Health and reproductive profiles of malaria antigen-producing transgenic goats derived by somatic cell nuclear transfer.

Nuclear transfer (NT) using transfected primary cells is an efficient approach for the generation of transgenic goats. However, reprogramming abnormalities associated with this process might result in compromised animals. We examined the health, reproductive performance, and milk production of four transgenic does derived from somatic cell NT. Goats were derived from two fetal cell lines, each transfected with a transgene expressing a different version of the MSP-1(42) malaria antigen, either glycosylated or non-glycosylated. Two female kids were produced per cell line. Health and growth of these NT animals were monitored and compared with four age-matched control does. There were no differences in birth and weaning weights between NT and control animals. The NT does were bred and produced a total of nine kids. The control does delivered five kids. The NT does expressing the glycosylated antigen lactated only briefly, probably as a result of over-expression of the MSP-1(42) protein. However, NT does expressing the non-glycosylated antigen had normal milk yields and produced the recombinant protein. These data demonstrated that the production of healthy transgenic founder goats by somatic cell NT is readily achievable and that these animals can be used successfully for the production of a candidate Malaria vaccine.

Cloned transgenic offspring resulting from somatic cell nuclear transfer in the goat: oocytes derived from both follicle-stimulating hormone-stimulated and nonstimulated abattoir-derived ovaries.

The use of nuclear transfer (NT) techniques to create transgenic offspring capable of producing valuable proteins may have a major impact on the pharmaceutical market. Our objective was to compare the in vivo developmental potential of NT embryos produced from the fusion of transgenic donor cells with cytoplasts prepared from either FSH-stimulated ovaries or nonstimulated abattoir-derived ovaries. Donor cells were prepared from a transgenic fetus carrying the gene for human antithrombin III as a marker and used within four to eight subpassages. Cells were serum deprived for 4 days prior to cytoplast transfer. Oocytes were enucleated by removing the metaphase plate using a DNA stain and epifluorescent illumination. Donor cells were fused to enucleated oocytes by electric pulse and then chemically activated. There was no difference in the number of transferable embryos produced from cytoplasts of FSH-stimulated ovaries or from the fusion of cytoplasts from abattoir ovaries, nor was there a difference in the number of pregnancies established per recipient with either treatment. All pregnancies from both groups culminated in the births of healthy female kids (five total). To our knowledge, this is the first report of cloned goats produced from NT using cytoplasts derived from abattoir ovaries.

Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperm injection.

To evaluate the potential for fertilization by sperm injection into fish eggs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological changes of gametes on the possible performance of intracytoplasmic sperm injection (ICSI), four different combinations of injection conditions were conducted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval stages, and another 3 (2%) developed normally and hatched. The highest fertilization rate (blastodisc formation) was achieved by injection of activated spermatozoa into nonactivated eggs (35%). Injections were most effective when performed within the first hour after egg collection. Flow cytometric analysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Our study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.

Comparing follicle stimulating hormone from two commercial sources for oocyte production from out-of-season dairy goats.

Until recently, two sources of follicle stimulating hormone (FSH-P; Schering-Plough; Kenilworth, NJ and Super-Ov; FSH-SOV; AUSA International, Tyler, TX) have been commercially available in the United States and routinely used for superovulation of ruminants. Because there have been no comparative follicle stimulating hormone studies on small ruminants, we determined the difference between the number of follicles induced and the number of oocytes that can subsequently be harvested from goats stimulated with either of these two follicle stimulating hormone products. Anestrous Saanen does were fitted with a progestin implant then randomly assigned to one of two ovarian stimulation groups. Starting 4 d after introducing the progestin implant, donors in treatment 1 were administered daily injections of FSH-P for 4 d. Does in treatment 2 were similarly treated but were administered FSH-SOV for 4 d. Follicle aspirations were performed by laparotomy in the morning of treatment d 8. In summary, no difference was detected between the two stimulatory agents for the number of follicles and quality of oocytes harvested from stimulated does, indicating that these two commercial FSH products could be used successfully for ovarian stimulation of anestrous dairy goats.

Development of in-vitro-derived bovine embryos cultured in 5% CO2 in air or in 5% O2, 5% CO2 and 90% N2.

To evaluate the effects of a three gas mixture of 5% O2, 5% CO2 and 90% N2 (OCN) on preimplantation embryo development, bovine in-vitro fertilization (IVF) oocytes were cultured in a defined medium (mBECM) with various supplements either under 5% CO2 in air or under OCN. When cultured in mBECM alone, embryo development was significantly stimulated in OCN compared to 5% CO2 in air (experiment 1). In the OCN atmosphere, blastocyst formation was further increased after addition of fetal bovine serum (FBS; 10%) or FBS + cumulus granulosa cells (CGC) to mBECM. The ratio of blastocysts to 8-cell embryos, number of hatched blastocysts and embryo diameter were markedly increased, and zona thickness was decreased after FBS addition. However, development up to the morula stage was fully supported by mBECM alone. There was no significant effect of beta-mercaptoethanol (ME; 10 microM) in OCN. In the 5% CO2 atmosphere, embryo development was significantly (P < 0.05) enhanced after addition of FBS + CGC + ME. In experiment 2, in OCN, FBS added at 60 h post-insemination was effective in stimulating blastocyst formation, but changes in medium volume per oocyte from 13.6 to 1.36 microliters had only a marginal effect. In conclusion, OCN gas mixture provides a suitable atmosphere for early embryo growth in vitro and mBECM + FBS in the optimal culture medium under this atmosphere.

Effects of reduced maternal lipoprotein-cholesterol availability on placental progesterone biosynthesis in the baboon.

Maternal low density lipoprotein (LDL) is the principal source of cholesterol substrate for progesterone biosynthesis in the primate placental syncytiotrophoblast. The relationship of LDL-cholesterol availability and other potential cholesterol-yielding pathways to placental progesterone production have not, however, been demonstrated in vivo in a nonhuman primate. Therefore, maternal peripheral lipoprotein-cholesterol and progesterone concentrations were determined in blood samples obtained by venipuncture, from day 72 until day 100, from pregnant baboons (Papio sp) that were either untreated (n = 4) or treated (n = 3) with the inhibitor of hepatic lipoprotein production, 4-aminopyrazolo [3-4-d]pyrimidine (4-APP, 10 mg/kg BW) on days 98-99 of pregnancy (term = 184 days). Although LDL-cholesterol and progesterone levels remained unchanged in untreated animals, LDL-cholesterol concentrations were 9-fold lower (P < 0.005) in baboons receiving 4-APP than in untreated baboons 2 days following initial administration. Commensurate progesterone levels were 3.5-fold lower (P < 0.03) in 4-APP-treated baboons than in untreated baboons. RT-PCR was used to approximate relative changes in transcription of messengers RNAs (mRNAs) for selected cholesterol-sensitive pathways in placental tissue collected on day 100. Thus, expression of mRNAs for LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase appeared enhanced, whereas acyl-coenzyme A:cholesterol acyl transferase (ACAT) mRNA was diminished in syncytiotrophoblast-enriched cell fractions as a result of 4-APP administration. No relative differences in mRNAs were apparent in whole placental villous tissue, however, as a result of 4-APP treatment. In summary, this experiment demonstrates a significant decline in progesterone production elicited by maternal LDL-cholesterol withdrawal, and attests to the efficacy of 4-APP administration during baboon pregnancy. These results also suggest a commensurate regulation of cholesterol-sensitive pathways in primate syncytiotrophoblast. However, no relative differences were apparent in mRNA levels for LDL receptor, HMG-CoA and ACAT in whole placental villous tissue as a result of LDL-cholesterol withdrawal, which may suggest potential disparities in the mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. those in proliferative nonendocrine placental constituents.

Perifusion culture system for bovine embryos: improvement of embryo development by use of bovine oviduct epithelial cells, an antioxidant and polyvinyl alcohol.

Three experiments were conducted in an attempt to improve a continuous flow-perifusion system capable of maintaining embryo development for long periods of time. Bovine embryos (8-16 cells) obtained from static co-culture with cumulus cells in a serum-free medium were perifused in an ACUSYST-S cell culture incubator. Culture chambers of the incubator consisted of a 0.2-mL unit (Chamber 1) connected to a 1.5-mL unit (Chamber 2), with the outflow from Chamber 1 routed to the inlet to Chamber 2. A bovine embryo culture medium supplemented with 3 mg mL-1 bovine serum albumin (BSA) and 25 mM HEPES was used as a perifusion culture medium (PCM). Embryos were perifused in Chamber 2 for 24, 48 and 72 h and further co-cultured in a static system up to 216 h after insemination. In Experiment 1, conditioning PCM with frozen-thawed bovine oviduct epithelial cells (BOEC) placed in Chamber 1 enhanced (P < 0.05) blastocyst formation of embryos in Chamber 2, after 24, 48 and 72 h of perifusion culture. The proportion of blastocysts was not further increased by placing BOEC in Chamber 2 along with the embryos. In Experiment 2, embryos were perifused with PCM conditioned with BOEC in Chamber 1 for 48 h or 72 h. A higher proportion of perifused embryos developed to the blastocyst stage after addition of 25 U mL-1 or 50 U mL-1 of superoxide dismutase (SOD) to PCM than in its absence. However, blastocyst formation of embryos perifused for 72 h was not increased after addition of 50 U mL-1 SOD compared with its absence. In Experiment 3, the proportions of morulae and blastocysts were not decreased by replacement of 3 mg mL-1 BSA with 1 mg mL-1 polyvinyl alcohol (PVA) in a BOEC-conditioned medium containing 50 U mL-1 SOD after perifusion for 48 h. In conclusion, PCM conditioning with BOEC and addition of an antioxidant to the perifusion medium improved the developmental capacity of perifused embryos. PVA is an adequate replacement for BSA in the perifusion medium.

Effects of pregnant human, nonpregnant human, and fetal bovine sera on human chorionic gonadotropin, estradiol, and progesterone release by cultured human trophoblast cells.

Explant and cell culture methodologies are frequently employed in the investigation of the mechanisms that mediate placental hormone production. Recent reports suggest the presence of unknown regulatory factors in maternal serum that may impact significantly on the regulation of these biosynthetic pathways. The present study, therefore, determined the effects of sera obtained from pregnant women in the second to third trimester (PWS), nonpregnant women (NPWS), and men (MS) as well as commercially prepared FBS on hCG, estradiol, and progesterone release into medium by cultured human trophoblast cells. Placental villous tissue was enzymatically dispersed, and cytotrophoblast cells were purified via density gradient centrifugation and cultured (37 C; 90% air-10% CO2) in DMEM with 10% PWS, NPWS, MS, or FBS. All cytotrophoblast cultures aggregated and progressed to syncytial forms, although cells cultured with PWS exhibited notably larger multinucleated syncytial elements by 48 h in culture than cells cultured with FBS. Significant increases (P < 0.05) occurred in hCG, estradiol, and progesterone release due to the progression of cytotrophoblasts into the syncytiotrophoblast phase in all cultures. The quantity of hCG release was unaffected by serum origin. Cells cultured with human serum released greater (P < 0.05) amounts of estradiol than cells cultured with FBS. Cells cultured with MS released more (P < 0.01) estradiol than cells cultured with either PWS or NPWS, in a ratio to the concentration of endogenous androgen precursor available. Progesterone release was greater (P < 0.01) for PWS-cultured cells than for FBS-cultured cells. Progesterone release by NPWS- and MS-cultured cells was intermediate. Syncytiotrophoblasts cultured with PWS released approximately 3-fold more (P < 0.01) progesterone than syncytiotrophoblasts cultured with FBS and low density lipoprotein cholesterol, although the concentrations of available cholesterol substrate were similar. Culture of cells in steroid-depleted or lipoprotein-depleted PWS or FBS resulted in similar decreases (P < 0.01) in estradiol and progesterone release, respectively. In summary, increased estradiol release by placental cells cultured in intact human serum was attributed to aromatizable androgens, whereas enhanced progesterone release by cells cultured in human serum could be only partially attributed to higher concentrations of low density lipoprotein cholesterol substrate in human serum. Evidence of increased syncytial maturity and progesterone release by PWS-cultured cells may indicate the presence of undefined serum-borne regulators, which is enhanced during pregnancy.

Association of endogenous steroid levels with high and low density lipoprotein concentrations in the human maternal-fetoplacental unit at term.

OBJECTIVE: To determine the concentrations of high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), estradiol (E2), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) in both maternal and venous cord blood upon delivery at term; to determine the degree of association between lipoprotein cholesterol concentrations and concentrations of selected steroid hormones, thus indicating correlations between regulatory mechanisms mediating steroid biosynthesis and cholesterol homeostasis in the maternal-fetoplacental unit; and to determine the efficacy of both heparin-manganese precipitation and agarose-gel electrophoresis techniques for quantitation of HDL-C and LDL-C in fetal and maternal plasma. STUDY DESIGNS: Steroids were quantitated by radioimmunoassay in maternal and umbilical cord venous blood collected from 56 patients upon delivery. Correlation analysis and ANOVA were utilized to determine the degree of association between quantitative parameters in the total study population and differences in regard to fetal sex, respectively. RESULTS: Values determined for all parameters measured fell within previously established normal ranges. HDL-C concentrations in the cord blood of female newborns were higher than those of males, while LDL-C concentrations were unchanged in regard to fetal sex. Concentrations of both LDL-C and all steroids measured in the maternal periphery were unchanged in regard to fetal sex, but HDL-C concentrations were higher in plasma of women bearing female fetuses. Further, LDL-C levels in cord blood positively correlated with maternal DHEAS levels and negatively correlated with maternal E2 levels. Cord E2 concentrations correlated highly with cord levels of both DHEA and DHEAS, and maternal E2 levels correlated, although to a lesser degree, with maternal DHEA concentrations. CONCLUSION: The results indicated both positive and negative correlations of lipoprotein cholesterol levels with concentrations of the various steroid hormones associated with human pregnancy and illustrate the complicated interplay of mechanisms regulating steroid/lipoprotein metabolism in the maternal-fetoplacental unit. Changes in lipoprotein levels in the maternal-fetoplacental unit may suggest changes in steroid metabolism associated with fetal sex. Both heparin-manganese precipitation and agarose-gel electrophoresis techniques proved adequate for the quantitation of plasma lipoprotein cholesterol in both maternal and fetal plasma.