THE EFFECT OF PROSTAGLANDIN F 2a ON LUTEINIZING HORMONE AND TESTOSTERONE SECRETION IN BULLS Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY TERRY E. KISER 1977 LIBRARY Michigan State University This is to certify that the thesis entitled THE EFFECT OF PROSTAGLANDIN an ON LUTEINIZING HORMONE AND TESTOSTERONE SECRETION IN BULLS presented by Terry E. Kiser has been accepted towards fulfillment of the requirements for Ph.D. degree in Dairy Scfince 4 7 Major professo Date February 234 1977 0-7639 ABSTRACT THE EFFECT OF PROSTAGLANDIN F2a ON LUTEINIZING HORMONE AND TESTOSTERONE SECRETION IN BULLS BY Terry E. Kiser The objective of these experiments was to determine the role of prostaglandin F a (PGFZa) on LH and testosterone secretion in bulls. 2 In the first experiment, each of five Holstein and three Guernsey bulls (average weight 301 1.21 kg) was given (so) saline or 20 mg PGF a Tham salt (14.9 mg free acid) in a two-period crossover 2 design. On the first day four bulls were given PGF and four bulls 2a were given saline, then the treatments were reversed on the second day. Blood serum LH averaged 1.1 :_O.l ng/ml before PGFZa; LH increased (P<.05) to 3.5 :_l.0 ng/ml at 30 minutes after PGFZa' peaked (3.9 i 0.9 ng/ml) at 45 minutes and declined to pre-injection values by 4 to 5 hours. Blood serum testosterone averaged 4.5 :_0.2 ng/ml before PGFZa; it increased (P<.Ol) synchronously in each of the eight bulls to 8.5 :_0.9 by 60 minutes after PGF peaked at 15 to 2a’ 16 ng/ml between 90 and 120 minutes, and then declined toward pre- injection concentrations by 180 minutes. The increase in testosterone was preceded by increased serum LH in each of the eight bulls. In contrast, an average of one episodic increase of blood serum LB (average peak 14.2; range 8 to 22.8 ng/ml) occurred apparently at random intervals during the 8-hour period after bulls were given Terry E. Kiser saline. An increase of blood serum LH (average peak 3.0; range 1.5 to 4.3 ng/ml) occurred about 30 minutes before each of these tes- tosterone surges. On the average, however, neither LH nor testos- terone changed significantly after saline. The second experiment utilized four Holstein bulls (average weight 355 i_19 kg) with cannulae inserted into both jugular veins, one for infusion of saline or PCP and the other for blood collec- 20 tion. Prostaglandin F a Tham salt was infused (0.2 mg/min; 0.15 mg 2 free acid) into two bulls and the other two bulls were given an equivalent quantity of saline vehicle for 20 hours. The infusion treatments were reversed beginning 28 hours after completion of the first infusion. Blood sampling was continued for 8 hours following the 20-hour treatment infusion. Blood plasma LH averaged 1.2 :_0.1 ng/ml before PGF a infusion; it doubled (P<.07) within 1.5 hours 2 after the infusion was started and peaked (P<.01) at 4.2 i_0.8 ng/ml at 6.5 hours before declining to basal concentrations before the end of the infusion. Blood plasma testosterone averaged 7.0 i_0.6 ng/ml during the 90 minutes before iv infusion of PGF it increased 2a; (P<.Ol) to 16.0 :_l.5 ng/ml by 2.5 hours after the start of the infusion, remained near this peak until 10 hours and then gradually returned toward pre-injection values by the end of the infusion. Luteinizing hormone started to decline at least 3 hours earlier than testosterone, preceding the decline of testosterone in each of the four bulls. Episodic surges of testosterone similar to those in untreated bulls resumed within 8 hours after the conclusion of the 20-hour infusion of PGF Two or three episodic surges of testos- 2o' terone occurred in each of the bulls during the 20—hour control infusion of saline; the peak (17.2 :_0.2 ng/ml) of these surges was Terry E. Kiser equivalent to the peak concentration of testosterone during infusion of PGan and 18 of 19 control surges of testosterone were preceded by increased blood LH (average peak 2.8 :_0.3 ng/ml). The third experiment consisted of a preliminary and a main experiment. In the preliminary experiment four Holstein bulls (average weight 355 1.19 kg) were used during a 3-day period. On the first day, starting at 0800 hours, jugular blood was taken at frequent intervals for 4 hours. On the second and third days each hull was fed 1.01 kg of feed containing 0.5 mg melengestrol acetate (MGA) at 0700 and again at 1900 hours. Then on the third day jugular blood was collected at frequent intervals, starting at 0800 hours and continuing for 4 hours. The average testosterone concentration during the 4-hour period before MGA pre-treatment was 8.5 :_l.1 ng/ml, significantly greater (P<0.0l) than average testosterone (1.8 :_0.1 ng/ml) during a 4-hour period after MGA pretreatment. The main experiment was conducted as a two-period crossover design with repeat measurement on four bulls (average weight 307 i 36 kg). Each bull was fed 1.0 mg of MBA daily (0.5 mg at 0700 and at 1900 hours) throughout the experiment. Starting 24 hours after the first feeding, two bulls were given 20 mg PGF Tham salt (so) 2d and two bulls were given saline (so). The treatments were reversed 10 hours later. Then 48 hours after the first feeding of MGA, each of the four bulls was given iv 200 ug NIH-LH-B8. The testosterone response from these bulls was compared with four control bulls (average weight 328 :_42 kg) given 200 ug LH after the same sequence of PGFZd treatment but without MGA pretreatment. After the four MGA-treated bulls were given saline, serum LH concentrations did not change significantly, ranging from 0.30 :_0.05 Terry E. Kiser to 0.40 :_0.05 ng/ml. Similarly, serum testosterone fluctuated very little (0.8 :_0.3 to 1.2 :_0.2 ng/ml) and did not change signifi- cantly. By comparison, serum LH averaged 0.40 :_0.01 ng/ml before MGA-treated bulls were given (so) 20 mg PGF it increased (P<.05) 2o; 5-fold at 45 minutes after PGan' peaked at 2.3 :_0.5 ng/ml at 60 minutes and declined to basal values between 4 and 5 hours. Serum testosterone averaged 0.8 :_0.3 ng/ml before PGF increased (P<.05) 2a' to 13.4 :_4.1 ng/ml at 75 minutes after PGF and reached a peak 2d concentration of 21.3 :_2.3 at 105 minutes. Serum testosterone then was maintained at a stable concentration until 3 hours after PGan’ and declined to basal concentrations at 7 hours after PGFZa' Pre— treatment of bulls with MGA did not influence testosterone response to exogenous LH treatment compared with control bulls not pre-treated with MGA. In the final experiment, each of five bulls was given intra- carotid infusion of 0 (saline vehicle), 20, 200 and 2,000 ng of PGan/min and intrajugular infusion of 2,000 ng PGFZa/min and 0.2 mg PGFZa/min during 3 days. The infusions were maintained for 3 hours at 10 ml/hour with 12-hour intervals between the start of con- sequentive treatments. Intracarotid infusion of saline, 20 and 200 ng PGFZa/min or jugular infusion of 2,000 ng PGFZa/min was ineffective in causing increased blood LH. In contrast, blood LH increased (P<.05) from 0.8 :_O.l ng/ml to a peak of 2.6 :_0.5 ng/ml within 1 hour after beginning jugular infusion of the largest dose of PGF (0.2 mg/min), 2d then declined to 1.4 :_0.3 ng/ml at the end of the 3-hour infusion. A similar increase (P<.05) (peak of 3.6 :_l.1 ng/ml) occurred during intracarotid infusion of 2,000 ng PGFZa/min, but LH remained elevated Terry E. Kiser throughout this 3-hour intracarotid infusion. In addition, the LH response during intracarotid infusion of 2,000 ng PGan/min was greater (P<.001) than the comparable response during intrajugular infusion of 0.2 mg PGFZa/min. Testosterone remained below 3 ng/ml during jugular infusion of 2,000 ng PGFZa/min, but the same dose of PGFZa infused into the carotid increased (P<.05) blood testosterone from 1.3 :_0.4 ng/ml before infusion to 7.2 :_2.2 ng/ml at 2 hours during infusion and testos— terone remained elevated until the intracarotid infusion was stopped. Episodic increases of testosterone occurred in two bulls during intracarotid infusion of saline, but duration of these testosterone surges was significantly shorter than duration of elevated testos— terone during intracarotid infusion of 2,000 ng PGFZa/min or jugular infusion of 0.2 mg PGFZa/min. The results from these four experiments demonstrate conclusively that PGFZa causes increased blood LH and testosterone secretion in bulls. Furthermore, increased LH preceded testosterone surges after administration of PGF2 , suggesting that increased LH was the pri- mary stimulus for causing increased testosterone secretion. A constant PGFZd stimulus via a 20-hour intravenous infusion caused a prolonged increase in LH and testosterone, but both hormones decreased to basal concentrations toward the end of the infusion, suggesting that a constant PGFZa stimulus results in hypothalamic or pituitary refractoriness. If a constant PGF20 stimulus caused depletion of hypothalamic LHRH or pituitary LH, the effect was short-lived because episodic secretion of LH and testosterone resumed again within 8 hours after the end of the infusion. Pre-treatment of bulls with MGA abolished episodic secretion of LH and testosterone, but PGFZa Terry E. Kiser overcame this inhibition, suggesting that feedback inhibition of episodic LH secretion possibly may be mediated through or involved with PGFZo' Finally, the data provide evidence that PGFZa was acting at the brain to cause release of LH. THE EFFECT OF PROSTAGLANDIN an ON LUTEINIZING HORMONE AND TESTOSTERONE SECRETION IN BULLS BY Terry E. Kiser A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1977 TO MY WIFE ii BIOGRAPHICAL SKETCH Terry E. Kiser was born on August 24, 1947, in Sumner, Illinois. He attended public schools in Lawrence County and graduated from Sumner High School in June 1965. Following two years in the agri- cultural curriculum at Vincennes University, he decided to follow his interest in animal agriculture at Southern Illinois University and received a Bachelor of Science degree in June 1969. After serving two years as an operations and intelligence specialist in the United States Army, he enrolled in the graduate school of the University of Wyoming. He received the Master of Science degree in December 1973, majoring in Animal Science. He then entered a Ph.D. program in the area of reproductive endocrinology and physiology in the Department of Dairy Science at Michigan State University under the joint directorship of Drs. W. D. Oxender and H. D. Hafs. He received the Doctor of Philosophy degree in March 1977 and accepted a position as assistant professor in the area of Bovine Reproductive Physiology, Department of Animal and Dairy Science, University of Georgia, at Athens. iii ACKNOWLEDGEMENTS I express my sincere appreciation to Drs. w. D. Oxender and H. D. Hafs for sharing the responsibility of directing my graduate program. They provided me guidance, support, encouragement and enthusiasm to complete my graduate program. I am also grateful for the advice and support of my committee members, Drs. John L. Gill, Joseph Meites and Harlan D. Ritchie and to Dr. N. B. Haynes for his help and cooperation during the course of this research. Appreciation is also expressed for the financial support pro- vided by the Department of Dairy Science through a Graduate Research Assistantship. This dissertation involved the help of many individuals in the Dairy Science Department who unselfishly gave their time. Their cooperation and conscientious work are gratefully appreciated. The gifts of hormones from the Endocrinology Study Section of the National Institutes of Health and the help of Dr. Jim Lauderdale of the Upjohn Company in providing the prostaglandin F necessary 2d for my research are appreciated. Finally, a most sincere appreciation is expressed to my wife, Gloria, and children, Sheri, Karen and Kevin, for tolerating and supporting my efforts as a graduate student. Also, a thanks to my parents, Mr. and Mrs. Merrill Kiser, and Gloria's parents, Mr. and Mrs. James Jones, for encouraging my educational efforts. iv TABLE OF CONTENTS Page INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O O O O 1 REVIEW OF LITERATURE. . . . . . . . . . . . . . . . . . . . . . 2 Hypothalamo-Hypophyseal-Testis Interactions. . . . . . . 2 A 0 General 0 O O O O C O O C O O O O O O O O O O 2 B. Gonadotropin Releasing Hormone and Gonado- tropin Secretion. . . . . . . . . . . . . . . 3 C. Relationship Between LH and Testosterone Secretion . . . . . . . . . . . . . . . . . . 5 D. Hormonal Regulation of Spermatogenesis. . . . 9 Prostaglandins and Reproductive Processes. . . . . . . . 10 A. Background. . . . . . . . . . . . . . . . . . 10 B. Effect of Prostaglandins on Gonadotropin and Steroid Secretion . . . . . . . . . . . . 13 EXPERIMENT 1 - THE TEMPORAL RELATIONSHIP BETWEEN BLOOD SERUM LUTEINIZING HORMONE AND TESTOSTERONE AFTER PGF OR SALINE IN BULLS. . . . . . . . . . . .I. . . .2? . . . . 21 Introduction . . . . . . . . . . . . . . . . . . . . . . 21 Materials and Methods. . . . . . . . . . . . . . . . . . 21 Results and Discussion . . . . . . . . . . . . . . . . . 23 EXPERIMENT 2 - THE EFFECT OF A CONTINUOUS INTRAVENOUS INFUSION OF PGan ON LUTEINIZING HORMONE AND TESTOSTERONE SECRE- TION IN BULLS. . . . . . . . . . . . . . . . . . . . . . 38 Introduction . . . . . . . . . . . . . . . . . . . . . . 38 Materials and Methods. . . . . . . . . . . . . . . . . . 39 Results and Discussion . . . . . . . . . . . . . . . . . 39 EXPERIMENT 3 - THE EFFECT OF EXOGENOUS LH OR PGFZa ON BLOOD LH AND TESTOSTERONE IN BULLS GIVEN MELENGESTROL ACETATE . . 54 Introduction . . . . . . . . . . . . . . . . . . . . . . 54 Materials and Methods. . . . .'. . . . . . . . . . . . . 55 Results and Discussion . . . . . . . . . . . . . . . . . 56 EXPERIMENT 4 - THE EFFECT OF CAROTID ADMINISTRATION ON BLOOD LH AND TESTOSTERONE IN BULLS. Introduction . . . . . . . . . . . . . Materials and Methods. . . . . . . . . Results and Discussion . . . . . . . . GENERAL DISCUSSION. . . . . . . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . . . . . LITERATURE CITED. . . . . . . . . . . . . . . vi Page 65 65 65 71 82 86 91 Table LIST OF TABLES Page Interval to and duration of first testosterone surge after PGFZG (20 mg, so) or saline in eight bulls . . . . 31 Average serum testosterone during a 4-hour interval before and after treatment with melengestrol acetate in four Holstein bulls . . . . . . . . . . . . . . . . . 57 Percent cross-reactions of rabbit antitestosterone (MSU #74) O O O O O O O I O O O O 0 O O O O O O O O O O O 69 Percent recovery of added testosterone after direct extraction or column chromatographic isolation from sera from an estrous cow or bull, or from steer plasma . 70 vii Figure 10 ll 12 13 LIST OF FIGURES The structure of prostaglandin FZa . . . . . . . . . . . Blood serum LH in eight bulls given saline or 20 mg PGFZG (SC) 0 O O O O O O O O O O O O O O O O O O O O O 0 Blood serum testosterone in eight bulls given saline or 20 mg PGde (so). . . . . . . . . . . . . . . . . . . Blood serum LH and testosterone in eight bulls given 20 “'9 PGan (SC) 0 o o o o o o o o o o o o o o o o o o 0 Blood serum LH and testosterone in eight bulls given saline . . . . . . . . . . . . . . . . . . . . . . . . . Blood plasma LH during iv infusion of saline or PGF . 2d (0.2 mg/mln) o o o o o o o o o o o o o o o o o o o o o 0 Blood plasma testosterone in four bulls during infusion (iv) of saline or PGFZo' . . . . . . . . . . . . . . . . Blood plasma LH and testosterone in four bulls during infusion of saline . . . . . . . . . . . . . . . . . . . Blood plasma LH and testosterone in four bulls during infusion (iv) of PGFZd (0.2 mg/min). . . . . . . . . . . Average blood plasma LH and testosterone in four bulls during infusion (iv) of PGFZo (0.2 mg/min) . . . . . . . Blood serum LH (top) and testosterone (bottom) in four bulls pre-treated with melengestrol acetate and given saline (sc) or PGFZa (20 mg, sc) . . . . . . . . . Blood serum LH (top) and testosterone (bottom) in four control bulls and four melengestrol acetate treated bulls after 200 ug LH (iv) . . . . . . . . . . . Blood plasma LH during a 3-hour infusion of PGFZu (0 ng/min [C], 2,000 ng/min [C], 2,000 ng/min [J], and, 0.2 mg/min [J]) into the carotid artery (C) or jugular vein (J) . . . . . . . . . . . . . . . . . . . . viii Page 12 25 30 34 36 41 44 46 49 51 60 64 74 Figure 14 15 Page Blood plasma testosterone during a 3-hour infusion of PGFZa (0 ng/min [C], 2,000 ng/min [C], 2,000 ng/min [J] and 0.2 mg/min [J]) into the carotid artery (C) or the jugular vein (J). . . . . . . . . . . . . . . . 77 Mean duration of LH and testosterone surges occurring prior to (control) and during infusion of PGFZa into . . . . 80 five bulls . . . . . . . . . . . . . . . . . . . ix INTRODUCTION With successful estrus and ovulation control through the use of prostaglandin in cattle, more emphasis will be placed on utilization of artificial insemination. As a result, fewer and better bulls should be used to sire the next generations of cattle. The bull is one factor affecting fertility of cattle, but little is known about the control of libido, sperm production and sperm quality in bulls, aside from general information to show that they are under the con- trol of the endocrine system (reviewed by Steinberger and Steinberger, 1973; Neaves, 1975). The purpose of this dissertation was to study the relationship between luteinizing hormone and testosterone secretion in bulls, with special attention to the possibility that prostaglandin F may 2d modulate this system. Specifically, the objectives of this dissertation research were to determine: 1. The temporal relationship between blood serum LH and tes- tosterone in bulls given a single subcutaneous injection of PGFZo' 2. Whether a continuous iv infusion of PGF2o can maintain elevated blood LH and testosterone in bulls. 3. Whether PGon causes LH release in bulls pre-treated with a progestogen. 4. If PGFZd acts centrally at the hypothalamo-pituitary axis to cause release of LH. REVIEW OF LITERATURE Hypothalame-Hypophyseal-Testis Interactions A. General Green and Harris (1947) were among the first to support the idea that the anterior pituitary was regulated by substances originating from the hypothalamus. And in 1948, Harris concluded that the essen- tial hypothalamo-adeno-hypophyseal linkage was vascular, not neural. Subsequent studies verified the hypothesis that the anterior pituitary was influenced by the hypothalamus, that the influence may be both stimulatory and inhibitory, and that the influence is media- ted by neurohumors which normally reach the anterior pituitary via the hypophyseal portal blood vessels. After this hypothesis was established, subsequent investigations were conducted to identify and isolate individual neurohumors, define their mechanism of action, and elucidate their physiological role (reviewed by Geschwind, 1970; Meites, 1970). The evidence from the neuroendocrine literature suggests at least nine releasing or inhibiting hormones exist in the hypothalamus. Thus far three have been isolated and synthesized: thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone/follicle stimulating hormone releasing hormone (LHRH/FSHRH) and somatotropin releasing inhibiting factor (SRIF). For the purpose of this review, only LHRH/FSHRH or the synthetic material commonly referred to as gonadotropin releasing hormone (GnRH) will be discussed. In the course of this discussion LHRH and GnRH will be used to denote the same substance. B. Gonadotropin Releasing Hormone and GonadotrOpin Secretion Synthetic gonadotropin releasing hormone causes increased LH in cattle (Golter et al., 1973; Zolman et al., 1973), sheep (Galloway et al., 1974; Reeves et al., 1972), pigs (Chakraborty et al., 1973), rats (Arimura et al., 1972), hamsters (Arimura et al., 1971) and humans (Roth, Grumbach and Kaplan, 1973; Arimura et al., 1973). Presumably, GnRH acts directly on the pituitary to cause release of LH, because Zolman et a1. (1973) reported a dose-related increase in LH release when bovine pituitary explants were exposed to purified porcine GnRH during superfusion with Medium 199. Zolman et a1. (1973) also demonstrated that LH release after GnRH differed between male and female cattle. The peak of LH in heifers during the luteal phase of the estrous cycle increased linearly with increasing dose of GnRH up to 80 ug. In contrast, the peak LH response in bulls increased in a linear manner with doses of GnRH up to 160 ug, the highest dose used. In heifers, the steroid environment appears to alter LH release in response to GnRH. Kaltenbach et al. (1974) suggested that the high concentration of progesterone in luteal phase of the estrous cycle in heifers may dampen LH release after administration of GnRH. In contrast, Zolman, Convey and Britt (1974) found no significant difference in LH release after GnRH on day 15 of the cycle when pro- gesterone was high, or on day 20 of the estrous cycle when proges- terone was low in heifers. However, in the same study the magnitude 4 of LH release in response to GnRH was significantly related to estra- diol and estrone concentrations at the time GnRH was administered. Increased concentrations of estradiol and estrone accentuated the LH response after GnRH. Similarly, Reeves et a1. (1971) showed that LH response to GnRH varied during the estrous cycle and was maximal around the period of estrus in ewes. Furthermore, LH response to GnRH was increased by pre-treatment with estradiol benzoate in ewes (Reeves et al., 1971). Conversely, daily injections of 20 mg progesterone for 14 days or infusion of 500 ug progesterone/hr for 76 hours suppressed the LH response to GnRH in anestrous and cyclic ewes, respectively (Hooley et al., 1974; Pant and Ward, 1974). In summary, LH response after GnRH in the female is influenced by ovarian steroids. Estradiol appears to facilitate and proges— terone to inhibit LH release after GnRH. After administration of GnRH to bulls, serum LH increased simi- larly in 2—, 4- and 6-month—old bulls (Mongkonpunya et al., 1975); however, a clear testosterone response was evident only in 6-month- old bulls. Thiber (1976) and Galloway et a1. (1974) also reported an increase in serum testosterone following administration of GnRH to post-pubertal bulls and rams, respectively. When GnRH was given to steers 14 days after castration, peak LH concentrations were higher than in bulls; however, the areas under the LH response curve did not differ significantly (Mongkonpunya et al., 1974). In Mongkonpunya's study, testosterone replacement in steers did not restore the magnitude of peak LH after GnRH to that characteristic of bulls. 5 A greater LH response occurred when purified porcine GnRH was given to wethers than in rams (Reeves et al., 1970), and the LH response was related to dose. More recently, Pelletier (1976) reported that peak magnitude of LH occurred sooner and was greater in wethers than in rams. These authors suggested that the enhanced LH response to GnRH in castrate animals was not due to higher pitui- tary content, but to endogenous testosterone inhibition at the level of the pituitary. Furthermore, Galloway et a1. (1974) suggested that pre-injection concentrations of endogenous testosterone could influence the amount of LH released after GnRH in rams; the lower basal testosterone led to greater LH response to a given dose of GnRH. Exogenous testosterone treatment reversed the post-castration increase in basal LH concentrations in bulls (McCarthy and Swanson, 1976; Mongkonpunya et al., 1974) and in rams (Galloway and Pelletier, 1975). Thus, in steers and wethers, exogenous testosterone lowers basal LH. In rams basal testosterone and the physiological state of the animal (castrate or intact) influences the LH response to GnRH. The published data suggest that exogenous testosterone does not influence the LH response to GnRH in steers, but further research will be necessary to delineate the complete steroid feedback relationship to gonadotropin secretion in the bovine male. C. Relationship_Between LH and Testosterone Secretion Studies concerning the relationship between LH and testosterone secretion in the male are complicated by short term episodic fluctua- tions in the serum concentrations of these hormones. This necessitates 6 frequent sampling to fully characterize the profiles of LH and tes- tosterone and their temporal relationship. For example, Mongkonpunya et a1. (1974) reported an average of 3.7 LH spikes daily in bulls. The magnitude of each episodic LH release was 2.7 :_0.6 ng/ml, and each LH release was followed by an increase in blood testosterone. Thus, the testis appears to be sensitive to small changes in serum LH concentrations. Similar temporal relationships between LH and testosterone exist in all species studied, including rams (Katongole, Naftolin and Short, 1974; Sanford, Winter, Palmer and Howland, 1974), rabbits (Moor and Younglai, 1975) and humans (Naftolin, Judd and Yen, 1973), and similar episodes of testosterone increases were observed in male rats (Bartke et al., 1973) and mice (Bartke and Dalterio, 1975). Other stimuli are known to alter testosterone secretion, but in general the alteration is mediated by LH. For example, pro- nounced seasonal changes of testosterone occur in rams (Gomes and Joyce, 1975; Katongole, Naftolin and Short, 1974; Purvis, Illius and Haynes, 1974; Schanbacher and Ford, 1976). In the report by Schanbacher and Ford (1976), baseline and peak concentrations of tes- tosterone were higher in September than in May. Although season (May or September) had no effect on the number and magnitude of LH peaks, basal concentrations were higher in September than in May. In addition, a temporal relationship existed between LH and testos- terone, indicating a cause and effect relationship, during both seasons. Sanwal, Sundley and Edqvist (1974) reported synchronous varia— tion of plasma concentrations of testosterone in bulls. In their study testosterone peaked synchronously at 0600, 1200 and 2000 hours 7 in four bulls. Presumably, the increase in testosterone was caused by release of LH, but the apparent synchrony of release remains open to question. Katongole et a1. (1971) suggested that rhythms of tes- tosterone were most likely due to some inherent central rhythm. In contrast, Smith et a1. (1973) found no apparent synchrony of LH and testosterone secretion in bulls; rather, the episodic burst of LH occurred at random intervals during periods of light and darkness. Other stimuli, such as exposure of bulls to estrous cows or ejaculation, cause increased testosterone (Smith et al., 1973). Similar changes occur in rats (Purvis and Haynes, 1974) and rabbits (Saginor and Horton, 1968; Haltmeyer and Eik-Nes, 1969) and rhesus monkeys (Rose, Gordon and Bernstein, 1972). But whether the increase in testosterone is mediated via increased LH is questionable. Katongole, Naftolin and Short (1971) suggested that sexual stimuli caused release of LH and testosterone in bulls, but neither Convey et al. (1971) nor Gombe et a1. (1973) were able to confirm the obser- vation that sexual stimuli caused increased LH. On the other hand, testosterone increased in six mature bulls (3 to 6 years old) and in two of six younger bulls (1.5 to 2.5 years old) at 30 minutes after ejaculation. The question remaining to be answered from these experiments is whether LH precedes the increased testosterone following sexual stimu- lation. The possibility exists that LH increased prior to the increase in testosterone but that the increase was of low peak mag- nitude and short duration, such that detection at hourly samplings was improbable. Alternatively, testosterone may have increased without a preceding increase in LH. For example, local regulation of testosterone may occur during sexual stimulation in bulls. Thus, 8 if the observation of Convey et a1. (1971) is correct, that testos- terone increases in the absence of increased LH after ejaculation, it represents the exception rather than the rule with regard to LH and testosterone relationships. Further understanding of the dynamic relationship between the pituitary and the testis has resulted by studying the effects of exogenous gonadotropins and androgen secretion in many species, including rats (El Safoury and Bartke, 1974; Moger and Armstrong, 1974; Purvis and Haynes, 1974), rabbits (Johnson and Ewing, 1971; Smith and Hafs, 1973), humans (Maver, Volkwein and Tamm, 1973; Weinstein et al., 1974), rhesus monkeys (Bennett et al., 1973), rams (Falvo et al., 1975), bulls (Katongole et al., 1971; Smith et al., 1973; Sundby, Tallman and Velle, 1975), and pigs (Andresen, 1975). In all species studied, injections of LH or HCG resulted in a rapid increase in testosterone concentrations. After iv infusion of LH into bulls, serum testosterone concen- trations doubled within 1 hour (Smith et al., 1973), and the concen- tration remained elevated 4 hours when the last sample was collected. In contrast, prolactin did not cause a significant testosterone increase and LH and prolactin together resulted in no greater increase in testosterone than LH alone. Others have reported increases in testosterone after HCG in bulls (Katongole et al., 1971; Lindner, 1969; Sundby, Tallman and Velle, 1975). Human chorionic gonadotropin concentrations remained elevated for 3 days after administration to bulls (Sundby, Tallman and Velle, 1975), but testosterone was elevated for 8 days after HCG. Thus, HCG appears to prolong the testosterone surge even when HCG was not detectable in the blood. 9 Luteinizing hormone appears to initiate a sequence of biochemi- cal events in the testes by first binding to specific membrane receptors on Leydig cells. The hormone-receptor complex causes an increase in the activity of adenylate cyclase which results in changes in the concentration of intracellular concentrations of cyclic AMP. Cyclic AMP modulates several metabolic events within the cell which together account for the action of LH (reviewed by Catt and Dufau, 1976; Means, Fakunding and Tindall, 1976; Marsh, 1976). Dorrington and Fritz (1974) reported that LH (not FSH) increased cyclic AMP production in isolated interstitial cells of Leydig. Conversely, in isolated seminiferous tubules freed of interstitial cells, FSH (not LH) stimulated adenyl cyclase activity. Furthermore, cyclic AMP was released by rat testis during LH stimulation (Catt, watanabe, and Dufau, 1973) and addition of cyclic AMP to slices of rat (Catt et al., 1973) and rabbit testis (Eik-Nes, 1971) stimulated testosterone production in vitro. D. Hormonal Regulation of Spermatogenesis That pituitary hormones are required for Spermatogenesis was demonstrated by the classic hypophysectomy experiments of Smith et a1. (1927, 1930). Luteinizing hormone, follicle stimulating hormone (FSH) and testosterone are all required for Spermatogenesis and sperm maturation (reviewed by Steinberger and Steinberger, 1973). As dis- cussed above, LH controls testosterone secretion. Since androgens maintain Spermatogenesis (Walsh et al., 1934), Nelson (1937) sug- gested that LH maintenance of Spermatogenesis was mediated via androgen secretion. 10 Greep and Fevold (1937) were the first to suggest LH control of Leydig cell function and FSH control of spermatogenesis. Follicle stimulating hormone appears necessary for the complete maintenance of Spermatogenesis, but the effects of FSH can be mimicked by andro- gens, indicating a functional relationship between FSH and androgens in the process of spermatogenesis. The discovery of a specific androgen binding protein (ABP) (French and Ritzen, 1973; Hansson et al., 1973a) produced by the Sertoli cell and stimulated by FSH (Hansson et al., 1973b) has added new insight into the hormonal control of spermatogenesis. Hansson et a1. (1975) proposed that androgen binding protein produced by the Sertoli cell served to concentrate active androgen in close proximity to target cells within the seminiferous tubule and epididymis. Prostaglandins and Reproductive Processes A. Background The early history of prostaglandins focused almost exclusively on the male. As early as 1930, Kurzrok and Lieb (1930) demonstrated that fresh human semen caused contractions of human uterine strips in vitro. Goldblatt (1933, 1935) and von Euler (1935) reported vasodepressor and smooth muscle stimulatory properties of alcoholic extracts of human seminal plasma. Furthermore, the active substance was soluble in organic solvents at acid pH (von Euler, 1936) and the substance was present in the semen and vesicular glands of humans and sheep. Von Euler (1935, 1939) named the substance "prostaglandin" apparently to distinguish it from other agents associated with the male reproductive tract, such as "vesiglandin" (von Euler, 1936), although prostaglandin is a misnomer since the greatest source of 11 the prostaglandins is the seminal vesicular glands, not the prostate gland as the name implies. Initially von Euler (1936, 1939) defined prostaglandins as a nitrogen-free unsaturated carboxylic acid containing hydroxyl groups, but it was not until the 1950's and 1960's that the structure and identity of prostaglandins were determined (reviewed by Bergstrom, Carlson and Weeks, 1968). The prostaglandins constitute some of the most biologically active substances ever discovered and undoubtedly participate in a wide variety of endocrine and metabolic systems. The natural prosta- glandins are unsaturated hydroxy acids with 20 carbon atoms and containing a cyclopentane ring with two carbon side chains, one of which has a terminal carboxyl group. Four main groups of naturally occurring prostaglandins are dis- tinguished as E, F, A and B, denoting differences in the cyclopentane ring. A trans double bond between carbons 13-14 and a hydroxyl group at carbon 15 are common to the four naturally occurring pros- taglandins. Prostaglandin E and F both contain a hydroxyl at carbon 11, but the E prostaglandins have a ketone, whereas F prostaglandins have a hydroxyl at carbon 9. Subscript numbers indicate the number of double bonds in the side chains. The terms a and 8 refer to the spatial configuration of the carbon 9 hydroxyl group. For example, the structure of PGFZa is shown in Figure l. Prostaglandins A and B are dehydration products of PGE. The relationship between the structure and the biological activity of the prostaglandins was complicated by diverse species differences and different physiological states of animals. For example, PGE causes relaxation of non-pregnant human uterus while 12 Figure 1. The structure of prostaglandin F20° PGF causes contraction, and both PGE and PGF cause contractions of pregnant myometrial tissue (Bygdeman, 1967). As an example of species difference, PGF G acts to decrease blood pressure in the rabbit and 2 cat, similar to the action generally attributed to PGE (Horton and Main, l965),but PGF G acts as a pressor in the dog and rat (DuCharme 2 and Weeks, 1967). In the intact animal, the structural activity relationship is difficult to assess, because prostaglandins are metabolized and inactivated very rapidly by the lungs (Nakano, 1973). Studies on structural activity suggest that E and F prostaglandins were the most biologically potent of the naturally occurring prostaglandins; that metabolism generally results in loss of activity (Kloeze, 1969); and altering the carboxyl side chain, the cyclopentane ring or the alkyl side chain changes the biological activity of a particular prostaglandin. One of the more important structural features of prostaglandins is the hydroxyl group at carbon 15. The metabolite 15-Keto-PGE, which lacks a hydroxyl group at carbon 15 position, exhibits no bio- logical activity in dogs and rats (Pike, Kupiecki and Weeks, 1967). 13 B. Effect of Prostaglandins on Gonado- tropin and Steroid Secretion Although the prostaglandins appear ubiquitous in mammalian tissue, the concentration present in most tissue is miniscule com- pared with the amounts found in the seminal vesicles. F and F have been detected However, prostaglandins E1, E2, la Zo in brain tissue (Ambache, 1966; Coceani, Pace-Asciak and Wolfe, 1968; Coceani and WOlfe, 1965; Holmes and Horton, 1967; Horton and Main, 1966, 1967), cerebellum (Coceani and Wolfe, 1965; Ramwell and Shaw, 1966), spinal cord (Coceani et al., 1968; Ramwell, Shaw and Jessup, 1966) and spinal fluid (Feldberg and Myers, 1966). In addi- tion, the prostaglandins were released spontaneously (Coceani and Wolfe, 1965; Ramwell and Shaw, 1966) after electrical stimulation, or after central nervous stimulation with picrotoxin, pentylene- tetrazol and strychnine (Coceani and Wolfe, 1965; Ramwell and Shaw, 1966). Evidence for a specific prostaglandin synthetase in the brain (Van Dorp, 1966), taken together with the fact that prosta- glandins are released after electrical stimulation, suggests that they perhaps have a role as a transmitter substance. Other evidence demonstrates that prostaglandins injected into the cerebral ventricles (Horton, 1964) or intravenously (Duda, Horton and McPherson, 1968) have a long duration of action at central neurons, a finding incompatible with rapid metabolism of transmitter substances. Coceani, Puglisi and Lavers (1971) suggested that the prostaglandins were unlikely to mediate synaptic transmission, but possibly may act as a modulator substance. For example, the prosta- glandins may regulate the release of transmitters or possibly act within the effector membrane to decrease or enhance the effectiveness 14 of a transmitter. In the latter cases, the prostaglandins may act as intracellular messengers by directly affecting an enzyme or by affecting the concentration of substances such as cyclic nucleotides or calcium ions. To my knowledge no reports are available linking prostaglandins to such systems in the brain; however, in other types of tissue the prostaglandins may directly influence the activity of enzymes such as adenylate kinase (Abdulla and McFarlane, 1971). adenylate cyclase (Ramwell and Shaw, 1970), phosphodiesterase (Amer and Marquis, 1972), and Na+/K+ activated ATPase (Johnson, Jessup and Ramwell, 1973). The prostaglandins may increase or decrease the synthesis of cyclic AMP by adenyl cyclase or the breakdown of cyclic AMP by phosphodiesterase (Hinman, 1972). Westermann and Stock (1970) sug— gested that prostaglandins interfere with binding of ATP to adenylate cyclase to decrease cAMP concentrations, and Blecher et a1. (1969) presented evidence that there are two agonist binding sites on adenylate cyclase and that PGE inhibits only one. For reviews on 1 this subject, see Daly (1976), Hittelman and Butcher (1973) and Kuehl (1974). That the prostaglandins may be involved in the hypothalamo- hypophyseal control of gonadotropin secretion remains to be determined. Harms, Ojeda and McCann (1973), Spies and Norman (1973) and Tsafriri et al. (1973) were among the first to suggest a role for prosta- glandins in control of pituitary hormone secretion. Harms et a1. (1973) reported that PGE injected into the third ventricle caused 2 a 4- to 5-fold increase in LH, 15 minutes after treatment. Prosta- glandin E caused a significant increase in prolactin, and PGF or 1 lo PGFZd had no effect on either LB or prolactin. In contrast to 15 intraventricular injection, PGE1 or PGE2 failed to alter LH or pro- lactin when injected directly into the anterior pituitary. Thus, the results from these data suggest a hypothalamic site of action for the prostaglandins. Spies and Norman (1973) also reported increased LH after intra- ventricular injection of prostaglandin; however, in contrast to Harms et al. (1973), PGE elicited LH release and was more effective 1 than either PGE2 or PGan in inducing ovulations. Similar to the results of Harms et a1. (1973), intrapituitary infusion of PGE1 was less effective than intraventricular infusion in causing LH release, suggesting that the primary site of action was in the central ner- vous system. Tsafriri and co-workers (1973) also concluded that PGE2 was effective in causing LH release. Several papers have confirmed that PGE1 and PGE2 cause release of pituitary hormones (Batta, Zanisi and Martini, 1974; Harms, Ojeda and McCann, 1974). Whereas Harms et a1. (1973) reported no increase in LH after PGFZd' recently Warberg, Eskay and Porter (1976) reported a marked increase in LH after infusion of PGFZa into a lateral ven- tricle. Furthermore, PGFZd caused an increase in serum LH in hamsters (Saksena, Lau and Chang, 1974), in sheep (Carlson, Barcikowski and McCracken, 1973) and in cattle (Hafs, 1975; Louis et al., 1974). In heifers, blood plasma LH, prolactin, growth hormone (GH) and glucocorticoids increased either after a single im injection or a constant iv infusion of PGF . In bulls as in cows, increases in 2a blood prolactin and glucocorticoids were related to the dose of PGFZa (Hafs, 1975). However, Haynes et a1. (1975) failed to prove that administration of PGFZa caused LH release. 16 Recently, infusion of PGE into a lateral ventricle resulted in 2 a 2- to 3-fold increase in LHRH in portal blood plasma (Eskay et al., 1975), suggesting that LH release was mediated at least in part by an increased secretion of LHRH after PGEZ. In agreement, Ojeda, Wheaton and McCann (1975) demonstrated that PGE2 stimulated release of LHRH. They suggested, therefore, that the hypothalamus was the principal site of action of P632 to cause LH release. Indirect support for this suggestion was provided by Chobsieng et a1. (1975) when they reported that antiserum to LHRH blocked PGEZ-induced release of LH in rats. In a recent report, Harms, Ojeda and McCann (1976) indicated that the effects of PGE2 on LH release were not mediated through adrenergic, dopaminergic, serotoninergic or cholinergic receptors. Antagonists which were previously shown to inhibit or block these receptors were unable to block PGE -induced release of LH. These 2 observations suggested that PGE acts directly on LHRH neurons or 2 other LHRH-secreting elements to stimulate release of LHRH into portal vessels. In my opinion, the published data collectively indicate that the principal site of action of PGE2 on LH release is on the central nervous system. As reported by Harms et a1. (1973, 1974), prosta- glandins may act indirectly at the pituitary, but the response was small relative to that after intraventricular administration of PGE2. Treatment of rats with inhibitors of prostaglandin synthesis suppressed ovulation (Armstrong and Grinwich, 1972; Behrman, Orczyke and Greep, 1972). Indomethacin appeared to exert at least part of its effect at the ovary (Armstrong and Grinwich, 1972; Tsafriri, Koch and Lindner, 1973). However, aspirin interfered with pituitary l7 gonadotropin release (Behrman, Orczyke and Greep, 1972). In the latter study, aspirin blockade of ovulation was reversed by adminis- tration of LH or GnRH. In contrast, ovulation blockade by indo- methacin was not reversed by either treatment. Similarly, indomethacin blocked the estradiol induced release of LH in anestrous sheep (Carlson et al., 1974). More recently, Saksena et al. (1975) reported that treatment of male rats with indomethacin caused decreased blood LH and testosterone. They suggested that the decrease in testosterone was mediated by decreased plasma LH concentrations. Since indomethacin is known to block synthesis of prostaglandins and prostaglandins administered into the brain cause LH release, these results suggest evidence of a role for endogenous prostaglandin synthesis in release of LH. How- ever, additional research will be necessary to determine the inter- relationship of the prostaglandins with adrenergic, dopaminergic, serotoninergic and cholinergic neurotransmitters which are known to be involved with pituitary hormone secretion (reviewed by Wilson, 1974). The presence of both E and F prostaglandins in the testis (Carpenter, 1974; Hargrove et al., 1973; Michael, 1973), the ability of the testis to synthesize prostaglandins (Carpenter et al., 1971) and the rapid metabolism of prostaglandins (Anggard, Larsson and Samuelsson, 1971; Nakano, Montague and Darrow, 1971; Nakano and Prancan, 1971) implicate these biologically active substances in a variety of testicular functions, including spermatozoal development and metabolism, the ability of sperm to fertilize, the ejaculatory process and steroidogenesis. These areas were recently reviewed by 18 Cenedella (1975). For purposes of this section, only the effects of prostaglandins on steroidogenesis will be discussed. The majority of evidence in the literature supports the hypothe- sis that the prostaglandins inhibit testosterone secretion. Chronic admdnistration of PGE2 or PGan (Bartke et al., 1973; Saksena, El Safoury and Bartke, 1973) or PGAl or A2 (Saksena, Lau and Bartke, 1974) to male rats and mice resulted in a decrease in accessory gland weights and plasma testosterone concentration. More recently, Bartke, Kupfer and Dalterio (1976) reported that production of testosterone by decapsulated mouse testes in vitro was inhibited by adding PGAl, PGA2 or PGE2 to the incubation medium. Behrman et a1. (1971) suggested that the inhibitory influence of prostaglandins in the rat ovary could be mediated via alterations in available esterified cholesterol. However, the decrease of ovarian cholesterol esters following administration of PGde to female rats (Behrman et al., 1971) was in direct contrast to the increase in cholesterol ester concentrations in mouse testes after PGFZa (Bartke et al., 1973), although steroidogenesis was inhibited in both cases after PGF Bartke suggested that this increase in 2a' testicular concentration of esterified cholesterol was due to inhi- bition by PGF of the utilization of esterified cholesterol. More 2d research will be needed in this area to clarify the mechanisms involved. Prostaglandins can have a dramatic effect on testicular blood flow. Free and Jaffe (1972) and Finer-Jensen and Soofi (1974) reported decreased testicular blood flow after administration of PGF to rats. Free and Jaffe (1972) reported that intratesticular 2a injections of PGF into rats significantly increased testis venous 20 19 pressure and reduced blood flow. Prostaglandin E1 and E2 also decreased blood flow, but PGF appeared to be the most potent. 20 Thus, decreased blood flow could explain the decrease in testosterone after the prostaglandins in the studies of Bartke et a1. (1973), Saksena, E1 Safoury and Bartke (1973) and Saksena, Lau and Bartke (1974). On the other hand, testosterone secretion increased significantly when the dog testis was infused with PGE via the spermatic artery 2 (Eik-Nes, 1969). Eik-Nes suggested that PGE might increase cyclic 2 AMP concentrations in preparations of rat testes incubations in vitro, a suggestion that Keichline and Hagen (1973) verified. Keichline and Hagen (1973) also demonstrated that LH caused equiva- lent increases in cyclic AMP. When testicular tissue was incubated with 7-oxa-13-prostynoic acid, a competitive inhibitor of prosta- glandins, neither LH nor PGE stimulated increases in cyclic AMP, 1 and the inhibition was not overcome by increasing the amount of LH added to the medium. Furthermore, addition of cyclic AMP to testicu- lar explants, in vitro, resulted in increased testosterone concentra- tions (Rommerts et al., 1972). Thus, PGE like LH, stimulated 1. testosterone secretion in explants from rat testis. The finding that 7-oxa-13-prostynoic acid, a competitive inhibitor of prosta- glandin action, blocked testosterone secretion in response to a LH stimulus suggests that PGE possibly may act as an intermediate in 1 the action of LH on testosterone secretion. Finally, the work of Barcikowski, Saksena and Bartke (1973) suggested that androgens may be involved in the control of plasma prostaglandin concentrations in rats. In their study, plasma prosta- glandins were significantly decreased 2 weeks after castration, and 20 administration of testosterone propionate restored plasma concentra- tions of PGan. In summary, this review provides evidence that LH is the pri- mary stimulator of testosterone secretion in the male. However, the complete physiological control of LH and testosterone secretion remains to be elucidated. The evidence is persuasive, at least in the rat, that the prostaglandins are implicated in LH release, but Haynes et a1. (1975) were unable to prove that PGan caused LH release in the bull. Furthermore, the majority of evidence in the literature supports the hypothesis that the prostaglandins inhibit testosterone secretion, but Haynes et a1. (1975) reported increased testosterone concentrations after PGFZa in bulls. Therefore, the objectives of this dissertation were to determine: 1. The temporal relationship between blood serum LH and tes- tosterone in bulls given a single subcutaneous injection of PGFZa' 2. Whether a continuous iv infusion of PGFZo can maintain elevated blood LH and testosterone in bulls. 3. Whether PGFZo causes LH release in bulls pre—treated with a progestogen. 4. If PGan acts centrally at the hypothalamo-pituitary axis to cause release of LH. Since four separate experiments are contained in this disserta— tion, the introduction, materials and methods and results and dis- cussion will be given for each experiment separately. EXPERIMENT 1 THE TEMPORAL RELATIONSHIP BETWEEN BLOOD SERUM LUTEINIZING HORMONE AND TESTOSTERONE AFTER PGFZa OR SALINE IN BULLS Introduction As a prelude to determining the effect of PGF d on sperm output 2 in bulls, Haynes et a1. (1975) conducted an experiment to define some endocrine changes in bulls given PGF They reported blood testos- 2a' terone increased to peaks at l to 2 hours after treatment with PGFZa' and the testosterone peaks were proportional to the dose of PGFZo' However, Haynes et a1. (1975) were unable to detect significant increases in serum LH after treatment with PGFZo' Since it is gen- erally assumed that normally LH is the stimulator for testosterone secretion in bulls (Mongkonpunya et al., 1974; Smith et al., 1973), the following experiment was conducted to determine the temporal relationship between serum LH and testosterone in bulls after a single administration of PGFZo' Materials and Methods Each of five Holstein and three Guernsey bulls (7 to 12 months and weighing 301 :_21 kg) was given (so) 0.9 percent saline or 20 mg PGFZa Tham salt (14.9 mg free acid) in a two-period crossover design, with repeated measurements on each bull. 0n the first day, four bulls were given PGF a and four were given saline, and the treatments were 2 reversed on the following day. 21 22 One day before the experiment, a jugular vein was punctured with a lZ-gauge thin wall needle (Becton-Dickinson, Rutherford, NJ, T462 LNR) and approximately 15 cm of intravenous polyvinyl tubing (Bo Lab, Derry, NH, 88317, Stock No. V10) was passed through the needle into the vein. Then the needle was removed and the exposed end of the tubing was fitted with a 16-gauge tubing adapter, flushed with 3.5 percent sodium citrate solution and sealed until use for blood col- lection. The cannula was held in a reclosable pouch made from adhesive tape and affixed to the neck with tag cement (Nasco, Fort Atkinson, WI, C2283 N-FO-6l7). To collect blood, approximately 3 to 5 m1 of blood and sodium citrate solution were withdrawn and discarded. Then, 10 m1 of blood was taken and transferred to a 15 x 85 mm test tube. The final step consisted of flushing the cannula with 4 ml of sodium citrate solution and resealing the cannula. Blood was taken from each bull for 1 hour prior to treatment and for 8 hours after treatment to characterize hormonal changes. To maximize the possibility of detecting changes in LH after either PGF20 or saline, blood was collected at 15-minute intervals for 1 hour prior and 2 hours after treatment, at 30-minute intervals for 2 hours and at hourly intervals for the remaining 4 hours. Blood was allowed to clot at room temperature for 2 hours and stored at 4 C for approximately 48 hours prior to centrifugation at 2500 xg for 20 minutes. Serum was decanted and stored at -20 C until assayed. Serum LH and testosterone were determined by specific radio- immunoassay previously described by Convey et a1. (1976) and Mongkonpunya et a1. (1975), respectively. The standards for the 23 assays reported were NIH-LH-BB (National Institutes of Health, Bethesda, MD) and testosterone (Sigma Chemical Company, St. Louis, M0). Serum hormone data from this two-period crossover experiment were analyzed by split-plot analysis of variance to account for correlation of errors arising out of repetitive measurement of individual animals (Gill and Hafs, 1971). Heterogeneous variance from one treatment to another was tested by Hartley's Fmax test (Hartley, 1950) and if the departure from homogeneous variance was significant, then a conserva- tive tabular value of F was used to test main effects and interactions (Gill and Hafs, 1971). Selected comparisons were made by Scheffé's procedure (Kirk, 1968). Results and Discussion In agreement with previously reported data (Mongkonpunya et al., 1974), basal concentrations of LH averaged 1.1 :_0.1 ng/ml in bulls given saline or PGF a (Figure 2) and no significant baseline differ- 2 ence was observed between the two treatments. After PGF LH 2d' increased (P<.05) to 3.5 :_l.0 ng/ml at 30 minutes, peaked (3.9 i 0.9 ng/ml) at 45 minutes and declined to pre-injection values by 4 to 5 hours. An unexplained increase in LH in one of the eight bulls caused a secondary increase in mean LH approximately 2 hours after PGFZo treatment. If the data from this bull were excluded, mean serum LH would have decreased to baseline within 2.5 to 3 hours. In contrast, average serum LH was not significantly increased during the 8 hours after saline (Figure 2); the average ranged between 0.6 :_0.2 and 1.4 :_0.4 ng/ml for the eight bulls. The difference between LH released after PGF2o and that released after saline declined with 24 Figure 2. Blood serum LH in eight bulls given saline or 20 P . mg GFZa (sc) 25 0‘ I LH (no/ml) m 080!an -| O l 2 3 4 5 6 7 8 Hours After Injections Figure 2 26 time, resulting in a significant (P<.005) interaction of treatment and time. The LH released in response to PGF is clear in the present Zd experiment, because LH increased in each of the eight bulls after PGF but in only one bull after saline. In retrospect, this result 2d' was dependent upon the good fortune that all of the bulls were in a nadir of LH at the time of PGFZd treatment; otherwise, interpretation would be complicated by the normal episodic release of LH. Further- more, if the releases of LH are influenced by external stimuli or a conditioned stimulus, then the treatment effects would be confounded with the external stimuli. Assuming that the normal release in LH occurs at random intervals, then one may increase the opportunity to reveal significant treatment differences by increasing the number of bulls used in the experiment, by reducing the number of episodic spikes of LH with exogenous steroids or inhibitors of LH release, or by prolonging the treatment response so that the episodic spikes are masked. The LH results of the present experiment are in contrast to those of Haynes et al. (1975). Although they observed increased serum LH concentrations after PGFZd' episodic secretion of LH in control bulls made interpretation of the data unclear. Possibly, differences between results from the present experiment and those from the experiment of Haynes et a1. (1975) reflect differences in age of bulls. They used mature bulls (5 to 9 years) which were rou- tinely used in a semen collection center, whereas bulls used in the present experiment were relatively young (7 to 12 months) and sexually inexperienced. Smith et a1. (1973) reported that testosterone increased consistently in mature bulls, but not in young bulls 27 following ejaculation. Katongole et a1. (1971) reported that stimuli such as the sight of a cow may elicit release of LH. Perhaps, in the experiment of Haynes, the presence of semen collection personnel or the subcutaneous sham injection could have caused a release of LH mediated by conditioned neuroreflex mechanism. Increased LH after PGFZa treatment has been reported previously. Carlson et a1. (1973) observed an increase in serum LH after intra- carotid infusion of PGF into diestrous but not anestrous ewes. In 2d addition, a single injection of PGF into estrogen pre-treated, 20 ovariectomized hamsters (Saksena et al., 1974) or into male rabbits (Agmo, 1975) and intraventricular administration of PGan in rats (warberg et al., 1976) were followed by increased blood LH. Further- more, treatment of male rats with indomethacin, an inhibitor of prosta- glandin synthesis, caused a significant decrease in the concentration of LH and testosterone (Saksena et al., 1975). Indomethacin also blocked the estradiol-induced release of LH in sheep (Carlson et al., 1974). Both of the latter studies suggest an obligatory role for prostaglandin synthesis in release of LH, but they do not provide definitive proof for PGan involvement in LH release because indo- methacin blocks synthesis of PGE as well as PGF. However, effects of indomethacin may be secondary because indomethacin may not only have a central effect at the head, but may affect physiological systems anywhere in the body. Serum testosterone concentrations did not change significantly during the 8 hours after saline treatment (Figure 3). Average testosterone concentrations ranged between 4.3 :_1.4 to 8.3 :_2.7 ng/ml. Episodic secretion of LH and testosterone occurred in tandem in each bull, but on the average neither LH nor testosterone changed 28 significantly after saline (Figures 2 and 3). Before PGF blood 2o' serum testosterone averaged 4.5 :_0.2 ng/ml (Figure 3); it increased (P<.01) synchronously in each of the eight bulls to 8.5 i 0.9 ng/ml by 60 minutes after PGF , peaked at 15 to 16 ng/ml between 90 and 2a 120 minutes, and then declined toward pre-injection concentrations by 180 minutes. The overall analysis of variance for the testosterone response indicated a significant (P<.06) treatment effect, a signifi- cant (P<.01) time effect and a significant (P<.001) treatment by time interaction. Bull effects and period effects were nonsignificant. The increase in blood serum testosterone after PGFZd reported in this study agrees well with that described by Haynes et a1. (1975) for mature bulls. In both studies synchronized peaks of testosterone occurred with maximal concentrations about 2 hours after treatment. As another means of evaluating the testosterone response, the interval to and duration of the first detectable increase in testos- terone after PGFZo or saline were determined. An increment of 2 standard deviations over pre-injection values was considered a surge. Injection of PGF a reduced (P<.01) the interval to first increase in 2 testosterone and prolonged (P<.05) the duration of the testosterone surge relative to those after saline (Table 1). Similar results were reported by Haynes et a1. (1975). Interpolating from their data, 20 mg PGF Tham salt administered to the bulls in the present study 20 would be a dose sufficient to elicit a maximal response, based on body weight of the bulls. The increase in testosterone which we report is in contrast to reduced testosterone at 3 and 12 hours after the last of a series of PGFZd injections in rats and mice (Bartke et al., 1973; Saksena et al., 1973). Aside from the possible species differences, it is 29 Figure 3. Blood serum testosterone in eight bulls given saline or 20 mg PGan (sc). Blood testosterone (ng/ml) 30 Saline IIIIIIIIIIII I I I I I I 2 3 4 5 6 7 8 Hours after saline or PGFZa Figure 3 31 Table 1. Interval to and duration of first testosterone surge after PGFZd (20 mg, so) or saline in eight bullsa Treatment Criteria Saline PGFZa Interval to surge 274 1.70 50 :.10 Duration of surge 56 + 14 173 + 86 a . o a o o I An increment of 2 standard dev1ations over pre-injection values was considered a surge. Values are means :_standard errors. possible that the reduced testosterone in rats and mice may have been associated with the chronic administration of PGFZa in amounts which were much greater (~40-fold) on a body weight basis than I gave to bulls. In the experiments of Bartke et a1. (1973) and Saksena et a1. (1973), chronic repeated injections may have depleted the hypothalamic stores of LHRH or possibly the pituitary releasable stores of LH. Previously Ojeda et a1. (1976) presented evidence that PGE2 acts on the LHRH neuron to induce discharge of LHRH into the hypophyseal portal vessels; then LHRH evokes release of LH. Thus, after chronic exposure to a prostaglandin stimulus, the ability of the hypothalamus to release LHRH or the capacity of the pituitary to release LH may be diminished. The net effect as shown by Bartke et a1. (1973) would be decreased plasma testosterone concentration and reduced accessory gland weights. Perhaps if LH is released by acute treatment with PGF , PGF 0. 2 a may cause increased plasma concentrations of testosterone 2 in rats as in bulls. Rippel, Johnson and White (1974) reported that the LH response was reduced following consecutive injections of GnRH at 24-hour intervals to anestrous and ovariectomized ewes. These 32 data support the hypothesis that the pituitary becomes refractory to repeated GnRH stimuli. In the present study, the temporal relationship of changes in blood LH and testosterone (Figure 4) after an injection of PGF20 resembles that in untreated bulls (Mongkonpunya et al., 1974) and that in bulls given synthetic GnRH (Mongkonpunya et al., 1975). For example, Mongkonpunya et al. (1974) reported an average of 3.7 I.0'3 testosterone surges per 24 hours with an average magnitude of change of 2.7 :_0.6 ng/ml. In agreement with the present study, each increase in LH was followed by an increase in testosterone concentration which averaged 12.0 :_0.7 ng/ml. Prostaglandin F a caused a synchronous 2 increase in serum LH in the present study; the first significant increase occurred at 45 minutes and preceded the first significant increase in testosterone which occurred at 60 minutes post-injection (Figure 4). Furthenmore, the average peak of LH after PGF was 3.9 I 0.9 2d ng/ml, comparable to peak episodic LH in Monkonpunya's experiment. Similarly, 20 mg PGF a administered to bulls in the present experi- 2 ment (a dose sufficient to elicit a maximal response based on Haynes et al., 1975, data) resulted in increases of LH within physiological limits when compared with control bulls. Although no significant temporal relationship exists between average LH and testosterone concentrations in bulls given saline (Figure 5), an average of one episodic surge of testosterone (average peak 14.2; range 8.0 to 22.8 ng/ml) occurred apparently at random intervals during the 8-hour period after bulls were given saline. Increased blood LH (average peak 3.0; range 1.5 to 4.3 ng/ml) occurred before each of these testosterone surges. However, because the 33 . Aomv cm mom 06 ON co>flm maasn unmflm cfl mcoumumoumou pom ma Esuwm ooon .v ousmflm 34 e 333 can. 56:22:35 tote 950: m n. m m .V m N _ O _I 1 u q T . q d O C C O . . . . m .V m 1 .1. GEOLO~QO~OOP Uflkmvl IO .N IV (nu/On) euomisotsel 10 H1 S2 33 . Aomv UN mom we om cw>fim maazn ucmflo ow odououmOOmou odd mg Esuom oooam .v ousmflm 34 v 0335 awn. 565.023...“— 356 950: m n m m e m m _ o T q d (1) 0 SI’ NO 2.22330... 0 (nu/bu) euomtsoisej, JO l-l'l I L :r. s $2 35 .mCHHom co>Hm maasn usmflo cw ocououmoummu one mu Eamon ooon .m museum 36 m madman 3:2,... tote 2:0: 0 0 .v m N _ O s. in m b 0:0..Opa0uaok I J 1 a) (D V N O (nu/bu ) euoaetsoisej, 10 H1 L 25.5 EN9 2 37 testosterone and LH surges after saline occurred at random, the average LH and testosterone concentrations did not change signifi- cantly during this 8-hour period. The major conclusion from this experiment is that administration of PGF2a to bulls causes increased blood LH, followed by increased blood testosterone. Both peaks and the temporal relationship of these LH and testosterone surges resemble the normal episodic releases of these hormones in bulls. EXPERIMENT 2 THE EFFECT OF A CONTINUOUS INTRAVENOUS INFUSION OF PGan ON LUTEINIZ- ING HORMONE AND TESTOSTERONE SECRETION IN BULLS Introduction The results of the first experiment clearly showed that PGan administered to bulls as a single subcutaneous injection caused an increase in both LH and testosterone in bulls. Furthermore, after PGFZa the first significant increase of LH preceded the first signifi- cant increase of testosterone, the same as the temporal relationship of LH and testosterone in control bulls. In addition, the peak and duration of elevated LH and testosterone were similar to those reported in control bulls. In the first experiment, I advanced the hypothesis that the decrease in testosterone observed by Bartke et al. (1973) and Saksena et a1. (1973) was the result of chronic treatment with PGFZa' possibly refractoriness or exhaustion of the hypothalamo-pituitary axis to a continuous prostaglandin stimulus. In view of these considerations, the objectives of the second experiment were: 1. To determine if a continuous prostaglandin stimulus would maintain elevated LH and testosterone secretion in bulls. 2. To determine the temporal relationship between LH and testos- terone following the conclusion of a continuous PGFZa stimulus in bulls. 38 39 Materials and Methods This experiment utilized four Holstein bulls (12 months old, weighing 355 :_19 kg) with cannulae inserted in both jugular veins as previously described for the first experiment. One cannula was used for infusion of saline or PGFZa Tham salt and the other for blood collection. A Harvard pump (Harvard Apparatus, Mills, MA) was used to infuse saline or PGFZa' Prostaglandin F20 Tham salt was infused (0.2 mg/min) into two bulls and the other two bulls were given an equivalent quantity of saline vehicle for 20 hours. The infusion treatments were reversed beginning 28 hours after completion of the first infusion. Thus, each bull was given 240 mg PGF a during 2 the 20-hour infusion. Blood was sampled at 30-minute intervals for 1 hour before infusion, during the 20-hour infusion, and for 8 hours afterward for determination of blood plasma LH and testosterone by radioimmunoassays described in Experiment 1. The data were analyzed by a split-plot analysis of variance (Gill and Hafs, 1971) and selected comparisons were made by Scheffé's procedure (Kirk, 1968). Results and Discussion Blood plasma LH (Figure 6) averaged 1.2 i 0.1 ng/ml before infusion of PGFZa; it doubled (P<.07) within 1.5 hours after the infusion of PGFZa was started and peaked (P<.01) at 4.2 :_0.8 ng/ml at 6.5 hours before declining to basal concentrations before the end of the infusion. By contrast, average LH concentration fluctuated between 0.6 i 0.1 to 1.8 :_1.1 ng/ml and did not change significantly during the infusion of saline. As shown in Figure 6, average LH con- centrations following the end of the infusion did not differ in bulls 4O .lcas\me N.oe UN mom no wCHHMm mo coflm5mcfl >fl mcauso ma mamoam ooon .o masons 41 (Iw/DU)H'I Infusion 20 IS IO 25 Hours Figure 6 4O .Icwexme N.oc UN mom Ho oCHHmm mo cofimSMCH >fl mcwuso ma campam ooon .o endows 0'6 .0" 0’ A' .‘r O .> :. .l - -0 0’ U ° --_ (Iw/fiulH'I Infusion IS 20 25 IO Hours Figure 6 42 given saline or PGF . The overall pattern of LH during infusion of 2d PGFZo was similar to the pattern of LH during a continuous infusion of GnRH to bulls of equivalent age (Haynes, personal communication). Luteinizing hormone increased to a peak, and then declined before the end of GnRH infusion. The interval to peak LH résponse was shorter, occurring at about 2 hours, and the peak of LH was much greater, averaging greater than 40 ng/ml during infusion of GnRH. Presumably the pituitary becomes refractory to continuous GnRH stimulus. Simi- larly, the decline of LH before the end of the PGF infusion may be 2a refractoriness of the hypothalamo-hypophyseal axis to a continuous stimulus. The testosterone profiles for each of the four bulls during infusion of saline or PGF20 are illustrated in Figure 7. Testosterone increased to greater than 10 ng/ml in each bull within 2.5 hours after the start of the PGFZa infusion, remained clearly elevated for 15 hours in three bulls and elevated for the entire infusion period in bull 2. Episodic surges of testosterone similar to those of con- trol bulls resumed well within 8 hours after the conclusion of the 20—hour infusion of PGFZd' Two or three episodic surges of testosterone occurred in each of the bulls during the 20-hour infusion of saline (Figure 7); the peak (17.2 :_0.2 ng/ml) of these surges was equivalent to the peak concentration of testosterone during infusion of PGF2a' The temporal relationship of LH and testosterone in bulls infused with saline (Figure 8) was in agreement with previously published data for untreated bulls (Mongkonpunya et al., 1974). In 18 of 19 cases, blood testosterone surges were preceded by an increase in LH (average peak 2.8 :_0.3 ng/ml). Furthermore, increases in LH of as little as 43 Figure 7. Blood plasma testosterone in four bulls during infusion (iv) of saline or PGan. No samples were collected during a 20-hour period between the first (left) and second (right) experi- mental periods. Testosterone (ng/ml) 20 IS IO l5 44 BULLI l fl. PGFZa famine BULL 2 K) 20 0 IO 20 Hours infusion of POI-‘20 (0.2 mg/min) or saline Figure 7 43 Figure 7. Blood plasma testosterone in four bulls during infusion (iv) of saline or PGFZQ. No samples were collected during a 20-hour period between the first (left) and second (right) experi- mental periods. Testosterone (ng/ml) 20 IS IO IS 44 BULLI I T? PGF-20 rDOIIDC BULL 2 I l I A‘I '7 N) 20 0 IO 20 Hours infusion of PGFZQ (0.2 mg/min) or saline Figure 7 45 Figure 8. Blood plasma LH and testosterone in four bulls during infusion of saline. LH (ng/rnl) 0--o 4 ’ o---oLH 46 lk Saline :I-l 20 e—e Testosterone 4p 3‘ 120 BULL-2 I 3- . : its 2. i 1‘.” I I ' .|o 'A..q1'iii'l.;b \5 [11.1.1 I 1111.1..41 1 1 1 I 1 1 1 1 I 1.4.11I'. 1 1 O 9 O 3 I5 «Is BULL-3 8 :I '0 2 e. o I ' . d "‘ ’o i : 3.. " ...o.ooo.00.o..0.ooo.0 0.. .0009...0...o .'e°°°e 15 o 1 1 1 1 44L 1 1 1 L4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 o 4’r Q j 2C) ;; BULL-4 25L :; 'l5 2) ' I '8 ' 1’ ‘ . ‘IO a". ". : q ,.‘ ,. I. o.... s .00. . °° 0.0. 0.. ' .°e o o. .': 0.0 5 I 590"”. ° .° ‘r-‘ o I 1 1 1 1 I4 1 1 1 I 1 1 1 1 I 1 1 1 1 L L 1 1 I l 1 800 I300 IBOO 2300 400 900 Hours Figure 8 TESTOSTERONE (no lml) o—-e 45 Figure 8. Blood plasma LH and testosterone in four bulls during infusion of saline. 46 .. Saline " 20 4 ’ o.-—OLH I o——a Testosterone BULL-l 3 D T '5 2 _ '9 . l0 I I ' 5 is o I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 0 4p 8‘ 120 BULL-2 : 3- I ~l5 -. I , ? ' : t I ' ‘ 3 52' I. :‘I‘. I9 ‘ ‘ "0 "I... A“ H' '2’ \f E "'eeo..e. .0..o.'o.. ..o. w .°°.04'o. 1 1 1 11 1 1 1 1 1 1 1 1 I 1 1 1 14 1 1 1 1 I 1 1 2° ' ‘ 9 ° 5 BULL’3 I? :1 2 ;-\ """'—' [1“lt , : 1“ . 3 q|() ' \ I. I.. : .00 I- ...00000.0 .0... as... 0.0 .0001...0. .0 .'o°°'e ‘5 O 1 1 1 1 1 l 1 1 1 111 1 1 1 141 1 1 I 1 1 1 1 l 1 1 o 4" Q 120 I'. ELL-.3 3r . I'. -.5 2 I ' I '8 I0 5 l - I . . I“ ' l e wt.- '- .0... .0. . .. 0.00 o . ..e o e. .': e I ”904’": “11° ‘II'-° 5 011 1 1 1 1 1 141 11 1 14 1 1 1 LI 1 I 1 1 1| 1 1 800 I300 I800 2300 400 900 Hours Figure 8 TESTOSTERONE (ng lml) H 47 1 ng/ml resulted in increased testosterone, but as illustrated for bull 4, the magnitude of change of LH was not proportionally related to the testosterone response. The temporal relationship between LH and testosterone in bulls infused with PGF was consistent with the hypothesis that LH is the 2d primary stimulus of testosterone secretion in bulls given PGFZa (Figure 9). For example, in all four bulls, blood LH increased prior to the increase in testosterone and, as blood LH declined, so did testosterone. Frequent "bursts" of LH were observed in the bulls during infus- ion of PGF in contrast to the less frequent "bursts" of LH in 2a' control bulls (Figure 7). The data suggest that, if PGFZa released LHRH in these bulls as was shown for the rat, the release of LHRH occurred as frequent "bursts" which then caused "bursts" of LH. In two bulls (l and 3) there was a prolonged period of elevated LH, but the LH patterns during these elevations of LH suggested that LH was released in short (”15 minute) "bursts" followed by periods when circulating concentrations declined. The frequent "bursts" of LH maintained what appears to be an elevation in baseline LB during infusion of PGF Certainly as long 2a° as there were "bursts" of LH, there was also increased concentration of blood testosterone. Thus, in overview, the increased concentration of LH during infusion of PGFZa was not the result of an abrupt increase of LH, as was frequently observed after administration of GnRH to bulls (Mongkonpunya et al., 1975); rather, it resulted from an increase in the frequency of bursts of LH. 48 Figure 9. Blood plasma LH and testosterone in four bulls during infusion (iv) of PGFZa (0.2 mg/min). LH (ng/ml)o—-o 49 2K 0- -o L H ‘ 9 e—o Testosterone 1 25 F . I . I\ . 2~ ' ail . .' I R «'0 IJUP ya! gxgykodRIU. :de ' l . - 5 h . .'.o o ‘31- .1.111.1.1.111.1.111.1.1.111.111.1.111.1.1.1.11 . .1.1 0 5 Iamfi 25 (BULLS ' .. ' I N, .m 3 .. . i - l5 . 5" . ‘5!’ IO.‘ .. '0 N 4 ~ oI1I1I1lll1l1l1I1I1I1I1I1I1I1I1I1I1I1I1I1I1I1I1I1I1I1I111l1l 4 . 3!!!! 4 Q .20 3 . I ’ I - l5 0 v . .0 ' I. 2 '0. . k8 " I0 ll- .. 5 0 0 800 I300 I800 2300 400 900 HOURS Figure 9 TESTOSTERONE (no/ml) e—e 48 Figure 9. Blood plasma LH and testosterone in four bulls during infusion (iv) of PGFZa (0.2 mg/min). LH (no/ml)o-o 49 .‘= PGF“ #1. s . ‘h BULL l ,' III o--0LH ‘ 30 5 _ : ‘ 9 e—o Testosierone . 25 I 4» ‘ri 0 O 4 . -2° 3 . . I ' I9 -I5 I I 2x I Ragga, .IO 0 ¢ . l- , .° +5 0800 I300 I800 2300 400 900 HOURS Figure 9 TESTOSTERONE (no/ml) H 50 UN .A:«E\mE N.ov mum mo A>HV COHm5mca mcflusp mHHDQ snow CH wcouwumoumwu Ucm mg mammHm COOHQ wmmnm>d .OH manage 51 (Ila/60) 3N033i$01$3i m. OH mousse $32.. 8 n. o. q u 4 .9 Seasewoa 2.9. 2378K N [O (nu/6") H1 52 When the LH and testosterone data for all four bulls were com- bined, as shown in Figure 10, average blood LH and testosterone increased and decreased in tandem. However, blood testosterone reached maximal concentrations sooner and the peak persisted longer than the comparable LH response. Perhaps the peak of testosterone at 2.5 hours after infusion of PGan damped the increase in LH, because peak LH concentration was not apparent until approximately 6 hours after the infusion was started. Notwithstanding this observation, blood LH progressively increased for at least 4 hours during the PGFZa infusion, in the face of high (about 15 ng/ml) testosterone (Figure 9). Then LH started to decline at least 3 hours earlier than testosterone, preceding the decline in testosterone in all four bulls. This decline in LH beginning about 8 hours after the start of infusion of PGFZa may be explained in several ways. If PGFZa was acting directly on LHRH neurons to cause release of LHRH, perhaps the hypothalamic content of LHRH was exhausted. To my knowledge, there are no reports to support this hypothesis, but if in fact the hypo- thalamic content of LHRH was depleted, the effect was not long term because episodic secretion of LH and testosterone resumed again within 8 hours after the end of the infusion of PGFZa' Perhaps a more plausible explanation is depletion of releasable stores of LH from the pituitary. In sheep, the LH response to consecutive injections of GnRH was reduced even when GnRH was administered at 24-hour intervals (Rippel et al., 1974). Alternatively, prolonged high concentrations of testosterone in the present experiment may begin to inhibit LH release at about 8 hours of PGFZa infusion, similar to testosterone feedback on LH 53 release after successive injections of GnRH in rams (Galloway et al., 1974). In summary, a constant infusion of PGF caused a prolonged 2a increase in LH and testosterone secretion in bulls, but neither LH nor testosterone remained elevated during the entire infusion period. The results of these data suggest that the hypothalamus or pituitary may become refractory to a continuous stimulus of PGF I speculate Za' that the mechanism(s) involved in causing decreased concentrations of LH and testosterone in this experiment could explain the decreased concentration of testosterone after chronic administration of PGan to rats and mice (Bartke et al., 1973; Saksena et al., 1973). EXPERIMENT 3 THE EFFECT OF EXOGENOUS LH OR PGFZa ON BLOOD LH AND TESTOSTERONE IN BULLS GIVEN MELENGESTROL ACETATE Introduction The results of the first two experiments demonstrated that PGFZa caused acute release of LH followed by increased testosterone secre- tion in bulls. The temporal relationship between changes in LH and testosterone as well as the magnitude of changes of these hormones were similar to changes observed in control bulls (Mongkonpunya et al., 1974). Similar surges of testosterone followed administration of LH (Smith et al., 1973) or GnRH (Mongkonpunya et al., 1975). Thus, LH normally induces testosterone secretion in bulls. To the extent that PGFZa stimulates acute release of LH, further experiments to determine if inhibitory effects of gonadal steroids or other compounds could be overcome by PGF u might add insight into 2 control of LB and testosterone by PGF in bulls. 2a In steers, basal LH was elevated by comparison to that in bulls (McCarthy and Swanson, 1976; Mongkonpunya et al., 1974) and testos- terone or estradiol replacement caused a decline in baseline LH. Similarly, a synthetic progestogen, melengestrol acetate (MGA), sup- pressed the ovulatory surge of L8 in cows (Hill et al., 1971). The high progestogenic potency and the ease of oral administration of MGA made this progestogen attractive in this preliminary experiment to 54 55 test whether PGFZa could override possible progestogen inhibition of LH release in bulls. Thus, the objectives of this experiment were to determine: 1. If MGA inhibited the episodic secretion of LH and testos— terone in bulls. 2. If administration of PGFZa can overcome the inhibitory effects of MGA on LH and testosterone secretion. 3. The effects of exogenous LE on testosterone secretion in bulls treated with MGA. Materials and Methods In a preliminary experiment four Holstein bulls (average weight 355 :_l9 kg) were used during a 3-day period. On the first day, jugular blood was taken at 0, 15, 30, 45, 60, 75, 90, l05, 120, 135, 150, 165, 180, 210 and 240 minutes relative to the start of sampling at 0800 hours. On the second day each bull was fed 1.01 kg of con- centrate feed containing 0.5 mg MGA at 0700 hours and again at 1900 hours. Blood samples were not taken on the second day. Starting at 0700 hours on the third day, each bull was fed 0.5 mg MGA, then jugu- lar blood was collected at frequent intervals as on the first day, starting at 0800 hours. Thus, each bull was bled before oral inges- tion of MGA and again starting 25 hours after beginning feeding of MGA. Blood serum testosterone was quantified by radioimmunoassay (Mongkonpunya et al., 1975). Differences in testosterone before and after MGA were compared using Student's tftest for paired observations. The main experiment was conducted as a two-period crossover design with repeat measurements on four bulls (average weight 307 i 36 kg). Each bull was fed 1.0 mg of MGA daily (0.5 mg at 0700 and 56 1900 hours) throughout this main experiment. Starting 24 hours after the first feeding, two bulls were given 20 mg PGF 0 Tham salt (so) 2 and two bulls were given saline (sc). The treatments were reversed 10 hours later. Blood was collected at lS-minute intervals for 1 hour prior to PGF a or saline, at 15-minute intervals for 2 hours 2 after treatment, at 30-minute intervals for 2 hours, and then hourly until 8 hours. Then 48 hours after the first feeding of MGA, each of the four bulls was given 200 ug NIH-LH—BB (iv). The testosterone response from these bulls was compared to four control bulls given 200 ug LH (average weight 328 :_42 kg) after the same sequence of PGFZa treatment, but without MGA. Data from the main experiment were analyzed by split—plot analysis of variance with repeated measurements on each bull (Gill and Hafs, 1971). Results and Discussion In the preliminary experiment, each bull exhibited one episode of increased serum testosterone (range 12.6 to 20.0 ng/ml) during the 4-hour blood sampling period before treatment with MGA. In contrast, surges of testosterone were abolished after the bulls were fed MGA; the highest concentration of testosterone was 2.5 ng/ml (Table 2). The average testosterone concentration before MGA was greater (P<.001) than that after feeding MGA (8.5 t 1.1 vs 1.8 :_0.1 ng/ml) (Table 2). Furthermore, variation in testosterone concentrations due principally to episodic surges of testosterone was greater (P<.001) before than after feeding MGA (SE = 1.1 vs 0.1 ng/ml). Although serum LH was not quantified in this preliminary experi- ment, the results indicate that MGA suppressed episodic secretion of 57 Table 2. Average serum testosterone during a 4-hour interval before and after treatment with melengestrol acetate in four Holstein bulls Before MGA After MGA Dif- Mean Mean fer- Bull :_SE Range :_SE Range ence sang/ml 69 11.5 i_1.6 3.9-20.0 1.6 i_0.1 1.0-2.1 9.9 70 8.8 :_1.3 2.4-16.3 1.8 i 0.1 1.3—2.0 7.0 71 6.4 :_1.3 1.6-12.6 2.0 :_0.1 1.5-2.5 4.4 72 7.3 :_l.0 2.3-14.4 1.8 :_0.1 1.3-2.2 5.5 Average 8.5 2'; 1.1 1.8 i 0.1 6.7 :a 1.2 ap<.001. LH, because in no instance in any of my experiments was testosterone increased without a preceding increase in LH. This conclusion assumes that exogenous MGA did not directly inhibit testosterone secretion at the testis. Hill et a1. (1971) provided evidence for this assumption, because they reported that MGA did not influence either the life span or progesterone secretion of corpora lutea (CL) in cows, but MGA pre— vented the ovulatory surge of LH. The objective of the main experiment was to determine if admin- istration of PGFZa stimulated release of LH and testosterone in bulls given MGA. The analysis of variance for LH revealed that treatment effects approached significance (P=.08); however, time effects and treatment 58 by time interaction were highly significant (P<.001). All other effects were nonsignificant. After bulls were given saline during MGA treatment (Figure 11, top), serum LH concentrations did not change significantly (P>.05). The average LH concentrations ranged from 0.30 :_0.05 to 0.40 :_0.05 ng/ml. Pretreatment means were comparable between bulls given saline or PGFZa' averaging 0.6 :_0.2 and 0.4 + 0.01 ng/ml, respectively, at the time of saline and PGF injection. After administration of Za PGF , serum LH increased (P<.05) to 2.0 :_0.5 ng/ml at 45 minutes, 2a peaked at 2.3 :_0.5 ng/ml at 60 minutes, and then declined to base- line between 4 and 5 hours (Figure 11, top). Similarly to blood LH in bulls treated with MGA after saline, serum testosterone fluctuated between 0.8 :_0.3 and 1.2 :_0.2 ng/ml, and did not change significantly during the sampling period in bulls given saline (Figure 11, bottom). In contrast, serum testosterone averaged 0.8 i 0.3 ng/ml before PGF increased (P<.05) to 13.4 i 20' 4.1 ng/ml at 75 minutes after PGFZd' and reached a peak concentration of 21.3 i 2.3 at 105 minutes. Serum testosterone then plateaued until 3 hours after PGFZd and declined to baseline concentrations at 7 hours after PGF The testosterone response appeared to be 2a' exaggerated compared to the response in Experiment 1. However, the prolonged LH response, illustrated in Figure 11 (top), after PGFZa probably accounted for the prolonged testosterone response. The results of this experiment establish that MGA inhibits epi— sodic secretion of LH and testosterone in bulls. More recently, in a similar study, infusion of testosterone also completely abolished episodic LH release in bulls (Haynes, personal communication). 59 Figure 11. Blood serum LH (top) and testosterone (bottom) in four bulls pre-treated with melengestrol acetate and given saline (sc) or PGFZa (20 mg, sc). d) S E 2 L- g: E E?» 25 ' .E 2 :I .J o 20 E \ l6 0 5 0 I2 C. g e ‘m' ?) 4 *— 0 6O PGF“ or Saline Figure 11 9‘ l- 0 I '1' bg/PGFZQ I I awn. I- , \ I I' Saline b“0.. so... " -| “6“”. e 3 4 5 .PGFZaor Saline 90-“ \ . I i \h PGF“ L )5 ‘s/ .' ‘K I ‘x I ‘x I 5 ,' Saline / “ -| 0 I 2 3 4 5 Hours 59 Figure 11. Blood serum LH (top) and testosterone (bottom) in four bulls pre-treated with melengestrol acetate and given saline (sc) or PGFZa (20 mg, sc). Luteinizin; hormone (ng ml) 20 IS Testosterone (ng / ml) 60 PGF“ or Saline 9. I. O \ :' 5/“an ! h ‘I °°v’°\‘ l- I \ I I' Saline 21‘0.“ O.“ 1" s -‘I‘“a‘”i 2 3 4 5 6 7 e -PGFZaor Saline 90.0.12 \ - I '1' \\ PGFZa _ <5 x/ I' h I, \\ .. , n \ i ' ,' Saline h“ -| 0 I 2 3 4 5 6 7 8 Hours Figure 11 61 Whether MGA acts directly at the pituitary or hypothalamus to inhibit release of LH remains to be determined. In the present experiment, administration of PGF a to bulls pre- 2 treated with MGA resulted in release of LH comparable to that after PGFZa in untreated bulls or episodic release of LH in control bulls observed in Experiment 1. If MGA inhibited release of LH at the level of the pituitary, one might expect no LH release after PGF , even if 2a LHRH was released by the prostaglandin stimulus, as has been demon- strated by Eskay et a1. (1975) in rats. McCarthy and Swanson (1976) concluded that testosterone feedback was not at the pituitary because the LH response to GnRH in steers pre-treated with testosterone was greater than in untreated steers. In support of this conclusion, intrahypothalamic implantation of crystalline testosterone in dogs (Davidson and Sawyer, 1961) and progesterone in rats (Smith, Weick and Davidson, 1969) exerted "negative feedback" on gonadotropic function. In the latter report, the effect of progesterone apparently was specific to the medial basal hypothalamic region, because implants placed in the anterior hypothalamic-medial preoptic region and in the anterior pituitary were ineffective. More recently, however, evidence was provided that progesterone can act on the pituitary to reduce but not block LH secretion after GnRH (Arimura and Schally, 1970). In the present experiment, PGF induced LH release in bulls in 2a which episodic release of LH was suppressed by MGA. Two explanations may be offered in this case, neither of which is definitive. Either PGFZa is acting directly at the pituitary to cause release of LH or PGFZa is overriding or superceding the inhibi- tory effects of MGA at a higher level (hypothalamus or higher centers). 62 The case for PGFZa acting at the pituitary lacks support from the literature because, in most reports, intrapituitary injection of prostaglandin results in low or no release of LH (Harms et al., 1973; Norman and Spies, 1973), although in one report (Warberg, Eskay and Porter, 1976) intraventricular PGFZa caused secretion of LH. I specu- late that MGA inhibited hypothalamic prostaglandin secretion and thereby abolished LHRH secretion and the episodic release of LH. This explanation is compatible with the observation that PGFZa caused release of LH in the face of MGA inhibition of LH in the present experiment. Prior to initiation of Experiment 3, the possibility of MGA interacting directly at the level of the testis to modulate testos- terone secretion was considered. However, as shown in Figure 12, MGA did not interfere with testosterone secretion after LH administra— tion, in agreement with the data of Hill et a1. (1971), who demon- strated that MGA did not alter progesterone secretion in cows. In summary, this experiment demonstrated that MGA suppressed episodic release of LH and testosterone in bulls, but the inhibitory effect of MGA was overcome by PGF The results suggest that MGA 2d° inhibition of episodic LH release was mediated by an action on prosta- glandin secretion. Finally, the results indicate that MGA does not interact directly at the level of the testis to alter testosterone secretion in bulls. 63 Figure 12. Blood serum LH (top) and testosterone (bottom) in four control bulls and four melengestrol acetate treated bulls after 200 ug LH (iv). 64 4.6 . +5 . 4 13 W M . .2 nu nm e 1 m ml. I 10 . ..... no a m 3.. o m m a 4 o :EBE 2635 30.8323... 956.6: 9.5533 Hours Figure 12 63 Figure 12. Blood serum LH (top) and testosterone (bottom) in four control bulls and four melengestrol acetate treated bulls after 200 pg LH (iv). 64 --9---9 Hours Figure 12 m A N G . . ‘.I I 1!. ”HHHI 1 Hll' I 1 L b b P b p n 8 6 4. 2 O 6 2 8 4. O 2535 26:35 9122.866... 9.959. 05552:... EXPERIMENT 4 THE EFFECT OF CAROTID ADMINISTRATION OF PGF2a ON BLOOD LH AND TESTOSTERONE IN BULLS Introduction Although the first three experiments demonstrated that PGFZa causes increased blood LH and testosterone in bulls, the locus of PGFZa action remained to be ascertained. In previous experiments, administration of PGFZa was by subcutaneous injection or intravenous infusion. Therefore, PGFZa could act at one or more of several sites in the periphery to cause release of LH and testosterone. For example, Hafs et al. (1975) convincingly demonstrated that the transitory increase in blood serum LH within 2 hours of PGFZa treatment was the result of a decrease in progesterone secretion, not a direct effect of PGFZa at the hypothalamo-pituitary axis in cows. An analogous possibility could not be excluded on the basis of my first three experiments. In other words, the possibility existed that PGFZa acted directly at the testis to modify testicular hormones, and the alteration in steroid feedback may have been responsible for the increase in LH and the subsequent testosterone surge. Therefore, the objective of my fourth experiment was to determine if PGFZQ acted directly on the brain to elicit release of LH in bulls. Materials and Methods In a 6 x 6 Latin square design, each of six yearling bulls (260 :_27 kg) was given intracarotid infusion of O (saline vehicle), 20, 65 66 200 and 2000 ng of PGan/minute (min) and 2000 ng and 0.2 mg of PGFZa/ min into a jugular vein. The infusion was maintained for 3 hours at 10 ml/hour by a Harvard pump, with lZ-hour intervals between the start of consequentive treatments. In a previous experiment (Experiment 2 of this dissertation), significant increases in LH accompanied infusion of 0.2 mg PGan/min into a jugular vein. This dose (0.2 mg/min) was therefore used as a positive control. On the assumption that 95 per- cent of the PGFZa is metabolized on one passage through the lungs (Piper and Vane, 1969) and 10 percent of the circulating blood passes through the head, then of the 0.2 mg PGFZa/min infusion into the jugular vein, only approximately 2000 ng PGFZa/min reaches the brain. If the major effect of PGF a in stimulating testosterone output is 2 mediated through secretion of LH, then a dose of 2000 hg PGFZa/min given via the carotid should give a LH and testosterone response equivalent to 0.2 mg PGFZa/min administered by jugular infusion and a significantly greater response than 2000 ng PGFZa/min given via the jugular. Thus, the LH and testosterone response was compared after carotid and jugular infusion of 2000 ng PGan/min. Lower intracarotid doses of PGFZa were included to investigate possible dose-response relationships. Blood samples were collected from jugular cannulae at 30-minute intervals starting immediately prior to the start of the infusions and continuing for 12 hours. To prepare the animals for carotid cannulation, each bull was given 1.5 g of SuritalR (Parke, Davis & Company, Allen Park, MI) and 30 g of GuaifenesinR (Ganes Chemical Works, 535 5th Avenue, NY) intra- . . . R venously and then maintained on halothane anestheSIa (Fluothane , Ayerst Laboratory, Inc., New York, NY) for the duration of surgery. 67 To expose the carotid artery for cannulation, a 10 cm incision through the skin was made parallel and ventral to the jugular vein. Then the carotid was located by blunt dissection ventral to the jugular vein, between the cleidomastoid and sternomandibular muscle, the carotid artery was freed from surrounding tissue, and silk suture was passed under the anterior and posterior end of the carotid to lift the artery and control blood flow during insertion of the cannula. Prior to insertion of the cannula, a pursestring suture was placed in the wall of the carotid around the area of insertion. Then a small incision was made through the carotid sheath and silastic cannula was placed into the carotid and secured with the pursestring suture. The incision was closed with silk suture and the remaining end of the cannula was positioned under the skin dorsally to the carotid and exposed to the outside 10 cm from the incision. The entire cannula- tion procedure required about 1 hour. The carotid cannula consisted of a 43 cm inner silastic cannula (.062" x .125", Dow Corning, Midland, MI) and an outer sheath of polyvinyl cannula (.156" x .25", Portex Limited, Hythe, Kent, England). The outer cannula was 30 cm in length and had a 1 cm x 1 cm x 0.5 cm silicone sponge (Bellco Glass, Inc., Vineland, NJ) attached around one end with silastic glue. The end with the silicone sponge was passed over the inner silastic cannula for 30 cm leaving 13 cm of the silastic cannula exposed. The silicone sponge surrounding the outer cannula was used to anchor the carotid cannula to the carotid sheath. A 48-hour interval was allowed after surgery before the start of the experiment. Blood serum LH was quantified by specific radioimmunoassay (Convey et al., 1976). Serum testosterone was quantified by 68 radioimmunoassay using MSU antitestosterone #74 raised against testosterone-3-oxime-human serum albumin. In the initial validation (Kiser et al., 1977), the antiserum was used at a final dilution of 1:50,000. Percent binding in the zero tube was 30 percent and 10 pg of testosterone resulted in a reduction in binding from 30 to 20 percent. Dihydrotestosterone and androstenedione cross-reacted 60 and 1.7 percent, respectively, but no other steroid or sterol tested cross-reacted more than 0.14 percent (Table 3). Zero, 0.05, 1.0, 5 and 10 ng testosterone added per milliliter of estrous cow serum, bull serum and blood plasma from an adrenalectomized steer were assayed with and without isolation of testosterone by Sephadex LH-20 chroma- tography. Testosterone recovery averaged 89 and 92 percent before and after chromatographic isolation (Table 4). Serum testosterone averaged 0.04, 8.7 and 0.05 ng/ml for estrous cow, bull and steer, respectively, and the quantity of testosterone determined with and without chromatographic isolation did not differ significantly. Finally, testosterone was assayed in a single sample of bull blood serum in quadruplicate with the present antitestosterone assay and with the previous antitestosterone assay (Mongkonpunya et al., 1975) in five separate assays. Average testosterone concentration with the present assay (9.0 :_0.9 ng/ml) was slightly less (P=.08) than with the previous assay (10.1 :_1.3 ng/ml), and the within and among assay coefficients of variation were 10.4 and 12.8 percent. An additional modification of this assay (Haynes et al., 1977) utilized sheep antirabbit gamma globulin to separate free- from antibody-bound testosterone. Using this separational technique, the antitestosterone serum was diluted 1:10,000, but this alteration did 69 Table 3. Percent cross-reactions of rabbit antitestosterone (M80 #74) Steroid or sterol Percent cross-reactiona Testosterone 100.00 Dihydrotestosterone 60.00 Androstenedione 1.70 Epiandrosterone 0.04 Dehydroepiandrosterone 0.02 Androstadiene-3,l7-dione 0.14 Estradiol-17B 0.02 Estriol 0.02 Estrone 0.04 Progesterone 0.02 20a-hydroxyprogesterone 0.02 l7a-hydroxyprogesterone 0.01 Cortisol 0.02 Corticosterone <0.01 Cholesterol <0.01 Cortisone 0.02 Aldosterone <0.01 a . . . Percent expressed as act1v1ty relative to testosterone. 70 Table 4. Percent recovery of added testosterone after direct extrac- tion or column chromatographic isolation from sera from an estrous cow or bull, or from steer plasma Added Direct extraction Chromatographic isolation testosterone Cow Bull Steer Cow Bull Steer (ng) % 0.05 56 90 50 80 99 111 1.00 93 101 101 79 86 90 5.00 96 99 99 90 107 94 10.00 93 103 91 82 94 95 not change validity criteria estimates of the assay (Haynes et al., 1977). Thus, quantification of serum testosterone in this experiment utilized a double antibody technique. Breifly, 50 ul of each unknown was placed in 2 disposable extraction tubes (15 x 85 mm). To account for procedural loss, 3,000 dpm 3H-testosterone (85-105 Ci/mmol) was added to a third aliquot from a representative number (10 to 20 within each BOO-tube assay) of unknowns. Each tube was vortexed with 2 m1 of benzene-hexane (1:2) for 30 seconds, then stored at ~20 C for at least 2 hours to freeze the aqueous phase. The organic solvent from each tube with 3H-testosterone was decanted into "mini" scintillation vials (#125503, Research Products, Inc., Elk Grove, IL) for recovery (extraction efficiency) determination.‘ The organic solvent from samples for quantification was decanted into 12 x 75 mm disposable culture tubes for radioimmunoassay. The solvent was evaporated and 200 pl of antitestosterone was added to each tube. 71 Sets of standard tubes containing 0.0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, 0.60, 0.80, and 1.00 ng testosterone were included in each assay and treated similarly to unknowns. After addition of antibody, each tube was vortexed 10 seconds and incubated at room temperature for 2 hours. Then approximately 5,000 cpm 3H—testosterone (1,2,6,7-3H-testosterone 85-105 Ci/mM) diluted in 200 ul of 0.1 per- cent gelatin in 0.1 M phosphate buffered saline (0.1 percent gel-PBS) was added to each tube and the tubes were vortexed 10 seconds and incubated at 4 C for approximately 24 hours. To separate free from antibody-bound testosterone, 400 ul of sheep antirabbit gamma globulin serum in an appropriate dilution to bind 80 percent of the 3H- testosterone in the zero tube was added to each tube, vortexed and incubated for about 38 hours at 4 C. Finally, each tube was centri- fuged for 30 minutes at 2,500 xg at 4 C. Free 3H-testosterone in 0.5 m1 of supernatant fluid was diluted to 5 ml with scintillation fluid (3a7OB, Research Products International, Inc., Elk Grove Village, IL) and counted in a liquid scintillation spectrometer. Statistical analyses were performed by split-plot analysis of variance (Gill and Hafs, 1971). Designed contrasts among treatments were made orthogonally and some non-orthogonal contrasts were analyzed using Bonferroni t (Miller, 1966). One-way analysis of variance with unequal numbers was used to test duration of LH and testosterone responses after treatments. Results and Discussion During the course of this experiment, it was determined that the carotid cannula of one bull was improperly placed; thus, the data 72 from this bull were excluded from statistical analyses. The data represent five bulls given six treatments. Plasma LH averaged 1.3 :_0.2 ng/ml for bulls in all 6 treatments before start of the infusions (Figure 13). After the start of infus- ions, both 0.2 mg PGFZa/min in the jugular and 2,000 ng PGFZa/min in the carotid caused an increase (P<.05) of plasma LH (Figure 13). However, release of LH after 2,000 ng PGFZa/min into the carotid was prolonged by comparison to the 0.2 mg PGFZa/min intrajugular treat- ment. In contrast, carotid administration of saline or jugular administration of 2,000 ng PGFZa/min treatments were ineffective in causing increased elevations in plasma LH. For clarity, since the patterns of LH after 20 and 200 ng/min of PGan were similar to saline treatment, these data were not represented in Figure 13. Closer inspection of the data reveals that plasma LH increased to a peak of 2.6 i_0.5 ng/ml within 1 hour after beginning jugular infusion of 0.2 mg PGFZa/min, then declined to 1.4 :_0.3 ng/ml at the end of the 3-hour infusion. A similar increase (3.6 :_1.1 ng/ml) occurred during carotid infusion of 2,000 ng PGFZa/min, but LH remained clearly elevated throughout the 3-hour infusion period. Statistical analysis of data for blood LH revealed a signifi- cant (P<.005) time effect and the different pattern of LH secretion following carotid infusion of 2,000 ng PGFZa/min and jugular infusion of 0.2 mg PGFZa/min, compared to the remaining treatments probably was responsible for the significant (P<.003) treatment by time interaction. The "a priori" expectation was that 0.2 mg/min jugular infusion and 2,000 ng/min carotid infusion would result in increased plasma LH. Indeed, orthogonal contrasts of these two treatments versus 73 Figure 13. Blood plasma LH during a 3-hour infusion of PGFZa (0 ng/min [C], 2,000 ng/min [C], 2,000 ng/min [J], and 0.2 mg/min [J]) into the carotid artery (C) or jugular vein (J). 74 r INFUSION i 2000 ng/min (C) r. A TE \ or 5 I _I o E m 2 0. 'o o 2 an 2000 ng/min(J) 0O I 2 3 4 Time (hr) Figure 13 75 jugular infusion of 2,000 ng/min and carotid saline infusion indicated a greater response after 0.2 mg/min jugular and 2,000 ng/min carotid treatments (P<.001). These data suggest that PGan acted at the brain to cause LH release, because the 2,000 ng PGan/min infused to the head via the carotid artery caused release of LH, while the same amount of PGan infused away from the head via the jugular vein caused no significant elevation in serum LH. Furthermore, the LH response after carotid saline infusion did not differ from the 2,000 ng/min dose of PGF 0 infused into the jugular. However, the LH 2 response after carotid infusion of 2,000 ng PGFZa/min was greater (P<.001) than after jugular infusion of 0.2 mg PGFZa/min. These data suggest that less than 1 percent of the 0.2 mg PGan infused via the jugular vein actually reached the head. Interpretation of the testosterone response was complicated first by the large variation in plasma testosterone (pooled SE = 16.1 ng/ml) and secondly by a nonsignificant increase in plasma testosterone in control bulls infused with saline (Figure 14). In two controls, episodic LH releases occurred immediately prior to the start of the saline infusion and caused increased testosterone secretion during the infusion period. The overall analysis of variance revealed a significant time effect (P<.005) and a significant treatment by time interaction (P<.001). Orthogonal contrasts revealed no significant difference between treatments. However, the interaction between treatment and time indicated a different pattern of testosterone concentrations between treatments. For example, plasma testosterone remained below 3 ng/ml during jugular infusion of 2,000ng/ndJIof PGF . In contrast, 2a after infusion of the same dose of PGan into the carotid, plasma 76 Figure 14. Blood plasma testosterone during a 3-hour infusion of PGan (0 ng/min [C], 2,000 ng/min [C], 2,000 ng/min [J] and 0.2 mg/min [J]) into the carotid artery (C) or the jugular vein (J). Blood Plasma Testosterone (ng/ml) 7 77 I INFUSION I I 4s. ZOOOng/min(C)-I 3’.‘ I o,”’ 3‘ I\ ' 43' I . . 0.2 mg/min (J) 4% “X \, \9.."Fi \ \‘1 \\ 5,11 \ \ - 5:! I .II'I / ' ‘x I \\ b-u-A / . (I, Ong mIn(C) Xx A I .' \ !/ ox \ l- / E , I! I I ’/ ZOOOng/minw) o I z 3 4 Time (hr) Figure 14 76 Figure 14. Blood plasma testosterone during a 3-hour infusion of PGFZG (0 ng/min [C], 2,000 ng/min [C], 2,000 ng/min [J] and 0.2 mg/min [J]) into the carotid artery (C) or the jugular vein (J). Blood Plasma Testosterone (ng/ml) 77 I INFUSION“ I 7 ' 2000ng/min(C)-.I \z’-‘ I "It ‘1‘. \ I . . Cliinmg/Tnh1(~” 4PL“\ \I \& AM (I. \ . \‘ CD I .’ ,/ 2000 ng/min (J) I L 0O l 2 3 4 Time (hr) Figure 14 78 testosterone increased (P<.05) from 1.3 :-.4 ng/ml before infusion to 7.2 :_2.2 ng/ml after 2 hours of infusion. In addition, although no differences existed between plasma testosterone prior to infusion of treatments, average plasma testosterone was greater (P<.05) at 2.5 hours of infusion with 2,000 ng PGFZa/min into the carotid than at 2.5 hours of infusion with the same dose of PGFZa into the jugular. This observation is consistent with the LH response during the same treatments. Treatment effects also were evaluated by determination of the duration of LH and testosterone response. Two independent referees estimated the duration of elevated LH and testosterone after each treatment in each bull. Prior to treatments, blood was sampled at frequent intervals for 24 hours to determine the duration of LH and testosterone surges. The average duration of LH and testosterone surges was 2.2 :_0.2 and 3.2 :_0.3 hours, respectively (Figure 15). By comparison, during carotid infusion of saline (0 ng [C]) only one bull exhibited increased LH (2.0 hour duration). In contrast, average duration of the LH surge (all five bulls) was 6.3 :_0.7 and 5.2 :_1.2 hours (Figure 15) during infusion of 2,000 ng [C] and 0.2 mg [J], respectively. Relative to bulls given saline, duration of LH was greater (P<.05) during the 2,000 ng [C] and 0.2 mg [J] treatments. In addition, average duration of LH response was greater after 2,000 ng [C] than that after 2,000 mg [J]. These observations were consistent with the view that PGF2a acted at the brain to elicit LH release. Similarly, the durations of the testosterone surges after 2,000 ng [C] and 0.2 mg [J] treatments were greater (P<.05) than those after 0 ng [C] or 2,000 ng [J]. Average duration of testosterone was 79 odN Imoumou cam mg mo Hogan: mgu ucmmoummn mumn on» cfinufi3 mumafisz mam mo :OAmnmcw mcfiuso maauuaooo mmmuam ocouou .maasn m>HM oucw ammwm mo COamaucw mcflusn was Aaouucoov ou Howum mcfiuusooo mwmusm oconmumoumou new :4 mo coflumuzc coo: .ma musmwm 80 W“, (mu-2'0 W ( p) bu 0003 \\\\\\\\\\\\\\\\\\\\\\\\\\m\\\\\\ , (O) bu 0003 ID 4w (0) m, 003 ,0 (0)60 oz ,, mono I! '- 7: D S |ouuoo «b .3 it .1: é. L o ('Ju) emu Figure 15 79 .am Imoummu cam ma mo umnfisc on» ucmmmumwu when on» segues muonsdz mom mo cowmnwcfi mcwuso mcauuaouo mmeSm wcoumu .mHHsn m>fiu once ammom mo cofimsucfl mcflusv one Aaouucoov ou uoflum mcfluusooo mwmusm wcoumumoumwu cam ma mo :oflumusc cows .mH musmflm 80 i M . Wm (r) z o M ( p) on 0003 x\\\\\\\\\\\\\\\\\\\\\\\\m\\\\\ ' (O) bu 0002 In W (a) 5.. 003 ,0 10) Du oz nmwmo I: p._J . I D S Iouuoo l L 1 l l l J 0 no «- :0 u — 0 Wu) mm Figure 15 81 6.7 :_O.7 and 5.8 :_0.4 hours for 2,000 ng [C] and 0.2 mg [J] treat- ments, respectively, compared to 2.6 :_0.3 and 2.5 :_0.5 hours for 0 ng [C] and 2,000 ng [J] treatments. In summary, infusion of PGFZa into a carotid in an amount that was ineffective when infused into the jugular vein caused rapid and prolonged increase in plasma LH and testosterone in bulls. These results provide persuasive evidence that PGF a acted directly at the 2 head to cause release of LH; presumably, the subsequent increase in plasma testosterone was in response to the elevated LH. Whether PGFZa acted directly at the level of the LHRH neurons to elicit release of LHRH which caused the increase in LH, as sug- gested by Eskay et a1. (1976), awaits further investigation. GENERAL DISCUSSION Haynes et al. (1975) reported that PGFZa caused increased tes- tosterone secretion in bulls, but they were unable to prove that PGFZa caused release of LH. Although these researchers acknowledged the possibility that PGF may have caused release of LH, which then 2a caused increased testosterone, they favored the hypothesis that PGFZa acted directly at the testis to increase testosterone secretion. This assumption was based on the report of Eik-Nes (1969) that PGE2 caused increased testosterone secretion in the perfused dog testis. Furthermore, Flack, Jessup and Ramwell (1969) found that prosta- glandins stimulated corticosteroid synthesis in superfused rat adrenals. Haynes et al. (1975) also ruled out alteration in peripheral blood flow because the predominant action of-PGFZG is vasoconstric- tion, which would decrease blood flow and, presumably, testosterone secretion. In support of this idea, Finer-Jensen and Soofi (1974) reported that intratesticular administration of PGan decreased blood flow in rats. Furthermore, Haynes et al. (1975) found no difference in heart rate or peripheral blood pressure after PGF compared to 2o controls. The experiments discussed in this dissertation provide, for the first time, conclusive evidence that PGF a is a potent releaser of 2 LH in bulls. Furthermore, the magnitude of LH release after PGan was similar to the peak magnitude of LH observed during episodic 82 83 secretion in bulls. In other words, PGFZa released LH in amounts that were in the physiological range for control bulls. In my experiments, pharmacological doses of PGFZG were adminis- tered, but these treatment doses were necessary in view of the rapid clearance of PGFZa in the peripheral blood. A closer evaluation of the amounts of PGFZa used in these experiments indicate that indeed PGFZa was a potent stimulus for LH release. For example, in the final experiment, the highest dose of PGF a (2,000 ng/min) adminis- 2 tered into the carotid interpolates to approximately 200 pg PGFZa/ml of blood actually reaching the brain area based on the approximate blood flow to the head (9L/min). Other researchers have demonstrated that PGE2 causes release of LH in rats, but until recently very little information was avail- able implicating PGF a to release of LH. Warberg et a1. (1976) 2 reported increased LH after PGF a in rats, in contrast to results 2 from Harms et al. (1973). The discrepancy between these reports remains to be resolved; however, Warberg et a1. (1976) used about 5-fold more PGFZa than did Harms et a1. (1973). In contrast to the rapid increase of'testosterone that Haynes et a1. (1975) observed after PGan and the increase of testosterone in the present series of experiments, the majority of reports indi- cate that PGFZa causes inhibition of testosterone, at least in rodents. Aside from the possible species differences, I speculate that the reduced testosterone in rats and mice may have been associated with the chronic administration of PGF20L in amounts which were much greater (~40-fold) on a body weight basis than I gave to bulls. In addition, these researchers measured testosterone at 3 and 12 hours after the last of a series of PGFZa injections. Based on the research 84 reported in this dissertation, sampling of blood would have to occur immediately after administration of PGF to maximize the possibility 2a of detecting increased testosterone secretion. Furthermore, my research data would indicate that increased testosterone secretion occurs acutely after PGan and possibly not after a chronic or con— stant PGFZa stimulus. Hafs, Louis and Stellflug (1974) demonstrated increased sperm output in rabbits and bulls. However, from a practical standpoint, it would appear contraindicated to administer PGF a to increase sperm 2 output in bulls and at the same time cause a decrease in blood tes- tosterone, an essential hormone for complete Spermatogenesis and sperm maturation. The results of the present experiments do not rule out a detrimental effect of PGFZd on testosterone secretion in bulls when used on.a chronic basis. In fact, the evidence suggests that chronic administration of PGFZa may cause a decrease in both LH and testosterone, because LH and testoSterone were declining at the end of a 20-hour infusion of PGFZa in Experiment 2. From a physiological point of view, it is difficult to reconcile the fact that, in addition to release of LH, PGFZa causes release of growth hormone (GH), prolactin and glucocorticoids in bulls (Hafs, 1975; Hafs et al., 1977). Prolactin, GH and glucocorticoids increased several-fold within 5 to 15 minutes after PGan treatment in cows (Louis et al., 1974) and bulls (Hafs, 1975; Hafs et al., 1977) alike, much more rapidly than LH release. Moreover, the release of LH after PGFZa in diestrous heifers is dependent upon rapid withdrawal of pro- gesterone, which typifies luteolysis induced by PGF (Hafs et al., 2a 1976). These observations suggest that the action of PGFZa to cause release of prolactin, GH and glucocorticoids may differ from the 85 action of PGFZa to cause LH release. Prostaglandin an may possibly act directly on the pituitary to cause the rapid release of prolactin, GH and glucocorticoids and at the same time act at the hypothalamus or other sites to elicit release of LH. Certainly a sex difference in PGFZa-induced release of LH is indicated from these observations. In bulls, feedback of testicular androgens may elevate the release threshold for LH compared with that for prolactin, a hormone for which feedback has not been demonstrated in cattle (Beck et al., 1976). The evidence suggests that prostaglandins may be a common inter- mediate in the control of secretion of anterior pituitary hormones and that specificity to one hormone arises out of local production and local utilization of the prostaglandin. Perhaps one also could speculate that dual control of hormone secretion exists. In one case, catecholamines or other intermediates, which appear to be established as controllers of hormone secretion (Wilson, 1974), may control synthesis of releasing factors and prostaglandins may control release. Alternatively, there may be a dual control mechanism for both synthesis and release and each may operate independently. That PGFZa caused release of LH and testosterone in bulls is clear from the data in the present series of experiments. However, whether PGF normally is a physiological mediator of LH release 2a remains unanswered. SUMMARY AND CONCLUSIONS The purpose of these experiments was to determine the effect of prostaglandin F (PGF 0) on secretion of LH and testosterone in bulls. 2a 2 The first experiment was designed to determine the temporal rela- tionship between blood serum LH and testosterone in bulls given a subcutaneous injection of PGFZa or saline. Before PGFZa' blood serum LH averaged 1.1 i_0.1 ng/ml; after PGF , LH increased 3-fold at 30 2a minutes, peaked (3.9 :_O.9 ng/ml) at 45 minutes and declined to pre- injection values after 4 or 5 hours. Blood serum testosterone averaged 4.5 :_0.2 ng/ml before PGFZa; it increased synchronously in each of the eight bulls to 8.5 :_0.9 ng/ml by 60 minutes after PGFZa' peaked at 15 to 16 ng/ml between 90 and 120 minutes, and then declined toward pre-injection values by 180 minutes. Thus, the tes- tosterone surge followed the PGF -induced LH surge by 30 to 60 minutes. 2a In contrast, an average of one episodic surge of testosterone (average peak 14.2; range 8.0 to 22.8 ng/ml) occurred apparently at random intervals during the 8 hours after bulls were given saline. An increase of blood serum LH (average peak 3.0; range 1.5 to 4.3 ng/ml) occurred about 30 minutes before each of these testosterone surges. 0n the average, neither LH nor testosterone changed signifi- cantly after saline. The major conclusion from this experiment is that administration of PGF to bulls causes increased blood LH, fol- 20 lowed by increased blood testosterone. Both peaks and the temporal 86 87 relationship of these changes in LH and testosterone were similar to the normal episodic releases of these hormones in bulls. The second experiment was designed to test whether a continuous iv infusion of PGFZa could maintain elevated blood LH and testosterone in bulls. Blood plasma LH averaged 1.2 :_0.1 ng/ml before PGan infus- ion; it doubled within 1.5 hours after the infusion was started and peaked approximately 4-fold higher than pre-infusion values at 6.5 hours before declining to basal concentrations before the end of the 20-hour infusion. Blood plasma testosterone averaged 7.0 :_O.6 ng/ml during the 90 minutes before infusion of PGFZa; it increased 2-fold by 2.5 hours after the start of the infusion, remained near this peak until 10 hours and then gradually returned toward pre-infusion values by the end of the infusion. Thus, LH and testosterone increased together after the start of the infusion, but the peak testosterone concentration occurred sooner and the duration of the peak testos- terone concentration persisted longer than the LH response. Luteiniz- ing hormone started to decline at least 3 hours earlier than testos— terone, preceding the decline of testosterone in all four bulls. Episodic release of LH and testosterone resumed within 8 hours after the end of the 20-hour PGan infusion. Two or three episodic surges of testosterone occurred in each of the bulls during the 20-hour control infusion of saline; the peak (17.2 :_O.2 ng/ml) of these surges was equivalent to the peak concentration of testosterone during infusion of PGFZa' and 18 of 19 control surges of testosterone were preceded by increased blood LH (average peak 2.8 :_0.3 ng/ml). In summary, constant infusion of PGan caused prolonged increases in LH and testosterone secretion in bulls, but both LH and testosterone 88 declined toward basal values before the end of the 20-hour infusion period. In the third experiment, the major objective was to determine if the inhibitory effects of melengestrol acetate (MGA) on episodic release of LH and testosterone could be overcome by PGF A pre- 2d' liminary experiment demonstrated that feeding 0.5 mg MGA twice daily (0700 and 1000 hours) abolished surges of testosterone. The average testosterone concentration during a 4-hour period before MGA was 8.5 i_l.1 ng/ml. During the period 1 to 5 hours after the third feeding of MGA, testosterone averaged only 1.8 i 0.1 ng/ml. In the main experiment, each bull was fed 1.0 mg of MGA daily (0.5 mg at 0700 and 1900 hours). After four MGA-treated bulls were given saline, blood LH concentrations did not change significantly, ranging from 0.30 i 0.05 to 0.40 i_0.05 ng/ml. Similarly, serum testosterone fluctuated between 0.8 :_0.3 to 1.2 :_0.2 ng/ml and did not change significantly. By comparison, serum LH averaged 0.40 i 0.01 ng/ml before MGA-treated bulls were given (sc) 20 mg PGF2 ; it increased S-fold at 45 minutes after PGFZa' peaked at 2.3 :_0.5 ng/ml at 60 minutes and declined to basal values between 4 and 5 hours. Serum testosterone averaged 0.8 :_0.3 ng/ml before PGF increased 2a' 25-fold to peak at 105 minutes after PGF , plateaued until 3 hours 2a after PGFZa and declined to baseline concentrations by 7 hours. In conclusion, 1) treatment of bulls with MGA abolished LH and testos- terone secretion in bulls and 2) PGF a caused release of LH and, 2 subsequently, testosterone in the face of MGA inhibition. Although the first three experiments demonstrated that PGFZo caused increased blood LH and testosterone, the site of action of PGFZa remained to be ascertained. Therefore, the objective of the 89 final experiment was to determine if PGF acted directly on the brain 2a to elicit release of LH in bulls. Prostaglandin F2a was administered by a 3-hour intracarotid (to affect the brain) or intrajugular (systemic treatment) infusion in five bulls. Intracarotid infusion of saline or jugular infusion of 2,000 ng PGFZa/minute did not cause increased blood LH; LB averaged 1.1 and 1.0 ng/ml during the 3-hour infusion for intracarotid saline and jugular infusion of 2,000 ng PGFZa/min, respectively. Blood LH increased from 0.8 :_0.1 ng/ml to a peak of 2.6 i 0.5 ng/ml within 1 hour after beginning jugular infusion of a large dose of PGF a (0.2 mg/min), then declined to 1.4 :_0.3 ng/ml 2 at the end of the 3-hour infusion. A similar increase (to a peak of 3.6 i 1.1 ng/ml) occurred during intracarotid infusion of 2,000 ng PGFZa/min, but LH clearly remained elevated throughout the 3-hour infusion period. The LH response during intracarotid infusion of 2,000 ng PGFZa/min was greater than that during intrajugular infusion of 0.2 mg PGFZa/min. The data indicate that PGFZa acted at the brain to cause LH release because the 2,000 ng/min dose of PGFZa infused to the head via the carotid caused release of LH, whereas the same amount of PGFZa infused away from the head via the jugular caused no significant elevation in LH. Testosterone remained below 3 ng/ml during jugular infusion of 2,000 ng PGFZa/min, but the same dose of PGFZa increased blood testosterone from 1.3 i_0.4 ng/ml before intra- carotid infusion of PGFZa to 7.2 :_2.2 ng/ml at 2 hours during infus- ion, and testosterone remained elevated until the intracarotid infusion of PGFZa was stopped. I conclude that PGFZa acted directly at the brain to cause release of LH; then LH caused the subsequent increase in plasma testosterone. 90 The results from this series of experiments provide conclusive evidence, the first report in bulls, that PGFZa causes increased blood LH. Luteinizing hormone increased when PGF was given as a 2d single subcutaneous injection. The temporal relationship of changes in LH and testosterone suggested that the increase in testosterone was caused by increased LH secretion. Secondly, a 20-hour intravenous infusion of PGFZa resulted in a prolonged increase of LH and testos- terone, but LH and testosterone decreased to basal concentrations toward the end of the 20-hour infusion. The results indicate that a continuous PGFZa stimulus results in hypothalamic or pituitary refractoriness, but the refractoriness was short-lived because epi- sodic secretion of LH and testosterone (similar to that observed in control bulls) resumed again within 8 hours after the end of the infusion. Thirdly, suppression of episodic secretion of LH and testosterone by MGA was overcome with a single sc injection of PGFZa' I speculate that MGA might possibly inhibit hypothalamic PGan secre- tion and thereby inhibit LH release. Lastly, intracarotid, but not intrajugular, infusion of 2,000 ng PGFZG/min caused an increase in LH and testosterone. These results provide persuasive evidence that PGan acts at the brain to cause release of LH. LITERATURE CITED LITERATURE CITED Agmo, A. 1975. Effect of prostaglandins E1 and F20‘ on serum luteinizing hormone concentration and on some sexual func- tions in male rabbits. Prostaglandins 9:451. Abdulla, Y. H. and E. McFarlane. 1971. Control of adenylate kinase by prostaglandins E2 and E3. Biochem. Pharmacol. 20:1726. Ambache, N. 1966. Biological characterization of, and structure- action studies on, smooth muscle contracting hydroxy-acids. Memoirs Society for Endocrinology 14:19. Amer, M. A. and N. R. Marquis. 1972. The effect of prostaglandins, epinephrine and aspirin on cyclic AMP and phosphodiesterase activity of human blood platelets and their aggregation. In: P. W. Ramwell and B. B. Pharriss (eds.), Prostaglandins in Cellular Biology, p. 93, Plenum Press, New York. Andresen, O. 1975. 5a-Androstenone in peripheral plasma in pigs, diurnal variation in boars, effect of intravenous HCG adminis- tration and castration. Acta Endocr., Copenh. 78:385. Anggard, E., C. Larsson and B. Samuelsson. 1971. The distribution of lS—hydroxy prostaglandin dehydrogenase and prostaglandin- A13—reductase in tissues of the swine. Acta Physiol. Scand. 81:396. Arimura, A., H. Matsuo, Y. Baba and A. V. Schally. 1971. Ovulation induced by synthetic luteinizing hormone—releasing hormone in the hamster. Science 174:511. Arimura. A., H. Matsuo, Y. Baba, L. Debeljuk, J. Sandow and A. v. Schally. 1972. Stimulation of release of LH by synthetic LHRH in viva. I. A Comparative study of natural and syn- thetic hormones. Endocrinol. 90:163. Arimura, A., M. Saito, Y. Yaoi, T. Kumasaka, H. Sato, T. Koyama, N. Nishi and A. J. Kastin. 1973. Comparison of the effects of subcutaneous and intravenous injection of synthetic LH releas- ing hormone (LH-RH) on serum LH and FSH in men. J. Clin. Endocrinol. Metab. 36:385. Arimura, A. and V. Schally. 1970. Progesterone suppression of LH- releasing hormone-induced stimulation of LH release in rats. Endocrinol. 87:653. 91 92 Armstrong, D. T. and D. L. Grinwich. 1972. Blockade of spontaneous and LH-induced ovulation in rats by indomethacin, an inhibitor of prostaglandin biosynthesis. Prostaglandins 1:21. Barcikowski, B., S. K. Saksena and A. Bartke. 1973. Androgenic regu- 1ation of plasma prostaglandin F levels in the rat. J. Reprod. Fert. 35:549. Bartke, A. and S. Dalterio. 1975. Evidence for episodic secretion of testosterone in laboratory mice. Steroids 26:749. Bartke, A., D. Kupfer and S. Dalterio. 1976. Prostaglandins inhibit testosterone secretion by mouse testes in vitro. Steroids 28:81. Bartke, A., N. Musto, B. V. Caldwell and H. R. Behrman. 1973. Effects of a cholesterol esterase inhibitor and of prostaglandin an on testis cholesterol and on plasma testosterone in mice. Prosta- glandins 3:97. Bartke, A., R. E. Steele, N. Musto and B. V. Caldwell. 1973. Fluctua- tions in plasma testosterone levels in adult male rats and mice. Endocrinol. 92:1223. Batta, S. R., M. Zanisi and L. Martini. 1974. Prostaglandins and gonadotropin secretion. Neuroendocrinol. 14:224. Beck, T. W., V. G. Smith, B. E. Seguin and E. M. Convey. 1976. Bovine serum LH, GH and prolactin following chronic implanta- tion of ovarian steroids and subsequent ovariectomy. J. Anim. Sci. 42:461. Behrman, H. R., G. T. MacDonald and R. O. Greep. 1971. Regulation of ovarian cholesterol esters: Evidence for the enzymatic sites of prostaglandin-induced loss of corpus luteum function. Lipids 6:791. Behrman, H. R., G. P. Orczyk and R. 0. Greep. 1972. Effect of syn- thetic gonadotropin-releasing hormone (Gn-RH) on ovulation blockade by aspirin and indomethacin. Prostaglandins 1:245. Bennett, W. I., M. L. Dufau, K. J. Catt and W. W. Tullner. 1973. Effect of human menOpausal gonadotropin upon spermatogenesis and testosterone production in juvenile rhesus monkeys. Endocrinol. 92:813. Bergstrom, S., L. A. Carlson and J. R. Weeks. 1968. The prosta- glandins: A family of biologically active lipids. Pharmacol. Reviews 20:1. Blecher. Mn N. s. Merlino, J. 'r. Ro'Ane and p. o. Flynn. 1969. Inde- pendence of the effects of epinephrine, glucogon and adreno— corticotropin on glucase utilization from those on lipolysis in isolated rat adipose cells. J. Biol. Chem. 244:3423. 93 Bygdeman, M. 1967. Studies of the effects of prostaglandins in seminal plasma on human myometrium in vitro. Proceedings of the Second Nobel Symposium, Stockholm, p. 71, Almqvest and Wiksell, Stockholm. Carlson, J. C., B. Barcikowski, V. Cargill and J. A. McCracken. 1974. The blockade of LH release by indomethacin. J. Clin. Endocrinol. Metab. 39:399. Carlson, J. C., B. Barcikowski and J. A. McCracken. 1973. Prosta- glandin F2“ and the release of LH in sheep. J. Reprod. Fert. 34:357. Carpenter, M. P. 1974. Prostaglandins of rat testis.. Lipids 9:397. Carpenter, M. P., L. Manning and B. Wiseman. 1971. Prostaglandin synthetase in rat testis. Fed. Proc. 30:1081 (abst.). Catt, K. J. and M. L. Dufau. 1976. Basic concepts of the mechanisms of action of peptide hormones. Biol. Reprod. 14:1. Catt, K. J., K. Watanabe and M. L. Dufau. 1973. Cyclic AMP released by rat testis during gonadotropin stimulation in vitro. Nature 239:280. Cenedella, R. J. 1975. Prostaglandins and male reproductive physiol- ogy. In: J. A. Thomas and R. L. Senghal (eds.), Advances Sex HOrmone Research, p. 325, University Park Press, Baltimore, Maryland. Chakraborty, P. K., J. J. Reeves, A. Arimura and A. V. Schally. 1973. Serum LH levels in prepubertal female pigs chronically treated with synthetic luteinizing hormone-releasing hormone/follicle stimulating hormone releasing hormone (LHRH/FSHRH). Endocrinol. 92:55. Chobsieng, P., Z. Naor, Y. Koch, U. Zor and H. R. Lindner. 1975. Stimulatory effect of prostaglandin E2 on LH release in the rat: Evidence for hypothalamic site of action. Neuroendocrinol. 17:12. Coceani, F., C. Pace-Asciak and L. S. Wolfe. 1968. Studies on the effect of nerve stimulation on prostaglandin formation and release in the rat stomach. In: P. W. Ramwell and J. E. Shaw (eds.), Prostaglandin Symposium of the WOrcester Foundation, p. 39, Interscience, New York. Coeani, F., L. Puglisi and B. Lavers. 1971. Prostaglandins and neuronal activity in spinal cord and cuneate nucleus. Ann. N.Y. Acad. Sci. 180:289. Coceani, F. and L. S. Wolfe. 1965. Prostaglandin in brain and the release of prostaglandin-like compounds from the cat cerebellar cortex.‘ Canadian J. Physiol. and Pharmacol. 43:445. Csmuen Conv 0a 94 Convey, E. M., W. E. Beal, B. E. Seguin, K. J. Tannen and Y. C. Lin. 1976. Gonadotropin releasing hormone induced luteinizing hor- mone release after prostaglandin F2a in heifers. Proc. Soc. Exptl. Biol. Med. 151:84. Convey, E. M., E. Bretschneider, H. D. Hafs and W. D. Oxender. 1971. Serum levels of LH, prolactin and growth hormone after ejacu- lation in bulls. Biol. Reprod. 5:20. Daly, J. W. 1976. The nature of receptors regulating the formation of cyclic AMP in brain tissue. Life Science 18:1349. Davidson, J. M. and C. A. Sawyer. 1961. Evidence for a hypothalamic focus of inhibition of gonadotropin by androgen in the male. Proc. Soc. Exptl. Biol. Med. 107:4. Dorrington, J. H. and I. B. Fritz. 1974. Effects of gonadotropins on cyclic AMP production by isolated seminiferous tubule and interstitial cell preparations. Endocrinol. 94:395. ' DuCharm, D. W. and J. R. Weeks. 1967. Cardiovascular pharmacology of prostaglandin F20, a unique pressor agent. Proceedings of the Second Nobel Symposium, Stockholm, p. 173, Almqvist and Wiksell, Stockholm. Duda, P., E. W. Horton and A. McPherson. 1968. The effects of prosta- glandins E1, Fla and an on monosynaptic reflexes. J. Physiol. (London) 196:151. Eik-Nes, K. B. 1969. Patterns of steroidogenesis in the vertebrate gonad. Gen. and Comp. Endocrinol. Suppl. 2:87. Eik-Nes, K. B. 1971. Production and secretion of testicular steroids. Rec. Prog. Horm. Res. 25:517. Einer-Jensen, N. and G. Soofi. 1974. Decreased blood flow through rat testis after intratesticular injection of PGFZa. Prosta- glandins 7:377. E1 Safoury, S. and A. Bartke. 1974. Effects of follicle-stimulating hormone and luteinizing hormone on plasma testosterone levels in hypophysectomized and in intact immature and adult male rats. J. Endocrinol. 61:193. Eskay, R., J.Warberg, R. S. Mical and J. C. Porter. 1975. Prosta- glandin Ez-induced release of LHRH into hypophyseal portal blood. Endocrinol. 97:816. Falvo, R. B., A. E. Buhl, T. J. Reimers, G. R. Foxcroft, M. H. Dunn and P. J. Dziuk. 1975. Diurnal fluctuations of testosterone and LH in the ram: Effect of HCG and gonadotrophin-releasing hormone. J. Reprod. Fert. 42:503. 9S Feldberg, W. and R. D. Myers. 1966. Appearance of 5-hydroxytryptamine and an unidentified pharmacologically active lipid acid in effluent from perfused cerebral ventricles. J. Physiol. (London) 184:837. Flack, J.D., R. Jessup and P. W. Ramwell. 1969. Prostaglandin stimu- lation of rat corticosteroidogenesis. Science 163:691. Free, M. J. and R. A. Jaffe. 1972. Effect of prostaglandins on blood flow and pressure in the conscious rat. Prostaglandins 1:483. French, F. S. and E. M. Ritzen. 1973. A high-affinity androgen- binding protein (ABP) in rat testis: Evidence for secretion into efferent duct fluid and absorption by epididymis. Endo- crinol. 93:88. Galloway, D. B., Y. Cotta, J. Pelletier and M. Terqui. 1974. Circu- lating luteinizing hormone and testosterone response in rams after luteinizing hormone releasing hormone treatment. Acta Endocrinol. 77:1. Galloway, D. B. and J. Pelletier. 1975. Luteinizing hormone release in entire and castrated rams following injection of synthetic luteinizing hormone releasing hormone and effect of testosterone propionate pre-treatment. J. Endocrinol. 64:7. Geschwind, I. I. 1970. Mechanism of action of hypothalamic adeno- hypophysiotropic factors. In: J. Meites (ed.), Hypophysio- tropic Hormones of the Hypothalamus: Assay and Chemistry, p. 298, The Williams and Wilkins Co., Baltimore. Gill, J. L. and H. D. Hafs. 1971. Analysis of repeated measurements of animals. J. Anim. Sci. 33:331. Goldblatt, M. W. 1933. A depressor substance in seminal fluid. J. Soc. Chem. and Ind. (London) 52:1056. Goldblatt, M. W. 1935. Properties of human seminal plasma. J. Physiol. (London) 84:208. Golter, T. D., J. J. Reeves, C. C. 0. Mary, A. Arimura and A. V. Schally. 1973. Serum LH levels in bulls treated with syn- thetic LH-RH/FSH-RH. J. Anim. Sci. 37:123. Gombe, S., W. C. Hall, K. McEntee, W. Hansel and B. W. Pickett. 1973. Regulation of blood levels of LH in bulls: Influence of age, breed, sexual stimulation and temporal fluctuations. J. Reprod. Fert. 35:493. Gomes, W. R. and M. C. Joyce. 1975. Seasonal changes in serum tes- tosterone in adult rams. J. Anim. Sci. 41:1373. Green, J. D. and G. W. Harris. 1947. The neurovascular link between the neurohypophysis and adenohypophysis. J. Endocrinol. 5:136. 96 Greep, R. 0. and H. L. Fevold. 1937. The spermatogenic and secretory function of the gonads of hypophysectomized adult rats treated with pituitary FSH and LH. Endocrinol. 21:611. Hafs, H. D. 1975. Prostaglandins and the control of anterior pitui- tary hormone secretion. In M. Motta, P. G. Crosignani and L. Martini (eds.), Hypothalamic Hormones, p. 183, Academic Press, New York. Hafs, H. D., T. E. Kiser, N. B. Haynes, J. S. Kesner and J. N. Stellflug. 1977. Release of pituitary hormones, cortisol, testosterone and insulin in response to prostaglandin an given during intracarotid infusion of somatostatin in bulls. J. Anim. Sci. 44:in press. Hafs, H. D., T. M. Louis, J. N. Stellflug, E. M. Convey and J. H. Britt. 1975. Blood LH after PGFZa in diestrous and ovariec- tomized cattle. Prostaglandins 10:1001. Hafs, H. D., T. M. Louis, R. J. Waters, J. N. Stellflug and N. B. Haynes. 1974. Increased sperm output of rabbits and bulls treated with PGFZo' Prostaglandins 8:417. Haltmeyer, G. C. and K. B. Eik-Nes. 1969. Plasma levels of testos- terone in male rabbits following copulation. J. Reprod. Fert. 19:273. Hansson, V., 0. Djoseland, E. Reusch, A. Attramadal and 0. Torgersen. 1973a. An androgen binding protein in the testis cytosol fraction of adult rats. Comparison with the androgen binding protein in the epididymis. Steroids 21:457. Hansson, V., E. Reusch, 0. Trygstad, O. Torgersen, E. M. Ritzen and F. 8. French. 1973b. FSH stimulation of testicular androgen binding protein. Nature New Biol. 246:56. Hansson, V., E. M. Ritzen, F. 5. French and S. N. Nayfeh. 1975. Androgen transport and receptor proteins in the testis and epididymis. In: R. 0. Greep and D. W. Hamilton (eds.), Handbook of Physiology V9, p. 173, Amer. Physiol. Society, Washington, D.C. Hargrove, J. L., R. R. Seeley, J. M. Johnson and L. C. Ellis. 1973. Prostaglandin-like substances: Initiation and maintenance of rabbit testicular contractions in vitro. Proc. Soc. Exptl. Biol. Med. 142:205. Harms, P. G., S. R. Ojeda and S. M. McCann. 1973. Prostaglandin involvement in hypothalamic control of gonadotropin and pro- lactin release. Science 181:760. Harms, P. G., S. R. Ojeda and S. M. McCann. 1974. Prostaglandin- induced release of pituitary gonadotropins: Central nervous system and pituitary sites of action. Endocrinol. 94:1459. 97 Harms, P. G., S. R. Ojeda and S. M. McCann. 1976. Failure of mono- aminergic and cholinergic receptor blockers to prevent prosta— glandin Ez-induced luteinizing hormone release. Endocrinol. 98:318. Harris, G. W. 1948. Neural control of the pituitary gland. Physiol. Rev. 28:139. Hartley, H. O. 1950. The maximum F-ratio as a short-cut test for heterogeneity of variance. Biometrika 37:308. Haynes, N. B., H. D. Hafs, R. J. Waters, J. G. Manns and A. Riley. 1975. Stimulatory effect of prostaglandin FZa on the plasma concentration of testosterone in bulls. J. Endocrinol. 66:329. Haynes, N. B., T. E. Kiser, H. D. Hafs, T. Carruthers, W. D. Oxender and M. S. McCarthy. 1977. The effect of carotid infusion of prostaglandin F23 on plasma LH, testosterone and glucocorticoid concentrations in bulls. J. Anim. Sci. In press. Hill, Jr., J. R., D. R. Lamond, D. M. Henricks, J. F. Dickey and G. D. Niswender. 1971. The effect of melengestrol acetate (MGA) on ovarian function and fertilization in beef heifers. Biol. Reprod. 4:16. Hinman, J.W. 1972. Prostaglandins. Ann. Rev. Biochem. 41:161. Hittelman, K. J. and R. W. Butcher. 1973. Cyclic AMP and the mechanism of action of the prostaglandins. In: M. F. Cuthbert (ed.), The Prostaglandins Pharmacological and Therapeutic Advances, p. 151, J. B. Lippincott Co., Philadelphia. Holmes, S. W. and E. W. Horton. 1967. The nature and distribution of prostaglandins in the central nervous system in the dog. J. Physiol. (London) 191:134. Hooley, R. D., R. W. Baxter, W. A. Chamley, I. A. Cumming, H.A. Jones and J. K. Findlay. 1974. FSH and LH response to gonadotropin releasing hormone during the ovine estrous cycle and following progesterone administration. Endocrinol. 95:937. Horton, E. W. 1964. Actions of prostaglandins E1, E2 and E3 on the central nervous system. Brit. J. Pharmacol. 22:189. Horton, E. W. and I. H. M. Main. 1965. A comparison of the actions of prostaglandin Egg and E1 on smooth muscle. Brit. J. Pharmacol. and Chemotherapy 24:470. Horton, E. W. and I. H. M. Main. 1966. The identification of prosta- glandins in central nervous tissues of the cat and the fowl. J. Physiol. (London) 185:36. Horton, E. W. and I. H. M. Main. 1967. Further observations on the central nervous actions of prostaglandin an and E1. Brit. J. Pharmacol. and Chemotherapy 30:568. 98 Johnson, B. H. and L. L. Ewing. 1971. Follicle—stimulating hormones and the regulation of testosterone secretion in rabbit testes. Science 173:635. Johnson, M., R. Jessup and P. W. Ramwell. 1973. Ultraviolet light modification of the prostaglandin receptor. Prostaglandins 4:593. Kaltenbach, C. C., T. G. Dunn, T. E. Kiser, L. R. Corah, A. M. Akbar and G. D. Niswender. 1974. Release of FSH and LH in beef heifers by synthetic gonadotropin releasing hormone. J. Anim. Sci. 38:357. Katongole, C. B., F. Naftolin and R. V. Short. 1971. Relationship between blood levels of luteinizing hormone and testosterone in bulls and the effect of sexual stimulation. J. Endocrinol. 50:457. Katongole, C. B., F. Naftolin and R. V. Short. 1974. Seasonal varia- tions in blood luteinizing hormone and testosterone levels in rams. J. Endocrinol. 60:101. Keichline, L. D. and A. A. Hagen. 1973. A comparison on the effects of prostaglandin E1 and luteinizing hormone on cAMP levels in the rat testis. Fed. Proc. 32:298 (abst.). Kirk, R. E. 1968. In Experimental Design: .Procedures for the .Behavioral Sciences, p. 90, wadsworthxpublishing Co., Belmont, California. Kiser, 'r. E., R. A. Milvae, H. D. Hafs, w. o. Oxender and T. M. Louis. 1977. Comparison of testosterone and androstenedione secretion induced by prostaglandin F2“ and luteinizing hormone in bulls. J. Anim. Sci. In press. Kloeze, J. 1969. Relationship between chemical structure and platelet- aggregation activity of prostaglandins. Biochem. Biophys. Acta 187:285. Kuehl, Jr., F. A. 1974. Prostaglandins, cyclic nucleotides and cell function. Prostaglandins 5:325. Kurzrok, R. and C. C. Lieb. 1930. Biochemical studies of human semen. II. The action of semen on the human uterus. Proc. Soc. Exptl. Biol. Med. 28:268. Lindner, H. R. 1969. The androgenic secretion of the testis in domestic ungulates. In: K. W. McKerns (ed.), The Gonads, p. 615, North Holland Publishing Co., Amsterdam. Louis. T. M., J. N. Stellflug, H. A. Tucker and H. D. Hafs. 1974. Plasma prolactin, growth hormone, luteinizing hormone and gluco- corticoids after prostaglandin F2a in heifers. Proc. Soc. Exptl. Med. 147:128. 99 Marsh, J. M. 1976. The role of cyclic AMP in gonadal steroidogenesis. Biol. Reprod. 14:30. Maver, 0L” U. Volkwein and J. Tamm. 1973. The effect of intravenously administered human chorionic gonadotrophin on plasma levels of testosterone and 5a-dihydrotestosterone in normal male subjects. Acta Endocrinol. 72:615. McCarthy, M. S. and L. V. Swanson. 1976. Serum LH concentration following castration, steroid hormone and gonadotropin releas- ing hormone treatment in the male bovine. J. Anim. Sci. 43:151. Means, A. R., J. L. Fakunding and D. J. Tindall. 1976. Follicle stim- ulating hormone regulation of protein kinase activity and protein synthesis in testes. Biol. Reprod. 14:54. Meites, J. 1970. Direct studies of the secretion of the hypothalamic hypophysiotropic hormones (HHH). In: J. Meites (ed.), Hypo- physiotropic Hormones of the Hypothalamus: Assay and Chemistry, p. 261, The Williams & Wilkins Co., Baltimore. Michael, C. M. 1973. Prostaglandins in swine testes. Lipids 8:92. Miller, Jr., R. G. 1966. Simultaneous Statistical Inference (2.1.5.2, 2.2, 2.2.3.1), McGraw-Hill, New York. Moger, W. H. and D. T. Armstrong. 1974. Changes in serum testosterone levels following acute LH treatment in immature and mature rats. Biol. Reprod. 11:1. Mongkonpunya, K., H. D. Hafs, E. M. Convey, W. D. Oxender and T. M. Louis. 1974. Luteinizing hormone release by gonadotropin releasing hormone before and after castration in bulls. Proc. Soc. Exptl. Biol. Med. 147:873. Mongkonpunya, K., H. D. Hafs, E. M. Convey, H. A. Tucker and W. D. Oxender. 1975. Serum luteinizing hormone, testosterone and androstenedione in pubertal and prepubertal bulls after gonado- tropin releasing hormone. J. Anim. Sci. 40:682. Mongkonpunya, K., Y. C. Lin, P. A. Noden, W. D. Oxender and H. D. Hafs. 1975. Androgens in the bovine fetus and dam. Proc. Soc. Exptl. Biol. Med. 148:489. Moor, B. C. and E. V. Younglai. 1975. Variations in peripheral levels of LH and testosterone in adult male rabbits. J. Reprod. Fert. 42:259. Naftolin, F., H. L. Judd and S. S. C. Yen. 1973. Pulsatile patterns of gonadotropins and testosterone in man: The effects of clomiphene with and without testosterone. J. Clin. Endocrinol. Metabol. 36:285. 100 Nakano, J. 1973. General pharmacology of prostaglandins. In: M. F. Cuthbert (ed.), The Prostaglandins: Pharmacological and Therapeutic Advances, p. 23, J. B. Lippincott Co., Philadelphia. Nakano, J., B. Montague and B. Darrow. 1971. Metabolism of prosta- glandin El in human plasma, uterus and placenta in swine ovary and in rat testicle. Biochem. Pharmacol. 20:2512. Nakano, J. and A. V. Prancan. 1971. Metabolic degradation of prosta- glandin E1 in the rat plasma and in rat brain, heart, lung, kidney and testicle homogenates. J. Pharm. Pharmacol. 23:231. Neaves, W. B. 1975. Leydig cells. Contraception 11:571. Nelson, W. 0. 1937. Maintenance of spermatogenesis in testis of hypophysectomized rats with sterol derivatives. Proc. Soc. Exptl. Biol. (NY) 36:825. Ojeda, S. R., J. E. Wheaton and S. M. McCann. 1975. Prostaglandin Ez-induced release of luteinizing hormone-releasing factor (LRF). Neuroendocrinol. 17:283. Pant, H. C. and W. R. Ward. 1974. Effect of intravenous infusion of oestradiol-17B with and without prior progesterone treatment on the plasma luteinizing hormone and follicle stimulating hormone concentrations in anoestrous ewes. J. Endocrinol. 61, V—VI. Pelletier, J. 1976. Influence of LH—RF on LH and FSH releases in domestic mammals. Ann. Biol. Anim. Bioch. Biophys. 16:213. Purvis, K. and N. B. Haynes. 1974. Short-term effects of copulation, human chorionic gonadotrOpin injection and non-tactile associa- tion with a female on testosterone levels in the male rat. J. Endocrinol. 60:429. Purvis, K., A. W. Illuis and N. B. Haynes. 1974. Plasma testosterone concentrations in the ram. J. Endocrinol. 61:241. Pike, J. E., F. P. Kupiecki and J. R. Weeks. 1967. Biological activity of the prostaglandins and related analogs. In: S. Bergstrom and B. Samuelsson (eds.), Prostaglandins, p. 161, Almqvist and Wiksell, Stockholm. Piper, P. J. and J. R. Vane. 1969. Release of additional factors in anaphylaxis and its antagonism by anti-inflammatory drugs. Nature 223:29. Ramwell, P. W. and J. E. Shaw. 1966. Spontaneous and evoked release of prostaglandins from the cerebral cortex of anesthetized cats. Amer. J. Physiol. 211:125. Ramwell, P. W. and J. E. Shaw. 1970. Biological significance of the prostaglandins. Recent Progress Hormone Res. 26:139. 101 Ramwell, P. W., J. E. Shaw and R. Jessup. 1966. Spontaneous and evoked release of prostaglandins from frog spinal cord. Amer. J. Physiol. 211:998. Reeves, J.J., A. Arimura and A. V. Schally. 1970. Studies on dose response relationship of luteinizing hormone releasing hormone (LH-RH) in sheep. J. Anim. Sci. 31:933. Reeves, J. J., A. Arimura and A. V. Schally. 1971a. Pituitary responsiveness to purified luteinizing hormone releasing hormone (LH-RH) at various stages of the estrous cycle. J. Anim. Sci. 32:123. Reeves, J. J., A. Arimura and A. V. Schally. 1971b. Changes in pituitary responsiveness to luteinizing hormone releasing hormone (LHRH) in anoestrous ewes pretreated with estradiol benzoate. Biol. Reprod. 4:88. Reeves, J. J., A. Arimura, A. V. Schally, C. L. Kragt, T. W. Beck and J. M. Casey. 1972. Effects of synthetic luteinizing hormone-releasing hormone/follicle stimulating hormone releasing hormone (LHRH/FSHRH) on serum LH, FSH and ovulation in anestrous ewes. J. Anim. Sci. 35:84. Rippel, R. H., E. S. Johnson and W. F. White. 1974. Effect of con- secutive injections of synthetic gonadotropin releasing hormone on LH release in the anestrous and ovariectomized ewe. J. Anim. Sci. 39:907. Rommerts, F. F. G., B. A. Cooke, J. W. C. M. Van Derkemp and H. J. Van Dermolen. 1972. Stimulation of 3'5'-cyc1ic AMP and tes- tosterone in rat testis in vitro. Fed. European Biochem. Societies 24:251. Rose, R. M., T. P. Gordon and I. S. Bernstein. 1972. Plasma testos— terone levels in the male rhesus: Influence of sexual and social stimuli. Science 178:643. Roth, J. C., M. M. Grumbach and S. L. Kaplan. 1973. Effect of syn- thetic luteinizing hormone-releasing factor on serum testosterone and gonadotropins in prepubertal, pubertal and adult males. J. Clin. Endocrinol. Metabol. 37:680. Saginor, M. and R. Horton. 1968. Reflex release of gonadotropin and increased plasma testosterone concentration in male rabbits during copulation. Endocrinol. 82:627. Saksena, S. K., I. F. Lau and A. Bartke. 1974. Prostaglandins A1 and A2 decrease testosterone levels in mice and rats. Endocrinol. 95:311. Saksena, S. K., I. F. Lau, A. Bartke and M. C. Chang. 1975. Effect of indomethacin on blood plasma levels of LH and testosterone in male rats. J. Reprod. Fert. 42:311. 102 Saksena, S. K., I. F. Lau and M. C. Chang. 1974. Prostaglandin an and LH release in female hamsters. J. Reprod. Fert. 41:215. Saksena, S. K., S. El Safoury and A. Bartke. 1973. Prostaglandin E2 and F20 decrease plasma testosterone levels in male rats. Prostaglandins 4:235. Sanford, L. M., J. S. D. Winter, W. M. Palmer and B. E. Howland. 1974. The profile of LH and testosterone secretion in the ram. Endocrinol. 95:627. Sanwal, P. C., A. Sundby and L. E. Edqvist. 1974. Diurnal variation of peripheral plasma levels of testosterone in bulls measured by a rapid radioimmunoassay procedure. Acta Vet. Scand. 15:90. Sato, T., M. Hirono, T. Jyujo, T. Iesaka, K. Taya and M. Igarashi. 1975. Direct action of prostaglandins on the rat pituitary. Endocrinol. 96:45. Schally, A. V., A. Arimura, A. J. Kastin, H. Matsuo, Y. Baba, T. w. Redding, R. M. G. Nair and L. Debeljuk. 1971. Gonadotropin- releasing hormone: one peptide regulates secretion of luteiniz— ing and follicle-stimulating hormones. Science 173:1036. Schanbacher, B. D. and J. J. Ford. 1976. Seasonal profiles of plasma luteinizing hormone, testosterone and estradiol in the ram. Endocrinol. 99:752. Smith, P. E. 1927. The disabilities caused by hypophysectomy and their repair. J. Amer. Med. Assoc. 88:158. Smith, P. E. 1930. Hypophysectomy and a replacement therapy in the rat. Amer. J. Anat. 45:205. Smith, 0. W. and H. D. Hafs. 1973. Competitive protein binding and radioimmunoassay for testosterone in bulls and rabbits: blood serum testosterone after injection of LH or prolactin. Proc. Soc. Exptl. Biol. Med. 142:804. Smith, 0. W., K. Mongkonpunya, H. D. Hafs, E. M. Convey and W. D. Oxender. 1973. Blood serum testosterone after sexual prepara- tion or ejaculation or after injection of LH or prolactin. J. Anim. Sci. 37:979. Smith, E. R., R. F. Weick and J. M. Davidson. 1969. Influence of intracerebral progesterone on the reproductive system in female rats. Endocrinol. 85:1129. Spies, H. G. and R. L. Norman. 1973. Luteinizing hormone release and ovulation induced by intraventricular infusion of prostaglandin E1 into pentobarbital-blocked rats. Prostaglandins 4:131. 103 Steinberger, A. and E. Steinberger. 1973. Hormonal control of mam- malian spermatogenesis. In: S. J. Segal, R. Crozier, P. A. Corfman and P. G. Condliff (eds.), The Regulation of Mammalian Reproduction, p. 139, Charles A. Thomas, Springfield, Illinois. Sundby, A., R. Tollman and W. Velle. 1975. Long term effects of HCG on plasma testosterone in bulls. J. Reprod. Fert. 45:249. Thibier, M. 1976. Effect of synthetic gonadotrophin releasing hor- mone (Gn-RH) on circulating luteinizing hormone (LH) and tes- tosterone in young post-pubertal bulls. Acta Endocrinol. 81:635. Tsafriri, A., Y. Koch and H. R. Lindner. 1973. Ovulation rate and serum LH levels in rats treated with indomethacin or prosta- glandin E2. Prostaglandins 3:461. van Dorp, D. A. 1966. The biosynthesis of prostaglandins. Memoirs Society for Endocrinol. 14:19. von Euler, U. S. 1935. A depressor substance in the vesicular gland. J. Physiol. (London) 84:21. von Euler, U. S. 1936. On the specific vasodilating and smooth muscle stimulating substance from accessory genital gland in man and certain animals (prostaglandin and vesiglandin). J. Physiol. (London) 88:213. von Euler, U. S. 1939. Weitere Untersuchungen uber prostaglandin, die physiolgisch aktive substany gewisser genitaldrusen. Skandinavica Arch. Physiologica 81:65. Walsh, E. L., W. K. Cuyler and D. R. McCullagh. 1934. Physiologic maintenance of male sex glands: Effect of androtin on hypo- physectomized rats. Amer. J. Physiol. 107:508. Warberg, J., R. L. Eskay and J. C. Porter. 1976. Prostaglandin- induced release of anterior pituitary hormones: Structure— activity relationships. Endocrinol. 98:1135. Weatherbee, P. S. and J. R. Lodge. 1976. Serum testosterone and estrogen concentrations in the Holstein-Friesian bull after successive ejaculations. Am. J. Vet. Res. 37:465. Weinstein, R. L., R. P. Kelch, M. R. Jenner, S. L. Kaplan and M. M. Grumbach. 1974. Secretion of unconjugated androgens and estrogens by the normal and abnormal human testes before and after human chorionic gonadotropin. The J. of Clin. Invest. 53:1. Westermann, E. and K. Stock. 1970. Inhibitors of lipolysis: Potency and mode of action of a- and B-adrenolytics, methoxamine derivatives, prostaglandin E1 and phenylisopropyl adenosine. Hormone Metab. Res. (Suppl.) 2:47. "7'11llilfllllif'lilflii'llir