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Seasonal variations in circulating levels of progesterone and estradiol in unmated adult female woodchucks (Marmota monax ) in captivity
Life Sciences, Vol. 49, pp. 353-359 Printed in the U.S.A.
Pergamon Press
S E A S O N A L V A R I A T I O N S IN CIRCULATING LEVELS OF P R O G E S T E R O N E AND ESTRADIOL IN UNMATED ADULT FEMALE W O O D C H U C K S (MARMOTA MONAX) IN CAPTIVITY A. P. Sinha Hikim L2, A. G. Amador 3, A. Woolf 2, A. Bartke l, and C. G r e m i l l i o n - S m i t h 2 D e p a r t m e n t of Physiology l, School of Medicine, and C o o p e r a t i v e W i l d l i f e R e s e a r c h Laboratory 2, Southern Illinois University, Carbondale, Illinois 62901-6512 D e p a r t m e n t of Obstetrics and G y n e c o l o g y 3, Southern Illinois University, School of Medicine, P.O. Box 19230, Springfield, Illinois 62794-9230 (Received in final form May 28, 1991) Summary
The annual profile of serum levels of p r o g e s t e r o n e (P) and estradiol-17~ (E2) was c h a r a c t e r i z e d in a s e a s o n a l l y breeding rodent, the w o o d c h u c k (Marmota monax). Hormonal levels were d e t e r m i n e d in serum samples taken at weekly or biweekly intervals from u n m a t e d female woodchucks m a i n t a i n e d all year indoors under c o n t r o l l e d conditions of p h o t o p e r i o d and temperature. Annual fluctuations included a rise of E 2 levels during late January through February, followed by a modest increase in plasma P c o n c e n t r a t i o n s by late March, the latter attaining peak values during April and May. A temporal d i s s o c i a t i o n of peak values of c i r c u l a t i n g levels of P and E 2 during the annual r e p r o d u c t i v e cycle was also detected. The timing of changes in serum levels of P and E 2 in these captive w o o d c h u c k s c o r r e s p o n d e d to reproductive events during the normal breeding season of the w o o d c h u c k in the southern part of its range. W o o d c h u c k s are large, seasonally b r e e d i n g sciurid rodents that in the wild typically mate during a 3-week period following emergence from h i b e r n a t i o n during late winter (1,2). In the wild, the timing of reproductive sequence is set by the timing of e m e r g e n c e of females from hibernation; adult males u s u a l l y emerge from h i b e r n a t i o n 1-3 weeks earlier than females and begin b r e e d i n g as soon as females emerge (1,3). At emergence, the ovaries are enlarged and contain multiple, mature follicles (3). H y p e r t r o p h y of many interstitial cells in the ovary along with the a c c u m u l a t i o n of lipids have also been reported (3). There are indications that this seasonal r e p r o d u c t i v e rhythm occurs also in captive woodchucks m a i n t a i n e d under a natural 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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photoperiod, (4,5) .
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even in the absence of prior winter h i b e r n a t i o n
However, little is known about the changes in annual p r o f i l e s of c i r c u l a t i n g levels of p r o g e s t e r o n e (P) and estradiol17~ (E2) d u r i n g the r e p r o d u c t i v e cycle of this species, or about hormonal and r e p r o d u c t i v e cycles under constant laboratory conditions. Serum P levels r e p o r t e d l y are e l e v a t e d d u r i n g p r e g n a n c y and lactation, and during the latter part of the b r e e d i n g season in unmated females (4,6). Temporal changes in serum levels of P and E 2 during the estrous cycle in a laboratory colony of w o o d c h u c k s have been recently d o c u m e n t e d (7). In that study, we noted elevated levels of E 2 a week before and during estrus, followed by a significant decline in E 2 levels one week after estrus. Serum levels of P, however, r e m a i n e d unchanged. The o b j e c t i v e of this study was to analyze the c h a n g i n g p a t t e r n s of c i r c u l a t i n g levels of P and E 2 during a complete annual r e p r o d u c t i v e cycle of w o o d c h u c k s in c a p t i v i t y with special emphasis on the b r e e d i n g season. Materials
& Methods
C o l o n y - h o u s e d adult (2 + yrs.), female w o o d c h u c k s used in this study were live trapped in the vicinity of C a r b o n d a l e (Jackson County), Illinois, during 1988 and 1989. They were h o u s e d i n d i v i d u a l l y in rabbit cages (0.61m x 0.60m x 0.90m) with wood chips or straw as nesting m a t e r i a l s in indoor v i v a r i u m for the p r e v i o u s 6-11 months under controlled laboratory conditions. T e m p e r a t u r e was m a i n t a i n e d at 22 ± 2 ° C and p h o t o p e r i o d at 12 h of light and 12 h of darkness. Rabbit chow (Teklad, Harlan S p r a g u e - D a w l e y Inc., Co., Madison, WI) s u p p l e m e n t e d with fresh v e g e t a b l e s and/or fruits (2-3 times/week) and w a t e r were p r o v i d e d ad libitum. None of the females h i b e r n a t e d in these conditions. B l o o d c o l l e c t i o n s and hormone assays. Blood samples were c o l l e c t e d at w e e k l y or biweekly intervals from 4 adult females e q u i p p e d with s u b c u t a n e o u s v a s c u l a r access ports (8) t h r o u g h o u t the year, except during the breeding season when these animals were bled twice or thrice weekly. Animals were r e s t r a i n e d in devices made of P o l y v i n y l c h l o r i d e pipe (40 cm long w i t h a d i a m e t e r of 15 cm) which avoided need for anesthesia. However, if ports failed, blood was obtained by v e n i p u n c t u r e under K e t a m i n e (Ketaset, Aveco Co., Inc., Fort Dodge, Iowa) - x y l a z i n e (Rompun, Haver, Mobay Corp., Kansas) anesthesia. W e e k l y samples were also obtained from 12 females from January 16 until March 31 under K e t a m i n e - x y l a z i n e anesthesia and from 4 a d d i t i o n a l females e q u i p p e d with s u b c u t a n e o u s vascular access ports during April through July. No s i g n i f i c a n t (P<0.05) d i f f e r e n c e s (student's t test) in the c i r c u l a t i n g levels of P or E 2 were noted between samples o b t a i n e d from females fitted with v a s c u l a r ports and samples o b t a i n e d from w o o d c h u c k s under k e t a m i n e - x y l a z i n e anesthesia. Therefore, hormonal data from all animals were c o m b i n e d for further statistical analysis and presentation. For each month, samples were divided into two groups, those c o l l e c t e d in the first half of the month, d e s i g n a t e d Jan I, Feb I, etc., and those c o l l e c t e d in the second half of the month, r e f e r r e d to as Jan II, Feb II, etc. Numbers of animals used for hormonal analysis in each time period are listed in Table i.
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S e r u m w a s o b t a i n e d by c e n t r i f u g a t i o n a f t e r a l l o w i n g t h e b l o o d to c l o t a n d t h e n f r o z e n (-20 ° C) u n t i l assayed. M e a s u r e m e n t s of s e r u m levels of P and E 2 w e r e p e r f o r m e d by s o l i d p h a s e RIAs, u s i n g k i t s w i t h p o l y p r o p y l e n e t u b e s c o a t e d w i t h a n t i P or a n t i - E 2 a n t i b o d i e s ( D i a g n o s t i c P r o d u c t s Corp., Los A n g e l e s , CA) as d e s c r i b e d p r e v i o u s l y (7). T h e t r a c e r s u s e d w e r e n5 I - P a n d n5 I - E2, r e s p e c t i v e l y . D a t a w e r e t h e n o b t a i n e d by R I A P L O T a n d R I A D O S E p r o g r a m s for R I A a n a l y s i s (9). Since these kits were d e s i g n e d for u s e w i t h h u m a n s a m p l e s a n d c o n t a i n a s t a n d a r d c u r v e b a s e d on h u m a n serum, t h e p a r a l l e l i s m b e t w e e n l o g - l o g i t c u r v e s m a d e by a d d i n g d i f f e r e n t v o l u m e s f r o m a p o o l of w o o d c h u c k s e r u m and t h e r e s p e c t i v e s t a n d a r d c u r v e s w e r e d e t e r m i n e d for P ( w o o d c h u c k serum: slope, -1.772; y - i n t e r c e p t , 0.414; r = - l . 0 0 0 ; standard: slope, -1.763; y - i n t e r c e p t , 0.417; r = - l . 0 0 0 ) and E 2 ( w o o d c h u c k serum: slope, -1.861; y - i n t e r c e p t , 4.186; r = - l . 0 0 0 ; standard: slope, -1.863; y - i n t e r c e p t , 4.188; r = - 0 . 9 9 4 ) . The d e t e c t i o n l i m i t s for P and E 2 w e r e 25 p g / m l and 4 pg/ml, respectively. Statistical analysis. D a t a are p r e s e n t e d as m e a n s ± SE. S t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d u s i n g SAS ( S t a t i s t i c a l A n a l y s i s S y s t e m I n s t i t u t e Inc., Carry, NC) program. Results were t e s t e d for s t a t i s t i c a l s i g n i f i c a n c e u s i n g D u n c a n ' s M u l t i p l e R a n g e T e s t f o l l o w i n g a o n e - w a y a n a l y s i s of v a r i a n c e (ANOVA). W h e n the F - t e s t f r o m A N O V A i n d i c a t e d s i g n i f i c a n c e , d i f f e r e n c e s of t h e m e a n s w e r e c a l c u l a t e d u s i n g D u n c a n ' s M u l t i p l e R a n g e Test. D i f f e r e n c e s w e r e c o n s i d e r e d s i g n i f i c a n t if P<0.05. Results C h a n g e s in the a n n u a l p r o f i l e of s e r u m l e v e l s of P a n d E 2 in a d u l t u n m a t e d f e m a l e w o o d c h u c k s m a i n t a i n e d in c a p t i v i t y are p r e s e n t e d in T a b l e i. G r a p h i c s u m m a r y of t h e s e d a t a p r o v i d e d in F i g u r e 1 s h o w the g e n e r a l t r e n d s in c i r c u l a t i n g l e v e l s of P (F=8.10; df=22; P < 0 . 0 0 1 and E 2 (F=2.75; df=22; P<0.002) d u r i n g the a n n u a l r e p r o d u c t i v e cycle. M e a n levels of c i r c u l a t i n g P w e r e e x t r e m e l y low (<0.2 ng/ml) f r o m A u g u s t u n t i l t h e e n d of F e b r u a r y . T h e y r o s e to 0.6 n g / m l by the s e c o n d h a l f of March, w e r e m a x i m a l (5.6-7.2 ng/ml) d u r i n g the p e r i o d of A p r i l and M a y b e f o r e d e c l i n i n g a g a i n in J u l y (0.5-0.7 ng/ml). V a l u e s for P r e p o r t e d d u r i n g A p r i l and M a y w e r e s i g n i f i c a n t l y (P<0.05) d i f f e r e n t t h a n the v a l u e s o b t a i n e d d u r i n g the r e s t of t h e year. However, the v a l u e s t h a t w e r e o b t a i n e d d u r i n g the m o n t h of J u n e w e r e not s i g n i f i c a n t l y d i f f e r e n t f r o m v a l u e s r e c o r d e d for A p r i l I a n d M a y I. D u r i n g t h e p e a k p e r i o d of P secretion, s e r u m l e v e l s r a n g e d f r o m 3.4 to 13.0 n g / m l and f r o m 4.2 to 19.5 n g / m l for t h e m o n t h s of A p r i l and May, r e s p e c t i v e l y in 5 out of 7 a n i m a l s studied. O n e f e m a l e h a d o n l y m i n o r e l e v a t i o n in P (0.3 - 1.6 ng/ml) d u r i n g A p r i l a n d a n o t h e r f e m a l e also h a d low levels of P (0.i - 0.4 ng/ml) d u r i n g the m o n t h of May. The m a x i m a l v a l u e s of P v a r i e d w i t h i n as w e l l as b e t w e e n females. U n l i k e the r e s u l t s for P, t h e r e w e r e o n l y m o d e s t s e a s o n a l c h a n g e s in c i r c u l a t i n g levels of E 2 d u r i n g t h e a n n u a l r e p r o d u c t i v e cycle. S e r u m E 2 levels w e r e low ( 1 5 . 9 - 2 6 . 0 pg/ml) d u r i n g m o s t of the y e a r (from M a r c h to J a n u a r y I), b e g a n to r i s e (29.3 pg/ml) at the o n s e t of the b r e e d i n g s e a s o n ( J a n u a r y II) a n d r e m a i n e d e l e v a t e d at or a b o v e t h i s level u n t i l late F e b r u a r y . V a l u e s o b t a i n e d d u r i n g F e b I (32.1 ± 1.7) w e r e s i g n i f i c a n t l y
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(P<0.05) higher than the values obtained during Jan I (20.7 ± 3.0 pg/ml). However, differences in m e a n v a l u e s o f E 2 b e t w e e n e a r l y ( 2 0 . 7 ± 3 . 0 p g / m l ) a n d l a t e (29.3 + 2 . 8 p g / m l ) J a n u a r y w e r e n o t statistically significant. A significant (P<0.05) decline in E 2 levels was noted between early February and late March. Serum concentrations of E2 were highly variable within as well as between females during our study periods. For example, variations in E 2 l e v e l s d u r i n g J a n II, F e b I a n d F e b II, r a n g e d from 19.3 to 44.6, from 21.1 to 44.8, and from 21.9 to 52.6 pg/ml, respectively. TABLE Annual Profile of in Woodchucks. animals. Sampling periods Jan Jan Feb Feb Mar Mar Apr Apr May May Jun Jun Jul Jul Aug Aug Sep Sep Oct Oct Nov Nov Dec
I II I II I II I II I II I II I II I II I II I II I II I
i.
Serum Levels of Progesterone and Estradiol Numbers in p a r e n t h e s i s indicate the number
(3) (14) (14) (15) (15) (15) (7) (7) (7) (7) (7) (7) (6) (6) (3) (3) (3) (3) (3) (3) (3) (3) (3)
* In each column, means significantly different
Progesterone (ng/ml) 0.06 0.07 0. i0 0.17 0.29 0.55 5.57 6.61 5 73 7 24 2 85 2 69 0 69 0 49 0 Ii 0 09 0 ii 0 09 0 i0 0 08 0 09 0 05 0.04 with
the
+ + + + + + + + + + + + + + + + + + + + + + +
-
Estradiol (pg/ml) 2 0 . 7 + 3 . 0 0 bcd 2 9 . 3 + 2 . 8 a~ 3 2 . 1 + 1.7 a 2 9 . 6 + 2 . 7 ~b 2 2 . 3 _+ 1.8 abcd 2 0 . 3 + 1.9 ~d 2 0 . 9 + 1.7 "~d 2 1 . 4 + 2.3 abed 2 3 . 5 + 3 . 0 a~d 2 0 . 2 + 2 . 4 ~d 1 5 . 9 + 1.7 d 2 0 . 0 + 1.4 ~d 2 2 . 0 + 2.2 a~d 1 9 . 2 + 1.8 ~d 2 3 . 0 + 2.8 a~d 2 0 . I + 7 . 1 bcd 2 3 . 5 + 3 . 0 a~d 2 2 . 4 + 4 . 7 a~d 2 6 . 0 + 5 . 0 a~d 2 0 . 1 + 4 . 3 bcd 1 7 . 8 + 2 . 1 cd 2 0 . 4 + 3 . 5 ~d 1 9 . 7 + 2 . 1 ~d
0 . 0 1 c" 0.01 c 0.03 c 0.08 c 0.12 c 0.24 c 1 . 1 8 ab 1.28 a 1 . 3 3 ab 2.51 a 0 . 9 4 bc 1 . 3 1 bc 0.41 c 0.23 c 0.03 c 0.03 c 0.04 c 0.01 c 0.01 c 0.02 c 0.02 c 0.01 c 0.01 c same
17 of
letter
are
not
Discussion Annual cycles of serum testosterone levels along with seasonal variations in t e s t i s s i z e h a v e b e e n e v a l u a t e d in a laboratory colony of male woodchucks (i0), b u t w e a r e u n a w a r e o f comparable d a t a f o r P a n d E 2 in t h e f e m a l e s . Recently, we described the temporal c h a n g e s in s e r u m l e v e l s o f P a n d E 2 d u r i n g t h e e s t r o u s c y c l e in t h i s s p e c i e s in c a p t i v i t y (7). In the present report, we analyzed the changing patterns of P and E2 l e v e l s in s e r u m s a m p l e s t a k e n f r o m u n m a t e d w o o d c h u c k s during the course of a whole year under a constant laboratory setting to substantiate and extend our previous findings describing changes in c i r c u l a t i n g levels of P and E2 during the breeding season.
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T h e h o r m o n a l c h a r a c t e r i s t i c s of t h e a n n u a l r e p r o d u c t i v e c y c l e of t h e f e m a l e w o o d c h u c k c a n be d e f i n e d b y a r i s e of E 2 l e v e l s (29.3 - 32.1 pg/ml) d u r i n g late J a n u a r y t h r o u g h F e b r u a r y , f o l l o w e d by a m o d e s t i n c r e a s e in P c o n c e n t r a t i o n s b y late March, w i t h p e a k P v a l u e s (5.6 - 7.2 ng/ml) d u r i n g A p r i l a n d May. Thus, t h e r e is a t e m p o r a l d i s s o c i a t i o n of p e a k v a l u e s of E 2 a n d P. The e l e v a t i o n in s e r u m P l e v e l s a f t e r the b r e e d i n g s e a s o n a p p e a r e d to c o i n c i d e w i t h a r e d u c t i o n in E 2 levels. The circulating levels of P a n d E 2 were, however, e x t r e m e l y low d u r i n g t h e r e s t of t h e year. T h e s e low l e v e l s of P a n d E 2 m e a s u r e d d u r i n g t h e r e m a i n d e r of t h e a n n u a l r e p r o d u c t i v e c y c l e m i g h t be of p r e d o m i n a n t l y a d r e n a l r a t h e r t h a n o v a r i a n o r i g i n as i n d i c a t e d by C o n c a n n o n et al. (6) a n d by o u r p i l o t s t u d i e s in o v a r i e c t o m i z e d a n i m a l s ( u n p u b l i s h e d data). It is of p a r t i c u l a r i n t e r e s t t h a t d a t a o b t a i n e d f r o m 4 f e m a l e s w h i c h w e r e s a m p l e d for t h e e n t i r e y e a r s h o w e d a s i m i l a r p a t t e r n of a n n u a l f l u c t u a t i o n s in s e r u m P a n d E 2 levels. S e r u m P levels w e r e e x t r e m e l y low (<0.3 ng/ml) f r o m A u g u s t u n t i l t h e b e g i n n i n g of the b r e e d i n g s e a s o n (Feb. I), r o s e to 1.0 n g / m l by t h e s e c o n d h a l f of March, w e r e m a x i m a l (6.8-9.2 ng/ml) d u r i n g t h e p e r i o d of A p r i l a n d May, a n d t h e n d e c l i n e d to 0.6 n g / m l b y July. Conversely, serum E2 levels were higher (28.9-31.7 pg/ml) at the o n s e t (Jan. II) a n d d u r i n g t h e b r e e d i n g s e a s o n t h a n in t h e r e s t of t h e year.
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A I I M I M I I JI JII JI JII AI All SI SII O I O I I N I Fig.
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s e a s o n a l f l u c t u a t i o n s of c i r c u l a t i n g l e v e l s of progesterone (o) a n d e s t r a d i o l (+) in u n m a t e d f e m a l e w o o d c h u c k s k e p t all y e a r u n d e r c o n t r o l l e d l a b o r a t o r y conditions. B l a c k bar i n d i c a t e s the n o r m a l b r e e d i n g s e a s o n of t h e w o o d c h u c k in t h e s o u t h e r n p a r t of its geographical distribution.
0 Nil DI
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B o t h b a s e l i n e a n d p e a k v a l u e s of E 2 l e v e l s v a r i e d w i t h i n as w e l l as b e t w e e n females. T h e p e a k l e v e l s of E 2 (29.3 - 32.1 pg/ml) n o t e d d u r i n g late J a n u a r y and F e b r u a r y in t h i s s t u d y are s i m i l a r to t h o s e r e p o r t e d p r e v i o u s l y (31.4 - 34.4 pg/ml) d u r i n g p r e - e s t r u s a n d e s t r u s (7). It s h o u l d be p o i n t e d o u t t h a t e s t r u s ( c h a r a c t e r i z e d by a c l e a r p r e d o m i n a n c e of c o r n i f i e d c e l l s in t h e v a g i n a l smear) in the m a j o r i t y of f e m a l e s we c a p t u r e d a n d h e l d w a s d e t e c t e d d u r i n g February. Thus, t h e a t t a i n m e n t of m a x i m u m E 2 l e v e l s c o u p l e d w i t h e x t r e m e l y low l e v e l s of P (<0.2 ng/ml) f r o m s e c o n d h a l f of J a n u a r y t h r o u g h F e b r u a r y is m o s t l i k e l y t h e c o n s e q u e n c e of r e s u m e d f o l l i c u l a r g r o w t h in p r e p a r a t i o n for and d u r i n g t h e e s t r o u s p e r i o d (3). C h a n g e s in c i r c u l a t i n g l e v e l s of P in i s o l a t e d c a p t i v e f e m a l e w o o d c h u c k s e x p o s e d to a n a t u r a l p h o t o p e r i o d h a v e b e e n e x a m i n e d p r e v i o u s l y d u r i n g s p r i n g and s u m m e r (4,5). These authors documented that among unmated woodchucks, P levels never r e a c h p e a k l e v e l s o b s e r v e d in p r e g n a n t and p o s t p a r t u m females, but s e r u m P l e v e l s do r i s e s p o n t a n e o u s l y a f t e r the b r e e d i n g s e a s o n and a t t a i n p e a k v a l u e s p r i o r to t h e s u m m e r s o l s t i c e (late A p r i l t h r o u g h e a r l y June). H i g h l e v e l s of s e r u m P o b s e r v e d d u r i n g p r e g n a n c y a n d a f t e r p a r t u r i t i o n are m o s t l y d u e to s e c r e t i o n of c o r p o r a lutea (CL) of p r e g n a n c y (4,6). Increased s e c r e t i o n of p r o g e s t e r o n e by the CL of p r e g n a n c y c o u l d r e f l e c t l u t e o t r o p i c i n f l u e n c e of t h e p l a c e n t a e . F r o m an a d a p t i v e p o i n t of view, t h e p r o l o n g e d s e c r e t i o n of P a f t e r the b r e e d i n g s e a s o n m a y h a v e long t e r m e f f e c t s on the p i t u i t a r y - h y p o t h a l a m i c axis l e a d i n g to a long p e r i o d of a n e s t r u s , t h u s l i m i t i n g t h e b r e e d i n g s e a s o n to a p a r t i c u l a r t i m e of the y e a r w h e n e n v i r o n m e n t a l c o n d i t i o n s are f a v o r a b l e for m a t i n g a n d for s u r v i v a l of t h e young. We o b s e r v e d a s i m i l a r p a t t e r n of s e r u m P c o n c e n t r a t i o n s in w o o d c h u c k s in the p r e s e n t study, e x c e p t t h e r e w a s an a d v a n c e m e n t of the o n s e t of P s u r g e by a p e r i o d of a p p r o x i m a t e l y 2 weeks. T h e s e f i n d i n g s lead us to b e l i e v e t h a t the s e a s o n a l c h a n g e s in c i r c u l a t i n g l e v e l s of P and E 2 m a y h a v e o c c u r r e d in t h e s e c a p t i v e w o o d c h u c k s i n d e p e n d e n t of p h o t o p e r i o d , t e m p e r a t u r e or p r i o r h i b e r n a t i o n , as s u g g e s t e d for a n o t h e r s p e c i e s of s c i u r i d rodent, the g o l d e n - m a n t l e d g r o u n d s q u i r r e l (ii). It s h o u l d be n o t e d t h a t o u r a n i m a l s w e r e c a p t u r e d and h e l d in t h e s o u t h e r n p a r t of t h e i r g e o g r a p h i c a l d i s t r i b u t i o n , w h i l e t h o s e s t u d i e d by C o n c a n n o n et al. (4,5) w e r e c a p t u r e d and h e l d n e a r t h e c e n t e r of their geographical distribution. T h u s t h e d i f f e r e n c e in t h e p r o f i l e of P l e v e l s p r e s u m a b l y r e f l e c t s a s h i f t of t h e n o r m a l b r e e d i n g s e a s o n of w o o d c h u c k s in t h e s o u t h e r n p a r t of t h e i r distribution (7,12). S p o n t a n e o u s e l e v a t i o n s of p l a s m a P l e v e l s a f t e r t h e b r e e d i n g s e a s o n h a v e a l s o b e e n r e p o r t e d in u n m a t e d f e m a l e s of t w o o t h e r s p e c i e s of s c i u r i d rodents, the p r a i r i e dog, C y n o m y s l u d o v i c i a n u s (13) and t h e C a l i f o r n i a g r o u n d squirrel, S p e r m o p h i l u s b e e c h e y i (14). Like woodchucks, f e m a l e s of b o t h t h e s e s p e c i e s a r e m o n o e s t r o u s s e a s o n a l b r e e d e r s and h a v e a single, s h o r t a n n u a l mating season during early spring beginning shortly after emergence from hibernation (13,14). S p o n t a n e o u s l u t e i n i z a t i o n of f o l l i c l e s a c c o m p a n i e d by i n c r e a s e d s e c r e t i o n of P f o l l o w i n g t h e b r e e d i n g s e a s o n w a s a l s o n o t e d in e a r l i e r s t u d i e s on u n m a t e d female woodchucks (4) and the C a l i f o r n i a g r o u n d s q u i r r e l (14). W h e t h e r t h e s e are p o s t - o v u l a t o r y f o l l i c l e s or n o n - o v u l a t o r y l u t e i n i z e d f o l l i c l e s r e m a i n s to be d e t e r m i n e d . The spontaneous
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e l e v a t i o n in c i r c u l a t i n g P after the b r e e d i n g season in u n m a t e d female w o o d c h u c k s w o u l d be consistent with s p o n t a n e o u s o v u l a t i o n in these females. We do know that such o v u l a t i o n can occur under the c o n d i t i o n s we used in the present study (15) but l u t e i n i z a t i o n of u n o v u l a t e d follicles is also possible. Studies aimed at d i s t i n g u i s h i n g between these two p o s s i b i l i t i e s are c u r r e n t l y in progress. In summary, isolated female w o o d c h u c k s m a i n t a i n e d in captivity, under a constant p h o t o p e r i o d and temperature, t h r o u g h o u t the year m a n i f e s t seasonal fluctuations of c i r c u l a t i n g levels of P and E 2 w h i c h correspond very closely to the timing of their normal breeding season in the same g e o g r a p h i c a l location. Acknowledgment This work was supported by NIH grant RR03372(A.W.). The t e c h n i c a l a s s i s t a n c e of Mr. Pat Conley and Mrs. C a t h e r i n e A. H e n e b r y was greatly appreciated. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.
W.J. HAMILTON, Ann. Carnegie Mus. 23, 85-178 (1934). R.L. SNYDER and J.J. CHRISTIAN, Ecology 41, 647-655 (1960). A. RASMUSSEN, E n d o c r i n o l o g y 2, 353-404 (1918). P. CONCANNON, B. BALDWIN, and B. TENNANT, Biol. Reprod. 30, 945-951 (1984). P.W. CONCANNON, L.A. FULLAM, B.H. BALDWIN, and B.C. TENNANT, Biol. Reprod. 41, 255-261 (1989). P. CONCANNON, B. BALDWIN, J. LAWLESS, W. HORNBUCKLE, and B. TENNANT, Biol. Reprod. 29, 1128-1134 (1983). A.P. SINHA HIKIM, A. WOOLF, A. BARTKE, and A.G. AMADOR, Biol. Reprod. 44, 733-738 (1991). A. WOOLF, C. JAMES, and C. GREMILLION-SMITH, Lab. Anim. Sci. 39, 620-622 (1989). A.G. A M A D O R and S.L. HODGES, Comput. Biol. Med., 19, 343-351 (1989). B.H. BALDWIN, B.C. TENNANT, T.J. REIMERS, R.G. COWAN, and P.W. CONCANNON, Biol. Reprod. 32, 804-812 (1985). G.J. KENAGY, J. Comp. Physiol. 135, 333-339 (1980). R.A. YOUNG and E.A.H. SIMS, Lab. Anim. Sci. 29, 770-780 (1979). D. FOREMAN and D. GARRIS, Gen. Comp. Endocrinol. 55, 315-322 (1984). K.E. HOLEKAMP, C. NUNES, and F. TALAMANTES, Biol. Reprod. 39, 1051-1059 (1988). A.G. AMADOR, C. GREMILLION-SMITH, AND A. WOOLF, Biol. Reprod. (suppl. i). 42, 49 (Abstr) (1990).
Pergamon Press
S E A S O N A L V A R I A T I O N S IN CIRCULATING LEVELS OF P R O G E S T E R O N E AND ESTRADIOL IN UNMATED ADULT FEMALE W O O D C H U C K S (MARMOTA MONAX) IN CAPTIVITY A. P. Sinha Hikim L2, A. G. Amador 3, A. Woolf 2, A. Bartke l, and C. G r e m i l l i o n - S m i t h 2 D e p a r t m e n t of Physiology l, School of Medicine, and C o o p e r a t i v e W i l d l i f e R e s e a r c h Laboratory 2, Southern Illinois University, Carbondale, Illinois 62901-6512 D e p a r t m e n t of Obstetrics and G y n e c o l o g y 3, Southern Illinois University, School of Medicine, P.O. Box 19230, Springfield, Illinois 62794-9230 (Received in final form May 28, 1991) Summary
The annual profile of serum levels of p r o g e s t e r o n e (P) and estradiol-17~ (E2) was c h a r a c t e r i z e d in a s e a s o n a l l y breeding rodent, the w o o d c h u c k (Marmota monax). Hormonal levels were d e t e r m i n e d in serum samples taken at weekly or biweekly intervals from u n m a t e d female woodchucks m a i n t a i n e d all year indoors under c o n t r o l l e d conditions of p h o t o p e r i o d and temperature. Annual fluctuations included a rise of E 2 levels during late January through February, followed by a modest increase in plasma P c o n c e n t r a t i o n s by late March, the latter attaining peak values during April and May. A temporal d i s s o c i a t i o n of peak values of c i r c u l a t i n g levels of P and E 2 during the annual r e p r o d u c t i v e cycle was also detected. The timing of changes in serum levels of P and E 2 in these captive w o o d c h u c k s c o r r e s p o n d e d to reproductive events during the normal breeding season of the w o o d c h u c k in the southern part of its range. W o o d c h u c k s are large, seasonally b r e e d i n g sciurid rodents that in the wild typically mate during a 3-week period following emergence from h i b e r n a t i o n during late winter (1,2). In the wild, the timing of reproductive sequence is set by the timing of e m e r g e n c e of females from hibernation; adult males u s u a l l y emerge from h i b e r n a t i o n 1-3 weeks earlier than females and begin b r e e d i n g as soon as females emerge (1,3). At emergence, the ovaries are enlarged and contain multiple, mature follicles (3). H y p e r t r o p h y of many interstitial cells in the ovary along with the a c c u m u l a t i o n of lipids have also been reported (3). There are indications that this seasonal r e p r o d u c t i v e rhythm occurs also in captive woodchucks m a i n t a i n e d under a natural 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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photoperiod, (4,5) .
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even in the absence of prior winter h i b e r n a t i o n
However, little is known about the changes in annual p r o f i l e s of c i r c u l a t i n g levels of p r o g e s t e r o n e (P) and estradiol17~ (E2) d u r i n g the r e p r o d u c t i v e cycle of this species, or about hormonal and r e p r o d u c t i v e cycles under constant laboratory conditions. Serum P levels r e p o r t e d l y are e l e v a t e d d u r i n g p r e g n a n c y and lactation, and during the latter part of the b r e e d i n g season in unmated females (4,6). Temporal changes in serum levels of P and E 2 during the estrous cycle in a laboratory colony of w o o d c h u c k s have been recently d o c u m e n t e d (7). In that study, we noted elevated levels of E 2 a week before and during estrus, followed by a significant decline in E 2 levels one week after estrus. Serum levels of P, however, r e m a i n e d unchanged. The o b j e c t i v e of this study was to analyze the c h a n g i n g p a t t e r n s of c i r c u l a t i n g levels of P and E 2 during a complete annual r e p r o d u c t i v e cycle of w o o d c h u c k s in c a p t i v i t y with special emphasis on the b r e e d i n g season. Materials
& Methods
C o l o n y - h o u s e d adult (2 + yrs.), female w o o d c h u c k s used in this study were live trapped in the vicinity of C a r b o n d a l e (Jackson County), Illinois, during 1988 and 1989. They were h o u s e d i n d i v i d u a l l y in rabbit cages (0.61m x 0.60m x 0.90m) with wood chips or straw as nesting m a t e r i a l s in indoor v i v a r i u m for the p r e v i o u s 6-11 months under controlled laboratory conditions. T e m p e r a t u r e was m a i n t a i n e d at 22 ± 2 ° C and p h o t o p e r i o d at 12 h of light and 12 h of darkness. Rabbit chow (Teklad, Harlan S p r a g u e - D a w l e y Inc., Co., Madison, WI) s u p p l e m e n t e d with fresh v e g e t a b l e s and/or fruits (2-3 times/week) and w a t e r were p r o v i d e d ad libitum. None of the females h i b e r n a t e d in these conditions. B l o o d c o l l e c t i o n s and hormone assays. Blood samples were c o l l e c t e d at w e e k l y or biweekly intervals from 4 adult females e q u i p p e d with s u b c u t a n e o u s v a s c u l a r access ports (8) t h r o u g h o u t the year, except during the breeding season when these animals were bled twice or thrice weekly. Animals were r e s t r a i n e d in devices made of P o l y v i n y l c h l o r i d e pipe (40 cm long w i t h a d i a m e t e r of 15 cm) which avoided need for anesthesia. However, if ports failed, blood was obtained by v e n i p u n c t u r e under K e t a m i n e (Ketaset, Aveco Co., Inc., Fort Dodge, Iowa) - x y l a z i n e (Rompun, Haver, Mobay Corp., Kansas) anesthesia. W e e k l y samples were also obtained from 12 females from January 16 until March 31 under K e t a m i n e - x y l a z i n e anesthesia and from 4 a d d i t i o n a l females e q u i p p e d with s u b c u t a n e o u s vascular access ports during April through July. No s i g n i f i c a n t (P<0.05) d i f f e r e n c e s (student's t test) in the c i r c u l a t i n g levels of P or E 2 were noted between samples o b t a i n e d from females fitted with v a s c u l a r ports and samples o b t a i n e d from w o o d c h u c k s under k e t a m i n e - x y l a z i n e anesthesia. Therefore, hormonal data from all animals were c o m b i n e d for further statistical analysis and presentation. For each month, samples were divided into two groups, those c o l l e c t e d in the first half of the month, d e s i g n a t e d Jan I, Feb I, etc., and those c o l l e c t e d in the second half of the month, r e f e r r e d to as Jan II, Feb II, etc. Numbers of animals used for hormonal analysis in each time period are listed in Table i.
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S e r u m w a s o b t a i n e d by c e n t r i f u g a t i o n a f t e r a l l o w i n g t h e b l o o d to c l o t a n d t h e n f r o z e n (-20 ° C) u n t i l assayed. M e a s u r e m e n t s of s e r u m levels of P and E 2 w e r e p e r f o r m e d by s o l i d p h a s e RIAs, u s i n g k i t s w i t h p o l y p r o p y l e n e t u b e s c o a t e d w i t h a n t i P or a n t i - E 2 a n t i b o d i e s ( D i a g n o s t i c P r o d u c t s Corp., Los A n g e l e s , CA) as d e s c r i b e d p r e v i o u s l y (7). T h e t r a c e r s u s e d w e r e n5 I - P a n d n5 I - E2, r e s p e c t i v e l y . D a t a w e r e t h e n o b t a i n e d by R I A P L O T a n d R I A D O S E p r o g r a m s for R I A a n a l y s i s (9). Since these kits were d e s i g n e d for u s e w i t h h u m a n s a m p l e s a n d c o n t a i n a s t a n d a r d c u r v e b a s e d on h u m a n serum, t h e p a r a l l e l i s m b e t w e e n l o g - l o g i t c u r v e s m a d e by a d d i n g d i f f e r e n t v o l u m e s f r o m a p o o l of w o o d c h u c k s e r u m and t h e r e s p e c t i v e s t a n d a r d c u r v e s w e r e d e t e r m i n e d for P ( w o o d c h u c k serum: slope, -1.772; y - i n t e r c e p t , 0.414; r = - l . 0 0 0 ; standard: slope, -1.763; y - i n t e r c e p t , 0.417; r = - l . 0 0 0 ) and E 2 ( w o o d c h u c k serum: slope, -1.861; y - i n t e r c e p t , 4.186; r = - l . 0 0 0 ; standard: slope, -1.863; y - i n t e r c e p t , 4.188; r = - 0 . 9 9 4 ) . The d e t e c t i o n l i m i t s for P and E 2 w e r e 25 p g / m l and 4 pg/ml, respectively. Statistical analysis. D a t a are p r e s e n t e d as m e a n s ± SE. S t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d u s i n g SAS ( S t a t i s t i c a l A n a l y s i s S y s t e m I n s t i t u t e Inc., Carry, NC) program. Results were t e s t e d for s t a t i s t i c a l s i g n i f i c a n c e u s i n g D u n c a n ' s M u l t i p l e R a n g e T e s t f o l l o w i n g a o n e - w a y a n a l y s i s of v a r i a n c e (ANOVA). W h e n the F - t e s t f r o m A N O V A i n d i c a t e d s i g n i f i c a n c e , d i f f e r e n c e s of t h e m e a n s w e r e c a l c u l a t e d u s i n g D u n c a n ' s M u l t i p l e R a n g e Test. D i f f e r e n c e s w e r e c o n s i d e r e d s i g n i f i c a n t if P<0.05. Results C h a n g e s in the a n n u a l p r o f i l e of s e r u m l e v e l s of P a n d E 2 in a d u l t u n m a t e d f e m a l e w o o d c h u c k s m a i n t a i n e d in c a p t i v i t y are p r e s e n t e d in T a b l e i. G r a p h i c s u m m a r y of t h e s e d a t a p r o v i d e d in F i g u r e 1 s h o w the g e n e r a l t r e n d s in c i r c u l a t i n g l e v e l s of P (F=8.10; df=22; P < 0 . 0 0 1 and E 2 (F=2.75; df=22; P<0.002) d u r i n g the a n n u a l r e p r o d u c t i v e cycle. M e a n levels of c i r c u l a t i n g P w e r e e x t r e m e l y low (<0.2 ng/ml) f r o m A u g u s t u n t i l t h e e n d of F e b r u a r y . T h e y r o s e to 0.6 n g / m l by the s e c o n d h a l f of March, w e r e m a x i m a l (5.6-7.2 ng/ml) d u r i n g the p e r i o d of A p r i l and M a y b e f o r e d e c l i n i n g a g a i n in J u l y (0.5-0.7 ng/ml). V a l u e s for P r e p o r t e d d u r i n g A p r i l and M a y w e r e s i g n i f i c a n t l y (P<0.05) d i f f e r e n t t h a n the v a l u e s o b t a i n e d d u r i n g the r e s t of t h e year. However, the v a l u e s t h a t w e r e o b t a i n e d d u r i n g the m o n t h of J u n e w e r e not s i g n i f i c a n t l y d i f f e r e n t f r o m v a l u e s r e c o r d e d for A p r i l I a n d M a y I. D u r i n g t h e p e a k p e r i o d of P secretion, s e r u m l e v e l s r a n g e d f r o m 3.4 to 13.0 n g / m l and f r o m 4.2 to 19.5 n g / m l for t h e m o n t h s of A p r i l and May, r e s p e c t i v e l y in 5 out of 7 a n i m a l s studied. O n e f e m a l e h a d o n l y m i n o r e l e v a t i o n in P (0.3 - 1.6 ng/ml) d u r i n g A p r i l a n d a n o t h e r f e m a l e also h a d low levels of P (0.i - 0.4 ng/ml) d u r i n g the m o n t h of May. The m a x i m a l v a l u e s of P v a r i e d w i t h i n as w e l l as b e t w e e n females. U n l i k e the r e s u l t s for P, t h e r e w e r e o n l y m o d e s t s e a s o n a l c h a n g e s in c i r c u l a t i n g levels of E 2 d u r i n g t h e a n n u a l r e p r o d u c t i v e cycle. S e r u m E 2 levels w e r e low ( 1 5 . 9 - 2 6 . 0 pg/ml) d u r i n g m o s t of the y e a r (from M a r c h to J a n u a r y I), b e g a n to r i s e (29.3 pg/ml) at the o n s e t of the b r e e d i n g s e a s o n ( J a n u a r y II) a n d r e m a i n e d e l e v a t e d at or a b o v e t h i s level u n t i l late F e b r u a r y . V a l u e s o b t a i n e d d u r i n g F e b I (32.1 ± 1.7) w e r e s i g n i f i c a n t l y
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(P<0.05) higher than the values obtained during Jan I (20.7 ± 3.0 pg/ml). However, differences in m e a n v a l u e s o f E 2 b e t w e e n e a r l y ( 2 0 . 7 ± 3 . 0 p g / m l ) a n d l a t e (29.3 + 2 . 8 p g / m l ) J a n u a r y w e r e n o t statistically significant. A significant (P<0.05) decline in E 2 levels was noted between early February and late March. Serum concentrations of E2 were highly variable within as well as between females during our study periods. For example, variations in E 2 l e v e l s d u r i n g J a n II, F e b I a n d F e b II, r a n g e d from 19.3 to 44.6, from 21.1 to 44.8, and from 21.9 to 52.6 pg/ml, respectively. TABLE Annual Profile of in Woodchucks. animals. Sampling periods Jan Jan Feb Feb Mar Mar Apr Apr May May Jun Jun Jul Jul Aug Aug Sep Sep Oct Oct Nov Nov Dec
I II I II I II I II I II I II I II I II I II I II I II I
i.
Serum Levels of Progesterone and Estradiol Numbers in p a r e n t h e s i s indicate the number
(3) (14) (14) (15) (15) (15) (7) (7) (7) (7) (7) (7) (6) (6) (3) (3) (3) (3) (3) (3) (3) (3) (3)
* In each column, means significantly different
Progesterone (ng/ml) 0.06 0.07 0. i0 0.17 0.29 0.55 5.57 6.61 5 73 7 24 2 85 2 69 0 69 0 49 0 Ii 0 09 0 ii 0 09 0 i0 0 08 0 09 0 05 0.04 with
the
+ + + + + + + + + + + + + + + + + + + + + + +
-
Estradiol (pg/ml) 2 0 . 7 + 3 . 0 0 bcd 2 9 . 3 + 2 . 8 a~ 3 2 . 1 + 1.7 a 2 9 . 6 + 2 . 7 ~b 2 2 . 3 _+ 1.8 abcd 2 0 . 3 + 1.9 ~d 2 0 . 9 + 1.7 "~d 2 1 . 4 + 2.3 abed 2 3 . 5 + 3 . 0 a~d 2 0 . 2 + 2 . 4 ~d 1 5 . 9 + 1.7 d 2 0 . 0 + 1.4 ~d 2 2 . 0 + 2.2 a~d 1 9 . 2 + 1.8 ~d 2 3 . 0 + 2.8 a~d 2 0 . I + 7 . 1 bcd 2 3 . 5 + 3 . 0 a~d 2 2 . 4 + 4 . 7 a~d 2 6 . 0 + 5 . 0 a~d 2 0 . 1 + 4 . 3 bcd 1 7 . 8 + 2 . 1 cd 2 0 . 4 + 3 . 5 ~d 1 9 . 7 + 2 . 1 ~d
0 . 0 1 c" 0.01 c 0.03 c 0.08 c 0.12 c 0.24 c 1 . 1 8 ab 1.28 a 1 . 3 3 ab 2.51 a 0 . 9 4 bc 1 . 3 1 bc 0.41 c 0.23 c 0.03 c 0.03 c 0.04 c 0.01 c 0.01 c 0.02 c 0.02 c 0.01 c 0.01 c same
17 of
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Discussion Annual cycles of serum testosterone levels along with seasonal variations in t e s t i s s i z e h a v e b e e n e v a l u a t e d in a laboratory colony of male woodchucks (i0), b u t w e a r e u n a w a r e o f comparable d a t a f o r P a n d E 2 in t h e f e m a l e s . Recently, we described the temporal c h a n g e s in s e r u m l e v e l s o f P a n d E 2 d u r i n g t h e e s t r o u s c y c l e in t h i s s p e c i e s in c a p t i v i t y (7). In the present report, we analyzed the changing patterns of P and E2 l e v e l s in s e r u m s a m p l e s t a k e n f r o m u n m a t e d w o o d c h u c k s during the course of a whole year under a constant laboratory setting to substantiate and extend our previous findings describing changes in c i r c u l a t i n g levels of P and E2 during the breeding season.
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T h e h o r m o n a l c h a r a c t e r i s t i c s of t h e a n n u a l r e p r o d u c t i v e c y c l e of t h e f e m a l e w o o d c h u c k c a n be d e f i n e d b y a r i s e of E 2 l e v e l s (29.3 - 32.1 pg/ml) d u r i n g late J a n u a r y t h r o u g h F e b r u a r y , f o l l o w e d by a m o d e s t i n c r e a s e in P c o n c e n t r a t i o n s b y late March, w i t h p e a k P v a l u e s (5.6 - 7.2 ng/ml) d u r i n g A p r i l a n d May. Thus, t h e r e is a t e m p o r a l d i s s o c i a t i o n of p e a k v a l u e s of E 2 a n d P. The e l e v a t i o n in s e r u m P l e v e l s a f t e r the b r e e d i n g s e a s o n a p p e a r e d to c o i n c i d e w i t h a r e d u c t i o n in E 2 levels. The circulating levels of P a n d E 2 were, however, e x t r e m e l y low d u r i n g t h e r e s t of t h e year. T h e s e low l e v e l s of P a n d E 2 m e a s u r e d d u r i n g t h e r e m a i n d e r of t h e a n n u a l r e p r o d u c t i v e c y c l e m i g h t be of p r e d o m i n a n t l y a d r e n a l r a t h e r t h a n o v a r i a n o r i g i n as i n d i c a t e d by C o n c a n n o n et al. (6) a n d by o u r p i l o t s t u d i e s in o v a r i e c t o m i z e d a n i m a l s ( u n p u b l i s h e d data). It is of p a r t i c u l a r i n t e r e s t t h a t d a t a o b t a i n e d f r o m 4 f e m a l e s w h i c h w e r e s a m p l e d for t h e e n t i r e y e a r s h o w e d a s i m i l a r p a t t e r n of a n n u a l f l u c t u a t i o n s in s e r u m P a n d E 2 levels. S e r u m P levels w e r e e x t r e m e l y low (<0.3 ng/ml) f r o m A u g u s t u n t i l t h e b e g i n n i n g of the b r e e d i n g s e a s o n (Feb. I), r o s e to 1.0 n g / m l by t h e s e c o n d h a l f of March, w e r e m a x i m a l (6.8-9.2 ng/ml) d u r i n g t h e p e r i o d of A p r i l a n d May, a n d t h e n d e c l i n e d to 0.6 n g / m l b y July. Conversely, serum E2 levels were higher (28.9-31.7 pg/ml) at the o n s e t (Jan. II) a n d d u r i n g t h e b r e e d i n g s e a s o n t h a n in t h e r e s t of t h e year.
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s e a s o n a l f l u c t u a t i o n s of c i r c u l a t i n g l e v e l s of progesterone (o) a n d e s t r a d i o l (+) in u n m a t e d f e m a l e w o o d c h u c k s k e p t all y e a r u n d e r c o n t r o l l e d l a b o r a t o r y conditions. B l a c k bar i n d i c a t e s the n o r m a l b r e e d i n g s e a s o n of t h e w o o d c h u c k in t h e s o u t h e r n p a r t of its geographical distribution.
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B o t h b a s e l i n e a n d p e a k v a l u e s of E 2 l e v e l s v a r i e d w i t h i n as w e l l as b e t w e e n females. T h e p e a k l e v e l s of E 2 (29.3 - 32.1 pg/ml) n o t e d d u r i n g late J a n u a r y and F e b r u a r y in t h i s s t u d y are s i m i l a r to t h o s e r e p o r t e d p r e v i o u s l y (31.4 - 34.4 pg/ml) d u r i n g p r e - e s t r u s a n d e s t r u s (7). It s h o u l d be p o i n t e d o u t t h a t e s t r u s ( c h a r a c t e r i z e d by a c l e a r p r e d o m i n a n c e of c o r n i f i e d c e l l s in t h e v a g i n a l smear) in the m a j o r i t y of f e m a l e s we c a p t u r e d a n d h e l d w a s d e t e c t e d d u r i n g February. Thus, t h e a t t a i n m e n t of m a x i m u m E 2 l e v e l s c o u p l e d w i t h e x t r e m e l y low l e v e l s of P (<0.2 ng/ml) f r o m s e c o n d h a l f of J a n u a r y t h r o u g h F e b r u a r y is m o s t l i k e l y t h e c o n s e q u e n c e of r e s u m e d f o l l i c u l a r g r o w t h in p r e p a r a t i o n for and d u r i n g t h e e s t r o u s p e r i o d (3). C h a n g e s in c i r c u l a t i n g l e v e l s of P in i s o l a t e d c a p t i v e f e m a l e w o o d c h u c k s e x p o s e d to a n a t u r a l p h o t o p e r i o d h a v e b e e n e x a m i n e d p r e v i o u s l y d u r i n g s p r i n g and s u m m e r (4,5). These authors documented that among unmated woodchucks, P levels never r e a c h p e a k l e v e l s o b s e r v e d in p r e g n a n t and p o s t p a r t u m females, but s e r u m P l e v e l s do r i s e s p o n t a n e o u s l y a f t e r the b r e e d i n g s e a s o n and a t t a i n p e a k v a l u e s p r i o r to t h e s u m m e r s o l s t i c e (late A p r i l t h r o u g h e a r l y June). H i g h l e v e l s of s e r u m P o b s e r v e d d u r i n g p r e g n a n c y a n d a f t e r p a r t u r i t i o n are m o s t l y d u e to s e c r e t i o n of c o r p o r a lutea (CL) of p r e g n a n c y (4,6). Increased s e c r e t i o n of p r o g e s t e r o n e by the CL of p r e g n a n c y c o u l d r e f l e c t l u t e o t r o p i c i n f l u e n c e of t h e p l a c e n t a e . F r o m an a d a p t i v e p o i n t of view, t h e p r o l o n g e d s e c r e t i o n of P a f t e r the b r e e d i n g s e a s o n m a y h a v e long t e r m e f f e c t s on the p i t u i t a r y - h y p o t h a l a m i c axis l e a d i n g to a long p e r i o d of a n e s t r u s , t h u s l i m i t i n g t h e b r e e d i n g s e a s o n to a p a r t i c u l a r t i m e of the y e a r w h e n e n v i r o n m e n t a l c o n d i t i o n s are f a v o r a b l e for m a t i n g a n d for s u r v i v a l of t h e young. We o b s e r v e d a s i m i l a r p a t t e r n of s e r u m P c o n c e n t r a t i o n s in w o o d c h u c k s in the p r e s e n t study, e x c e p t t h e r e w a s an a d v a n c e m e n t of the o n s e t of P s u r g e by a p e r i o d of a p p r o x i m a t e l y 2 weeks. T h e s e f i n d i n g s lead us to b e l i e v e t h a t the s e a s o n a l c h a n g e s in c i r c u l a t i n g l e v e l s of P and E 2 m a y h a v e o c c u r r e d in t h e s e c a p t i v e w o o d c h u c k s i n d e p e n d e n t of p h o t o p e r i o d , t e m p e r a t u r e or p r i o r h i b e r n a t i o n , as s u g g e s t e d for a n o t h e r s p e c i e s of s c i u r i d rodent, the g o l d e n - m a n t l e d g r o u n d s q u i r r e l (ii). It s h o u l d be n o t e d t h a t o u r a n i m a l s w e r e c a p t u r e d and h e l d in t h e s o u t h e r n p a r t of t h e i r g e o g r a p h i c a l d i s t r i b u t i o n , w h i l e t h o s e s t u d i e d by C o n c a n n o n et al. (4,5) w e r e c a p t u r e d and h e l d n e a r t h e c e n t e r of their geographical distribution. T h u s t h e d i f f e r e n c e in t h e p r o f i l e of P l e v e l s p r e s u m a b l y r e f l e c t s a s h i f t of t h e n o r m a l b r e e d i n g s e a s o n of w o o d c h u c k s in t h e s o u t h e r n p a r t of t h e i r distribution (7,12). S p o n t a n e o u s e l e v a t i o n s of p l a s m a P l e v e l s a f t e r t h e b r e e d i n g s e a s o n h a v e a l s o b e e n r e p o r t e d in u n m a t e d f e m a l e s of t w o o t h e r s p e c i e s of s c i u r i d rodents, the p r a i r i e dog, C y n o m y s l u d o v i c i a n u s (13) and t h e C a l i f o r n i a g r o u n d squirrel, S p e r m o p h i l u s b e e c h e y i (14). Like woodchucks, f e m a l e s of b o t h t h e s e s p e c i e s a r e m o n o e s t r o u s s e a s o n a l b r e e d e r s and h a v e a single, s h o r t a n n u a l mating season during early spring beginning shortly after emergence from hibernation (13,14). S p o n t a n e o u s l u t e i n i z a t i o n of f o l l i c l e s a c c o m p a n i e d by i n c r e a s e d s e c r e t i o n of P f o l l o w i n g t h e b r e e d i n g s e a s o n w a s a l s o n o t e d in e a r l i e r s t u d i e s on u n m a t e d female woodchucks (4) and the C a l i f o r n i a g r o u n d s q u i r r e l (14). W h e t h e r t h e s e are p o s t - o v u l a t o r y f o l l i c l e s or n o n - o v u l a t o r y l u t e i n i z e d f o l l i c l e s r e m a i n s to be d e t e r m i n e d . The spontaneous
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e l e v a t i o n in c i r c u l a t i n g P after the b r e e d i n g season in u n m a t e d female w o o d c h u c k s w o u l d be consistent with s p o n t a n e o u s o v u l a t i o n in these females. We do know that such o v u l a t i o n can occur under the c o n d i t i o n s we used in the present study (15) but l u t e i n i z a t i o n of u n o v u l a t e d follicles is also possible. Studies aimed at d i s t i n g u i s h i n g between these two p o s s i b i l i t i e s are c u r r e n t l y in progress. In summary, isolated female w o o d c h u c k s m a i n t a i n e d in captivity, under a constant p h o t o p e r i o d and temperature, t h r o u g h o u t the year m a n i f e s t seasonal fluctuations of c i r c u l a t i n g levels of P and E 2 w h i c h correspond very closely to the timing of their normal breeding season in the same g e o g r a p h i c a l location. Acknowledgment This work was supported by NIH grant RR03372(A.W.). The t e c h n i c a l a s s i s t a n c e of Mr. Pat Conley and Mrs. C a t h e r i n e A. H e n e b r y was greatly appreciated. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.
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