Typescript (photocopy). / Digitized by Kansas Correctional Industries

Multiplex polymerase chain reaction (PCR) assays were designed that incorporated primer pairs for the sex determining region on the Y chromosome, Sry, and one or two control sequences. A triplex and a duplex assay were created involving Sry and Dax, a single copy X chromosome gene which is involved in the female sex determination pathway. The third sequence in the triplex assay was a repetitive Y chromosome sequence, YR. A minimum of 2.5 x 10-3 to 2.5 x 10 -4 m g/ m L of male DNA and 2.5 x 10-5 m g/ m L of female DNA was required if a single multiplex PCR was performed. To demonstrate that sex determination of preimplantation porcine embryos is possible, morulae were collected 5 d post insemination from pregnant mare serum gonadotrophin (PMSG)/human chorionic gonadotrophin (hCG)-treated 60--70 kg prepubertal gilts. Embryos were biopsied using a micromanipulator and cells were placed into individual microcentrifuge tubes for further analysis. Embryos were then cultured to the blastocyst stage. A total of 315 embryos were sexed via the PCR assay with a resultant female/male (%) ratio of 73/27. When 94 embryos from heavier gilts (90--100 kg) were sexed in a similar assay, the resultant female/male ratio was 60/40. Attempts were made to correlate these results with karyotypes. Five transfers of sexed embryos into synchronized recipients were attempted. None of these resulted in pregnancies; although return to estrus was delayed by two to eight days, in four out of the five recipients. Our findings suggest that PCR amplification of the Sry gene can be a reliable method for sexing porcine embryos. It does appear that embryo quality is critical for both the PCR assay and subsequent successful culture. (Abstract shortened by UMI.)

Currently more than 300 million adults are obese and 1 billion are overweight throughout the world. Obesity is frequently accompanied by an array of health conditions such as hyperglycemia, hyperlipidemia, hypertension, cardiovascular diseases, and diabetes which are all considered to be part of what is now known as the metabolic syndrome. The role of adipose tissue as an endocrine organ has been emphasized by the characterization of its hormones: leptin, adiponectin and resistin. All three proteins regulate energy utilization. Over the past decade, leptin and resistin have also been shown to affect the reproductive system. This suggests that other adipocytokines, such as adiponectin, may also affect reproduction. This relationship was investigated using a porcine in vitro maturation system. When porcine cumulus oocyte complexes were matured in the presence of 30mug/mL of recombinant adiponectin an improvement in the meiotic maturation was observed. Moreover, maturation of denuded oocytes revealed that adiponectin acts through the cumulus cells to improve meiotic maturation of porcine oocytes. Finally, maturation of cumulus-oocyte complexes in the presence of MAPK pathway inhibitors suggested that adiponectin acts at or downstream of MEK1/2 and 38MAPK. This study shows, for the first time, an effect of adiponectin on porcine oogenesis. Further investigation will determine whether adiponectin also affects embryo development.

No description available.

No description available.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Graduation date: 1997

The objective of this study was to investigate whether or not the treatment of nuclear donor cells with inhibitors of protein synthesis and cyclin-dependent kinases (CDKs) affect the development of swine embryos produced by somatic cell nuclear transfer. Host oocytes were derived from pre-pubertal females and matured in vitro for 42-46 h under standard conditions. Nuclear donor cells were fetal fibroblasts maintained in culture for 2 to 6 passages. Oocytes were reconstructed with cells treated for 22-24 hours with cycloheximide (CHX; 10mug/ml), roscovitine (ROS; 25 muM), the combination of CHX + ROS (CR), or untreated cells. Two hours after reconstruction, the oocytes were activated using ionomycin (15muM/5 min) and strontium chloride (10mM/4h), maintained for 6 h in the presence of cytochalasin B (7.5mug/ml) and CHX (10mug/ml), and then cultured in porcine zygote medium (PZM3) for 6 days. The cleavage rate, 63.7% (n=318), 55.2% (n=99), 56.7% (n=107) and 60.6% (n=347), at 48 h post-fusion were not significantly different between embryos derived from ROS, CHX, CR and control cells, respectively. Developmental rate to blastocyst stage was higher for embryos reconstructed with ROS (12.2%) and untreated cells (12.1%) when compared to CHX (5.7%) and CR (4.9%). Blastocysts produced with ROS treated cells had similar number of nuclei compared to embryos reconstructed with untreated donor cells (30.9+/-10.4 vs. 32.2+/-8.0). Phosphorylated H2A.X (gammaH2A.X) was highly expressed in donor cells treated with CR compared to non treated cells, but it was similarly expressed in most of 1-cell stage embryos reconstructed with control or treated cells. Flow cytometry analysis showed that the majority of the fibroblasts were at G 0/G1 phase of the cell cycle at the time of nuclear transfer. It was concluded that the treatment of nuclear donor cells with inhibitors of protein synthesis and CDKs did not improve the in vitro development of somatic cell nuclear transfer embryos in pigs.

No description available.

Graduation date: 1996