0013-7227/90/1262-0773$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 2 Printed in U.S.A.
Discordant Effects of Aging on Prolactin and Luteinizing Hormone-/? Messenger Ribonucleic Acid Levels in the Female Rat* DAVID A. STEWART, MARC R. BLACKMAN, MARY ANN KOWATCH, DAVID B. DANNER, AND GEORGE S. ROTH Laboratory of Molecular Genetics (D.A.S., D.B.D.) and Endocrinology Section (M.R.B.), Laboratory of Clinical Physiology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224; the Department of Medicine, Francis Scott Key Medical Center and the Johns Hopkins University School of Medicine (M.R.B.), and the Molecular Physiology and Genetics Section (M.A.K., G.S.R.), Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224
ABSTRACT. To examine the molecular genetic basis for the age-related increase in PRL secretion and decrease in LH production in the rat, we measured steady state levels of PRL and LH/3 mRNA in pituitary homogenates and cell lysates from monolayer adenohypophyseal cultures. These mRNA levels were compared with the corresponding levels of immunoreactive PRL and LH in sera and culture media. Paired groups (n = 4-10/ group) of intact and 4-week ovariectomized mature (6-7 months old) and old (23-25 months old) female Wistar rats were studied. Serum PRL levels were 550% higher in intact old us. mature rats (P < 0.001), whereas the corresponding pituitary homogenate levels of PRL mRNA were similar (P > 0.4). Medium PRL concentrations were 230% greater (P < 0.006) whereas cell lysate concentrations of PRL mRNA were unaltered (P > 0.2) in monolayer cultures from intact old vs. mature rats. Serum PRL levels were 650% higher (P < 0.003) and pituitary homogenate PRL mRNA levels were slightly increased (P < 0.04) in ovari-
A
ectomized old us. mature rats. Neither serum LH values (P > 0.07) nor pituitary homogenate LH/3 mRNA levels (P > 0.1) differed in intact old and mature rats, whereas the corresponding medium concentrations of LH were reduced (P < 0.001). Ovariectomized old us. mature rats exhibited reductions in serum (P < 0.02) and medium (P < 0.001) LH concentrations, as well as in pituitary homogenate (P < 0.002) and cell lysate (P < 0.006) LH0 mRNA levels. Thus, these data revealed coordinate decreases with age in LH/3 mRNA and LH secretion, particularly in ovariectomized rats, suggesting an age-related alteration at or before LH/3 gene transcription. These findings parallel observations on other genes whose products change with age. In contrast, the observation that the increased secretion of PRL in old rats is accompanied by little or no increase in PRL mRNA is novel and suggests that age-related alterations in PRL gene expression proceed through a posttranscriptional mechanism. {Endocrinology 126: 773-778, 1990)
related changes in PRL and LH secretion cannot be explained by changes at the pituitary receptor level (1416) or significant alterations in the proportions of responsive PRL-secreting (lactotropic) or LH-secreting (gonadotropic) cells (17-19). Thus, differential age changes in the expression of PRL and LH genes may occur. Physiological modulation of PRL and LH production is primarily regulated by transcription of the genes for PRL (20-23) and the hormone-specific /3-subunit of LH (24-27), respectively. We, therefore, investigated whether steady state mRNA levels for PRL and LH/? are altered in aging female rats to an extent that would account for the observed differences in PRL and LH secretion.
LTERATIONS in gene expression (1-4) and neuroendocrine function (5, 6) are two well documented manifestations of the aging process, yet little is known about the effects of aging on the molecular genetic mechanisms regulating hypothalamic -pituitary hormone production. One particularly well studied paradigm of endocrine aging has been reproductive senescence in aging female rats (5-8). Numerous investigators have reported that both in vivo and in vitro basal and stimulated PRL levels are increased (8-11) while basal and estrogen-or GnRH-modulated levels of LH are decreased (8-10, 12, 13) from adenohypophyses of intact or ovariectomized old female rats. In healthy rats, these ageReceived July 31,1989. Address requests for reprints to: Dr. M. R. Blackman, Endocrinology Section, Gerontology Research Center, Room 2B-20, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224. * This work was supported in part by the MacArthur Foundation Research Program on Successful Aging.
Materials and Methods Experimental animals All experiments were conducted in healthy intact or ovariectomized mature (6-7 months old) and old (23-25 months old) 773
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EFFECTS OF AGING ON PRL AND LH0
774
female Wistar rats obtained from the Gerontology Research Center (GRC) Animal Colony. The GRC Animal Colony is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Animals were housed one or two per cage in a temperature-controlled environment with a 12-h day, 12-h night cycle and were fed Purina rat chow and tap water ad libitum. Intact mature rats (n = 16) were studied on the first or second day of diestrus, whereas the intact old rats (n = 12) were in a state of constant diestrus, as shown by vaginal smears obtained biweekly for 2 weeks before experiments (28). Ovariectomized mature (n = 18) and old (n = 11) rats were studied 4 weeks after bilateral gonadectomy. Because aging in the female rat is known to be associated with an increased occurrence of PRL-secreting pituitary tumors (17, 18), rats with visible pituitary tumors at the time of death or exhibiting serum PRL levels in excess of 200 ng/ml were excluded from study. Approximately 25-30% of old rats were thus excluded. To ascertain whether rats with microprolactinomas might be missed by the above screening procedure, we performed a series of preliminary experiments wherein anterior pituitary glands from individual intact old female rats with normal serum PRL levels were quartered, and the PRL mRNA levels compared among the quarters. No significant differences were detected in PRL mRNA levels in pituitary quarters obtained from individual rats (data not shown). Pituitary cell cultures Paired groups (n = 4-10 rats/group) of intact or ovariectomized mature and old rats were rapidly decapitated between 0900-1000 h. Trunk blood was obtained, and serum separated by centrifugation and stored at —80 C until assayed. Anterior pituitary glands were removed and divided into quarters. Three quarters of each gland were used for preparation of primary monolayer adenohypophyseal cultures. For the intact rats, individual pituitary cell cultures were obtained by a mechanical and enzymatic digestion procedure (10, 11). Cells were then incubated overnight in Dulbecco's Modified Eagle's Medium supplemented with dextran-coated charcoal (DCC) and subsequently maintained in duplicate or triplicate cultures for 96 h in either the absence or presence of DCC-treated rat/fetal calf serum, as previously described (10,11). For the ovariectomized rats, pituitary cells were pooled from each age group, digested and preincubated overnight as above, and maintained in quadruplicate or sextuplicate in serum-containing Dulbecco's Modified Eagle's Medium for 96 h. RNA slot blots The remaining quarter of each pituitary gland was immediately extracted for total cytoplasmic RNA by a modification of a previously described method (24, 25). Pooled aqueous layers from pituitary homogenate extracts were reextracted with phenol-chloroform-isoamyl alcohol. Ethanol precipitation was performed for 16 h at -20 C. After centrifugation at 13,000 X g for 30 min, total RNA was resuspended in 200 n\ sterile water and immediately denatured by adding 600 jul 10 X SSC (1 x SSC = 0.15 M NaCl/0.015 M Na citrate) and 6.5 M formaldehyde and heating at 65 C for 15 min; samples were stored at -80 C.
Endo • 1990 Vol 126 • No 2
Steady state levels of PRL and LH/? mRNA were quantified by slot blot analyses (24-27) of in vivo material (pituitary membrane homogenates) and in vitro material (pituitary cell lysates from primary monolayer adenohypophyseal cell cultures). Each denatured sample was applied in triplicate to GeneScreen Plus (DuPont, Wilmington, DE) filter paper using a Scleicher and Schuell Minifold II Apparatus (Keene, NH), allowed to sit on the filter for 30 min, passed through under vacuum, washed with 10 X SSC, and air dried. The formaldehyde reaction was reversed by baking at 80 C under vacuum for 2 h. Filters were prehybridized for more than 30 min at 42 C in roller bottles. Prehybridization buffer contained 50% formaldehyde, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), and 10% dextran sulfate. For hybridization, randomly primed cDNA probe plus 200 Mg/ml heat-denatured herring sperm DNA were added to the prehybridization buffer (final concentration,
Vol. 126, No. 2 Printed in U.S.A.
Discordant Effects of Aging on Prolactin and Luteinizing Hormone-/? Messenger Ribonucleic Acid Levels in the Female Rat* DAVID A. STEWART, MARC R. BLACKMAN, MARY ANN KOWATCH, DAVID B. DANNER, AND GEORGE S. ROTH Laboratory of Molecular Genetics (D.A.S., D.B.D.) and Endocrinology Section (M.R.B.), Laboratory of Clinical Physiology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224; the Department of Medicine, Francis Scott Key Medical Center and the Johns Hopkins University School of Medicine (M.R.B.), and the Molecular Physiology and Genetics Section (M.A.K., G.S.R.), Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224
ABSTRACT. To examine the molecular genetic basis for the age-related increase in PRL secretion and decrease in LH production in the rat, we measured steady state levels of PRL and LH/3 mRNA in pituitary homogenates and cell lysates from monolayer adenohypophyseal cultures. These mRNA levels were compared with the corresponding levels of immunoreactive PRL and LH in sera and culture media. Paired groups (n = 4-10/ group) of intact and 4-week ovariectomized mature (6-7 months old) and old (23-25 months old) female Wistar rats were studied. Serum PRL levels were 550% higher in intact old us. mature rats (P < 0.001), whereas the corresponding pituitary homogenate levels of PRL mRNA were similar (P > 0.4). Medium PRL concentrations were 230% greater (P < 0.006) whereas cell lysate concentrations of PRL mRNA were unaltered (P > 0.2) in monolayer cultures from intact old vs. mature rats. Serum PRL levels were 650% higher (P < 0.003) and pituitary homogenate PRL mRNA levels were slightly increased (P < 0.04) in ovari-
A
ectomized old us. mature rats. Neither serum LH values (P > 0.07) nor pituitary homogenate LH/3 mRNA levels (P > 0.1) differed in intact old and mature rats, whereas the corresponding medium concentrations of LH were reduced (P < 0.001). Ovariectomized old us. mature rats exhibited reductions in serum (P < 0.02) and medium (P < 0.001) LH concentrations, as well as in pituitary homogenate (P < 0.002) and cell lysate (P < 0.006) LH0 mRNA levels. Thus, these data revealed coordinate decreases with age in LH/3 mRNA and LH secretion, particularly in ovariectomized rats, suggesting an age-related alteration at or before LH/3 gene transcription. These findings parallel observations on other genes whose products change with age. In contrast, the observation that the increased secretion of PRL in old rats is accompanied by little or no increase in PRL mRNA is novel and suggests that age-related alterations in PRL gene expression proceed through a posttranscriptional mechanism. {Endocrinology 126: 773-778, 1990)
related changes in PRL and LH secretion cannot be explained by changes at the pituitary receptor level (1416) or significant alterations in the proportions of responsive PRL-secreting (lactotropic) or LH-secreting (gonadotropic) cells (17-19). Thus, differential age changes in the expression of PRL and LH genes may occur. Physiological modulation of PRL and LH production is primarily regulated by transcription of the genes for PRL (20-23) and the hormone-specific /3-subunit of LH (24-27), respectively. We, therefore, investigated whether steady state mRNA levels for PRL and LH/? are altered in aging female rats to an extent that would account for the observed differences in PRL and LH secretion.
LTERATIONS in gene expression (1-4) and neuroendocrine function (5, 6) are two well documented manifestations of the aging process, yet little is known about the effects of aging on the molecular genetic mechanisms regulating hypothalamic -pituitary hormone production. One particularly well studied paradigm of endocrine aging has been reproductive senescence in aging female rats (5-8). Numerous investigators have reported that both in vivo and in vitro basal and stimulated PRL levels are increased (8-11) while basal and estrogen-or GnRH-modulated levels of LH are decreased (8-10, 12, 13) from adenohypophyses of intact or ovariectomized old female rats. In healthy rats, these ageReceived July 31,1989. Address requests for reprints to: Dr. M. R. Blackman, Endocrinology Section, Gerontology Research Center, Room 2B-20, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224. * This work was supported in part by the MacArthur Foundation Research Program on Successful Aging.
Materials and Methods Experimental animals All experiments were conducted in healthy intact or ovariectomized mature (6-7 months old) and old (23-25 months old) 773
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 November 2015. at 17:29 For personal use only. No other uses without permission. . All rights reserved.
EFFECTS OF AGING ON PRL AND LH0
774
female Wistar rats obtained from the Gerontology Research Center (GRC) Animal Colony. The GRC Animal Colony is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Animals were housed one or two per cage in a temperature-controlled environment with a 12-h day, 12-h night cycle and were fed Purina rat chow and tap water ad libitum. Intact mature rats (n = 16) were studied on the first or second day of diestrus, whereas the intact old rats (n = 12) were in a state of constant diestrus, as shown by vaginal smears obtained biweekly for 2 weeks before experiments (28). Ovariectomized mature (n = 18) and old (n = 11) rats were studied 4 weeks after bilateral gonadectomy. Because aging in the female rat is known to be associated with an increased occurrence of PRL-secreting pituitary tumors (17, 18), rats with visible pituitary tumors at the time of death or exhibiting serum PRL levels in excess of 200 ng/ml were excluded from study. Approximately 25-30% of old rats were thus excluded. To ascertain whether rats with microprolactinomas might be missed by the above screening procedure, we performed a series of preliminary experiments wherein anterior pituitary glands from individual intact old female rats with normal serum PRL levels were quartered, and the PRL mRNA levels compared among the quarters. No significant differences were detected in PRL mRNA levels in pituitary quarters obtained from individual rats (data not shown). Pituitary cell cultures Paired groups (n = 4-10 rats/group) of intact or ovariectomized mature and old rats were rapidly decapitated between 0900-1000 h. Trunk blood was obtained, and serum separated by centrifugation and stored at —80 C until assayed. Anterior pituitary glands were removed and divided into quarters. Three quarters of each gland were used for preparation of primary monolayer adenohypophyseal cultures. For the intact rats, individual pituitary cell cultures were obtained by a mechanical and enzymatic digestion procedure (10, 11). Cells were then incubated overnight in Dulbecco's Modified Eagle's Medium supplemented with dextran-coated charcoal (DCC) and subsequently maintained in duplicate or triplicate cultures for 96 h in either the absence or presence of DCC-treated rat/fetal calf serum, as previously described (10,11). For the ovariectomized rats, pituitary cells were pooled from each age group, digested and preincubated overnight as above, and maintained in quadruplicate or sextuplicate in serum-containing Dulbecco's Modified Eagle's Medium for 96 h. RNA slot blots The remaining quarter of each pituitary gland was immediately extracted for total cytoplasmic RNA by a modification of a previously described method (24, 25). Pooled aqueous layers from pituitary homogenate extracts were reextracted with phenol-chloroform-isoamyl alcohol. Ethanol precipitation was performed for 16 h at -20 C. After centrifugation at 13,000 X g for 30 min, total RNA was resuspended in 200 n\ sterile water and immediately denatured by adding 600 jul 10 X SSC (1 x SSC = 0.15 M NaCl/0.015 M Na citrate) and 6.5 M formaldehyde and heating at 65 C for 15 min; samples were stored at -80 C.
Endo • 1990 Vol 126 • No 2
Steady state levels of PRL and LH/? mRNA were quantified by slot blot analyses (24-27) of in vivo material (pituitary membrane homogenates) and in vitro material (pituitary cell lysates from primary monolayer adenohypophyseal cell cultures). Each denatured sample was applied in triplicate to GeneScreen Plus (DuPont, Wilmington, DE) filter paper using a Scleicher and Schuell Minifold II Apparatus (Keene, NH), allowed to sit on the filter for 30 min, passed through under vacuum, washed with 10 X SSC, and air dried. The formaldehyde reaction was reversed by baking at 80 C under vacuum for 2 h. Filters were prehybridized for more than 30 min at 42 C in roller bottles. Prehybridization buffer contained 50% formaldehyde, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), and 10% dextran sulfate. For hybridization, randomly primed cDNA probe plus 200 Mg/ml heat-denatured herring sperm DNA were added to the prehybridization buffer (final concentration,
