INTRODUCTION

The feeding behavior of the rat changes from suckling to chewing between 14 and 28 days after birth (Maeda et al., 1981a). During this postnatal period, morphological, biochemical, and functional changes in the masseter muscle reportedly occur. In this process, the masseter myofibers grow rapidly and markedly (Maeda et al., 1981b; Miyata et al., 1996); expression profiles of myosin heavy chains markedly change (Saito et al., 2002a); the diameters of motoneurons innervating the masseter muscle increase and the pattern of electromyography changes markedly (Kubota et al., 1988; Miyata et al., 1996); and the transition of nicotinic acetylcholine receptors (nAChRs) from embryonic to adult types and the elimination of nAChRs are completed. (Saito et al., 2002b).

It is well known that the insulin-like growth factors (IGF)-I and IGF-II are essential for the embryonic development of skeletal muscle, including its proliferation, differentiation, and synaptogenesis (Ishii, 1989; Florini et al., 1996; Yamane et al., 2000a). IGFs are also reported to play important roles in the postnatal growth of skeletal muscle (Beck et al., 1988; Alexandrides et al., 1989) and the change in the mass and phenotype of adult skeletal muscle (Yang et al., 1997; Lalani et al., 2000; Olson and Williams, 2000). These signals of IGFs are mediated via IGF receptor 1 (IGFR1) (Ewton et al., 1987; Quinn et al., 1994; Navarro et al., 1997), while IGF receptor 2 (IGFR2) serves as a factor for the turnover IGF-II protein in skeletal muscle (Kiess et al., 1987; Wang et al., 1994; Ludwig et al., 1996). The IGF actions are regulated and modified by a family of six high-affinity IGF binding proteins (IGFBP), designated as IGFBP1 to IGFBP6 (Jones Clemmons, 1995). These findings suggest that the IGFs, IGFRs, and IGFBPs are closely related to the morphological, biochemical, and functional changes in masseter muscle in association with the shift of feeding behavior from suckling to biting, and that their expressions change markedly during the postnatal development of masseter muscle.

However, there are, to date and to the best of our knowledge, no published data on the expressions of IGFs, IGFRs, and IGFBPs during the postnatal development of rat masseter muscle. We analyzed the expressions of IGF, IGFR, and IGFBP mRNAs in rat masseter muscle between 0 and 70 days after birth using the competitive, reverse transcriptase-polymerase chain reaction (RT-PCR) method. We then related our findings with changes in the rat masseter muscle during postnatal development.

MATERIALS AND METHODS

Experimental animals

Ten pregnant Wistar rats with similar birth windows were purchased (Nippon Clea, Tokyo, Japan) and then housed in individual cages. After birth, pups (each litter containing 10–12 pups) were randomly selected for study at 1, 7, 14, and 19 days of age (n=6 each). At 19 days of age, only male pups were removed from their dams, and fed a pellet diet (CE-2, CLEA Inc., Tokyo, Japan) until they reached 70 days of age. They were then randomly selected for study at 28, 49, and 70 days of age (n=6 each). All animals were weighed and killed by exsanguination under anesthesia with pento-barbital sodium (Nembutal; Abbott Laboratories, Chicago, IL, USA) at a fetal overdose of 50 mg/kg. The body weight of the animals increased gradually with development (Fig. 1). The central portion of the superficial masseter muscles was immediately dissected, rapidly frozen in liquid nitrogen, and stored at −85°C until subsequent analyses. The protocols used in the present study were approved by the Institutional Animal Care Committee of Tsurumi University School of Dental Medicine.

Fig. 1

Changes in rat body weight. Each point and its vertical bar represent the mean ± SD of six rats. The diagonal rectangle shows the period for the shift of rat feeding behavior from suckling to chewing.

RNA extraction, reverse transcription, and competitive polymerase chain reaction (competitive RT - PCR) amplification.

Total RNA extraction, reverse transcription, and competitive PCR amplification were performed as described previously (Ohnuki et al., 2000; Yamane et al., 1998; Yamane et al., 2000b). Briefly, total RNA was isolated from individual masseter samples (30–50 mg in wet weight) using FastRNA^TM Kit-GREEN according to the manufacturer's specifications (BIO 101, Vista, CA, USA). The RNA was treated with 2 unit of ribonuclease-free deoxyribonuclease I (Life Technologies, Inc., Rockville, MD, USA), and was then reverse-transcribed to cDNA with 200 unit of reverse-transcriptase (SuperScript II; Life Technologies).

Table 1 shows the primer sequences and product sizes for IGFs, IGFRs, and IGFBPs; those for the house keeping gene S16 (ribosomal protein) were previously reported (Ohnuki et al., 2000). In the competitive PCR, target cDNA (reverse-transcribed target mRNA) existing in the total cDNA (reverse-transcribed total RNA) was coamplified together with the externally added DNA fragment (competitor), which had identical primer-annealing sites to those of the target cDNA. The competitor was constructed by using a PCR MIMIC construction kit (Clontech Laboratory, Inc., Palo Alto, CA, USA) or Competitive DNA Construction Kit (TaKaRa Biochemicals, Shiga, Japan). The PCR products were separated by electrophoresis in a 3% agarose gel containing ethidium bromide to visualize the PCR products as bands (Fig. 2). The fluorescent intensity of each band was measured with an image analyzer (FLA-3000, Fujifilm, Tokyo, Japan). To normalize the variations in the yield of total RNA and efficiency of reverse transcription, we performed PCR amplification for S16 together with its competitor, and also obtained the S16 standard curve. Then, the quantity of endogenous S16 mRNA was quantified to normalize the quantity of target mRNA relative to the quantity of S16 mRNA, as in our previous study (Ohnuki et al., 2000). Here, the quantity of target mRNA was represented by the ratio of the target mRNA to S16 mRNA quantities (target mRNA/S16 mRNA).

Table 1

Sequences of target gene-specific PCR primers and, target and Competitor sizes

Fig. 2

Typical example of a gel electrophoretic pattern for IGF-II competitive PCR products of rat masseter muscle at 1, 7, 14, 19, 28, 49, and 70 days of age.

RESULTS

Figs. 3A and 3B show the data for IGF-I and IGF-II mRNA expressions, respectively. The quantity of IGF-I mRNA drastically decreased between 1 and 19 days of age and reached 31% (p<0.0001) of the 1 day value at 19 days. The rate of decrease (8% per a day) between 14 and 19 days was approximately 4-times greater than that (2.1% per a day) between 1 and 14 days. After 19 days, the quantity of IGF-I mRNA increased and recovered to 52% (p<0.05) of the 1 day value at 28 days, then decreased again gradually up to 70 days of age. The quantity of IGF-II mRNA, though transient, increased to 246% (p<0.0001) of the 1 day value between 1 and 14 days. After that, the quantity sharply fell and became only 66% (p<0.0001) of the 1 day value at 19 days. Thereafter, the level of IGF-II mRNA expression was quite constant up to 70 days.

Fig. 3

Relative changes in the quantities of IGF-I (A) and IGF-II (B) mRNAs in the rat masseter muscle at 1, 7, 14, 19, 28, 49, and 70 days of age assessed by using competitive RT-PCR. Each point with its vertical bar represents the mean ± SD of six samples. The vertical axis is expressed as a ratio of the target mRNA to S16 mRNA quantities. The diagonal rectangle shows the period for the shift of rat feeding behavior from suckling to chewing.

Figs. 4A and 4B show the data for IGFR1 and IGFR2 mRNA expressions, respectively. IGFR1 mRNA was highly expressed between 1 and 14 days, then drastically fell by 19 days to 46% of the value at 1 day (p<0.0001). After that, the quantity of IGFR1 mRNA did not change significantly. The quantity of IGFR2 mRNA markedly and transiently increased to 204% of the 1 day value between 1 and 14 days (p<0.0001). After that, the quantity sharply fell and became only 69% (p<0.0001) of the 1 day value at 19 days. After 19 days, the quantity of IGFR2 mRNA increased and reached 128% (p<0.01) of the 1 day value at 49 days. Thereafter, the quantity did not change significantly up to 70 days.

Fig. 4

Relative changes in the quantities of IGFR1 (A) and IGFR2 (B) mRNAs in the rat masseter muscle at 1, 7, 14, 19, 28, 49, and 70 days of age assessed by using competitive RT-PCR. Each point with its vertical bar represents the mean ± SD of six samples. The vertical axis is expressed as a ratio of the target mRNA to S16 mRNA quantities. The diagonal rectangle shows the period for the shift of rat feeding behavior from suckling to chewing.

Figs. 5A, 5B, 5C, and 5D show the data for IGFBP3, IGFBP4, IGFBP5, and IGFBP6 mRNA expressions, respectively. No expression of IGFBP1 and IGFBP2 mRNAs was detected throughout the experimental period according to this PCR approach. IGFBP3 mRNA was highly expressed between 1 and 7 days of age, then began to decrease in the quantity at 14 days. The quantity sharply fell between 14 and 19 days, and became only 35% (p<0.001) of the 1 day value at 19 days. After that, the quantity gradually decreased up to 70 days. The quantity of IGFBP4 mRNA linearly decreased between 1 and 14 days, and became only 7.5% of the 1 day value (p<0.0001) at 14 days. However, the quantity increased transiently between 14 and 28 days and recovered to 53% of the 1 day value (p<0.05) at 28 days. After that, the quantity decreased again and reached a negligible level at 49 days of age. IGFBP5 mRNA expression increased to 414% (p<0.0001) of the 1 day value between 1 and 14 days, decreased to 184% (p<0.01) of the 1 day value at 19 days, then increased again to 409% (p<0.01) of 1 day value at 28 days. After that, the high expression level of IGFBP5 mRNA continued up to 70 days. IGFBP6 mRNA was expressed quite constantly throughout the experimental period, except for during a transient reduction and recovery between 14 and 28 days.

Fig. 5

Relative changes in the quantities of IGFBP3 (A), IGFBP4 (B), IGFBP5(C), and IGFBP6 (D) mRNAs in the rat masseter muscle at 1, 7, 14, 19, 28, 49, and 70 days of age assessed by using competitive RT-PCR. Each point with its vertical bar represents the mean ± SD of six samples. The vertical axis is expressed as a ratio of the target mRNA to S16 mRNA quantities. The diagonal rectangle shows the period for the shift of rat feeding behavior from suckling to chewing.

DISCUSSION

In the present study, the shift of rat feeding behavior from suckling to chewing occurred between 14 and 19 days of age, since we observed that the pups took residues of a pellet diet which had been dropped in a cage after 14 days of age, and since pups were removed from the dam and fed on a pellet diet at 19 days of age. During this period, we observed sharp falls in the quantity of all gene mRNAs, which were examined and measurable, except for IGFBP4, although the quantity of IGFBP4 mRNA rose sharply during the same period. Since proteins, not mRNA, are responsible for biological functions of these molecules, it seems to be necessary to analyze the level of protein expression in order to understand the biological meaning of these drastic changes in the quantities of IGF, IGFR, and IGFBP mRNAs in this period. However, since the period for these drastic changes appears to be consistent with the period for the shift of rat feeding behavior from suckling to chewing (14 ∼ 19 days of age), these changes may be involved in the shift of rat feeding behavior.

The roles of both IGF-I and IGF-II in the embryonic development of skeletal muscle have been studied extensively, but no marked difference in the roles has been found between IGF-I and IGF-II except that the effect of exogenous IGF-II on the differentiation of cultured myoblasts is more potent than that of exogenous IGF-I (Ewton et al., 1994; Florini et al., 1996). In the present study, we observed much higher expression level of IGF-I mRNA than that of IGF-II mRNA and the different expression profiles of IGF-I and IGF-II mRNAs except for the sharp fall between 14 and 19 days during the postnatal development of rat masseter muscle (Fig. 3). These suggest that IGF-I plays a main role distinct from that of IGF-II in the postnatal development of rat masseter muscle.

IGFR2 is reported to be involved in the breakdown of IGF-II protein (Kiess et al., 1987; Wang et al., 1994; Ludwig et al., 1996). In the present study, the quantities of IGF-II and IGFR2 mRNAs rose sharply and then fell between 1 and 19 days of age, suggesting that very active synthesis and breakdown of IGF-II occur during this period. This active metabolism of IGF-II may be involved in the suckling movement of masseter muscle and the shift of rat feeding behavior.

It is reported that all six IGFBPs are expressed in skeletal muscles and that IGFBP4, 5, and 6 play important roles in the embryonic development of skeletal muscle cells such as proliferation and differentiation (Ferguson et al., 1992; Florini et al., 1996). IGFBP4, 5, and 6 inhibit the proliferation and differentiation of cultured myogenic cell lines such as C2 and L6, although IGFBP5 has the additional capability of stimulating differentiation of these cell lines under the proper conditions (James et al., 1993; Ewton and Florini, 1995; Rotwein et al., 1995; Silverman et al., 1995; Ewton et al., 1998). However, the roles of these IGFBPs in the postnatal development of skeletal muscle are unclear. In the present study, we observed that the expression level of IGFBP5 mRNA was highest in all 4 IGFBPs during the whole period of post-natal development and that the high level of expression continued until 70 days of age (Fig. 5). These results suggest that IGFBP5 may play a major role in the postnatal development of rat masseter muscle.