Concentrations of LH, prolactin and progesterone in early-pregnant and oestradiol-treated pigs

Ammal Reproductwn Scwnce, 10 (1986) 215--224

215

Elsewer Scmnce Pubhshers B V , Amsterdam -- Printed m The Netherlands

CONCENTRATIONS OF LH, PROLACTIN AND PROGESTERONE IN EARLY-PREGNANT AND OESTRADIOL-TREATED PIGS*

A ZIECIK l, T E R E S A DOBOSZYNSKA ~ and L U I Z A DUSZA 1

Department of Ammal Physwlogy and 2Department o f Animal Anatomy, Unwerszty o f Agrwulture and Technology, 10-718 Olsztyn (Poland) (Accepted 15 April 1985)

ABffrRACT Zmclk, A , Doboszynska, T and Dusza, L , 1986 Concentratmns o f LH, prolactm and progesterone m early-pregnant and oestradml-treated pigs Atom Reprod Sc~, 10 215--224 Fifteen gilts (n = 5 per group) were used to study plasma LH, prolactm and progesterone concentrations on days 13--19 after oestrus m early-pregnant, oestradlol-treated (5 mg, admlmstered on days 11--15) and control cychng pigs Peripheral blood samples were taken without stress at one-hour intervals for 12 h on days 13--14, 15--16 and 18--19 There was no difference amongst groups m LH levels on days 13--14 and 15--16 The LH levels m the cychng untreated pigs was lower (P < 0 05) than m pregnant gilts on days 18--19 Concentratmns o f prolactm m oestradmltreated pigs were 7--20 times higher than m pregnant pigs The greatest differences m progesterone concentratmns were recorded on days 18--19 Progesterone levels were less (P < 0 0 1 ) m oestrogen-treated gilts (14 54 -+ 1 09 ng/ml) when compared with pregnant gilts (24 23 -+ 4 10) A comparison o f the secretmn patterns for the three hormones showed that mjectmns o f oestradlol given to the cychng gilt did not result m patterns which fully imitate the lmplantatmn period o f natural pregnancy m the pig

INTRODUCTION

Admlmstratlon of oestrogens d u n n g the mld-luteal phase of the oestrous cycle prolongs luteal function In p ~ s (Kidder et al, 1955, Gardner et a l , 1963, Kraellng et a l , 1975), b u t the mechamsms o f this process have not been fully explamed. It is suggested that the antl-luteolytlc effect o f oestrogen depends u p o n the reduction o f prostaglandln F2a release from the endom e t n u m into the clrculatmn (Frank et al., 1977, Bazer and Thatcher, 1977, Guthrle and Rexroad, 1981), preventing luteolysls of the corpora lutea On the other hand the luteolytlc effect o f the uterus m a y be limited by an increase m u t e n n e blood flow, m response to administered oestrogen, leading *Parts o f this paper were presented at 5th International Symposium on " I m m u n o l o g y of R e p r o d u c t i o n " , Varna, Bulgaria, 1982

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216 to a decrease m t he c o n c e n t r a t m n o f PGF2a m uterine venous blood by dilut m n (Ford et a l , 1982) Recently Garvenck et al (1982) r epor t e d t hat admmlstratmn of oestrogen leads to an increase m luteal LH r e c e p t o r levels and this evidence, m connection with studms m wvo (Anderson et a l , 1967) and m vitro (Watson and Maule Walker, 1978) whmh d e m o n s t r a t e d a lack o f the direct oestradml effect on progesterone secretmn suggests t hat LH may be revolved m the luteotrophm actmn of oestrogens An e x p e r i m e n t was designed t o com pa r e t he patterns o f LH, prolactm and progesterone secretmn m pregnant, oestrogen-treated (pseudopregnant) and cychng pigs MATERIALS A N D METHODS Animals

The e x p e r i m e n t was c o n d u c t e d with 15 crossbred cychng gilts weighing 100--110 kg Gilts were assigned to 3 groups o f 5 ammals each Pigs m Group 1 were intact -- u n m a t e d c ont r ol Ammals m Group 2 received 5 mg oestradlol benzoate by intramuscular m j e c t m n on days 11--15 o f t he oestrous cycle. Group 3 consisted o f pregnant gilts Three to four days before samphng was mltmted, a cannula was inserted t hrough the cephahc veto into the a n t e n o r vena cava and e xt e r l ons e d by passage under the skin to t he back Blood was collected at one-hour intervals for 12 h (09 00--15 00 h, 23.00--05 00 h) on days 13--14, 15--16 and 18--19 after t he first day o f oestrus (day 0) Blood samples were collected m heparmlzed tubes and the plasma stored at - 2 0 ° C until assay T he ammals m Groups 2 and 3 were killed o n days 22--28 after oestrus The reproductive tracts were collected and pseudopregnancy and pregnancy venfmd E m bryos with a detectable heartbeat and o f a size comparable to their contemporaries were judged to be normal R a d lo ~mm u n o a s s a y s

The concentrations o f LH were estimated by a homologous double-antib o d y radloxmmunoassay using porcine LH GPZ-1 (blo-potency 0.63 × NIHLH S1) as a standard and for the lodmatlon, as described by Zmclk et al (1978) Antl-LH serum (Zmclk et a l , 1979) was used at a final dilution of 1 160 000 The sensitivity o f the assay was 0 02 ng L H / t u b e Pooled withinassay coeffmmnt o f variation was 6 7% and t he between-assay coeffmmnt was 11 3% Prolactm c o n c e n t r a t i o n was estimated by t he double-antibody m e t h o d described b y Dusza and K r z y m o w s k a (1979) using t he porcine prolactm preparation KK-2 with blologmal p o t e n c y o f about 30 1 u / m g The sensitivity o f this assay was 0 015 ng/tube The mtra- and rater-assay variations were 3 7%

217 and 9.8%, respectively The plasma progesterone concentratmn was measured by the radlodmmunoassay described by Hotchkms et al {1971) using specffm antiserum (Stupmckl et a l , 1975} The sensltlwty o f the assay was 35 pg/ml, recovery o f progesterone added to plasma was 93 0% and the pooled lntra- and rater-assay coeffmlents were 5 2% and 10 0%, respectively

Stattstteal analysts The differences among the t r e a t m e n t groups m mean hormones concentratmns were examined by analysm of vanance RESULTS

Corpora lutea were mmntmned m all pigs receiving 1 m rejections of oestrogen The mean + SEM number o f the corpora lutea were 11 8 -+ 0 6 and vaned from 11 to 14 Pregnant pigs possessed 9 8 + 1 1 normally developing embryos m both u t e n n e horns and the number of the corpora lutea per gilt were 12 3 + 0 8 The concentrations of LH did n o t differ between the groups d u n n g days 13--14 and 15--16 (Figs 1 and 2) The LH levels m cychng untreated pigs on days 18--19 (0 81 + 0 03) were lower (P < 0 05) than m pregnant gilts (1 47 -+ 0 15 ng/ml) In untreated and oestradlol-treated animals the concentration of LH decreased on days 18--19 when compared with days 13--14 and 15--16 but a slgmfmant fall (P < 0 05) was found only m cychng pigs (1 61 -+ 0 24 versus 0 81 -+ 0 03 ng/ml) The concentration o f prolactm m the oestradlol-treated animals was approximately 7--20 tunes higher than m pregnant animals, whose values remamed low d u n n g the entire e x p e n m e n t (1.89 -+ 0 34, 2 02 + 0 45, 0 97 + 0 17 ng/ml, on days 13--14, 15--16 and 18--19, respectively). The largest fluctuation m prolactm levels was observed m cychng ammals where plasma concentratmns increased from 7 89 + 2.16 {days 13--14) to 15 10 + 5 61 (days 15--16, P < 0 05) and then decreased to 11.88 + 4 18 ng/ml on days 18--19 Plasma progesterone concentratmn on days 13--14 did n o t differ slgmflcantly among the three groups and vaned from 23 6 to 30 1 ng/ml A decrease (P < 0 01) m progesterone concentratmns was found when days 15-16 untreated anLmals were compared with days 13--14 as well with the pregnant and oestradlol-treated groups (P < 0 01 and P < 0 05, respectively) The plasma c o n c e n t r a t m n of progesterone m oestradml-treated gilts on days 15--16 at mght were lower (P ~ 0 05) than the levels of pregnant ammals The progesterone level was elevated (P < 0 01) when compared with cychng pigs (Fig 2) Day 18--19 progesterone concentratmns exhibited the greatest differences across groups Oestrogen-treated animals (14 54 + 1 09 ng/ml) progesterone values were higher (P < 0 01) than cychng-untreated gilts (7 80 -+ 0 42 ng/ml) but m u c h lower (P < 0 01) than those observed m pregnant

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221

gilts (24.23 + 4.10 ng/ml) Wlthm the oestradml-treated group a large decrease ( P < 0.01) was found relative to day 15--16 progesterone concentratmns (22 08 + 2 05 ng/ml). DISCUSSION

The present study confirmed that it is possible to prolong the hfe-span o f the corpora lutea b y multiple rejections of oestrad:ol Oestradlol benzoate treatment from day 11 to day 15 of the oestrous cycle has been reported to extend luteal function for 70--300 days (Bazer et al, 1982). It is also possible to mmntam the hfe-span o f the corpora lutea by a smgle rejection of 10 mg (Guthne and Rexroad, 1981) or 5 mg oestradlol benzoate (Garvenck et a l , 1982) This prolongatmn of luteal function m pigs by oestradlol became the starting p o m t for the t h e o r y o f maternal recogmtlon of pregnancy proposed by Bazer and Thatcher {1977) A comparison o f the patterns o f secret:on o f the three hormones m our study showed, however, that the state of pseudopregnancy revoked b y oestradlol rejections does not m:mlc fully the period o f implantation during natural pregnancy (days 13--19) The last important difference between oestradlol-treated and pregnant ammals is shown m their pattern o f LH secretmn It should be noted that the blood collection started 43 h after the first m]ectmn of oestradml benzoate An mltml drop and then a rise m LH concentratmn 36 h after oestrad]ol rejection was observed m ovanectomlzed gilts (Cox and Bntt, 1982}. Garvenck et al (1982) did not fmd a swmlar change m plasma LH levels after oestradml benzoate mjectmn m pigs with functional corpora lutea In general, our results confirmed earher observatmns wh:ch demonstrated that the level o f LH m early pregnancy m:m:cs LH concentratmns m the mldluteal phase of the oestrous cycle (Z:eclk et a l , 1983). Plasma progesterone levels on days 13--14 d:d not differ slgmfmantly among the three groups and these results partially confirmed the study o f Webel et al (1975) who found no difference m mean level of progesterone between pregnant and nonpregnant gilts on days 5--14 after oestrus Consldenng the fact that the corpora lutea of pregnant and pseudopregnant pigs do not show histological signs of regress:on (Doboszynska and Zleclk, 1986), :t was surprising that the plasma progesterone concentrations decreased :n the oestradml-treated pigs (Figs 1, 2 and 3) Similar results were obtmned by G u t h n e and Rexroad (1981) Perhaps the large doses of oestradml benzoate (10--25 mg} used m both stud:es for provoking the state of pseudopregnancy reduce the p r o d u c t m n or secretmn o f progesterone by luteal cells It was shown that oestradml-17#3 reduced the effect of LH on progesterone synthesis b y isolated porcine luteal cells {A.P F Fhnt, personal c o m m u m c a t m n , 1982), the bovine corpus luteum {Ursey and Leymane, 1979) and by rabbit corpora lutea m wvo (Day and Blrnbaumer, 1980) Garvermk et al (1982) concluded that oestrogens may have a direct effect

222 on the corpus luteum m the pig since oestradlol-17~ raised luteal LH receptor levels b y a mechanism independent o f the uterus This suggests that endogenous oestrogen secreted b y the conceptus into the systematm circulation could be the reason for the rise m luteal LH receptors observed between days 20 and 30 o f gestation (Zmclk et al, 1980) A direct stlmulatory effect of oestrogen on progesterone secretion by granulosa cells has been shown in vitro (Goldenberg et a l , 1972, Veldhuls et a l , 1981) Nevertheless, a direct effect of oestradlol on progesterone synthesis by the corpora lutea was not observed m vlvo (Anderson et a l , 1967) or by luteal cells in vitro (Watson and Maule Walker, 1978) Klrchmk and Blrnbaumer (1981) reported that oestradlol treatment suppressed the LH-responslve luteal adenyl cyclase m rabbits They suggested that exogenous oestradlol either causes a decrease m LH receptor number or afflmty or results in a partml uncouphng o f the LH receptor from the adenyl cyclase system The reduction of progesterone secretmn during pseudopregnancy in the pig seems to be caused by a partial blockade of the synthesis o f this hormone, probably due to lower adenyl cyclase activity rather than by a decrease m LH receptor number or affinity (Garvenck et al, 1982). One cannot exclude an mdyrect effect of exogenous oestradlol on the decrease of progesterone secretmn in pseudopregnant pigs through elevated prolactm levels (Figs 1--3) This hormone has been shown to suppress progesterone synthesis m porcine luteal cells m wtro (Przala et al., 1984) Prolactm is not luteotrophm m the pig d u n n g the cycle (Du Mesnll du Bulsson, 1973) and its mcreased level on days 14--16 of the oestrous cycle (Dusza and Krzymowska, 1979, Van de Wml et al, 1981) were confirmed m our studms It is worth noting the high plasma prolactm concentratmns in oestradml-treated animals (Figs 1--3) compared with pregnant pigs (Figs 1--3, Dusza and Krzymowska, 1981), as it might be the cause of mhlbltmn of progesterone secretion. There may be t w o reasons why there is not an mcrease m prolactm concentrations dunng early pregnancy such as reduced by oestradlol mjectmns blood levels of oestradml-17~ are relatively lower (Robertson and King, 1974) when compared to pseudopregnant gilts (approx 100 pg/ml of plasma, A Zmclk, unpubhshed observatmn, 1982} and/or the fact that the circulating oestrogen during early pregnancy is m the form of sulphoconjugate--oestrous sulphate (Robertson and King, 1974, Robertson et al, 1978). On the other hand, prolactm concentratmns m pregnant gilts are low because this hormone is not important for corpus luteum functmn during early pregnancy m the pig (Spies et al, 1967) ACKNOWLEDGEMENTS

This work was supported by grant No II 10 from the Ministry of Scmnce, Higher Education and Technology, Warsaw

223 REFERENCES Anderson, L L , Dyck, G.W, Morn, H , Henricks, D M and Melampy, R M , 1967 Ovaman function m pigs following hypophysial stalk transection or hypophysectomy Am J Physlol, 212 1188--1194 Bazer, F W and Thatcher, W W, 1977 Theory of maternal recogmtlon of pregnancy m swine based on estrogen controlled endocrine versus exocrme secretion of prostaglandm F2a by the uterine endometrmm Prostaglandms, 14 397--401 Bazer, F W, Gelsert, R D , Thatcher, W W and Roberts, R M , 1982 The establishment and maintenance of pregnancy In D J A Cole and G R Foxcroft (Editors), Control of Pig Reproduction Butterworths, London, pp 227--252 Cox, N M and Britt, J H , 1982 Effect of estradlol on hypothalamlc GnRH and pituitary and serum LH and FSH in ovarlectomIzed pigs J Atom Scl, 55 901--908 Day, S L and Blrnbaumer, L , 1980 The effect of estradiol on hormonally stlmulable adenylyl cyclase activity and regressing corpora lutea from control and hCG-treated pseudopregnant rabbits Endocrinology, 106 375--381 Doboszynska, T and Zmclk, A , 1986 Histomorphologmal comparison of the ovaries in early-pregnant and oestradlol treated-pigs Anita Reprod Scl, 10 000--000 Du Mesnll du Buisson, F , 1973 Facteurs luteotropes chez la trine In R Denamur and A Netter (Editors), Le Corps Jaune Masson, Paris, pp 225--237 Dusza, L and Krzymowska, H , 1979 Plasma prolactin concentrations during the oestrous cycle of sows J Reprod FertIl, 57 511--514 Dusza, L and Krzymowska, H , 1981 Plasma prolactin levels in sows during pregnancy, parturition and early lactation J Reprod Ferti1,61 131--134 Ford, S P , Chmstenson, R K and Ford, J J , 1982 Uterine blood flow and uterine arterial, venous and luminal concentratmns of oestrogens on Days 11, 13 and 15 after oestrus m pregnant and non-pregnant sows J Reprod Ferti1,64 185--190 Frank, M F , Bazer, F W , Thatcher, W W and Wilcox, C J , 1977 A study of prostaglandin F2~ as the luteolysln in swine HI Effect of estradlol valerate on prostaglandln F, progestins, estrone and estradiol concentrations in the utero-ovarian vein of nonpregnant gilts Prostaglandins, 14 1183--1196 Gardner, H L , First, N L and Casida, L E , 1963 Effect of exogenous estrogens on corpus luteum maintenance m gilts J Atom Scl, 22 132--134 Garverick, H A , Polge, C and Fhnt, A.P F , 1982 Oestradml admmlstratmn rames luteal LH receptor levels m intact and hysterectomlzed pigs J Reprod Fertfl, 66 371-377 Goldenberg, R L , Bndson, W E and Kohler, P O , 1972 Estrogen stlmulatmn of progesterone synthesls by porcine granulosa cells in culture Biochem Blophys Res C o m m u n , 48 101--107 Guthrm, H D and Rexroad, C E, Jr, 1981 Endometrlal prostaglandin F release m wtro and plasma 13,14-dlhydro-15-keto-prostaglandm F2~ m plgs with luteolysls blocked by pregnancy, estradml benzoate, or human chormmc gonadotropm J Atom Scl, 52 330--339 Hotchkms, J , Atkmson, L E and Knobil, E , 1971 Time course of serum estrogen and lutemlzmg hormone (LH) concentratmn during the menstrual cycle of the rhesus monkey Endocrinology, 89 177--183 Kidder, H E , Caslda, L E and Grummer, K H , 1955 Some effects of estrogen mjectmns on the estrual cycle of gilts J Atom Scl, 14 470--474 Klrchlck, H J and Blrnbaumer, L , 1981 Prostaglandms do not appear to play a role m hCG-mduced regressmn or desensltizatmn of rabbit corpora lutea Bml Reprod, 24 1006--1012 Kraehng, R R , Barb, C R and Daws, B J , 1975 Prostaglandm reduced regressmn of porcine corpora lutea maintained by estrogen Prostaglandms, 9 459--462

224 Przala, J , Grazul, A and Wmsak, T , 1984 The influence of prolactm on progesterone synthesis by porcine luteal cells m vitro Atom Reprod Scl, 7 351--362 Robertson, H A and King, G J , 1974 Plasma concentrahons of progesterone, strone oestradlol-17~ and oestrone sulphate m the pig at implantation, during pregnancy and at partumtlon J Reprod F e r h l , 40 133--141 Robertson, H A , King, G J and Dyck, G W , 1978 The appearance of oestrone sulphate m the peripheral plasma of the pig early m pregnancy J Reprod Fertll, 52 337-338 Spies, H G , Slyter, A L and Quadrl, S K , 1967 Regression of corpora lutea m pregnant gdts admmmtered antlovme LH rabbit serum J Atom Scl, 26 768--771 Stupnlckl, R , Zajac, H and Made], A , 1975 Direct radlolmmunoassay of progesterone m plasma of farm animals Endokrmologm, 66 145--151 Ursey, J and Leymarm, P , 1979 Varying response to lutemmmg hormone of two luteal cell types isolated from bovine corpus luteum J Endocrmol, 83 303--310 Van de Wml, D F M , Erkens, J , Koops, W , Wos, E and Van Landeghem, A A J , 1981 Perlestrus and mldluteal time courses of circulating LH, FSH, prolactm, estradlol-17~ and progesterone m the domestic pig Bml Reprod, 25 223--233 Veldhuls, J D , Klase, P A and Hammond, J H , 1981 Direct achons of 17~-estradlol and progesterone productmn by highly dlfferentmted porcine granulosa cells m vitro II Regulatory mteractmns of estradlol with lutemlzmg hormone and cyclic nucleotides Endocrinology, 109 433--442 Watson, J and Maule Walker, F M , 1978 Progesterone secretion by the cropus luteum of the early pregnant pig during superfusmn m wtro with PGF2a, LH and oestradlol J Reprod F e r t d , 5 2 209--212 Webel, S K , ReImers, T J and Dzmk, P J , 1975 The lack of relationship between plasma progesterone levels and number of embryos and their surwval m the pig Blol R e p r o d , 13 1 7 7 - 1 8 6 Zmcik, A , Goralska, M , Krzymowskh T and Pogorzelskh K , 1978 Isolation and pumfication of porcine LH for radmlmmunoassay and radloreceptor assay Bull Acad Scl Polon Ser Scl Blol, 26 739--744 Zmclk, A , Krzymowska, H and Sobczak, B , 1979 Production of anhbodms against the porcine lutemmmg hormone by two immumzatmn methods Endokrmologm, 74 304--308 Zmclk, A , Shaw, H J and Flint, A P F , 1980 Luteal LH receptors dunng the oestrous cycle and early pregnancy in the pig J Reprod F e r h l , 60 129---137 Zmcik, A , Tflton, J E , Welgl, R and Wdhams, G L , 1983 Plasma luteimzmg hormone during pregnancy m the pig Atom Reprod Scl, 5 213--218