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Changes in thymosin β4 and gonadal function during puberty in boars and gilts immunized against estrone
Anima/Repr~~icfionSc:enc~, 24 (1991) 139-152 Elsevier Science Publishers B.V.. Amsterdam
139
Changes in thymosin ,B4 and gonadal function. during puberty in boars and gilts immunized against estrone T. Wise’, J. Klindt, J.J. Ford and R.K. Christeuson L’SLLI~, Agricultural Research Service, Roman L. Hruska U.S. Meat Animal Research Center. P.O. Box 166, Clay Center. NE bBY33 (U.S.A.) (Aceepwd 16 July 1990)
ABSTRACT Wise. T., Klindt. J., Ford, J.J. and Christenson. R.K.. 1991. Changes in thymosin #4 and gonadal function during puberly in boars and gills immunized against estrone. Anim. Rep&. Sci.. 24: 139152. Thymosin gS. a thymic secretory peptide, may have an imponant integmdve role in gonadal funcdon by prom&q the release of gonadolmphin releasing hormone (GnRii ). To investigate possible thymic endocrine relationships to pnadal function in swine, wum changes in thymosin /?4 were monitored throughoa the pnpubenal period in gilts and bosrs immunized Pgainst either keyhole limpet hemocysnin (KLH-cuntrols; &IS 68, boars 6) or KLH conjugated 1o eswone (and-El; gilts 76. boars 6). Immunization was initiated at 5 weeks of age and animals were blood sampled a14-wk intervals until puberty. Gilts were bred at the second estrus. Af 30 (~61) and 70 days (n=83) of pregnancy. ovarian we&h& c’orpom lulea wei@ and numbers, and feud numbem were collected. A( 24 weeks of a& b+an were killed and the weights of testes and ~eeessoryglands collected. As boars nod gills matured, serum coneenlrations of thymosin ,?4 dsreased (PcO.05) wilh age. Gilts that almined puberty (eslrus) had lower concentrations ofthymosin,% (PxO.05) than animals thar demonstrated no eswous activity. No differences were detected between controls or and-E 1 gilts in nlalion 10 any repmducriw parameters monitored, gonadal steroids or thymosinfl concentrations. AntiEl boars had lowerthymonin j74(PcO.05) throughoulthe pnpubenal period than controls, and tended lo have lower testicular, epididymal, and prostate weights; ooly seminal vesicle weight was significantly decreased (103.2?21.2 vs. 173.2f 15.2 g P-zO.05). Boars immunized against estrone tended 10 have lower LeydiE cell numbers (P-zO.09) and seminiferous tubule diameter (PcO.09). but increwd Leydig cell size (PcO.01). A rexually dimorphic rerpoox was noted 1o immunization a8aiW estmne. in that lhymoain ,%I concentrations were decreased in boars, buf unchanged in gilts.
INTRODUCTION Initial reports have linked the function of thymosinp (43 amino acid peptide) to various immunological processes, induction of terminal deoxynu*Author to whom correspondence should he addressed. “Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warranty by Ihe USDA and does not imply its approval to the exclusion of other products that may also be suitable.
037S-4320/91/$03.50
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cleotidyltransferase in thymocytes (Low et al., 1981), inhibition of macrophage migration (Thurman et al., 1981) and the development of humoral immunity (Gondo et al., 1987). Later reports on thymic peptides and thymosin 84 in GnRH release (Rebar et al., 198I; Rebar, 1984; Cieslak et al., i 550) have renewed interest in the integrative relationships of the immune system and reproductive function. The thymus gland has androgen and estrogen receptors (Grossman et al., 1979; McCruden and Stimson, 1981) and, although steroid actions on thymic functions are poorly defined, it has been proposed that androgens depress and estrogens stimulate immune function (Grossman, 1985, 1989). Sexual differences in endocrine and reproductive relationships are common across species, and sexually dimorphic differences of the immuno-reproductive axis are now being elucidated (Farookhi et al., 1588; Grossman, 1589). Sexual dimorphism in immune response is presumably based on differences in the predominant types or ratios of circulating sex steroids (androgenor estrogen;Grossman, 1985,1989). Mostgonadal-thymic relationships have been proposed from information established in rodents, and little is known in relation to farm animals. To establish possible thymic endocrine relationships to gonadal function in swine, changes in thymosin j?4 concentrations were monitored during the period of pubertal development in gilts and boars, and in gilts and boars immunized against estrone (E, ) during pubertal development. Since E and estrone sulfate (EtS) concentrations are important steroids in both porcine sexes (Robertsnn and King, 1974: Ford, 1983), low titer immunization against estrone could potentiate possible differences in thymic-gonadal relationships during pubertal development since steroidal feedback mechanisms may be different due to primary secretion of androgens or estrogens by the gonads of males or females. MATERIALS AND METHODS
To study the relationships of puberty and thymosin 84, crossbred gilts ( I / 4 Yorkshire l/4 Landrace I /4 Large White I /4 Chester White) were immunized against Keyhole limpet hemocyanin (KLH) (controls, n=68). or E, conjugated at the “three &sition” to KLH.(antiiEi; n=76). This allowed production of antibodies cross-reactive with E, and E,S, but not with estradiol (Er) or testosterone (T). Starting at 5 weeks of age, the immunization regimen consisted of an initial intradermul iniection of I ml of Freund’s complete adjuvant (SO0 l(~ antigen) on the vet&al surface, followed by three boosters ( 100 fug) at four-weekly intervals. This regimen was specitically da signed to produce low antibody titers ( 1: lOa), because higher titers ( > I : 1000) produce gonadal dysfunction and acyclicity (Christenson and Wise, 1985). Animals were bled at four-weekly intervals (IO ml) until the
THYMOSN PCHANGESDURING
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141
first detected estrus. Starting at 20 weeks of age, 8ilts were monitored for estrus daily in the presence of mature boars, and mated at second cstrus. Gilts were slaughtered at 30 ( n= 6 1) or 70 days (n = 83) of pregnancy, when embryo number, uterine weight, ovarian we&t, and both corpora lutea weight and number were collected to assess reproductive function for any treatment differences. Contemporary gilts (littermates; x=254) lo the treated animals were monitored and bled with the same regimen to provide a group of animals that did not attain puberty (no estrus demonstrated; n = 25). In conjunction with the gilts, I2 crossbred boars (6 controls and 6 immunized against estrone) were immunized at 5 weeks of age with the same protocol utilized for gilts. An immunization regimen was established to attain titers of I : 108 after the last booster. Boars were bled approximately every 30 days after the initiation ofthe experiment, cannulated at 23 weeks ofage, and blood ( IO ml) cokcted every I5 min for 8 h. All blood samples were placed on ice immediately after collection, serum harvested the next day by refrigerated centrifu8atioo, then frozen ( - 20°C) until hormonal analysis was performed. Boars were killed at 26 weeks of age and testes and accessory glands were weighed. One testis was Axed for histolo8ical analysis and the other frozen ( - 20” C ) for steroidal analysis. lmmunoassays
Antiserum binding titers in animals immunized against E, were determined by dextran- charcoal radioassay with afiquots of diluted serum, amniotic or allantoic fluid ( 1: 10, I : 100. 1: 1000 in phosphate-buffered saline) incubated with 20,000 dpm of [2,4,6,7]estrone (SA 108 Ci mmol-‘; New England Nuclear Corp., Boston, MA) (Wise and Schanbacher, 1983; Wise and Femil, 1984). Concentrations of thytnosing4 were measured by doubleantibody radioimmunoassay (Naylor et al., 1984) asadapted for swine (Vakharia et al., 1986a. b). Duplicate samples ( 150 @) of serum were analyzed. The assay limit of detection was 250 pg per tube and the standard curve ranged front 200 pg to 200 ag. Iatra-assay and inter-assay coefficients of variation were 12% and 16% respectively. Concentrations of luteioizing hormone (LH) were determined by the method of Niswender et al. ( 1970) as modified by 9llrich et al. (1982). The limit of sensitivity of the LH assay was 25 pg ml- ‘, with an inter-assay coefficient of variation of 14%. Growth hormone (GH) and prolactin were quantified by radioimmunoassays (Ohlson et al., 1981; Klindt et al., 1983). Limits of sensitivity for GH md prolactin were I50 and 70 pg ml-‘, with coefficients of variation of 123%and 14.446,respectively. Blood serum and placental fluid steroids (T, dehydroepiandrosterone sulfate (DHEAS), E,, Ez and ES) were measured by established radioimmunoassays (Allrich et al., 1982, Ford, 1983, Wise, 1987). The limits of sensitivity
T.WSEEKAL.
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for T, DHEAS, E,, E2 and E,S assays were 10, 10, 5, 5, and 5 pg ml-‘, respectively, with respective inter-assay coefficients of variation of 10.8, 10.0, 8.7, 14.0 and 12.3%. Testicular tissue (250 mg) was ether extracted, submitted to double development thin layer chromatography (Linear-K, Whatman; hexane:ethyl ether:acetic acid 90: 10: I, v/v, and then chloroform:ethanol 99: 1, v/v). Progesterone (P), T, and E, were quantitated by radioimmunoassay (Wise, 1987), the limits of sensitivity being 100, 10 and 5 pg ml-‘, respectively, with intra-assay variations of 8.5.9.0 and 12.5%. Statistical
analysis
Data representing multiple blood samplings from animals were analyzed by split-plot analysis of variance (MS, 1979) to determine effects of treatment (control vs. anti-El ), time, and time by treatment interaction (Figs. I, 2 and 3). Significant main effects or interactions were subsequently submit-
37
65
92
120 1LR 176 2136
Age (days1
tbl
=
9 10
I 8O
37
65
92
120 118 176 20~
Fig. I ~(a ) Serum estrone cotvxnlralions (tncsr8+ SEM ) in prepubxtal gilts immunized against KLH (open bars). and gills immunized against estrone (hatched bars). No differences were noted b~lwrcn ~re;untcntor over time. Artwe indicate rimes of immunization. (b) Serum estrodiol conuentmtions in prepukrtal canlrol gilts immunized against KLH (cpcn bars). and $ilts immunized ngainsl estrone conjugated to KLH (halched bars). No treattnentdifferences wc~~detected hut cstndinl incwased as puhwty was atrninedand animals demonstrated estrw.
THYMOSlN84CHANGESDURlNG
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143
ted to means comparison by f-test. Other endocrine and weight comparisons were analyzed by one-way analysis of variance where treatment (control vs. anti-El ) was considered a fixed effect (Tables I and 2). RESULTS
Control animals bound less than 1% ‘H-estrone at 1: IO or 1: 100 serum dilutions. Anti-El gilts bound 23% ‘H-estrone and boars bound 5% after the third booster ( 1: 100 dilution). Peripheral E, and Ez monitored at 90-200 days of age (Fig. i ), or E,, E,S and E2 at slaughter (30 days pregnant, Table 1) were the same in serum and placental fluids. No binding of 3H-estrone was detected in amnioiic or allantoic fluids, indicating that the antibody did not cross the placenta at these stages of pregnancy. TABLE I Comparisons of tcpmductive tissua and gonadal steroids in gilts immunized against keyhole limpet hemwynnin ) and Keyhole limpet hemqanin conjugated to e?.tm”e (Anti-El )
(contml
Gilts
30 days prq”a”t CO”W”l. “~28 Anti-El. n=33 70 days pregnant Contml. n=40 Anti-El. Iv=43
OVWil” weight’
corpora lutea number
Uterine weight2
Embryo number
Il.8iO.5
4.1 to.1
12.3tO.7
2.0fO.15
10.8?0.6
12.0+0.3
4.2tO.2
12.1 to.4
2.Sf0.19
10.7+0.6
14.2tO.3
4.9ro.1
13.220.3
2.2+0.06
11.1+0.3
4.9to.1
13.8kO.4
2.4kO.09
14.6tO.S
11.3*0.4
Swum esttvgensJ
Estrone’
EStI”“e sulfate’
Estradiol’
Control. n=28 Anti-El. 1x=33
26.3Al.8 24.2+ 1.7
2.5tO.3 2.0-fo.2
6.8fl.l 6.5fl.O
PlBX”l”l estrogens’~
Allantoic enrone
Allantoic estmne sulfate’
Amniotic estmne’
Control. II= 28 Anti-El.“=33
3.5io.5 3.4io.5
35.lt2.6 32.8k2.5
459.62 143.7 554.52 132.5
‘h4canfSEM.g *Meanf SEM, kg. ‘At 30 day pregnancy. “Mean+SEM.pgm!-‘. ‘Man+SEM,“gml-‘.
T. WlSE ET
.a.
100 t 0 40
: 60
: 60
: 1W
120
IL0
160
160
200
2a
c 264
Fig. 2. (a) TP conccnlrations (LS means) and regxssion line depictingdecreases in T/74 prior lo puhcrly (dny O=dayofcsIrus) inconlrolgills (circles. immunizedagainst KLH,n=68) and gills immunized againsl c~lmnr (squares. anti-El, n=76). Data were normalized so Day 0 ~~P~~scII~sthe day oflirsl eslrus (220 daysprior IO pubcrly is approximalcly 40 days of age). No differences were dulccled between trcalmcnls. and the line represents the “best fit” polynomial rc6rcssion line for pooled data. (b) T&I concentrations in gills (n=25) in which no cslrus was detected. Tp conccnlralions were slgnificanlly incrcati over gilts that allained pubcrly (delcclable cslrus and prcgmmcy 1. Standardcrrcw are indicatedal e&h meantime.
14s
9l
Icy ,d!$)
.166
lL? Contrd’0 Anti-Eq:m lb1
0s
e; f
-.a
-em.
lm
. .
_ _. . . . . .
-.;.-
,*-0
: l.: . . -.;--a-,
.
Fig. 3. (a) Tfl concentrations (mean+SEM) in boars immunized against KLH (controls. circles, n=6) or KLH conjugated to estrone (squares. n=b) during the period of prepubertal development. Animals immunized against estrone had lower Tm concentrations than controls ( I13 vs. 150 ng ml-‘: P
T WISEETAL.
146 TABLE 2
Changes in reproductive tissue weights and gonadal steroids in boars immunized against Keyhole limpet hcmocyanin (control) or keyhole Iimpel hemocyanin conjugated to estrone (Anti-E
I)
COIlld. n=6 Anti-El, n=6
Epididymis
Seminal vesicles
Prostate
Bulbourethal
239.62
20.9
42.9f2.0
173.2t15.2
5.9 + 0.8
106.9+
8.1
196.6f
13.4
37.02 2.8
103.2+21.2*
4.8? 1.0
110.8f
17.8
15.1~1.6 20.1+3.1
44.8f 15.4 72.72 27.2
5.8 f 3.0 2.6 2 7.0
Serum hormones’
T~iOStC~O~C
Dehydrwpiandrostcmne SIllfate
Estmne sulfa1c
Control. “~6 Anti-El.n=6
I2.6 f
14.312.1 2.0
34.5fS.l 26.92 3.9
5.9+_1.8 4.6f 1.0
Conlml. Anti-El,
n=6 n=O
‘hteall i SE,+,, 8. *P
‘ngg-’ wet weight. ‘Eight-hour
intensive sampling mean+ SEM. ng ml- ‘.
In gilts, thymosin/B concentrations showed no significant treatment or timextreatment effects, but decrease 1 in concentration from 6 weeks of age until puberty (kO.01, Fig. 2a). In contemporary gilts that did not attain puberty (n = 25), thymositQ4 concentrations remained elevated over animals that exhibited estrus (200-250 ng ml-‘; Kg. 2b. P-zO.05). At slaughter (280 days ofage),it was determined that 52% (13/25) of the anestrousanimals were prepubertal (small uteri and nonfunctional ovaries), and 48% ( 12/ 25 ) were of behavioral anestrous status (functional ovaries but no characteristics of estrus). There were no differences in the concentrations of thymosing4 in prepubertal(255.8 2 4.8 ng ml-‘) or behaviorally anestrous animals (257.9 + 5.0 ng ml-‘) thus, data were pooled for analysis. Immunization of gilts against E had no effect on reproductive function in relation to age at lirst estrus ( 18324 or 18624 days,controlsand anti-El, respectively), ovulation rate, corpora lutea weight, or fetal numbers (Table I ). Thymosin@ concentrations in control boars decreased with maturity (Fig. 3a; P
tensive sampling period (PC 0.05; Fig. 3b), but time by treatment interaction was not significant (P> 0.1). A trend towards lower testicular, epididymal and accessory gland weights was noted in anti-E I boars, but only seminal vesicle weights were significantly decreased (173.2 vs. 103.2 a. P-=0.05: Table 2). Total Leydig ceil numbers tended to be reduced in a&El boars compared to those of controls (20.022.8x IO9vs. 27.122.8x IO”;P-=0.09) in conjunction with reduced diameter of seminiferous tubules in anti-El testes (24.6 2 5.0 vs. 25.8 2 5.0 pm, PC 0.09). Leydig cell volume was increased in the testes of anti-E I boars compared to those of controls (3 142 + 108/rm’ vs. 23212 149 pm3, PeO.01) and was accompanied by an increase in the size and number of intra-cellular organelles (data not presented). Levels of serum and testicular steroids were not different in control or anti-El boars (Table 2).
Thymosin/34 has teen shown to release GnRH in the rodent, both in vitro and in vivo (Rebar, 1984). but knowledge of the reproductive relationships with thymosin/&i in other species is scant. Thymosin 84 concentrations were found to decrease during the prepubertal period (PcO.05) both in gilts and boars as they matured and reached puberty, consistent with steroid feedback effects on thymic function as gonadal function is established at puberty. It is known that as heifers mature, attain puberiy and become pregnant, peripheral thymositQ4 concentrations also decrease; but in animals that did not become pregnant (noncyclic), the level remained elevated (Wolfe et al., 1989). A similar pattern was noted in noncyclic gilts (Fig. 2b)..Overall, the immbnization against E had no effect on reproductive function (Table I ) or thymosin/34 concentrations in gilts. In sheep immunized against estrogens, ov& lation rate and fecundity are significantly increased (Scaramuzzi et al., 1980; Smith, 1985). but no alteration of ovulation rate or embryo numbers was noted in anti-El gilts (Table I ). Serum binding titers were generally lower in the swine utilized in this study when compared to titers in sheep that responded with increased lambing rates, but a negative aspect of attaining high titer responses (> I : 1000) is competition for circulating El by antibodies that yields less antigen (E, ) available to target cell receptors and sterility (Christenson and Wise, 1985). Also, maternal antibodies to E, did not cross the placental barrier and thus had no influence on fetal-placental steroids !%a! I ) that are critically important at implantation (Robertson and King, Thk effects of estrogens upon thymic function is both complex and conflicting (Glucksman and Cherry, 1968; Grossman et al., 1982; Berczi, 1986). Variable responses to estrogens may be due to pharmacological dosages administered (Raveche and Steinberg, 1986). Chronicestrogen therapy can reduce thymosinfl concentrations in women (Sub et al., 1985), and administration of thymosin fraction V (from which thymosin/I4 is derived) can
148
T. WISE ET AL
increase plasma estrogen levels in mice (Michael et al., 1981). Contrary to the lack of treatment differences noted in gilts (control vs. anti-El), boars immunized against E, (even at low titers) demonstrated significant decreases in thymosi@ (Fig. 3), which was accompanied by lowered testicular and accessory gland weights, decreased Leydig cell numbers and increased Leydig cell size. No differences were noted in steroid concentrations (Table 2) between control and anti-El boars, even though testicular weights tended to be decreased. This may he due to an increase in Leydig cell size and volume in anti-El boars (Lunstra et al., 1988). which compensates for decreased testes weight. Responses to immunization against steroids are thought to be related to two physiological mechanisms, (1) establishment of either high binding titers ( > I : 1000)or high affmity constants in which the antibody effectively competes with cellular receptors and deprives target tissues of steroids, and (2) reponses to low titers ( -Z1: lOOO),in which the antibody may act as a steroid-bound reservoir and decrease clearance, thus providing increased steroid to target tissues (Pardridge, 1981: Wise and Ferrell, 1984). Interpretation of the mechanism of ste:oid immunization is complex, particularly in a species like swine, where the testes produce large amounts of estrogens and estrogen sulfates (Booth, 1980; Ford, 1983; Raeside. 1983), to which the antibody (in this study) would bind. The antigen in this study was synthesized to bind both E, and E,S because of the known predominance of sulfated estrogens in the male and female of this species (Orava et al., 1985; Ruokonen and Vihko, 1974). Depression of accessory gland weight and function during exogenous estrogen administration is thought to be due to decreasesin T production which occur via decreases in LH stimulation and local effects on T synthesis (Kalla et al., 1980; Price and Williams-Ashman, 1961; Ronco et al., 1987). The observed decreases in thymosiQ4, accessory gland weights, testicular weights, and Leydigcell numbers in anti-El boars (titer 5% binding at I : 100dilution) support the concept of an overall increase of stemid (estrogen) available to target tissue. With the high concentrations of El and E,S in the boar, it is hard to imagine immunoneutralization and competition with cellular receptors at a 1: 100titer. Although thymosinpt concentrations were lower in the anti-El boars, LH concentrations (mean, amplitude and basal; data not presented) did not differ. This would appear to challenge the concept of thymosinfi involvement in GnRH release and subsequent LH discharge from the pituitary. However, changes in gonadotrophin heterogeneity and biological activity may not have been detected with the radioimmunoassay used in our studies ( Wakabagashi, 1977; Grotjan and Steinberger, 1978). Administration of EZ is known to change the pattern of pituitary LH isohormones, which could result in the secretion of gonadotrophin lower in biological activity in sheep (Keel et al., 1987). Thus, lack of differences in LH concentrations between treatments as established by radioimmunoassay may bc due to production of LH isohor-
THYMOStN~CHANoESoVR,NGPUsERTY
149
IN sO*RSANoolLTS
manes of less biological activity whit’? would be reSected in altered testicular weight, Leydigcell size, and Leydig r.:ll numbers. Alternatively, recent cDNA probe analysis for thymosin/%t (Gor. ez-Marquez et al., 1989; has established the presence of thymosin~ mRNA in most t&tics $idxy, lung, testis, ovary, muscle, etc.), although immunological tissues possessed the highest concentratioc of message (tbymus, spleen). The common presence of mRNA for thymosin /94in many tissues indicates an important but common tissue function for thymosinfl, though possibly not as an endocrine regulator (GomexMarquez et al., 1989). It is not known which tissues produce the thymosinp4 present in the peripheral circulation, but circulating concentrations can be altered by various endocrine inputs (Suh et al., 1985;Wise et al., 1987,199(J). In conclusion, circulating tbymosinfl in swine decreased with maturity, with the lowest concentrations detected at puberty. Only in anti-El boars were thymosing4 concentrations reduced, thus providing evidence of a sexually dimorphic response in which gilts showed no effects. Testes of anti-El boars were altered on a gross and histological basis; however, on an endocrine basis, the testis seems to have compensated in function. The differences noted in thymosin@4 concentrations as animals matured and attained puberty, when considered in conjunction with the sexually dimorphic response noted between anti-E 1 boars and gilts, demonstrates that circulating concentrations of thymosinw are under some aspect of control. Whether thymosir@ has any direct function in control of the gonad has yet to be ascertained. ACKNOWLEDGMENTS
The authors thank D.J. Taubenheim, S. Reece, J. Crisp, A. Kruger, and L. Pamell for their technical and clerical assistance. Dr. Don Lunstra provided the histological information on boar testes.
REFERENCES Allrich. R.D., Christenson, R.K., Ford, J.J. and Zimmernun, D.R., 1982. Pubertal develop ment of the boar: Testosterone. estradiol-17fl. cortisnl and LH conccslrations before and al various ages. J. Anim. Sci., 55: 1139-l 146. Berczi, I., 1986. Immunoregulation by pituitary hormones. In: I. fterczi (Editor). Piluitary Function and Immunity. CRC Press, Etoca Raton, FL, p. 233. Booth, W.D.. 1980. A study ofsome major testicular steroids in the pig in relation to theireffect on the development of male characteristics in the prepubwtally castrated hoar. 1. Reprod. Fenil.. 59: 155-l 62. Christenson, R.K. and Wise, T.H., 1985. Effect of active immunization against estradiol-17,9 on estrous activity in sow. J. Anim. Sci., 61 (Suppl. I I2 (abstract). Cieslak, D.. Kane+Radek, J. and Pawlikowski, M., 1990. Evidence for prostagJandin mediation of the stimulation of luteinizing hormone releasing hormone release by calf thymus extract. Thymus, 15: 193-198.
aftercastration
I ):
Famokhi, R., Wesolowski, E., Trader. J.M. and Robaire, B., 198% Wudulation by neonatal thymeemmy of the reproductive axis in male and fern& rats during development. Biol. Reprod.. 38: 91-99. Ford, J.J., 1983. Serum estmgen concentrations during postnatal development in male pigs. Proc. Sot. =.:@.Bml. Med., 174: 160-164. Glucksmann, A.-and Cherry. C.P., 1968. The cl&I of castration. o-estrogens. IestosIerone and the oestrous cycle on the cortical epithelium of the thymus in male and female milts.1. Anal., 103: 113-133. Gomez-Marquez, I., Dosil, M., Semde, F.. Bus1e1o.X.R. Pichel, J.G., Dominguez. F. anct Freire, M., 1989. Thymosin@l gene: PreliminarychanctcrizaIion and expression in tissues, thymic cellsand lymphocytes. 1. Immunol.. 143: 2740-2744. Gondo, H.. Kudo. J., White, J.W., Barr, C., Selvanayagam, P. andSaunders, G.F.. 1987. Differential expression of the human Ihymosin ~?dgene in lymphocytes, macrophages. and granulocyles. J. Immunol., 139: 3840-3848. Grossman. C.J., 1985. lnteractions beween the gonadal steroids and the immune system. Science, 227: 257-261. Grossman. C.J.. 1989. Possible underlying mechanisms of sexual dimorphism in the immune response, facl and hypothesis. J. Steroid Biochem., 34: 241-251. Grossman. C.J., Sholiton, L.J. and Nathan. P.. 1979. Rat thymic estrogen receptor. I. Prepamlion, location and physiochemical pmperties. J. Steroid Biochem., I I: 1233-1240. Grossman. C.J., Sholiron, L.J. and Roselle, GA.. 1982. Estradiol rgulation of thymic function in the 1x1:Medialion by serum Ihymic factors. 1. Steroid B&hem.. 16: 683-690. Grotjan. H.E. Jr. and SIeinbergcr. E.. 1978. On the relative biological and immunoreactive PO_ tencies of luteinizing hormone in the cestradiol benmate treated male rat. Acta Endocrinol. (Copenhagen). 89: 538-550. Kalla. N.R.. Nisula. B.C.. Menard, R. and Loriaux, D.L., 1980. The cl&xl of estradiol on IesIicular tesIos1emne biosynthesis. Endocrinol~, 106: 35-39. Keel. B.A.. Schanbacher, B.D. and Gnjsn. H.E.. 1987. Ovine luteinking hormone. 1. Effects of castrarion and steroid administration on charge hetcmgeneiIy of pituitary luteinking hormone. Biol. Reprod.. 36: 1102-l 113. Klindt, J.. Ford, J.J., &rardh&elli. J.G. and Anderson. L.L., 1983. Gmwth hormone secretion after hypophysial stalk transec,ion in pigs. hoc. Sot. Exp. Biol. Med., 172: 508-513. Low. T.L.K.. Hu, S.K. and Goldstein. A.L., 1981. Compktc amino acid sequence of thymosin /?4: A Ihymic hormone Ihal induces terminal deoxynuckotidal transfer& activity-in thy. mocyte woulations. Prrx. NatI. Acad. Sci.. 78: 1162-I 166. Lunstra; DID:. Ford. J.J., Klindi, J. and Wi&, T.H.. riS8. Effect of immrt!i?ation against estmne on boar Leydigcell structunsnd&nction. Biol. Repmd. 38. (Suppi. I ):82 (absIrac1). McCruden, A.M. and Stimson. W.H.. 1981. Andmgen bindingcytosol receptors in the mt thymus: Physiochemical pmpwties. specificity and location. Thymus. 3: 105-I 17. Michael. SD.. Allen. L.S.. McClure. J.E., Goldalein. AL and Barkley. MS.. 1981. Interaction belween estmdiol and thymosin levels in Ihc female mouse. 63rd Annu. Mest. Endow Sw.. p. IS9 (Abstract 308). Naylor. P.H.. McCiure, J.E., Spangelo. B.L.. Low. T.K.L. and Goldstein, A.L.. 1981. Radioimmuuoassay ofthymosin w. I...: :nOpha”naCOlOgy.7: 9-16. Niswcndet. G.D.. Reichen. L.E. and Zimmerman. D.R.. 1970. Radioimmunoassay of serum levels of lureinizing hormone throughout the eslmus cycle in pigs. Endocrinology. 87: 576580. Ohlson. D.H.. Spacer, L.J. and Davis. S.L.. 1981. Use ofactive immunization against pmlaclin to study the influewx of pmlactin on gmwlh and repreduction in the ram. J. Anim. Sci.. 52: 1350-1359. Own. M.. Haour. F.. Leinonen. P., Ruokonen, A. and Vihko, R.. 1985. Relationships between
tutconjugated and sulphated steroids in porcine primary B&hem.. 22: 507-512.
Leydig cell culture. J. Steroid
Pardridge, W.M.. 198!. Transport of protein-bound hormones into tissues in viva. Endocrine Rev.. 2: 103-123. Price. D. and Williams-Ashman. H.G.. 1961. The accessory reproductivegiands of mammals. in: WC. Young (Editor), Sex and internal Secretions. Vol. I. Williams and Wilkins. Baltimore. MD. p. 366. Raeside, J.I.. 1983. The fomta1ion of unconjugated and suifoeonjugated estrogens by Leydig ceils of boar testis. Can. J. BiocLem. Ceil Biol.. 61: 790-795. Raveche, ES. and Steinberg, A.D.. 1986. Sex hormones in auto-immunity. in: I. Betczi (Editor), Pituitary Function and Immunity. CRC Press. Boca Ralon, FL, p. 287. Rebar, R.W., 1984. Effects of thymic peptides on hypothalamic-pituitary function. in: A.L. Goldstein (Editor), Thymic Hormones and Lymphokines. Basic Chemistry and Clinical Applications. Pienum Press. New York, pp. 325-334. Rebar, R.W.. Miyake, A.. Low. T.L.K. and Goldstein, A.L., 1981. Thymosin stimulates secretion ofiuteinizing hormone-releasing factor. Science, 214: 669-671. Robertson, H.A. and King. G.J.. 1974. Plasma concentrations of progesterone, oestrone, oestradioi-17gand oeslmne suiphate in the pig at impiantalion, during pregnancy and at parturition. 1. Repmd. Fet-tii.. 40: 133-141. Ronco, A.M.. Pino. A.M. and Vailadares, L.E.. 1987. RNA synthesis in the Leydig ceils of the mature rat: EtTect ofestradioi. J. Endocrinoi.. i ib: 387-392. Ruokonen. A.T. and Vihko. R.. 1974. Stemid metabolism in testis tissue: Concentrations of unconjugated and sulfated neulrai slemids in boar testis. J. Steroid B&hem., 5: 33-38. SAS. 1979. Users Guide: Stttistical Ar+sis System. SAS fnstitttte Inc.. Gary, NC. Searamuzzi, R.J.. Martensz, N.D. and VanLook. P.F.A., 1980. Ovarian morphology and the concentration of steroids, and ofgonadotrophins during the breeding season in ewes activeiy immunired againsluestradioi-17flor oestrone. J. Repwd. Fertil., 59: 303-310. Smith, J.F.. 1985. immunisatian of ewes against steroids: A review. Pmt. New Zealand Sot. Anim. Prod.45 171-177. Suh. B.Y.. Naylor, P.H.. Goldstein. A.L. and Rebar. R.W.. 1985. Modulation of thymosin-/?4 by estrogen. Am. J. Obstet. Gyneeoi., 544-549. Thurman, G.B., Low. T.L.K.. Rossio. J.L. and Goldstein, A.L.. 1981. Specific and nonspecific macrophage migration inhibition. in: A.L. Goldstein and M. Chirigos (Editors), Lymphokinesand Thymic Facron. Their Potentiai in Cancer Therapeutics. Raven Press, New York, p. 145.
1.5 I:
Vakhrria. D.D.. Kinder, J.E. and Wise, T.H.. 1986a. Thymosin (ThB4) levels in serutn and foliicular fluid during estrous cycle (EC) in swine. Fed. Proc., 45: 405 (abstract). Vakharia. D.D., Kinder, J.E. and Wise, T.H., 1986b. Thymosikai in the ovary and the levels of thymosin-al and T lymphocytes during estrous cycle in swine. in: M.E. Tumbieson (Editor). Swine in Biomedical Research, Vol. 3. Plenum Pubiishine Comoration. New York. pp. 1925-1936. Wakabagashi. K.. 1977. Heterogeneity of rat iuteinizing hormone revealed by radioimmunoassay and eiectrofocusingstudies. Endocrinoi. Jpn., 24: 473-485. Wise. T.. 1987. Biochemical analysisof bovine foiiicuiar fluid: Albumin, total protein, iysosomai enzymes, ions, steroids and ascorbic acid eontent in relation to foliicuiar size, rank, alresia classification, and day of estrous cycle. J. Anim. Sei., 64: 153-I 169. Wise.T.H. and Ferreli,C.L., 1984. Effectsofimmunizttionofheifersagainslestradioiongrowh, reproductive trails. and carcass characteristics. Pmt. Sot. Exp. Bioi. Med., 176: 243-248. Wise. T., Kiindt. J.. Macdonaid, G.J. and Ford, J.J., 1990. Effects of neonatal sexual differentiation, growth homtone and lestostemne on thymic weigltts and thymosin @4 in hypophysectomized rats. J. Reprod. immunoi., in press,
I
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Wise, T.H. and Schanbacher. B.D.. 1983. Reproductive effects of immunizing heifers against androaenedione and oestradiol-l7P. I. Reprod. Fenil., 69: 605-612. Wise, T.H., Siiss, U. and Maurer, R.R., 1987. Relationships of thymorin /34 in superovulated beef heifers with and without Norgestomct implants. J. Anim. Sci.. 65 {Sqp:. I j: 368 (abstract). Wolfe, M.W., Vakharia, D.D., Wise, T.H. and Kinder, J.E., 1989. Characterization ofsecretion of thymosin alpha and beta 4, during prepubeny. estrtts and pregnancy in the bawine female. Dom. Attim. Endocrinol.. 6: 71-78.
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139
Changes in thymosin ,B4 and gonadal function. during puberty in boars and gilts immunized against estrone T. Wise’, J. Klindt, J.J. Ford and R.K. Christeuson L’SLLI~, Agricultural Research Service, Roman L. Hruska U.S. Meat Animal Research Center. P.O. Box 166, Clay Center. NE bBY33 (U.S.A.) (Aceepwd 16 July 1990)
ABSTRACT Wise. T., Klindt. J., Ford, J.J. and Christenson. R.K.. 1991. Changes in thymosin #4 and gonadal function during puberly in boars and gills immunized against estrone. Anim. Rep&. Sci.. 24: 139152. Thymosin gS. a thymic secretory peptide, may have an imponant integmdve role in gonadal funcdon by prom&q the release of gonadolmphin releasing hormone (GnRii ). To investigate possible thymic endocrine relationships to pnadal function in swine, wum changes in thymosin /?4 were monitored throughoa the pnpubenal period in gilts and bosrs immunized Pgainst either keyhole limpet hemocysnin (KLH-cuntrols; &IS 68, boars 6) or KLH conjugated 1o eswone (and-El; gilts 76. boars 6). Immunization was initiated at 5 weeks of age and animals were blood sampled a14-wk intervals until puberty. Gilts were bred at the second estrus. Af 30 (~61) and 70 days (n=83) of pregnancy. ovarian we&h& c’orpom lulea wei@ and numbers, and feud numbem were collected. A( 24 weeks of a& b+an were killed and the weights of testes and ~eeessoryglands collected. As boars nod gills matured, serum coneenlrations of thymosin ,?4 dsreased (PcO.05) wilh age. Gilts that almined puberty (eslrus) had lower concentrations ofthymosin,% (PxO.05) than animals thar demonstrated no eswous activity. No differences were detected between controls or and-E 1 gilts in nlalion 10 any repmducriw parameters monitored, gonadal steroids or thymosinfl concentrations. AntiEl boars had lowerthymonin j74(PcO.05) throughoulthe pnpubenal period than controls, and tended lo have lower testicular, epididymal, and prostate weights; ooly seminal vesicle weight was significantly decreased (103.2?21.2 vs. 173.2f 15.2 g P-zO.05). Boars immunized against estrone tended 10 have lower LeydiE cell numbers (P-zO.09) and seminiferous tubule diameter (PcO.09). but increwd Leydig cell size (PcO.01). A rexually dimorphic rerpoox was noted 1o immunization a8aiW estmne. in that lhymoain ,%I concentrations were decreased in boars, buf unchanged in gilts.
INTRODUCTION Initial reports have linked the function of thymosinp (43 amino acid peptide) to various immunological processes, induction of terminal deoxynu*Author to whom correspondence should he addressed. “Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warranty by Ihe USDA and does not imply its approval to the exclusion of other products that may also be suitable.
037S-4320/91/$03.50
0 1991 -
Eisevier Science Publishers B.V.
140
T. VISE ET AI
cleotidyltransferase in thymocytes (Low et al., 1981), inhibition of macrophage migration (Thurman et al., 1981) and the development of humoral immunity (Gondo et al., 1987). Later reports on thymic peptides and thymosin 84 in GnRH release (Rebar et al., 198I; Rebar, 1984; Cieslak et al., i 550) have renewed interest in the integrative relationships of the immune system and reproductive function. The thymus gland has androgen and estrogen receptors (Grossman et al., 1979; McCruden and Stimson, 1981) and, although steroid actions on thymic functions are poorly defined, it has been proposed that androgens depress and estrogens stimulate immune function (Grossman, 1985, 1989). Sexual differences in endocrine and reproductive relationships are common across species, and sexually dimorphic differences of the immuno-reproductive axis are now being elucidated (Farookhi et al., 1588; Grossman, 1589). Sexual dimorphism in immune response is presumably based on differences in the predominant types or ratios of circulating sex steroids (androgenor estrogen;Grossman, 1985,1989). Mostgonadal-thymic relationships have been proposed from information established in rodents, and little is known in relation to farm animals. To establish possible thymic endocrine relationships to gonadal function in swine, changes in thymosin j?4 concentrations were monitored during the period of pubertal development in gilts and boars, and in gilts and boars immunized against estrone (E, ) during pubertal development. Since E and estrone sulfate (EtS) concentrations are important steroids in both porcine sexes (Robertsnn and King, 1974: Ford, 1983), low titer immunization against estrone could potentiate possible differences in thymic-gonadal relationships during pubertal development since steroidal feedback mechanisms may be different due to primary secretion of androgens or estrogens by the gonads of males or females. MATERIALS AND METHODS
To study the relationships of puberty and thymosin 84, crossbred gilts ( I / 4 Yorkshire l/4 Landrace I /4 Large White I /4 Chester White) were immunized against Keyhole limpet hemocyanin (KLH) (controls, n=68). or E, conjugated at the “three &sition” to KLH.(antiiEi; n=76). This allowed production of antibodies cross-reactive with E, and E,S, but not with estradiol (Er) or testosterone (T). Starting at 5 weeks of age, the immunization regimen consisted of an initial intradermul iniection of I ml of Freund’s complete adjuvant (SO0 l(~ antigen) on the vet&al surface, followed by three boosters ( 100 fug) at four-weekly intervals. This regimen was specitically da signed to produce low antibody titers ( 1: lOa), because higher titers ( > I : 1000) produce gonadal dysfunction and acyclicity (Christenson and Wise, 1985). Animals were bled at four-weekly intervals (IO ml) until the
THYMOSN PCHANGESDURING
PUBERTY IN ROARSANDGILTS
141
first detected estrus. Starting at 20 weeks of age, 8ilts were monitored for estrus daily in the presence of mature boars, and mated at second cstrus. Gilts were slaughtered at 30 ( n= 6 1) or 70 days (n = 83) of pregnancy, when embryo number, uterine weight, ovarian we&t, and both corpora lutea weight and number were collected to assess reproductive function for any treatment differences. Contemporary gilts (littermates; x=254) lo the treated animals were monitored and bled with the same regimen to provide a group of animals that did not attain puberty (no estrus demonstrated; n = 25). In conjunction with the gilts, I2 crossbred boars (6 controls and 6 immunized against estrone) were immunized at 5 weeks of age with the same protocol utilized for gilts. An immunization regimen was established to attain titers of I : 108 after the last booster. Boars were bled approximately every 30 days after the initiation ofthe experiment, cannulated at 23 weeks ofage, and blood ( IO ml) cokcted every I5 min for 8 h. All blood samples were placed on ice immediately after collection, serum harvested the next day by refrigerated centrifu8atioo, then frozen ( - 20°C) until hormonal analysis was performed. Boars were killed at 26 weeks of age and testes and accessory glands were weighed. One testis was Axed for histolo8ical analysis and the other frozen ( - 20” C ) for steroidal analysis. lmmunoassays
Antiserum binding titers in animals immunized against E, were determined by dextran- charcoal radioassay with afiquots of diluted serum, amniotic or allantoic fluid ( 1: 10, I : 100. 1: 1000 in phosphate-buffered saline) incubated with 20,000 dpm of [2,4,6,7]estrone (SA 108 Ci mmol-‘; New England Nuclear Corp., Boston, MA) (Wise and Schanbacher, 1983; Wise and Femil, 1984). Concentrations of thytnosing4 were measured by doubleantibody radioimmunoassay (Naylor et al., 1984) asadapted for swine (Vakharia et al., 1986a. b). Duplicate samples ( 150 @) of serum were analyzed. The assay limit of detection was 250 pg per tube and the standard curve ranged front 200 pg to 200 ag. Iatra-assay and inter-assay coefficients of variation were 12% and 16% respectively. Concentrations of luteioizing hormone (LH) were determined by the method of Niswender et al. ( 1970) as modified by 9llrich et al. (1982). The limit of sensitivity of the LH assay was 25 pg ml- ‘, with an inter-assay coefficient of variation of 14%. Growth hormone (GH) and prolactin were quantified by radioimmunoassays (Ohlson et al., 1981; Klindt et al., 1983). Limits of sensitivity for GH md prolactin were I50 and 70 pg ml-‘, with coefficients of variation of 123%and 14.446,respectively. Blood serum and placental fluid steroids (T, dehydroepiandrosterone sulfate (DHEAS), E,, Ez and ES) were measured by established radioimmunoassays (Allrich et al., 1982, Ford, 1983, Wise, 1987). The limits of sensitivity
T.WSEEKAL.
142
for T, DHEAS, E,, E2 and E,S assays were 10, 10, 5, 5, and 5 pg ml-‘, respectively, with respective inter-assay coefficients of variation of 10.8, 10.0, 8.7, 14.0 and 12.3%. Testicular tissue (250 mg) was ether extracted, submitted to double development thin layer chromatography (Linear-K, Whatman; hexane:ethyl ether:acetic acid 90: 10: I, v/v, and then chloroform:ethanol 99: 1, v/v). Progesterone (P), T, and E, were quantitated by radioimmunoassay (Wise, 1987), the limits of sensitivity being 100, 10 and 5 pg ml-‘, respectively, with intra-assay variations of 8.5.9.0 and 12.5%. Statistical
analysis
Data representing multiple blood samplings from animals were analyzed by split-plot analysis of variance (MS, 1979) to determine effects of treatment (control vs. anti-El ), time, and time by treatment interaction (Figs. I, 2 and 3). Significant main effects or interactions were subsequently submit-
37
65
92
120 1LR 176 2136
Age (days1
tbl
=
9 10
I 8O
37
65
92
120 118 176 20~
Fig. I ~(a ) Serum estrone cotvxnlralions (tncsr8+ SEM ) in prepubxtal gilts immunized against KLH (open bars). and gills immunized against estrone (hatched bars). No differences were noted b~lwrcn ~re;untcntor over time. Artwe indicate rimes of immunization. (b) Serum estrodiol conuentmtions in prepukrtal canlrol gilts immunized against KLH (cpcn bars). and $ilts immunized ngainsl estrone conjugated to KLH (halched bars). No treattnentdifferences wc~~detected hut cstndinl incwased as puhwty was atrninedand animals demonstrated estrw.
THYMOSlN84CHANGESDURlNG
PUBERTY IN BOARSANDG,lTS
143
ted to means comparison by f-test. Other endocrine and weight comparisons were analyzed by one-way analysis of variance where treatment (control vs. anti-El ) was considered a fixed effect (Tables I and 2). RESULTS
Control animals bound less than 1% ‘H-estrone at 1: IO or 1: 100 serum dilutions. Anti-El gilts bound 23% ‘H-estrone and boars bound 5% after the third booster ( 1: 100 dilution). Peripheral E, and Ez monitored at 90-200 days of age (Fig. i ), or E,, E,S and E2 at slaughter (30 days pregnant, Table 1) were the same in serum and placental fluids. No binding of 3H-estrone was detected in amnioiic or allantoic fluids, indicating that the antibody did not cross the placenta at these stages of pregnancy. TABLE I Comparisons of tcpmductive tissua and gonadal steroids in gilts immunized against keyhole limpet hemwynnin ) and Keyhole limpet hemqanin conjugated to e?.tm”e (Anti-El )
(contml
Gilts
30 days prq”a”t CO”W”l. “~28 Anti-El. n=33 70 days pregnant Contml. n=40 Anti-El. Iv=43
OVWil” weight’
corpora lutea number
Uterine weight2
Embryo number
Il.8iO.5
4.1 to.1
12.3tO.7
2.0fO.15
10.8?0.6
12.0+0.3
4.2tO.2
12.1 to.4
2.Sf0.19
10.7+0.6
14.2tO.3
4.9ro.1
13.220.3
2.2+0.06
11.1+0.3
4.9to.1
13.8kO.4
2.4kO.09
14.6tO.S
11.3*0.4
Swum esttvgensJ
Estrone’
EStI”“e sulfate’
Estradiol’
Control. n=28 Anti-El. 1x=33
26.3Al.8 24.2+ 1.7
2.5tO.3 2.0-fo.2
6.8fl.l 6.5fl.O
PlBX”l”l estrogens’~
Allantoic enrone
Allantoic estmne sulfate’
Amniotic estmne’
Control. II= 28 Anti-El.“=33
3.5io.5 3.4io.5
35.lt2.6 32.8k2.5
459.62 143.7 554.52 132.5
‘h4canfSEM.g *Meanf SEM, kg. ‘At 30 day pregnancy. “Mean+SEM.pgm!-‘. ‘Man+SEM,“gml-‘.
T. WlSE ET
.a.
100 t 0 40
: 60
: 60
: 1W
120
IL0
160
160
200
2a
c 264
Fig. 2. (a) TP conccnlrations (LS means) and regxssion line depictingdecreases in T/74 prior lo puhcrly (dny O=dayofcsIrus) inconlrolgills (circles. immunizedagainst KLH,n=68) and gills immunized againsl c~lmnr (squares. anti-El, n=76). Data were normalized so Day 0 ~~P~~scII~sthe day oflirsl eslrus (220 daysprior IO pubcrly is approximalcly 40 days of age). No differences were dulccled between trcalmcnls. and the line represents the “best fit” polynomial rc6rcssion line for pooled data. (b) T&I concentrations in gills (n=25) in which no cslrus was detected. Tp conccnlralions were slgnificanlly incrcati over gilts that allained pubcrly (delcclable cslrus and prcgmmcy 1. Standardcrrcw are indicatedal e&h meantime.
14s
9l
Icy ,d!$)
.166
lL? Contrd’0 Anti-Eq:m lb1
0s
e; f
-.a
-em.
lm
. .
_ _. . . . . .
-.;.-
,*-0
: l.: . . -.;--a-,
.
Fig. 3. (a) Tfl concentrations (mean+SEM) in boars immunized against KLH (controls. circles, n=6) or KLH conjugated to estrone (squares. n=b) during the period of prepubertal development. Animals immunized against estrone had lower Tm concentrations than controls ( I13 vs. 150 ng ml-‘: P
T WISEETAL.
146 TABLE 2
Changes in reproductive tissue weights and gonadal steroids in boars immunized against Keyhole limpet hcmocyanin (control) or keyhole Iimpel hemocyanin conjugated to estrone (Anti-E
I)
COIlld. n=6 Anti-El, n=6
Epididymis
Seminal vesicles
Prostate
Bulbourethal
239.62
20.9
42.9f2.0
173.2t15.2
5.9 + 0.8
106.9+
8.1
196.6f
13.4
37.02 2.8
103.2+21.2*
4.8? 1.0
110.8f
17.8
15.1~1.6 20.1+3.1
44.8f 15.4 72.72 27.2
5.8 f 3.0 2.6 2 7.0
Serum hormones’
T~iOStC~O~C
Dehydrwpiandrostcmne SIllfate
Estmne sulfa1c
Control. “~6 Anti-El.n=6
I2.6 f
14.312.1 2.0
34.5fS.l 26.92 3.9
5.9+_1.8 4.6f 1.0
Conlml. Anti-El,
n=6 n=O
‘hteall i SE,+,, 8. *P
‘ngg-’ wet weight. ‘Eight-hour
intensive sampling mean+ SEM. ng ml- ‘.
In gilts, thymosin/B concentrations showed no significant treatment or timextreatment effects, but decrease 1 in concentration from 6 weeks of age until puberty (kO.01, Fig. 2a). In contemporary gilts that did not attain puberty (n = 25), thymositQ4 concentrations remained elevated over animals that exhibited estrus (200-250 ng ml-‘; Kg. 2b. P-zO.05). At slaughter (280 days ofage),it was determined that 52% (13/25) of the anestrousanimals were prepubertal (small uteri and nonfunctional ovaries), and 48% ( 12/ 25 ) were of behavioral anestrous status (functional ovaries but no characteristics of estrus). There were no differences in the concentrations of thymosing4 in prepubertal(255.8 2 4.8 ng ml-‘) or behaviorally anestrous animals (257.9 + 5.0 ng ml-‘) thus, data were pooled for analysis. Immunization of gilts against E had no effect on reproductive function in relation to age at lirst estrus ( 18324 or 18624 days,controlsand anti-El, respectively), ovulation rate, corpora lutea weight, or fetal numbers (Table I ). Thymosin@ concentrations in control boars decreased with maturity (Fig. 3a; P
tensive sampling period (PC 0.05; Fig. 3b), but time by treatment interaction was not significant (P> 0.1). A trend towards lower testicular, epididymal and accessory gland weights was noted in anti-E I boars, but only seminal vesicle weights were significantly decreased (173.2 vs. 103.2 a. P-=0.05: Table 2). Total Leydig ceil numbers tended to be reduced in a&El boars compared to those of controls (20.022.8x IO9vs. 27.122.8x IO”;P-=0.09) in conjunction with reduced diameter of seminiferous tubules in anti-El testes (24.6 2 5.0 vs. 25.8 2 5.0 pm, PC 0.09). Leydig cell volume was increased in the testes of anti-E I boars compared to those of controls (3 142 + 108/rm’ vs. 23212 149 pm3, PeO.01) and was accompanied by an increase in the size and number of intra-cellular organelles (data not presented). Levels of serum and testicular steroids were not different in control or anti-El boars (Table 2).
Thymosin/34 has teen shown to release GnRH in the rodent, both in vitro and in vivo (Rebar, 1984). but knowledge of the reproductive relationships with thymosin/&i in other species is scant. Thymosin 84 concentrations were found to decrease during the prepubertal period (PcO.05) both in gilts and boars as they matured and reached puberty, consistent with steroid feedback effects on thymic function as gonadal function is established at puberty. It is known that as heifers mature, attain puberiy and become pregnant, peripheral thymositQ4 concentrations also decrease; but in animals that did not become pregnant (noncyclic), the level remained elevated (Wolfe et al., 1989). A similar pattern was noted in noncyclic gilts (Fig. 2b)..Overall, the immbnization against E had no effect on reproductive function (Table I ) or thymosin/34 concentrations in gilts. In sheep immunized against estrogens, ov& lation rate and fecundity are significantly increased (Scaramuzzi et al., 1980; Smith, 1985). but no alteration of ovulation rate or embryo numbers was noted in anti-El gilts (Table I ). Serum binding titers were generally lower in the swine utilized in this study when compared to titers in sheep that responded with increased lambing rates, but a negative aspect of attaining high titer responses (> I : 1000) is competition for circulating El by antibodies that yields less antigen (E, ) available to target cell receptors and sterility (Christenson and Wise, 1985). Also, maternal antibodies to E, did not cross the placental barrier and thus had no influence on fetal-placental steroids !%a! I ) that are critically important at implantation (Robertson and King, Thk effects of estrogens upon thymic function is both complex and conflicting (Glucksman and Cherry, 1968; Grossman et al., 1982; Berczi, 1986). Variable responses to estrogens may be due to pharmacological dosages administered (Raveche and Steinberg, 1986). Chronicestrogen therapy can reduce thymosinfl concentrations in women (Sub et al., 1985), and administration of thymosin fraction V (from which thymosin/I4 is derived) can
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increase plasma estrogen levels in mice (Michael et al., 1981). Contrary to the lack of treatment differences noted in gilts (control vs. anti-El), boars immunized against E, (even at low titers) demonstrated significant decreases in thymosi@ (Fig. 3), which was accompanied by lowered testicular and accessory gland weights, decreased Leydig cell numbers and increased Leydig cell size. No differences were noted in steroid concentrations (Table 2) between control and anti-El boars, even though testicular weights tended to be decreased. This may he due to an increase in Leydig cell size and volume in anti-El boars (Lunstra et al., 1988). which compensates for decreased testes weight. Responses to immunization against steroids are thought to be related to two physiological mechanisms, (1) establishment of either high binding titers ( > I : 1000)or high affmity constants in which the antibody effectively competes with cellular receptors and deprives target tissues of steroids, and (2) reponses to low titers ( -Z1: lOOO),in which the antibody may act as a steroid-bound reservoir and decrease clearance, thus providing increased steroid to target tissues (Pardridge, 1981: Wise and Ferrell, 1984). Interpretation of the mechanism of ste:oid immunization is complex, particularly in a species like swine, where the testes produce large amounts of estrogens and estrogen sulfates (Booth, 1980; Ford, 1983; Raeside. 1983), to which the antibody (in this study) would bind. The antigen in this study was synthesized to bind both E, and E,S because of the known predominance of sulfated estrogens in the male and female of this species (Orava et al., 1985; Ruokonen and Vihko, 1974). Depression of accessory gland weight and function during exogenous estrogen administration is thought to be due to decreasesin T production which occur via decreases in LH stimulation and local effects on T synthesis (Kalla et al., 1980; Price and Williams-Ashman, 1961; Ronco et al., 1987). The observed decreases in thymosiQ4, accessory gland weights, testicular weights, and Leydigcell numbers in anti-El boars (titer 5% binding at I : 100dilution) support the concept of an overall increase of stemid (estrogen) available to target tissue. With the high concentrations of El and E,S in the boar, it is hard to imagine immunoneutralization and competition with cellular receptors at a 1: 100titer. Although thymosinpt concentrations were lower in the anti-El boars, LH concentrations (mean, amplitude and basal; data not presented) did not differ. This would appear to challenge the concept of thymosinfi involvement in GnRH release and subsequent LH discharge from the pituitary. However, changes in gonadotrophin heterogeneity and biological activity may not have been detected with the radioimmunoassay used in our studies ( Wakabagashi, 1977; Grotjan and Steinberger, 1978). Administration of EZ is known to change the pattern of pituitary LH isohormones, which could result in the secretion of gonadotrophin lower in biological activity in sheep (Keel et al., 1987). Thus, lack of differences in LH concentrations between treatments as established by radioimmunoassay may bc due to production of LH isohor-
THYMOStN~CHANoESoVR,NGPUsERTY
149
IN sO*RSANoolLTS
manes of less biological activity whit’? would be reSected in altered testicular weight, Leydigcell size, and Leydig r.:ll numbers. Alternatively, recent cDNA probe analysis for thymosin/%t (Gor. ez-Marquez et al., 1989; has established the presence of thymosin~ mRNA in most t&tics $idxy, lung, testis, ovary, muscle, etc.), although immunological tissues possessed the highest concentratioc of message (tbymus, spleen). The common presence of mRNA for thymosin /94in many tissues indicates an important but common tissue function for thymosinfl, though possibly not as an endocrine regulator (GomexMarquez et al., 1989). It is not known which tissues produce the thymosinp4 present in the peripheral circulation, but circulating concentrations can be altered by various endocrine inputs (Suh et al., 1985;Wise et al., 1987,199(J). In conclusion, circulating tbymosinfl in swine decreased with maturity, with the lowest concentrations detected at puberty. Only in anti-El boars were thymosing4 concentrations reduced, thus providing evidence of a sexually dimorphic response in which gilts showed no effects. Testes of anti-El boars were altered on a gross and histological basis; however, on an endocrine basis, the testis seems to have compensated in function. The differences noted in thymosin@4 concentrations as animals matured and attained puberty, when considered in conjunction with the sexually dimorphic response noted between anti-E 1 boars and gilts, demonstrates that circulating concentrations of thymosinw are under some aspect of control. Whether thymosir@ has any direct function in control of the gonad has yet to be ascertained. ACKNOWLEDGMENTS
The authors thank D.J. Taubenheim, S. Reece, J. Crisp, A. Kruger, and L. Pamell for their technical and clerical assistance. Dr. Don Lunstra provided the histological information on boar testes.
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Vakhrria. D.D.. Kinder, J.E. and Wise, T.H.. 1986a. Thymosin (ThB4) levels in serutn and foliicular fluid during estrous cycle (EC) in swine. Fed. Proc., 45: 405 (abstract). Vakharia. D.D., Kinder, J.E. and Wise, T.H., 1986b. Thymosikai in the ovary and the levels of thymosin-al and T lymphocytes during estrous cycle in swine. in: M.E. Tumbieson (Editor). Swine in Biomedical Research, Vol. 3. Plenum Pubiishine Comoration. New York. pp. 1925-1936. Wakabagashi. K.. 1977. Heterogeneity of rat iuteinizing hormone revealed by radioimmunoassay and eiectrofocusingstudies. Endocrinoi. Jpn., 24: 473-485. Wise. T.. 1987. Biochemical analysisof bovine foiiicuiar fluid: Albumin, total protein, iysosomai enzymes, ions, steroids and ascorbic acid eontent in relation to foliicuiar size, rank, alresia classification, and day of estrous cycle. J. Anim. Sei., 64: 153-I 169. Wise.T.H. and Ferreli,C.L., 1984. Effectsofimmunizttionofheifersagainslestradioiongrowh, reproductive trails. and carcass characteristics. Pmt. Sot. Exp. Bioi. Med., 176: 243-248. Wise. T., Kiindt. J.. Macdonaid, G.J. and Ford, J.J., 1990. Effects of neonatal sexual differentiation, growth homtone and lestostemne on thymic weigltts and thymosin @4 in hypophysectomized rats. J. Reprod. immunoi., in press,
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Wise, T.H. and Schanbacher. B.D.. 1983. Reproductive effects of immunizing heifers against androaenedione and oestradiol-l7P. I. Reprod. Fenil., 69: 605-612. Wise, T.H., Siiss, U. and Maurer, R.R., 1987. Relationships of thymorin /34 in superovulated beef heifers with and without Norgestomct implants. J. Anim. Sci.. 65 {Sqp:. I j: 368 (abstract). Wolfe, M.W., Vakharia, D.D., Wise, T.H. and Kinder, J.E., 1989. Characterization ofsecretion of thymosin alpha and beta 4, during prepubeny. estrtts and pregnancy in the bawine female. Dom. Attim. Endocrinol.. 6: 71-78.
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