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Biology of Reproduction 60, 1172-1182 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Tissue-Specific Expression of Messenger Ribonucleic Acids for Insulin-Like Growth Factors and Insulin-Like Growth Factor-Binding Proteins during Perinatal Development of the Rat Uterus1

Y. Gua, W.S. Branhama, D.M. Sheehana, P.J. Webba, C.L. Molanda, and R.D. Streck2,a

a Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The rodent uterus undergoes extensive cellular proliferation and differentiation during the first few weeks of postnatal life. During this critical developmental period, uterine weight increases markedly, longitudinal and circular layers of uterine muscle begin to differentiate, luminal epithelial cells subsequently invaginate into the stroma to form glands, and estrogen receptor levels increase [1–5]. Coincident with this uterine development, the ovaries begin to secrete estrogens, resulting in elevated serum estradiol levels beginning at about Day 9 of postnatal life [6]. Although uterine development during the postnatal period has been shown not to be mediated solely by estrogens [7], the response of the uterus to estrogen, particularly administration of exogenous estrogens, is one of the most dramatic growth responses demonstrated in the postnatal animal. It has been proposed that estrogen-induced uterine growth involves activation of a variety of genes including protooncogenes and genes encoding trans-acting factors, growth factors, and their receptors. These biologically active molecules mediate stromal-epithelial cell interactions and facilitate the growth response through paracrine and/or autocrine pathways [8–11].

The insulin-like growth factor system consists of insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGF receptors (IGFRs). IGFs are pleiotropic in bioactivity and promote mitosis and differentiation in a variety of cell types in virtually every organ system of the body [12]. IGFBPs constitute a family of structurally related secreted proteins with high affinity for IGFs and are present in nearly all body fluids [13]. IGFBPs can either inhibit or enhance IGF activity and can also act independently of the IGFs [13]. The IGF system in many tissues mediates the response to steroid hormones, including the dramatic and complex response of the uterus to estrogen in immature rodents [8, 9]. A recent study, in which homologous recombination was used to "knock out" IGF-I gene expression, demonstrated that IGF-I is essential for normal development of the reproductive systems of both sexes in rodents [14]. For example, in IGF-I null female mice, severe reductions in uterine weight and size, and dramatic myometrial hypoplasia, are observed [14, 15]. IGF-I is also required for estrogen-induced uterine growth in cycling female rodents. Adesanya and colleagues [16] have reported a greater than 4-fold uterine mitotic index reduction in response to estradiol in mutant mice that lack a functional IGF-I gene compared to wild-type mice. This evidence strongly supports the notion that the mitogenic action of estrogen in a variety of tissues, especially in uterine tissues, is mediated at least in part through locally produced IGFs. However, a systematic study of uterine IGF and IGFBP mRNA expression during rodent perinatal development has yet to be reported. Here, we examined the developmental expression patterns and localization of IGF and IGFBP mRNAs in the rat uterus during late fetal and early postnatal development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All experiments were conducted in accordance with the principles and procedures of the NIH Guide for the Care and Use of Laboratory Animals and the National Center for Toxicological Research Institutional Animal Care and Use Committee. Breeding, housing, and feeding procedures for the Sprague-Dawley rats used here have been previously described [2]. In the present study, the day of vaginal plug discovery is designated as gestational day (GD)-0, and the day of birth is designated as postnatal day (PND)-1. Uteri were removed from female animals at age GD-20 and PND-5,-10,-14,-20, and -24. Each age group contained 3–5 animals obtained from different litters.

Preparation of Frozen Sections

Pregnant dams and female pups were killed by ether overdose. Uteri were immediately removed and fixed in PBS-buffered 1% paraformaldehyde for 12 h at 4°C. After fixation, uteri were rinsed with PBS and stored in 20% sucrose in PBS containing 0.02% sodium azide at 4°C until embedding. Fixation, embedding, and sectioning procedures have been described in detail previously [5]. Three blocks were prepared for sectioning, in each of which were coembedded one randomly selected representative uterus from each of the six age groups (GD-20, PND-5, -10, -14, -20, and -24). Each block was cryostat sectioned at 4 µm. Therefore, each block was treated as a single unit after embedding, and thus all uteri in a given block were processed under identical conditions, including sectioning, slide storage, hybridization, washing, autoradiography, developing, and photography. By eliminating procedural variability, this method allows direct visual comparison of relative mRNA abundance among uteri from different age groups in addition to localization [5].

Riboprobe Preparation and In Situ Hybridization

35S-Labeled riboprobes were transcribed from cDNA-containing plasmids pRBP1-501 (corresponding to rat IGFBP-1) [17], pG3-2-11 (corresponding to rat IGFBP-2) [18], pRBP3-AR (corresponding to rat IGFBP-3) [19], pRBP4-SH (corresponding to rat IGFBP-4) [20], pGEM3Z/BP5,2-3 (corresponding to mouse IGFBP-5) [21], pRBP6-PP (corresponding to rat IGFBP-6) [22], the pGEM-2 plasmid described by Lowe et al. (corresponding to rat IGF-I) [23], and pGrIGF-II-7a (corresponding to rat IGF-II) [24]. Labeled RNA probes were generated by incubating linearized plasmid in the presence of ATP, CTP, GTP (Riboprobe Gemini System II, Promega, Madison, WI), [35S]UTP (NEN Life Science, Boston, MA), and either T3, T7, or SP6 polymerase (Promega). The labeled riboprobes were purified on a Sephadex G-50 spin column (Boehringer-Mannheim, Indianapolis, IN). Riboprobe specificity, demonstrated in the original studies cited above, has been confirmed by the results of a number of previous in situ hybridization studies using identical conditions, in which these IGF-I and IGF-II riboprobes yielded distinct hybridization patterns in rat and mouse tissues despite the fact that IGF-I and IGF-II genes are more closely related to each other than to any other rat genes. Similarly, each of these IGFBP riboprobes has been characterized in previous studies as yielding a unique hybridization pattern in spite of the close sequence homologies among members of the IGFBP gene family [25–29].

In situ hybridization procedures were performed as previously described [26]. In brief, sections were treated with hybridization solution in the absence of probe to minimize nonspecific hybridization and then were hybridized with specific RNA probes overnight at high stringency (50°C and 50% formamide; Boehringer-Mannheim). After hybridization, sections were washed in 50% formamide in single-strength SSC buffer (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) containing 10 mM dithiothreitol for 30 min at 50°C, treated with RNase A (Sigma, St. Louis, MO) to remove unhybridized probe, and washed in 0.2-strength SSC at 60°C for 2 h. After autoradiography and development of NTB-2 emulsion-coated (Eastman Kodak, Rochester, NY) slides, sections were counterstained with hematoxylin and eosin (H-E; Sigma).

The hybridization signals in each section were visualized using darkfield illumination. Sections were photographed using a Leitz DMRB microscope (Leica, Deerfield, IL). All photographs of individual uteri within a single section were taken at identical camera settings and magnification. With each probe, five independent observers scored relative signal intensities for each age (- [not detectable]; + [low]; ++ [medium]; +++ [high]). Mean scores, rounded to the nearest whole value, are presented in Table 1. The signal intensity was compared within individual uteri (to infer tissue-specific differences in transcript levels), among the uteri within the same section (to infer developmental changes in transcript levels), and with adjacent sections hybridized with IGF-I and IGF-II sense probes (to define the background level of nonspecific labeling). One set of darkfield photographs from a single representative section was selected for each probe and is presented below. The specific results discussed below were consistently obtained using sections from all blocks.


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  		TABLE 1. Relative levels of uterine hybridization signals during perinatal development for each IGF and IGFBP mRNA species.a


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In order to visualize the expression patterns and localization of each IGF and IGFBP mRNA in the perinatal rat uterus, we examined both H-E-stained sections (Fig. 1A) and adjacent hybridized uterine sections. In Figure 1A the uterine structures at different developmental stages are identified. By GD-20, a simple cuboidal to low columnar luminal epithelium is apparent, while tissues derived from underlying mesenchyme are not yet fully differentiated. Between PND-5 and PND-10, luminal epithelium began to invaginate into the adjacent mesenchyme, where it formed distinct uterine glands between PND-14 and PND-20. Uterine myometrium became structurally distinguishable on PND-5. By PND-20, uterine structural organization was complete.



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  		FIG. 1. A) Brightfield micrographs of staged uteri from a single section reveal uterine tissue morphology and structures at each age. B) Darkfield micrographs of staged uteri (from the section shown in A) reveal localization of uterine IGF-I mRNA at each stage. C) Darkfield micrographs of staged uteri reveal very low background levels of nonspecific hybridization at each stage. Sections are of rat uterine horns from GD-20 to PND-24. All six uterine horns had been coembedded in a single block, and each block was cryostat sectioned at 4 µm. Sections of each block were hybridized with 35S-labeled antisense RNA complementary to IGF-I mRNA (A and B) or with 35S-labeled IGF-I sense RNA (C) and counterstained with H-E. Specific activity, probe concentrations during hybridization, exposure time of NTB-2 emulsion, and photographic conditions were identical for IGF-I sense and IGF-I antisense probes. Silver grains, visualized as bright spots in darkfield illumination, indicate hybridization. The highest IGF-I mRNA signal was observed on GD-20 with a diffuse distribution throughout the entire uterus. Arrowheads, luminal epithelium; arrows, glands; m, myometrium; s, stroma. Bar = 100 µm.

The developmental expression patterns and localization of each IGF and IGFBP mRNA in the perinatal rat uterus were examined by in situ hybridization. The combination of tissue specificity and developmental timing of mRNA expression was unique for each of the genes of the IGF system examined. IGF-I mRNA was detected in all uteri examined, and the signal was distributed in a diffuse pattern throughout the stroma and myometrium (Fig. 1B). The highest IGF-I mRNA level was seen at GD-20, and then it appeared to decline by PND-5. In contrast, labeling of perinatal uterine sections using IGF-I sense probe was very low and showed no obvious change during development, implying that the level of nonspecific hybridization was low and constant throughout this period (Fig. 1C).

As with IGF-I, IGFBP-1 mRNA was expressed throughout all uterine tissues during the period of GD-20 to PND-24. IGFBP-1 mRNA levels seemed to be similar in newly formed glandular tissue and surrounding stroma on PND-20 (Fig. 2). In contrast to observations for IGF-I, the IGFBP-1 mRNA signal did not appear to decline between GD-20 and PND-5, but it appeared to increase moderately by PND-20 to 24 (Fig. 2). Although IGFBP-3 mRNA was below the level of detection of our assay on GD-20, it became detectable by PND-5, and the mRNA level appeared to increase between PND-5 and PND-24 (Fig. 3). The IGFBP-3 gene is unique among the IGF and IGFBP genes we examined in demonstrating a pattern of developmental regulation such that mRNA expression began around the time of birth. The IGFBP-3 mRNA signal was distributed throughout uterine tissues (Fig. 3). IGFBP-4 mRNA demonstrated a unique pattern of tissue-specific expression, with abundant expression in stromal tissue, especially in the region surrounding the uterine luminal epithelium (Fig. 4). The intensity of IGFBP-4 mRNA signal appeared to be highest at PNDs-10 and -14. IGFBP-4 mRNA signal in the myometrium was very weak. In contrast to IGFBP-4 mRNA expression, IGFBP-5 mRNA was abundant in the myometrium, while little or no IGFBP-5 mRNA was detectable in stromal tissue (Fig. 5). The intensity of IGFBP-5 mRNA signal was correlated with uterine muscle differentiation, with the lowest IGFBP-5 mRNA signal on GD-20 prior to muscle differentiation (Fig. 1A, GD-20, and Fig. 5). IGFBP-6 mRNA signal was detected in both stroma and myometrium with a diffuse pattern similar to that seen for IGF-I mRNA (Figs. 1B and 6). Between GD-20 and PND-24, the IGFBP-6 mRNA signal intensity increased moderately throughout the uterus (Fig. 6). These results are summarized in Table 1.



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  		FIG. 2. Localization of IGFBP-1 mRNA in uteri from rats aged from GD-20 to PND-24. This section, from the same block used in Figure 1, was hybridized with 35S-labeled antisense RNA complementary to IGFBP-1 mRNA. IGFBP-1 mRNA signal was distributed in a generally diffuse pattern throughout uterine tissues, and the signal intensity appeared to increase with uterine development. IGFBP-1 mRNA signal appeared to be at least as high in glandular tissue as in stroma on PND-20. Arrowheads, luminal epithelium; arrows, glands; m, myometrium; s, stroma. Bar = 100 µm.



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  		FIG. 3. Localization of IGFBP-3 mRNA in uteri from rats aged from GD-20 to PND-24. This section, from the same block used in previous figures, was hybridized with 35S-labeled antisense RNA complementary to IGFBP-3 mRNA. IGFBP-3 mRNA was not detectable on GD-20 by in situ hybridization but became detectable by PND-5. The IGFBP-3 mRNA level appeared to increase between PND-5 and PND-24. IGFBP-3 mRNA signal was more evident in muscle layers from PND-10. Arrowheads, luminal epithelium; m, myometrium; s, stroma. Bar = 100 µm.



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  		FIG. 4. Localization of IGFBP-4 mRNA in uteri from rats aged from GD-20 to PND-24. This section was hybridized with 35S-labeled antisense RNA complementary to IGFBP-4 mRNA. Intensity of IGFBP-4 mRNA signal appeared to be highest at PND-10 and PND-14. IGFBP-4 mRNA signal was primarily in stromal tissue, particularly in the stroma surrounding the luminal epithelium. Arrowheads, luminal epithelium; m, myometrium; s, stroma. Bar = 100 µm.



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  		FIG. 5. Localization of IGFBP-5 mRNA in uteri from rats aged from GD-20 to PND-24. This section, from the same block used in Figure 4, was hybridized with 35S-labeled antisense RNA complementary to IGFBP-5 mRNA. The IGFBP-5 mRNA signal was relatively low on GD-20. A more intense IGFBP-5 mRNA signal was characteristic of the myometrium at PND-5 (as it began to differentiate) and throughout postnatal development. The tissue section of PND-20 is folded at its right edge. Arrowheads, luminal epithelium; m, myometrium; s, stroma. Bar = 100 µm.



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  		FIG. 6. Localization of IGFBP-6 mRNA in uteri from rats aged from GD-20 to PND-24. This section, from the same block used in Figures 4 and 5, was hybridized with 35S-labeled antisense RNA complementary to IGFBP-6 mRNA. The IGFBP-6 mRNA signal was distributed throughout all uterine tissues in a diffuse pattern. The signal intensity remained fairly constant during perinatal development. Arrowheads, luminal epithelium; m, myometrium; s, stroma. Bar = 100 µm.

In the present study, uterine IGF-II and IGFBP-2 mRNA signals in perinatal rat uteri were indistinguishable from those in background controls hybridized with sense probes (results not shown). In agreement with these results, RT-PCR assays also failed to detect either IGF-II or IGFBP-2 mRNAs in perinatal rat uteri (unpublished results).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The IGF system is involved in the development and function of the mammalian reproductive system [30–32]. Ghahary et al. [33] previously reported detectable IGF-I mRNA expression in the immature rat uterus. In our study, IGF-I mRNA expression was widespread in all uterine tissues at each perinatal developmental stage examined, including the muscle layer once myometrium began to differentiate. This is consistent with the multiple roles of IGF-I illustrated through the analysis of IGF-I knock-out mice, which exhibited disproportionately small uterine size and severe muscle hypoplasia [14]. Uterine IGF-I mRNA is dramatically up-regulated in immature rats by the administration of exogenous estrogen [9, 34]. Estrogen receptor-{alpha} mRNA is expressed in the perinatal rat uterus as early as GD-20 [5, 35]. The relatively high IGF-I mRNA levels observed in the GD-20 uterus are likely due to elevated estrogen levels in circulation of late fetuses (due to diffusion of maternally derived estrogen) relative to postnatal levels [6]. While maternally derived estrogen is largely bound to serum alpha-fetoprotein and is thought to be unavailable to fetal estrogen target tissues, the bioavailability of estrogen under conditions of a dynamic equilibrium remains a complex question [36, 37]. Our findings that uterine IGF-I mRNA levels did not precisely reflect the changing neonatal serum estrogen levels [6] suggest that the level of endogenous circulating estrogen is not adequate to regulate uterine IGF-I expression during postnatal development in rats.

It is not surprising that we failed to detect any IGF-II mRNA signal in the postnatal rat uterus, since it has been proposed that IGF-II functions primarily as a fetal growth factor in rodents and its mRNA is undetectable in most adult tissues [38, 39].

IGFBP-1 mRNA expression was detectable throughout all uterine tissues of perinatal rats. IGFBP-1 mRNA has been reported to be highly expressed in the glandular components of decidualized and preimplantation uteri analyzed by in situ hybridization [28, 40]. However, we did not observe this expression pattern in the uterus of perinatal rats (GD-20 to PND-24). Ghahary et al. [41] demonstrated by Northern analysis that IGFBP-1 mRNA expression varies throughout the estrous cycle in a manner suggesting that IGFBP-1 is down-regulated by estrogen. However, the increase in postnatal IGFBP-1 mRNA levels did not reflect the changing postnatal serum estrogen levels [6], which suggests, again, that the level of endogenous circulating estrogen may not be sufficient to regulate uterine IGFBP-1 expression during this developmental period in rats. It has been proposed that IGFBP-1 acts primarily in an autocrine/paracrine manner in the uterus [42], and the colocalization of IGFBP-1 and IGF-I mRNA expression in uterine tissues during perinatal development in our study supports this hypothesis.

Girvigian et al. [29] reported the detection of IGFBP-2 mRNA expression restricted to the uterine luminal epithelium of cycling adult rats. IGFBP-2 mRNA expression has been identified in rat decidual tissue and myometrium at mid and late pregnancy by in situ hybridization [28, 43]. However, we failed to detect any IGFBP-2 mRNA signal in uteri at any of the perinatal developmental stages examined. As a positive control for our IGFBP-2 probe, we demonstrated that it yielded strong specific hybridization signals in rat fetal tissues previously reported to express IGFBP-2 (e.g., fetal brain and liver [27]). The variation in uterine IGFBP-2 expression in perinatal and cycling adult and pregnant rats indicates that uterine IGFBP-2 mRNA expression may be differentially activated and regulated in different developmental and hormonal situations.

Depending on circumstances, IGFBP-3 can either enhance or inhibit IGF-I activity [13]. IGFBP-3 potentiation of IGF-I activity has been demonstrated to involve the association of IGFBP-3 with cell surfaces [13]. Although the function of uterine IGFBP-3 is not clear at present, it might have a role in mediating IGF-I effects on postnatal uterine development, since its expression is similarly widespread in stromal tissues and myometrium. It is interesting to note that at GD-20, IGFBP-3 mRNA was not detectable in the uterus while IGF-I mRNA was highly expressed. In contrast to the apparent decrease in IGF-I mRNA levels between GD-20 and PND-5, IGFBP-3 mRNA levels appeared to increase. It may be that IGFBP-3 potentiates IGF-I activity in the rat uterus such that increasing IGFBP-3 expression functions to maintain IGF bioactivity at a high level even as IGF-I expression declines.

In contrast to the IGF-potentiating activity frequently exhibited by IGFBP-3, IGFBP-4 is generally a very potent inhibitor of IGF-I activity [13]. In our study, a strong IGFBP-4 mRNA signal was detected in stromal tissue, especially the area surrounding the uterine luminal epithelium, in contrast to a very weak signal for IGFBP-4 mRNA in myometrium. Assuming a similar expression pattern in mice, this might explain why the complete loss of IGF-I expression in IGF-I null mice has little effect on postnatal uterine stromal development, while leading to dramatic myometrial hypoplasia [14].

The expression of IGFBP-5 mRNA in our study was predominantly in uterine myometrium from the beginning of muscle differentiation at PND-5. IGFBP-5 was previously demonstrated to be associated with myoblast differentiation and to be highly expressed in developing embryonic muscles [25, 44]. The specific spatial and temporal expression patterns in adult cycling females and pregnant animals suggest that IGFBP-5 may promote myometrial proliferation [28, 29]. Recently, it has been shown that IGFBP-5 has the property of adhering tightly to the extracellular matrix (ECM) of fibroblasts. This association with ECM potentiates the mitogenic action of IGF-I on fibroblasts [45, 46]. Since IGFBP-5 expression was predominantly in uterine myometrium and IGF-I mRNA was also abundantly colocalized in the same tissue, it is tempting to speculate that IGFBP-5 may act together with IGF-I to regulate uterine myometrial development. However, determining whether IGFBP-5 acts in a fashion in uterine myometrium similar to how it acts on fibroblasts through the association with ECM will require future experiments. It is also not clear whether IGFBP-5 inhibits or enhances IGF-I activity in neonatal uterine myometrial proliferation.

Reports on the role of uterine IGFBP-6 are very limited. Girvigian et al. [29] have demonstrated that IGFBP-6 is prominently expressed in myometrium in cycling adult rodents. Recently, Cerro and Pintar [28] suggested that IGFBP-6 may play a functional role in myometrial proliferation during pregnancy, as they detected an elevated level of IGFBP-6 mRNA in myometrium at mid and late pregnancy. However, the expression of IGFBP-6 mRNA not only was detected in myometrium but was also widespread in uterine stromal tissues of perinatal rats. Therefore, IGFBP-6 may play a role for both uterine myometrial and endometrial development during perinatal development in the rat.

Each component of the IGF system exhibits a specific temporal and spatial expression pattern in the adult uterus, likely corresponding to its physiological functions. The spatial and temporal expression patterns of IGFs and IGFBPs in the present study, although different from that seen in adult animals, suggest that the IGF system may play equally important physiological roles during early postnatal uterine development in the rat. The effects of IGF-I on each cell type may be a function of the subset of IGFBPs being produced locally as well as the level of local IGF-I expression. However, the specific biological activity and function of each component of the IGF system on uterine development and physiology remain to be defined. The development of the reproductive tract is increasingly well characterized. By combining this knowledge with characterization of the dynamically changing, tissue-specific differential expression of each of the components of the IGF system, a comprehensive model for their individual roles in uterine development may emerge.



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  		FIG. 1. Continued.


   ACKNOWLEDGMENTS
 
The authors are pleased to thank Dr. C. Roberts for the IGF-I clone; Dr. M. Rechler for the IGF-II and IGFBP-2 clones; Dr. S. Shimasaki and the Scripps Research Institute for the IGFBP-1, IGFBP-3, IGFBP-4, and IGFBP-6 clones; and Dr. P. Rotwein for the IGFBP-5 clone.


   FOOTNOTES
 
1 Y.G. was supported by a Postgraduate Research Fellowship administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. Back

2 Correspondence: R.D. Streck, Division of Genetic and Reproductive Toxicology, HFT-130, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079–9502. FAX: 870 543 7682; rstreck{at}nctr.fda.gov Back

Accepted: December 11, 1998.

Received: June 23, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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