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Pregnant Mare Serum and Human Chorionic Gonadotropin Stimulate Ovarian ∆5-3β-Hydroxysteroid Dehydrogenase in Aged Mice
Vol. 28, No.7, July 1977 Printed in U.s.A.
FERTILITY AND STERILITY Copyright' 1977 The American Fertility Society
PREGNANT MARE SERUM AND HUMAN CHORIONIC GONADOTROPIN STIMULATE OVARIAN il 5 -3,B-HYDROXYSTEROID DEHYDROGENASE IN AGED MICE
EUGENE D. ALBRECHT, PH.D.* ROBERT D. KOOS, M.S.t SHELDON F. GOTTLIEB, PH.D. Biological Sciences Department, Purdue University, Fort Wayne, Indiana 46805
Aged (12- to 14-month-old) estrous and diestrous C57BL mice exhibited lower histochemically demonstrable ovarian tJ.5-3f3-hydroxysteroid dehydrogenase (3f3-HSD) activity in thecal, luteal, and interstitial cells, and lower (P < 0.01) ovarian 3f3-HSD concentration and total content than did young (3-month-old) estrous animals. Administration of pregnant mare serum (PMS, 10 lU subcutaneously), followed in 40 hours by human chorionic gonadotropin (HCG, 5 lU subcutaneously) or HCG (2 lU daily for 4 days) alone, restored luteal and interstitiaI3f3-HSD in aged mice. Follicular, luteal, and interstitiaI3f3-HSD activity was increased in aged mice by a single PMS injection (10 lU). The total ovarian dehydrogenase concentration was increased 100% in aged animals by PMS and/or HCG administration. Restoration of histochemically demonstrable ovarian 3f3-HSD and total enzyme content in aged mice by PMS and/or HCG indicates ovarian sensitivity to gonadotropin and subnormal tropic hormone stimulation of the ovary in situ.
MATERIALS AND METHODS
A decline in mammalian reproductive function occurs with advancing age and may be related in part to impaired hypothalamic-hypophyseal regulation of gonadotropin secretion. 1-3 Recently we reported that aging in nonpregnant C57BL mice in estrus was associated with a decline in activity of ovarian il5-3,B-hydroxysteroid dehydrogenase (3,B-RSD),4.5 an enzyme necessary for il 4 -3-ketosteroid synthesis. Since ovarian 3,B-RSD activity is maintained by gonadotropin6 • 7 the present study was designed to determine whether aged mice would respond to pregnant mare serum (PMS) and/or human chorionic gonadotropin (RCG) with an increase in ovarian 3,B-RSD activity.
Female C57BLl6J mice (Jackson strain) reared in this laboratory were weaned at 21 days of age and housed five per cage. Animals were maintained in air-conditioned quarters (22" C) with a controlled lighting schedule (lights on at 6 A.M., off at 6 P.M.) and had access to laboratory chow (Ralston Purina Co., St. Louis, Mo.) and water ad libitum. Vaginal smears of 3-month-old (young) and 12- to 14-month-old (aged) females were examined daily, and animals in estrus and diestrus were studied. Additional young and aged mice were treated without regard to the stage of the estrous cycle with (1) PMS-RCG, a single subcutaneous injection of 10 IU of PMS (gonadotropin; Sigma Chemical Co., St. Louis, Mo.) between 8 A.M. and 10 A.M. followed 40 hours later by a single subcutaneous injection of 5 IU of RCG (chorionic gonadotropin; Sigma Chemical Co.), both in 0.1 ml of saline; animals were killed 24 hours after RCG injection; (2) PMS, a single subcutaneous injection of10 IU ofPMS in 0.1 mlof
Received January 19, 1977: accepted February 17, 1977. * Present address and address for reprint requests: Pregnancy Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014. tPresent address: Department of Animal Sciences, Cornell University, Ithaca, N. Y. 14850.
762
Vol. 28, No.7
763
PMS, HCG, AND OVARIAN 3f:l-HSD IN AGED MICE
saline between 8 A.M. and 10 A.M.; animals were killed 40 hours later; (3) HCG, four daily subcutaneous injections of 2 IU of HCG in 0.1 ml of saline between 8 A.M. and 10 A.M.; animals were killed 24 hours after the last injection. All animals were killed by cervical dislocation; ovaries were , immediately removed, dissected free of adhering tissue, weighed, and stored frozen on solid CO 2 • Ovarian 3p-HSD was assayed histochemically by the method of Levy et aLB Both ovaries were sectioned at 12 IJ-m in a cryostat; dehydroepiandrosterone (Sigma Chemical Co.) was used as the substrate, and alternate sections were incubated for 1 hour at 3'r C. Activity of3p-HSD was rated on an arbitrary scale of 0 to 5+. Alternate ovarian sections were stained with hematoxylin and eosin for histologic examination. Ovaries were pooled (two or three pairs per determination), and 3p-HSD was determined biochemicallY'· 9 within 24 hours by using dehydroepiandrosterone as substrate. Activity is expressed as micrograms of androstenedione formed per minute per milligram of tissue, per minute per milligram of protein,IO or per minute per pair of ovaries. RESULTS
3P-HSD Histochemistry. Estrous cycles were exhibited by mice of both ages, although prolonged (but not constant) estrus and diestrus commonly occurred in older animals. The concentration of histochemically demonstrable ovarian 3p-HSD was lower in the theca interna of vesicular follicles
and in corpora lutea of aged estrous and diestrous mice than in young mice (Table 1). Orange-yellow pigmented material comprised much of the interstitial tissue of aged animals, particularly those in diestrus; therefore, 3P-HSD activity over-all in this tissue in aged diestrous mice was noticeably lower than that in young animals. Ovaries of young and aged animals given PMSHCG contained numerous corpora lutea with similar dehydrogenase activity of moderate intensity (Table 1). Vesicular follicles were rare, but, in those observed, 3p-HSD activity was low in the granulosa and absent in the theca interna at both ages. PMS-HCG enhanced the amount of enzymatically active interstitial tissue of aged animals, largely by reducing the proportion of pigmented material. Numerous, large, well-developed, preovulatory follicles with moderately intense granulosa 3PHSD activity were observed in animals of both age~ given PMS alone. PMS increased dehydrogenase activity in corpora lutea and interstitial tissue of aged animals to a level similar to that in young mice. Young and aged females given HCG alone exhibited numerous corpora lutea and interstitial cells with similar 3P-HSD activity of moderate intensity. 3p-HSD Biochemistry. The concentration, specific activity, and total content of ovarian 3P-HSD in aged mice in estrus or diestrus were lower(P
TABLE 1. Effect of Aging and Pregnant Mare Serum and/or Human Chorionic Gonadotropin on Ovarian tJ.5-3f3-Hydroxysteroid Dehydrogenase Histochemistry in C57BL Mice a Vesicular follicles' Stage of estrous cycle
Treatment
No. of mice
Age Theca interna
Granulosa
Corpora lutea'
Interstitial tissue'
rno
Estrus
8 8
3-4 12-14
++ +
+ 0
+++ +
++ +
Diestrus
8 9
3-4 12-14
++ +
+ +
++ +
+++ +
PMS-HCGc
8 9
3-4 12-14
0 0
+ +
++ +++
++ ++
PMSd
8 8
3-4 12-14
++ +
++ ++
+++ +++
++ ++
HCGP
8 8
3-4 12-14
++ +
+ ++
++ +++
++ ++
aTissues were incubated at 37° C for 1 hour with dehydroepiandrosterone as substrate. bHistochemical activity is presented as relative (0 to 5+ ) intensity of formazan precipitation. C Animals received a single subcutaneous injection of 10 IU of PMS followed 40 hours later by a single subcutaneous injection of 5 IU of HCG and were killed 24 hours later. d Animals received a single subcutaneous injection of 10 IU of PMS and were killed 40 hours later. e Animals received four daily subcutaneous injections of 2 IU of HCG and were killed 24 hours later.
764
Stage of estrous cycle
ALBRECHT ET AL.
July 1977
TABLE 2. Effect of Aging and Pregnant Mare Serum and/or Human Chorionic Gonadotropin on Ovarian !J.5-3f3-Hydroxysteroid Dehydrogenase Activity in C57BL Mice" p.g a4 -Androstenedione/minl Treatment
No. of analysesb
Estrus
Age
Ovarian wt
mo
mg
Mg
Mg protein
Organ pair
10 11
3-4 12-14
6.23 ± 0.16 6.18 ± 0.17
0.159 ± 0.013 0.102 ± 0.004 e
1.74 ± 0.14 1.08 ± 0.05 e
1.01 ± 0.11 0.60 ± 0.05 e
7 7
3-4 12-14
4.92 ± 0.22e 4.65 ± 0.18 e
0.119 ± 0.009 0.100 ± 0.013 e
1.19 ± 0.08 e 0.94 ± 0.10e
0.60 ± 0.06 e 0.41 ± 0.05 e
PMS-HCGd
9 7
3-4 12-14
12.27 ± 0.58 e 11.03 ± 0.83 e
0.118 ± 0.010 0.106 ± 0.00g e
1.15 ± O.l1e 1.02 ± 0.0g e
1.51 ± 0.14" 1.22 ± 0.12
PMSd
7 7
3-4 12-14
9.15 ± 0.42e 11.40 ± 0.68 e
0.113 ± 0.006 0.103 ± 0.007 c
1.12 ± 0.08 e 1.04 ± 0.06 e
1.00 ± 0.08 1.38 ± 0.15
HCGd
8 7
3-4 12-14
9.88 ± 0.37 e 11.00 ± 0.63 e
0.128 ± 0.010 0.096 ± 0.010 e
1.35 ± 0.11 0.97 ± O.l1e
1.32 ± 0.13 1.12 ± 0.09
Diestrus
"Values are means ± standard error of the mean. bOvaries of one to four mice were pooled for a single analysis. eSignificantly different (P < 0.01) from untreated 3- to 4-month-old estrous animals. dSe e footnotes to Table 1 for description of treatment.
genase specific activity and total content
DISCUSSION
Ovaries of estrous and diestrous mice 12 to 14 months of age possessed corpora lutea and follicles in various stages of maturation, both with subnormal levels of 3,B-HSD activity. Aged rats exhibit an alteration in ovarian 3,B-HSD substrate specificity,12 also suggesting changes in ovarian function with advancing age. The decline in histo-
chemically demonstrable 3,B-HSD in aged mice was correlated with significant decreases in ovarian dehydrogenase concentration, specific activity, and total content, which agrees with our previous findings 4, 5 and with similar observations in aged rats. 13 The decline in ovarian 3,B-HSD activity in aged mice indicates a reduced capacity of older animals to produce ovarian ,:l4-3-ketosteroids. Follicular development is impaired and corpora lutea show hyalinization and gelatinization only after 15 months of age in C57BL mice 14; therefore, the decrease in ovarian 3,B-HSD activity in aging estrous and diestrous mice precedes degenerative changes in ovarian morphology. Superovulation was induced and histochemically demonstrable 3,B-HSD activity was restored in aged mice by PMS-HCG, as evidenced by numerous corpora lutea and interstitial tissue, both with dehydrogenase activity of moderate intensity. Administration of PMS alone to aged mice enhanced follicular maturation and increased follicular, luteal, and interstitial 3,B-HSD activity, while HCG alone stimulated luteal and interstitial dehydrogenase activity in aged mice. Ovarian weight was comparably elevated in young and aged mice by gonadotropin treatment, and total ovarian 3,B-HSD content was elevated 100% in aged animals by the administration of PMS and/or HCG. Stimulation of ovarian 3,B-HSD activity in aged mice by PMS and HCG presumably reflects follicle-stimulating hormone and luteinizing hormone activities (which these hormones possess), since histochemically demonstrable ovarian 3,B-HSD activity was also restored in aged mice by a regimen of follicle-stimulating
Vol. 28, No.7
PMB, HCG, AND OVARIAN 3/3-HSD IN AGED MICE
hormone, luteinizing hormone, and prolactin. IS Ovarian 3,8-HSD activity is maintained by gonadotropin, since enzyme activity in mature rats is depleted or substantially reduced by hypophysectomy6.7 and restored by HCG.6 The restoration of histochemically demonstrable ovarian 3,8-HSD and the increase in ovarian weight and total 3,8-HSD content in aged mice by gonadotropin indicate responsiveness to gonadotropin and suggest that subnormal ovarian 3,8-HSD activity in mice 12 to 14 months old is related to inadequate gonadotropic stimulation of the ovary. Studies in aged rats have indicated impaired hypothalamichypophyseal regulation of gonadotropin secretion. l -3 Although histochemically demonstrable 3,8HSD activity and total dehydrogenase content were restored in aged mice by the administration of gonadotropin, concentration and specific activity remained subnormal. However, gonadotropin adminis.tration to young mice (Table 2) or rats 16 also did not elevate, but significantly reduced, 3,8-HSD concentration and specific activity; histochemically demonstrable ovarian 3,8-HSD was largely unaltered. The reduction in enzyme concentration in young mice and the maintenance of subnormal concentrations in aged mice that received exogenous gonadotropin may re1lect tissue dilution by antral1luid of vesicular follicles formed by this treatment. Acknowledgments. The authors appreciate the assistance of Dr. Gerald J. Pepe in reviewing the paper and Miss Jana Crowl in typing the manuscript.
765
REFERENCES 1. Ingram DL: The vaginal smear of senile laboratory rats.
J Endocrinol 19:182, 1959 2. Clemens J A, Amenomori Y, Jenkins T, Meites J: Effects of hypothalamic stimulation, hormones, and drugs on ovarian function in old female rats. Proc Soc Exp BioI Med 132:561, 1969 3. Watkins BE, Meites J, Riegle GD: Age-related changes in pituitary responsiveness to LHRH in the female rat. Endocrinology 97:543,1975 4. Albrecht ED, Koos RD, Wehrenberg WB: Ovarian t,5-3f3hydroxysteroid dehydrogenase and cholesterol in the aged mouse during pregnancy. BioI Reprod 13:158, 1975 5. Wehrenberg WB, Gottlieb SF, Albrecht ED: Ageing and ovarian t,5-3f3-hydroxysteroid dehydrogenase in the pregnant mouse. J Endocrinol 70:183, 1976 6. Taylor FB: Histochemical changes in the ovaries of normal and experimentally treated rats. Acta Endocrinol (Kbh) 36:361, 1961 7. Turolla E, Gaetani M, Baldratti G, Aguggini G: Histochemistry of ovarian 20a-hydroxysteroid dehydrogenase in mature hypophysectomized rats. Experienta 24:345, 1968 8. Levy H, Deane HW, Rubin BL: Visualization of steroid3f3-ol-dehydrogenase activity in tissues of intact and hypophysectomized rats. Endocrinology 65:932,1959 9. Rubin BL, Deane HW, Hamilton JA, Driks EC: Changes in t,5-3f3-hydroxysteroid dehydrogenase activity in the ovaries of maturing rats. Endocrinology 72:924, 1963 10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chern 193:265, 1951 11. Hunter JS, Hunter F: Ovarian-adrenal-liver interactions in the estrous cycle in the rat. Endokrinologie 58:355, 1971 12. Leathem JH, Murono EP: Ovarian t,5-3f3-hydroxysteroid dehydrogenase in aging rats. Fertil Steril 26:996, 1975 13. Leathem JH, Shapiro BH: Aging and ovarian t,'-3f3hydroxysteroid dehydrogenase in rats. Proc Soc Exp BioI Med 148:793, 1975 14. Loeb L: Aging processes in the ovaries of mice belonging to strains differing in the incidence of mammary carcinoma. Arch Pathol 46:401, 1948 15. Albrecht ED, Koos RD, Gottlieb SF: Unpublished data 16. Rubin BL, Deane HW: Effects of superovulation on ovarian t,5-3f3-hydroxysteroid dehydrogenase activity in rats of different ages. Endocrinology 76:382, 1965
FERTILITY AND STERILITY Copyright' 1977 The American Fertility Society
PREGNANT MARE SERUM AND HUMAN CHORIONIC GONADOTROPIN STIMULATE OVARIAN il 5 -3,B-HYDROXYSTEROID DEHYDROGENASE IN AGED MICE
EUGENE D. ALBRECHT, PH.D.* ROBERT D. KOOS, M.S.t SHELDON F. GOTTLIEB, PH.D. Biological Sciences Department, Purdue University, Fort Wayne, Indiana 46805
Aged (12- to 14-month-old) estrous and diestrous C57BL mice exhibited lower histochemically demonstrable ovarian tJ.5-3f3-hydroxysteroid dehydrogenase (3f3-HSD) activity in thecal, luteal, and interstitial cells, and lower (P < 0.01) ovarian 3f3-HSD concentration and total content than did young (3-month-old) estrous animals. Administration of pregnant mare serum (PMS, 10 lU subcutaneously), followed in 40 hours by human chorionic gonadotropin (HCG, 5 lU subcutaneously) or HCG (2 lU daily for 4 days) alone, restored luteal and interstitiaI3f3-HSD in aged mice. Follicular, luteal, and interstitiaI3f3-HSD activity was increased in aged mice by a single PMS injection (10 lU). The total ovarian dehydrogenase concentration was increased 100% in aged animals by PMS and/or HCG administration. Restoration of histochemically demonstrable ovarian 3f3-HSD and total enzyme content in aged mice by PMS and/or HCG indicates ovarian sensitivity to gonadotropin and subnormal tropic hormone stimulation of the ovary in situ.
MATERIALS AND METHODS
A decline in mammalian reproductive function occurs with advancing age and may be related in part to impaired hypothalamic-hypophyseal regulation of gonadotropin secretion. 1-3 Recently we reported that aging in nonpregnant C57BL mice in estrus was associated with a decline in activity of ovarian il5-3,B-hydroxysteroid dehydrogenase (3,B-RSD),4.5 an enzyme necessary for il 4 -3-ketosteroid synthesis. Since ovarian 3,B-RSD activity is maintained by gonadotropin6 • 7 the present study was designed to determine whether aged mice would respond to pregnant mare serum (PMS) and/or human chorionic gonadotropin (RCG) with an increase in ovarian 3,B-RSD activity.
Female C57BLl6J mice (Jackson strain) reared in this laboratory were weaned at 21 days of age and housed five per cage. Animals were maintained in air-conditioned quarters (22" C) with a controlled lighting schedule (lights on at 6 A.M., off at 6 P.M.) and had access to laboratory chow (Ralston Purina Co., St. Louis, Mo.) and water ad libitum. Vaginal smears of 3-month-old (young) and 12- to 14-month-old (aged) females were examined daily, and animals in estrus and diestrus were studied. Additional young and aged mice were treated without regard to the stage of the estrous cycle with (1) PMS-RCG, a single subcutaneous injection of 10 IU of PMS (gonadotropin; Sigma Chemical Co., St. Louis, Mo.) between 8 A.M. and 10 A.M. followed 40 hours later by a single subcutaneous injection of 5 IU of RCG (chorionic gonadotropin; Sigma Chemical Co.), both in 0.1 ml of saline; animals were killed 24 hours after RCG injection; (2) PMS, a single subcutaneous injection of10 IU ofPMS in 0.1 mlof
Received January 19, 1977: accepted February 17, 1977. * Present address and address for reprint requests: Pregnancy Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014. tPresent address: Department of Animal Sciences, Cornell University, Ithaca, N. Y. 14850.
762
Vol. 28, No.7
763
PMS, HCG, AND OVARIAN 3f:l-HSD IN AGED MICE
saline between 8 A.M. and 10 A.M.; animals were killed 40 hours later; (3) HCG, four daily subcutaneous injections of 2 IU of HCG in 0.1 ml of saline between 8 A.M. and 10 A.M.; animals were killed 24 hours after the last injection. All animals were killed by cervical dislocation; ovaries were , immediately removed, dissected free of adhering tissue, weighed, and stored frozen on solid CO 2 • Ovarian 3p-HSD was assayed histochemically by the method of Levy et aLB Both ovaries were sectioned at 12 IJ-m in a cryostat; dehydroepiandrosterone (Sigma Chemical Co.) was used as the substrate, and alternate sections were incubated for 1 hour at 3'r C. Activity of3p-HSD was rated on an arbitrary scale of 0 to 5+. Alternate ovarian sections were stained with hematoxylin and eosin for histologic examination. Ovaries were pooled (two or three pairs per determination), and 3p-HSD was determined biochemicallY'· 9 within 24 hours by using dehydroepiandrosterone as substrate. Activity is expressed as micrograms of androstenedione formed per minute per milligram of tissue, per minute per milligram of protein,IO or per minute per pair of ovaries. RESULTS
3P-HSD Histochemistry. Estrous cycles were exhibited by mice of both ages, although prolonged (but not constant) estrus and diestrus commonly occurred in older animals. The concentration of histochemically demonstrable ovarian 3p-HSD was lower in the theca interna of vesicular follicles
and in corpora lutea of aged estrous and diestrous mice than in young mice (Table 1). Orange-yellow pigmented material comprised much of the interstitial tissue of aged animals, particularly those in diestrus; therefore, 3P-HSD activity over-all in this tissue in aged diestrous mice was noticeably lower than that in young animals. Ovaries of young and aged animals given PMSHCG contained numerous corpora lutea with similar dehydrogenase activity of moderate intensity (Table 1). Vesicular follicles were rare, but, in those observed, 3p-HSD activity was low in the granulosa and absent in the theca interna at both ages. PMS-HCG enhanced the amount of enzymatically active interstitial tissue of aged animals, largely by reducing the proportion of pigmented material. Numerous, large, well-developed, preovulatory follicles with moderately intense granulosa 3PHSD activity were observed in animals of both age~ given PMS alone. PMS increased dehydrogenase activity in corpora lutea and interstitial tissue of aged animals to a level similar to that in young mice. Young and aged females given HCG alone exhibited numerous corpora lutea and interstitial cells with similar 3P-HSD activity of moderate intensity. 3p-HSD Biochemistry. The concentration, specific activity, and total content of ovarian 3P-HSD in aged mice in estrus or diestrus were lower(P
TABLE 1. Effect of Aging and Pregnant Mare Serum and/or Human Chorionic Gonadotropin on Ovarian tJ.5-3f3-Hydroxysteroid Dehydrogenase Histochemistry in C57BL Mice a Vesicular follicles' Stage of estrous cycle
Treatment
No. of mice
Age Theca interna
Granulosa
Corpora lutea'
Interstitial tissue'
rno
Estrus
8 8
3-4 12-14
++ +
+ 0
+++ +
++ +
Diestrus
8 9
3-4 12-14
++ +
+ +
++ +
+++ +
PMS-HCGc
8 9
3-4 12-14
0 0
+ +
++ +++
++ ++
PMSd
8 8
3-4 12-14
++ +
++ ++
+++ +++
++ ++
HCGP
8 8
3-4 12-14
++ +
+ ++
++ +++
++ ++
aTissues were incubated at 37° C for 1 hour with dehydroepiandrosterone as substrate. bHistochemical activity is presented as relative (0 to 5+ ) intensity of formazan precipitation. C Animals received a single subcutaneous injection of 10 IU of PMS followed 40 hours later by a single subcutaneous injection of 5 IU of HCG and were killed 24 hours later. d Animals received a single subcutaneous injection of 10 IU of PMS and were killed 40 hours later. e Animals received four daily subcutaneous injections of 2 IU of HCG and were killed 24 hours later.
764
Stage of estrous cycle
ALBRECHT ET AL.
July 1977
TABLE 2. Effect of Aging and Pregnant Mare Serum and/or Human Chorionic Gonadotropin on Ovarian !J.5-3f3-Hydroxysteroid Dehydrogenase Activity in C57BL Mice" p.g a4 -Androstenedione/minl Treatment
No. of analysesb
Estrus
Age
Ovarian wt
mo
mg
Mg
Mg protein
Organ pair
10 11
3-4 12-14
6.23 ± 0.16 6.18 ± 0.17
0.159 ± 0.013 0.102 ± 0.004 e
1.74 ± 0.14 1.08 ± 0.05 e
1.01 ± 0.11 0.60 ± 0.05 e
7 7
3-4 12-14
4.92 ± 0.22e 4.65 ± 0.18 e
0.119 ± 0.009 0.100 ± 0.013 e
1.19 ± 0.08 e 0.94 ± 0.10e
0.60 ± 0.06 e 0.41 ± 0.05 e
PMS-HCGd
9 7
3-4 12-14
12.27 ± 0.58 e 11.03 ± 0.83 e
0.118 ± 0.010 0.106 ± 0.00g e
1.15 ± O.l1e 1.02 ± 0.0g e
1.51 ± 0.14" 1.22 ± 0.12
PMSd
7 7
3-4 12-14
9.15 ± 0.42e 11.40 ± 0.68 e
0.113 ± 0.006 0.103 ± 0.007 c
1.12 ± 0.08 e 1.04 ± 0.06 e
1.00 ± 0.08 1.38 ± 0.15
HCGd
8 7
3-4 12-14
9.88 ± 0.37 e 11.00 ± 0.63 e
0.128 ± 0.010 0.096 ± 0.010 e
1.35 ± 0.11 0.97 ± O.l1e
1.32 ± 0.13 1.12 ± 0.09
Diestrus
"Values are means ± standard error of the mean. bOvaries of one to four mice were pooled for a single analysis. eSignificantly different (P < 0.01) from untreated 3- to 4-month-old estrous animals. dSe e footnotes to Table 1 for description of treatment.
genase specific activity and total content
DISCUSSION
Ovaries of estrous and diestrous mice 12 to 14 months of age possessed corpora lutea and follicles in various stages of maturation, both with subnormal levels of 3,B-HSD activity. Aged rats exhibit an alteration in ovarian 3,B-HSD substrate specificity,12 also suggesting changes in ovarian function with advancing age. The decline in histo-
chemically demonstrable 3,B-HSD in aged mice was correlated with significant decreases in ovarian dehydrogenase concentration, specific activity, and total content, which agrees with our previous findings 4, 5 and with similar observations in aged rats. 13 The decline in ovarian 3,B-HSD activity in aged mice indicates a reduced capacity of older animals to produce ovarian ,:l4-3-ketosteroids. Follicular development is impaired and corpora lutea show hyalinization and gelatinization only after 15 months of age in C57BL mice 14; therefore, the decrease in ovarian 3,B-HSD activity in aging estrous and diestrous mice precedes degenerative changes in ovarian morphology. Superovulation was induced and histochemically demonstrable 3,B-HSD activity was restored in aged mice by PMS-HCG, as evidenced by numerous corpora lutea and interstitial tissue, both with dehydrogenase activity of moderate intensity. Administration of PMS alone to aged mice enhanced follicular maturation and increased follicular, luteal, and interstitial 3,B-HSD activity, while HCG alone stimulated luteal and interstitial dehydrogenase activity in aged mice. Ovarian weight was comparably elevated in young and aged mice by gonadotropin treatment, and total ovarian 3,B-HSD content was elevated 100% in aged animals by the administration of PMS and/or HCG. Stimulation of ovarian 3,B-HSD activity in aged mice by PMS and HCG presumably reflects follicle-stimulating hormone and luteinizing hormone activities (which these hormones possess), since histochemically demonstrable ovarian 3,B-HSD activity was also restored in aged mice by a regimen of follicle-stimulating
Vol. 28, No.7
PMB, HCG, AND OVARIAN 3/3-HSD IN AGED MICE
hormone, luteinizing hormone, and prolactin. IS Ovarian 3,8-HSD activity is maintained by gonadotropin, since enzyme activity in mature rats is depleted or substantially reduced by hypophysectomy6.7 and restored by HCG.6 The restoration of histochemically demonstrable ovarian 3,8-HSD and the increase in ovarian weight and total 3,8-HSD content in aged mice by gonadotropin indicate responsiveness to gonadotropin and suggest that subnormal ovarian 3,8-HSD activity in mice 12 to 14 months old is related to inadequate gonadotropic stimulation of the ovary. Studies in aged rats have indicated impaired hypothalamichypophyseal regulation of gonadotropin secretion. l -3 Although histochemically demonstrable 3,8HSD activity and total dehydrogenase content were restored in aged mice by the administration of gonadotropin, concentration and specific activity remained subnormal. However, gonadotropin adminis.tration to young mice (Table 2) or rats 16 also did not elevate, but significantly reduced, 3,8-HSD concentration and specific activity; histochemically demonstrable ovarian 3,8-HSD was largely unaltered. The reduction in enzyme concentration in young mice and the maintenance of subnormal concentrations in aged mice that received exogenous gonadotropin may re1lect tissue dilution by antral1luid of vesicular follicles formed by this treatment. Acknowledgments. The authors appreciate the assistance of Dr. Gerald J. Pepe in reviewing the paper and Miss Jana Crowl in typing the manuscript.
765
REFERENCES 1. Ingram DL: The vaginal smear of senile laboratory rats.
J Endocrinol 19:182, 1959 2. Clemens J A, Amenomori Y, Jenkins T, Meites J: Effects of hypothalamic stimulation, hormones, and drugs on ovarian function in old female rats. Proc Soc Exp BioI Med 132:561, 1969 3. Watkins BE, Meites J, Riegle GD: Age-related changes in pituitary responsiveness to LHRH in the female rat. Endocrinology 97:543,1975 4. Albrecht ED, Koos RD, Wehrenberg WB: Ovarian t,5-3f3hydroxysteroid dehydrogenase and cholesterol in the aged mouse during pregnancy. BioI Reprod 13:158, 1975 5. Wehrenberg WB, Gottlieb SF, Albrecht ED: Ageing and ovarian t,5-3f3-hydroxysteroid dehydrogenase in the pregnant mouse. J Endocrinol 70:183, 1976 6. Taylor FB: Histochemical changes in the ovaries of normal and experimentally treated rats. Acta Endocrinol (Kbh) 36:361, 1961 7. Turolla E, Gaetani M, Baldratti G, Aguggini G: Histochemistry of ovarian 20a-hydroxysteroid dehydrogenase in mature hypophysectomized rats. Experienta 24:345, 1968 8. Levy H, Deane HW, Rubin BL: Visualization of steroid3f3-ol-dehydrogenase activity in tissues of intact and hypophysectomized rats. Endocrinology 65:932,1959 9. Rubin BL, Deane HW, Hamilton JA, Driks EC: Changes in t,5-3f3-hydroxysteroid dehydrogenase activity in the ovaries of maturing rats. Endocrinology 72:924, 1963 10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chern 193:265, 1951 11. Hunter JS, Hunter F: Ovarian-adrenal-liver interactions in the estrous cycle in the rat. Endokrinologie 58:355, 1971 12. Leathem JH, Murono EP: Ovarian t,5-3f3-hydroxysteroid dehydrogenase in aging rats. Fertil Steril 26:996, 1975 13. Leathem JH, Shapiro BH: Aging and ovarian t,'-3f3hydroxysteroid dehydrogenase in rats. Proc Soc Exp BioI Med 148:793, 1975 14. Loeb L: Aging processes in the ovaries of mice belonging to strains differing in the incidence of mammary carcinoma. Arch Pathol 46:401, 1948 15. Albrecht ED, Koos RD, Gottlieb SF: Unpublished data 16. Rubin BL, Deane HW: Effects of superovulation on ovarian t,5-3f3-hydroxysteroid dehydrogenase activity in rats of different ages. Endocrinology 76:382, 1965