The embryo influences adenylate cyclase activity and hormonal response in rabbit myometrium during early pregnancy

Life Sciences, Vol. 43, pp. 1653-1662 Printed in the U.S.A.

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(xcLA!n

Pergamon Press

ACTIVITY MD HORMol?ALRESPONSE

IN NABBIT MYOMETUHMDUIUMGEANLYFREGMAMCY Boulet, A.P.1, Fortier, M.A.2,' and Lambert, R.D. Ontogenie et Reproduction,Centre de Recherche du CHUL, Lava1 University Medical Center, 2705 Laurier Blvd, Ste-Foy (Quebec)GlV 4G2, CANADA (Received in final form September 20, 1988)

The effect of pseudopregnancy(PSPG; days: 0 (estrus),1, 6.5, 9 and 15) and pregnancy (PG; days: 6.5, 9 and 15) on adenylate cyclase (AC) activity was verified in rabbit myometrium.During PSPG, there was a time related decline in basal activity from 71 f 16.2 (D 0) to 13.1 f 1.6 (PSPG-D9)pmoles CAMP formed& prot-min. Stimulation of the enzyme by GTP, Isoproterenol(ISO), ProstaglandinE, (PGE,) or Sodium Fluoride (NaF) followed a similar pattern. AC activity was compared in myometrial tissues of pregnant animals (PG) separated into embryonic (ES) and interembryonic(IES) sites. On days 6.5 and 9, AC activity measured in tissues from PG rabbits (ES and IES) was always higher than that found in tissues from PSPG animals on correspondingdays. On day 6.5, AC activity was slightly higher (p < 0.01) in ES than in IES. This was confirmed on day 9 where basal as well as GTP, IS0 and PGE, stimulatedactivitieswere higher in ES than in IES (p
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necessitates"effecters"interactingprobably like "hormones receptors-second messengers" during intracellularcommunication(8). Indeed in the uterus, CAMP, a well known and widely spread second messenger, increases followinga stimulationby a decidual stimulus or the embryo (28). Other changes in uterine CAMP levels have been described in associationwith physiological status (3,4,20,30). So far, the concept of receptivityhas been used exclusivelyfor endometrium. Uterine smooth muscle however, may play a leading part not only at parturition and during transportationof gametes and embryo, but also just prior to implantationto immobilizethe conceptus. Between early implantation and parturition,the myometrium must undergo a relative quiescence. It has been shown that during pregnancy, uterine contractilityis regulatedby sex steroids (33). The latter exert their control over differentmechanisms: membrane electricalproperties (211, gap junctions (261, uterine innervation (23) and availabilityof contractileagents and their receptors (11,X,24, 29). However, data describing the relation that could exist between the embryo and the myometrium at early stages of pregnancy are not available. In this tissue, CAMP appears to be the mediator of relaxation following g-adrenergic stimulationof adenylate cyclase (17). Since, at early pregnancy, the embryo interactswith endometriwnto induce a cascade of mechanisms that allow the implantationand growth of the conceptus, it should not be excluded that a similar local effect may reach the myometrium to control muscular movements and contribute to implantation. The aim of the present study was to investigatethe relationshipthat could exist between the embryo and the myometrial AC activity in early pregnancy. In the first place, we have investigatedthe effect of pseudopregnancy (PSPG; days: 0 (estrus), 1, 6.5, 9 and 15) on AC activity. The results were then compared to the AC activity in rabbit myometrium found during early and mid pregnancy (PG; days: 6.5, 9 and 15). Moreover, we have studied the local effect of the embryo after separationof myometrial tissue into implantation and interimplantationsites. MATERIALS

AND HEllIODS

Materials

Human chorionic gonadotropin (hCG1 was purchased from Ayerst Laboratories (Montreal,Canada). Guanosine triphosphate (GTP) was purchased from BoehringerManheim (W. Germany). a-["P]ATP and [aH]cAMPwere purchased from New England Nuclear (Boston,MA, USA). Other reagents were purchased from Sigma (St-Louis,MO, USA). Animals

Mature New-Zealandwhite rabbits weighing 3-3.5 kg were used throughout this study. Animals in estrus, detected by the presence of edematous and purple vulvae, were injected (i.v.1with 75 IU of hCG to induce pseudopregnancy (PSPG) or were injected with hCG and allowed to copulate to induce pregnancy (PG). Animals from the PSPG group were sacrificed 18 hours (PSPG-D11and 6.5 (PSPG-D6.51,9 (PSPG-D9)or 15 (PSPG-D15)days after gonadotropin injection. Animals from PG group were killed on days 6.5, 9 or 15. Animals in estrus were sacrificedwithout injection to form the day 0 group (D 0). Corpora hemorrhagiaor corpora lutea were counted and the uteri were excised. The tissue was placed in a Petri dish containing freshly gassed (5% CO,; 95% 0,) incompleteHank's buffered salt solution (IHBSS; Ca++ and Mg*+ free; pH 7.5) and kept at 4OC for all processing. Uterine horns from non-pregnantanimals (DO: n=4; PSPG-Dl: n=3; PSPG06.5: n=4; PSPG-D9: n=5; PSPG-D15: n=5) were cut longitudinallyand endometrium was separated from myometrium by scraping. The remainingmuscle tissue

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was then processed for determinationof adenylate cyclase (AC) activity. Horns from pregnant animals (PG-D6.5:n=4; PG-D9: n=5; PG-DlS: n=4) were cut transverselyat one extremity of the implantationsites and the embryos were removed with the endometrium. Uterine segments holding embryos were defined as embryonic sites (ES) and segments between embryos as interembryonicsites (IES). To maximize the potential differencebetween ES and IES, 0.5 cm of tissue, proximal to the implantationarea, was removed from IES and discarded. Uterine ne segments (IES and ES) were then processed for AC assay. On day 15 of pregnancy, it was usually not possible to obtain interembryonictissue because the growing embryos used the entire length of the uterus. In one experiment,a female had only 3 embryos interspacedin one uterine horn and in that case it was possible to obtain IES to compare with ES. &termination of adenylate cyclase activity AC activity was measured as described by Krall et al. (18,19). It was defined as the enzymatic conversionof a['aP]ATP to [3PP1cAMPin 5 min in broken cell preparationsfrom fresh tissues. Tissues (estrus,PSPG and PG animals) were minced and diluted in 10 ml of cyclase homogenizationbuffer (CHB; pH 7.6) composed of 0.001 M ethyleneglycol-bis(p-amino-ethylether) N,N'-tetraaceticacid (EGTA), 0.05 M NJ-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES) and 10% dimethylsulfoxide(DMSO) at 4'C. The suspensions were homogenizedwith a polytron tissue disintegrator(Brinkman;PT 10) at a setting of 5 for 20 seconds. The homogenateswere filtered through a nylon mesh (0.1 mma) and centrifugedat 30,000 X g for 20 min at 4'C. The pellets were washed twice, resuspendedin CHB to obtain a final protein concentration of about 2 mg/ml and AC was assayed on fresh preparations. An aliquot (100 pl), from each preparation,was taken for protein determination according to the method of Lowry et al. (21). Adenylate cyclase activity was measured without stimulation (basal) and after addition of GTP (75 pM) or NaP (10 mM). Beta-adrenergicresponse which plays a major role in uterine relaxationwas estimatedby a dose response (1 nM to 0.1 mM) to the pa-agonistisoproterenol(ISO). ProstaglandinE, (PGE,) response was estimated with the same concentrationrange. Both agonist responses were measured in presence of GTP (75 @f). Statisticalanalysis Data from experimentsin which two experimentalgroups were to be compared were subjected to two-way analysis of variance (repeatedmeasurements;two-way ANOVA). Data from experimentsin which more than two experimentalgroups were to be compared were subjected to Duncan's new multiple range test. Comparisons within the same treatment group or between implant and nonimplant regions were made by student'spaired t test. a) Significantlydifferent from DO females, Duncan's test, p < 0.05; b) Significantlydifferent from DO and PSPG-Dl females, Duncan's test, p < 0.01; c) Significantlydifferent from PSPG-D9 females, two way ANOVA, p < 0.01; 1) GTP increased significantlyAC activity compared to Basal of same group, Student's test, p < 0.01; 2) IS0 and PGE, increased significantlyAC activity compared to GTP of same group, Student's test, p < 0.01. RESULTS

Adenylate cyclase activity was measured in broken cell preparationsof freshly excised myometrium from animals under various physiologicalstatus. During pseudopregnancy(Fig. 1) there was a progressive decline in basal activity from 71 f 16.2 pmoles CAMP formed/mgprot-min on day 0 to 13.1 f 1.6 on day 9. On day 15, there was a recovery of adenylate cyclase activity (21.8 f 4.4) compared to D-9 (p < 0.01). When the enzyme activity was stimulated with GTP (75 pM: Fig. 1B). IS0 (100 pM; Fig. lC), PGE, (100 @l; Fig.

A

1007 c E : ij B

60-

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h

a

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kc 20 -

%

C

O-

PSPG-01

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0 DO

PSPG-Dl

PSPG-D6.5

PSPG-D9

PSPG-D15

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PSPG-Dl

PSPG-D6.5

PSPG-D9

Fig. 1: Influence of pseudopregnancyon adenylate cyclase activity at different stimulationlevels. Uterine horns from estrus or pseudopregnant(inducedby the injection of 751U of hCG in estrous animals on day 0) animals were collected on indicated days. The tissue was homogenizedin cold (4°C) cyclase homogenizationbuffer, filtered through a nylon mesh and membranes were recovered by centrifugationat 30,000 X g for 20 min. The pellet was washed twice and resuspendedat 2 mg/ml protein concentration. The preparation was used fresh for determinationof adenylate cyclase (AC) activity defined as the conversionof a-['ZPIATP into [3aPlcAMPduring a 5 min period. AC activity was measured in each preparationat five different stimulationlevels A: Basal (no stimulation),B: Guanosine triphosphate (GTP, 75 @f), C: GTP + Isoproterenol(ISO, 10 W, D: GTP + ProstaglandinE, (PGE,, 10 uhM). Results are the mean f SE of 3 to 5 experimentsrun in triplicate.

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1D) or NaF (10 mM; not shown) comparable alterationswere observed throughout PSPG. Interestingly,even though overall enzyme activity declined during PSPG, the responsivenessto stimulatingagonists was maintained. Compared to basal, mean stimulationfor all days studied ranked in the following order: GTP (2.4 fold) < GTP + IS0 (10 pM; 3.2 fold) = GTP + PGE, (10 MM; 3.2 fold) < NaF (10 mM 14.6 fold). During pregnancy, adenylate cyclase activity measured on day 6.5 or 9 either from IES or ES (Fig. 2) was always higher (p < 0.0011 than in corresponding days of PSPG. On day 6.5 and to a greater extent on day 9 of pregnancy, significantdifferences (p < 0.01) were detected between IES and ES in basal (Fig. 2A) as well as in GTP (Fig. 2B1, IS0 (Fig. 2C1, PGE, (Fig. 2D1 and NaF (not shown) stimulatedactivities. On day 15, activity in ES (IES not available)was similar to that measured on PG-D6.5 or IES PG-D9. In the experimentwhere comparisonwas possible, there was no difference in AC activity or hormone response between IES and ES. Dose responses were altered in several ways during pseudo-pregnancy (Fig. 3A, Table Il. The most apparent effect was a striking diminution in response on day 9 followed by a recovery on day 15. Sensitivity (EC,,) of AC to 8-adrenergicstimulationwas similar on days 0 (0.60 pM), 1 (0.50 pM), 6.5 (0.95 pMM)and 15 (0.40 nM) but diminished to a level that prevented its determinationon day 9 (> 1 mM). The hormone response at threshold level (Table I) shows that isoproterenolstimulatedAC activity at comparable levels on day 0, 1, 6.5 and 15 but that no respone could be detected on day 9. During pregnancy IS0 response was higher in ES than in IES (p < 0.01) on day 6.5 and to a greater extent on day 9 (Fig. 3B). Response at threshold level (Table I), an indication of sensitivitywas significantlyhigher in ES D-9 than in other groups PG or PSPG. EC,,s of hormone stimulationwere not affected by the day of pregnancy or by the presence of the embryo. Dose responses to PGE, showed a pattern of response similar to that observed with IS0 (resultsnot shown) in pseudopregnantand pregnant animals.

TABLE

1.

INFLmcEoFPsEuDoPRE~cYmPRE~cYm~ REsPoIisETo ISOPRDTERENOL GROUPS

PSPG ESTRUS (DO) DAY 1 DAY 6.5 DAY 9

Responsiveness115.4 113.3 at 0.1 pM (a) + 2.0 + 0.6 (% of GTP)

112.5 f 4.0

Apparent Vmax (b) (% of GTP)

130.0

129.5 128.5

PG-DAY 6.5 DAY 15

104.0* 119.8 f 4.3 + 3.4

124.5

ADElrfLATEcYcLAsE

IES

ES-_

PG-DAY 9 IES

ES

107.5 108.5 114.5 128.7** f 3.0 + 3.3 f 5.8 f 6.4

140.5 127.7 143.5 132.0 150.0

(a) Responsivenessat lo-rM was calculated as described in Materials and Methods: % of GTP = total response (IS0 + GTPl/GTP. (b) Apparent Vmax was the maximal level of stimulationobserved on the dose response curves. * Significantlydifferent from other groups: Duncan's test, p < 0.05. ** Significantlydifferent from all other groups; Duncan's test, p < 0.01.

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a,b

ab l-

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IES

ES

ES

ES

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ES

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200

150

100

50

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IES

ES

ES

PG-D6.5

ES

IES

ES

IES

ES

ES

-cPG-D9

PG-Dl5

PGdI6.5

PG-D9

PG-D15

Fig. 2: Influence of pregnancy on adenylate cyclase activity at different stimulation levels. Animals in estrus (DO) were injected (i.v.) with 751U of hCG and allowed to copulate to induce pregnancy. Uterine horns were collected on indicated days and embryos were flushed with incomplete Hank's buffered salt solution. Uterine segments holding embryos were defined as embryonic sites (ES) while segments between embryos as interembryonic sites (IES). Tissues were processed for AC assay as described in Figure 1. AC activity was measured in each preparationat four different stimulationlevels: A) Basal (no stimulation); B) GTP (75 pM); C) GTP + IS0 (75 @l, 10 @f); D) GTP + PGE, (75 @l, 10 pM). Results are the mean f SE of 4 or 5 experimentsrun in triplicate. a) Implant value significantlydifferent from non-implantvalue of same group, Student's test, p < 0.01; b) Implant value of D9 significantlydifferent from implant or non-implantvalue of other groups, Duncan's test, p < 0.01; 1) GTP increased significantlyAC activity compared to Basal of same group, Student's test, p < 0.01.; 2) IS0 and PGE, increased significantlyAC activity compared to GTP of same groups, Student's test, p < 0.01.

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l8O-A

90

’

I

-9

-8

-7

-6

-5

-4

-9

-8

-7

-6

-5

IS0 (LOG M)

Fig. 3: Influence of pseudopregnancy(A) and pregnancy (B) on adenylate cyclase response to Isoproterenol.Dose responses to IS0 (1 nM to 100 pM) were determined in presence of GTP (75 pM) on tissues prepared as described in figures 1 and 2. Individualpoints represent the hormone response defined as % of GTP alone = (GTP t ISO)/GTP. A) Results are the mean f SD of 4 experimentsrun in tri licate: q , DO (estrus); A , PSPG-Dl; A , PSPG-D6.5; k PSPG-D9; . , PSPG-D15. B) Results are the mean f.SD of 5 e' ES; ~~i~~~:~s~un i “r$‘~~“‘ei;t~~tio~ ~~;~;~“~~d ~~~;‘responses are summarized'inTable I. a) Maximal response was higher in PSPG-D15 than in other days of PSPG, Duncan's test, p < 0.05; b) Maximal response was significantlyhigher in ES than in IES on day 6.5 and 9. Student's test, p < 0.01.

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DISCDSSIDD In the present study, we have found that adenylate cyclase activity in general and #3-adrenergic (or PGE,) response in particularwere modulated during pseudopregnancyand pregnancy. During PSPG, AC activity decreased progressivelyuntil day 9 and exhibited a slight recovery on day 15. Similar results have been observed in the rat. For instance,uterine AC (3) and incorporationof "H-methylgroups into epithelialphospholipids (26) showed a progressive decrease and minimal values on day 6 of PSPG. The decrease in activity reported in the present study was found at all levels of stimulation but responses to GTP, ISO, PGE, or NaF were still measurable when activity was the lowest. Since during pseudopregnancyuterine weight and protein content increase considerably (Fortier,unpublished observations),the low levels of activity measured may result from dilution of AC with cellular protein. Expression of dose responses as percentage of GTP (Table I), which compensates for variation in protein content, showed that IS0 was still able to induce CAMP formation at similar levels during pseudopregnancyuntil day 6.5. This suggested that protein content was the primary candidate to explain the decline of adenylate cyclase activity. However, on day 9, a reduction in hormonal response and sensitivitywas observed in addition to that of overall adenylate cyclase activity. The reduction in response at threshold level (0.1 pH ISO, Table I) from 115% on day 0 to 104% on day 9 may not appear of physiologicalimportance. However, the absence of response on day 9 (104% was not significantlydifferent from 100% (GTP alone)) compared to other days of PSPG appeared as the important factor associatedwith the lower sensitivityof AC to isoproterenolon day 9. Those results indicate that on day 9 additional factors are involved in conjunctionwith protein content. Since on that day, sensitivitywas reduced, the coupling between the subunits of the enzyme and the receptors may be altered. Indeed, sex steroids have been shown to affect enzyme turnover via their effect on protein synthesis (3). Results obtained on PSPG-DAY 15 support the possible role of steroid in the regulation of adenylate cyclase properties during pseudopregnancy. At that time, the uterine protein content appear similar to that found on days 6 and 9 (2). The recovery of enzyme activity may be explained by an estradiol surge due to the return to estrus (10). The regulationof adenylate cyclase activity and hormonal response in myometrium during PSPG may be related to uterine receptivityfor the embryo. The regulationof AC activity during pregnancy was most interesting. Indeed we have found differencesboth between pregnant and pseudopregnantanimals of correspondingdays and between implantationand inter-implantation sites in pregnant animals. On days 6.5 and 9, the overall AC activity was higher in pregnant than in pseudopregnantanimals. Since plasma and tissue levels of estradiol (E,) and progesterone (P) do not differ among pregnant and pseudopregnant rabbits at that time (7,14,31),the changes in AC activity reported in the present study, must be related to the presence of the conceptus. Thorbert et al. (32) have reported similar discrepanciesbetween pregnancy and pseudopregnancyin the rabbit when NE levels were measured in the genital tract. They have proposed that the differenceswere related to the presence of the embryo but to date the nature of the embryonic signal responsiblefor this action and the physiologicalsignificanceof the differencesbetween PG and PSPG on days 6.5 and 9 are not known. On days 6.5 and 9, AC activity in general and g-adrenergicresponse in particular were higher in ES than in IES. We have reported previously that it was possible to modify g-adrenergic response in cultured myometrial cells by altering the E, to P ratio (5). The change in g-adrenergicresponse at the ES may thus result from a local alteration in the E,/P ratio due to E, contributionfrom the embryo (11) or P metabolism by the endometrium (34). Other factors such as histamine (9) and

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gonadotropins (13) released by the embryo may also evoke functionalchanges in the uterus. Finally, these or other factors released by the endometrium upon the action of the embryo or released by the conceptus itself, may reach the myometrium and influence enzyme activation as early as on day 6. The movements of embryo along the entire length of the uterus until day 6 may be responsible for the higher AC activity in PG 6.5 compared to PSPG 6.5. The marked increase in AC activity observed on day 9 may result from the rapid expansion of the conceptus between day 7 and 10. At that time, there is a dramatic increase in implantationdome and the ratio of cell surface to cell volume is optimized to favor metabolic exchanges between the conceptus and environmentaltissues (25). How a signal could be transferredacross the endometriumall the way to the myometrium appears as a tricky problem. Interestingly,when we have found that AC response to IS0 or PGE, was enhanced at ES, the translocation of prostaglandinsthrough the uterine wall was also found to be increased (6). It is thus possible that a similar mechanism is involved for embryonic signals. The effect of the embryo on myometrial AC activity appeared however to be transient. Indeed, on day 15, AC activity in ES was comparableto that of PG day 6.5. Moreover, in one experiment,the particular spacing of embryos in one uterine horn allowed the preparationof myometrial tissue from IES. In that experiment,no difference in AC activity was found between IES and ES at any level of stimulation. In mid-pregnancy,since the uterus exhibits its lowest contractileactivity at the time we observe a diminished g-adrenergic response, other mechanisms such as a decrease in the number of a-adrenergic receptors (34) and elevated levels of circulatingrelaxin (1) may contribute to the maintenance of a relaxed state. The embryo may thus exert a transient and local action on myometrial activity until placentationis completed. A more generalizedmechanism regulated by steroid levels may then take over the control of uterine contractility. In summary, myometrial AC activity decreased progressivelyduring PSPG and recovered as progesteronelevel decreased following regressionof corpora lutea. During pregnancy, the presence of embryos exerted a general increase in AC activity which was more pronounced at implantation (ES) than at non implantation (IES) sites, Our results on alterationof adenylate cyclase activity together with the observationof Kanda and Kuriyama (17) on electrical activity suggest that the presence of the conceptus influences locally uterine contractility. This appears to be mediated by a multi-component mechanism involvingphysicochemicaland hormonal factors which acted only transientlyuntil placentationwas completed. Our results suggest that the concept of receptivityapplied to the endometrium,relative to implantation, could be extended to the myometrium.

Supported by grant UlJO389and # A2788 from Natural Sciences and EngineeringResearch Council of Canada. The authors wish to express their gratitute to Mrs. Nicole Breton for her excellent technical assistance.

1. 2. 3.

L.L. ANDERSON In: Biology of the uterus. Eds. Wynn, R.M., 587-651, Plenum Press, New York (1977). R. ARNOLD and C.D. SHOREY, Acta Anat. 127 119-124 (1986). A.M. BBKAIRI, R.B. SANDERS and J.M. YOCHIM. Biol Reprod 31 742-751 (1984a).

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

29. 30. 31. 32. 33. 34. 35.

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A.M. BEKAIRI, R.B. SANDERS, F.S. ABULABAM and J.M. YOCHIM, Biol. Reprod. 31 752-758 (1984b). 'iiTP. BOULET and M.A. FORTIER, Life Sci. 42 829-840 (1988). 2. CAO, M.A. JONES and M.J.K. HARPER, Biol. Reprod. 31 505-519 (1984). J.R.G. CHALLIS, I.J. DAVIS and K.J. RYAN, Endocrinology93 971-976 (1973). W.Y. CHBUNG, Science 207 19-27 (1980). S.K. DEY, D. JOHNSON and J. SANTOS, Biol. Reprod. 21 1169-1173 (1979). B. FISHER, E. WINTERHAGER and L.C.J. BUSH, J. Reprod. Fertil. 78 529-540 (1986). A.R. FUSCH, S. PERIYASAMY,M. ALEKANDROVA and M.S. SOLOFF, Endocrinology 113 742-749 (1983). g. GEORGE and J. WILSON, Science 199 200-201 (1978). F. HAOUR and B. SAKENA, Science 185 444-445 (1974). F.E. HARRINGTON and J.D. ROTHERMEL, Life Sci 20 1333-1340 (1977). L. JACOBSON, R.K. RIEMER, D. GOLDFINB, P.K. SIITERI and J.M. ROBERTS, Endocrinology120 1184-1189 (1987). S. KANDA and H. KURIYAMA, J. Physiol. 299 127-144 (1980). S.G. KORENMAN and J.F. KRALL, Biol. Reprod. 16 1-17 (1977). J.F. KRALL and S.G. KORENMAN, Biochem. Pharmacol.2 2771-2775 (1979). J.F. KRALL, J.D. BARRETT and S.G. KORENMAN, Biol. Reprod. -24 859-866 (1981). F.A. KUEHL, E.A. HAM, M.E. ZANETII, C.H. SANFORD, S.E. NICOL and N.D. GOLDBERG, Proc. Nat1 Acad. Sci. 71 1866-1870 (1974). H. KIJRIYAMAand H. SUZUKI, J. PhFiol. 260 315-333 (1976). O.H. LOWRY, N.T. ROSEBROUGH,A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). J.M. MARSHALL, Ergebn. Physiol. 62 6-67 (1970). R.C. MERCADO-SIMMEN,G.D. BRYANT.-GREENWOOD and F.C. GREENWOOD, Endocrinology110 220-226 (1982). J.A. MITCHELL, G. DRIESSEN, H. SCHEIDT and H.W. DENKER, Biol. Reprod. 36 669-675 (1987). B.C. MOULTON and B.B. KOENIG, Endocrinology118 244-249 (1986). C.P. PURI and R.E. GARFIELD, Biol. Reprod. 27 967-975 (1982). J.C. RANKIN, B.E. LEDFORD and B. BAGGET, in Cellular and Molecular Aspects of Implantation,eds: S.R. Glasser and D.W. Bullock, pp. 428-430, Plenum Press, New York (1981). J.M. ROBERTS, P.A. INSEL and A. GOLDFIEN, Mol. Pharmacol.20 52-58 (1981). B.M. SANBORN, R.C. BHALLA and S.G. KORENMAN, Endocrinology92 494-499 (1973). C.H. SPILMAN and J.W. WICKS, Proc. Sot. Exp. Biol. Med. -151 726-729 (1976). G. THORBERT, S. BATRA, C.K. OWMAN, E. ROSENBERG and N.D. SJOBERG, Endocrinology99 1207-1212 (1976). G.D. THORNBURN and J.R.G. CHALLIS, Physiol. Rev. 59 863-918 (1979). J. VALLIERES,M. FORTIER and L. BUKOWIECKI,Biol. Reprod. 19 318-325 (1978). T. WISE and R.B. HEAP, Biol. Reprod. 28 1097-1106 (1983).