Effects of Exogenous Thyroid Hormone on the Postnatal Morphogenesis of the Rat Parotid Gland.код для вставкиСкачать
THE ANATOMICAL RECORD 291:94–104 (2007) Effects of Exogenous Thyroid Hormone on the Postnatal Morphogenesis of the Rat Parotid Gland RIE IKEDA,1* SHIGEO AIYAMA,1 AND ROBERT S. REDMAN2 1 The Nippon Dental University, Tokyo, Japan 2 Department of Veterans Affairs Medical Center, Washington, DC ABSTRACT Administration of thyroid hormone has been shown to accelerate the early postnatal development of the rat parotid gland, but these studies have dwelt almost entirely on biochemical changes. The objective of this study was to describe the effects of exogenous thyroid hormone on morphologic aspects of the developing parotid gland, in particular the transient appearance of scattered mucous cells in this otherwise serous gland. Pups were given a daily subcutaneous injection of thyroxine (T4) of 0.1, 0.5, or 5.0 mg/g body weight, vehicle only (injection control), or no injection (normal control) beginning at 4 days, and killed for the collection of blood and parotid glands at intervals through 15 days. The serum was analyzed for T4 and the glands were examined by light and electron microscopy. The results indicated that both serum T4 and the pace of gland development were proportional to the dose of T4. In particular, T4 accelerated decreases in acinar size and gland area occupied by stroma and translocation of a subset of cells with small secretory granules, deeply stained with periodic acid–Schiff, from acini to intercalated ducts. However, the chronology of mucous cell disappearance was indifferent to treatment. In addition, signs of toxicity, including slower gain in body weight and greatly increased apoptosis and vacuoles in the glands, occurred with the higher doses of T4. Anat Rec, 291:94–104, 2007. Ó 2007 Wiley-Liss, Inc. Key words: apoptosis; morphogenesis; parotid gland; rats; thyroid; hormone The mature parotid gland of the rat is classiﬁed as serous by Young and van Lennep (1978), with the carbohydrates of the secretory glycoproteins being almost entirely of the neutral variety. Accordingly, in sections of formaldehyde-ﬁxed, parafﬁn-embedded tissues, the acinar secretory granules are compact and mainly eosinophilic, stain modestly with the periodic acid–Schiff procedure, and not at all with Alcian blue. By transmission electron microscopy (TEM), the secretory granules are electron-dense. The secretory granules contain acidic epididymal glycoprotein (AEG), parotid secretory protein (PSP), and proline-rich proteins; several digestive enzymes, for example, a-amylase, deoxyribonuclease I, and alkaline ribonuclease; and other enzymes, for example, peroxidase (reviewed by Ball, 1993). Several workers have described the transient appearance of occasional cells with mucous secretory granules Ó 2007 WILEY-LISS, INC. in the developing rat parotid gland at ages 1 through 8 days after birth (Lawson, 1970; Taga and Sesso, 1979; Ikeda and Aiyama, 1997, 1999). These secretory granules stain intensely with both periodic acid–Schiff and Grant sponsor (RI): The Nippon Dental University, Tokyo, Japan. Grant sponsor (RSR): The National Institute of Dental and Craniofacial Research, The National Institutes of Health, Bethesda, MD; Grant number: DE 14995. *Correspondence to: Rie Ikeda, Department of Histology, School of Life Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-Ku, Tokyo 102-8159 Japan. Fax: 81-3-3264-8399. E-mail: email@example.com Received 11 April 2007; Accepted 15 September 2007 DOI 10.1002/ar.20620 Published online in Wiley InterScience (www.interscience.wiley. com). T4 EFFECTS ON RAT PAROTID MORPHOGENESIS Alcian blue, indicating that they are rich in both neutral and acidic glycoproteins. By TEM, these secretory granules have bipartite or tripartite structures of mostly electron-lucent material, with not only a ﬁne ﬁbrillar substructure consistent with mucins, but also eccentrically located electron-dense cores. It is noteworthy that the secretory granules of the mucous anterior buccal gland, which develops as a branch of the parotid duct in rat, have a similar ultrastructural pattern (Nicholas and Redman, 1985). It is not clear what governs the appearance and disappearance of the parotid mucous cells. Apoptosis is rare in the developing rat parotid gland (Kruse and Redman, 1999; Tsujimura et al., 2006). In the developing mouse parotid gland, amylase has been immunohistochemically localized to the mucous secretory granules, especially the electron-dense cores, which gradually displace the electron-lucent areas with age of pups (Takada et al., 2001). These observations suggest that the mucous cells redifferentiate into serous cells. Johnson et al. (1987) reported that the parotid secretory granules of thyroxine-treated mature rats showed a markedly different morphology from that of the vehicle-injected controls. These secretory granules were electron-lucent with an amorphous, rather dense core, superﬁcially resembling those of the early postnatal mucous cells. Although this ﬁnding suggests that the additional thyroxine may have brought about the re-appearance of mucous cells, the secretory granules were not analyzed for mucins. In a preliminary study (Ikeda and Aiyama, unpublished data), rat pups were given daily injections of T4 (5 mg/g body weight) beginning at age 5 days. Mucous cells were observed in the parotid glands of two of the T4-treated pups at 11 days, the oldest in the experiment. The purpose of this study was to determine the inﬂuence of exogenous thyroid hormone on the morphogenesis of the rat parotid gland in the early postnatal period, with special attention given to the possibility that it would prolong mucous cell survival. MATERIALS AND METHODS All experimental procedures were approved by the appropriate review boards of The Nippon Dental University and the Washington, DC, Veterans Affairs Medical Center. Experimental Animals Sprague-Dawley rats (Rattus norvegicus albinus; certiﬁed viral pathogen free) were obtained from Harlan (Indianapolis, IN), arriving on day 14 of gestation. Pups were considered to be age 0 days on the day of birth. Each rat dam and her litter were housed in a plastic cage with shredded corncob bedding in a quiet room with controlled temperature and humidity, lights on at 7:00 AM and off at 7:00 PM, and a commercial pelleted diet and water available ad libitum. Litters were adjusted to 10 pups at 2 days to eliminate runts. All pups were weighed daily. Two pups in each litter were given a daily subcutaneous injection of thyroxine (T4) of 0.1, 0.5, or 5.0 mg/g body weight (Low, Medium, and High doses groups, respectively), vehicle only (injection control; Vehicle group), or no injection (Normal Control group) between 8:00 AM and 9:00 AM beginning at age 4 95 days after birth. As much as possible, the treatments were equalized by sex. Serum samples were collected 3 hr after the last injection of T4 from four litters each (total of 8 pups per treatment or control group) at ages 4, 7, 10, and 15 days (12 days for the High dose T4). Additional samples of parotid glands were taken from noninjected pups at ages 1, 3, 5, and 21 days, to determine whether the mucous cells appear and disappear in these rats on a schedule similar to that previously reported. Pups were deeply anesthetized with halothane and the abdominal wall and thoracic cage opened. Blood was collected from the right atrium and allowed to clot at room temperature for 10 min, then centrifuged at 700 3 g for 10 min at 48C. The serum (supernate) was stored in cryogenic vials at 2708C for subsequent assays. Assays The serum concentration of total T4 was assayed in duplicate for each sample by chemiluminescent enzyme immunoassay (CLEIA; Access1 Total T4) by means of an Access1 Discrete Analyzer (Beckman Coulter, Fullerton, CA). Tissue Preparation The left parotid gland from each rat was cut into small pieces and prepared for TEM by immersion in a mixture of 1% glutaraldehyde and 4% formaldehyde (prepared from paraformaldehyde) in 0.05 M phosphate buffer (PB) at pH 7.4 for 6 hr. Tissues were rinsed in PB overnight and post-ﬁxed in sodium cacodylate-buffered 1% osmium tetroxide for 2 hr, dehydrated with ethanol and propylene oxide, and embedded in Embed-812 (Electron Microscopy Sciences, Fort Washington, PA). The right parotid glands were ﬁxed whole (small glands) or hemisected (larger glands) as above but without glutaraldehyde and osmium, dehydrated with ethanol and xylol, and embedded in parafﬁn. Light Microscopy Parafﬁn sections were cut at 5 mm, stained with periodic acid–Schiff or Alcian blue 8GX at pH 2.5, and lightly counterstained with hematoxylin (PAS-H and AB-H, respectively) by the methods of Mowry (1963), or with Mayer’s mucicarmine (Luna, 1968). Mucous cells were counted in at least two parafﬁn sections (one stained with AB-H; the other, mucicarmine) from each gland by two of us (R.I., R.S.R.) independently. Discrepancies were resolved by conferring on those slides with a two-headed microscope. Histometrics Sections stained with PAS-H at ages 4, 7, 10, and 15 days (12 for High dose T4) were assessed for the proportion (percent) of gland area occupied by acini, the several types of duct, and stroma, by contact with intersections in a grid superimposed by means of an eyepiece. Areas occupied by individual proﬁles of the parenchymal structures were calculated (mm2) by analysis of tracings of projected images (SigmaScan1, Jandel Scientiﬁc, Chicago, IL). Details of these methods have been presented previously (O’Connell et al., 1999). 96 IKEDA ET AL. Electron Microscopy For selection and orientation of specimens for TEM, thick (ca. 1 mm) sections were stained with 1.0% toluidine blue O in 1.0 % sodium borate (Todd et al., 2005). Thin (ca. 70 nm) sections were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined in a JEOL-2000EX-II electron microscope. Analysis of Apoptosis by Means of TUNEL TUNEL (terminal deoxynucleotide transfer method UTP/nick end-labeling) reactions were performed using an ApopTag1Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA). Per the kit instructions, parafﬁn sections were treated with 3% H2O2/PBS after proteinase K treatment, then incubated with terminal deoxynucleotidyl transferase (TdT) and deoxyuridine triphosphate–digoxigenin. After further incubation with anti-digoxigenin peroxidase, the reaction was developed using the DAB method, and the sections were counterstained with hematoxylin. Negative control sections were incubated with distilled water in the absence of TdT. Statistical Evaluation The presence or absence of mucous cells was subjected to w2 analysis (Ball et al., 2003). Body weight and histometric data were subjected to one-way analysis of variance using the SPSS version 14 software package (SPSS Japan, Tokyo) and are presented as means 6 SE. With both methods, null hypotheses were rejected and signiﬁcance was accepted when P < 0.05. RESULTS Serum Total T4 The serum T4 values are presented by age and treatment group in Figure 1. The serum concentration of T4 did not differ by sex or litter within treatment groups at the same age, so these data were combined for each group. Serum T4 also did not differ between the Normal and Vehicle pups at each age, so these data also were combined into one group, labeled ‘‘Vehicle/Normal.’’ There was a rise in serum T4 (mean mg/dL 6 S.D.) in the Vehicle/Normal pups from 1.68 6 0.16 at 4 days to 8.45 6 0.84 at 15 days. The serum T4 concentrations were proportional to the doses injected at each age. Peak values for the Low, Medium, and High dose groups were 67.4 6 14.5 at 10 days, 141.6 6 22.0 at 7 days, and 637.6 6 42.8 at 4 days, respectively. Curiously, the value for the Medium dose declined after 7 days and the value for the High dose declined after 4 days and remained almost constant for the Low dose, indicating that not only was there no day-to-day accumulation of T4, but Fig. 2. Photomicrographs of sections of parotid glands of Normal or Vehicle-injected rats. A: At 4 days, Alcian blue and hematoxylin stains. Several mucous cells (arrows), including one with a mitotic ﬁgure, show ‘‘robin’s egg blue’’ secretory granules. B: At 6 days, mucicarmine. Mucous cells contain magenta secretory granules (arrow). C: At 4 days. Periodic acid–Schiff and hematoxylin (PAS-H) stains in this and subsequent light micrographs. Cells with deep magenta secretory granules Fig. 1. Serum total T4 (mg/dL) by age and treatment. The scale for the Vehicle/Normal (vehicle-injected and noninjected) pups is on the right multiplied 340, as the increase with age would appear barely above baseline if set to the scale of the T4-treated pups. that as the pups developed, they became more efﬁcient in eliminating the excess T4 between injections. Growth and Development The body weights of the pups by age and treatment group are shown in Table 1. Growth of the pups in the High dose group slowed between 7 and 10 days and in the Medium dose group between 10 and 15 days. By 11 days, the health of the High dose group was deteriorating and one pup had died. Therefore, all other pups in this group were killed at age 12 days. Consistent with reports by others (Gamborino et al., 2001), the growth of fur, opening of eyes, and eruption of incisors were noticeably accelerated in the High and Medium dose pups. The body weights and general development of may be mucus, as most other cells have lighter staining. D: At 10 days. Acinar secretory granules vary from deeply (arrow) to moderately PASpositive. E: At 15 days. Cells containing small, strongly PAS-positive granules (arrow) are predominantly localized in intercalated ducts. F: At 21 days. Acinar secretory granules are weakly or moderately PAS-positive, while the smaller granules of the intercalated ducts (arrow) are strongly stained. Magniﬁcation 5 A, 3500, B, 3450; C–F, 3410. 97 T4 EFFECTS ON RAT PAROTID MORPHOGENESIS TABLE 1. Body weights (mean g 6 SE) by age and T4 treatmenta Age (days) Treatment High T4 Medium T4 Low T4 Vehicle/Normal a 4 10.8 10.8 10.3 10.2 6 6 6 6 7 0.2 0.2 0.2 0.2 16.1 16.6 16.3 15.8 6 6 6 6 10 0.3 0.3 0.4 0.3 19.1 21.9 22.8 22.7 6 6 6 6 12 0.7* 0.5 0.6 0.8 20.1 25.1 29.0 29.2 6 6 6 6 15 1.7* 0.9* 0.6 1.0 Values marked with an asterisk differ from those not so marked in the same age column (P < 0.05). Figure 2. 28.0 35.6 37.9 6 6 6 6 0.9* 0.6 0.7 98 IKEDA ET AL. Vehicle/Normal group, were not signiﬁcant. Thus, there was no consistent pattern of the mucous cells staying longer or disappearing more rapidly with any of the treatment groups. Morphogenesis Fig. 3. Electron micrograph of mucous cells. At 4 days, Normal. Secretory granules have a ﬁbrillar substructure and electron-dense cores. Magniﬁcation, 327,000. TABLE 2. Number of pups with parotid mucous cells present or absent by age and T4 treatment Age (days) 1 2 4 5 7 10 12 15 21 Treatment High T4 Mucous cells Present Absent Present Medium T4 Absent Present Low T4 Absent Vehicle/Normal Present Absent 3 0 3 0 3 0 6 0 3 0 5 1 3 0 4 3 4 1 5 1 8 0 1 5 0 6 2 5 3 5 0 6 - 0 4 1 4 0 8 0 2 pups in the Low dose group did not differ from those of the Normal and Vehicle groups. Mucous Cells Examples of mucous cells in the parotid glands of early postnatal pups in parafﬁn sections stained with AB-H, mucicarmine, and PAS-H are shown in Figure 2A–C, and in an electron micrograph in Figure 3. The secretory granules of mucous cells stained intensely with PAS, AB, and mucicarmine, indicating that they are rich in acidic and neutral glycoproteins. By TEM, the mucous granules had a bipartite or tripartite substructure with dense cores. Mucous cells were observed beginning at age 1 day (Table 2). Mitotic ﬁgures were seen in a few mucous cells in the younger pups (Fig. 2A). The number of pups with mucous cells peaked at 4 days and declined thereafter, the decline being signiﬁcant for all groups collectively (i.e., all T4 and all T4 plus Vehicle/Normal) and individually. Only one pup, in the Low dose T4 group, had mucous cells after 10 days. Differences among the T4 groups, and between the T4 groups, both collectively (High 1 Medium dose, and 1 High 1 Medium 1Low dose) and individually, and the The sections stained with PAS-H were used to assess the effects of T4 on several aspects of morphogenesis. No differences were seen among the Normal, Vehicle and T4 groups at age 4 days, nor between the Normal and Vehicle groups at any age. Representative photomicrographs of glands from Normal pups are shown in Figure 2C–F and from T4-treated groups in Figure 4. In the Normal and Vehicle groups, the secretory granules in the developing acini were moderately to strongly PAS-positive through age 7 days, after which there developed a dichotomy of larger, weakly PAS-positive granules and much smaller, intensely PASstained but AB-negative granules. The cells with the smaller granules became localized to the acinar-intercalated duct junctions between 10 and 15 days, and by 21 days all of these were localized to the granular segments of the intercalated ducts. It is not clear whether these cells migrate away from the acini or vice versa. In the Low dose groups, development of the dichotomy of larger, weakly PAS-positive secretory granules in acini, and smaller, densely PAS-positive, AB-negative granules in intercalated ducts began at age 10 days (Fig. 4A) and was virtually complete by 15 days (Fig. 4B). This pattern developed between 7 and 10 days in the Medium (Fig. 4C,D) and High (Fig. 4E,F) dose groups. The weakly PAS-positive acinar secretory granules were greatly diminished by 10 and 15 days in the High and Medium dose T4 groups, respectively. Scattered vacuoles appeared in the acini of the High dose group at 10 days and were extremely numerous at 12 days (Fig. 4F). At age 15 days, the vacuoles were common in the Medium dose group (Fig. 4D) and uncommon in the Low dose group. Ultrastructurally (Fig. 5), the vacuoles had a sparse substructure and were membrane-bounded. Histometrics The proportional areas (mean percentage of total area per gland) occupied by parenchymal structures and stroma are presented in Table 3. There were no signiﬁcant differences in the proportional areas of parenchyma and stroma among all groups at age 4 days, nor between the Normal and Vehicle groups at any age. The proportional acinar areas increased progressively with age while the proportional stromal areas decreased, with the largest changes occurring between 10 and 15 days. Both changes were accelerated in all of the T4 groups at 7 and 10 days except for High dose T4 at 10 days. Proportional intercalated duct areas also increased in the Low and Medium dose T4 groups at 10 days and in all T4 groups at 12 and 15 days. Otherwise, the proportional areas of all ducts changed little with age. The mean areas of individual parenchymal elements are shown in Table 4. Mean acinar areas decreased progressively with age between 4 and 15 days in the Normal, Vehicle, and Low dose T4 pups, and even more in the Medium and High dose T4 pups. The mean areas of the ducts changed very little with age in all groups. T4 EFFECTS ON RAT PAROTID MORPHOGENESIS 99 Fig. 4. Photomicrographs of sections of parotid glands of T4treated rats, periodic acid–Schiff and hematoxylin (PAS-H) stains. A: At 10 days, Low dose T4. Small, strongly PAS-positive secretory granules are partly in intercalated ducts. B: At 15 days, Low dose T4. The gland is similar to the 21-day normal, with strongly PAS-positive secretory granules localized in intercalated ducts and lightly stained granules in acini. C: At 10 days, Medium dose T4. Many secretory granules in the acini have a weaker positive reaction with PAS compared with Normal day 10. Small, densely PAS-positive secretory granules are seen in intercalated ducts and adjacent acini. D: At 15 days, Medium dose T4. Vacuoles are seen in many acinar cells. Acinar secretory granules are decreased, and densely PAS-positive secretory granules are located in intercalated ducts. E: At 7 days, High dose T4. Densely PAS-positive secretory granules are located partly in acini and partly in intercalated ducts. F: At 12 days, High dose T4. Secretory granules are largely depleted in both acini and intercalated ducts, and vacuoles are very numerous in acini and striated ducts. Magniﬁcation (all), 3410. Apoptosis normal and vehicle-injected groups, and at age 4 days in all groups. TUNEL-positive cells, mostly acinar, were noticeably more numerous (10–40 cells per 200 3 ﬁeld) at 10 days in the Medium dose group, and strikingly increased (50–100 cells per 200 3 ﬁeld) in the High dose Apoptosis, as indicated by the TUNEL reaction (Fig. 6), of all cell types was very rare (one to four TUNELpositive parenchymal cells per section) at all ages in the 100 IKEDA ET AL. trend for the exogenous T4 to hasten the exit of the mucous cells, this effect was not consistent. Serum thyroid hormone (T4) concentration is low in the fetus and newborn rat, increases to twice the adult level by 16 days of age, and returns to the adult level by 30 days (Clos et al., 1974; Porterﬁeld et al., 1981; Dussault et al., 1982; Porterﬁeld, 1985; Farwell and DubordTomasetti, 1999). The rise in serum T4 in the Vehicle/ Normal group is consistent with these ﬁgures. Fetal serum corticosterone concentration is at the adult level during the last stage of gestation, rapidly declines after birth, and increases back to the adult level between 15 and 25 days of age (Takeuchi et al., 1977a, 1978). Maturation of the parotid acini, as assessd by a-amylase per unit of tissue, is delayed when the postnatal rise in either thyroid or corticosteroid hormones are surgically or chemically prevented (Takeuchi et al., 1977b) and precocious when either hormone is injected at 6 to 8 days of age (Sasaki et al., 1976; Takeuchi et al. 1978; Takuma et al., 1978; Kumegawa et al., 1980). It should be noted that there are parallel effects on general development, e.g., eye opening and tooth eruption (Bakke et al., 1975; Froelich and Meserve, 1982; Gamborino et al., 2001) that may inﬂuence parotid acinar maturation by means of early change from liquid to solid diet (Redman, 1987). Maintenance of mature rat parotid acini also depends on adequate levels of both hormones (Johnson et al., 1987). As cited previously (Johnson et al., 1987), the parotid secretory granules of thyroxine-treated mature rats were electron-lucent with an amorphous, rather dense core, somewhat like those of the early postnatal mucous cells. This ﬁnding raises the possibilty that the extra thyroxine may have brought about the re-appearance of the mucous cells. In addition, Takuma et al. (1978) demonstrated electron-lucent secretory granules with dense cores in parotid glands of mice at age 11 days after daily injections of hydrocortisone starting at 6 days. This sug- group at 10 and 12 days and the Medium dose group at 15 days. DISCUSSION Mucous Cells There were as many glands (3 glands) with mucous cells among the 8 glands of the Normal and Vehicle groups as there were among the 19 glands of the three T4-treated groups at 10 days, but the only mucous cells seen in any of the glands at 15 days were in one pup in the Low T4 group (Table 2). Thus, although there was a Fig. 5. Electron micrograph, at 12 days, acinar cell, High dose T4. Vacuoles are membrane-bounded and have a sparse substructure. Magniﬁcation 326,000. TABLE 3. Proportions of gland area (mean % 6 SE) occupied by parenchymal structures and stroma by age and T4 treatment Age (days) 4 7 10 15 Treatment High Medium Low Normal Vehicle High Medium Low Normal Vehicle High Medium Low Normal Vehicle Highc Medium Low Normal Vehicle Acini 12.0 11.5 7.1 13.0 11.3 32.6 28.7 28.1 19.8 19.8 36.0 62.6 54.7 26.5 35.1 66.7 59.4 65.2 54.6 62.0 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 1.8 1.3 0.8 2.1 1.8 1.5a,b 1.5a,b 1.9a,b 1.3 1.2 2.9 2.6a,b 3.0a,b 2.1 4.4 3.9 3.4 2.7 6.2 3.7 I.D. 1.0 1.6 1.2 1.1 1.5 3.6 4.2 3.4 4.2 3.5 3.9 5.1 4.1 2.0 3.0 7.1 10.6 7.7 4.1 3.9 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 I.D., intercalated duct; S.D., striated duct; E.D., excretory duct. Signiﬁcant difference from Normal group at this age. b Signiﬁcant difference from Vehicle group at this age. c High T4 pups in this row were age 12 days. a 0.2 0.2 0.2 0.2 0.1 0.1 0.4 0.5 0.5 0.4 0.6 0.5a,b 0.4a 0.3 0.3 0.3a,b 0.3a,b 0.6a,b 0.5 0.3 S.D. 1.9 1.6 1.2 1.4 1.4 3.7 3.4 3.2 3.3 3.4 2.8 3.5 3.1 2.6 2.4 4.2 5.8 3.6 3.1 3.7 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 0.2 0.4 0.3 0.4 0.3 0.4 0.5 0.5 0.3 0.5 0.4 0.6 0.6 0.5 0.5 0.7 0.8 0.8 0.9 0.6 E.D. 1.7 2.4 2.0 3.5 3.4 3.1 4.0 3.4 5.5 3.5 3.5 3.4 4.1 2.3 5.4 10.3 7.9 5.8 4.6 4.9 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Stroma 0.6 0.8 0.3 1.6 0.6 0.5 0.5 1.1 0.6 0.7 0.7 0.8 0.9 0.3 1.6 2.4 1.6 0.8 1.3 1.1 83.6 83.2 88.8 81.8 82.4 57.1 59.7 62.4 67.2 70.5 54.7 25.8 34.0 66.6 54.8 14.4 16.3 17.6 33.6 26.2 6 1.9 6 1.5 6 0.9 6 1.9 6 1.7 6 1.3a,b 6 2.3a,b 6 2.0b 6 1.2 6 1.9 6 3.4 6 3.1a,b 6 3.1a,b 6 1.8 6 2.9 6 2.7 6 2.1 62.6 6 5.7 6 2.9 101 T4 EFFECTS ON RAT PAROTID MORPHOGENESIS TABLE 4. Areas (Mean mm2 6 SE) of parenchymal structures by age and T4 treatment Age (days) 4 7 10 15 21 Treatment High Medium Low Normal Vehicle High Medium Low Normal Vehicle High Medium Low Normal Vehicle Highd Medium Low Normal Vehicle Normal Acini 81.0 76.3 72.7 76.4 81.2 51.4 53.7 54.7 64.7 55.1 31.2 37.3 42.2 42.5 45.7 28.2 31.5 44.2 39.4 39.8 50.6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 9.7 10.9 4.0 6.9 3.3 6.5 3.3 1.1 1.1 7.7 0.7b,c 2.0c 1.9 2.1 2.3 1.1b,c 1.6b,c 1.4 2.0 1.6 2.3 I.D. 40.5 39.9 39.3 35.9 38.1 35.4 33.6 31.3 33.9 30.4 31.2 33.9 33.9 33.8 35.8 26.2 34.8 35.4 31.6 34.1 36.1 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 S.D. 4.4 3.3 3.0 3.3 4.2 0.8 2.3 2.2 1.2 1.3 1.9 0.6 1.1 1.3 2.4 2.7 1.3 0.4 1.0 1.4 1.5 105.9 98.4 112.9 107.8 100.4 100.2 111.6 106.7 117.7 105.6 107.7 108.8 102.5 105.7 108.0 98.1 105.6 103.3 102.1 106.1 112.3 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 E.D. 2.9 9.8 8.0 6.2 1.8 4.3 6.8 2.1 7.5 2.6 2.8 1.6 3.6 3.8 2.5 2.1 3.6 2.4 4.3 3.5 2.9 225.3 249.3 234.1 414.5 253.0 256.2 371.4 266.6 457.0 352.4 373.9 411.5 320.6 371.8 314.6 435.1 351.4 293.4 377.5 435.3 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 49.1 32.9 44.3 28.3 37.3 22.1 21.6 23.6 50.0 22.3 36.9 48.2 31.1 14.3 27.3 25.4 I.D., intercalated duct; S.D., striated duct; E.D., excretory duct. Excretory ducts were present in only one sample of some groups at ages 4 and 7 days; hence, the values for these groups have no SE. b Signiﬁcant difference from Normal group at this age. c Signiﬁcant difference from Vehicle group at this age. d High T4 pups in this row were age 12 days. a gests that the hydrocortisone maintained these cells past their usual disappearance by 10 days (Takada et al., 2001). Furthermore, one might speculate that the cited (Takeuchi et al., 1977a, 1978) adult serum corticosteroid concentration before and at birth is necessary for the differentiation of the parotid mucous cells, and the low concentration after birth is insufﬁcient to sustain them past 8 days. Administering exogenous corticosteroid to attain adult serum levels before their disappearance thus might sustain the mucous cells for awhile. Unfortunately, however, no mucous stains were used in either study, and it is possible that the electron-lucent areas in the secretory granules were due to other substances. Furthermore, the disappearance of mucous granules at 8 to 10 days coincides with the normal rise in thyroid hormone, but precedes the rise in corticosterone (Takeuchi et al., 1977a, 1978). Thus, it is possible that the normal thyroid hormone rise triggers the mucous cell disappearance and that exogenous T4 may accelerate, rather than retard, this process. On the other hand, administration of thyroid hormone to rat pups induces a precocious rise in serum corticosterone and corticosteroid-binding globulin (D’Agostino and Henning, 1982) and, in rat pancreas, in glucocorticoid receptors (Lu et al., 1988). Thus, the lack of a consistent effect of the additional T4 on the chronology of mucous cell reduction observed in the present study might be due to opposing effects of the T4 and the corticosteroid factors which it induces. Morphogenesis The replacement of stroma by parenchymal elements in the developing rat parotid gland can occur by proportional increases in either the areas or numbers of individual parenchymal structures, or both. That no parenchymal structures increased in individual area, and acini actually decreased in individual area, indicate that, in all groups, the decrease in stromal area was due almost entirely to a proportional increase in the number of parenchymal structures. However, there also are changes in the composition of the stroma during this period (Cutler, 1990; Lazowski et al., 1994) that may result in its being less hydrophilic during histologic processing, and thus may artifactually reduce the areas in histologic sections. Enzymes involved in these changes have been shown to be increased by exogenous thyroxine (Imai et al., 1982). In any event, the processes involved in the replacement of stroma by parenchyma were accelerated in proportion to increased T4. Between 10 and 14 days, a subset of cells in the parotid acini become localized near the acinar/intercalated duct junction, and by 21 days all are in the granular segments of the intercalated ducts (Redman and Field, 1993; Sivakumar et al., 1998). The secretory granules of these cells can be distinguished from those in the mature acini by a lack of salivary peroxidase (Redman and Field, 1993) and expression of CSP-1 and SMGB (Sivakumar et al., 1998). These granules are smaller and stain more intensely with PAS than those of the mature acini, and we relied on PAS-H to follow the effects of T4 on overall differentiation. The aspects of differentiation as outlined above, especially the change in location of the cells with small, strongly PAS-positive secretory granules from acini to intercalated ducts, were accelerated in proportion to increased T4. All doses of T4 initially accelerated the maturation of acinar cells, as evidenced by the earlier and greater accumulation of moderately PAS-positive secretory granules and relocalization of cells with small, intensely PAS-positive 102 IKEDA ET AL. Fig. 6. Photomicrographs of sections of parotid glands of T4-treated rats, TUNEL (terminal deoxynucleotide transfer method UTP/nick end-labeling) procedure (DAB chromogen). Cells with brown nuclei are in various stages of apoptosis. A: At 10 days, Low dose T4. B: At 15 days, Low dose T4. C: At 10 days, Medium dose T4. D: At 15 days, Medium dose T4. E: At 7 days, High dose T4. F: At 10 days, High dose T4. Magniﬁcation (all), 3370. T4 EFFECTS ON RAT PAROTID MORPHOGENESIS secretory granules from acini into intercalated ducts. However, the number of secretory granules per acinus was greatly diminished by 10 days in the Medium and High dose T4 groups as compared to the Vehicle/Normal and Low dose groups. Apoptosis, Vacuoles, and Toxicity The paucity of apoptotic cells at all ages in the Normal and Vehicle groups is consistent with previous reports (e.g., Kruse and Redman, 1999; Tsujimura et al., 2006). Therefore, their much greater number in the Medium and High dose groups clearly is abnormal. The vacuoles that developed in the Medium and High dose T4 pups are similar to the ‘‘watery vacuoles’’ described in rat parotid gland after strong secretory stimulation (Garrett, 1978; Garrett et al., 1978). Exogenous thyroxine has been shown to induce hypersecretion by rat salivary glands by means of adrenergic nerve (Tumilasci et al., 1982; Medina et al., 1984), 5-hydroxytryptamine (Ostuni et al., 2003), substance P (Tumilasci et al., 1986a), and vasoactive intestinal peptide (Tumilasci et al., 1986b) routes, and indirectly by increased Na1, K1 -dependent adenosine triphosphatase activity (Saito et al., 1982). The observed appearance of vacuoles and reduction of acinar size thus are most likely due to salivary hypersecretion caused by the induced hyperthyroidism. Hyperthyroidism-increased metabolic rate, and an associated inability to obtain sufﬁcient nourishment (as indicated by slowing of gain in body weight), may also have contributed to reduced acinar size. To some extent, the numerous vacuoles seen with the two higher doses could be considered a physiologic response to the increased thyroid hormone. On the other hand, the slowing of the rate of growth, the greatly increased number of apoptotic cells in the Medium and High dose groups beginning at 10 days, and the death in the High dose group at 11 days, indicate that the pups administered the two higher doses developed a considerable degree of toxicity. In this study, the serum T4 was measured 3 hr after injection, and precise comparison with the normal, endogenous T4 would require taking multiple samples to obtain a 24 hour average of serum and tissue T4. Considering this, the Low dose T4 induced a precocious increase in serum T4 that came closest to approximating the normal increase that occurs 6–10 days later. The High and Medium doses of T4 produced, respectively, severe and moderate hyperthyroidism, the resulting toxicity limiting their usefulness. In several other studies on the effects of T4 on various aspects of rat growth and development, the amounts administered have ranged from 0.05 to 2.0 mg/g body weight (Dussault et al., 1982; D’Agostino and Henning, 1982; Fitch et al., 1999; Mutapcic et al., 2005). In general, doses of 1.0 or more were detrimental to the organ or tissue being studied, but little or no mention was made regarding general effects on the pups. The observations presented here indicate that, for rats, daily doses greater than 0.1 mg/g body weight beginning at 4 days after birth will exceed that which is needed to produce a precocious increase in serum thyroid hormone similar to that occurring at 10– 15 days. 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