1 , 1.“ .m‘ ,,,H1.1...'v',..‘1.. ',-.'., w ‘r-' ,1,» .. w... .m w...“ V110." . ”WI-1.- 1....1‘. Tilly-"1‘." ,_ an" ,1. -' r. .1» 131-1. '7. y .5 - "I . 1Av‘wy1-Dw 11- m 1 AL: .l-‘HHI-xlAb‘ n '1 "G v 11~[«.~1v 1 i u “m... . . « - .- - '- W11 . 1 . ' ""H";"'H" ""1 1‘ .1 IM~~1-1.-~um11.,1...11...”. V ., H ..11~...».1~.,1. """"11""""" "31%" r: 1 . .. ...'.1..u-:—---: ..,.. w. 'V‘ru. ,' .3 '1 "E51" r1171- TH ESVS 3" LIBRAP" 3 Michigan St. 1 M‘ University .;_amamm ‘ l M This is to certify that the I 1 thesis entitled \ ROLE OF THE HYPOTHALAMUS IN PITUITARY HORMONE FEEDBACK, MAMMARY CANCER, PUBERTY AND AGING IN THE RAT presented bg James Allen Clemens has been accepted towards fulfillment of the requiremenls for Ph D degree inwh i0 gy nr nrn‘pccnr Date August 8, 1968 07169 ABSTRACT ROLE OF THE HYPOTHALAMUS IN PITUITARY HORMONE FEEDBACK, MAMMARY CANCER, PUBERTY AND AGING IN THE RAT by James Allen Clemens 1. Single stereotaxic implants of a prolactin—cocoa butter mixture were made in the median eminence of 10 mature and 6 ovariectomized female Sprague-Dawley rats! A similar number of control rats were implanted with cocoa butter alone. The animals were killed 6 days after implan- tation. The prolactin implanted rats showed marked mammary regression when compared with those of the control intact or control ovariectomized rats, nearly a 3 fold increase in hypothalamic content of prolactin inhibiting factor (PIF) in both intact and ovariectomized rats, and a 40% decrease in pituitary prolactin concentration in the intact rats. The ovaries of intact prolactin-implanted rats were charac- terized by many well developed follicles and few corpora lutea while the ovaries from the corresponding controls showed mainly well developed corpora lutea. The former showed regular cycling while the latter appeared to be pseudopregnant, as judged by daily vaginal smears. These James Allen Clemens observations suggest that implanted prolactin inhibits pituitary prolactin synthesis and release by a direct feedback action on hypothalamic PIF, and this results in depressing pituitary prolactin concentration, mammary growth and luteal function. 2. Twenty—five lactating female Sprague-Dawley rats were divided into 3 groups on the 4th day postpartum, and were each implanted stereotaxically with 23 gauge glass tubing containing the following substances: group I, controls, cocoa butter; group II, mixture of prolactin and cocoa butter; group III, prolactin, ACTH and cocoa butter. All implanted tubes were fixed rigidly in place by means of dental cement and skull screws. On the day of implantation (day 0) litter size was reduced to 6 pups. On the 4th day after implantation, average weight gains for 6 pups were as follows: group I, 40.4i2.7g; group II, 18.8i3.5g; group III, 8.7i3.6g. Daily vaginal smears on all implanted rats showed that rats in group I remained in diestrus, whereas the rats in group II and group III came into estrus and began to cycle. These results demonstrate that prolactin implants into the median eminence can counteract the stimulating effects of suck— ling on prolactin release by the pituitary, thereby causing a significant impairment of lactation and regres- sion of the corpora lutea of lactation. Combining James Allen Clemens prolactin with ACTH resulted in an even greater impairment of lactation, by inhibiting pituitary secretion of these two most essential hormones for the lactation process. 3. Median eminence (ME) lesions were placed before and after carcinogen (DMBA) administration in female rats. Intact and ovariectomized Sprague—Dawley rats with ME lesions placed prior to treatment with DMBA, showed a 30 and 0% mammary tumor incidence, respectively, in contrast to a 95 and 54% mammary tumor incidence in the respective non—lesioned control groups. Intact and ovariectomized rats lesioned 75 days after DMBA treatment showed a 120 and 80% increase, respectively, in the number of palpable mammary tumors by 10 days after treatment. In contrast the non—lesioned intact and ovariectomized control groups showed a 19% increase and a 27% decrease in number of palpable mammary tumors, respectively. After 25 days, stimulation of mammary tumor growth was still marked in the intact—lesioned group but regression occurred in the ovariectomized—lesioned group. These results indicate that ME lesions placed before DMBA treatment inhibit mammary tumorigenesis, presumably by increasing prolactin secretion and stimulating mammary gland development, thereby rendering it refractory to the carcinogen. The dramatic increase in number of tumors and tumor growth when rats were lesioned after carcinogen administration, James Allen Clemens is believed to be due primarily to stimulation of already formed tumors by increased prolactin secretion. 4. Administration of prolactin (NIH-P-Bl and ZNIH—P-S7) significantly advanced the onset of puberty .in.immature rats in 3 separate trials. Treatment of :meature rats with anterior pituitary hormone impurities :specified to be present as contaminants in the NIH pro- jlactin preparations did not advance the onset of puberty. Ifledian eminence implants of prolactin were found to hasten Iguberty by an average of 6.6 days, whereas subcutaneous .implants containing the same amount of prolactin or median enninence implants of prolactin contaminants had no effect lactin dependent processes, mammary growth, lactation and pregnancy. Even though the brain is the primary regulator of the: endocrine system, no studies have yet been reported on its; possible relationship to hormone dependent cancers. One: such cancer is the chemically induced rat mammary cxuucer. Previous reports from our and other laboratories have demonstrated the importance of prolactin in mammary cancer development, and have strongly suggested that pro- lactin is a primary factor in development of mammary cancer. Since it has been well established that lesions in the median eminence area severly impair secretion of all anterior pituitary hormones except prolactin secretion, I decided to produce median eminence lesions in rats before and after carcinogen administration. With this type of lesion the only pituitary hormone secreted in significant quantities is prolactin. By this means we can see what «affect prolactin alone has on mammary cancer development and growth. Another physiological process under brain control is; the estrous cycle. Currently it is thought some neural exnent in the CNS initiates the estrous cycle at puberty; .hcnnever, the mechanism responsible for initiation of cynclicity remains unknown and is currently the subject of mulch.controversy. Even less is known about the mechanisms ccnncerned with the loss of the estrous cycle and concom- ituant infertility in old female rats. Since many of the erqperimental treatments that cause precocious puberty also CEUJSG prolactin release it was of interest to determine the effects of exogenous prolactin on the onset of puberty. It aJAso seemed possible that loss of cyclicity in aging andJnals may be due to changes in the brain and not to citrect failure of the ovaries. Therefore, studies were also initiated in infertile, old rats to try to understand Why reproductive functions were lost in these animals. REVIEW OF LITERATURE I. Anatomy of the Hypothalamus Many of the early studies on the hypothalamus have been devoted to studying the configuration of some of its nurilear masses (Malone, 1910, 1914; Sutkowaja, 1928; priegel, 1928; Grunthal, 1929, 1930; Rioch, 1929; Morgan, ]£X30; Loo, 1931). All of these studies were performed usiJng animals other than the rat. One of the first studies 1irflrat.hypothalamus defining its nuclei and outlining its fiber tracts. The nomenclature of the various hypothalamic nuclei has varied greatly from one Species to another, but even within one and the same species various authors employ dif— ferent terms. To alleviate the resulting confusion, Rioch, Wislocki and O'Leary (1940) have established a uniform terminology applicable to all mammalian species. All the following work in this thesis uses this terminology. According to Zeman and Innes (1963) the hypo- thalamus can be divided into an anterior part containing a fenfl myelinated fibers and many unmyelinated fibers, represented by the tuber cinereum and the nuclei in the lerteral walls of the third ventricle as well as the pre- ogrtic area. Included in this part of the hypothalamus are true infundibulum and the posterior lobe of the pituitary. Ir: contrast, the posterior wall of the hypothalamus, com— prxising the mammillary complex contains a wealth of Inywalinated fiber systems. The poorly myelinated part of tflne hypothalamus appears to be the final common pathway fcn: neural influences on the pituitary, whereas mammillary kxodies apparently exert no direct control over this Gandocrine gland. This poorly myelinated part is termed tile vegetative hypothalamus. An excellent stereotaxic artlas for the rat hypothalamus has been developed by Che Groot (1959). This atlas shows the location of all INJclei and most of the fiber pathways in the hypothalamus 61nd was the atlas used in these studies. III. Physiology of the Hypothalamo-Hypophyseal System A. Anatomical connections of the anterior_pitu- itary and their functional significance The anterior pituitary is connected to the hypo— ‘thalamus by means of portal blood vessels which originate in the capillary beds in the median eminence and upper infundibular stem. Popa and Fielding (1930a,b) described portal vessels running along the pituitary stalk and ccnnnecting it with nervous tissue of the hypothalamus. Truey concluded from their studies that blood flowed from true pituitary gland upward to the hypothalamus. These stnadies led to much work on the blood supply to the pitu- itxary, and it soon became apparent that the blood in the Exxrtal vessels of the pituitary stalk did not flow from time gland to the brain, but in the Opposite direction. vmislocki and King (1936) came to the conclusion on morpho— lcxgical grounds alone, that the direction of blood flow nuist be from the capillary bed to the pars distalis. The Ctirection of blood flow along the portal vessels of the Eftalk was observed in living animals first by Houssay at El. (1935) and later by Green (1947). However, as pointed out 13y Flerko (1966), the pituitary circulation is not as Ettmple as revealed in most textbooks and monographs on Jfieuroendocrinology. On the basis of observations by Torok (1954, 1962, 31964), Szentagothai et_al. (1957) and Duvernoy (1960), a knackflow of blood from the dense vascular plexus between tflne median eminence and pars tuberalis toward the hypothal- Ennus has been postulated. The pars tuberalis contains Significant amounts of anterior pituitary hormones and Shows clear signs of secretory activity. Blood could bring Pituitary hormones from here to the hypothalamus. In addition Torok (1954) has described veins on the posterior SUJrface of the pars distalis which have an upward dijsection of flow. These observations provide the anatom- icxal basis for a direct feedback of anterior pituitary hormones on the hypothalamus. B. Hypothalamic releasing and inhibiting factors There are many indirect lines of evidence for héqoothalamic releasing and inhibiting factors, which have bexen reviewed scores of times. Here I will present only CtLrect evidence, obtained by the use of hypothalamic enctracts, for releasing and inhibiting factors concerned With this thesis . Definitive evidence for the existence of a pro— liactin inhibiting factor (PIF) in acid extracts of hy- EXDthalamus was presented by Meites and his co—workers Ufleites et_al., 1961, 1962; Talwalker at al., 1963). Acid Enitracts of beef, sheep and pig hypothalami were also shown tKD have PIF activity (Talwalker e: 31., 1963; Shally 3: 31., 1965). Convincing in yitrg evidence for the existence of a Sirowth hormone releasing factor (GRF) was first presented lmy Deuben and Meites (1963) and later confirmed (Deuben and.Meites, 1964; Shally et_al., 1965). Igarashi and McCann (1964) presented evidence for the presence of a follicle—stimulating hormone—releasing factor (FSH-RF) in rat hypothalamus using the HCG augmented rmoiise uterine weight assay which later proved to be .inxralid as a specific bioassay. Therefore, the first (iilrect, valid, demonstration of an FSH-RF was by Mittler and Meites (1964) . A luteinizing hormone releasing factor CLI{F) was probably first demonstrated by McCann et 21. (1A960) by using intravenous infusions of acid extracts of :rad: median eminence tissue. These results were confirmed larter by Courrier et a1. (1961). They found that hypothal- anuic extracts from both rats and sheep can induce release of? LH from the pituitary. C. Hormone feedback 1. Conventional type-target organ hormone feedback a. Gonadotrophin-sex steroid The conventional feedback is that of target organ hcunnones exerting a negative feedback on the hypothalamus arMEI/or pituitary gland to inhibit secretion of their trophic hormones . Estrogen may inhibit FSH release by a direct action at-iihe pituitary level. Rose and Nelson (1957) perfused estrogen into the hypophyseal fossa and inhibited the CaStration reaction. Bogdanove (1963) concluded that there could be a direct inhibitory effect of estrogen on the pituitary. However, these experiments do not demon- strate unequivocally the existance of such a direct iJnrlibitory action. Most experimental evidence supports tilea view that gonadal steroids feed back on the hypothal— anuas to reduce gonadotrophin secretion. Flerko (1957) demonstrated the importance of the arrtemior hypothalamus as a site of estrogen feedback. He irflnibited the effects of estrogen on the castration induced rixse of FSH in rats with anterior hypothalamic lesions. FJJerko and Szentagothai (1957) showed that estrogen re— ltaased from small fragments of ovarian tissue autotrans- pfiLanted into the anterior hypothalamus inhibits FSH seucretion. Similar ovarian implants into the anterior gxituitary or liver tissue grafts into the anterior hjqoothalamus did not produce this effect. Hohlweg and Daxume (1959) found that injections of estrogen into the aurterior hypothalamus of rats has an anti-FSH effect 1255 times that of estrogen administered subcutaneously. The above data support the hypothesis that nervous elements located in the anterior hypothalamus play some r0113 in the feedback mechanism by which FSH release is inhibited by a slight elevation in blood sex steroid level. MEi‘ites et_a1, (1966) reported that gonadal steroids can alter hypothalamic FSH—RF and LH-RF, and indicate that this is the major pathway by which gonadal steroids exert their negative feedback on anterior pituitary release of gonadotrophins. 10 There is much data which indicate that neural stxructures in the medial basal hypothalamus are affected lb}? sex steroids in a negative feedback fashion. Davidson arnfl.Sawyer (1961) implanted estradiol benzoate into various remgions of the brain in the female rabbit. Failure of (scapulation—induced ovulation and ovarian atrOphy followed irmplants in the basal tuberal area near the posterior Inexiian eminence. Implants in the anterior pituitary and Inaunmillary bodies had no effect. These findings were eoctended by Kanematsu and Sawyer (1963). Kanematsu and Ehawyer found also that these implants caused decreased aunounts of LH in the plasma of ovariectomized rabbits. SiJnilarly Ramirez et 31. (1964) found that implants of enstradiol in the median eminence prevented the rise in Eilasma LH which occurs after ovariectomy in rats. Lisk (1960) implanted estradiol benzoate into the hYINothalamus in male and female rats. The sensitive area, jJIWhich implants caused atrophy of male and female tracts anti gonads included the arcuate nucleus, surrounding ven- ‘tral.hypothalamus and mammillary bodies. Lisk (1963) fcDund that estradiol implants into the arcuate nucleus of Spayed rats prevented the characteristic changes in pitu- itary cytology which occur following gonadectomy. Implants in the mammillary bodies and anterior lobe of the pituitary did not alter the castration reaction. Control substances 11 also were ineffective. Lisk and Newlon (1963) found that estradiol implants in the arcuate nucleus caused decreased size of nucleoli in the cell bodies accompanied by atrophy <3f ovaries, uteri, testes, etc. Lisk (1962) implanted testosterone into brains of Inale and female rats. Atrophy of ovaries, uteri, testes, sneminal vesicles and prostates was obtained 30 days later :Erom implants into the median eminence and other parts of 'the basal tuberal region. Davidson and Sawyer (1961) :melanted testosterone propionate into the brains of male ciogs. Testis atrophy resulted from implants in the pos- 'terior median eminence and posterior tuberal regions. These findings conflict sharply with those of IBogdanove (1963) who found that ovarian or estradiol ciipropionate implants were effective directly on the pitu- istary. Such implants produced localized regression of czastration cells in ovariectomized female rats. In eaddition, Ramirez g3 g1. (1964) found that estradiol janlants in the anterior pituitaries of female rats pre— \nented the rise in plasma LH after ovariectomy. Kanematsu and Sawyer (1964), in contrast to some of their earlier 'work reported an increase in plasma LH following estradiol implants in the anterior pituitaries of ovariectomized rabbits. 12 b. Corticotrophin-adrenal-cortical steroids The dilemma of site of action of adrenal steroids in the feedback control mechanism of pituitary ACTH secre— 'tion is perhaps as inconclusive as that of the sex steroids jllst mentioned. A major function of circulating adrenal sateroids is the feedback control of pituitary ACTH secre— 1:ion, and there is strong evidence that this action of the cxorticoids is exerted at least in part indirectly via the Irypothalamus. In a careful study on dogs Eik—Nes and IBrizzee (1965) discovered that intravenously injected :radioactive cortisol was rapidly concentrated in and slowly lreleased from hypothalamic nuclei; only small amounts of 1:he steroid were taken up by the pituitary gland or (zerebral cortex. The question of the site of action in the feedback (nontrol mechanism has also been investigated extensively vvith micro—injections or implantations of the steroids ciirectly into the hypothalamus and pituitary gland. IEndroczi, Lissak and Tekeres (1961) reported that intra- llypothalamic and mid—brain implants of cortisone in agar f cortisol into the rat hippo-campus or mid-brain .inhibited the diurnal changes in adrenal and plasma <30rticosteroids, but only implants into the median Eflninence depressed adrenal steroid levels and induced adrenal atrOphy. Atrophy of the adrenals has also been Observed following median eminence implants of cortisol by Davidson and Feldman (1963) and Chowers gE g1. (1963). In the larger hypothalamus of the rabbit, Smelik and Sawyer (1962) noted that implants of cortisol into the l4 anterior median eminence and postoptic area blocked the stress—induced rise in adrenal steroids without causing adrenal atrophy. If the negative feedback action of the corticoids is exerted primarily at the hypothalamic level one would expect a rise in hypothalamic corticotrophin releasing factor (CRF) following adrenalectomy. Such was found to be the case by Vernikos-Danellis (1965). However, DeWeid (1964) has found that pretreatment with dexamethasone prevents the ACTH releasing effect of hypothalamic extracts containing CRF; probably the steroids exert both direct and indirect effects on the pituitary. Support for this view is provided by experiments by Chowers g2 g1. (1967) in which they found that both median eminence and pituitary implants of dexamethasone reduced pituitary ACTH release, while only median eminence dexamethasone implants de- creased CRF content. In View of these different observations it does not Seem unreasonable to propose three levels of feedback for the adrenal corticosteroids. Corticosteroids may act on structures of the limbic system to modify diurnal rhythms; they may act on the hypothalamus to decrease base levels of CRF, and they may act directly on the pituitary to decrease ACTH. 15 c. Special case of prolactin and growth hormone The question that immediately poses itself is how are prolactin and growth hormone regulated by a feedback control when there are no target organ hormones to exert a feedback? It would appear that the answer to this is that prolactin and growth hormone exert a feedback action upon the hypothalamus to control their own production, however this is an instance of hypothalamic feedback which will be discussed in the next section. Although there are no target organ hormones for prolactin there exist numerous hormones and pharmacological agents which stimulate pro- lactin secretion (see Meites and Nicoll, 1965, 1966). 2. Feedback of pituitary hormones on the hypothalamus a. CorticotrOphin-ACTH There is considerable evidence that ACTH can exert a feedback influence on its own secretion by acting on the hypothalamus. The existence of this hypothalamic feedback mechanism is suggested by the observation that treatment 0f adrenalectomized animals with exogeneous ACTH may reduce Pituitary weight (Eriksson, 1959; Gemzell and Heijkenskjold, 1957) and block the fall of pituitary corticotrophin usually induced by stress (Kitay g3 31., 1959; Stark g3|gl., 1953). Furthermore, in adrenalectomized rats submitted to stressful stimuli, a greater increase in blood ACTH occurs 16 when the initial blood levels of the hormone are low than when they are high (Hodges and Vernikos, 1958, 1959). More direct evidence for hypothalamic feedback is provided by Halasz and Szentagothai (1960) who implanted anterior pituitary tissue in the infundibular recess of the third ventricle of the rat and found a depressing effect of such implants on adrenal function. Motta g3 g1. (1965) made stereotaxic implantations of solid ACTH into several areas of the brain and into the pituitary of normal male rats. In these studies, ACTH implants into the median eminence 'Jere effective in significantly depressing blood corticos- terone levels. A significant decrease in pituitary weight occurred, but there was no change in adrenal weight. Implants of ACTH elsewhere were completely ineffective. Legori g3 g1. (1965) have shown that implants of ACTH in adrenalectomized rats significantly reduce pituitary ACTH content. The work of Torok (1954, 1964) showing the exist— ence of a two directional blood flow in the hypophyseal Portal system from and to the hypothalamus, has demon- strated one anatomical route by which pituitary hormones might pass from the pituitary to the hypothalamus. b. Gonadotrophins-—FSH and LH A hypothalamic feedback in gonadotrophic functions has independently been suggested by Sawyer and Kawakami 17 (1961) based on indirect evidence. They noted EEG changes associated with the behavorial after—reaction following coitus. At first it was thought that the EEG after- reaction was in fact a correlate of the nervous activation of the hypOphysis (Sawyer and Kawakami, 1959). It soon became apparent, however, that the time of these events was too much delayed for such a relationship. Therefore, it seemed more probable that the EEG after—reaction was due to the discharge process of the pituitary hormones, or perhaps a direct feedback action of the released hormones on the nervous system. Since the reaction also occurred in spayed rabbits it was evident that ovarian steroids could not be concerned with the feedback (Sawyer and Kawakami, 1959a). Attempts were made to induce the Spontaneous EEG after-reaction in the absence of vaginal stimulation or coitus (Kawakami and Sawyer, 1959b). This was successful with purified preparation of pituitary LH, chorionic gonadotrophin (HCG) and pregnant mare's serum (PMS) and also with prolactin and neurohypophyseal hormones. Negative results were obtained with other hormones. It is quite interesting to note that all of the pituitary hormones Whidh produced an EEG after-reaction are released in re- SPOnSe to coitus in the rabbit. Thus, it is evident that the Pituitary hormones, released after coitus, exert a feedback action on the hypothalamus. Recent evidence by l8 (:cxrbin and Cohen (1966a, 1966b) and David, 23.3l' (1966), .leznd the concept of short 100p feedback strong support by showing that median eminence implants of tubes containing JLEI in a cholesterol or cocoa butter vehicle depress the JLEI content of rat pituitary and plasma. Recently Corbin and Story (1967) have demonstrated a hypothalamic feedback fcxr FSH by using the above mentioned implant technique, arnd for the first time have shown that a pituitary hormone can decrease production of its own releasing factor by acrting directly on the hypothalamus. c. Prolactin and growth hormone At the time research work was begun on this thesis thexre was no direct evidence for a hypothalamic feedback for: prolactin, except for the hypothesis of Meites and Signouris (1953) that prolactin injections decrease pitu- itétry prolactin content. Recently indirect evidence for a.1rypotha1amic feedback influence by prolactin and growth hOInnone was provided by MacLeod, EE.§i' (1966). In these Stxuiies they found that subcutaneous transplants into iJTtact rats of MtTW5 pituitary tumors secreting large 'wmounts of prolactin and growth hormone result in reduced Pituitary weight and decrease the prolactin and growth hormone content of the animal's own pituitary. These Observations have recently been confirmed by MacLeod, §£_gl. (1968), who in addition found a decrease in l9 Exituitary TSH content. The initial findings of MacLeod rmegarding pituitary prolactin content were confirmed using 51 Inore quantitative technique by Chen gE g1. (1967) and e>lactin-imp1anted rats. These observations can be regyarded as the most definite proof for the hypothalamic feedback and its possible physiological significance, becxause target organ responses were observed which showed fOI' the first time that the lowered pituitary hormone le\nals meant decreased hormone release into the systemic Circulation. In further pursuit of the hypothesis of a hypothal- amic feedback for prolactin, Clemens g3 g1. (1968) found that median eminence implants of prolactin inhibit Postpartum lactation in rats. In addition they found 20 that when ACTH was added to the prolactin implant there was a greater inhibition of lactation noted than with the prolactin alone. The only published evidence regarding a hypothal- amic feedback for growth hormone is the abstract by Katz gE_gl. (1967) indicating that median eminence implants of growth hormone in a cholesterol pellet reduce pituitary growth hormone content. III. Expgrimental Production of Mammary Tumors in Rats A. Methods of induction Mammary tumors may be induced readily in a high percentage of animals by a number of means. The mammary tiSSue of the rat appears to be highly susceptible to malignant changes following treatment with aminofluorene cCanounds, benzpyrine, 3—methy1cholanthrene (3-MCA) and 7, lZ-dimethylbenz (a) anthracene (DMBA). Noble and (hitts (1959) presented an extensive review of mammary tUHuars of the rat and methods of tumor induction. The tWtD most widely used carcinogens are 3-MCA and DMBA. The irlduction of mammary tumors by these carcinogens apparently require both anterior pituitary hormones and ovarian hOrmones. Thus, rats ovariectomized or hypophysectomized Prior to carcinogen administration develop few or no mammary tumors (Dao, 1964). 21 B. Hormonal dependence The carcinogen induced mammary tumors are generally adenocarcinomas which are sensitive to hormone manipula- tion. The hormonal dependency of mammary tumors in rats ‘was described by Noble and Collip (1941) after treatment xvith estrogen pellets. The adenocarcinomas rapidly re- gressed in the rats when the estrogen pellet was surgically removed. Most of the studies demonstrating the hormone dependency of rat mammary adenocarcinomas used ablation of certain endocrine organs. In some cases endocrine organs are removed before carcinogen administration in order to ascertain endocrine influences on tumor induction, and.in other cases endocrine organs are ablated some period <15 time after carcinogen administration to ascertain effects CHI tumor growth. In other experiments, hormones are administered either before or after appearance of the mannnary tumors. Tumor induction and growth were studied in ovariec— ttunized and in hypophysectomized Sprague—Dawley rats by Huggins and Briziarelli (1959) and Huggins g’g EL (1959) . Theii'findings indicate that ovariectomy delays the onset and inhibits tumor growth, while hypOphysectomy completely inhibits the formation of tumors. Hypophysectomy after tumor formation caused regression of tumors. The effects 22 of ovariectomy were largely mediated through estrogen, since administration of 0.1-1.0 ug estradiol per day completely nullified the effects of ovariectomy on tumor formation and growth. According to the findings of Dao (1962) ovariectomy sufficiently in advance of carcinogen administration (30 days) completely inhibits tumor formation. Dao concluded from his study that the presence of estrogen is necessary for the formation of mammary tumors. This conclusion may be premature, since it is entirely possible that the observed inhibition of tumor incidence may be due to lack of prolactin. Estrogen has been shown to maintain or stimulate growth or mammary tumors only in the presence of the pituitary (Sterental EE.E£°I 1963). In an excellent study Talwalker EE.El° (1964) have shown that even in the absence of the ovaries treatment with the pituitary hor- mones, prolactin and growth hormone, can induce mammary tumors in rats given a single feeding of DMBA. This study suggests that the pituitary hormones, prolactin and growth hormone, are of primary importance in mammary carcinogen- esis in rats. IV} figural Control of Puberty in Female Rats A. General review There is agreement that increased pituitary gonadOtrOphin secretion is responsible for the onset of 23 puberty (Donovan and Van der Werff ten Bosch, 1965; Van der Werff ten Bosch, 1966; Critchlow, 1967). The majority of recent investigations concerning control of puberty are centered around "trigger" mechanisms respon— sible for this surge in gonadotrophin secretion before puberty. Puberty occurs in female Sprague—Dawley rats at approximately 38 days of age. In the young rat the ovaries have been shown to be refractory to gonadotrophin stimula- tion until about the twenty—second day (Leonard and Smith, 1934; Gaarenstroom g3 g1., 1960; McCormack and Meyer, 1964). In order to obtain an understanding of the neural mecha— nisms, investigators have tried to hasten or delay puberty by various manipulations. These studies will be reviewed in the ensuing sections. B. Experimental production of precocious puberty Thirty-six years ago Hohlweg and Junkmann (1932) ingeniously arrived at the conclusion that a neural sex Center was involved in the control of puberty and reproductive cycles. Unfortunately this brilliant theory was almost totally obscured by the popular theory of Moore and Price (1932) which considered pituitary—gonadal inter— actions sufficient to explain phenomena associated with reproductive functions. It was 20 years later that Harris and Jacobsohn (1952) demonstrated that the immature rat ¥ 24 hypophyses transplanted under the median eminence of hypophysectomized adult female rats restored vaginal cyclicity and ovulation. This study furnished the major stimulus for once again considering the nervous system in relation to puberty, and demonstrated the passive role of the pituitary in the initiation of puberty. This study also suggested that the portion of the prepubertal period which follows the attainment of gonadal responsitivity to gonadotrophins may result from inhibition or inactivity of hypothalamic mechanisms regulating pituitary—gonad func- tions in the adult. Donovan and Van der Werff ten Bosch (1956, 1959) found that electrolytic lesions in the anterior hypothalamus of rats resulted in precocious puberty as ascertained by vaginal opening and ovulation. These studies supported the concept of neural inhibition of gonadotrophin secretion in the prepubertal animal and have been confirmed (Bogdanove and Schoen, 1959; Elwers and Critchlow, 1960; Horowitz and Van der Werff ten Bosch, 1962; Schiavi, 1964). Attempts to localize and identify the hypothalamic structures involved in the control of puberty have not been fruitful. In addition to anterior hypothalamic lesions advancing puberty Schiavi (1964) found that posteriorly placed electrolytic lesions were equally effective. In Contrast to the above findings Gellert and Ganong (1960) ¥ 25 found posterior tuberal, not anterior hypothalamic destruction, effective in advancing puberty. In addition to hypothalamic mechanisms controlling sexual development, experimental evidence by Elwers and Critchlow (1960) demonstrated that lesions placed in the amygdaloid complex advanced puberty. Other treatments which are probably mediated through the central nervous system and hasten onset of puberty are mild stress (Morton g2 g1. 1963) and electrical stimulation of the uterus (Swingle g: 31. 1951). C. Experimental delay of pubeggy Whereas the treatments described above resulted in acceleration of sexual development in female rats, destruction localized to the region of the ventromedial and arcuate nuclei prevented or delayed puberty in rabbits (Bustamante, 1942; Bustamante g5 g1., 1942). A similar delay in sexual maturation was recently reported in con— junction with electrolytic lesions placed in the region Of the ventromedial—mamillary body area of female rats (Corbin and Schottelius, 1960, 1961). These observations were interpreted as evidence for the presence of a gonado— trophin—stimulating area in this part of the brain. Recently Bar—Sela and Critchlow (1966) reported that Stimulation of the amygdaloid nuclear complex significantly delayed puberty in female rats. ‘_ 26 D. Possible neural mechanism involved in the onset of puberty The original idea of Hohlweg and Junkmann (1932), that neural structures are involved in control of repro— ductive functions are in accord with present day concepts. Tings Donovan and Van der Werff ten Bosch (1959), while ccnusidering the effects of neural lesions, suggested that tlie main difference between the immature and adult animal :is the extreme sensitivity of the brain of the former to gcnuadal hormones. Similarly, Ramirez and McCann (1963) arni McCann and Ramirez (1964), on the basis of differences ir1 the supressive effects of estrogen and androgen on pitu— iizary and plasma LH levels in gonadectomized rats, suggested tliat the onset of puberty results from resetting of a h§ipothalamic "gonadostat." Because of this proposed change iri threshold, circulating levels of gonadal hormones which arwe effective in restraining gonadotrophin secretion in the inunature animal are rendered ineffective at puberty. Thus, the current concept is that gonadotrophin— regtflating mechanisms in the CNS of the immature animal are markedly more sensitive to the negative feedback influences of the sex steroids, and that a decrease in sensitivity may be responsible for the onset of puberty. 27 V. Loss of Reproductive Cycligg Activity With Aging A. Age changes in the pituitary The first detailed study of the senile decline of the human pituitary gland was made by Cooper (1925). She studied the pituitary from fetal life to old age and Exointed out that in the early stages of life the pituitary \vas oval in sagittal section but in old age it became spherical. The cleft between the anterior pituitary and pnosterior that could be seen in fetal life became much snnaller after puberty, and in early adult life it dis- alppeared from View. She also noted that the maximum vas- c111arity in the gland occurred at puberty and that this \rascularity persisted until the fifth or sixth decade; aiiter that period vascularity became reduced, at least ill the anterior lobe. In the case of the posterior pitu— istary, without such a rich blood supply, there was no apuparent change with increasing age. At birth the epithelial cells of the anterior lobe Vnere well separated and as age advanced they became more denisely packed. According to Cooper, the acidophil cells were the first ones to be differentiated, and she found that they were present in the third month of intra-uterine life. She found that they rapidly increased in childhood until puberty. The basophilic cells, she said, differen- tiated a little later and occurred in the greatest number 28 in old subjects, and it is possible that this correlates with the increased gonadotropin secretion in older people. The chromophobes did not appear until about 7 months. She also noted that Herring bodies were absent in the fetus but were present in the second year of age and that they increased steadily in number and size until puberty they ceased to increase any further. Weiss and Jansing (1953) made an electron micro- scopic study of the aging pituitary gland. They used Swiss albino mice and found that in the pituitary of normal young animals the chromophobes, acidophils, and basophils contained granules, double membrane systems and mitochondria in varying amounts, and that the nuclear membrane appeared to be double. With the onset of aging, however, the nuclear membrane was found to become irregu— lar in outline, the mitochondria enlarged and became vacuolated and the double membrane system became fragmented. Thus, it is clear that there are fundamental cytophysiolog— ical changes taking place in the pituitary cells with the onset of aging. Spagnoli and Charipper (1955) found that in old male golden hamster pituitaries, there was an increase in degranulation and an occurrence of pyknotic nuclei and vacuolated basophils. Chromophobic and colloid—filled acini appeared among the cell cords in both males and 29 females and these increased slightly in number with increasing age. Chromophobic networks appeared in the glands of the animals over 12 months of age. There was a decrease in the number of acidophils in older males and an increase in basophils in older females. There seemed also to be some increase in the density, and some dis- organization of the reticular framework. Assays of pituitary hormones reflect the function— ing of the aging gland. In the human, Becker and Albert (1965) found urinary excretion of FSH increased 11 times and LH increased 9 times indicating high production of these hormones in post-menopausal women. In the rat evidence concerning FSH and LH levels is scanty and very unconvincing. Lausen g2 g1. (1939) studied pituitaries of rats of different ages and measured gonadotrophic activity b}? the uterine assay. Throughout sexual life the activity rennained at the puberal level, however in EhEgg females 2.5 years of age a remarkable increase in total gonado- tr<>phic content was found. Similar assays were carried Curt by Saxton and Greene (1939), Solomon and Shock (1950), Dollcan g3 g1. (1952), Blumenthal (1955) and Ceresa and Lacroix (1951) and gii concluded that there were gg differences between the content of gonadotrophin of the hypophyses of young, mature and old animals. This View Was adopted by Korenchevsky (1961), Hollandbeck g3 g1. 30 (1956), and Jones and Krohn, (1959). Evidence to the contrary will be presented in this thesis. Meites g5 31. (1961) presented evidence indicating that as rats become older the pituitary prolactin content increases and Aschheim and Pasteels (1963) have indirect and scanty evidence that prolactin secretion may be in- creased in old, senile constant estrous rats. No signifi- cant differences have been reported in STH, TSH and ACTH content of pituitaries from young and old animals. B. Age changes in the ovary Since it appears that the decline of ovarian function is of a different etiology in humans than in other mammals, age changes in human ovaries will be re- viewed separately. 1. Human ovary In women the decline of fertility and of endocrine ovarian function are associated with a decrease in number of follicles, which has led to the assumption that meno— pause occurs when the last oocyte is spent (Engle, 1951, 1955; Zuckerman 1956). This view is contradicted by poSt—menopausal ovaries which still contain follicles and probably produce estrogen as well. Yet such cases may not be incompatible with the primary role of follicular depletion in ovarian senesence (Comfort 1956). The 31 menopause marks only a relative failure in ovarian function, the decline of which had started long before this point is reached. In addition to menopause there are other events that accompany the gradual downhill slope of ovarian involution. An early symptom is the occurrence of anovu- latory cycles, i.e. the failure of ovulation and corpus luteum formation. Anovulatory cycles may, of course, cxzcur at all ages but their frequency is highest in young adrflescent and near—climacteric women. At both age levels, fluoreover, even when corpora lutea are formed, they may be lesss stable functionally than those formed between the agges of 25 and 40, as was demonstrated by Collett, WEErtenberger and Fiske (1954), using basal temperature CLLrves. It appears that both the period during which ovwarian function gets into its stride, and the years when it. is slowly regressing, are characterized by deficient prwagestational activity. Follicular growth, however, and tilea concomitant production of estrogens are manifest over a Inuch longer period, from before puberty to long after true menopause. The urinary excretion of estrogens has beeni shown to decrease gradually and does not reach its JINvest level until about the age of 60 (Pincus, 1955). SOHEB estrogens are still excreted, although the site of their: elaboration is disputed. Vaginal smears may also 32 demonstrate this estrogenic activity (Berger and Keller, 1954) which has been claimed to be cyclic (Bourg and Pundel, 1951). Continued estrogenic activity during the climac- teric, unopposed by progesterone secretion, has been related to the frequency, about this age, of endomentrial hyperpla- sia (Novack and Richardson, 1941). It is of considerable interest that even after 60 such hyperplasia is not uncommon. ITiis again raises the question, as did the continued urinary esrtrogen excretion, whether at this age these hormones can st:ill be ascribed to the ovaries. Except in cases of exxogenous hormone influences of hormone-producing ovarian ttunors, most authors tend to ascribe late estrogenic acrtivity to the adrenal gland, which has even been called "tlue gonad of the aged" (Hamblen, 1945). Although the addrenals may be the main source of sex hormones in senile ‘WOrnen, the ovaries should not be ruled out as a contribu— txblry factor. Besides follicles which may be found up to 6 §rears after the menopause, patches of ovarian stromal hYIDerplasia (Hertig, 1944) and hilus cells have been Susggested as possible sources of estrogens in senile OVEiries. In this connection the work of Paulsen g3 g1. (1955) deserves attention. These authors found that, in Pest—menopausal women, the administration of gonadotropic hermcnaes could lead to an increase in urinary estrogen 33 excretion, while ovariectomy could be followed by a decrease. A recent study (Becker and Albert, 1965) has shown that compared to normal men and women, in post— menopausal women FSH and LH secretions were increased 11 and 9 fold respectively due to the decreased feedback from the ovary. 2. Other mammals Information on ovarian changes in old animals is scarce, but it is well known that fertility decreases with irlcreasing age (Ekstein, 1955; Engle, 1944). The general innpression is that ovarian involution in most animals takes a slower course, relative to the life span, than in women (Iakstein, 1955) and that consequently estrogenic activity 311d follicular growth still occur in many senile animals, exren in monkeys (Engle, 1944; Krohn, 1955). Most available déita are concerned with laboratory or domesticated animals. Mcxre is known about mice and rats than about any other S£>ewies. The literature indicates that in mice fertility deacflines with increasing age (Bittner, 1935; Murray, 1934) bLIt follicular growth and ovulation may still occur in SeIIile animals, although the number of ovulated oocytes is 10“? and most of these are abnormal. Vaginal cycles may alSC) continue up to extreme ages but there are quantitative Straiiidifferences in this respect. Cycles may become lrreg'ular with increasing diestrous intervals which merge 34 into a final anestrous condition, while in other cases periods of continuous estrogenic stimulation intervene (Thung g3 gl. 1956). It has been suggested that in rats and mice the decline in ovarian function is secondary to hypophyseal failure, while in man ovarian involution pre— cedes the senile changes of other organs (Krohn, 1955). The unimpaired sensitivity of old mouse ovaries to hypophyseal hormones was demonstrated by Zondek and Aschheim (1927). Similar experiments (Hoffman, 1931; Romeris, 1931; Zc>ndek, 1929) reported reactivation of the ovaries of old Hnlce by hypophyseal implants or extracts. Similar results luave been reported for rats (Engelhart and Hauser, 1937; VVesisner, 1932). The above results would seem to indicate tkiat there may be changes in the hypophysis with age or Inc>re fundamentally changes in the central nervous system. Tilese results suggest that the effects produced by pitu- it:ary implants were due to the residual amount of gonado- tlcopin remaining in the pituitary, and that the effects 013 aging on the ovaries in rats and mice are initiated by fllndamental changes in the hypothalamus or higher centers, pC>ssib1y the limbic forebrain system. This center has kxaen shown to be intimately connected with activity of the hyrxathalamus (Nauta, 1963). 35 C. Changes in the estrous cycle with age Estrous cycles in aging rats exhibit several different patterns. Studies by Aschheim (1961) and Mandl (1961) demonstrate 3 different patterns of cycling in senile rats. The most prevalent pattern was repeated periods of pseudo—pregnancy evidenced by prolonged periods of diestrus and ability to produce a decidual reaction. The second most frequent pattern was manifested by persist- erit vaginal cornification. This constant estrous condition lasted for many months, usually uninterrupted. Finally the tliird pattern was characterized by total irregularity in tile pattern of cycles; no regular, cyclic sequence of exrents could be established. Aschheim, (1965) suggested triat it was not the ovary that failed in aging but possibly tlie hypothalamus or the portal circulation, since LH irijections into old constant estrous rats caused regular es trous cycles . MATERIALS AND METHODS I. Animals Mature and immature intact female Sprague-Dawley rats from Spartan Animal Farms (Haslett, Michigan) and Holtzman Company (Madison, Wisconsin) were used in all experiments. The animals were housed in a temperature controlled (75i1°F) and light controlled (l4 hr/day) room. The animals were maintained on a diet of Wayne Lab Blox (Allied Mills, Chicago, Illinois) and tap water. Female hypophysectomized rats, 21 days old, were obtained from Hormone Assay Labs., Chicago, Illinois, for the puberty study. White King Squabs (Cascade Squab Farm, Grand Rapids, Michigan) 4 to 8 weeks old, were used for prolactin assays. II . Stereotaxic Technique The stereotaxic instrument used in these studies Was the Stoelting, Stellar (Fig. 1) model, obtained from C. H. Stoelting Company, Chicago, Illinois. The stereo- taxic instrument is constructed in such a manner that any given point in the brain may be located with reference to the three cartesian planes in space. The animal's head is 30 held in the instrument that these three planes are relatively constant from animal to animal. 36 37 Fig. 1. Stoelting, Stellar model, stereo- taxic instrument used in implant studies. 38 The three planes are called the horizontal, coronal and sagittal. The horizontal planes are parallel to that plane which passes through the inferior orbital margins and the centers of the external auditory meatus. The coronal planes are perpendicular to the horizontal planes, the principal one of which corresponds to a line connecting the centers of the two external auditory meatuses. The sagittal planes are perpendicular to both the horizontal and coronal planes and parallel to the mid—sagittal plane to the animals head, i.e., corresponding to the interhemispheric fissure. Rats used in these studies were adult female rats of the Sprague—Dawley strain (Holtzman Co., Madison, Wisc.) weighing about 200 grams. Rats of this size have a fairly constant head shape and size. At about 200 grams there is little change in the brain or head growth; since mature albino rats were used, modifications were not necessary. The best anesthetic for all types of work turned out to be ether. It was difficult to establish a standard dose of pentobarbital. Some rats were much more sensitive to it than others. Anesthesia was regarded as sufficiently deep when the animal just failed to respond to a paw pinch. The top and the sides of the head were shaved, and the head was washed with 70% ethanol. The ear plugs were then inserted. Rats' ears are usually clean and the ear plugs are pointed, so that the necessity of cleaning the ears is 39 probably not as great as in other animals. The ears must be inspected for abnormalities, however, so as to prevent any unnecessary error. Cold sterilization of the ear plugs in a cold non-corrosive germicidal solution was carried out periodically. The ear plug on one side is inserted into the rat's ear and seated against the side of the rat's skull. The head is then held firmly against this plug while the other is placed in the opposite canal. The ear plugs are then placed into the ear bars (Fig. 2) and are secured by tightening the set-screws. The head is observed to assure that a line joining the two eyes is parallel to the ear bars which hold the two plugs. If the lines are not parallel the rat is not in the instrument properly and, the entire procedure must be repeated. Great care must be taken in placing the ear plugs in the rat's ears since it is possible to puncture and fracture the rat's skull and irreparably damage the animal for experimental work. The same precautions are necessary when the ear bars are placed in the ear plugs; however a firm setting must be obtained if accuracy is to be main— tained. The incisor bar was positioned behind the upper incisor so that its top surface was 5.0 mm above the interaural line. This measurement was necessary in order for the rat's brain to be fixed in the same plane as the 40 Fig. 2. Ear plugs held in place by the ear bars. 41 brain in the brain atlas. The atlas used was that of de Groot (1959). A cut is made from the occiput forward to a line between the eyes. The skin is retracted and the galea and the periosteum are scraped from the bone. All ruptured blood vessels are then cauterized so that the skull becomes dry allowing one to see the suture lines. The bregma of the rat's skull was used to determine the mid-sagittal plane. This is the point of intersection of the sagittal suture and the coronal suture. Since the suture lines in the rat, as in other animals, are not straight lines, it was frequently necessary to estimate the center of this point. The horizontal bar of the elec— trode carrier was moved until the electrode tip was exactly over the bregma. The reading thus obtained on the horizon- tal bar was used as the zero reference for all para-sagittal settings. After the coordinates were adjusted according to those in the atlas for the median eminence and recorded, a hole was drilled through the skull with a dental drill. Care was taken not to damage the brain. The stereotaxic instrument used in the lesion studies was a Neuman and Lab-Tronics Stereotaxic instru- ment (Fig. 3) (Neuman Company, Chicago, Illinois). Much of the preparation of the animal and operational techniques are the same for use of this instrument and the Stoelting instrument previously described. There are some critical differences however and these will now be mentioned. 42 Fig. 3. Neuman and Lab Tronics stereo— taxic instrument used in lesion studies. 43 The major part of the instrument consists of a yoke with two horizontal side bars. A removable posterior cross brace is placed between these to prevent any angular deviation after the animal is in place. Attached to the undersurface of the yoke is a plate containing the jaw clamps. On the undersurface of each horizontal side bar, is an ear bar support, in each of which is an ear bar. The ear plugs are fastened to the bar that joins them by set-screws. It is easier to place the rat between the ear plugs if one plug remains fixed on the bar. This plug is inserted into the rat's ear and seated against the side of the rat's skull. The head is then held firmly against this plug while the other is placed in the opposite canal and external auditory meatus by sliding it along the connect- ing bar. It is secured by tightening the set-screws. The head is observed to assure that a line joining the two eyes parallel to the bar which holds the two plugs. The rela- tive position of the ear plugs from the end of the bar is not essential, since the bar serves only as a support and has nothing to do with aligning the rat's head in the mid— sagittal plane. Once the ear plugs are seated, they and the rat can be placed in position between the two ear bars of the instrument. The ear bars can be slipped into the apprOpriate holes in the plugs and adjusted to align the rat's head in the center between the two horizontal side bars. 44 Examination of the upper incisor bar reveals that there is an expanded area in the center with a sharp ridge around its circumference. This expanded area goes directly behind the upper incisors and the sharp ridge is fitted between the two teeth. The assembly is moved back toward the animal until the rat's upper incisor teeth can just be placed over the bar with the expanded area located as above. When the incisor bar clamp is tightened care should be taken that undue pressure is not exerted against the upper incisor teeth as it is possible to crush them. The verti- cally adjustable incisor bar should be set at the approxi- mate position which is applicable for the atlas employed to avoid any undue motion of the head up and down after the plate and the incisor bar have been firmly fixed. Any lindue deviation of the head after this may pull the animals knead forward and cause distortion. The atlas used was that of de Groot. This atlas calls for placing the tOp of the incisor bar 5 mm above the line between the ear plugs. Other atlases differ. III. MakingfLesioning Electrodes Most of the lesioning electrodes are made from #00 insect pins which can be purchased from any book store. The black coating was scraped off the pins using fine steel wool. The pins were then washed in xylene to remove any 45 grease. The pins were dipped in epoxylite resin very slowly and then baked in a vacuum oven at 190°C for one hour. The dipping and subsequent baking is repeated 3 more times. Before the electrodes can be used insulation is scraped off at the tip. IV. Production of Lesions The electrode tips are usually sharp and will readily pierce the dura very easily. The lesion is made with the anode and the indifferent cathode is placed in the rectum. The amount of current used depends on the size and location of the lesion to be produced. Using 0.15 mm stainless steel electrodes and a current of 5 milliamperes for 15 seconds, a 1.0 mm diameter lesion can be produced in the hypothalamus. A Stoelting direct current lesion maker with a regulated power supply was used to produce all neural lesions. V. Preparation of Implantation and Securing Hormone Implants In this study all substances to be implanted were placed inside 23-gauge glass tubing. Measured portions of hormones and cocoa butter were thoroughly mixed, and used to implant into the experimental groups. Cocoa twitter alone was implanted into the control groups. Tubes to be implanted were prepared by tamping the glass tubes 46 into the mixture to be implanted, and after an adequate amount entered the tube the opposite end was sealed with bone wax in order to prevent a back pressure from pushing the hormone mixture up and out of the tube when it was lowered into the brain. The glass tubes were placed into the chuck of the stereotaxic instrument (Fig. 4) and lowered into the brain through the hole previously drilled (Fig. 5). The glass tube was lowered until the tip touched the floor of the cranial cavity and then raised 1.0 mm so that the tip would lie in the middle of the anterior median eminence. Skull screws were placed into holes previously drilled along side of the hole into which the implant was lowered. Silk thread was tied around the implanted tube and fastened to the skull screws. The entire area was then covered with dental cement. The skull screws served to anchor the implanted tube and cement to the skull and the silk thread serves a purpose analogous to the steel rods in reinforced concrete. VI. Incubation Technique Rats used in these experiments were killed by guillcfiine, and the anterior pituitaries and hypothalami ‘were removed quickly. The posterior lobe was discarded and the anterior lobe was weighed, frozen and stored at -20°C until assayed. The hypothalamus and median eminence 47 Fig. 4. Glass tube, filled with hormone, secured in chuck of stereotaxic instrument. 48 Fig. 5. Glass tube, filled with hormone, being lowered through hole drilled in skull. 49 were collected in chilled o.lN HCl (1.0 hypothalamus/0.1ml) and frozen at —20°C until used for incubation. A. Preparation of hypothalamic extract On the day of incubation the hypothalami were thawed and homogenized in a ground glass homogenizer, and centrifuged at 12,000 g for 40 minutes at 4°C. The super- natant was placed in protein free medium 199 (Difco Lab., Detroit, Michigan), and the pH was adjusted to 7.4 by adding drop by drop 1N NAOH and testing with glass elect— rodes. The hypothalamic extract was neutralized immediately before use. B. Incubation Adult male rats of the Sprague—Dawley strain were killed by guillotine, the anterior pituitaries were re- moved immediately and placed in a petri dish over a filter paper moistened with medium 199. Each pituitary was hemisected, one—half being placed in a control flask (25 m1 Erlenmeyer) containing 2.0 ml of medium 199 and the other half in the experimental flask also containing 2.0 ml of medium 199. The equivalent of 4 anterior pituitaries (8 halves) was placed into each flask. The neutralized hypothalamic extract (equivalent of 0.25 or 1 hypothalamus per pituitary incubated) was added rapidly to each flask. Incubations were carried out in a Dubnoff metabolic Shaker (60 cycles/min.) under constant gassing with 95% 50 02—5% CO2 at 37i0.5°C for 4 hours. At the termination of the incubation, the pituitary halves were weighed and the medium was centrifuged at 2,500 g for 10 minutes, and the supernatant was frozen at —20°C until assayed. VII. Bioassays A. Prolactin The incubation medium was assayed for prolactin activity by the intradermal pigeon—crop technique of Lyons (1937) as modified by Reece and Turner (1937). A dose of 0.2 ml was injected daily for 4 days into each bird. A direct comparison was made between samples by injecting one sample over one side of the crop sac and the other sample over the other side of the crop of the same bird. Anterior pituitaries were homogenized in normal saline. Each injection was in a 0.1 m1 volume and an equivalent of 3.0 mg of pituitary was injected into each bird for 4 days. The prolactin responses in each bird were expressed in Reece-Turner units (RTU), and these units were converted into international units (IU) by use of a standard dose response curve established in the same breed of pigeons With NIH prolactin (Nicoll and Meites, 1963). B. Follicle stimulating hormone (FSH) The method of Steelman and Pohley (1953) as modified by Parlow and Reichert (1963) was used to measure 51 FSH. Pituitaries were ground in a glass homogenizer and extracted with physiological saline solution. The solu- tions were centrifuged and stored at 4°C during the assay. C. Lutenizing hormone (LH) LH assays were done by the ascorbic acid depletion method of Parlow (1961). All materials were assayed at four-fold differences in concentration. The media were assayed in doses equivalent to 2.5 and 10.0 mg of incubated pituitary. Pituitary LH content was assayed by injecting 1.0 and 4.0 mg of pituitary tissue, respectively. VIII. Statistical Analysis Statistical analysis for prolactin was performed by the "t" test for paired observations. Bioassays for FSH and LH were analyzed according to Bliss (1952). EXPERIMENTAL I. Direct Hypothalamic Feedback for Prolactin A. Effects of median eminence implants of prolactin on hypothalamic PIF content, pituitary prolactin concentration, mammary glands andiovaries Objectives There is evidence that anterior pituitary hormones participate in their own feedback control by acting directly on the hypothalamus. Thus it has been reported that FSH (Corbin and Story, 1967), ACTH (Legori gE_gl., 1965), LH (Corbin and Cohen, 1966) and GH (Katz gE_gl., 1967) implants into the hypothalamus reduce pituitary concentration of each of these hormones in the rat. The present experiment was designed to determine the effects of prolactin implants in the median eminence of rats on hypothalamic content of prolactin inhibiting factor (PIF), pituitary prolactin con— centration and on the mammary glands and ovaries. Materials and Methods A total of 35 mature female Sprague—Dawley rats, approximately 3 months old, were used in this study. Single stereotaxic implantations of approximately 250 ug prcflactin (NIH-P-S7) in cocoa butter were made in the 52 .Ihlilfltfllf ... I.‘.iT.L I .. .llu I ll. u I l :I -.ll ‘. VI .6. '1 - . ‘Ic‘ - . : bl .. 53 median eminence of 13 mature intact and 6 ovariectomized rats. Implants in the intact rats were all made on the day of estrus. A total of 10 intact and 6 ovariectomized control rats were implanted with cocoa butter alone. Glass 23-gauge tubing was tamped into the prolactin-cocoa butter mixture, implanted and left ig_gi£g, In intact rats, vaginal smears were taken daily beginning 10 days prior to implantation and continued until the rats were sacrificed. Ovariectomy was performed 7 days prior to implantation. All animals were killed 6 days after implantation, and the ventral surface of the brain was exposed. The exact location of the implant was ascertained with the aid of a dissecting microscope. The hypothalami were removed and placed in 0.1 N HCl, frozen and stored at -20°C. On the day of PIF assay, the hypothalami were homogenized in 0.1 N HCl and the homogenate was boiled for 12 minutes in order to destroy any prolactin which may have been present, although prolactin has not been detected in rat hypothal- amic extracts Talwalker g3 g1., (1963). The hypothalamic extract was incubated with male rat pituitary according to the method of Kragt and Meites (1965), and the medium was assayed for prolactin by the method of Lyons (1937) as modified by Reece and Turner (1937). 54 Results The results in Table 1 show that pituitary incubated with hypothalamic extract from intact prolactin implanted rats released significantly less prolactin (p < .05) than that from sham-implanted rats. A similar difference was noted between the prolactin-implanted and sham operated ovariectomized groups (p < .01). This indicates that hypothalamic PIF content was increased by the prolactin implants. There was also a significant reduction in pituitary prolactin concentration in the intact (p < .01) and ovariectomized (p < .05) prolactin- implanted rats when compared with the non-prolactin im- planted controls. Anterior pituitary weight was also significantly depressed by the prolactin implants (p < .05). Mammary glands from intact control rats implanted with cocoa butter alone showed well developed ducts and some lobulo—alveolar development (Fig. 6). Implantation of prolactin in the hypothalamus produced marked mammary gland atrOphy (Fig. 7) characterized by only bare ducts and no alveolar elements. These glands resembled those from long term ovariectomized rats even though the rats were still cycling. The control ovariectomized rats with cocoa butter implants also showed much better structural maintenance of the mammary glands (Fig. 8) than the ova— riectomized rats with prolactin implants (Fig. 9). mm g game. .ooNHEouomHum>o n x>oH ma.onmm.o mm.onmm.m m.onm.m w oopcmHmEH cfluomaoud x>o 5 ov.onmo.a mm.onam.m m.onm.HH o mHonuqoo 5 Hx>o eH.onHm.o o~.onmm.H e.onm.o OH smusnfiaas cflpomaoud pomch «om.owhh.a om.owom.m N.ona.m ma maoupcoo oompaH and mm ooa\DH me ooa\DH me much ucwfiummne ouufl> CH emmmmamn .ocoo #coflmz mo .02 cflpomaoum mn pom coflumuwcoocoo cflpUMHonm mumuflspflm no mommcHEm nuance wcp oucfl whomHmEH cfluomaoum mo mpommmm .H mqmda 56 Fig. 6. Mammary gland of intact control rat implanted with cocoa butter. Note well developed ducts and some alveolar growth (x30). 57 Fig. 7. Mammary gland of intact rat implanted with prolactin. Note marked regression of ducts and absence of alveoli (x30). 58 Fig. 8. Mammary gland from control ovariectomized rat implanted with cocoa butter. Less development is evident as compared to intact control rats (x30). 59 Mammary gland from ovariec— tomized rat with a prolactin implant. Note marked regres- sion and breaking up of ductal system (x30). 60 .5 Ten of the 13 cocoa butter-implanted control rats showed persistent diestrous smears indicative of pseudo— pregnancy, whereas they all exhibited regular 4 or 5 day cycles prior to implantation. The ovaries of these rats showed mainly well developed corpora lutea (Fig. 10). The ovaries of the intact prolactin—implanted rats had many well developed follicles and corpora lutea in various stages of development (Fig. 11), typical of cycling rats. Discussion These observations demonstrate that prolactin can exert a feedback action on the hypothalamus to regulate its own production. This negative feedback by the hypo- thalamic implants of prolactin apparently is exerted by increasing PIF synthesis and release in the hypothalamus, which in turn depresses prolactin secretion by the pitu- itary. The possibility that prolactin may act directly on the pituitary cannot be ruled out at this time. It is interesting that most of the rats given sham implants became pseudopregnant presumably due to leaving the glass tube in the median eminence and perhaps also to the stress of the operation. On the other hand all of the prolactin—implanted animals demonstrated normal estrous cycles indicating that the hormone implant exerted a primary influence on pituitary prolactin secretion. This 61 Fig. 10. Ovary from intact control rat implanted with cocoa butter. Note predominance of well developed corpora lutea (x15). 62 Fig. 11. Ovary from prolactin implanted rat. Note well developed follicles (x15). 63 inhibition of prolactin secretion can be attributed to the enhanced PIF in the hypothalamus. The rapid regression of the mammary glands observed in this study indicates that constant secretion of pro— lactin is necessary to maintain the growth and structural integrity of the mammary gland. The findings in the ova— riectomized rats indicate that even in the absence of ovarian hormones, pituitary prolactin still helps to main- tain some structural integrity of the mammary glands, and that inhibition of prolactin secretion results in mammary regression. We have observed mammary regression as early as 4 days after prolactin implantation into the median eminence (Unpublished observations). Recent reports indicate that transplants into intact rats of pituitary tumors secreting large amounts of prolactin and GH result in reduced pituitary weight and prolactin concentration (MacLeod gE gl., 1966; Chen gE_gl., 1967), and increased hypothalamic PIF content (Chen g3 g£., 1967). These observations are in agreement with the results on pituitary weight, pituitary prolactin concentration and hypothalamic PIF content found in this experiment. To what extent prolactin in the circulation under normal physiological states may exert a negative feedback on pituitary prolactin secretion remains to be determined. 64 B. Effects of median eminence implants of prolactin and ACTH on postpartum lactation in rats Objectives Since prolactin and the ACTH-adrenal cortical system are both essential for maintenance of lactation (Meites, 1959, 1966), it was of interest to determine the effects of ME implants of prolactin and ACTH on postpartum lactation in rats. Materials and Methods Fifty lactating Sprague—Dawley rats (Spartan Animal Farms, Haslett, Michigan) approximately 3 months old, were divided into 3 groups on the 4th day postpartum and each received stereotaxic implants in the anterior ME of the following substances: group I, controls, cocoa butter; group II, a mixture of NIH-P-S8 prolactin and cocoa butter; group III, a mixture of prolactin, ACTH (Nutritional Biochemicals) and cocoa butter. The cocoa butter or hormone mixtures were tamped into the ends of 23 gauge glass tubing, implanted into the ME and fixed rigidly in place by means of dental cement and skull screws. It is estimated that the rats were implanted with about 250—300 ug of each hormone. On the day of implantation (day 0) litter size was reduced to 6 pups, and individual pup weights were recorded on days 1, 3, 5 and 7 after implantation. Some 65 of the rats were killed on the 5th day after implantation, and the mammary glands were removed for weighing and his- tological examination. The other rats were killed 7 days post implantation in order to more adequately ascertain the effects of the implants on the estrous cycle. When the animals were killed the ventral part of the skull was removed to expose the hypothalamus, and the exact location of the implant was determined with a dissecting microscope. When the implants were not found in the anterior ME, the rats were not included in the experiments presented here. The data were subjected to analysis of variance, and significance of differences between means were assessed by Duncan's (1956) multiple range test. Results Prolactin implants elicited a significant decrease in litter weight gains when compared with the controls (Table 2), whereas implants containing both ACTH and pro- lactin resulted in litter weight gains significantly below those of rats given prolactin alone. The effects of the implants on litter weight gains were essentially similar at 5 and 7 days after implantation. The mammary glands from rats 5 days after implanta- tion of prolactin showed a significant decrease in weight as compared to the controls, whereas a combination of pro- lactin and ACTH reduced mammary gland weights even below _ 66 .meEflcm mo .02 u A v .mo. v m uocuo roam Eoum pamHmMMHc mapamoHMflcmHm ohm mpmfluomuomSm uGoMoMMHo mcfl>mn mosam> oen.m AmVOvH.0HNv.H Amoco.hfim.wa Am VUm.MHb.w Hmppoo moooo + meo< .qfluomHonm Amcnma.onmm.a Amvnm.mnm.mm leacnm.mnm.mfi saunas noooo + afioomHOHm Revama.onsm.m Amva.onn.on AHHVMn.NHe.ov “mouse moooo .maoupcoo m o m coflumuquQEfl pmom nofluMpcmamSH #mom coqupcmamEH “mom mmmc m .pz cacao whmp h mood m he made m mmsm w an humeamfi .>¢ cflmo .pz .>< sfimo .pB .>< #GOfiMQHB muonum oumpcmum n wcmwfi mum meHm> .mpnmflmz cameo humEEmE cam mcflmo pnoflws Hoppfla no oocwcHEw cmflome map cfi meom one cauomHOHm mo mquHQEH mo mpommwm .N Mdm<fi 67 the values obtained with prolactin alone. A significant regression was observed in the mammary glands of the pro— lactin implanted rats (Fig. 12) as compared to the controls (Fig. 13). Control lactating rats were all in diestrus, whereas all animals implanted with prolactin or prolactin and ACTH came into estrus and began to cycle. The ovaries of the control rats showed follicular inhibition and large corpora lutea (Fig. 14), whereas the ovaries from both experimental groups showed follicles in different stages of development (Fig. 15). Ova were detected in the ovi— ducts of the cycling rats. Discussion The present study shows that implants of prolactin or prolactin and ACTH into the anterior ME resulted in a significant impairment of lactation. The implanted hor— mones decreased their own secretion apparently by acting on the hypothalamic neurons responsible for secreting PIF and CRF. We recently reported that ME implants of pro- lactin into mature cycling female rats significantly increased PIF content in the hypothalamus, decreased pituitary prolactin concentration and induced involution of the mammary glands (Clemens and Meites, 1967, 1968). ME implants of ACTH were reported to decrease pituitary ACTH secretion (Legori g3 g1., 1965). 68 Fig. 12. Mammary gland from lactating rat implanted with prolactin and cocoa butter. Note atrophy and little milk in alveoli (x70). 69 Fig. 13. Mammary gland from lactating control rat implanted with cocoa butter alone. Note compact alveoli filled with milk (x70). Fig. 14. 70 Ovary from lactating control rat implanted with cocoa butter alone. Note predom- inance of corpora lutea of lactation (x15). 71 Fig. 15. Ovary from lactating rat implanted with prolactin and cocoa butter. Note large preovulatory follicles as well as corpora lutea (x15). 72 In the rat, prolactin stimulates mammary growth and milk secretion, and also maintains the corpora lutea of lactation. The suckling stimulus serves to maintain lactation by evoking release of prolactin and ACTH—adrenal cortical hormones (Meites, 1966). ME implants of prolactin and ACTH apparently counteracted the ability of the suck- ling stimulus to elicit release of these two hormones from the pituitary, and resulted in reduced milk secretion and loss of luteal function. This provides further evidence that prolactin is essential in maintaining milk secretion as well as luteal function in the rat, and that the feed- back of ME implants of prolactin and ACTH are powerful enough to suppress lactation. C. Effects of median eminence implants of prolactin on pregnancygin rats Object Recent experiments by Clemens and Meites (1967, 1968) have demonstrated the existence of a hypothalamic feedback for prolactin, and have been extended by Clemens g5 31. (1968) to show that the prolactin dependent process of lactation can be inhibited by median eminence prolactin implants. In addition to inhibition of lactation they found that the corpora lutea of lactation disappeared as a result of the prolactin implant. Since these implants appear to terminate luteal function, it was of interest 73 to determine the effects of a prolactin implants in the median eminence on pregnancy in the rat. Materials and Methods Adult, female, 3 month old Holtzman rats (Madison, Wisc.) were used in this study. The rats were housed with males until pregnancy had occurred as ascertained by the appearance of spermatozoa in the vaginal smear. The day spermatozoa were detected in the vaginal smear was desig- nated as day 1 of pregnancy. Single glass tubes containing a prolactin (NIH-P-S7)—cocoa butter mixture were stereotaxically implanted into the median eminence of each of one group of pregnant rats. Another group (controls) received implants of glass tubes containing cocoa butter alone into the median eminence. Six rats were implanted with tubes containing prolactin and cocoa butter, and 6 control rats were implanted with cocoa butter alone each day from day 1 of pregnancy until day 8 of pregnancy. A total of 48 rats received implant of the prolactin-cocoa butter mixture, and 48 rats received cocoa butter alone. On the 4th day of pregnancy 12 rats were divided equally into 2 groups. The first group of rats served as controls and received implants of cocoa butter and daily injections of 2 mg of progesterone from day 4 until day 11 (midpregnancy). The second group received implants of the 74 prolactin-cocoa butter mixture on the 4th day of pregnancy and injections of progesterone from day 4 until day 11. Results Table 3 shows that when prolactin was implanted into pregnant rats from days 1—6, pregnancy was terminated in all cases. Laporatomies were performed on day 15 and no fetuses were found in these rats. Control implants of cocoa butter had no effect regardless of the day of preg- nancy on which they were implanted, and at laparotomy both number and size of fetuses appeared to be normal. When prolactin was implanted on the 4th day of pregnancy and 2 mg progesterone were administered daily, pregnancy was maintained. However, when progesterone treatment was withdrawn on day 11, all of the prolactin-implanted rats aborted by day 15 while cocoa butter—implanted control rats remained pregnant (Table 4). Discussion The results of this experiment demonstrate that prolactin secretion is necessary for maintenance of preg— nancy, at least for the first 6 days. The probably deCreased prolactin secretion from the implant after the 5th day did not appear to have any effect on pregnancy. This may be due to secretion of luteotrophin by the newly formed placenta. The prolactin implant appeared to exert 75 m m w = o N. m .— m m m = w m m .- o v m = m m o = m N m = m H w pcmHmEH Mopusn moooo m w w = m h o = o m o = o m o = o e m = o m m = o m o = o H e passage snuonaonm pcmcmwnm ooomHm ucmadafl moan pawfirmoue mGHGHMEwH wow“ .02 mocmqomnm mo who .02 when cfl mosmcmwum so moqmcfifio scheme map SH mpqmamfifi cflpomHoud mo muoommm .m mamme 76 m o v mop Hod ocouopmwooum ma N can ocmamfifl Morgue $0000 0 o v map Hod mcoumhmwooum 08 N can pcmHmEH afloomaonm HH map so ocououmooond HH amp so Goflpmunmamafi pcmfiumoue mo Hmsmuonufls Hopmm pcmcomnm mo mmo ma woo no pcmcmonm mpmu .02 who“ .02 hocmcowud so coflumupmHGflEom ocouopmmmOHm waflmp can wocmcmoum mo amp She may no ooomam mucmHQEH cflpomHoum mo whoommm .v mqmda 77 its effect by causing luteal regression, resulting in a decrease in progesterone production. Evidence to support this hypothesis stems from the observation that daily administration of 2 mg of progesterone maintained pregnancy in rats given implants of prolactin. When the progesterone treatments were discontinued the animals immediately aborted. This indicates that the prolactin implant caused the corpora lutea to regress and resulted in a concomitant decrease in progesterone secretion, while the exogenous progesterone maintained the pregnant state. Since the rats aborted when progesterone was withdrawn after the 11th day, it appears that once the corpora lutea have regressed as a result of the prolactin implant they cannot be reactivated by placental luteotrophin. Thus, it appears that pituitary prolactin secretion is essential during the early stages of pregnancy and that prolactin exerts its effects through the corpus luteum by stimulating proges— terone secretion. II. Hypothalamic Control of Mammary Carcinogenesis and Tumor Growth Object The onset and growth of mammary adenocarcinomas in female rats given carcinogens apparently require both anterior pituitary and ovarian hormones. Thus rats ovar- iectomized or hypophysectomized prior to carcinogen treat- ment develop few or no mammary tumors (Dao, 1964), and 78 ovariectomy or hypophysectomy after mammary tumor development can result in tumor regression (Huggins g3 g1., 1959). Further evidence of the importance of anterior pituitary hormones, particularly prolactin, is provided by experiments showing that single or multiple pituitary transplants can increase the incidence of mammary tumors in mice (Muhlbock and Boot, 1959). Prolonged injections of prolactin into intact female mice also resulted in mammary tumors (Boot g3 g1., 1962), as did transplantation of pituitary "mammotropic" tumors in mice (Haran—Ghera, 1961) and rats (Meites and Sinha, 1966). Placement of lesions in the median eminence (ME) of the tuber cinereum results in enhanced release of pro- lactin and in marked reduction in release of all other anterior pituitary hormones (Meites, g3 g1., 1963). It was of interest therefore, to study the effects of ME lesions on the induction and growth of mammary tumors in intact and ovariectomized rats treated with a carcinogen. Materials and Methods Placement of ME lesions before DMBA Seventy—five virgin female Sprague-Dawley rats (Holtzman, Madison, Wisc.) all uniform in body weight were divided randomly into four groups. At 56 days of age all animals were given a single intravenous injection of 1 m1 of a lipid emulsion containing 5 mg of 7, 79 12-dimethylbenzanthracene (DMBA). Additional treatments were as follows: group I, intact controls, no treatment; group II, intact, bilateral lesions placed in the ME at 50 days of age; group III, ovariectomized at 64 days of age and no further treatment; group IV, placement of bilateral lesions in the ME at 50 days of age and ovariec- tomized at 64 days of age. After carcinogen treatment all animals were examined once weekly for a period of 6 months for development of mammary tumors. A11 ME lesions were produced by passing a direct current of 2 mA for 7 sec through epoxylite coated steel electrodes prepared from insect pins. The ME was located with the aid of a Stoelting sterotaxic instrument and de Groot's atlas of the rat brain. At the time of autopsy the brains were removed, fixed in neutral buffered formalin, sectioned at 10 u and stained with cresyl violet and luxol fast blue. The location of the ME lesions (Fig. 16) was con— firmed. Placement of ME lesions after DMBA Fifty—seven virgin female Sprague—Dawley rats were divided randomly into four groups. At 55 days of age all animals were given a single intravenous injection Of 5 mg of DMBA. Additional treatments were as follows: group I, controls, no treatment; group II, bilateral 51' " Fig. 16. 80 Histological section through the hypothalamus of a rat with a median eminence lesion. Arrow points toward lesion (x25). 81 lesions placed in the ME 75 days after DMBA treatment; group III, ovariectomized 75 days after DMBA treatment; group IV, ovariectomized and bilateral lesions placed in the ME 75 days after DMBA treatment. All animals were examined at O, 10 and 25 days after ovariectomy and/or placement of lesions for palpable mammary tumors. Tumors larger than 1 cm in diameter were measured with a vernier caliper. A11 tumors were removed at the end of each experiment, fixed in Bouin's fluid, sectioned at 4 p and stained with hematoxylin and eosin. They were all ad- enocarcinomas, similar to those described by Huggins g5 gi. (1959) in Sprague-Dawley rats treated with DMBA. Results Placement of ME lesions before DMBA (Table 5) The results show that ME lesions had a definite inhibitory effect on mammary tumor induction. In the intact control rats (group I), 95% of the rats had mammary tumors when the experiment was terminated 6 months after DMBA treatment, whereas intact rats with bilateral lesions in the ME (group II) had only a 30% incidence of mammary tnnnors. The ovariectomized controls (group III) showed a 54% tumor incidence 6 months after DMBA treatment whereas OVariectomized rats with bilateral lesions of the ME (group IV) were completely free of mammary tumors. The aVerage number of tumors per tumor bearing rat was 82 .vm map so ooEHomuom m80powflum>om .om amp co cocoad zoflmwa mzo .mdouo 30mm Mom corm0flocfl we name use wo Hound cumocmpm .Amo. u do hocpo roam Eon“ pamumMMAp Mapcmoflmanoflm ohm mumflhowHoQSm pcmquMHp mcflmmwmmOQ mmsouw to.o.h .om map so anmpmflcflfiom «mzom on o . o Ao v o om omNflEOSUmHHm>o pmcoflmoq .>H DAB Homav omHINOH Omv.0Hmm.N Avmv n ma waNflfiopomHHm>o Houpcoo .HHH UAVHHVOHV hbfilmw Umo.onoH.H Aomv m cm whompcfl pocoflqu .HH him “we v msfinoe nHH.on H.m Ammo Hm mm pounce Honusoo .H Ammmov emu ocflumeIHOEdu muofioh memo mo pamfiommup can dsouw coeuom moqumH \muoaop mo .02 .>< spas menu .02 Hopoa some pom omnmm mo w com .oz #520 Sp flB pcwEumep ouowwn mpws ca ooomam meHme wocwsHEm cmflomE mo muommmm .m mqmfle 83 significantly lower in the lesioned groups than in either of the control groups. The number of rats with tumors and average number of tumors per rat were significantly fewer in the ovariectomized controls (group III) than in the intact controls (group I). Placement of ME lesions after DMBA (Table 6) Prior to placement of the ME lesions most tumors were barely palpable. The ME lesions resulted in rapid growth stimulation and in appearance of a greater number of mammary tumors. At the end of 10 days, average mean diameter of measurable tumors in the lesioned intact group (group II) increased by 7.1 mm as compared to an increase of 3.3 mm in the control rats (group I). Ten days after ovariectomy (group III) the average mean diameter of measurable tumors was reduced by 6.5 mm, whereas there was an increase of 2.1 mm in the average mean diameter of tumors in the lesioned ovariectomized rats (group IV). The lesioned intact rats (group II) showed a 120% increase in number of palpable mammary tumors 10 days after treat- ment, whereas the intact controls (group I) had only a 19% increase in tumors during the same period. The stimulatory action of the ME lesions was still apparent 25 days after the lesions were placed. Ovariectomy alone resulted in significant tumor regression 10 days after surgery, and in even greater 84 .Gmmfi mflnv m0 WmQ .ucmfiumonp fimzo Hmpmm mmwo mm Mamumefixoummm poCOAme H0\osm owNHEOpomHHm>o who? mono map .mom mo mmmp mm on oouopmHQHEUm mus «mzom mm I o.onm.N om + H.Hnm.m H.Hnm.v ma cmNHEOpooHum>o cocoammq .>H mm I m.one.a hN I m.onm.N n.0Ho.v ma poNHEO#ooHum>o Houusoo .HHH mma+ P.HHH.OH 0NH+ m.NH>.b N.Hnm.m NH humane pocofimoq .HH mm + Qw.owm.e ma + Qm.onw.m Q>.onN.m ma pomocfl Hosucoo .H Awe unwEumoHp Awe ucoEumoup HMHpHcH much mo mocospwwnp can moouo omqmro “comm omqmco “comm .oz Hmpoa mane mm mane OH you Mom mHOEou manomfimm mo .02 .>< o mo Homco Hawspmmup .>< .>< pm wow .>< mod mums onEom oHsHmEEH mo mprmeB ocHHmuo can cmHum>o so can apnonsm mo mEHH orp co mocofiuon porno can GHHomHOHm mo mHoommm .m mqmo usofiummnu .>¢ .>¢ mos mumu omNHEOHommmsmomms cH mpanoS mcHHops pom :mHHm>o co monofiuor Monro can cHHOMHOHm mo mpoommm .w mqmge 94 o I QMO mm + 2+ .Ho. v m». o.NHH.mm h pcMHmEH cHHOMHoum moowcmpoonsm .HHH +H.NHN.wm wH HGMHQEH mmHHHHomEH cHHOMHOHm .HH rm.HHo.Hm mH HGMHQEH QHHowHoum .H Amhmov moon ucmfiuwoup can doouo munonsm mo Homco mo .02 pm wow .>¢ when onEmm cH wuuoosm mo Hmmco so wmcosuos Ho mHGMHdEH moooQMHDUQSm Ugo ooqocHEw cchoE HoHHopcm mo muommmm .m momma 95 when compared with rats receiving median eminence implants of prolactin impurities (group II) or given subcutaneous prolactin implants (group III). Discussion The results of this study demonstrate that NIH—P—Bl and NIH-P—S7 prolactins significantly advanced the onset of puberty in rats in 3 separate trials. A direct action on the ovaries can be ruled out because of the inability of prolactin to stimulate the ovary or uterus of the hypophy- sectomized rats. The significant increase in uterine weight in intact rats injected with prolactin suggests that this may have been a consequence of estrogen production by the ovaries. However, the amounts of FSH and LH reported to be present in the 2 prolactin preparation had no effect on the onset of puberty in these rats. Uterine stimulation by prolactin in the intact immature rats and the inability of prolactin injections to stimulate the uterus in hypophysectomized rats suggests that prolactin acted centrally in the immature rats to stimulate production of gonadotrophins by the pituitary. This hypothesis is strengthened by the implantation experi— ment, since median eminence implants of prolactin were found to hasten puberty by an average of 6.6 days. Since subcutaneous implants of pellets containing the same 96 amount of prolactin as the median eminence implants elicited no effect on the onset of puberty, the observed advancement of puberty in the rats with median eminence prolactin implants is believed to be due to the local effect on the hypothalamus. Prolactin implants may stimu- late FSH-RF release by the hypothalamus and result in an increased pituitary FSH production. Recently our labora— tory obtained evidence that median eminence prolactin implants in immature rats resulted in increased FSH secre- tion. It is possible that the decrease in pituitary prolactin level which results from prolactin implants (Clemens and Meites, 1967, 1968) may allow the pituitary to utilize more of its cellular machinery in synthesis of gonadotrophins. Clemens EE.§l°I (1968) recently observed that in postpartum lactating rats with suckling litters, median eminence implants of prolactin induce regression of corpora lutea of lactation and reinitiate follicular growth and cycling. Studies in our laboratory by Minaguchi g£_gl,, (1968) on the changes in pituitary prolactin content and concentration in female rats before and after the onset of puberty showed that prolactin levels remained uniformly low prior to puberty and increased significantly only after the onset of puberty. The increase in prolactin 'after puberty was attributed to estrogen secretion. This 97 suggests that prolactin may have no role in the onset of puberty in the rat except in a negative sense. There are many physiological states in which there appears to be a divergence between prolactin and gonado— trophin secretion. These data suggest that there may be a complex interaction among the anterior pituitary hormones whereby secretion of one hormone may influence the produc— tion and/or release of another. Some investigators who implanted anterior pituitary hormones into the hypothalamus to demonstrate a hypothalamic feedback (Corbin and Cohen, 1966; David gEkg£., 1966) suggested that a specific anterior pituitary hormone influences pituitary production of that hormone only. Our present results suggest that this hypothesis may not be correct, since hypothalamic implants or injection of prolactin appears to stimulate pituitary FSH release in the pre-pubertal rat. IV. A Neuroendocrine Profile of Reproductive Functions in the Old Rat A. Analysis of pituitary prolactin, FSH, LH and hypothalamic FSH-RF and PIF in old constant estrous rats Opjective In general there is agreement in the literature regarding gonadotrophin secretion in the aged animal. Unlike the senile human, in which gonadotrOphin secretion is high, most animals appear to show little change in 98 gonadotrophin secretion as they become old (Korenchevsky, 1961). Since the hormone assay methods used in arriving at these conclusions are now outdated and considered to be unreliable, the question of anterior pituitary hormone secretion in the aged animal once again poses itself. It is well known that reproductive cycling activity is a good index of anterior pituitary function, and in old rats normal estrous cycles rarely occur (Aschheim, 1961; Mandl, 1961). Vaginal smears from old rats indicate 3 different abnormal patterns of cyclic activity. The first is characterized by constant vaginal cornification (constant estrus), the second by repeated pseudopregnancies and the third by no pattern at all (Mandl, 1961). Changes in pituitary gonadotrophin secretion could account for the age changes observed in the estrous cycle and ovary; how- ever Zuckerman (1956) argues that loss of reproductive function is due merely to exhuastion of the ovary. In spite of this argument, ovaries from old anovulatory rats nevertheless contain mature follicles capable of ovulating (Aschheim, 1965). The present study was undertaken to determine the changes in anterior pituitary LH, prolactin and FSH levels and in hypothalamic FSH—RF and PIF content in the senile constant estrous rat. 99 Materials and Methods The animals used in this study were discard Sprague-Dawley breeding rats obtained from Spartan Animal Farms, Haslett, Michigan and from Holtzman Co., Madison, Wisc. Upon arrival in our laboratory the rats were approximately 12—15 months of age. The rats were housed in steel cages, fed the standard lab diet and received oxytetracycline in their drinking water (0.2 mg/ml) once weekly as a prOphylactic measure against respiratory infections which are common in old rats. Experiments were not begun until the rats reached 20 months of age. At this time daily vaginal smears were taken and recorded. When it was ascertained that a rat was not cycling, but in constant estrus for 1 month or more, it was selected for experimentation. Rats found to be in constant estrus were killed by guillotine and the hypothalami and anterior pituitaries were removed. The hypothalami were placed in ice cold 0.1N HCl (0.1 ml per hypothalamus) and stored at —20°C. The pituitaries were weighed individually and stored at -20°C. A few days later the hypothalami were assayed for FSH-RF and PIF, and the pituitaries were assayed for FSH, prolactin and LH by methods described previously. The values obtained for the above hormones were compared with values obtained from 3 month old, normally cycling, female rats killed on the day of estrus. 100 The results of the FSH-RF, FSH and LH assays were analyzed for statistical significance by the methods of Bliss (1952), while the "t" test for paired observations was used for PIF and prolactin. Ovaries and uteri were cleaned, weighed and processed for histological study. Differences in organ weights were analyzed by Student's "t" test. Results Effect of aging on hypothalamic FSH-RF and pituitary FSH levels Anterior pituitary halves incubated with hypothal- amic extracts from aged rats released significantly more FSH into the medium than the corresponding anterior pitu- itary halves incubated with hypothalamic extracts from young control animals killed in estrus (Table 10). This indicates that the FSH-RF content of the hypothalamus increases with age. It can be seen in Table 11 that pitu- itary content and concentration of FSH were also higher in old than in young rats. Effect of aging on hypothalamic PIF and pituitary prolactin and LH levels When the male rat pituitary was incubated with hypothalamic extracts from old and young rats, similar amounts of prolactin were released into the media, indi- cating no difference in hypothalamic PIF content between Young and old rats (Table 12). In contrast Table 12 shows lOl .nonHomHm mo xooaHo .mpHEHH mosmpquoo wmm can cmmzn .mmlmmmImHz mo mocmHm>H5go on mm ommmmumxmm Amm.o I ms.mv mH.m mN.o AONV moonpmm :H mono HHSpm ocsow mNH.o Aom.mH I em.sv om.OH mN.o AONV moms moonpmm ucmumcoo mHHcom oi noisnouHsqu mo oa\msv muouHsqu news mo .02 ommmemH 3mm couchsocH\chme>Hsom one coHHHocoo OHEMHMSHommm usmucoo mmImmm OHEmHmsuomha so oGHom mo Hommmm .OH momma 102 .conHoon Mo xmcho h .mmlmmmImHz mo mucmHm>H5co ms mm tmmmmumxmm .muHEHH mocooncoo wmm was com: Aow.o I oo.mv mm.v Aomv mouumm CH moon HHSUM masow wNH. Awm.m I ms.Hv om.N Acme mums msouumo unopmcoo oHHGmm OK poi humpHopHm Hos mo oE\oov moms mo .oz mmm tam SOHpHpcoo .GOHHMHpsmosoo mmm mumuHsuHm no osHmm mo roommm .HH momma 103 .mo. v mm .mpcoEHummxm omHHmd How ummu so: ochs an nonsmEoo Hoo. v mN .Houum oumocmpm can now: .MHMHHSHHQ HoHkucm u mgH om.onom.m AOHV msuuwo GH OCHHomo ossow mes.OHMH.o AOHV msuumm pneumaoo oHo N Hm.onHo.H ee.onom.H AoHv mourns sH osHHoso moses ov.oHom.H NsN.HHNm.m AOHV monumo ucwumcoo oHo H leg as ooH\oH ma ooH\oH nuns non mass .02 OHHH> gH pommmHmH .ocoo mo .02 .mxm CHHUMHOHQ msv QHHOMHOMQ usopcoo mHm Hie moonumm mo moo one so moms HHoom mono» momHm> mums mooupmm Hampmcoo oHHcom mo usmucoo mHm oHEMHmsuommr pom coHumuucoocoo cHHOMHoum mnmuHouHm .NH mHmo.n.m .mm H cowsi os.HHnomm 0H.Nnoo.oo oso.onmm.o ms nsouuno .mssow no.m~HooH nmm.mnom.mm noH.onon.mH oe msouunm usoumooo .oHo os.senmos on.sHom.mm omm.onmo.MH oH usosomndoosmmm .oHo AmEV .uz Aofiv .HB Amav .uB whom you mo coHHHocou mcHHouD GMHHM>O smumoHouHm mo .02 mSHHmm :H mums HHSUM mono» momHm> mum“ HomcmoHQOUDomm tHo pom mpmu moonumo unnumaoo oHo mo .muanoa quHous can GMHum>o .mumuHsuHm .mH mqmde 108 Fig. 19. Ovary from old constant estrous rat. Note absence of corpora lutea (x15). 109 Fig. 20. Ovary from old rat which exhibited repeated periods of pseudopregnancy. Note predominance of corpora lutea (x15). .GOHmHomHm mo xoocHo .mpHEHH oocmonaoo wmm can Gonzo .HmImHImHz Mo chmHm>Hsoo ms mm commoumxmm 110 ANm.NH I oo.mc Hm.m AmHv msupmm :H maHHomo HHSpm endow msH.o Aom.v I so.Nv so.m AmHv moon mooupmm panamaoo oHo 04 QmAMHMHHsqu Hos mo mE\osv mums Hon Mo :OHHHUGOO EH WHO .02 . monumo mo amp oru no mpHsow mono» momuo> mums msouumw pcmumcoo oHo mo cOHpmnucooooo mg mnmuHoon .vH flames lll aging, which may account for the loss of reproductive function observed at this period of life. Since Aschheim (1965) could reactivate the ovaries of senile rats with gonadotrophin injections, we can no longer accept the pre— vious conclusion that loss of reproductive cycles is due to aging of the ovary. These results suggest that the constant estrous state observed in old rats is a result of a malfunction arising in the hypothalamus, since the FSH-RF content of the hypothalamus was observed to be very high. The lack of ovulation in these animals can be attributed to the very low levels of LH. The low level of LH observed in these constant estrous rats may be due to death of neurons in the brain which are excitatory to LRF production or else possibly due to a lack of the neural transmitter substance responsible for stimulating neurons that secrete LRF. Such a lack of transmitter substance in old rats has been demonstrated by Frolkis (1966). Since these animals are unable to ovulate, they will remain in constant estrus. The high pituitary prolactin content paralleled the hyper— plasia noted in many of the mammary glands. Although no hormone assays were performed on old rats demonstrating repeated periods of pseudopregnancy, the many corpora lutea found in the ovaries of these rats and the well developed 112 mammary glands suggest that prolactin secretion may have been high. The high prolactin levels found in the pituitaries of the old rats is in agreement with the previous observa- tions reported by Meites g2 g£., (1961). The increase in pituitary prolactin is probably due, at least in part, to the estrogen secreted by the follicles of the constant estrous rats. This increased prolactin together with the continuous estrogen secretion by the ovaries may account in part for the increased incidence of mammary tumors found in old rats. More work must be done in this area to determine changes in pituitary hormone secretion in rats with advancing age. In addition it would be helpful to explore possible changes in other age related endocrine changes from the standpoint of their neural control centers. B. Effects of antiandrogen, progesterone, epingphrine, cervical stimulation, resperine and preoptic stimulation on the estrous cycle anddon ovulation in the old constant estrous rat Objective Observations by Aschheim (1964-65) demonstrated that transplantation of young ovaries into old rats did not result in initiation of estrous cycles, providing evidence that the loss of reproductive cycling in old rats did not originate at the ovarian level. Furthermore, 113 Aschheim showed that ovulation and vaginal cycles could be restored in old constant estrous rats by means of LH administration. This suggested that the primary cause of ovarian dysfunction was a change in anterior pituitary hormone secretion. Since anterior pituitary hormone secretion has been shown to be controlled by the hypothalamus, it is possible that changes within the hypothalamus may account for the loss of reproductive cycles. In the present study this possibility was investigated by the use of centrally acting drugs and hormones and direct electrical stimulation of the preoptic area of the hypothalamus. The preoptic hypothalamus was stimulated, since Everett (1966) has shown that this area controls ovulation. In addition, thyroxine, and antiandrogen and castration were used in an attempt to ascertain their effects on reproductive functions in old rats. Materials and Methods Rats similar to those used in the previous study were used in this study. General treatment of the animals was also the same. Progesterone, antiandrogen (Cyproterone acetate, Schering) and epinephrine were dissolved in sesame Oil. Reserpine and thyroxine were injected in aqueous Solutions. Progesterone, 4.0 mg was administered sub- cutaneously daily for 3 days. Other hormones were 114 administered subcutaneously daily for 14 days at the following dosages: antiandrogen, 10 mg; epinephrine, 0.25 mg; reserpine 50 mg and thyroxine at l, 5 and 10 ug. The preoptic hypothalamus was electrolytically stimulated by passing monophasic square wave pulses, 150 u amperes intensity, 1.0 msec. duration and 100 per sec. for 2 minutes through stainless steel electrodes. While stimulation was in progress, current and waveform were continuously monitered on Tektronics 564 oscilloscope. The electrodes were composed of wire 0.05 mm thick and were bipolar. After electrodes were implanted a laparotomy was performed, and the ovaries were examined for corpora 1dtes. No corpora lutea were observed in any of the senile constant estrous rats. One week after stimulation a laparotomy was again performed, and the ovaries were again checked for corpora lutea to determine whether ovulation had occurred. Results The results of all treatments are shown in Table 15. Treatment with progesterone, which is known to act centrally in facilitating ovulation, caused 6 out of 10 animals to ovulate. Epinephrine which is also known to act centrally in many aspects of neuroendocrine func- tion, produced ovulation in 11 out of 22 rats. Unlike progesterone, which only produced a few days of diestrus 115 AmH\mHv monumo unnumcou O NH mHHmo on OH Ho m .H OGonquB cumupmm HMHsmmHHH an poBOHH0m m m mSEMHmsuomms 0Hpmooum Am\mv msuummHo mane 3mm mo cOHumHDEHum Ao\oc nsspmm usounsoo o oH mHHoo ma oH cmoonocMHucd Ao\ov monumo Hawumqoo o o coHHmHSEHpm H60H>Hoo Ao\ov msuumm panamaoo o o mHHmo OE om ochHomom Am\mv monummcm unnumsoo o m mEouooHHm>o Amm\oHc mmHoso Hesse: News HH mm NHHoo ms mm.o mcHusmmsHmm AOH\mv monpmm unopmcoo an m CH mmmo m UOSOHHOH msuummHU mmmo son now MHHmp me a ocoumpwomoum moHomo moonpmm om>ummno soHuwHo>o moon usmfiumwue Coz moon moouvmm unmomcoo whoumH5>ocw .oHo cH mmHomo msouumm can ooHHwH5>o co GHMHQ map mo mono UHHQOmHm Ho coHumHSEHpm pom GOHHMHHmmo .mocoEHos .moono mSOHHm> mo mpoommm .mH mqmge 116 after administration, epinephrine administration caused many normal appearing estrous cycles in 16 out of 22 rats. Treatments which exerted no observable effects were Cyproterone acetate administration, cervical stimula— tion, reserpine treatment and daily administration of various doses of thyroxine. Ovariectomy resulted in a permanent anestrous condition demonstrating that estrogen secretion from the ovaries was responsible for the constant estrus. In 3 out of 5 very old females (2 years old) with atrophic appearing ovaries and a history of 3 months of constant estrus, stimulation of the preoptic area of the hypothalamus caused ovulation. Implantation of electrodes without stimulation did not cause ovulation. Discussion The production of ovulation in old rats by the administration of centrally acting hormones and by elec— trical stimulation of one of the neural centers controlling ovulation, suggests that changes in brain function as a result of aging, possibly in the preoptic area, are at least in part responsible for the senile decline of ovarian function. We cannot entirely rule out aging of the ovary as a contributory factor to its own senescence. However, the fact remains that old ovaries in rats can be reactivated 117 and that ovarian failure is at a level higher than the ovaries. The inability of the antiandrogen (Cyproterone acetate) to counteract the constant estrus observed in these animals eliminated the possibility of androgens causing the constant estrus. The anestrous condition ob— served after ovariectomy showed that the adrenals, which may be capable of secreting estrogens with advancing age (Verzar, 1966) was not responsible for the estrogen secre- tion. In addition it does not appear that decreased thyroid function can be the cause of ovarian failure, since administration of thyroxine in this experiment had no effect. Thus, it is evident that possible changes in other endocrine organs have little to do with the decline of ovarian function. Since the ovary is entirely dependent upon pitu- itary gonadotrophin secretion for its functions, it is easy to conceive how a change in gonadotrophin secretion could severely impair its function. If a loss of gonado- trophin secretion were allowed to continue for a long period of time, severe ovarian atrophy could result. This may explain why not all animals ovulated in response to treatment with progesterone, epinephrine or preoptic Stimulation. It appears from this and the related study in the preceeding experiment that the altered secretion of 118 releasing factors by the hypothalamus and changes in anterior pituitary gonadotrophin secretion are probably due to some fundamental change in neural activity in the brain. It is not at all clear what the nature of this neural change is that accompanies the "menopause" in the rat or if it is located in the hypothalamus or in areas impinging on the hypothalamus. It may be possible that a fundamental neural change takes place with aging similar to that which takes place at puberty. The possibility exists that there may be a decrease in a neural transmitter substance for the release of LRF. Frolkis (1966) has ob— served a decrease in neural transmitter substances with aging. This possibility appears likely since electrical stimulation caused ovulation. Dead neurons could not respond. The exact nature of these neural changes awaits further investigation. GENERAL DISCUSSION In this thesis an attempt was made to determine the role of the hypothalamus in the feedback of prolactin and in various other physiological states. The evidence presented in the implant studies demonstrates that the hypothalamus is a site of pituitary prolactin feedback. The negative feedback effect of a prolactin implant in the hypothalamus is great enough to inhibit many normal physiological processes. The processes of pseudopregnancy, pregnancy, mammary development and lactation have been shown to be either terminated or severely impaired by these prolactin implants. From the data presented in this thesis there is little doubt that this negative feedback action by prolactin on pituitary prolactin secretion arises from increased hypothalamic PIF production. A rise in hypothalamic PIF content was noted in both intact and ovariectomized rats receiving prolactin implants. Since prolactin implants could produce an increase in hypothal- amic PIF production in the absence of the ovaries, it is reasonable to assume that estrogen and progesterone are not needed for the operation of this feedback of prolactin upon the hypothalamus. 119 120 There have been numerous experiments demonstrating that the sex steroids, estrogen, testosterone and proges- terone stimulate prolactin secretion; however none of these hormones has been shown to increase hypothalamic PIF content and on the contrary, they decrease PIF content. There are no reports suggesting that any hormone, with the exception of prolactin can increase hypothalamic PIF content and act as a negative feedback control for prolactin. The results of these studies therefore indicate that prolactin may constitute the major negative feedback control for pro- lactin secretion. Anatomical basis for the short loop feedback is provided by the observations of Torok (1964) who reported that some portal vessels actually carry blood from the pituitary to the hypothalamus. It is easy to see how these vessles could play a role in the hypothalamic feed- back, because they would contain a higher concentration of prolactin in the blood than in any other vessels. This upward flow of portal blood has not yet been confirmed by other workers. We do not yet know whether the prolactin levels in the systemic circulation normally become high enough to exert a negative feedback action upon the hypo- thalamus. Further work is necessary to establish to what extent the normal circulating levels of prolactin act as a negative feedback on pituitary prolactin secretion and 121 interfere with agents which may stimulate pituitary prolactin secretion, i.e. steroids, drugs, etc. Another hypothalamic mechanism which may involve prolactin is that which determines the time of onset of puberty. At this time we can only speculate regarding this mechanism. The current hypothesis is that the hypo— thalamus becomes less sensitive to sex steroid feedback, and this results in greater quantities of gonadotrophin released and leads to the onset of puberty. This is an attractive theory, but it does not reveal the root of the problem. The basic, theoretical question is: what causes the hypothalamus to become less sensitive to sex steroid feedback? The results presented in this thesis indicate that when prolactin is implanted into the hypothalamus of immature female rats, the onset of puberty is significantly advanced. This may explain why many nonspecific mildly stressful stimuli are able to advance puberty, since they can also elicit prolactin secretion. These results also indicate that there is a complex interaction of anterior pituitary hormones at the hypothalamic level, because the prolactin implant also resulted in stimulation of gonado- trOphin secretion. Therefore, it is possible that inter- actions among anterior pituitary hormones at the level of the hypothalamus may be partially responsible for puberty. The‘possibility cannot be ruled out that pituitary hormones may also act directly on the pituitary. 122 Neural changes responsible for the onset of puberty may be similar in some ways to those occurring at the time of the loss of cycling activity. From the experiments reported in this thesis it appears that loss of reproduc- tive functions in rats is due to changes occurring in the hypothalamus. Much work remains to be done in elucidating the mechanisms producing these changes. It appears that these mechanisms are neural in origin since centrally acting drugs caused ovulation in the old rats with constant estrus. The fundamental site of failure-to ovulate in old rats may be located in the preoptic area, since this area appears to control LH secretion and electrical stimulation of this area caused ovulation. Thus, it is apparent that the brain plays a major role in the initiation of reproduc- tive activity in the young immature animal, and in loss of reproductive functions in old animals. The results on the effects of hypothalamic lesions on development and growth of carcinogen induced mammary tumors in rats emphasizes the importance of the CNS in this process. It is probable that the increased release of prolactin resulting from the median eminence lesions was the principal factor influencing mammary tumorogenesis and growth. The fact that transplantation of extra pitu- itaries before and after carcinogen treatment of rats results in essentially similar decreases and increases in 123 mammary tumorogenesis, respectively, is in agreement with this view. Pituitaries transplanted to extra-hypothalamic sites are known to secrete mainly prolactin, and only small amounts of other hormones. The possibility cannot be ex- cluded however, that other effects arising from the median eminence lesions may also influence mammary tumorogenesis. This appears to be particularly evident in the rats given median eminence lesions after tumors were already estab- lished by the carcinogen, when the tumors grew extremely rapidly. Further work with brain lesions is indicated. The inhibitory effects of prolactin implants in the median eminence on mammary development also suggests that this may be a fertile approach for treating mammary tumors. In other words, for such experiments to be suc- cessful, it will be necessary to devise methods to insure that the effects of prolactin implants will be long-lasting. At the present time, the implants produce effects which last up to 10 days at the most. When means are devised for prolonging the prolactin effects, it should be possible to inhibit growth and develOpment of mammary tumors. As an alternative, when purified PIF becomes available, adminis— tration of this neurohormone should similarly result in mammary tumor regression . B I BL IOGRAPHY Aschheim, P. (1964, 1965). Resultats fournis par la greffe heterochrone des ovaries dans l'etude de laregula- tion hypothalamo—hypophyso-ovarienne de la ratte senile. Gerontologia. 10: 65-75. Aschheim, P. (1965). 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The regenerative capacity of ovarian tissue. In: CIBA Foundation Colloquia on Aging. Vol. 2. APPENDIX I CURRICULUM VITAE AND LIST OF PUBLICATIONS DURING GRADUATE STUDIES AT MICHIGAN STATE UNIVERSITY IN WHICH WRITER WAS AUTHOR OR CO-AUTHOR 137 138 CURRICULUM VITAE NAME: Clemens, James Allen DATE OF BIRTH: February 4, 1941 PLACE OF BIRTH: Windsor, Pennsylvania, U.S.A. NATIONALITY: American SEX: Male MARITAL STATUS: Married PRESENT ADDRESS: Department of Physiology ' Michigan State University East Lansing, Michigan. FUTURE ADDRESS: Department of Anatomy and Brain Research Institute University of California at Los Angeles. EDUCATION: Major Field Degree Year Institution of Study B.S. 1963 Pennsylvania State University Zoology - Bio— chemistry M.S. 1965 Pennsylvania State University Zoology Ph.D. 1968 Michigan State University Physiology HONORS: (a) Elected associate member Sigma Xi, 1964. (b) Elected full member Sigma Xi, 1967. (c) Elected member of Phi Sigma Society, 1964. (d) Outstanding graduate student award, Dept. of Physiology, Michigan State University, 1967. (e) Graduate Student Research Award, Sigma Xi, 1967. (f) NIH Postdoctoral Fellowship Award to begin September, 1968. 139 POSITIONS HELD: (a) Teaching Assistant, Department of Zoology, Pennsylvania State University, 1963-1965. (b) Instructor in Anatomy and Physiology, Pennsylvania State University, 1965. (c) National Institutes of Health Trainee, Michigan State University, 1965-1968. 140 Curriculum.Vitae Clemens, James Allen TALKS PRESENTED AT SCIENTIFIC MEETINGS: Meeting Date Topic 1. Ann. Meeting of Mich. 1967 Effects of hypothal- Acad. of Science, amic lesions on DMBA Brooklodge, Michigan induced mammary tumorigenesis in rats. 2. Slst Ann. Meeting of 1967 Inhibition by hypothal- Fed. Am. Soc. Exptl. amic lesions of mammary Biol., Chicago, tumorigenesis induced Illinois by DMBA in rats. 3. Fall Meeting of Am. 1967 Prolactin implant into Physiol. 800., the median eminence Washington, D.C. inhibits pituitary pro- lactin secretion mammary growth and luteal function 4. 52nd Ann. Meeting of 1968 Inhibition of lactation Fed. Am. Soc. Exptl. by median eminence Biol., Atlantic City, implant of prolactin N. J. and ACTH. 5. Co-author of paper 1968 Growth and development presented at 3rd of carcinogen induced Internat. Cong. of mammary tumors as Endocrinol., influenced by hypothal- Mexico City, Mexico amic lesions 6. 24th Internat. Cong. 1968 Termination of pregnancy of Physiological by median eminence Sciences, Washington, implants of prolactin. D.C. 141 Curriculum Vitae Clemens, James Allen RESEARCH PUBLICATIONS (During graduate studies at Michigan State University) 1. Clemens, J. A., and J. Meites. (1968). Inhibition by hypothalamic prolactin implants of prolactin secretion, mammary growth and luteal function. Endocrinology. 82: 878-881. 2. Minaguchi, H., J. Clemens and J. Meites. (1968). Changes in pituitary prolactin levels in rats from weaning to adulthood. Endocrinology. 82: 555-558. 3. Clemens, J. A. and J. Meites. (1967). Prolactin implant into the median eminence inhibits pitu- itary prolactin secretion, mammary growth and luteal function. The Physiologist. 10: 144, (Abstract). 4. Clemens, J. A., C. W. Welsch and J. Meites. (1968). Effects of hypothalamic lesions on incidence and growth of mammary tumors in carcinogen treated rats. Proc. Soc. Exp. Biol. Med. 127: 969-972. 5. Clemens, J. A., and J. Meites. (1967). Inhibition by hypothalamic lesions of mammary tumorigenesis induced by DMBA in rats. Fed. Proc. 26: 366, (Abstract). 6. Clemens, J. A., M. Bar and J. Meites. (1968). Termination of pregnancy by prolactin implants in median eminence of rats. To be presented at XXIV International Congress of Physiological Science, August, 1968. (Abstract in proceedings of Society). 7. Clemens, J. A., M. Sar and J. Meites. (1968). Inhibition of lactation by median eminence implants of prolactin and ACTH. Fed. Proc. 27: 269, (Abstract). 8. Welsch, C. W., J. A. Clemens and J. Meites. (1968). Effects of multiple pituitary homografts or progesterone on DMBA induced mammary tumors in rats. J. Natl. Cancer Inst. in press. 10. 11. 12. 142 Welsch, C. W., J. A. Clemens and J. Meites. (1968). The effects of hypothalamic lesions on growth and development of DMBA induced mammary tumors in rats. Proc. Amer. Assoc. Cancer Res. 9: 76. Welsch, C. W., J. A. Clemens and J. Meites. (1968). Growth and development of carcinogen induced mammary tumors as influenced by hypothalamic lesions. Experta Medica. 157: 203. Welsch, C. W., M. Sar, J. A. Clemens and J. Meites. (1968). Effects of estrogen on pituitary pro— lactin levels of female rats bearing median eminence implants of prolactin. Proc. Soc. Exp. Biol. Med. In Press. Clemens, J. A., H. Minaguchi and J. Meites. (1968). Relation of the local circulation between the ovaries and uterus on the life span of corpora lutea. Proc. Soc. Exp. Biol. Med. 127: 1248— 1251. 143 Curriculum Vitae Clemens, James Allen MANUSCRIPTS IN PREPARATION Clemens, J. A., M. Sar and J. Meites. (1968). Inhibition of lactation by prolactin and ACTH implants in the median eminence. Clemens, J. A., M. Sar and J. Meites. (1968). Termination of pregnancy in rats by prolactin implants in the median eminence. Clemens, J. A., H. Minaguchi, R. Storey, J. Voogt and J. Meites. (1968). Advancement of puberty in rats by prolactin injections and hypothalamic prolactin implants. Voogt, J., J. Clemens and J. Meites. (1968) Stimula- tion of FSH secretion in immature rats by median eminence prolactin implants. Welsch, C., J. Clemens and J. Meites. (1968). The effect of median eminence, preoptic and amygdaloid lesions on development and growth of carcinogen induced mammary tumors in female rats. Clemens, J. A., Y. Amenomori and J. Meites. (1968). Analysis of pituitary prolactin, FSH, LH and hypothalamic FSH-RF and PIF in senile constant estrous rats. Clemens, J. A., Y. Amenomori, T. Jenkins and J. Meites. (1968). Effects of drug and hormone administration and preoptic stimulation on estrous cycles and ovulation in senile constant estrous rats. Clemens, J. A., T. Jenkins, C. Welsch and J. Meites. (1968). Effects of estradiol implants in the median eminence and constant light on carcinogen induced mammary tumors. 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