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
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
