PHYSIOLOGICAL CHARACTERIZATION OF PINEAL ANTIGONADOTROPIN

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Authors Wray, Mary Jane Matthews

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University Microfilms International 300 North Zeeb Road Ann Arbor. Michigan 48106 USA St. John's Road. Tyler's Green High Wycombe, Bucks, England HP10 8HR 7901725

WRAY. MARY JANE MATTHEWS PHYSIOLOGICAL CHARACTERIZATION OF PINEAL ANTIGONADOTROPIN.

UNIVERSITY OF ARIZONA, PH.D., 1978

University Microfilms International, Ann Arbor, Michigan 48106 PHYSIOLOGICAL CHARACTEREZATION OF

PINEAL ANTIGONADOTROPIN

by

Mary Jane Matthews Wray

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF ANATOMY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 7 8 THE UNIVERSITY OF ARIZONA.

GRADUATE COLLEGE

1 hereby reconmend that this dissertation prepared under my direction by Mary Jane Wray entitled Physiological Characterization, of

Pineal Antigonadotropin be accepted as fulfilling the dissertation requirement for the degree of Doctor of Philosophy

yi,'?7r )issartation Director Dat

As members of the Final Examination Committee, we certify that we have read this dissertation and agree that it may be presented for final defense.

ft 6-C^ / T jruU, /°/7-2 zr('SU4 ms;

(f-19 ^3 C\J;, IT7X

Final approval and acceptance of this dissertation Is contingent on the candidate's adequate performance and defense thereof at the final oral examination. STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission/ provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: . . rr\ oJOcUnw^ — a* ACKNOWLEDGMENTS

My warmest and deepest appreciation is extended to Dr. Bryant

Benson who has been my close friend and colleague for the eight years

that I either directly or indirectly have been associated with this

project. He has contributed more than any other person to my intel­

lectual as well as my personal growth and I will always remain

indebted to him. I also thank Dr. Alvin Rodin who served as my major

advisor for my master's work. I am grateful to the members of my

committee: Dr. Philip Krutzsch, Dr. Jay Angevine, Jr., Dr. Richard

Stouffer, and Dr. Eldon Braun. These men were all kind and patient with me in this long endeavor.

The other members of our laboratory also made important contributions to this project. I wish to thank especially Debbie and

Bruce Richardson, Carla LaPorte, Peggy Krasovich, Carmen Bria, and Dr.

Brent Larsen. I also am very grateful to April McFarland, Toni Foster, and Lois Bucherer for their help with the typing.

For both their helpful advice and support I also thank Dr. Mac

Hadley of the Department of Biology and Dr. Victor Hruby of the

Department of Chemistry.

I am thankful to my mother and father, Vera and Buck Matthews, in all that X do. Lastly I thank my husband, Vernon, for all the new horizons that he opened in my now rich and meaningful life.

iii TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS vii

LIST OF TABLES ix

LIST OF ABBREVIATIONS X

ABSTRACT xiii

HISTORICAL REVIEW * 1

Introduction 1 The Effects of Light on Reproduction 4 Effects on Reproduction 4 Effects on the Pineal Gland 10 Diurnal Pineal Rhythms 11 Non-Reproductive Effects of Light 12 Pinealectomy 13 Reproductive Effects 14 Non-Reproductive Effects on Endocrine Organs 19 Effects of Extracts . 22 Pineal Substances 29 Indoles and Melatonin 29 Pineal Enzymes 37 Pineal Peptides 42 Morphological Correlation 52 Pineal Incubation 60 Human Pineal Relations 62 Conclusion 64

STATEMENT OF THE PROBLEM 65

Site of Action of Pineal Antigonadotropin 65 Effects of Pineal Antigonadotropin in the Hamster 66 Studies on the Effects of Arginine Vasotocin 68

MATERIALS AND METHODS 69

Extraction of Pineal Glands 69 Organic Solvent Extraction 69 Aqueous Extraction 70 Radioimmunoassay (RIA) Procedures 70 Testosterone RIA 70 Luteinizing Hormone and Prolactin RIA 72

iv V

TABLE OF CONTENTS—Continued

Page

Site of Action of Pineal Antigonadotropin 76 Dose Response of Immature Mouse Ovaries to Pregnant Mare Serum Gonadotropin 76 Dose Response of Immature Mouse Ventral Prostates to Human Chorionic Gonadotropin 79 Effect of Pineal Extract on Pregnant Mare Serum Gonadotropin Stimulation of Ovaries in Immature Female Mice 79 Effect of Pineal Extract on Human Chorionic Gonadotropin Stimulation in Immature Male Mice .... 80 Dose Response of Castrate Mouse Ventral Prostates to Prolactin 81 Effect of Pineal Extract on Luteinizing Hormone Release from Luteinizing Hormone-Releasing Hormone-Stimulated Rat Anterior Pituitary Glands In Vitro 81 Effect of Microinjection of Pineal Extract into the Rat Pituitary and Median Eminence on Luteinizing Hormone and Prolactin 82 Effects of Pineal Antigonadotropin in the Hamster 84 Effect of Short-Term Treatment of Male Hamsters .... 84 Effects of Long-Term Treatment of Male Hamsters .... 85 Effect of Extract on the 24-Hour Post-Castration Rise of Serum Luteinizing Hormone in Male Hamsters . . 86 Effect of Pineal Extract on the Luteinizing Hormone in Long-Term Castrate Hamsters 86 Effect of Pineal Extract on Luteinizing Hormone Surge in Female Hamsters 86 Effect of Prolactin and Anterior Pituitary Grafts on Gonadal Atrophy in Blinded Male Hamsters 87 Studies on the Effect of Arginine Vasotocin 88 Statistical Methods 89

RESULTS 90

Site of Action of Pineal Antigonadotropin 90 Direct Gonadal Effects of Pineal Antigonadotropin ... 90 Effect on Pituitary Glands In Vitro 90 Effect of Microinjection of Pineal Extract 97 Dose Response of Castrate Mouse Ventral Prostate to Prolactin 97 Effect of Pineal Antigonadotropin in the Hamster 97 Effect of Short-Term Treatment on Male Hamsters .... 97 Effect of Long-Term Treatment on Male Hamsters 100 Effect on the 24-Hour Post-Castration Serum Luteinizing Hormone Rise in Male Hamsters 107 vi

TABLE OF CONTENTS—Continued

Page

Effect of Pineal Extract on Long-Term Castrate Hamsters 107 Effect of Pineal Extract on Luteinizing Hormone Surge in Female Hamsters 107 Effect of Prolactin and Anterior Pituitary Grafts on Gonadal Atrophy in Blinded Male Hamsters 107 Effects of Arginine Vasotocin 112

DISCUSSION 122

Site of Action of Pineal Antigonadotropin 123 Effect of Pineal Antigonadotropin in the Hamster 125 Studies on the Effects of AVT 130

CONCLUSION 133

LITERATURE CITED 134 LIST OF ILLUSTRATIONS

Figure Page

1. Standard curve for testosterone radioimmunoassay 73

2. Iodination of LH for radioimmunoassay 75

3. Standard curve for LH radioimmunoassay 77

4. Standard curve for prolactin radioimmunoassay 78

5. Dose response of immature mouse ovaries to PMS 91

6. Dose response of immature mouse ventral prostate to HCG . 92

7. Effect of pineal extract on ventral prostate in mature mice 93

8. Lack of an effect of pineal extract on PMS stimulation of immature mouse ovaries 94

9. Lack of an effect of pineal extract on HCG stimulation of immature mouse ventral prostates 95

10. Absence of an effect of pineal extract on LH output from LHRH-stimulated rat anterior pituitaries 96

11. Effect of pineal extract on ventral prostate weight in hamsters 101

12. Effect of pineal extract on serum testosterone in hamsters 103

13. Effect of long-term treatment with pineal extract on ventral prostate weight in intact and blinded hamsters 104

14. Lack of an effect of pineal extract on the LH surge in female hamsters 110

15. Effect of either anterior pituitary (pit) or kidney homografts on testicular weights in blinded hamsters . . 113

16. Effect of either anterior pituitary or kidney homografts on ventral prostate and seminal vesicle weights in blinded hamsters 114

vii viii

LIST OF ILLUSTRATIONS—Continued

Figure Page

17. Lack of an effect of AVT on ventral prostate weight in adult mice 117

IS. Lack of an effect of bovine pineal stalk extract on ventral prostate weight 121 LIST OF TABLES

Table Page

1. Effect of pineal extracts on rat LH after intra- pituitary and median eminence microinjection under ether anesthesia 98

2. Effect of testosterone plus prolactin on ventral prostate weight in castrate mice 99

3. Effect of pineal extracts on seminal vesicle and testicular weight in hamsters 102

4. Effect of pineal extracts on seminal vesicle and testicular weight in blinded and intact hamsters .... 105

5. Effect of pineal extracts on LH in intact and blinded male hamsters 106

6. Effect of pineal extracts on the 24-hour post- castration rise in serum LH in rats and hamsters .... 108

7. Effect of pineal extract on LH in long-term castrate hamsters 109

8. Effect of exogenous prolactin on gonad and accessory organ weight in blinded hamsters Ill

9. Effect of either anterior pituitary or kidney homografts on testicular and accessory organ weights in blinded hamsters 115

10. Effect of prolactin injection and anterior pituitary homografts on prolactin in the blinded hamster 116

11. Effect of AVT in immature female mice 119

12. Effect of AVT in immature male mice 120

ix LIST OF ABBREVIATIONS

ACTH adrenocorticotropic hormone

AMP adenosine monophosphate

AVT arginine vasotocin

BOBX bilateral olfactory bulbectomy cAMP 3*,5'-cyclic adenosine monophosphate

°C degrees Celsius cm centimeter

COH compensatory ovarian hypertrophy cmp counts per minute

CSF cerebrospinal fluid db-cAMP 3",5'-dibutyryl cyclic adenosine monophosphate

DNA deoxyribonucleic acid

ER endoplasmic reticulum

Fig figure

FSH follicle-stimulating hormone gm gram

HCG human chorionic gonadotropin

HIOMT hydroxyindole-O-methyl transferase

I iodide ip intraperitoneally

IU International Unit

Km Michaelis-Menton constant

KRB Krebs-Ringer buffer

x xi

LIST OF ABBREVIATIONS—Continued

L:D light to dark ratio

LH luteinizing hormone

LHRH luteinizing hormone-releasing hormone

M molar

MB median eminence

MFB medial forebrain bundle

mg milligram

min minute

ml milliliter

MW molecular weight

yl microliter

pg microgram

N normal

n number of animals per experimental group

NAD nicotinamide-adenosine-dinucleotide

NADP phosphonicotinamide-adenosine-dinucleotide

NAT N-acetyltransferase

NE norepinephrine ng nanogram

nm nanometer

NS not significant at 5% probability level

P phosphorus

PAG pineal antigonadotropin pg picograms

PMS pregnant mare serum gonadotropin Xll

LIST OF ABBREVIATIONS—Continued

RIA radioimmunoassay

RNA ribonucleic acid sc subcutaneously

SCG superior cervical ganglion

SCGX bilateral superior cervical ganglionectomy

TLC thin layer chromatography

TRH thyrotropin-releasing hormone

UM05R UM05 residue

UO unilateral ovariectomy wt weight

X g times gravity ABSTRACT

Pineal extracts contain antigonadotropic activity which can be separated from melatonin by gel filtration. Since this activity is eluted in a ninhydrin-positive peak on sephadex gels and is inactivated by proteolytic enzymes, it has been proposed that the pineal gland contains a small peptide hormone called pineal antigonadotropin (PAG).

This non-melatonin pineal factor has been found in bovine, ovine, rat, and human glands. Observed effects of its administration have been reduction of ovarian and ventral prostate weight in mature mice, reduction of gonad and accessory organ weight in immature mice, inhibi­ tion of the compensatory ovarian hypertrophy which follows unilateral ovariectomy in mice, diminution of the post-castration rise in luteinizing hormone (LH) which occurs in rats and reduction in fertility of mice and rats. PAG was further characterized in this study by investigation of its site of action and studying its effects upon the pineal-mediated control of reproduction in the hamster.

The site of action of PAG was investigated by testing the effects of extracts on the gonad, the pituitary and the hypothalamus.

Extracts containing PAG, which were effective in reducing ventral prostate weight in mature mice, did not reduce the stimulation produced by exogenous gonadotropins in immature mice. Extracts containing PAG were ineffective in reducing LH secretion from the rat pituitary gland.

Pineal extracts have recently been shown to possess factors that both stimulate and inhibit prolactin release from the rat pituitary in

xiii xiv vitro, but the portion of the extract containing PAG was ineffective on prolactin release.

Since it was felt that PAG did not act on the gonad or pituitary directly, the hypothesis was considered that PAG works in the hypothalamus. Accordingly, extracts containing PAG were microinjected into the median eminence or pituitary of rats. Most of these experi­ ments gave negative results, but they are very difficult to perform.

Nevertheless it yas found that PAG significemtly increased the LH in the pituitary gland when injected into the median eminence, but not the pituitary. These data suggest a hypothalamic site of action but other studies must be done before this question can be answered.

Hamsters blinded or placed in less than 12.5 hours of lighting a day undergo gonadal atrophy completely preventable by prior pinealectomy. We began our studies with the working hypothesis that pineal extracts would reduce LH in this species and thus terminate sexual function. It was observed that pineal extracts containing PAG reduced the ventral prostate weights in hamsters just as is seen in the mouse. There was also an inconsistent but significant reduction in serum testosterone. In contrast, LH tended to be increased (definitely not reduced) in the sera of treated hamsters. And astounding: a pineal extract which significantly reduced the post-castration rise in LH 24 hours after surgery in rats significantly increased this surge in hamsters1 It was concluded that PAG did not work through a reduction of LH in the hamster.

Since it has recently been observed that intracarotid injection of PAG into rats reduced prolactin within one hour, we investigated the XV effect of prolactin on gonadal atrophy in hamsters. Both injection of prolactin and prolactin output from pituitary homografts placed beneath the kidney capsule maintained gonads and accessory organs in a func­ tional state in blinded male hamsters. It was concluded that the effects of the pineal gland in the hamster may be mediated through a reduction in prolactin.

These results are as important to interpreting prolactin's role in reproduction as they are to evaluating the function of the pineal gland. There are many questions demanding clarification, but these studies have brought us closer to an understanding of pineal gland function. HISTORICAL REVIEW

Introduction

In many large mammals, including man, the pineal body or epiphysis cerebri is a structure which lies, near the habenular and posterior commissure. In the rat, mouse, hamster, and certain other rodents, the pineal gland becomes attached to the meninges of the dorsal cranium and lies contiguous with the skull plate immediately inferior to the lambda (Quay 1974a). Although the stalk of the gland is in close proximity to the third ventricle, its chief cells or pinealocytes do not appear to contact the cerebrospinal fluid (CSF).

Paradoxically, pinealocytes have been described by some authors as being similar to certain neurons with processes that contact the CSF

(Vigh and Vigh-Teichmann 1974, 1975). Nerve fibers from the posterior and habenular commissures enter the glandular pineal body but appear to exit again without evidence of synapses (Quay 1974a). The sensory input into the pineal gland appears to begin in the retina and branches from the optic tract anterior to the chiasm as the accessory optic tract (Ariens-Kappers 1960). These fibers enter the hypothalamus through the medial forebrain bundle (MFB) and innervate their terminal nuclei in the midbrain tegmentum. Certain of these fibers also apparently synapse in the suprachiasmatic nucleus which may act as a control center for certain biological rhythms (Moore and Eichler 1976).

Fibers originating from the above mentioned nuclei make their way by an unknown route to the superior cervical ganglia (SCG). It is assumed

1 2 that the nerve fibers travel in the spinal cord and synapse with certain neuronal somata located in the intermediolateral cell column whose axons reach the SCG. From the SCG the fibers enter the cranium again by way of the carotid canal along with the sympathetic fibers of the carotid plexus. Collectively they travel along the tentorium cerebelli to the pineal gland as the nervi conarii which enter the posterior portion of the gland. From the carotid plexus, sympathetic fibers also follow blood vessels into the pineal parenchyma (Ariens-Kappers 1960).

The arterial supply to the pineal gland is derived from branches of the posterior choroid arteries which in turn branch from the posterior cerebral arteries. The pineal parenchyma is rich in vasculature which in many species ends as fenestrated capillaries with perivascular spaces (Arstila, Kalimo, and HyyppS 1971). Generally the pinealocyte cell bodies are located some distance from these perivascular spaces, but cytoplasmic extensions (termed polar processes) end in perivascular terminals and provide a route for transport of secretory product to the perivascular space (Quay 1974a). Two types of secretory vesicles have been identified. Small clear vesicles are present in the cytoplasm, and have been assumed to contain indoleamines. In addition, there are large granular structures surrounded by a membrane which form in association with the Golgi apparatus and appear to be discharged at the pericapillary terminal. The contents of these vesicles are thought to be of proteinaceous or peptidic nature (Pevet 1977). These morpho­ logical observations suggest that the pineal gland is an endocrine organ which discharges its secretory products into the blood-vascular system. 3

Venous drainage from the pineal gland appears to enter the systemic circulation. In man, pineal veins join the great vein of Galen which terminates in the straight sinus. In rats, the pineal veins lead to the superior sagittal sinus, where it is hypothesized that reversal of blood flow might carry the secretory product directly to brain structures. This speculation has not been proved (Quay 1974a).

In the classical sense, the criteria of endocrine function have been fulfilled for the pineal gland, since the injection of pineal extracts results in inhibition (Engel 1935c) and pinealectomy produces the opposite effect, i.e., stimulation of reproductive organs (Kitay

1954a). The effects of pinealectomy can be reversed by transplantation of the organ (Gittes and Chu 1965). The hormonal substance(s) responsible for these observations is(are) still unknown. Within the last two decades the discovery of melatonin and its postulated role as the pineal hormone became the hope of pinealogists. Disappointingly, pharamcologic amounts of melatonin are required to produce effects on reproductive organs (Wurtman and Axelrod 1965) and in contrast, peptidic pineal substances discovered within the last ten years appear to be active in physiologic quantities {Ebels, Benson, and Matthews 1973).

Only time will tell whether peptide hormones are the answer to the riddle of the pineal gland or whether the honor of the pineal principle will pass to some other substance. At the present time, investigation of peptidic compounds is most relevant to pineal research.

A function for the pineal gland, though vague, persists as a hypothesis even in the face of the suggestion that the structure is a functionless vestige. A large amount of evidence suggests that this 4 gland inhibits sexual function in response to decreased photoperiod.

Blinding or shortening of photoperiod appear to stimulate secretion of a pineal substance which reduces weight and activity of reproductive organs. Three promising areas for the study of these phenomena are the effects of altered lighting conditions, pinealectomy, and the injection of extracts on reproductive function.

The Effects of Light on Reproduction

The positive effects of light on sexual function and diurnal rhythms have been linked convincingly to pineal activity, since these phenomena often can be abolished by pinealectomy. While many experi­ ments relating to pineal function are questionable, these remain as stable observations. In this section reproductive activities will be discussed, as well as certain non-reproductive functions which have been linked by experimentation to light.

Effects on Reproduction

The effects of lighting on reproductive organs was first demonstrated in the classical experiments by Benoit (1961), who showed that testicular weight of ducks was markedly increased in spring and decreased in winter. These atrophic "wintertime" testes could be stimulated to springtime weights by exposure to 16 hours of lighting and eight hours of darkness a day and returned to the atrophic state by hypophysectomy. Blinding alone did not abolish the variation in weight with lighting, but hoods placed over the cranium in conjunction with blinding did result in atrophy of the testes. Benoit felt that these phenomena were mediated through the hypothalamus and showed that a 5 lighted glass rod placed in the third ventricle was stimulatory to the genital organs.

Pats placed in constant light demonstrated an increase in the percentage of estrous smears associated with an increase in pituitary luteinizing hormone (LH; Chu 1965; Moore et al. 1968; Marie, Matsuyama, and Lloyd 1965; Lawton and Schwartz 1965). This response is referred to as persistent estrus. Hypertrophy of the ovaries was reported as a concomitant finding in rats (Wurtroan et al. 1961), but decreased ovarian weights were also observed (Marie et al. 1965). A rise in pituitary and a decrease in plasma prolactin concentration were reported by Relkin

(1972a). This response was reversed by constant darkness or prevented by pinealectomy (Relkin 1972b). Pregnant mare serum gonadotropin (PMS) and luteinizing hormone-releasing hormone (LHRH)-induced increases in

LH were both enhanced in rats placed in constant light (Steger, Peluso, and Hafez 1976).

Whether the effects of constant light are mediated totally by the pineal gland is a controversial issue. Either blinding or bilateral removal of the superior cervical ganglia (SCGX) blocked the persistent estrus produced by constant illumination (Wurtman, Axelrod, Chu, and

Fischer 1964). Transection of the optic tract, however, had no effect, indicating a prechiasmatic departure of the retinal-pineal gland pathway (Critchlow and deGroot 1960). Pinealectomized, sham- operated, and intact rats placed in constant light developed persistent estrus at the same rate, began normal cycles at the same interval when returned to diurnal lighting, and achieved persistent estrus similarly once again when placed in constant light a second time (Hoffman and 6

Cullin 1975). In contrast to the results obtained by Wurtman, Axelrod,

Chu, and Fischer (1964) in the superior cervical ganglionectomized rat,

Brown-Grant and 6stberg (1974) found no differences in the rate of achievement of persistent estrus, ovarian weights, sexual receptivity, the incidence of ovulation, or the number of ovulations between sham or superior cervical ganglionectomized rats. The before-mentioned effect on prolactin appears to be the only undisputed pineal-mediated effect of constant light.

Pats kept in constant darkness showed delayed vaginal opening time and decreased LH as measured by bioassay (Fiske 1941). Hoffman

(1967) found that rats blinded before pubertal onset had normal estrous cycles, but when blinded after the establishment of estrous cycles prolonged cycles resulted and 25% of the animals became anestrous.

Uterine, ovarian, adrenal, and pituitary weights were lower in these anestrous animals. Certain effects of blinding were enhanced by the simultaneous removal of the olfactory bulbs, and these combined effects were reversed by pinealectomy, SCGX, or bilateral transection of the

MFB (Reiter, Sorrentino, and Jarrow 1971). Five weeks after blinding and bilateral olfactory bulbectomy (BOBX), young female rats demon­ strated significantly depressed body, anterior pituitary, uterine, and ovarian weights. Pituitary LH and serum prolactin were elevated, while pituitary prolactin and serum LH were depressed. The effects of blinding/BOBX were essentially reversed by pinealectomy (Blask and

Reiter 1975a, 1975b; Kjrfnnekleiv and McCann 1975a, 1975b).

Blinded male rats showed marked atrophy of accessory sex organs and significant reduction in testicular weights (Otoh, Takahashi, and 7

Sakai 1962). In this work, accessory organs refer to the seminal

vesicle-coagulating gland complex and the ventral prostate. Similar results were obtained in constant darkness, and reinstatement of the photoperiod restored function and brought about a rise in serum

testosterone (Takahashi, Choi, and Suzuki 1971). Atrophy of accessory organs was reversed by the administration of LH (Sorrentino and Benson

1970). These authors found no significant effect of blinding in females except for a reduction in compensatory ovarian hypertrophy

(COH) six days after unilateral ovariectomy (U0). Unlike intact controls, blinded rats showed no diurnal variation in serum testosterone

{Kinson and Liu 1973), yet pinealectomy restored the diurnal variation and, in addition, effected an overall increase. Blinding/BOBX in male rats was followed by retarded body development, reduced gonad and accessory organ weight, and depressed pituitary LH and prolactin. All of these changes were negated by pinealectomy (Reiter, Blask, and

Vaughan 1975). From the above experiments, it is clear that altera­ tions in lighting affect reproduction in rats, and that this effect is pineal-mediated in some instances.

When male hamsters were blinded or placed in shortened photo- periods (less than 12.5 hours of light per day), their testes underwent complete atrophy by ten weeks and regenerated spontaneously after 30 weeks even when maintained in short photoperiods. Following this response, the testes of most, but not all the hamsters, became refrac­ tory to the inhibitory effects of short photoperiods. BDinded males demonstrated an inconsistent reduction in LH and a consistent reduction in prolactin in the anterior pituitary gland, but there were no 8

detectable changes in plasma values of these hormones. These effects of

blinding or reduced photoperiods were reversed by pinealectomy or SCGX

(Hoffman and Reiter 1965; Gaston and Menaker 1967; Reiter 1969a, 1972a,

1975b; Reiter, Sorrentino, and Hoffman 1970; Reiter and Johnson 1974a;

Zucker and Morin 1977). The testicular atrophy observed in hamsters

placed in short photoperiods was partially prevented by the daily

injection of prolactin for 17 days, beginning eight weeks after place­

ment in light.-dark (L:D) = 5.19 (Bartke, Croft, and Dalterio 1975).

Hamsters kept in an outdoor environment in which they were exposed to

natural photoperiodic changes underwent gonadal atrophy and displayed

decreased plasma LH during the winter months. These changes were

prevented by pinealectomy (Reiter 1973). The effects of light depriva- Y \t;ion were also prevented by severing the nervi conarii (Reiter 1972b)

or by isolation of the medial basal hypothalamus (Reiter and

Sorrentino 1972). Hamsters placed in shortened photoperiod and

reduced temperatures hibernate, but if gonadal atrophy was prevented

by testosterone administration, hibernation did not occur (Smit-Vis

1972). It is not understood how maintenance of the testes prevents

hibernation in this species, but is assumed that this event may result

in some feedback mechanism which is yet unknown. Electron microscopy

of the Leydig cells in hibernating hamsters showed increased rough

endoplasmic reticulum (ER) and decreased smooth ER in comparison to

non-hibernating animals. Smooth ER is associated generally with

steroid hormone synthesis. Spermatids showed assymetric head caps,

bizarre acrosomes, and damaged nuclear envelopes not seen in non-

hibernating animals (Reznik-Schttller and Reznik 1974). Studies suggest 9

also that the pineal gland may be involved in certain metabolic changes

associated with hibernation, as well as in the control of seasonal

breeding (Reiter 1974b).

Blinding or reduced photoperiod decreased uterine weight,

reduced follicles and corpora lutea, and terminated cyclic vaginal

phenomenon in the hamster (Reiter 1968, 1969b; Sorrentino and Reiter

1970; Reiter and Johnson 1974b). In contrast to the male, pituitary

m was increased and prolactin decreased (Reiter and Johnson 1974b,

1974c; Reiter 1976). The ovaries of blinded hamsters showed corpora

lutea and follicles after the injection of follicle-stimulating hormone

(FSH) and LH (Reiter 1967a). Changes similar to those induced by

shortened photoperiod also occurred in winter when hamsters were kept

outdoors (Reiter 1974a). Pinealectomy reversed all effects of light

deprivation on the reproductive system (Reiter 1969b).

Ferrets normally come into estrus in the spring, but the

timing of this event was changed to December or January by exposure to

artificial long days in the winter. This light-induced advancement of

estrus was prevented by pinealectomy or SCGX, but estrus still occurred

at the usual time in the spring of the following year after these

procedures. Pineal indoles (melatonin, 5-methoxytryptamine, or 5-

methoxytryptophol), administered in pharmacologic amounts, were not

effective in restoring the ability of extra light to advance estrus

after pinealectomy (Herbert 1971). After the period of normal estrus in the spring following pinealectomy, the subsequent estrus was sig­ nificantly delayed and remained so for subsequent years. It is assumed

that the animals had already received the signal for the next estrus 10 when pinealectomy was performed {Herbert 1972). Blinding abolished the estrus occurring the following spring* but in subsequent years .the animals returned to normal cycles {Thompson 1954). No effects on estrus were observed with optic tract lesions placed posterior to the chiasm (Clark, McKeown, and Zuckerman 1939). Electrolytic lesions in the nuclei of the accessory optic tract, or in the MFB did not affect estrus (Thorpe and Herbert 1976a, 1976b).

Alterations in lighting also produced reproductive changes in the mink (Murphy and James 1974), rabbit {Kerenyi et al. 1972, Lincoln

1976), canary (Nicholls 1974), Japanese quail (Oishi and Lauber 1974), and duck (Hisano et al. 1972), as well as in other animals (Reiter

1974b).

In conclusion, lighting apparently influences reproductive function, and in some cases the effects are clearly mediated through the pineal gland.

Effects on the Pineal Gland

Continuous light produced a decrease in the weight of the pineal gland in rats (Fiske, Bryant, and Putnam 1960; Fiske, Pound, and

Putnam 1962). The metabolic rate of the gland, as determined by per cent nuclear volume, was greater during the day than at night (Roth

1965). Constant light produced a reduction in the size of the parenchymal cells (Upson, Benson, and Satterfield 1976), fewer nucleoli and diminished cytoplasmic basophilia (Roth, Wurtman, and Altschule

1962). Blinded rats showed shrunken pinealocytes with decreased numbers of lipid granules and mitochondria (Satodate, Katsura, and Ota 11

1973; Upson and Benson 1977). Furthermore, reductions in lipid and glycogen content, succinic dehydrogenase, and oxygen were observed by

Quay (1961, 1963). Recently, a diurnal rhythm of pineal glycogen content was discovered, which was abolished by constant darkness or constant light and reversed by inverse lighting (Kachi, Matsushima, and

Ito 1974, 1975). In the rat, continuous light enhanced the norepinephrine (NE)-stimulated increase in pineal adenyl cyclase

(Weiss 1969). The circadian rhythm in pineal NE was abolished when rats were blinded or kept in either constant illumination or darkness

(Wurtman et al. 1967).

In addition, there are marked effects of light on pineal enzymes, proteins, and indoles which will be discussed in those respective sections.

Diurnal Pineal Rhythms

There is a 24-hour rhythm in NE (see List of Abbreviations) turnover in sympathetic nerves innervating the pineal gland in rats.

This rhythm persisted in blinded animals, but was suppressed by light

(Brownstein and Axelrod 1974). A diurnal rhythm in pineal protein content was observed, with a peak at 1600 hours and a trough at 2400

(Nir, Hirschmann, and Sulman 1971), and a similar variation in pineal ribonucleic acid (RNA) was seen (Novakova et al. 1971). The anti­ diuretic activity of pineal glands of the rat also showed diurnal variation, with the maximum value three hours after the onset of dark­ ness and the minimum at midnight (Konig and Meyer 1971). Presumably this fluctuation was due to variation in the content of arginine 12 vasotocin (AVT), a cyclic octapeptide isolated from pineal extracts.

In the rat, pineal taurine content also showed a diurnal variation, with increasing amounts throughout the dark period and a sudden decrease during the light phase. Blinding or constant light had no effect on pineal taurine at the sample times reported, but SCGX decreased taurine content (Benson et al. 1977). A 24-hour rhythm in rat pineal mitotic activity, increasing during the day and falling at night was reported (Quay and Renzoni 1966). Dense-cored vesicles were also observed to accumulate during the day and to be released shortly after the onset of the dark phase (Benson and Krasovich

1977a, 1977b).

Variations in pineal enzymes and indoles will be discussed in those sections.

Non-Reproductive Effects of Light

Exposure of female hamsters to darkness caused a reduction in hypertrophy of the thyroid in response to goitrogens, and this effect was not reversed by pinealectamy. The adrenal glands, ovaries, and uteri were also reduced in size; and in female hamsters, housed in normal light, induced hypothyroidism produced similar effects on the weights of these organs (Reiter, Hoffman, and Hester 1966). The thyroid glands of rats in constant light weighed more than controls and less 131 when in constant darkness. Changes in the uptake of I were con­ sistent with these changes in weight (De Prospo et al. 1969).

Blinded female rats showed an absence of diurnal rhythm in their corticosterone secretion until the time of vaginal membrane 13

opening, when the diurnal variation returned to that of intact controls

(Ramaley 1974). Dopamine beta-hydroxylase activity in the adrenal

medulla showed a diurnal rhythm in male rats with a six-fold increase at

the onset of darkness. This nocturnal rise did not occur when lights

were left on, and was abolished in pinealectomized animals (Banerji

and Quay 1976).

Female rats showed depressed epiphyseal cartilage growth when

kept in either constant light or darkness. Pinealectomy reversed the

decrease seen in darkness, but did not alter the effects of constant

light (Nir et al. 1972). This phenomenon is a recurrent theme: various

effects of blinding are generally reversed by pinealectomy, while

effects of constant light, although partially pineal-dependent, appear

to be mediated through additional neural mechanisms.

Adult male hamsters preferred five per cent ethanol over water

when kept in diurnal lighting (L:D = 14:10). Pinealectomy abolished

this ethanol preference. Blinded hamsters also preferred ethanol,

and this was reduced by pinealectomy or SCGX (Reiter, Blum, et al.

1974).

Pinealectomy

Although many studies in which pinealectomy was performed have yielded equivocal results, there is sufficient evidence to support the view that removal of the gland produces a change in the reproductive

function of the organism. In general, such removal is stimulatory to

reproduction. 14

Reproductive Effects

Pinealectomy in castrate male rats stimulated the synthesis of

pituitary LH, as measured by ovarian ascorbic acid depletion. Further­

more, pineal tissue and crystals of melatonin implanted in the median

eminence (ME; but not in the pituitary) decreased pituitary LH

(Fraschini, Mess, and Martini 1968). Although the pituitary content

of LH was increased after pinealectomy, the ultrastructure of the

gonadotropin-producing cells was unchanged (Clementi, Devirgiliis, and

Mess 1969). When pinealectomy was performed before puberty, premature

sexual development occurred in rats. This effect was prevented by

pineal extracts (Thieblot and Blaise 1963, 1965).

Male rats showed increased testicular and seminal vesicle

weights at one, two, and three months after pinealectomy; females

showed increased ovarian and uterine weights (Simonnet and Thieblot

1951). Kind and Benagiano (1967) found no effect of pinealectomy on

reproduction in rats; however, the time of vaginal membrane opening was

observed to be accelerated. Izawa (1926) observed accelerated growth

of testes, accessory organs, uteri, and ovaries in pinealectomized rats,

whereas growth of the pituitary appeared retarded.

In female rats, pinealectomy produced acceleration of puberty

and massive luteinization of the ovary (Simonnet, Thieblot, and Melik

1951); ovarian hypertrophy after 50 days (Kitay 1954a); ovarian, pituitary, and adrenal hypertrophy which could be reversed by pineal

extract (Wurtman, Altschule, and Holmgren 1959); and a temporary

decrease in pituitary LH content between the thirteenth and twenty-first day of age (Slama-Scemama 1976). Fewer fetuses were present in rats 15 pinealectomized ori the eleventh day of pregnancy than from control or sham-operated animals (Guerra et al. 1973). This latter study is paradoxical since the pineal gland is proposed to be an antireproductive organ, but pr©reproductive substances have also been discovered in this gland as will be discussed.

Experimental lesions placed bilaterally in the suprachiasmatic area produced persistent estrus and polyfollicular ovaries in rats; pinealectomy was then followed by luteinization of the ovaries and a return to irregular estrous cycles (Mess et al. 1971}. Rats, showing a persistent anovulatory syndrome secondary.to forebrain deafferenta- tion, ovulated following pinealectomy. This phenomenon was blocked by the injection of serotonin, and ovulation was restored by SCGX (Tima,

Trentini, and Mess 1973). In half of these animals, serum LH was elevated on the day of proestrus; in the other half ovulation proceeded without variation in serum LH (Trentini et al. 1976). Rats in which persistent anovulatory syndrome had been induced by neonatal testo­ sterone treatment showed marked thecal luteinization after pinealectomy or SCGX. The sensitivity of the pituitary to LHRH (see List of

Abbreviations) was also decreased in these animals (Ruzsas, Trentini, and Mess 1977).

Dunaway (1969) observed that the timing of PMS-stimulated ovulation after pinealectomy was earlier, which effect he presumed was due to the removal of some inhibitor to LH release or action. After pinealectomy, rats kept in normal lighting changed from five- to four-

i day ovulatory cycles, although the persistent estrus induced by constant light occurred at the same time in these animals (Simonovitf et al. 1976). 16

Many authors have found no changes following pinealectomy in female rats

(DeFronzo and Roth 1971/ Tigchelaar and Nalbandov 1975, Blake 1976,

Takeo et al. 1975).

Gittes and Chu (1965) observed increased numbers of estrous

smears after pinealectomy, and reversal of this phenomenon with

transplantation of ten to twenty pineal glands either intramuscularly or under the renal capsule. Interestingly, when these transplanted

glands were assayed for the enzyme which produces melatonin, a very low level was found. On this basis it was postulated that a pineal substance other than melatonin reversed the effects of pinealectomy.

In this regard, however, Reiter (1967b) could not induce gonadal involution in blinded, pinealectomized hamsters with pineals trans­ planted under the renal capsule.

In male rats, pinealectomy resulted in premature sexual develop­ ment (Martin and Davis 1941). In adults, an increase in accessory organ weight and/or testicular weights was observed (Hofmann 1925;

Motta, Fraschini, and Martini 1967). Augmentation of androgens in the rat testicle was seen (Timmermans and Devecerski 1972a), along with an increase in dehydrogenases and isoenzymes of the seminal vesicle

(Timmermans and Devecerski 1972b) which decreased to normal as the rat adapted (Devecerski 1972). Baum (1968) found that male rats pinealec­ tomized at three days of age mounted more often and showed pelvic thrusting at an earlier age. There was no difference at 42 days of age.

Hemicastration at 7-28 days of age caused compensatory testicular hypertrophy in rats and a rise in pituitary LH content.

These effects were not prevented by pinealectomy, but plasma LH was 17 slightly increased in animals that had been both pinealectomized and hemicastrated (Hus-Citheral, Roseau, and Zurburg 1977).

R^nnekleiv, Krulich, and McCann (1973) observed a diurnal peak and trough in serum prolactin in rats, and the rise was abolished by pinealectomy. The nadir of this diurnal rhythm was at 1130 hours and the zenith was at 2130 hours. The rhythm persisted after placement of the animals for two weeks in constcint darkness and then reversed, so that the nadir was then at 2130 hours. SCGX or the injection of 6- hydroxydopamine into the lateral ventricle also reversed the low and high points. The injection of 6-hydroxydopamine into the third ventricle decreased serum prolactin, but did not reverse the rhythm.

The third ventricular injection led to depleted dopamine and NE in the hypothalamus, as did injection into the lateral ventricle, but injection via the lateral ventricular route also resulted in decreased pineal dopamine and NE. Pinealectomy slightly increased early morning serum prolactin, but had no effect on diurnal variation, which may suggest a constant inhibition of prolactin by the pineal gland (Kizer et al.

1975). Daytime prolactin was also lower in pinealectomized rats and in anosmic rats. Pinealectomy and anosmia lowered late nocturnal serum prolactin only. Blinding lowered nocturnal prolactin initially and dampened the rhythm, but pinealectomy had no effect on serum prolactin in blinded animals- Pituitary prolactin content was significantly lowered in blinded animals and restored to normal by pinealectomy. It was concluded that pineal secretions elevated serum prolactin, particu­ larly at night, and that loss of input from olfactory and to a lesser 18

extent from visual pathways resulted in a decreased titer of the hormone

(Rjrfnnekleiv and McCann 1975a, 1975b).

Mice showed transient increases in ventral prostate and

coagulating gland weights 12-24 days postpinealectomy (Vaughan 1971,

Vaughan and Reiter 1971). A prolongation of estrus has also been

observed (Mesaki 1939). Adult male house mice became less aggressive

following pinealectomy (McKinney, Vaughan, and Reiter 1975).

When male hamsters were pinealectomized while in a state of

gonadal regression and placed in either long or short photoperiods, the

acceleration of gonadal development due to long photoperiods was

inhibited by pinealectomy (Hoffman and Kuderling 1975). The placement

of female hamsters in reduced photoperiods (L:D = 1:23) produced

gonadal atrophy and decreased prolactin in the anterior pituitary.

These effects were prevented by pinealectomy (Reiter 1975a).

Ducks blinded by bilateral optic enucleation showed a decrease

in ovarian and oviductal weight following pinealectomy (Cuello, Hisano,

and Tramezzani 1972). Male ducks pinealectomized in the spring showed

decreased testicular weight and concomitant reduction in serum

testosterone and synthesis of steroids ill vitro. These changes were

reversed after one season (Cardinali et al. 1971).

Pinealectomized chickens showed early development of sexual

* characteristics, although most of the animals did not survive the

surgery (Izawa 1923). In similar experiments, Shellaberger and

Breneman (1950) reported an early decrease in testicular weight in

pinealectomized chickens, followed by an increase. The decrease could

be reversed by injection of acetone-dried pineal extract (Shellaberger 19

1952). In contrast, Badertscher (1924) found no change in pinealec-

tomized chickens.

Estrous ferrets became anestrous on exposure to short photo- periods, and this effect was prevented by pinealectoiny (Thorpe and

Herbert 1976a). Conversely, the testes and seminal vesicles of pinealectomized or SCGX weanling or adult voles were heavier than those of appropriate controls {Charlton, Grocock, and Cstberg 1976).

All of the above experiments show that the pineal gland may inhibit sexual function and that stimulation is the usual result of its removal- In addition, numerous non-reproductive effects have been observed.

Non-Reproductive Effects on Endocrine Organs

The pineal gland has been reported to affect the thyroid, adrenal, parathyroid, and thymus glands, as well as various neural structures. Insulin-like activity has also been found in the pineal gland. The adrenal gland has been the most thoroughly studied structure for non-reproductive pineal gland effects.

The adrenal gland weights of female hamsters were reduced in pinealectomized animals placed in L:D = 16:8, while those in L:D = 1:23 were increased (Hoffman and Reiter 1966). This finding is in contrast to the testis in which steroidogenesis appears to be increased by pinealec- tomy. The RNA to deoxyribonucleic acid (DMA) ratio was increased in adrenal tissue following pinealectomy in rats (DeFronzo and Roth 1972).

A decreased secretion of all corticosteroids was seen three to five weeks after surgery (Jouan and Samperez 1965). In contrast to these 20 findings# Kinson, Wahid, and Singer (1967) found increased secretion of aldosterone and corticosterone one month after pinealectomy. Nir,

Schmidt, et al. (1971) also observed elevated corticosterone levels, but this increase was significant only in light-deprived animals.

Golikov and Lebedev (1975) found that pinealectomy produced reductions in serum aldosterone, increases in corticosterone, and increased transcortin-binding capacity in metestrous female rats. Pinealectomy diminished the postcastration rise in adrenal 5-alpha-reductase and enhanced the secretion of corticosterone by adrenal slices in vitro

(Ogle and Kitay 1976). In this same study, pinealectomy diminished the secretion in vivo of dihydro- and tetrahydrocorticosterone (reduced metabolites of corticosterone). In mice, pinealectomy resulted in adrenal hypertrophy, an effect reversed by treatment with synthetic melatonin (Vaughan et al. 1972). 131 Thyroids of pinealectomized rats showed a 50% increase in I uptake (Csaba, Kiss, and Bodoky 1968). Ishibashi et al. (1966) observed increased food consumption following pinealectomy in rats, and this effect was abolished by melatonin. A transient increase in thyroid weight was found by Pazo et al. (1968), but no change in ^*1 uptake or iodinated amino acids was observed, indicating no increase in synthesis of thyroid hormone. Parafollicular cells of the thyroid gland showed an increase in both number and granulosity following pinealectomy (Csaba and Barath 1974). Pinealectomized rats showed increased plasma phosphorus and reduced urinary output of this substance. There was also reduced incorporation of methionine, a 21

constituent of parathormone, into the parathyroid glands (Kiss,

Banhegyi, and Csaba 1969).

If parathyroidectomized rats were pinealectomized, 80% developed

intermittent seizures and frequently died (Reiter, Blask, et al.

1973). Seizure medication was without effect; and neither melatonin,

beta-carboline, calcium gluconate, nor aqueous bovine pineal extracts

prevented the seizures (Reiter, Morgan, and Talbot 1973). Other

surgical procedures (such as opening the skull, olfactory bulbectomy,

unilateral transection of the nervi conarii, or 5CGX) did not cause

convulsions when combined with parathyroidectomy; however, if

ganglionectomized rats were pinealectomized, they developed the

seizures (Reiter and Morgan 1972). Development of seizures was

dependent on the timing of the surgery (Pomerantz and Reiter 1973).

Pargyline, a monoamine oxidase inhibitor, prevented the seizures in parathyroidectomized, pinealectomized rats (Herrera, Morgan, and

Reiter 1975). 3 Compared with control animals, the hypothalamic uptake of H- leucine was increased in blinded and pinealectomized, immature rats, and the degree of uptake increased with maturity of the animals.

Melatonin injections did not alter this increased uptake (Chazov et al. 1976a, 1976b). Rat subcommissural organs showed morphological and histoenzymatical evidence of increased activity one month after pinealectomy (Ziegels and Devecerski 1976).

Hypertension has been observed in rats within five weeks following pinealectomy (Zanoboni and Zanoboni-Mucaiccia 1976, Karppanen and Vapaatalo 1971). Plasma renin activity of pinealectomized rats 22

was elevated, but aldosterone output from the adrenal glands in vitro

was unaffected. It was proposed that the increase in renin was due to

increased sympathetic activity (Karppanen et al. 1975). •

Pinealectomy in rats decreased the primary, and eventually the

secondary, immune response (Csaba et al. 1966). If combined with

thymectomy, there was augmentation of lymphocytes in the blood (Lapin

1972).

Bovine pineal extracts produced an insulin-like effect, and

pinealectomy had an anti-insulin effect. Glucose-tolerance was

decreased in pinealectomized animals and insulin-like activity was

increased, while plasma immunoreactive insulin was decreased (Milcu,

Nanu-Ionescu, and Milcu 1971).

Effects of Extracts

Compared to the other areas of pineal research, little defini­

tive work has actually been done with pineal extracts. It appears

that data so obtained were difficult to duplicate and results were

often disappointing. These problems result from the presence of

multiple active pineal gland factors, exerjbing both stimulatory and

inhibitory effects on reproductive activity, the net effect of which may

vary with photoperiod or seasons.

The first person to use pineal extracts effectively was Engel,

a pre-World War II Viennese physician who discovered two factors in

bovine pineal glands: one synergistic to gonadotropins and one antagonis­

tic to them. Engel*s favorite bioassay was based on the ability of pineal fractions to enhance or delay vaginal membrane opening time (an 23

indication of the achievement of puberty) in mice. Fischer (1938, 1943)

published Engel's methods of extraction in English. Initially, acetone-

dried pineal powder was extracted with 30% ammonium hydroxide. When

injected, this extract exhibited either advancement or retardation of

vaginal opening time. Precipitation with 50% ethanol removed the

synergistic factor, but a linear dose response was still not obtained

with the inhibitor. Precipitation with picric acid consistently

removed the synergist. Engel {1934a, 1934b) found that when the alkaline

extract of acetone-dried pineal powder was injected into infantile mice or rats, both luteinization and ovulation were retarded in the adult

animals. Extracts from cows, pigs, and chickens retarded growth of the cock's comb, a testosterone-dependent structure, but an extract of human

glands was ineffective. Reduction of anterior pituitary hormones as measured by bioassay was observed in immature rats (Engel 1935a). This impure extract enhanced vaginal membrane opening in mice, but a purified extract produced delay in a dose response fashion (Engel 1939). High doses produced follicular stimulation but no corpora lutea, while lower doses caused both follicular stimulation and luteinization. Filtra­ tion resolved this extract into a higher molecular weight estrogenic factor and a lower antigonadotropic factor (Engel 1935b). It is probable that Engel was dealing with pineal antigonadotropin (PAG) and pineal LHRH at this time.. The antigonadotropic hormone was described as antiluteotropic by him (Engel 1935c), and it could not be isolated from bovine blood (Engel 1935a, 1935e), although extraction of human blood did result in slight antigonadotropic activity (Engel 1935c). As a measure of estrogenic activity (Silberstein and Engel 1933), the 24 substance was evaluated with the Allen-Doisy test, which measures stimulation of vaginal membrane opening time in mice ovariectomized before puberty. The alteration in the vaginal membrane was the same as that produced by FSH (Engel 1935f). Stimulation of vaginal membrane opening time in ovariectomized mice was produced with subcutaneous implants of human pineal glands (Engel 1934c). Human pineal extract retarded growth of the anterior pituitary in response to growth hormone

(Engel 1934d), but did not influence the effect of growth hormone on callus formation in the broken ribs of rats (Engel 1935g). There was some suggestion that pineal extract retarded the effect of prolactin on the rabbit ovary (Engel 1934e). In 1937 Engel concluded that the pineal was antagonistic to the endocrine function of the pituitary.

Reiss et al. (1963) also found stimulatory and inhibitory 32 factors in bovine pineal glands which decreased P uptake into all organs of adult rats but increased uptake in all but the adrenals of immature animals. Hypertrophy of chick testes and comb was blocked by 32 pineal extract and P uptake into these structures was decreased.

Later the antigonadotropic substance was proposed by Thieblot,

Alassimone, and Blaise (1966) to be a peptide which retained activity at a stage of purification at which seven ninhydrin-positive components were present upon electrophoresis.

Moszkowska, Kordon, and Ebels (1971) found two active fractions following gel filtration of an aqueous extract of ovine pineals on

Sephadex G-25. Fraction "F2" was stimulatory to FSH production by rat pituitaries iji vitro and "F3" was inhibitory. FSH secretion into the medium was tested using the Steelman-Pohley bioassay for FSH. In vivo, 25

F3 inhibited the growth of gonads and accessory organs, but it did not

inhibit the effects of exogenous LH. LH production ill vitro and

effects of exogenous FSH were not affected. activity was also

found in extracts of cerebrum, but the F3 activity seemed to be specific

for the pineal body and was thought to be related to catecholamines

(Moszkowska and Ebels 1971). Glands that had once been inhibitory

became stimulatory after being stored (lsbels, Moszkowska, and Scemama

1970).

The gel-filtered extracts were further separated with Diaflo

ultrafiltration membranes. The UM2 ultrafiltration membrane retains

substances of molecular weight (MW) greater than 1000, and the UM05

membrane substances with MW greater than-500. Fractions of ovine

pineal glands separated into inhibitory and stimulatory fractions

were tested in a hypothalamic-hypophyseal system in vitro. Fractions

were incubated with hypothalami, and LHRH release into the incubation

medium tested by incubating rat anterior pituitary halves in the media in which the hypothalami had been incubated. Gonadotropin release was

tested by determining the effect of the media on ovarian and uterine weights in mice {Ebels 1975). The UM2 residue (MW > 1000) produced a decreased secretion of hypophysiotropic hormones from incubated mouse

hypothalami and the UM05 residue (UMOSR; MW 500-1000) produced in­

creased hypophysiotropic hormone activity. The UM05 filtrate, which contains substances of MW less than 500, decreased gonadotropin output from mouse pituitaries (Ebels, Hus-Citharel, and Moszkowska 1975;

Ebels.1976; Moszkowska et al. 1976). When UM05 filtrate was separated on Sephadex G-10, mass spectral analysis showed 6-biopterin, but 26 biological activity of this fraction was equivocal (Van der Have-

Kirchberg et al. 1977). Five-methoxytryptophol, 5-hydroxytryptophol, melatonin, 5-hydroxyindole acetic acid, and 5-methoxyindole acetic acid have also been identified in ovine pineal extracts and were undetectable in these active fractions (Zurburg and Ebels 1975, Ebels and Horwitz-

Bresser 1976). The work done in Moszkowska's laboratory, though extensive, suffers from the lack of verification by RIA. In addition, change in LHRH in incubated hypothalami is the basis of many of their assays. This is an experimental design of questionable validity in the light of recent discoveries (Sunberg and Knigge 1978).

Engel (1935a) found that stimulatory and inhibitory factors were extractable into basic solutions, but were not present in acidic solutions. In contrast, Altschule and co-workers found that acidic extracts of bovine pineal glands interrupted the persistent estrus which develops in rats in middle age and also decreased ovarian weights

(Meyer et al. 1961).

In high dosages Engel*s extract was reported to reduce total sperm counts in rat seminal vesicles and augment the counts at low dosages (Milcu and Pitis 1944), which is a questionable observation because the seminal vesicles are generally known to contain no sperm.

Bovine pineal extracts reduced mating behavior in chronically treated rats and diminished growth of their offspring in following generations.

Sexual differentiation and development were increased, producing an appearance of precocious puberty similar to that produced in humans by 27

pineal tumors (Rowntree et al. 1936a, 1936b). Altschule's extract was

found to inhibit the spermatazoan response to human chorionic gonado­

tropin (HCG) in male frogs (Juszkiewicz and Rakalska 1963).

In addition to these positive reports, there are many negative

reports on the lack of an effect of pineal extracts on reproduction.

As stated above, such contradictory findings are probably because the

crude extracts contain both inhibitory and stimulatory activity which

may negate each other (Dixon and Halliburton 1909, Weinberg and Fletcher

1930, Wade 1937, Debeljuk 1970).

Pineal extracts have also often been reported to increase blood pressure (Malmejac and Donnet 1937, Bergmann 1940), and sometimes to decrease it (Eyster and Jordan 1911, Jordan and Eyster 1911).

Bovine acetone-dried pineal powder which was extracted in aqueous media produced a reduction in compensatory adrenal hypertrophy following unilateral adrenalectomy (Dickson and Hasty 1972). A similarly prepared extract powerfully inhibited adrenocorticotropic hormone

(ACTH)-mediated corticosterone release by isolated adrenal cells.

Melatonin was ineffective in this regard (Porter and Heiman 1977).

Extracts were also found to inhibit thyroid gland function in rats (Milcu and Pitis 1946, 1947). Pineal extracts reduced the accumula­ tion of blood urea after amino acid infusion, indicating a reduction in nitrogen metabolism (Milcu et al. 1969). Bovine pineal extract also decreased growth, in a dose response fashion, of donor pituitaries transplanted into hypophysectomized rats (Grachev, Usanova, and

Seliverstov 1972). 28

It is evident that the pineal gland may contain multiple sub­

stances with various biological activities, and that many of these

have not been investigated thoroughly. It is possible to develop the

history of pineal extracts without mention of the pineal indole,

melatonin. Before the discovery of melatonin, the effects of extracts

were studied without understanding of substances present, and after

the discovery of this indole, studies were basically done with synthetic

melatonin without use of actual pineal material. Suffice it to say

that, in the United States, all of the effects observed with pineal

extracts were assumed to be due to the presence of this indole.

Benson, Matthews, and Rodin (1971, 1972) removed melatonin from

bovine pineal extracts ^nd found that the extracts retained the ability

to inhibit COH (see List of Abbreviations) in unilaterally ovariectomized

mice. This same non-melatonin activity was found also in human pineal

extracts (Matthews, Benson, and Rodin 1971). Partially purified

melatonin-free extracts of bovine and ovine glcinds were found to reduce

ventral prostate and ovarian weights in both mature and immature mice.

The ovine pineal extract delayed vaginal membrane opening time in mice

(Benson, Matthews, and Smith 1971; Ebels et al. 1973). Furthermore, melatonin-free bovine pineal extract inhibited the 24-, 48-, and 96- hour post-castration rise in LH in male rats (Orts and Benson 1973;

Orts, Benson, and Cook 1974).

Melatonin was removed from these antigonadotropic extracts by separation on Sephadex G-25 columns, on which melatonin is significantly retarded because of its hydrophobicity. Three such fractions of bovine pineal glcinds were found to be antigonadotropic in male rats by Orts 29 in 1977. Fractions F^, F^, and F^ significantly reduced serum LH when injected into rats for five days, and F^ reduced serum testosterone and testicular weight. An organic solvent extract of bovine pineal glands produced a fraction which was found to diminish the probability of pregnancy in rats when injected through one estrous cycle. An aqueous extract was found to contain two fractions, which blocked pregnancy and inhibited COH (Orts, Kocan, and Yonushonis 1975). The incidence of pregnancy and the mean number of fetuses were reduced in mice after treatment with similar extracts {Benson and Matthews 1974; Orts, Kocan, and Johnson 1977). The latter authors also found a reduction in the preovulatory surge of LH and the mean number of ova shed.

Since the effect of pineal extracts are numerous, one can confidently propose the presence of numerous substances responsible for these various activities.

Pineal Substances

Indoles and Melatonin

It is possible that the discovery of melatonin (Lerner et al.

1958) was a setback to the overall understanding of pineal function.

The enzyme involved in the synthesis of this substance, hydroxyindole-

O-methyltransferase (HIOMT), was discovered in pineal gland tissue and was at first thought to be localized to this structure. The assumption that melatonin was unique to the pineal gland was the major basis for its emergence as the proposed pineal hormone. Today it is known that

HIOMT, and hence melatonin, are found in the retina (Cardinali and

Rosner 1971), the Harderian gland (a modified lacrimal gland present in 30 some species; Vlahakes and Wurtman 1972), and peripheral nerves (Lerner et al. 1959, Barchas and Lerner 1964). Most recently it has been found to be distributed throughout the system of enterochromaffin cells

(Raikhlin, Knetnoy, and Tolkachev 1975; Raikhlin et al. 1976). These cells are morphologically similar to pinealocytes.

Until recently the only method of measuring melatonin was by quantitating its blanching effects on the dermal melanophores of the

Hana pipiens tadpole. Although this test is specific and sensitive, a radioimmunoassay (RIA) is now available which was reported to be superior {Arendt, Paunier, and Sizonenko 1975).

It is germane to note that melatonin has a concentration of -5 2 x 10 gram (gm)/gm of tissue in the pineal gland itself; in -7 peripheral nerves it is only 2 x 10 gm/gm. Immunohistochemical techniques for n-acetylindoles, viz. n-acetylserotonin and melatonin, localized these compounds to the pineal gland, retina, and the cerebellar granule and Golgi type II cells. Pinealectomy did not diminish the reaction in these extra-pineal sites, a finding indicating synthesis outside the pineal gland (Bubenik et al. 1974). Melatonin was found in the sera of rats, and showed a diurnal rhythm—being high at the mid- dark period and low at mid-light as it was in the pineal gland itself.

In the above study melatonin was measued by the tadpole bioassay and was found to disappear from the sera of pinealectomized rats. Castration had no effect on serum or pineal gland concentration (Pang and Ralph

1975). In contrast, Ozaki and Lynch (1976), using the tadpole bioassay,

RIA, and thin layer chromatography (TLC), found melatonin in the serum and urine of pinealectomized rats. The amounts were 20% of control 31

values# and the light-dark rhythm disappeared. Employing bioassay and

RIA, Ozaki, Lynch, and Wurtman (1976) showed that melatonin was greater

during the dark period in rats and that the RIA was not specific for

melatonin extracted from urine. Pretreatment with chlorpromazine, a

major tranquilizer, increased the concentration of melatonin in pineal

gland and plasma in addition to slowing the rate disappearance of

melatonin from plasma. The promazines are called major tranquilizers

because of their wide effect on neurotransmitters unlike the minor tranquilizers which are simple sedatives.

The concentration of melatonin varied also in the pineal glands of chickens, being higher in gland, blood, and brain during the night

(Ralph 1976). Studies in free-running chickens showed that peak melatonin values occurred during the period of low locomotor activity

(Ralph et al. 1976, Binkley et al. 1975). Melatonin was undetectable in the serum of pinealectomized chickens (Pelham 1975).

A mid-dark melatonin peak has been found in the serum and urine of humans (Lynch et al. 1975, Ralph 1976, Arendt et al. 1977).

Melatonin was lower in cerebrospinal fluid than in serum (Arendt et al.

1977).

Continuous plasma sampling of ewes revealed a diurnal rhythm in melatonin levels with a peak during the dark. After pinealectomy the daytime value was increased, so that there was no difference between day and night (Matthews et al. 1977).

Melatonin, 5-methoxyindole acetic acid, and 5-hydroxyindole acetic acid were identified in sheep pineal extracts (Ebels, Balemans, and Verkley 1972). More importantly, the gland contained large amounts 32 of serotonin which appeared to be taken up by pineal nerves (Owman

1964). Serotonin also showed a 24-hour rhythm in rat pineal glands which could be altered by hooding, removal of the Harderian glands,- or

SCGX, but not blinding (Snyder, Zweig, and Axelrod 1964; Zweig, Snyder, and Axelrod 1966; Machado, Machado, and Wragg 1969; Wetterberg, Geller, and Yuwiler 1970; Illnerova 1971). Destruction of the optic chiasm of monkeys abolished this rhythm (Kenny et al. 1973).

Owman (1968) pointed out that the large amount of serotonin and low amount of melatonin in the pineal gland would suggest a search for other mechanisms for pineal control and function than melatonin alone.

It is possible that serotonin is the neurotransmitter of the pineal gland, and that it causes release of active peptides or other hormones with melatonin synthesis serving as a pathway for serotonin breakdown to a less active product.

Melatonin did have multiple effects on reproduction and other organs, but dosages required were so high that the small amount found in the pineal gland cannot possibly be producing such effects (Quay 1974a).

Only Wurtman and Axelrod (1965) reported effects of melatonin at a more nearly physiological dosage, i.e., one yg of melatonin per day reduced ovarian weight in mice, but the paper was written ambiguously and the table gave the dose as one mc[ per day. It was also observed that cyrstals, constituting a physiological amount of melatonin, decreased serum LH (Fraschini et al. 1968) as well as FSH (Martini 1974) when implanted in the center of the median eminence. Further studies showed that 3H-melatonin administered into the cisterna magna of rats decreased 33 microtubular protein in the median eminence and hypothalamus (Cardinali and Freire 1975). The meaning of this finding is not clear.

The hamster, which has been used in a model of choice designed to delineate pineal gland function (Hoffman and Reiter 1965), has also become the source of new information on the function of melatonin. For years, one of the caveats related to acceptance of melatonin as the active pineal principle was that the pineal-mediated gonadal atrophy which occurs in hamsters could not be reproduced by treatment of animals with this substance. More alarming, melatonin implanted sub- cutaneously in beeswax pellets weekly prevented the gonadal atrophy that normally occurs in blinded hamsters (Reiter, Vaughan, et al. 1974).

Melatonin administered.in such pellets also increased plasma LH in male hamsters, and increased plasma and pituitary LH in females (Reiter,

Vaughan, and Waring 1977). Blinding produced regression of reproductive organs, elevation of pituitary LH concentration, and depression of pituitary prolactin in female hamsters; these effects were reversed by both pinealectomy and melatonin treatment via beeswax implants

(Reiter, Vaughan, Rudeen, et al. 1975). In male hamsters that have undergone gonadal atrophy, both pituitary LH and prolactin are depressed

{Reiter 1976). In dark-exposed male hamsters that had undergone gonadal atrophy, melatonin pellets implanted subcutaneously caused testicular regeneration and elevated pituitary prolactin. When these same animals were removed from darkness for a period and then returned, gonadal atrophy was not induced (Reiter, Vaughan, et al. 1976). It was eventually found that as little as 3.6 yg melatonin per day (implanted in beeswax pellets) counteracted the inhibitory effect of darkness 34

(Reiter, Vaughan, and Waring 1975). Pinealocytes of hamsters with melatonin implants had reduced nuclear diameters and decreased smooth

ER and lipid, indicating reduced activity (Barratt, Nadakavukaren, and

Frehn 1977). Injection of LHRH into male hamsters living in 23 hours of dark per day did not prevent gonadal atrophy, but further reduced LH stores (Reiter, Vaughan, Blask, and Johnson 1975). An implant of 6- hydroxymelatonin also prevented gonadal regression and the decrease in pituitary prolactin produced by blinding; n-acetylserotonin and 5- hydroxytryptophol were ineffective in blocking these two phenomena

(Reiter and Vaughan 1975).

These progonadotropic actions of melatonin were explained by proposing that the hamster differed from the white laboratory rat, the most commonly utilized animal of reproductive hormone research. Then came the discovery that subcutaneous implants of melatonin prevented the effects produced by blinding and anosmia in male rats. The retarded body development, reduced gonadal and accessory organ weight, and depressed pituitary LH and prolactin were not observed in animals bearing implants (Reiter, Blask, and Vaughan 1975). It was also shown that melatonin or pinealectomy partially restored reproductive organ weight in rats treated neonatally with testosterone and blinded (Banks and Reiter 1975). It appeared that melatonin was also progonadal in rats.

In 1975, Turek, Desjardins, and Menaker discovered that melatonin caused marked testicular regression in hamsters maintained in L:D =

14:10. Animals in L:D = 6:18, a schedule which should have produced testicular atrophy, did not show regression when treated with small 35 amounts (50 yg/day) of melatonin, but did show testicular atrophy when larger amounts (75-100 yg/day) were used. Later it was found that melatonin injected into male hamsters in the afternoon daily for six weeks produced testicular regression and lower levels of serum LH and

FSH after six weeks. Injections made in the morning were ineffective.

Females became acyclic and showed no diurnal variation in LH secretion when melatonin was injected in the afternoon (Tamarkin et al. 1976;

Tamarkin, Hollister, et al. 1977; Tamarkin, Lefebvre, et al. 1977).

Female and male hamsters, maintained on L:D = 16:8 and treated with

10 yg of melatonin daily, ceased ovulatory cycles or exhibited reduced testicular weight, respectively, if injected at 2000 or 0400 hours but showed no responses when injected at 2400 or 0100 hours'(Tamarkin,

Lefebvre, et al. 1977). When melatonin was administered by silastic capsules implanted subcutaneously into male hamsters and grasshopper mice, there was reduction in testicular weight and spermatogenesis. No effects were observed in the rat or house mouse (Turek, Desjardins, and

Menaker 1976; Brackman 1977). Reiter, Blask, et al. (1976) confirmed the report that 25 yg of melatonin per day reduced testicular weight in male hamsters when injected in the afternoon. It was also shown that melatonin was ineffective after pinealectomy, SCGX, decentralization of the SCG, or anterior deafferentation of the pineal gland. In another study (Sackman et al. 1977), 25 yg of either melatonin, 5- methoxytryptophol, n-acetylserotonin, or 6-hydroxymelatonin was given daily to hamsters at 1900 hours for 50 days; Melatonin alone sig­ nificantly elevated body weight and depressed the growth of testes and accessory organs as well as pituitary prolactin levels. In 36

pinealeeternized hamsters, this effect did not occur. Five-

methoxytryptophol and n-acetylserotonin were slightly active, and

their effects were also negated by pinealectomy whereas 6-

hydroxymelatonin was ineffective.

When adult male hamsters were immunized with a melatonin-

thyroglobulin conjugate, the testes of antibody-bearing hamsters

exhibited atrophy. Blinding exacerbated the degree of involution

(Knigge and Sheridan 1976). These authors concluded that the pineal

gland may secrete a substance which, under conditions of diminished or

absent circulating levels of biologically active melatonin, is anti-

gonadotropic. It was also assumed that.the secretion of this substance

is enhanced by dark-induced stimulation of the pineal gland. In other

experiments (Brown et al. 1976), hamsters immunized with N-

acetylserotonin produced antibodies that bound melatonin and N-

acetylserotonin equally. When these animals were exposed to short

photoperiods, the gonads regressed and plasma testosterone fell,

indicating that circulating melatonin does not mediate the effects of

short photoperiods. Blinding, performed alone or in conjunction with

either pinealectomy or immunization against N-acetyserotonin, did not

alter resting prolactin levels. Pinealectomy alone slightly lowered

prolactin, but did not affect its diurnal rhythm. Immunization alone

caused a significant reduction in prolactin levels, but the diurnal

rhythm persisted (Niles, Brown, and Grota 1977). In summary, the

pineal gland appears to contain a non-melatonin substance which is

inhibitory to prolactin. The secretion or action of this unknown compound may be under the influence of pineal melatonin. 37

In contrast to Reiter's finding, Tamarkin, Hollister, et al.

(1977) found that small doses of melatonin injected at the appropriate time caused gonadal regression in the pinealectomized hamsters. These authors have attempted to duplicate their experiments in Reiter's laboratory without success (Blask 1978). Turek (1977) has also observed antigonadal effects of melatonin in pinealectomized hamsters.

Melatonin's role in the pineal gland remains to be delineated, but the accumulated data suggest that it is an unlikely candidate for the pineal antigonadotropic hormone.

Pineal Enzymes

Pineal enzymes involved in indole synthesis have been thoroughly studied, but others have largely been ignored. HIOMT has been most widely studied; it has been the basis for a great deal of pineal research, particularly in the United States. Melatonin is synthesized from tryptophan in the pineal gland and in other tissues that contain

HIOMT. The outline of the biosynthesis is given below:

Tryptophan L Aromatic Amino Tryptophan • 6-Hydroxy tryptophan • Hydroxylase Acid Decarboxylase

N-acetyl- HIOMT Serotonin N-acetylserotonin Melatonin transferase

HIOMT varies with the photoperiod, being higher during the dark phase in rats and guinea pigs (Yochim and Wallen 1974a, Farriss and

Botz 1975). There was also variation with the estrous cycle of rats, 38

with HIOMT being highest at diestrus and lowest at estrus (Wurtman et

al. 1965, Wallen and Yochim 1974a). The wavelength of lighting to

which rats were exposed had an effect: HIOMT was lowest in animals

maintained in green light, while red light produced no change (Cardinal!,

Larin, and Wurtman 1972a, 1972b). This finding was the opposite in sparrows in which HIOMT was lowest in red light. In this species,

HIOMT varied throughout the season but testicular weight increased at

the beginning of the mating season before HIOMT declined, suggesting that HIOMT and the resulting melatonin production are not the mediators of this phenomenon (Barfuss and Ellis 1971). A seven-day rhythmic activity in pineal HIOMT was observed in rats {Vollrath, Kantarjian, and Howe 1975). There was also a sudden rise in sheep pineal HIOMT in the last four to five days of pregnancy (Kennaway and Seamark 1975).

Injections of exogenous FSH, LH, and prolactin increased HIOMT activity in the pineal glands of castrated male rats which had been sham superior cervical ganglionectomized; this increase was insignifi­ cant, however, when the SCG were actually removed (Cardinali, Nagle, and Rosner 1976). The light-induced changes in HIOMT in rat pineal glands were abolished by transection of the medial forebrain bundle

(Axelrod et al. 1966). HIOMT was decreased by ovariectomy and the induction of pseudopregnancy (Yochim and Wallen 1974b); on the other hand, estrogen administration in castrate male and female rats increased it (Houssay and Barcelo 1972a). Injection of testosterone also increased HIOMT, although castration produced no change (Houssay and

Barcelo 1972b). In contrast, testosterone and dihydrotestosterone decreased pineal monamine oxidase and HIOMT activity, while estradiol 39 stimulated the activity of both enzymes (Urry et al. 1976). In one study (Mizobe and Kurokawa 1976), HIOMT and DNA-dependent RNA polymerase were measured in pineal glands from ovariectomized rats in vitro, and it was found that 17-beta-estradiol increased HIOMT activity in this system; this effect was abolished by inhibitors of protein and RNA synthesis. Clomiphene citrate, which inhibits binding of steroid hormones to receptors/ also abolished this increase, suggesting that

HIOMT activity is controlled both by lighting and hormonal factors.

The original hypothesis suggesting that HIOMT was confined to the pineal gland has proved to be false, since it has also been found in the Harderian gland of rats. HIOMT is probably present as well in other melatonin-containing structures; however, Harderian gland HIOMT differs from pineal HIOMT in that it is markedly stimulated by divalent cations (Vlahakes and Wurtman 1972). i Klein, Weller, and Moore (1971) found that the rate-limiting step for melatonin synthesis actually involved N-acetyltransferase (NAT) rather than the methylating enzyme HIOMT. Like HIOMT, NAT was also found to show a diurnal variation; it appeared on the fourth postnatal day in rats and achieved adult levels by three weeks (Klein and Weller

1970; Ellison, Weller, and Klein 1972; Rudeen and Reiter 1977). NAT rhythm in the rat was found to be free-running after blinding, and was abolished by partial hypothalamic deafferentation (Deguchi 1975, Moore and Traynor 1976). NAT activity was inhibited by light in a sensitive, dose-response fashion (Minneman, Lynch, and Wurtman 1974), and its rhythm was inversed by reverse lighting (Nir et al. 1974). The rhythm 40 in chickens and rats persisted in constant darkness, but was lost in constant light (Binkley 1976).

Changes in NAT activity in the rat pineal were found to be mediated through a beta-adrenergic receptor (Deguchi and Axelrod 1972;

Romero, Zatz, and Axelrod 1975; Zatz et al. 1976). Stress enhanced the amount of this enzyme (Parfitt and Klein 1976). Pineal NAT levels were stimulated by isoproterenol and dibutyryl cyclic adenosine monophosphate

(db-cAMP); these effects varied diurnally (Romero 1976). Nuclear protein appeared to be,phosphorylated at the early stages of this induction (Winters et al. 1977). NAT activation by isoproterenol was enhanced in spontaneously hypertensive rats (IllnerovS and Albrecht

1975).

Pinealocytes were found to contain enzymes connected with the hexose monophosphate shunt and polypeptide synthesis. Phospho- nicotinamide-adenosine-dinucleotide (NADP) was required in this pathway

(Botticelli et al. 1972).

Cyclic adenosine monophosphate (cAMP) phosphodiesterase activity in the rat pineal gland showed a 24-hour rhythm, with highest values during the dark period. SCGX (see List of Abbreviations) blocked this dark-induced increase (Minneman and Iversen 1976). Pineal cAMP phospho­ diesterase existed as two enzymes, one with a high Michaelis-Menton constant (Km) and the other with a low Km value. Isoproterenol increased the low Km phosphodiesterase (Oleshansky and Neff 1975).

Organ culture of rat pineal tissue produced four cell types: one capable of migration and considered a fibroblast, two with oxidative enzymes and presumed to be the two types of parenchymal cells (light and 41

dark) , and a fourth type with lytic enzymes and thought to be a

macrophage (Huxley and Tapp 1972). The seven enzymes found in the

parenchymal cells were: succinic and lactic dehydrogenase, glucose-6-

phosphate dehydrogenase, nicotinamide-adenosine-dinucleotide (NAD)

diaphorase, leucine aminopeptidase, cytochrome oxidase, and monoamine

oxidase. Looking at this list, the reader should note that Niemi and

Xkoken (1960:928) once stated, "The existence of leucine aminopeptidase

activity can be taken to suggest secretion of some agent of a protein

nature by the rat pineal gland." Recent studies localized this enzyme

to connective tissue around blood vessels, with lower levels in the

parenchymal cells. The high levels of oxidative enzymes in the list

suggest a high metabolic activity; these enzymes were closely associated

with lipid droplets, suggesting that these structures are important in

pineal metabolism (.Tapp, Huxley, and Davies 1973). Cholinesterase

activity in bovine pineal glands varied significantly with sex and age

(Karppanen, Klinge, and Aro 1972), and carbonic anhydrase varied with

lighting in rat pineal glands (Quay 1972), as did catechol-0-

methytransferase (BackstrSm and Wetterberg 1972).

Recently a neuraminidase which deactivated HCG by removing sialic

acid was isolated from bovine pineal glands; this may account for the

inhibition of HCG-induced ovulation by some pineal extracts (Cheesman

and Forsham 1974). A peptidase capable of destroying arginine vasotocin

(AVT) was also found in the pineal gland (Reinharz and Vallotton 1974). 42

Pineal Peptides

A very active current area of pineal research is the charac­

terization of active peptide fractions. The rapid synthesis of amino

acids and presence of rough ER and Golgi in pinealocytes has led some

authors to propose secretion of a peptide or protein hormone (Hellman

and Owman 1961, Thieblot and Blaise 1966). In accord with such pro­ posals, histochemical procedures for the identification of polypeptide secretion in bovine and monkey pineal glands showed droplets of presump­ tive secretory products (Lukaszyk and Reiter 1975a, 1975b).

The active pineal principle was proposed to be a peptide after the finding (Benson, Matthews, and Rodin 1971, 1972) that the fraction from bovine, human, rat, and ovine pineal extracts which inhibited COH and reduced ventral prostate weight in mice was eluted on Sephadex G-25 in a fraction different from that containing melatonin. Whereas melatonin is retarded on Sephadex because of its hydrophobicity, the active fraction was found in the polypeptide range. Further evidence for the polypeptidic nature of this compound was obtained by showing that these extracts were inactivated by trypsin and chymotrypsin

(Matthews and Benson 1973).

The active portion was assumed to contain pineal antigonado- tropin (PAG), an antigonadotropic substance different from melatonin.

Partially purified PAG inhibited the COH seen in mice (Benson,

Matthews, and Rodin 1971, 1972) and hamsters (Little 1971). In addi­ tion, the ventral prostates and ovaries of intact adult mice were sig­ nificantly reduced in wt (Benson, Matthews, and Smith 1971). Histo­ logical examination showed reduction of epithelial height in the ventral 43 prostate and apparent central lysis of corpora lutea in the ovary.

Fertility was significantly reduced in mice with partially purified extracts containing PAG (Benson, Matthews, and Hruby 1976). These same extracts from both bovine and rat glands inhibited the post-castration rise in serum LH in male rats {Orts and Benson 1973, Orts et al. 1974).

In further purification studies, PAG was isolated from aqueous extracts of bovine and ovine pineal glands by gel-filtration of the crude extract on Sephadex G-25. The void volume, protein-containing, and low molecular weight portions were all discarded, while the fraction in the range of polypeptides was retained. Melatonin was retarded (and actually eluted far behind the amino acids and salts), so it was widely separated from the putative PAG fractions. The peptide portion was passed through two Diaflo membranes, one (UM2) retaining substances of

MW greater than 1000 and the other (UM05) holding substances of MW greater than 500. The active fraction was retained by the UM05 membrane, and was thus referred to as the UM05 residue (UM05R). Since melatonin has a molecular weight of approximately 250, any remaining following gel filtration would be separated from the active fraction by the UM05 membrane. The UM05R was then gel-filtered on Sephadex G-10; peaks were discerned at 280 nanometers (nm). Activity was found in the third and fourth peaks which were referred to as fractions "F^" and These peaks were further purified by descending paper chromatography and Rp values for the active substance in five different solvent systems obtained (Ebels et al. 1973). This activity had always been found in a ninydrin-positive spot, but the substance was eventually purified on ion-exchange resins and TLC to the point that sensitivity to ninhydrin 44

was lost. At the present time, further purification with high pressure, reverse phase liquid'chromatography is being performed in order that a purified PAG fraction can be obtained for complete characterization of this substance (Benson 1978).

Studying the above fractions, Orts, Kocan, and Wilson (1975) showed that melatonin implanted subcutaneously in beeswax pellets blocked the ability of the fourth peak from Sephadex G-25 to inhibit

COH, while the third peak was still active. This may indicate the presence of two antigonadotropic substances, only one of which is inhibited by melatonin.

The F2 and F3 fractions of Moszkowska and Ebels (1971) could also be peptides, since they were obtained from ninhydrin-positive peaks on Sephadex. The inhibitor affected FSH, while PAG appeared to inhibit only LH-dependent structures. Neascu (1972) extracted a peptide from bovine pineal glands which inhibited the effects of FSH and LH on frog spermatogenesis. An amino acid analysis was reported for this peptide, but no follow-up studies have been reported.

Most studies implicating peptides were done on acetone-insoluble fractions of aqueous pineal extracts. An antigonadotropic substance which inhibited COH in mice was isolated from the acetone-soluble portion of bovine pineal glands by Bensinger, Vaughan, and Klein in

1973. The chemical nature of this substance was not studied, but it was presumed to be a peptide. A substance with antigonadotropic properties has also been isolated from acetone-defatted powder from bovine pineal glands (Benson, Matthews, and Rodin 1972). This powder was extracted with aqueous methanol and precipitated with ammonium sulfate. The 45 resulting crystals were dissolved in 0.05 molar (M) ammonium hydroxide and partitioned into isobutanol. After evaporation/ the isobutanol residue behaved similarly to PAG, had a similar elution pattern on

Sephadex G-25, and was retained by the UM05 filter. Its behavior on

Sephadex G-10 was erratic, but biologically it appeared to be identical to PAG. It is interesting to note that the extraction method was modified from one developed by Cheesman and Fariss (1970) for the isolation of arginine vasotocin (AVT) from bovine pineal glands.

A polypeptide with the properties of AVT was found in bovine pineal glands (Milcu, Pavel, and Neascu 1963) and structural confirma­ tion proved by mass spectral analysis (Cheesman and Fariss 1970).

Also, leucine vasotocin was identified chromatographically in the pig pineal gland (Pavel 1965). In recent years AVT has become commercially available, and numerous studies have been carried out with this neurohypophysial hormone. AVT blocked the stimulatory action of PMS on mouse uteri and ovaries (Pavel and Petrescu 1966) and the HCG- induced stimulation of ovaries and uterus (Vaughan, Vaughan, and Reiter

1975, 1976). Daily injections of either two micrograms (Vg) of AVT or

100 yg melatonin significantly prolonged the estrous cycle of mice. If administered daily throughout pregnancy, none of the AVT-treated mice delivered viable pups (Vaughan, Reiter, and Vaughan 1976). AVT retarded ventral prostate growth in Swiss-Webster mice, wild house mice, and hamsters while oxytocin and vasopressin had no effect (Vaughan, Reiter, et al. 1974). When AVT was injected into mice neonatally, there was increased growth of the reproductive organs in adulthood. Injection of

AVT after the neonatal period resulted in the opposite effect. COH in mice was inhibited in a dose-response fashion by AVT administered intra-

peritoneally and also by microinjection of very reduced amounts into the

third ventricle. Arginine vasopressin, lysine vasopressin, and 4-

leucine AVT also produced this response (Pavel, Petrescu, and Vicoleanu -4 1973; Vaughan, Vaughan, Blask, et al. 1976); 2 x 10 picograms of AVT, injected as two microliters (pi) into the third ventricle of mice on the day of pinealectomy, prevented the increase in pituitary prolactin which occurred one week post-pinealectomy (Pavel, Calb, and Georgescu

1975); prolactin was bioassayed by the pigeon crop sac method. AVT reduced uterine weight in intact and hypophysectomized animals, a result indicating a gonadal site of action, and had little effect on ovarian weight (Moszkowska and Ebels 1968).

In the presence of AVT, the LH and prolactin released from

LHRH-stimulated rat hemi-pituitaries were elevated. AVT was not effec­ tive in the absence of LHRH (Vaughan, Blask, et al. 1975)- Both AVT and bovine pineal extract were shown to cause prolactin release from rat pituitaries in vitro and in vivo, yet prolactin-inhibiting activity was also found in pineal gland extracts (Blask et al. 1976; Vaughan,

Blask et al. 1976; Blask, Vaughan, and Reiter 1977).

Through use of the rat antidiuretic and oxytocic assays and frog bladder assays for AVT (Pavel 1973), melatonin injected intra- ventricularly was implicated in the release of AVT into the third ventricle of cats. As little as 10 ^ picograms of AVT (six molecules) injected into the third ventricle of cats induced slow-wave sleep and decreased plasma Cortisol. AVT partially purified from the pineal gland was equally as effective as synthetic AVT, but vasopressin and 47 oxytocin were ineffective (Pavel, Psatta, and Goldstein 1977; Pavel,

Cristovcanu, et al. 1977). If one considers the interaction of hormones with their receptors as a sound hypothesis, such effects produced with as little as six molecules would be extremely difficult to explain.

These results are of questionable validity. Xntracarotid injection of synthetic and bovine melanocyte-inhibiting factor induced AVT release into the CSF of cats and decreased pineal AVT activity (.Pavel, Goldstein,

Gheorghiu, and Calb 1977).

Pavel (1971) found that AVT activity, as detected by frog bladder assay, was confined to the stalks of bovine glands from adult animals but was spread throughout the fetal glands. Since the stalk contains only ependyma, which extends into the parenchyma of fetal animals but not of adults, it was hypothesized that AVT is an ependymo- secretion. Neurohypophysial hormone activity was found also in the culture media from human fetal pineal glands (Pavel, Dorcescu, et al.

1973). Trypsin, tryosinase, and sodium thioglycollate abolished COH inhibition by this medium (Pavel et al. 1973-74). Rat pineal AVT, measured by rat antidiuretic and frog bladder assays, markedly decreased from fetal stages through the newborn to adult ages, and this decrease correlated with the regression of pineal secretory ependymal cells from the glandular tissue (Pavel> Goldstein, and Calb 1975). Cultured pineal ependymal cells from rat fetuses released a substance into the media which was physiologically and chemically similar to AVT (Pavel,

Goldstein, Ghinea, and Calb 1977).

In an attempt to ascertain whether the antigonadotropic activity of the bovine pineal gland was in the same location as AVT, bovine 48 pineal stalks were separated grossly from the parenchymatous tissue before extraction. Vasotocic activity, measured by a milk ejection assay in mice, was confined to the stalks while antigonadotropic activity was found in the pineal parenchyma itself (Benson, Matthews,

Hadley, et al. 1976). In contrast, immunocytochemical staining of rat pineal glands for AVT showed fluorescence diffusely throughout the parenchyma (Bowie and Herbert 1976). AVT was measured in the rat pineal by RXA (Rosenbloom and Fisher 1975b), and it had a diurnal variation, with a peak at noon and a low at midnight. Pineal AVT content was decreased by constant light and increased by constant darkness (Calb, Goldstein, and Pavel 1977). Neurophysin-like proteins, proposed to be carriers for AVT, have also been isolated from bovine and human pineal glands (Reinharz, Czernichow, and Vallotton 1974,

1975; Legros, Louis, and Grotschel-Stewart 1975; Legros et al. 1976;

Reinharz and Vallotton 1977).

Paradoxically, Pavel (1971) supported strongly the ependymal origin of AVT, but proposed it as the antigonadotropic hormone of the pineal parenchyma. Although AVT has potent antigonadotropic effects

(see below), it may be present in pineal extracts simply because the stalk is pulled loose and remains attached to the pineal gland as the latter is removed from the skull. It is logical to assume that this substance is present throughout the third ventricle, as well as in the pineal stalk, because of its contiguous locations. AVT was also found in the anterior pituitary gland of the cockeral (Jackson and Nalbandov

1969) and in the rabbit subcommissural organ (Rosenbloom and Fisher

1975a). 49

Congparisons of biologically active PAG and synthetic AVT showed

dissimilar ultraviolet absorptions and fluorescent maxima, different

mobilities on thin layer electrophoresis/ and different amino acid

compositions (Rosenblum, Benson, and Hruby 1976). It can be concluded

that PAG differs from AVT.

Another possible pineal peptide is a substance isolated from

urine, gonadotrophin-inhibiting substance (GIS), which blocks the

effects of exogenous gonadotropins {Ota, Dronkert, and Gates 1968a). It

was found consistently in the urine of children under six years of age

(Landau, Schwartz, and Soffer 1960) and sometimes in hypogonadotropic-

hypogonadic adults (Landau, Landau, and Schwartz 1965). It reduced the

gonadotropin content of castrate rat pituitaries, and was proposed to

be a polypeptide based on the manner of its separation on Sephadex

G-25 (Thieblot, Berthelay, and Blaise 1966). GIS prevented the action

of LH on the ventral prostates of immature, intact, and hypophysectomized

rats and on the uteri of immature mice (Futterweit et al. 1963). The

number of ova induced by PMS and HCG were reduced by GIS (Ota, Obara,

and Dronkert 1967). Histological examination showed atretic and enlarged

ovarian follicles, as if the LH present were insufficient to produce ovulation (Ota, Dronkert, and Gates 1968b). GIS activity disappeared

from the urine of pinealectomized rats (Ota, Hsieh, and Obara 1971).

Further, GIS reduced fertility in mice (Landau et al. 1969), and PMS- and HCG-induced ovulation in immature mice was suppressed by GIS isolated from acetone-defatted bovine pineal glands. This substance was believed to be different from melatonin, AVT, and PAG (Ota, Horiuchi, and Obara 1975). An anti-LH substance, partially purified from human 50 urine, was found to be a sialic acid containing glycoprotein of MW

162,000, but it was not clear whether or not this substance was GIS

(Banerji, Kothari, and Shah 1977). Recently a thermostable inhibitor of PMS and HCG, as well as a heat-labile inhibitor, have been isolated from human urine by ultrafiltration. These compounds possessed MW's greater than 10,000 and less than 1,000 respectively {Ota, Sato, and

Obara 1977). It is not known whether they are responsible for the GIS activity or not.

White et al. (1974) found high concentrations of LHRH in bovine, ovine, and porcine pineal glands. When the hypothalamus of a single ewe was compared with the pineal gland from the same ewe, four to ten times more LHRH was found in the gland. Thyrotropin-releasing hormone was also present in pineal glands, in amounts equivalent to that present in hypothalamic tissue. In contrast, Duraiswami et al. (1976) found LHRH to be 11.2 yg/100 gm of acetone powder from bovine pineal glands, as measured by RIA, and concluded that this amount is much less than that in the hypothalamus. Similarly, Gradwell, Miller, and

Symington (1976) tested bovine pineal glands for LHRH, by bioassay in vitro and by RIA, and found far less in the gland than in the hypo­ thalamus. A possible reason for this conflict was soon discovered;

Joseph (1976) reported that LHRH in rat pineal glands was extremely low in winter months and very high in the spring. LHRH content of the median eminence and hypothalamus, in contrast, exhibited no seasonal variation. Another seasonally varying releasing factor has been found

(Jackson, Saperstein, and Reichlin 1977): in early winter, midwinter, and spring, thyrotropin-releasing hormone (TRH) in frog pineal glands 51

was much less than the amount present in autumn. Comparable hypothala­

mic TRH was lower in spring than in autumn; neither constant light nor

constant dark affected these values. The reason for the presence of

these releasing factors in the pineal gland is unknown.

The pituitary and pineal gland contained higher levels of renin activity than other brain tissues. This renin-concentrating

ability decreased in salt-loaded animals, while non-glandular brain tissue showed an increase (Haulica et al. 1975).

Steroid hormones appear to affect the pineal gland and its peptide synthesis. Estrogen and androgen receptors were present and were controlled by catecholamine transmitters (Cardinali, Nagle, and

Rosner 1975b). Testosterone and estradiol enhanced protein synthesis by the rat pineal gland, perhaps increasing the secretion of peptide substances (Cardinali, Nagle, and Rosner 1974a; Nagle, Cardinali, and

Rosner 1975). SCGX diminished high-affinity binding of estradiol to the pineal cytosol of female rats and of testosterone to that of males.

This procedure also blocked the increased incorporation of tritiated leucine into pineal proteins which resulted from treatment with estradiol and testosterone (Cardinali, Gomez, and Rosner 1976). Pineal denervation abolished the 8S protein receptor for steroids and blocked the effects of androgen on tritiated leucine uptake (Cardinali, Nagle, and Rosner 1975a, 1975b; Nagle et al. 1975).

These results indicate that peptides are important to pineal gland function and that steroids may play a role in peptide synthesis and/or secretion. 52

Morphological Correlation

Morphological descriptions of the pineal gland have been biased by the need of numerous investigators to justify indoleamine secretion.

Essentially, the chief cells—the pinealocytes—resembDe cells which secrete peptide hormones. In the rat pineal gland, Arstila et al.

(1971) found rough EH and prominent Golgi apparatus with associated dense-cored granulated vesicles. These vesicles were said to be characteristic of monoamines (Oksche et al. 1972), but such compounds are not secreted by the Golgi and monoamines were not detected in them with immunohistochemical techniques. Fluorescent antibodies to melatonin were found localized in the marginal zone of parenchymal cells in rats killed at night (Freund, Arendt, and Vollrath 1977).

Beginning at the onset of the dark phase, the granular vesicles in rat pinealocytes decreased markedly until midnight, and increased again at the end of the dark period, whereas agranular vesicles remained un­ changed (Matsushima and Ito 1972). After castration, male rats showed an increase in pinealocytic rough ER, Golgi and associated granules, lipid droplets, and lysosomes. These changes, described as consistent with increased protein synthesis, were enhanced by LHRH (Karasek,

Pawlikowski, Ariens-Kappers, and St^pien' 1967).

The mouse pineal gland also contains rough ER, prominent Golgi, and dense-cored granulated vesicles, the latter apparently secreting into the pericapillary space from adjoining terminals of pinealocytes, which are long, protoplasmic extensions directed toward the capillaries.

After exposure to constant light, there was a reduction in pinealocyte size, as well as in the Golgi, and in the number of lipid droplets. 53

The number of granulated vesicles per pericapillary terminal and the number of terminals were also reduced {Upson et al. 1976). One month after bilateral optic enucleation, mouse pineal glands showed a

55% increase in dense-cored vesicles, an increase in agranular vesicles, and hypertrophy of the Golgi (Upson and Benson 1977). There appeared to be a diurnal rhythm in the number of granulated vesicles in the mouse pineal gland: vesicles were more numerous during the light period and less so during the dark. SCGX prevented the increment during the light period and a single intraperitoneal injection of melatonin decreased the number of granulated vesicles (Benson and Krasovich 1977a,

1977b).

In some mammalian species two different populations of pinealocytes have been observed. One type (dark pinealocyte) produced a secretory material in granulated vesicles which originated from the

Golgi; the other (light pinealocyte) formed agranular vesicles directly from cisterns of the rough ER. In animals with one type of pinealocyte

(mole, hedgehog, and rat), both types of secretory activity were present in each cell. It was theorized that.pinealocytes may be similar to the parafollicular cells of the bat thyroid which were found to contain a polypeptide (thyrocalcitonin) and an indoleamine (serotonin) bound to 3 each other (Pevet 1977). In rabbit pineal glands, H-5-hydroxytrypto­ phan, a melatonin precursor, was found diffusely in the cytosol of light pinealocytes after incubation. From this, it was concluded that indoleamine synthesis occurs in the cytosol rather than in the organelles

(Romijn, Mud, and Wolters 1977). 54

Eight weeks after blinding, hamster pineal glands showed marked hypertrophy of the smooth ER, with dense-cored vesicles appearing in the pinealocyte reticulum; SCGX produced atrophied pinealocytes (Lin, Hwang, and Tsang 1975). The smooth ER has been associated with production of steroids or indoles, and the dense-cored granules are considered to be secretory product. The pineal gland of a rodent is characteristically composed of the parenchyma and a sac continuous with the choroid plexus of the third ventricle. The sac does not abut the parenchyma, and probably cannot be a route for secretion. In a study by Gregorek,

Seibel, and Reiter (1977), venous drainage in the rodent appeared to be into the superior sagittal sinus by means of the vena cerebri magna.

The pineal of the penguin has a lobular structure with the predominant cell type displaying prominent Golgi, free ribosomes, both clear and granular vesicles, and lysosomes (Piezzi and Gutierrez 1975).

In pinealocytes of the mole, a prominent Golgi apparatus with associated clear vesicles was observed. Secretory granules were always rare, but were most numerous from June until January. Peculiar paracrystalline structures, not described in other species, were found in the rough ER and were believed to be related to protein synthesis. There was an increase in the number of these structures and in the size of the Golgi during the period of high sexual activity in the male and during estrus, gestation, and lactation in the female (Pevet and Smith 1975a, 1975b).

The number of dense-cored vesicles surrounding the Golgi complex in the rabbit was highest at noon and in the evening (Romijn, Mud, and

Wolters 1976). When rabbits were superior cervical ganglionectomized or placed in constant illumination, similar changes occurred in the 55

pinealocytes: loss of ribosomes from the rough ER, loss of NE from nerve

terminals# and conversion of the terminals into whorls (Romijn 1975a,

1975b, 1976a). There was also a decrease in dense-cored vesicles and

transformation of the smooth ER from a tubular to a vesicular character

(Romijn 1976a).

Bats have two distinct types of pinealocytes; one type contained

granular vesicles, apparently of Golgi origin, and the other showed

glycogen granules and a large system of vacuoles. Accordingly, two

t secretory products were proposed (Pevet, Ari&ns-Kappers, and Vodte

1977). In these animals, pinealocyte nuclear and nucleolar diameters

varied with season of the year. Peak nucleolar size occurred in March,

when hibernation was ending, and the trough occurred in fall, just

before hibernation began (Quay 1976).

Certain morphological characteristics suggest that the pineal

gland may secrete peptidic substances. Histochemical techniques applied

to monkey pineal glands demonstrated neurosecretory features similar to

those seen in polypeptide hormone secretion (Lukaszyk and Reiter 1975a,

1975b). The lizard (Lacerta) pineal organ was examined after

argentiaffin and chromaffin reactions; such reactions took place in

dense-cored granules. Tritiated 5-hydroxytryptamine was taken up by

I these same granules. It was suggested that in lizards this compound coexists with a proteinaceous material in these granules (Collin,

Juillard, and Falcon 1977).

A variety of functional properties has been reported in the rat pineal gland. Pinealocytes transplanted beneath the kidney capsule showed characteristics of high protein or peptide secretory activity 56

(Aguado et al, 1977). Parenchymal perfusion of pineal glands with India ink brought out an extracellular and intracellular network of canaliculi,

the diameter of which varied diurnally (a peak diameter was seen near

the end of the light phase and a trough during the dark; Quay 1974b).

With the scanning electron microscope, a three-dimensional network of

anastomosing intracellular spaces was observed. All pineal cells appear to be in contact with this system (Krstic 1975). The function of these structures has not been discovered.

In contrast to reports which indicate protein or peptide secre­ tion from the pinealocyte, certain investigators suggested that a lipid secretory product, compatible with indoleamines, is present. Lipid droplets were observed to migrate through the rat pinealocytes to the terminal enlargements, where they were apparently secreted into the pericapillary spaces. It was theorized that these droplets contain indoleamines (Gonzales and Blazquez 1975). When male rats were pharma­ cologically castrated by administration of an inhibitor of androgen synthesis, cyproterone, light pinealocytes showed increased activity three weeks after treatment. Liposomes were especially enlarged, and bundles of microtubules were seen. Surgical castration did not produce such outstanding changes, possibly because the adrenals of these animals were still producing androgens (Gusek 1976). Administration of p- chlorophenylalanine and p-chloroamphetamine, which inhibit indoleamine synthesis, reduced the smooth ER. of the pinealocytes of rabbits, suggesting that these structures are involved in the synthesis of

* indoleamines (Romijn 1976b). 57

Pineal morphology varies markedly from species to species. The pineal gland of the ground squirrel differed from that of the rat by a more pronounced differentiation into cortical and medullary zones.

Hibernation in this species was accompanied by reduced pineal serotonin and by reduction of the differences in cortical and medullary zones.

These changes were similar'to those following denervation (Popova,

Kolaeva, and Diansva 1975). The ground squirrel also has perivascular and intervascular spaces surrounding pinealocytes (Povlishock, Kriebel, and Seibel 1975). The endothelial cells were observed to be fenestrated in rat, mouse, and squirrel, but non-fenestrated in the chinchilla, with marked differences in pinealocyte vesicles (Matsushima and Reiter

1975).

Other structures visible in pinealocytes are synaptic ribbons: two parallel fibers lined with clear vesicles; Extension of the light period in guinea pigs reduced the increase in synaptic ribbons which occurred normally during the dark period, while prolongation of the dark period inhibited the decrease which usually occurred during the light period (Vollrath 1976). Synaptic ribbons of rat pinealocytes seemed to be neither cholinergic nor adrenergic when tested chemically, and though they did not originate from the ER, they appeared to be proteinaceous with some chemical resemblance to microtubules (.Krstic

1976a). Karasek (1976) showed that pinealocytes removed from castrated rats or cultured in vitro had increased numbers ,of synaptic ribbons.

He proposed that microtubules may be precursors of these structures.

Synaptic ribbons were rare in humans, but one was reported in the pineal gland of a three-year-old girl at autopsy (Kurumado and Mori 1976). 58

In the continuing search for indoleamines through the use of immunohistochemical techniques, an autofluorescent material was found in the rat pineal gland. This material was believed to be a tryptophan- rich protein, and was observed to be increased following castration

(Karasek, Pawlikowski, Pevet, and Stjpien' 1976). Pineal serotonin was not influenced by treatment with exogenous gonadotropin/ but the autofluorescent material increased (Smith et al. 1976, Pevet et al.

1975). This material was also present in the mole (Pevet et al. 1976).

Two types of postganglionic nerve endings were described in the rabbit pineal gland. Some endings contained dense-cored vesicles that were absent after SCGX and therefore were assumed to be noradrenergic.

Other endings contained clear vesicles and were considered to be cholinergic parasympathetic fibers; their presence was abolished by severing the facial nerves (Romijn 1975a).

Calcium granules were found in pinealocytes of some species, but these have only rarely been studied. Gerbils possess vaculated pinealocytes containing calcareous granules. Both vacuolation and calcium deposits were found to be absent when the gland was denervated

(Reiter, Welsh, and Vaughan 1976). These granules were most striking in the human pineal gland. Under the scanning electron microscope, they were mulberry-shaped; chemically they resembled hydroxyapatite

(Krstic 1976b).

Recent questions about pineal morphology have centered on the ependyma of the pineal recess, since this aperture has been suggested as a possible route of pineal secretion. Vigh, Vigh-Teichmann, and Aros 59

(1975) found that pinealocytes and cereborspinal fluid-contacting neurons were ultrastructurally similar, but not identical.

In sheep and rabbit fetuses, a pineal nerve extends from the pineal gland to the subcommissural organ, a ependymal structure. This nerve was not found in adult rabbits (Miller, M^llgard, and Kimble

1975). In intact or pinealectomized frogs, secretory activity in the subcommissural organ was altered by light and darkness. The pineal gland seemed to have an overall inhibitory influence on this organ

(Diederan 1975). Although one might hypothesize that pinealocytes make contact with fibers of neurons of the nearby habenular nucleus, or with the ependyma of the pineal recess, no such synaptic connections or cell junctions were observed in studies by Nielsen and Miller (1975) and

MjzJller (1976).

Morphological evidence indicated that melatonin acts on the pineal gland itself, since its administration, or a comparable exposure of rats to darkness for two weeks, both induced changes in pinealocyte ultrastructure compatible with activation of the gland. HIOMT and NAT were increased by melatonin, but NE was not affected. It was concluded that the pineal is a target organ for melatonin (Freire and Cardinali

1975). Prominent production of dense-cored vesicles by the Golgi apparatus was noted in rabbit pineal glands cultured vitro for eight days. Melatonin added to the medium increased pineal HIOMT, a result indicating that the gland may be a target organ (Romijn and Gelsema

1976). Further, melatonin appeared to release the material from the dense-cored vesicles in mice (Benson and Krasovich 1977a, 1977b). 60

In summary, pinealocytes have the morphological characteristics

of cells that secrete proteinaceous material as well as indoleamines.

The nature of its secretory product or products cannot be ascertained

at this time/ but it is very clear from its cellular fine structure

that the pineal gland is actively engaged in secretion.

Pineal Incubation

Pineal glands have been maintained in organ culture, and

metabolic and pharmacologic studies carried out. Rat pineal glands 32 incorporated p into phospholipids, an effect enhanced by NE. On the

other hand, parachlor©phenylalanine, which blocks melatonin precursor 32 synthesis, did not affect P uptake (Berg and Klein 1972).

In cultures bo.th cAMP and NE stimulated the conversion of

tritiated tryptophan to melatonin (Klein et al. 1970). When pineal

glands from two-day-old rats were cultured and then examined with the

electron microscope, the pinealocytes were gathered in nests and were

seen to contain vesicle-crowned rodlets and cilia. Treatment with NE

or db-cAMP increased pineal cAMP in such cultures, but this stimulation

was blocked by propranolol (Rowe et al. 1977). When pineal cells were

cultured in monolayers, db-cAMP caused a rapid and striking elaboration

of cell processes, a phenomenon reversed by removing the cyclic

dinucleotide (Wilkinson 1975). NE stimulated accumulation of cAMP

in incubated rat pineal glands; this effect was inhibited by TRH but not by an inactive analog of TRH. LHRH was less effective than TRH,

and somatostatin-release-inhibiting factor had effects only at high 61

concentration (Tsand and Martin 1976). The function of the pineal gland

appears to be mediated through NE with cAMP as a second messenger.

Ultrastructure of the rat pineal gland in vitro was similar to

the ultrastructure iji situ. Glands cultured with either NE/ db-cAMP, or

adenohypophysis were similarly stimulated; all showed an increase in the dense-cored granules, rough ER, and pinealocyte Golgi apparatus

{Karasek 1974). It is noteworthy that Karasek was convinced that the dense-cored granules contained indoleamines, and was perplexed because they were so similar to the neurosecretory vesicles which contain peptide hormones in the median eminence.

Testosterone was readily taken up and retained by rat pineal glands in vitro, where it is converted to 5-alpha-reduced metabolites

(Cardinal!, Nagle, and Rosner 1974b). When pineal glands from castrated rats were incubated, it was found that treatment with testosterone propionate elevated tritiated leucine incorporation into pineal protein by 79%. The magnitude of the results depended on the time of adminis­ tration of testosterone, treatment at 0600 hours being associated with a 150% increase. SCGX blocked this increase (Nagle et al. 1975).

Sucrose gradient of cytosols from rat pineal glands incubated with titriated estradiol or tritiated testosterone showed an 8S peak which disappeared after SCGX or if the incubation was done with excess unlabeled hormone (Cardinal! et al. 1975a).

Cultured rat pineal glands elaborated a potent antigonadotropic substance which inhibited COH. This substance was deactivated by trypsin and was assumed to be proteinaceous (Benson, Matthews, and Orts 62

1972). It is possible that this substance is PAG, but AVT could also be responsible for such activity.

The medium from 100 bovine pineal glands incubated in Krebs-

Ringer buffer showed five indoles and six unknown substances when subjected to TLC. Polypeptides were also present. Fractions were inactive, however, when tested on LH output from anterior pituitary

(Gradwell, Candy, and Symington 1975).

Human Pineal Relations

It was once thought that the human pineal involuted with age, but measurement of the activity of HIOMT, monoamine oxidase and histidine-N-methyltransferase in human pineal glands in individuals from three to 70 years old showed no correlation between activity and age (Wurtman, Axelrod, and Barchas 1964). Histologic examination showed no atrophy or involution with age, and the calcium deposits were in the interstitium (Tapp and Huxley 1972). When calcifications were studied by scanning electron microscopy and by detailed chemical analysis, they were found to be mulberry in shape and more common than realized (Michotte et al. 1977). There was no significant difference in pineal weight, calcium, or amine content in glands of age-matched groups of patients dying suddenly. There was significant correlation, however, between pineal weight and calcium content in patients over 60.

Amine levels varied in all groups, and dopamine was most abundant in the pineal glands of patients who died of malignancy. Calcium, on the other hand, was highest in patients who died of renal disease associated with hypertension (Hinterberger and Pickering 1976). 63

The most convincing evidence for human pineal function has been the documentation of precocious puberty in children with destructive, non-parenchymal pineal tumors. This condition was hypothesized by Bing,

Globus, and Simon (1938) to be due to pressure on contiguous brain structures. Kitay (1954b) reported that extrapineal tumors compressing the proper structure may result in precocity. The tendency of opinion, however, is away from this concept, because precocious puberty so often precedes the onset of increased intracranial pressure.

Pineal extracts were used therapeutically on a number of occasions: for excessive masturbation, nymphomania, and precocious puberty (Rohleder 1928), mental retardation (Berkeley 1920), and schizophrenia (Altschule 1957, Bigelow 1974). Although the effects on most of the above conditions have proved questionable, the beneficial effects on schizophrenia were confirmed, and Altschule's extract is presently being used for this condition. It is interesting that these extracts are melatonin-free.

Melatonin was found in the urine of humans where it exhibited a daily rhythm (Lynch et al. 1975). A 24-hour rhythm of melatonin, with a peak during the dark phase, was also found in single human pineal glands taken at autopsy (Greiner and Chan 1978).

Blindness in humans accelerated sexual maturation, as measured by the onset of menarche (Zacharias and Wurtman 1964). This finding was in contrast to the presumed antireproductive activity of the pineal gland, which is supposedly stimulated by blinding. Additional evidence indicated that women living near the equator experience menarche earlier than those in more northern or southern latitudes (Reiter 1972a). 64

A study of 747 human pineal glands provided another interesting finding:

a seasonal variation in weight which was increased in winter (Legait

and Legait 1977).

It seems probable that the pineal gland plays a role in the

human throughout life, but such function has not been thoroughly investi­

gated. A most interesting observation was the report by Dr. Cook, a

physician who went to the Narth Pole during its initial exploration.

Cook found that Eskimos stopped their sexual activity and that menstrual

cycles ceased during the winter night. Eskimos now have artificial

light, and these reproductive hiatuses are no longer observed today

(Reiter 1972c).

A very important finding is that melatonin-free extracts of

human pineal glands inhibited COH as did such extracts from the glands

of other species. This fact suggests that PAG is also present in human

pineal glands (Matthews et al. 1971).

It is evident that human pineal function is a highly neglected

area.

Conclusion

The pineal gland clearly has apparent inhibitory effects on

reproduction, but also may have stimulatory actions and be involved in

non-reproductive events. The mechanism through which these phenomena operate and the substances involved remain mysteries. It appears that

this gland contains multiple factors which have a wide range of effects.

Further investigations into almost every area of pineal activity are obviously necessary. STATEMENT OF THE PROBLEM

Site of Action of Pineal Antigonadotropin

The basic assumption for the experiments reported in this

dissertation was that there is a peptide-containing hormone present in

the pineal gland that possesses antigonadotropic properties, i.e., a

pineal antigonadotropin (PAG). It was hypothesized that PAG might work

directly on the gonad, on the pituitary gland, or centrally in the

brain—perhaps in the hypothalamus. The possibility of multiple sites

of action was also considered. In order to test direct gonadal effects,

the extract considered to contain PAG was injected simultaneously by

different routes (subcutaneously and intraperitoneally) with the

exogenous gonadotropins, PMS and HCG, into immature animals. PMS has a

predominantly FSH-like activity, while HCG produces an LH-like effect.

If PAG worked directly on the gonad, it should inhibit the stimulation

produced by these hormones in immature mice, which are accepted as

lacking endogenous gonadotropins. A similar experiment to test the

direct gonadal effects of PAG on exogenous prolactin, which acts

synergistically with testosterone on the ventral prostate of castrate

male mice, has been planned.

The putative PAG was also tested experimentally for direct action on the pituitary gland by both in vitro and in vivo experiments.

PAG was incubated with rat pituitary halves, which were then stimulated with LHRH; and the resulting LH release into the media was compared with controls. Effects on the pituitary iii vivo were measured by

65 66 micro injecting PAG directly into the gland and then measuring LH and prolactin in serum and pituitary. These in vivo studies utilized both long- and short-term castrate rats. In the long-term castrate, LH and prolactin changes were measured at one-hour intervals after injection.

In short-term experiments, microinjections were made at the time of castration and LH and prolactin were measured 24 hours later.

Hypothalamic effects of PAG were tested by microinjecting extract into the median eminence in long- and short-term castrate rats and comparing LH and prolactin to the similarly performed intra- pituitary microinj ections.

Effects of Pineal Antigonadotropin in the Hamster

Since the pineal gland produces marked gonadal atrophy in the hamster, we wondered if PAG might be responsible for this antireproduc- tive effect. Male golden Syrian hamsters were treated for ten days or eight weeks with extracts containing PAG and their reproductive organ weights and serum testosterone and LH levels compared with saline- treated controls. Blinded hamsters were also treated for eight weeks with PAG to see.if testicular atrophy produced by blinding could be further enhanced with PAG.

Since PAG has been shown to decrease the 24-hour post-castration rise in serum LH in rats (Orts and Benson 1973), the effect of PAG on this rise in the hamster was also studied. In addition, LH was measured in long-term castrate hamsters that had been treated with either PAG or saline. The effects of PAG on the LH surge in female hamsters was 67

measured since PAG appears to be antagonistic to rising serum LH in

male rats.

The results of these experiments showed that# although PAG

decreased ventral prostate weight in hamsters as it does in mice, it

did not decrease serum or pituitary LH. Blinded or light-deprived

hamsters show a decrease in both pituitary LH and prolactin, so it was

decided to test the effects of prolactin on the pineal-mediated gonadal

atrophy observed in the blinded hamster. The idea was that if pineal

extracts do not work on LH in the hamsters, and injection of LHRH will

not prevent this atrophy# maybe these effects are mediated through

prolactin.

In one experiment, exogenous prolactin was injected daily into

blinded hamsters to see if the gonadal regression could be prevented.

In a second experiment, anterior pituitary homografts which had been placed underneath the kidney capsule were used to maintain serum prolactin at a normal level in the blinded hamster, prolactin output from rat pituitary glands increases markedly when the glands are placed in a non-sellar site because they are removed from the effect of prolactin-inhibiting factor in the hypothalamus. Conversely, other anterior pituitary hormones disappear, because the trophic hormones from the hypothalamus are missing. Although this phenomenon has not been studied in the hamster, it is probable that a similar mechanism exists in this species. 68

Studies on the Effects of Arginine Vasotocin

Despite the fact that arginine vasotocin has been shown to be

chemically different from PAG (Kosenblum et al. 1976), it is still

questioned whether the activity of PAG is due to contamination with AVT.

The effects of different amounts of AVT were measured on ventral

prostate weight in male mice in a manner similar to that used to test

PAG.

Vaughan, Vaughan, and Klein (1974) found reduction of ventral

prostate and ovarian weight in immature mice treated with AVT, and this

experiment was repeated in our laboratory with synthetic AVT.

Finally/ an organic solvent extract of bovine pineal stalks shown to contain most of the cyclic octapeptide activity (Benson,

Matthews, Hadley, et al. 1976) was tested by evaluating its effect upon ventral prostate weight in mice in the same manner that parenchymal

extracts are tested. This approach was considered to be a good way to clarify whether the AVT present in extracts reduces ventral prostate weight. MATERIALS AND METHODS

Extraction of Pineal Glands

Bovine pineal glands were obtained from Pelfreeze, East St.

Louis, Illinois; Endocrine Research and Supply Company (ERASCO), San

Mateo, California; and Roth Co., Philadelphia, Pennsylvania. The glands were frozen at the abbatoir and shipped in solid carbon dioxide. On arrival they were stored at -40°C until extraction. Two types of extracts were employed, "organic" and "aqueous." The details of these extractions follow:

Organic Solvent Extraction

Approximately 60 gm of bovine pineal glands were homogenized in an equal volume of acetone for ten minutes in a Virtis Model #45 homog- enizer. The homogenate was brought to a total volume of one liter with acetone and stirred for seven hours at 4°C, The acetone-insoluble material was isolated on a Buchner funnel and blended into 240 ml of 33% methanol in water (volume/volume). The mixture was stirred for six hours at 50°C. After centrifugation at 12,400 times gravity (x g) for

20 minutes (min), the supernatant portion was saturated at 25°C with ammonium sulfate and stored overnight at 4°C, The resultant white crystals were isolated on a Buchner funnel fitted with Whatman #41 filter paper and dissolved in 40 ml of 0,05 molar (M) ammonium hydrox­ ide. This solution was extracted twice with 40 ml portions of iso- butanol. The organic phase was centrifuged (500 x g) for ten minutes

69 70

and removed from the small amount of lower aqueous phase. The iso-

butanol was rotary-evaporated to dryness at 50°C, the residue dissolved

In 15 ml water, and dialyzed through UM2 and UM05 Diaflo membranes under

75 millimeters of mercury nitrogen pressure. As stated earlier, the former ultrafiltration membranes retain substances of MW >1000 and the latter MW>500. The UM05 residue (MW 500-1000) was lyophilized and used

for testing.

Aqueous Extraction

One hundred grams of freshly thawed bovine pineal glands were placed in 100 ml of 0.2 normal (N) acetic acid and heated to 80°C in a water bath for ten minutes. The mixture was quickly chilled in an ice bath and homogenized for five minutes in a Virtis Model #45 homogenizer.

The solution was centrifuged at 12,000 x g for one hour and the super­ natant portion placed on a 4 x 60 centimeter (cm) Sephadex G-25 column.

The column eluate was collected at a rate of three ml/min. The first

400 ml were discarded and the second 400 ml collected for ultrafiltra­ tion. The first 400 ml contained the void volume of the column and a peak which showed ultraviolet absorption at 280 nm. The second 400 ml, probably corresponding to the elution of peptides, was ultracentrifuged through UM2 and UM05 membranes under 75 millimeters of mercury nitrogen pressure. The UM05 residue was lyophilized and used for testing.

Radioimmunoassay (RIA) Procedures

Testosterone RIA

A modification of two different assay procedures was used for testosterone radioimmunoassay (New England Nuclear Testosterone RIA kit; 71 Lox, Christian, and Heine 1974). The standard preparation of testoster- 3 one was obtained from Sigma Chemical Company and tritiated 1,2- H- testosterone (specific activity = 43.5 Curies/mi11imole) from New

England Nuclear. The first antibody was graciously supplied by Dr.

Charles Lox, Texas Tech University Medical Center, Lubbock, Texas. This antibody exhibited 100% cross-reactivity with testosterone, 66% with I dihydrotestosterone, and 0% with other steroids. I In order to calculate recovery from the extraction, tritiated testosterone was added to one ml serum samples in an amount totaling

1,000 counts per minute (cpm). The samples were allowed to stand at room temperature for 30 minutes, so that the tritiated testosterone could equilibrate with serum-binding proteins. A ten-fold volume of methylene chloride was added to each sample, and the mixture vortexed for 30 seconds. The samples were centrifuged for ten minutes at 2000 x g and the aqueous phase carefully removed with a Pasteur pipette. One ml of 0.1 N sodium hydroxide was added to the orgcinic phase to wash out interfering substances, and this base was then vortexed and removed.

The process was repeated with one ml of 0.1 N acetic acid, followed by one ml of water. The methylene chloride'was transferred to a scintil- I lation counting vial and evaporated under nitrogen while in a 40°C water bath. Steroid assay buffer (2,2 ml) was added to each vial, which was i then vortexed for three minutes. The contents of the vials were added to borosilicate glass tubes in 0.1, 0.2, and 0,4 ml duplicate aliquots for RIA, while to another scintillation vial was added 0.5 ml for the purpose of determining the per cent recovery of testosterone. No other reagents were added to the vial containing 0.5 ml. To each of the other 72

tubes, 0.1 ml of a 1:400 dilution of anti-testosterone gamma globulin

(first antibody) was added, followed by 0.1 ml of tritiated testosterone

(4,000 cpm) ,

The tubes were incubated at 4°C overnight. The next day 0.2 ml of dextran-charcoal solution was added to each, and the tubes were

allowed to sit for 20 min at 4°C, Then they were centrifuged at 2,000 x g for 10 min, and the supernatant portion, containing the bound testosterone, decanted into scintillation vials. Toluene-based scintillation cocktail was added, and each vial counted for 20 min in a

Beckman LS-15 scintillation counter. For various increasing concentra­ tions of the standard {10-500 pg/ml), bound-over total testosterone was obtained. The linear portion of the curve was programmed for linear regression with a Hewlett':Packard model #65 computer. Serum testosterone values were determined by this program and means for groups compared. A representative standard curve is shown in Fig. 1.

Luteinizing Hormone and .Prolactin RIA

The LH RIA was a method provided by the National Institutes of

Arthritis, Metabolism, and Digestive Diseases with materials supplied by Dr. A. F. Parlow through that organization. The method has been modified by Niswender et al, (1968). Ovine LH (LER'LH #1056-C2), kindly supplied by Dr. Leo Rsichert, Department of Biochemistry, Emory Uni- 125 versity, was iodinated with I (New England Nuclear). One millicurie 125 of I was added to a small disposable polyethylene vial, followed by

25 yl of 0,5 M phosphate buffer, pH 7.6, and 25 pg containing two yg of ovine LH. Ten pi of a chloramine-T solution (35 mg/10 ml buffer) were 73

JOO

90

80

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60

50

40

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20

20 50 100 200 400 TESTOSTERONE pg/ml

Fig. 1. Standard curve for testosterone radioimmunoassay — B/T = Bound labelled testosterone over total testosterone bound when the antibody was saturated with labelled testosterone. 74 added, and the vial agitated for 60 seconds, ihe reaction was termi^ nated by adding 25 yl of sodium metabisulfate (35 mg/10 ml), then the entire mixture was placed on a 1 x 10 cm Sephadex G-75 column which had been equilibrated with 0.01 M phosphosaline buffer at pH 7,6 and coated with 50 yl of 2% bovine serum albumin in buffer. Fractions (0.5 ml) were collected in borosilicate glass test tubes and a 10 pi aliquot from each fraction was counted in a Packard gamma counter. Two peaks of radioactivity were obtained. The first consisted primarily of the iodinated hormone, the second of free iodide. The undamaged hormone, based on immunoactivity, was in the second portion of the first peak after the apex, This portion was diluted with phosphosaline buffer, so that each 0.1 ml contained approximately 40,000 cpm, and used for the assay. These peaks are illustrated in Fig, 2,

The RIA was performed with a micromedic automatic pipeter.

Serum or diluted pituitary homogenate was aspirated by the pump in amounts less than 0.4 ml and diluted to a total volume of 0,8 ml with 1% bovine serum albumin-phosphosaline buffer. Simultaneously, the pump added 0.2 ml of a 1:10,000 dilution of anti-LH (#15; first antibody) and 0.1 ml of the iodinated LH. The mixture was placed in borosilicate glass test tubes and stored at 4°C for five days, and the fifth day ovine- anti-rabbit gamma globulin (sedond antibody) was added to each tube.

The tubes were kept overnight at 4°C, and the next day they were centri- fuged for 30 min at 2,000 x g and the supernatant portion aspirated.

The precipitate was counted in the Packard gamma counter, m first antibody was supplied by Dr, Gordon Niswender (Department of Biochemis­ try, University of Colorado), and the second antibody by Dr. Charles Lox. 75

CPM X 10'

I 5 10 15 20 25 30 35 40 45 I I TUBES USED IN RIA TUBE NO.

Fig. 2.| lodination of LH for radioimmunoassay. 76

As a standard, NIAMD-Rat LH-RP-1 was used at a concentration

ranging frcm 1.25-500 ng. Pooled hamster pituitary extract was also run

through the procedure at various dilutions for the purpose of validating

the assay for hamsters. The amount of LH was determined with the linear

regression equation and the Hewlett Packard computer.

The RIA procedure for prolactin was almost identical to that

for LH. The kit provided by the National Institutes for Arthritis,

Metabolism, and Digestive Diseases with materials made by Dr. A, F.

Parlow was utilized. The method is that of Niswender et al, (1969),

During iodination, the prolactin (NIAMD-Rat Prolactin-I-3) was agitated

for only 30 seconds. The immunoassay samples were treated in the same manner, except that the rabbit-anti-rat prolactin antibody (NIAMD-Anti-

Rat Prolactin Serum 4, 5, or 6; first antibody) was diluted to 1:2,500, while 0.2 ml was added to each tube instead of 0.1 ml. Standard values from 0.125-25 ng of prolactin (NIAMD-Rat Prolactin-RP-1) were used.

Representative standard curves for both LH and prolactin are shown in

Figs, 3 and 4.

Site of Action of Pineal Antigonadotropin

Dose Response of Immature Mouse Ovaries to Pregnant Mare serum Gonadotropin

Charles River CD-I mice were housed under controlled conditions of temperature (25-26°C) and lighting (L:D = 14:10), Immature females were weaned at 21 days of age and experiments begun on day 24. Groups of eight mice were treated subcutaneously (sc) with Sigma pregnant mare serum gonadotropin (PMS) in amounts of 2.5, 5,0, or 10,0 International 100

90 -

80 -

70 -

60 -

B/T 50 -

40 -

30 -

20 -

10 -

0 _l -l l I 125 2.5 5 10 20 50 125 250

LH ng/ml

Fig. 3. Standard curve for LH radioimmunoassay B/T = Bound over total LH.

•j •o 100

90 80

70

60 «i

50

40

30 20

.25 .5 1.25 2.5 5 10 15 25 PROLACTIN ng/ml

Fig. 4. Stcindard curve for prolactin radioimmunoassay — B/T = Bound over total prolactin.

03 79 Units (IU) total dosage per mouse. The injections were begun in the

evening of the first day, with one-fifth of the total dosage in 0,1 ml

given morning and evening for three days (five injections total). On

the morning of the fourth day, the animals were sacrificed and the

ovaries weighed to the nearest 0.1 mg on a torsion balance. Mean

weights for each group were compared with saline-treated controls.

Dose Response of Immature Mouse Ventral Prostates to Human Chorionic Gonadotropin

Immature male mice were similarly treated for this experiment.

A total dosage of HCG was injected as above. Dosages used were 5.0,

10.0, and 20.0 IU. On the fourth day the ventral prostates were removed

and weighed to the nearest 0.1 mg on a torsion balance. Comparisons were made between groups and saline-treated controls.

The experimental design for the dose response curves for PMS and

HCG was developed through trial and error with different amounts of the

hormones. The dosages have been confirmed for mice (Vaughan, Vaughan,

Blask, et al. 1976).

Effect of Pineal Extract on Pregnant Mare Serum Gonadotropin Stimulation of Ovaries in Immature Female Mice

Sixty gm of bovine pineal glands were prepared by the organic solvent extraction method. Half of the resulting material was divided into five equal parts, dissolved in saline, and injected daily intra- peritoneally (ip) into groups of eight 45-day-old mature mice for the purpose of testing the activity of the extract. On the sixth day, the

animals were sacrificed and their ventral prostates weighed to the 80

nearest 0,1 mg on a torsion balance. Mean ventral prostate weights were

compared to those of a group of saline-treated controls. This portion

of the extract gave 0.78 gm of starting material/mouse/day.

The 24-day-old immature female mice-were divided into three

groups of eight mice each. The first group received 0.1 ml saline sc

and 0,2 saline ip morning and evening for three days, beginning on the evening of day one (five total injections). The second group, injected on the same time schedule, received 0.2 ml saline ip and 1.0 IU PMS sc per injection. This amount gave a total dose of-5,0 IU PMSf a value near the middle of the established dose response curve. The third group received a total of 5,0 IU PMS sc and 0.2 cc of a solution con­ taining the same organic solvent extract injected into the mature mice.

One quarter of that extract was used for the eight mice. On the fourth day the mice- were sacrificed and their ovaries weighed to the nearest

0.1 mg on a torsion balance. Mean ovarian weights for each group were compared. These mice were treated with only 0.38 gm of starting material/mouse/day but they weighed approximately only 15 gm while the mature mice weighed 30-40 gm.

Effect of Pineal Extract on Human Chorionic Gonadotropin Stimulation in Immature Male Mice

The foregoing experiment was done similarly for immature male micQ, which were 24 days old. The first group received saline sc and ip. The second group received saline ip and a total dose of 10,0 IU of HCG sc. This dose was near the middle of the dose response curve.

The third group received 10,0 IU HCG sc and one quarter of the extract 81 prepared, for^these experiments, ip. On the fourth day the animals were sacrificed and the ventral prostates weighed to the nearest 0.1 mg on a torsion balance. The means for each group were compared.

Dose Response of Castrate Mouse Ventral Prostates to Prolactin

Mature male mice, eight weeks old, were castrated under ether anesthesia. Six days later, 1.5 pg/gm body weight of testosterone pro­ pionate was injected sc in corn oil, morning and evening, starting on the evening of the first day. Injections were continued for three days with five injections. Control castrates received corn oil sc and saline ip. Three additional groups received the testosterone and either 0.2,

0.4, 0.8, or 1.0 lu/gm body wt of ovine prolactin (Sigma, 32 IU/mg) ip per injection simultaneously. Animals were sacrificed on the fourth day and their ventral prostates weighed to the nearest 0.1 mg on a Cahn model #7000 electrobalance.

Effect of Pineal Extract on Luteinizing Hormone Release from Luteinizing Hormone-Releasing Hormone-Stimulated Rat Anterior Pituitary Glands In Vitro

Charles River CD rats (200 gin) were sacrificed and pituitaries taken for incubation. Half pituitaries were placed in polyethylene scintillation vials in a Dubnoff shaking incubator at 37°C. Each vial contained two ml of Krebs-Ringer buffer (KRB) equilibrated with oxygen.

The glands were preincubated for fifteen minutes, and were then placed in flasks containing fresh buffer. In five of the flasks, 0,2 ml of

UM05R from an aqueous extract (10 mg/ml) was placed. A 20 p equivalent portion of this extract had been active in ventral prostate reduction 82 in eight mice (0.5 gnj/mouse/day). The flasks were incubated for 30 min, and then 50 ng of LHRH in 0.1 ml KRB was added to each flask. For each half pituitary incubated with UM05R and LHPH, a half pituitary from the same rat was incubated without UM05R and with synthetic LHRH, The

LHRH was graciously supplied by Dr. Victor Hruby, Department of

Chemistry, The University of Arizona. Additional amounts of LHRH {25,

50, and 100 ng) were added to additional half pituitaries to obtain a dose response curve. Two half pituitaries served as untreated controls.

Incubation was carried out for three additional hours and the media were stored for LH RIA.

Effect of Microinjection of Pineal Extract into the Rat Pituitary and Median Eminence on Luteinizing Hormone and Prolactin

Extract. Thirty gm of bovine pineal glands were subjected to aqueous extraction and ultrafiltration. A 20 gm equivalent portion was lyophilized and used for testing on ventral prostate reduction in mice.

The remaining ten gm equivalents were divided into ten one gm aliquots to be used for microinjection. These were dissolved in 0.5 ml saline for use in each microinjection experiment.

Animals. Charles River CD male rats were kept under controlled conditions of temperature (25-27°C) and lighting(LiD = 12:12). The long- term castrated animals were gonadectomized under ether anesthesia at 24 days of age and microinjected one month later. The short-term castrated animals were gonadectomized at the time of microinjection, which was done at the age of nine weeks. 83

Microinjection Technique. Glass cannulas were pulled from capillary tubes and cemented to the stubs of 19 gauge needles. The rats were placed in a Knopf sterotaxic instrument equipped with a micrometer driven syringe for microinjection. The syringe was filled with water and solutions for injections were drawn into the cannula by reversing the micrometer. A bubble was drawn into the cannula followed by one pi of toluidine blue stain, another bubble, and finally one pi of either pineal extract (2 ng starting material/injection) or saline. A bone flap was removed from the center of the skull with a Reiter-Hoffman pinealectomy trephine attached to a dental drill. The cannula was in­ serted 0.2 mm lateral to the superior sagittal sinus. For intra- pituitary injections insertion was 3 mm anterior to the interaural line, whereas tp inject the median eminence insertion was 5 mm anterior

{DeGroot 1959)j the incisor bar was set 5.0 mm above the horizontal interaural line. The cannula was advanced downward until it was ob­ served to bend slightly as it contacted the base of the skull. It'was then raised until it straightened and microinjection was begun by turning the micrometer slowly over a period of one to two minutes. One gm equivalent of extract in 0,5 ml of saline was prepared fresh daily for each group of extract-treated rats.

Sampling and Sacrifice. The long-term castrates were anesthe­ tized with urethane {0.2 gm/100 gm body wt) sc. One ml of blood was taken from the internal jugular vein at 0, 5, 15, and 30 min post- microinjection. At 60 min blood was removed by cardiac puncture per­ formed at thoracotomy. The pituitary and brain were removed and the 84

location of the dye was mapped in either the pituitary or median

eminence with the aid of a dissecting microscope. The serum and

pituitary extract were stored at -20°C for eventual IH and prolactin

RIA. The 24-hour castrate rats were anesthetized with ether, and blood and pituitaries were obtained at sacrifice as described for the 60 min sample above. LH.and prolactin for each group were compared with saline-treated controls.

Effects of Pineal flntigonadotropin in the Hamster

Effect of Short-Term Treatment of Male Hamsters

The extract used in this experiment was modified as follows:

Bovine glands were individually separated from their stalks, a technique which has been shown to remove the oxytocic activity, ostensibly due to the presence of AVT (Benson, Matthews, Hadley, et al. 1976). Forty and

100 gin portions were subjected to the organic solvent extraction described previously, with the exception that the amount of aqueous methanol was increased to 330 ml in order to adjust for the increased amount of starting material (100 gm). The 40 gm extract was prepared and divided into five equal portions for daily injection into a group of eight male golden Syrian hamsters. The second 100 gm extract was used for injec­ tion into this same group for five additional days (19 gm total starting material/hamster). On the 11th day, when the hamsters were 12 weeks old, they were sacrificed; their ventral prostates, seminal vescicles, and testes were weighed to the nearest 0.1 mg on a torsion balance. 85

Blood was taken for testosterone RIA and means for all values were com­

pared with those of saline-treated controls.

Effects of Long-Term Treatment of Male Hamsters

The effects of long-term treatment with bovine pineal gland extract were assessed in male hamsters which were either intact or blinded by optic enucleation under ether anesthesia. The intact animals received ether anesthesia without surgery. Treatment began the day after surgery, and was continued for eight weeks while the animals were maintained on L:D = 14:10. The first group of five animals re­

mained intact and was treated daily with 0.2 ml saline ip. The second group was blinded and similarly treated. The third group was blinded and treated daily with an organic solvent extract of glands with stalks removed which had not been ultrafiltered. A 20 gm extract was prepared freshly each week (0.3 gm starting material/injection). The fourth group remained intact and received the extract daily. At the end of eight weeks the animals were sacrificed and their ventral prostates,' seminal vesicles, and testes weighed to the nearest 0.1 mg on a torsion balance. After thoracotomy under ether anesthesia, blood was collected by cardiac puncture and the serum prepared for IH RIA, After weighing to the nearest 0.1 mg on a Mettler Model H51AR balance, the anterior pituitaries were homogenized in two ml 0.01 M phosphosaline buffer pH

7.6, Both blood and pituitary homogenate were stored at -20°C for eventual LH RIA. 86

Effect of Extract on the 24-Hour Post-Castration Rise of Serum Luteinizing Hormone in Male Hamsters

Fifty gm of pineals, with stalks removed, were extracted with

organic solvent without ultracentrifugation, Five male golden Syrian

hamsters (eight weeks old) were castrated under ether anesthesia and on the same day received half of the extract (5 gm starting material/

animal). The other half was injected into each of a group of five eight-week-old Charles River CD rats on the day of castration in order

to test the activity of PA G Additional groups of five animals were treated with saline ip on the day of castration. Twenty-four hours later the animals were sacrificed and sera and anterior pituitaries collected for LH RIA.

Effect of Pineal Extract on the Luteinizing Hormone in Long-Term Castrate Hamsters

Five-week-old, male hamsters were castrated under ether anesthesia and treatment begun eight weeks postoperatively, A group of five animals was treated daily for five days with equal portions of a

40 gm organic solvent extract of bovine pineal glands with stalks removed (1 gm starting material/hamster/day). Five control castrates received saline. On the sixth day, both groups were sacrificed, and blood and pituitaries were taken for LH RIA.

Effect of Pineal Extract on Luteinizing Hormone Surge in Female Hamsters

The estrous cycle of six-week-old female hamsters was monitored through two cycles by observation of vaginal mucus. On the day of 87 estrus of the second cycle, a group of seven animals received ip in­ jections of one quarter of a 40 gm organic solvent extract of bovine glands with stalks removed. Another group of seven received saline ip at the same time. The other three quarters of the extract was used for an injection on the mornings of the following metestrus, diestrus, and proestrus. The hamsters were sacrificed from 1430-1530 hours on the day

of proestrus theoretically at the height of the LH surge (.approximately

six hours after the last injection). A group of five animals was also

sacrificed at 0800 when theoretically no LH surge was occurring. Serum

and pituitaries were saved for LH RIA.

Effect of Prolactin and Anterior Pituitary Grafts on Gonadal Atrophy in Blinded Male Hamsters

Two groups of ten male golden Syrian hamsters were blinded at

11-12 weeks of age under ether anesthesia. Three to four weeks post­ operatively, injections were begun with 3,2 IU of ovine prolactin

(Sigma) per hamster per day given sc. Control blinded hamsters received daily saline injections. Ten weeks postoperatively, the animals were sacrificed and their ventral prostates weighed to the nearest 0.1 mg on a Cahn #7000 electrobalance. Seminal vesicles and testes were weighed to the nearest 0,1 mg on a torsion balance. Blood and pituitaries were taken for prolactin and RIA,

In a similar experiment,' 6.4 IU of prolactin per hamster per day was begun on the day of blinding and then was given daily for seven weeks (including the morning of sacrifice). 88

Groups of 13 male golden Syrian hamsters were blinded under ether anesthesia at nine weeks of age. The first group simultaneously received two anterior pituitaries taken from donor hamsters (not litter-

mates) and pushed underneath the right kidney capsule from a posterior approach. The second group received two comparable-sized pieces of ! kidney tissuej from donor hamsters, A group of five hamsters served as unoperated, intact controls. Seven weeks postoperatively, the hamsters were sacrificed and their organs were treated as described above. This experiment was repeated with the use of three pituitaries.

Studies on the Effect of Arginine Vasotocin

j The effects of synthetic AVT (supplied by Dr. Victor Hruby) were tested in tenj-week-old male CD-I mice. Groups of eight mice were in­ jected ip with either saline or 1.55, 3.1, 6.25, or 12,5 yg AVT/mouse/ day for five days. On the sixth day the animals were sacrificed and the ventral prostates weighed to the nearest 0,1 mg on a torsion balance.

Synthetic AVT was also injected into groups of eleven male and female Charles River CD-I mice which were 22 days old. controls re j ceived water ip, while AVT-treated animals received one pg/mouse/day.

The females were injected for three days, the males for four days. The | animals were sacrificed 24 hours after the last injection. Ovaries, uteri, ventral prostates, seminal vesicle-coagulating glands, and testes were weighed to the nearest 0,01 mg on a Mettler analytical balance.

Comparisons were made between AVT-treated animals and controls.

Since AVT has been localized to the stalk of the bovine pineal gland (Benson. Matthews, Hadley, et al. 19-76) , 50 gm of stalks were 89 extracted without ultrafiltration by the organic solvent method. The resulting powder was lyophilized in five equal portions for injection daily for five days into eight-week-old mice. Groups of eight mice received the extract or saline. On the sixth day, the animals were sacrificed and ventral prostates weighed to the nearest 0.1 mg on a torsion balance.

Statistical Methods

All data were analyzed for mean, standard deviation, and standard error with a Hewlett-Packard model 65 computer. Significant differences between means were determined by a computer program for the one-way analysis of variance. RESULTS

Site of Action of Pineal Antigonadotropin

Direct Gonadal Effects of Pineal Antigonadotropin

PMS was found to stimulate ovarian weight in immature mice in a dose response fashion, A dosage of 5 IU stimulated ovarian weight over saline-treated controls and at the same time did not produce maximal stimulation. The results are illustrated in Fig. 5. Likewise HCG was observed to stimulate ventral prostate weight in immature male mice in a dose response fashion with 10 IU being an intermediate dose (Fig, 6).

The organic solvent extract used for this experiment signifi­ cantly reduced ventral prostate-weight in mature, eight-week-old mice

(Fig. 7).

When this same extract was injected into immature mice simul­ taneously with 5.0 IU PMS, no significant effect was seen on the stimu­ lation of ovarian weight by the exogenous hormone (Fig. 8). This extract also had no effect on ventral prostate weight when injected simultaneously with exogenous HCG (10 IU; Fig, 9),

Effect on Pituitary Glands In_ vitro

Synthetic LHRH was found to stimulate IHRH release from rat pituitary halves in a dose response fashion. PAG had no effect on IH output from LHRH-stimulated pituitaries (Fig. 10},

90 91

NUMERICAL 3.0 3.6 VALUES + + •7e 15

10 OVARIAN WT (mg)

L L 2.5 5 10 PMS (IU)

Fig. 5. Dose response of immature mouse ovaries to PMS — a = Mean ± standard error of the mean (SEM); b = p < 0.001 compared to saline-treated controls; n (number of animals per experi­ mental group) - 8. NUMERICAL 11.5° I7.8b 20 Ab 24.3^ VALUES + + + + 0.6 1.2 0.9 L9 25 T b "T" 20 VENTRAL PROSTATE 15 WT (mg/IOOgm body wt) 10

5

0 L L 10 20

HCG (III)

Fig. 6. Dose response of immature mouse ventral prostate to HCG • a = Mean ± SEM; b = p < 0.001 compared to saline-treated controls; n = 8. 27.1° b NUMERICAL I9.6 + 4-• VALUES ITI u

30 — VENTRAL a PROSTATE 20

WT 1 (mg/IOOgm b body wt) 10

o 1 saline pineal extract

Fig. 7. Effect of pineal extract on ventral prostate in mature mice a = Mean ± SEM; b = p < 0.001 compared to saline-treated controls; n = 8. 94

3.0 ll.9b NUMERICAL + + VALUES 0^25 K2 I5r

10 OVARIAN WT {mg)

L L saline saline pineal extract PMS + PMS

Fig. 8. Lack of ail effect of pineal extract on PMS stimulation of immature mouse ovaries — a = Mean ± SEM; b = p < 0.001 when compared to saline-treated controls; c = not significant (NS) when compared to saline + PMS; n = 8. 95

10.1 " 22.2 19.7c NUMERICAL + + VALUES L3 r.2 0?8

30 VENTRAL PROSTATE 20 ,, WT b b (mg/IOOgm c body wt) 10 a

OL- L1 i saline saline pineal extract HCG + HCG

Fig. 9. Lack of an effect of pineal extract on HCG stimulation of immature mouse ventral prostates — a = Mean ± SEM; b = p < 0.001 when compared to saline-treated controls; c = NS when compared to saline + HCG; n = 8. NUMERICAL 15.7 73.2 85.2 111.69 89.8 + + 80.5 VALUES + + t . + I7I 12 l?3 0T3 172 16.7

125 100 X MEDIA 75 I LH (ng/ml) 50

25

0 - 0 25 ng 50 ng OOng 50ni50ng 50 ng LHRH LHRH LHRH LHRH LHRH LHRH + + saline pineal extract

Fig. 10. Absence of an effect of pineal extract on LH output from LHRH-stimulated rat anterior pituitaries — a = Mean ± SEM; n = 3 for standard curve and 5 for saline versus pineal extract.

cn 97 Effect of Microinjection of Pineal Extract

In rats sacrificed 24 hours after microinjection and castration,

there was a significaht increase in pituitary LH content in animals in­

jected in the median eminence compared to saline-treated controls, but

no significant change in serum LH. Intrapituitary injection produced

no significant effect. The results are shown in Table 1. There was no

effect on prolactin following median eminence or intrapituitary injec­

tion with UM05R; this extract, however, produced a non-significant re­

duction in ventral prostate weight in mice, so its activity is in

question. In the long-term castrate rat, prolactin was decreased in

serum and pituitary in the first hour with any manipulation of the

animal, so these results could not be interpreted.

Dose Response of Castrate Mouse Ventral Prostate to Prolactin

The weights of the ventral prostate of castrate mice, which had been treated with either testosterone alone or testosterone plus

prolactin, were significantly different from each other only at the highest prolactin dosage (1.0 lU/gm body wt/injection). The result is shown in Table 2.

Effect of Pineal flntigonadotropin in the Hamster

Effect of Short-Term Treatment on Male Hamsters

After ten days of treatment with the organic solvent extract,

hamsters demonstrated significant reduction in the weights of the ventral prostates compared to saline-treated controls. The seminal Table 1. Effect of pineal extracts on rat LH after intrapituitary and median eminence micro­ injection under ether anesthesia.

Serum LH Pituitary LH Pituitary Wt Group (ng/ml) (Total yg) (yg/mg) (mg)

Pituitary microinjection: Saline 539.7 + 113.7a 588.4 + 52.6 122.1 + 10.8 4.7 + 0.31

Extract 648.0 + 122.2 684.1 + 47.8b 123.8 + 5.6 4.8 0.18

ME microinjection: Saline 500.3 + 124.7 682.3 + 13.8 116.0 6.5 6.0 + 0.33

Extract 962.8 ± 259.lb 736.8 + 20.9° 136.2 + 7.0b 5.5 + 0.29

aMean ± SEM.

NS when compared to saline-treated controls.

Qp < 0.05 when compared to saline-treated controls,

n = 10. 99

Table 2. Effect of testosterone plus prolactin on ventral prostate weight in castrate mice.

Ventral prostate wt Treatment (mg/100 gm body wt)

Corn Oil + Saline 13.4 ± 1.3

Testosterone + Saline 16.6 ± 1.4

Testosterone + Prolactin 22.8 + l.lb aMean ± SEM. kp « o.OOl when compared to testosterone + saline animals. n = 8. 100

vesicle-coagulating glands and the testes were not significantly changed

in weight. The results of duplicate experiments are shown in Fig, 11

and Table 3.

When serum testosterone was measured in those animals with sig­

nificantly reduced ventral prostates, there was a significant reduction

in serum testosterone in the first experiment. However, serum

testosterone was not significantly changed in the second experiment

(Fig. 12).

Effect of Long-Term Treatment on Male Hamsters t When male hamsters, both blind and intact, were treated with organic solvent extract for eight weeks, weight of the ventral prostates was reduced in both groups over saline-treated controls. The reduction was significant in the blind, extract-treated group, but not in the intact group (F.ig. 13). Seminal vesicle and testicular weights were not

significantly changed (Table 4).

Blinding significantly reduced hamster anterior pituitary weight, serum LH, total pituitary LH, and pituitary LH concentration, when results were compared with intact controls. There was no sig­ nificant difference between saline- and extract-treated blinded animals. Treatment with extract increased serum LH and decreased pituitary LH and LH concentration in intact animals, but these values were not significant (Table 5). 46.9° 33.2b 43.3 25.5' NUMERICAL + + 4- + VALUES 2?6 371 2~e z.i 50

40 VENTRAL PROSTATE 30 WT (mg/IOOgm 20 body wt)

10

0L L saline pineal saline pineal extract extract

Fig. 11. Effect of pineal extract on ventral prostate weight in hamsters — a Mean ± SEM; b = p < 0.001 when compared to saline-treated controls; n = 10.

H O H 102

Table 3. Effect of pineal extracts on seminal vesicle and testicular weight in hamsters.

Seminal Vesicle Wt Testes Wt Exp # Treatment (mg/100 gm body wt) (mg/100 gm body wt)

1 Saline 329 ± 25a 2419 ± 235

Pineal Extract 297 + iob 2758 ± 87b

2 Saline 325 ± 11 2987 ± 57

Pineal Extract 290 ± 14b 3168 ± 119b aMean ± SEM. bNS when compared to saline-treated controls, n = 10. 103

2037° 506b 1357 1444 NUMERICAL + + i - VALUES 612 134 278 303 3 000

2000 SERUM TESTOSTERONE (pg/ml)

1000

OL L saline pineal saline pineal extract extract

Fig. 12. Effect of pineal extract on serum testosterone in hamsters — a = Mean ± SEM; b = p < 0.05 when compared with saline-treated controls; n = 10. 104

NUMERICAL 42.9° 34.7 34.4 25.3' + + + + VALUES 573 L2 2~6 L7 50

40 VENTRAL PROSTATE 30 WT (mg/IOO gm 20 body wt)

10

oL L intact intact blind blind saline pineal saline pineal extract extract

Fig. 13. Effect of long-term treatment with pineal extract on ventral prostate weight in intact and blinded hamsters — a = Mean ± SEM; b = p < 0.025 when compared to saline-treated controls; n = 5. 105

Table 4. Effect of pineal extracts on seminal vesicle and testicular weight in blinded and intact hamsters.

Seminal Vesicle Wt Testes Wt Group (mg/100 gm body wt) (mg/100 gm body wt)

Intact Saline 429 + 3ia 2612 + 116

Intact Pineal Extract 434 + 33 2128 + 263

Blind Saline 228 + 32 1465 + 394

Blind Pineal Extract 190 + 31 1270 ± 393

aMean ± SEM. n = 5. 106

Table 5. Effect of pineal extracts on LH in intact and blinded male hamsters.

Serum LH Pituitary LH Pituitary Wt Group (ng/ml) (total yg) {yg/mg) (mg)

Intact Saline 144.6 ± 41.6 a 242.9 ± 56.9 92.4 ± 19.1 2.6 ± 0.13

Intact Extract 274.7 ± 49.6 202.3 ± 57.2 73.9 ± 15. 3 2.6 ± 0.21

Blind Saline 14.5 ± 1.4 b 136.4 ± 37.2 71.2 ± 17.3 1.8 ± 0.19°

Blind Extract 9.8 ± 2.6 b 131.7 ± 29.2 65.7 ± 11.7 1.9 ± 0.14° aMean ± SEM. kp < 0.001 when compared to intact saline-treated controls,

p < 0.01 when compared to intact saline-treated controls. 107

Effect on the 24-Hour Post-Castration Serum Luteinizing Hormone Rise in Male Hamsters

One-half of the organic solvent extract which significantly re­ duced the 24-hour post-castration rise in LH in male rats significantly elevated the 24-hour post-castration rise in LH in the male hamster I

Pituitary LH was not significantly affected (Table 6). In contrast, the pituitary weight in extract-treated rats was significantly reduced» however, there was not a significant effect on pituitary LH content or concentration.

Effect of Pineal Extract on Long-Term Castrate Hamsters

Organic solvent extract, injected for five days into male hamsters eight weeks after castration, tended to increase serum LH, pituitary LH content and concentration, and pituitary weight, but none of these values were significant (Table 7).

Effect of Pineal Extract on Luteinizing Hormone Surge in Female Hamsters

Organic solvent extract injected throughout the previous estrous cycle did not significantly change the magnitude of the LH surge in female hamsters (Fig. 14).

Effect of Prolactin and Anterior Pituitary Grafts on Gonadal Atrophy in Blinded Male Hamsters

Daily injections of prolactin, in doses of either 3.2 or 6.4 lU/day, partially blocked the gonadal atrophy observed in blinded hamsters when compared to blinded saline-treated controls. The decrease in ventral prostate and seminal vesicle weight was also reduced (Table 8). Table 6. Effect of pineal extracts on the 24-hour post-castration rise in serum LH in rats and hamsters.

Serum LH Pituitary LH Pituitary Wt Group (ng/ml) (total yg) (yg/mg) (mg)

Rats 24 hour post-sham saline treated 38.5 ± 7-4 258.8 + 11.9 37.5 ± 1.1 6.9 + 0.49

Rats 24 hour post-gonadectomy saline treated 2129.7 ± 155.9 303.1 ± 41.2 43.8 ± 4.9 6.7 ± 0.21

Rats 24 hour post-gonadectomy extract treated 1301.5 ± 165.4b 242.6 ± 31.0 43.1 ± 3.6 5.6 ± 0.40C

Hamsters 24 hour post-gonadectomy saline treated 602 ± 98.9 194.9 ± 38.8 86.4 ± 17.2 2.3 ± 0.11

Hamsters 24 hour post-gonadectomy extract treated 1019 ± 69.8b 247.6 ± 48.1 109.7 ± 19.3 2.2 ± 0.21

Mean ± SEM.

bp < 0.01 compared to saline-treated controls,

p < 0.05 compared to saline-treated controls,

n = 5.

o 00 Table 7. Effect of pineal extract on LH in long-term castrate hamsters

Serum LH Pituitary LH Pituitary Wt Group (ng/ml) (total yg) (yg/mg) (nig)

Saline Treated 625.2 ± 93.7a 1265.8 ± 387.4 267.9 ± 58.7 4.3 ± 0.51

Extract Treated 852.7 ± 222.6 1784.4 ± 158.6 320.7 ± 13.7 5.5 ± 0.28 110

2683 2583 NUMERICAL + VALUES ± 403 216

4000

uj _j 3000 m SERUM 2 LH o UJ (ng/ml) 2000 t— aUJ z Z3 1000

J 0830 1500 1500 proestrus proestrus proestrus saline pineal extract

Fig. 14. Lack of an effect of pineal extract on the LH surge in female hamsters — a = Mean ± SEM; n = 7. Table 8. Effect of exogenous prolactin on gonad and accessory organ weight in blinded hamsters.

Ventral Prostate Wt Seminal Vesicle Wt Testes Wt Group (mg/100 gm body wt) (mg/100 gm body wt) (mg/100 gm body wt)

Blinded Saline 20-8 + 1.4a 108 + 8 223 19

Blinded Prolactin K (3.2 IU/day) 28.0 + 2.6 148 ± 14 806 + 205°

Blinded Saline 15.9 + 1.0 107 + 9 244 + 18

Blinded Prolactin A a (6.4 IU/day) 28.9 + 1.6 176 ± 14 764 + 124 aMean ± SEM.

p < 0.05 when compeared to saline-treated controls.

Cp < 0.025 when compared to saline-treated controls.

dp « 0.001 when compared to saline-treated controls,

n = 10. 112

Hamsters receiving kidney tissue grafted under the kidney capsule at the time of blinding exhibited gonadal atrophy, as has been previously reported. Two anterior pituitaries grafted under the kidney capsule almost completely inhibited the effects of blinding. The weight of testes and seminal vesicles was significantly decreased in hamsters with pituitary transplants, but these organs appeared to be functional as evidenced by presence of seminal and epididymal fluid, large seminiferous tubules, and sperm on testicular smears. Ventral prostates were increased in weight compared to unblinded controls, but this difference was not significant. Results for the testes are shown in Fig. 15, those for ventral prostates and seminal vesicles in Fig. 16,

When three anterior pituitary grafts were used in a repeat experiment, the testes were completely maintained and their weights were even above unblinded control values, but this difference was not significant

(Table 9).

Prolactin was significantly reduced in the sera of blinded animals when compared to intact controls, except for those blinded hamsters receiving anterior pituitary homografts in which serum prolactin was normal. Pituitary prolactin was also reduced in blinded animals compared to intact controls (Table 10). Prolactin was not sig­ nificantly elevated in the sera of animals receiving daily injections of prolactin compared to blinded animals receiving saline.

Effects of Arginine Vasotocin

Synthetic AVT had no effect on ventral prostate weight of adult male mice at any concentration used (Fig, 17). 113

Numerical Values

3000

Testes Wt (mg/IOOgm 2000 body wt) 1000 -

intact blind blind controls kidney pit graft graft

Fig. 15. Effect of either anterior pituitary (pit) or kidney homografts on testicular weights in blinded hamsters — a = Mean ± SEM; b = p « 0.001 compared to blinded hamsters with kidney grafts; c = p < 0.05 compared to intact controls; n = 13 for grafted hamsters and 6 for intact controls. Numerical 42.2 28.2 51.1 355 178 269c Values + + + + + + 4?6 2.5 3?0 30 14 15

50 JL X 40 400 Ventral Seminal Prostate Vesicle 30 Wt 300 Wt (mg/IOOgm b 20 c (mg/IOOgm body wt) 200 50{Jy Wf)

10 100

JL intact blind blind intact blind blind controls kidney pit controls kidney pit graft graft graft graft

Fig. 16. Effect of either anterior pituitary or kidney homografts on ventral prostate and seminal • vesicle weights in blinded hamsters — a = Mean ± SEM; b = p « 0.001 compared to blinded hamsters with kidney grafts; c = p < 0.025 compared to intact controls; n = 13 for grafted hamsters and 6 for intact controls.

H H Table 9. Effect of either anterior pituitary or kidney homografts on testicular and accessory organ weights in blinded hamsters.

Testes Wt Ventral Prostate Wt Seminal Vesicle Wt Treatment Group (mg/100 gm body wt) (mg/100 gm body wt) (mg/100 gm body wt)

Unoperated, intact Control 2132 ± 14ia 40.0 ± 3.7 505 ± 21

Kidney Graft/ Blinded 1299 ± 219b 42.6 ± 2.6 353 ± 22b

Anterior Pituitary Graft, Blinded 2580 ± 187° 53.5 ± 5.2d 463 ± 44°

3Mean ± SEM.

p « 0.001 compared to unblinded controls. c NS compared to unblinded controls.

^p < 0.05 compared to unblinded controls.

n = 10 for grafted hamsters and 6 for intact controls. 1X6

Table 10. Effect of prolactin injection and anterior pituitary homografts on prolactin in the blinded hamster.

Serum Prolactin Pituitary Prolactin Treatment Group (ng/ml) (yg/mg)

Unoperated, Intact Control 21.5 ± 2.4a 0.55 ± 0.10

Kidney Graft, Blinded 11.9 ± 0.5b 0.37 ± 0.05

Anterior Pituitary Graft/ Blinded 22.8 ± 1.3 0.32 ± 0.03

Saline Control, Blinded 11.9 ±0.5 0.28 ± 0.03

Prolactin-treated Blinded 12.7 ± 0.4 0.28 ± 0.03 aMean ± SEM. kp « 0.001 compared to intact controls. 117

37.8 34.8 40.0 365 37.9 NUMERICAL + + + + + VALUES 276 3?4 2?9 L3 23

40 JL VENTRAL 30 PROSTATE WT (mg/IOOgm 20 body wt) 10

L L saline l.55;jg 3.ljug 6.25jjg I2.5;jg AVT/day AVT/day AVT/day AVT/day

Fig. 17. Lack of an effect of AVT on ventral prostate weight in adult mice — a = Mean ± SEM; n = 8. 118

When AVT was injected at a dosajge of one yg/day into 22-day-old female and male mice, there were significant decreases in ventral prostate and accessory organ weights when the values were corrected for body weight. These decreases were not significant when uncorrected weights were used. The previously reported decrease in ovarian weight was not observed (Table 11 and 12).

When mature male mice were treaited with an organic solvent i extract of bovine pineal stalks, no significant reduction of ventral [ prostate weight was observed (Fig. 18). 119

Table 11. Effect of AVT in iinmature female mice.

Body Wt Ovarian Wt Uterine Wt Group (gm) (mg) (mg/100 gm) (mg)

Water 10.7 ± 0.6a 4.16 ± 0.35 39.2 ± 3.2 6.96 ± 0.71

AVT 10.7 ± 0.7 3.99 ± 0.39 37.0 ± 2.3 7.06 ± 0.73

3Mean ± SEM. n = 11. Table 12. Effect of AVT in immature male mice.

Body Wt Ventral Prostate Wt Seminal Vesicle Wt Testis Wt Group tgm) (mg) (mg/100 gm)' (mg) (mg/100 gm) (mg) (mg/100 gm)

Water 12.1 ± 0.94a 1.52 ± 0.19 12.4 ± 0.84 7.3 ± 1.1 58.5 ± 4.4 62.5 ± 5.9 514 ± 19

AVT 11.6 ± 0.87 1.06 ± 0.12 9.1 ± 0.79b 5.5 ± 0.9 44.9 ± 4.1° 53.5 ± 6.4 451 ± 25 aMean ± SEM. kp < 0.01 when compared to water-treated controls.

p < 0.05 when compared to water-treated controls. n = 11. 121

49.0 45.7 NUMERICAL + + VALUES 4~l 3~7 60

50

40 VENTRAL PROSTATE 30 WT (mg/IOOgm body wt) 20

10

L L pineal saline stalk extract

Fig. 18. Lack of an effect of bovine pineal stalk extract on ventral prostate weight — a = Mean ± SEM; n = 8. DISCUSSION

Previous studies in our laboratory strongly suggest that bovine, ovine, human, and rat pineal extracts contain an antigonadotropic sub­ stance which can be separated from the melatonin by gel-filtration

(Benson, Matthews, and Rodin 1971, 1972> Matthews et al. 1971; Ebels et al. 1973). The substance was eluted in a ninhydrin-positive peak on

Sephodex G-25 and G-10, and is in the range of polypeptides (Benson,

Matthews, and Rodin 1972) Ebels et al. 1973), Further evidence for its polypeptide nature was the fact that it was deactivated by proteolytic enzymes (Matthews and Benson 1973). We have called this substance

"pineal antigonadotropin" (PAG). Extracts containing this compound have been shown to reduce ventral prostate and ovarian weights in mice

(Benson, Matthews, and Smith 1971), inhibit compensatory ovarian hyper­ trophy following unilateral ovariectomy in mice (Benson, Matthews, and

Rodin 1971), reduce gonad and accessory organ weight in immature mice

(Ebels et al. 1973) , reduce the 24-hour post-castration rise in LH in rats (Orts and Benson 1973), and reduce fertility in mice (Benson,

Matthews, and Hruby 1976). The work reported in this dissertation was an attempt to identify the site(s) of action of PAG, to test its effects in the hamster, and to further differentiate its activity from that of the cyclic octapeptide, arginine vasotocin (AVT).

122 123

Site of Action of Pineal Antigonadotropin

PAG's site of action was studied by testing the effects of bovine pineal extracts directly on the gonad, pituitary, and hypo­ thalamus. An extract which reduced the ventral prostate weight of mature male mice was found to have no effect on the stimulation produced by PMS or HCG in immature mice. PMS has FSH activity, and was used to stimulate ovarian weight in immature mice. An amount of PMS which had previously produced moderate response was selected for use in testing the extract containing PAG to avoid supramaximal stimulation, This amount of PMS actually produced stimulation of ovarian weight to approximately 13 mg. Although this was near the highest value on the dose response curve, stimulation up to 25 mg has been observed, so this effect clearly did not represent supramaximal stimulation. Similarly,

PAG did not affect the stimulation of the ventral prostate observed after injection of HOG, From these results it was concluded that PAG did not block the effects of exogenous gonadotropins on the ovary or ventral prostate, although it was effective in animals whose hypothalamo-hypophyseal system was functioning.

An attempt was made to study the effect of PAG on exogenous prolactin, which was used synergistically with testosterone to stimulate the ventral prostates of castrate mice. This synergistic effect of prolactin and testosterone was confirmed in these studies, There was a significant difference between the weights of the ventral prostates of animals receiving testosterone alone and those of animals treated with both testosterone and prolactin. If PAG works directly on the ventral prostate by blocking the action of prolactin, one should be able by the 124 injection of PAG to block the synergistic action and limit the effect.to that of testosterone alone. Unfortunately we had no active extract to use, but the design of the experiment is there:; for use when extracts become available. We expect that PAG will have no effect in this experiment, because intracarotid injections of it resulted in decreased prolactin within 30 minutes (Benson, Larsen, and Richardson 1978), a finding which suggests a central action in the brain or pituitary.

In vitro studies with the rat pituitary showed that pineal extracts containing PAG had no effect on the LHEH-stimulated release of

LH from anterior pituitary glands. Pineal extracts have recently been observed to contain both prolactin-releasing and release-inhibiting factors, but the UMOSR containing PAG had no effect on prolactin release from the rat pituitary in vitro (Change and Ebels 1978).

All these studies indirectly suggest that PAG mediates its action in the hypothalamus. To test this hypothesis further, extracts containing PAG were injected directly into either the pituitary or medicin eminence of rats that had either been castrated for four weeks or were castrated at the time of microinjection. The effects of in vivo injection of PAG into the pituitary and median eminence on serum and pituitary prolactin and LH were tested. In one experiment, total pituitary LH increased 24 hours after castration, when PAG was injected into the median eminencet serum LH and pituitary LH concentration also increased but these values were not significant. These data suggest an increase in both the synthesis and release of LH after injection into the medicin eminence, but not the pituitary; no significant effect was 125 observed by intrapituitary injection, further supporting a hypothalamic mechanism of action.

The effects of median eminence and intrapituitary injections on prolactin were also measured. In the long-term castrate, this experi­ ment was impossible to interpret, because injection of both saline and extract into either pituitary or median eminence resulted in inhibition of prolactin release in the first hour, Intrapituitary and median eminence injection of extract did not affect prolactin 24 hours after castration, but the extract used in this particular experiment did not produce significant reduction in ventral prostate weight in adult mice.

The extract was probably only borderline in its activity.

An active extract appeared to work in the median eminence or hypothalamus; we have observed no extract that seemed to work on the gonad or at the pituitary level. We suspect that PAG works in the hypothalamus, and are still engaged in experiments to provide conclu­ sive evidence.

Effect of Pineal Antigonadotropin in the Hamster

Since pineal extracts containing PAG have been observed to reduce ventral prostate weight and to produce lysis of corpora lutea in mice, it was concluded that LH-dependent structures are affected. This plus the fact that similar extracts have been observed to reduce the post-castration rise in serum LH in rats led us to hypothesize that the pineal gland decreases pituitary and thus serum LH. Decreased LH was assumed to reduce gonad and accessory organ weight and function, leading to the gonadal quiescence observed in this species following blinding 126 or shortened photoperiod. As has been observed in the mouse.(Benson,

Matthews, and Smith 1971), short-term treatment with pineal extracts decreased ventral prostate weight in hamsters, without effects on the weights of seminal vesicle or testis. The reduction seemed to be accentuated in animals which had been both blinded and treated for a long period.

In the present study, however, even after eight weeks of daily injections of extract, ventral prostate reduction was the only ob­ servable effect on the reproductive organs. Moreover, this effect was not as pronounced as that observed in animals treated short-term for ten days. This could be explained by postulating the induction of PAG antibodies with long-term treatment or induction of a suppressive feed­ back mechanism. In animals with reduced ventral prostate weights as a result of treatment with PAG, serum and pituitary LH were not different from saline-treated controls. Blinding, however, reduced both serum and pituitary LH to a highly significant degree at ten weeks. Although such reductions in the blinded hamster have been inconsistent (Reiter and

Johnson 1974a), these two hormones were always significantly reduced when measured in hamsters blinded in our laboratory. Blask (1978) thinks that Reiter';s values are at the lower limit'of detectability, and are read using the logit transformation, while ours seem to lie on the linear portion of the curve. This interpretation may explain the incon­ sistent results obtained by Reiter's group.

In one experiment, there was a significant decrease in serum testosterone in animals with reduced ventral prostates following short- terra treatment, but this effect was not substantiated in a repeat 127

experiment, These variations are not unexpected, since serum testoster­

one fluctuates markedly with any alteration of hormonal or emotional

state and shows significant change even throughout the 24-hour period.

One may conclude that the decreased testosterone in the one experiment

has some meaning,but its exact significance is unclear. If testosterone

is significantly reduced only at certain times, this could produce marked

anti-reproductive effects,

Further negative anti-LH effects were observed when an extract

containing PAG was injected into hamsters. The extract, which signifi­

cantly reduced the 24-hour post-castration rise of LH in rats, sig­

nificantly elevated the 24-hour post-castration rise of LH in hamsters,

PAG also had no effect on the LH surge in female hamsters, nor did it

elevate serum and pituitary LH in long term castrate hamsters.

Although these results do not confirm our original hypothesis,

they are in keeping with previous experiments that show that gonadal

function in hamsters housed in L:D = Is23 was not maintained by in­

jection of IHRH (Reiter, Vaughan, Blask, and Johnson 1975). The

pituitary hormones which appeared to be consistently decreased in the light-deprived hamster in gonadal quiescence were LH and prolactin

(Reiter 1972a, 1975b; Reiter and Johnson 1974a}. In our laboratory, serum values of LH and prolactin are always significantly decreased 8-10 weeks following blinding. Since the results did not indicate a PAG-mediated decrease in LH, we decided to test whether the effects of the pineal gland in the hamster were mediated through prolactin. 128

Bartke et al. (1975) showed that injection of prolactin partially restored gonad and accessory organ weights in intact hamsters maintained in short photoperiods. In the present studies, we observed this same phenomenon in the blinded hamster. The partial response was assumed to result from the single daily injection, Which probably has the disadvantage of producing a solitary daily rise in blood levels with a subsequent low at a sub-normal level. This assumption was sup­ ported by our RIA data, in which serum and pituitary prolactin levels for both blind, saline-treated and blind, prolactin-treated hamsters were the same. Certainly, however, these values could also be due to lack of cross-reactivity of the ovine prolactin in the rat RIA.

Homoplastic anterior pituitary grafts were utilized in an attempt to overcome this problem. In rats, viable pituitary homografts have the advantage of maintaining prolactin at a steady blood level, since transplantation of the anterior pituitary to a nonsellar site removes it from inhibitory control by the hypothalamus. Such grafts are thought not to produce other pituitary hormones that are under the control of hypothalamic trophic factors. In the hypophysectomized, ovariectomized rat, blood levels of prolactin returned to normal with transplantation of one pituitary gland beneath the kidney capsule.

Furthermore, blood levels of prolactin increased progressively with in­ creasing numbers of homografts. The transplantation of four pituitaries, for example, to a single animal increased the value to that seen in pseudopregnancy (Chen et al. 1970). Transplantation of the anterior pituitary has not been studied in the hamster, but it can be assumed that similar behavior would occur in this species. 129 The homoplastic anterior pituitary grafts were observed to main­ tain ventral prostate, testicular, and seminal vesicle-coagulating gland weights at functional levels in the blinded hamster. These functional states were evidenced by the presence of seminal and

epididymal fluid, large seminiferous tubules, and sperm on testicular smears-—none of which were seen in blinded hamsters bearing grafts of kidney fragments. Two grafts were observed to restore serum prolactin to normal levels and to partially restore gonad and accessory organ weight, while three grafts completely restored the weights of these organs. Serum and pituitary prolactin have been shown to fluctuate in a circadian rhythm, and it is possible that the chronic level produced by the two anterior pituitary grafts may not have reached a peak as high as that for the normal hamster. From these results, it may be hypothesized, that a fall in blood prolactin levels occurs as a primary event in gonadal atrophy following blinding in this species.

To recapitulate material covered earlier, the.effects of pineal extracts observed in our laboratory include reduction of ventral prostate and ovarian weights in mice, inhibition of COH following uni­ lateral ovariectomy in mice, reduction of gonad and accessory organ weight in immature mice, reduction of ventral prostate weight in hamsters, reduction of the 24 hour post-castration rise in LH in rats, reduction of fertility in mice, and blockage of ovulation in rats

(Benson, Matthews, and Smith 1971; Benson, Matthews, and Hruby 1976).

Microscopically, a reduction in the height of ventral prostate and seminal vesicle epithelium, as well as lysis of corpora lutea, was ob­ served in mice (Benson, Matthews, and Smith 1971). In retrospect, 130 these effects, which were considered initially to be anti-LH actions, might better be explained by a reduction in prolactin synthesis and/or release. Prolactin is the luteotropic hormone of mice and rats, and recently it has been reported that this hormone acted synergistically with testosterone in the maintenance of ventral prostate and was synergistic to LH-stimulated steroidogenesis in the corpus luteum

(Armstrong, Miller, and Knudsen 1969), Additionally, prolactin was observed to increase testicular sensitivity to LK in rats (Bartke and

Dalterio 1976).

In conclusion, the primary "antigonadotropic" effect of pineal extracts could be a reduction in prolactin secretion. Recent experiments have shown the presence of prolactin-releasing and release-inhibiting factors in pineal extracts (Blask et al 1976), These findings further support the hypothesis that pineal antigonadotropic activity may be mediated through effects on the synthesis and/or release of prolactin.

The next important experiment to test this hypothesis would be, of course, to evaluate the effects of extracts containing PAG on prolactin in the hamster.

Studies en the Effects of AVT

Although Pavel (1971) found that AVT, as detected by the frog bladder assay, was confined to the stalks of bovine glands, he continued to propose that AVT is the major pineal antigonadotropic substance. No argument for this paradoxical position was given in any of his papers.

Since the pineal stalk is made up of ependyma, he has proposed that this is an ependymal secretion (Pavel, Goldstein, and Calb 1975). The 131 localization of AVT in the stalk of the bovine pineal gland was con­ firmed by Benson, Matthews, and Hadley (1976), but antigonadotropic activity was simultaneously confined to the non-stalk portion of the gland. These data, plus the findings of AVT in other ependymal struc­ tures not contiguous to the pineal parenchyma (such as the subcommis­ sural organ of the rabbitj Rosenbloom and Fisher 1975a), would seem sufficient to warrant a characterization of AVT as an ependymosecretion unrelated to pineal secretory activity.

AVT has been proven to differ from PAG in absorption spectra, thin layer electrophoresis, ion-exchange chromatography, and amino acid analysis (Rosenblum et al, 1976), Despite these findings, the question persists: Are some of the effects of pineal extracts due to the AVT which they contain? In responding to this question, one must remember that in these studies the stalks were cut away from the parenchyma of each pineal gland. In addition, the UM2 filter has been shown to retain

AVT, so AVT would not be in the UM05R that was used. Further, AVT (in the small amount of 1.55 yg/day to the large amount of 12.5 yg/day) was tested by ventral prostate reduction in mice in a manner exactly like that used for PAG. The fact that no ventral prostate reduction at any concentration was observed is highly significant. The AVT used in the above study had been synthesized in the laboratory of Dr. V. J. Hruby and had acceptable oxytocic activity when tested in the laboratory of

Dr. M. E. Hadley (Benson, Matthews, Hadley, et al. 1976). From all this, it is safe to declare that AVT does not contribute to the reduc­ tion of ventral prostate weight that we have observed with pineal extracts. 132

Vaughan, Vaughan, and Klein (1974) have reported reduction in

accessory organ weight in male and ovarian weight in female 25-day-old mice treated with one yg of AVT/day for three or four days. This experiment was repeated in the present study exactly as these authors described. Reduction in ventral prostate and seminal vesicle weight was seen, but the reported effect on ovarian weight did not occur. It is i germane to this discussion to note that the ventral] prostate of a 25^ i | day-old mouse weighs one to two mg; and to realize that we are talking about a reduction from 1.52 ± 0.19 to 1.06 ±0.12. j (Our Mettler balance requires a three mg load in order to be accurate.) Similarly, seminal vesicles were reduced from 7.32 ± 1.1 to 5.47 ± 0.9. Since these are very small values, we feel that the validity of these experiments may be in question. It is also significant that AVT had no effect on the ventral prostates of mature animals. ! Localization of AVT to the stalk of the bovine pineal gland is | even more certain when one looks at the results of the present work, j An organic solvent extract of stalks from pineal glands was not effec­ tive in reducing ventral prostate weight in mature male mice, although i the extract from the parenchymal portion of these glands had been active | in the same way. The antigonadotropic activity of ,the pineal- gland was not found in the same place as AVT. j

j In summary, all studies here listed show conclusively that the pineal antigonadotropic hormone differs from AVT, CONCLUSION

The original hypothesis that the antigonadal effects of the pineal gland are mediated through reduction in serum LH was not substantiated by these studies. Although pineal extracts appeared to reduce the function of LH-dependent structures, such organs are also stimulated by prolactin so reduction in this hormone may be the mechanism of pineal function- The observation that prolactin maintained gonad and accessory organ weight in the blinded hamster indicates that pineal regulation of prolactin may be of paramount importance in reproductive physiology.

Pineal extracts containing PAG did not interfere with the effect of exogenous gonadotropins {PMS and HCG) in immature animals. Similarly, they did not affect the release of LH from the pituitary gland in vitro.

They did, however, appear to affect LH when extracts were injected into the median eminence (but not into the pituitary) iii vivo. These find­ ings suggest a central nervous system site of action for PAG.

Finally, it is once again concluded that pineal antigonadotropin differs from arginine vasotocin and that the latter's antigonadal effects are greatly overrated.

The mechanism of pineal function is still a mystery, but these studies along with other recent works, have helped in directing our attention to avenues that appear to lead to ultimate solution. The most exciting of these avenues is the investigation of the possible regula­ tion of prolactin by the pineal gland.

133 LITERATURE CITED

Aguado, L. I., G. A. Benelbaz, L. S. Gutierrez, and E. M. Rodriquez 1777 Ultrastructure of the rat pineal gland grafted under the kidney capsule. Cell Tiss. Res. 3/76:131-142.

Altschule, M. D. 1957 Some effects of aqueous extracts of acetone- dried beef pineal substance in chronic schizophrenia. New Eng. J. Med. 257:919-922.

Arendt, J., L. Paunier, and P. C. Sizonenko 1975 Melatonin radio­ immunoassay. J. Clin. Endocr. Metab. 40:347-350.

Arendt, J., L. Weterberg, T. Heyden, P. C. Sizonenko, and L. Paunier 1977 Radioimmunoassay of melatonin: Human serum and cerebrospinal fluid. Horm. Res. jB:65-75.

AriSns-Kappers, J. 1960 The development, topographical relations and innervation of the epiphysis cerebri in the albino rat. Z. Zellforsch. 52:163-215.

Armstrong, D. T., L. V. Miller, and K. A. Knudsen 1969 Regulation of lipid metabolism and progesterone production in rat corpora lutea and ovarian interstitial elements by prolactin and luteinizing hormone. Endocrinology 85:393-401.

Arstila, A. U., H. 0. Kalimo, and M. Hyyppa 1971 Secretory organelles of the rat pineal gland; electron microscopic and histochemical studies in vivo and in vitro. In: Ciba Foundation Symposium on the Pineal Gland. G. E. W. Wolstenholme and J. Knight (eds.) J. & A. Churchill: London. 147-175.

Axelrod, J., S. H. Snyder, A. Heller, and R. Y. Moore 1966 Light- induced changes in pineal hydroxyindole-O-methyltransferase: Abolition by lateral hypothalamic lesions. Science 154:898-899.

Backstrom, M., and L. Wetterberg 1972 Catechol-O-methyltransferase, histamine-N-methyltransferase and methanol forming enzymes in the rat pineal gland. Life Sci. I 11:293-299.

Badertscher, J. A. 1924 Results following the extirpation of the pineal gland in newly hatched chicks. Anat. Rec. 2IB: 177-197.

Banerji, A. P., L. S. Kothari, and P. N. Shah 1977 Some observations on gonadotropin inhibitor (anti-LH) from human urine—with dis­ cussion on its possible source of origin. Endokrinologie 69:273- 281.

134 135

Banerji, T, K., and W. B. Quay 1976 Adrenal dopamine-beta-hydroxylase activity: 24-hour rhythmicity and evidence for pineal control. Experientia 32;253-254.

Banks, A. F-, and R. J. Reiter 1975 Melatonin inhibition of pineal anti-gonadotropic activity in male rats. Horm. Res. 6^:351-356.

Barchas, J D., and A. B. Lerner 1964 Localization of melatonin in the nervous system. J. Neurochem. 11:489-491. i Bar fuss, D! W., and L. C. Ellis 1971 Seasonal cycles in melatonin synthesis by the pineal glands as related to testicular function in|the house sparrow (Passer domesticus). Gen. Comp, Endocr. 17 183-193.

Barratt, Gj F., M. J. Nadakavukaren, and J. L. Frehn 1977 Effect of melatonin implants on gonadal weights and pineal gland fine structure of the golden hamster. Tiss. Cell 9:335-347.

Bartke, B.'. T., B. T. Croft, and S. Dalterio 1975 Prolactin restores plasma testosterone levels and stimulates testicular growth in hamsters exposed to short day length. Endocrinology 97:1601- 1604.

Bartke, A.j and S. Dalterio 1976 Effect of prolactin on the sensi­ tivity of the testis to LH. Biol. Reprod. 15:90-93.

Baum, M. 1968 Pineal gland: Influence on development of copulation inimale rats. Science 162:586-587. I I Benoit, J.j 1961 Opto-sexual reflex in the duck: Physiological and histological aspects. Yale J. Biol. Med. 34:97-116.

Bensinger,j R., M. K. Vaughan, and D. C. Klein 1973 Isolation of a non- melatonin lipophylic antigonadotrophic factor from the bovine pineal gland. Fed. Proc. 32:252.

Benson, B.j 1978 Professor and Head, Department of Anatomy, University of[Arizona Health Sciences Center, Tucson, unpublished observa­ tions.

Benson, B.J D. S. Grosso, I. Y. Rosenblum, and R. Bressler 1977 Circadian rhythm in pineal taurine levels in the rat: Effects ofjoptic enucleation, constant light or superior cervical ganglionectomy. Abs. Ann. Endocr. Soc. 59:491. | Benson, B.j and M. Krasovich 1977a Circadian rhythm in the number of granulated vesicles in mouse pinealocytes: Effects of denerva­ tion or melatonin treatment. Anat. Rec. 187:536. 136

Benson, B., and M. Krasovich 1977b Circadian rhythm in the number of granulated vesicles in the pinealocytes of mice: Effects of sympathectomy and melatonin treatment. Cell Tiss. Res. 184: 499-507.

Benson, B., B. Larsen, and D. L. Richardson 1978 Effect of intra- carotid injection of pineal extract on prolactin in the rat. In preparation.

Benson, B., and M. J. Matthews 1974 Reduction in fertility in mice treated with bovine pineal antigonadotropin. Anat. Rec. 178: 309-310.

Benson, B., M. J. Matthews, M. E. Hadley, S. Powers, and V. J. Hruby 1976 Differential localization of antigonadotropic and vasotocic activities in bovine and rat pineal. Life Sci. 19:747-754.

Benson, B., M.J. Matthews, and V. J. Hruby 1976 Characterization and effects of a bovine pineal antigonadotropic peptide. Amer. Zool. 16:17-24.

Benson, B., M. J. Matthews, and R. J. Orts 1972 Presence of an antigonadotropic substance in rat pineal incubation media. Life Sci. I 11:669-677.

Benson, B., M. J. Matthews, and A. E. Rodin 1971 A melatonin-free extract of bovine pineal with antigonadotropic activity. Life Sci. I 10:607-612.

Benson, B., M. J. Matthews, and A. E. Rodin 1972 Studies on a non- melatonin pineal antigonadotropin. Acta Endocr. 69:257-266.

Benson, B., M. J. Matthews, and M. L. Smith 1971 Effects of a pcirtially purified pineal antigonadotropin in male and female mice. Workshop on the Pineal Gland, NIH. 32 Berg, G. R., and D. C. Klein 1972 Norepinephrine increases P labelling of a specific phospholipid fraction of post­ synaptic pineal membranes. J. Neurochem. 19:2519.

Bergmann, W. 1940 WIrkung von Pinealis extracten auf das Gefassystem des Frosches. Arch. Neerl. Physiol. 24:391-397.

Berkeley, W. N. 1920 Comments on the function and clinical use of the pineal gland. Med. Rec. 97:12-14.

Bigelow, L. B. 1974 Effects of aqueous pineal extract in chronic schizophrenia. Biol. Psychiatry' 8^5-15. 137

BInkley, S. 1976 Pineal gland biorhythms: N-acetyl-transferase in chickens and rats. Fed. Proc. 35:2347-2352.

Binkley, S., S. E. MacBride, D. C. Klein, and C. L. Ralph 1975 Regulation of pineal rhythms in chickens: Refractory period and nonvisual light perception. Endocrinology 96;848-853.

Bing, J. F., J. H. Globus, and H. Simon 1938 Pubertas praecox; survey of reported cases and verified anatomical findings with particular reference to tumors of the pineal body. J. Mt. Sinai Hosp. 4^:935-965

Blake, C. A. 1976 Effects of pinealectomy on the rat oestrous cycle and pituitary gonadotrophin release. J. Endocr. 69:67-75.

Blask, D. E. 1978 Post-Doctoral Fellow, Department of Anatomy, University of Texas Health Sciences Center, San Antonio, personal communication.

Blask, D. E., and R. J. Reiter 1975a Pituitary and plasma LH and prolactin levels in female rats rendered blind and anosmic: Influence of the pineal gland. Biol. Reprod. 12:329-334.

Blask, D. E., and R. J. Reiter 1975b The pineal gland of the blind- anosmic female rat: Its influence on medial basal hypothalamic LRH, PIF and/or PRF activity in vivo. Neuroendocrinology 17: 362-374.

Blask, D. E., M. K. Vaughan, and R. J. Reiter 1977 Modification by adrenergic blocking agents of arginine vasotocin-induced prolactin secretion in vitro. Neuroscience Letters 6:91-95.

Blask, D. E., M. K. Vaughan, R. J. Reiter, L. Y. Johnson, and G. M. Vaughan 1976 Prolactin-releasing and release-inhibiting factor activities in the bovine, rat, and human pineal gland: In vitro and ill vivo studies. Endocrinology 99:152-162.

Botticelli, A., G. P. Trentini, B. Barbolini, and C. F. deGaetani 1972 Histoenzymology of the circum-ventricular organs of the rat. V. The oxidoreductases, the transferases and the hydrolases of the pineal gland. Acta Histochem. (Jena) 44^288-299.

Bowie, E. P., and D. C. Herbert 1976 Immunocytochemical evidence for the presence of arginine vasotocin in the rat pineal gland. Nature 261:66.

Brackman, M. 1977 Melatonin delays puberty in the Djungarian hamster. Die Naturwissenschaften 64:642-643. 138

Brown, G. M., J. Basinka, G. Bubenik, D. Sibony, L. J. Grota, and H. C. Stancer 1976 Gonadal effects of pinealectomy and immunization against N-acetyl-indolealkylamines in the hamster. Neuroendocrinology 22;289-298.

Brown-Grant, K., and A. J. C. Ostberg 1974 Lack of effect of pineal denervation on the responses of the female albino rat to exposure to constant light. J. Endocr. 62:45-50.

Brownstein, M., and J. Axelrod 1974 Pineal gland: 24-hour rhythm in norepinephrine turnover. Science 184:163-165.

Bubenik, G. A., G. M. Brown, I. Uhlir, and L. J. Grota 1974 immuno- histological localization of N-acetylindolealkylamines in pineal gland, retina and cerebellum. Brain Res. 81^233-243.

Calb, M., R. Goldstein, and S. Pavel 1977 Diurnal rhythm of vasotocin in the pineal of the male rat. Acta Endocr. 84: 523-526.

Cardinali, D. P., A. E. Cuello, J. H. Tramezzani, and J. M. Rosner 1971 Effects of pinealectomy on the testicular function of the adult male duck. Endocrinology 89:1082-1093.

Cardinali, D. P., and F. Freire 1975 Melatonin effects on brain. Interaction with microtubule protein, inhibition of fast axoplasmic flow and induction of crystaloid and tubular formations in the hypothalamus. Mol. Cell. Endocr. 2^:317-330.

Cardinali, D. P., E. Gomez, and J. M. Rosner 1976 Changes in (^H) leucine incorporation into pineal proteins following estradiol or testosterone administration: Involvement of the sympathetic superior cervical ganglion. Endocrinology 98:849-858.

Cardinali, D. P., F. Larin, and R. J. Wurtman 1972a Action spectra for effects of light on hydroxyindole-O-methyltransferases in rat pineal, retina and Harderian gland. Endocrinology 91: 877-886.

Cardinali, D. P., F. Larin, and R. J. Wurtman 1972b Control of the rat pineal gland by light spectra. Proc- Nat. Acad. Sci. USA 69:2003-2005.

Cardinali, D. P., C. A. Nagle, and J. M. Rosner 1974a Effects of estradiol on melatonin and protein synthesis in the rat pineal organ. Hor. Res. 5:304-311. 139

Cardinal!, D. P., C. A. Nagle, and J. M. Rosner 1974b Metabolic fate of androgens in the pineal organ: Uptake, binding to cytoplasmic proteins and conversion of testosterone to 5-a- reduced metabolites. Endocrinology 95;179-187.

Cardinali, D. P., C. A. Nagle, and J. M. Rosner 1975a The rat pineal gland. A model for studying the interactions between sex steroids and the sympathetic nervous system. Acta Physiol. Lat. Amer. 25:241-244.

Cardinali, D. P., C. A. Nagle, and J. M. Rosner 1975b Control of estrogen and androgen receptors in the rat pineal gland by catecholamine transmitter. Life Sci. 16:93-106.

Cardinali, D. P., C. A. Nagle, and J. M. Rosner 1976 Gonadotrophin- and prolactin-induced increase of rat pineal hydroxyindole-0- methyl transferase involvement of the sympathetic nervous system. J. Endocr. 68:341-342.

Cardinali, D. P., and J. M. Rosner 1971 Retinal localization of hydroxyindole-O-methyltransferase in the rat. Endocrinology 89:301.

Chang,. N., and I. Ebels 1978 Studies on the separation of prolactin- releasing factor (PRF) and prolactin-release-inhibiting factor (PIF) activities in bovine pineal glands. Anat. Rec. 190:361.

Charlton, H. M., C. A. Grocock, and A. Ostberg 1976 The effects of pinealectomy and superior cervical ganglionectomy on the testis of the vole, Microtus argrestic. J. Reprod. Fert. 48:377-379.

Chazov, E. I., S. P. Veselova, O. G. Krivosheev, and V. A. Isachenkov 1976a The relationship between the pineal gland and the hypothalamo-hypophyseal complex. I. The effect of pinealectomy, blinding and continuous illumination on 3H-leucine incorporation in the nucleus of the anterior, middle and posterior hypothalamus of prepubertal and sexually mature rats. Probl. Endokr. (Mosk.) 22:33-39.

Chazov, E. I., S. P. Veselova, O. G. Krivosheev, and V. A. Isachenkov 1976b Interrelationship of the pineal body with the hypothalamo-hypophyseal complex: II. The effect of melatonin on the incorporation of *^H-leucine in the anterior and medial hypothalamic nuclei of pinealectomized blinded rats. Probl. Endokr. (Mosk.) 22:63-65.

Cheesman, D. W., and B. L. Fariss 1970 Isolation and characterization of a gonadotropin-inhibiting substance from the bovine pineal gland. Proc. Soc. Exp. Biol. Med. 133:1254-1256. 140

Cheesman, D. W., and P. H. Forsham 1974 Action of a gonadotropin inhibitor from the bovine pineal gland. Proc. Soc. Exp. Biol. Med. 147:438-439.

Chen, C. L., Y. Amenomori, K. H. Lu, J. L. Voogt, and J. Meites 1970 Serum prolactin levels in rats with pituitary transplants and hypothalamic lesions. Neuroendocrinology 6^:220-227.

Chu, E. W. 1965 Effect of environmental illumination on estrous cycles of rodents. Acta cytol. J9:221-227.

Clark, W. E. L., T. McKeown, and S. Zuckerman 1939 Visual pathways concerned in gonadal stimulation. Proc. Roy. Soc. Ser. B 126: 449-468.

Clementi, F., G. Devirgiliis, and B. Mess 1969 Influence of pineal gland principles on gonadotrophin-producing cell of the rat anterior pituitary gland: An electron microscope study. Endocrinology 44:241-246.

Collin, J.-P, M.-T Juillard, and J. Falcon 1977 Localization of 5- hydroxytryptamine and protein(s) in the secretion granules of the rudimentary photoreceptor cells in the pineal of Lacerta. J. Neurocytol. 6^:541-554.

Critchlow, V., and J. deGroot 1960 Experimental investigation of pathways involved in light-induced constant estrus in the rat. Anat. Rec. 136:179.

Csaba, G., and P. Barath 1974 The effect of pinealectomy on the parafollicular cells of the rat thyroid gland. Acta Anat. 88: 137-147.

Csaba, G., M. Bodoky, J. Fischer, and T. Aes 1966 The effect of pinealectomy and thymectomy on the immune capacity of the rat. Experientia 22:168-169.

Csaba, G., J. Kiss, and M. Bodoky 1968 Uptake of radioactive iodine by the thyroid after pinealectomy. Acta Biol. Acad. Sci. Hung. 19:34-41.

Cuello, A. C., N. Hisano, and J. H. Tramezzani 1972 The pineal gland and the photosexual reflex in female ducks. Gen. Comp. Endocr. 18:162-168.

Debeljuk, L. 1970 Lack of gonadotrophin-inhibiting activity of ovine pineal extracts. J. Reprod. Fert. 23:161-163. 141

DePronzo, R. A., and W. D. Roth 1971 Lack of effect of pinealectomy on ovarian function: Evidence for a time-dependent action. Proc. Soc. Exp. Biol. Med. 138;1058-1061.

DePronzo, R. A., and W. D. Roth 1972 Evidence for existence of a pineal-adrenal and pineal-thyroid axis. Acta Endocr. 70:35-42.

DeGroot, J. 1959 The Rat Forebrain in Stereotaxic Coordinates. N. V. Noord-Hollandsche Uitgevers Maatschappj: Amsterdam.

Deguchi, T. 1975 Shift of circadian rhythm of serotonin: Acetyl co­ enzyme A N-acetyl transferase activity in pineal gland of rat in continuous darkness or in the blinded rat. J. Neurochem. 25:91-93.

Deguchi, T., and J. Axelrod. 1972 Control of circadian change of serotonin N-acetyltransferase activity in the pineal organ by the |3-adrenergic receptor. Proc. Nat. Acad. Sci. USA 69: 2547-2550.

DeProspo, N. D., R. J. Safinski, L. J. DeMartino, and E. T. McGuinness 1969 Melatonin and its precursors' effects on ^31j uptake by the thyroid gland under different photic conditions. Life Sci. 8^:837-842.

Devecerski, V. 1972 Etude histoenzymologique de la glande pineale du rat en adaptation au froid. C. R. Soc. Biol. (Paris) 166: 970-972.

Dickson, K. L., and D. L. Hasty 1972 Effects of pineal gland in unilaterally adrenalectomized rats. Acta Endocr. 70:438-444.

Diederan, J. H. 1975 A possible functional relationship between the subcommissural organ and the pineal complex and lateral eyes in Rana esculenta and Rana temporaria. Cell Tiss. Res. 158: 37-60.

Dixon, W. E., and W. D. Halliburton 1909 The pineal body. Quart. J. Esq?. Physiol. 2^:283-285.

Dunaway, J. E. 1969 Alterations in the timing of PMS-induced ovula­ tion following pinealectomy. Neuroendocrinology 5^:281-289.

Duraiswami, S., P. Franchimont, D. Boucher, and H. Thieblot 1976 Immunoreactive luteinizing hormone-releasing hormone (LH-RH) in the bovine pineal gland. Hor. Metab. Res. 8^:232-233.

Ebels, I. 1975 Pineal factors other than melatonin. Gen. Comp. Endocr. 25:189-198. 142

Ebels, I. 1976 Isolation of avian and mammalian pineal indoles and antigonadotropic factors. Amer. Zool. 16:5-15.

Ebels, I., M. G. M. Balemans, and A. J. .Verkley 1972 Separation of pineal extracts on Sephadex G-10. II. A spectrofluorimetric and thin layer chromatographic study of indoles in a sheep pineal extract. J. Neurovisc. Rel. 32:270-281.

Ebels, I., B. Benson, and M. J. Matthews 1973 Localization of a sheep pineal antigonadotropin. Anal. Biochem. 56:546-565.

Ebels, I., and A. E. M. Horwitz-Bresser 1976 Separation of pineal extracts by gel filtration. IV. Isolation, location and identification from sheep pineals of three indoles identical with 5-hydroxytryptophol, 5-methoxytryptophol and melatonin. J. Neural. Transm. 38:31-41.

Ebels, I., A. Hus-Citharel, and A. Moszkowska 1975 Separation of pineal extracts by gel filtration. III. Sheep pineal factors acting either on the hypothalamus, or on the anterior hypophysis of mice and rats in in vitro experiments. J. Neural. Transm. 36:281-302.

Ebels, I., A. Moszkowska, and A. Scemama 1970 An attempt to separate a sheep pineal fraction showing antigonadotropic activity. J. Neurovisc. Rel. 32:1-10.

Ellison, N., J. L. Weller, and D. C. Klein 1972 Development of a circadian rhythm in the activity of pineal serotonin N- acetyltransferase. J. Neurochem. 19:1335-1341.

Engel, P. 1934a Zirbeldriise und gonadotropes Hormon. Ztschr. f. d. Ges. Exp. Med. 94:333-345.

Engel, P. 1934b Untersuchungen iiber die Wirkung der Zirbeldruse. Ztschr. f. d. Ges. Exp. Med. 93:69-79.

Engel, P. 1934c iiber das vorkommen einer wasserloslischen, Allen- Doisy Testgeben den Substanz in menschlichen Blute. Wein Klin. Wchnschr. 47:847-848.

Engel, P. 1934d Zirbeldruse und hypophysares Wachstum. Klin. Wchnschr. 13:1248-1249.

Engel, P. 1934e Uber den Einfluss von Epiphysenextracten auf die Wirkung der Hypophysenvorderlappenhormone. Klin. Wchnschr. 13:266-267. 143

Engel, P. 1935a Weitere Untersuchunger uber die biologischen und chemischen Eigenschaften des antigonadotropen Hormones der Zirbeldruse. Ztschr. f. d. Ges. Exp. Med. 96;328-336.

Engel, P. 1935b Uber die antigonadotrope wirkung des Epiphysans. Wein Klin. Wochschr. 48:1160-1161.

Engel, P. 1935c Uber die hormonalen Eigenschaften der Zirbeldruse. Wein Klin. Wchnschr. 48:481-486.

Engel, P. 1935d Gegenhormone und Zirbeldruse. Klin. Wchnschr. 14: 970-971.

Engel, P. 1935e Antigonadotropes Hormon in Zirbeldruse, Blut und Organ. Ztschr. f. d. Ges. Exp. Med. 95:441-457.

Engel, P. 1935f tfoer die veranderungen der Nagerscheide durch Zirbelextracte. Klin. Wchnschr. 14:830-831.

Engel, P. 1935g Uber die Beeinflussing der Callusbildung durch Hormone, Wachstumshormon und Zirbeldriisenextracte. Deutsche Ztschr. f. d. Chir. 244:591-592.

Engel, P. 1937 Uber den heutigen Stand unseres Wissens von der Zirbelfunktion. Wein Klin. Wchnshr. 50:1219-1222.

Engel, P. 1939 Assay of pineal extracts. Endocrinology 25:144-145.

Eyster, J. A. E., and H. E. Jordan 1911 Effect of intraveinous injection of extracts of the pineal body. Proc. Amer. Physiol. Soc. 27:33-35.

Farriss, B. L., and L. D. Botz 1975 Comparison of hydroxyindole-0- methyltransferase levels in pineal glands of normal, blinded and congenitally anophthalmia guinea pigs. Endocrinology 96: 1595-1596.

Fischer, E. 1938 Isolation and biological assay of an effecaceous fraction of the pineal gland. Arch. Internat. Pharm. Therap. 49:340-344.

Fischer, E. 1943 Preparation of an active fraction of the pineal gland. Endocrinology 33:116-117.

Fiske, V. M. 1941 Effect of light on sexual maturation, estrous cycles and anterior pituitary of the rat. Endocrinology 29: 187-196. 144

Fiske, V. M., G. K. Bryant, and J. Putnam. 1960 Effect of light on the weight of the pineal in the rat. Endocrinology 66:489-491.

Fiske, V. M., J. Pound, and J. Putnam 1962 Effect of light on the weight of the pineal organ in hypophysectomized, gonadeeternized, adrenalectomized or thiouracil fed rats. Endocrinology 71:130- 133.

Fraschini, F., B. Mess, and L. Martini 1968 Pineal gland, melatonin and the control of luteinizing hormone secretion. Endocrinology 82:919-924.

Freire, F., and D. P. Cardinali 1975 Effects of melatonin treatment and environmental lighting on the ultrastructural appearance, melatonin synthesis, norepinephrine turnover and microtubule content of the rat pineal gland. J. Neural. Transm. 37;237- 257.

Freund, D., J. Arendt, and L. Vollrath 1977 Tentative immunohisto- chemical demonstration of melatonin in the rat pineal gland. Cell Tiss. Res. 181:239-245.

Futterweit, w., S. A. Margolis, L. J. Soffer, and R. I. Dorfman 1963 Effect of urinary gonadotrophin inhibitor on the mouse uterine response to various gonadotrophins. Endocrinology 72:903-907.

Gaston, S., and M. Menaker 1967 Photoperiodic control of hamster testis. Science 158:925-928.

Gittes, R. F., and E. W. Chu 1965 Reversal of the effect of pinealectomy in female rats by multiple isogeneic pineal transplants. Endocrinology 77:106-107.

Golikov, P. O., and B. S. Lebedev 1975 The effect of epiphysectomy on the concentration of aldosterone, corticosterone and transcortin in the peripheral blood. Probl. Endokrinol. (Mosk.) 21:100-105.

Gonzales, G., and E. Blazques 1975 Ultrastructural evidence of a secretory process in the rat pineal gland. Eaqaerientia 31: 969-971.

Grachev, I. I., R. I. Usanova, and U. A. Seliverstov 1972 The influence of the pineal extract on the growth activity of the rat. Fiziol. Zh. SSSR 58:272-275.

Gradwell, P. B., E. J. Candy, and R. B. Symington 1975 Identification of some substances secreted by the bovine pineal body and their antigonadotropic activity in an iji vitro system. S. Afr. J. Med. Sci. 40:73-82. 145

Gradwell, P. B., R. B. Miller, and R. B. Symington 1976 Failure to demonstrate high concentrations of luteinizing hormone- releasing hormone in the bovine pineal body. S. Afr. Med. J. 50:217-219.

Gregorek, J. C., H. R. Seibel, and R. J. Reiter 1977 The pineal complex and its relationship to other epithalamic structures. Acta Anat. 99;425-435.

Greiner, A. C., and S. C. Chan 1978 Melatonin content of the human pineal gland. Science 199:83-85.

Guerra, M. 0., N. O. G. Silva, C. S. Guimaraes, J. P. Souza, and A. T. L. Andrade 1973 Pinealectomy and blindness during pregnancy in the rat. Amer. J. Obstet. Gyn. 115:582-583.

Gusek, W. 1976 Die Feinstruktur der Rattenzirbel und ihr Verhalter unter Einfluss von Antiandrogen und nach Kastration. Endokrinologie 67:129-151.

Haulica, I., D. D. Branisteanu, V. Rosea, A. Stratone, V. Berbeleu, G. Balan, and L. Ionescu 1975 A renin-like activity in pineal gland and hypophysis. Endocrinology 96:508-510.

Hellman, R., and C. Owman 1961 Utilization of uniformly labelled glucose in the pineal body of goats. Acta Endocr. 38; 353-360.

Herbert, J. 1971 The role of the pineal gland in the control by light of the reproductive cycle of the ferret. In: Ciba Foundation Symposium on the Pineal Gland. G. E. W. Wolstenholme and J. Knight (eds.). J. and A. Churchill: London. 303-327.

Herbert J. 1972 Initial observations on pinealectomized ferrets kept for long periods in either daylight or artificial illumination. J. Endocr. 55:591-598.

Herrera, H. H., W. W. Morgan, and R. J. Reiter 1975 Brain norepinephrine levels in parathyroidectomized rats induced to convulse by pinealectomy. Exp. Neurol. 48:595-600.

Hinterberger, H., and J. Pickering 1976 Catecholamine, indolealkylamine and calcium levels of human pineal glands in various clinical conditions. Pathology £1:221-229.

Hisano, N., D. P. Cardinali, J. M. Rosner, C. A. Nagel, and J. H. Tramezza 1972 Pineal role in duck extraretinal photoreception. Endocrinology 91:1318-1322. 146

Hoffman, J. C. 1967 Effects of light deprivation on the rat estrous cycle. Neuroendocrinology 2^:1-10.

Hoffman, J. C., and A.-M. Cullin 1975 Effects of pinealectomy and constant light on the estrous cycles of female rats. Neuroendocrinology 17;167-174.

Hoffman, K., and I. Kuderling 1975 Pinealectomy inhibits stimulation of testicular development by long photoperiods in a hamster (Phodopus sungorus). Experientia 31:122-123.

Hoffman, R. A., and R. J. Reiter 1965 Pineal gland: Influence on gonads of male hamsters. Science 148:1609-1611.

Hoffman, R. A., and R. J. Reiter 1966 Responses of some endocrine organs of female hamsters to pinealectomy and light. Life Sci. _5:1147-1151.

Hofmann, E. 1925 Zur Frage der inneren Sekretion der Zirbeldruse bei der Ratte. Pflugers Arch. ges. Physipl. 209:685-692.

Houssay, A. B., and A. C. Barcelo 1972a Effects of estrogens and progesterone upon the biosynthesis of melatonin by the pineal gland. Experientia 28:478-479.

Houssay, A. B., and A. C. Barcelo 1972b Effects of testosterone upon the biosynthesis of melatonin by the pineal gland. Acta. Physiol. Lat. Amer. 22:274-275.

Hus-Citharel, A., S. Roseau, and W. Zurburg 197.7 Effects of precocious pinealectomy and hemicastration on pituitaryand plasma LH levels in immature male rats. J. Neural. Transm. 40:33-46.

Huxley, M., and E. Tapp 1972 Rat pineal gland in tissue culture— Morphology and enzyme histochemistry. Arch. Path. 94:519-522.

Illnerovlt, H. 1971 Effect of light on the serotonin content of the pineal gland. Life Sci. I 10:955-960.

IllnerovS, H., and I. Albrecht 1975 Isoproterenol induction of pineal serotonin N-acetyl transferase in normotensive and spontaneously hypertensive rats. Experientia 31 .-95-96.

Ishibashi, T., D. W. Hahn, L. Srivastava, P. Kumaresan, and C. W. Turner 1966 Effect of pinealectomy and melatonin on feed consumption and thyroid hormone secretion rate. Proc. Soc. Exp. Biol. Med. 122:644-647.

Izawa, Y. 1923 A contribution to the study of the pineal body. Amer. J. Med. Sci. 166:185-196. 147

Izawa, Y. 1926 On some anatomical changes which follow removal of the pineal body from both sexes of the immature albino rat. flmer. J. Physiol. 77;126-139.

Jackson, G. L., and A. V. Nalbandov 1969 A substance resembling arginine vasotocin in the anterior pituitary gland of the cockeral. Endocrinology 84;1218-1223.

Jackson, I. M. D., R. Saperstein, and S. Reichlin 1977 Thyrotropin releasing hormone (TRH) in pineal and hypothalamus of the frog: Effect of season and illumination. Endocrinology 100: 97-100.

Jordan, H., and J. A. E. Eyster 1911 The physiological action of extracts of the pineal body. Amer. J. Physiol. 29:115-123.

Joseph, S. A. 1976 Seasonal variation in luteinizing hormone releasing hormone (LHRH) content of rat pineal gland. Anat. Rec. 184:439.

Jouan, P., and S. Samperez 1965 Etude de la secretion des corticosteroides et de 1*hormone adrenocorticotrope hypophysaire chez le rat 4piphysectomis£. Prog. Brain Res. 10:604-611.

Juszkiewicz, T., and Z. Rakalska 1963 Anti-estrogenic effects of bovine pineal glands. Nature 200:1329-1330.

Kachi, T., S. Matsushima, and T. Ito 1974 Effect of continuous dark­ ness on diurnal rhythm in glycogen content in pineal cells of the mouse: A semiquantitative histochemical study. Anat. Rec. 179:405.

Kachi, T., S. Matsushima, and T. Ito 1975 Postnatal observations on the diurnal rhythm and the light-responsiveness in the pineal glycogen content in mice. Anat. Rec. 183:39-46.

Karasek, M. 1974 Ultrastructure of rat pineal gland in organ culture; influence of norepinephrine, dibutyryl cyclic adenosine 3', 5'- monophosphate and adenohypophysis. Endokrinologie 64:106-115.

Karasek, M. 1976 Quantitative changes in number of "synaptic" ribbons in rat pinealocytes after orchidectomy and in organ culture. J. Neural. Transm. 38:149-157.

Karasek, M., M. Pawlikowski, J. Ariens-Kappers, and H. St^pien' 1976 Influence of castration followed by administration of LH-RH on the ultrastructure of rat pinealocytes. Cell Tiss. Res. 167: 325-339. 148

Karasek, M., M. Pawlikowski, P. Pevet, and H. Stfpien' 1976 Ultra- structural and fluorescence histochemical studies of the rat pineal gland after castration. Ann. Med. Sect. Pol. Anat. Sci. 21:57-58.

Karppanen, H., E. Klinge, and S. Aro 1972 Age and sex dependence of cholinesterase activities in bovine pineal gland. Scand. J. Clin. Invest. 29:41.

Karppanen, H., S. Lahovaara, P. Mannisto, and H. Vapaatalo 1975 Plasma renin activity and in vitro synthesis of aldosterone by the adrenal glands of rats with spontaneous renal or pinealectomy induced hypertension. Acta Physiol. Scand. 94:184-188.

Karppanen, H., and H. Vapaatalo 1971 Effects of an aldosterone antagonist, Spironolactone, on pinealectomized rats. Pharmacology 6^:257-264.

Kennaway, D. J., and R. F. Seamark 1975 The occurrence of hydroxyindole-O-methyl transferase activity in fetal sheep pineal tissue and its relationship to preparturient endocrine changes. J. Reprod. Fert. 45:529-531.

Kenny, G. C. T., C. J. Louis, K. C. Bradley, R. M. Anderson, and N. B. Walden 1973 Effect of optic chiasmotomy upon serotonin concentration in the pineal gland of the monkey. Amer. J. Physiol. Anthropol. 38:399-402.

Kerenyi, N. A., D. J. Lucis, J. Borkow, and R. Lucis 1972 Effect of constant darkness on pineal gland and gonads. Gen. Camp. Endocr. 18:600.

Kind, F. A., and G. Benagiano 1967 The failure of the pineal gland removal in neonatal animals to influence reproduction. Acta Endocr. 54:189-192.

Kinson, G. A., and Chung-Ching Liu 1973 Effects of blinding and pinealectomy on diurnal variations in plasma testosterone. Experientia 29:1415-1417.

Kinson, G. A., A. K. Wahid, and B. Singer 1967 Effect of chronic pinealectomy on adrenocortical hormone secretion rates in normal and hypertensive rats. Gen. Comp. Endocr. jl:445-454.

Kiss, J., D. Banhegyi, and G. Csaba 1969 Endocrine regulation of blood calcium level. II. Relationship between the pineal body and the parathyroid glands. Acta Med. Acad. Sci. Hung. 26:363-370. 149

Kitay/ J. I. 1954a Effect of pinealectomy on ovary weight in immature rats. Endocrinology 54:114-116.

Kitay, J. I. 1954b Pineal lesions and precocious puberty: A review. J. Clin. Endocr. Metab. 14:622-625.

Kizer, J. S., J. A. Zivin, D. M. Jacobowitz, and I. J. Kopin 1975 The nyctohemeral rhythm of plasma prolactin: Effects of ganglionectomy pinealectomy, constant light, constant darkness or 6-OH-dopamine administration. Endocrinology 96:1230-1240.

Klein, D. C., G. R. Berg, J. Weller, and W. Glinsmann 1970 Pineal gland: Dibutyryl cyclic adensoine monophosphate stimulation of labeled melatonin production. Science 167:1738-1740.

Klein, D. C., and J. L. Weller 1970 Indole metabolism in the pineal gland: A circadian rhythm in N-acetyltransferase. Science 169: 1093-1095.

Klein, D. C., J. L. Weller, and R. Y. Moore 1971 Melatonin metabolism: Neural regulation of pineal serotonin: acetyl coenzyme A N- acetyltransferase activity. Proc. Nat. Acad. Aci. USA 68: 3107-3156.

rau-yytj, M., and M. N. Sheridan 1976 Pineal function in hamsters bearing melatonin antibodies. Life Sci. 19:1235-1238.

Konig, A., and A. Meyer 1971 The effect of illumination on the circadian rhythm of the antidiuretic activity of the rat pineal. J. Interdiscipl. Cycle Res. 2:255-262.

Krstic, R. 1975 Scanning electron microscope observations of the canaliculi in the rat pineal gland. Experientia 31:1072-1074.

Krstic, R. 1976a Ultracytochemistry of the synaptic ribbons in the rat pineal organ. Cell Tiss. Res. 166:135-143.

Krstic, R. 1976b Combined scanning and transmission electron microscope study and electron probe microanalysis of human pineal acervuli. Cell Tiss. Res. 174:129-137.

Kurumado, K., and W. Mori 1976 Synaptic ribbon in the human pinealocyte. Acta Path. Jap. 26:381-384.

Landau, B., N. Ayalon, I. Lewis, and Y. Grunfeld 1969 Interruption of pregnancy in mice by gonadotropin inhibiting factor. Fert. Ster. 20:1023-1028. 150

Landau, B., R. Landau, and A. D. Schwartz 1965 . Presence of a gonadotrophin inhibiting factor in the urine of hypogonadotrophic-hypogonadal patients and further studies on its properties. J. Clin. Endocr. Metab. 25:339-342.

Landau, B., H. S. Schwartz, and L. J. Soffer 1960 Presence of a gonadotrophin inhibiting factor in urine of young children. Metab. Clin. Exp. 9^85-87.

Lapin, von V. 1972 Zur Wirkung sinultaner Thymektomie und Pinealktomie bei neugeborenen Ratten. Exp. Path. (Jena) ^7: 342-344.

Lawton, I. E., and N. B. Schwartz 1965 Pituitary LH content in rats exposed to continuous illumination. Endocrinology 77;1140- 1142.

Legait, H., and E. Legait 1977 Contribution a l'etude de la glande pineale humaine etude faite a l'aide de 747 glandes. Bull. Assoc. Anat. 61:107-129.

Legros, J. J., F. Louis, A. Demoulin, and P. Franchimont 1976 Immunoreactive neurophysins and vasotocin in human foetal pineal glands. J. Endocr. 69;289-290.

Legros, J. J., F. Louis, and U. Grotschel-Stewart 1975 Presence of immunoreactive neurophysin-like material in human target organs and pineal gland; Physiological meaning. Ann. N. Y. Acad. Sci. 248:157-171.

Lerner, A. B., J. D. Case, W. Mori, and M. R. Wright 1959 Melatonin in the peripheral nerve. Nature 183:1821.

Lerner, A. B., J. D. Case, Y. Takahashi, T. H. Lee, and W. Mori 1958 Isolation of melatonin, the pineal gland factor that lightens melanocytes. J. Amer. Chem. Soc. 80:2587.

Lin, H.S., B.-H. Hwang, and C.-Y. Tsang 1975 Fine structural changes in the hamster pineal gland after blinding and superior cervical ganglionectomy. Cell Tiss. Res. 158:285-299.

Lincoln, G. A. 1976 Seasonal changes in the pineal gland related to the reproductive cycle in the male hare, Lepus europaeus. J. Reprod. Fer. 46:489-491.

Little, J. C. 1971 Pineal modification of compensatory ovarian hypertrophy in the golden Hamster. Ph.D. dissertation, University of Texas Medical Branch, Galveston. 151

Lox, C. D., C. D. Christian, and M. W. Heine 1974 A simple radio­ immunoassay for testosterone. Amer. J. Obst. Gyn. 118;114-118.

Lukaszyk, A., and R. J. Reiter 1975a Hi3tophysiological evidence for the secretion of polypeptides by the pineal gland. Amer. J. Anat. 143:451-464.

Lukaszyk, A., and R. J. Reiter 1975b Morfologiczne podstawy dla sekrecji hormonow polipeptydowych w szyszynce matpy (Maccacus rhesus). Endokr. Pol. 26:603-611.

Lynch, H. J., R. J. Wurtman, M. A. Moskowitz, M. C. Archer, and M. H. Ho 1975 Daily rhythm in human urinary melatonin. Science 187:169-171.

Machado, C. R. S., A. B. M. Machado, and L. E. Wragg 1969 Circadian serotonin rhythm control: Sympathetic and non-sympathetic pathways in rat pineals of different ages. Endocrinology 85: 846-848.

Malmejac, J., and V. Donnet 1937 Sur 1'action vaso-motrice centrale des extraits epiphysaires. C. R. Soc. Biol. 126:370-372.

Marie, D. K., E. Matsuyama, and C. W. Lloyd 1965 Gonadotrophin content of pituitaries of rats in constant estrus induced by continuous illumination. Endocrinology 77;529-536.

Martin, J., and L. Davis 1941 Syndrome of destruction of the pineal gland: Experimental and clinical observations. Arch. Int. Med. 67:1119-1128.

Martini, L. 1974 Circadian rhythms and the role of the pineal gland. Chronobiologia First Supp. 1^:224-236.

Matsushima, S., and T. Ito 1972 Diurnal changes in sympathetic nerve endings in the mouse pineal: Semiquantitative electron micro­ scopic observations. J. Neural. Transm. 33:275-288.

Matsushima, S., and R. J. Reiter 1975 Ultrastructural observation of pineal gland capillaries in four rodent species. Amer. J. Anat. 143:265-281.

"Matthews, D. C.f D. J. Kennaway, R. G. Frith, G. Phillipou, A. LeCornu, and R. F. Seamark 1977 Plasma melatonin values in main and some domestic animals: Initial observations on the effects of pregnancy in man and pinealectomy in sheep. J. Endocr. 73: 41P-42P. •

Matthews, M. J., and B. Benson 1973 inactivation of pineal anti- gonadotropin by proteolytic enzymes. J. Endocr. 56:339-340. 152

Matthews, M. J., B. Benson, and A. E. Rodin 1971 Antigonadotropic activity in a melatonin-free extract of human pineal glands. Life Sci. I 10:1375-1379.

McKinney, T. D., M. K. Vaughan, and R. J. Reiter 1975 Pineal influence on intermale agression in adult house mice. Physiol. Behav. 15:213-216.

Mesaki, T. 1939 Experimental study on effect of pineal function on sexual cycle and sexual hormones. Jap. J. Obst. Gyn. 22: 26-37.

Mess, B. A., A. Heizer, A. Toth, and L. Tima 1971 Luteinization induced by pinealectomy in the polyfollicular ovaries of rats bearing anterior hypothalamic lesions. In: Ciba Foundation Symposium on the Pineal Gland. G. E. W. Wolstenholme and J. Knight (eds.). J. and A. Churchill: London 229-240.

Meyer, C. J., R. J. Wurtman, M. D. Altschule, and E. A. Lazo-Wasem 1961 The arrest of prolonged estrus in "middle-aged" rats by pineal gland extract. Endocrinology 69:795-800.

Michotte, Y., A. Lowenthal, L. Knaepen, M. Collard, and D. L. Massart 1977 A morphological and chemical study of calcification of the pineal gland. J. Neurol. 215:209-220.

Milcu, I., L. Nanu-Ionescu, R. Marcean, and V. lonescu 1969 The influence of the pineal gland on nitrogen metabolism. J. Endocr. 45:175-181.

Milcu, S. M., L. Nanu-Ionescu, and I. Milcu 1971 The effect of pinealectomy on plasma insulin in rats. In: Ciba Foundation Symposium on the Pineal Gland. G. E. W. Wolstenholme and J. Knight (eds.). J. and A. Churchill: London 345-360.

Milcu, S. M., S. Pavel, and C. Neascu 1963 Biological and chromato­ graphic characterization of a polypeptide from the bovine pineal gland. Endocrinology 72:563-566.

Milcu, S. M., and M. Pitis 1944 Technique pour le titrage de l'hormone inhibitrice sexuelle de l'epiphyse. Acta Endocr. 10:44-49.

Milcu, S. M., cind M. Pitis 1946 Action de l'epiphyse sur la glande thyroide. Acta Endocr. 12:52-58.

Milcu, S. M., and M. Pitis 1947 L'action de l'epiphyshormone sur la thyrotrophine. Acta Endocr. 13:7-9.

Minneman, K. P., and L. L. Iversen 1976 Diurnal rhythm in rat pineal cyclic nucleotide phosphodiesterase activity. Nature 260:59-61. 153

Minneman, K. P., H. Lynch, and R. J. Wurtman 1974 Relationship between environmental light intensity and retina-mediated supression of rat pineal serotonin-N-acetyl transferase. Life Sci. 15;1791- 1796.

Mizobe, F., and M. Kurokawa 1976 Enhancement of hydroxyindole-0- methyltransferase and DNA-dependent RNA polymerase activities induced by oestradiol in rat pineals in culture. Eur. J. Biochem. 66:193-199.

Miller, M. 1976 The ultrastructure of the human fetal pineal gland. II. INnervation and cell junctions. Cell Tiss. Res. 169:7-21.

Miller, M., K. MpJllgard, and J. E. Kimble 1975 presence of a pineal nerve in sheep and rabbit fetuses. Cell Tiss. Res. 158:451-459.

Moore, R. Y., and V. B. Eichler 1976 Central neural mechanism in diurnal rhythm regulation and neuroendocrine responses to light. Psychoneuroendocrinology 1^: 265-279.

Moore, R. Y., A. Heller, R. K. Bhatnager, R. J. Wurtman, and J. Axelrod 1968 Central control of the pineal gland: Visual pathways. Arch. Neurol. 18:208-218.

Moore, R. Y., and M. E. Traynor 1976 Diurnal rhythms in pineal N- acetyl transferase and hippocampal norepinephrine: Effects of water deprivation, blinding and hypothalamic lesions. Neuroendocrinology 20:250-259.

Moszkowska, A., and I. Ebels 1968 A study of the antigonadotropic action of synthetic arginine vasotocin. Experientia 24:610-611.

Moszkowska, A., and I. Ebels 1971 The influence of the pineal body on the gonadotropic function of the hypophysis. J. Neuro-visc. Rel. Suppl. Ji 160-176.

Moszkowska, A., A. Hus-Citheral, A. l'Heritier, W. Zurburg, and I. Ebels 1976 Separation of pineal extracts by gel filtration V. Location by paper chromatography of a sheep pineal principle inhibiting hypophyseal gonadotropic activity. J. Neural. Trans. 3B:239-247.

Moszkowska, A., C. Kordon, and I. Ebels 1971 Biochemical fractions and mechanisms involved in the pineal modulation of pituitary gonadotropin release. In: Ciba Foundation Symposium on the Pineal Gland. G. E. W. Wolstenholme and J. Knight (eds.). J. and A. Churchill: London 241-258.

Motta, M.,. F. Fraschini, and L. Martini 1967 Endocrine effects of pineal gland and of melatonin. Proc. Soc. Exp. Biol. Med. 126:431-435. 154

Murphy, B. D., and D. A. James 1974 The effects of light and sympathetic innervation to the head on nidation in mink. J. Exp. Zool. 187;267-276.

Nagler C. A., D. P. Cardinali, and J. M. Rosner 1975 Testosterone effects on protein synthesis in the rat pineal gland: Modula­ tion by the sympathetic nervous system. Life Sci. 16:81-92.

Neascu, C. 1972 The mechanism of antigonadotropic action of a polypeptide extracted from a bovine pineal gland. Rev. Roum. Physiol. j):161-169.

Nicholls, T. J. 1974 Changes in plasma LH levels during a photo- periodically controlled reproductive cycle in the canary (Serinus canarius). Gen. Comp. Endocr. 24:442.

Nielsen, J. T., and M. Miller 1975 Nervous connections between the brain and the pineal gland in the cat (Felis catus) and the monkey (Cercopithecus aethiops). Cell Tiss. Res. 161:293-301.

Niemi, M., and M. Ikoken 1960 Histochemical evidence of aminopeptidase activity in the rat pineal gland. Nature 185:928.

Niles, L. P., G. M. Brown, and L. J. Grota 1977 Effects of neutraliza­ tion of circulating melatonin and N-acetyl serotonin on plasma prolactin levels. Neuroendocrinology 22k 14-23.

Nir, I., N. Hirschmann, N. Kremer, and S. G. Sulman 1974' Inversion of pineal N-acetyltransferase rhythm by reversed environmental lighting. Neuroendocrinology 15:231-236.

Nir, I., N. Hirschmann, and F. G. Sulman 1971 Diurnal rhythms of pineal nucleic acids and proteins. Neuroendocrinology 7^:271- 277.

Nir, I., U. Schmidt, N. Hirschmann, and F. G. Sulman 1971 The effect of pinealectomy on rat plasma cortocosterone levels under various conditions of light. Life Sci. 10:317-324.

Nir, I., J. Shani (Mishkinsky), D. Locker, and F. G. Sulman 1972 Effect of light and pinealectomy on body weight and tibia cartilage of female rats. Life Sci. 11:41-49.

Niswender, G. D., C. L. Chen, A. R. Midgley, Jr., J. Meites, and S. Ellis 1969 Radioimmunoassay for rat prolactin. Proc. Soc. Exp. Biol. Med. 130:793-797.

Niswender, G. D-, A. R. Midgley, S. E. Monroe, and L. E. Reichert 1968 Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-^31I. Proc. Soc. Exp. Biol. Med. 128:807- 811. 155

Novakova, v., J. Sterc, W. Sandritter, and J. Krecek 1971 The day- night differences in total RNA content of peurenchymal cells of rat pineal gland. Beitr. Path. Bd. 144:211—215.

Ogle, T. F.f and J. I. Kitay 1976 Effects of pinealectomy on adrenal function in vivo and in vitro in female rats. Endocrinology 98:20-24.

Oishi, T., and J. K. Lauber 1974 Pineal control of photo-endocrine responses in growing Japanese quail. Endocrinology 94:1731- 1734.

Oksche, A., H. Kirschstein, H. Kobayashi, and D. S. Farner 1972 Electron microscopic and experimental studies of the pineal organ in the wite-crownea sparrow, Zonotrichia leucophrys gambelii. Z. Zellforsch. 124:247-274.

Oleshansky, M. A., and N. H. Neff 1975 Rat pineal adenosine cyclic 3*,5'-monophosphate phosphodiesterase activity: Modulation in vivo by a beta adrenergic receptor. Mol. Pharm. 11:552-557.

Orts, R. J. 1977 Reduction of serum LH and testosterone in male rats by a partially purified bovine pineal extract. Biol. Reprod. 16:249-254.

Orts, R. J., and B. Benson 1973 Inhibition effects on serum and pituitary LH by a melatonin-free extract of bovine pineal glands. Life Sci. II 12:513-519.

Orts, R. J., B. Benson, and B. F. Cook 1974 LH inhibiting properties of aqueous extracts of rat pineal glands. Life Sci. 14;1501- 1510.

Orts, R. J., K. M. Kocan, and R. Y. Johnson 1977 Antifertility properties of bovine pineal extracts, reduction of ovulation and pre-ovulatory luteinizing hormone in the rat. Acta Endocr. 85:225-235.

Orts, R. J., K. M. Kocan, and I. B. Wilson 1975 Inhibitory action of melatonin on a pineal antigonadotropin. Life Sci. 17:845-850.

Orts, R. J., K. M. Kocan, and W. P. Yonushonis 1975 Fertility control in female rats by bovine pineal gland extracts. Life Sci. 17:531-537.

Ota, M., A. Dronkert, and A. H. Gates 1968a The presence of a gonadotrophin inhibiting substance in human urine. Fert. Ster. 19:100-109. 156

Ota, M., A. Dronkert, and A. H. Gates 1968b Effect of a gonadotrophin- inhibiting substance on the ovarian histology in mice. Metab. Clin. Exp. 1*7:181-188.

Ota, M., S. HOriuchi, and K. Obara 1975 Inhibition of ovulation induced with PMS and HCG by a melatonin-free extract of bovine pineal powder. Neuroendocrinology 18;311-321.

Ota, M., K. S. Hsieh, and K. Obara 1971 Absence of gonadotropin inhibiting substance in the urine of pinealectomized rats. Endocrinology 88:816-820.

Ota, M., K. Obara, and A. Dronkert 1967 The effect of a gonadotropin- inhibiting substance on supra-ovulation induced by pregnant mare serum gonadotrophin and human chorionic gonadotrophin. Endocr. Jap. 14:308-312.

Ota, M., N. Sato, and K. Obara 1977 Further studies on gonadotropin- inhibiting substances excreted in human urine. Endocr. Jap. 24; 545-551.

Otoh, S., H. Takahashi, and T. Sakai 1962 Atrophy of reproductive organs in male rats housed in continuous darkness. J. Reprod. Fert. _4:233-244.

Owman, C. 1964 Sympathetic nerves probably storing two types of monoamines in the rat pineal gland. Int. J. Neuropharm. 2\ 105-112.

Owman, C. 1968 On the significance of the 5-hydroxytryptamine stores in the pineal gland. Adv. Phar. 6^:167-169.

Ozaki, Y., and H. J. Lynch 1976 Presence of melatonin in plasma and urine of pinealectomized rats. Endocrinology 99;641-644.

Ozaki, Y., H. J. Lynch, and R. J. Wurtman 1976 Melatonin in rat pineal, plasma, and urine: 24-hour rhythmicity and effect of chlorpromazine. Endocrinology 98^1418-1424.

Pang, S. F., and C. L. Ralph 1975 Pineal and serum melatonin at midday and midnight following pinealectomy or castration in male rats. J. Exp. Zool. 193:275-280.

Parfitt, A. G., and D. C. Klein 1976 Sympathetic nerve endings in the pineal gland protect against acute stress-induced increase in N-acetyltransferase {EC 2.3.1.5) activity. Endocrinology 99: 840-851.

Pavel, S. 1965 Evidence for the presence of lysine vasotocin in the pig pineal gland. Endocrinology 77:812-817. 157

Pavel, S. 1971 Evidence for the ependymal origin of arginine vasotocin in the bovine pineal gland. Endocrinology 89:613-614.

Pavel/ S. 1973 Arginine vasotocin release into cerebrospinal fluid of cats induced by melatonin. Nature 246:183-184.

Pavel, S., M. calb, and M. Georgescu 1975 Reversal of the effects of pinealectomy on the pituitary prolactin content in mice by very low concentrations of vasotocin injected into the third cerebral ventricle. J. Endocr. 66:289-290.

Pavel, S., A. Cristoveanu, R. Goldstein, and M. Calb 1977 Inhibition of release of corticotropin releasing hormone in cats by extremely small amounts of vasotocin injected into third ventricle of brain—Evidence for involvement of 5- hydroxytryptamine-containing neurons. Endocrinology 101: 672-679.

Pavel, S., M. Dorescu, R. Petrescu-Holban, and E. Ghinea 1973 Biosynthesis of a vasotocin-like peptide in cell cultures from pineal glands of human fetuses. Science 181:1252-1253.

Pavel, S., I. Dumitru, I. Klepsh, and M. Dorcescu 1973-74 A gonadotropin inhibiting principle in the pineal of human fetuses: Evidence for its identity with arginine vasotocin. Neuroendocrinology 13:41-46.

Pavel, S., R. Goldstein, and M. Calb 1975 Vasotocin content in the pineal gland of foetal, newborn and adult male rats. J. Endocr. 66:283-284.

Pavel, S., R. Goldstein, C. Gheorghiu, and M. Calb 1977 Pineal vasotocin: Release into cat cerebrospinal fluid by melanocyte- stimulating hormone release-inhibiting factor. Science 197: 179-180.

Pavel, S., R. Goldstein, E. Ghinea, and M. Calb 1977 Chromatographic evidence for vasotocin biosynthesis by cultured pineal ependymal cells from rat fetuses. Endocrinology 100:205-208.

Pavel, S., and S. Petrescu 1966 Inhibition of gonadotrophin by highly purified pineal peptide and by synthetic arginine vasotocin. Nature 212:1054.

Pavel, S., M. Petrescu, and N. Vicoleanu 1973 Evidence of central • gonadotropin inhibiting activity of arginine vasotocin in the female mouse. Neuroendocrinology 11:370-374. 158

Pavel, S., D. Psatta, and R. Goldstein 1977 Slow-wave sleep induced in cats by extremely small amounts of synthetic and pineal vasotocin injected into the third ventricle of the brain. Brain Res. Bull. 2^:251-255.

Pazo, J. H., A. B. Houssay, T. A. Davison, and R. J. Chait 1968 On the mechanism of the thyroid hypertrophy in pinealectomized rats. Acta Physiol. Lat. Amer. 18:332-340.

Pelham, R. W. 1975 A serum melatonin rhythm in chickens and its abolition by pinealectomy. Endocrinology 96:543-546.

Pevet, P. 1977 Presence of different populations of pinealocytes in mammalian pineal gland. J. Neural. Trans. 40:289-304.

Pevet, P., J. Ariens-Kappers, and A. M. Vovite 1977 The pineal gland of nocturnal mammals I. The pinealocytes of the bat (Nyctalus noctula, Schreber). J. Neural. Trans. 40:47-68.

Pevet, P., M. J. Juillard, A. R. Smith, and J. Ariens-Kappers 1976 The pineal gland of the mole (Talpa europaea L.) III. A fluorescence histochemical study. Cell Tiss. Res. 165:297-306.

Pevet, P., and A. R. Smith 1975a Proceedings: The mole pinealocytes. J. Endocr. 64:64P.

Pevet, P., and A. R. Smith 1975b The pineal gland of the mole (Talpa europaea L.) II. Ultrastructural variations observed in the pinealocytes during different parts of the sexual cycle. J. Neural. Trans. 36:227-248.

Pevet, P., A. R. Smith, L. Van de Kar, and H. Van Bronswijk 1975 Effect of castration on the rat pineal gland: A fluorescence histochemical and biochemical study. Experientia 31:1237-1239.

Piezzi, R. S., and L. S. Gutierrez 1975 Electron microscopic studies on the pineal organ of the Antartic penguin (Pygoscelis papua). Cell Tiss. Res. 164:559-570.

Pomerantz, G., and R. J. Reiter 1973 Age dependent changes in the susceptibility of parathyroidectomized, pinealectomized rats to seizures. Exp. Neurol. 40:254-257.

Popova, N. K., S. G. Kolaeva, and I. I. Diansva 1975 State of the epiphysis during winter hibernation. Biull. Eksp. Biol. Med. 79:116-117. 159

Porter, J. R., and M. Heiman 1977 The effect of pineal indoles and a crude aqueous pineal extract on ACTH mediated corticosterone release by isolated adrenal cells. Life Sci. 2Q;1363-1372.

Povlishock, J. T., R. M. Kriebel, and H. R. Seibel 1975 A light and electron microscopic study of the pineal gland of the ground squirrel, Citellus tridecemlineatus. Amer. J. Anat. 143: 465-484.

Quay, W. B. 1961 Reduction of mammalian pineal weight and lipid during continuous light. Gen. Comp. Endocr. 1_:211-217.

Quay, W. B. 1963 Cytologic and metabolic parameters of pineal inhibi­ tion by continuous light in the rat. Zeitschrift. Zellforsch. 60:479-490.

Quay, W. B. 1972 24-hour rhythmicity in carbonic-anhydrase activities of choroid plexuses and pineal-gland. Anat. Rec. 174:279-287.

Quay, W. B. 1974a Pineal Chemistry in Cellular and Physiological Mechanisms. Charles Thomas: Springfield.

Quay, W. B. 1974b Pineal canaliculi: Demonstration, 24-hour rhythmicity and experimental modification. Amer. J. Anat. 139:81-93.

Quay, W. B. 1976 Seasonal cycle and physiologic correlates of pinealocytes nuclear and nucleolar diameters in the bats, Myotis lucifugus and Myotis sodalis. Gen. Comp. Endocr. 29: 369-375.

Quay, W. B., and A. Renzoni 1966 Twenty-four-hour rhythms in pineal mitotic activity and nuclear and nucleolar dimensions. Growth 30:315-324.

Raikhlin, N. T., I. M. Knetnoy, Z. G. Kadagidze, and A. V. Sokolov 1976 Immunohistochemical demonstration of the localization of melatonin and N-acetyl serotonin in enterochromaffin cells. Biull. Eksp. Biol. Med. 82^1400-1401.

Raikhlin, N. T., I. M. Knetnoy, and V. N. Tolkachev 1975 Melatonin may be synthesized in enterochromaffin cells. Nature 255: 344-345.

Ralph, C. L. 1976 Correlation of melatonin content in pineal gland, blood, and brain of some birds and mammals. Amer. Zool. 16: 35-43. 160

Ralph, C. L., S. Binkley, S. E. MacBride, and D. C. Klein 1975 Regulation of pineal rhythms in chickens: Effects of blinding, constant light, constant dark, and superior cervical ganglionectomy. Endocrinology 97:1373-1378.

Ramaley, J. A. 1974 The changes in basal corticosterone secretion in rats blinded at birth. Experientia 30:827.

Reinharz, A. c., P. Czernichow, and M. B. Vallotton 1974 Neurophysin- like protein in bovine pineal gland. J. Endocr. 62:35-44.

Reinharz, A. C., P. Czernichow, and M. B. Vallotton 1975 Neurophysins I and II from the bovine posterior pituitary lobe and neurophysin-like proteins from bovine pineal gland. Ann. N. Y. Acad. Sci. 248:172-183.

Reinharz, A. C., and M. B. Vallotton 1974 Presence of peptidase in bovine pineal gland. Experientia 30:691.

Reinharz, A. C., and M. B. Vallotton 1977 Presence of two neurophysins in the human pineal gland. Endocrinology 100:994-1001.

Reiss, M., R. H. Davis, M. B. Sideman, I. Mauer, and E. S. Plichta 1963 Action of pineal extracts on the gonads and their function. J. Endocr. 27:107-118.

Reiter, R. J. 1967a Failure of the pineal gland to prevent gonadotrophin-induced ovarian stimulation in blinded hamsters. J. Endocr. 38:199-200.

Reiter, R. J. 1967b The effect of pineal grafts, pinealectomy and denervation of the pineal gland on the reproductive organs of male hamsters. Neuroendocrinology 2^:138-146.

Reiter, R. J. 1968 Changes in the reproductive organs of cold-exposed and light-deprived female hamsters (Mesocricetus auratus). J. Reprod. Fert. 16:217-222.

Reiter, R. J. 1969a Pineal function in longterm blinded male and female golden hamsters. Gen. Comp. Endocr. 12:460-468.

Reiter, R. J. 1969b Growth of the endocrine organs of female hamsters blinded at 25 days of age. Experientia 25:751-752.

Reiter, R. J. 1972a Evidence for refractoriness of pituitary-gonadal axis to pineal-gland in golden hamsters and its possible implications in annual reproductive rhythms. Anat. Rec. 173: 365-371. 161

Reiter, R. J. 1972b Surgical procedures involving the pineal gland which prevent gonadal degeneration in adult male hamsters. Ann. Endocr. 33:571-581.

Reiter, R. J. 1972c The role of the pineal in reproduction. In: Reproductive Biology. H. Balin and S. Glosser (eds.). Excerpta Medica: Amsterdam 71-114.

Reiter, R. J. 1973 Pineal control of a seasonal reproductive rhythm in male golden hamsters exposed to natural daylight and tempera­ ture. Endocrinology 92^423-430.

Reiter, R. J. 1974a Pineal-mediated regression of the reproductive organs of female hamsters exposed to natural photoperiods during the winter months. Amer. J. Obst. Gyn. 118;878-880.

Reiter, R. J. 1974b Circannual reproductive rhythms in mammals related to photoperiod and pineal function: a review. Chronobiologia 1:365-395.

Reiter, R. J. 1975a Changes in pituitary prolactin levels of female hamsters as a function of age, photoperiod, and pinealectomy. Acta Endocr. 79:43-50.

Reiter, R. J. 1975b Exogenous and endogenous control of the annual reproductive cycle in the male golden hamster: Participation of the pineal gland. J. Exp. Zool. 191:111-120.

Reiter, R. J. 1976 Regulation of pituitary gonadotropins by the mammalian pineal gland. In: Neuroendocrine Regulation of Fertility. T. C. Anand-Kumar (ed.). Karger: Basel 215-226.

Reiter, R. J., D. E. Blask, L. Y. Johnson, P. K. Rudeen, M. K. Vaughan, and P. J. Waring 1976 Melatonin inhibition of reproduction in the male hamster: Its dependence on time of day of adminis­ tration and on an intact and sympathetically innervated pineal gland. Neuroendocrinology 22:10*7-116.

Reiter, R. J., D. E. Blask, J. A. Talbot, and M. P. Barnett 1973 Nature and the time course of seizures associated with surgical removal of the pineal gland from parathyroidectomized rats. Exp. Neurol. 38:386-397.

Reiter, R. J., D. E. Blask, and M. K. Vaughan 1975 A counter anti- gonadotropic effect of melatonin in male rats. Neuroendo­ crinology 19:72-80.

Reiter, R. J., K. Blum, J. E. Wallace, and J. H. Merritt 1974 Pineal gland: Evidence for an influence on ethanol preference in male Syrian hamsters. Comp. Biochem. Physiol. 47:11-16. 162

Reiter, R. J., R. A. Hoffman# and R. J. Hester 1966 The effects of thiourea, photoperiod and the pineal gland on the thyroid, adrenal and reproductive organs of female hamsters. J. Exp. Zool. 162:263-268.

Reiter, R. J., and L. Y. Johnson 1974a Depressant action of the pineal gland on pituitary LH and prolactin in male hamsters. Hor. Res. _5:311-320.

Reiter, R. J., and L. Y. Johnson 1974b Elevated pituitary LH and depressed prolactin levels in female hamsters with pineal- induced gonadal atrophy and the effects of chronic treatment with synthetic LRF. Neuroendocrinology 14;310-315.

Reiter, R. J., and L. Y. Johnson 1974c Pineal regulation of iiranuno- reactive LH and prolactin in light deprived female hamsters. Fert. Ster. 125:958-965.

Reiter, R. J., and W. W. Morgan 1972 Attempts to characterize the convulsive response of parathyroidectomized rats to pineal gland removal. Physiol. Behav. 9^:203-208.

Reiter, R. J., W. W. Morgan, and J. A. Talbot 1973 Seizures in rats induced by pinealectomy: Influence of diazepam, chlordiazepoxide, diphenyldydantoin and pineal substances. Arch. Int. Pharmcodyn. Ther. 202:219-230.

Reiter, R. J., and S. Sorrentino 1972 Prevention of pineal-mediated reproductive responses in light-deprived hamsters by partial or total isolation of the medial basal hypothalamus. J. Neuro-visc. Rel. 32:355-367.

Reiter, R. J., S. Sorrentino, and R. A. Hoffman 1970 Early photo­ periodic conditions and pineal antigonadal function in male hamsters. Int. J. Fert. 15:163-170.

Reiter, R. J., S. Sorrentino, and E. L. Jarrow 1971 Central and peripheral neural pathways necessary for pineal function in the adult female rat. Neuroendocrinology jJ: 321-333.

Reiter, R. J., and M. K. Vaughan 1975 A study of indoles which inhibit pineal antigonadotropic activity in male hamsters. Endocr. Res. Comm. 2^:299-308.

Reiter, R. J., M. K. Vaughan, D. E. Blask, and L. Y. Johnson 1974 Melatonin: Its inhibition of .pineal antigonadotropic activity in male hamsters. Science 185:1169-1171. 163

Reiter, R. J., M. K. Vaughan, D. E. Blask, and L. Y. Johnson 1975 Pineal methoxyindoles: New evidence concerning their function in the control of pineal-mediated changes in the reproductive physiology of male golden hamsters. Endocrinology 96:206-213.

Reiter, R. J., m. K. Vaughan, R. K. Rudeen, and R. C. Philo 1976 Melatonin induction of testicular recrudescence in hamsters and its subsequent inhibitory action on the ant igonadotropic influence of darkness on the pituitary-gonadal axis. Amer. J. Anat. 147;235-242.

Reiter, R. J., M. K. Vaughan, P. K. Rudeen, G. M. Vaughan, and P. J. Waring 1975 Melatonin-pineal relationships in female golden hamsters. Proc. Soc. Exp. Biol. Med. 149:290-293.

Reiter, R. J., M. K. Vaughan, and P. J. Waring 1975 Studies on the minimal dosage of melatonin required to inhibit pineal anti- gonadotrophic activity in male golden hamsters. Hor. Res. 6z258-267.

Reiter, R. J., M. K. Vaughan, and P. J. Waring 1977 Prevention by melatonin of short day induced atrophy of the reproductive systems of male and female hamsters. Acta Endocr. 84:410-418.

Reiter, R. J., M. G. Welsh, and M. K. Vaughan 1976 Age related changes in the intact and sympathetically denervated gerbil pineal gland. Amer. J. Anat. 146:427-431.

Relkin, R. 1972a Effects of variations in environmental lighting on pituitary and plasma prolactin levels in the rat. Neuro- endocrinology 9^:278-284. ——

Relkin# R. 1972b Effects of variation in environmental lighting on pituitary and plasma prolactin levels in the rat. Neuro- endocrinology jJ:278-284.

Reznik-Schliller, H., and G. Reznik 1974 The influence of hibernation upon the ultrastructure of the Leydig cells and spermatids of the European hamster. Fert. Ster. 25:621-636.

Rohleder, H. 1928 Practiklaisch Therapie der Masturbation. D. Med. Woch. 54:189-190.

Romero, J. A. 1976 Influence of diurnal cycles on biochemical parameters of drug sensitivity: The pineal gland as a model. Fed. Proc. 35:1157-1161. 164

Romero, J. A-, M. Zatz, and J. Axelrod 1975 Beta-adrenergic stimula­ tion of pineal N-acetyltransferase: adenosine 3', 5*-cyclic monophosphate stimulates both RNA and protein synthesis. Proc. Nat. Acad. Sci. 72:2107-2111.

Romijn, H. J. 1975a Structure and innervation of the pineal gland of the rabbit, Oryctolagus cuniculus (L). III. An electron microscopic investigation of the innervation. Cell Tiss. Res. 157;25-51.

Romijn, H. J. 1975b The ultrastructure of the rabbit pineal gland after sympathectomy, parasympathectomy, continuous illumination, and continuous darkness. J. Neural. Trans. 36:183-194.

Romijn, H. J. 1976a Proceedings: Electron microscopy of endocrine structures in the rabbit pineal gland. Acta Morph. Neerl. Scand. 14:99-100.

Romijn, H. J. 1976b The influence of some sympatholytic, para­ sympatholytic, and serotonin-synthesis-inhibiting agents on the ultrastructure of the rabbit pineal organ. Cell Tiss. Res. 167:167-177.

Romijn, H. J., and A. J. Gelsema 1976 Electron microscopy of the rabbit pineal organ in vitro. Evidence of norepinephrine- stimulated secretory activity of the Golgi apparatus. Cell Tiss. Res. 172:365-377.

Rimijn, H. J., M. T. Mud, and P. S. V7olters 1976 Diurnal variations in number of Golgi dense-core vesicles in light pinealocytes of the rabbit. J. Neural. Trans. 38:231-237.

Romijn, H. J., M. T. Mud, and P. S. Wolters 1977 A pharmacological and autoradiographic study on the ultrastructural localization of indoleamine synthesis in the rabbit pineal gland. Cell Tiss. Res. 185:199-215.

RpSnnekleiv, O. K., L. Krulich, and S. M. McCann 1973 An early morning surge of prolactin in the male rat and its abolition by pinealectomy. Endocrinology jJ2:1339-1342.

Rjrfnnekleiv, O. K., and S. M. McCann 1975a Effects of pinealectomy, cinosmia and blinding on serum and pituitary prolactin in intact and castrated male rats. Neuroendocrinology 17:340-353.

Rjrfrmekleiv, 0. K., and S. M. McCann 1975b Effects of pinealectomy, anosmia and blinding alone or in combination on gonadotropin secretion and pituitary and target gland weight in intact and castrated male rats. Neuroendocrinology 19:97-114. 165

Rosenbloom, A. A., and D. A. Fisher 1975a Arginine vasotocin in the rabbit subcommissural organ. Endocrinology 96:1038-1040.

Rosenbloom, A. A., and D. A. Fisher 1975b JRadioimmunoassayable AVT and AVP in adult mammalian brain tissue: Comparison of normal and Brattleboro rats. Neuroendocrinology 17;354-361.

Rosenblum, X. Y., B. Benson, and V. J. Hruby 1976 Chemical differences between bovine pineal antigonadotropin and arginine vasotocin. Life Sci. 18:1367-1374.

Roth, W. D. 1965 MEtabolic and morphologic studies on the rat pineal organ during puberty. Prog. Brain Res. 10:552-561.

Roth, W. D., R. J. Wurtman, and M. D. Altschule 1962 Morphologic changes in the pineal parenchyma cells of rats exposed to continuous light or darkness. Endocrinology 71:888-892.

Rowe, V., E. A. Neale, L. Avins, G. Guroff, and B. K. Sdhrier 1977 Pineal gland cells in culture. Morphology, biochemistry, differentiation and co-culture with sympathetic neurons. Exp. Cell. Res. 104:345-356.

Rowntree, L. G., J. H. Clark, A. Steinberg, and A. M. Hanson 1936a Biological effects of pineal extract (Hanson); accruing retardation in growth and accruing acceleration in the develop­ ment in successive generations of rats under continuous treat­ ment with pineal extract. Endocrinology 20;348-354.

Rowntree, L. G., J. H. Clark, A. Steinberg, and A. M. Hanson 1936b Biologic effects of pineal extract (Hanson): amplification of effects in young resulting from treatment of successive genera­ tions of parent rats. JAMA 106:370-373.

Rudeen, P. K., and R. J. Reiter 1977 Effect of shortened photoperiods on pineal serotonin N-acetyltransferase activity and rhythmicity. J. Interdisc. Cycle Res. ji:47-55.

Ruzsas, C., G. P. Trentini, and B. Mess 1977 The role of the pineal gland in the regulation of XJi-release in rats with different types of the anovolutory syndrome. Endokrinologie 70:142-150.

Sackman, J. W., J. C. Little, P. K. Rudeen, P. J. Waring, and R. J. Reiter 1977 The effect of pineal indoles given late in the light period on reproductive organs and pituitary prolactin levels in male golden hamsters. Hor. Res. _8:84-92.

Satodate, R., S. Katsura, and M. Ota 1973 Structural changes of the pineals of blinded rats fed long term. Experientia 29;1414- 1415. 166

Shellabarger, C. J. 1952 Pinealectomy with pineal injection in the young cockerel. Endocrinology 51:152-154.

Shellabarger, C. J., and W. R. Breneman 1950 The effects of pineal­ ectomy on white leghorn cockerels. Proc. Indiana Acad. Sci. 59:299.

Silberstein, F., and P. Engel 1933 Uber das Vorkommen einer oestragenen Substanz in der Epiphyse. Klin. Wchnschr. 12; 908-910.

Simonnet, H., and L. Thieblot 1951 Recherches esqperimentales sur la physiologie de la glande pineale. Acta Endocr. 7^:306-320.

Simonnet, H., L. Thieblot, and T. Melik 1951 Influence de 1'epiphyse sur l'ovaire de la jeune rate. Ann. Endocr. 12:202-205.

Simonovic, I., L. Krsmanovic, S. Stojikovic, and D. Marie 1976 Effects of pinealectomy on normal and constant light induced persistent estrus in rats. Rev. Res. Sci. - Univ. Novi Sad 6^:327-334.

Slama-Scemama, A. 1976 Effect of pinealectomy on gonadotropins in immature female rats. J. Neural. Trans. 39:251-256.

Smith, A. R., P. Pevet, L. van de Kar, and R. V. Oosteron 1975 Effect of gonadotropic hormones on the rat pineal gland: A fluorescent histochemical and biochemical study. J. Neural. Trans. 36: 217-226.

Smit-Vis, J. H. 1972 The effect of pinealectomy and of testosterone administration on the occurrence of hibernation in adult male golden hamsters. Acta Morphol. Neerl. Scand. 10:269-282.

Snyder, S. H., M. Zweig, and J. Axelrod 1964 Control of circadian rhythm in the serotonin content of the rat pineal gland. Life Sci. 211175-1179-

Sorrentino, S., and B. Benson 1970 Effects of blinding and pinealectomy on the reproductive organs of adult male and female rats. Gen. Comp. Endocr. 15:222-236.

Sorrentino, S., and R. J. Reiter 1970 Pineal-induced alteration of estrous cycles in blinded hamsters. Gen. Comp. Endocr. 15: 39-42.

Steger, R. W., J. J. Peluso, and E. S. E. Hafez 1976 Effect of photo- period on gonadotropin releasing hormone-induced LH release in the immature rat. Neuroendocrinology 21:68-73. 167

Sunberg, D. K., and K. M. Knigge 1978 Luteinizing hormone-releasing hormone (LHRH) production and degradation by rat medial basal hypothalami in vitro. Brain Res. 139:89-101.

Takahashi, M., K. Choi, and Y. Suzuki 1971 Light activation of gonadal function restrained by continuous dark treatment in male rats. Endocr. Jap. 18:195-203.

Takeo, Y.r M. Anazawa, K. Shirama, K. Shimizu, and K. Maekawa 1975 Pinealectomy and sexual rhythm in female rats. Endocr. Jap. 22:219-224.

Tamarkin, L., C. W. Hollister, N. G. Lefebvre, and B. D. Goldman 1977 Melatonin induction of gonadal quiescence in pinealectomized Syrian hamsters. Science 88:953-955.

Tamarkin, L., N. G. Lefebvre, C. W. Hollister, and B. D. Goldman 1977 Effect of melatonin administered during the night on reproductive function in the Syrian hamster. Endocrinology 101:631-634.

Tamarkin, L., W. K. Westrom, A. I. Hamill, and B. D. Goldman 1976 Effect of melatonin on the reproductive systems of male and female Syrian hamsters: A diurnal rhythm in sensitivity to melatonin. Endocrinology 99:1534-1541.

Tapp, E., and M. Huxley 1972 The histological appearance of the human pineal from puberty to old age. J. Path. 108:137-144.

Tapp, E., M. Huxley, and S. Davies 1973 The enzyme histochemistry of the pineal gland in rats. Histochem. J. 5^49-56.

Thieblot, L., A. Alassimone, and S. Blaise 1966 Etude chromatographique et electrophoretique du facteur antigonadotrope de la glands pin&ale. Ann. d'Endocr. 27:861-866.

Thieblot, L., J. Berthelay, and S. Blaise 1966 Isolement d'unprincipe antigonadotrope dans 1'urine d'enfants impuberes. C. R. Soc. Biol. 160:1871-1874.

Thieblot, L., and S. Blaise 1963 Influence de la glands pineale sur les gonades. Ann. Endocr. 24:270-286.

Thieblot, L., and S. Blaise 1965 Influence de la glande pineale sur la sphere genitale. Prog. Brain Res. 10:577-584.

Thieblot, L., and S. Blaise 1966 Etude biochemique du principle pineal antigonadotrope. Probl. Actuel. Endocr. Natr. 10:257-275. 168

Thompson, A. p. D. 1954 The onset of estrus in normal and blinded ferrets. Proc. Roy. Soc. B142:126-135.

Thorpe, P. A., and J. Herbert 1976a Studies on the duration of the breeding season and photorefractoriness in female ferrets pinealectomized or treated with melatonin. J. Endocr. 70; 255-262.

Thorpe, P. A., and J. Herbert 1976b The effect of lesions of the accessory optic tract terminal nuclei on the gonadal response to light in ferrets. Neuroendocrinology 22^250-259.

Tigchelaar, P. V., and A. V. Nalbandov 1975 The effect of the pineal gland on ovulation and pregnancy in the rat. Biol. Reprod. 13:461-469.

Tima, L., C. P. Trentini, and B. Mess 1973 Effect of serotonin on ovulation induced by pinealectomy in anovulatory frontal- deafferented rats. Neuroendocrinology 12:149-152.

Timmermans, L.f and V. Devecerski 1972a Repercussions cytolenzymo- logiques de 1'epiphysectomie sur le testicule du rat. C. R. Soc. Biol. 166:733-736.

Timmermans, L., and V. Devecerski 1972b Influence de 1'epiphysectomie sur la structure et le metabolisme de la paroi vesicale du rat. C. R- Soc. Biol. 166:968-970.

Trentini, G. P., C. F. De Gaetani, L. Martini, and B. Mess 1976 Effect on pinealectomy and of bilateral cervical ganglionectomy on Serum LH levels in constant estrous-anovulatory rats. Proc. Soc. Exp. Biol. Med. 153:490-494.

Tsang, D., and J. B. Martin 1976 Effect of hypothalamic hormones on the concentration of adenosine 3', 5'-monophosphate in incubated rat pineal glands. Life Sci. 19:911-917.

Turek, F. W. 1977 Antigonadal effect of melatonin in pinealectomized and intact male hamsters. Proc. Soc. Exp. Biol. Med. 155: 31-79.

Turek, F. W., C. Desjardins, and M. Menaker 1975 Melatonin: Antigonadal and progonadal effects in male golden hamsters. Science 190: 280-282.

Turek, F. W., C. Desjardins, and M. Menaker 1976 Differential effects of melatonin on the testes of photoperiodic and nonphotoperiodic rodents. Biol. Reprod. 15:94-97. 169

Upson, R. H., and B. Benson 1977 Effects of blinding on the ultra- structure of mouse pinealocytes with particular emphasis on the dense-cored vesicles. Cell Tiss. Res. 183;491-499.

Upson, R. H., B. Benson, and V. Satterfield 1976 Quantitation of ultrastructural changes in the mouse pineal in response to continuous illumination. Anat. Rec. 184;311-323. j Urry, R. L., K. A. Dougherty, J. L. Frehn, and L. C. Ellis 1976 Factors other than light affecting the pineal gland: Hypophysectomy, testosterone; dihydrotestosterone, estradiol, cryptorchidism, and stress. Amer. Zool. 16:79-91.

Van der Have-Kirchberg, M. L. L., A. de Mor£e, J. F. Van Laar, G. -J. Gerwig, C. Versluis, I. Ebels, A. Hus-Citharel, A. l'Heritier, S. Roseau, W. Zurburg, and Aj Moszkowska 1977 Separation of pineal extracts by gel filtration. VI. Isolation and identification from sheep pineals of biopterin; comparison of the isolated compound with some synthetic pteridines and the biological activity in in vitro and in vivo bioassays. J. Neural. Trans. 40;205-220.

Vaughan, M. K. 1971 Effects on accessory organs, adrenals and thymus after pinealectomy in male mice. Anat. Rec. 169:446.

Vaughan, M. K., D. E. Blask, L. Y. Johnson, and R.J. Reiter 1975 Prolactin-releasing activity of arginine vasotocin in vitro. Hor. Res. jS:342-350. II Vaughan, M. K., D. E. Blask, G. M. Vaughan, and R. J. Reiter 1976 Dose dependent prolactin releasing activity of arginine vasotocin in intact and pinealectomized estrogen-progesterone treated adult male rats. Endocrinology j)9s 1319-1322.

Vaughan, M. K., and R. J. Reiter 1971 Transient hypertrophy of the ventral prostate and coagulating glands and accelerated thymic involution following pinealectomy in the mouse. Tex. Rep. Biol. Med. 29:579-586.

Vaughan, M. K. , R. J. Reiter, T. McKinney, and G. M. Vaughan 1974 Inhibition of growth of gonadal dependent structures by arginine vasotocin and purified bovine pineal fractions in immature mice and hamsters. Int. J. Fert. 19:103.

Vaughan, M. K. , R. J. Reiter, and G. M. Vaughan 1976 Fertility patterns in female mice following treatment with arginine vasotocin or melatonin. IntJ J. Fert. 21:65-68. 170

Vaughan, M. K., G. M. Vaughan, D. E. Blask, M. P. Barnett, and R. J. Reiter 1976 Arginine vasotocin: Structural-activity relation­ ships and influence on gonadal growth and function, Amer. Zool. 16;25-34.

Vaughan, M. K., G. M. Vaughan, and D. C. Klein 1974 Arginine Vasotocin: Effects on development of reproductive organs. Science 186:938-939.

Vaughan, M. K., G. M. Vaughan, and R. J. Reiter 1975 Inhibition of HCG-induced ovarian and uterine growth in the mouse by synthetic arginine vasotocin. Experientia 31:862-863.

Vaughan, M. K., G. M. Vaughan, and R. J. Reiter 1976 Inhibition of human chorionic gonadotrophin-induced hypertrophy of the ovaries and uterus in immature mice by some pineal indoles, 6-hydroxymelatonin and arginine vasotocin. J. Endocr. 68: 397-400.

Vaughan, M. K., G. M. Vaughan, R. J. Reiter, and B. Benson 1972 Effect of melatonin and other pineal indoles on adrenal en­ largement produced in male and female mice by pinealectomy, unilateral adrenalectomy, castration and cold stress. Neuroendocrinology 10:139.

Vigh, B., and I. Vigh-Teichmann 1974 Vergleich der Ultrastruktur der Liquorkontaktneurone und Pinealozyten. Vehr. Anat. Ges. 68: 433-443.

Vigh, B., and I. Vigh-Teichmann 1975 Vergleich der Ultrastruktur der Liquorkontakneurone und Pinealozyten der Saugetiere. Vehr. Anat. Ges. 69:453-461.

Vigh, B., I. Vigh-Teichmann, and B. Aros 1975 Comparative ultra- structure of cerebrospinal fluid-contacting neurons and pinealocytes. Cell Tiss. Res. 158:409-424. +2 Vlahakes, G. J., and R. J. WUrtman 1972 A Mg dependent HIOMT in rat Harderian gland. Biophys. Acta 261:194.

Vollrath, L. 1976 Light and drug induced changes of epiphyseal synaptic ribbons. Cell Tiss. Res. 165:383-390.

Vollrath, L., A. Kantarjian, and C. Howe 1975 Mammalian pineal gland: 7-day rhythmic activity? Experientia 31:458-460.

Wade, N. J. 1937 Studies on the function of pineal gland. Endo­ crinology 21:681-683.

t 171

Wallen, E. P., and J. M. Yochim 1974 Rhythmic function of pineal HIOMT during the estrous cycle: An Analysis. Biol. Reprod. 10:461-467.

Weinberg, S. J., and R. V. Fletcher 1930 Effects of-injected extracts of fresh pineal glands of young calf on growth of immature white mice; effects on sexual apparatus. Proc. Soc. Exp. Biol. Med. 28:323.

Weiss, B. 1969 Effects of environmental lighting and chronic denervation on the activation of adenyl cyclase of rat pineal gland by norepinephrine and sodium fluoride. J. Pharm. Exp. Ther. 168:146-152.

Wetterberg, L., E. Geller, and A. Yuwiler 1970 Harderian gland: An extra-retinal photoreceptor influencing the pineal gland in neonatal rats? Science 167:884-885.

White, W. F., M. T. Hedlund, G. F. Weber, R. H. Rippel, E. S. Johnson, and J. F. Wilber 1974 The pineal gland: A supplemental source of hypothalamic releasing hormones. Endocrinology 94:1422-1426.

Wilkinson, M. 1975 Proceedings: Growth and morphological differen­ tiation of rat pineal cells in monolayer cultures. J. Endocr. 64:61P.

Winters, K. E., J. J. Morrissey, P. J. Loos, and W. Lovenberg 1977 Pineal protein phosphorylation during serotonin N- acetyltransferase induction. Proc. Nat. Acad. Sci. 74:1928- 1932.

Wurtman, R. J., M. D. Altschule, and U. Holmgren 1959 Effects of pinealectomy and of bovine pineal extract in rats. Amer. J. Physiol. 197:108-110.

Wurtman, R. J., and J. Axelrod 1965 The formation, metabolism and physiologic effects of melatonin in mammals. Prog. Brain Res. 10:520-529.

Wurtman, R. J. , J. Axelrod, and J. D. Barchas 1964 Age and enzyme activity in the human pineal. J. Clin. Endocr. Metab- 24: 299-301.

Wurtman, R. J., J- Axelrod, E. W. Chu, and J. E. Fischer 1964 Media­ tion of some of effects of illumination on the rat estrous cycle by the sympathetic nervous system. Endocrinology 75:266-272. 172

Wurtman, R. J., J. Axelrod, G. Sedvall, and R. Y. Moore 1967 Photic and neural control of the 24-hour norepinephrine rhythm in the rat pineal gland. J. Pharm. Exp. Ther. 157:487-492.

Wurtman, R. J., J. Axelrod, S. H. Snyder, and E. W. Chu 1965 Changes in the enzymatic synthesis of melatonin in the pineal during the estrous cycle. Endocrinology 76:798-800.

Wurtman, R. J., w. Roth, M. D. Altschule, and J. J. Wurtman 1961 Interactions of the pineal and exposure to continuous light on organ weights of female rats. Acta Endocr. 36:617-624.

Yochim, J. M., and E. P. Wallen 1974a Photoperiod, pineal HIOMT activity and reproductive function in the rat: A proposed model. Biol. Reprod. 11:125-133.

Yochim, J. M., and E. P. Wallen 1974b Function of the pineal gland of the rat during progestation: Alterations in HIOMT rhythmicity. Biol. Reprod. 10:480-487.

Zacharias, L., and R. J. Wurtman 1964 Blindness: Its relation to age of menarche. Science 144:1154-1155.

Zanoboni, A., and A. Zanoboni-Mucaiccia 1967 Experimental hypertension in pinealectomized rats. Life Sci. jS: 2327-2332.

Zatz, M., J. W. Kebabian, J. A. Romero, R. J. Lefkowitz, and J. Axelrod 1976 Pineal beta-adrenergic receptor: Correlation of binding of ^H-l-alprenolol with stimulation of adenylate cyclase. J. Pharm. Exp. Ther. 196:714-722.

Ziegels, J., and V. Devecerski 1976 Effects of epiphysectomy on rat subcommissural body. Arch. Biol. 87:129-136.

Zucker, I., and M. P. Morin 1977 Photoperiodic influences on testicular regression, recrudescence and the induction of scotorefractoriness in male golden hamsters. Biol. Reprod. 17:493-498.

Zurburg, W., and I. Ebels 1975 Separation of pineal extracts by gel filtration II. Identification and isolation of two indoles from sheep pineal glands. J. Neural. Trans. 36:59-69.

Zweig, M., S. H. Snyder, and J. Axelrod 1966 Evidence for a non- retinal pathway of light to the pineal gland of newborn rats. Proc. Nat. Acad. Sci. 56:515-520.