Maternally-derived transcripts and proteins support early bovine and ovine embryo development until the 8- to 16-cell stage, at which time embryonic transcripts become essential for continued development. One purported mechanism for the switch from maternal to zygotic control of development (maternal to zygotic genome activation; MZGA) is the appearance of transcription factors that activate specific genes in the embryonic genome. Members of the E26 transformation specific (Ets) family are unique transcription factors involved in development, differentiation, and protease regulation. This study was undertaken to evaluate expression and function of the Ets transcription factors, Ets-1, Ets-2, and Elf-1, in early bovine and ovine embryos from the one-cell stage to Day 15 of pregnancy (Day 0 onset of estrus). In the first experiment, bovine embryos from the one- to 16-cell stages were derived by in vitro maturation, fertilization, and culture. Days 6, 8, 10, 12, and 14 embryos were collected nonsurgically from estrous synchronized and superovulated cows. RNA was extracted at the appropriate time interval and reverse transcribed. The resultant cDNA was amplified by PCR using primers designed for Ets-1, Elf-1, and Ets-2. Ets-1 transcripts were present in both primary and matured oocytes, cleavage stage embryos, and Days 10, 12, and 14 embryos, as well as in the positive control, bovine ovary. Elf-1 transcripts were detected in the matured oocyte, cleavage stage embryos, and Days 6, 10, and 14 embryos. Ets-2 transcripts were not observed in the embryonic stages investigated or the bovine ovary. Ovine embryos were surgically collected from synchronized and superovulated ewes and similarly analyzed for Ets-1 and Elf-1 expression using the same RNA extraction and RT-PCR technique. Embryos expressed both transcripts at Days 13 and 15, but did not show expression at any of the earlier stages evaluated. The second experiment was designed to determine if inhibition of ETS-1 translation would interfere with development and plasminogen activator (PA) production in bovine embryos. Plasminogen activator production was evaluated in Days 5 and 6 embryos nonsurgically collected from superovulated cows and cultured in 1, 2.5, 5, or 10 ��M concentrations of sense or antisense Ets-1 oligonucleotides. In preliminary experiments, 1 ��M antisense was ineffective in suppressing PA production, and 10 ��M oligonucleotides were detrimental to development. Day 5 embryos treated with 2.5 ��M oligonucleotides inhibited developmental effect and total PA production was (P<0.05) lower in antisense treatments when compared to either control or sense treatments. No difference (P>0.10) in PA production was observed between Day 6 embryos treated with 2.5 or 5 ��M sense and antisense oligonucleotides. A significant time effect on PA production was observed in both Day 5 and Day 6 embryos cultured in either 2.5 or 5 ��M concentrations of oligonucleotides. Based on these results, it is unlikely that Elf-1 and Ets-2 are involved in MZGA because the former is constituitively expressed throughout development, and the latter was not observed. There is some uncertainty regarding the expression of Ets-1 during MZGA. This factor may be expressed after MZGA for controlling PA production and other proteases involved in extracellular matrix turnover and early germ layer formation. / Graduation date: 2003

Two trials were conducted over consecutive years to examine the effects of ewe body condition and post-mating nutrition on ovulation rates and embryo survival. Trial 1 used 146 Polypay ewes ranging in age from 5 to 8 years in a 3 x 2 factorial array of pre-mating (high-H, low flushed-LF and low unflushed-L) and post-mating nutrition (high and low) treatments. Trial 2 was a 2 x 2 x 2 factorial array of 60 Polypay and 60 Coopworth x Polypay (C x P) three year old ewes, two pre-mating (H and LF) and two post-mating (high and low) treatments. To estimate timing and extent of embryo loss, two methods of embryo detection were employed during Trial 2. Real-time ultrasound was performed on all ewes at 21, 28, 34 and 45 days post-mating. Blood samples were also collected on these days for analysis of Pregnancy-specific Protein B (PSPB) levels. There was no effect of pre-mating treatment or ewe age on ovulation or conception rates for Trial 1. Pre-mating treatment was significant in Trial 2, with H ewes having higher ovulation rates than LF ewes. Pre-mating treatment, post-mating treatment, ewe age (for Trial 1) and genotype (for Trial 2) had no effect on mean litter size in either trial. Analysis of litter size among ewes with twin and triple ovulations showed pre-mating treatment to be significant among triple ovulators in Trial 1, where L ewes had much lower litter size (1.86) than LF (2.37) or H (2.60) ewes. In Trial 2, post-mating treatment was significant among the ewes with triple ovulations, with ewes on low nutrition having lower litter size (2.01) than ewes on high nutrition (2.59). Two-way interactions were significant among twin and triple ovulators in Trial 2. Breed x pre-mating interaction among twin ovulating ewes showed C x P LF ewes to have lower litter size (1.43) than the H ewes (1.87), while the opposite was true for Polypay ewes. Pre- x post-mating treatment interaction among triple ovulators showed LF ewes on low post-mating nutrition had lower mean litter size. This effect is largely due to lower litter size in the C x P triple ovulators on low post-mating nutrition. Data available from the ultrasound diagnosis showed little indication of treatment effects on amount or time of embryo loss. Ewes bearing single or multiple embryos differed in PSPB level at day 45, but not at earlier times. However, there were no differences in PSPB levels in ewes with twin or triple embryos at any of the trial days. Assay for PSPB failed to facilitate detection of the amount or time of embryo loss, as determined by ultrasound. / Graduation date: 1991

Most embryonal mortality occurs during early embryonic development. Two experiments were designed to study aspects of early embryonic development. Experiment 1 was to determine if early ovine embryos were capable of metabolizing arachidonic acid. Cyclic ewes were estrous synchronized with 6⍺-methyl-17β-hydroxy progesterone acetate (MPA) pessaries, superovulated with follicle stimulating hormone (FSH) and bred artificially. Embryos were collected on d 4, 8, 10, 12 or 14 of pregnancy and incubated with 1 μCi of [¹⁴C] arachidonic acid in an atmosphere of 5% CO₂, 45% O₂ and 50% N at 37°C for 24 h. Embryos from all days of pregnancy metabolized arachidonic acid to a number of compounds. Embryos produced primarily an unidentified polar compound, 6-keto-prostaglandin F₁⍺ (6-keto-PGF₁⍺), prostaglandin F₂⍺ (PGF₂⍺), prostaglandin E₂ (PGE₂), 13,14-dihydro-15-keto prostaglandin F₂⍺ (PGFM), prostaglandin B₂ (PGB₂) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Experiment 2 was to determine whether prostaglandins have a role in embryo hatching from the zona pellucida. Ewes were superovulated and bred artificially, and embryos were collected on d 7 of pregnancy. Embryos were incubated with ethanol (control), indomethacin, PGE₂ or indomethacin and PGE₂ in an atmosphere of 5% CO₂ and 95% air at 37°C for 24 h. Indomethacin appeared to decrease embryo hatching rate (indomethacin, 34.5% vs control, 46.4%). Prostaglandin E₂ appeared to increase embryo hatching rate (PGE₂, 60.0% vs. control, 46.4%). However, hatching rates for indomethacin and PGE₂ treatment groups were not different from control (P > .05). When compared to any group with indomethacin treatment, PGE₂ increased (P < .05) embryo hatching rate. The results of this study indicated that early ovine embryos can convert arachidonic acid to various compounds in vitro. Although not conclusive, indomethacin may decrease and PGE₂ may increase embryo hatching rate. Therefore, embryo-produced prostaglandins may be involved in hatching of sheep embryos from the zona pellucida. / Master of Science

This thesis reports the effects of an altered intrauterine environment on the offspring???s renal, cardiovascular and renin angiotensin systems. After a midgestational asphyxial episode in fetal sheep (30 min total umbilical cord occlusion at 90 days; term 150 days) the hydrops that resulted had not completely resolved by 130 days. While the heart and kidneys were apparently unaffected, the brain and lung weights were 37% and 50% lower than sham values, respectively and there were joint contractures. The effects of maternal renal disease on the offspring were investigated. Although in utero fetuses of subtotally nephrectomised ewes (STNx) had altered urine flow rates, sodium excretion, haematocrits, plasma chloride and plasma renin levels, by 1-2 weeks after birth these values in the lambs (STNxL) were similar to controls (ConL) under baseline conditions. Body weight and the weights of most organs were similar, including the kidney, in which glomerular number was normal. In the neonatal period, the lambs were subjected to four challenges: furosemide (2 mg/kg intravenous bolus), infusion of angiotensin II and phenylephrine, intravenous infusion of 0.15M saline (50 ml/kg over 30 min) and haemorrhage (20% estimated blood volume over 10 min). These challenges revealed evidence of programming of several aspects of the renal, cardiovascular and renin angiotensin systems in the STNx offspring. As young adults at 6 months of age, male and female offspring of STNx ewes were normotensive and had normal renal function. On a high salt diet (HSD, 0.17M NaCl in 8L water for 5-7days), female offspring of both groups did not become hypertensive. However, the STNx offspring must have retained salt and water as plasma sodium was increased and haematocrit was decreased. In the STNx offspring only, there was a relationship between glomerular filtration rate (GFR) and mean arterial pressure, indicating an inability to maintain a constant GFR in response to changes in arterial pressure.

This thesis reports the effects of an altered intrauterine environment on the offspring???s renal, cardiovascular and renin angiotensin systems. After a midgestational asphyxial episode in fetal sheep (30 min total umbilical cord occlusion at 90 days; term 150 days) the hydrops that resulted had not completely resolved by 130 days. While the heart and kidneys were apparently unaffected, the brain and lung weights were 37% and 50% lower than sham values, respectively and there were joint contractures. The effects of maternal renal disease on the offspring were investigated. Although in utero fetuses of subtotally nephrectomised ewes (STNx) had altered urine flow rates, sodium excretion, haematocrits, plasma chloride and plasma renin levels, by 1-2 weeks after birth these values in the lambs (STNxL) were similar to controls (ConL) under baseline conditions. Body weight and the weights of most organs were similar, including the kidney, in which glomerular number was normal. In the neonatal period, the lambs were subjected to four challenges: furosemide (2 mg/kg intravenous bolus), infusion of angiotensin II and phenylephrine, intravenous infusion of 0.15M saline (50 ml/kg over 30 min) and haemorrhage (20% estimated blood volume over 10 min). These challenges revealed evidence of programming of several aspects of the renal, cardiovascular and renin angiotensin systems in the STNx offspring. As young adults at 6 months of age, male and female offspring of STNx ewes were normotensive and had normal renal function. On a high salt diet (HSD, 0.17M NaCl in 8L water for 5-7days), female offspring of both groups did not become hypertensive. However, the STNx offspring must have retained salt and water as plasma sodium was increased and haematocrit was decreased. In the STNx offspring only, there was a relationship between glomerular filtration rate (GFR) and mean arterial pressure, indicating an inability to maintain a constant GFR in response to changes in arterial pressure.

This thesis reports the effects of an altered intrauterine environment on the offspring???s renal, cardiovascular and renin angiotensin systems. After a midgestational asphyxial episode in fetal sheep (30 min total umbilical cord occlusion at 90 days; term 150 days) the hydrops that resulted had not completely resolved by 130 days. While the heart and kidneys were apparently unaffected, the brain and lung weights were 37% and 50% lower than sham values, respectively and there were joint contractures. The effects of maternal renal disease on the offspring were investigated. Although in utero fetuses of subtotally nephrectomised ewes (STNx) had altered urine flow rates, sodium excretion, haematocrits, plasma chloride and plasma renin levels, by 1-2 weeks after birth these values in the lambs (STNxL) were similar to controls (ConL) under baseline conditions. Body weight and the weights of most organs were similar, including the kidney, in which glomerular number was normal. In the neonatal period, the lambs were subjected to four challenges: furosemide (2 mg/kg intravenous bolus), infusion of angiotensin II and phenylephrine, intravenous infusion of 0.15M saline (50 ml/kg over 30 min) and haemorrhage (20% estimated blood volume over 10 min). These challenges revealed evidence of programming of several aspects of the renal, cardiovascular and renin angiotensin systems in the STNx offspring. As young adults at 6 months of age, male and female offspring of STNx ewes were normotensive and had normal renal function. On a high salt diet (HSD, 0.17M NaCl in 8L water for 5-7days), female offspring of both groups did not become hypertensive. However, the STNx offspring must have retained salt and water as plasma sodium was increased and haematocrit was decreased. In the STNx offspring only, there was a relationship between glomerular filtration rate (GFR) and mean arterial pressure, indicating an inability to maintain a constant GFR in response to changes in arterial pressure.

Graduation date: 1996

This thesis reports the effects of an altered intrauterine environment on the offspring???s renal, cardiovascular and renin angiotensin systems. After a midgestational asphyxial episode in fetal sheep (30 min total umbilical cord occlusion at 90 days; term 150 days) the hydrops that resulted had not completely resolved by 130 days. While the heart and kidneys were apparently unaffected, the brain and lung weights were 37% and 50% lower than sham values, respectively and there were joint contractures. The effects of maternal renal disease on the offspring were investigated. Although in utero fetuses of subtotally nephrectomised ewes (STNx) had altered urine flow rates, sodium excretion, haematocrits, plasma chloride and plasma renin levels, by 1-2 weeks after birth these values in the lambs (STNxL) were similar to controls (ConL) under baseline conditions. Body weight and the weights of most organs were similar, including the kidney, in which glomerular number was normal. In the neonatal period, the lambs were subjected to four challenges: furosemide (2 mg/kg intravenous bolus), infusion of angiotensin II and phenylephrine, intravenous infusion of 0.15M saline (50 ml/kg over 30 min) and haemorrhage (20% estimated blood volume over 10 min). These challenges revealed evidence of programming of several aspects of the renal, cardiovascular and renin angiotensin systems in the STNx offspring. As young adults at 6 months of age, male and female offspring of STNx ewes were normotensive and had normal renal function. On a high salt diet (HSD, 0.17M NaCl in 8L water for 5-7days), female offspring of both groups did not become hypertensive. However, the STNx offspring must have retained salt and water as plasma sodium was increased and haematocrit was decreased. In the STNx offspring only, there was a relationship between glomerular filtration rate (GFR) and mean arterial pressure, indicating an inability to maintain a constant GFR in response to changes in arterial pressure.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Premature luteal regression occurs on average in 30% of superovulated sheep ewes. This phenomenon occurs early in the cycle before the embryo’s can be collected and is a major contributor to failure in embryo transfer programs. This research was done to understand the physiological mechanisms involved. Chapter two provides a general background of the physiology of natural luteolysis and the maternal recognition of pregnancy. The chapter introduces some new concepts on the topic of cell death and provides a recent literature review on research done on the phenomenon of premature luteal regression. This chapter forms the base of ideas and arguments that follows in the two studies containing new original work in this field. The research contained in this thesis comprises of two in vivo studies. The first study (Chapter 3) compare premature luteal regression to Prostaglandin F2α (PGF2α) induced regression with emphasis on the changes in levels of the steroid hormones progesterone (P4) and estradiol - 17β (E2-17β) and changes in structure and ultra structure. The following conclusions were made: 1. Premature luteal regression is not merely inadequate luteal support, but indeed early luteal regression, since seasonal influences could merely be nutritional influences, and a definitive increase in P4 were recorded in animals exhibiting the phenomena. 2. Nutritional influences could play a role, but the type and quality of nutrients and mechanism involved, is still unclear. 3. PGF2α-induced regression differs from premature luteal regression in that: a) The progression of functional and structural regression in PGF2α -induced regression is slower than in premature luteal regression. b) Regressed corpora lutea do not occur with normal functioning corpora lutea. 4. There is a distinct second E2-17β peak preceding the decline in P4 in animals that exhibits signs of premature luteal regression. A threshold initiating premature luteal regression was not established. The second study (Chapter 4) compares the changes in the ovine β estradiol - 17 β receptor (oERβ) between premature luteal regression and PGF2α induced regression. The study concludes that there could be a potential role for oERβ in premature luteal regression. The findings of these two studies raise some questions about the conventional perception that early release of PGF2α is the cause of premature luteal regression. The thesis concludes in a hypothesis (Chapter 4) explaining the phenomenon. / AFRIKAANSE OPSOMMING: Premature luteale regressie kom gemiddeld in 30% van gesuperovuleerde skaap-ooie voor. Die verskynsel kom vroeg in die siklus voor, voor die embrios gekollekteer kan word, en is een van die belangrikste oorsaake van mislukkings in ‘n embrio-oorplaasingsprogram. Die huidige navorsing poog om die fisiologiese meganismes betrokke by premature luteale regressie te verstaan. Hoofstuk twee verskaf ‘n algemene agtergrond van die fisiologiese aspekte betrokke by natuurlike luteale regressie en maternale herkenning van swangerskap. Die hoofstuk stel nuwe konsepte voor oor sel afsterwing en verskaf ‘n opgedateerde literatuuroorsig met betrekking tot die navorsing wat in die veld oor die verskynsel gedoen is. Die hoofstuk vorm die basis vir die idees en argumente, wat volg in die twee studies en wat oorspronklike nuwe navorsing bevat oor die onderwerp. Die navorsing in die tesis bestaan uit twee in vivo studies. Die eerste studie (Hoofstuk 3) vergelyk premature luteale regressie en prostaglandien F2α (PGF2α) ge-induseerde regressie met ‘n klem op die vlakke van die steröiedhormone progesteroon (P4) en estradiol - 17β (E2-17β) en veranderinge in die mikroskopiese struktuur en ultra struktuur van die corpus luteum. Die studie bevind: 1. Premature luteale regressie is nie slegs onvoldoende luteale funksie nie, maar vroë luteale regressie aangesien seisoenale invloede eitlik voedings invloede kan wees en P4 gestyg het in diere waar die verskynsel voorgekom het. 2. Voeding kan ‘n rol speel maar die tiepe en gehalte van die voedingstowwe en die meganismes betrokke is nie duidelik nie. 3. PGF2α - ge-induseerde regressie verskil van premature regressie in dat: a) Die verloop van funksionele en strukturele regressie is stadiger in PGF2α - ge-induseerde regressie in vergelyking met premature luteale regressie. b) Corpora lutea wat regressie ondergaan het kom nie voor saam met corpora lutea wat normal voorkom nie. 4. Daar die ‘n duidelike tweede piek van E2-17β gaan die afname in P4 vooraf in diere waar premature regressie voorkom. 5. Daar is nie geslaag om ‘n drempel vas te stel waar premature regressie ge-inisieer word nie. Die tweede studie vergelyk die veranderinge in estradiol-17β reseptore (oERβ) in die skaap tydens premature luteale regressie en PGF2α geinduseerde regressie. Die studie bevind dat daar ‘n moontlike rol is vir ERβ in premature luteale regressie. Die bevindinge van die twee studies bevraagteken die konvensionele opvatting dat vroë vrystelling van PGF2α verantwoordelik is vir premature luteale regressie. Die tesis sluit af met ‘n nuwe hipotese om die verskynsel te verduidelik.

Studies of humans and domestic animals have shown that there is a linkage between the neonatal and post-natal health of an individual and its uterine environment during gestation. However, very little information exists for sheep and there have been no studies that have directly examined the stage of gestation at which such effects could be introduced to the conceptus. In the present study, pure-breed embryos were transferred within and reciprocally between large (Suffolk: S) and small (Cheviot: C) breeds of sheep to establish different uterine environments; SinS (large control), SinC (restricted environment), CinS (luxurious environment) and CinC (small control) and their effects upon embryonic, fetal, neonatal and post-natal development and glucose metabolism of lambs were examined. By Day 19 of gestation, conceptuses (embryo and trophoblast) developing in a restricted uterine environment (SinC) were smaller (P<0.05) than in control (SinS). The head length of SinC fetuses was smaller (P<0.05) than in SinS fetuses on Day 55 of gestation and SinC lambs were lighter and smaller (P<0.05) than SinS lambs at birth. During subsequent post-natal life, there was no difference (P>0.05) in the growth rate of SinC and SinS lambs. The liveweight and body dimensions of SinC lambs were lower (P<0.05) than SinS lambs until 9 weeks and 12 weeks of age, respectively. Day 19 peri-implantation embryos and trophoblasts that developed in a luxurious environment were bigger than in control (CinC). However, CinS fetal size did not differ (P>0.05) from CinC fetuses by Day 55 of gestation. There was no difference (P>0.05) in the birthweight and body dimensions of lambs born from these two groups. Dimension of the placentas of SinC and SinS or CinS and CinC did not differ (P<0.05) during gestation or at lambing. Concentrations of ovine placental lactogen (oPL), progesterone, insulin-like growth factor-1 (IGF-1), glucose and free fatty acid (FFA) differed between uterine environments. During glucose challenge tests, there were no differences in the concentrations of glucose and insulin, between SinC and SinS female lambs, however, glucose concentrations declined more rapidly (P<0.05) in CinS than CinC female lambs at one year of age. It was concluded that restricted uterine environment affects embryonic, fetal and neonatal development of lambs, and that these effects perpetuates until at least one year of age; but there was no effect upon glucose metabolism. Conversely, a luxurious uterine environment enhances the early development of embryos but had no effects upon subsequent fetal, neonatal and post-natal development; however glucose metabolism of post-natal female lambs was improved. It appears that these effects of uterine environment were mediated through the trophoblast during the early embryonic period and via the placenta during subsequent gestation. oPL, progesterone, IGF-1, glucose and FFA were implicated in feto-maternal dialogue. These results suggest that uterine environment significantly influences the biology of young sheep with possible economic consequences.