G.B. Anderson

Birth of large calves that developed from in vitro-derived bovine embryos.

High birth weights were observed in calves that developed from bovine embryos produced by in vitro maturation (IVM) and in vitro fertilization (IVF) procedures. After IVM and IVF, embryos were either co-cultured in vitro with oviductal epithelial cells or transferred into the sheep oviduct for development to the blastocyst stage. Blastocysts were transferred to the reproductive tracts of recipient heifers and cows for development to term. Birth weights and gestation periods were compared between calves that developed from in vitro-derived embryos and calves born after artificial insemination (AI) of cows in the herd from which recipient females were selected. Gestation periods were not different among the groups (P > 0.05), but calves that developed from IVM/IVF-derived embryos co-cultured in vitro were larger at birth than calves born from IVM/IVF-derived embryos that developed into blastocysts in the sheep oviduct and calves born from AI (P < 0.001). Dystocia and calf mortality were associated with large calf size at birth. These data were collected from an experiment designed for other purposes, and confounding variables and small sample size could have influenced the observed differences in birth weights. Nevertheless, the extreme birth weights of some calves suggest that abnormal prenatal growth occurs in some IVM/IVF-derived bovine embryos and that conditions for co-culture to the blastocyst stage may exacerbate the problem.

On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos.

The efficiency of isolation and the characteristics of embryo-derived cell lines from murine, porcine, and ovine embryos cultured on STO feeders or homologous embryonic fibroblasts (HEF) feeders were compared. While murine isolated ICM or intact embryos plated on STO or HEF feeders gave rise to cell lines with embryonic stem cell-like (ES-like) morphology, ovine embryos did not. Cell lines with ES-like morphology were isolated from porcine intact embryos and isolated ICM when plated on STO feeders but not when plated on HEF. Neither murine nor porcine ES-like cell lines expressed cytokeratin 18 or vimentin. Unlike murine ES-like cell lines, porcine ES-like cells did not undergo observable differentiation in vitro or in vivo. Cell lines with epithelial-like morphology were isolated from porcine and ovine embryos. Both porcine and ovine epithelial-like cell kines expressed cytokeratin 18. When induced to differentiate in vitro, porcine and ovine epithelial-like cell lines formed vesicular structures. Electron microscopy revealed that the porcine vesicles were composed of polarized epithelial cells, each with a basally-located nucleus and an apical border containing numerous microvilli with a well organized microfilament core. The results of this study show that conditions which allow isolation of ES cells from murine embryos allow the isolation of porcine embryo-derived cell lines sharing some, but not all, the characteristics of murine ES cells.

Morphology and morphometry of in vivo- and in vitro-produced bovine concepti from early pregnancy to term and association with high birth weights

This study was designed to characterize conceptus development based on pre- and postnatal measurements of in vivo- and in vitro-derived bovine pregnancies. In vivo-produced embryos were obtained after superovulation, whereas in vitro-produced embryos were derived from established procedures for bovine IVM, IVF and IVC. Blastocysts were transferred to recipients to obtain pregnancies of single (in vivo/singleton or in vitro/singleton groups) or twin fetuses (in vitro/twins group). Ultrasonographic examinations were performed weekly, from Day 30 of gestation through term. Videotaped images were digitized, and still-frames were used for the measurement of conceptus traits. Calves and fetal membranes (FM) were examined and measured upon delivery. In vitro-produced fetuses were smaller than in vivo controls (P < 0.05) during early pregnancy (Day 37 to Day 58), but in vitro/singletons presented significantly higher weights at birth than in vivo/control and in vitro/twin calves (P < 0.05). From late first trimester of pregnancy (Day 72 to Day 93), placentomes surrounding in vitro-derived singleton fetuses were longer and thinner than controls (P < 0.05). At term, the presence of giant cotyledons in the fetal membranes in the in vitro group was associated with a larger cotyledonary surface area in the fetal horn (P < 0.05). The biphasic growth pattern seen in in vitro-produced pregnancies was characterized by conceptus growth retardation during early pregnancy, followed by changes in the development of the placental tissue. Resulting high birth weights may be a consequence of aberrant placental development due to the disruption of the placental restraint on fetal growth toward the end of pregnancy.

ESTABLISHMENT OF PLURIPOTENT CELL LINES FROM PORCINE PREIMPLANTATION EMBRYOS

Embryonic stem (ES) cells are pluripotent cells isolated from in vitro culture of preimplantation embryos. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotent, porcine embryo-derived cell lines could be isolated. Cell lines were established from in vitro culture of intact, porcine early hatched blastocysts and isolated inner cell masses (ICM) from intermediate and late hatched blastocysts on feeder layers prepared from permanent mouse embryonic fibroblasts (STO). The cells of these porcine embryo-derived cell lines had a morphology similar to that of murine ES cells, but colony morphology was more epithelial-like. The cell lines retained a normal diploid karyotype, consistently expressed alkaline phosphatase activity, and survived cryopreservation. When subjected to in vitro differentiation, either spontaneous or induced, the embryo-derived cell lines differentiated extensively into a wide range of cell types representing the 3 embryonic germ layers. In vivo pluripotency of the cells was demonstrated by birth of a chimeric piglet, documented by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. Such pluripotent embryo-derived cells provide a potential route for porcine genetic manipulation.

Age of ovary determines remaining life expectancy in old ovariectomized mice.

We investigated the capacity of young ovaries, transplanted into old ovariectomized CBA mice, to improve remaining life expectancy of the hosts. Donor females were sexually mature 2‐month‐olds; recipients were prepubertally ovariectomized at 3 weeks and received transplants at 5, 8 or 11 months of age. Relative to ovariectomized control females, life expectancy at 11 months was increased by 60% in 11‐month recipient females and by 40% relative to intact control females. Only 20% of the 11‐month transplant females died in the 300‐day period following ovarian transplantation, whereas nearly 65% of the ovariectomized control females died during this same period. The 11‐month‐old recipient females resumed oestrus and continued to cycle up to several months beyond the age of control female reproductive senescence. Across the three recipient age groups, transplantation of young ovaries increased life expectancy in proportion to the relative youth of the ovary. Our results relate to recent findings on the gonadal input upon aging in Caenorhabditis elegans and may suggest how the mammalian gonad, including that of humans, could regulate aging and determine longevity.

Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines

Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to 8-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), STO combined with BRL at a 9:1 and 1:1 ratio, STO with BRL-conditioned medium (STO + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:1 and 1:1 ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the STO and the STO with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (1:1), PEF:BRL (1:1), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by STO and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epithelial-like) cell lines over that of STO feeders.

The placenta as a contributor to production of large calves.

Unusually high birth weights frequently occur in calves born from cultured bovine embryos. The mechanism(s) through which in vitro manipulations during early cleavage are translated to enhanced fetal growth is (are) incompletely understood. Accelerated growth is primarily prenatal, and the placenta of an in vitro-derived conceptus could account for abnormal fetal growth. Results from a systematic comparison of placental morphology and function in bovine concepti produced in vitro versus in vivo are discussed.

Transplantation of Young Ovaries to Old Mice Increased Life Span in Transplant Recipients

Previously we reported that prepubertally ovariectomized mice that received young transplanted ovaries at a postreproductive age showed a 40% increase in life expectancy. To study this phenomenon in greater detail, 11-month-old ovariectomized and ovary-intact CBA/J mice underwent ovarian transplantation with 60-day-old ovaries or a sham surgery. Results from observations on transplant recipients in the current study extended our previous results. Whereas intact control mice lived an average of 726 days, transplant recipients lived an average of 770 days (i.e., 780 days for intact recipients and 757 days for ovariectomized recipients). If intact recipients had ceased reproductive cycling by the time of transplant, we observed a further increase in mean life span to 811 days. These results demonstrate that young ovaries enhanced longevity when transplanted to old mice and that ovarian status, examined by means of ovariectomy and ovarian transplantation, clearly influenced the potential of young transplanted ovaries to positively impact longevity.

Survival of porcine inner cell masses in culture and after injection into blastocysts

Isolation of embryonic stem cells has been documented only in the mouse and perhaps the hamster and cow. We report results of experiments designed to determine the effect of age of porcine embryos (6 through 10 d after the first day of estrus) on isolation of cell lines with embryonic stem cell-like morphology. The capacity of fresh and short-term cultured inner cell mass (ICM) cells to differentiate into normal tissues after injection into blastocysts was also measured. Few Day-6 ICM survived in culture to the first passage onto fresh feeder cells, but cell lines with embryonic stem cell-like morphology developed from Day-7 through Day-10 ICM. Isolation of embryonic stem cell-like colonies was achieved at a higher frequency from ICM isolated from older embryos, but embryonic stem cell-like colonies from older embryos also tended to differentiate spontaneously in culture. Viable porcine chimeras were born after injection of fresh ICM into blastocysts that were transferred to recipients for development to term; no chimeras were born from blastocysts injected with ICM subjected to short-term (1 to 6 d) culture. Germ-cell chimerism was confirmed in one of the chimeras. These results document that undifferentiated cells can be removed from porcine blastocysts, transplanted to other embryos, and contribute to development of normal differentiated tissues, including germ cells. Cells with embryonic stem-like morphology can be isolated in culture from ICM at various embryonic ages, but ICM from young blastocysts (e.g., Day-7 embryos) yield embryonic stem cell-like colonies at lower frequency than do ICM from older blastocysts (e.g., Day-10 embryos).

The limitation of conventional breeding programs and the need and promise of assisted reproduction in nondomestic species

The traditional concepts of captive propagation as a means to preserve biodiversity have limitations that are now well recognized. Too little space and a limited understanding of how to create optimal artificial environments prevent all but a few traditional breeding programs from meeting their ultimate goal of safeguarding targeted species. In order to achieve reasonable success we must be prepared to integrate new technologies in assisted reproduction with traditional propagation strategies and to view captive and noncaptive animals as a single resource. Methods such as cryogenics, artificial insemination and the transfer of in vivo- and in vitro-derived embryos have already been demonstrated for a limited number of endangered species. These techniques will likely be the foundation of broadly-based breeding programs for the conservation of many nondomestic animals in the future.