oMt1a-oGH Transgenic Mice Research Articles

Transgene transmission to progeny by oMt1a-oGH transgenic mice

Most studies utilizing transgenic technology focus on the impact to traits of interest, rather than propagation of the transgene to offspring. In animals containing growth hormone constructs, transgene transmission to progeny follows a Mendelian pattern of inheritance in the first few generations following generation of a founder animal, but decreases in subsequent generations. In the present study, the ovine metallothionein 1a-ovine growth hormone (oMt1a-oGH) transgenic mouse was used to determine whether transgene transmission rate to progeny was affected by overexpression of ovine growth hormone in the transgenic parent. The oMt1a-oGH mouse is a useful model for assessing transgene transmission, as the construct is easily regulatable and transgene inactivation results in a return of plasma GH to basal levels. Male and female hemizygous oMt1a-oGH mice were assigned to 1 of 3 treatment groups: (1) mice never actively expressing the transgene, (2) mice actively expressing the transgene from 3 weeks of age, and (3) mice actively expressing the transgene from 3 to 11 (males) or 3 to 8 (females) weeks of age. Transgenic mice were mated to wild type animals and the resulting progeny were genotyped. Males never actively expressing the transgene passed on the transgene to progeny in a Mendelian fashion, while males actively expressing the transgene transmitted the transgene to a smaller than expected number of progeny. However, following inactivation of the oMt1a-oGH construct in transgenic males, subsequent offspring demonstrated Mendelian inheritance of the transgene. In contrast, females expressing the transgene from 3 to 8 weeks of age were able to pass on the oMt1a-oGH construct in a Mendelian fashion, but females from other treatment groups were not. In oMt1a-oGH males, reduced transgene transmission appears to be due to selection against transgenic gametes. In females, however, selection against the transgenic genotype likely occurs at the embryonic level.

Leptin modulates fertility under the influence of elevated growth hormone as modeled in oMt1a-oGH transgenic mice.

Elevated growth hormone (GH) concentrations suppress reproductive function in a variety of species, although it is unclear whether GH directly suppresses reproductive performance, or whether GH activates other pathways to achieve these effects. The ovine metallothionein 1a-ovine GH (oMt1a-oGH) transgenic mouse has been used to model the effects of GH on both body composition and reproductive function. A recent report has documented increased leptin levels in obese oMt1a-oGH mice. Given the importance of leptin in modulation of the reproductive endocrine axis, as well as the reports documenting reduced leptin signal transduction in animals with elevated leptin levels, we hypothesized that high leptin concentrations in response to elevated GH would reduce fertility. To determine the effects of high circulating leptin levels on the reproductive endocrine axis, we assessed hypothalamic neuropeptide Y (NPY) and GnRH expression. At weaning, oMt1a-oGH transgenic (TG) and wild-type (WT) female mice were allocated to one of four treatment groups: oMt1a-oGH females chronically expressing the transgene (TG ON); oMt1a-oGH females expressing the transgene from 3 to 8 weeks of age (TG ON/OFF); WT females receiving the transgene stimulus from 3 to 8 weeks of age (WT ON/OFF); and WT females never receiving the transgene stimulus (WT OFF). Eight-week-old females were housed with males for a 2-week period, after which females were isolated from males and allowed to carry pregnancies to term. Body and gonadal fat pad (GFP) weights, along with plasma leptin concentrations, estrous cyclicity, pregnancy rate and litter characteristics, were recorded for each female. Chronic expression of the oMt1a-oGH transgene resulted in larger leaner mice, and inactivation of the transgene produced obese females. Pregnancy rate was reduced in TG ON females when compared with all other groups, and infertility was associated with elevated leptin levels. In addition, high leptin levels were associated with increased NPY expression, suggesting reduced leptin-signaling capacity, which may contribute to suppression of the reproductive axis in oGH animals.

Growth hormone and fertility in oMt1a-oGH transgenic mice

Female mice carrying a regulatable growth hormone transgene (oMt1a-oGH) are subfertile when the transgene is actively expressed. This study was designed to characterize subfertility caused by increased concentrations of growth hormone. In particular, this study aimed to: (i) determine the effects of transgene activation and inactivation on mating, conception, maintenance of pregnancy, ovulation rate, litter characteristics and embryonic survival at day 17 of pregnancy, (ii) characterize oestrous cyclicity in transgenic versus wild-type female mice, and (iii) correlate corticosterone concentrations with transgene expression and reproductive performance. Transgenic and wild-type female mice were allocated randomly to one of four treatment groups at weaning: (i) transgenic female mice that always express the transgene, (ii) transgenic female mice that never express the transgene, (iii) transgenic female mice that express the transgene for up to 8 weeks of age and (iv) non-transgenic wild-type female mice receiving the transgene stimulus until 8 weeks of age. Activation followed by inactivation of the transgene resulted in an increased incidence of remating, resulting in an extended interval to establish pregnancy in comparison with all other treatment groups. Transgenic mice that always expressed the transgene and those that expressed the transgene for up to 8 weeks of age had lower pregnancy rates and higher ovulation rates compared with mice from other treatment groups. Both embryonic survival and the duration of the oestrous cycle did not differ among treatment groups. Active expression of the transgene resulted in an increase in the plasma concentration of corticosterone, which was associated with reduced fertility. These data indicate that the presence of a high growth hormone concentration impedes the establishment and maintenance of pregnancy. Increased plasma corticosterone concentrations may interfere with implantation as well as potentiate leptin resistance, which has been reported previously in studies with these mice.

Growth hormone and fertility in oMt1a-oGH transgenic mice.

Female mice carrying a regulatable growth hormone transgene (oMt1a-oGH) are subfertile when the transgene is actively expressed. This study was designed to characterize subfertility caused by increased concentrations of growth hormone. In particular, this study aimed to: (i) determine the effects of transgene activation and inactivation on mating, conception, maintenance of pregnancy, ovulation rate, litter characteristics and embryonic survival at day 17 of pregnancy, (ii) characterize oestrous cyclicity in transgenic versus wild-type female mice, and (iii) correlate corticosterone concentrations with transgene expression and reproductive performance. Transgenic and wild-type female mice were allocated randomly to one of four treatment groups at weaning: (i) transgenic female mice that always express the transgene, (ii) transgenic female mice that never express the transgene, (iii) transgenic female mice that express the transgene for up to 8 weeks of age and (iv) non-transgenic wild-type female mice receiving the transgene stimulus until 8 weeks of age. Activation followed by inactivation of the transgene resulted in an increased incidence of remating, resulting in an extended interval to establish pregnancy in comparison with all other treatment groups. Transgenic mice that always expressed the transgene and those that expressed the transgene for up to 8 weeks of age had lower pregnancy rates and higher ovulation rates compared with mice from other treatment groups. Both embryonic survival and the duration of the oestrous cycle did not differ among treatment groups. Active expression of the transgene resulted in an increase in the plasma concentration of corticosterone, which was associated with reduced fertility. These data indicate that the presence of a high growth hormone concentration impedes the establishment and maintenance of pregnancy. Increased plasma corticosterone concentrations may interfere with implantation as well as potentiate leptin resistance, which has been reported previously in studies with these mice.

Segregation ratios and growth rate in inactive ovine metallothionein 1a-ovine growth hormone transgenic mice.

Objectives were to determine whether the oMt1a-oGH transgene shows normal Mendelian segregation and whether oMt1a-oGH mice exhibit normal growth without the zinc supplementation required to increase plasma oGH levels and stimulate growth. Transgenic mice were reciprocally backcrossed for four generations to high growth and control lines to form lines GM and GR, respectively. In the fifth generation, hemizygous transgenic mice (T/-) were crossed within each line. Pooled across backcross generations, there was a deficit (P < 0.001) of T/- progeny in lines GM (31.6%) and GR (22.2%) compared with expected (50%). In the T/- x T/- cross, the combined percentage of homozygous (T/T) and hemizygous transgenic mice was less (P < 0.001) than expected (75%) in both GM (44.2%) and GR (38.5%). Backcross T/- mice had lower (P < 0.05) 3-wk BW and lower (P < 0.001) 6-wk BW and 3- to 6-wk postweaning gains than nontransgenic mice. Similar genotypic differences were found in the T/- x T/- cross. No significant growth differences were found between T/T and T/- progeny. Using segregation ratios from the T/- x T/- mating, the relative fitness estimates of T/T, T/-, and -/- (nontransgenic) mice were 0.345, 0.223, and 1.0, respectively, in line GM and 0.218, 0.205, and 1.0 in line GR. Fitness estimates in the back-cross for T/- and -/- were 0.463 and 1.0 in line GM and 0.285 and 1.0 in line GR. Abnormal segregation ratios may be due to germline mosaicism or reduced fitness due to differential embryo survival. Reduced growth of oMt1a-oGH transgenic mice when the transgene is switched off suggests a subtle developmental abnormality, which may contribute to a reduction in fitness.

Correlated responses to selection for large body size in oMt1a-oGH transgenic mice: organ traits

The objective of the present study was to compare correlated responses in liver, spleen, kidney, heart and testis absolute weights and as a percentage of 8-week body weight following selection for large 8-week body weight in twice-replicated nontransgenic and transgene-carrier lines of mice from two genetic backgrounds. The transgene was an ovine metallothionein 1a-ovine growth hormone (oMt1a-oGH) construct, which was activated by adding 25 mM ZnSO4 to the drinking water. Lines NM and NC were nontransgenic lines derived from a high-growth and randomly selected background, respectively. Lines TM and TC were transgene-carrier lines formed from the respective genetic backgrounds. Line CC was a nontransgenic control from the randomly selected background. At weaning, male mice from each line were assigned to either zinc supplemented or control drinking water. Toe-clips were assayed by PCR for the presence or absence of the transgene. Correlated responses of absolute weights of all organs in nontransgenic lines indicated moderately high genetic correlations of organ weights with body weight, but on a percentage of body weight basis, the correlated responses were much lower. The correlated responses in visceral organ weights were lower in the presence of the inactivated oMt1a-oGH transgene than in its absence. The presence of the activated oMt1a-oGH combined with the effects of selection for growth increase had a greater impact on increasing the size of the splanchnic organs than did selection for large body weight in the absence of the transgene.

Correlated responses to selection for large body size in oMt1a-oGH transgenic mice: reproductive traits

Correlated responses in female reproductive performance were evaluated following short-term selection within full-sib families for increased 8-week body weight in two replicates of four lines of mice: two ovine metallothionein-ovine growth hormone (oMt1a-oGH) transgene-carrier lines, one from a high-growth background (TM) and one from a control background (TC), and two non-transgenic lines, one from each of these genetic backgrounds (NM and NC, respectively). A fifth line (CC), not containing the transgene, served as a randomly selected control. The initial frequency of the oMt1a-oGH transgene construct in the TM and TC lines was 0.5. The frequency of transgenic females sampled at generations 7 and 8 of selection was 84.0% and 6.1% in the TC and TM lines, respectively. No significant female infertility differences were detected between transgene-carrier and non-transgenic lines or between transgenic and non-transgenic mice within carrier lines, whereas high-growth background lines had a higher infertility than control background lines (P < 0.05). Correlated responses in the TC transgene-carrier line were suggestive of reduced reproductive performance as indicated by increased post-implantation mortality (P < 0.05), number of dead fetuses plus implants (P < 0.05), and loss of fetuses from day 16 to parturition (P < 0.001). For the first two traits, the negative correlated responses were accounted for by the reduced performance of transgenic compared with non-transgenic females. Embryos carrying the transgene may also have a lower viability. In contrast, the NC non-transgenic line did not exhibit reduced reproductive performance for these traits. The low frequency of the transgene in the high-growth background TM line was associated with reduced fitness and a lower additive effect for 8-week body weight compared with the control background TC line.

Correlated changes in fertility and fitness traits in lines of oMt1a-oGH transgenic mice selected for increased 8-week body weight *

Correlated changes in fertility and fitness traits in lines of oMt1a-oGH transgenic mice selected for increased 8-week body weight *

Correlated changes in fertility and fitness traits in lines of oMt1a-oGH transgenic mice selected for increased 8-week body weight

Correlated responses in fitness and fertility traits were compared in transgenic and nontransgenic lines of mice selected for increased 8-week body weight. Two replicates of lines which either carried or did not carry the sheep metallothionein-1a sheep growth hormone transgene (oMt1a-oGH) were established. Host lines had been previously selected for rapid growth or selected randomly. Within-litter selection was carried out for 13 generations, and a randomly selected control line was kept for each set of replicate lines. Mice were genotyped every generation for the presence of the transgene, but this information was not used in selection decisions. The oMt1a-oGH construct was activated by adding 25 m m ZnSO₄ to the drinking water from 3 weeks (weaning) until 8 weeks of age. Zinc stimulation of the transgene was not carried out during mating, gestation and lactation. Correlated responses in fitness traits were measured by regression of least-squares means (as deviations from the control lines) on generation number. Two fitness indexes were defined to combine the information on individual fitness traits. The proportion of infertile matings was higher in generations 7 to 13 than in generations 0 to 6. Correlated responses to selection showed an increase in the cohabitation to littering interval in nontransgenic lines and an increase in litter sizes in lines from the selected background. Preweaning pup survival did not change over generations. Overall fitness increased in the transgenic line from the selection background whereas no changes were observed in the transgenic line from the control background. The initial frequency of 0.5 of the transgene was reduced to less than 0.10 in the selected background, but increased to an average of 0.62 in the control lines. The comparison of specific mating groups involving transgenic and nontransgenic mates revealed that the only consistent disadvantage in having a transgenic parent was the increase in the length of the cohabitation to littering interval. Major fitness problems were not associated with the oMt1a-oGH transgene, which makes this construct a potential choice for use in livestock breeding programmes.

Overexpression of growth hormone affects alternatively spliced IGF-I mRNA expression in oMt1a-oGH transgenic mice.

Restorative growth hormone (GH) treatment of hypophysectomized rats differentially enhances the transcription of alternative IGF-I mRNA classes in liver. The goal of the present study was to determine the effects of GH overexpression on various classes of hepatic IGF-I mRNA in GH transgenic mice. Unstimulated oMt1a-oGH transgenic mice had low levels of transgene expression, and therefore were used to determine the effects of long-term, slightly elevated GH levels on the abundance on each alternative IGF-I mRNA class. The acute effects of high GH levels on the expression of alternative IGF-I mRNA were studied by gavaging transgenic mice with 25 mM zinc sulfate to activate oMt1a-oGH transgene expression. Long-term, low levels of oGH transgene expression in unstimulated transgenic mice resulted in a 73% down regulation of IGF-I 2Ea mRNA but not 1Ea and 2Eb mRNA. Acute stimulation of transgene expression triggered a rapid, 240% increase in 1Ea mRNA levels within 4 hours of transgene expression while 2Ea mRNA was down regulated to nearly non-detectable levels by 6 hours. IGF-I 2Eb mRNA was not affected by the short-term GH elevation. Our results showed that IGF-I 1Ea and 2Ea mRNA were differentially regulated by chronic low or acute high levels of GH. These results suggest that the regulation of IGF-I 1Ea and 2Ea mRNA transcription involve different postreceptor molecules and/or feedback mechanisms.

Development of obesity following inactivation of a growth hormone transgene in mice

Mice with a temporally regulatable ovine metallothionein 1a--ovine growth hormone transgene (oMT1a-oGH) were utilized to study the effects of withdrawal of elevated circulating levels of growth hormone (GH) on growth and body composition. The transgene was activated from 21-42 days of age by provision of zinc sulfate in the drinking water. At 42 days, mice were allocated to either activated transgenic (remain on zinc sulfate) or inactivated transgenic (removal of zinc sulfate) groups, and to receive either ad libitum or restricted (80-90% of ad libitum) access to feed. Non-transgenic control mice were treated similarly. Body weights and intakes were recorded weekly. Mice were killed at 70 d and epididymal and subcutaneous fat pads, trimmed hind carcass and various organs were weighed. The main findings of this study are: (1) food-restricted mice possessing an activated oMT1a-oGH transgene fail to demonstrate increased growth, but exhibit significantly reduced levels of fat (P < 0.05) relative to all other genotype x feed level combinations; and (2) inactivation of the oMT1a-oGH transgene, following a period of elevated GH levels, leads to development of obesity as evidenced by two to three fold increases in epididymal and subcutaneous fat pad weights (P < 0.01) relative to both activated transgenic and non-transgenic control mice. These large increases in fat deposition also occurred when intake was restricted to 80-90% of ad libitum levels, indicating that metabolic changes independent of intake occur in these inactivated transgenic mice. It is possible that highly elevated production of GH in activated oMT1a-oGH transgenic mice leads to (1) enhanced promotion of preadipocyte differentiation, leading to increased numbers of adipocytes that, upon cessation of oGH production, are available for lipid deposition resulting in obesity, or (2) alterations in production of or responsiveness to insulin, leading to increased fat deposition upon removal of the chronic anti-lipogenic actions of GH. The oMT1a-oGH transgenic mouse line should provide a new genetic model with which to investigate the mechanisms by which growth hormone affects obesity.

Rescue of pregnancy and maintenance of corpora lutea in infertile transgenic mice expressing an ovine metallothionein 1a-ovine growth hormone fusion gene.

Transgenic female mice expressing a temporally regulatable ovine metallothionein 1a-ovine growth hormone (oMT1a-oGH) fusion gene can cycle, mate, and support early embryonic development; but they fail to maintain pregnancy or, in most cases, to exhibit signs of pseudopregnancy. The present study was designed to determine whether or not the infertility of oMT1a-oGH transgenic female mice is due to luteal insufficiency. A series of five experiments was conducted in which various hormonal therapies were utilized in an attempt to overcome this infertility. Provision of 1 mg progesterone to oMT1a-oGH females from Days 1 to 17 of gestation rescued pregnancy in 19 of 20 females with an increased mean litter size (p < 0.01) compared to that of wild-type control females. Supplementation with progesterone also appeared on the basis of gross anatomical appearance to maintain CI. Provision of 2 mg progesterone in combination with 25 ng estradiol-17 beta from Days 4 to 17 rescued pregnancy in 14 of 24 oMT1a-oGH females, while provision of 100 or 25 micrograms ovine prolactin from Days 3 to 17 failed to overcome oMT1a-oGH infertility. Transgenic females with rescued pregnancies lactated and raised heavier pups than wild-type females by Day 15 postnatal (p < 0.05). Results indicate that infertility of oMT1a-oGH transgenic mice is caused by luteal insufficiency. Supplementation of transgenic females with progesterone appeared to maintain morphologically normal CL and pregnancy with subsequently increased litter size and normal lactation.

Expression of an ovine growth hormone transgene in mice increases archidonic acid in cellular membranes

The degree of unsaturation of membrane lipids has been implicated in a number of physiological disorders, yet its regulation remains poorly understood, especially the regulation of the synthesis and distribution of arachidonic acid levels, the most abundant long chain polyunsaturated fatty acid in membranes. Transgenic mice expressing the ovine metallothionein 1a-ovine growth hormone (oMt1a-oGH) fusion gene exhibited significantly elevated levels of a number of long chain polyunsaturated fatty acids in serum, including arachidonic acid. In oMt1a-oGH transgenic mice the products of all three desaturation pathways are affected by the expression of the ovine growth hormone transgene. The essential precursors of membrane long chain polyunsaturated fatty acids, 18:2n-6 and 18:3n-3, were reduced in transgenics relative to controls, and their desaturation and elongation products, arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3), were elevated. As rare intermediate long chain polyunsaturated fatty acids such as eicosatrienoic acid (20:3n-9) were also significantly elevated, we conclude that these observations reflect increased activity of the delta-5 and delta-6 desaturase enzymes. In contrast, the products of the stearoyl CoA or delta-9 desaturase, were significantly reduced in oMt1a-oGH expressing transgenics relative to their levels in control mice.