Mechanisms of Ageing and Development, 56 (1990) 77--88
77
Elsevier Scientific Publishers Ireland Ltd.
G O N A D A L FUNCTION IN A G I N G RATS AND ITS R E L A T I O N TO PITUITARY AND MAMMARY P A T H O L O G Y
RODOLFO G. GOYA',*, J O H N K.H. LU b and J O S E P H MEITES a •Department of Physiology, Michigan State University, East Lansing, All 48824 and bDepartment of Obstetrics~Gynecology and Anatomy, University of California at Los Angeles School of Medicine, Los Angeles, CA 90024 (U.S.A.) (Received May 19th, 1990)
SUMMARY
In the female rat, aging is characterized by a high incidence of prolactin (Prl)secreting pituitary adenomas and mammary tumors. In contrast to this, old males show only a moderate to low incidence o f pituitary and mammary pathology. Since gonadal steroids and Prl are thought to be key factors in the genesis of the above neoplastic pathologies, it was of interest to compare the serum levels of progesterone (P), estradiol (E2), testosterone (T) and Prl with the incidence of pituitary and mammary tumors in aging male and female rats. Young (3--4-month; YF), old (25month; OF) and senescent (33--35-month; SF) female and young (3--4-month; YM) and old (24--26-month; OM) male Sprague--Dawley rats were killed by decapitation and their pituitaries weighed. Serum sex steroids and Prl were measured by RIA. The average life span o f females but not males was markedly extended by systematic removal of mammary tumors. Females showed a rising incidence o f mammary tumors after 14 months of age. In males, this pathology which began to appear at 16 months, had a much lower incidence than in females at all ages. Serum levels of E 2 were (.~_ S.E.M.)22.0 ± 1.6; 18.9 ± 0.8; 32.9 + 2.5; 37.3 ± 2.0 and 32.2 _+ 3.0 pg/ml for YM, OM, YF, OF and SF, respectively. Serum P was 1.4 ± 0.3; 1.6 ± 0.2; 10.4 ± 2.2; 9.7 ± 3.3 and 6.8 ± 0.8 ng/ml for YM, OM, YF, OF and SF, respectively. Serum T was 1578.9 ± 188.7; 807.6 ± 103.0; 197.5 ± ll.8; 223.7 _ 25.5 and 176.9 ± 20.7 pg/ml for YM, OM, YF, OF and SF, respectively. Finally, serum Prl was 14.9 ± 1.7; 21.9 ± 4.0; 15.9 ± 1.4; 52.4 ± 9.4 and 170.8 ± 31.1 ng/ml for YM, OM, YF, OF and SF, respectively. A strong correlation was found between serum Prl and anterior pituitary weight in OM, OF and SF, but not between serum Prl and sex steroid levels or sex steroid ratios. We conclude that, although the sex-related differences in mammary and pituitary tumor incidence during aging in *Present address and to whom all correspondence should be sent: Centro de Estudios End6crinos, Facultad de Medicina, UNLP, casilla de correo 455, (1900) La Plata, Argentina. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
78
rats can be partially accounted for by the different serum profiles of Prl and gonadal steroids in each sex, sex-associated differences in target tissue susceptibility should also be considered as an important determinant of the level of tumor incidence.
Key words: Aging; Gonadal steroids; Prolactin; Pituitary adenomas; M a m m a r y tumors
INTRODUCTION
In the female laboratory rat aging is associated with a marked increase in pituitary and m a m m a r y tumors [1--5]. The incidence of these pathologies begins to rise shortly after the first year of life, an age at which most females show irregular estrous cycles [6,7] frequently characterized by extended periods of vaginal cornification which are associated with sustained estrogen secretion and low levels of circulating progestagens [8,9]. It is believed that in rats and mice continuous exposure to moderately increased or medium levels of estrogens unopposed by progesterone (P) lead initially to increased prolactin (Prl) secretion and later to the development of Prl-secreting pituitary adenomas [10--12] as well as Pri-induced m a m m a r y tumors [13]. Male rats also develop pituitary and m a m m a r y tumors with age [14--16]. Although the incidence of these pathologies is generally lower in males than in females, comparable levels of pituitary tumor incidence in both sexes have been reported for some rat strains [14,17,18]. It should be noted however that even within the same strain, figures may vary considerably from study to study. Since little is known about the age changes in the secretory patterns of estrogens and progestagens in male rats, it has not been possible to establish whether the development of pituitary and m a m m a r y pathology in old males is, like in females, chronologically associated with alterations in the serum profiles of sex steroids. The aim of the present study was to compare the incidence of pituitary and m a m m a r y tumors with the serum levels of estradioi (E2), P, testosterone (T) and Prl in male and female rats of different ages and to assess the relationship between age changes in gonadal steroid secretion and the development of pituitary tumors in each sex. MATERIALS AND METHODS
Animals Female and male S p r a g u e - - D a w l e y rats were obtained from Harlan Industries (Indianapolis, IN). Four separate lots of animals, received over a period of 16 months, were used in the longitudinal studies described below. Lots F-I and F-2 consisted of 79 and 55 females, respectively, while lots M-I and M-2 consisted of 42 and 35 males, respectively. All of the above animals were purchased at 8 - - 1 0 months of age as retired breeders. The animals used as young controls in the hor-
79 mone measurement studies were 3w4-month-old virgin males and females. All animals were housed in a temperature-controlled room (22 ± 2°C) on a 14:10 h light/ dark cycle. Food (Teklad Rat Diet, Winfield, IA) and water were available ad libitum. Mammary tumor removal In lots F-2, M-1 and M-2 mammary tumor removal was systematically performed throughout the study. In lot F-I this intervention was started at the 22nd month of life. Mammary tumors were removed when they became clearly palpable (major diameter approximately 1 cm). Surgery was performed under light ether anesthesia. Usually, animals recovered completely within a few hours. Hormone assays Animals were killed b y decapitation between 1000 h and 1300 h, trunk blood was collected and the serum obtained stored at - 25 °C until ready. For hormone determinations by radioimmunoassay (RIA). Anterior pituitaries (AP) were rapidly dissected and weighed. Prl was assayed in duplicate with the reagents provided by the NIADDK. Iodination grade Prl was radiolabeled by a lactoperoxidase-glucose oxidase method. The iodinated hormone was purified on a 1.5 × 40 cm Sephacryl S200 column equilibrated with 0.01 M phosphate buffered saline at pH 7.6. Ig Gsorb (Protein A, Enzyme Center, Maiden, MA) was used to separate bound from free hormone. Rat Prl RP-3 was used as standard, the average intra-and inter-assay coefficients of variation (CV) being 8.5 and 11.9, respectively. Steroid hormone assays Serum concentrations of P, T and E 2 were measured by RIA's using previously described procedures [12]. Prior to the RIAs, serum was extracted with diethyl ether and P, T and E 2 fractions were separated from other steroids by Celite column chromatography [19]. Known amounts (in cpm) of 3H-P, 3H-T and 3H-E2 were added to the serum before the extraction to estimate the recovery percentage. The final concentration of each steroid in the serum was corrected with the calculated recovery percentage. Statistical and survival analyses All statistical analyses were performed with a HP-41 CX computer (HewlettPackard, Corvallis, OR) loaded with specific programs for one-way ANOVA [20], Duncan's multiple range test [21] and single linear regression [22]. In the correlation analyses, each coefficient of correlation (R) is given with the level o f significance (P) with which the null hypothesis H0) 0 = 0 is rejected; if P is greater than 0.05, R is considered to be not significantly different from zero, which is indicated by NS. Survival analysis o f each cohort was performed by means o f a computer program which uses the actuarial life-table method [23]. The actuarial interval taken was 1 month. Standard life-tables were constructed recording three population variables
80
for each interval: number of rats at risk at start of interval (Ox); number of rats censored (wx) and number o f animals dying (dx) during interval. The types of censoring performed included: (1) withdrawal o f animals for hormone measurements and morphological studies; (2) death during mammary tumor removal; (3) animals surviving at the end of the observation period. For each interval, the following parameters were calculated: qx, the probability of death for interval x; px, the probability o f surviving during interval x (mathematically, p x = 1 - qx); Px, the cumulative probability of surviving; S.E. (Px), the standard error of Px. Survival curves were constructed by plotting P x (°7o) vs. age (months). RESULTS
Incidence o f m a m m a r y and pituitary tumors during aging Figure 1 shows the cumulative mammary tumor incidence during aging in the four cohorts o f rats studied. Mammary tumor incidence began to rise after the 13th --14th month of life in females and after the 20th-21st month in males. At all ages the incidence of mammary pathology was higher in females than in males. Males never developed more than one mammary tumor per animal while it was not uncommon to observe senescent females with a history o f 2 and 3 spontaneous mammary
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77
Elsevier Scientific Publishers Ireland Ltd.
G O N A D A L FUNCTION IN A G I N G RATS AND ITS R E L A T I O N TO PITUITARY AND MAMMARY P A T H O L O G Y
RODOLFO G. GOYA',*, J O H N K.H. LU b and J O S E P H MEITES a •Department of Physiology, Michigan State University, East Lansing, All 48824 and bDepartment of Obstetrics~Gynecology and Anatomy, University of California at Los Angeles School of Medicine, Los Angeles, CA 90024 (U.S.A.) (Received May 19th, 1990)
SUMMARY
In the female rat, aging is characterized by a high incidence of prolactin (Prl)secreting pituitary adenomas and mammary tumors. In contrast to this, old males show only a moderate to low incidence o f pituitary and mammary pathology. Since gonadal steroids and Prl are thought to be key factors in the genesis of the above neoplastic pathologies, it was of interest to compare the serum levels of progesterone (P), estradiol (E2), testosterone (T) and Prl with the incidence of pituitary and mammary tumors in aging male and female rats. Young (3--4-month; YF), old (25month; OF) and senescent (33--35-month; SF) female and young (3--4-month; YM) and old (24--26-month; OM) male Sprague--Dawley rats were killed by decapitation and their pituitaries weighed. Serum sex steroids and Prl were measured by RIA. The average life span o f females but not males was markedly extended by systematic removal of mammary tumors. Females showed a rising incidence o f mammary tumors after 14 months of age. In males, this pathology which began to appear at 16 months, had a much lower incidence than in females at all ages. Serum levels of E 2 were (.~_ S.E.M.)22.0 ± 1.6; 18.9 ± 0.8; 32.9 + 2.5; 37.3 ± 2.0 and 32.2 _+ 3.0 pg/ml for YM, OM, YF, OF and SF, respectively. Serum P was 1.4 ± 0.3; 1.6 ± 0.2; 10.4 ± 2.2; 9.7 ± 3.3 and 6.8 ± 0.8 ng/ml for YM, OM, YF, OF and SF, respectively. Serum T was 1578.9 ± 188.7; 807.6 ± 103.0; 197.5 ± ll.8; 223.7 _ 25.5 and 176.9 ± 20.7 pg/ml for YM, OM, YF, OF and SF, respectively. Finally, serum Prl was 14.9 ± 1.7; 21.9 ± 4.0; 15.9 ± 1.4; 52.4 ± 9.4 and 170.8 ± 31.1 ng/ml for YM, OM, YF, OF and SF, respectively. A strong correlation was found between serum Prl and anterior pituitary weight in OM, OF and SF, but not between serum Prl and sex steroid levels or sex steroid ratios. We conclude that, although the sex-related differences in mammary and pituitary tumor incidence during aging in *Present address and to whom all correspondence should be sent: Centro de Estudios End6crinos, Facultad de Medicina, UNLP, casilla de correo 455, (1900) La Plata, Argentina. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
78
rats can be partially accounted for by the different serum profiles of Prl and gonadal steroids in each sex, sex-associated differences in target tissue susceptibility should also be considered as an important determinant of the level of tumor incidence.
Key words: Aging; Gonadal steroids; Prolactin; Pituitary adenomas; M a m m a r y tumors
INTRODUCTION
In the female laboratory rat aging is associated with a marked increase in pituitary and m a m m a r y tumors [1--5]. The incidence of these pathologies begins to rise shortly after the first year of life, an age at which most females show irregular estrous cycles [6,7] frequently characterized by extended periods of vaginal cornification which are associated with sustained estrogen secretion and low levels of circulating progestagens [8,9]. It is believed that in rats and mice continuous exposure to moderately increased or medium levels of estrogens unopposed by progesterone (P) lead initially to increased prolactin (Prl) secretion and later to the development of Prl-secreting pituitary adenomas [10--12] as well as Pri-induced m a m m a r y tumors [13]. Male rats also develop pituitary and m a m m a r y tumors with age [14--16]. Although the incidence of these pathologies is generally lower in males than in females, comparable levels of pituitary tumor incidence in both sexes have been reported for some rat strains [14,17,18]. It should be noted however that even within the same strain, figures may vary considerably from study to study. Since little is known about the age changes in the secretory patterns of estrogens and progestagens in male rats, it has not been possible to establish whether the development of pituitary and m a m m a r y pathology in old males is, like in females, chronologically associated with alterations in the serum profiles of sex steroids. The aim of the present study was to compare the incidence of pituitary and m a m m a r y tumors with the serum levels of estradioi (E2), P, testosterone (T) and Prl in male and female rats of different ages and to assess the relationship between age changes in gonadal steroid secretion and the development of pituitary tumors in each sex. MATERIALS AND METHODS
Animals Female and male S p r a g u e - - D a w l e y rats were obtained from Harlan Industries (Indianapolis, IN). Four separate lots of animals, received over a period of 16 months, were used in the longitudinal studies described below. Lots F-I and F-2 consisted of 79 and 55 females, respectively, while lots M-I and M-2 consisted of 42 and 35 males, respectively. All of the above animals were purchased at 8 - - 1 0 months of age as retired breeders. The animals used as young controls in the hor-
79 mone measurement studies were 3w4-month-old virgin males and females. All animals were housed in a temperature-controlled room (22 ± 2°C) on a 14:10 h light/ dark cycle. Food (Teklad Rat Diet, Winfield, IA) and water were available ad libitum. Mammary tumor removal In lots F-2, M-1 and M-2 mammary tumor removal was systematically performed throughout the study. In lot F-I this intervention was started at the 22nd month of life. Mammary tumors were removed when they became clearly palpable (major diameter approximately 1 cm). Surgery was performed under light ether anesthesia. Usually, animals recovered completely within a few hours. Hormone assays Animals were killed b y decapitation between 1000 h and 1300 h, trunk blood was collected and the serum obtained stored at - 25 °C until ready. For hormone determinations by radioimmunoassay (RIA). Anterior pituitaries (AP) were rapidly dissected and weighed. Prl was assayed in duplicate with the reagents provided by the NIADDK. Iodination grade Prl was radiolabeled by a lactoperoxidase-glucose oxidase method. The iodinated hormone was purified on a 1.5 × 40 cm Sephacryl S200 column equilibrated with 0.01 M phosphate buffered saline at pH 7.6. Ig Gsorb (Protein A, Enzyme Center, Maiden, MA) was used to separate bound from free hormone. Rat Prl RP-3 was used as standard, the average intra-and inter-assay coefficients of variation (CV) being 8.5 and 11.9, respectively. Steroid hormone assays Serum concentrations of P, T and E 2 were measured by RIA's using previously described procedures [12]. Prior to the RIAs, serum was extracted with diethyl ether and P, T and E 2 fractions were separated from other steroids by Celite column chromatography [19]. Known amounts (in cpm) of 3H-P, 3H-T and 3H-E2 were added to the serum before the extraction to estimate the recovery percentage. The final concentration of each steroid in the serum was corrected with the calculated recovery percentage. Statistical and survival analyses All statistical analyses were performed with a HP-41 CX computer (HewlettPackard, Corvallis, OR) loaded with specific programs for one-way ANOVA [20], Duncan's multiple range test [21] and single linear regression [22]. In the correlation analyses, each coefficient of correlation (R) is given with the level o f significance (P) with which the null hypothesis H0) 0 = 0 is rejected; if P is greater than 0.05, R is considered to be not significantly different from zero, which is indicated by NS. Survival analysis o f each cohort was performed by means o f a computer program which uses the actuarial life-table method [23]. The actuarial interval taken was 1 month. Standard life-tables were constructed recording three population variables
80
for each interval: number of rats at risk at start of interval (Ox); number of rats censored (wx) and number o f animals dying (dx) during interval. The types of censoring performed included: (1) withdrawal o f animals for hormone measurements and morphological studies; (2) death during mammary tumor removal; (3) animals surviving at the end of the observation period. For each interval, the following parameters were calculated: qx, the probability of death for interval x; px, the probability o f surviving during interval x (mathematically, p x = 1 - qx); Px, the cumulative probability of surviving; S.E. (Px), the standard error of Px. Survival curves were constructed by plotting P x (°7o) vs. age (months). RESULTS
Incidence o f m a m m a r y and pituitary tumors during aging Figure 1 shows the cumulative mammary tumor incidence during aging in the four cohorts o f rats studied. Mammary tumor incidence began to rise after the 13th --14th month of life in females and after the 20th-21st month in males. At all ages the incidence of mammary pathology was higher in females than in males. Males never developed more than one mammary tumor per animal while it was not uncommon to observe senescent females with a history o f 2 and 3 spontaneous mammary
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