Exp. Geront. Vol. 13, pp. 107-114. © Pergamon Press Ltd. 1978. Printed in Great Britain.

0531-5565178/0801-0107502.00/0

P H A R M A C O L O G I C A L I N D U C T I O N OF O V U L A T I O N IN OLD A N D N E O N A T A L L Y ANDROGENIZED RATS* JAMES R. LEHMAN,'~ DELORES A. MCARTHUR'~ a n d SHELTON E. HENDRICKS Department of Psychology, University of Nebraska at Omaha, Omaha, NB 68101, U.S.A. (Received 6 August 1977)

INTRODUCTION RECENT findings suggest that changes in the quantity or availability of brain monoamines may be a key factor in the aging of reproductive function in the female rat. The aging female rat exhibits an irregularity of the normal estrous cycle which is reflected in the disruption of the vaginal smear pattern. Typically, only the estrous smear appears and the condition is designated as persistent vaginal estrous (PVE). This state is highly correlated with sterility and is the first definitive sign of reproductive senescence in about 80--90 ~ of female rats. Quadri et al. (1973) showed that the administration of iproniazid phosphate (a monoamine oxidase inhibitor) or L-DOPA (a catecholamine precursor) could reinstate vaginal cyclicity in old rats exhibiting PVE. This effect of L-DOPA was replicated by Huang and Meites (1975), and Huang, et al. (1976). These latter studies also showed that L-DOPA could induce ovulation in the old PVE rat as indicated by fresh corpora lutea (CL) in the ovaries. Furthermore, Huang et al. (1976) reported that progesterone and, in some instances, stress could induce ovulation in reproductively senescent female rats. The infertility and PVE, characteristic of the aged female rat, are also seen in young female rats which have received androgen within a few days of birth (Barraclough and Gorski, 1961). While a large dose of androgen may prevent even the initial pubertal ovulation (Gorski, 1968), a small dose may result in ovulatory cycles occurring for a variable period before the anovulatory condition with PVE is established (Swanson and van der Werff ten Bosch, 1964). Swanson and van der Werff ten Bosch (1964) suggest that the neonatally administered androgen causes a premature aging of neural systems which control ovarian physiology. If such is the case, treatments that have been shown to reinstate ovulation and vaginal cyclicity in old female rats might have comparable effects in the young female sterilized by neonatally administered androgen. Treatments analogous to those reported to restore ovulation in senile rats have been applied to the neonatally androgenized female. Barraclough and Gorski (1962) and Barraclough et al. (1964) failed to induce ovulation by the administration of progesterone to female rats which had received 1.25 mg testosterone propionate (TP) neonatally. Tima and Flerko (1974) injected norepinephrine intraventricularly in female rats made sterile by 250 lag TP injected neonatally. They found that 55 ~ of the subjects exhibited fresh CL 48 h after the norepinephrine treatment. Tournigant et al. * Supported by Research Grant NS 10027 from the National Institute of Neurological and Communicative Disorders and Strokes. A preliminary report of this work was presented at the Eastern Regional Conference on Reproductive Behavior, Saratoga Springs, New York, 1976, and at the American Aging Association Conference in Washington, DC, September, 1976. Steroid compounds were provided by Dr. Irving Tabachnick of the Schering Corporation and L-DOPA was provided by Dr. W. E. Scott, Hoffman-LaRoche. 1"In partial fulfillment of the M.A. degree. 107

108

JAMES R. LEHMAN, DELORES A. MCARTHUR AND SHELTON E. HENDRICKS

(1974) f o u n d that intrahypothalmic implants o f norepinephrine, dopamine or serotoni n could interrupt the PVE pattern in rats injected neonatally with 50 lag TP. The present studies were designed to assess the capacity o f pharmacological manipulations to induce ovulation in y o u n g female rats sterilized by various doses o f neonatally administered TP. Further, these same manipulations, previously f o u n d to be effective in the old female, were re-evaluated to provide a direct c o m p a r i s o n with the y o u n g female. A similarity o f response would suggest the comparability o f the old female with the young, andorgenized animal and the usefulness o f the neonatally androgenized female as a model for the study o f aging in the reproductive system. METHODS AND PROCEDURES EXPERIMENT I Subjects were 142 female rats of the Harlan Wistar strain, born in our laboratory. Animals were maintained in a temperature controlled room with a reverse (0700 h off; 1700 h on) light--dark cycle. Subsequent to weaning at 25 days of age, animals were housed four to a cage with food and water available ad libitum. At five days of age (birth=Day 1), pups were injected with 50 lag TP, 10 lag TP, 5 lag TP or the corn oil vehicle in 0.05 ml volumes. Daily vaginal smears were taken from 45 to 58 days of age, 73 to 86 days, 101 to 114 days and 129 to 142 days. Unilateral laparotomies were performed under ether anesthesia at 90 and 130 days of age. The ovaries were lifted, examined for the presence of CL and then replaced in situ. Drug treatments were initiated at 150 days of age. Daily vaginal smears were taken during the 30 days of drug administration and for 30 days after treatments stopped. Each of four neontal treatment groups was subdivided further for administration of the three drug treatments. These treatments were: (1) twice daily i.p. injections of 25 mg L-DOPA (L-B-3,4 dihydroxyphenylalanine) dissolved in 2 ml 0.05 N HC1; (2) a daily i.p. injection of iproniazid phosphate (10 mg/100 g body weight) in 2 ml of isotonic saline; or (3) twice daily control injections of 0"05 N HC1. The resulting twelve treatment groups consisted of subjects injected neontally with oil and receiving control (N=I2), L-DOPA ( N = l l ) or iproniazid (N=I2) injections; subjects injected neontally with 5 pg TP and receiving control (N=ll), L-DOPA (N=13) or iproniazid (N=I0) injections; subjects injected neontally with 10 lag TP and receiving control (N=I2), L-DOPA (N = 9), iproniazid (N = 13); or subjects injected neonatally with 50 lag TP and receiving control (N = 11), LDOPA (N=10) or iproniazid (N=I2) injections. At 180 days of age injections were discontinued and one ovary was removed from each subject under light ether anesthesia. The remaining ovary was removed at 210 days of age and microscopically examined for the presence of CL. RESULTS EXPERIMENT

I

The percentage o f subjects in each neonatal treatment g r o u p exhibiting vaginal cyclicity as a function o f age is plotted in Fig. 1. It is clear f r o m Fig. 1 that the capacity o f these animals to maintain normal vaginal cyclicity was an inverse function o f the dose o f T P given neonatally and age. Fisher's Exact Probability Test showed that at 58 days o f age the three TPinjected groups had significantly fewer animals cycling than the oil group (p----0.000). Also, a significantly greater precentage o f the animals injected with 5 lag T P exhibited cyclicity than ithose injected with 10 lag T P ( p = 0 . 0 3 ) . G r o u p s receiving 10 lag T P also had significantly m o r e cycling animals than the groups injected with 50 lag T P neonatally (p =0"02). By 86 days o f age, the 10-lag T P g r o u p showed a marked drop in the percentage o f cycling animals. With this decline, the 10-lag T P group was no longer significantly different f r o m the 50-lag T P group. This condition did not change for the remainder o f the pre-drug period. By 142 days o f age, the 5-lag T P g r o u p did n o t differ significantly f r o m the 50-lag T P group. The percentages o f animals in each g r o u p exhibiting C L at four different ages are shown in Fig. 2. A n inverse relationship between the neonatal dose o f T P and the percentage o f animals exhibiting C L can be seen in the 90 day old animals. Fisher's Exact Probability Test revealed a significantly greater percentage o f the 5 lag T P animals exhibiting C L as c o m p a r e d to those receiving 10 lag T P ( p = 0 . 0 4 7 ) ; groups receiving 10 lag TP also revealed more C L than groups injected with 50 lag T P (p=0.003). By 130 days there were no significant differ-

P H A R M A C O L O G I C A L I N D U C T I O N OF O V U L A T I O N I N O L D A N D N E O N A T A L L Y A N D R O G E N I Z E D R A T S

I0 0 -

cm c: -6 u u,

80

0

.~-



109

\

In 6040I-- X ~

c,

0;I



5/zg

X lO~g 0 50,u.g

"6 g

~9 8

ID /f~8

86

114

142.

Age FIG. 1. Cumulative percentages of subjects in each of the neonatal treatment groups of Experiment I exhibiting vaginal cyclicity. Data are plotted as a function of age. ences a m o n g the groups receiving various dosages of T P neonatally. N o significant effect of either i p r o n i a z i d or L - D O P A o n the percentage o f a n i m a l s exhibiting CL appeared immediately after drug a d m i n i s t r a t i o n (180 days o f age). However, at 210 days of age (30 days after the t e r m i n a t i o n o f drug treatments), the a n i m a l s receiving 5 lag T P n e o n a t a l l y showed a significantly greater percentage of C L after L - D O P A t r e a t m e n t as c o m p a r e d to those which received the c o n t r o l substance ( p = 0 . 0 3 ) . A similar b u t non-signficant trend ( p = 0 . 1 7 ) was observed in the a n i m a l s which received 10 lag T P neonatally. A significant increase ( p = 0 - 0 2 ) in the percentage of 5 lag T P a n i m a l s exhibiting C L also was seen 30 days after iproniazid injections were terminated. While iproniazid injections did produce a n increase in the percentage of subjects exhibiting C L in the 10-lag T P animals, the effect only a p p r o a c h e d statistical significance (p=0"06). Neither L - D O P A n o r i p r o n i a z i d had any effect on the percentage of subjects exhibiting C L in the groups given 50 lag T P neonatally. METHODS AND PROCEDURES

E X P E R I M E N T 11

Two kinds of animals were used in this experiment. Animals referred to as the 'old' rats were 83 female retired-breeders of the Long-Evans strain. These animals had successfully delivered and weaned three litters and were approximately one year of age when received in the laboratory. About one quarter of these animals arrived pregnant and their 88 female offspring constituted the 'young' rats. Environmental conditions were the same as described in Experiment I. The litters containing the young females were reduced in size to eight by the elimination of excess male pups. At five days of age, half of the female pups received s.c. injections of 5 tag TP in 0.05 ml corn oil while the other half received 0.05 ml of the corn oil vehicle. The young rats were weaned at 25 days and housed three per wire mesh cage for the duration of the study. The old rats were housed similarly. Vaginal smears were taken every other day from the young rats beginning at 50 days of age and from the old rats at 14 months of age. When two consecutive estrous smears were observed, daily vaginal smears were taken from that animal. After showing 20 consecutive days of estrous smears, a subject was classified as exhibiting PVE. Upon the establishment of PVE in the old and the young androgenized animal, 21 days of drug treatment were begun. For each young rat which received androgen neonatally, an animal which received oil neonatally was designated as a yoked control. The yoked control animal received the same drug treatment concurrently with the androgenized animal. Drug treatments consisted of twice daily i.p. injections

110

J A M E S R . L E H M A N , D E L O R E S A, M C A R T H U R

A N D S H E L T O N E. H E N D R I C K S

0

0il



5 #g

X

I O/.z g

rl 5 0 / ~ g Control I00

o-

80

60 40 ZO

0

g

I

.

I

I__

r__

L- Dopa .c: I0O 80

60 40

~

2o

~

x

o

j

~ Zproniazid

n =00

~

'

O

80

60 40 20

° 0 90

130

210

~80 t.

I

Age

FIG. 2. Percentage of subjects in Experiment I exhibiting CL at four ages. Groups were injected with oil, 5, 10 or 50 lag TP at 5 days of age. The bracket below the abscissa indicates the period during which animals received daily injections of the control vehicle, L-DOPA or iproniazid phosphate. of 25 mg L-DOPA suspended in 0.5 ml carboxy-methyl-cellulose (CMC), a daily i.p. injection of iproniazid phosphate (10 mg/100 g body weight) dissolved in 0'5 ml CMC buffered with NaOH to a pH of 6.3, or a daily i.m. injection of 500 tag progesterone dissolved in 0"05 ml corn oil. Control injections were either a daily i.p. injection of 0'5 ml CMC or an i.m. injection of 0"05 ml corn oil. At the termination of drug treatments, one ovary was removed under ether anesthesia. The remaining ovary was removed 30 days later. The ovarian tissue was microscopically examined to determine if CL were present. RESULTS EXPERIMENT

II

Figure 3 shows the cumulative percentages o f the neonatally androgenized and the old rats exhibiting PVE as a function o f age. All o f the androgenized animals exhibited P V E by 260 days o f age. O f the original 83 old females, seven were not included as they died prior to exhibiting PVE. Seventeen subjects did not exhibit P V E by 600 days o f age and were not exposed to the drug treatments. While they are represented in Fig. 3, they are not included in subsequent data analyses. The percentage o f subjects in each group which, 30 days subse-

PHARMACOLOGICAL INDUCTION OF OVULATION IN OLD AND NF~NATALLY ANDROGENIZED RATS

111

quent to drug or control treatments, exhibited CL are depicted in Fig. 4. The final cell sizes for these treatment groups were 11 for all young female groups and 14 and old, control; 15 for old, iproniazid; 16 for old, L-DOPA; and 14 for old, progesterone. It is apparent in Fig. 4 that the drug treatments did affect the appearance of CL in the ovaries of old and neonatally androgenized rats. Application of the Fisher Exact Probability Test to these data revealed significant effects attributable to the experimental treatments. In old females, the percentage of subjects exhibiting CL was significantly increased by the administration of iproniazid (p = 0-005) or progesterone (p = 0.002). L-DOPA also resulted in an increase in the percentage of subjects exhibiting CL. However, the L-DOPA group was not significantly different from the old females receiving only a control treatment (p=0.76). In the young neonatally-androgenized group progesterone significantly increased the percentage of subjects exhibiting CL as compared to the control treatment (p=0.045). While none of the control, androgenized females exhibited CL, 30 ~ of those treated with iproniazid and 20 of those treated with L-DOPA did exhibit CL. However, the affects attributable to iproniazid (p----0.107) or L-DOPA (p=0.238) did not attain statistical significance.

, TP females 1013

80 13_ 60

I

._.

/ e~

o Old females

/

-

-/

40

20

0

I

60

~

120

I

180

1

240

I

300 Age ,

I

I

3f~:) 420

t

480

J

J

r

54.8 600 660

days

FIG.3. Cumulativepercentagesof old and youngandrogenizedfemalesin ExperimentII exhibitingPVE as a function of age. DISCUSSION The findings of the present study confirm previous reports that L-DOPA, iproniazid phosphate or progesterone can induce ovulation in old PVE female rats (Quadri et al., 1973 ; Huang and Meites, 1975; Huang et al., 1976). Generally, the proportions of animals exhibiting CL in response to treatments were comparable to the proportions reported in these previous studies. While 3 of the 16 old, PVE rats injected with L-DOPA exhibited CL, this was not significantly different from that observed in the controls. Huang et al. (1976) observed CL in 3 of 12 females treated with L-DOPA. Thus, the present findings with respect to L-DOPA, while not statistically significant, are similar to previous observations. The mechanism(s) by which these treatments induce ovulation in old, sterile females are yet to be specified. Huang et aL (1976) induced ovulation in old, PVE females by ether stress

112

JAMES R. LEHMAN, DELORES A. MCARTHUR AND SHELTON E. HENDRICKS

[]

C- Control

[] [] []

D- L-Dopa IP-Iprcniazid phosphate P - Proges'terone

ioo 90--

'~

iili

j

8o-

u

7o-

__

il

-

5o-

g

4o-

~i!iz

iiii:

2o

iili

I0

L__J_LJ~LmtJ

0

Old

g

o

Young TP

Young oil

FIG. 4. Percentages of subjects in Experiment I[ exhibiting CL 30 days after drug treatment.

or the injection of ACTH as well as with L-DOPA or progesterone. Progesterone was the most effective treatment. They suggest that all of those treatments may exert their influence on ovulation by affecting brain catecholamine levels. However, it is possible that all are mediated by rising progesterone titers. ACTH, ether stress and either the influence of L-DOPA on A C T H release or the stress of its systemic injection might lead to the release of sufficient progesterone from the adrenal to induce ovulation. The efficacy of these treatments in old, adrenalectomized animals is yet to be determined. L-DOPA, iproniazid phosphate and progesterone were all effective in inducing ovulation in females which were injected with low doses of androgen neonatally. Previous studies (Barraclough and Gorski, 1962; Barraclough et al., 1964) have reported that progesterone injections are ineffective in inducing ovulation in neonatally androgenized rats. However, in these studies high dosages of TP were used. While the effect of L-DOPA was not statistically significant in Experiment II, two of eleven subjects did exhibit CL while no CL were observed in the ovaries of the eleven control animals. Interestingly, in Experiment I the effectiveness of L-DOPA and iproniazid to induce ovulation decreased drastically with increasing doses of neonatally administered TP. It is difficult to reconcile the present findings with the reports of the induction of ovulation by the direct application of monoamines to the brains of heavily androgenized females (Tima and Flerko, 1974; Tournigant et al., 1974). Perhaps it is simply a matter of the difference in the routes of administration. Alternatively, the direct application of the monoamines does not require the activity of relevant enzymes to raise monoamine levels as does the systemic administration of a monoamine precursor or a monoamine oxidase inhibitor. Any fundamental changes in monoamine synthesis which might be produced by high doses of neonatally administered androgen might be circumvented by the direct application of the monoamines. Still to be identified are the mechanism(s) responsible for the presence of CL 30 days after drug treatment but not immediately following cessation of drug administration. If catecholamine levels control the 4-5 day ovulatory cycle in the rat, the possibility exists that a regimen of injections every four or five days might have resulted in CL being present in the

PHARMACOLOGICAL INDUCTION OF OVULkTION IN OLD AND NEONATALLY ANDROGENIZED RATS

113

first ovary at the termination of drug administration. The efficacy of this regimen, however, has yet to be determined. A major objective of the present work was to determine the comparability of the neonatally androgenized and the reproductively senescent female. With respect to the sensitivity of the ovulatory mechanism to the present treatments, it would appear that such comparability does exist at a phenotypical level. However, it is limited to the 5 lag TP dosage and even here the sensitivity is less than in the aged female. The androgenized and the aged female rat share other characteristics which distinguish them from the reproductively competent female. The aged and the minimally androgenized animals exhibit normal or near normal levels of female sexual behaviour in response to ovarian hormones, (Hendricks and Duffy, 1974; Gerall and Dunlap, 1977"; Cooper and Linnoila, 1977). Female rats receiving higher doses of TP neonatally are much less sensitive to ovarian steriods, particularly progesterone (Clemens et al., 1970). Thus, the capacity to exhibit ovulatory cycles at some previous time is correlated with continued behavioural responsivitiy to ovarian hormones. Supporting the hypothesis that changes in monoamine function might mediate age-related changes in reproductive physiology are reports of decreasing brain levels and turnover rates of catecholamines and increasing turnover rates of indolamines in aging rats (Finch, 1976; Simpkins et al., 1977). Similarly, it has been found that neonatal androgenization modifies catecholamine levels in selected brain regions of female rats (Crowley et al., 1977"). In old females exhibiting PVE, decreased uptake of tritiated estradiol has been observed in the anterior hypothalamus, adenohypophysis and uterus (Peng and Peng, 1973) which is similar to the observed uptake in the same regions of neonatally androgenized females (Green et al., 1969). Thus aging and neonatal androgenization produce animals which exhibit similarities in reproductive behaviour, and show parallel changes in monoamine levels and reveal similar hormone receptor characteristics; the latter two of which might constitute the basic functional change resulting in sterility. It would appear that neonatal androgenization, specifically with low doses of androgen, is a useful model for the study of the aging process. SUMMARY Pharmacological manipulations previously reported to induce ovulation in aged female rats were administered to females sterilized by neonatal androgen. L-DOPA or iproniazid phosphate were found to induce ovulation in some females which had received 5 lag testosterone propionate (TP) at 5 days of age but not in females exposed to higher dosages of TP. A second experiment directly compared the effectiveness of progesterone as well as L-DOPA and iproniazid phosphate to induce ovulation in aged and androgenized females. While the effects are similar, it appears that the aged female is more responsive to these treatments than the young female exposed to 5 lag TP neonatally. It is concluded that age related changes in reproductive physiology might be accounted for by changes in the activity of brain monoamines or brain steroid receptor mechanisms or both. The neonatally androgenized female would appear to be a useful preparation for the study of age-related changes in reproductive function. REFERENCES BARRACLOUGH,C. A. and GORSKI,R. A. (1961) Endocrinology 68, 69. BARRACLOUGH,C. A. and GORSKI,R. A. (1962) J. Endocr. 25, 175. *Reported at the Eastern Conferenceon ReproductiveBehavior, Storrs, Conn. 1977.

114

JAMES R. LEHMAN, DELORES A. MCARTHUR AND SHEI~TON E. HENDRICKS

BARRACLOUGH,C. A., YRARRAZAVAL,S. and HATTON,R. (1964) Endocrinology 75, 838. CLEMENS,L. G., SHRYNE,J. and GORSKI,R. A. (1970) Physiol. Behav. 5, 673. COOPER, R. L. and LINNOILA,M. (1977) Physiol. Behav. 18, 573. FINCH, C. E. (1976) Q. Rev. Biol. 51, 49. GORSKI,R. A. (1968) Endocrinology 82, 1001. GREEN,R., LUTTAGEN,W. G. and WHALEN,R. E. (1969) Endocrinology 85, 373. HENDRICKS,S. E. and Durrv, J. A. (1974) Devl. Psychobiol. 7, 297. HUANG, H. H., MARSHALL,S. and MEI~S, J. (1976) Neuroendocrinology 20, 21. HUANG, H. H. and MEtTES,J. (1975) Neuroendocrinology 17, 289. PENG, M. and PENG, Y. (1973) Fertility and Sterility 24, 534. QUADRI, S. K., KLEDZII~,G. S. and MEIrES,J. (1973) Neuroendocrinology 11, 248. SIMPKINS,J. W., MUELI.ER,G. P., HUANG,H. H. and MEtrES, J. (1977) Endocrinology 100, 1672. SWANSON,H. E. and VANDER WERrr TEN BOSCH,J. J. (1964) Acta endocr. 45, 1. TIMA, L. and FLERKO,B. (1974) Neoroendocrinology 15, 364. TOURNIGANT,J., ARNAUD,O. and PASSOUANT-FONTAINE,T. (1974) C. r. Soc. Biol. 169, 113.

Pharmacological induction of ovulation in old and neonatally androgenized rats.

Exp. Geront. Vol. 13, pp. 107-114. © Pergamon Press Ltd. 1978. Printed in Great Britain. 0531-5565178/0801-0107502.00/0 P H A R M A C O L O G I C A...
497KB Sizes 0 Downloads 0 Views