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Nonhereditary Enhancement of Progeny Growth

Center for Cell and Gene Therapy (A.K., L.A.H, K.K.C., R.D.A.), Children’s Nutrition Research Center (M.F.), Department of Molecular and Cellular Biology (P.B.M., D.P., R.J.S., R.G.S., R.D.A.), and Huffington Center on Aging (R.G.S.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Ruxandra Draghia-Akli, Advisys, Inc., 2700 Research Forest Drive, Suite 180, The Woodlands, Texas 77381. E-mail: ruxandradraghia{at}advisys.net.

	Abstract

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The im electroporated injection of a protease-resistant GH-releasing hormone cDNA into rat dams at 16 d gestation resulted in enhanced long-term growth of the F₁ offspring. The offspring were significantly heavier by 2 wk of age, and the difference was sustained to 10 wk of age. Consistent with their augmented growth, the plasma IGF-I concentration of the F₁ progeny was increased significantly. The pituitary gland of the offspring was significantly heavier and contained an increased number of somatotrophs and PRL-secreting cells, which is indicative of modification of cell lineage differentiation. These unique findings demonstrate that enhanced GH-releasing hormone expression in pregnant dams can result in intergenerational growth promotion by altering development of the pituitary gland in the offspring.

	Introduction

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PITUITARY DEVELOPMENT, including the regulation and differentiation of somatotrophs, depends upon paracrine processes within the pituitary itself and involves several growth factors and neuropeptides, such as vasoactive intestinal peptide (1), angiotensin 2, endothelin (2), and activin (3). Secretion of GH from the pituitary is stimulated by GH-releasing hormone (GHRH) and is inhibited by somatostatin (4). In healthy adult mammals, GH is released in a highly regulated, distinctive pulsatile pattern, which occurs when the stimulatory properties of GHRH are enabled by the diminution or withdrawal of somatostatin secretion. This episodic pattern of GH secretion has profound importance for its biological activity (5) and is required for the induction of its physiological effects at the peripheral level (6). Regulated GH secretion is essential for optimal linear growth, for homeostasis of carbohydrate, protein, and fat metabolism, and for promoting a positive nitrogen balance (7). These effects are mediated largely by its downstream effector, IGF-I. GH secretion also is influenced by ligands for the GH secretagogue receptor, which are dependent on GHRH (8) for their GH secretory activity (9).

Hypothalamic tissue-specific expression of the GHRH gene is not required for its biological activity, as indicated by the biological activity of extracranially secreted GHRH (10, 11). Recently, we showed that in pigs, ectopic expression of a novel, serum protease-resistant porcine GHRH driven by a synthetic muscle-specific promoter could elicit robust GH and IGF-I responses after its in vivo administration by im injection and subsequent electroporation (12). In the rat model, GHRH administration is effective in inducing pituitary GH mRNA expression and increasing GH content as well as somatic growth (13). Although the intergenerational effects on the offspring of pregnant animals with sustained GHRH expression are as yet unknown, studies in adult animals indicate a potential plasticity of the somatotrophs in response to GHRH. Pathological GHRH stimulation (regardless of its source, from transgenic models to pancreatic tumors) of GH secretion can result in proliferation, hyperplasia, and adenomas of the adenohypophysial cells (14, 15). Therefore, we reasoned that GHRH delivered by plasmid DNA gene transfer to pregnant rats during the last trimester of gestation would alter the lineage specification of the anterior pituitary gland of the developing pups, favoring differentiation toward somatotrophs and lactotrophs (16). This treatment of pregnant rats should then produce long-term enhanced growth in their offspring in the complete absence of other exogenous growth stimulants.

	Materials and Methods

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DNA constructs
The plasmid pSPc5–12 contained a 360-bp SacI/BamHI fragment of the SPc5–12 synthetic promoter (17) in the SacI/BamHI sites of the pSK-GHRH backbone (18). The mutated GHRH cDNA was obtained by site-directed mutagenesis of human GHRH cDNA (Altered Sites II In Vitro Mutagenesis System, Promega Corp., Madison, WI) (19). This fragment was cloned into the BamHI/HindIII sites of pSK-GHRH. hGH pA is a 3'-untranslated region and polyadenylase signal from the human GH gene. Control plasmid contained the Escherichia coli ß-galactosidase gene under the same muscle-specific promoter, pSP-ß-gal. Plasmids were grown in E. coli DH5 (Life Technologies, Inc., Carlsbad, CA). Endotoxin-free plasmid (QIAGEN, Chatsworth, CA) preparations were diluted to 1 mg/ml in PBS, pH 7.4, and stored at -80 C before use.

In vivo protocols
Timed pregnant Wistar female rats (Taconic Farms, Inc., Germantown, NY) were housed and cared for in the animal facility of Baylor College of Medicine (Houston, TX). Dams were individually housed in polycarbonate cages with free access to food and water. Animals were maintained under environmental conditions of 10 h of light/14 h of darkness in accordance with NIH Guide, USDA, and Animal Welfare Act guidelines, and the protocol was approved by the institutional animal care and use committee.

Intramuscular injection of plasmid and electroporation
The experiment was repeated twice. On d 16 of gestation, dams (n = 20/group) were weighed and anesthetized using a combination of 42.8 mg/ml ketamine, 8.2 mg/ml xylazine, and 0.7 mg/ml acepromazine, administered im at a dose of 0.5–0.7 ml/kg. The left tibialis anterior muscle was injected with 30 µg pSP-HV-GHRH in 100 µl PBS using 0.3-ml insulin syringes (BD Biosciences, Franklin Lakes, NJ). Control animals were injected with 30 µg pSP-ß-gal. For both groups, the injection was followed by electroporation (19). Briefly, 2 min after injection, the rat leg was placed between a two-needle electrode (1 cm in length, 26 gauge, 1 cm between needles; Genetronics, San Diego, CA), and electric pulses were applied. Three 60-msec pulses at a voltage of 100 V/cm were applied in one orientation, then the electric field was reversed, and three more pulses were applied in the opposite direction. The pulses were generated with a T-820 Electro Square Porator (Genetronics). Blood was collected from the dams before injection, on delivery day, and then at the same time points as in the offspring analysis.

Offspring studies
All injected dams gave birth at 21–22 d gestation. At birth, litter size was adjusted to 10 pups/dam. Pups were weaned at 3 wk of age. In the first study 240 offspring and in the second study 60 offspring were analyzed at 3, 6, 10, 12, 16, and 24 wk of age. At least 2 randomly selected animals of each sex from each litter were analyzed at each time point (20–24 animals/sex/time point). Body weights were recorded at these time points and at 2 wk of age using the same calibrated balance. At the end of the experiment animals were anesthetized, blood was collected by cardiac puncture and centrifuged immediately at 4 C, and plasma was stored at -80 C before analysis. Organs [heart, liver, spleen, kidney, pituitary, brain, adrenals, skeletal muscles: tibialis anterior (TA), gastrocnemius, soleus, and extensor digitorum longus] from the offspring of treated and control dams were removed, weighed on an analytical balance, and snap-frozen in liquid nitrogen. The tibia was dissected, and length was measured to the nearest 0.1 mm using calipers.

Northern blot analysis of mRNA from the pituitary
Pituitaries from five animals per group that had been snap-frozen were pooled, homogenized, and extracted for RNA. Total RNA was treated with deoxyribonuclease I, and 20 µg of the DNA-free RNA were size separated in 1.5% agarose-formaldehyde gel and transferred to nylon membrane. The membranes were hybridized with specific GHRH, GH (gift from Dr. Kelly Mayo), and PRL (gift from Dr. Kathleen Mahon) ³²P-labeled, cDNA riboprobes.

Rat IGF-I RIA
Rat IGF-I was measured by specific RIA (Diagnostic Systems Laboratories, Inc., Webster, TX). The sensitivity of the assay was 0.8 ng/ml; intra- and interassay coefficients of variation were 2.4% and 4.1%, respectively.

Biochemistry
Glucose and other metabolites were analyzed by an independent specialized veterinary laboratory (Antech, Irvine, CA).

Immunohistochemistry
Pituitary glands were fixed immediately after dissection in 3% paraformaldehyde in PBS overnight. After fixation, samples were washed and stored in 70% ethanol until analyzed. Pituitary glands were paraffin embedded, and 5-µm-thick sections were cut, deparaffinized, and washed in PBS. Sections were blocked using a solution of 5% normal goat serum, 1% BSA, and 0.05% Tween 20 in PBS for 1 h at room temperature. The sections then were incubated for 2 h at room temperature with the primary antibodies, rabbit antirat GH (AFP5672099Rb, National Hormone and Peptide Program), rabbit antirat PRL (AFP4251091, National Hormone and Peptide Program), and rabbit antirat ACTH (AFP15612489, National Hormone and Peptide Program) diluted 1:2,000, 1:10,000, and 1:5,000, respectively. After washing off the primary antibodies, secondary peroxidase-conjugated goat antirabbit IgG antibody (Sigma, St. Louis, MO) at a 1:5,000 dilution was subsequently applied for 30 min at room temperature. Slides were washed in distilled water in between every step of the procedure. Peroxidase activity was revealed using a diaminobenzidene substrate applied for 4 min (Vector Laboratories, Inc., Burlingame, CA). Slides were counterstained with hematoxylin to visualize cell morphology and nuclei. For each animal, the number of immunoreactive cells per total number of cells was counted in at least five fields for each antibody, and the values were averaged; the analyzer was blinded to animal group. A total of five animals were analyzed per group. Digital images of the slides were captured using a CoolSnap digital color camera (Roper Scientific, Tucson, AZ) with MetaMorph software (Universal Imaging Corp., Downington, PA) and a Axioplan 2 microscope with a x40 objective (Carl Zeiss, New York, NY; numerical aperture, 0.75 plan).

Statistical analysis
Each experiment was carried out twice, with a total 20–24 pups/sex/time point. Values shown in the figures are the mean ± SE. Statistical significance was assessed using t test or ANOVA. P < 0.05 was set as the level of statistical significance.

	Results

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Pups from treated dams had enhanced growth and muscle hypertrophy
Pregnant rats at 16 d gestation were injected with 30 µg plasmid DNA pSP-HV-GHRH or pSP-ßgal in the tibialis anterior muscle (Fig. 1A). The injection was followed by electroporation to enhance plasmid uptake and GHRH gene expression. All rats gave birth at 21–22 d gestation. The average number of pups born per litter was similar between groups [pregnant rats treated with GHRH (T), 10.8 ± 0.75 pups/litter; controls (C), 11.75 ± 0.5 pups/litter]. At 2 wk of age, the average pup weight was 9% greater for the offspring of T compared with C dams (T, 31.5 ± 0.5 g/pup; C, 28.9 ± 0.8 g/pup; P < 0.014).

View larger version (24K): [in this window] [in a new window]   Figure 1. In vivo study of pSP-HV-GHRH expression vectors in pregnant rats. A, Thirty micrograms of pSP-HV-GHRH plasmid were delivered into the TA of rat dams at 16 d gestation. Control dams were injected with a similar construct driving the reporter, ß-galactosidase. The injection was followed by electroporation. B, Increased postnatal growth in offspring of GHRH-treated animals. Significant weight differences for both sexes were recorded at 3 and 10 wk of age (*, P < 0.05). CF, Female offspring of control dams; CM, male offspring of control dams. C, Muscle hypertrophy in the offspring of treated dams. Offspring of both sexes from treated animals had muscle hypertrophy at 3 wk of age. G/wt, Gastrocnemius weight/body weight; TA/wt, tibialis anterior weight/body weight; S/wt, soleus weight/total body weight. *, P < 0.02; , P < 0.008; , P < 0.01. At 24 wk of age TF maintained their muscle hypertrophy, whereas TM were similar to controls. , P < 0.007.

Pituitary composition is changed in offspring of T dams
Pituitary glands were dissected and weighed within the first few minutes postmortem. Pituitary weight adjusted for body (or brain) weight was significantly increased at least until 12 wk of age; this difference was more prominent for TF (Fig. 2A). Both the mRNA (Fig. 2B) and immunohistochemical (Fig. 3) results suggest that the increase in pituitary weight is due to hyperplasia of the somatotrophs and lactotrophs (20, 21). At 3 wk of age, Northern blot analysis of RNA extracted from the pituitaries of the progeny from T dams showed a 2.5-fold increase in GH and PRL mRNA expression compared with the levels in the progeny of C dams. This difference in response was associated with a 20% diminution in the endogenous rat GHRH mRNA levels. At the same age, sections immunostained with a rat GH-specific antibody (Fig. 3A), depicted an increased number of GH-immunoreactive cells (76% vs. 39% in C; Fig. 3C), with an increased amount of GH per immunoreactive somatotroph. Similarly, sections stained with a rat PRL-specific antibody (Fig. 3B), showed an increase in the number of PRL-producing cells (25% vs. 9% in C; Fig. 3C). Adrenocorticotrophs were present in equal proportion in T and C progeny.

View larger version (22K): [in this window] [in a new window]   Figure 2. GHRH has a trophic effect on the pituitary in vivo. A, Offspring of both sexes from treated dams had pituitary hypertrophy at all time points examined (wk 3 and 12 shown). B, Northern blot analysis of pituitary tissue at 3 wk (3w) and 10 wk (10w) of age show that GHRH acts as a growth factor on the pituitary gland. 18S, 18S rRNA, loading marker; GHRH, specific rat GHRH cDNA probe; GH, specific rat GH cDNA probe; PRL, specific rat PRL cDNA probe. The intensity of the bands was determined using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and associated software.

View larger version (85K): [in this window] [in a new window]   Figure 3. Immunostaining of pituitary glands from 3-wk-old progeny. A, Rat GH-specific staining. B, Rat PRL-specific staining. CP, Anterior pituitary from progeny control dams; TP, anterior pituitary from offspring of treated dams; NC, negative staining control, no primary antibody was added to the reaction. C, Percentage of immunoreactive cells for GH and PRL in control and offspring of treated dams.

View larger version (14K): [in this window] [in a new window]   Figure 4. IGF-I levels in offspring of injected pregnant rats at 3, 12, and 24 wk of age. Circulating IGF-I levels were measured by specific rat RIA. The graph depicts the fold increase in IGF-I levels over those in age- and sex-matched controls. *, P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The enhanced growth and the alterations in pituitary cellular composition of the progeny from T dams in the perinatal period are likely to be multifactorial. In the rat, there is an increase in plasma GH concentration in late pregnancy that results from increased pituitary GH gene transcription. This increase in circulating GH is associated, however, with a reduction in circulating IGF-I that is believed to occur because liver GH receptor expression is down-regulated during late pregnancy. Together with the development of peripheral insulin resistance, both of these responses that occur in late gestation facilitate the transfer of nutrient from the mother to the fetus (22). Thus, increased maternal GHRH production, by stimulating GH secretion and exacerbating insulin resistance, could enhance this process. This possibility is not supported by those studies that have observed no changes in placental size, fetal growth, or fetal IGF-I levels when rat dams were administered GH during pregnancy (23). Furthermore, plasma glucose levels were similar in C and T dams.

An alternative possibility is that maternal GHRH, when present at high concentrations as in the T dams, can cross the placenta in sufficient quantity to directly promote fetal growth. In utero growth of the wild-type fetuses of female transgenic mice that overexpress the GHRH gene (encoding for a 107-amino acid GHRH precursor) was normal (24). Nevertheless, in this instance the precursor molecule was processed to different smaller immunoreactive molecules, some of which are biologically inactive (25). Although the transplacental transmission of polypeptides is likely to be minimal, small biologically active growth factors of maternal origin have been identified in the fetuses (26, 27).

The efficiency of nutrient use for growth in newborn animals is very high. Thus, variations in postnatal weight gain in healthy newborn rats are dictated primarily by the amount of milk consumed (28). This is illustrated by the effect of litter size on weight gain; by 2 wk of age, rat pups suckled from birth in litters of 4 are 30% larger than pups suckled in litters of 10 (28). Even though GH and IGF-I are not as lactogenic in rodents as in ruminants, the increased weight of the progeny from T dams by 2 wk of age suggests that the sustained ectopic GHRH expression may have improved the volume and/or composition of their milk.

Milk, and colostrum especially, contain a variety of peptide growth factors that in rodents can be absorbed intact across the intestinal epithelium of the pups in the early neonatal period (29). These peptides include GHRH and PRL and thus, if present at higher concentrations in the milk of T dams, could function to modify pituitary differentiation (30). Additionally, given the greater sensitivity of the neonatal rat pituitary to the stimulatory effects of GHRH (31) and the responsiveness of growth to GH in newborn rat pups (32), stimulation of weight gain during the suckling period would be anticipated. The difference in weight between the progeny of T and C dams was maintained to adulthood. Thus, factors regulating development in both the prenatal and immediate postnatal periods require further study to define the mechanisms by which maternal changes in GHRH expression can reprogram the growth of the offspring.

The difference in weight between the progeny of treated and untreated dams was associated with enhanced postnatal growth of the musculature, which comprises the largest single tissue of lean body mass. In female offspring, the larger muscle mass was maintained for the entire period of the study (24 wk), whereas in male progeny the higher muscle to body weight ratio was present only to puberty. This difference might be explained by gender differences in the hormonal profiles. Males and females have similar concentrations of circulating testosterone before puberty, at which time testosterone levels increase dramatically in males (33). It is well known that postpubertal gonadal steroid environment plays an important role in determining anterior pituitary hormone synthesis and cellular composition (34). Thus, the high testosterone levels in postpubertal male rodents may promote further skeletal muscle growth and mitigate the difference associated with the early skeletal muscle growth enhancement in TM. In males, therefore, the early exposure to elevated GHRH may serve primarily to accelerate the time required to attain their maximum size.

Additionally, TF, unlike TM, sustained higher plasma IGF-I levels, as evidenced by the gender differences at 24 wk of age. This sexual dimorphism in plasma IGF-I concentrations is probably due to gender differences in the sensitivity of the hypothalamus and/or pituitary to IGF-I feedback. In female rats, ovarian hormones are thought to regulate the availability of IGF-I to hypothalamic and pituitary regions (35). The IGF system is under tight regulation during postnatal life when this gland continues to develop (36). The distinct temporal expression and its modification under our treatment of the IGF system probably determine further changes in the development and physiology of the anterior pituitary gland. The relatively increased levels of IGF-I may also explain the maintenance of muscle hypertrophy at 24 wk in TF.

Studies in GHRH transgenic animals have observed a specific pattern of progression, in which pituitary weight increases mainly after the first 6 months of life. Additionally, 70% of the glands examined contained grossly visible adenomas that stained positively for GH, whereas only some showed scattered PRL staining (37). This contrasts with our paradigm for enhanced GHRH production, in which the rat dams were treated in the last trimester of gestation. Thus, the pituitaries of the affected animals were exposed to high levels of hormone for only a limited period of time (4 wk at most) during a critical period of pituitary development. Evidently this afforded a change in pituitary cell lineage, causing increased concentrations of somatotrophs and lactotrophs, without neoplastic changes.

The use of GHRH, the upstream stimulator of GH, may be an alternate strategy to increase not only growth performance and milk production in animals, but, more importantly, the efficiency of production from both economical and metabolic perspectives (38, 39). We predict that, similar to treatments with recombinant GH or PRL during the immediate postnatal period, this treatment will mitigate specifically the deposition of body fat in later life (40) while enhancing lean tissue deposition. Animals would have a growth advantage without being individually treated with an exogenous growth promoter. Also, the high cost of repeatedly treating food animals with recombinant peptides, such as GH and IGF-I, limits widespread use for improving the productivity of livestock. These major drawbacks can be obviated by using a plasmid-mediated approach to direct the ectopic production of GHRH.

	Acknowledgments

 
The authors thank the National Hormone and Peptide Program, and Dr. A. F. Parlow for the rabbit antirat GH (AFP5672099Rb) and rabbit antirat PRL (AFP4251091) antibodies used in these experiments, Dr. Kathleen Mahon for the rat PRL and GHRH cDNA riboprobes, Dr. Kelly Mayo for the rat GH cDNA probe, Mrs. Brenda Johnson for the special care of the animals, and Mr. Michael Aron for data entry and correction of the manuscript. The authors also thank Hank Adams and Dr. Michael Mancini of the Integrated Microscopy Core Laboratory, Department of Molecular and Cellular Biology, Baylor College of Medicine.

	Footnotes

 
This work was supported by the Center for Cell and Gene Therapy, Baylor College of Medicine (Houston, TX), Dr. Malcolm Brenner (to R.D.-A.), and NASA (to R.J.S.), USDA/ARS (to M.L.F.), and NIH Postdoctoral Award DK-07696 (to A.S.K.).

Abbreviations: C, Controls; GHRH, GH-releasing hormone; T, treated; TA, tibialis anterior; TF, female offspring of treated dams; TM, male offspring of treated dams.

Received March 8, 2002.

Accepted for publication May 29, 2002.

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