SERUM GONADOTROPIN CONCENTRATIONS AND OVARIAN RESPONSE IN EWES TREATED WITH ANALOGS TO LH-RH/FSH-RH l,2,3

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1 SERUM GONADOTROPIN CONCENTRATIONS AND OVARIAN RESPONSE IN EWES TREATED WITH ANALOGS TO LH-RH/FSH-RH l,2,3 James E. Kinder a,s, Thomas E. Adams 4,6, Torrence M. Nett 7, David H. Coy 8, Andrew V. Schally 8 and Jerry J. Reeves 4 Wasbington State University 4, Pullman WA 99163, Colorado State University 7, Fort Collins, CO 80521, VA Hospital and Tulane University School of Medicine 8, New Orleans, LA SUMMARY One-hundred-twelve anestrous Columbia ewes were used in three experiments to evaluate biological potency of three analogs to LH-RH/FSH-RH. Either one or a combination of the following characteristics were utilized in the different experiments to evaluate biological potency of these analogs: Serum LH, serum FSH, ovulation rate or conception. In experiment 1, 32 ewes were assigned randomly to a 2 4 factorial experiment. Each ewe received either.5, 5, 50 or 500/ag of des Gly ~ ~ ethylamide or LH-RH/FSH-RH. Peak serum LH concentration was significantly greater in ewes treated with.5 gtg analog than in the ewes treated with.5 gtg LH-RH/FSH-RH. The peak serum LH concentrations did not differ significantly between the analog or LH-RH/FSH-RH treated ewes in any of the other treatment groups. Peak concentrations of FSH did not differ among the groups of ewes treated with ~Scientific Paper 4412, College of Agriculture Research Center, Washington State University. Project The authors would like to thank Dr. L. Reichert for the ovine LH and FSH used for iodination and also the Endocrine Study Section NIH for LH standards and Dr. G. D. Niswender for the facilities to conduct the FSH radioimmunoassay. The technical assistance of G. K. Tarnavsky, P. K. Chakraborty, J. J. Noonan and B. Adams are greatly appreciated. 3This study was supported in part by USPHS Grant AM-6192 to J JR. 4 Department of Animal Sciences. s Present address: Department of Animal Sciences, Ohio State University, Columbus, Ohio Present address: Department of Physiology and Biophysics, Colorado State University, Fort Collins Department of Physiology and Biophysics. Endocrine and Polypeptide Labs. LH-RH/FSH-RH or analog at any of the respective doses. However, there was a greater number (P<.05) of ovulations in the ewes treated with des glyl~ than in ewes treated with LH-RH/FSH-RH. Experiment 2 consisted of five ewes treated with 500 gig des-gly l~ and five ewes treated with saline. All ewes were artificially inseminated 12 hr post-injection. There was a greater number of ovulations (P<.05) in the ewes treated with the analog than in the ewes treated with saline. However, none of these ewes conceived. In experiment 3, 70 ewes were used to compare the effects of the analogs D-Ala 6 des Gly I ~ ethylamide and D- Leu 6 des Gly 1 ~ with those of LH-RH/FSH-RH. Comparisons indicated an increase (P<.05) in total LH released, mean peak LH and duration of the LH release in ewes treated with either.5, 5, 50 /ag D-Ala 6 and D-Leu 6 analog as compared to ewes receiving corresponding doses of LH-RH/FSH-RH. However, total LH released was not significantly different when the ewes treated with 500/~g of either analog were compared with those treated with LH-RH/FSH-RH. The amount of D-AIa 6, D-Leu 6 or LH-RH/FSH-RH which released 50% of the total LH released by the 500 /lg dose (effective dose 50) was calculted as 2.4, 5.4 and 100 /~g, respectively. The analogs were nine, four, and three times more potent in total LH releasing ability when compared to LH-RH/FSH-RH at the.5, 5 and 50 gg doses, respectively. These results indicated that the maximal pituitary response was obtained with 50/~g of the D-AIa 6 or D-Leu 6 analog but that 500//g of LH-RH/FSH-RH was required before maximal LH release was attained. (Key Words: Ewe, LH-RH/FSH-RH, LH, FSH, Ovulation, Analogs.) 1220 JOURNAL OF ANIMAL SCIENCE, Vol. 42, No. 5, 1976

2 LH-RH/FSH-RH ANALOGS 1221 INTRODUCTION Fujino et al. (1972) reported an analog of LH-RH/FSH-RH, (Pyro) Glu-His-Trp-Ser-Tyr- Gly- Le u- Arg-Pr o- NH-CH2-CH3 (des- GIyI~ ethylamide) which has greater biological activity than synthetic LH-RH/FSH-RH. Biological activity was evaluated as the ability to induce ovulation in the adult female rat. Other studies utilizing this analog in rats have substantiated the fact that it does have increased gonadotropin-releasing activity (Rippel et al., 1973; Arimura et al., 1974). Coy et al. (1974) and Fujino et al. (1974a) have reported a second alteration to this molecule which gives it a greater gonadotropin-releasing activity than the previously reported analog. The structure of this analog (D-AIa 6, des Glyl0_LH_RH_ethylamide) is (Pyro) Glu-His-Trp-Ser-Tyr-D-Ala- Leu-Arg-Pro- NH-CH2-CH3. Vilchez-Martinez et al. (1974) and Fujino et al. (1974b) have found that a similar analog with D-leucine in the six position has a gonadotropin-releasing activity similar to the D-alanine analog. It is important that analogs of LH-RH/FSH-RH which may increase ovulation by inducing LH and FSH release from the pituitary be evaluated in studies utilizing farm animals. The present study was designed to compare biological activity of these three analogs to the natural hormone in the anestrous ewe. MATERIALS AND METHODS Hormone Assays. Serum concentrations of LH were measured using a validated double antibody radioimmunoassay specific for ovine LH (Adams et al., 1975). Concentrations of LH were expressed either in terms of N1H-LH-S16 or NIH-LH-S17. Serum samples were analyzed for FSH using the double antibody radioimmunoassay described by L-'Hermite et al. (1972) and FSH concentrations were expressed in terms of NIH-FSH-S4. Experimental Design. Experiment 1 consisted of 32 mature Columbia ewes in mid-anestrus being assigned in equal numbers to eight groups in a 2 4 factorial design. The weight of these ewes ranged between 65 to 85 kilograms. Each ewe in the group treated with LH-RH/FSH-RH received a single intramuscular (im) injection of.5, 5, 50 or 500/ag of the hormone in 1 ml of acidified (.01 N acetic acid) saline., Likewise, each ewe treated with des-gly I ~ ethylamide received a single injection of.5, 5, 50 or 500 gig of this analog in 1 ml of acidified saline. Blood samples were collected immediately prior to the injection, at 15-min intervals for 6-hr and at 30-min intervals for the second 6-hr period following the injection. The resulting serum was stored frozen until assayed. Twenty-four hours prior to and after treatment laparotomies were performed according to the technique described by Hulet and Foote (1968). The ovulation data were analyzed by analysis of variance and comparison of means were made using Student's t test. Peak serum LH and FSH concentrations were analyzed by analysis of variance. Experiment 2 was performed utilizing 10 mature anestrous Columbia ewes which were randomly assigned in equal numbers to two groups and treated as described during late anestrus. One group received 1 ml of acidified saline (ira) and the other group received 500 gig of des GIyI~ ethylamide in 1 ml of acidified saline. Laparotomies were performed at the same times as described in the previous study and the ovulation data were analyzed by the Student's t test. No blood samples were collected in this study but all ewes were artificially inseminated 12 hr following treatment. In experiment 3, 70 mature Columbia ewes in mid-anestrus were randomly assigned to 12 groups in a 3 x 4 factorial design. Each ewe in the group treated with LH-RH/FSH-RH received a single intramuscular (im) injection of either.5, 5, 50 or 500 gtg LH-RH/FSH-RH in 1 ml acidified saline. Each ewe in the analog treatment groups received an im injection of either.5, 5, 50 or 500 gtg of the D-Ala6-des Glyl 0_LH_RH ethylamide or D-Leu6-des Glyl~ ethylamide in 1 rnl acidified saline. Laparotomies were performed to determine ovulation rate 48 hr before and 48 hr after the treatment. Blood samples were collected immediately prior to the injection and at 30-min intervals for 12 hr following the injection. Comparisons of the amounts of total LH release in the serum were made by determining the area under the peak of LH released from the time of injection until the time the serum LH returned to the basal concentration. A compensating polar planimeter was used to determine the area under the curve in square centimeters, and an analysis of variance was performed at all four dose levels for comparison of the treatment groups. Mean peak serum LH concentrations and number of ovulations were evaluated statistically by use of analysis of

3 1222 KINDER ET AL. TABLE 1. AVERAGE NUMBER OF OVULATIONS IN EWES TREATED WITH LH-RH/FSH-RH AND DES-GLV ~ ~ ETHVLAMIDE LH-RH/ Des-Gly ~ ~ Dosage #g FSH-RH ethylamide " *P<.05. variance and comparisons of means were made using Duncan's new multiple-range test (Steel and Torrie, 1960). RESULTS AND DISCUSSION In experiment 1 there was a greater number (P<.05) of ovulations in the des-gly ~ ~ ethylamide treated ewes ( %) than the LH--RH/FSH-RH treated ewes ( %). Analysis of variance indicated a dosage effect (P<.O1) and a hormone dosage interaction (P<.01) so that mean ovulation data for all eight treatments were compared. There was also a hormone effect (P<.05). There was a greater number of ovulations (P<.05) in the group treated with 500 #g of analog than in any other treatment group (table 1). All four ewes in this particular group ovulated. Figure 1 shows the pattern of serum LH release for the ewes in all treatment groups. Mean peak serum LH concen- trations were higher (P<.01) in the ewes treated with.5 #g of the analog ( ng/ml) as compared to ewes treated with.5 #g of LH-RH/FSH-RH ( ng/ml). There was a trend toward a higher (P<.10) peak serum LH concentration in the ewes treated with 5 ~tg of des.glyl 0.LH.RH ethylamide ( ng/ml) as compared to the peak serum LH concentration in the ewes treated with 5 #g LH-RH/FSH-RH ( ng/ml). The mean peak serum LH concentrations did not differ between the groups treated with 50 or 500 #g of the analog or LH-RH/FSH-RH. Mean peak serum FSH concentrations did not differ in the ewes treated with LH-RH/FSH-RH or the ethylamide analog at any of the four dosage levels (figure 2). In experiment 2 there was an increase (P<.05) in the number of ovulations in the analog-treated ewes ( ) as compared to no ovulations in the saline-treated ewes. None of the ewes conceived when artificially inseminated 12 hr subsequent to the injections. Several factors could have contributed to the lack of conception. The time of artificial insemination may not have been optimal for fertilization of ova to occur. Crighton et al. (1973) have reported that corpora lutea in anestrous ewes treated with LH-RH/FSH-RH secrete very low levels of progesterone. Therefore, progesterone levels following LH-RH/FSH-RH or analog administration may not be high enough to maintain pregnancy. 250,.5,u 9 dose.% T..-~. I O ~ 150 _ , 50 ' :-": 20 5J~ d~se K)O O~ Des Gy ~~ LH-~-I ethyiom~de g 9 30O 250. ~. 5~q dose ' %~ ~- 7c! ~Ep6 C, ~00 ~ dose 5( ,ug dose 40 m ~ ~'~9~ ~a 500 * ' 0 L ~, _ 500 ~g dose g 400" 400 ~ --~/b c, " ;~. ~ 50 ~g dose ~ ' to, ~0 ",ff 2O(> 200~ w 0 ) B 9 ~ HOURS POST TREATI~NT Figure 1. Serum LH concentrations following injections of LH-RH/FSH-RH o--o and 9 9 Des- Gly, O.LH_RH.ethylamide (Mean 4- SE). TIME (HR) AFTER HORMONE TREATMENT Figure 2. Serum FSH concentrations following injections of LH-RH/FSH-RH and Des-Gly I ~ Ethylamide (Mean + SE).

4 LH-RH/FS H-RH ANALOGS 1223 O-&lo 6 des GIy 1o LH-Rdet~ylomtde.. [~t.d~ 6 ae~ Gly ~o LH-RHothytomldr o o LH-RH/FSH-RH v ag dose 80 o" 5 ~g ao~ 20. u~,(..,,..,, 0f m 200 ~ (> ~n~: 140 9o.,,' 50,ug dose 140 ~k 500,g dose 60. R " %L a*~ ~.o.,o-,*.~ol 60 " ~.*,~ st.o.,~-,~.2~ o 9 ~'~-. o ~ ~'~ TIME (HR) AFTER HORMONE TREATMENT Figure 3. Serum LH concentrations in ewes treated with LH-RH/FSH-RH and the analogs D-Ala s and D-Leu 6. Segerson et al. (1974) theorized that cycling ewes which had been induced to ovulate prematurely with LH-RH/FSH-RH did not have adequate estrogen levels to allow for normal sperm transport. If lowered estrogen levels cause reduced sperm transport because of reduced uterine motility in cycling ewes one could easily theorize that low estrogen levels in anestrous ewes could have the same result. The patterns of LH release for the ewes in experiment 3 are shown in figure 3. The mean peak serum LH concentrations were higher (P<.05) for the ewes treated with the D-AIa 6 and D-Leu 6 analogs as compared to the LH-RH/FSH-RH treated ewes at all dosages except 500 micrograms. As shown in figure 3, the duration of LH release was longer in both groups treated with the D-AIa 6 and D-Leu 6 analogs at all doses as compared to the respective doses of LH-RH/FSH-RH. Concentrations of LH were still higher 12 hr following treatment (P<.05) than initial basal LH concentration in ewes treated with either 50 or 500/ag of either the D-AIa 6 or D-Leu 6 analogs. There was no difference in LH release in ewes as a result of treatment with either 50 or 500/lg of either D-AIa 6 or D-Leu 6 analogs. This would indicate that the maximum amount of releasable LH from the pituitary can be obtained with a dose between 5 and 50 #g of the D-AIa 6 and D-Leu 6 analogs. However, there was an increase (P<.05) in mean peak LH and total LH released when the ewes treated with 50 or 500 #g of LH-RH/FSH-RH were compared. Total amount of LH released takes into account both the peak of LH release and the duration of LH release which should be superior to just evaluating peak concentrations of serum LH (table 2). Analysis of variance indicated that there was a highly significant difference in the total LH released with the.5, 5 and 50 /ag dose. Comparisons of means indicated that the total amount of LH released was higher (P<.05)in the two groups of ewes treated with analogs than the groups treated with.5, 5 or 50 #g of LH-RH/FSH-RH. In addition the ewes treated with 50/zg of the D-AIa 6 analog released more LH (P<.05) than the ewes treated with 50/ag of the D-Leu 6 analog. The total amount of LH released did not differ between the groups of ewes injected with 500/~g of either of the three hormones. The ratio of total LH released was calculated by comparing the concentration of total LH released in the ewes treated with LH-RH/FSH-RH to the concentration of total LH released in the ewes treated with the TABLE 2. COMPARISON OF THE TOTAL AMOUNT OF LH RELEASED BY CALCULATING THE AREA (CM 2) UNDER THE CURVE OF LH RELEASE (MEAN STANDARD ERROR) Dosage of hormone (ug) ,0 LH-RH/FSH-RH.6 a.1 D-AIa 6, des Gly I O.LH.RH ethylamide D-Leu s, des Gly 1 ~ ethylamide Exact probability of F occurring just due to chance a 8.2a b asignificantly lower than the release by the D-AIa 6 and D-Leu 6 Analogs (P<.05). bsignificantly greater than the release by the D-Leu 6 Analog (P<.05).

5 1224 KINDER ET AL I!o- I ~ 80- ~ 70- o3 ~ 60- z: 50- _1 '~ ~: 20- I0- I I./" i i/'" i.. t./' LOG DOSE (#g) ///,~.i~,~! ~- ~ m~,, LH-RH elhylomide,' c*-o LH-RH/FSH-RH,t :1 Figure 4. Amount of LH-RH/FSH-RH or analog required to release 50% of the total LH released by 500 #g (100% = average amount of total LH released by 500 #g for the three hormones). analogs at their respective doses. It should be noted that the ratio of total LH released declines as the dose increased, from 1:9 at.5 /~g, 1:4 at 5 /ag; 1:3 at 50 gg to 1:1 in the ewes treated with 500 micrograms. Thus with lower doses a greater increase in LH release was noted between LH-RH/FSH-RH and the two analogs. This increased LH concentration was lower than those reported by Rippel et al. (1975) who found the D-Leu 6 analog in the anestrous ewe to be approximately 50 times more potent in LH releasing activity than LH-RH/FSH-RH. The reason for the discrepancy between these results is difficult to evaluate, but could reflect differences in doses used and the different methods used for the dose comparison. A second method was used to compare the LH releasing potency of LH-RH/FSH-RH and the analogs in experiment 3. The average amount of total LH released at the 500/ag dose was considered to be 100% and all other concentrations of total LH released at the other three doses were calculated as a percentage of the average LH concentration of the 500 /ag doses. The dosage of LH-RH/FSH-RH and analogs required to release 50% (effective dose 50) was calculated to be 2.4, 5.4 and 100/~g for the D-AIa 6 analog, D-Leu 6 analog and LH-RH/FSH-RH, respectively (figure 4). Effective dose 50 values indicate that the D-AIa 6 analog is 2 times more potent than the D-Leu 6 analog and 42 times more potent than LH-RH/FSH-RH. The D-Leu 6 analog is 18 times more potent than LH-RH/FSH-RH according to effective dose 50 values. Analysis of variance of the ovulation data in experiment 3 indicated a dosage effect (P<.01) and there was a trend (P<.10) toward a hormonal effect (table 3). There was no hormone dosage interaction based on the ovulation data in the present study; however, a significant hormone (LH) dosage interaction was found in experiment 1. The ovulation rate was lower in the ewes treated with.5 #g when ewes from all three hormone-treated-groups were combined and compared to the corresponding ewes treated with 5, 50 or 500 micrograms. Fujino et al. (1974a) have reported a greatly increased number of ovulations in rats treated with the D-Ala 6 analog than in rats treated with LH-RH/FSH-RH. The ovulation data from experiment 3 fails to show this dramatic increase in ovulation rate in the ewe. The explanation for the differences noted in the present study and the earlier work (Fujino et al., 1974a) may be due to the fact that the rat has a much higher ovulation rate per estrus than the ewe, TABLE 3. AVERAGE NUMBER OF OVULATIONS IN EWES TREATED WITH LH-RH/FSH-RH AND THE ANALOGS D-ALA 6 AND D-LEU 6 (MEAN -+ STANDARD ERROR) Dosage in #g D-Ala 6, des D-Leu 6, des LH-RH/ Glyl 0.LH.RH Glyl 0_LH_RH FS H- RH ethylamide ethylamide

6 LH-RH/FSH-RH ANALOGS 1225 therefore species differences may play an important role in the ovarian response to the analogs. In experiment 1, des GIyI~ ethylamide was shown to consistently induce ovulation in anestrous ewes at the 500 #g dose, whereas the 500 #g dose of LH-RH/FSH-RH and lower doses of the ethylamide analog failed to do so. The evidence in experiment 3 indicates that there was no difference in the ovulation-inducing ability of the D-AIa 6 and D-Leu 6 analogs when compared to LH-RH/FSH-RH. One possible answer to this discrepancy might be the difference in the time the ovaries were observed subsequent to the injections in the two studies. In experiment t the ovaries were observed 24 hr subsequent to the injection and in experiment 3 they were observed 48 hr subsequent to the injection. It might be that the ewes treated with the lower dose levels of analog did in fact ovulate in experiment 1 but ovulated later than 24 hr after the injection. If this is in fact the case it would appear that dose size may be effecting the time of ovulation with larger doses inducing ovulation earlier than smaller doses. In experiment I no increase in the release of LH or FSH in the ewes treated with 500 /ag analog as compared to the ewes treated with 500 tag LH-RH/FSH-RH was detected even though this was the group of ewes with the greater number of ovulations. It has been suggested that there is a change in the FSH molecule in the monkey and rat upon castration (Knobil et al., 1972; Bogdanove et al., 1973) and there is evidence for two FSH molecules with different biological potencies within the same species of animal. It could thus be possible that the FSH concentration we have measured is of varying biological potency under different physiological conditions which might explain the increased ovulation rate without increased FSH concentration. However, we neither favor nor have proof of this possibility. There is some evidence which indicates that LH-releasing activity in vivo is less than the ovulation inducing potency of the D-AIa 6 and D-Leu 6 analogs in rats (Fujino et al., 1974b). The data from experiment 3 does not substantiate these findings in the ewe. The data from experiment 3 show that the ratio of the difference in LH release with the analogs when compared to LH-RH/FSH-RH is much larger with smaller doses than with larger doses. This could explain the discrepancy that may arise between laboratories on percent increase in LH release between a particular analog and LH-RH/FSH-RH. Therefore, when investigators make statements about the increased potency of the analogs over LH-RH/FSH-RH with regard to their LH-releasing activity they should also indicate what dose they are using and of course what species tested. LITERATURE CITED Adams, T. E., J. E. Kinder, P. K. Chakraborty and J. J. Reeves Ewe luteal function influenced by putsatile administration of synthetic LH-RH/FSH- RH. Endocrinol. 97:1460. Arimura, A., J. A. Vilchez-Martinez and A. V. Schally In Vitro Comparison of LH-RH and FSH-RH activities of (Des-gly 10) (Pro9.Ethylamide).LH" RH, (Des-Gly 1~ (Prog-Propylamide)-LH-RH, and LH-RH using immature male rats. Proc. Soc. Exp. Biol. Med. 146:17. Bogdanove, E. M., G. T. Campbell, G. Grossman and D. Blair Prolonged "stop entry" survival time of endogenous serum FSH in androgen-treated orchidectomized rats. Fifty-fifth Annual Meeting Endocrine Society. Abstr Coy, D. H., E. J. Coy, A. V. Schally. J- Vilchez-Martinez, Y. Hirotsu and A. Arimura Synthesis and biological properties of (d-ala-6, des-gly-nh=- IO)-LH-RH ethylamide, a peptide with gready enhanced LH- and FSH-releasing activity. Biochem. Biophys. Res. Comm. 57:335. Cfighton, D. B., J. P. Foster, W. Haresign, N. B. Hayes and G. E. Lamming The effects of a synthetic preparation of gonadotropin releasing factor on pituitary and ovarian function in anoestrous ewes. J. Physiol. 231:98. Fujino, M., S. Kobayashi, M. Obayashi, S. Shinagawa, T. Fukuda, C. Kitada, R. Nakayama, I. Yamazaki, W. F. White and R. F. Rippel Structure-activity relationships in the c-terminal part of luteinizing hormone releasing hormone (LH-RH). Biochem. Biophys. Res. Comm. 49:863. Fujino, M., I. Yamazaki, S. Kobayashi, T. Fukuda, S. Shanagawa, R. Nakayama, W. F. White and R. H. Rippel. 1974a. Some analogs of luteinizing hormone releasing hormone (LH-RH) having intense ovulation-inducing activity. Biochem. Biophys. Res. Comm. 57:1248. Fujino, M., T. Fukuda, S. Shinagawa, S. Kobayashi, I. Yamazaki, R. Nakayama, J. H. Seely, W. F. White and R. H. Rippel. 1974b. Synthetic analogs of luteinizing hormone releasing hormone (LH-RH). Substituted in position 6 and 10. Biochem. Biophys. Res. Comm. 60:406. Hulet, C. V. and W. C. Foote A rapid technique for observing the reproductive tract of living ewes. J. Anim. Sci. 27:142. Knobil, E., T. Yamaji, W. D. Peckham and D. J. Dierschke Alterations in biological and physical properties of endogenous FSH consequent on castration. Intern. Congress of Endocrinol. Abstr L'Hermite, M., G. D. Niswender, L. E. Reichert and A. R. Midgley Serum follicle stimulating hor-

7 1226 KINDER ET AL. mone in sheep as measured by radioimmunoassay. Biol. Reprod. 6: 325. Rippel, R. H., E. S. Johnson, W. F. White, M. Fujino, J. Yamazaki and R. Nakayama Ovulating and LH-releasing activity of a highly potent analog of synthetic gonadotropin-releasing hormone. Endocrinol. 93:1449. Rippel, R. H., E. S. Johnson, W. F. White, M. Fujino, T. Fukuda and S. Kobayashi Ovulation and gonadotropin releasing activity of (D-Leu 6, Desgly_NH2,0, Pro.ethylamide 9)_Gn_RH. Proc. Soc. Exp. Biol. Med. (Accepted for publication). Segerson, E. C., L. C. Ulberg, J. E. Martin and R. E. Fellows Fertility in ewes treated with luteinizing hormone releasing factor. Proc. Soc. Exp. Biol. Med. 146:518. Steel, R. G. and J. H. Torrie Principles and procedures of statistics. McGraw-Hill Book Co., New York. Vilchez-Martinez, J. A., D. H. Coy, A. Arimura, E. J. Coy, Y. Hirotsu and A. V. Schally Synthesis and biological properties of (Leu-6)-LH-RH and (D-Leu-6, Des GIy-NH 210)_LH.RH ethylamide. Biochem. Biophys. Res. Comm. 59:1226.