follicle-stimulating hormone (FSH). PMSG was administered 72 h before withdrawal of CIDR devices and FSH was given in eight 0.063 unit injections at 12-hourly intervals starting at the time of PMSG administration. All embryo donor animals were inseminated, by laparoscopy in both uterine horns, 36 h after withdrawal of CIDR devices with 25 x 10(6) fresh spermatozoa collected from Mesopotamian fallow deer (Dama dama mesopotamica). Embryos were recovered by laparotomy on Day 3 (n = 6) or Day 6 (n = 6) after withdrawal of CIDR devices and the ovarian response was determined. In total, 22 embryos were transferred into the oviduct (2-4-cell stage, n = 14) or uterine horn (morula stage, n = 8) on Day 3 or Day 6 after withdrawal of CIDR devices respectively. The overall means (+/- s.e.m.) of total follicular response and corpora lutea were 24.2 +/- 3.5 and 14.1 +/- 3.6 respectively. The mean number of large unruptured follicles was higher on Day 6 than on Day 3 (13.5 +/- 2.9 v. 6.7 +/- 1.3, P < 0.05). The overall embryo recovery rate was 45.8%. There was no difference in pregnancy rate following transfer of embryos on either Day 3 or Day 6 (7/14 v. 5/8 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro fertilization and embryo development in the marmoset monkey (Callithrix jacchus).

Oocytes aspirated from preovulatory (i.e. > or = 2 mm) follicles of marmoset monkeys were graded for maturity according to the degree of cumulus expansion, grade I being most mature and grade IV least mature. After preincubation for 2-5, 9-11 or 21-29 h, 82% of oocytes could be fertilized using epididymal spermatozoa and only 2.3% were polyspermic. Fertilization rate was lowest (60%) in grade IV oocytes and all oocytes preincubated for 2-5 h (53%). Fertilization rate increased to 92% in oocytes preincubated for 21-29 h. Embryos developed in vitro to a mean of eight cells. Embryo development was unaffected by oocyte maturity but correlated with preincubation time. Oocytes preincubated for 2-5 h developed into embryos with significantly fewer cells than those preincubated for 9-11 or 21-29 h (P < 0.001). Fifty-six per cent of embryos showed delayed cleavage and these had fewer cells than non-delayed embryos (P < 0.001). When oocytes were preincubated for 2-5 h, development of all resulting embryos was delayed. However, only 17 and 58% of embryos developing from oocytes preincubated for 9-11 and 21-29 h, respectively, were delayed and this was independent of oocyte maturity.