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Endocrinology Vol. 143, No. 3 868-876
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Overexpression of Human Stanniocalcin Affects Growth and Reproduction in Transgenic Mice

Robin Varghese, Anthony D. Gagliardi, Peter E. Bialek, Siu-Pok Yee, Graham F. Wagner and Gabriel E. Dimattia

Departments of Biochemistry (R.V., P.E.B., S.P.Y., G.E.D.), Oncology (A.D.G., S.P.Y., G.E.D.), Physiology (G.F.W.), and Obstetrics/Gynecology (G.E.D.), Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 4L6

Address all correspondence and requests for reprints to: Dr. Gabriel DiMattia, London Regional Cancer Center, 790 Commissioners Road, London, Ontario, Canada N6A 4L6. E-mail: . dimattia{at}uwo.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In mammals stanniocalcin (STC) is widely expressed, and in the kidney and gut it regulates serum calcium levels by promoting phosphate reabsorption. To shed further light on its functional significance in mammals we have created several lines of mice that express a human STC (hSTC) transgene. Three lines expressed the hSTC transgene, but only two lines exhibited high expression and contained circulating hSTC, and in these animals there was a reduction in postnatal growth (30–50%) that persisted after weaning. Moreover, even wild-type pups exhibited a growth retardation phenotype when nursed by a transgenic foster mother, and this implies that hSTC overexpression deleteriously affects maternal behavior and/or lactation. The reproductive potential of female transgenic mice was also compromised, as evidenced by significantly smaller litter sizes, but transgenic male fertility was unchanged even though the transgene was most highly expressed in testes. Interestingly, transgene-derived serum hSTC increased significantly after puberty and was severalfold higher in females than in males, suggesting a gender-specific mechanism for maintaining elevated circulating levels of STC. Blood analysis revealed that both transgenic lines had elevated phosphate and decreased alkaline phosphatase levels, indicative of altered kidney and bone metabolism. These studies provide the first evidence that STC is involved in growth and reproduction and reaffirm its role in mineral homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STANNIOCALCIN (STC) is a glycoprotein hormone that was first identified in fish as a major secretory product of the corpuscles of Stannius, an organ unique to bony fish (1, 2, 3). In fish, STC is secreted into the blood in response to elevated serum calcium and acts on the gills, gut, and kidneys to regulate calcium absorption and phosphate excretion, thus aiding in the maintenance of calcium homeostasis. Because of its unique site of synthesis in fish, a mammalian homolog was not immediately predictable. Evidence for STC in mammals was first obtained by demonstrating specific immunoreactivity in human serum when examined with a salmon STC RIA. Moreover, proteins similar in size to fish STC were identified in human kidney extracts by Western blotting, and the antiserum produced specific staining in a distinct population of cells in human kidney tubules (4). The human STC (hSTC) cDNA was subsequently isolated as an established sequence tag (5) with significant nucleotide sequence homology to the salmon STC cDNA (6). Similarly, others identified STC as an overexpressed mRNA from simian virus 40-transformed cells (7). In contrast to its restricted expression in bony fish, mammalian STC is expressed in a wide variety of tissues and does not normally circulate in the blood (7, 8, 9). Furthermore, unlike its well defined role in regulating serum calcium in fish, little is known about its function in mammals.

To date, studies have focused on elucidating the cell-specific expression and localization pattern of STC in mammalian tissues, including kidney (10, 11, 12), bone (13, 14), and brain (15), and during mouse development (16, 17). Recently, STC has been implicated in different physiological processes, including wound healing (18), atherogenesis (19), and angiogenesis (20). Significant changes in STC mRNA levels have also been correlated with neuronal cell differentiation (21), ischemic insult to neurons (22), glucose responsivity in the MIN6 pancreatic ß-cell line (23), and the response of MDCK cells to osmotic stress (24). Functional analysis of STC in mammals has centered on showing that its role in regulating blood calcium has been conserved from fishes. In vivo studies using the rat kidney indicated that STC may regulate blood calcium by increasing reabsorption of phosphate (25), and in vitro experiments on mammalian intestine have shown that STC can reduce the movement of calcium while increasing absorption of phosphate across the gut (26). In both models the predicted net effect would be a decrease in circulating levels of calcium, thereby acting as an antihypercalcemic factor.

Recently, we reported the unexpected finding that the ovary is the site of greatest STC expression in sexually mature rodents (8). In situ hybridization and immunohistochemical studies showed that STC expression is strongest in thecal-interstitial cells, and that ovarian STC expression begins as early as 5 d after birth. Interestingly, STC is concentrated in the egg and corpus luteum, suggesting that these cells sequester STC produced by the adjacent thecal-interstitial cells. Furthermore, ovarian STC production is significantly increased during pregnancy and lactation, such that it becomes detectable in the circulation, with a peak serum concentration at midpregnancy (27). The ovarian expression of STC during lactation can be modulated by the presence or absence of a nursing litter, and this is an unusual characteristic that has not been previously described for ovarian secreted peptides. Collectively, these observations suggest that STC plays a previously unrecognized role in ovarian physiology, pregnancy, and lactation.

To determine the role of STC in mammals, we have generated transgenic mice that ectopically overexpress hSTC. The bioactivity of human STC in rodents was assured by the fact that hSTC is 96% identical to the mouse protein (8, 28) and that it functions in rodents (5). We chose the mouse metallothionein I (MT-I) minimal promoter to drive expression of hSTC transgenes because it has been used extensively for ectopic overexpression studies of secreted and intracellular proteins (29, 30, 31, 32, 33, 34, 35). Previous studies have shown that when used to drive transgene expression, the MT-I promoter displays strong preferential expression in liver, kidney, brain, and intestine and lower activity in numerous other tissues (36, 37, 38). In this way, we expected that high levels of transgene expression would translate into high circulating levels of hSTC from late development to maturity, thus optimizing the possibility of obtaining a phenotype(s) that would help reveal novel physiological roles for STC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of hSTC transgenic mice
The transgene was created by fusing the mouse MT-I 653-bp minimal promoter to 3 kb of an hSTC genomic clone (8) containing 93 bp of the 5'-untranslated region (5'UTR), exon I, intron I, exon II, and intron II. This, in turn, was fused to an hSTC cDNA fragment containing exon III and 573 bp of exon IV. The remaining 2 kb of hSTC 3'UTR were replaced by 800 bp of the human GH 3'UTR. This construct was designed to yield a 2-kb transcript, which could be easily distinguished from the endogenous 4-kb mouse STC mRNA by Northern analysis. The MT-I/hSTC transgene was microinjected into the pronuclei of fertilized C57B/6xCBA oocytes, as described previously (39). Transgenic mice were genotyped by genomic DNA dot-blot hybridization using an hSTC cDNA probe (40). Southern blot analysis was performed on DNA isolated from tail clippings at 3–4 wk of age to confirm intact integration of the transgene. Transgenic mouse lines were maintained on a C57B/6xCBA background. Unless otherwise indicated, all studies were performed with mice hemizygous for the MT-1/hSTC transgene. Mice were housed and used in accordance with protocols approved by the university council on animal care at University of Western Ontario.

Serum STC determination and blood chemistry
Whole blood was taken from transgenic and wild-type mice, immediately after CO2 asphyxiation, by opening the abdomen and collecting blood from the caudal vena cava. Trunk blood was collected from mice at 5 d of age. Blood was allowed to coagulate, and serum was collected as the supernatant from two consecutive centrifugations. Serum hSTC was characterized by Western blotting (1:40,000 dilution, primary antibody) and measured by RIA as previously described using a well characterized hSTC antiserum (9). It should be noted that mouse STC is not normally detectable in serum (9), and we are unable to detect mouse STC in mouse tissues homogenates by Western blot. The size of the transgene-derived protein was compared with STC produced naturally by the HT1080 human fibrosarcoma cell line and AtT-20 mouse corticotrope cells (41) by Western blotting (4). Serum IGF-I was measured with a rat IGF-I RIA (Diagnostics Systems Laboratories, Inc., Webster, TX) after acid-ethanol extraction of 50-µl serum samples from 13.5- to 16-wk-old wild-type and hSTC transgenic mice. Serum GH determinations were made with a rat GH RIA (Biocode S.A., Liege, Belgium) using 100-µl adult serum samples. All wild-type serum samples were measured in duplicate. Recovery of serum from human STC transgenic mice was generally less than that from wild-type mice because they are dwarves with smaller blood volumes. We routinely obtained 100–250 µl serum from hSTC transgenic mice, and as a result some of the transgenic mouse sera could not be assayed for GH in duplicate. For serum GH and IGF-I measurements, the data are presented as the mean ± SEM. Blood chemistry determinations were carried out using the Synchron clinical system CX7 and LX20 (Beckman Coulter, Inc., Brea, CA) at the London Health Sciences Center (London, Canada).

Northern hybridization
To determine the tissue expression pattern of the hSTC minigene, mice were killed, and tissues were removed and extracted in TRIzol (Life Technologies, Inc., Grand Island, NY) for the isolation of total RNA. All RNA samples were subjected to Northern blot analysis using a 32P random primer-labeled human cDNA encoding amino acids 43–180 of the mature protein (8). For transgene expression studies, total RNA was pooled from at least 5 animals or embryos. The steady state level of pituitary hormone mRNAs was examined using RNA isolated from 10–20 female or male pooled pituitaries. Pituitary total RNA was blotted and hybridized with 460-bp mouse {alpha}-glycoprotein subunit cDNA, a 300-bp rat LH ß-subunit cDNA fragment, 420-bp mouse TSH ß-subunit cDNA, 500-bp mPOMC cDNA, 770-bp mGH cDNA, 850-bp rat PRL, and 800-bp rat FSH ß-subunit cDNA. To normalize for RNA loading and to determine fold changes in steady state mRNA levels, blots were hybridized to 18S ribosomal DNA and quantitated using PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA), and data were analyzed as STC/18S ratios.

Analysis of transgenic phenotype
It was apparent early on that transgenic pups were smaller than their wild-type littermates and appeared to gain weight at a reduced rate. To explore this in greater detail, growth studies were conducted on each transgenic line using wild-type littermates as controls. Heterozygous transgenic male mice were bred with wild-type females to generate mixed litters of wild-type and transgenic pups. Pups were numbered at birth with a surgical marker and weighed on d 2, 5, 10, 15, and 21. They were then weaned and genotyped as described above. The mice were weighed once more at 45 d of age to determine sexually mature body weight.

Studies were also carried out to assess the possible effects of the maternal genotype on postnatal weight gain in both wild-type and transgenic pups. The transgenic line chosen for these experiments (line 1A) had a growth phenotype and high serum levels of hSTC. Pups were generated by mating wild-type mice and homozygous transgenic line 1A males with wild-type females to generate pools of wild-type and line 1A pups, respectively. Wild-type males were also mated with wild-type or line 1A virgin females to generate recipient wild-type and transgenic foster mothers, each of which received wild-type or transgenic line 1A pups. Foster mothers were restricted to only six pups to reduce the effect of competition within litters. Pups were numbered and weighed as described above.

Statistical analysis
The analysis of postnatal weight gain was carried out using repeated measures ANOVA with a multivariant or univariant module, followed by Bonferroni post hoc analysis to compare means at each time point. Blood chemistry and serum hSTC, IGF-I, and GH levels were analyzed by unpaired t test, and ANOVA was performed using PRISM 3.0a (GraphPad Software, Inc., San Diego, CA). Statistical significance was assumed at P < 0.05 for all experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of hSTC transgene expression
The transgene shown in Fig. 1Go was used to generate two transgenic founders that gave rise to lines 1 and 2. Two independent integrations of the transgene occurred in line 1. These were segregated by mating to wild-type mice, and the progeny were established as independent lines 1A and 1B. Based on genomic DNA dot-blot analysis (data not shown), line 1B contains only one copy of the transgene, whereas lines 1A and 2 contain approximately 10 and 24 copies of the transgene, respectively. Northern analysis confirmed expression of the transgene-derived 2-kb transcript in each line, with the highest levels of expression being detected in liver, heart, and brain (Fig. 2Go). In reproductive tissues of line 2 animals, expression of the transgene was highest in testis and mammary gland. However, the high level of expression in mammary gland could be associated with the mammary fat pad because adipose tissue had equally high levels of hSTC transgene mRNA (Fig. 2Go). In lines 1A and 2, transgene mRNA levels in heart and liver were consistently 2- and 5-fold higher in females, respectively, than those in males (Fig. 2Go). To assess transgene expression during development, RNA was isolated from line 1A embryos, and Northern analysis showed strong expression beginning between 12.5 and 14.5 d gestation and continuing until parturition (Fig. 3Go). Endogenous mSTC gene expression during development, as determined by Northern blot analysis, was not altered by hSTC transgene expression in line 1A hemizygous transgenic embryos from embryonic d 8.5–18.5 (data not shown). The level of mouse STC gene expression in wild-type mice was barely detectable in all tissues except ovary (8). We assessed endogenous mouse STC mRNA expression in pooled line 1A ovaries and found a 4-fold reduction compared with wild-type littermates (data not shown). Down-regulation of the corresponding endogenous gene in transgenic overexpression mouse models has been described previously (42, 43, 44, 45).



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  		Figure 1. MT-1/hSTC transgene structure. Schematic representation of the transgene. The mouse MT promoter was fused to an hSTC minigene containing hSTC introns I and II with the 3'UTR in exon 4 truncated and fused to the 3'UTR from the hGH gene. The transgene was microinjected into the pronuclei of fertilized eggs, resulting in three independent lines of transgenic mice.

 


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  		Figure 2. Expression of the MT-1/hSTC transgene in adult mouse tissues. Northern blot analysis of total RNA pooled from three to five mature transgenic mice (30 µg/lane). The tissues analyzed are indicated above each lane, and the transgenic line from which they were collected is given at the left of each panel. Male and female kidney, heart, liver, spleen, and brain were processed separately to examine sex-specific differences in transgene expression. All blots were rehybridized with an 18S ribosomal DNA to confirm equal loading of lanes (data not shown) and to normalize hybridization signal intensities for comparison of transgene expression levels between tissues. Mamm., Mammary gland; Sem. Ves., seminal vesicles.

 


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  		Figure 3. Expression of the MT-1/hSTC transgene during development. A Northern blot is depicted containing line 1A pooled whole embryo total RNA, with the day postcoitum indicated above each lane (30 µg/lane). The lane labeled L contains transgenic mouse liver RNA as a positive control. This blot was rehybridized with an 18S ribosomal DNA to confirm equal loading of lanes (data not shown).

 
The level of transgene expression in these lines was such that hSTC could be easily detected by Western blotting (Fig. 4Go). This demonstrated that hSTC in transgenic mouse serum is the same size as that produced by human HT1080 cells, but is somewhat larger than that elaborated by mouse AtT-20 cells (Fig. 4AGo). In transgenic tissues, the relative levels of transgene-derived hSTC protein were highest in liver (Fig. 4BGo) and testis (data not shown) and were undetectable in brain and spleen. Interestingly, even though heart and liver had comparable levels of transgene expression, the hSTC protein content was much greater in liver.



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  		Figure 4. Western blot analyses of MT-1/hSTC transgene-derived STC. A, Serum from line 1A and line 2 transgenic mice and from human HT1080 and mouse AtT-20 cell lines were reduced with a final concentration of 8% ß-mercaptoethanol and resolved by SDS-PAGE. Note the molecular size difference between human and mouse STC. B, Western blot analysis of line 2 tissue extracts. Even though brain, liver, and heart express the transgene, only liver and heart contain sufficient hSTC to be detectable by this method. Each lane contained 25 µg total protein. Similar results were obtained using line 1A tissue extracts.

 
In normal mouse serum, STC was not measurable except during pregnancy (27), but was readily detectable in hSTC transgenic animals. Moreover, serum levels of hSTC were directly proportional to the level of transgene expression. For instance, line 1B mice had the lowest level of transgene expression and, like wild-type mice, contained no radioimmunoassayable hSTC in their serum (detection limit, 0.4 ng/ml). Line 1A mice, on the other hand, contained more than 100 ng hSTC/ml serum, whereas line 2 mice, with the highest level of transgene expression, contained more than 700 ng hSTC/ml serum. Serum hSTC levels were also significantly higher in mature females compared with males of the same age in lines 1A and 2 (Fig. 5Go; P < 0.01); this may be the result of 5- and 11-fold elevated liver transgene expression levels in line 1A and 2 females, respectively, compared with males (Fig. 2Go). The serum hSTC level in sexually mature line 2 mice was also significantly greater than that in line 1A; this may reflect the 40-fold higher steady state level of liver hSTC mRNA in line 2 compared with line 1A (data not shown). In contrast to mature transgenic mice, a sexual dimorphism in serum hSTC levels was not observed in prepubertal transgenic mice. Although, we detected hSTC in the serum of transgenic mice as early as 5 d postnatally (Fig. 5Go), serum hSTC levels in line 2 were at least 7-fold lower in prepubertal mice compared with the levels in sexually mature mice. Therefore, physiological changes associated with sexual maturation appear to be responsible for the overall large increase and sexual dimorphism in serum hSTC levels in transgenic animals (Fig. 5Go).



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  		Figure 5. Serum hSTC levels in transgenic mice. The age at which blood samples were taken is indicated below each set of bars. The numbers of animals tested for d 5, 15, and 23 were 9, 11, 20, respectively, and the number of adult samples assayed is given in Table 2Go. Serum hSTC levels in line 2 prepubertal transgenic mice were significantly lower than those in adult mice. Circulating hSTC was greatly elevated in adult female mice from both lines compared with males. Differences in circulating hSTC in adult mice appear to coincide with sexual maturation, as juvenile mice did not exhibit a significant difference between sexes. Line 1B and wild-type adult mice did not have RIA-detectable levels of serum STC (data not shown).

 
hSTC overexpression results in growth retardation
An overt phenotype, reduced body size, was immediately apparent for some line 1A and 2 neonatal pups, all of which subsequently proved to be transgenic through genotyping. This phenotype was maintained through the prepubertal period in hSTC-overexpressing transgenic mice and resulted in a significant reduction in weight and overall size of both line 1A and 2 mice compared with their wild-type littermates (Fig. 6AGo). At 45 d, line 1A mice had achieved about 70% the body weight of wild-type littermates, while line 2 mice only achieved about 50% of wild-type weight. In contrast, the low transgene-expressing line 1B mice had no overt size phenotype and no measurable changes in growth rate (Fig. 6AGo). Line 1A and line 2 pups nursed by wild-type mothers also exhibited significantly reduced growth rates compared with wild-type pups, as early as d 5 (P < 0.01; Fig. 6BGo). Furthermore, the fact that line 2 pups weighed significantly less than line 1A pups from d 15 onward (P < 0.01) suggests that the higher level of transgene expression in line 2 accentuates the dwarf phenotype (Fig. 6BGo). Moreover, transgenic mice did not recover from their dwarf phenotype after weaning (Fig. 6AGo).



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  		Figure 6. hSTC-expressing transgenic mice exhibit decreased weight gain. A, Representative age-matched adult mice from each transgenic line are shown with mean adult body weights (±SEM) given below each mouse. B, Weight gain of transgenic and wild-type mice born to and nursed by wild-type mothers. C, Weight gain of wild-type pups nursed by either wild-type or transgenic mothers. D, Weight gain of transgenic pups nursed by either wild-type or transgenic mothers. The number of mice in each group varied between 18–54 mice. Weight gains of both lines of transgenic mice were significantly less than those for wild-type littermates. Nursing by transgenic foster mothers resulted in significantly reduced weight gain by both transgenic and wild-type pups, compared with transgenic and wild-type mice nursed by wild-type foster mothers. *, P < 0.01 between transgenic and wild-type mice; {dagger}, P < 0.01 between line 1A and line 2.

 
We also obtained evidence for a direct maternal influence on hSTC-induced dwarfism. For instance, when wild-type pups were nursed by wild-type mothers they gained significantly more weight than those nursed by transgenic mothers (P < 0.01; Fig. 6CGo). Similar results were obtained for transgenic pups nursed by transgenic or wild-type mothers (P < 0.01; Fig. 6DGo). Wild-type and transgenic pups were 10% and 25% larger, respectively, when nursed by wild-type mice, compared with those nursed by transgenic mothers. Thus, in addition to an inherent defect in hSTC transgenic pups, which independently results in growth retardation, it is apparent that maternal transgene expression also confers a separate and distinctly deleterious affect on postnatal growth.

To determine whether the hSTC-induced dwarfism was due to reduced levels of GH expression, we isolated RNA from pools of male and female line 2 mouse pituitaries. The steady state mRNA levels of the trophic hormones were compared with those in age-matched wild-type littermates. No significant differences were detected when each pituitary hormone mRNA hybridization signal was quantitated and normalized to the 18S rRNA hybridization signal (Fig. 7Go). Circulating GH levels were also measured in sexually mature line 2 transgenic animals and compared with those in age- and sex-matched wild-type littermates. In females, wild-type (43.38 ± 9.57 ng/ml; n = 10) and hSTC transgenic (32.02 ± 4.811 ng/ml; n = 13) serum GH levels were not significantly different. Wild-type male (19.07 ± 4.756 ng/ml; n = 13) GH levels were not statistically different from those of transgenic littermates (31.67 ± 6.707 ng/ml; n = 12). Serum IGF-I levels were analyzed in hSTC transgenic line 2 and wild-type mice as a downstream mediator of GH action. No significant difference was observed in IGF-I levels between wild-type (1354 ± 116.9 ng/ml; n = 7) and line 2 transgenic females (1604 ± 149.5 ng/ml; n = 8). Similar results were obtained with male serum from wild-type (1377 ± 85.18 ng/ml; n = 7) and line 2 transgenic males (1537 ± 58.92 ng/ml; n = 7). This indicates that pituitary function in hSTC transgenic mice was not significantly altered and does not account for the decrease in growth or reduction in reproductive capacity, as described below.



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  		Figure 7. Pituitary hormone mRNA levels in MT-1/hSTC line 2 and wild-type mice. Each panel represents a Northern blot containing 6 µg pituitary total RNA pooled from 10–20 male or female mice and hybridized with the cDNA fragment corresponding to one of the pituitary hormones indicated at the left of each panel. The bottom panel in each column shows representative hybridization results when the above blots were hybridized with 18S ribosomal DNA to normalize for RNA loading and to measure changes between wild-type (WT) and transgenic mouse pituitaries. Apparent differences in the hybridization signal between transgenic and wild-type animals were due to differences in RNA loading, because normalized values indicated no difference between these groups. {alpha}GSU, {alpha}-Glycoprotein subunit; ßLH, LH ß-subunit; TSHß, TSH ß-subunit; ßFSH, FSH ß-subunit.

 
hSTC overexpression reduces reproductive capacity
The overproduction of hSTC also resulted in significantly reduced litter sizes (P < 0.01; Table 1Go). To estimate the degree of embryonic exposure to transgene-derived hSTC (assuming that maternally derived hormone did not cross the placenta), we also determined the level of transgene expression in both embryos and placentas. To determine whether the transgenic maternal decidual and/or the fetal portion of the placenta expressed the transgene, we collected placentas from wild-type and transgenic embryos produced by mating transgenic females to wild-type males. Northern blotting of these RNAs from different days of development indicated that the MT-1 promoter used in our transgene was not active in the embryonic or maternal portions of the placenta (data not shown). However, similarly analyzed RNA from the corresponding transgenic embryos indicated that the MT-1/hSTC transgene was expressed at high levels in embryos after midgestation (Fig. 3Go).


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  		Table 1. Litter sizes from wild-type and MT-1/hSTC transgenic mice

 
Serum calcium, phosphate, and alkaline phosphatase analysis
The fact that STC regulates epithelial calcium and phosphate transport prompted us to measure serum electrolytes in hSTC transgenic mice. Although serum sodium, chloride, and anion gap values were not significantly affected by overexpression of hSTC (data not shown), males and females from both transgenic lines exhibited significantly higher levels of serum phosphate and significantly lower alkaline phosphatase levels compared with wild-type mice (Table 2Go). With regard to serum calcium, a significant change (increase) was only found in the line 2 females in which we observed the highest circulating levels of hSTC (Table 2Go). Overall, serum phosphate and alkaline phosphatase levels appeared to be the most sensitive to high circulating levels of hSTC, whereas serum calcium was generally refractory to higher than normal STC levels.


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  		Table 2. Serum calcium, alkaline phosphatase, phosphate, and STC in MT-1/hSTC transgenic and wild-type mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study widespread transgenic overexpression of hSTC in mice was employed to identify physiological systems that would be responsive to higher than normal levels of the hormone in the circulation and in tissues. Interestingly, ectopic expression of hSTC affected growth and reproduction, as manifested by dwarfism and reduced litter size, respectively. Additionally, maternal transgene expression negatively affected weight gain of pups, a condition that was more pronounced in transgenic pups. Both sexes of transgenic mice were severely compromised with regard to postnatal weight gain and body size, neither of which achieved normal levels after sexual maturity. These data demonstrate for the first time that STC can act as a homeostatic regulator of growth and female fecundity.

Numerous growth-retarded mouse models have been reported in the literature, the majority of which result from perturbations of GH or IGF-I levels. Although it has been shown that the effects of GH on postnatal growth are mediated through IGF-I (46), the importance of liver-derived IGF-I has been recently questioned because targeted disruption of liver IGF-I does not result in dwarfism (47, 48), and it seems likely that nonhepatic IGF-I is sufficient for normal growth in mice. To address the possible mechanism of hSTC-induced dwarfism, pituitary trophic hormone steady state mRNA levels were examined, and no significant difference was found between transgenic and wild-type mice. Stimulation of GH secretion has been shown to correlate with elevated transcription of the GH gene (49, 50, 51, 52), and we found that serum levels of GH and IGF-I in our MT-I/hSTC transgenic mice were not significantly different from those in wild-type mice as seen in other dwarf mouse mutants (53, 54).

At birth, transgenic mice were visibly smaller than wild-type mice, suggesting that embryonic transgene expression contributes to the dwarf phenotype. Moreover, we were unable to detect transgene expression in the placenta, and dwarf neonates were observed when transgenic pups were born to wild-type mothers. Therefore, the dwarf phenotype must result from embryo transgene expression and an associated developmental defect. Gross inspection of transgenic placentas did not reveal any abnormalities like those seen in GH receptor null mice (55), and it is likely that hSTC affects growth of the embryo directly.

Work by our group and others has shown STC expression in regions of bone formation and chondrocyte differentiation in the developing mouse embryo (14, 56). The distribution of STC gene expression and protein localization in the embryo overlaps with that described for PTHrP (57), a hypercalcemic hormone that affects numerous tissues, including bone (58). Furthermore, PTHrP-null mice exhibited accelerated hypertrophic chondrocyte differentiation (59, 60), and the expression of endogenous STC, a putative hypocalcemic hormone in mammals, at sites similar to PTHrP, suggests that dwarfism exhibited in hSTC-overexpressing mice may also be the result of defects in bone formation.

In addition to the effects of hSTC overexpression on early postnatal growth, it appears to influence maternal development or behavior. Transgenic mothers exhibited behavioral or physiological shortcomings that prevented even wild-type pups from attaining their full size potential during the nursing period. We have shown that the transgene was highly expressed in the mammary gland and may have had deleterious effects on mammary gland structure and function. The mechanisms underlying this defect are unclear and require a detailed analysis of mammary gland histology, milk chemistry, and possible behavioral abnormalities to elucidate the causes of this unique phenotype. As ovarian STC expression is significantly up-regulated during gestation and nursing, it is perhaps not surprising that a perturbation of normal STC production could compromise maternal function.

We previously reported that STC was dynamically regulated in the ovary and may act as a regulatory molecule in mammalian reproduction (8, 27). Not surprisingly, the fecundity of hSTC overexpressing mice was compromised, as demonstrated by reduced litter size. Although the precise mechanism underlying this phenotype is unclear, it may involve a defect in ovulation similar to that reported in IGF-binding protein-1-overexpressing transgenic dwarf mice (61, 62). Alternatively, as blastocyst implantation is accompanied by dramatic and precise shifts in uterine STC gene expression (17), this carefully tuned program may be derailed in transgenic mothers. We have also assessed the steady state mRNA levels of pituitary FSH ß-subunit, LH ß-subunit, and {alpha}-glycoprotein hormone subunit and found no change in the levels of these transcripts, suggesting that hormone production is not altered in transgenic mice. In light of our results, it is tempting to speculate that STC may regulate ovarian function through changes in calcium and/or phosphate levels, as recently described for vitamin D modulation of ovarian aromatase gene expression, in part through changes in calcium levels (63). In contrast, reproductive function of male mice was unaffected even though testicular expression of the hSTC transgene was severalfold greater than that in ovary. Moreover, although we detected robust levels of hSTC protein in the testis, circulating levels of the hormone were lower in males than females. Therefore, it is likely that hSTC produced at this site remains behind the blood-testis barrier and fails to enter the circulation, a phenomenon similar to that reported for transgenic mice expressing human IGF-I driven by the cytomegalovirus promoter (64). Alternatively, serum hSTC may be more rapidly degraded in males than females, which would help explain why it accumulates in the serum of females during pregnancy and lactation.

Factors associated with sexual maturation are probably responsible for the 7-fold elevation in serum hSTC levels in adult mice. The further 3-fold elevation in sexually mature females may be the result of hormone response elements present in the introns of the hSTC minigene, sex-specific transgene mRNA stabilization, or hSTC half-life, as mentioned above. Numerous transgenic mouse lines have been generated using the MT-I promoter, and none has been reported to result in sexually dimorphic expression or responsiveness to sex steroids (65, 66). The unexpected rise in circulating hSTC after sexual maturation and the further increase seen in female transgenic mice is perhaps not surprising given the fact that STC expression is highest in ovary and is up-regulated during gestation and lactation (27).

In transgenic mice, elevated serum phosphate levels indicate that overexpression of hSTC induces an imbalance in the mechanisms controlling mineral metabolism. The effect of high circulating levels of hSTC on serum phosphate is in accordance with previously described roles for STC in stimulating phosphate uptake by both kidney and gut. This degree of hyperphosphatemia did not result in a reduction in serum levels of calcium; in fact, line 2 females showed a slight, but significant, increase in serum calcium. It is notable that line 2 females contain the highest circulating level of transgene-derived hSTC, suggesting that only very high levels of hSTC can overcome the normal physiological compensatory mechanisms for the maintenance of normocalcemia. Further studies can now be performed to delineate the mechanism by which hyperphosphatemia is induced by exposure to chronically high levels of hSTC.

High levels of serum hSTC may also have produced alterations in bone remodeling. A function for STC in bone physiology has yet to be clearly established, but STC is expressed in both osteoblasts and chondrocytes (13). Synthetic fragments of fish STC have been reported to counteract the effects of PTH on osteoclast recruitment and bone resorption in fetal rat calvariae (67). Furthermore, recombinant hSTC has recently been reported to promote bone nodule formation in fetal rat calvariae (68). Hence, the studies to date are clearly contradictory. Our findings of lower serum alkaline phosphatase levels in MT-1/hSTC transgenic animals support the idea that STC somehow slows the rate of bone mineralization and/or growth. Such an interpretation is also more in keeping with the role of STC in fish, where it restricts the entry of calcium into the animal from the gut and the aquatic environment. As such, it is viewed as a negative regulator of growth (69), and this could also contribute to the dwarf phenotype attained as a result of hSTC overproduction. To our knowledge no other mutant mouse models exhibit hyperphosphatemia with no change in serum calcium; therefore, MT-1/hSTC mice may provide a unique model for studies of the pathologies and treatments relevant to inappropriately elevated serum PO4.

In summary, our data implicate STC as a regulator of postnatal growth, female reproductive potential, and mineral metabolism. It is tempting to speculate that STC can regulate bone remodeling and that ectopic expression results in accelerated differentiation of chondrocytes to osteoblasts, resulting in a dwarf mouse. Continued work with this new mouse model will allow us to identify cells and molecular processes that are sensitive to regulation by STC and impact reproduction and growth. In doing so, it will be possible to identify specific targets of STC action and define its physiological role(s) in mammals.


   Acknowledgments
 
We thank James Lee, Kathy James, Marc Paciga, and Sanda Raulic for technical assistance. We extend special thanks to Dr. G. Hammond for critical reading of and helpful suggestions for the manuscript.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research, London Health Sciences Research, Inc., and The Plunkett Foundation of London (Ontario, Canada).

Abbreviations: hSTC, Human stanniocalcin; MT-I, metallothionein I; STC, stanniocalcin; UTR, untranslated region.

Received July 24, 2001.

Accepted for publication November 1, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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