Physiology & Behavior, Vol. 18, pp. 573--576. Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.
Sexual Behavior in Aged, Noncycling Female Rats RALPH L. COOPER AND M A R K K U LINNOILA
Center for the Study o f Aging and Human Development Duke University Medical Center, Durham NC 27710 (Received 6 March 1976) COOPER, R. L. AND M. LINNOILA. Sexual behavior in aged, noncycling female rats. PHYSIOL. BEHAV. 18(4) 573-576, 1977. - Sexual behavior of 19 month old, noncycling, female rats was evaluated for a period of 8 days. The sexual behavior of 11 aged females revealing constant vaginal cornification (CVC) for 30 days prior to, and during the observation period was variable. Eight of the CVC females showed consistently high lordosis quotients (LQ), whereas 3 showed no lordosis. A significant negative correlation was found between food intake and LQ over the 8 day period. In 8 other aged females, the vaginal smear was characterized by a mixture of both leucocytes and cornified cells, each day, for a period of 30 days prior to, and during the observation period. Lordosis behavior was absent in all females revealing this mixed type (MX) smear when they were tested. At random times after the test period, 3 CVC and 2 MX females showed a vaginal cycle. As the vaginal cycle was observed each female was again tested for sexual behavior. All 5 females were highly receptive when placed with the male during the evening of vaginal proestrus, but not at other times. It is concluded that despite disruptions of regular ovarian cyclicity, aged female rats axe still capable of showing a lordosis response. Sexual behavior
Age
Constant estrus
FOLLOWING the onset of puberty, the female rat exhibits recurrent, cyclical changes in her sexual behavior which are inextricably linked to the secretory pattern of the pituitary and ovarian hormones involved in ovulation. The recurrent estrous cycles last 4 or 5 days depending on the individual rat. Under normal conditions, the peak level of sexual receptivity occurs during the evening of vaginal proestrus and precedes ovulation by several hours [7]. At this time, the female will respond to the mounting attempts of the male by displaying the lordosis response. With increasing age, there is a well recognized decline in the reproductive capacity of the female rat [9]. One primary reason for this reduced capacity appears to be the spontaneous occurrence of irregular or prolonged ovarian cycles - and fewer ovulations. More specifically, between the ages of 1 0 - 1 2 months the 4 or 5 day estrous cycle is interrupted by prolonged nonovulatory periods and the vaginal smear is characterized by one of the three following patterns: (1) constant cornification, (2) prolonged periods during which the daily smear reveals a mixture of cornified cells and leucocytes (often termed pseudopregnancy [8, 13, 14]), or (3) changes in cell types which occur with no predictable pattern [5, 8, 10]. The ovaries of the old constant vaginal estrous (CVC) females contain several well developed follicles, but no corpora lutea, whereas those of the females showing the second pattern described above, having vaginal smears with a mixture of cell types (MX), contain many well developed corpora lutea [ 8]. The pattern of ovarian estrogen and progesterone secretion which accompanies these ovarian conditions
remains to be determined. In addition, there is little information available describing the sexual behavior of the aged, female rat. Ingram [10] reported that the sexual behavior of the aged females was rare and spasmodic and could not be correlated consistently with the vaginal states. This apparent lack of synchrony between the behavioral and physiological events related to the reproductive process in the old, female rat could further contribute to a decreased probability of fertilization. However, in his study Ingram [10] used the presence of sperm in the vaginal smear as the measure of sexual receptivity. He did not report the actual behavior of the animal. The purpose of this study is to compare the sexual activity of the aged, noncycling female with that of the young, adult female to determine whether the former do show a decrease in sexual behavior and whether such a decrease may be related to the ovarian state of the female. METHOD
Animals and Procedure Twenty multiparous female Long Evans rats, 8 - 1 0 months of age, were obtained from Marland Breeding Farms, Hewitt, New Jersey. Upon arrival, each female was housed individually in a 17.5 × 24.5 × 18 cm stainless steel cage with mesh front and floor. The room lighting was controlled artificially on a 14 hour light and 10 hour dark schedule (lights off at 3 p.m.). Room temperature was maintained at a constant 72 degrees F. All rats were provided with Purina chow and tap water ad lib.
1This research was supported by NIH Training Grant HD 00164 from the National Institute of Child Health and Human Development. 573
574
COOPER AND LINNOILA
W h e n the rats r e a c h e d 16 m o n t h s of age, daily vaginal smears were t a k e n j u s t p r i o r to t h e o n s e t of t h e dark period. Daily f o o d i n t a k e was also m e a s u r e d in all females as a part of a n o t h e r e x p e r i m e n t [ 3 ] . Vaginal smears were classified as diestrus ( p r e d o m i n a n c e of l e u c o c y t e s ) , proestrus (all r o u n d , n u c l e a t e d epithelial cells w i t h c l u m p i n g ) , or estrus (all c o r n i f i e d cells). In a d d i t i o n , the aged females were f u r t h e r classified as showing e i t h e r c o n s t a n t vaginal c o r n i f i c a t i o n or a m i x e d smear c h a r a c t e r i z e d b y the presence of b o t h l e u c o c y t e s and cornified cells simu l t a n e o u s l y , for p r o l o n g e d periods. Eight 3 - 4 m o n t h old Long Evans female rats served as controls. This g r o u p was selected f r o m an available, larger p o p u l a t i o n of 29 females showing e i t h e r 4- or 5-day cycles. T h e vaginal smears of these 29 females were e x a m i n e d daily for a t w o week period prior to o b s e r v a t i o n . Regular vaginal cycles were observed in all the y o u n g females. Only females s h o w i n g regular 4-day cycles were used in this s t u d y because the s h o r t e r cycle l e n g t h was m o r e c o n v e n i e n t . In the 8 c o n t r o l females, the vaginal smears were also e x a m i n e d daily d u r i n g the test period. All the aged females were 19 m o n t h s old at the o n s e t of the o b s e r v a t i o n period. T h e vaginal smears of the aged females were followed daily t h r o u g h o u t the o b s e r v a t i o n p e r i o d and for 30 a d d i t i o n a l days. All aged females were tested daily with an active male ( 4 - 6 m o n t h s old) for 8 c o n s e c u t i v e days. The y o u n g females were tested daily over 2 c o n s e c u t i v e vaginal cycles ( c o m m e n c i n g on diestrus day 1, Cycle 1). The y o u n g females were also tested o n the n e x t t w o days of diestrus (Cycle 3) to d e t e r m i n e the possible effects of r e p e a t e d testing o n sexual behavior. Thus, the y o u n g females were tested for a t o t a l of 10 days. O b s e r v a t i o n s were m a d e b e t w e e n 1.5 a n d 3.5 h o u r s a f t e r the o n s e t of the dark period. Behavioral tests were c o n d u c t e d b y placing t h e female in the m a l e ' s h o m e cage, c o n s t r u c t e d of Plexiglas, curled a n d b o l t e d to form a c y l i n d e r 15 in. high a n d 17 in. in d i a m e t e r . Each female was left w i t h the male for 15 m i n or u n t i l 10 m o u n t s with t h r u s t were observed. If a given stimulus male did n o t show the r e q u i r e d 10 m o u n t s d u r i n g the 15 m i n u t e s , the female was placed w i t h a n o t h e r male. Sexual r e c e p t i v i t y was expressed as t h e lordosis q u o t i e n t
[ 1 2 ] . F o r each female, for each test, the lordosis q u o t i e n t was c o m p u t e d b y dividing the t o t a l n u m b e r of lordosis responses by the t o t a l n u m b e r of m o u n t s and m u l t i p l y i n g b y 100. In a d d i t i o n to the lordosis q u o t i e n t , the f r e q u e n c y of ear wiggling, darting and h o p p i n g displayed b y each female was also n o t e d . T h e S t u d e n t ' s t-test was used to d e t e r m i n e d i f f e r e n c e s b e t w e e n g r o u p m e a n s for each test day. T h e Pearson p r o d u c t - m o m e n t c o e f f i c i e n t was used to d e t e r m i n e the c o r r e l a t i o n b e t w e e n the m e a s u r e s of f o o d i n t a k e and sexual behavior. RESULTS
Observations on Vaginal Smears T h e vaginal smears of the 20 females o b s e r v e d b e t w e e n the ages of 16.0 and 20.5 m o n t h s revealed a progressive decrease in the o c c u r r e n c e of cycles. F o r the 30 day period p r i o r to testing ( b e t w e e n 1 8 - 1 9 m o n t h s ) the vaginal smears o f 11 females revealed a p a t t e r n of c o n s t a n t vaginal c o r n i f i c a t i o n . T h e vaginal smears of 8 females s h o w e d a m i x t u r e of cell t y p e s in t h a t b o t h l e u c o c y t e s and c o r n i f i e d cells were p r e s e n t each day. A vaginal cycle was o b s e r v e d in o n e female during this 30 day p e r i o d and she was e x c l u d e d f r o m behavioral testing. N o n e of the 19 females t e s t e d cycled during the test period. However, at r a n d o m t i m e s d u r i n g the p o s t o b s e r v a t i o n period, 5 females did show a vaginal cycle. During these cycles the females were again tested for sexual b e h a v i o r as described below.
Sexual Behavior in Young Adult Females The display of sexual b e h a v i o r in the y o u n g females varied p r e d i c t a b l y over t h e 2 c o n s e c u t i v e vaginal cycles (Table 1). W h e n tested during the evening o f vaginal proestrus, the m e a n lordosis q u o t i e n t for this g r o u p was m a x i m u m being 87.5 (+ 6.75) for the first cycle and 89 (+ 6.66) for t h e second cycle. On the evening of vaginal estrus, some of the females also r e s p o n d e d t o the m o u n t i n g a t t e m p t s of the male and the m e a n lordosis q u o t i e n t for this day was 30 (* 15.12) and 36 (+ 15.8) for the t w o
TABLE1 SEXUAL RECEPTIVITY OF YOUNG AND OLD FEMALE RATS TESTED ON EIGHT CONSECUTIVE DAYS
Day 1 2 3 4 5 6 7 8 9 10
Young Females v.s. LQ _+ SEM d d P E d d P E d d
0.0 0.0 87.5 30.0 0.0 0.0 89.0 36.3 0.0 0.0
0.00 0.00 6.75 15.12 0.00 0.00 6.66 15.80 0.00 0.00
v.s. CVC CVC CVC CVC CVC CVC CVC CVC
All Old Females LQ + SEM t
p
v.s.
60.9 56.4 58.2 60.0 63.6 62.3 67.3 62.7
* * ns ns * * ns ns
CVC CVC CVC CVC CVC CVC CVC CVC
13.58 14.60 12.78 12.21 13.40 13.30 13.40 12.70
3.79 3.37 1.82 1.56 4.21 4.31 1.28 1.30
Responding Old Females LQ +_ SEM t 83.8 77.5 80.0 82.5 87.5 92.5 92.5 86.3
9.40 12.64 8.45 5.26 7.26 3.13 4.12 4.98
8.87 6.13 .69 3.28 12.06 29.52 .48 3.02
p * * ns * * * ns *
Mean daily lordosis quotient (LQ) and standard error of the mean (___S EM) of eight young females, all 19 month old CVC females (n = 11) and the subgroup of responding old CVC females (n = 8). Young females were tested for two additional diestrus days. See text for details, v.s. = vaginal smear, d = diestrus, P = proestrus, E = estrus, CVC = constant vaginal cornification, t = value of Student's t obtained when the mean LQ of the old females were compared with the mean LQ of the young females on each test day,* = p
Sexual Behavior in Aged, Noncycling Female Rats RALPH L. COOPER AND M A R K K U LINNOILA
Center for the Study o f Aging and Human Development Duke University Medical Center, Durham NC 27710 (Received 6 March 1976) COOPER, R. L. AND M. LINNOILA. Sexual behavior in aged, noncycling female rats. PHYSIOL. BEHAV. 18(4) 573-576, 1977. - Sexual behavior of 19 month old, noncycling, female rats was evaluated for a period of 8 days. The sexual behavior of 11 aged females revealing constant vaginal cornification (CVC) for 30 days prior to, and during the observation period was variable. Eight of the CVC females showed consistently high lordosis quotients (LQ), whereas 3 showed no lordosis. A significant negative correlation was found between food intake and LQ over the 8 day period. In 8 other aged females, the vaginal smear was characterized by a mixture of both leucocytes and cornified cells, each day, for a period of 30 days prior to, and during the observation period. Lordosis behavior was absent in all females revealing this mixed type (MX) smear when they were tested. At random times after the test period, 3 CVC and 2 MX females showed a vaginal cycle. As the vaginal cycle was observed each female was again tested for sexual behavior. All 5 females were highly receptive when placed with the male during the evening of vaginal proestrus, but not at other times. It is concluded that despite disruptions of regular ovarian cyclicity, aged female rats axe still capable of showing a lordosis response. Sexual behavior
Age
Constant estrus
FOLLOWING the onset of puberty, the female rat exhibits recurrent, cyclical changes in her sexual behavior which are inextricably linked to the secretory pattern of the pituitary and ovarian hormones involved in ovulation. The recurrent estrous cycles last 4 or 5 days depending on the individual rat. Under normal conditions, the peak level of sexual receptivity occurs during the evening of vaginal proestrus and precedes ovulation by several hours [7]. At this time, the female will respond to the mounting attempts of the male by displaying the lordosis response. With increasing age, there is a well recognized decline in the reproductive capacity of the female rat [9]. One primary reason for this reduced capacity appears to be the spontaneous occurrence of irregular or prolonged ovarian cycles - and fewer ovulations. More specifically, between the ages of 1 0 - 1 2 months the 4 or 5 day estrous cycle is interrupted by prolonged nonovulatory periods and the vaginal smear is characterized by one of the three following patterns: (1) constant cornification, (2) prolonged periods during which the daily smear reveals a mixture of cornified cells and leucocytes (often termed pseudopregnancy [8, 13, 14]), or (3) changes in cell types which occur with no predictable pattern [5, 8, 10]. The ovaries of the old constant vaginal estrous (CVC) females contain several well developed follicles, but no corpora lutea, whereas those of the females showing the second pattern described above, having vaginal smears with a mixture of cell types (MX), contain many well developed corpora lutea [ 8]. The pattern of ovarian estrogen and progesterone secretion which accompanies these ovarian conditions
remains to be determined. In addition, there is little information available describing the sexual behavior of the aged, female rat. Ingram [10] reported that the sexual behavior of the aged females was rare and spasmodic and could not be correlated consistently with the vaginal states. This apparent lack of synchrony between the behavioral and physiological events related to the reproductive process in the old, female rat could further contribute to a decreased probability of fertilization. However, in his study Ingram [10] used the presence of sperm in the vaginal smear as the measure of sexual receptivity. He did not report the actual behavior of the animal. The purpose of this study is to compare the sexual activity of the aged, noncycling female with that of the young, adult female to determine whether the former do show a decrease in sexual behavior and whether such a decrease may be related to the ovarian state of the female. METHOD
Animals and Procedure Twenty multiparous female Long Evans rats, 8 - 1 0 months of age, were obtained from Marland Breeding Farms, Hewitt, New Jersey. Upon arrival, each female was housed individually in a 17.5 × 24.5 × 18 cm stainless steel cage with mesh front and floor. The room lighting was controlled artificially on a 14 hour light and 10 hour dark schedule (lights off at 3 p.m.). Room temperature was maintained at a constant 72 degrees F. All rats were provided with Purina chow and tap water ad lib.
1This research was supported by NIH Training Grant HD 00164 from the National Institute of Child Health and Human Development. 573
574
COOPER AND LINNOILA
W h e n the rats r e a c h e d 16 m o n t h s of age, daily vaginal smears were t a k e n j u s t p r i o r to t h e o n s e t of t h e dark period. Daily f o o d i n t a k e was also m e a s u r e d in all females as a part of a n o t h e r e x p e r i m e n t [ 3 ] . Vaginal smears were classified as diestrus ( p r e d o m i n a n c e of l e u c o c y t e s ) , proestrus (all r o u n d , n u c l e a t e d epithelial cells w i t h c l u m p i n g ) , or estrus (all c o r n i f i e d cells). In a d d i t i o n , the aged females were f u r t h e r classified as showing e i t h e r c o n s t a n t vaginal c o r n i f i c a t i o n or a m i x e d smear c h a r a c t e r i z e d b y the presence of b o t h l e u c o c y t e s and cornified cells simu l t a n e o u s l y , for p r o l o n g e d periods. Eight 3 - 4 m o n t h old Long Evans female rats served as controls. This g r o u p was selected f r o m an available, larger p o p u l a t i o n of 29 females showing e i t h e r 4- or 5-day cycles. T h e vaginal smears of these 29 females were e x a m i n e d daily for a t w o week period prior to o b s e r v a t i o n . Regular vaginal cycles were observed in all the y o u n g females. Only females s h o w i n g regular 4-day cycles were used in this s t u d y because the s h o r t e r cycle l e n g t h was m o r e c o n v e n i e n t . In the 8 c o n t r o l females, the vaginal smears were also e x a m i n e d daily d u r i n g the test period. All the aged females were 19 m o n t h s old at the o n s e t of the o b s e r v a t i o n period. T h e vaginal smears of the aged females were followed daily t h r o u g h o u t the o b s e r v a t i o n p e r i o d and for 30 a d d i t i o n a l days. All aged females were tested daily with an active male ( 4 - 6 m o n t h s old) for 8 c o n s e c u t i v e days. The y o u n g females were tested daily over 2 c o n s e c u t i v e vaginal cycles ( c o m m e n c i n g on diestrus day 1, Cycle 1). The y o u n g females were also tested o n the n e x t t w o days of diestrus (Cycle 3) to d e t e r m i n e the possible effects of r e p e a t e d testing o n sexual behavior. Thus, the y o u n g females were tested for a t o t a l of 10 days. O b s e r v a t i o n s were m a d e b e t w e e n 1.5 a n d 3.5 h o u r s a f t e r the o n s e t of the dark period. Behavioral tests were c o n d u c t e d b y placing t h e female in the m a l e ' s h o m e cage, c o n s t r u c t e d of Plexiglas, curled a n d b o l t e d to form a c y l i n d e r 15 in. high a n d 17 in. in d i a m e t e r . Each female was left w i t h the male for 15 m i n or u n t i l 10 m o u n t s with t h r u s t were observed. If a given stimulus male did n o t show the r e q u i r e d 10 m o u n t s d u r i n g the 15 m i n u t e s , the female was placed w i t h a n o t h e r male. Sexual r e c e p t i v i t y was expressed as t h e lordosis q u o t i e n t
[ 1 2 ] . F o r each female, for each test, the lordosis q u o t i e n t was c o m p u t e d b y dividing the t o t a l n u m b e r of lordosis responses by the t o t a l n u m b e r of m o u n t s and m u l t i p l y i n g b y 100. In a d d i t i o n to the lordosis q u o t i e n t , the f r e q u e n c y of ear wiggling, darting and h o p p i n g displayed b y each female was also n o t e d . T h e S t u d e n t ' s t-test was used to d e t e r m i n e d i f f e r e n c e s b e t w e e n g r o u p m e a n s for each test day. T h e Pearson p r o d u c t - m o m e n t c o e f f i c i e n t was used to d e t e r m i n e the c o r r e l a t i o n b e t w e e n the m e a s u r e s of f o o d i n t a k e and sexual behavior. RESULTS
Observations on Vaginal Smears T h e vaginal smears of the 20 females o b s e r v e d b e t w e e n the ages of 16.0 and 20.5 m o n t h s revealed a progressive decrease in the o c c u r r e n c e of cycles. F o r the 30 day period p r i o r to testing ( b e t w e e n 1 8 - 1 9 m o n t h s ) the vaginal smears o f 11 females revealed a p a t t e r n of c o n s t a n t vaginal c o r n i f i c a t i o n . T h e vaginal smears of 8 females s h o w e d a m i x t u r e of cell t y p e s in t h a t b o t h l e u c o c y t e s and c o r n i f i e d cells were p r e s e n t each day. A vaginal cycle was o b s e r v e d in o n e female during this 30 day p e r i o d and she was e x c l u d e d f r o m behavioral testing. N o n e of the 19 females t e s t e d cycled during the test period. However, at r a n d o m t i m e s d u r i n g the p o s t o b s e r v a t i o n period, 5 females did show a vaginal cycle. During these cycles the females were again tested for sexual b e h a v i o r as described below.
Sexual Behavior in Young Adult Females The display of sexual b e h a v i o r in the y o u n g females varied p r e d i c t a b l y over t h e 2 c o n s e c u t i v e vaginal cycles (Table 1). W h e n tested during the evening o f vaginal proestrus, the m e a n lordosis q u o t i e n t for this g r o u p was m a x i m u m being 87.5 (+ 6.75) for the first cycle and 89 (+ 6.66) for t h e second cycle. On the evening of vaginal estrus, some of the females also r e s p o n d e d t o the m o u n t i n g a t t e m p t s of the male and the m e a n lordosis q u o t i e n t for this day was 30 (* 15.12) and 36 (+ 15.8) for the t w o
TABLE1 SEXUAL RECEPTIVITY OF YOUNG AND OLD FEMALE RATS TESTED ON EIGHT CONSECUTIVE DAYS
Day 1 2 3 4 5 6 7 8 9 10
Young Females v.s. LQ _+ SEM d d P E d d P E d d
0.0 0.0 87.5 30.0 0.0 0.0 89.0 36.3 0.0 0.0
0.00 0.00 6.75 15.12 0.00 0.00 6.66 15.80 0.00 0.00
v.s. CVC CVC CVC CVC CVC CVC CVC CVC
All Old Females LQ + SEM t
p
v.s.
60.9 56.4 58.2 60.0 63.6 62.3 67.3 62.7
* * ns ns * * ns ns
CVC CVC CVC CVC CVC CVC CVC CVC
13.58 14.60 12.78 12.21 13.40 13.30 13.40 12.70
3.79 3.37 1.82 1.56 4.21 4.31 1.28 1.30
Responding Old Females LQ +_ SEM t 83.8 77.5 80.0 82.5 87.5 92.5 92.5 86.3
9.40 12.64 8.45 5.26 7.26 3.13 4.12 4.98
8.87 6.13 .69 3.28 12.06 29.52 .48 3.02
p * * ns * * * ns *
Mean daily lordosis quotient (LQ) and standard error of the mean (___S EM) of eight young females, all 19 month old CVC females (n = 11) and the subgroup of responding old CVC females (n = 8). Young females were tested for two additional diestrus days. See text for details, v.s. = vaginal smear, d = diestrus, P = proestrus, E = estrus, CVC = constant vaginal cornification, t = value of Student's t obtained when the mean LQ of the old females were compared with the mean LQ of the young females on each test day,* = p
