Decomposing genomic variance using information from GWA, GWE and eQTL analysis.

A commonly used procedure in genome-wide association (GWA), genome-wide expression (GWE) and expression quantitative trait locus (eQTL) analyses is based on a bottom-up experimental approach that attempts to individually associate molecular variants with complex traits. Top-down modeling of the entire set of genomic data and partitioning of the overall variance into subcomponents may provide further insight into the genetic basis of complex traits. To test this approach, we performed a whole-genome variance components analysis and partitioned the genomic variance using information from GWA, GWE and eQTL analyses of growth-related traits in a mouse F2 population. We characterized the mouse trait genetic architecture by ordering single nucleotide polymorphisms (SNPs) based on their P-values and studying the areas under the curve (AUCs). The observed traits were found to have a genomic variance profile that differed significantly from that expected of a trait under an infinitesimal model. This situation was particularly true for both body weight and body fat, for which the AUCs were much higher compared with that of glucose. In addition, SNPs with a high degree of trait-specific regulatory potential (SNPs associated with subset of transcripts that significantly associated with a specific trait) explained a larger proportion of the genomic variance than did SNPs with high overall regulatory potential (SNPs associated with transcripts using traditional eQTL analysis). We introduced AUC measures of genomic variance profiles that can be used to quantify relative importance of SNPs as well as degree of deviation of a trait's inheritance from an infinitesimal model. The shape of the curve aids global understanding of traits: The steeper the left-hand side of the curve, the fewer the number of SNPs controlling most of the phenotypic variance.

Genetic influences on growth and body composition in mice: multilocus interactions.

BACKGROUND: The genetic architecture of body weight and body composition is complex because these traits are normally influenced by multiple genes and their interactions, even after controlling for the environment. Bayesian methodology provides an efficient way of estimating these interactions. SUBJECTS AND MEASUREMENTS: We used Bayesian model selection techniques to simultaneously estimate the main effects, epistasis and gene-sex interactions on age-related body weight (at 3, 6 and 10 weeks, denoted as WT3wk, WT6wk and WT10wk) and body composition (organ weights and fat-related traits) in an F(2) sample obtained from a cross between high-growth (M16i) mice and low-growth (L6) mice. RESULTS: We observed epistatic and main-effect quantitative trait loci (QTL) that controlled both body weight and body composition. Epistatic effects were generally more significant for WT6wk than WT10wk. Chromosomes 5 and 13 interacted strongly to control body weight at 3 weeks. A pleiotropic QTL on chromosome 2 was associated with body weight and some body composition phenotypes. Testis weight was regulated by a QTL on chromosome 13 with a significantly large main effect (2log(e)BF approximately 15). CONCLUSION: By analyzing epistatic interactions, we detected QTL not found in a previous analysis of this mouse population. Hence, the detection of gene-gene interactions may provide new information about the genetic architecture of complex obesity-related traits and may lead to the detection of additional obesity genes.

Quantitative genetics of transgenic mice: components of phenotypic variation in body weights and weight gains.

Transgenic mice possessing an ovine growth hormone gene were used to study the effects of elevated growth hormone on quantitative genetic variation. Males hemizygous for the transgene were mated to wild-type females to produce half- and full-sib families in which approximately half the progeny were transgenic and half were wild type. Analyses of body weights at 3-10 weeks, and weight gains from 3 to 6, and 6 to 10 weeks produced estimates of the proportion of total variance due to additive genetic effects (h2) and common litter effects (c2), and the genetic correlation between transgenic and wild-type expression of each trait. At 10 weeks, body weight of transgenics exceeded that of wild types by 26 and 49% in males and females, respectively. Estimated genetic variances in the transgenic group were significantly greater than zero for body weights at most ages and for both measurements of gain. Common litter effects accounted for a similar proportion of variation in the wild-type and transgenic groups. Additive genetic correlations between wild-type and transgenic expression of body weights tended to decline with age, indicating that a partially different array of genes may have begun to affect body weight in the transgenic group.

The impact of maternal uterine genotype on postnatal growth and adult body size in mice.

Embryo transfers were used to demonstrate that the genotype of the mother providing the uterine developmental environment significantly influences postnatal growth and adult body size of her progeny. Irrespective of their own genotype, mouse embryos transferred into the uterus of an inbred strain with large body size (C3H) had greater body weights, longer tails and higher growth rates than those transferred into the uterus of a strain with small body size (SWR). Uterine heterosis on body size was smaller than progeny heterosis, and both progeny and uterine heterosis persisted in adult mice. Uterine litter size was significantly negatively associated with body weight, tail length, growth rate and the timing of developmental events. The inbred SWR strain was more sensitive to the embryo transfer procedure than the C3H strain, but effects due to embryo transfer were moderate. Prenatal uterine effects have ramifications for biotechnologies utilizing embryo transfer as well as predictions about evolutionary change by selection.

Identification of quantitative trait loci influencing traits related to energy balance in selection and inbred lines of mice.

Energy balance is a complex trait with relevance to the study of human obesity and maintenance energy requirements of livestock. The objective of this study was to identify, using unique mouse models, quantitative trait loci (QTL) influencing traits that contribute to variation in energy balance. Two F2 resource populations were created from lines of mice differing in heat loss measured by direct calorimetry as an indicator of energy expenditure. The HB F2 resource population originated from a cross between a noninbred line selected for high heat loss and an inbred line with low heat loss. Evidence for significant QTL influencing heat loss was found on chromosomes 1, 2, 3, and 7. Significant QTL influencing body weight and percentage gonadal fat, brown fat, liver, and heart were also identified. The LH F2 resource population originated from noninbred lines of mice that had undergone divergent selection for heat loss. Chromosomes 1 and 3 were evaluated. The QTL for heat loss identified on chromosome 1 in the HB population was confirmed in the LH population, although the effect was smaller. The presence of a QTL influencing 6-wk weight was also confirmed. Suggestive evidence for additional QTL influencing heat loss, percentage subcutaneous fat, and percentage heart was found for chromosome 1.

Gene expression in hypothalamus and brown adipose tissue of mice divergently selected for heat loss.

Gene expression was evaluated in mice divergently selected for 16 generations for heat loss, measured by direct calorimetry. The high (MH) heat loss line has approximately 50% greater heat loss, approximately 35% less body fat, approximately 20% greater feed intake, and twofold greater activity levels than the low (ML) heat loss line. At 11 wk, inbred males (developed from MH and ML) were euthanized 3 h after dark for dissection of tissues and extraction of RNA. Differential display PCR (DD-PCR) was used to evaluate transcriptional differences between lines in hypothalamus and brown adipose tissue (BAT). Evaluation was replicated within and across lines, using family pools of mRNA. Two genes were confirmed by competitive RT-PCR and/or Northern analysis to have greater levels of mRNA present in ML relative to MH mice. In both hypothalamus and BAT, the ribosomal protein L3 (RPL3) gene was expressed at higher levels in ML, whereas an unknown expressed sequence tag (EST) was also found at higher levels in the hypothalamus of ML mice. These results implicate RPL3 in regulation of energy balance and extend the genetic dissection of response to selection to the transcriptional level.

Lower sodium lactate in Whitten's medium improves in vitro developmental capacity of one-cell mouse embryos.

A modification of Whitten's medium, involving a reduced content of Na-lactate syrup (0.2 ml/100 ml; 11.65 mM) and osmolarity (251 mOsm), was compared with normal Whitten's medium (0.37 ml/100 ml; 21.6 mM) for ability to support mouse embryonic development in vitro from one-cell to the blastocyst stage. In a pilot study utilizing 10 ICR donor female mice, in vitro developmental capacity (IVDC; percentage of fertilized one-cell embryos developing to blastocysts in vitro per female donor) was significantly enhanced by the modified medium (68.0 versus 24.0%; P<0.001). In the main study, utilizing 134 ICR and 17 ICR x C57BL/6J F(1) donor females, the modified medium supported increased IVDC for both ICR (67.9 versus 51.1%; P<0.001) and F(1) females (98.5 versus 89.4%; P<0.05). A large degree of among donor-female variation in IVDC was observed for both media in the ICR stock (SD = 30.0). The beneficial role of the reduction of Na-lactate in Whitten's medium may be related to an improved provision of energy requirements for first cleavage and/or a more suitable osmolarity for development.

Genetic variation in reproductive responses to a high-energy diet in mice.

Effects of a high-energy diet on reproduction were studied in 300 mice from lines selected for litter size and(or) 6-wk BW (L+, increased litter size; W+, increased body weight; L+W-, increased litter size and decreased body weight; L-W+, decreased litter size and increased body weight; and K, randomly selected control). Mice received a high-energy diet (HED; 3.8 kcal/g of ME) or a standard diet (STD; 3.3 kcal/g of ME) from 8 to 11 wk of age and were then mated and evaluated for ovulation rate and embryo survival through 17 d of gestation. The HED increased ovulation rate in all lines (P less than .05). The line x diet interaction was significant, with increased ovulation rate due to HED ranging from 9.9% in W+ to 24.2% in L-W+. Within-line regression coefficients of ovulation rate on ME intake (kilocalories from 10 to 11 wk) varied from .08 +/- .04 (P less than .05) in L+W- to .177 +/- .05 (P less than .01) in L+. In contrast, nonsignificant increases were observed in litter size (live fetuses at 17 d of gestation) due to HED. Effects of HED on embryo survival rate were significantly negative in L+ and L+W-; the decrease in L+ was a result of preimplantation losses, and the decrease in L+W- was due to postimplantation losses. The line x diet interaction was significant for postimplantation embryo survival. The results indicate significant genetic variation in reproductive responses to a high-energy diet in mice.

Feasibility of the grandprogeny design for quantitative trait loci (QTL) detection in purebred beef cattle.

The grandprogeny design (GPD) was developed for dairy cattle to use existing pedigreed populations for quantitative trait locus (QTL) detection. Marker genotypes of grandsires and sons are determined, and trait phenotypic data from grandprogeny are analyzed. The objective of this study was to investigate the potential application of GPD in purebred beef cattle populations. Pedigree structures of Angus (n = 123,319), Hereford (n = 107,778), Brangus (n = 14,449), and Gelbvieh (n = 8,114) sire evaluation reports were analyzed to identify potentially useful families. Power of QTL detection was calculated for a range of QTL effects (.1 to .5 SD) and two Type I error rates (.01 and .001). Reasonable power (> .75) could be achieved using GPD in Angus and Hereford for QTL having moderate effects (.3 SD) on weaning weight and large effects (.4 to .5 SD) on birth, yearling, and maternal weaning weights by genotyping 500 animals. Existing Gelbvieh and Brangus families useful for GPD were limited, and reasonable power could be expected only for QTL having large effects on weaning or birth weights. Although family structures suitable for GPD exist in purebred beef populations, large amounts of genotyping would be required to achieve reasonable power, and only QTL having moderate to large effects could be expected to be identified.

Cytoplasmic effects on selection response for increased growth rate in mice.

To determine if cytoplasmic effects have contributed to long-term selection response for increased growth rate in mice, reciprocal cross matings were made between an unselected control line (ICR) and a line (M16) derived from ICR by long-term selection for high postweaning weight gain from 3 to 6 wk of age. Embryos were recovered 2 to 4 d following mating and transferred to pseudopregnant F1 (DBA/2NCrlBR X C57BL/6NCrlBR) females. Thus, all embryos developed in similar uterine and postnatal maternal environments. A total of 122 M16 X ICR and 123 ICR X M16 mice was produced, representing 19 litters from each cross. Litters were standardized at birth to five to seven pups. Litter weights at birth and 1 wk were recorded. Body weights at 2, 3, 4, 5 and 6 wk and weight gain from 3 to 6 wk were obtained. Weights of liver, kidneys, and sc and epididymal fat pads of males were obtained at 6 wk. Females were mated at 8 wk, and litter size at birth was recorded. Least-squares procedures were used to test for differences between reciprocal crosses for all traits. Body weight at 4 wk was higher (P less than .05) for mice with ICR cytoplasm. No other significant differences were detected. There was no evidence that cytoplasmic effects influenced direct or correlated responses to long-term selection for increased postweaning weight gain.

Characterization of DNA polymorphisms in three populations of hereford cattle and their associations with growth and maternal EPD in line 1 herefords.

Three populations of Hereford cattle differing in inbreeding levels and genetic potential for growth were genotyped for seven DNA polymorphisms. The populations were compared to determine differences in allele frequency and genetic variation. Significant differences in allele frequency among the populations were found at six of the seven polymorphisms genotyped, and average genetic variation differed as expected when inbreeding levels were considered. Effects of several polymorphisms on growth and maternal EPD were evaluated for one population (Miles City Line 1 Herefords) using regression analysis. Substitution of a B allele for an A allele of the kappa-casein polymorphism accounted for significant decreases in direct birth weight and maternal 180-d gain from birth to weaning EPD explaining 15% and 8%, respectively, of EPD variability. Several other significant effects accounting for small portions of EPD variability were also detected.

Evaluation of gene expression in pigs selected for enhanced reproduction using differential display PCR and human microarrays: I. Ovarian follicles.

Differential display PCR (ddPCR) and complementary DNA microarray analyses were used to evaluate gene expression differences in porcine ovarian follicles between a line of pigs selected for an index of ovulation rate and embryo survival (Line I) and its randomly selected control line (Line C). Follicles (4.0 to 7.0 mm) were dissected from ovaries of multiparous sows (n = 27) at either 2 or 4 d following PGF2alpha analog injection on d 12 to 14 of the estrous cycle. Using ddPCR, differentially expressed bands (n = 282) were excised from gels and 107 were sequenced, yielding 84 unique porcine follicle expressed sequence tags. Northern hybridization confirmed differential expression (between lines, days, or follicle sizes) for messenger RNA representing the calpain I light subunit, cytochrome C oxidase subunit III, cytochrome P450 aromatase, and cytochrome P450 side chain cleavage genes. For microarray analysis, two mRNA pools representing follicles (d 2; 4.50 to 4.75 mm) from Line I and Line C sows were hybridized to the Incyte UniGEM V1.0 human chip (approximately 7,000 gene probes). A second analysis was performed using mRNA from follicles (d 2; 4.50 to 5.00 mm) hybridized to the Incyte UniGEM V2.0 human chip (approximately 9,100 gene probes). A total of 33 and 21 genes were identified with significant expression differences using UniGEM V1.0 and V2.0, respectively (twofold or greater relative expression following adjustment for expression of control probes). However, there was little overlap between results of the two hybridizations. Expression differences between lines for two genes, follistatin and nuclear receptor subfamily 4, group A, member 1, were confirmed using Northern hybridization. These results demonstrate changes in follicular gene expression as the result of long-term selection for enhanced reproduction. These correlated responses may directly represent allelic variation utilized by selection (e.g., quantitative trait loci), or more likely, transcriptional changes in other genes that interact with reproductive QTL. This work represents one of the first applications of gene expression analysis to evaluate long-term selection response in livestock populations.

Evaluation of gene expression in pigs selected for enhanced reproduction using differential display PCR: II. Anterior pituitary.

The objective of this study was to identify differentially expressed genes in the anterior pituitary (AP) of sows selected for enhanced reproductive phenotypes. Selection in the Index (I) line was based on an index of ovulation rate and embryo survival, whereas random selection was used in the Control (C) line. Average numbers of fully formed piglets at birth were 12.5 +/- 1.5 and 9.9 +/- 2.0 for Line I and C sows used in this study, respectively. In order to induce luteolysis and synchronize follicle development, sows were injected (i.m.) with 2 mL of prostaglandin F2alpha analog between d 12 and 14 of the estrous cycle. Tissue was harvested 2 d (d2) or 4 d (d4) after injection, resulting in four experimental groups: Cd2 (n = 6), Cd4 (n = 4), Id2 (n = 6), and Id4 (n = 7). Differential display PCR (ddPCR) was used to search for transcriptional changes between selection lines in the AP, using samples within line but pooled across days. Northern hybridization was used to confirm ddPCR results. For ddPCR, two pools were used from each line (C and I). Three genes were confirmed to be differentially expressed between Lines I and C: G-beta like protein, ferritin heavy-chain, and follicle stimulating hormone beta subunit, whereas many other expressed sequence tags were observed to be differentially expressed but still require confirmation. Our findings indicate that long-term selection to increase ovulation rate and decrease embryo mortality has altered transcriptional patterns in the anterior pituitary, most likely as correlated responses.

Candidate gene analysis for loci affecting litter size and ovulation rate in swine.

A candidate gene approach was used to determine whether specific loci explain responses in ovulation rate (OR) and number of fully formed (FF), live (NBA), stillborn, and mummified pigs at birth observed in two lines selected for ovulation rate and litter size compared with a randomly selected control line. Line IOL was selected for an index of OR and embryonic survival for eight generations, followed by eight generations of two-stage selection for OR and litter size. Line C was selected at random for 16 generations. Line COL, derived from line C at Generation 8, underwent eight generations of two-stage selection. Lines IOL and C differed in mean EBV by 6.1 ova and 4.7 FF, whereas lines COL and C differed by 2.2 ova and 2.9 FF. Pigs of Generation 7 of two-stage selection lines were genotyped for the retinol binding protein 4 (RBP4, n = 190) and epidermal growth factor (EGF, n = 189) loci, whereas pigs of Generations 7 and 8 were genotyped for the estrogen receptor (ESR, n = 523), prolactin receptor (PRLR, n = 524), follicle-stimulating hormone beta (FSHbeta, n = 520), and prostaglandin-endoperoxide synthase 2 (PTGS2, n = 523) loci. Based on chi-square analysis for homogeneity of genotypic frequencies, distributions for PRLR, FSHbeta, and PTGS2 were different among lines (P < 0.005). Differences in gene frequencies between IOL vs C and COL vs C were 0.33 +/- 0.25 and 0.16 +/- 0.26 for PRLR, 0.35 +/- 0.20 and 0.15 +/- 0.24 for FSHbeta, and 0.16 +/- 0.16 and 0.08 +/- 0.18 for PTGS2. Although these differences are consistent with a model of selection acting on these loci, estimates of additive and dominance effects at these loci did not differ from zero (P > 0.05), and several of them had signs inconsistent with the changes in allele frequencies. We were not able to find significant associations between the polymorphic markers and phenotypes studied; however, we cannot rule out that other genetic variation within these candidate genes has an effect on the traits studied.

Sex identification in mammals with polymerase chain reaction and its use to examine sex effects on diameter of day-10 or -11 pig embryos.

The objectives of this study were to develop a rapid method for sex determination for several mammalian species using polymerase chain reaction (PCR) and to use this method to determine whether there is a significant developmental difference in spherical diameter between male and female d-10 or -11 porcine embryos. The PCR system was developed and verified using genomic DNA from pigs of known sex, then it was tested with genomic DNA from several other mammalian species. Sex is determined by amplification of two genes in a single reaction. The presence or absence of a region of the Sry (sex-determining region Y) gene determines sex, and amplification of the Zfy (male) or Zfx (female) genes acts as a positive control for PCR. Sex determination was successful for all animals tested, including pigs, cattle, sheep, goats, llamas, horses, humans, baboons, dogs, cats, rats, and mice. A total of 209 embryos were collected from 21 crossbred gilts on d 10 or 11 of gestation, and their diameters were measured. No significant difference in embryo diameter was detected between male and female embryos, indicating that sexual dimorphism in embryonic growth in pigs does not occur before the period of rapid embryo elongation. The present sexing technique using PCR is rapid (approximately 6 h from receipt of embryos to results), and it may be useful for examining the effects of sex on any trait of interest in early porcine embryos and embryos from several other mammals.

Observations on the cooling and cryopreservation of pig oocytes at the germinal vesicle stage.

This study examined the viability of pig oocytes at the germinal vesicle stage following cooling or cryopreservation. Cumulus-intact oocytes (n = 641) were collected from slaughterhouse pig ovaries and used in two experiments. In Exp. I the viability of 1) control, 2) cryoprotectant control (CC, 1.5 M glycerol/.5 M sucrose), 3) cooled (0 degrees C) and 4) cryopreserved (-196 degrees C) oocytes was assessed after no incubation or a 24-h incubation. Survivability was judged by morphological appearance, trypan blue exclusion and fluorescein diacetate staining. Survival rate of control oocytes (90%; based primarily on morphological appearance of the cumulus) incubated 0 h was greater (P less than .05) than that of all other groups, whereas survival rate of -196 degrees C oocytes (57%) was less (P less than .05) than that of all other groups. However, vital staining of 0 degrees C and -196 degrees C oocytes showed 0% survival rate as evidenced by trypan blue uptake and lack of fluorescence. The cumulus cells surrounding oocytes that were stored at 0 degrees C or -196 degrees C survived freezing as evidenced by trypan blue exclusion and intense fluorescence. Similar differences among treatment groups were found for oocytes incubated 24 h. Exp. 2 examined the temperature at which oocytes became sensitive to cooling. Oocyte death occurred when oocytes were cooled to 15 degrees C or lower. These results demonstrate that pig oocytes at the germinal vesicle stage did not survive cooling to 15 degrees C or below. When assessing the viability of cryopreserved cumulus enclosed oocytes it is important to use vital stains in conjunction with morphological appearance.

Identification of quantitative trait loci affecting reproduction in pigs.

The objective of this research was to identify chromosomal regions harboring QTL affecting reproduction in pigs. A three-generation resource population was developed by crossing low-indexing pigs from a randomly selected control line (C) with high-indexing pigs of a line selected for increased index of ovulation rate and embryonic survival (I). Differences between Lines I and C at Generation 10 were 6.7 ova and 3.3 fetuses at 50 d of gestation and 3.1 fully formed and 1.6 live pigs at birth. Phenotypic data were collected on F2 females, born in three replicates, for ovulation rate (n = 423), age at puberty (n = 295), litter size (n = 370), and number of nipples (n = 428). Litter-size data included number of fully formed, live, stillborn, and mummified pigs. Grandparent, F1, and F2 animals were genotyped for 151 microsatellite markers distributed across all 18 autosomes and the X chromosome. Genotypic data were available on 423 F2 females. Average spacing between markers was 19.3 Kosambi centimorgans. Calculations of logarithms of odds (LOD) scores were by least squares, and fixed effects for sire-dam combination and replicate were included in the models. Genome-wide significance level thresholds of 5% and 10% were calculated using a permutation approach. There was evidence (P < 0.05) for QTL affecting ovulation rate on SSC9, age at puberty on SSC7 and SSC8, number of nipples on SSC8 and SSC11, number of stillborn pigs on SSC5 and SSC13, and number of fully formed pigs on SSC11. There was evidence (P < 0.10) for additional QTL affecting age at puberty on SSC7, SSC8, and SSC12, number born live on SSC11, and number of nipples on SSC1, SSC6, and SSC7. Litter size is lowly heritable and sex-limited. Therefore, accuracy of selection for litter size may be enhanced by marker-assisted selection. Ovulation rate and age at puberty are laborious to measure, and thus marker-assisted selection may provide a practical and efficient method of selection.

Dependence of increased linear bone growth on age at oMT1a-oGH transgene expression in mice.

We have previously shown that growth hormone (GH) accelerates the rate and extends the duration of linear bone growth in a GH-transgenic mouse model. To determine if this GH effect was temporally regulated, bone lengths and growth plate widths were determined in transgenic mice carrying an ovine metallothionein 1a-ovine growth hormone (oMT1a-oGH) transgene. Transgene expression was initiated in oMT1a-oGH hemizygous transgenic mice by addition of 25 mM ZnSO4 to the water at 21 d of age; littermate control mice were also ZnSO4 supplemented. These mice were maintained on ZnSO4 until 70 d of age at which time the mice were killed and the ulna, humerus, and tibia were collected. Additional transgenic and control mice were stimulated at 21 d and the ZnSO4 stimulus withdrawn after 21 d of treatment and killed at 70 d or stimulated at 28 d with the stimulus withdrawn after 28 d and killed at 70 d. Continuously stimulated transgenic mice had longer bones than those of comparable control mice. The increased bone length in the transgenics was correlated with wider growth plates. Transgene expression initiated at 21 d of age increased bone length to the same or greater extent than that observed for mice with transgene expression initiated at 28 d but maintained for a longer interval thereby indicating that the age of GH initiation is more critical in bone elongation than the duration of GH exposure. The elevated GH effects were independent of circulating thyroid hormones and attainment of puberty. Elevated GH also increased the widths of all growth plate zones to the same extent by increasing cell numbers, rather than enhancing matrix production or individual cellular area.

The epigenetic influence of growth hormone on skeletal development.

We studied the epigenetic effect of growth hormone using mice that were transgenic for a sheep metallothionein 1a-sheep growth hormone, which was expressed beginning at 21 days postnatal age. The impact of exogenous growth hormone (GH) on various skeletal traits with special emphasis on the mandible was examined by conventional statistical analysis and finite element scaling analysis. In long bones, growth hormone enhances the proliferation rate of cartilage cells in the growth plate and should thus lead to increased lengths. Further, growth hormone is known to increase muscle mass. Our results are consistent with these developmental considerations. We found that the lengths of long bones increased in the transgenic mice compared to the control mice, while the differences in long bone width were less pronounced. In the mandible and skull, the impact of GH is most pronounced in areas of major muscle attachment, i.e., the proximal part of the mandible and the occipital and malar bones in the skull.

Body composition of inactivated growth hormone (oMt1a-oGH) transgenic mice: generation of an obese phenotype.

The consequences of a 42 d exposure to elevated growth hormone (GH) on adipose tissue were assessed using the regulatable ovine metallothionein- ovine GH (oMt1a-oGH) transgene in male and female GH transgenic (TG) mice. Activation of transgene expression at 21 d of age followed by inactivation of transgene expression at 63 d of age (TG-on/off) increased individual white adipose tissue (WAT) depots and total body lipid stores in both males and females. WAT, expressed as a percentage of fasted body weight, did not differ in wildtype (WT) and continuously activated TG males and females up to 105 d of age, but was increased approximately 270% following inactivation of the transgene. Inguinal depot adipocytes were more numerous in both male and female TG +/- relative to WT or TG animals. The ensuring obesity was not accompanied by a decrease in thermogenic capacity of brown adipose tissue, as indexed by uncoupling protein quantity. GH transgene expression was accompanied by elevated insulin levels that were restored to WT levels upon cessation of transgene expression (p > 0.1). Early, transient exposure to elevated GH increased total body lipid by nearly threefold independent of gender; the increased lipid content was sustained and reflected WAT hypertrophy and hyperplasia. The oMt1a-oGH mouse provides a novel model of induced obesity in response to inactivation of a GH-transgene by the withdrawal of the transgene stimulus.