Department of Endocrinology. Medical College of Georgia. Augusta, Georgia ABSTRACT

Transcription

1 BOLOGY OF REPRODUCTON 3, (1984) Peptidase Activity in the Hypothalamus and Pituitary of the Rat: Fluctuations and Possible Regulatory Role of Luteinizing Hormone Releasing Hormone-Degrading Activity During the Estrous Cycle JAMES L. O CONNER, CAROL A. LAPP and VRENDRA B. MAHESH Department of Endocrinology Medical College of Georgia August Georgia 3912 ABSTRACT Peptidase activity capable of inactivating uteinizing hormone (LHRH) may have a physiological role in partially determining hypothalamic LHRH levels as well as LHRH levels at the gonadotrope. n our previous work (Lapp and O Conner, 1984, companion paper), use of the synthetic substrate leucine-p-nitroanilide (Leu-p-NA) to assay LHRH-degradative activity was validated by several methods. The current studies were conducted in order to monitor peptidase activity in the hypothalamus and pituitary throughout the rat 4-day estrous cycle. Activity in both tissues was significantly decreased during proestrus and diestrus. t seems possible that the proestrous reduction in peptidase activity represents a permissive period necessary for the induction of the LHRH and L- surges. The decreased degradative activity in the pituitary on diestrus may be involved in inducing the pituitary LHRH receptors which are reportedly synthesized prior to proestrus. The peptidase exhibits positive cooperativity with Leu-p-NA, and the degree of this cooperativity also fluctuates during the estrous cycle. Estradiol and progesterone given alone or mn combination to prepubertal castrate animals increased the activity of the hypothalamic peptidase in vitro. The degree of positive cooperativity with which the enzyme functioned was also apparently altered by these gonadal steroids. NTRODUCTON The regulation of gonadotropin secretion during the estrous cycle requires a delicate balance between complex hormonal interactions. One component known to be an important influence is luteinizing hormone releasing hormone (LHRH). Changes in the rates of LHRH synthesis and release, as we!! as the rate of degradation of this hormone, have been proposed as additional factors which modify its role in influencing gonadotropin release (Kuhl et a!., 1974, 1977; Advis et al., 1982, 1983). A prvious manuscript (Lapp and O Conner, 1984, companion paper) validated the use of the synthetic substrate leucine-pnitroanilide (Leu-p-NA) as a tool for the study of enzymatic activity capable of degrading LHRH. The present manuscript describes the variations in this enzymatic activity in the hypothalamus and pituitary throughout the 4-day estrous cycle of the adult rat. n addition to enzyme activity, the LHRH content of the Accepted January 19, Received September 2, Reprint requests. mediobasal hypothalamus and serum were estimated. Since others (Griffiths and Hooper, 1972; Kuhl et a!., 1974, 1979) have reported stimulatory effects of sex steroids on LHRHdegrading enzyme activity, the coexisting serum levels of estrogen and progesterone were also determined in the present study. njections of luteinizing hormone (LH) have been reported to stimulate LHRH inactivation (Kuhl and Taubert, 1975); thus serum gonadotropin levels were simultaneously estimated. Even though LHRH, steroid and gonadotropin levels have been reported in other studies, these were determined by radioimmunoassay (RA) at identical time points in the present studies in order to facilitate interpretation of results. n the second part of this work, a well-defined animal mode! (McPherson and Mahesh, 1979) was used to investigate whether exogenously administered steroids were capable of modulating the velocity of the enzymatic activity seen with the synthetic substrate and whether the hypothalamus and pituitary were affected similarly. Using the immature, estrogen-primed ovariectomized rat model, Smanik et a!. (1983) have shown significant progesterone-mediated differences between the hypothalamus and pituitary in receptor-mediated responses to 855 Downloaded from on 24 November 217

2 856 O CONNER ET AL. estradiol. n their experiments, tissue-specific differences were apparent within 2 h of progesterone administration. Therefore, to allow suffkient time for a receptor-mediated response to occur in the present studies, peptidase activity was examined 3 h after progesterone was given. MATERALS AND METHODS Estrous Cycle Fluctuations of Peptidase Activity Preparation of animals and collection of samples. Female Sprague-Dawley rats were monitored through 3 successive 4-day estrous cycles before sacrifice at 68-7 days of age. Animals were decapitated every 3 h starting at 9 h on proestrus, and at 9, 18, and 24 hon estrus, diestrus and!. The mediobasal hypothalamus (MBH), anterior pituitary and trunk blood samples were rapidly collected. Every effort was made to subject the animals to minimal stress prior to sacrifice. Each point required 6 animals. Assay system for leucine-p-nitroanilide degradation. Details of the assay system were as previously described (Lapp and O Conner, 1984, companion paper). MBH and pituitaries were individually homogenized in 1.5 ml.5 M Tris-HC buffer (with.25 M sucrose,.3 mm CaCl3,.1 mm dithiothreitol, ph 7.3) on ice; homogenates were centrifuged at 8 X g for 1 h. Supernatants were frozen at -4#{176}C until assay. Hypothalamic supernatants were diluted 1:5 with Tris-HC buffer before assay; pituitary supernatants were not diluted further. Preliminary experiments had determined the substrate preference and optimum substrate concentration range for the enzyme activity found in the hypothalamus and pituitary. The in-assay substrate concentration range for each enzyme source was 37, 56, 84,12,18 and 28MM for hypothalamic preparations and 12, 18, 28, 42, 6 and 96MM for pituitary preparations. Hypothalamic incubations were for 45 mm and pituitary for 9 mm (all at 3 7#{176}C). Tissues from each rat were prepared and assayed individually and the data for each animal were calculated separately. Mean and SEM were then determined for each group and the enzyme velocity was expressed as pg p-nmtroanilmne (p-na) per tmssue per mm. The data were then plotted as velocity versus substrate concentration on double reciprocal plots (Lineweaver and Burk, 1934) and also analyzed by Hill plots (191). RA system for gonadotropins. A double-antibody RA using NAMDD reagents was used to estimate levels of LH and follicle-stimulating hormone (FSH) (O Conner et a!., 198). Purified rat hormones were iodinated with 12S by the chloramine-t method (Bolton, 1977). The first antibodies used for LH and FSH. respectively, were anti-rat LH (rabbit) NAMDD- A-rat LH-S-2 and anti-rat-fsh (rabbit) N!AMDD-A-rat FSH S-7. The interassay and intraassay coefficients of variation were 1% and 15%, respectively, for both hormones over a range of ng/tube (n#{176}18). RA system for LHRH. Hypothalamic tissues destined for LHRH determination by RA were extracted with methanol immediately upon collection in order to minimize the enzymatic inactivation of LHRH. The hypothalami were collected on ice and were quickly homogenized in 1 ml.1 M phosphatebuffered saline (PBS) +.1% gel (ph 7.4) with a glass homogenizer (Dual 21), whereupon the homogenates were extracted in 3 volumes cold methanol. Following centrifugation, the methanol extract was dried under N3 and resuspended in the same buffer prior to freezing at -4#{176}C until LHRH assay. Blood destined for LHRH RA was collected in heparinized tubes on ice and centrifuged at 15 X g for 15 mm. The plasma was then extracted with 3 volumes cold methanol, dried under N2 and resuspended in 2. ml.1 M PBS +.1% gel. The extracted hypothalami and plasma were then subjected to LHRH RA by the method described previously (Lapp and O Conner, 1984). Assay systems for eat radiol and progesterone. Estradiol (E2) RA was performed by the method of Melner and Abney (198). The sensitivity of the R!A was 5 pg and the linear range of the standard curve was 5-16 pg. The interassay and intraassay coefficients of variation were 7.1% and 1.2%, respectively. Progesterone was estimated by the RA of Mills and Osteen (1977). The sensitivity of the assay was 9.5 pg, and the linear range was pg. nterassay and intraassay coefficients of variation were 2.8% and 12.8%, respectively. n Vivo Steroid Effects on Peptidase Activity Preparation of animals and tissues. Rats were ovariectomized on Day 26, and s.c. injections of.1 g E2/kg BW in corn oil, or vehicle alone, were begun at 163 h the same day. After 4 days of E3 priming, two groups of rats (6 per group) received.8 mg/kg BW or 3.2 mg/kg BW doses of progesterone (P) in corn oil at 93 h. Another group of unprimed castrate animals received only.8 mg P/kg BW at the same time. All animals were sacrificed starting at 123 h. Hypothalami and pituitaries were removed to 1 ml iced.5 M Tris-HC buffer, then frozen until the day of enzyme assay. Tissues were individually homogenized (Duall 21) in 1 ml Tris-HC buffer and centrifuged at 8 X g for 1 h. Supernatants were diluted to 5 ml with the same buffer. n order that these assays might be terminated by heat inactivation rather than by trichloracetic acid (TCA) precipitation, the assay volumes were increased by 1.5-fold. ncubation times also were decreased to 1 5 mm for the hypothalamus and 3 mm for pituitary. The reaction was stopped by heating tubes to 1#{176}Cfor 3 mm. Velocity (g p-na! mm per mg protein) was estimated for each rat by absorbance at 45 nm; protein content of the supernatants was estimated by absorbance at 28 nm. Statistical Evaluations Sequential points were analyzed for significant differences according to Duncan s new multi-range test (Steel and Torrie, 198). The minimal acceptable significant difference was at P=.5. For the LHRH R!A, tests of significance were made with the unpaired t test. RESULTS Estrous Cycle Pep tidase Activity Figures 1A and B illustrate the variations in enzymatic activity observed in crude hypothal- Downloaded from on 24 November 217

3 PEPTDASE ACTVTY DURNG THE ESTROUS CYCLE 857 a4 ae z.2 &.1 A *, a o on o ),- C ),-C,-.--C J)N ) - Hours Diestrus Diestrus Proestrus Estrus FG. 1. Activity of hypothalamic and pituitary supernatant toward the synthetic substrate Leu-p-NA during the 4-day estrous cycle. A) Hypothalamic supernatant incubated for 45 mm with 28 MM Leu-p-NA. B) Pituitary supernatant incubated for 9 mm with 96 pm Leu-p-NA. The points represent mean (± SEM) of 4 rats assayed in dplicate. Horizontal bars indicate hours of darkness. Value significantly different (P<.5) from preceding value; b and c indicate pairs of values which differ significantly from each other. amic and pituitary supernatants during the 4-day estrous cycle. Although each tissue was assayed at six different substrate concentrations, only the activity data with maximum substrate (28 pm for hypothalamus and 96 pm for pituitary) have been illustrated;points represent mean ± SEM for 4 animals. Peptidase activity showed a significant depression between 9 and 15 h on proestrus in both the hypothalamus (Fig. 1A) and pituitary (Fig. 1B). Simultaneously, hypothalamic and circulating LHRH (Fig. 2A) and serum LH (Fig. 2B) rose to their highest levels during the cycle. As the proestrous decrease in peptidase activity began, E2 levels were high; at the end of proestrus when peptidase activity was restored, E2 levels were generally decreasing and P levels were rising (Fig. 2C). On diestrus, a significant decrease in enzymatic activity was again noted in both tissues, although at slightly different time points. When pituitary activity was falling (9-18 h), P and plasma LHRH were on the increase and MBH LHRH was very low. Hypothalamic activity fell between 18 and 24 on diestrus. Cooperativity in the Estrous Cycle For each tissue and each time point, data from the full range of substrate concentrations was graphically examined with double reciprocal and Hill plots. These plots indicated the presence of positive cooperativiry throughout the estrous cycle in both the hypothalamic and pituitary preparations. llustrative of this cooperativity is Fig. 3, which presents the hypothalamic enzyme activity from one group (9 h proestrus) plotted according to the double reciprocal (left) and Hill plot (right). Positive cooperativity was indicated by the upwardly concave double reciprocal plot and a Hill plot slope which was greater than 1.. Similar indications of positive cooperativity were seen upon analysis of the pituitary data. A plot of the Hill coefficient (NH) values in the 2 tissues throughout the cycle is presented in Fig. 4. The hypothalamic enzyme had NH values from 1.8 ±.21 to 3.4 ±.8 (mean + SEM) with peak values at 9 and 18 h on proestrus, and 24 h on diestrus. The pituitary. a.. -j E C -J g 2 1 A o oo ) fl ) OX flo.o ) ( fl.-, < Hours Diestrus Diestrus Proestrus Estrus #{176} -J #{149} #{149}15 12 #{149}9 6 #{149}3 FG. 2. Estrous cycle fluctuations in: A) LHRH levels hypothalamus (solid line); plasma (dotted line)] ; B) serum gonadotropmns!lh (solid line); FSH (dotted line)! ; and C) serum gonadal steroids lp (solid line); E2 (dotted line)], n A and B, 4 animals were used per point while in C, 2 animals were used per point. E C U, U. E. N Li Downloaded from on 24 November 217

4 858 O CONNER ET AL. enzyme exhibited NH values with a range of 1.11 ±.18 to 2.35 ±.49, with a broad peak from 12 to 18 h on proestrus and another elevation during estrus. t can be seen that the hypothalamic and pituitary Nu declined significantly (P<.1) between 18 and 21 h on proestrus, and that both tissues showed some increase in cooperativity from 18 to 24 h on diestrus. n Vivo Steroid Effects aa. C Results of the studies conducted to determine if E2 or P administration in vivo altered the velocity or cooperativity of peptidase activity in the hypothalamus or pituitary are shown in Fig. 5 and Table 1. Estrogen alone or P alone, or the two steroids in combination, induced a significant (P<.5) increase in MBH peptidase velocity in comparison to control or castrate unprimed animals (Fig. 5). Enzyme activity in the pituitary was not significantly changed by any treatment. Table 1 shows that in the hypothalamus, E2 priming plus the.8 mg/kg P dose caused a significant increase in cooperativity in comparison to control or castrate + vehicle group; the 3.2 mg/kg dose increased cooperativity significantly above all groups except the E2 plus.8 mg/kg P-treated group. n the pituitary, P in either E2-primed or unprimed animals caused a significant decrease in cooperativity in comparison to control animals. t will be noted that the standard errors were generally larger in the pituitary l(s)mm > K 5 E > > -J 2. NH P Log (S) FG. 3. ndications of positive cooperativity from the kinetic data from hypothalamic supernatant at 9 h proestrus. Peptidase activity was assayed at 6 substrate concentrations as detailed in Materials and Methods; values plotted represent mean (± SEM) of 4 animals assayed in duplicate. n the left panel, data are plotted by double reciprocal method, while in the right panel the same data are plotted according to Hill method. U C) a * Hypo(----) Pt (-) e - N.- N O,.-.-e4N - N Hours Diestrus Diestrus Proestrus Estrus FG. 4. Changes in the Hill coefficient in pituitary (solid line) and hypothalamus (dotted line) during the estrous cycle. Points represent the mean (± SEM) of 4 animals assayed in uplicate. Horizontal bars indicate hours of darkness. Value significantly different from preceding value; b and c indicate pairs of values significantly different from each other. enzyme study; this greater variability was also seen in the unusual biphasic shapes of the direct plots of some of these groups. DSCUSSON Pep tidase Activity During Proestrus Prior to the proestrous LHRH and LH surges, which in this experiment occurred at 15 h, peptidase activity in the MBH underwent marked fluctuations (Fig. 1A). The significant decrease in hypothalamic activity from 9 to 12 h occurred as serum E2 levels were at peak values. From 12 to 15 h, as plasma LHRH and LH were increasing, hypothalamic enzyme activity remained depressed. n conjunction with decreasing E2 and increasing P in serum between 15 and 18 h, hypotha!- amic activity began to increase. Everett and Sawyer (195) demonstrated that hypothalamic LHRH was no longer required for initiation of the LH surge after 16 h on proestrus. Thus the decreased enzyme activity from 9 to 15 h could serve a permissive function, allowing the LHRH levels to build sufficiently to trigger the proestrous gonadotropin release. The transient decrease in LHRH degradation observed by Advis et a!. (1982) prior to the first LH surge in pubertal rats probably represents the same phenomenon. n a similar manner, pituitary enzymatic activity was significantly decreased (P<.5) between 9-15 h on proestrus, while plasma LHRH levels were increasing. Unlike the Downloaded from on 24 November 217

5 PEPTDASE ACTVTY DURNG THE ESTROUS CYCLE Hypothalamus second-highest level in the cycle at 18 h. The significance of this sharp decrease in hypothalamic activity is unknown. However, the enzymatic events in the pituitary on diestrus might be explained in light of the phenomenon of self-priming. Clayton et al. (1982) and Pieper et al. (1982) have demonstrated that the induction of pituitary receptors for LHRH is dependent upon hypothalamic secretion of LHRH. Clayton et a!. (198) showed that the LHRH receptor content of pituitaries at 18 h diestrus was near its nadir in the estrous cycle, and that a significant increase in receptors occurred between 18 h diestrus and 18 h diestrus!. t is suggested that the increase in plasma LHRH at 18 h, coupled with the sharp decrease in LHRH degradation by the pituitary enzyme at this time, again represents a permissive effect which may lead eventually to the induction of LHRH receptors in the pituitary. ndeed, Clayton (1982) reported pituitary receptor induction in animals with very low serum LHRH (<4 pg/m) which was close to the value of 6 pg LHRH/ml serum seen at 18 h on diestrus in this experiment. Thus the observed reduction in LHRH degradation occurs just when the pituitary may be exposed to a potentially sensitizing dose of LHRH, thereby perhaps facilitating LHRH receptor formation in preparation for proestrus. Steroidal Alterations in Peptidase Activity C 9 9+E29+P9+E29+E. +.8P +3.2P FG. 5. Effects of exogenous steroids on the velocity of peptidase activity in hypothalamic and pituitary supernatants in control and castrate females. Bar represents mean (± SEM) of 6 animals. 5Significant increase (P<.5) above control or castrate + vehicle animals. hypothalamic enzyme, the peak of pituitary activity was not reached until 9 h estrus. Peptidase Activity During Diestrus Although hypothalamic-pituitary interaction during proestrus has been extensively studied, this interrelationship during diestrus is less understood. Figure 1 shows that the diestrous enzymatic activity from MBH underwent a significant decrease (P<.5) between 18 and 24 h. Serum LHRH showed a smaller peak at 18 h, and serum P also reached its The prepubertal animal mode! experiment seemed to confirm that steroids may play some part in modulation of this peptidase activity. A significant increase in degradation by the hypothalamic enzyme occurred under the influence of either or both of the gonada! steroids which were given at subphysiologica! levels (Fig. 5). This confirms the work of Griffiths and Hooper (1972), who reported that an LHRH-degrading hypothalamic activity was stimulated to the same extent by E2 and P, alone or in several combinations. That hypotha!- amic and pituitary enzyme activities were not always similarly affected by serum steroid levels was observed by Krause et a!. (1981); progestinmetabolizing enzyme activities from the pituitary fluctuated throughout the estrous cycle while such activities from the hypothalamus did not. Recently, Smanik et a!. (1983) found that P significantly reduced nuclear estrogen receptor binding in the anterior pituitary, but not in the hypothalamus of the immature, castrate, E2-primed rat model. Such a phenomenon Downloaded from on 24 November 217

6 86 O CONNER ET AL. TABLE 1. Effects of exogenous steroids on the Hill coefficient (NH) calculated from Leu-p-NA degradation (mean ± SEM, N=6). Hill coeffic ient (NH) Treatment Hypothalamus Pituitary Control 2.27 ± ±.9 Castrate + vehicle 2.27 ± ±.9 Castrate + estradiol 2.35 ± ±.8 Castrate +.8 mg/kg BW 2.32 ± ± 2c progesterone Castrate + estradiol ± o.ooa 1.3 ±.8 mg/kg BW progesterone Castrate + estradiol ± 1.6 ± 3.2 mg/kg BW progesterone 5Significantly (P<.5) than control or castrate + vehicle. bsignificantly larger (P<.5) than castrate + estradiol or castrate +.8 mg progesterone. csignificantly smaller (P<.5) than control. might provide a mechanistic explanation for cyclic fluctuations in protein. i.e., enzyme synthesis. Advis et a!. (1983) have shown that 3 h after a large dose of P was administered to E2 -primed, ovariectomized adult rats, total LHRH-degrading activity in the median eminence (ME) was significantly reduced, while LHRH in ME was increased. However, McPherson and Mahesh (1979) demonstrated that P may have stimulatory or inhibitory effects on LH and FSH release in prepuberta! animals, depending upon the steroid concentrations used. The doses of steroids used in these experiments were many times lower than those used by Advis et a!. (1983); thus the stimulation of the hypothalamic enzyme by E2 and/or P here does not necessarily conflict with the P inhibition seen by Advis group. The hypothalamic-pituitary axis in these prepubertal animals is extremely sensitive to low doses of steroids (McPherson and Mahesh, 1979). Hence, these animal models should probably not be compared, except to note that, in each case, the gonada! steroids did modulate enzyme activity. Manipulation of endogenous steroid levels also caused significant changes in the Hill coefficients observed in each tissue (Table 1). The hypothalamic enzyme operated with increased cooperativity when P was administered to E2-primed animals. n contrast, the NH of the pituitary enzyme was significantly reduced by P in the immature rats, from a value of 1.3 in controls to a value near 1.. This was close to the pituitary NH seen during diestrus with the cycling rats, at which time P was slightly elevated and E2 was low. This diestrous steroidal combination produced an increased NH in the hypothalamic enzyme. A progesterone effect on cultured pituitary cells was reported by Drouin and Labrie (1981), who observed that P alone could increase both the basal release and the LHRH-stimulated release of FSH. LH response was not affected by P alone in these cultures. The results from immature animals here seemed to demonstrate a P effect on cooperativity, although this acute effect was not manifested in a change in peptidase velocity. Cooperativity changes due to P were only seen in the E2 -primed hypothalamic tissue. The relationship between fluctuations in positive cooperativity and the velocity of LHRH degradation is not yet clear. The variability in NH may indicate that the enzyme is masked or unmasked, rather than synthesized, in response to steroidal influences. n a system where the substrate (i.e., LHRH) is released in a pulsatile manner, there would seem to be an advantage to degradation via an enzyme exhibiting positive cooperativity. Normally, the intracellular substrate level would be near the Km for a given enzyme (Sega!, 1976); but where the substrate is presented as a pulse, an enzyme which exhibited positive cooperativity would allow a system to respond quickly to a substrate challenge without the necessity of synthesizing new enzyme molecules. Such a system would be more conservative of cellular energy than one relying on inducible en- Downloaded from on 24 November 217

7 PEPTDASE ACTVTY DURNG THE ESTROUS CYCLE 861 zymes. t is probable, however, that LHRH is not the only substrate for this enzyme. n summary, a peptidase activity thought to degrade LHRH fluctuates during the estrous cycle in the rat hypothalamus and pituitary. The decreased activity during the hours surrounding the LHRH and LH surges on proestrus suggests that this peptidase is inhibited by some factor during this significant period. t is suggested that a similar decrease in pituitary degrading activity during diestrus may be important in the self-priming process by which LHRH induces the formation of its own receptors in the pituitary. The gonada! steroids do seem to be capable of modulating the activity and the degree of positive cooperativity displayed by this enzymatic activity. ACKNOWLEDGMENTS LH and FSH RA kits were provided by the NAMDD. This work represents partial fulfillment by C.L. of the requirements for the degree of Doctor of Philosophy at the Medical College of Georgi Dept. of Endocrinology. This research was supported in part by Grants #HD to JO. and #HD to V. M. REFERENCES Advis, J. P., Krause, J. F. and McKelvy, J. F. (1982). Luteinizing hormone-releasing hormone peptidase activities in discrete hypothalamic regions and anterior pituitary of the rat: apparent regulation during the pre-pubertal period and first estrus cycle of puberty. Endocrinology 11; Advis, J. P., Krause, J. E. and McKelvy, J. F. (1983). Evidence that endopeptidase-catalyzed luteinizing hormone-releasing hormone cleavage contributes to the regulation of median eminence LHRH levels during positive steroid feedback. Endocrinology 112: Bolton, A. (1977). Experimental protocols for the radioiodination of proteins and other compounds. n: Radioiodination Techniques Review 18 (A. Bolton, ed). Amersham-Searle, Arlmngton Heights, L, p. 45. 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8 862 O CONNER ET AL. pituitary cells to luteinizing hormone-releasing hormone: retention in pituitary cell culture. Endocrinology 16: Pieper, D. R., Gal R. R., Regiani, S. K. and Marshall, J. C. (1982). Dependence of pituitary gonadotropin-releasing hormone (GnRH) receptors on GnRH secretion from the hypothalamus. Endocrinology 11: Segal, J. (1976). Biochemical Calculations, 2nd ed. John Wiley & Sons, New York, pp Smanik, E. J., Young, H. K., Muldoon, T. G. and Mahesh, V. B. (1983). Analysis of the effect of progesterone in vivo on estrogen receptor distribution of the rat anterior pituitary and hypothalamus. Endocrinology 113:1-8. Steel, R. G. and Torrie, J. H. (198). Principles and Procedures of Statistics, 2nd ed. McGraw-Hill, New York, pp Downloaded from on 24 November 217