MOLECULAR REPRODUCTION AND DEVELOPMENT 293374341 (1991)

Rabbit Blastocysts Accumulate [3H]Quinuclidinyl Benzilate In Vitro M.A. JONES AND M.J.K. HARPER2 'Department of Chemistry, Illinois State University, Normal, Illinois; 2Department of Obstetrics and Gynecology, The University of Texas Health Science Center at Sun Antonio, San Antonio, Texas

ABSTRACT Day6 rabbit blastocysts were able to accumulate [3H]quinuclidinyl benzilate (QNB) from their environment. This accumulation was reduced approximately 50%in the presence of 1.5 x lop4 M atropine (an accepted antagonist for ligands which bind to muscarinic cholinergic receptors). The accumulation of QNB was sensitive to temperature and was apparently saturable. In the presence of 2 nM QNB, Day6 blastocysts accumulated 30.3 2 2.0 fmoles per blastocyst. When the cellular elements alone were examined, lesser amounts of specific binding were detected. Owing to the complexity of this multicompartmental system, Scatchard analysis did not provide meaningful results. This accumulation appears higher than that reported for other tissues such as rabbit heart homogenates or rabbit uterine endometrial cells. This muscarinic cholinergic accumulation may have some role in blastocyst-maternal recognition. Key Words: Muscarinic cholinergic receptors,Atropine

INTRODUCTION Maternal recognition of pre-implantation blastocysts is a critical event for which various molecular mechanisms have been proposed. Proteins, such as ovine trophoblast protein-1, released from sheep embryos (Vallet et al., 1988), oxytocin (Flint et al., 19861, histamine (Dey et al., 1978), and various lipids, such as blastocyst-derived estrogens (Bazer and Thatcher, 19771, prostaglandins (PGs) (Kennedy, 1977; Phillips and Poyser, 19801, leukotrienes (Gupta et al., 19891, and platelet-activating factor (PAF) (Harper, 1989) have been studied as chemical signals involved in this recognition. However, there have been few studies involving molecules that bind to receptors traditionally considered to bind neurotransmitters (the adrenergic and the cholinergic receptors). Agonists which bind to muscarinic cholinergic receptors have been demonstrated t o stimulate G-protein activation, which, in turn, can activate phospholipase C and stimulate phosphatidyl-inositol 4,5-bisphosphate (P1P2)breakdown. This, in turn, can affect arachidonic acid liberation and subsequent PG synthesis (Abdel-Latif, 1986; Yousufzai et al., 1987). Marnet et al. (1987) reported that sheep myometrium contained alpha adrenergic receptors which were estrogen dependent. Richelsen and Ped0 1991 WILEY-LISS, INC.

ersen (1985) found that prostaglandin E, receptors were sensitive to regulation by a beta-adrenergic agonist (isoproterenol). Johns and Riehl (1982) showed that a cell suspension from guinea pig uterus contained specific binding sites for [3Hl-l-quinuclidinyl benzilate (QNB), a ligand used to study muscarinic cholinergic receptors. The Kd values were reported to be 0.11 nM, and the non-specific binding was very low (approximately 2% of total binding). Jones and Harper (1983) isolated uterine endometrial e ithelial cells from Day-6 pregnant rabbits and studied [! HIQNB binding to these cells. These cells were able to bind L3H1QNB both specifically (atropine displaced) and non-specifically (not displaced by atropine). Atropine is an antagonist often used in the study of muscarinic cholinergic receptors (Johns and Riehl, 1982; Gil and Wolfe, 1985). Muscarinic cholinergic agonists have been shown to have a wide variety of physiological responses in target tissues. Jones et al. (1982) reviewed the activation of muscarinic receptors which can be correlated with a calcium-independent decrease in the concentration of inositol phospholipids. Gil and Wolfe (1985) also demonstrated a muscarinic receptor-mediated phosphoinositide breakdown and inhibition of adenylate cyclase activity using rat brain, parotid gland, and heart. Hsia et al. (1985) reported that the muscarinic agonist oxotremorine inhibited adenylate cyclase activity in cultured human fibroblasts while the beta-adrenergic agonist isoproterenol activated the enzyme. It appears well established that neurotransmitters can affect both hormone-sensitive adenylate cyclase and other important receptor-mediated systems such as phosphatidylinositol turnover. We, therefore, wished to test if embryos could also interact with neurotransmitters which may be derived from the maternal environment. If the embryos can interact with the neurotransmitters and respond, via a receptor-mediated process, this would strongly suggest one important way for the maternalembryonic systems to communicate.

Received July 25, 1990; accepted January 21, 1991. Address reprints to Michael J.K. Harper, Department of Obstetrics and Gynecology, The University of Texas Science Center at San Antonio, San Antonio, TX 78284-7836. Presented in part a t the 32nd Annual Meeting of the Society for Gynecological Investigation, Phoenix, Arizona, 1985.

338

M.A. JONES AND M.J.K. HARPER

MATERIALS AND METHODS Materials l-[3H]quinuclidinyl benzilate (QNB) (32 Ci/mmol) was purchased from Amersham Corp., Arlington Heights, IL. Atropine sulfate, human chorionic gonadotrophin (hCG), and follicle-stimulating hormone (FSH) were purchased from Sigma Chemical Company, St. Louis, MO. Glass fiber filters (GF/F) were purchased from Whatman, Clifton, NJ. Methods Mature New Zealand White-Cambridge female rabbits (body weight > 3.0 kg) were caged individually in a controlled environment as previously reported (Jones and Harper, 1983). To obtain embryos, estrous rabbits were treated for 3 days with FSH, then inseminated with 0.5 ml of a mixed sperm suspension collected immediately before use (Cao et al., 1984). The females were then injected intravenously with 100 IU of hCG. On Day-6 after insemination, the rabbits were sacrificed using an overdose of sodium pentobarbital (Fort Dodge Laboratories, Fort Dodge, IA). The uteri were rapidly removed and flushed with Krebs Ringer bicarbonate buffer (KRB) (Jones et al., 19861, pH 7.4, to remove the blastocysts. The blastocysts were pooled from six rabbits for each experiment and washed three times in KRB. Day-6 blastocysts randomly selected from the pool were incubated with L3H1QNB under a variety of conditions. The reaction was stopped by filtration using GF/F glass fiber filters and rinsing with 20 ml of ice-cold KRB following the procedure of Johns and Coons (1983). Following the washing, the filters were removed, the number of intact blastocysts determined, and the radioactivity associated with the filter and blastocysts evaluated by liquid scintillation spectroscopy. Counting efficiency was determined using L3H] toluene as an internal standard. Non-specific binding was determined in the presence of 1.5 x M atropine. In some experiments, the intact blastocysts were incubated in microcentrifuge tubes (Jones and Harper, 1984) with [3H]QNB, then washed ( 4 ~with ) cold KRB buffer. The blastocysts were ruptured, using a 22-gauge needle. The tubes were then centrifuged, using an Eppendorf table top centrifuge (Model 5412) at 12,800g for 2 min. This resulted in a pellet (blastomeres) and a supernatant fraction (blastocoelic fluid). In these experiments, each fraction was evaluated separately for radioactivity (Jones and Harper, 1984). Data are reported as the mean k SEM for the indicated number of experiments.

- I , , 0

20

40

,

,

,

,

1

60

80

100

120

140

Time (minutes)

Fig. 1. Mean ? SEM [3H]QNB accumulation by Day6 rabbit blastocysts as a function of in vitro incubation time in KRB at 37°C. The concentration of [3H]QNB in the incubation was 2 nM. n = number of experiments with three to four blastocysts each. No standard error bar is shown when it is smaller than the symbol for the mean.

Since these data might indicate equilibrium at 90 min rather than saturation, the experiment was repeated using a concentration of 12 nM L3H1QNB.These results are shown in Figure 2, where it appears that at this higher ligand concentration saturation was clearly reached by 90 min. Incubation times of 90 min were used in subsequent experiments. [3H]QNB binding by blastocysts as a function of number of blastocysts per incubate was also determined. This was felt to be an important experiment since the pool of blastocysts obtained from several rabbits always shows some variation in the embryo size (Jones and Harper, 1988). Figure 3 shows the results from experiments, each with a designated number of blastocysts per incubation. As shown, the amount of ligand bound (fmol [3H]QNB per incubate) increased apparently linearly with increase in number of blastocysts over the range examined. The (n) is the number of experiments. The correlation coefficient (r)determined by linear regression analysis of these data is 0.9968. The amount of [3H]QNBaccumulated by Day-6 blastocysts under these experimental conditions was calculated to be 30.3 ? 2.0 fmoles per blastocyst (mean t SEM, for 54 blastocysts). Assuming for comparative purposes that each blastocyst contains on average 0.02 mg protein (Jones and Harper, 1988), then each blastocyst can accumulate approximately 1.5 pmol L3HlQNB per mg protein. In fact, since there is considerable variation in blastocyst size, individual protein values also vary. This value is approximately 18-fold higher than that reported by Fields et al. (1978) using rabbit heart homogenates (57.2 fmol t3H1QNB per mg RESULTS AND DISCUSSION protein) and also higher than the values reported by To test for apparent saturation of [3H]QNB binding, Jones and Harper (1983) for specific binding of QNB to blastocysts were incubated in 2 nM r3H1QNB at 37°C isolated uterine endometrial cells (approximately 200 for 5 to 120 min. The data are shown in Figure 1. Each fmol/mg protein). The values reported here are also time point represents the mean ? SEM for (n) number higher than those reported using hypothalamic regions of experiments with each incubation containing three of rat brains and in the same range as values reported to four blastocysts. Apparent binding saturation oc- using parietal cortex (Rainbow et al., 1984). The accumulation of l3H1QNB was temperature decurred by 60 min incubation under these conditions.

BLASTOCYSTS ACCUMULATE QNB

339

TABLE 1. Effect of Incubation Temperature on [3H]QNB Accumulation by Day-6 Blastocysts Temperature ("C) fmol [3H]QNB per blastocyst 37 31.5 f 4.8 (8)a 22 46.9 k 10.0(3)a 4 13.7 1.8 (3)b Values a r e reported as m e a n k SEM and (n) = n u m b e r of experiments w i t h 2 to 4 blastocysts per experiment. Values w i t h t h e s a m e letter are n o t significantly different

+

(Student's t-test). Time (minutes)

Fig. 2. Mean 2 SEM r3H1QNB accumulation by Day-6 rabbit blastocysts as a function of in vitro incubation time in KRB at 37°C. The concentration of L3H1QNB in the incubation was 12 nM. n = number of experiments with three to four blastocysts each.

G

m

* L

Total

250-

2. 0

200-

o Non-specific + 1 5 I 10% atropine A

Specific

150-

al CL m 100-

z

-

E"

v-

L

50-

4 0 0

120

2

4

6

8

10

12

14

nM fH] QNB

(I)

LI 1601

*

-

0

40

3

c L

0

1

2

3

4

5

6

Fig. 4. Mean SEM [3H]QNB accumulation by Day-6 rabbit blastocysts in relation to concentration of the radioligand during a 90 rnin in vitro incubation in KRB at 37°C. No standard error bar is Total shown when it is smaller than the symbol for the mean. (M) non-specific ) binding; (A-A) specific binding. binding; (M

Number of Blastocysts

Fig. 3. Mean ? SEM [3H]QNB accumulation by Day-6 rabbit blastocysts as a function of number of blastocysts/tubeduring a 90 rnin in vitro incubation in KRB at 37°C. The concentration of r3H1QNB in the incubation was 2 nM. No standard error bar is shown when it is smaller than the symbol for the mean. n = number of experiments; r = linear regression coefficient.

pendent, as shown in Table 1. Blastocysts were incubated for 90 min at the designated temperatures, in 2 nM [3H]QNB.There was an increase of accumulation of ligand as a temperature function, but there was no apparent Qlo effect (a doubling of accumulation with a lo" rise in temperature), which implies a saturation effect. The effect of l3HIQNB concentration and incubation in the absence and presence of 1.5 x l o p 4M atropine is shown in Figure 4. Blastocysts were incubated in KRB for 90 min at 37°C. Each incubate contained three to four blastocysts, and the values reported are the mean 2 SEM for three experiments. Total accumulation of I3H]QNB increased from 1.5 2 0.3 fmol [3H]QNB per blastocyst at 0.13 nM ligand to 219.7 k 72.7 fmol [3H]QNBper blastocyst at 12 nM ligand concentration. However, when specific accumulation was calculated (total accumulation - accumulation in the presence of atropine), this curve a peared t o reach a saturation at approximately 6 nM [ HIQNB. The non-specific accu-

P

mulation of ligand, however, did not appear to saturate and was approximately 50% of the total accumulation measured. Therefore, the percent accumulated was very near that reported by Jones and Harper (1983) using isolated uterine endometrial cells. However, when the saturation data were evaluated by Scatchard analysis to determine an apparent Kd value, the data did not plot as a meaningful linear Scatchard line. This experiment was then repeated using only ruptured blastocysts to remove the complication of the fluid compartment. The results shown in Figure 5 are very similar to those in Figure 4,with the specific binding curve showing saturation at 4 nM. The total amount of binding at 4 nM in the absence of the blastocoel is about one-quarter of that of the whole blastocyst. Even using these data, a meaningful Scatchard plot could not be generated, probably because the cellular elements comprised trophoblastic cells, inner cell mass, and blastocyst investments. Uptake of [3HlQNB by blastocysts is, however, displaceable by atropine. Blastocysts were incubated in 12 nM [3H]QNBfor 90 min before addition of 1.5 x l o p 4M atropine. The blastocysts were washed and evaluated for remaining radioactivity. The data are shown as % of I3H]QNBremaining after atropine addition. By 20 min, approximately 49% of the radioactivity had been displaced (Fig. 6), and this did not change over a further

340

M.A. JONES AND M.J.K. HARPER 0

Total

0

Non-specific +15

x IO'M alrapme

A Specific

~

.......

5

10

~

6

*

5 15

nM [3H] Q N B

Fig. 5. L3H]QNB accumulation by cellular elements of Day-6 rabbit blastocysts in relation to concentration of the radioligand during a 90 Total binding; min in vitro incubation in KRB at 37°C. (-) (w) non-specific binding; (A-A) specific binding.

40 min. This result again demonstrates the multicompartmental nature of the blastocyst. To attempt to define the contribution of the cellular material to the binding of [3H]QNB to the whole blastocyst, we incubated blastocysts in microcentrifuge tubes (three to four blastocysts/tube) for 90 min in the presence of 12 nM L3H]QNB with and without 1.5 x lop4 M atropine. The blastocysts were washed four times with 1 ml of KRB buffer and then pierced with a 20-gauge needle and centrifuged for 3 rnin at 12,800g to separate supernatant (blastocoel fluid) and pellet (cellular material). Each fraction was evaluated for radioactivity separately. There was a decrease in the % of radioactivity in the cellular elements in the presence and absence of atropine of about 55% (from 10.9 2 1.6 to 4.9 2 2.7 fmolhuptured blastocyst), while in the fluid, only about 25% of the L3H1QNB was displaced by atropine (from 16.6 ? 5.4 to 12.4 ? 2.8 fmol/original number of blastocysts). Thus, binding of [3H]QNB to blastocyst cellular material is more atropine-sensitive than is the transport mechanism involved in [3H]QNB uptake to the blastocoel. In this experiment, the percentage of label in the blastomeres was 39.6%, and in the blastocoel, 60.6%. In previous experiments (five experiments with three blastocysts per incubate) using a concentration of only 2 nM L3H]&NBfor 90 min, the same compartments accumulated 53.7 2 3.6 and 46.3 2 3.6%, respectively, of the total uptake. These percentages were much different from those found when blastocysts were incubated with [3H]prostaglandins, when approximately 90% of the label was found in the blastocoel (Jones and Harper, 1984). This demonstrates the large difference in accumulation (transport) process for prostaglandins and QNB in Day-6 blastocysts. From the experiments reported here, it is clear that Day-6 rabbit blastocysts can accumulate [3H]QNBfrom their environment. That the process, however, is not a simple one, is evident from the saturation and distribution data. The blastocysts may contain both a receptor system for binding muscarinic cholinergic ligands

0

1'0

20

30

40

50

60

70

Time (minutes) After Atropine Addition

Fig. 6. Displacement of [3H]QNB accumulated by Day-6 rabbi) blastocysts in a 90 min pre-incubation in KRB at 37°C by addition of 1.5 x M atropine. The concentration of L3H1QNB in the incubation was 12 nM. There were three to four blastocysts per time point. The data show % of [3H]QNB remaining (100% a t zero time) after atropine addition over a 60 min period.

as well as a transport system for such ligands. Whether the blastocysts can respond, in vivo, to maternally derived neurotransmitters is not known. However, Day-6 rabbit blastocyst prostaglandin production was not stimulated or inhibited by exposure of the blastocysts to atropine or carbachol(O.15 mM) for 1h at 37°C in vitro (Harper et al., 1989).

ACKNOWLEDGMENTS The authors wish to thank Dr. Anthony Johns for the kind gift of the L3H1QNB and Cate Norris and Elizabeth Hemmick for their technical assistance with the rabbits. This study was supported by NIH grants HD 14048 and HD25224.

REFERENCES Abdel-Latif AA (1986): Calcium-mobilizing receptors, polyphosphoinositides and the generation of second messengers. Pharmacol Rev 381227-272. Bazer FW, Thatcher WW (1977): Theory of maternal recognition of pregnancy in swine based on estrogen controlled endocrine versus exocrine secretion of prostaglandin F,, by the uterine endometrium. Prostaglandins 14:397-401. Cao Z-d, Jones MA, Harper MJK (1984): Prostaglandin translocation from the lumen of the rabbit uterus in vitro in relation to day of pregnancy or pseudopregnancy. Biol Reprod 31:505-519. Dey SK, Villanueva C, Chien SM, Crist RD (1978): The role of histamine in implantation in the rabbit. J Reprod Fertil 53:23-26. Fields JZ, Roeske WR, Morkin E, Yamamura HI (1978): Cardiac muscarinic cholinergic receptors. J Biol Chem 253:3251-3258. Flint AP, Leat WM, Sheldrick EL, Stewart H J (1986): Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium. Biochem J 237:797-805. Gil DW, Wolfe BB (1985): Pirenzepine distinguishes between muscarinic receptor-mediated phosphoinositide breakdown and inhibition of adenylate cyclase. J Pharmacol Exp Ther 232:608-616. Gupta A, Huet YM, Dey SK (1989): Evidence for prostaglandins and leukotrienes as mediators of phase I of estrogen action in implantation in the mouse. Endocrinology 124:54&548. Harper MJK (1989): Platelet-activating factor: A paracrine factor in preimplantation stages of reproduction. Biol Reprod 40:907-913.

BLASTOCYSTS ACCUMULATE QNB Harper MJK, Jones MA, Norris CJ, Woodard DS (1989): Prostaglandin synthesis by Day-6 rabbit blastocysts in uitro. J Reprod Fertil 86:315325. Hsia JA, Hewlett EL, Moss J (1985): Heterologous desensitization of adenylate cyclase with prostaglandin E, alters sensitivity to inhibitory as well as stimulatory agonists. J Biol Chem 260:4922-4926. Johns A, Coons LW (1983): An inexpensive filtration unit and manifold for radioligand binding studies. J Pharmacol Methods 9:263-267. Johns A, Riehl RM (1982): A simple method for preparing single cell suspensions of heart and smooth muscle for radioreceptor labeling studies. J Pharmacol Methods 7:153-159. Jones MA, Harper MJK (1983): Prostaglandin accumulation by isolated uterine endometrial epithelial cells from six-day pregnant rabbits. Biol Reprod 29:1201-1209. Jones MA, Harper MJK (1984): Rabbit blastocysts accumulate L3H1 prostaglandins in uitro. Endocrinology 115:817-823. Jones MA, Harper MJK (1988): Effects of Iloprost, a stable prostacyclin analog, PGE, and PGF,, on rabbit blastocysts. Gamete Res 20:203-213. Jones LM, Kirk Ch J , Michell RH (1982): Molecular events following activation of muscarinic receptors: The role of inositol phospholipids. Scand J Gastroenterol [Suppl 721 17:3340. Jones MA, Cao Z-d, Anderson W, Norris C, Harper MJK (1986): Capillary permeability changes in the uteri of recipient rabbits

341

after transfer of blastocysts from indomethacin-treated donors. J Reprod Fertil 78:261-273. Kennedy TG (1977): Evidence for a role of prostaglandins in the initiation of blastocyst implantation in the rat. Biol Reprod 16:286291. Marnet PG, Garcia-Villar R, Laurentie MP, Toutain PL (1987):In vivo pharmacological characterization of alpha adrenergic receptors in sheep myometrium and their physiological meaning. Biol Reprod 37:241-248. Phillips CA, Poyser NL (1980): Prostaglandins and implantation in the rat. Adv Prostaglandin Thromboxane Res 8:1391-1394. Rainbow TC, Biegon A, Berck DJ (1984): Quantitative receptor autoradiography with tritium-labeled ligands: Comparison of biochemical and densitometric measurements. J Neurosci Methods 11:231-241. Richelsen R, Pedersen 0 (1985): P-Adrenergic regulation of prostaglandin E, receptors in human and rat adipocytes. Endocrinology 116:1182-1188. Vallet JL, Bazer FW, Fliss MFV, Thatcher WW (1988):Effect of ovine conceptus secretory proteins and purified ovine trophoblast protein1 on interoestrous interval and plasma concentrations of prostaglandins F-2a and E and of 13,14-dihydro-15-ketoprostaglandin F-2a in cyclic ewes. J Reprod Fertil84:493-504. Yousufzai SYK, Honkanen RE, Abdel-Latif AA (1987): Muscarinic cholinergic induced subsensitivity in rabbit iris-ciliary body. Invest Ophthalmol Vis Sci 28:1630-1638.

Rabbit blastocysts accumulate [3H]quinuclidinyl benzilate in vitro.

Day-6 rabbit blastocysts were able to accumulate [3H]quinuclidinyl benzilate (QNB) from their environment. This accumulation was reduced approximately...
511KB Sizes 0 Downloads 0 Views