i | w l M W '1 u i t ' u I 5 WW \ l 3 U \ l I — ___—_7_ k,_____— — ,’_‘____ ‘ | ‘ EFFECYS CF S‘TARVATEQH; THEOURACIL THYRQWECTOMY AND" TWROXINE ON HYPOTHALAMiC CONTENT OF "SQMATOTRQPEN RELEASENG FACTOR" AND PETU‘ETARY GRQWTH HQE‘EMQNE CQRT'ENT The“: for H10 Degree of: Mo 5‘. MCEIEA‘L‘I STA-TE UE‘SWEESETY Nicholm J. Fiel 1:965 THESIS f) LIBRARY Michlgan State University ROOM USE ONLY ABSTRACT EFFECTS OF STARVATION, THIOURACIL,THYROIDECTOMY AND THYROXINE ON HYPOTHALAMIC CONTENT OF "SOMATOTROPIN RELEASING FACTOR" AND PITUITARY GROWTH HORMONE CONTENT by Nicholas J” Fiel The effects of acute starvation on hypothalamic content of “somatotropin releasing factor” (SRF) and on pituitary growth hormone content were studied in rats. After complete food removal for 5-7 days or after ad libitgm feeding, adult male donor rats were decapitated and their hypothalami were removed and extracted with cold 0.1N HClc The neutralized extract was injected into the left common carotid artery of adult male recipient rats and 50 minutes later the rats were sacrificed and their pituitaries were removed. The pituitaries were assayed for somatotropin (STH) by the standard tibia test in young hypophysectomized rats. A logmdose relationship was demonstrated between the dose of hypothalamic extract injected and the amount of STH released by the recipient‘s pituitary, indicating that rat hypothalamic extract contains a factor which stimulates STH release. When the hypothalami of starved rats were compared with hypothalami of ad_libitum fed controls in 2 separate experiments, it was found that starvation markedly reduced Nicholas J. Fiel the hypothalamic content of SRF. The hypothalami of the ad libitum fed rats showed approximately 5 times as much capacity to release pituitary STH as the hypothalami of the starved rats. Starvation also reduced the pituitary content of STH by 40-50%. Similar experiments were conducted using thyroidecto- mized donor rats and donor rats treated with thiouracil and thyroxine. The hypothalamic extracts from these rats were then injected intracarotidly into recipient rats and the pituitaries of the latter assayed for growth hormone as described above. The results showed that, over a two week period, thio‘ uracil and thyroidectomy had no effect on hypothalamic SRF content since the pituitaries of the recipient rats, injected with these hypothalami}had the same amount of STH as the pituitaries of recipient rats injected with control hypothalami. However. a dose of 5 ug of therXine per 100 g body weight. injected over a period of two weeks, approximately doubled the hypothalamic content of SRF as measured by the decrease in the STH content of the pituitaries of the recipient rats. These results suggest that acute starvation in rats results in decreased synthesis and release of SRF by the hypothalamus, and this in turn results in depressed production and release of STH by the pituitary. Also, that thiouracil Nicholas J. Fiel and thyroidectomy, over a two week period, have no effect on the synthesis of hypothalamic SRF, whereas thyroxine, in a dose of 5 ug per 100 g body weight, about doubles the synthe- sis of hypothalamic SRF. EFFECTS OF STARVATION, THIOURACIL,THYROIDECTOMY AND THYROXINE ON HYPOTHALAMIC CONTENT OF "SOMATOTROPIN RELEASING FACTOR" AND PITUITARY GROWTH HORMONE CONTENT BY Nicholas J. Fiel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1965 \“\Ll:~';‘3: Dedicated to Bro. C. C. Chudd, Ph. D. ii TABLE OF CONTENTS INTRODUCT ION O O O O O O O O O O O O O O O O O O 0 REVIEW OF LITERATURE . . . . . . . . . . . . . . . Relation of Hypothalamus to Somatotropin Secretion . . . . . . . . . . . . . . . . . Effects of Reduced Food Intake on Pituitary STH Secretion . . . . . . . . . . . . . . . Effect of Hypo- and Hyperthyroidism on STH Secretion . . . . . . . . . . . . . . . . . EXPERIMENTAL METHODS AND MATERIALS . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . Animals . . . . . . . . . . . . . . . . . . . . Hypothalamic Extracts . . . . . . . . . . . . . Intracarotid Injections . . . . . . . . . . . . Somatotropin Assay and Statistical Analysis . . RESULTS AND DISCUSSION . . . . . . . . . . . . . . Experiment 1: Effectsof Different Doses of Hypothalamic Extract on Pituitary STH Release . . . . . . . . . . . . . . . . . . Experiment 2: Effect of Starvation on Hypotha- lamic SRF Content . . . . . . . . . . . . . Experiment 3: Relative Content of Hypothalamic SRF in Starved and Ad Libitum Fed Rats . . . Experiment 4: Relative Content of STH in Anter- ior Pituitaries of Starved and Ad Libitum Fed Rats . . . . . . . . . . . . . . . . . . Experiment 5: Effect of Thyroxine and Thioura- cil on Hypothalamic SRF Content . . . . . . Experiment 6: Effects of Thyroxine and Thyroid- ectomy on Hypothalamic SRF Content . . . . . Experiment 7: Effects of Different Doses of Thyroxine and Thyroidectomy on Hypothalamic SRF Content . . . . . . . . . . . . . . . . Experiment 8: Effects of Injection of Pituitary Extracts on the Adrenal and Thyroid Glands of Hypophysectomized Assay Rats . . . . . . GENERAL DISCUSSION . . . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . iii Page 10 10 10 11 ll 12 l3 13 15 l7 l9 19 21 23 27 29 34 TABLE LIST OF TABLES Effect of Intracarotid Injections of Different Doses of Hypothalamic Extract on Pituitary STH Content . . . . . . . . . . . . . . . . . . . . Effects of Intracarotid Injections of Hypotha— lamic Extract from Starved and 5g Libitum Fed Rats on Pituitary STH Content . . . . . . . . . Effects of Intracarotid Injections of Hypotha- lamic Extract from Starved and Ad Libitum Fed Rats on Pituitary STH Content . . . . . . . . . Effects of Starvation on Pituitary STH Content . Effects of Intracarotid Injections of Hypotha- lamic Extracts from Control, Thyroxine and Thio- uracil Treated Rats on Pituitary STH Content . . Effects of Intracarotid Injections of Hypotha- lamic Extracts from Intact Control, Thyroidecto- mized and Thyroidectomized Rats Treated with Thyroxine on Pituitary STH Content . . . . . . . Effects of Intracarotid Injections of Hypotha— lamic Extracts from Intact Control, Thyroidecto- mized and Thyroidectomized Rats Treated with Two Different Doses of Thyroxine on Pituitary STH Content . . . . . . . . . . . . . . . . . . . . Effects of Injections of Pituitary Extracts from Recipient Rats on the Adrenal and Thyroid Glands of Hypophysectomized Assay Rats . . . . . . . . iv Page 14 16 18 20 22 24 26 28 INTRODUCTION A great deal of speculation on brain-hypophyseal con- nections has occurred because of the embryological develop- ment of the pituitary gland and its close proximity to the cerebral cortex. The work of Popa and Fielding (1950) first demonstrated the presence of a portal vascular system con- necting the hypothalamus and anterior pituitary. Unfortunate“ ly they erred (Popa and Fielding, 1950, 1955) in describing the direction of blood flow, which they asserted was from the anterior pituitary to the hypothalamus. This was cor- rected by Wislocki g£_al. (1956, 1957, 1958) who clearly showed that the course was actually the reverse, from the hypothalamus down to the anterior lobe of the pituitary gland. This was corroborated by the definitive work of Green and Harris (1947) and Harris (1955), and led to the hypothesis that the hypothalamus secretes neurohumors which traverse the portal vessels and stimulate the anterior pituitary to release its hormones. This initiated a tremendous surge of investigation into the field of neuro-endocrinology, but it was nearly twenty years later before Saffran and Schally (1955) demonstrated, ig_yit£g, that a hypothalamic neurohumor (corticotropin releasing factor, CRF) existed which produced release of an anterior pituitary hormone (adrenocorticotropic hormone, ACTH). .In vitro and in_yiyg_work on CRF (Munsor1_§§_al., 1955; Smelik and D. de Wied, 1958; Grindeland et al., 1962; Casentini g£_a1., 1959) continued, but it was not until later that Vernikos-Danellis (1965, 1964, 1965a, 1965b) demonstrated a physiological role for CRF and elucidated the principal factors involved in stimulating CRF and ACTH synthesis and release. Since 1960, neurolunors have been extracted from the hypothalamus which have been reported to induce release of luteinizing hormone (McCann, 1962), thyrotropin (Campbell §£_§l., 1960; Guillemin, §t_§l., 1965), follicle-stimulating hormone (Igarachi and McCann, 1964; Mittler and Meites, 1964) and growth hormone (Franz §§_al,, 1962; Deuben and Meites, 1964, 1965); a prolactin inhibiting factor has also been extracted (Talwalker §£_§1,, 1965; Pasteels, 1965). The objectives of the work reported here were to attempt to demonstrate a physiological role for the somatotropin releasing factor (SRF) of the hypothalamus. Specifically, the effects of starvation and of hyper~ and hypothyroidism were determined on the concentration of SRF in the hypo— thalamus, and attempts were made to correlate this work with somatotrOpin (STH) release by the anterior pituitary. REVIEW OF LITERATURE Relation of Hypothalamus to Somatotropin Secretion The necessity of an intact hypothalamo-hypophyseal relationship for proper growth has been determined. Greep (1936) showed that when 1-5 pituitaries were transplanted into the sella turcica of rats just hypophysectomized, growth curves of these animals only reached 50—65% of those for similar non—operated controls. He did not determine whether the portal circulation had been re-established. Hertz (1959) got comparable results by transplanting 4 pituitaries beneath the kidney capsule of young hypophy- sectomized rats, Smith (1961) delayed transplantation of the anterior pituitary for 60 and 150 days respectively, and found that he could re—establish growth, although at a reduced rate. The 150 day transplant induced growth equal to only 1/4-1/3 of that in normal rats. Swelheim and WOlthuis (1963) showed that hypophysectomized rats given single pituitary transplants under the kidney capsule, or pituitary implants into the abdominal cavity, showed greater body weight gains than hypophysectomized controls, but significantly less than intact rats. Meites and Kragt (1964) transplanted single pituitaries subcutaneously into young hypophysecto- mized rats (10 days post-operation)and observed body growth equal to 47% of that of intact controls. Hypothalamo—hypophyseal relationships to body growth and STH secretion have also been studied by means of stero- taxic placement of electrolytic lesions in the hypothalamus. Cahane and Cahane (1958) placed lesions in the hypothalamus of rats and observed a reduction of body growth. Later Bogdanove and Lipner (1952) observed substantial reduction in growth rates of rats, together with degranulation of anterior pituitary acidophils, following placement of large . hypothalamic lesions. Reichlin (1960a) demonstrated that lesions involving the median eminence and primary portal plexus of the stalk, significantly reduced the growth rate of rats judged by nose-tail length and tibial width. In a subsequent study Reichlin (1960b) injected replacement doses of thyroxine, testosterone and pitressin into hypothalamic lesioned rats, but growth still did not return to control levels. In another study, Reichlin (1961) showed that massive ventral lesions in the hypothalamus reduced the growth hormone content of the pituitary to 15% of control values when assayed by the standard tibia test of Greenspan et al. (1950). Franz g£_al. (1962) were the first to report the presence of a hypothalamic factor stimulating synthesis of STH by the anterior pituitary. They observed that the addition of acetic acid extracts of hog hypothalami to in— cubated rat pituitary tissue increased the total amount of STH released as compared to controls. However, their results are unconvincing since they did not use standard assay procedures and the statistical significance of their data is questionable. In an independent study Deuben and Meites (1964) showed that anterior pituitaries cultured for 18 days released 167% more STH into the media than was originally present in the fresh anterior pituitary tissue. They also demonstrated that addition of hypothalamic extract to a cul- ture medium increased STH release 4-6 fold. In vitro rem initiation of STH release by rat AP tissue by hypothalamic extract was also reported after such release had ceased (Deuben and Meites, 1965). Franz et al. (1962) claimed that injections of hog hypothalamic extracts into rats over a period of several weeks significantly increased growth rate as compared to uninjected controls. However, this was not confirmed by Sinha and Meites (unpublished) who injected the equivalent of 7 sheep hypothalami daily into mature female rats for a period of ten days° Pecile gtmalf (1964) confirmed the presence of a hypothalamic SRF by intrae carotid injections of neutralized extracts of rat hypothalami into rats and within 10 minutes observed a significant rem duction in STH content of the pituitary. Effects of Reduced Food Intake on Pituitary STH Secretion Inadequate diets or complete starvation have been shown to influence all of the endocrine organs. Numerous investigators have demonstrated ovarian and uterine atrophy as a result of starvation (Jackson, 1917; Papanicolaou and Stockard, 1920; Selye and Collip, 1956; Mulinos and Pomerantz, 1940). Ovarian hypofunction apparently is not the direct result of inanition but is due to the decrease in circulating gonadotropins. This has been proven by re- establishing ovarian function and weight in starved animals by injecting hypophyseal or chorionic gonadatropins (Mulinos .EE_31°' 1959; Drill and Burril, 1944; Rinaldini, 1949). I The effects of starvation are similar in the male animal (Siperstein, 1921; Mason, 1955; Mulinos and Pomerantz, 1941). The thyroid gland has also been shown to atrophy I during starvation (Jackson, 1916; Mulinos and Pomerantz, 1940; Stephens, 1940). Also, Grassie and Turner (1962) demonstrated that a 48% reduction in food intake significantly reduces the thyroxine secretion rate of rats. Alteration in the structure of the thyroid gland (Smith, 1950), increased sensitivity of the gland to thyrotropin (Stephens, 1940; Anderson and Collip, 1954) and reduced uptake of radio- active iodine in underfed or starved rats (Meites, 1949: Meites and Wolterink, 1950), indicate that thyroid atrophy during starvation is caused by a decrease in circulating thyrotropin. The adrenal gland has been shown to increase in size during severe starvation (Selye, 1956; Seyle g£_al., 1940; Quimby, 1948). The adrenal hypertrOphy is mainly in the two inner cortical zones, and histological studies by D'Angelo_§t_al. (1948) suggest there is increased secretory activity. These findings are in agreement with Selye's hypothesis of a "general adaptation syndrome" (1946). In milder undernutrition ACTH and adrenal cortical function show reduced activity, in common with other endocrine glands (Mulinos and Pomerantz, 1941). When immature animals are given an insufficient diet, growth as measured by body weight and skeletal elongation, is decreased. However, a positive nitrogen balance and increased growth rate can be obtained by administering somatotropin (Lee, 1958; Li et al., 1949). Srebnik et al. (1959) demonstrated that when rats were placed on a protein—free diet they lost considerable body weight compared to controls and also showed a significant reduction in pituitary STH content. Here again the reduction in body weight and growth would appear to be the result of decreased pituitary activity. Effect of Hypo— and Hyperthyroidism on STH Secretion Investigation of the relationship of the thyroid gland to pituitary STH has been undertaken by only a few re- searchers. Solomon and Greep (1959) demonstrated that thy- roidectomy significantly reduced the pituitary content of STH of rats 42 days after the operation. However, if they gave 0.7% desiccated thyroid powder beginning 45 days after thyroidectomy body growth was restored together with an increase in pituitary acidophils. Contopoulos et_al. (1958) performed a similar experiment which results in a decrease in pituitary STH content 21 or 56 days after thyroidectomy. Knigge (1958) showed that thyroidectomy reduced the pituitary acidophils almost to zero in 7 days, but the STH content of the pituitary was still normal after 14 days; also, stress increased pituitary STH content and acidophil count 14 days after surgery. But 100 days after thyroidectomy there was essentially no STH in the pituitary and no acidophils, even after the animals were stressed. Eartly and Leblond (1954) hypophysectomized rats and compared their growth rate to normal rats, and rats hypophy- sectomized and given thyroxine. The hypophysectomized and hypophysectomized—thyroidectomized groups showed an immedi- ate leveling off in the growth curve. This experiment was then repeated and two additional groups were added; a thyroidectomized group and a thyroidectomized group given thyroxine. The results were as follows: 1. The hypophysectomized and hypophysectomized- thyroxine groups stopped growingimmediately following surgery. 2. The thyroidectomized group grew for 10 days after surgery and then stOpped. 3. The thyroidectomized group given thyroxine had a normal growth curve. Meites and Kragt (1964) showed that the growth rate of hypophysectomized rats could be returned to 46.6% of normal by implanting a pituitary subcutaneously. When these rats were given thyroxine injections, in addition to the trans— plant, growth was increased to 75.5% of normal. When thy- roxine was injected into hypophysectomized rats not given a pituitary transplant it had no significant effect on body growth. Stasilli g§_al. (1961) treated rats with thiouracil and observed depressed growth rates after 2 weeks and virtual cessation of growth after 6-7 weeks. When thyroxine was administered, growth was re—established. Griesbach gt_§l, (1963) and Asling and Evans (1963) demonstrated that admin- istration of KI, following thyroidectomy, maintained pituit— ary acidophils and some body growth. Presumably this was due to formation of thyroactive compounds elsewhere in the body. Demokidova (1958) thyroidectomized rats, and 35—37 days later gave daily injections of STH and observed normal body growth. The above evidence suggests that thyroxine has a dual action on STH: one is to increase production and release of STH by the anterior pituitary, and the other is to act synergisti— cally with STH to promote body growth. EXPERIMENTAL METHODS AND MATERIALS Introduction The basic experimental procedure was to subject donor rats to various treatments. Following this their hypothalami were removed and injected into recipient rats. The growth hormone content of the pituitaries of the recipient rats was then determined and in this manner the amount of SRF in the donor hypothalamic could be estimated. Animals Mature male rats of the Carworth CFN strain (Carworth Farms, New City, New York) were used in all experiments unless otherwise stated. They were housed in a light (14 hrs. per day) and temperature-controlled (75 i 1°F) room, and fed Wayne Lab Blox pellets. Immature female Sprague-Dawley rats (Hormone Assay Labs, Chicago, Illinois) were hypophysectomized at 26-28 days of age and Shipped to us 10 days later. Three days later they were used for assay of growth hormone by the standard tibia test of Greenspan g£_gl. (1950). Each rat was injected intraperitoneally for 4 days and the rats were killed on the fifth day. The diet of the hypophysectomized rats was supplemented daily with whole milk, bread, orange slices, carrots and sugar cubes. 10 11 Hypothalamic Extracts After decapitation, the hypothalamus of each donor rat was immediately removed and placed in cold 0.1N HCl. The posterior pituitary was discarded and the anterior pituitary was weighed, frozen immediately and stored at -200 C until assayed. On the day the rats were killed, the hypo- thalami were homogenized with a ground glass homogenizer and centrifuged at 20,000 x g for 50 minutes at 40C. The super- natant was decanted and stored in a refrigerator overnight. On the following day, 1N NaOH was added dropwise to the extract until neutralization was achieved, as indicated by a pH meter. The extract was then diluted to the desired volume with 0.85% NaCl. Intracarotid Injections Each recipient rat was anesthetized with ether, injected in the left common carotid artery with 1 ml. of hypothalamic extract and decapitated 50 minutes later. The anterior pituitary was removed immediately, weighed and placed in a freezer at -200C until ready for STH assay. The intracarotid method of assaying hypothalamic extracts was first used by Pecile et al. (1964), and more recently was employed by Vernikos-Danellis (1964) for assaying median eminence extracts for CRF. 12 Somatotropin Assay and Statistical Analysis The anterior pituitary was assayed for STH by the standard tibia test of Greenspan et al. (1950), using a 6-point assay procedure. The pituitary tissue was injected intraperitoneally, as an aqueous suspension, once daily for 4 days. Irwin's analysis, as described by Pugsley (1946), was used to compare the relative potencies of the experi- mental with the control groups. SlOpes and significant differences between elevations of regression lines were determined by analysis of covariance. All statistical analyses were performed with the aid of a 5600 Control Data Corporation Computer. RESULTS AND DISCUSSION Experiment 1 Effects of Different Doses of Hypothalamic Extract on Pituitary STH Release The purposes of this experiment was to determine whether a dose—response relationship could be demonstrated between the concentration of rat hypothalamic extract injec- ted and the amount of pituitary STH released. After two pre- liminary trials, the following experiment was performed. Three groups of 7-week old male rats were used as donors for hypothalamic tissue. The hypothalamic extracts from each group were prepared as previously described, and injected into recipient rats at 3 dose levels: 0.8 hypothalami/ml., 1.6 hypothalami/ml. and 3.2 hypothalami/ml. Intracarotid injections of rat cerebral cortical extract, prepared in the same manner as the hypothalami, were given to a control group of recipient rats in an amount equivalent to the highest dose of hypothalamic extract used. It can be seen (Table I) that the largest dose of hypothalamic extract (Group 4) induced a greater decrease in pituitary STH content than the smaller doses of hypotha- lamic extract (Groups 2 and 3). The cerebral cortical extract (Group 1) apparently elicited no depletion of pituitary STH. There was a linear reduction in pituitary l3 .ao.o V m "d Ucm m cmmBqu ummpIB .UmNHEODUmm>SQOQ>£ u xommmm 14 .xmunoo Hmunmumo u UU.fl mHouucoo mHOma III m I SMmmm Nxomhm m mefinfi m\H m d .HE\E N.m d mflmom m\a m m .HE\m w.a m whamm m\a m m .HE\m m.o N qumupxm Hoo nuflz mfiammm m\fi m m .bodfldflv dabbbcoo 5 mm H S mwmo w mumm Samm< mumm UmpomflcH msouo Adv QDUHB \umm mmmm< mew ucmflmfloom Amv HEmHMEDOQSm Hmflnfle .>¢ \dd mmoa mo .oz mo .02 mo .02 ucmwcou mam humuflsuflm co uomuuxm UHEMmeuom>m mo mmmoo ucmumwwflo mo mcofluothH UHDOHMUMHDCH mo uommmm .a mHQMH 15 STH content in response to the logarithm of the dose of hypothalamic extract injected. The index of precision was 0.50 and the slope (88.7 i.16.1) was significant (p<0.01). These results indicated that (a) the equivalent of 5 rat hypothalami, injected intracarotidly, produced a signifi- cant decrease in pituitary STH content, and (b) this was a suitable dose to employ for studying the effects of starva- tion on hypothalamic SRF content. Experiment 2 Effect of Starvation on Hypothalamic SRF Content Food but not water was removed from 6-week old male rats for 5 days, and similar control rats were fed ag_libitum. On the 6th day the rats were killed and the hypothalami were extracted and injected into recipient 9-week old male rats. In another experiment, begun at the same time, 10-week old male donor rats were starved for 7 days and their hypothalami were similarly assayed for SRF in 9-week old recipient males. The starved rats lost an average of 26 and 60 g per animal, respectively, whereas the §g_libitum fed controls gained an average of 25 and 50 g, reSpectively. It can be seen (Table 2) that the pituitaries of the rats injected with hypothalami from starved rats (Groups 2 and 4) contained more STH and hence elicited a signifi~ cantly greater increase in tibial epiphyseal width than the pituitaries from rats injected with hypothalamic extract 16 .mo.o v m “d Use m cmmBDwQ ”No.0 V m .m .UmNflEouomm>£mom%£ n UGO H COOBDOQ wmm#!B xomhmm .mmHmE UHO xmmz 0 mo Umumamcou d one m mmsouwd mHouucou mflama III m III III an >mmm¢ mxomhm m mdwmom m\a m wma mam Om apoom oz w in: mm flawmwa m\fi m «em dam Om Ummnmaowucou m «fiflmmm m\a m mma ama ma boom oz m .03 dd. oawmma m\a m mma Owd ma meamHOHDCOO H mm H 2 mmmo w mumm >mmm< m m mumm ucmEpmmuH msouo Adv tubes \bmm mmmmd mew Hmcflm HmHquH mo . HMHQHB .>¢ \md wwom mo .02 usmHmB hpom .>< ucmbcoo 38m xnmuasuHm do mumm pom ESDHQHfi.mm pew Um>umum Eoum pomuuxm UHEmHmzuommm mo mcoaubmmcH UHDOHMUMLDCH mo mpommmm mfiflme 17 from the ad libitum fed controls (Groups 1 and 5). This indicates that starvation markedly decreased the capacity of the rat hypothalamus to evoke pituitary STH release. Experiment 5 Relative Content of Hypothalamic SRF in Starved and Ad Libitum Fed Rats To further determine whether starvation could reduce hypothalamic SRF content, an experiment employing a 6-point assay was performed. Food was withheld from 8-week old male rats for 7 days and a comparable control was fed _ad libitum. The starved rats lost an average of 45 g per" rat and the_ad libitum fed controls gained an average of 55 g per rat. Nine week old male rats were used for the intracarotid injections. In this experiment, each assay rat was injected with the same weight of pituitary tissue, rather than with the same fraction of a pituitary. The results in Table 5 show that the pituitaries of the rats injected with hypothalamic extract from the starved rats (Group 2) contained 5 times as much STH as the pituitaries of the rats injected with hypothalamic extract from the _ad libitum fed rats (Group 2). This suggests that acute starvation for 7 days markedly depleted the rat hypo- thalamus of SRF. 18 .8. v d Wm 6cm H cmmzumfl mosmHmMMHU mo mocmoHMchflm .ucmuwmeU SHDCMUHMHcmHm Doc muoz meHH COHmmmummH mnu mo mmmon .UmNHEOuommwsmom%£ u xomxma mHouucou III MHONH in: H Inn in! >mmw< axomhm m QHONN o.m m Hmw hflmmm m.H m mwmnH m.o m de mmH UOOM 02 m OHHMON o.m m Ii. OOH wnmmH m.H m .QHH Um ¢H>MH m.o m «mm mmH bowlmaoupcoo H .XHOMu mm H 2 ma mumm Semm< m m ucwEummwB Qsouw adoo Adv tube: mate a mem Htcfim HmeHdH do & Hmflnfls .>< \bmm mmmmd mo .02 profits Atom .>< \dd mmom Ucm Um>nmum Eoum uomuuxm UHEMHMLDOQ>E Mo mcoHDomHCH pauoumomuuCH mo muommmm bcmudoo mam mumbflsudm co mbmm 6mm adbdnflu mm .m OHQMH 19 Experiment 4 Relative Content of STH in Anterior Pituitaries of Starved and Ag Libitum Fed Rats Nine-week old male rats were starved for 6 days and a comparable control group was fed ad_libitum. The starved rats lost an average of 58 g each and the ag_1ibitum fed controls gained an average of 24 g each. The rats were sacrificed by decapitation, and their anterior pituitaries were removed and frozen until assayed. The same amount of pituitary tissue was injected into each assay rat. The results (Table 4) show a 40% decrease in STH content of the pituitaries from the starved rats as compared to those of the ad libitum fed con- trols on a per mg. basis, and a 50% decrease in STH content on a per pituitary basis. Experiment 5 Effects of Thyroxine and Thiouracil on Hypothalamic SRF Content This experiment was designed to determine if treatment of rats with thyroxine or thiouracil would effect the hyp0w thalamic content of SRF. Nine-week old male rats were used as donors and divided into three groups; a control group, a group injected with 5 ug thyroxine per 100 g body weight per day, and a third group given feed containing 0.1% thiouracil. On the 7th day the rats were sacrificed and their hypothalami were extracted and injected into 9-week old male recipient rats. The control rats gained an average of 22 g each and 20 .HO. v Q ”N Ocm H cmmBqu museumMMHp mo mocmoHMHCOHm .UmNHEouommhsmomxn u xomxma maowucou III HHONH run A In: :11 tau >mmm¢ axomhm m HH©ON Om.m H OO OHOOH m>.H H HHHOH 5.0 H 0.0 OmH HHN boom oz N OHOHN Om.m m II OOH NHNOH mm.H H .QHH .pm OHNOH >.O H m.> Omm mHm bowlmaouusoo H Houp mm H E OE mumm >Mmm< OE O O ucmEummHH moouw icoo Adv nubfiz mate H mew mumm Hmcfim HmHuHcH mo & HMHQHB .>¢ \umm kamwfl mo .02 mo .pz ugOHmz xpom .>< \m< mmoo .uHm .>< Dcmucou 38m >HMDHDDHO co COHum>umum mo muommmm .H mHQwH 21 the two experimental groups each gained an average of 24 g per rat. It can be seen (Table 5) that the pituitaries of the recipient rats injected with the hypothalamic extract from the controls and the thiouracil treated rats (Groups 1 and 5) contained approximately twice as much STH as the pituitaries of the rats injected with the hypothalamic extract from the thyroxine treated rats (Group 2). This suggests that thyroxine increases the hypothalamic content of SRF and that thiouracil has no effect on the content of hypothalamic SRF. Experiment 6 Effects of Thyroxine and Thyroidectomy on Hypothalamic SRF Content Eight-week old CFN female rats were used as donors and divided into three groups: an untreated intact control group, a thyroidectomized group, and a third group which was thy- roidectomized and injected subcutaneously with 5 ug thyroxine per 100 g body weight per day. The rats were killed on the 16th day of the experiment and their hypothalami were ex- tracted and injected into 10-week old CFN female recipient rats. The intact controls gained an average of 28 g per rat, the thyroidectomized rats gained an average of 14 g per rat and the thyroidectomized rats treated with thyroxine gained 18 g per rat. The slopes of the regression lines from the assay of the pituitaries injected with the hypothalamic extracts 22 058.5: u H... ... .mO. V Q ”m UGO N COO3¥OQ OUCOHOMMHU m0 OUCMUHMHCWHm .mcoc um paw H cmmBqu mocmummmHU mo mocmoHMHCOHm .OO. V m "m pew H cmmemQ mocmanMHU mo mocmoHMHCOHm .UmNHEouommhnmomwn u xomxmH maouucoo In: OHNNH nan A III In: In: wmmm< axomxm H OHNOH 0.0 O OO OHONN O.H O Ummm CH HHHNON 0.0 O 0.0 OHN Omm HHomusoflnu RH.O m OHHOOH 0.0 O OH Hfiboam m.fi m smb\.3.m mabomm o.m b H.m omm bmm mooafite m: m m NHHNOH m.O m ucmEummHu OOH NHOON O.H O H.O OHm Omm 0c I mflouucoo H HHHHON O.m O Houu mm H 2 OE mumm wmwm< OE O O ucmaumwne msouw udoo Adv numflz mama H mew mubm Hmcflm HmauHcH Mo & HMHQHB .>< \umm wmwmé mo .02 mo .uz unOsz mwom .>4 \m< mwoo .uflm .>< ucmpcoo mew mumuHSDHm co mumm Umpmmue HHUMpSOHxfi Ucm OConnwze .Houpcou Eoum mwomupxm UHEMHMSpomhm mo mcoHuommmH UHuowmomuusH mo muommmm .m mHQME \ 25 from the controls and thyroidectomized rats (Groups 1 and 2) are significantly different and therefore cannot be compared. However, it can be seen (Table 6) that the pituitaries of the rats injected with the hypothalamic extract from the intact control rats (Group 1) again contained about twice as much STH as the pituitaries of the rats injected with hypo- thalamic extract from thyroidectomized rats treated with thyroxine (Group 5). Also, the pituitaries of the rats in- jected with the hypothalamic extract from the thyroidectomized rats (Group 2) contained approximately twice as much STH as the pituitaries injected with the hypothalamic extract from the thyroidectomized rats treated with thyroxine (Group 5). This experiment therefore compares favorably with the previous one, and again indicates that thyroxine, in a dose of 5 ug per 100 g body weight per day, approximately doubles the hypothalamic content of SRF. Experiment 7 Effects of Different Doses of Thyroxine and Thyroidectomy on Hypothalamic SRF Content One final experiment was performed to substantiate the effect of the absence of the thyroid gland on hypothalamic SRF content, and to test the effect of two different doses of thyroxine on the content of hypothalamic SRF. Nine-week old Sprague-Dawley rats (Spartan Labs, East Lansing, Michigan) were used as donor rats and divided into four groups: 24 O. V m ”m paw N cmm3umn wocmHOMMHU mo mocmonHCOHm O. V O Wm paw H cmm3umfl OUCOHOMMHU mo mocmoHMHCOHm .ucmHOMMHU mum mwmon mmsmown UmummEoo mg pocsmo Mm pew H cmmBDOQ mUCOHOMMHp mo mocmoHMHcOHm .H .m UmNHEOpomm%LQOQ%£ H xomxm m OmNHEOuumpHOH>£u u ompHOHOLBH maoupcoo III HHHNH III 0H III III III Ebmmd mxommm H QHHNOH O.H O >mb\.3.m am NHHEOH o.m H s ooa\.e mam + maflmom O.H m 0.0 OOH AHA HodEHOHEEB m OHNOH O.H m III HHHOOH O.m H OHENN O.H H 0.0 HOH EHH HompHouwnH m OHEEH O.H m ucmEummuH OOH mHmOH O.m H O.H OEH EHH o: I mHouucou HHHOON O.H m powwcH H Houu mm H E OE mumm mmwm< OE O O pcmEpmmHB msouw Icoo Adv SDUHS mmmo H EEO mumm Hmch HMHQHCH O0 H Edfinfle .>< \bmm Hmmmd mo .02 mo .bg ubmflmz Atom .>< \mfl mmoo .pHm .>< DCOHEOU mbm humuHsuHm co mconuwsE SwHB pmpmwuB mumm pmNHEODUOUHOE>LH pcm pmNanuompHouwza .Houpcou Domucm Eouw wuomnuxm UHEmHmspom%m mo mcoHDUOHCH OHuoumomMDCH mo muomwmm . O (11 P“ Q {U [H 25 1. Intact controls. 2. Thyroidectomized controls. 5. Thyroidectomized + 2.5 ug thyroxine per 100 g body weight per day. 4. Thyroidectomized + 5.0 ug thyroxine per 100 g body weight per day. The rats were on experiment for 15 days after thyroidectomy and were then sacrificed. Their hypothalami were extracted and injected into 11—week-old Sprague-Dawley male recipient rats. Each group showed the following average body weight gain: Group 1, 72 g per rat; Group 2, 60 g per rat; Group 5, 51 g per rat; and Group 4, 46 g per rat. In Table 7, it can be seen that there was no signifi— cant difference in the STH content of the pituitaries of the rats injected with hypothalamic extracts from the intact controls, the thyroidectomized group and the thyroidectomized group given 2.5 ug thyroxine per 100 g body weight per day. Also, all these groups had approximately twice as much STH as the pituitaries of the rats injected with the hypothalamic extract from the thyroidectomized rats treated with 5.0 ug thyroxine per 100 g body weight per day. Therefore, this experiment substantiates the previous two. A dose of 5.0 ug thyroxine per 100 g body weight per day approximately doubled the hypothalamic content of SRF, and thyroidectomy had no effect on the hypothalamic content of SRF, at least not within 2 weeks. 26 .H . V m ”H Ucm m paw “H paw m “H ppm H cmmSqu mocmummOHp mo mocmoHMHCOHO .mcoc Hm pew m pew “O p m H Hm pcm H cmmemQ mocmummme mo mocmonHcOHm UmNHEOuUmm>£QOQ>£ n xomxmm pmNHEOpomUHouwcu n UOUHOHOLHH mHonucoo III MHONH III OH III III III >mmm< mxommm m hub HHNOH O.H H \.3.m m OOH OO HHOHH O.H H \H9 m: o.O + OHEOH O.H O H.> OHN OOm HompHou>LH H hmp OHOOH O.H O \.z.m m 00H Om EHEOH O.m O \HH O: m.m + HHHONN O.H H O.H Omm OOH HomUHoumnfi n NHOOH O.H m mHH NHHEOH O.m H . HHNON O.H H O.H OOH mmH HombHOHHOH m OOH NHmOH O.H m #EmEumme OHEOH_ o.m H O.> Hum OOH o: I mHouuqou OHHNN O.H O pumch H Houu _ mm H.z OE mumm wmmmé OE O O HcmEummHB QnOHO Idoo Adv rusz mmmq H OHO mbmm Hmch HmeHcH wo & HOHQHE .>< \umm >mmm¢ mo .02 mo .uz “LOHmz hpom .>4 \m4 mmoo .pHm .>< ucmpcou EEO %umDHfiuHm co mcHx0H>LH mo mmmom usmummmHQ 038 LHHB pmrmwub mumm pmNHEoHomp»onhgh pcm pmNHEouompHoM%LP .Houucow womucH Eoum muommuxm UHEmHmLHOQOm mo wroauommcu OHHoumomuunH mo muommwm .. mHQmF 27 Experiment 8 Effects of Injection of Pituitary Extracts on the Adrenal and Thyroid Glands of Hypophysectomized Assay Rats One final experiment was performed to determine whether the pituitary glands of the recipient rats had any effects on the adrenal and thyroid glands of the hypophysectomized assay rats. This was considered to be of possible importance, since the recipient pituitaries might contain sufficient TSH and ACTH to influence the tibia test (Greenspan et al., 1950). Therefore, when the tibias were removed from the assay rats in Experiment 7, their adrenal and thyroid glands were also removed, trimmed, and weighed to the nearest mg. The identification of the different groups of assay rats in Table 8 refers to the treatment given the donor rats whose hypothalami were injected into the recipient rats and whose pituitaries were in turn injected into the assay rats. As can be seen, only in one instance was there a signifi- cant difference between the organ weights of the nonuinjected assay controls and the assay animals receiving crude pituitary extracts. The thyroid glands were slightly heavier in the assay animals injected with the pituitaries of the recipient rats given hypothalami from the untreated control donor rats. However, in reviewing the results of Experiment 7, this slight difference in thyroid weight apparently had no effect upon the tibia test. 28 HO.O V Q “N paw H mpHouxzu cmmzumfl mocmummep mo wocmonHCOHm .mco: "O paw H “H paw H "O paw H OUHou>nu cmmBHmQ mocmummme mo mocmonHcOHm .mcoc "O Ucm H “H can H “O paw H “N paw H mHmsmem cmmzymfl mocmHmOMHU mo mocmonHcOHm OmNHEOHUmUHOHOQu H ompHouxnhm UmNHEouowmhnmommn n xommma >m©\.3.m m OOH\He m: o.O + O.HHO.O O.HHO.NH O mbocow modOHoumge O >m©\.3.m .m OOH\HH m: O.H + H.HHO.0H O.HHO.NH H muocow momOHOMEEH H H.0Ho.m O.HHH.NH O mybddb modeobHEH O pcmEummuu o: O.HHH.NH 0.0HO.HH O muocop Houucoo m .UmuomHCH won I mHouucoo O.on.m m.oHH.OH m Htmmm axedmm H mm H 2 mm H S mumm >mmm< pcmEpmmHB msouw .OE CH .OE CH mew OUHOHOLB mo mHmcmHU< Mo mo .02 .umz .>< .umz .>< mpmm mammd OwNHEouommwnmomhm mo mpcmHO pHouhne Ocm Hmcmuwm mnu co wumm ucmHmHomm Eoum muomuwxm xumuHspHm mo OCOHuomncH mo muomwmm .O mHQmB GENERAL DISCUSSION The inanition experiments provide evidence that a dietary regimen can alter the content of a neurohumor in the hypothalamus and presumably thereby alter the pituitary con- tent of STH. These data indicate that acute starvation in rats results in marked depletion of hypothalamic content of SRF as well as in a significant reduction in pituitary STH content. Almost 5 times as much growth hormone remained in the pituitaries of the rats injected with hypothalamic extract from starved as in the pituitaries of rats given hypothalamic extract from §g_libitum fed rats. It is possible that the hypothalami of the starved rats were completely depleted of SRF. In confirmation of part of these results, Friedman and Reichlin (1965) recently reported that starvation in rats reduced the pituitary content of STH. A possible alternate explanation of these observations is that the decreased SRF content of the hypothalamus and reduced STH level in the pituitary, are indicative of inn creased release of SRF into the portal vessels and of en- hanced STH release into the blood. This would be consistent with the reports of Roth et al. (1965a, 1965b) that fasting in humans results in increased STH content in the plasma, as determined by a radioimmunological assay procedure. 29 50 However, the work which is believed to most closely corre - qnnd to our experimental conditions is that of Srebnik et a1. (1959), who noted that rats fed a protein-free diet showed a marked loss of body weight, accompanied by a sig- nificant reduction in pituitary and plasma STH levels. These and the related observations already cited (Lee, 1958; Li et al., 1949) suggest that acute starvation in rats leads to reduced STH release into the blood. It has been suggested that the mechanism by which underfeeding depresses the secretion of anterior pituitary hormones is by reducing availability of amino acids and other nutrients in the blood, which directly limits the capacity of the anterior pituitary to synthesize hormones (Ershoff, 1952). However, it has been observed during severe or chronic starvation, that ACTH secretion by the anterior pituitary is actually increased (Quimby, 1948; D'Angelo et al., 1948; Selye, 1956), indicating there is no deficiency of blood precursors for the synthesis or release of this hormone. Also, the capacity of the pituitary of rats to produce greater amounts of gonadotropins as a re— sult of ovariectomy is not impaired by feeding a protein- deficient diet (Srebnik et al., 1961), nor does reduced food intake interfere with release of prolactin and other pituitary hormones in response to the suckling stimulus during postpartum lactation in rats (Ratner and Meites, 1965). These observations suggest that reduced availability vi 'th‘ Enj'h- ‘- ' V r -|I. u 51 of nutrients to the pituitary, as a result of decreased food intake, is probably not the principal mechanism responsible for depressed function of the anterior pituitary. It is more probably due to a failure of hypothalamic stimu- lation of the anterior pituitary. The thyroid experiments suggest that thyroxine can increase the hypothalamic content of SRF and that thiouracil or thyroidectomy have no effect on hypothalamic SRF. In three successive experiments 48-55% less growth hormone re— mained in the pituitaries of the rats injected with hypo~ thalamic extract from rats treated with 5.0 ug thyroxine per 100 g body weight per day as compared to rats given hypothalamic extract from control rats. (A dose of 2.5 ug of thyroxine apparently had no effect on hypothalamic SRF and neither did treatment of rats with thiouracil or thy~ roidectomy. These latter results would appear to be in conflict with the work of Solomon and Greep (1959) and Contopoulos et al. (1958), who noted a reduction of pituitary STH content following thyroidectomy. However, they dew termined the pituitary content of STH 45 and 21 days, respectively, after surgery, whereas the experiment of long- est duration described here was 15 days. Also, Knigge (1958) found the pituitary content of STH in thyroidectomized rats to be at normal levels two weeks after the operation, al~ though there were virtually no pituitary acidophils remain- ing. It would appear then, that if a longer period of time, 52 i.e., 5-4 weeks, were allowed to elapse following thyroid- ectomy, a decrease in both pituitary STH and in hypothalamic SRF might have been found. A possible complication in interpreting the results presented here is the presence of other "releasing factors" in addition to SRF in the hypothalamic extracts. Since acid extracts of rat hypothalamus presumably contain all the factors which act on the anterior pituitary, it is possible that the corticotropin releasing factor and thyrotropin releasing factor in the hypothalami of the experimental donor rats were not present in equal amounts and induced differential release of ACTH and TSH by the pituitaries of the recipient rats. However, the last experiment clearly demonstrated that the adrenal and thyroid glands of the hypophysectomized assay rats were not significantly different in weight except in one instance. Although the thyroid glands were slightly enlarged, there was no apparent change in hypothalamic SRF. The log-dose relationship between the dose of hypo~ thalamic extract injected intracarotidly and the reduction in pituitary growth hormone content, suggests that the rat hypothalamus contains a specific SRF which can induce rapid release of growth hormone. This as well as the recent reports by Pecile §£_al. (1965) and Deuben and Meites (1964), provides ip.yiyg evidence for the existence of a hypothalamic SRF. '13 vitro experiments indicate that rat hypothalamic extracts could induce release of 4-6 times more growth hormone from 55 the rat pituitary during a 6-day culture period than was originally present in fresh rat anterior pituitary (Deuben and Meites, 1964). This suggests that SRF elicits synthesis as well as release of pituitary STH. The data reported here support the concept that environ- mental agents (internal and external) can influence anterior pituitary function by altering production of hypothalamic neurohumors which regulate synthesis and release of anterior pituitary hormones. Pecile et al. (1964) observed that the hypothalami of old rats contained much less SRF than the hypo~ thalami of young rats. The suckling stimulus, estradiol and reserpine were recently reported to deplete the rat hypo- thalamus of prolactin inhibiting factor (Ratner and Meites, 1964), thereby permitting increased synthesis and release of prolactin by the pituitary. Vernikos-Danellis (1964) noted that ether stress followed by sham unilateral adrenalectomy in rats, resulted in a rapid and marked increase in cortico~ tropin releasing factor in median eminence extracts. Preliminary observations suggest there may be an increase in FSH and LH releasing activity in the hypothalamus following ovariectomy (Mittler and Meites, 1964; Piacsek, unpublished). How environmental or other agents can alter synthesis or release of neurohumors by the hypothalamus which regulate anterior pituitary function remains to be clarified. LITERATURE CITED Anderson, E. M. and Collip, J. B. 1954. Studies on the physiology of the thyrotropic hormone of the anterior pituitary. J. Phypiol. 82:11-25. Asling, C. W. and Evans, E. S. 1965. Maintenance of skeletal growth and maturation in thyroidectomized rats by in- jection of iodide. Endocrinology. 72:285-291. Bogdanove, E. M. and Lipner, H. J. 1952. Intestinal ab- sorption of glucose in hypothalamic obesity. Proc. Soc. Exper. Biol. and Med. 81:410n412. Cahane, M. and Cahane, T. 1958. Sur le r61e du diéncephale dans le developpement somatique. Rev. franc. d' endocrinol. 16:181-184. Campbell, H. J., George, R. and Harris, G. W. 1960. The acute effects of injections of thyrotrophic hormone or of electrical stimulation of the hypothalamus on thyroid activity. J. Physiol. 152:527~544. Casentini, S., DePoli, A., Hukovic, S. and Martini, L. 1959. Studies of the control on corticotrophin release. Endocrinology. 64:485—495. Contopoulos, A. N., Simpson, M. E. and Koneff, A. A. 1958. Pituitary function in the thyroidectomized rat. Endocrinology. 65:642-655. D'Angelo, S. A., Gordon, A. S. and Charipper, H. A. 1948. The effect of inanition on the anterior pituitary- adrenocortical interrelationship in the guinea pig. Endocrinology. 42:599-411. Demokidova, N. K. 1958. A trial of growth hormone prepara- tions in rats whose thyroid function has been blocked with methylthiouracil. Bull. Exper. Biol. and Med. 45:747-750. Deuben, R. R. and Meites, J. 1964. Stimulation of pituitary growth hormone release by a hypothalamic extract pip vitro. Endocrinology. 74:408-414. 54 55 Deuben, R. and Meites, J. 1965. Ip_yi££p reinitiation of pituitary somatotropin release by an acid extract of hypothalamus. Proc. Soc. Exper. Biol. and Med. 118:409-412. Drill, V. A. and Burrill, M. W. 1944. Effect of thiamin deficiency and controlled inanition on ovarian func- tion. Endocrinology. 55:187-192. Eartly, H. and Leblond, C. P. 1954. Identification of the ”effects of thyroxine mediated by the hypophysis. ' Endocrinology. 54:2492271. Ershoff, B. H. 1952. Nutrition and the anterior pituitary with special reference to the general adaptation syndrome. Vitamins and Hormones. 10:79-141. Franz, J., Haselbach, C. H. and Libert, O. 1962. Studies of the effect of hypothalamic extracts on somatotrophic pituitary function. Acta Endocrin. 41:556-550. Friedman, R. C. and Reichlin, S. 1965. Growth hormone con- tent of the pituitary gland of starved rats. Endocrinology. 76:787-788. Green, J. D. and Harris, G. W. 1947. The neurovascular link between the neurohypophysis and adenohypophysis. J. Endocrinol. 53156—147. Greenspan, F. S., Li, C. H., Simpson, M. E. and Evans, H. M. 1950. In Hormone Assay. C. W. Emmens (Ed.). Academic Press, New York. Greep, R. O. 1956. Functional pituitary grafts in rats. Proc. Soc. Exper. Biol. and Med. 54:754-755. Griesbach, W. E., Evans, E. S. and Chaikoff, I. L. 1965. Does iodide produce a significant regranulation of pituitary acidophils in thyroidectomized rats? Endocrinology. 72:474-479. Grindeland, R. E., Wherry, F. E. and Anderson, E. 1962. Vasopressin and ACTH Release. Proc. Soc. Exper. Biol. and Med. 110:577—580. Grossie, J. and Turner, C. W. 1962. Thyroxine secretion rates during food restriction in rats. Eggc. Spg. Exper. Biol. and Med. 110:651—655. Guillemin, R., Yamacaki, E., Gard, D. A., Jutisz, M. and Sakiz, E. 1965. lp_vitro secretion of thyrotropin (TSH): stimulation by a hypothalamic peptide (TRF). Endocrinology. 75:564-572. 56 Harris, G. W. 1955. In Neural Control of the Pituitary gland. Arnold, London. Hertz, R. 1959. Growth in the hypophysectomized rat sus~ tained by pituitary grafts. Endocrinology. 65:926m951- Igarashi, M. and McCann, S. M. 1964. A hypothalamic follicle stimulating hormone—releasing factor. Endocrinology. 74:446-452. Jackson, C. M. 1916. Effects of inanition upon the structure of the thyroid and parathyroid glands of the albino rat. Amer. J. Anat. 19:505-552. Jackson, C. M. 1917. Effects of inanition and refeeding upon the growth and structure of the hypophysis in the albino rat. Amer. J. Anat. 21:521-558. Knigge, K. M. 1958. Cytology and growth hormone content of rat's pituitary gland following thyroidectomy and stress. Anat. Record. 150:545-551. Lee, M. O. 1958. Relation of the anterior pituitary growth hormone to protein metabolism. Association Research Nervous and Mental Diseases. 17:195-221. Li, C. H., Simpson, M. E. and Evans, H. M. 1949. Influence of growth and adrenocorticotropic hormones on the body composition of hypophysectomized rats. Endocrinology. 44:71u75. Mason, K. E. 1955. Differences in testis injury and repair after vitamin Amdeficiency, vitamin E-deficiency and inanition. Amer. J. Anat. 52:155-259. McCann, S. M. 1962. A hypothalmic luteinizing~hormonem releasing factor. Amer. J. Physiol. 202:595—400. Meites, J. 1949. Effects of starvation in rats and mice on thyroid secretion rate as indicated by uptake of radi0w active iodine and thiouracil action. J. Animal Science. 8:642. Meites, J. and Kragt, C. L. 1964. Effects of pituitary homotransplant and thyroxine on body and mammary growth in immature hypophysectomized rats. Epdocrinology. 75:565-570. Meites, J. and Wolterink, L. F. 1950. Uptake of radioactive iodine by the thyroids of underfed rats. Science. 111:175m176. 57 Mittler, J. C. and Meites, J. 1964. IQ vitro stimulation of pituitary follicle-stimulating—hormone release by hypo~ thalamic extract. Proc. Soc. Exper. Biol. and Med. 117:509-515. Mulinos, M. G. and Pomerantz, L. 1940. Pseudo-hypophysectomy. A condition resembling hypophysectomy produced by ma1~ nutrition. J. Nutrition. 19:495~504. Mulinos, M. G. and Pomerantz, L. 1941. The reproductive organs in malnutrition. Endocrinology. 29:267~275. Mulinos, M. G., Pomerantz, L., Smelzer, J. and Kurzrok, R. 1959. Estrus-inhibiting effects of inanition. £399. Soc. Exper. Biol. and Med. 40:79-85. Munson, P. L. and Briggs, F. N. 1955. The mechanism of stimulation of ACTH secretion. Rec. Prog. Hormone Res. 11:85-117. Papanicolaou, G. N. and Stockard, C. R. 1920. Effect of underfeeding on ovulation and the oestrus rhythm in guinea~pigs. Proc. Soc. Exper. Biol. and Med. 17: 145-144. Pasteels, J. L. 1965. Recherches morphologiques et experi— mentales sur la secretion de prolactine. Extrait des Archives de Biologie. 74:440w555. Pecile, A., Muller, E., Falconi, G. and Martini, L. 1964. Growth hormone releasing activity of hypothalamic extracts at different ages. 46th Meeting, The Endocrine Society. 152. Popa, G. and Fielding, U. 1950. A portal circulation from the pituitary to the hypothalamic region. J;_Apag. 65:88-91. Popa, G. and Fielding, U. 1955. HypOphysio-portal vessels and their colloid accompaniment. J. Anat. 67:227-252. Pugsley, L. I. 1946. The application of the principles of statistical analysis to the biological assay of hor- mones. Endocrinology. 59:161a176. Quimby, F. H. 1948. Organ weights of rats receiving hormone supplements during recovery from chronic starvation. Endocrinology. 42:265‘272. Ratner, A. and Meites, J. 1965. Effects of hormone adminiSH tration on milk production of underfed rats. Amer. J. Physiol. 204:268—270. 58 Ratner, A. and Meites, J. 1964. Depletion of prolactin- inhibiting activity of rat hypothalamus by estradiol or suckling stimulus. Endocrinology. 75:577-582. Reichlin, S. 1960. Thyroid function, body temperature regum lation and growth in rats with hypothalamic lesions. Endocrinology. 66:540-554. Reichlin, S. 1960. Growth and the hypothalamus. Endocrinology. 67:760-775. Reichlin, S. 1961. Growth hormone content of pituitaries from rats with hypothalamic lesions. Endocrinology. 69:225-250. Rinaldini, L. M. 1949. Effect of chronic inanition on the gonadotrophic content of the pituitary gland. J. Endocrinology. 6:54-65. Roth, J., Glick, S. M., Yalow, R. S. and Berson, S. A. 1965. Secretion of human growth hormone: physiologic and experimental modification. Metab. Clin. and Exper. 12:577-579. Roth, J., Glock, S. M., Yalow, R. S. and Berson, S. A. 1965. Hypoglycemia: a potent stimulus to secretion of growth hormone. Science. 140:987-988. Saffran, M. and Schally, A. V. 1955. The release of corticoH trOphin by anterior pituitary tissue i3 vitro. Canadian J. Biochem. Physiol. 55:408-415. Selye, H. 1956. Thymus and adrenals in the response of the organism to injuries and intoxication. British J. Exper. Path. 17:254-248. Selye, H. 1946. The general adaptation syndrome and the diseases of adaptation. J. Clin. Endocrin. 6:117—250. Selye, H. and Collip, J. B. 1956. Fundamental factors in the interpretation of stimuli influencing endocrine glands. Endocrinology. 20:667~672. Selye, H., Dosne, C., Bassett, L. and Whittaker, J. 1940. On the therapeutic value of adrenal cortical hormones in traumatic shock and allied conditions. Canadiap Med. Assoc. J. 45:1-8. Siperstein, D. M. 1921. The effects of acute and chronic inanition upon the development and structure of the testis in the albino rat. Anat. Record. 20:555—581. 59 Smelik, P. G. and Wied, D. de. 1958. Corticotropin-releas~ ing action of adrenaline, serotonin and pitressin. Experientia. 14:17-18. Smith, P. E. 1950. Hypophysectomy and a replacement therapy in the rat. Amer. J. Anat. 45:205-274. Smith, P. E. 1961. Postponed homotransplants of the hypo- physis into the region of the median eminence in hypophysectomized male rats. Endocrinology. 68:150-145. Solomon, J. and Greep, R. O. 1959. The effects of altera- tions in thyroid function on the pituitary growth horn mone content and acidophilcytology. Endocrinology. 65:158-164. Srebnik, H. H., Nelson, M. M. and Simpson, M. E. 1959. Reduced growth hormone content in anterior pituitaries of rats on protein-free diets. Proc. Soc. Exper. Biol. and Med. 101:97-99. Srebnik, H. H., Nelson, M. M. and Simpson, M. E. 1961. Follicle-stimulating hormone (FSH) and interstitialm cell~stimulating hormone (ICSH) in pituitary and plasma of intact and ovariectomized proteinmdeficient rats. Endocrinology. 68:517-526. Stasilli, N. R., Kroc, R. L. and Nemith, P. J. 1961. Effects of thyroxine and triiodothyro-propionic acid on growth of thiouracil-treated rats. Endocrinology. 68:1068n 1071. ‘ Stephens, D. J. 1940. The effect of the thyrotropic princi- ple of the anterior pituitary on the thyroid of the undernourished quinea pig. Endocrinology. 26:485-492. Swelheim, T. and Wolthuis, O. L. 1965. On the growth hormone production by pituitary transplants. Acta Physiol. et Pharm Neerlandica. 11:545-549. Talwaker, P. K., Ratner, A. and Meites, J. 1965. 'Ip vitro inhibition of pituitary prolactin synthesis and release by hypothalamic extract. Amer. J. Physiol. 2052215- 218. Vernikos-Danellis, J. 1965. Effect of acute stress on the pituitary gland: changes in blood and pituitary ACTH concentration. Endocrinolpgy. 72:574-520. Vernikos-Danellis, J. 1964. Estimation of corticotropinw releasing activity of rat hypothalamus and neuro~ hypophysis before and after stress. Endocrinology. 75:514-520. 40 Vernikos-Danellis, J. 1965. Effect of stress, adrenalectomy, hypophysectomy and hydrocortisone on the corticotropin- releasing activity of rat median eminence. Endocrinology. 76:122-126. Vernikos-Danellis, J. 1965. Effect of rat median eminence extracts on pituitary ACTH content in normal and adrenalectomized rats. Endocrinology. 76:240-245. Wislocki, G. B. 1957. The vascular supply of the hypophysis cerebri of the cat. Anat. Record. 69:561-587. Wislocki, G. B. 1958. Further observations on the blood supply of the hypophysis of the rhesus monkey. Anat. Record. 72:157-150. Wislocki, G. B. and King, L. S. 1956. The permeability of the hypophysis and the hypothalamus to vital dyes, with a study of the hypOphyseal vascular supply. 58:421-472. Amer. J. Anat. MICHIGAN STATE UNIVERSITY LI JIHI Hlll lllllll BRARIES 1 3 1293 03056 444