GROWTH HORMONE,’ PROLACTIN, LUTEINIZING HORMONE AND GLUCOCORTICOID RESPONSES TO PROSTAGLAND'IN F2a IN CATTLE Dissertation for'the Degree of Ph. D. ' I MICHIGAN STATE UNIVERSITY JOHN NORMAN STELLFL‘UG ' 1976 w II3IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII This is to certify that the thesis entitled GROWTH HORMONE , PROLACTIN , LUTEINIZING HORMONE AND GLUCOCORTICOID RESPONSES TO PROSTAGLANDIN Fed IN CATTLE presented by has been accepted towards fulfillment of the requirements for PhD. Dairy Science degree in Major professor Date November 12, 1976 0-7 639 John Norman Stellflug l l l 35751, 6/032 ABSTRACT GROWTH HORMONE, PROLACTIN, LUTEINIZING HORMONE AND GLUCOCORTICOID RESPONSES TO PROSTAGLANDIN an IN CATTLE By John Norman Stellflug The purpose of this thesis was to determine if prolactin (PRL), growth hormone (GH), luteinizing hormone (LH) and glucocorti- coids were released after luteolytic doses of PGFZa in cattle. After administration of l5, 30 or 60 mg PGFZO to diestrous heifers (n=4, 6, 6, respectively), (l) PRL increased (P<.0l) more than 3—fold, within 10 minutes, to a peak 6-fold above basal values, and returned to pre- injection-values within 4 hours, (2) growth hormone increased (P<0.05) in a dose-related manner, peaking at 30 minutes and remaining above pre-injection values for over 1 hour, (3) LH increased (P<0.05) 2-fold or greater above pre-injection values, within 1.5 to 6 hours, and (4) glucocorticoid (indicator of ACTH release) increased (P<0.0l) more than 6-fold at 30 minutes, and returned to pre-injection values by 4 hours. A second experiment was conducted to determine the site of action of PGFZQ on glucocorticoid release. PGan (25 mg) and saline were given im 7 days aftera pretreatment with triamcinolone acetonide (TA) to suppress serum glucocorticoid to less than 0.5 ng/ml within 24 hours. In saline-treated heifers not given TA, glucocorticoid fluctuated between 10 and 20 ng/ml without relation to the saline John Norman Stellflug injection whereas it increased (P<0.01) 5-fold by 30 minutes after PGFZa and returned to pre-injection values by 3 to 4 hours. In TA- pretreated heifers, peak glucocorticoid response was depressed 50 per- cent after ACTH and 88 percent after PGan in comparison with heifers not given TA. Because glucocorticoid response after ACTH in TA- pretreated heifers was partially inhibited, another experiment was con- ducted to minimize possible adrenal regression after TA, and to maximize the effectiveness of TA. Submaximal doses of porcine ACTH (200 IU) and PGFZa (5mg) were administered to heifers 6 hours after TA pretreatment when glucocorticoid was maximally inhibited by TA. In animals not pre- treated with TA, the first 30 minutes of glucocorticoid response to PGan resembled that after ACTH, but the peak response to ACTH was much greater (P<0.0l), and the duration of response to ACTH was much more prolonged (P<0.01) than that after PGan. TA-pretreatment reduced the glucocorticoid response to ACTH by 50 percent, but it essentially abolished the response to PGan. Three added treatments consisting of simultaneous or sequential administration of PGF 6 and ACTH were included in this third experiment. Glucocorticoid response was more prolonged (P<0.01) when PGFZd and ACTH were injected simultaneously, or when PGFZQ followed ACTH-treatment by comparison to that after ACTH alone. In contrast, when PGan was administered before ACTH, peak glucocorticoid response to ACTH was suppressed by (P<0.05) by comparison to that after ACTH alone. In conclusion, the secretion of PRL, GH, LH, and glucocorticoid after administration of PGFZQ may represent relatively specific action John Norman Stellflug at the hypothalamus or pituitary. No evidence was derived from these experiments for the site Of action of PGan on GH, PRL, or LH secretion, but on the basis of the last two experiments, I suggest PGFZa acts on the hypothalamo-pituitary axis to cause glucocorticoid secretion. Further research is required to distinguish between these sites of PGan action and to determine the physiological importance of PGFZa in A pituitary hormone secretion in cattle. Regardless of whether or not the diversified hormone release after PGan affects the decision of prosta- glandin use for control of ovulation, the results from this research raise the possibility of using prostaglandins to regulate intermediary metabolic hormones in food producing animals. GROWTH HORMONE, PROLACTIN, LUTEINIZING HORMONE AND GLUCOCORTICOID RESPONSES TO PROSTAGLANDIN F IN CATTLE 2d By John Norman Stellflug A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1976 TO SHERYL ii ACKNOWLEDGMENTS The Department of Dairy Science at Michigan State University provided facilities, faculty and financial support for this graduate program. I express my sincerest thanks and appreciation to my major advisor and friend, Dr. H. D. Hafs, for his enthusiastic leadership and encouragement. The participation and consent of the other members of my guidance committee, Drs. J. L. Gill, J. Meites and w. L. Smith, also are gratefully acknowledged. I also wish to thank Dr. R. Neitzel for his assistance with statistical and hormone assay programs. I wish to thank my fellow colleagues, N. Beck, Dr. T. Beck, D. Bolenbaugh, Dr. R. Gorewit, R. Kittok, T. Kiser, Dr. T. Louis, Dr. K. Mongkonpunya, J. Plog and D. Vines, for their assistance at the barn and with laboratory tasks. For their assistance with radio- immunassay procedures, I thank E. Bostwich, P. Kaneshiro, II. Mapes, J. Martin, C. Rankin and J. Yonker. I express a very special note of gratitude to Dr. E. L. Moody whose interest and enthusiasm for reproductive biology was fundamental in leading to my decision to pursue a career in reproductive endocri- nology. To my wife, Sheryl, to whom this dissertation is dedicated, I express my gratitude for her patience, understanding and support throughout my doctoral program. BIOGRAPHICAL SKETCH John Norman Stellflug was born on May 24, 1947, in Glasgow, Montana. He attended Newton School for 6 years prior to entering Glasgow High School and graduated in June, 1965. Pursuing his interest in agriculture, he completed requirements for a Bachelor of Science degree at Montana State University in December, 1969. He accepted a graduate assistantship in the Animal Science and Range Management Department at Montana State University in January, 1970. From February to June, 1970, he completed his active training in the United States Army Reserve before reinitiating his graduate studies and received the Master of Science degree in December, 1972. His thesis was entitled "Periparturient Estrogen Levels in the Plasma of Beef Cows." He then enrolled at Michigan State University studying under the directorship of Dr. Harold D. Hafs. He completed the requirements for the Ph.D. in December, 1976, and accepted a position as a Research Physiologist with the Agricultural Research Service at the United States Sheep Experiment Station near Dubois, Idaho. iv TABLE OF CONTENTS DEDICATION . . ACKNOWLEDGMENTS BIOGRAPHICAL SKETCH . LIST OF FIGURES INTRODUCTION LITERATURE REVIEW Endocrine Events During the Normal Bovine Estrous Cycle . Prostaglandins Historical Review Nomenclature . Temporal Endocrine Events During the Bovine Estrous Cycle Initiated by Prostaglandin an Anterior Pituitary Hormone Responses after Prostaglandin Prolactin . . . . . Growth Hormone Gonadotropins Glucocorticoid Response after Prostaglandins Site and Mechanism of Action of Prostaglandins on Hormone Release . Prolactin . Growth Hormone Gonadotropins . . . ACTH and Glucocorticoid MATERIALS AND METHODS Experimental Design . . . . . . . . , . . . Experiment I: Anterior Pituitary Response to PGFZQ . Experiment II: Site of action of PGan-induced Glucocorticoid Release . Page ii iv vii NVO‘ (A) 00 Experiment III: PGFZQt - Versus ACTH- Induced Gluco- corticoid Release . . . Statistical Analysis RESULTS AND DISCUSSION Experiment I: Anterior Pituitary Hormone Response to PGFZa' Prolactin Growth Hormone LH . . Glucocortiocoid Experiment II: Site of Action of PGan-Induced Glucocorticoid Release . Experiment III: PGan—versus ACTH-Induced Glucocorticoid SUMMARY AND CONCLUSIONS . BIBLIOGRAPHY vi Page 45 53 74 77 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10. 11. 12. LIST OF FIGURES Plasma PRL after 15(2x) (o --o) 30 (A-.-.A) or 60 (0---0) mg PGFZa (im) . . . . . . Plasma GH after 15 (2x) (a no) 30 (A-.-.A) or 60 (0---0) mg PGan (im) . . . . . . Plasma LH after 15 (2x) (a --o) 30 (A-.-.A) or 60 (0—--0) mg PGFZQ (im) . . . . . . Plasma glucocorticoid after 15(2x) (o --o) 30 (A-.-.—A) or 60 (0---0) mg PGan (im) Blood glucocorticoid after sc injection of Triamcinolone Acetonide (22 mg, sc) in heifers Blood glucocorticoid after PGFga (25 mg, so) of saline treatment in heifers with or without Triamcinolone Acetonide (TA) pretreatment Blood glucocorticoid after sc injection of Triamcinolone Acetonide (20 mg) in heifers . Blood glucocorticoid (n=3) after injection (iv) of saline with or without pretreatment with 20 mg Triamcinolone Acetonide (TA, sc) in heifers Blood glucocorticoid (n=3) after injection (iv) of 5 mg PGan or 200 IU ACTH in control heifers Blood glucocorticoid (n=3) in response to injection (iv) of 5 mg PGFZ or 200 IU ACTH 6 hrs. after pretreatment 0% heifers with 20 mg Triamcinolone Acetonide (TA, sc) Blood glucocorticoid (n=3) after 5 mg PGan 200 IU ACTH (+, iv) with or without pretreatment of heifers with 20 mg Triamcinolone Acetonide (TA,sc) Blood glucocorticoid (n=3 ) after injection (iv) of 5 mg PGFZa (+) with or without 200 IU ACTH (l) at 0. 5 hr in heifers . . . vii Page 28 34 38 44 47 50 55 57 60 62 65 67 Figure 13. Figure 14. Blood glucocorticoid (n= 3) after injection (iv) of 200 IU ACTH (t) with or without 5 mg PGan (4) at 2 hr in heifers . Blood glucocorticoid (n= 3) after injection (iv) of 200 IU ACTH (i) with or without 5 mg PGan (A) at 2 hr in heifers viii Page 70 72 INTRODUCTION Artificial insemination to obtain genetic improvement has been hindered by mandatory detection of estrus. This has resulted in development of techniques to sychronize ovulation. One method of ovulation control in cattle is to administer prostaglandin F2a twice at 12-day intervals and inseminate at 80 hours after the second injection of PGan without estrus detection. During the developmental stages of this research other investigators reported that prostaglandins of the E series stimulate release of growth hormone, prolactin, and adreno- corticotropin in several species. In addition, the F series of prostaglandins release luteinizing hormone in sheep,eunigrowth hormone and cortisol but not LH, FSH or TSH in humans. Because PGan shows potential for reproductive control in farm animals, and because of the known diversified actions of PGE'S and of PGF'S, the first study in this thesis was to evaluate the responses of anterior pituitary hormones in cattle to luteolytic doses of PGan. Two additional experiments were executed to determine the site of action of PGFZd on glucocorticoid release. This research has two kinds of practical significance. First, PGan already has been approved for commercial ovulation control in cattle and horses in several countries, and it may be approved in the United States as early as 1977. Whether or not PGan causes release of pituitary hormones, at the time of its use Uncontrol ovulation, may affect decisions on the extent of its use. Secondly, if PGan causes release of intermediary metabolic hormones, such as growth hormone, prolactin and glucocorticoid, perhaps out of such a discovery new methods may arise to regulate these hormones to improve efficiency of food producing animals. LITERATURE REVIEW This literature review initially is a brief review of repro- ductive endocrine events during a normal estrous cycle. The second and larger part is a description of endocrine patterns after luteolytic doses of prostaglandin an to provide a basis for comparison with the normal changes. Endocrine Events During the Normal Bovine Estrous Cycle Days of the estrous cycle in this discussion are numbered from the day of estrus (day 0), which is a 12- to 18-hour period of sexual receptivity. In sequence, the remaining three categories of a normal estrous cycle are: (1) metestrus, the interval from day l to day 4 during luteal development; (2) diestrus, the interim of luteal function which persiSts from day 4 to day 17 or 18; and (3) proestrus, the period from about day 18 to day 21 or 22 during luteal regression and follicular growth. The most recent comprehensive description of the progesterone, estradiol and LH interrelationships from late diestrus until ovulation in the cow was published by Chenault et_al, (1975). They integrated and referenced numerous endocrine studies on the bovine estrous cycle. Blood serum progestins averaged 5.6 ng/ml on day 15 and declined to less than 1 ng/ml by day 19 (Chenault et_al,, 1975). Progesterone normally may begin to decline in individual cows at any time from day 16 to day 19 (Hansel et_al,, 1973), and progesterone synthesis fails rapidly once luteolysis begins. Progestins monitored at 6-hour intervals during the late luteal phase and early proestrus revealed a precipitous decline to less than 50 percent of diestrus concentration within 12 to 18 hours after onset of luteolysis (Chenault gt_al,, 1975). Progestins remained near 0.5 ng/ml thoughout proestrous,estrus, and early metestrus (Kazama and Hansel, 1970; Chenault et al., 1975). Most reports showed no evidence of a proestrus pro- gesterone surge in the cow as was suggested by Ayalon and Shemesh (1974). Luteolysis or death of the corpus luteum, indicated by declining blood progesterone, is initiated by production of the luteolytic factor from the uterus (reviewed by Anderson et_al,, 1969). There are several kinds of evidence for uterine control of luteolysis. For example, ligation of the uterine vein prevents normal luteal regression in sheep (McCracken and Baird, 1969). In addition, ovarian transplants to the neck in sheep (McCracken et_al,, 1971) prolonged the life span of the corpus luteum, but estrous cycles were normal when the uterus was transplanted with the ovary to the neck (McCracken et_al,, 1971); this suggested that a proximal anatomical relationship is required between the uterus and the ovary for normal luteal regression. Follow- ing the suggestion of Babcock (1966) that prostaglandin may be the uterine luteolytic factor, Hansel et_al, (1975) isolated a precursor of prostaglandin an, arachadonic acid, from uterine extracts and found it to be luteolytic in pseudopregnant hysterectomized hamsters. Although prostaglandin Féa has received extensive attention, and Goding et_al, (1972) and McCracken et_al, (1972) concluded that PGF20c is the uterine luteolytic factor in sheep, more evidence is required to confirm this role of PGan in sheep and cattle. Once progesterone declines during proestrus, a wave of follicu- lar growth occurs (Rajakowski 1960; Cole gt_al, 1969) which is thought to be controlled by follicle stimulating hormone (FSH). In support of this notion, Hackett and Hafs (1969) found the decrease of pituitary content of FSH from day 19 to day 0 indicative of FSH release. However, FSH was not elevated in the blood plasma until near the onset of estrus concident with the ovulatory LH surge (Akbar gt_al,, 1974). Follicle stimulating hormone induces formation of FSH receptors in the rat follicle, while FSH and LH jointly are thought to be necessary for estrogen production (Richards and Midgley, 1976). Estrogen increases estradiol receptors in the follicle and estrogen plus FSH induce LH receptors (Richards and Midgley, 1976). However, the control of follicular growth and its temporal relationship with FSH secretion in cattle must await further clarification. In the cow, serum estrogen increases gradually during proestrus, then increases abruptly before estrus (Chenault gt_al,, 1975). This high estrogen stimulates the preovulatory surge of LH (Hobson and Hansel, 1972). Once this LH surge occurs, estrogen decreases pre- cipitously to basal values before the end of estrus (Chenault gt_al,, 1975). In addition to the estradiol increase at proestrus, smaller increases of estradiol may occur (Glencross et_al,, 1973) during metestrus and diestrus. Hansel and Echternkamp (1972) found smaller increases of estrone instead of estradiol during diestrus. The small estrogen increase during diestrus coincides with midcycle follicular development which lasts about 7 days (Cole et_al,, 1969). During the last day of proestrus, LH increases abruptly from 1 ng/ml to between 10 or 20 ng/ml, before it returns to basal concentra- tions (0.5 to 1 ng/ml) within 6 to 12 hours (Swanson and Hafs, 1971). This surge of LH not only stimulates final maturation of the follicle and oocyte, but it also initiates corpus luteum formation. The corpus luteum starts producing singificant amounts of progesterone at the end of metestrus (day 3-4) and then proliferates dramatically, secreting its highest progesterone concentrations by day 15 (Christensen et_al,, 1974). Prolactin is another hormone reported to increase in the blood of cattle near estrus (Sinha and Tucker, 1969; Swanson and Hafs, 1971), but Shams and Karg (1970) found no relationship of serum prolactin to stages of the bovine estrous cycle. Perhaps prolactin increases during proestrus in cattle in response to high estrogen, as reported in rats (Ojeda and McCann, 1974), or it may reflect increased physical activity during behavioral estrus. The functional role of prolactin in relation to estrus, ovulation, and luteal growth in cattle is unclear. Prostaglandins AS a prelude to the subsequent discussion of the endocrine events after PGan-induced luteolysis, here follows a brief background on prostaglandins. Historical Review Prostaglandin, the generic name for a family of biologically active lipids, was first discovered by two gynecologists (Kurzrok and Lieb, 1930). They reported contraction and relaxation of strips of human uterus after applying human seminal fluid. Goldblatt (1933) and Von Euler (1935) described similar muscle contractile properties of human seminal fluid, and Von Euler (1935) related this property to a lipid he named prostaglandin. It was not until the late 1950's that mass spectrophotometric and chromatographic techniques were developed to allow isolation and chemical identification of prostaglandins. Bergstrom et_al, (1962) stimulated new interest in the physiological and pharmacological properties of prostaglandin when he elucidated the chemital structures of three naturally occurring prostaglandins. Nomenclature Prostaglandins (PG'S) are analogs of prostanoic acid, the hypothetical parent 20-carbon fatty acid with a cyclopentane ring at C-8 to C-12. All naturally occurring prostaglandins have a hydroxyl group at C-15 and a 13, 14 transdouble bond. The four major subdivisions of prostaglandins are designated A, B, E, and F which identify groups on the cyclopentane ring. For example, PGan has an a-hydroxyl group which lies below the plane of the ring while beta (8) indicates a group above the plane. The numerical subscripts, which follow the letter, indicate the degree of unsaturation in the alkyl and carboxyl side chains. Temporal Endocrine Events During the Bovine Estrous Cycle Initiated by Prostaglandin F°d Following the initial discovery by Phariss and Wyngarden (1969) that PGan was luteolytic in pseudopregnant rats, Louis et_gl, (1972) and Rowson §t_al, (1972) reported that PGan induced luteal regression in cattle. These results and an increasing supply of synthetic PGFZa stimulated numerous investigations on its luteolytic action. The most recent review article is by Thatcher and Chenault (1976). They charac- terized plasma progestin, estradiol and LH changes from injection of PGFZQ until the time of ovulation. Progesterone decreased 50 percent within 7 hours after PGFZa’ similar to previous studies (Hafs et_al,, 1974; Stellflug et_al,, 1975). When progesterone was measured at frequent intervals after 5, 10 or 15 mg PGan given to six heifers during three consecutive estrous cycles, Stellflug et_al, (1976) found progesterone decreased for the first hour and then rebounded slightly at 2 to 3 hours, as Hixon and Hansel (1974) reported. Then progesterone decreased to less than 1 ng/ml by 24 hours and remained below 1 ng/ml until day 3 to 5. Similarly to Chenault et_gl, (1975) and Thatcher and Chenault (1976, Stellflug gt_al, (1976) found no evidence for the preovulatory progesterone surge reported by Ayalon and Shemesh (1972). Estradiol increased linearly from 2.7 pg/ml at time of PGan injection to 4.6 pg/ml at 76 hours post-treatment (Thatcher and Chenault, 1976). This estradiol increase induced a preovulatory LH surge like the response during normal proestrus. The LH surge was centered at 65 :.5 hours after PGan, coincident with onset of estrus (Louis et_al,, 1974). Louis et_al, (1974), Chanault et_al, (1975), and Thatcher and Chenault (1976) found little difference in magnitude of the peak and duration of the LH surges which occur during normal or PGan-induced luteolysis. LH peaked between 10 to 20 ng/ml, per- sisted for 11.2 :_7.5 hours (i :_sd) and follicles ovulated on the average 21 hours after the LH peak (Thatcher and Chenault, 1976). This is similar to the interval from peak LH to ovulation reviewed by Hafs gt 31. (1974) after PGan and described by Chenault gt_gl, (1975) during a normal estrous cycle. I conclude that endocrine patterns and interrelationships of progesterone, estrogen, and lutenizing hormone after PGFaiin the cow are strikingly similar to these events during a normal cycle. Unfortu- nately, the studies on normal events have not been as intensive as those on events after PGFZd‘ Some further evidence for the similarity of PGan-induced estrus to the normal estrus, although indirect at best, is that conception rate from artificial insemination of cattle treated with PGFZa is not reduced when compared to controls (Lauderdale gt_al,, 1974, and Hafs et_al, 1975). Anterior Pituitary Hormone Responses after Prostaglandin PGan has promising potential for control of ovulation in farm animals if it does not cause undesirable Side effects. If PGan causes release of metabolic hormones, it may provide a means to regulate these important hormones. Previous studies provided intri- guing evidence that prostaglandins of the E series stimulated prolactin, growth hormone, gonadotropin and adenocorticotropin (ACTH) release in 10 several species, but hormone responses to the F series of prostaglandins were equivocal. Prolactin PGFZu caused prolactin (PRL) release as well as lactogenesis and abortion on day 18 of pregnancy in the rat (Vermouth and Deis, 1972). Infusion of PGE1 into the third ventricle of ovariectomized rats also released PRL by 15 minutes, but equal amounts of PGE2, PGFla or PGan failed to release PRL (Harms et_al,, 1973). In contrast, no PRL synthesis or release occurred during incubation of rat pituitaries with PGE PGE PGA or PGF MacLoed and Lehmeyer, 1970). 2’ 1 2d ( Thus, previous literature supports an jn_vivo release of PRL in ‘l’ ovariectomized rats induced only by PGE1 and in pregnant rats during PGan-induced lactogenesis and abortion, but suggests no jn_vitro pituitary response to prostaglandins. Growth Hormone Prostaglandins E1 and E2 induced growth hormone (GH) release jn_vitrg, For example, PGE} (5 x 10'6M) added to the incubation media of heifer pituitary slices stimulated 30 percent more GH production than controls. By monitoring incorporation of 4,5-H3 leucine into GH and its subsequent release from bisected rat pituitaries into medium 199, PGElincreased GH release 165 percent with normal amounts of GH still present in the pituitary tissue (MacLoed and Lehmeyer, 1970). In addition, PGE2 caused similar GH release while PGA1 increased GH synthesis but not release, and PGFZO had no effect on synthesis or release. In agreement with this study, Hertelendy (1971) found 11 increased GH release into media 148 percent after infusion of male rat anterior pituitaries with PGE], but PGE2 stimulated GH to a lesser extent. In vivo studies with sheep and rats also indicated a prostaglan- din induced GH release. When PGE1 was infused iv into male castrate sheep, GH increased from 2.0 to 14 ng/ml by 20 minutes and decreased to 2.1 ng/ml by 60 minutes (Hertelendy gt_al,, 1972). However, this GH response to PGE1 was abolished when PGE1 was administered during infusion of epinephrine. GH peaked at 60 to 90 minutes after a 30-minute iv infusion of 50, 100 or 140 pg of PGE1/kg/min into two adult men per treatment dose (Ito et_al,, 1971). Coudert and Faiman (1973) also observed a slight increase in GH after 2.0 pg of PGFZa/kg/min iv infusion for 30 minutes to 5 men (23-35 years of age). In addition, blood GH increased over 4-fold to a maximum by 10 to 20 minutes after PGE1 (5 pg) injection into the carotid of male rats anesthetized with pentobarbital (Hertelendy et_al,, 1972). In general, jn_vitro studies indicate PGE1 and PGE2 stimulate GH release and PGA1 appears to stimulate only synthesis, while PGFZa has no effect on GH release. In_yjyg_studies also reveal that PGE1 induces GH release and suggest that PGFZd also causes slight stimu- lation of GH release. 12 Gonadotropins Prostaglandin involvement in gonadotropin release is equivocal. Although as little as 0.1 pg/ml PGE1 stimulated rat anterior pituitary cyclic AMP (cAMP) concentrations, 20 pg/ml of PGE had little if any 1 effect on LH release during a 20-minute incubation (Zor et al,, 1970). 1, PGB1 and PGF1é,which augmented cyclic AMP to a lesser extent than PGE], failed to release LH. Infusion of PGan (iv) at Similarily, PGA 0.05, 0.20 or 2.0 pg/kg/min caused no changes in serum LH or FSH in adult men (Coudert and Faiman, 1973). And infusion of 5 pg PGF1a or PGan into the third ventricle of rats caused no significant changes in blood concentrations of LH or FSH (Harms §t_gl,,l973). 0n the other hand, PGE2 given iv early in the afternoon of proestrus elicited a rise in blood LH in rats in which the ovulatory LH-surge had been blocked by pentobarbital (Tsafriri et_al,, 1973). For instance, the LH response after PGE2 was 470 :_87, versus 106 :_17 ng/ml (mean :_se) for controls, and PGE2 induced ovulation in 66 percent of the pentobarbital blocked rats. More convincingly, infusion of 5, 10 and 20 pg of PGE1 into the third ventricle of cycling rats caused release of LH half as high as the peak and half as long in duration as the ovulatory LH surge in rmwnml proestrus females (Spies and Norman, 1973). Concurrently, 5 pg PGE2 induced a 4- to 5-fold increase in blood LH at 15 minutes and a slight increase of FSH at 15, 30 and 60 minutes after infusion into the third ventricle (Harms et_al,, 1973). Also, Carlsonjgtal, (1973) reported increased LH after intracarotid injection of PGF a into diestrous sheep. 13 Thus, as of 1973, the role of prostaglandins in gonadotropin release remained uncertain. Glucocorticoid Response after Prostaglandins Prostaglandins of the E and F series have been reported to affect adrenal steroidogenesis in rats, cattle and humans. For example, PGE], PGEZ, PGF1a, ACTH, and cAMP increased corticosterone during superfusion of rat adrenals (Flack et_al,, 1969), but the most effective prostaglandin (PGEZ) stimulated corticosterone production 1 hour less in duration than the response after ACTH or cAMP. PGE1 and PGE2 also released aldosterone, corticosterone and cortisol from slices of beef adrenals, but PGA], PGF1a and PGan were ineffective (Saruta and Kaplan, 1972). In contrast, infusion (If 25 ng PGan (iv) at 50 pg/min into women (ages 21-40) increased daily urinary cortisol output 3-fold, and plasma cortisol remained high throughout the 8-hour interval (Wentz gt_al, (1973). Coudert and Faiman (1973) also observed a slight in- crease in cortisol concentration after infusion of 2.0 pg PGan/kg/min into five men (23-35 years old). In overview, PGE1 and E2 induced adrenal steroidogenesis in superfused rat and beef adrenals while PGF1a stimulated steroidogenesis only in rat adrenals, and PGan was ineffective in both species. However, PGFZa induced cortisol production in men and women. 14 Site and Mechanism of Action of Prostaglandins on-Hormone Release Prolactin Prostaglandin E1 apparently acts on the hypothalamus and not at the pituitary to cause PRL release, since Harms gt_§l, (1973) found PRL release after intraventricular injections but failed to observe a PRL response after equivalent injections directly into each lobe of the anterior pituitary. In support of PGE1 ndt acting at the pituitary, MacLeod and Lehmeyer (1970), reported no prolactin synthesis or release during incubation of rat pituitaries with PGE], PGEZ, PGA1 or PGFZa' Because prostaglandins have been implicated in transmission across adrenergic synapses in superior cervical ganglion and in the cerebellum (Hoffer §t_al, 1970) Harms et a1, (1973) suggests prostaglandins may act by mediating or modulating the action of synaptic transmitters such as norepinephrin and dopamine, which already have been shown to possess capabilities of altering PRL release (Hoffer gt_§l,, 1970). Harms gt_al, (1973) also presumed that PGE1 may stimulate prolactin releasing factor (PRF) or inhibit prolactin inhibiting factor (PIF). Although further research is required to determine the mechanism of action of prostaglandin-induced PRL release most evidence suggests a hypothalamic site of action. Growth Hormone The site of action for PGE1- and PGE2 -induced growth hormone release is thought to be the pituitary. The most substantial evidence 15 for this is that PGE1 and PGE2 stimulated GH release during 1! vitgg pituitary incubation (Schofield, 1970; Hertelendy, 1971). The mechanism of action might involve cAMP, since PGE1 in- creased cAMP concentration in pituitary cultures (Zor gt_gl,, 1969). Moreover, theophylline, a cyclic nucleotide phosphodiesterase inhibitor, potentiated PGE1 (Schofield, 1970) and PGE, and PGE2 stimulation of GH (Cooper gt_gl,, 1972), suggesting that this increased GH release was a result of increased cyclic AMP content. Adenyl cyclase also increased in the anterior pituitary after PGE], PGEZ, PGA, and PGFZa treatment, but GH synthesis and release was observed only with the PGE's and not after PGan, while PGA1 increased synthesis but not release of GH (MacLoed and Lehymer, 1970). When 7-oxa-13-prosynoic acid, a prostaglandin synthesis anta- gonist, was added to pituitaries jn_vitrg_it blocked PGE1 stimulation of GH release and cyclic AMP accumulation but failed to block the stimulatory action of theophylline and dibutryl cyclic AMP (chMP), substantiating involvement of PGE1 with cyclic AMP (Ratner gt_gl,, 1973). On the other hand, according to Hertelendy gt_al, (1972), PGE1 stimula- tion of GH release was abolished when PGE1 was given to castrated rams and anesthetized rats during epinephrin infusion and neither alpha- nor beta-adrenergic blocking drugs could overcome this block. They suggested that the site of epinephrin block was beyond the adenyl cyclase-CAMP step and argue against the idea that PGE], in this instance may, act as a mediator of releasing factors, or that releasing factors first activate biosynthesis which converts fatty acids to prostaglandins which activate adrenyl cyclase. 16 Thus, further investigation is required to determine the mechanism of action of PGEI-induced GH release. PGE1 probably acts directly on the pituitary, independently of adrenergic receptors. Gonadotropins The sites and mechanism of action of prostaglandins on gonado- tropin release are less well defined than those for prolactin and growth hormone. The general consensus up to 1974 was that prostaglandins of the E series cause release of LH jn_ijg; but not jn_vitrg, The E series of prostaglandin did not cause release of LH jn_vjtrg_nor'in_yjyg_ except in sheep (Carlson et_al, 1973). The negative results from in_ 11339 pituitary incubation (Zor gt_al,, 1970) may not contradict the positive jn_yivg_results because infusion of prostaglandins directly into the anterior pituitary did not increase blood LH, but infusion of the same doses of PGE1 and PGE2 into the third ventricle of the rat increaseleiconcentrations significantly (Spies and Norman, 1973 and Harms et_al,, 1973). From the demonstration that the PGE's stimulate release of gonadotropins only when put into the third ventricle, Spies and Norman (1973) and Harms et_al, (1973) suggested that PGE1 and PGE2 activate a neurally controlled gonadotropin releasing mechanism. PGE'S may be an intermediate step in release of hypothalamic releasing factors, or may act by mediating or modulating the action of synaptic transmitters such as norepinephrin or dopamine which are capable of altering releasing factors (Harms §t_al,, 1973). Tsafriri et a1, (1973) also suggested that PGE2 has a central effect and that indomethacin (a prostaglandin biosynthesis inhibitor) 17 does not block LH release, but rather it interferes with ovulation at the level of the ovary. Carlson et_al, (1973) suggested that PGan stimulates LH release in ewes during day 5 to 10 of the estrous cycle, probably by action primarily at the hypothalamus, but action at the pituitary could not be ruled out. In overview, most of the literature supports a PGE- but not a PGF-induced LH release at the hypothalamus, but verification of this stimulatory action, the site of action, and its physiological signifi- cance will require further research. ACTH and Glucocorticoid Two schools of thought exist on the site and mechanism of action of prostaglandins on adrenal steroidogenesis. Initially, corticosterone synthesis increased with addition of PGE], PGE2 or PGan during jn_vjtrg_adrenal culture; this increase was mimiced by ACTH and cAMP (Flack et_al:, 1969). Cycloheximide, which inhibits formation of proteins controlling the adrenal steroidogenic pathway, inhibited increase of corticosterone jn_yitrg, indicating that these stimulating substances induce synthesis as well as release of glucocorticoids. In addition, preliminary in_yjyg_studies portrayed the stimulatory action of PGE2 to be similar to that of ACTH and CAMP, because corticosterone concentrations in plasma and the adrenal increased 70 and 40 percent in response to PGEZ, respectively (Flack et_al,, 1969). Pursuing this concept of similarity, Flack and Ramwell (1972) demonstrated that the initial rate of synthesis of corticosterone was equivalent during PGE2 and ACTH incubation, with peak production occurring at 60 minutes, l8 considerably sooner than the peaks after cAMP (90 min) and dibutryl cyclic AMP (dbcAMP, 150 min). Corticosterone responses to ACTH, cAMP and dbcAMP decayed at comparably slow rates, but the response to PGE2 decreased more rapidly indicating another marked difference from ACTH. They concluded that PGE2 stimulates corticosteroidogenesis by direct action on the adrenal cortex. Saruta and Kaplan (1972) reported results similar to those from Flack and Ramwell (1972) for PGE1 and PGE2 in beef adrenals. In their jn_vitrg_system, PGE1 and PGE2 significantly induced synthesis of aldosterone, corticosterone and cortisol, while PGA, PGF1a and PGan were ineffective. PGE1 stimulated steroidogenesis in a manner similar to that of ACTH. For example, PGE1 required calcium, was inhibited by puromycin but not actinomycin D, and increased CAMP concentrations, and its effects on endogenous cAMP were not additive with those from ACTH. However, PGE] did not have additive effects with maximal or submaximal concentrations of ACTH. These findings support the hypothesis that PGE1 shares receptor sites on the plasma membrane with ACTH (Saruta and Kaplan, 1972). Others hypothesize a specific role of PGE1 and PGE2 (in the regulation of adrenal steroidogenesis) on the central nervous system (CNS) instead of a direct action on the adrenals. PGE1 and PGE2 were the only substances of low molecular weight found in the brain and capable of stimulating ACTH discharge in a number of assay systems that are commonly used as CRF (CorticotrOpin releasing factor) assays (rats pretreated with pentobarbital and chlorpromazine or pentobarbital and dexamethasone, deWied et a1. 1969). In agreement with deWied gt_al, 19 (1969), PGE1 failed to alter adrenal ascorbic acid in cortisol- pretreated hypophysectomized rats, indicating PGE1 has no ACTH-like effect on the adrenal but acts by stimulating ACTH release (Peng et_al,, 1970). This stimulation could be at the site of the pituitary or hypothalamus because cortisol may inhibit adrenal steroidogenesis at either or both sites (Ganong, 1963). Similarly, the plasma cortisol increase observed throughout infusion of PGan into women was eliminated by dexamethasone pretreatment, suggesting that PGan does not act directly on the adrenal to stimulate cortisol biosynthesis, but probably operates through induction of ACTH release (Wentz §t_al,, 1973). More convincingly, Peng et_al, (1970) inhibited the adrenal ascorbic acid response to PGE1 in intact rats anesthetized with sodium pento- barbital by pretreating them with morphine, which inhibits secretion of CRF. Hedge (1972) defined more clearly the effects of prostaglandins on ACTH secretion; rats were anesthetized with sodium pentobarbital and pretreated with dexamethasone. PGE], and PGE1a and PGan (1.0, 0.5 and 0.5 pg, respectively) increased ACTH comparably when injected into the median eminence, butwere ineffective when injected into nearby regions of the basal hypothalamus, the anterior pituitary or the tail vein. In addition, the ACTH responses to PG's were abolished by pretreatment with mOrphine and were partially inhibited by a higher dose of dexamethasone. The stimulatory effect of PGE1a was abolished by incubation in rat plasma at 37°C for only 1 minute before injection but the effect of PGE1 was unaltered. Hedge (1972) also concluded that prostaglandins stimulate ACTH secretion indirectly by acting at 20 the median eminence presumably via CRF, but the mechanism, as well as the physiological significance, remains unanswered. In overview, the evidence for prostaglandins mediating adrenal steroidogenesis through ACTH release is more convincing to me, but a direct action of prostaglandins on the adrenal cannot be ruled out completely. MATERIALS AND METHODS One experiment was conducted to determine if anterior pituitary hormones were released in response to luteolytic doses of prostaglandin F2“ (PGan). Glucocorticoids (indicator of adrenocorticotropin, ACTH, release), luteinizing hormone (LH), prolactin (PRL) and growth hor- mone (GH) were monitored for 18 hours after intramuscular (im) in- jections of PGFZQ. As the results will indicate, this first experiment permitted description of surges of anterior pituitary hormones and glucocorticoid in cattle after PGan treatment. Consequently, two additional experiments were designed to determine the site(s) where PGan acts to cause the surge of glucocorticoid. Prior to the first of these two experiments, heifers in a perliminary trial were given a long-acting synthetic glucocorticoid (triamcinolone acetonide, TA), and were bled daily via tail vein puncture, for 1 week to verify that TA inhibited glucocorticoid production. Then, TA-pretreated heifers and control heifers were given saline or PGan to identify the site of PGan action. Based upon the results from the second experiment, there was concern that the adrenals may have atrophied by 1 week after TA, retarding corticoid response to ACTH and PGan. Therefore, another preliminary trial was conducted to ascertain precisely how rapidly TA decreased glucocorticoid production to basal concentrations. Then, 21 22 a third experiment was conducted to provide more information about the site of action of PGFZa on glucocorticoid release. Detailed descrip- tions of these three main experiments follow. Experimental Design Experiment I: Anterior Pituitary Hormone Response to PGFOa Because im injection of 0.85 percent sodium chloride did not alter (P>0.05) glucocorticoid, prolactin (PRL), or growth hormone (GH) concentrations during a 6-hour post-injection interval, controls (given saline vehicle) were not included in Experiment I. Three luteolytic doses of PGFZQ tham salt diluted in 2 m1 saline were injected im to evaluate their effects on blood plasma PRL, GH, LH and glucocorticoids. Diestrous heifers were given (1) 15 mg PGan at time 0 and 6 hours later (n=4), (2) 30 mg PGF2 (n=6) or (3) 60 mg PGan (n=6). Blood was put into heparinized tubes in an ice bath immediately after collection through jugular cannulae. Blood was sampled just before treatment (time 0), at lO-minute intervals for 1 hour and then at 1.5, 2, 4, 6, 12, and 18 hours. After the second lS-mg PGFZd injection, blood was collected at the same frequency as after the injections at time 0. The blood was centrifuged and the plasma was decanted and frozen within 4 to 6 hours after collection. Plasma glucocorticoids were measured by protein-binding assay described by Smith et_a1, (1973). GH, LH, and PRL were analyzed by radio immunoassays (RIA) described by Purchas et a1, (1970), Oxender et_al, (1972), and Tucker (1971), respectively. 23 Experiment II: Site of Action of PGEzqunduced Glucocorticoid Release The experimental animals consisted of 9 diestrus and 9 ovari- ectomized heifers. To ensure each diestrus heifer had a functional corpus—luteum, each intact heifer was given 25 mg PGFZOI (Tham salt, im) 11 days before beginning experimental treatments. This pretreatment was intended to induce luteolysis and to provide a 7- to 9-day corpus luteum when treatments were given. TA (22 mg) was injected (sc) into 3 intact and 3 ovariectomized heifers, and the heifers were bled (tail vein puncture) daily for 1 week before they were given (im) the experimental dose (25 mg) of PGFZQ tham salt. The remaining 12 animals were not pretreated with TA; 3 diestrus and 3 ovariectomized heifers were given (im) 2 ml saline and 3 of each were given (im) 25 mg PGan. The 25-mg im dose of PGan was chosen because it was known to cause luteolysis in heifers. The experimental blood samples were collected through jugular cannulae at half-hour intervals for 4 hours before saline or PGan injections, then at 5—minute intervals for 30 minutes after the experi- mental treatments, followed by lO-minute samples for another 30 minutes, then half-hour samples for another 11 hours. At 12 hours after saline or PGan, 2 of the 3 heifers within each treatment group were given (sc) 5000 IU ACTH. This ACTH treatment was intended to insure that the heifers could produce glucocorticoids. Blood sample collection con- tinued at half-hour intervals for 4 hours after ACTH treatment. Blood samples clotted at 25°C for 2 to 4 hours before they were stored at 24 4°C for 8 to 12 hours. Then, samples were centrifuged and serum was decanted and stored at -20°C prior to analysis for glucocorticoids, as in Experiment I. Experiment III: PGF?” - Versus ACTH-Induced Glucocorticoid Release Results from Experiment II suggested that the TA pretreatment may have caused some atrophy of the adrenal cortex within 7 days after TA was given. Consequently, a preliminary trial was conducted to determine the minimal period required for TA to maximally suppress glucocorticoid production. Each of two diestrous heifers was given 20 mg of TA (so) and bled at 3-hour intervals for 12 hours, then at 16 and 24 hours. Inspection of these preliminary data indicated that TA maximally suppressed glucocorticoid within 6 hours. Consequently, treatments were given 6 hours after TA pretreatment in the following experiment. Eighteen heifers were given 25 mg PGF2 (im) 11 days before a. administration of experimental treatments, to obtain diestrous heifers at the time Of treatment. Nine of the 18 diestrous animals were pre- treated with 20 mg TA (sc) 6 hours before treatments then, 2 ml of saline, 5 mg of PGFZQ and ACTH were chosen to stimulate a submaximal glucocorticoid response to enable observation of any additional release induced by an added stimulus. Blood samples were collected through jugular cannulae at 6, 3, l, 0.5, 0.25 and 0 hours before treatment. After treatment, samples were taken at 15-minute intervals for 3 hours, followed by 30 minute intervals to 4 hours post-treatment, and then 25 hourly for 6 hours. Serum glucocorticoid was monitored as described under Experiment I. Statistical Analysis The hormone data from the three principal experiments were analyzed by the split-plot method described by Gill and Hafs (1971) because of repeated measurements within animals. Due to this repeat measurement, the probability of type 1 error may be artificially low because of the high correlation of errors if there is heterogeneity of the variance and covariance of samples taken at several times from one animal. Thus, the type 1 error may exceed its actual value if measurements at close intervals of time are more significantly correlated than those more distant in time. A conservative F-test (Gill and Hafs, 1971) was used to confirm results that were marginally significiant. Thus, analysis of all three experiments was a variation of the split-plot theme. The first experiment was a split-plot design with time as a factor and selected contrasts were compared via Scheffés procedure (Kirk, 1968). Both the second and third experiments were split-plot designs where animals were assigned to two pretreatment and three treatment combinations with time as a factor, and selected con- trasts were compared via Bonferroni T-test (Miller, 1968). RESULTS AND DISCUSSION Experiment I: Anterior Pituitary Hormone Response to PGFoa The objectives of the first experiment were to monitor anterior pituitary hormones and glucocorticoid in blood plasma after luteolytic dOses (15, 30 or 60 mg) of PGan given intramsucularly. The results for each hormone will be discussed separately. Prolactin ‘ Prolactin (PRL) response did not differ (P<0.05) among the three PGFZa doses during the first 6 hours after injection. On the average, plasma PRL increased (P<0.01) from 26 to 81 ng/ml within 10 minutes after PGFZO injection and remained above pre-injection concen- trations for approximately 4 hours (figure 1). A similar PRL response occurred after the second lS-mg injection of PGan given 6 hours after the first injection in hour heifers. These PRL surges were: (1) greater in magnitude than those normally observed in response to milking (Tucker, 1971); (2) similar to the PRL release induced by iv injection of thyrotropin releasing hormone (Convey et_gl,, 1973); and (3) considerably less than those which normally occur at parturition in cows (Ingalls §t_al,, 1973). Earlier, PGan had been reported to induce release of PRL when administered to pregnant rats; it also caused lactogenesis and abortion suggesting that decreasing progesterone (precipitated by PGan-induced 26 27 Figure l.--P1asma PRL after 15 (2x) @--0) 30 (A-.-.A) or 60 0---0) mg PGan (im). Standard errors of means ranged from 1 to 27 ng/ml and were generally proportional to the mean. 28 33.2 s Sham 5. Loco czosoi 0635 owned .23 950... Q_N___O.mwb wm¢mm_ arm - _ _ 7311111441. _ _ qJfid-_-dfi '~ 100. ION. . . 103 a . a row. sauce I 9:9 85a 8. I o m (|uI/OU)u1;OO|OJd 29 luteolysis) might stimulate the increase in PRL (Vermouth and Deis, 1972). In partial agreement with this concept, Vermouth and Deis (1972) found delayed onset of lactogenesis and parturition with pro- gesterone replacement therapy in PGan-treated pregnant rats, but this only partially blocked PRL release. In addition, no PRL release occurred after intraventricular injection of PGFZO into ovariectomized rats (Harms gt_al,, 1973) or after iv administration of PGFZa into adult men (Coudert and Faiman, 1973). To my knowledge, at the outset of my research, the only other reported increase of PRL after prosta- glandin occurred after PGE1 infusion into the third ventricle of ovariectomized rats, but not after an identical dose was infused into the pituitary (Harms et_al,, 1973). This specific PGE -induced PRL 1 release suggests a hypothalamic site of action. During the course of my research, more evidence has accumulated concerning the role of prostaglandins in PRL release. Yue et_gl, (1974) observed a greater increase of serum PRL concentration after 20 intra- uterine injections of 500-1500 pg PGFZa at intervals of l to 2 hours or intra-amniotic injection of 30 mg PGan during induction of abortions than after normal birth or hypertonic saline-induced abortions in women. In contrast, five post-menopausal women had no increase of serum PRL after intravenous infusion of 18 mg PGan over a 6-hour period (Vanderheyden et_al,, 1974). Some further information on release of PRL by PGFZa is available from studies with rats. Serum PRL increased 10 to 60 minutes after a single iv injection of PGFZO’ PGE1 or PGE2 (670 pg/rat) 30 into ovariectomized rats primed with estrogen and progesterone (Ojeda et_al,, 1974a). In fact, as little as 20 pg of PGF 2 pg of PGE 2d’ 1 or PGE2 increased blood PRL in the same rats, confirming that the PRL release is stimulated by some prostaglandins of the E and F series. In contrast (unlike the response to PGEl), PGFZa’ PGFla and PGE2 failed to increase plasma PRL when infused into the third ventricle of ovariectomized estrogen-pretreated rats (Ojeda gt_al,, 1974a). In the male rat, PGE2 increased PRL when it was infused into the lateral ventricle but not when infused into the hypophyseal portal vessels (Eskay et_al,, 1975). Similarly, PRL increased 6- to 7—fold at 30 minutes after infusion of 5 pg PGE2 or 20 pg PGF28 (3- to 4-fold greater than controls) into the lateral ventricle but, 20 pg of PGFZa’ PGF PGB PBG did not alter the a PGF1B, PGA], PGA or 10 pg of PGE 1 2’ 1’ 2 l basal secretion of PRL in male rats anesthetized with sodium pento- barbital (Warbert et al., 1976). In further support of the PRL release induced by PGFZa in heifers, release of PRL in bulls was related directly to the dose of PGan injected im (Hafs, 1975). In addition, plasma PRL increased 25- fold after an intracarotid injection of 50 pg PGFga into mature bulls (Stellflug, unpublished data). The PRL release after systemic administration of PGFZa is contradicted by the lack of PRL release after intraventricular infusion of PGFZd‘ But this contradiction might be expected since the rat possesses a reversible enzyme (PGE2-9-Keto-reductase) which converts PGE2 to PGan (Leslie and Levine, 1973). And, their preliminary experiments have shown this activity to be present in heart homogenates 31 of chickens, rabbits, cats, cattle and guinea pigs and in rabbit kidney and guinea pig liver homogenates. Enzyme location may explain how PGan induces PRL release when administered systemically and not when infused into the third ventricle. However, caution must be taken when comparing species; for instance, an enzyme present in the cellular fraction of sheep blood reduces PGE2 to PGan but is not reversible or coenzyme-dependent like the enzyme in rats (Hensby, 1974). In contrast to PGF PGE and PGE stimulated PRL release when infused into the 2d’ 1 2 third ventricle but not when PGE1 wasinfused into the pituitary and not when PGE2 was infused into the hypophysial portal vessel--sug- gesting action on the central nervous system, possibly the hypothalamus. However, some evidence indicates that under certain conditions (ovari- ectomized rats), PGE1 may also stimulate a small PRL release by action on the adenohypophysis but this PRL increase was less than when PGE was infused in the third ventricle and it could be abolished by estrogen pretreatment unlike the PRL release after intraventricular infusion (Ojeda gt_al,, 1974). Regarding the mechanism of action for PRL release induced by E prostaglandins, Ojeda et_al, (1974b) suggested a possible role of CAMP mediation and PGE1 modulation'hi the dopaminergic control of PRL release. In summary, the PGan-induced PRL release in heifers (figure 1) is supported by jn_vjyg_studies on pregnant rats and women, on ovariectomized rats primed with estrogen and progesterone, on adult men given PGan intravenously, and on bulls after intramuscular or intracarotid injections. In contrast, PRL was not released when PGan 32 was (1) infused into the third ventricle of ovariectomized estrogen- treated rats and adult male rats, (2) after infusion directly into the anterior pituitary of rats or (3) injected iv into post-menopausal women. These results suggest that the steroid environment modulates release of PRL after PGan in the female, but further studies will be required to verify this hypothesis especially since it is based on information from several species. Growth Hormone Before PGan, plasma GH averaged 3 ng/ml (figure 2). Although GH at least doubled after each of the two lS-mg im injections of PGan, these increases did not differ from pre-injection values (P>0.05). GH increased (P<0.01) to 35 and 51 ng/ml within 30 minutes after 30- and 60-mg PGFga’ respectively, and remained above pre-injection values for at least 1 hour. The 30-minute peak plasma GH concentrations were linearly related to the log of the dose of PGan (P<0.05). This GH increase in our heifers was greater than that induced by TRH in lactating cows (Convey et_al,, 1973), but less than those which occur normally at parturition (Ingalls et_al,, 1973). Previously, only one study had indicated GH release after PGFZa Coudert and Faiman (1972) reported a slight increase in CH after 2.0 pg of PGan/kg/min in five men, but no increase occurred after 0.2 pg of PGan/kg/min or less. To my knowledge, just two other studies monitored GH after PGan and both reported no effect on GH release in_ yjtgg; one incubated heifer pituitary slices (Cooper et_al,, 1972) and the other cultured bisected male and female rat pituitaries 33 Figure 2.--P1asma GH after 15 (2x) (one) 30 (A-.0.A) or 60 (0---0) mg PGFZQ (im). Standard errors of means ranged from 1 to 14 ng/ml and were generally proportional to the mean. 34 tea; 5 ab... .5 5:0 .16 2:85 canon 3:0 8:0: 0.N_=O_mmh @DVDN_ 0 IT. a - WIVNE-fi. 44.¢_-_ I¢/./ >Aw’ S. 1 .I.05) between diestrous and ovariectomized heifers. A similar, prolonged decrease in glucocorticoids also was observed in people given TA. For example, plasma cortisol in women did not in- crease above 1.2 ng/ml until 11 days after a 25-mg TA injection (Cunningham et__l,, 1975). That is greatly lower than normal average cortisol value of 72 ng/ml (Czeisler §t_a1,, 1976). Seven days after the TA pretreatment, the experimental doses of 25 mg PGFZd or saline were given im. Since glucOcorticoid re- sponses to PGFZO treatments did not differ (P>0.5) between diestrous and ovariectomized heifers, in these results, treatment glucocorti- coid values are averaged. Glucocorticoid response to the three treatments (figure 6) differed significantly (P<0.001) from each other. Blood glucocorticoid increased (P<0.01) from 10 to almost 50 ng/ml by 30 minutes after PGan, and remained above pre-injection values for 3 to 4 hours in heifers without TA pretreatment. In con- trast, blood glucocorticoid increased (P<0.01) from 0.5 to 7 ng/ml at 30 minutes after PGFZO and remained above pre-injection concen- tration for 4 to 5 hours in TA pretreated heifers. Thus, peak glucocorticoid response in TA pretreated heifers was much less (P<0.001) than that in heifers not given TA. In saline treated controls, blood glucocorticoid fluctuated between 10 and 20 ng/ml during the 12-hour observation period; saline injection induced no glucocorticoid response like those observed after PGFZOI‘ In sumnary, increased glucocorticoid (P<0.01) in heifers without TA pretreatment (figure 6) resembled the increase observed after PGan in experiment 49 Figure 6.-—Blood Glucocorticoid after PGan (25 mg, sc) or Saline Treatment in Heifers with or without Triamcinolone Acetonide (TA) Pretreatment. The standard errors of the means ranged from 9.2 to 1.3 ng/ml (NOTA, Saline), 5.7 to 1.0 ng/ml (NOTA, PGFga), 1.4 to 0.1 ng/ml (TA, PGan) and were generally proportional to the mean. 60 5O 4O 30 20 GLUCOCORTICOID (no / ml ) 50 1 Pretreot PG F20 no yes 0—-O (n=6) no no Ann-A (n=6) TA yes H (n=6) "0 TA, PGE-20 . / O - 1 _ _ -2 O 2 4 6 8 IO l2 HOURS FROM PGF“ (251119.80) BLOOD GLUCOCORTICOID AFTER PGF“ (251119.86) OR SALINE TREATMENT IN HEIFERS WITH OR WITHOUT TRIAMCINOLONE ACETONIDE I TA) PRETREATMENT 51 I, and TA partially blocked (P<0.001) the PGFZa-induced peak gluco- corticoid response in these heifers. In addition to the main part of experiment II, 5000 IU ACTH was administered iv to two of the three heifers in each state (diestrous or ovariectomized) of each treatment group at 12 hours after the injection of saline or PGFZa' The purpose of the ACTH in- jection was to test whether the heifers could respond with glucocorti- coid secretion 7 days after TA pretreatment. Glucocorticoid increased (P<0.01) in all ACTH-treated heifers without TA from 8.6 :_2.9 ng/ml to a peak of 57.3’ i 4.6 ng/ml (32 : SE), but glucocorticoid response to ACTH in the TA pretreated heifers was reduced (P<0.01) to 21.7 1; 5.4 ng/ml in comparison with that for heifers not receiving TA. The inhibition of glucocorticoid by TA illustrated in figure 6, is similar to inhibition of the PGan-induced cortisol response by dexamethasone in women (Wentz §t_al,, 1973). Dexamethasone pre- treatment also blocked PGE -, PGEZ- or PGan- induced glucocorticoid release when these PG's were injected into the basal hypothalamus, the anterior pituitary or a tail vein but not after injection into the median eminence of rats anesthetized with pentobarbital (Hedge, 1972). Thus, the results of the present study agree with previous research. However, TA did not completely abolish the glucocorticoid response to PGFZO in my heifers. This incomplete inhibition might indicate that TA lost some inhibitory action within 7 days. In support of this notion, glucocorticoid increased slightly although not 52 significantly between day 6 and 7 in the ovariectomized heifers and between days 1 and 6 in the diestrous animals after TA pretreatment (figure 6), however, the glucocorticoid values in the TA pretreated heifers given saline did not increase (P<0.01) throughout the entire 8 day sampling period. Cortisol apparently did not increase within 11 days after TA treatment in humans as mentioned previously (Cunningham et_al,, 1975), but extrapolation across species may be unjustified and to my knowledge there are no other reports on glucocorticoids after TA in cattle. Similarly, larger doses of TA might be required to completely suppress glucocorticoid in cattle, since inhibition of glucocorticoid was proportional to the amount of dexamethasone in rats (Hedge, 1972). Another explanation could be that TA pretreatment prevented action by some facilitating factors required for full glucocorticoid response to PGFZa‘ For example, an interaction of anterior pituitary hormones is a possibility because both basal secretion and hypoglycemia-induced release of prolactin, ACTH, and growth hormone are suppressable by glucocorticoids (Copinschi §t_al,, 1975). Dexamethasone also blocked stress-induced prolactin release in a dose dependent manner (Harms at al,, 1975). The reduction of glucocorticoid response to ACTH appears anomalous since synthetic corticoids are thought to inhibit glucocorti- coid production by inhibiting ACTH release (Kendall et_al,, 1966 and Arim ir a et_al,, 1969). However, dexamethasone, another glucocorti- coid, accelerated adrenal protein and RNA degradation and this rate of degradation appeared to be correlated with the degree of dexamethasone-induced atrophy (Ichii et_al,, 1974). Thus, 53 dexamethasone not only acts on the pituitary and CNS to inhibit ACTH secretion, but it also may inhibit steroidogenesis by direct action on the adrenals, especially if one waits for long periods of time after pretreatment with a synthetic glucocorticoid because the rate of degradation appeared to be correlated with the degree of synthetic glucocorticoid-induced atrophy. This factor could prevent one from observing the direct action of a prostaglandin on the adrenal so per- haps PGFZa does have some direct action on the adrenal. Consequently, another experiment was conducted to minimize possible adrenal atrophy and to maximize the effectiveness of TA. Experiment III: PGF2a_- Versus ACTH- Induced Glucocorticoid Release. In the preliminary experiment with two heifers, serum gluco- corticoid declined (P<0.01) abruptly from 10.6 to 0.2 ng/ml, within 6 hours after sc injection of 20 mg TA, and remained below 0.5 ng/ml throughout a 24-hour observation period (figure 7). Therefore, 6 hours was selected as the interval between TA pretreatment and the time of injection (iv) of 5 mg PGFZa or 200 iu ACTH in experiment III. The split plot analysis of this experiment revealed signifi- canttxeatment(P<0.0l) and pretreatment effects (P<0.05). Serum glucocorticoid for the saline injected controls was significantly higher (P<0.01; figure 8) than that for saline-injected heifers pretreated with TA. This response to TA resembled that in the pre- liminary trial and verifies the inhibition of glucocorticoid secretion in response to TA observed in experiment II. 54 Figure 7.——Blood Glucocorticoid after sc Injection of Triamcinolone Acetonide (20 mg) in Heifers. The standard errors of the means ranged from 0.7 to 0.1 ng/ml and were generally proportional to the mean. GLUCOCORTICOID (no/ml) 55 L O O 6 l2 l8 HOURS AFTER TRIAMCINOLONE ACETONIDE BLOOD GLUCOCORTICOID AFTER sc INJECTION OF TRIAMCINOLONE ACETONIDE (20mg) IN HEIFERS 24 56 Figure 8.--Blood Glucocorticoid (n=3) after Injection (iv) of Saline with or without Pretreatment with 20 mg Triamcinolone Acetonide (TA, sc) in Heifers. The Standard errors of the means ranged from 4.1 to 0.7 ng/ml (Sham) and 2.1 to 0.7 ng/ml (TA, Saline) and were generally proportional to the mean. 57 l2ISham )fl E'°'(,/ I‘ Ifil £86111 I!) 1“ 11 EB— }? § §”\ IR 3.. I; 11 11 VS 4" g 2f SalIne (9 -6-3 -10 2 4 6 81021 Hours from injection BLOOD GLUCOCORTICOID (n=3) AFTER INJECTION (iv) OF SALINE WITH OR WITHOUT PRETREATMENT WITH 20 mg TRIAMCINOLONE ACETONIDE (TA,sc) IN HEIFERS 58 In heifers not given TA, the glucocorticoid response after ACTH was greater (P<0.01) than that after PGan. Glucocorticoid increased (P<0.01) to 30 ng/ml within 30 minutes after PGF and ACTH 2a alike (figure 9), but glucocorticoid continued to increase (P<0.01) to over 60 ng/ml at 2 hours after ACTH, whereas it began to fall (P<0.01) 30 minutes after PGFthreatment toward, pre-injection values. Glucocorticoid remained above pre-injection values for about 2 hours after PGan, much less (P<0.01) than the lO-hour period of elevated glucocorticoid after ACTH injection (figure 9). In other words, the initial glucocorticoid response to PGFZd resembled that from ACTH, but the duration of the response to the porcine ACTH was much more prolonged than that after PGF The differences in ACTH- and PGF 2a' 2&- induced glucocorticoid release may indicate that the dose of PGFZa was smaller than the dose of ACTH relative to their glucocorticoid stimulatory activity. More likely, perhaps PGanwas cleared more rapidly than the porcine ACTH; PGan is known to have a metabolic clearance rate of 17 liters/minute in cattle (Stellflug et_al,, 1975). The simultaneous increases of glucocorticoid after PGFZa and ACTH suggest that the iv PGanmay have caused rapid ACTH release. If this hypothesis is correct, one might expect prolonged glucocorticoid elevation during infusion of PGFZa for several hours to prolong its action. In heifers pretreated with TA (figure 10), peak glucocorticoid response to ACTH (30 ng/ml) was much greater (P<0.01) than that after PGan (2ng/m1). In fact, the glucocorticoid response to PGan was not significant (P>0.05) in TA-pretreated animals. In other words, 59 Figure 9.--Blood Glucocorticoid (n=3) after Injection (iv) of 5 mg PGan or 200 IU ACTH in Control Heifers. The standard errors of the means ranged from 0.2 to 6.2 ng/ml (PGF2 ) and 0.3 to 19.4 ng/ml (ACTH) and were generale proportional to the mean. J Glucocorticoid (ng/rnl) 6O 60 01 O A 0 OJ 0 N 0 IO i. M 0.1; $71-10 2 4 6 e 10"2'1" Hours from injection BLOOD GLUCOCORTICOID (n=3) AFTER INJECTION (iv) OF 5 mg PGF“ OR 200 IU ACTH IN CONTROL HEIFERS 61 Figure lO.--Blood Glucocorticoid (n=3) in Response to Injection (iv) of 5 mg PGan or 200 IU ACTH 6 Hr after Pretreatment of Heifers with 20 mg Triamcinolone Acetonide (TA, sc). The standard errors of the means ranged from 0.1 to 0.3 ng/ml (PGan) and 0.3 to 24.3 ng/ml (ACTH) and were generally proportional to the mean. 3O — N N 01 (3 (fl Glucocorticoid (ng/ml) 5 62 /PGF2a o \Méf‘ -6 -3 -I O 2 4 6 8 Hours from injection BLOOD GLUCOCORTICOID (n=3) IN RESPONSE TO INJECTION (iv) OF 5 mg PGan OR 200 IU ACTH 6 HR AFTER PRETREATMENT OF HEIFERS WITH 20 mg TRIAMCINOLONE ACETONIDE. (TA,sc) 63 TA pretreatment reduced (P<0.01) from 64 to 27 ng/ml peak gluco- corticoid response to ACTH; by contrast, glucocorticoid did not respond significantly to PGan after TA (figure 10). To facilitate comparisons of the glucocorticoid responses, all ACTH and PGFZO treatments are illustrated in figure 11. Glucocorti- coid was averaged for the six TA-pretreated heifers and for the six non-TA-pretreated heifers before PGan or ACTH was injected. In overview, the peak glucocorticoid response to ACTH after TA was about 50 percent of that i‘ncontrols;'by comparison peak g1 ucocorticoid response to PGFZa in'non-TA-pretreated heifers was over 30 ng/ml, but it was not significant in TA-pretreated heifers. In other words, while TA reduced the glucocorticoid response to ACTH it essentially abolished the response to PGFZa supporting the results from experiment II that the major site of PGan-induced glucocorticoid release is at the hypothalamopituitary axis, presumably to release ACTH. Three additional treatments on three heifers each were in- cluded as an adjunct to the principal experiment. The first treatment consisted of a simultaneous (iv) injection of 200 IU ACTH and 5 mg PGF2a° The submaximal dose of ACTH was chosen so an additional sti- mulus released by PGde (presumably ACTH) could be monitored by an increase of glucocorticoid. Thus, the more prolonged glucocorticoid response (P<0.01) after the simultaneous injection of ACTH and PGan then that after ACTH alone (figure 12) also supports an ACTH release after PGan. The results from this simultaneous injection do not rule out a direct action on the adrenals, however, the almost complete 64 Figure ll.--Blood Glucocorticoid (n=3) after 5 mg PGF2a or 200 IU ACTH (T, iv) with or without Pre- treatment of Heifers with 20 mg Triamcinolone Acetonide (TA, sc). Standard errors of the means are listed on figures 9 and 10. Glucocorticoid (ng/rnl) 65 60 t I one ACTI-I H ACTH after TA 1' °--O PGFZQ A | 0—0 PGF“ after TA 50 - I I Perm or P I ACTH T k 40 _ I I 1 ‘1 I 1 3O 1- " K o‘ . \ I ’1 20 A 1 13A Sham IA \ A 1. ‘3 lo '- \b. I Y \ I\ -. v TA - (SQ -. Q 0"0‘0-0—0'0 0 Pl 1 l l . lllllll l l I 1 l h . 1 ”F— -6-3 0 2 4 6 810721 Hours from injection BLOOD GLUCOCORTICOID (n=3) AFTER 5mg PGFg on 200 Iu ACTH (1.1v) wITI-I OR wIT I-I'OUT PRETREATMENT or HEIFERS mm 2’0 mg TRIAMCINOLONE ACETONIDE SC 66 Figure 12.--Blood Glucocorticoid (n=3) after Injection (iv) of 200 IU ACTH with or without 5 mg PGan in Heifers. The standard errors of the means ranged from 0.3 to 19.4 ng/ml (ACTH) and 0.6 to 30.1 ng/ml (ACTH and PGan) were generally proportional to the mean. Glucocorticoid (ng/ml) 60 r- 50: +4: I. #:1831011“ :15 _ j ’1. .0. : .../.\ 20A ' \\ IO \4 § ,u... 0 1 J I‘IIIIIIIIIIIIII I I I I I I I ’ -l O 2 4 6 8 IO 21 Hours from injection -e 45’ BLOOD GLUCOCORTICOID (n=3) AFTER INJECTION (iv) OF 200 IU ACTH WITH OR WITHOUT 5mg PGFZa IN HEIFERS 68 inhibition of PGFZa-induced glucocorticoid release by TA provide strong evidence that the major action of PGan is not at the adrenal. The other two treatments were included to determine if the sequential order of ACTH and PGan administration altered the duration of glucocorticoid response in comparison to when the initial stimulus was injected alone. The intervals between sequential treatments were chosen so the second injection would be near the peak glucocorticoid response induced by the first injection. ACTH was injected at time 0 and PGan 2 hours later, glucocorticoid response was more prolonged (P<0.01) than the response after ACTH alone (figure 13). Again the glucocorticoid response to these submaximal doses of PGan appears to be additive and is consistant with the notion that PGan acts to increase glucocorticoid secretion by causing ACTH release but does not competely rule out a direct action on the adrenals. In addition when PGFZa was injected at time 0 and ACTH 30 minutes later, gluco- corticoid response was prolonged (P0.05) in comparison to that after ACTH alone (figure 9) even though the duration of response was similar. Perhaps the PGFZa injection modified sterol precursor stores in the adrenal, curtailing gluco- corticoid production in response to the subsequent ACTH injection. More probable, the PGFZa—induced glucocorticoid release might resemble injection of synthetic corticoid to partially inhibit the ACTH- induced glucocorticoid release, as observed in the main body of this experiment and experiment II. In addition, ACTH increases the half- lives of adrenal protein and RNA (Ichii gt_al:, 1974). This effect 69 Figure 13.-—Blood Glucocorticoid (n=3) after Injection (iv) of 200 IU ACTH (t) with or without 5 mg PGFZQ (I) at 2 Hr in Heifers. The standard errors of the means ranged from 0.3 to 19.4 ng/ml (ACTH) and 0.3 to 18.0 ng/ml (ACTH and PGFga at 2 hr) and were generally proportional to the mean. 7O 7O - 60 P 2 5° ' I ._ACTH and g . T. PGF“ at 2 hr .3 I I t ' 1 . I 8 so - , A o g I /\ (9 P ACTH \ 20 1' M 10 - \ \ j \r A O 1 4 71M ‘f -6-3 -IO 2 4 6 8 IO 21 Hours from ACTH BLOOD GLUCOCORTICOID (n=3) AFTER INJECTION (iv) or 20010 ACTH (1) mm oR WITHOUT 5 mg PGF“ (II AT 2 HR IN HEIFERS 71 Figure 14.--Blood Glucocorticoid (n=3) after Injection (iv) of 5 mg PGan (t) with or without 200 IU ACTH (I) at 0.5 Hr in Heifers. The standard errors of the mean ranged from 0.2 to 6.2 ng/ml (PGF a) and 1.0 to 7.2 ng/ml (PGan.and ACTH at 0.5 hrT and were generally proportional to the mean. Glucocorticoid (ng/ml) A O 5 O 72 M R 11 «A A 1.— PGFZa and 1 )I T ACTH at 0.5 hr -6 377-10 2 4 6 8 10’21' Hours from PGFZQ BLOOD GLUCOCORTICOID (n=3) AFTER INJECTION (iv) OF 5mg 1:6an I 1) WITH OR WITHOUT zoom ACTH (1) AT 0.5 HR 1N HEIFERS Wei .L..a . bani. 73 of ACTH might account for the prolonged glucocorticoid release after ACTH in figure 11 in comparison to the reduced response when PGan was given before ACTH. That is, perhaps, glucocorticoids released in response to PGan suppressed the protein and RNA sustaining action of ACTH given 30 minutes later. In overview of the results from experiements II and III, I believe the surge of glucocorticoid which follows PGan treatment in heifers represents an action of PGFZQ on the pituitary of hypothalamus. One cannot determine from my data whether the action is on the pituitary,cn~the hypothalamus, because dexamethasone (a corticoid similar to TA) suppressed the synthesis and release of ACTH through action on the pituitary in viyg_(Kendall et_al,, 1966 and Yasuda gt_al,, l976)and jn_yjtrg_(Arimura et a1., 1969). Glucocorticoid receptor Sites in the pituitary presumably facilitate this inhibitory action of dexamethasone, but in addition the dorsal hippocampus contains glucocorticoid receptor sites in rats (Rotsztein et_al,, 1975). In other words, glucocorticoid also inhibits ACTH secretion by action on the CNS. To determine whether the PGFZO acts on the pituitary or the hypothalamus in cattle, future experiments might include (1) treatments with small doses (If PGFZO given into brain ventricles or into the pituitary, (2) jn_yitrg_incubations of pituitaries with PGFZO with and without CRF extracts from median eminence, and (3) treatments such as morphine to block CRF production before PGFZO treatment. SUMMARY AND CONCLUSIONS Prostaglandin F2a(PGF2a) potentially may be used widely for control of ovulation in farm animals. Some other prostaglandins altered secretion of important metabolic hormones. Thus, the purpose of the three main experiments in this thesis was to determine if anterior pituitary hormones were released after luteolytic doses of PGan in cattle. In the first experiment after administration of 15, 30 or 60 mg PGF2 to diestrous heifers, prolactin increased over 3-fold within 10 minutes and returned to pre-injection values within 4 hours; growth hormone increased in a dose-related manner, peaking at 30 minutes and remaining above pre-injection values for 1 hour; LH in- creased at least 2-fold over pre-injection values within 1.5 to 6 hours; glucocorticoids (indicator of ACTH release) increased more than 6-fold at 30 minutes and returned to pre-injection values by 4 hours. A second experiment was conducted to determine the site of action of PGan on glucocorticoid release. PGan (25 mg) or saline was given im 7 days after a triamcinolone acetonide (TA) pretreatment which decreased serum glucocorticoid to less than 0.5 ng/ml within 24 hours. In saline-treated heifers not given TA, blood glucocorticoid fluctuated at random whereas glucocorticoid increased from 10 to 50 ng/ ml by 30 minutes after PGan and returned to pre-injection values 4 74 75 hours later in heifers without TA. However, in TA-pretreated heifers peak glucocorticoid response to PGan was depressed to 12 percent of that in heifers not given TA. The results suggested that the TA inhibition might have been partially lost and that the adrenals might have been partially regressed by 7 days after TA, thereby reducing the glucocorticoid response to a stimulus. Consequently, another experiment was conducted to minimize possible adrenal regression and to maximize the effectiveness of TA. Submaximal doses of porcine ACTH (200 IU) and PGFZO (5mg) were administered to heifers 6 hours after TA-pretreatment, when gluco- corticoid secretion was fully inhibited by TA. In animals not pre- treated with TA, the first 0.5 hours of glucocorticoid response to pGde resembled that after ACTH, but the peak response to ACTH was much greater and the duration of response to ACTH was much more pro- longed than that after PGFZO' The TA-pretreatment reduced the gluco- corticoid response to ACTH by 50 percent but it essentially abolished the response to PGFZO’ Three added treatments which consisted of simultaneous or sequential administration of the same amount of PGFZO and ACTH were included in this third experiment. The glucocorticoid response was more prolonged when PGFZO and ACTH were injected simul- taneously or when PGFZO followed ACTH-treatment by 2 hours. In con- trast when PGFZO was administered 30 minutes before ACTH, peak glucocorticoid response to ACTH was suppressed by comparison to that after ACTH alone. IrIconclusion, prolactin, growth hormone, luteinizing hormone and glucocorticoids are secreted transitorily in relatively large 76 amounts in response to treatment of heifers with luteolytic doses of PGan. Review of the literature for other species justifies the hypothesis that prostaglandins may normally mediate pituitary hormone secretion. Consequently, the pituitary hormone releases reported in response to PGFZO in this thesis may represent relatively specific action at the hypothalamus or pituitary. I have no evidence in these three experiments for a pituitary-hypothalamic site of PGFZQ action for growth hormone, prolactin or LH secretion. However, on the basis of the last two experiments, I believe PGan acts primarily on the hypothalamus or the pituitary to cause glucocorticoid secretion. Further research is required to discriminate between these sites of PGan action, and to determine whether PGan normally participates in pituitary hormone secretion in cattle. Whether or not it does, the results from this thesis raise the possibility of using prostaglandins to regulate intermediary metabolic hormones in food-producing animals. BIBLIOGRAPHY 77 BIBLIOGRAPHY Akbar, A.M., L.E. Reichert, Jr., T.G. Dunn, C.C. Kaltenbach and 6.0. Niswender. 1974. Serum levels of follicle stimulating hormone during the bovine estrous cycle. J. Anim. Sci. 39:360-365. Anderson, L.L., K.P. Bland, and R.M. Melampy. 1969. Comparative aspects of uterine-luteal relationships. Rec. Prog. Hormone Res. 25:57. Arimura, A., C.Y. Bowers, A.V. Schally, M. Saito and M.C. Miller III. 1969. Effect of corticotropin-releasing factor, dexamethasone and actinomycin D on the release of ACTH from rat pituitaries in vivo and in vitro. Endocrinol. 85:300. Ayalon,lt and M. Shemesh. 1974. Pro-oestrous surge in plasma pro- gesterone in the cow. J. Reprod. Fertil. 36:239. Babcock, J.C. 1966. Luteotrophic and luteolytic mechanisms in bovine corpora lutea. In: "Ovarian regulatory mechanisms." J. Reprod. Fertil. Suppl. 1, p. 47. Bergstrom, S., R. Ryhage, B. Samuelsson and J. Stoval. 1962. The structure of prostaglandin E], F1 and F2. Acta Chem. Scand. 16:501. Carlson, J.C., B. Barcikowski and J.A. McCracken. 1973. Prosta- glandin F23 and the release of LH in sheep. J. Reprod. Fertil. 34:357. Chenault, J.R., W.W. Thatcher, P.S. Kalra, R.M. Abrams and C.J. Wilcox. 1975. Transitory changes in plasma progestins, estradiol, and luteinizing hormone approaching ovulation in the bovine. J. Dairy Sci. 58:709. Christensen, D.S., M.L. Hopwood and J.N. Wiltbank. 1974. Levels of hormones in the serum of cycling beef cows. J. Anim. Sci. 38:577. Cole, H.H. and R.B. Snook. 1964. Proc. 5th Intern. Congr. Animal Reprod., Trento II, 143. Convey, E.M., H.A. Tucker, V.G. Smith, and J. Zolman. 1973. Bovine prolactin, growth hormone, thyroxine and corticoid response to thyrotropin-releasing hormone. Endocrinol. 92:471. 78 79 Cooper, R.H., M. McPherson and J.G. Schofield. 1972. The effect of prostaglandins on 0x pituitary content of adenosine 3':5'- cyclic monophosphate and the release of growth hormone. Biochem. J. 127:143. Copinschi, G., M. L'Hermite, R. Leclercq, J. Golstein, L. Vanhaelst, E. Virasoro, and C. Robyn. 1975. Effects of glucocorticoids on pituitary hormonal responses to hypoglycemia. Inhibition of prolactin release. J. Clin. Endocrinol. Metab. 40:442. Coudert, S.P., and C. Faiman. 1973. Effect of prostaglandin F2a on anterior pituitary function in man. Prostaglandins 3:89. Cunningham, G.R., E:M. Caperton, Jr., and J.N. Goldzieher. 1975. Antiovulatory activity of synthetic corticoids. J. Clin. Endocrinol. Metab. 40:265. Czeisler, C.A., M.C.M. Ede, Q.R. Regestein, E.S. Kisch, V.S. Fang and E.N. Ehrlich. 1976. Episodic 24-hour cortisol secretory patterns in patients awaiting elective cardiac surgery. J. Clin. Endocrinol. Metab. 42:273-283. de Wied, D., A. Witter, D.H.G. Versteeg, and A.H. Mulder. 1969. Release of ACTH by substances of central nervous system origin. Endocrinol. 85:561. Eskay, R.L., J. Warberg, R.S. Mical, and J.C. Porter. 1975. Prosta- glandin Ez-Induced Release of LHRH into hypophysial portal blood. Endocrinol. 97:816. Flack, J.D. and P.W. Ramwell. 1972. A comparison of the effects of ACTH, cyclic AMP, dibutyryl cyclic AMP, and PGE2 on cortico- steroidogenesis in vitro. Endocrinol. 90:371. Flack, J.D., R. Jessup, and P. W. Ramwell. 1969. Prostaglandin stimulation of rat corticosteroidogenesis. Science 163:691. Ganong, W.F., In Nalbandov, A.V. (ed.). 1963. Advances in Neuro- endocrinology, Univ. Ill. Press, Urbana, p. 92. Gill, J.L., H.D. Hafs. 1971. Analysis of repeated measurements of animals. J. Anim..Sci. 33:331. Glencross, R.G., I.B. Munro, B.E. Senior, and G.S. Pope. 1973. Con- centrations of oestradiol-17B, oestrone, and progesterone, in jugular venous plasma of cows during the oestrous cycle and in early pregnancy. Acta Endocrinol. 73:374. Goding, J.R., M.D. Cain, J. Cerini, M. Cerini, W.A. Chamley, I.A. Cumming. 1972. Prostaglandin an "the" luteolytic hormone in the ewe. J. Reprod. Fertil. 28:146. Goldblatt, M.W. 1933. A depressor substance in seminal fluid. J. Soc. Chem. Ind., Lond. 52:1056. 80 Hackett, A.J., H.D. Hafs. 1969. Pituitary and hypothalmic endocrine changes during the bovine estrous cycle. J. Anim. Sci. 28:531. Hafs, H.D., T.E. Kiser, N.B. Haynes, J.S. Kesner, J.N. Stellflug. 1976. Release of pituitary hormones, cortisol, testesterone and insulin in response to prostaglandin an given during intracarotid infusion of somatostatin in bulls. J. Anim. Sci. In press. Hafs, H.D., T.M. Louis, P.A. Noden, and W.D. Oxender. 1974. Control of the estrous cycle with prostaglandin F20 in cattle and horses. J. Anim. Sci. Suppl. 1, p. 10. Hafs, H.D. 1975. Prostaglandins and the control of anterior pituitary hormone secretion. In Hypothalamic Hormones. Eds., M. Motta, P.G. Crosignani and L. Martini. Academic Press, New York, p. 183. Hafs, H.D., J.G. Manns and B. Drew. 1975. Onset of oestrus and fertility of dairy heifers and suckled beef cows treated with prostaglandin F2“. Anim. Prod. 21:13. Hafs, H.D., T.M. Louis, J.N. Stellflug, E.M. Convey, and J.H. Britt. 1975. Blood LH after PGFZG in diestrous and ovariectomized cattle. Prostaglandins 10:1001. Hansel, W., P.W. Concannon, and J.H. Lukaszewska. 1973. Corpora lutea of the large domestic animals. Biol. Reprod. 8:222. Hansel, W., S.E. Echternkamp. 1972. Control of ovarian function in domestic animals. Am. 2001. 12:225. Hansel, W., M. Shemesh, J. Hixon, and J. Lukaszewska. 1975. Extrac- tion, isolation and identification of a luteolytic substance from bovine endometrium. Biol. Reprod. 13:30. Harms, P.G., P. Langlier, and S.M. McCann. 1975. Modification of stress-induced prolactin release by dexamethasone or adrenalectomy. Endocrinol. 96:475. Harms, P.G., S.R. Ojeda, and S.M. McCann. 1973. Prostaglandin involvement in hypothalamic control of gonadotropin and prolactin 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. Harms, P.G., S.R. Ojeda, S.M. McCann. 1976. Failure of mono- aminergic and cholinergic receptor blockers to prevent prostaglandin Ez-induced luteinizing hormone release. Endocrinol. 98:318. 7 mm. . 1*— a 81 Hedge, G.A. 1972. The effects of prostaglandins on ACTH secretion. Endocrinol. 91:925. Hertelendy, F. 1971. Studies on growth hormone secretion 11. Stimulation by prostaglandin in vitro. Acta Endocrinol. 68:355. Hertelendy, F., G. Peake, and H. Todd. 1971 Studies on growth hormone secretion: Inhibition of prostaglandin, theophylline and cyclic AMP stimulated growth hormone release by valino- mycin in vitro. Biochem. Biophys. Res. Commun. 44:253. Hertelendy, F., H. Todd, K. Ehrhart and R. Blute. 1972. Studies on growth hormone secretion: IV. In vivo effects of prosta- glandin E]. Prostaglandins 2:79. Hensby, C.N. 1974. Reduction of prostaglandin E2 to prostaglandin F2a by an enzyme in sheep blood. Biochim1ca et Biophysica Acta 348:145. Hixon, J.E. and W. Hansel. 1974. Evidence for preferential transfer of prostaglandin F26 to the ovarian artery following intra- uterine administration in cattle. Biol. Reprod. 11:543-552. Hobson, W.C. and W. Hansel. 1972. Plasma LH levels after ovari- ectomy, corpus luteum removal and estradiol administration in cattle. Endocrinol. 91:185. Hoffer, B.S., G.R. Siggins, and F.E. Bloom. 1970. Possible cyclic AMP—mediated adrenergic synapses to rat cerebellar purkinje cells: Combined structural, physiological, and pharmaco- logical analyses. In Advan. Biochem. Psychopharmacol. Eds. P. Greengard and E. Costa. Raven Press, N.Y., p. 349. Ichii, S., N. Murakami, and M. Izawa. 1974. Effect of ACTH and dexamethasone on the rate of protein and RNA degradation in rat adrenal glands. Endocrinol. Japan 21:465. Ingalls, W.G., E.M. Convey, and H.D. Hafs. 1973. .Bovine serum LH, GH, and prolactin during late pregnancy, parturition and early lactation. Proc. Soc. Exp. Biol. and Med. 143:161. Ito, H., G. Momose, T. Katayama, H. Takagishi, L. Ito, H. Nakajima, and Y. Takei. 1971. Effect of prostaglandin on the secre- tion of human growth hormone. J. Clin. Endocrinol. and Metab. 32:857. Kato, Y., J. Dupre, and J.C. Beck. 1973. Plasma growth hormone in the anesthetized rat: Effects of dibutyryl cyclic AMP, prostaglandin E], adrenergic agents, vasopressin,.chlorpro- mazine, amphetamine L—dopa. Endocrinology 93:135. 82 Kazama, N. and W. Hansel. 1970. Preovulatory Changes in progesterone level of bovine peripheral blood plasma. Endocrinol. 86:1252. Kendall, J.W., A.K. Stott, C. Allen, and M.A. Greer. 1966. Evidence for ACTH secretion and ACTH suppressibility in hypophysecto- mized rats with multiple heterotropic pituitaries. Endocrinol. 78:533. Kirk, R.E. 1968. Experimental Design: Procedures for the Behavioral Sciences. Brooks/Cole Pub. Co., Belmont, Calif., p. 90. Kiser, T. E, H. D. Hafs, and H.D.Oxender.1976. Increased blood LH and testosterone after administration of prostaglandin an in bulls. Prostaglandins 11:545. Kurzrok, R., and C.C. Lieb. 1930. Biochemical studies of human semen. II. The action of semen on the human uterus. Proc. Soc. Exp. Biol. Med. 26:268. Lauderdale, J.W., B.E. Seguin, J.N. Stellflug, J.R. Chenault, W.W. Thatcher, C. K. Vincent, and A. F. Loyancano. 1974. Fertility of cattle following PGFZO injection. J. Anim. Sci. 38: 964. Leslie, C.A., and L. Levine. 1973. Evidence for the presence of a prostaglandin Eza-Q-Keto reductase in rat organs. Biochem. and Biophys. Res. Comm. 52:717. Louis, T.M., H.D. Hafs, and D.A. Morrow. 1972. Estrus and ovulation after PGFZG in cows. J. Anim. Sci. 35:1121 (Abstr.). Louis, T.M., H.D. Hafs, and D.A. Morrow. 1974. Intrauterine administration of prostaglandin F in cows: Progesterone, estrogen, LH, estrus and ovulatioH? J. Anim. Sci. 38:347. MaCLeod, R.M. and J.E. Lehmeyer. 1970. Release of pituitary growth hormone in prostaglandins and dibutyryl adenosine cyclic 3':5'-monophosphate in the absence of protein synthésis. Proc. Natn. Acad. Sci. U.S.A. 67:1172. McCracken, J.A. and D.T. Baird. 1969. The study of ovarian function by means of transplantation of the ovary in the ewe. The Gonads, K. MCKerns (ed.), Appleton-Century-Crofts. New York. Chap. 7, p. 175. McCracken, J.A., D. T. Baird, and J. R. Goding. 1971. Factors affecting the secretion of steroids by the transplanted ovary in the sheep. Rec. Progr. Hormone Res. 27. 83 McCracken, J.A., J.C. Carlson, M.E. Glew, J.R. Goding, D.T. Baird, K. Green, and B. Samuelsson. 1972. Prostaglandin an identified as a luteolytic hormone in sheep. Nature New Biol. 238:129. Miller, R.G., Jr. 1966. Simultaneous Statistical Inference. McGraw Hill Book Co. New York, p. 67. Ojeda, S.R., P.G. Harms, and S.M. McCann. 1974a. Central effect of prostaglandin E1 (PGEl) on prolactin release. Endocrinol. 95:613. Ojeda, S.R., P.G. Harms, and S.M. McCann. 1974b. Possible role of cyclic AMP and prostaglandin E1 in the dopaminergic control of prolactin release. Endocrinol. 95:1694. Ojeda, S.R., and S.M. McCann. 1974. Development of Dopaminergic and estrogenic control of prolactin release in the female rat. Endocrinol. 95:1499. Oxender, H.D., H.D. Hafs, and L.A. Edgerton. 1972. Serum growth hormone, LH and prolactin in the pregnant cow. J. Anim. Sci. 35:51. Peng, T.C., K.M. Six, and P.L. Munson. 1970. Effects of prosta- glandin E] on the hypothalamO-hypophyseal-adrenocortical axis in rats. Endocrinol. 86:202. Pharriss, 8.3., and L.J. Wyngarden. 1969. The effect of prosta- glandin an on the progestin content of ovaries from pseudo- pregnant rats. Proc. Soc. Exp. Biol. Med. 130:92. Purchas, R.W., K.L. Macmillan, and H.D. Hafs. 1970. Pituitary and * plasma growth hormone levels in bulls from birth to one year of age. J. Anim. Sci. 31:358. Rajakowski, E. 1960. The ovarian follicular system in sexually mature heifers with special reference to seasonal, cyclical and left-right variations. Acta Endocrinol. (Kbh.), 34 (Suppl. 52):l-68. Ratner, A., M.C. Wilson, and G.T. Peake. 1973. Antagonism of prostaglandin-promoted pituitary cyclic AMP accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid. Prostaglandins 3:413. Richards, J.S., and A.R. Midgley, Jr. 1975. Protein hormone action: A key to understanding ovarian follicular and luteal cell development. Biol. Reprod. 14:82. 84 Rotsztefit W.H., M. Normand, J. LaLonde, and C. Fortier. 1975. Relationship between ACTH release and corticosterone binding by the receptor sites of the adenohypophysis and dorsal hippocampus following infusion of corticosterone at a constant rate in the adrenalectomized rat. Endocrinol. 97:223. Rowson, L.E.A., R. Tervit, and A. Brand. 1972. The use of prosta- glandins for synchronization of oestrus in cattle. J. Reprod. Fertil. 29:145. Saruta, T., and N.M. Kaplan. 1972. Adrenocortical steroidogenesis: The effects of prostaglandins. J. Clin. Invest. 51:2246. 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. Sato, T., T. Iesaka, T. Jyujo, K. Taya, J. Ishikawa, and M. Igarashi. 1974. Prostaglandin-induced ovarian ascorbic acid depletion. Endocrinol. 95:417. Schams, 0., and H. Karg. 1970. Untersuchungen uber prolactin im rinderblut mit einer radio-immunologischen bestimmungs- methode. 2 b1. Vet. Med. 17:193. Schofield, J.G. 1970. Prostaglandin E] and the release of growth hormone in vitro. Nature 228:179. Sinha, Y.N., and H.A. Tucker. 1969. Mammary development and pituitary prolactin level of heifers from birth through puberty and during the estrous cycle. J. Dairy Sci. 52:507. Smith, V.G., L.A. Edgerton, H.D. Hafs, and E.M. Convey. 1973. Bovine serum estrogens, progestins and glucocorticoids during late pregnancy, parturition and early lactation. J. Anim. Sci. 36:391. Spies, H.G., and R.L. Norman. 1973. Luteinizing hormone release and ovulation induced by the intraventricular infusion of prostaglandin E into pentobarbital blocked rats. Prostaglandins :131. Stellflug, J.N., T.M. Louis, R.C. Gorewit, W.D. Oxender, and H.D. Hafs. 1976. Luteolysis induced by prostaglandin F a before and after hysterectomy in heifers. Biol. Reprod. n Press. Stellflug, J.N., T.M. Louis, H.D. Hafs, and B.E. Seguin. 1975. Luteolysis, estrus and ovulation, and blood prostaglandin F after intramuscular administration of 15, 30 or 60 mg prostaglandin an. Prostaglandins 9:609. 85 Sundberg, D.K., C.P. Fawcett, P. Illner, and S.M. McCann. 1975. The effect of various prostaglandins and a prostaglandin synthetase inhibitor on rat anterior pituitary cyclic AMP levels and hormone release in Vitro. Proc. Soc. Exp. Biol. and Med. 148:54-59. Swanson, L.V., and H.D. Hafs. 1971. LH and prolactin in blood serum from estrus to ovulation in Holstein heifers. J. Anim. Sci. 33:1038. Szabo, M., and L.A. Frohman. 1975. Effects of porcine stalk median eminence and prostaglandin E2 on rat growth hormone secretion .ifl vivo and their inhibition by somatostatin. Endocrinol. 96:955. Tucker, H.A. 1971. Hormonal response to milking. J. Anim. Sci. 32, Suppl. 1:137. Tucker, H.A., D. T. Vines, J. N. Stellflug, and E.M. Convey. 1975. Milking, thyrotropin-releasing hormone and prostaglandin induced release of prolactin and growth hormone in cows. Soc. Exp. Biol. Med. Proc. 149:462. Thatcher, W.H., and J.R. Chenault. 1976. Reproductive physiological responses of cattle to exogenous prostaglandin FZO- J. Dairy Sci. 59:1366. Tsafriri, A., Y. Koch, and H.R. Lindner. 1973. Ovulation rate and serum LH levels in rats treated with indomethacin or prostaglandin E2. Prostaglandins 3:461. Vanderheyden, K., M. Dhont, D. Vandekerckhove, A. Vermuelen, and M. Thiery. 1974. Prostaglandin F2“: Effect on cortisol, growth hormone, luteinizing hormone, follicle-stimulating hormone, prolactin and thyroid-stimulating hormone. Obstet. and Gynecol. 2:1164. Vermouth, N.T., and R.P. Deis. 1972. Prolactin release induced by prostaglandin an in pregnant rats. Nature New Biol. 238:248. Von Euler, V.S. 1935. A depressor substance in the vesicular gland. J. Physiol., Lond. 84:21. Warberg, J., R.L. Eskay, and J.C. Porter. 1976. Prostaglandin- induced release of anterior pituitary hormones: Structure- activity relationships. Endocrinol. 98:1135. Wentz, A.C., G.S. Jones, and T. Bledsoe. 1973. Effects of PGF a infusion on human cortisol biosynthesis. Prostaglanding 3:155. 86 Yasuda, N., K. Takeba, and M.A. Greer. 1976. Studies on ACTH dynamics in cultured adenohypophyseal cells: Effect of adrenalectomy or dexamethasone in vivo. Endocrinol. 98: 717. Yue, D.K., I.D. Smith, J.R. Turtle, and R.P. Shearman. 1974. Effect of PGF on the secretion of human prolactin. Prostaglandin308:387. Zor, U., T. Kaneko, H.P.G. Schneider, S.M. McCann, and J.B. Field. 1970. Further studies of stimulation of anterior pituitary cyclic adenosine 3':5'-monophosphate formation by hypothalamic extract and prostaglandins. J. Biol. Chem. 245:2883. ”llIIIIIIIIIIIIIIT