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Calretinin and Calbindin-D28K in Male Rats During Postnatal Development
Neurobiology of Aging, Vol. 19, No. 3, pp. 253–257, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0197-4580/98 $19.00 1 .00
PII:S0197-4580(98)00060-8
Calretinin and Calbindin-D28K in Male Rats During Postnatal Development E. D. LEPHART,1 H. TAYLOR, N. A. JACOBSON, AND M. A. WATSON Department of Zoology, Cellular Biology Division, Brigham Young University, Provo, Utah 84602, USA Received December 5, 1997; Revised February 23, 1998; Accepted March 12, 1998 LEPHART, E. D., H. TAYLOR, N. A. JACOBSON, AND M. A. WATSON. Calretinin and calbindin-D28K in male rats during postnatal development. NEUROBIOL AGING 19(3) 253–257, 1998.—Calcium-binding proteins play potentially important roles in neurogenesis and neuroprotective mechanism(s). Some evidence exists that brain calbindin-D28K (CALB) is regulated by androgens. In the present study, calretinin (CALRET) and CALB patterns were determined by Western analysis in the medial basal hypothalamus (MBH) from male rats along with assaying plasma testosterone levels during postnatal development. Testosterone levels were very low in 7-, 10-, and 30-day-old animals (' 0.5 ng/mL), increased in a stair-step fashion to peak levels at 90 days (' 3.8 ng/mL), then declined with increasing age to very low levels at 300 days of age (' 0.3 ng/mL). At 7 and 10 days, MBH CALRET and CALB levels were low; however, at Day 30 a significant twofold increased was observed. Thereafter, in 60-, 120-, 180-, and 300-day-old animals MBH CALRET and CALB levels were, in general, comparable to 30-day-old values. These findings suggest that there is not a clear correspondence between the androgen status in male rats and the calcium-binding proteins (CALRET & CALB) expressed in the MBH. Therefore, it appears that brain CALRET and CALB are regulated in a developmental fashion with significant increases in expression occurring around the 4th postnatal week. © 1998 Elsevier Science Inc. Calretinin Calbindin-D28K Postnatal development Rat
Calcium-binding proteins
Hypothalamus
CALRETININ (CALRET) and Calbindin-D28K (CALB) are calcium-binding proteins that are members of a large family of proteins containing the EF-hand calcium-binding motif (2,6,10). Both calcium-binding proteins serve potentially important roles in the development and function of the central nervous system (CNS) (2,11,13,14,21,24). However, CALB represents one of the most abundant proteins found in certain brain regions (2,15). The buffering of excess calcium levels by calcium-binding proteins appear to influence neuronal maturation and survival (13–15,21– 24). Neuronal degeneration associated with Parkinson’s and Alzheimer’s disease and the normal aging process is thought to be mediated by toxic intracellular levels of free calcium (11–15,23, 24). It has been proposed that brain sites that have calcium-binding proteins present experience less neurodegeneration then areas where CALB and CALRET are less abundant (11,15,22–24). Previous studies from our laboratory suggest that the dimorphic expression of CALB in the medial basal hypothalamus (MBH) may play a supportive role in the neurogenesis of sexually dimorphic brain structures (17). In this case, during perinatal development, males displayed significantly higher CALB levels compared to females implying a protective role in preventing neuronal cell loss, along with the influence of sex steroids, that may account for larger sexual dimorphic nuclear volumes of specific brain areas in males vs. females (17,18). In studies examining the regulation of CALB, glucocorticoids have been reported to directly influence the abundance of brain CALRET or 1
Western analysis
CALB during prenatal and postnatal development (12,19,30). In connection with the activation of the hypothalamic-pituitaryadrenal axis, excess glucocorticoid production appears to increase neurotoxic levels in the hippocampus, presumably, by elevating excitatory amino acids and subsequent calcium influx to toxic concentrations that may exceed the protective effects of calciumbinding proteins in their buffering effects (3,29). Conversely, in addition to glucocorticoids, androgens also appear to modulate MBH CALB expression during prenatal development; where flutamide, an androgen receptor blocker, significantly decreased, whereas, testosterone treatment significantly increased MBH CALB levels (20). Therefore, in order to test the hypothesis that androgens influence calcium-binding proteins during postnatal development, we determined the changes in circulating testosterone levels and characterized the patterns of CALRET and CALB (by Western analysis) in MBH tissue homogenates from male Sprague-Dawley rats at 7, 10, 30, 60, 120, 180, and 300 days of age. MATERIALS AND METHODS
Western Analysis Male Sprague-Dawley rats [tissue samples were collected on postnatal 7, 10, 30, 60, 120, 180, and 300 days] were obtained from Charles River Research Laboratories (Wilmington, MA). During infantile development (i.e., 7 or 10 days old) animals were
Address correspondence to: Edwin D. Lephart, Department of Zoology, Cellular Biology Division, Brigham Young University, Provo, Utah 84602.
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obtained from lactating litters and males were determined using a dissecting microscope (identifying the presence of testes in the pelvic cavity of each animal). The medial basal hypothalamic (MBH) brain region (from 6 to 9 male animals) was collected using landmark boundaries, as previously described (17,19). The MBH tissue samples were pooled, homogenized in Tris buffered saline (TBS) and further processed by a micro-ultrasonic cell disrupter. Samples (homogenized aliquots) were then quantified for total protein content by the Lowry protein assay. The Western analysis method has been reported in detailed elsewhere (17,19). However, in brief, the homogenate protein samples (30 mg) were resolved on 14% Tris/glycine gels and then transferred to Millipore Immunobilon-P membranes (Millipore Corp., Bedford, MA) by electroblotting. The membranes were blocked with 6% dried milk (in TBS) for 45–55 min. Calbindin (CALB) levels were determined using a rat calbindin antibody [that does not cross-react with calretinin, dilution 1:50,000; kindly provided by AM Iacopino, Baylor School of Dentistry, Dallas, TX]. The calretinin (CALRET) antibody was purchased from Swant Antibodies, Bellinzona, Switzerland, dilution 1:10,000. The primary antibodies (utilized in separate experiments) were incubated with the membranes for 30 – 40 min., washed twice in TBS, then incubated with a secondary antibody (goat anti-rabbit IgG conjugated with horseradish peroxide, Bio-Rad, Inc., Hercules, CA; dilution 1:5,000). The bound antibody-complex for each sample on the immunoblots was detected with the enhanced chemiluminescence (ECL) Western blotting system (Amersham Corp., Arlington Heights, IL). The Western analysis, represents 4 to 6 independent immunoblots. The immunoreactive bands were quantified by measuring optical density using an imaging analysis system (Fotodyne, Inc., Hartland, WI). For each immunoblot, the lowest intensity band was assigned an arbitrary value of one (i.e., the Day 7 sample where no variance is shown). All other bands were then expressed as a fraction of this value (for the CALRET and CALB results). A representative immunoblot for CALRET is shown in Figure 2 and for CALB in Figure 3. Composite results of the quantified immunoblots are shown in Figures 4 and 5, respectively, for CALRET and CALB.
FIG. 1. Serum testosterone levels in male rats at 7, 10, 30, 45, 60, 90, 120, 180, and 300 days old. Testosterone levels were determined by radioimmunoassay. The expected significant increase after Day 45 or significant decreases after Day 90 in testosterone levels are not noted. The number of animals per age assayed was between 6 to 9 rats.
than 0.5 ng/mL), remained low in 10-day-old animals (just above 0.5 ng/mL), and in 30-day-old animals testosterone levels were comparable to 7-day-old levels (around ' 0.5 ng/mL). Thereafter, testosterone levels increased in a stair-step fashion as a function of increasing age until 90 days of age where peak levels of testosterone were observed (at ' 3.8 ng/mL, or more than threefold higher than Day 45 levels). After 90 days, testosterone levels decreased as a function of increasing age where in 300-day-old animals very low testosterone levels were recorded (around Day 7 levels; ' 0.3 ng/mL). Western analysis of the presence and abundance for CALRET and CALB in MBH brain samples are depicted in Figures 2 and 3. In general, similar patterns were observed for the profile and abundance of MBH CALB and CALRET in male rats during the
Testosterone Radioimmunoassay Serum testosterone levels were determined (from trunk blood) by radioimmunoassay obtained from male Sprague-Dawley rats at 7, 10, 30, 45, 60, 90, 120, 180, and 300 days old [using a coated-tube RIA, Diagnostic Systems Lab., (Webester, TX) kit for testosterone]. The high and low internal controls were within normal range(s) while the intra-assay coefficient of variation was less than 4%. Note: MBH tissue samples were not collected in 45or 90-day-old animals for the quantification of CALB or CALRET levels, therefore, these data are not shown in the immunoblots. Statistical Analysis To compare CALRET or CALB protein abundance, the quantified immunoblot (and testosterone) data were tested by analysis of variance (ANOVA), followed by pairwise comparison (via Tukey’s analysis) to determine significant differences between ages for male samples (p , 0.05). To determine whether a relationship exists between testosterone levels and CALRET or CALB protein abundance, a Pearson’s Correlation was performed. RESULTS
To determine the postnatal androgen hormonal profile in aging male rats, testosterone levels were measured in 7-, 10-, 30-, 45-, 60-, 90-, 120-, 180-, and 300-day-old animals. As displayed in Figure 1, testosterone levels were very low in 7-day-old rats (less
FIG. 2. Calretinin protein abundance determined by Western analysis (of a representative immunoblot) for medial basal hypothalamic (MBH) tissue samples in male rats at 7, 10, 30, 60, 120, 180, and 300 days old. Prestained molecular weight (kDalton) standards (Mr 3 103) are indicated by the horizontal bars on the left above. In preliminary studies, rat cerebellum homogenate samples were used as positive controls, however, this sample is not shown in this exposure.
CALCIUM-BINDING PROTEINS IN MALE RATS
255 remained relatively high in 60-, 120-, 180-, and 300-day-old animals (see Fig. 4). For the MBH CALB profile, the lowest levels were detected in 7-day-old animals, then the pattern increased in a stair-step fashion until Day 30 (see Fig. 5). At Day 10, CALB levels significantly increased twofold (over Day 7 values). In 30-day-old animals CALB levels significantly increased twofold (over Day 10 values). Thereafter, CALB levels remained high in 60-day-old animals, but then gradually declined in 120- and 180-day-old males, until moderate MBH CALB levels were recorded in 300-day-old animals. To determine whether a positive relationship exists between testosterone levels and the abundance of hypothalamic calciumbinding proteins, correlation analysis was evaluated. There was not a clear correspondence between testosterone and CALRET, r 5 0.23, t(5) 5 0.52, p 5 0.62, or CALB levels, r 5 0.48, t(5) 5 1.23, p 5 0.28.
FIG. 3. Calbindin-D28K protein abundance determined by Western analysis (of a representative immunoblot) for medial basal hypothalamic (MBH) tissue samples in male rats at 7, 10, 30, 60, 120, 180, and 300 days old. Prestained molecular weight (kDalton) standards (Mr 3 103) are indicated by the horizontal bars on the left above. In preliminary studies, rat cerebellum homogenate samples were used as positive controls, however, this sample is not shown in this exposure.
postnatal aging intervals examined. Low levels of calcium-binding proteins were seen at Days 7 and 10. However, at Day 30 for both CALRET and CALB, a notable increase in immunoreactivity was observed. Thereafter, 60-, 120-, and 180-day-old animals displayed immunoreactive CALRET and CALB levels comparable to Day 30 animals. In 300-day-old animals there was a slight but notable decline in the abundance of the calcium-binding proteins. After the MBH CALRET and CALB immunoblots were scanned and quantified the following results were obtained (see Figs. 4 and 5). In the MBH, males displayed low CALRET levels at 7 and 10 days of age. At 30 days, CALRET levels significantly increased by approximately twofold. Thereafter, CALRET levels
DISCUSSION
Calcium-binding proteins appear to play an important role in the development and function of central nervous system structures, where calcium levels regulate cellular proliferation, nuclear and cytoplasmic function, programmed cell death, and neurotoxicity (1–3,8,9,11,20 –27). The medial basal hypothalamus (MBH) represents an important brain region where physiologic and neuroendocrine functions are regulated (17,18). We have previously shown during perinatal development that CALB expression is dimorphic in the MBH, where males display higher levels of this calcium-binding protein compared to females (17). Additionally, our laboratory has provided some evidence for the regulation of CALB by androgens during prenatal development (20). In the present study, we examined the relationship of androgen status with the expression of the calcium-binding proteins, CALRET and CALB in MBH tissue from infantile to aged male rats. To quantify androgen status plasma testosterone levels were determined in the male rats throughout postnatal development. In general, the profile for plasma testosterone levels in male rats (obtained in the present study) during the aging process is in agreement with that reported
FIG. 4. Calretinin: Densitometric analysis of the medial basal hypothalamic (MBH) Western autoradiograms of male samples shown in Figure 2. For each immunoblot, the lowest band was assigned an arbitrary number of 1 and all other band intensities were expressed as a fraction of this value (four immunblots were analyzed). * Significant increase in calretinin levels in males at 30 days of age compared to 7- or 10-day-old animals.
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FIG. 5. Calbindin-D28K: Densitometric analysis of the medial basal hypothalamic (MBH) Western autoradiograms of male samples shown in Figure 3. For each immunoblot, the lowest band was assigned an arbitrary number of 1 and all other band intensities were expressed as a fraction of this value (six immunblots were analyzed). Œ: Significantly less calbindin in males on Day 7 compared to males 10 days of age. F: Significantly lower calbindin levels in males on Day 10 compared to males 30, 60, 120, and 180 days of age.
by other investigators (7,28); [i.e., before Day 30 androgen levels are very low, increase significantly after Day 45, peaking around 90 to 120 days of age, then testosterone concentrations decline with increasing postnatal age to prepubertal levels]. When the calcium-binding proteins were characterized during postnatal development, a similar expression pattern was observed for MBH CALRET and CALB. CALRET and CALB levels were low in infantile animals (at 7 and 10 days of age), whereas, both calcium-binding proteins significantly increased in abundance at 30 days of age. Thereafter, MBH CALRET and CALB levels remained relatively high in 60- and 120-day-old animals, while in 180- and 300-day-old animals the calcium-binding proteins were at high to moderate levels of expression. Finally, in general, the relative abundance of the CALRET and CALB immunopositive reactions by Western analysis is similar to that previously reported by our laboratory during the prenatal or perinatal developmental interval (17,19). Upon analysis of the collected parameters (by correlation analysis), there was not a clear-cut correspondence between androgen status of the male rats during postnatal development and the calcium-binding proteins expressed in the MBH brain site. While both parameters were low in 7- and 10-day-old animals, the most notable change in the profiles for MBH CALRET and CALB was a significant increase in these calcium-binding proteins at 30 days of age when testosterone levels in these animals were very low. There was not a clear correlation between the rise in the calcium-binding proteins and the pattern of plasma testosterone concentrations during early postnatal development. This is in contrast to the prenatal developmental interval where blocking androgen hormone action with flutamide significantly decreased MBH CALB levels (20). In this respect, prenatal androgen regulation of MBH CALB may be mediated by its conversion to
estrogens via the aromatase enzyme (17,18,20), because estrogen modulation of calbindin gene expression has been recently reported (9). Based upon these findings, it would appear that androgens during postnatal development do not significantly modulate calcium-binding protein(s) expression in the MBH brain region. However, the present data displayed a similar postnatal developmental pattern that overlaps with results reported by other investigators where CALB expression in various central nervous system (CNS) structures, such as, the cerebral cortex, cerebellum, and brainstem reached adult levels by the 4th to 5th postnatal week (16,31). Thereafter, CNS CALB levels in the adult rat were not significantly affected by age (16,31). On the other hand, the gradual decrease in the calcium-binding proteins with age in the present study is similar to that seen for the renal CALB profile in aged rats (4). Finally, some evidence does exist that CALB levels are regulated by TNF and NGF in vitro and in vivo or by IGF-1 in embryonic neurons in vitro (5,22,25,32); nevertheless, it is not known whether these factors are involved in altering basal levels of brain CALB as a function of increasing postnatal age. Therefore, the calcium-binding proteins (CALRET and CALB) appear to be developmentally regulated in several different brain regions independent of the hormonal status in male rats during postnatal maturation. However, due to the importance of these molecules, further research is required to determined the factor(s) or agent(s) which regulate these calcium-binding proteins during postnatal development. ACKNOWLEDGEMENTS
We thank Lori Mathias for her technical assistance. This work was supported, in part, by NSF grant IBN-9507972.
CALCIUM-BINDING PROTEINS IN MALE RATS
257 REFERENCES
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K. Developmental and age-dependent changes of 28-kDa calbindin-D in the central nervous system tissue determined with a sensitive immunoassay method. J. Neurochem. 58:128 –134; 1992. Lephart, E. D. Dimorphic expression of calbindin-D28k in the medial basal hypothalamus from perinatal male and female rats. Dev. Brain Res. 96:281–284; 1996. Lephart, E. D. A review of brain aromatase cytochrome P450. Brain Res. Rev. 22:1–26; 1996. Lephart, E. D.; Watson, M. A.; Jacobson, N. A.; Rhees, R. W.; Ladle, D. R. Calbindin-D28K is regulated by adrenal steroids in hypothalamic tissue during prenatal development. Dev. Brain Res. 100:117–120; 1997. Lephart, E. D.; Watson, M. A.; Mathias, L.; Rhees, R. W.; Diano, S.; Horvath, T. L. Co-localization of aromatase cytochrome P450 and calbindin-D28K and androgen regulation of calbindin-D28k during perinatal development. Soc. Neurosci. 23:139. 10; 1997. Liang, C. L.; Sinton, C. M.; German D. C. Midbrain dopaminergic neurons in the mouse: co-localization with calbindin-D28K and calretinin. Neurosci. 75:523–533; 1996. Mattson, M. P. Components of neurite outgrowth that determine neural cytoarchitecture: Influence of calcium and the growth substrate. J. Neurosci. Res. 20:331–345; 1988. Mattson, M. P. Calcium as sculptor and destroyer of neural circuitry. Exp. Gerontol. 27:29 – 49; 1992. Mattson, M. P.; Rydel, R. E.; Lieberburg. I.; Smith-Swintosky, V. L. Altered calcium signaling and neuronal injury: stroke and Alzheimer’s disease as examples. Ann. N. Y. Acad. Sci. 679:1–21; 1993. Mattson, M. P.; Cheng, B.; Baldwin, S. A.; Smith-Swintosky, V. L.; Keller, J.; Geddes, J. W.; Scheff, S. W.; Christakos, S. Brain injury and TNFs induce calbindin-D28k in astrocytes: Evidence for a cytoprotective response. J. Neurosci. Res. 42:357–370; 1995. McDonald, A. J. Calretinin immunoreactive neurons in the basolateral amygdala of the rat and monkey. Brain Res. 667:238 –242; 1994. McMahon, A.; Lephart, E. D.; Chi, S.; Lee, M.; Iacopino, A. M.; German, D. C. Calbindin-D28K in the nucleus of nerve growth factor-treated PC12 cells. NeuroReport 7:2419 –2422; 1996. Paz, G. F.; Winter, J. S. D.; Reyes, F. I.; Faiman, C. Developmental pattern of testosterone production by the rat testis. Steroids 36:675– 688; 1980. Sapolski, R. M. Stress, the aging brain, and the mechanism of neuron death. Cambridge, MA: MIT Press; 1992:181–186 and 219 –220. Strauss, K. I.; Schulkin, J.; Jacobowitz, D. M. Corticosterone effects on rat calretinin mRNA in discrete brain nuclei and the testes. Mol. Brain Res. 28:81– 86; 1995. Varghese, S.; Lee, S.; Huang, Y. C.; Christakos, S. Analysis of rat vitamin D-dependent calbindin-D28k gene expression. J. Biol. Chem. 263:9776 –9784; 1988. Yamaguchi, T.; Keino, K.; Fukuda, J. The effect of insulin and insulin-like growth factor-1 on the expression of calretinin and calbindin D-28K in rat embryonic neurons in culture. Neurochem. Int. 26:255–262; 1995.
PII:S0197-4580(98)00060-8
Calretinin and Calbindin-D28K in Male Rats During Postnatal Development E. D. LEPHART,1 H. TAYLOR, N. A. JACOBSON, AND M. A. WATSON Department of Zoology, Cellular Biology Division, Brigham Young University, Provo, Utah 84602, USA Received December 5, 1997; Revised February 23, 1998; Accepted March 12, 1998 LEPHART, E. D., H. TAYLOR, N. A. JACOBSON, AND M. A. WATSON. Calretinin and calbindin-D28K in male rats during postnatal development. NEUROBIOL AGING 19(3) 253–257, 1998.—Calcium-binding proteins play potentially important roles in neurogenesis and neuroprotective mechanism(s). Some evidence exists that brain calbindin-D28K (CALB) is regulated by androgens. In the present study, calretinin (CALRET) and CALB patterns were determined by Western analysis in the medial basal hypothalamus (MBH) from male rats along with assaying plasma testosterone levels during postnatal development. Testosterone levels were very low in 7-, 10-, and 30-day-old animals (' 0.5 ng/mL), increased in a stair-step fashion to peak levels at 90 days (' 3.8 ng/mL), then declined with increasing age to very low levels at 300 days of age (' 0.3 ng/mL). At 7 and 10 days, MBH CALRET and CALB levels were low; however, at Day 30 a significant twofold increased was observed. Thereafter, in 60-, 120-, 180-, and 300-day-old animals MBH CALRET and CALB levels were, in general, comparable to 30-day-old values. These findings suggest that there is not a clear correspondence between the androgen status in male rats and the calcium-binding proteins (CALRET & CALB) expressed in the MBH. Therefore, it appears that brain CALRET and CALB are regulated in a developmental fashion with significant increases in expression occurring around the 4th postnatal week. © 1998 Elsevier Science Inc. Calretinin Calbindin-D28K Postnatal development Rat
Calcium-binding proteins
Hypothalamus
CALRETININ (CALRET) and Calbindin-D28K (CALB) are calcium-binding proteins that are members of a large family of proteins containing the EF-hand calcium-binding motif (2,6,10). Both calcium-binding proteins serve potentially important roles in the development and function of the central nervous system (CNS) (2,11,13,14,21,24). However, CALB represents one of the most abundant proteins found in certain brain regions (2,15). The buffering of excess calcium levels by calcium-binding proteins appear to influence neuronal maturation and survival (13–15,21– 24). Neuronal degeneration associated with Parkinson’s and Alzheimer’s disease and the normal aging process is thought to be mediated by toxic intracellular levels of free calcium (11–15,23, 24). It has been proposed that brain sites that have calcium-binding proteins present experience less neurodegeneration then areas where CALB and CALRET are less abundant (11,15,22–24). Previous studies from our laboratory suggest that the dimorphic expression of CALB in the medial basal hypothalamus (MBH) may play a supportive role in the neurogenesis of sexually dimorphic brain structures (17). In this case, during perinatal development, males displayed significantly higher CALB levels compared to females implying a protective role in preventing neuronal cell loss, along with the influence of sex steroids, that may account for larger sexual dimorphic nuclear volumes of specific brain areas in males vs. females (17,18). In studies examining the regulation of CALB, glucocorticoids have been reported to directly influence the abundance of brain CALRET or 1
Western analysis
CALB during prenatal and postnatal development (12,19,30). In connection with the activation of the hypothalamic-pituitaryadrenal axis, excess glucocorticoid production appears to increase neurotoxic levels in the hippocampus, presumably, by elevating excitatory amino acids and subsequent calcium influx to toxic concentrations that may exceed the protective effects of calciumbinding proteins in their buffering effects (3,29). Conversely, in addition to glucocorticoids, androgens also appear to modulate MBH CALB expression during prenatal development; where flutamide, an androgen receptor blocker, significantly decreased, whereas, testosterone treatment significantly increased MBH CALB levels (20). Therefore, in order to test the hypothesis that androgens influence calcium-binding proteins during postnatal development, we determined the changes in circulating testosterone levels and characterized the patterns of CALRET and CALB (by Western analysis) in MBH tissue homogenates from male Sprague-Dawley rats at 7, 10, 30, 60, 120, 180, and 300 days of age. MATERIALS AND METHODS
Western Analysis Male Sprague-Dawley rats [tissue samples were collected on postnatal 7, 10, 30, 60, 120, 180, and 300 days] were obtained from Charles River Research Laboratories (Wilmington, MA). During infantile development (i.e., 7 or 10 days old) animals were
Address correspondence to: Edwin D. Lephart, Department of Zoology, Cellular Biology Division, Brigham Young University, Provo, Utah 84602.
253
254
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obtained from lactating litters and males were determined using a dissecting microscope (identifying the presence of testes in the pelvic cavity of each animal). The medial basal hypothalamic (MBH) brain region (from 6 to 9 male animals) was collected using landmark boundaries, as previously described (17,19). The MBH tissue samples were pooled, homogenized in Tris buffered saline (TBS) and further processed by a micro-ultrasonic cell disrupter. Samples (homogenized aliquots) were then quantified for total protein content by the Lowry protein assay. The Western analysis method has been reported in detailed elsewhere (17,19). However, in brief, the homogenate protein samples (30 mg) were resolved on 14% Tris/glycine gels and then transferred to Millipore Immunobilon-P membranes (Millipore Corp., Bedford, MA) by electroblotting. The membranes were blocked with 6% dried milk (in TBS) for 45–55 min. Calbindin (CALB) levels were determined using a rat calbindin antibody [that does not cross-react with calretinin, dilution 1:50,000; kindly provided by AM Iacopino, Baylor School of Dentistry, Dallas, TX]. The calretinin (CALRET) antibody was purchased from Swant Antibodies, Bellinzona, Switzerland, dilution 1:10,000. The primary antibodies (utilized in separate experiments) were incubated with the membranes for 30 – 40 min., washed twice in TBS, then incubated with a secondary antibody (goat anti-rabbit IgG conjugated with horseradish peroxide, Bio-Rad, Inc., Hercules, CA; dilution 1:5,000). The bound antibody-complex for each sample on the immunoblots was detected with the enhanced chemiluminescence (ECL) Western blotting system (Amersham Corp., Arlington Heights, IL). The Western analysis, represents 4 to 6 independent immunoblots. The immunoreactive bands were quantified by measuring optical density using an imaging analysis system (Fotodyne, Inc., Hartland, WI). For each immunoblot, the lowest intensity band was assigned an arbitrary value of one (i.e., the Day 7 sample where no variance is shown). All other bands were then expressed as a fraction of this value (for the CALRET and CALB results). A representative immunoblot for CALRET is shown in Figure 2 and for CALB in Figure 3. Composite results of the quantified immunoblots are shown in Figures 4 and 5, respectively, for CALRET and CALB.
FIG. 1. Serum testosterone levels in male rats at 7, 10, 30, 45, 60, 90, 120, 180, and 300 days old. Testosterone levels were determined by radioimmunoassay. The expected significant increase after Day 45 or significant decreases after Day 90 in testosterone levels are not noted. The number of animals per age assayed was between 6 to 9 rats.
than 0.5 ng/mL), remained low in 10-day-old animals (just above 0.5 ng/mL), and in 30-day-old animals testosterone levels were comparable to 7-day-old levels (around ' 0.5 ng/mL). Thereafter, testosterone levels increased in a stair-step fashion as a function of increasing age until 90 days of age where peak levels of testosterone were observed (at ' 3.8 ng/mL, or more than threefold higher than Day 45 levels). After 90 days, testosterone levels decreased as a function of increasing age where in 300-day-old animals very low testosterone levels were recorded (around Day 7 levels; ' 0.3 ng/mL). Western analysis of the presence and abundance for CALRET and CALB in MBH brain samples are depicted in Figures 2 and 3. In general, similar patterns were observed for the profile and abundance of MBH CALB and CALRET in male rats during the
Testosterone Radioimmunoassay Serum testosterone levels were determined (from trunk blood) by radioimmunoassay obtained from male Sprague-Dawley rats at 7, 10, 30, 45, 60, 90, 120, 180, and 300 days old [using a coated-tube RIA, Diagnostic Systems Lab., (Webester, TX) kit for testosterone]. The high and low internal controls were within normal range(s) while the intra-assay coefficient of variation was less than 4%. Note: MBH tissue samples were not collected in 45or 90-day-old animals for the quantification of CALB or CALRET levels, therefore, these data are not shown in the immunoblots. Statistical Analysis To compare CALRET or CALB protein abundance, the quantified immunoblot (and testosterone) data were tested by analysis of variance (ANOVA), followed by pairwise comparison (via Tukey’s analysis) to determine significant differences between ages for male samples (p , 0.05). To determine whether a relationship exists between testosterone levels and CALRET or CALB protein abundance, a Pearson’s Correlation was performed. RESULTS
To determine the postnatal androgen hormonal profile in aging male rats, testosterone levels were measured in 7-, 10-, 30-, 45-, 60-, 90-, 120-, 180-, and 300-day-old animals. As displayed in Figure 1, testosterone levels were very low in 7-day-old rats (less
FIG. 2. Calretinin protein abundance determined by Western analysis (of a representative immunoblot) for medial basal hypothalamic (MBH) tissue samples in male rats at 7, 10, 30, 60, 120, 180, and 300 days old. Prestained molecular weight (kDalton) standards (Mr 3 103) are indicated by the horizontal bars on the left above. In preliminary studies, rat cerebellum homogenate samples were used as positive controls, however, this sample is not shown in this exposure.
CALCIUM-BINDING PROTEINS IN MALE RATS
255 remained relatively high in 60-, 120-, 180-, and 300-day-old animals (see Fig. 4). For the MBH CALB profile, the lowest levels were detected in 7-day-old animals, then the pattern increased in a stair-step fashion until Day 30 (see Fig. 5). At Day 10, CALB levels significantly increased twofold (over Day 7 values). In 30-day-old animals CALB levels significantly increased twofold (over Day 10 values). Thereafter, CALB levels remained high in 60-day-old animals, but then gradually declined in 120- and 180-day-old males, until moderate MBH CALB levels were recorded in 300-day-old animals. To determine whether a positive relationship exists between testosterone levels and the abundance of hypothalamic calciumbinding proteins, correlation analysis was evaluated. There was not a clear correspondence between testosterone and CALRET, r 5 0.23, t(5) 5 0.52, p 5 0.62, or CALB levels, r 5 0.48, t(5) 5 1.23, p 5 0.28.
FIG. 3. Calbindin-D28K protein abundance determined by Western analysis (of a representative immunoblot) for medial basal hypothalamic (MBH) tissue samples in male rats at 7, 10, 30, 60, 120, 180, and 300 days old. Prestained molecular weight (kDalton) standards (Mr 3 103) are indicated by the horizontal bars on the left above. In preliminary studies, rat cerebellum homogenate samples were used as positive controls, however, this sample is not shown in this exposure.
postnatal aging intervals examined. Low levels of calcium-binding proteins were seen at Days 7 and 10. However, at Day 30 for both CALRET and CALB, a notable increase in immunoreactivity was observed. Thereafter, 60-, 120-, and 180-day-old animals displayed immunoreactive CALRET and CALB levels comparable to Day 30 animals. In 300-day-old animals there was a slight but notable decline in the abundance of the calcium-binding proteins. After the MBH CALRET and CALB immunoblots were scanned and quantified the following results were obtained (see Figs. 4 and 5). In the MBH, males displayed low CALRET levels at 7 and 10 days of age. At 30 days, CALRET levels significantly increased by approximately twofold. Thereafter, CALRET levels
DISCUSSION
Calcium-binding proteins appear to play an important role in the development and function of central nervous system structures, where calcium levels regulate cellular proliferation, nuclear and cytoplasmic function, programmed cell death, and neurotoxicity (1–3,8,9,11,20 –27). The medial basal hypothalamus (MBH) represents an important brain region where physiologic and neuroendocrine functions are regulated (17,18). We have previously shown during perinatal development that CALB expression is dimorphic in the MBH, where males display higher levels of this calcium-binding protein compared to females (17). Additionally, our laboratory has provided some evidence for the regulation of CALB by androgens during prenatal development (20). In the present study, we examined the relationship of androgen status with the expression of the calcium-binding proteins, CALRET and CALB in MBH tissue from infantile to aged male rats. To quantify androgen status plasma testosterone levels were determined in the male rats throughout postnatal development. In general, the profile for plasma testosterone levels in male rats (obtained in the present study) during the aging process is in agreement with that reported
FIG. 4. Calretinin: Densitometric analysis of the medial basal hypothalamic (MBH) Western autoradiograms of male samples shown in Figure 2. For each immunoblot, the lowest band was assigned an arbitrary number of 1 and all other band intensities were expressed as a fraction of this value (four immunblots were analyzed). * Significant increase in calretinin levels in males at 30 days of age compared to 7- or 10-day-old animals.
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FIG. 5. Calbindin-D28K: Densitometric analysis of the medial basal hypothalamic (MBH) Western autoradiograms of male samples shown in Figure 3. For each immunoblot, the lowest band was assigned an arbitrary number of 1 and all other band intensities were expressed as a fraction of this value (six immunblots were analyzed). Œ: Significantly less calbindin in males on Day 7 compared to males 10 days of age. F: Significantly lower calbindin levels in males on Day 10 compared to males 30, 60, 120, and 180 days of age.
by other investigators (7,28); [i.e., before Day 30 androgen levels are very low, increase significantly after Day 45, peaking around 90 to 120 days of age, then testosterone concentrations decline with increasing postnatal age to prepubertal levels]. When the calcium-binding proteins were characterized during postnatal development, a similar expression pattern was observed for MBH CALRET and CALB. CALRET and CALB levels were low in infantile animals (at 7 and 10 days of age), whereas, both calcium-binding proteins significantly increased in abundance at 30 days of age. Thereafter, MBH CALRET and CALB levels remained relatively high in 60- and 120-day-old animals, while in 180- and 300-day-old animals the calcium-binding proteins were at high to moderate levels of expression. Finally, in general, the relative abundance of the CALRET and CALB immunopositive reactions by Western analysis is similar to that previously reported by our laboratory during the prenatal or perinatal developmental interval (17,19). Upon analysis of the collected parameters (by correlation analysis), there was not a clear-cut correspondence between androgen status of the male rats during postnatal development and the calcium-binding proteins expressed in the MBH brain site. While both parameters were low in 7- and 10-day-old animals, the most notable change in the profiles for MBH CALRET and CALB was a significant increase in these calcium-binding proteins at 30 days of age when testosterone levels in these animals were very low. There was not a clear correlation between the rise in the calcium-binding proteins and the pattern of plasma testosterone concentrations during early postnatal development. This is in contrast to the prenatal developmental interval where blocking androgen hormone action with flutamide significantly decreased MBH CALB levels (20). In this respect, prenatal androgen regulation of MBH CALB may be mediated by its conversion to
estrogens via the aromatase enzyme (17,18,20), because estrogen modulation of calbindin gene expression has been recently reported (9). Based upon these findings, it would appear that androgens during postnatal development do not significantly modulate calcium-binding protein(s) expression in the MBH brain region. However, the present data displayed a similar postnatal developmental pattern that overlaps with results reported by other investigators where CALB expression in various central nervous system (CNS) structures, such as, the cerebral cortex, cerebellum, and brainstem reached adult levels by the 4th to 5th postnatal week (16,31). Thereafter, CNS CALB levels in the adult rat were not significantly affected by age (16,31). On the other hand, the gradual decrease in the calcium-binding proteins with age in the present study is similar to that seen for the renal CALB profile in aged rats (4). Finally, some evidence does exist that CALB levels are regulated by TNF and NGF in vitro and in vivo or by IGF-1 in embryonic neurons in vitro (5,22,25,32); nevertheless, it is not known whether these factors are involved in altering basal levels of brain CALB as a function of increasing postnatal age. Therefore, the calcium-binding proteins (CALRET and CALB) appear to be developmentally regulated in several different brain regions independent of the hormonal status in male rats during postnatal maturation. However, due to the importance of these molecules, further research is required to determined the factor(s) or agent(s) which regulate these calcium-binding proteins during postnatal development. ACKNOWLEDGEMENTS
We thank Lori Mathias for her technical assistance. This work was supported, in part, by NSF grant IBN-9507972.
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