Differences in the cyclic nucleotide mediation of luteinizing hormone-releasing hormone action on the rat and hamster anterior pituitary gland

Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,

55 (1988) 173-182 Ltd.

173

MCE 01788

Differences in the cyclic nucleotide mediation of luteinizing hormone-releasing hormone action on the rat and hamster anterior pituitary gland Wan-Song A. Wun I, Albert S. Berkowitz 2 and James P. Preslock ’ ’ Department of Obstetrics and Gynecolop, Obstetrics,

Tulane Unrversity School of Medicine, New Orleans, LA 70112, U.S.A., and ’ Department Gynecology and Reproductrve Sciences, Unrversity of Texas Medical School at Houston, Houston, TX 77030, U.S.A. (Received

11 May 1987: accepted

Key words: Luteinizing hormone-releasing hormone; system; (Male rat; Male hamster)

Cyclic

nucleotide;

18 September

of

1987)

Gonadotrophin;

Anterior

pituitary

gland;

Superfusion

Summary

A continuous flow superfusion system which was previously developed in our laboratory was utilized to study the modulation of LH and FSH release by cyclic nucleotides and LHRH from anterior pituitary glands (APG) obtained from rats or hamsters. There was a transient increase in LH and FSH secretion from superfused rat APG in response to superfusion with 1 X lop3 M l-methyl-3-isobutyl-xanthine (MIX), while 1 X lop4 MIX M had no effect. Furthermore, a dose of 5 X lop5 M MIX did not potentiate the gonadotrophin-releasing effect of 1 X lo- ” M LHRH. Neither 1 X lo-’ M 8-Br-CAMP nor 1 X 10m3 M 8-Br-cGMP mimicked the gonadotrophin-releasing effects of 1 X 10m9 M LHRH. In the experiments utilizing hamster APG, FSH release gradually increased during superfusion with 1 x lo-’ M MIX or 1 x lop4 M MIX, while LH release was transient but significantly increased in response to superfusion with both doses of MIX. A dose of 5 x 10m5 M MIX potentiated the effect of a low dose of LHRH (1 X lOpi M) upon both LH and FSH secretion. 1 X lo-’ M 8-Br-CAMP mimicked the effect of LHRH upon LH and FSH released from superfused hamster APG, while 1 X 10-j M 8-Br-cGMP was inhibitory. These results suggest that cyclic nucleotides are involved in the mediation of the LHRH-induced release of gonadotropins from the anterior pituitary gland of the hamster, but do not mediate the LHRH-induced release of gonadotropins from the rat anterior pituitary gland.

Introduction

The role of adenosine 3’,5’-cyclic monophosphate (CAMP) in the mediation of luteinizing hormone-releasing hormone (LHRH) stimulation of gonadotrophin release from the rat anterior

Address for correspondence: Wan-Song A. Wun, Department of Obstetrics and Gynecology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112, U.S.A. 0303-7207/88/$03.50

0 1988 Elsevier Scientific

Publishers

Ireland,

pituitary gland has been studied for more than a decade. In spite of a large body of information obtained from experiments utilizing rat pituitaries, the effect of CAMP remains unclear. The evidence in favor of CAMP as the second messenger of LHRH includes the observations that (1) LHRH activated adenyl cyclase activity in both crude homogenates (Decry and Howell, 1973) and plasma membrane fractions of rat pituitaries (Spona, 1975); (2) LHRH stimulation elevated intracellular CAMP levels (Borgeat et al., 1972, Ltd.

174

1974; Labrie et al., 1973; Makino, 1973); (3) exogenous CAMP stimulated LH release (Ratner. 1970; Labrie et al., 1973; Makino, 1973); and (4) phosphodiesterase inhibitors such as theophylline and l-methyl-3-isobutyl-xanthine (MIX) induced LH release and potentiated the stimulatory effect of LHRH (Ratner, 1970; Makino, 1973; Tang and Spies, 1976). Evidence negating the CAMP mediation of LHRH’s effect upon the rat anterior pituitary gland includes the observations that (1) LHRH could not stimulate adenyl cyclase in either crude homogenates (Berault et al., 1980) or plasma membrane fractions (Clayton et al., 1970); (2) LHRH did not cause a rise in intracellular levels of CAMP (Noar et al., 1975; Ratner et al., 1976; Conn et al., 1979); (3) exogenous CAMP derivatives did not stimulate release of LH (Sundberg et al., 1976; Tang and Spies, 1976; Stern and Conn, 1981); (4) phosphodiesterase inhibitors neither induced LH release nor potentiated the effect of LHRH (Ratner et al., 1976). Furthermore, it has been suggested that guanosine 3’,5’-cyclic monophosphate (cGMP) may be involved in the mechanism of LHRH action upon the rat anterior pituitary gland both in vitro (Noar et al., 1978a, b) and in vivo (Kawakami and Kimura, 1980). LHRH stimulates the release of LH and FSH from hamster anterior pituitary glands (Wun et al., 1986a, b). However, the pattern of the seasonal breeding of this species suggests that the hamster anterior pituitary gland may be regulated by LHRH through a mechanism different from the rat anterior pituitary gland. The purpose of this study was to investigate the cyclic nucleotide mediation of the action of LHRH upon LH and FSH release from the anterior pituitary glands of both rats and hamsters. In this study we utilized an in vitro system in which fresh anterior pituitary glands from rats or hamsters were continuously superfused with the test substances for a period of 3 h (Wun et al., 1986a, b). Materials and methods Animals

Three-month-old male Long-Evans rats (250280 g) were obtained from a random-bred in-house colony, and 2-month-old (91~-100 g) male golden

hamsters (~~~~cr~~e~~ auratus) were obtained from Engle Laboratory Animals, Farmersburg, IN. All animals were housed at 22°C and maintained in an LD 14: 10 photoperiod (lights on at 05.00 h). Food (Purina Lab Chow) and water were provided ad libitum. The animals were sacrificed by decapitation at 08.30-09.00 h. The pituitary glands were dissected free from the sella turcica, the posterior lobes removed, and the anterior lobes were bisected at the isthmus.

The continuous flow system for the superfusion of anterior pituitary glands has been validated in our laboratory and described previously (Wun et al., 1986a, b). In brief, the system consists of a superfusion chamber of 80 ~1 capacity containing four hemi-anterior pituitary glands (APG) from four separate animals. Medium 199 buffered with 0.017 M NaHCO, and aerated with 95% 0,/5% CO, is delivered continuously by a 4-channel peristaltic pump (Gilson Volumetric) at a rate of 1 ml/.5 min. The superfusate is collected in 3 ml fractions, frozen, and stored at - 20” C until assay. Radioimmunoassay procedures The LH and FSH concentrations in the superfusate were determined by double-antibody radioimmunoassays validated in our laboratory. Hamster LH was measured by a heterologous radioimmunoassay which was developed by Blake et al. (1973) and Bast and Greenwald (1974). The assay utilizes anti-ovine-LH GDN No. 15 (Niswender) antiserum and ovine LER 1056 C* LH (Reichert) for iodination. The reference preparation for the hamster LH assay was NIAMDD-rat LH-RP-1. Displacements between rat LH-RP-1 and hamster LH in superfusate, serum, and pituitary homogenates were not significantly different. The intraassay coefficient of variation (CV) was 5.2% and the interassay CV was 7.2% in the hamster LH assay. Rat LH was measured with the materials supplied and procedures outlined by the NIAMDD pituitary distribution program. The intraassay CV was 5.8% and the interassay CV was 10.1% for the rat LH assay. Rat and hamster FSH were measured with NIAMDD rat FSH reagents and protocol (Bast and Greenwald, 1974). The intraassay CV

175

was 6.3% and the interassay CV was 8.7% for the FSH assay. Radioimmunoassay results were calculated on a Hewlett Packard desktop calculator using a 4parameter logistic curve fitting program (Grotjan and Steinberger, 1977). Reagents l-Methyl-3-isobutyl-xanthine (MIX; lot UOF0097), 8-bromo-adenosine-3’,5’-cyclic monophosphoric acid (8-Br-CAMP, lot 150F-7042) and 8bromo-guanosine-3’,5’-cyclic monophosphoric acid (8-Br-cGMP, lot 69c-7330) were purchased from Sigma Chemical Company (St. Louis, MO). LHRH was obtained from Boehringer Mannheim (Indianapolis, IN). Medium 199 with Earle’s salts was obtained from Gibco (Grand Island, NY). The total amounts of LH and FSH release (above basal levels) in response to continuous superfusion of the test substances was calculated by summing the amount of hormone in the 15 min fractions from the initiation of superfusion with the test substances until the cessation of superfusion 3 h later. Experimental procedures As a general protocol the APG from adult male rats or hamsters were superfused with medium 199 alone for the first 2 h, with the test substances for 3 h, and again with medium 199 alone for 1 h. As a control, APG were superfused with medium 199 alone for the entire 6 h. The experiments were designed to examine: (1) the effect of MIX on gonadotropin release; (2) the possible potentiation by MIX of the effect of LHRH; and (3) the ability of 8-Br-CAMP or 8-Br-cGMP to mimic the effect of LHRH. Statistical analysis The analysis of the patterns of FSH and LH release was evaluated by the nonparametric Sign Test (Daniel, 1978) and Runs Test (Zar, 1974). The Sign Test is a statistical test to determine the significance of differences between control and experimental groups by generating a series of signs ( f or - ) and compares whether the appearing frequency of these signs is significantly different. The Runs Test examines the aggregate pattern of the positive and negative signs along with the time

of culture. The levels of significance for these tests in the present studies is at least P I 0.05. Results Each set of experiments was replicated three times and the results which were obtained were similar for all replicates. For brevity, onIy one representative set of results is demonstrated. The effect of MIX on the release of LH and FSH In the experiments utilizing rat APG, 1 X 10v4 M MIX had no detectable effect upon the release rate of LH (Fig. 1A) or FSH (Fig. 2.4). 1 X lo-’ M MIX induced only a transient increase in the release rate of LH and FSH from rat APG in that there was an approximately 20% increase in the release rate of LH that lasted for 30 min (Fig. IA), while for FSH there was an approximate 10% increase in the release rate which lasted for 2 h (Fig. 2A). The patterns of the release rates of LH and FSH were not significantly different by the Sign Test or Runs Test. In the experiments using hamster APG, MIX significantly stimulated the release rate of both LH and FSH in a dose-dependent manner (Figs. 3A and 4A). During 90 min of superfusion with 1 x IOb3 M MIX, the release rate of LH gradually increased until it was 2-fold the basal release rate: the release rate then returned to baseline even though superfusion with 1 x lop3 M MIX continued. The effect of 1 x lop4 M MIX upon the LH release rate was similar to the effect of 1 x low3 M MIX except for a 1 h latent period and a peak release rate of only approximately 1.6-fold the basal release rate. In contrast to the profile of LH release, 1 X 10M3 M MIX induced a significant increase in the FSH release rate that plateaued at approximately 1 h after the initiation of superfusion with MIX, and remained elevated despite the return to superfusion with medium 199 alone. The FSH release rate showed a gradual and continuous increase during the 4 h superfusion with 1 X 10e4 M MIX, and continued to increase after the cessation of superfusion with MIX. The potentiating effect of MIX on LHRH action Superfusion of rat APG with 5 X 10e5 M MIX did not increase the release rate of LH (Fig. 1B)

176

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min Fig. 1. Release rate profiles of rat LH. In each channel, four hemipituitary glands obtained from four adult male rats were with medium 199 at a rate of 1 ml/5 min. The first 90 min of superfusion was the period of pre-equilibration; the collected during this period was discarded. The dark bar represents the period of superfusion of the hemipituitaries with agents, Each panel is a representative of three similar experiments. (A) The effect of MIX; (B) the potentiating effect LHRH action; (C) the effect of g-Br-CAMP and 8-Br-cGMP upon the release rate of rat LH.

superfused superfusate the testing of MIX on

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Fig. 2. The release rate profiles of rat FSH. See the legend to Fig. 1. (A) The effect of MIX; (B) the potentiating LHRH action; (C) the effect of 8-Br-CAMP and 8-Br-cGMP on the release rate of rat FSH.

effect of MIX on

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180

nor of FSH (Fig. 2B). 1 x lo-‘” M LHRH significantly increased the release rate of LH and FSH by approximately 1.3-fold (Fig. 1H) and l.l-fold (Fig. 28), respectively. Superfusion of the rat APG simultaneously with 1 X 10-i’ M LHRH and 5 X lo-’ M MIX did not increase the gonadotropin release rate above that obtained by 1 X lo-” M LHRH alone. In the experiments utilizing hamster APG, 5 X 1O-5 M MIX had no effect upon the LH or FSH release rates (Figs. 38 and 48) while 1 x lo-” M LHRH significantly increased the LH release rate 1.8-fold over basal release (Fig. 3B). The superfusion of hamster APG with 1 x 10-i” M LHRH and 5 X lop5 M MIX significantly increased the release rate of LH by 3.5-fold (Fig. 3B). 1 x lo- ” M LHRH did not have a significant stimulatory effect upon the release rate of hamster FSH (Fig. 4B); however, superfusion of hamster APG with 1 x lo-‘* M LHRH and 5 X lo-i0 M MIX significantly increased the release rate of FSH by 2-fold (Fig. 4B).

10-” M 8-Br-CAMP nor 1 x lo-’ M 8-Br-cGMP stimulated gonadotropin release, while 1 x lo-” M LHRH significantly stimulated the release rate of both LH (Fig. 1C) and FSH (Fig. 2C) by 1.5-fold. Superfusion of hamster APG with I x lo- ’ M 8-Br-CAMP resulted in a significant 5-fold increase in the release rate of LH (Fig. 3C), while 1 X IOW3 M 8-Br-cGMP significantly decreased the release rate of LH by 50% (Fig. 3C). 1 x lo-’ M 8-Br-CAMP significantly increased the release rate of FSH 3-fold over basal release (Fig. 4C), while 1 X lo-’ M 8-Br-cGMP significantly decreased the release rate of FSH by approximately 30% (Fig. 4C). 1 x lo-’ M LHRH significantly increased the release rate of both LH and FSH from hamster APG by 4-fold and 2-fold, respectively. Discussion These results suggest that the mediation of the action of LHRH on the pituitary gland of the rat and the hamster are substantially different. In the experiments using rat anterior pituitary glands, MIX did not stimulate gonadotropin release, a finding which is in agreement with results previously reported by Noar et al. (1978). Furthermore, superfusion of rat anterior pituitary glands with 1 x 10m3 M dibutyryl CAMP did not increase the gonadotropin release rate (unpublished results). The inability of these analogues of CAMP to stimulate gonadotropin release from superfused rat anterior pituitary glands is similar to previous reports utilizing organ or cell culture systems (Sundberg et al., 1976; Tang and Spies, 1976; Conn et al., 1979; Stern and Conn, 1981). Also,

The total increments of LH and FSH released from rat and hamster APG during superfusion with 1 x 10-‘” M LHRH or with 1 X lo-‘* M LHRH and 5 x 1O--sM MIX are summarized in Table 1. The Student t-test for the difference between the means of these two treatments demonstrated that MIX significantly potentiated the effect of LHRH upon hamster APG, but no significant potentiation could be demonstrated by MIX upon rat APG.

The effect of 8-Br-CAMP and FSH

and S-Br-cGMP

upon LH

release

In experiments

utilizing

rat APG,

neither

1X

TABLE

1

TOTAL FUSED

LH AND FSH INCREMENTS FROM RAT AND WITH LHRH ALONE OR WITH LHRH + MIX

HAMSTER

LH fng/mg 1 x lo-”

M LHRH

1 x lO_” M LHRH plus 5x10.-’ M MIX a Standard deviation. * P 5 0.05.

230 * 21

PITUITARY

(pit)

GLANDS

SUPER-

Hamster

Rat

198?15

ANTERIOR

a

piit

FSH (ng/mg 140111

152*15

pit)

LH (pg/mg 5.5 f0.33

23.5 + 3.2 *

pit)

FSH (ng/mg 1oi:

914+93

1

*

pit)

181

the inability of 8-Br-cGMP to stimulate gonadotropin release agrees with the results obtained by Noar and Catt (1980) and supports the hypothesis that in the rat LHRH does not exert its gonadotropin-releasing effect through the mediation of cyclic nucleotides. It is interesting to note that early reports suggested that CAMP mediated the effect of LHRH upon the rat anterior pituitary gland (Borgeat et al., 1972, 1974; Labrie et al., 1973; Makino, 1973). However, more recent reports consistently deny the role of CAMP in the action of LHRH (Noar et al., 1975; Conn et al., 1979). This discrepancy between the early studies and the more recent studies is not clear. However, the age and sex of the rats and the specific experimental systems utilized may account for these differences. Calcium is currently receiving support as a primary factor mediating the effect of LHRH upon the rat anterior pituitary gland (Bourne and Baldwin, 1980; Conn et al., 1980). In the experiments utilizing superfused hamster anterior pituitary glands, MIX induced gonadotropin release and potentiated the effect of LHRH, and 8-Br-CAMP mimicked the effect of LHRH. These results support the hypothesis that cyclic nucleotides may be the intracellular mediator of the action of LHRH upon hamster anterior pituitary glands. The inhibitory effect of 1 mM 8-BrcGMP upon the release of LH and FSH is similar to that which was recently reported for melatonin (Wun et al., 1986a, b). The mechanism whereby these substances manifest an inhibitory effect upon LH and FSH secretion is currently unknown. The interpretation of the effect of MIX upon gonadotropin secretion from the anterior pituitary gland must be made with extreme caution. It has been recently reported that MIX can compete with adenosine for adenosine receptors with an inhibition constant of l-2 PM (Martinson et al., 1987). Adenosine receptors may be divided into two subtypes, A, and A,, which respectively inhibit or stimulate adenyl cyclase (Martinson et al., 1987). Although the distribution of A, and A, adenosine receptors in gonadotrophs is not clear, MIX appears to be nearly equipotent as a phosphodiesterase inhibitor or antagonist of the adenosine receptors (Cooper and Londos, 1979). In the liver, MIX increased cytosolic Ca2+ to 250 PM or higher (Gabbay and Lardy, 1986). It is

possible that the transient increase in LH and FSH release is due to an increase in cytosolic Ca2+ by the high concentration of MIX. It is also possible that MIX inhibits both phosphodiesterase for CAMP and phosphodiesterase for cGMP (Wells and Kramer, 1981). It is an inherent characteristic of MIX that its effects are highly nonspecific, and its effects should be considered in conjunction with other parameters such as the potentiation of the effect of LHRH and the mimic effect of cyclic nucleotides upon LHRH. The dissimilarity of the mediation messenger(s) of LHRH in the rat and hamster cannot be attributed solely to the difference between seasonally and nonseasonally breeding species. Although the domestic pig is a nonseasonal breeder as is the rat, results from porcine pituitary cell culture experiments demonstrated that phosphodiesterase inhibitors potentiated both the basal and the LHRH-stimulated release of LH (Walker and Hopkins, 1978). LHRH induced the release of LH with an accumulation of CAMP which was dosedependent and parallel to the increase in LH. Exogenous CAMP derivatives and nonspecific adenyl cyclase activators (prostaglandin and cholera toxin) also stimulated the release of LH. Experiments utilizing pituitary glands from subhuman primates demonstrated that neither a phosphodiesterase inhibitor nor exogenous CAMP derivatives stimulated LH release (Tang and Spies, 1974). In the chicken, Bonney and Cunningham (1977) reported that a phosphodiesterase inhibitor did not stimulate the basal release of LH, but did potentiate action of LHRH, while an exogenous CAMP derivative could not consistently stimulate LH release from anterior pituitary cells in culture. In ovine anterior pituitary cells, LHRH stimulated the release of CAMP, MIX potentiated the effect of LHRH, and an exogenous CAMP derivative stimulated LH release (Adams et al., 1979). These results suggest that the mediation of the action of LHRH upon the anterior pituitary gland is a phenomenon which is species-specific. References Adams, T.W., Wagner, T.O.F.. Sawyer, H.R. and Nett. T.M. (1979) Biol. Reprod. 21, 735-747. Bast, J.D. and Greenwald, G.S. (1974) 1295-1299.

Endocrinology

94,

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Berault, A., Theoleyre, M. and Jutisz, M. (1980) in Pituitary Plasma Membranes, Cyclic AMP and LH Release. I. Synthesis and Release of Adenohypophyseal Hormones (M. Jutisz and K.W. McKems, eds.), pp. 44-462, Plenum Press, New York. Blake, CA., Norman, R.L. and Sawyer, C.H. (1973) Biol. Reprod. 8, 299-305. Bonney, R.C. and Cunningham, F.J. (1977) Mol. Cell. Endocrinol. 7, 233-244. Borgeat, P., Chavaney, G., DuPont, A., Labrie, F., Arimura, A. and Schally, A.F. (1972) Proc. Nat]. Acad. Sci. U.S.A. 69, 2677. Borgeat, P., Labrie, F., Cote, J.. Rue], F., Schally, A.V., Coy, D.H., Coy, E.J. and Yanailhara, N. (1974) Mol. Cell. Endocrinol. 1, 7-20. Bourne. G. and Baldwin, D. (1980) ~ndoc~nology 107, 780-788. Clayton, R.N., Shakespear, R.A. and Marshall, J.C. (1970) Mol. Cell Endocrinol. 11, 63-78. Conn, P.M., Morrell, D.V., Dufau, M.L. and Catt, K.J. (1979) Endocrinology 104,448-453. Conn, P.M., Marian, J., McMillian, M. and Rogers, D. (1980) Cell Calcium 1, 7-20. Cooper, D.M.F. and Londos, C. (1979) J. Cyclic Nucleotide Res. 5, 289-302. Daniel. W.W. (1978) Biostatistics: A Foundation for Analysis in Health Sciences, 2nd edn., pp. 376-383, John Wiley& Sons, New York. Decry, D.J. and Howell, S.L. (1973) Biochim. Biophys. Acta 329, 17-22. Gabbay, R.A. and Lardy, H.A. (1986) J. Biol. Chem. 261, 4002-4007. Grotjan, Jr., E.H. and Steinberger, E. (1977) Comput. Biol. Med. 7, 159-163. Kawakami, M. and Kimura, F. (1980) Endocrinology 106, 626-630.

Labrie, F., Pelletier, G., Lemay, A., Borgeat, P., Barden, N., DuPont, A., Avary, M., Cote, J. and Boucher, R. (1973) Acta Endocrinol. Suppl. 180, 301-340. Makino, T. (1973) Am. J. Obstet. Gynecol. 115, 606-614. Martinson, E.A., Johnson, R.A. and Wells, J.N. (1987) Mol. Pharmacol. 31, 247-252. Noar, Z. and Catt. K.J. (1980) J. Biol. Chem. 255, 342-344. Near, Z., Koch, Y., Chobsieng, P. and Zor, U. (1975) FEBS Lett. 58, 318-321. Noar, Z., Fawcett, C.P. and McCann, SM. (1978a) Am. J. Physiol. 235, E586-E590. Noar, Z., Snyder, G., Fawcett, C.P. and McCann, SM. (1987b) J. Cyclic Nucleotide Res. 4, 475-486. Ratner, A. (1970) Life Sci. 9, 1221-1226. Ratner, A., Wilson, M.C., Srivastava, L. and Peke, G.T. (1976) Neur~nd~~nology 20, 35-42. Spona, J. (1975) Endocrinol. Exp. 9, 27-33. Stern, J.E. and COM, P.M. (1981) Am. J. Physiol. 240, E504-E509. Sundberg, D.K., Fawcett, C.P. and McCann, S.M. (1976) Proc. Sot. Exp. Biol. Med. 151, 149-154. Tang, L.K.L. and Spies, H.G. (1974) Endocrinology 94, 1016-1021. Tang, L.K.L. and Spies, H.G. (1976) Proc. Sot. Exp. Biol. Med. 151, 189-192. Walker. A.M. and Hopkins, CR. (1978) Mol. Cell. Endocrinol. 12, 177-183. Well, J.N. and Kramer, G.L. (1981) Mol. Cell. Endocrinol. 23, l-9. Wun, W.S.A., Berkowitz, A.S. and Preslock, J.P. (1986a) Mol. Cell. Endocrinol. 46, 215-225. Wun, W.S.A., Jackson, F.L., Preslock, J.P. and Berkowitz, A.S. (1986b) Mol. Cell. Endocrinoi. 46, 227-234. Zar, J.H. (1974) Biostatistical Analysis, pp. 551-559, PrenticeHall, New Jersey.