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Effects of estradiol on secretion of LH, hypothalamic function and testicular development in bull calves
DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 5(4):307-316, 1988
EFFECTS OF ESTRADIOL ON SECRETION OF LH, HYPOTHALAMIC FUNCTION AND TESTICULAR DEVELOPMENT IN BULL CALVES 1 D.R. Deaver2, J.D. Glass 3, D.M. Grieger4 and J.J. Reeves4 Department of Dairy and Animal Sciences, Penn State University2 Department of Biological Sciences, Kent State University3 Department of Animal Science, Washington State University4 Received April 25, 1988 ABSTRACT Two experiments were conducted in order to determine the effects of estradiol (E2) on the development of the hypothalamic-pituitary-testicular axis in bull calves. In experiment 1, calves were assigned randomly to one of the following groups: 1) intact, 2) intact E2-treated, 3) castrated, or 4) castrated E2-treated. Treatments began when the calves were 7.5 wk of age and continued for 16.5 wk. Samples of blood were collected once a week from 3 to 14 wk of age and every 10 rain for 6 hr at 8, 12 and 16 wk of age. Concentrations of E 2 in plasma decreased between 3 and 4 wk of age and were further reduced by castration. Maximum concentrations of E2 (24.3 pg/ml) were observed 72 hr after insertion of E2 implants, however, plasma E2 stablized at 5-9 pg/ml by 2 wk after insertion of implants. Treatment with E 2 eliminated the pulsatile secretion of LH in intact and castrated calves and retarded testicular growth. In experiment 2, calves were assigned to a control (n----4) or E2-treated ( n = 6 ) group. Implants of E 2 w e r e inserted at 7.5 wk of age. At 24 wk of age, calves were bled and then sacrificed to collect hypothalamic and pituitary tissues. Age-related changes in testicular weight and secretion of LH were blocked by E 2. Neither the morphology nor the intensity of immunostaining of GnRH nerve cell bodies in the preoptic area (POA) were affected by E2. However, the density of GnRI-I fibers and beads in the stalk median eminence (SME), and concentrations of pituitary GnRH receptors were greater (P<.01) in E2treated compared to control calves. In addition, concentrations of norepinephrine (NE) in the SME were lower in E2-treated calves when compared to controls. Based on these observations, it is concluded that administeration of E 2 a t 7.5 wk of age causes profound alterations in hypothalamic function including, changes in metabolism of NE and suppression of GnRH release. INTRODUCTION The t r a n s i t i o n b e t w e e n t h e i n f a n t i l e a n d p r e p u b e r t a l stages o f d e v e l o p m e n t is c h a r a c t e r i z e d b y a d r a m a t i c i n c r e a s e in t h e p u l s a t i l e s e c r e t i o n of LH in f e m a l e ( 1 , 2 ) a n d m a l e ( 3 , 4 ) r u m i n a n t s . In r a m l a m b s i n c r e a s e d s e c r e t i o n of LH o c c u r s p r i o r to 5 w k o f age, w h i l e in c a l v e s a m a r k e d i n c r e a s e in e p i s o d i c s e c r e t i o n o c c u r s b e t w e e n 8 a n d 12 w k of age. This c h a n g e in t h e f r e q u e n c y o f LH r e l e a s e is t h o u g h t to i n i t i a t e d i f f e r e n t i a t i o n a n d d e v e l o p m e n t o f t e s t i c u l a r c e l l s t h a t p a r t i c i p a t e in s p e r m a t o g e n e s i s ( 5 ) . C a s t r a t i o n of t h e n e o n a t a l l a m b or c a l f c a u s e s p r e c o c i o u s d e v e l o p m e n t of p u l s a t i l e LH r e l e a s e ( 1 , 3 ) . H o w e v e r , t h e t i m e r e q u i r e d to o b s e r v e an i n c r e a s e in LH p u l s e f r e q u e n c y f o l l o w i n g c a s t r a t i o n is l o n g e r for i n f a n t i l e than p e r i p u b e r t a l or a d u l t r u m i n a n t s ( 1 , 6 , 7 ) . It a p p e a r s that a d e c r e a s e in s e n s i t i v i t y o f t h e h y p o t h a l a m u s to n e g a t i v e f e e d b a c k effects o f E 2 p r e c e d e s t h e i n c r e a s e d s e c r e t i o n of IM in e w e l a m b s ( 2 ) a n d h e i f e r s ( 8 ) . A m a n n et al ( 9 ) have r e p o r t e d t h a t c i r c u l a t i n g c o n c e n t r a t i o n s of E 2 in p l a s m a a n d n u m b e r s of h y p o t h a l a m i c r e c e p t o r s for E 2 d e c r e a s e b e f o r e t h e i n c r e a s e d s e c r e t i o n of LH in b u l l calves.
Copyright© 1988by DOMENDO,INC.
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In contrast, in a n o t h e r study (10) plasma concentrations of Ez w e r e not observed to change p r i o r to the initial rise in LH. Although the administration of E2 has b e e n s h o w n to inhibit the s e c r e t i o n of LH in p e r i p u b e r t a l bulls ( 1 1 , 1 2 ) , t r e a t m e n t o f infantile calves w i t h E2 has b e e n r e p o r t e d to inhibit (1 O) or have no effect (13) on the secretion of LH. Two e x p e r i m e n t s w e r e c o n d u c t e d to investigate the possible role(s) of E2 in controlling the secretion of LH in infantile and p r e p u b e r t a l calves. In the first e x p e r i m e n t the objectives were: 1) to d e t e r m i n e if c o n c e n t r a t i o n s of E2 changed with age and following castration and 2) to evaluate the effects of estradiol on testicular d e v e l o p m e n t and the o n t o g e n y of pulsatile secretion of LH in intact and castrated calves. The p u r p o s e of the s e c o n d e x p e r i m e n t was to examine changes in the function o f GnRH and m o n o a m i n e r g i c h y p o t h a l a m i c neurons and pituitary concentrations of GnRH r e c e p t o r s during treatment w i t h Ez. MATERIALS AND METHODS E x p e r i m e n t 1. T w e n t y Holstein bull calves w e r e p u r c h a s e d at 3-5 days of age and raised at the Dairy Breeding Research Center as previously described ( 1 4 ) . At 7 w k o f age, calves w e r e assigned at r a n d o m to one o f four treatments: 1) intact ( c o n t r o l ) , 2) intact and E2, 3) castrated or 4) castrated and E2. Castrations w e r e p e r f o r m e d a n d / o r a 30 m m C o m p u d o s e T M implant containing E2 was inserted into the right ear at 7.5 w k of age. Testes from castrated calves w e r e w e i g h e d for c o m p a r i s o n w i t h those obtained at 24 w k of age. Between 3 and 14 wk, o f age samples o f b l o o d (10 ml) were c o l l e c t e d b y jugular v e n i p u n c t u r e every 7 days. In addition, b l o o d samples w e r e c o l l e c t e d using jugular catheters every 10 min for 6 hr at 8, 12 and 16 w k o f age from all calves and at 24 w k o f age from control and castrated calves receiving E2. Plasma was c o l l e c t e d and stored at - - 2 0 C until assayed for E 2 o r LH. At 24 w k o f age intact animals w e r e castrated and testicular weights recorded. E x p e r i m e n t 2. Holstein calves used for this study w e r e purchased at 3-5 days o f age and raised as in e x p e r i m e n t 1. Calves w e r e assigned at r a n d o m to a c o n t r o l ( n = 4 ) or Ez-treated ( n = 6 ) group. At 7.5 wk o f age, a C o m p u d o s e T M implant was cut to a length o f 15 m m and inserted into the right ear of a p p r o p r i a t e calves. W h e n calves w e r e 24 wk old b l o o d samples w e r e obtained e v e r y 10 min for 6 hr using i n d w e l l i n g jugular catheters; they w e r e sacrified the f o l l o w i n g day by exsanquination. The POA-hypothalamic tissues (including the SME) w e r e bissected along the midline. The right side of the SME was o b t a i n e d and stored in liquid N2 and the left POA, hypothalamus and SME w e r e p l a c e d in modified Zamboni's fixative, h e l d at 4C for two days, transferred to .01 M p h o s p h a t e buffer and stored at 4C until p r o c e s s e d for quantitative i m m u n o c y t o c h e m i c a l p r o c e d u r e s as d e s c r i b e d below. A s s a y P r o c e d u r e s . Concentrations of LH in plasma w e r e d e t e r m i n e d using a p r e v i o u s l y d e s c r i b e d RIA ( 1 5 ) . The sensitivity o f the assay was 25 p g / t u b e (USDA-bLH-B-5) and samples w e r e assayed at a v o l u m e of 100 gtl. The interand intra-assay coefficients of variation w e r e less than 10%. C o n c e n t r a t i o n s of plasma estradiol w e r e d e t e r m i n e d using immunoreagents o b t a i n e d from Serono Diagonstics, Inc. (Norwell, MA). Plasma ( 2 0 0 Ixl) was e x t r a c t e d t w i c e w i t h 2 ml of diethyl ether. Ether extracts w e r e placed into 12 )< 75 borosilicate tubes and e v a p o r a t e d to dryness. The residue was rec o n s t i t u t e d w i t h buffer ( 2 0 0 ~tl) and tubes v o x t e x e d vigorously. After vortexing, 50 gtl of 125I-E2 (containing a p p r o x i m a t e l y 15,000 d p m ) and 50 ~tl of rabbit anti-E2 antisera ( w h i c h b o u n d 40% of the total counts) w e r e a d d e d to each
HYPOTHALAMO-HYPOPHYSEAL-TESTICULAR AXIS IN CALVES
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tube. These reagents were incubated at room temperature for 3 hr then sheep anti-rabbit serum (100 !11) and 20% polyethyleneglycol (1 ml) were added to precipitate the bound fraction. After incubating for 20 min, the tubes were centrifuged for 20 min at 1200 X g and the supernatant aspirated and the radioactivity in pellet counted. The sensitivity of the assay was 0.1 pg/tube (calculated as 3 X the standard deviation of Bo counts) or 0.5 pg/ml. Recovery of 5 pg of exogenous E 2 added to 200 III of steer plasma averaged over 90%. Samples ranging in concentrations from 2 to 15 pg/ m l were extracted at volumes o f 100, 200 and 400 gtl and each individual dose response was parallel to the standard curve. The intra- and inter-assay coefficients of variation were less than 9%. A high pressure liquid chromatograph e q u i p p e d with an electrochemical detector (Bioanalytical Systems, Lafayette, IN) was used to determine concentraitons of tissue nor e pi ne phr i ne (NE), dopamine (DA), 3,4-diphenylhydroxyacetic acid (DOPAC), 4-hydroxy-3-methoxy-phenylaceticacid (HVA), serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA). The assay was established and validated as previously described (16) with minor modifications. Tissues were homogenized using a Brinkman polytron (3 sec at a setting of 6) in 500 gtl of .05 M HCI04 containing .1% cysteine. Homogenates were centrifuged at 11,000 X g and then filtered through .2 gM filter (Gilman ARCO LC13, Thomas Scientific Co., Philadelphia, PA) prior to injection onto a 5-gin C-18 column (ODS, Biophase 250 X 4.6 mm, Bioanalytical Systems). Amines used for standards were obtained from Sigma Chemical Co (St. Louis, MO) and all results were expressed as free-base equivalents. The mobile phase consisted of 14.2 g monochloroacetic acid (MCAA), 4.68 g NaOH, 200 mg sodiumoctyl sulfate in 1 L of distilled H20. Mobile phase was adjusted to a pH of 3.0 using solid NaOH or MCAA, filtered through a 0.2 gM filter and degassed prior to use. Acetonitrile (35 ml/L) and tetrahydrofuran (18 ml/L) were added to the mobile phase after degassing. A glassy carbon eletrode, set at a potential of 750 mV relative to a AgCI reference electrode, was used as the detector. The flow rate o f the mobile phase was 1.0 ml/min. Immuno-reactive GnRH neurons were localized using the method previously described (17). Serial 100 ~tm-thick sagittal sections were made 2mm lateral from the midline to the ventricular wall using a vibratome (La.ncer Ted Pella Inc., Justin, CA). Due to the dense connective tissue within the SME, this structure was separated from the basal hypothalamus and embedded in gelatin for sectioning. The SME was sectioned longitudinally at 100 gtm thickness, and every section was saved for immunochemical staining for GnRH. Uniformity of staining was optimized by processing the sections in 24-well cell culture plates (Primaria, Becton Dickinson, Balto, Md.). Sections were incubated with primary antiserum GnRH (Nett ~tR38; 1:1000), secondary antiserum (sheep antirabbit gamma globulin; 1:15) and then with rabbit peroxidase-antiperoxidase c o m p l e x (Sigma Chem Co., St. Louis; 1:100). Sections were reacted with diaminobenzidine containing H202 and counter stained with methylene blue. The GnRH neuronal system was then mapped and the morphology and OD of immunostaining determined according to Glass (18). Optical density of cell bodies (an index of content of GnRH) was measured using an optical densitometer attached to the microscope. The density of GnRH fibers in the SME was determined by grid analysis of the number of intersections of positive fibers with the reticules of a 10 X 10 mm ocular grid placed over the SME. Concentration of GnRH receptors in the anterior pituitary were determined as previously described (19).
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S t a t i s t i c a l A n a l y s e s . T h e f r e q u e n c y a n d a m p l i t u d e o f LH discharges w e r e d e t e r m i n e d using the PULSAR p r o g r a m (Merriam and Wachter, 1982). Effects o f t r e a t m e n t ( m a i n p l o t ) , age and the i n t e r a c t i o n o f age X t r e a t m e n t ( s u b p l o t effects; e x p e r i m e n t 1) or t r e a t m e n t ( e x p e r i m e n t 2) on variables e x a m i n e d w e r e tested for using the general linear m o d e l p r o c e d u r e of SAS (16); differences a m o n g main-effect m e a n s w e r e f u r t h e r e v a l u a t e d using the PDIFF p r o c e d u r e . RESULTS E x p e r i m e n t 1. B e t w e e n 3 a n d 4 w k o f age, c o n c e n t r a t i o n s o f E2 d e c r e a s e d in p l a s m a (Figure 1; P < . 0 1 ) . C o n c e n t r a t i o n s o f E2 in jugular p l a s m a averaged 24 p g / m l in s a m p l e s c o l l e c t e d 72 hr after insertion of E2-implants (8 w k of age). H o w e v e r , w i t h i n 2 wk, E2 in jugular p l a s m a stablized at a p p r o x i m a t e l y 5-9 p g / m l . B e t w e e n 8 and 14 w k o f age, p l a s m a E2 was higher in E2-treated calves t h a n in intact (1.5 +_ 0.3) or castrated (0.6 _+ .2 p g / m l ) calves not g i v e n implants. The c o n c e n t r a t i o n of E2 was significantly l o w e r ( P < . 0 5 ) in castrated calves w h e n c o m p a r e d to the intact calves not r e c e i v i n g estradiol. Effects o f age and castration on s e c r e t i o n o f LH are s h o w n in Table 1. The f r e q u e n c y o f e p i s o d i c LH release in intact and castrated calves was similar at 8 w k o f age ( 7 2 hr post-castration). H o w e v e r , at 12 and 16 w k of age the f r e q u e n c y o f pulsatile LH release was g r e a t e r in castrates than in controls. As a result o f the higher LH p u l s e f r e q u e n c y , the average c o n c e n t r a t i o n of LH in p l a s m a was g r e a t e r in castrated than c o n t r o l calves at 12 and 16 w k of age. Castration did not affect LH p u l s e a m p l i t u d e at any age. Administration of E 2 e l i m i n a t e d the pulsatile s e c r e t i o n o f LH in b o t h intact and castrated calves; c o n c e n t r a t i o n s o f LH b e i n g b e l o w the l i m i t of sensitivity of the assay in the m a j o r i t y o f the s a m p l e s c o l l e c t e d at 8, 12 and 16 w k o f age. At 24 w k of age, LH p u l s e f r e q u e n c y a v e r a g e d less than 1 p u l s e / 6 hr in c o n t r o l and castrated calves r e c e i v i n g E2. H o w e v e r , o n e castrated E2-treated calf had 5 pulses of LH in 6 hr a n d a p p e a r e d to have e s c a p e d f r o m the i n h i b i t o r y effect o f estradiol. T r e a t m e n t o f intact calves w i t h E2 b l o c k e d testicular d e v e l o p m e n t (Figure 2). E x p e r i m e n t 2. Within the b o v i n e P O A - h y p o t h a l a m i c unit, the majority of the G n R H - i m m u n o r e a c t i v e cell b o d i e s w e r e associated w i t h the diagonal b a n d o f Broca a n d the m e d i a l POA (Figure 3). Cell b o d i e s e x t e n d e d dorsoventrally
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AGE (wks) Fig. 1. Changes in plasma E2 with age and after treatment with E2-implants. Concentrations of E2 decreased (P<.O1; N-20; pool SEM = 0.2) between 3 and 4 weeks of age. The age X treatment interaction was significant (P<.001) between 8 and 14 weeks of age.
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TABLE 1. EFFECTS OF CASTRATION ON SECRETION OF LH IN BULL CALVES" Age (Weeks) 8 12 16
Frequency (pulses/6 hr)Average Amplitude (n~/ml) I n t a c t vs Castrated I n t a c t vs C a s t ra t e d 3.2 -+ .2 2.2 - .4 2.8 +- .6
4.0 _+ .4 5.2 _+ .5 b 6.2 +_ .6 b
1.4 + .2 1.4 + .2 1.7 _+ .4
1.8 _+ .2 1.4 _+ .2 1.7 -+ .1
Mean ( n a / m l ) Int a c t vs Castrated .7 +_ .1 .7 -+ .1 .9 -+ .1
1.1 +_ .2 1.5 -+ .2 b 1.7 _+ .1 b
"Mean -~ SEM. bEffect o f c a s t r a t i o n s i g n i f i c a n t P < . 0 5 .
f r o m the level of the a n t e r i o r c o m m i s s u r e to the ventral b o r d e r of the POA, m o s t b e i n g l o c a t e d w i t h 2 m m o f the m i d l i n e and caudal to the anterior c o m m i s s u r e . H o w e v e r , a small n u m b e r o f cell b o d i e s w e r e f o u n d in the lateral POA and rostral to the anterior c o m m i s s u r e . No cell b o d i e s w e r e o b s e r v e d w i t h i n the a n t e r i o r or p o s t e r i o r h y p o t h a l a m u s . Average d i m e n s i o n s of i m m u n o s t a i n e d cells w e r e 11 X 35 pma. Most of the n e u r o n s c o n t a i n i n g i m m u n o reactive-GnRH w e r e b i p o l a r or t r i p o l a r (Figure 4A), h o w e v e r , s o m e u n i p o l a r n e u r o n s w e r e present. Fibers c o n t a i n i n g i m m u n o r e a c t i v e - G n R H f o r m e d a cons p i c u o u s p l e x u s in the SME w h i c h p r o j e c t e d t h r o u g h o u t the length o f the i n f u n d i b u l a r stalk (Figure 4B). Fibers w e r e d e n s e l y stained, b e a d e d in app e a r a n c e a n d varied f r o m less than 2 ~m to 3 ~tm in diameter. These fibers o r i g i n a t e d a l m o s t e x c l u s i v e l y f r o m cell b o d i e s l o c a t e d in the POA. Administration o f E2 affected the density of fibers c o n t a i n i n g i m m u n o r e a c t i v e GnRH in the SME, the c o n c e n t r a t i o n o f NE w i t h i n the SME and the c o n c e n t r a t i o n of GnRH r e c e p t o r s in the pituitary (Table 2). T r e a t m e n t w i t h E2 increased the c o n t e n t of the G n R H - i m m u n o - r e a c t i v e material w i t h i n the SME as r e f e l c t e d b y the i n c r e a s e d o p t i c a l density of fibers and beads. In contrast, E2 did not influence the m o r p h o l o g y ( p r o p o r t i o n of b i o p o l a r and t r i p o l a r cells), n u m b e r s or trajectories of i m m u n o r e a c t i v e - G n R H fibers n o r the intensity of i m m u n o s t a i n i n g of GnRH n e r v e cell b o d i e s (data not s h o w n ) . The c o n c e n t r a t i o n o f NE was l o w e r ( P = . 0 6 ) in the SME of bulls treated w i t h E2 than in intact controls. No differences in c o n c e n t r a t i o n s o f DA, DOPAC, HVA, 5-HT or 5-HIAA w e r e n o t e d b e t w e e n g r o u p s in the SME. Pituitary c o n c e n t r a t i o n s of GnRH r e c e p t o r s w e r e h i g h e r (P<.O1) in E2-treated calves t h e n in controls. A single p u l s e of LH was d e t e c t e d in 3 o f 4 c o n t r o l calves d u r i n g the f r e q u e n t b l e e d p e r i o d the day p r i o r to sacrifice. H o w e v e r , no pulses of LH w e r e o b s e r v e d in the calves i m p l a n t e d w i t h Ez d u r i n g the s a m e period. Paired-testes weights
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AGE (wks) Fig. 2. I n i t i a l t e s t i c u l a r w e i g h t (7.5 w e e k s of age; N = 10) a n d t e s t i c u l a r w e i g h t s of i nt a c t c o n t r o l and Ez-treated calves at 24 w e e k s of age. A d m i n i s t r a t i o n of E2 r e d u c e d (P<.OO1) t e s t i c u l a r size.
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~A
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Fig. 3. Diagrammatic sagittal sections of the bovine diencephalon showing the distribution of GnRH-immunoreactive cell bodies (O) and fibers (thin lines). The distance of each section from the midline is shown above the drawings. A, anterior commissure; AH, anterior hypothalamus; DB, diagonal band of Broca; ME, median eminence; O, optic chiasma; PH, posterior hypothalamus; POA, preoptic area. Broken lines are where tissues were trimmed for sectioning.
w e r e 74.0 g _ respectively.
10.2 vs 24.4 g +_ 2.2 in control and E2-treated calves, DISCUSSION
In bull calves, an increase in the secretion of LH has b e e n observed b e t w e e n 7 and 9 w k of age (3,4) and it has b e e n suggested that this increase is r e q u i r e d for normal testicular growth and d e v e l o p m e n t . Results from the present study, and those r e c e n t l y r e p o r t e d b y Schanbacher (12), clearly demonstrate that administration o f E2 to infantile or p e r i p u b e r t a l calves inhibits testicular dev e l o p m e n t . O t h e r important findings from the present e x p e r i m e n t s include: 1) s e c r e t i o n o f E2 decreases during the infantile period; 2) castration results in a small but significant decrease in E2; 3 ) administration of E2 may result in
Fig. 4. A. Bipolar GnRH-immunoreactive cell body in the diagonal band of Broca. Note the presence of beaded GnRI-I fibers that project to the SME. B. Longitudinal section of the infundibular stalk that contains a rich innervation of GnRH fibers.
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Txacr 2. EFFECTSOF ESTRADIOLON THE HYPOTHALAMIc-PITUITARYAXISOF BULLCALVES* Treatment Variable Control (n=4)vs Estradiol (n=6) Probability Optical density of GnRH 42.8 ± 2.9 41.6 ± 0.3 NS cell bodies in POA Density of GnRH fibers/Grid 8.6 ± 2.4 13.5 ± 2.1 P<.05 in the SME % GnRH cell bodies tripolar 52 51 NS Concentration of NE in SME 1.61 ± .4 .78 ± .2 P=.06 Pituitary receptors for GnRH .53 ± .1 .98 ± .1 P<.O1 (fmole/mg tissue) *Mean + SEM.
a d e c r e a s e d release o f GnRH, as the density o f GnRH staining in the SME neural fibers i n c r e a s e d in r e s p o n s e to E2; 4) t r e a t m e n t w i t h E2 b l o c k e d age-related c h a n g e s in c o n c e n t r a t i o n s of NE in the SME; and 5) the c o n c e n t r a t i o n of p i t u i t a r y r e c e p t o r s for GnRH w e r e higher in calves r e c e i v i n g E2 c o m p a r e d to c o n t r o l animals. A m a i n o b j e c t i v e o f this s t u d y was to investigate a p o s s i b l e role of E 2 in r e g u l a t i o n of LH s e c r e t i o n d u r i n g the infantile a n d p r e p u b e r t a l stages o f d e v e l o p m e n t . In a g r e e m e n t w i t h an earlier r e p o r t (9) the c o n c e n t r a t i o n of E2 in b l o o d d e c r e a s e d several w k p r i o r to the increase in p r e p u b e r t a l pulsatile s e c r e t i o n of LH; h o w e v e r , the decreases in E2 o c c u r r e d earlier in this study. In e x p e r i m e n t 1, the i n c r e a s e d pulsatile s e c r e t i o n of LH also o c c u r r e d earlier t h a n p r e v i o u s l y r e p o r t e d ( 4 , 1 5 ) and this m a y have b e e n d u e to the earlier d e c r e a s e in E2. Wise et al ( 1 0 ) have s u g g e s t e d that a testicular p r o d u c t o t h e r t h a n E2 is r e s p o n s i b l e for age a n d castration-related c h a n g e s in LH. This c o n c l u s i o n w a s b a s e d on t h e i r o b s e r v a t i o n that t h e r e was no c h a n g e in the c o n c e n t r a t i o n of E2 in p l a s m a f o l l o w i n g castration. In the p r e s e n t study, a slight b u t significant d e c r e a s e in E 2 was o b s e r v e d f o l l o w i n g castration. W h i l e the i m p o r t a n c e of testicular s e c r e t i o n s o t h e r t h a n estradiol can not be r u l e d out, it is p o s s i b l e that E2 exerts i m p o r t a n t effects o n the h y p o t h a l a m i c - p i t u i t a r y axis d u r i n g the infantile a n d p r e p u b e r t a l p e r i o d o f males. This c o n c l u s i o n is b a s e d o n the relationships b e t w e e n age and c a s t r a t i o n - d e p e n d e n t changes in E2 a n d the i n c r e a s e d pulsatile LH release o b s e r v e d in this s t u d y and by others ( 9 ) . It is p r o b a b l e that d u r i n g the infantile stage o f life the h y p o t h a l a m i c p i t u i t a r y axis of bulls is e x q u i s i t e l y sensitive to E2, b e c a u s e the d r a m a t i c increase in LH p u l s e f r e q u e n c y was associated w i t h o n l y a slight d e c l i n e in
E2. It is c l e a r that a d m i n i s t r a t i o n o f E2 b l o c k e d t e s t i c u l a r d e v e l o p m e n t . At p r e s e n t it is u n c e r t a i n if this effect o f E2 is related o n l y to i n h i b i t i o n of LH s e c r e t i o n or r e d u c e d LH and follicle s t i m u l a t i n g h o r m o n e (FSH) secretion. Recently, S c h a n b a c h e r et al ( 1 3 ) r e p o r t e d that a d m i n i s t r a t i o n o f E2 to calves i n h i b i t e d s e c r e t i o n o f FSH b u t not LH. H o w e v e r , in that s t u d y the s a m p l i n g f r e q u e n c y u s e d w a s n o t a d e q u a t e to judge the effect o f E2 on pulsatile release o f LH. T h e d r a m a t i c d e c r e a s e in LH s e c r e t i o n n o t e d in this s t u d y and that b y Wise et al ( 1 0 ) d e m o n s t r a t e s the E 2 can i n h i b i t the s e c r e t i o n o f LH as w e l l as FSH ( 1 3 ) . It is p o s s i b l e that a d e q u a t e s e c r e t i o n o f b o t h g o n a d o t r o p i n s is necessary for n o r m a l testicular d e v e l o p m e n t . T h e a n a t o m i c a l localization o f b o v i n e GnRH n e u r o n s is similar to that r e p o r t e d for s h e e p ( 2 1 ) and a v a r i e t y of o t h e r l a b o r a t o r y animals ( 2 2 ) . The majority of the i m m u n o s t a i n e d cell b o d i e s w e r e in the m e d i a l POA, and these n e u r o n s
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are likely the m a i n s o u r c e of GnRH i n n e r v a t i o n to the SME. The large m u l t i p o l a r p r o p o r t i o n of GnRH cell b o d i e s in the bull calf is similar to that r e p o r t e d for the o v i n e ( 2 1 ) , h o w e v e r , this is different than in primates, bats, rats (22) and m i c e ( 1 7 ) w h e r e m o s t GnRH n e u r o n s are uni- or bipolar. It is i m p o r t a n t to n o t e that areas o f the brain outside the POA-hypothalamic region w e r e not e x a m i n e d in this study. Thus the possibility exists that there are extra-POA p o p u l a t i o n s of n e u r o n s c o n t a i n i n g i m m u n o r e a c t i v e - G n R H . T r e a t m e n t w i t h E2 increased the density of i m m u n o r e a c t i v e - G n R H e l e m e n t s in the SME. This i n c r e a s e d intensity o f staining is b e l i e v e d to b e caused b y an a c c u m u l a t i o n of GnRH in these fibers ( 1 8 ) . As the antisera to GnRH does not cross-react w i t h the p r o - G n R H m o l e c u l e ( 2 2 ) and t r e a t m e n t w i t h E2 did not affect the intensity o f staining of GnRH cell bodies w e p r o p o s e that synthesis, p r o c e s s i n g and t r a n s p o r t of the GnRH p r e c u r s o r w e r e not affected by Ez, b u t that the release of GnRH into the h y p o t h a l a m i c portal vessels was attenuated. This suggestion is f u r t h e r s u p p o r t e d b y a p r e v i o u s study (12) w h e r e administration of e x o g e n o u s GnRH re-initiated testicular g r o w t h in estradiol-treated p e r i p u b e r t a l bulls. A r e d u c e d rate o f GnRH release m i g h t also have c o n t r i b u t e d to the h i g h e r c o n c e n t r a t i o n of p i t u i t a r y GnRH r e c e p t o r s f o u n d in the E2-treated calves. In addition, since t r e a t m e n t w i t h E2 has b e e n s h o w n to increase the n u m b e r s o f GnRH r e c e p t o r s in o v a r i e c t o m i z e d s h e e p ( 2 4 ) and m o n k e y s ( 2 5 ) it is p o s s i b l e that E2 acted d i r e c t l y on the pituitary to e n h a n c e the synthesis of GnRH r e c e p t o r s . Previously, w e have r e p o r t e d that c o n c e n t r a t i o n s of NE and DA within the SME i n c r e a s e d w i t h age and w e r e p o s i t i v e l y c o r r e l a t e d w i t h p l a s m a testosterone ( 1 5 ) . In e x p e r i m e n t 2, c o n c e n t r a t i o n s of NE, b u t not DA, w e r e affected b y t r e a t m e n t w i t h Ez and the values r e p o r t e d are similar to those p r e v i o u s l y f o u n d in the SME of 8 and 24 w k calves (.84 and 1.59 n g / m g , r e s p e c t i v e l y ) . Based on these findings, it is p o s s i b l e that age-related changes in NE are d e p e n d e n t on c h a n g e s in s e c r e t i o n o f testosterone. The i m p o r t a n c e of n o r a d r e n e r g i c neurons, t e r m i n a t i n g the SME, in r e g u l a t i n g GnRH release and LH secretion is not unclear. Administration o f NE to m a t u r e s h e e p ( 2 6 , 2 7 ) and cattle ( 2 8 ) has b e e n s h o w n to inhibit the s e c r e t i o n o f LH. Since SME c o n c e n t r a t i o n s of NE are l o w in y o u n g and Ez-treated calves, it is p o s s i b l e that it is not a m a j o r i n h i b i t o r of GnRH and LH s e c r e t i o n until after the o n s e t of testicular secretion of testosterone. Alternatively, age-related increases in c o n c e n t r a t i o n s of NE w i t h i n the SME m i g h t reflect a d e c r e a s e d sensitivity of GnRH fibers to the i n h i b i t o r y effects o f NE. Since p l a s m a c o n c e n t r a t i o n s of E2 w e r e higher in i m p l a n t e d than c o n t r o l calves, it is possible that GnRH n e u r o n s r e m a i n e d e x t r e m e l y sensitive to i n h i b i t o r y effects o f NE and c o n s e q u e n t l y e l i m i n a t e d the n e e d for age-related c h a n g e s in NE m e t a b o l i s m w i t h i n the SME. In s u m m a r y , a d m i n i s t r a t i o n of E2 causes p r o f o u n d changes in h y p o t h a l a m i c function, i n c l u d i n g an a p p a r e n t r e d u c t i o n in the release of GnRH and alterations in synthesis o f NE. These changes likely c o n t r i b u t e significantly to the r e d u c e d s e c r e t i o n of g o n a d o t r o p i n s and i n h i b i t i o n of testicular d e v e l o p m e n t observed in calves t r e a t e d w i t h Ez. FOOTNOTES 1 This paper was authorized for publication as paper No. 7904 in the journal series of the Pennsylvania Agricultural Experiment Station. Address correspondence and requests for reprints to Dr. Daniel R. Deaver, Dariy Breeding Research Center, Pennsylvania State University., University Park, PA 16802
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Vol. 5(4):307-316, 1988
EFFECTS OF ESTRADIOL ON SECRETION OF LH, HYPOTHALAMIC FUNCTION AND TESTICULAR DEVELOPMENT IN BULL CALVES 1 D.R. Deaver2, J.D. Glass 3, D.M. Grieger4 and J.J. Reeves4 Department of Dairy and Animal Sciences, Penn State University2 Department of Biological Sciences, Kent State University3 Department of Animal Science, Washington State University4 Received April 25, 1988 ABSTRACT Two experiments were conducted in order to determine the effects of estradiol (E2) on the development of the hypothalamic-pituitary-testicular axis in bull calves. In experiment 1, calves were assigned randomly to one of the following groups: 1) intact, 2) intact E2-treated, 3) castrated, or 4) castrated E2-treated. Treatments began when the calves were 7.5 wk of age and continued for 16.5 wk. Samples of blood were collected once a week from 3 to 14 wk of age and every 10 rain for 6 hr at 8, 12 and 16 wk of age. Concentrations of E 2 in plasma decreased between 3 and 4 wk of age and were further reduced by castration. Maximum concentrations of E2 (24.3 pg/ml) were observed 72 hr after insertion of E2 implants, however, plasma E2 stablized at 5-9 pg/ml by 2 wk after insertion of implants. Treatment with E 2 eliminated the pulsatile secretion of LH in intact and castrated calves and retarded testicular growth. In experiment 2, calves were assigned to a control (n----4) or E2-treated ( n = 6 ) group. Implants of E 2 w e r e inserted at 7.5 wk of age. At 24 wk of age, calves were bled and then sacrificed to collect hypothalamic and pituitary tissues. Age-related changes in testicular weight and secretion of LH were blocked by E 2. Neither the morphology nor the intensity of immunostaining of GnRH nerve cell bodies in the preoptic area (POA) were affected by E2. However, the density of GnRI-I fibers and beads in the stalk median eminence (SME), and concentrations of pituitary GnRH receptors were greater (P<.01) in E2treated compared to control calves. In addition, concentrations of norepinephrine (NE) in the SME were lower in E2-treated calves when compared to controls. Based on these observations, it is concluded that administeration of E 2 a t 7.5 wk of age causes profound alterations in hypothalamic function including, changes in metabolism of NE and suppression of GnRH release. INTRODUCTION The t r a n s i t i o n b e t w e e n t h e i n f a n t i l e a n d p r e p u b e r t a l stages o f d e v e l o p m e n t is c h a r a c t e r i z e d b y a d r a m a t i c i n c r e a s e in t h e p u l s a t i l e s e c r e t i o n of LH in f e m a l e ( 1 , 2 ) a n d m a l e ( 3 , 4 ) r u m i n a n t s . In r a m l a m b s i n c r e a s e d s e c r e t i o n of LH o c c u r s p r i o r to 5 w k o f age, w h i l e in c a l v e s a m a r k e d i n c r e a s e in e p i s o d i c s e c r e t i o n o c c u r s b e t w e e n 8 a n d 12 w k of age. This c h a n g e in t h e f r e q u e n c y o f LH r e l e a s e is t h o u g h t to i n i t i a t e d i f f e r e n t i a t i o n a n d d e v e l o p m e n t o f t e s t i c u l a r c e l l s t h a t p a r t i c i p a t e in s p e r m a t o g e n e s i s ( 5 ) . C a s t r a t i o n of t h e n e o n a t a l l a m b or c a l f c a u s e s p r e c o c i o u s d e v e l o p m e n t of p u l s a t i l e LH r e l e a s e ( 1 , 3 ) . H o w e v e r , t h e t i m e r e q u i r e d to o b s e r v e an i n c r e a s e in LH p u l s e f r e q u e n c y f o l l o w i n g c a s t r a t i o n is l o n g e r for i n f a n t i l e than p e r i p u b e r t a l or a d u l t r u m i n a n t s ( 1 , 6 , 7 ) . It a p p e a r s that a d e c r e a s e in s e n s i t i v i t y o f t h e h y p o t h a l a m u s to n e g a t i v e f e e d b a c k effects o f E 2 p r e c e d e s t h e i n c r e a s e d s e c r e t i o n of IM in e w e l a m b s ( 2 ) a n d h e i f e r s ( 8 ) . A m a n n et al ( 9 ) have r e p o r t e d t h a t c i r c u l a t i n g c o n c e n t r a t i o n s of E 2 in p l a s m a a n d n u m b e r s of h y p o t h a l a m i c r e c e p t o r s for E 2 d e c r e a s e b e f o r e t h e i n c r e a s e d s e c r e t i o n of LH in b u l l calves.
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In contrast, in a n o t h e r study (10) plasma concentrations of Ez w e r e not observed to change p r i o r to the initial rise in LH. Although the administration of E2 has b e e n s h o w n to inhibit the s e c r e t i o n of LH in p e r i p u b e r t a l bulls ( 1 1 , 1 2 ) , t r e a t m e n t o f infantile calves w i t h E2 has b e e n r e p o r t e d to inhibit (1 O) or have no effect (13) on the secretion of LH. Two e x p e r i m e n t s w e r e c o n d u c t e d to investigate the possible role(s) of E2 in controlling the secretion of LH in infantile and p r e p u b e r t a l calves. In the first e x p e r i m e n t the objectives were: 1) to d e t e r m i n e if c o n c e n t r a t i o n s of E2 changed with age and following castration and 2) to evaluate the effects of estradiol on testicular d e v e l o p m e n t and the o n t o g e n y of pulsatile secretion of LH in intact and castrated calves. The p u r p o s e of the s e c o n d e x p e r i m e n t was to examine changes in the function o f GnRH and m o n o a m i n e r g i c h y p o t h a l a m i c neurons and pituitary concentrations of GnRH r e c e p t o r s during treatment w i t h Ez. MATERIALS AND METHODS E x p e r i m e n t 1. T w e n t y Holstein bull calves w e r e p u r c h a s e d at 3-5 days of age and raised at the Dairy Breeding Research Center as previously described ( 1 4 ) . At 7 w k o f age, calves w e r e assigned at r a n d o m to one o f four treatments: 1) intact ( c o n t r o l ) , 2) intact and E2, 3) castrated or 4) castrated and E2. Castrations w e r e p e r f o r m e d a n d / o r a 30 m m C o m p u d o s e T M implant containing E2 was inserted into the right ear at 7.5 w k of age. Testes from castrated calves w e r e w e i g h e d for c o m p a r i s o n w i t h those obtained at 24 w k of age. Between 3 and 14 wk, o f age samples o f b l o o d (10 ml) were c o l l e c t e d b y jugular v e n i p u n c t u r e every 7 days. In addition, b l o o d samples w e r e c o l l e c t e d using jugular catheters every 10 min for 6 hr at 8, 12 and 16 w k o f age from all calves and at 24 w k o f age from control and castrated calves receiving E2. Plasma was c o l l e c t e d and stored at - - 2 0 C until assayed for E 2 o r LH. At 24 w k o f age intact animals w e r e castrated and testicular weights recorded. E x p e r i m e n t 2. Holstein calves used for this study w e r e purchased at 3-5 days o f age and raised as in e x p e r i m e n t 1. Calves w e r e assigned at r a n d o m to a c o n t r o l ( n = 4 ) or Ez-treated ( n = 6 ) group. At 7.5 wk o f age, a C o m p u d o s e T M implant was cut to a length o f 15 m m and inserted into the right ear of a p p r o p r i a t e calves. W h e n calves w e r e 24 wk old b l o o d samples w e r e obtained e v e r y 10 min for 6 hr using i n d w e l l i n g jugular catheters; they w e r e sacrified the f o l l o w i n g day by exsanquination. The POA-hypothalamic tissues (including the SME) w e r e bissected along the midline. The right side of the SME was o b t a i n e d and stored in liquid N2 and the left POA, hypothalamus and SME w e r e p l a c e d in modified Zamboni's fixative, h e l d at 4C for two days, transferred to .01 M p h o s p h a t e buffer and stored at 4C until p r o c e s s e d for quantitative i m m u n o c y t o c h e m i c a l p r o c e d u r e s as d e s c r i b e d below. A s s a y P r o c e d u r e s . Concentrations of LH in plasma w e r e d e t e r m i n e d using a p r e v i o u s l y d e s c r i b e d RIA ( 1 5 ) . The sensitivity o f the assay was 25 p g / t u b e (USDA-bLH-B-5) and samples w e r e assayed at a v o l u m e of 100 gtl. The interand intra-assay coefficients of variation w e r e less than 10%. C o n c e n t r a t i o n s of plasma estradiol w e r e d e t e r m i n e d using immunoreagents o b t a i n e d from Serono Diagonstics, Inc. (Norwell, MA). Plasma ( 2 0 0 Ixl) was e x t r a c t e d t w i c e w i t h 2 ml of diethyl ether. Ether extracts w e r e placed into 12 )< 75 borosilicate tubes and e v a p o r a t e d to dryness. The residue was rec o n s t i t u t e d w i t h buffer ( 2 0 0 ~tl) and tubes v o x t e x e d vigorously. After vortexing, 50 gtl of 125I-E2 (containing a p p r o x i m a t e l y 15,000 d p m ) and 50 ~tl of rabbit anti-E2 antisera ( w h i c h b o u n d 40% of the total counts) w e r e a d d e d to each
HYPOTHALAMO-HYPOPHYSEAL-TESTICULAR AXIS IN CALVES
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tube. These reagents were incubated at room temperature for 3 hr then sheep anti-rabbit serum (100 !11) and 20% polyethyleneglycol (1 ml) were added to precipitate the bound fraction. After incubating for 20 min, the tubes were centrifuged for 20 min at 1200 X g and the supernatant aspirated and the radioactivity in pellet counted. The sensitivity of the assay was 0.1 pg/tube (calculated as 3 X the standard deviation of Bo counts) or 0.5 pg/ml. Recovery of 5 pg of exogenous E 2 added to 200 III of steer plasma averaged over 90%. Samples ranging in concentrations from 2 to 15 pg/ m l were extracted at volumes o f 100, 200 and 400 gtl and each individual dose response was parallel to the standard curve. The intra- and inter-assay coefficients of variation were less than 9%. A high pressure liquid chromatograph e q u i p p e d with an electrochemical detector (Bioanalytical Systems, Lafayette, IN) was used to determine concentraitons of tissue nor e pi ne phr i ne (NE), dopamine (DA), 3,4-diphenylhydroxyacetic acid (DOPAC), 4-hydroxy-3-methoxy-phenylaceticacid (HVA), serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA). The assay was established and validated as previously described (16) with minor modifications. Tissues were homogenized using a Brinkman polytron (3 sec at a setting of 6) in 500 gtl of .05 M HCI04 containing .1% cysteine. Homogenates were centrifuged at 11,000 X g and then filtered through .2 gM filter (Gilman ARCO LC13, Thomas Scientific Co., Philadelphia, PA) prior to injection onto a 5-gin C-18 column (ODS, Biophase 250 X 4.6 mm, Bioanalytical Systems). Amines used for standards were obtained from Sigma Chemical Co (St. Louis, MO) and all results were expressed as free-base equivalents. The mobile phase consisted of 14.2 g monochloroacetic acid (MCAA), 4.68 g NaOH, 200 mg sodiumoctyl sulfate in 1 L of distilled H20. Mobile phase was adjusted to a pH of 3.0 using solid NaOH or MCAA, filtered through a 0.2 gM filter and degassed prior to use. Acetonitrile (35 ml/L) and tetrahydrofuran (18 ml/L) were added to the mobile phase after degassing. A glassy carbon eletrode, set at a potential of 750 mV relative to a AgCI reference electrode, was used as the detector. The flow rate o f the mobile phase was 1.0 ml/min. Immuno-reactive GnRH neurons were localized using the method previously described (17). Serial 100 ~tm-thick sagittal sections were made 2mm lateral from the midline to the ventricular wall using a vibratome (La.ncer Ted Pella Inc., Justin, CA). Due to the dense connective tissue within the SME, this structure was separated from the basal hypothalamus and embedded in gelatin for sectioning. The SME was sectioned longitudinally at 100 gtm thickness, and every section was saved for immunochemical staining for GnRH. Uniformity of staining was optimized by processing the sections in 24-well cell culture plates (Primaria, Becton Dickinson, Balto, Md.). Sections were incubated with primary antiserum GnRH (Nett ~tR38; 1:1000), secondary antiserum (sheep antirabbit gamma globulin; 1:15) and then with rabbit peroxidase-antiperoxidase c o m p l e x (Sigma Chem Co., St. Louis; 1:100). Sections were reacted with diaminobenzidine containing H202 and counter stained with methylene blue. The GnRH neuronal system was then mapped and the morphology and OD of immunostaining determined according to Glass (18). Optical density of cell bodies (an index of content of GnRH) was measured using an optical densitometer attached to the microscope. The density of GnRH fibers in the SME was determined by grid analysis of the number of intersections of positive fibers with the reticules of a 10 X 10 mm ocular grid placed over the SME. Concentration of GnRH receptors in the anterior pituitary were determined as previously described (19).
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S t a t i s t i c a l A n a l y s e s . T h e f r e q u e n c y a n d a m p l i t u d e o f LH discharges w e r e d e t e r m i n e d using the PULSAR p r o g r a m (Merriam and Wachter, 1982). Effects o f t r e a t m e n t ( m a i n p l o t ) , age and the i n t e r a c t i o n o f age X t r e a t m e n t ( s u b p l o t effects; e x p e r i m e n t 1) or t r e a t m e n t ( e x p e r i m e n t 2) on variables e x a m i n e d w e r e tested for using the general linear m o d e l p r o c e d u r e of SAS (16); differences a m o n g main-effect m e a n s w e r e f u r t h e r e v a l u a t e d using the PDIFF p r o c e d u r e . RESULTS E x p e r i m e n t 1. B e t w e e n 3 a n d 4 w k o f age, c o n c e n t r a t i o n s o f E2 d e c r e a s e d in p l a s m a (Figure 1; P < . 0 1 ) . C o n c e n t r a t i o n s o f E2 in jugular p l a s m a averaged 24 p g / m l in s a m p l e s c o l l e c t e d 72 hr after insertion of E2-implants (8 w k of age). H o w e v e r , w i t h i n 2 wk, E2 in jugular p l a s m a stablized at a p p r o x i m a t e l y 5-9 p g / m l . B e t w e e n 8 and 14 w k o f age, p l a s m a E2 was higher in E2-treated calves t h a n in intact (1.5 +_ 0.3) or castrated (0.6 _+ .2 p g / m l ) calves not g i v e n implants. The c o n c e n t r a t i o n of E2 was significantly l o w e r ( P < . 0 5 ) in castrated calves w h e n c o m p a r e d to the intact calves not r e c e i v i n g estradiol. Effects o f age and castration on s e c r e t i o n o f LH are s h o w n in Table 1. The f r e q u e n c y o f e p i s o d i c LH release in intact and castrated calves was similar at 8 w k o f age ( 7 2 hr post-castration). H o w e v e r , at 12 and 16 w k of age the f r e q u e n c y o f pulsatile LH release was g r e a t e r in castrates than in controls. As a result o f the higher LH p u l s e f r e q u e n c y , the average c o n c e n t r a t i o n of LH in p l a s m a was g r e a t e r in castrated than c o n t r o l calves at 12 and 16 w k of age. Castration did not affect LH p u l s e a m p l i t u d e at any age. Administration of E 2 e l i m i n a t e d the pulsatile s e c r e t i o n o f LH in b o t h intact and castrated calves; c o n c e n t r a t i o n s o f LH b e i n g b e l o w the l i m i t of sensitivity of the assay in the m a j o r i t y o f the s a m p l e s c o l l e c t e d at 8, 12 and 16 w k o f age. At 24 w k of age, LH p u l s e f r e q u e n c y a v e r a g e d less than 1 p u l s e / 6 hr in c o n t r o l and castrated calves r e c e i v i n g E2. H o w e v e r , o n e castrated E2-treated calf had 5 pulses of LH in 6 hr a n d a p p e a r e d to have e s c a p e d f r o m the i n h i b i t o r y effect o f estradiol. T r e a t m e n t o f intact calves w i t h E2 b l o c k e d testicular d e v e l o p m e n t (Figure 2). E x p e r i m e n t 2. Within the b o v i n e P O A - h y p o t h a l a m i c unit, the majority of the G n R H - i m m u n o r e a c t i v e cell b o d i e s w e r e associated w i t h the diagonal b a n d o f Broca a n d the m e d i a l POA (Figure 3). Cell b o d i e s e x t e n d e d dorsoventrally
'10.0.
0--0 0--0
A--A A--A
t~ O.
1.0-
A~ t
Intact ~IIL. Castrated ~,/_k Intact + E2 A X A \ ./ l i , A I Caatrated + g2 A
0 O-O ~0~0~0-0 \ /0~010-0 o~o~O'O.o~O~O
i,t
0.1
2
4
6
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AGE (wks) Fig. 1. Changes in plasma E2 with age and after treatment with E2-implants. Concentrations of E2 decreased (P<.O1; N-20; pool SEM = 0.2) between 3 and 4 weeks of age. The age X treatment interaction was significant (P<.001) between 8 and 14 weeks of age.
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TABLE 1. EFFECTS OF CASTRATION ON SECRETION OF LH IN BULL CALVES" Age (Weeks) 8 12 16
Frequency (pulses/6 hr)Average Amplitude (n~/ml) I n t a c t vs Castrated I n t a c t vs C a s t ra t e d 3.2 -+ .2 2.2 - .4 2.8 +- .6
4.0 _+ .4 5.2 _+ .5 b 6.2 +_ .6 b
1.4 + .2 1.4 + .2 1.7 _+ .4
1.8 _+ .2 1.4 _+ .2 1.7 -+ .1
Mean ( n a / m l ) Int a c t vs Castrated .7 +_ .1 .7 -+ .1 .9 -+ .1
1.1 +_ .2 1.5 -+ .2 b 1.7 _+ .1 b
"Mean -~ SEM. bEffect o f c a s t r a t i o n s i g n i f i c a n t P < . 0 5 .
f r o m the level of the a n t e r i o r c o m m i s s u r e to the ventral b o r d e r of the POA, m o s t b e i n g l o c a t e d w i t h 2 m m o f the m i d l i n e and caudal to the anterior c o m m i s s u r e . H o w e v e r , a small n u m b e r o f cell b o d i e s w e r e f o u n d in the lateral POA and rostral to the anterior c o m m i s s u r e . No cell b o d i e s w e r e o b s e r v e d w i t h i n the a n t e r i o r or p o s t e r i o r h y p o t h a l a m u s . Average d i m e n s i o n s of i m m u n o s t a i n e d cells w e r e 11 X 35 pma. Most of the n e u r o n s c o n t a i n i n g i m m u n o reactive-GnRH w e r e b i p o l a r or t r i p o l a r (Figure 4A), h o w e v e r , s o m e u n i p o l a r n e u r o n s w e r e present. Fibers c o n t a i n i n g i m m u n o r e a c t i v e - G n R H f o r m e d a cons p i c u o u s p l e x u s in the SME w h i c h p r o j e c t e d t h r o u g h o u t the length o f the i n f u n d i b u l a r stalk (Figure 4B). Fibers w e r e d e n s e l y stained, b e a d e d in app e a r a n c e a n d varied f r o m less than 2 ~m to 3 ~tm in diameter. These fibers o r i g i n a t e d a l m o s t e x c l u s i v e l y f r o m cell b o d i e s l o c a t e d in the POA. Administration o f E2 affected the density of fibers c o n t a i n i n g i m m u n o r e a c t i v e GnRH in the SME, the c o n c e n t r a t i o n o f NE w i t h i n the SME and the c o n c e n t r a t i o n of GnRH r e c e p t o r s in the pituitary (Table 2). T r e a t m e n t w i t h E2 increased the c o n t e n t of the G n R H - i m m u n o - r e a c t i v e material w i t h i n the SME as r e f e l c t e d b y the i n c r e a s e d o p t i c a l density of fibers and beads. In contrast, E2 did not influence the m o r p h o l o g y ( p r o p o r t i o n of b i o p o l a r and t r i p o l a r cells), n u m b e r s or trajectories of i m m u n o r e a c t i v e - G n R H fibers n o r the intensity of i m m u n o s t a i n i n g of GnRH n e r v e cell b o d i e s (data not s h o w n ) . The c o n c e n t r a t i o n o f NE was l o w e r ( P = . 0 6 ) in the SME of bulls treated w i t h E2 than in intact controls. No differences in c o n c e n t r a t i o n s o f DA, DOPAC, HVA, 5-HT or 5-HIAA w e r e n o t e d b e t w e e n g r o u p s in the SME. Pituitary c o n c e n t r a t i o n s of GnRH r e c e p t o r s w e r e h i g h e r (P<.O1) in E2-treated calves t h e n in controls. A single p u l s e of LH was d e t e c t e d in 3 o f 4 c o n t r o l calves d u r i n g the f r e q u e n t b l e e d p e r i o d the day p r i o r to sacrifice. H o w e v e r , no pulses of LH w e r e o b s e r v e d in the calves i m p l a n t e d w i t h Ez d u r i n g the s a m e period. Paired-testes weights
._.'~16° t I--
~ 120 ~
[X-R-I E2 B2E] Control
8O
m 4O XX~
7.5
24
24
AGE (wks) Fig. 2. I n i t i a l t e s t i c u l a r w e i g h t (7.5 w e e k s of age; N = 10) a n d t e s t i c u l a r w e i g h t s of i nt a c t c o n t r o l and Ez-treated calves at 24 w e e k s of age. A d m i n i s t r a t i o n of E2 r e d u c e d (P<.OO1) t e s t i c u l a r size.
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~A
',
~ ......
, ~
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Fig. 3. Diagrammatic sagittal sections of the bovine diencephalon showing the distribution of GnRH-immunoreactive cell bodies (O) and fibers (thin lines). The distance of each section from the midline is shown above the drawings. A, anterior commissure; AH, anterior hypothalamus; DB, diagonal band of Broca; ME, median eminence; O, optic chiasma; PH, posterior hypothalamus; POA, preoptic area. Broken lines are where tissues were trimmed for sectioning.
w e r e 74.0 g _ respectively.
10.2 vs 24.4 g +_ 2.2 in control and E2-treated calves, DISCUSSION
In bull calves, an increase in the secretion of LH has b e e n observed b e t w e e n 7 and 9 w k of age (3,4) and it has b e e n suggested that this increase is r e q u i r e d for normal testicular growth and d e v e l o p m e n t . Results from the present study, and those r e c e n t l y r e p o r t e d b y Schanbacher (12), clearly demonstrate that administration o f E2 to infantile or p e r i p u b e r t a l calves inhibits testicular dev e l o p m e n t . O t h e r important findings from the present e x p e r i m e n t s include: 1) s e c r e t i o n o f E2 decreases during the infantile period; 2) castration results in a small but significant decrease in E2; 3 ) administration of E2 may result in
Fig. 4. A. Bipolar GnRH-immunoreactive cell body in the diagonal band of Broca. Note the presence of beaded GnRI-I fibers that project to the SME. B. Longitudinal section of the infundibular stalk that contains a rich innervation of GnRH fibers.
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Txacr 2. EFFECTSOF ESTRADIOLON THE HYPOTHALAMIc-PITUITARYAXISOF BULLCALVES* Treatment Variable Control (n=4)vs Estradiol (n=6) Probability Optical density of GnRH 42.8 ± 2.9 41.6 ± 0.3 NS cell bodies in POA Density of GnRH fibers/Grid 8.6 ± 2.4 13.5 ± 2.1 P<.05 in the SME % GnRH cell bodies tripolar 52 51 NS Concentration of NE in SME 1.61 ± .4 .78 ± .2 P=.06 Pituitary receptors for GnRH .53 ± .1 .98 ± .1 P<.O1 (fmole/mg tissue) *Mean + SEM.
a d e c r e a s e d release o f GnRH, as the density o f GnRH staining in the SME neural fibers i n c r e a s e d in r e s p o n s e to E2; 4) t r e a t m e n t w i t h E2 b l o c k e d age-related c h a n g e s in c o n c e n t r a t i o n s of NE in the SME; and 5) the c o n c e n t r a t i o n of p i t u i t a r y r e c e p t o r s for GnRH w e r e higher in calves r e c e i v i n g E2 c o m p a r e d to c o n t r o l animals. A m a i n o b j e c t i v e o f this s t u d y was to investigate a p o s s i b l e role of E 2 in r e g u l a t i o n of LH s e c r e t i o n d u r i n g the infantile a n d p r e p u b e r t a l stages o f d e v e l o p m e n t . In a g r e e m e n t w i t h an earlier r e p o r t (9) the c o n c e n t r a t i o n of E2 in b l o o d d e c r e a s e d several w k p r i o r to the increase in p r e p u b e r t a l pulsatile s e c r e t i o n of LH; h o w e v e r , the decreases in E2 o c c u r r e d earlier in this study. In e x p e r i m e n t 1, the i n c r e a s e d pulsatile s e c r e t i o n of LH also o c c u r r e d earlier t h a n p r e v i o u s l y r e p o r t e d ( 4 , 1 5 ) and this m a y have b e e n d u e to the earlier d e c r e a s e in E2. Wise et al ( 1 0 ) have s u g g e s t e d that a testicular p r o d u c t o t h e r t h a n E2 is r e s p o n s i b l e for age a n d castration-related c h a n g e s in LH. This c o n c l u s i o n w a s b a s e d on t h e i r o b s e r v a t i o n that t h e r e was no c h a n g e in the c o n c e n t r a t i o n of E2 in p l a s m a f o l l o w i n g castration. In the p r e s e n t study, a slight b u t significant d e c r e a s e in E 2 was o b s e r v e d f o l l o w i n g castration. W h i l e the i m p o r t a n c e of testicular s e c r e t i o n s o t h e r t h a n estradiol can not be r u l e d out, it is p o s s i b l e that E2 exerts i m p o r t a n t effects o n the h y p o t h a l a m i c - p i t u i t a r y axis d u r i n g the infantile a n d p r e p u b e r t a l p e r i o d o f males. This c o n c l u s i o n is b a s e d o n the relationships b e t w e e n age and c a s t r a t i o n - d e p e n d e n t changes in E2 a n d the i n c r e a s e d pulsatile LH release o b s e r v e d in this s t u d y and by others ( 9 ) . It is p r o b a b l e that d u r i n g the infantile stage o f life the h y p o t h a l a m i c p i t u i t a r y axis of bulls is e x q u i s i t e l y sensitive to E2, b e c a u s e the d r a m a t i c increase in LH p u l s e f r e q u e n c y was associated w i t h o n l y a slight d e c l i n e in
E2. It is c l e a r that a d m i n i s t r a t i o n o f E2 b l o c k e d t e s t i c u l a r d e v e l o p m e n t . At p r e s e n t it is u n c e r t a i n if this effect o f E2 is related o n l y to i n h i b i t i o n of LH s e c r e t i o n or r e d u c e d LH and follicle s t i m u l a t i n g h o r m o n e (FSH) secretion. Recently, S c h a n b a c h e r et al ( 1 3 ) r e p o r t e d that a d m i n i s t r a t i o n o f E2 to calves i n h i b i t e d s e c r e t i o n o f FSH b u t not LH. H o w e v e r , in that s t u d y the s a m p l i n g f r e q u e n c y u s e d w a s n o t a d e q u a t e to judge the effect o f E2 on pulsatile release o f LH. T h e d r a m a t i c d e c r e a s e in LH s e c r e t i o n n o t e d in this s t u d y and that b y Wise et al ( 1 0 ) d e m o n s t r a t e s the E 2 can i n h i b i t the s e c r e t i o n o f LH as w e l l as FSH ( 1 3 ) . It is p o s s i b l e that a d e q u a t e s e c r e t i o n o f b o t h g o n a d o t r o p i n s is necessary for n o r m a l testicular d e v e l o p m e n t . T h e a n a t o m i c a l localization o f b o v i n e GnRH n e u r o n s is similar to that r e p o r t e d for s h e e p ( 2 1 ) and a v a r i e t y of o t h e r l a b o r a t o r y animals ( 2 2 ) . The majority of the i m m u n o s t a i n e d cell b o d i e s w e r e in the m e d i a l POA, and these n e u r o n s
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are likely the m a i n s o u r c e of GnRH i n n e r v a t i o n to the SME. The large m u l t i p o l a r p r o p o r t i o n of GnRH cell b o d i e s in the bull calf is similar to that r e p o r t e d for the o v i n e ( 2 1 ) , h o w e v e r , this is different than in primates, bats, rats (22) and m i c e ( 1 7 ) w h e r e m o s t GnRH n e u r o n s are uni- or bipolar. It is i m p o r t a n t to n o t e that areas o f the brain outside the POA-hypothalamic region w e r e not e x a m i n e d in this study. Thus the possibility exists that there are extra-POA p o p u l a t i o n s of n e u r o n s c o n t a i n i n g i m m u n o r e a c t i v e - G n R H . T r e a t m e n t w i t h E2 increased the density of i m m u n o r e a c t i v e - G n R H e l e m e n t s in the SME. This i n c r e a s e d intensity o f staining is b e l i e v e d to b e caused b y an a c c u m u l a t i o n of GnRH in these fibers ( 1 8 ) . As the antisera to GnRH does not cross-react w i t h the p r o - G n R H m o l e c u l e ( 2 2 ) and t r e a t m e n t w i t h E2 did not affect the intensity o f staining of GnRH cell bodies w e p r o p o s e that synthesis, p r o c e s s i n g and t r a n s p o r t of the GnRH p r e c u r s o r w e r e not affected by Ez, b u t that the release of GnRH into the h y p o t h a l a m i c portal vessels was attenuated. This suggestion is f u r t h e r s u p p o r t e d b y a p r e v i o u s study (12) w h e r e administration of e x o g e n o u s GnRH re-initiated testicular g r o w t h in estradiol-treated p e r i p u b e r t a l bulls. A r e d u c e d rate o f GnRH release m i g h t also have c o n t r i b u t e d to the h i g h e r c o n c e n t r a t i o n of p i t u i t a r y GnRH r e c e p t o r s f o u n d in the E2-treated calves. In addition, since t r e a t m e n t w i t h E2 has b e e n s h o w n to increase the n u m b e r s o f GnRH r e c e p t o r s in o v a r i e c t o m i z e d s h e e p ( 2 4 ) and m o n k e y s ( 2 5 ) it is p o s s i b l e that E2 acted d i r e c t l y on the pituitary to e n h a n c e the synthesis of GnRH r e c e p t o r s . Previously, w e have r e p o r t e d that c o n c e n t r a t i o n s of NE and DA within the SME i n c r e a s e d w i t h age and w e r e p o s i t i v e l y c o r r e l a t e d w i t h p l a s m a testosterone ( 1 5 ) . In e x p e r i m e n t 2, c o n c e n t r a t i o n s of NE, b u t not DA, w e r e affected b y t r e a t m e n t w i t h Ez and the values r e p o r t e d are similar to those p r e v i o u s l y f o u n d in the SME of 8 and 24 w k calves (.84 and 1.59 n g / m g , r e s p e c t i v e l y ) . Based on these findings, it is p o s s i b l e that age-related changes in NE are d e p e n d e n t on c h a n g e s in s e c r e t i o n o f testosterone. The i m p o r t a n c e of n o r a d r e n e r g i c neurons, t e r m i n a t i n g the SME, in r e g u l a t i n g GnRH release and LH secretion is not unclear. Administration o f NE to m a t u r e s h e e p ( 2 6 , 2 7 ) and cattle ( 2 8 ) has b e e n s h o w n to inhibit the s e c r e t i o n o f LH. Since SME c o n c e n t r a t i o n s of NE are l o w in y o u n g and Ez-treated calves, it is p o s s i b l e that it is not a m a j o r i n h i b i t o r of GnRH and LH s e c r e t i o n until after the o n s e t of testicular secretion of testosterone. Alternatively, age-related increases in c o n c e n t r a t i o n s of NE w i t h i n the SME m i g h t reflect a d e c r e a s e d sensitivity of GnRH fibers to the i n h i b i t o r y effects o f NE. Since p l a s m a c o n c e n t r a t i o n s of E2 w e r e higher in i m p l a n t e d than c o n t r o l calves, it is possible that GnRH n e u r o n s r e m a i n e d e x t r e m e l y sensitive to i n h i b i t o r y effects o f NE and c o n s e q u e n t l y e l i m i n a t e d the n e e d for age-related c h a n g e s in NE m e t a b o l i s m w i t h i n the SME. In s u m m a r y , a d m i n i s t r a t i o n of E2 causes p r o f o u n d changes in h y p o t h a l a m i c function, i n c l u d i n g an a p p a r e n t r e d u c t i o n in the release of GnRH and alterations in synthesis o f NE. These changes likely c o n t r i b u t e significantly to the r e d u c e d s e c r e t i o n of g o n a d o t r o p i n s and i n h i b i t i o n of testicular d e v e l o p m e n t observed in calves t r e a t e d w i t h Ez. FOOTNOTES 1 This paper was authorized for publication as paper No. 7904 in the journal series of the Pennsylvania Agricultural Experiment Station. Address correspondence and requests for reprints to Dr. Daniel R. Deaver, Dariy Breeding Research Center, Pennsylvania State University., University Park, PA 16802
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