Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.

Seven transgenic Merino sheep have been produced by the technique of pronuclear microinjection. Two different Sheep Metallothionein-1a-Sheep Growth Hormone fusion genes were used. Four of the transgenic sheep, all of which contained the gene MTsGH5, did not express the transgene. The remaining three sheep carrying the second fusion gene, MTsGH9, expressed the gene at high levels in a variety of tissues and had elevated blood levels of sheep growth hormone.

To learn why some zygotes remain viable after gene transfer while others lyse, we injected DNA into fertilized eggs and compared those that lysed within 1 h of injection with others that retained a normal appearance. Using scanning electron microscopy, we found open holes on the surface of lysed eggs, indicating failure of the plasma membrane to reseal after microinjection. No holes were seen in unlysed eggs, but many of them had membrane alterations suggestive of healed punctures. We also examined aspects of the gene transfer procedure that might influence survival such as the size of injection pipettes and their taper relative to zygote diameter, possible toxicity of the injection medium, the timing of injection, and immediate vs. delayed pipette withdrawal. The only factors that significantly affected cell viability were pipette size and taper, and timing of injection in relation to first cleavage. This suggests that zygote viability correlates inversely with the size of the hole produced by the injection pipette and that damage to the membrane is less successfully repaired as the fertilized egg readies itself for division.

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Pregnant Merino ewes were treated with 90 8g murine epidermal growth factor (EGF) per kg body weight at either 25 (n = 80), 50 (n= 40) or 75 (n =40) days of gestation. Untreated control ewes were included at each gestational age (n=20, 12 and 12 respectively). Fifteen and twenty per cent of the ewes treated with EGF at days 25 and 50 respectively failed to lamb, significantly more (P < 0.01 ) than in ewes treated at day 75, where only one ewe failed to lamb, and in control ewes which all lambed. These differences were not reflected in significant differences between the overall percentage of lambs born in each group, as the incidence of abortion in single-bearing ewes was higher than in ewes carrying multiple fetuses. All lambs born alive to EGF-treated ewes appeared normal. Plasma progesterone concentrations measured before treatment and at 8, 24 and 48 h after EGF injection fell significantly in treated ewes relative to controls (P<0.01 at day 25; P<0.05 at days 50 and 75) and concentrations were lowest at 8 and 24 h after injection in those ewes which aborted. Following EGF treatment at days 25 and 50 of gestation, abortion occurred in all ewes with very low plasma progesterone concentrations 8 to 48 h after EGF injection, probably as a result of EGF-induced luteolysis. In other ewes plasma progesterone concentrations returned to pretreatment values by 48 h, indicating incomplete luteolysis. The delayed abortion observed in some of these ewes further suggests that other mechanisms of action are involved in EGF-induced abortion.