the permission of the Author.

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

STUDIES OF NEUROENDOCRINE HECHANISMS INFLUENCING SEASONAL VARIATIONS IN SE�lEN PRODUCTION AND PLASHA

HORMONE LEVELS IN RAHS

A thesis presented in partial fulfilment of the requirements for the

Degree of Doctor of Philosophy at Massey University

GRAHAM KEITH BARRELL

1976

Abstract of a thesis presented in partial fulfilment of the re1uirements for the Degree of Doctor of Philosophy

STUDIES OF NEUROENDOCRINE NECHANISHS

INFLUENCING SEASONAL VARIATIONS IN SEMEN PRODUCTION AND PLASHA HORMONE LEVELS ^{IN RAI1S}

by Graham Keith Barrell

Many workers have shown that sheep are seasonal breeders with peak reproductive activity occurring during the autumn months. The initial experiment in this thesis was designed to define the seasonality of reproduction in rams of the N.Z� Romney breed as determined by repeated measurements of semen characterist- ics and of plasma hormone levels. These parameters were studied

for ₁₆ months in six N.Z. Romney rams on pasture, with five Merino and four Polled Dorset rams included for comparison.

Semen from all three breeds showed relatively regular seas­

onal changes in ejaculate volumes and seminal fructose levels _with peak values being recorded during March. Likewise, monthly

hormone levels VRried in a regular manner with plas�a LH, testost­

�rone and prolactin concentration being elevated during the summer months. Many of the other semen parameters measured showed little tendency for seasonal v�riations, however a change in semen collect­

ion technique, from predominantly artificial vagina to entirely electro-ejaculation, may have masked some seasonal changes. All three breeds showed similar seasonal changes in the parameters studied although semen from the Polled Dorsets did not exhibit regular seasonal variations in fructose levels.

iii

Further experiments were carried out to define the neuroendoc-

rine mechanisms which regulate the seasonal reproductive changes in N.Z. Romney rams. Three ol factory bulbectomized rams, three

cranial cervical ganglionectomized rams and four rams which had undergone both of these surgical modifications, were grazed to-

gether with the rams mentioned above. These surgical treatments

disrupted the regular seasonal changes in plasma levels o f LH and prolactin , but not, o f testosterone. Spermatozoal concentrations

in ejaculates from operated rams were higher than those from un- operated controls, whereas mean fructose concentrations were lower;

however the pattern o f seasonal chan ges in seminal fructose levels

was similar in all groups o f rams. Cranial cervical ganglionectomy reduced hydroxyindole-0-methyl trans ferase activity and cell vol-

umes in the pineal glands, so it was concluded that disrupted seasonal patterns o f changes in _plasma LH and prolactin levels , plus the altered semen production in the surgically treated rams , resulted from modi fied pineal gland and/or ol facto�y system activity.

A preliminary investigation into the role of changes in daily

photoperiod as the stimulus for seasonality o f reproduction, was

carried out by placing rams in light-controlled rooms at the time

o f the March equinox. Over the following nine months rams expose^d to a phase-reversed annual lighting cycle showed earlier elevations

o f seminal fructose and plasma testosterone levels than rams on

I .

either the normal annual or a constant equinoctal lighting regime.

�

In all three groups plasma prolactin levels were directly related to the length of daily photoperiod.

The findings of the above experiments were extended by a final study in which both pinealectomized and sham-operated rams were exposed to normal or reversed annual lig^{hting cycles.}

Effects of lighting on plasma testosterone and prolactin levels, and on seminal fructose levels, were diminished by pinealectomy.

Autopsy data related to gonadal and accessory sex gland function showed significant operations x light_ing regime_s interactions, which supported the conclusion that in rams pineal gland function mediates endocrine ann gonadal responses to changes in daily photoperiod.

Three short-term investigations of hormonal secretion pro­

files conducted during the latter experiment, showed that major fluctuations in the release of LH, testosterone, prolactin and cortisol occurred irregularly during the day. A nocturnal elevat- ion of plasma prolactin levels was abolished by pinealectomy.

These acute studies tended to confirm the findings of the latter experiment, but in particular they highlighted the pulsatile nature of hormonal secretion.

ACKNOWLEDGMENTS

I wish to express my gratitude for the help ful advice and generous assistance provided by my chie f supervisor , Dr K.R.

Lapwood , who also proposed the area o f research covered i n this thesis.

V

Further thanks are due to Dr R.M. Greenway for his role as supervisor a nd provider o f encoura gement, and to Pro f. R.E. Mun­

ford for ge nerously providing facilities in the Department o f Physiology a nd Anatomy a nd for his major contributio n to the statistical a nalyses o f the experimental data.

The financial assistance o f the Veterinary Research Fund o f Massey University is grate fully acknowledged, and the National I nstitute o f Arthritis, Metabolism and Digestive Diseases, Nation­

al I nstitutes of Health, U.S.A. is acknowledged for provision o f reagents for hormone assays. Dr G.D. Niswender o f Colorado State

U niversity, U.S.A. is acknowledged for supplying antisera to testostero ne and ovine-LH, so also is Dr L.E. Reichert, Jr, Emory U niversity , U.S.A. for donating the highly puri fied ovine prolactin and LH for radioimmunoassay , and Pro f. D.S. Flux for donati n g

a ntisera to bovine prolactin. Dr D.C. Thurley and sta f f at

Wallaceville Animal Research Centre, Upper Hutt, are thanked for per formin g cortisol assays.

I am i ndebted to Mr M.J. Birtles for carrying out the histo­

lo gical processin g and to Mr T.G. Law for the photographs in this thesis. The enthusiastic and un fla gging technical assista nce o f

Mrs H. Carter, Mr R.N. Ward and _Mr R.H. Telfer is warmly appreciated , a nd I wish to thank Mrs D.P. de Wiele, Mrs H.E. Walker a nd Miss C.

Howell for their co ntrib11tions to the preparation o f this ma nuscript.

Finally, I thank my wi fe Rosali nd and daughters, Snrah and Rebecca, for their e ncouragement, support a nd perserverance whilst I was e ngaged in these studies.

In these studies,the candidate made the major contribution

to the conception and execution o f the experiments. The chief supervisor assisted with the ex perimental design and in the dev•

elopment of surgical techniques and hormone assays. Otherwise, apart from the execution o f cortisol assays and histological processing per formed by others, a nd the development o f a n ovine­

LH radioimmunoassay where the candidate's contribution was

minimal, all remaining work described in this thesis i nvolved the full participation o f the candidate.

CHAPTER I: INTRODUCTION

TABLE OF CONT�NTS

l. SeasonBlity of reuroduction ( 1) General

(2) Manifest�tions of reprorluctive seasonality in female animals

(a) ^Gen^era^l (b) Sheep

(3) Manifestations of reproductive seasonality in

vii

PAGE

1 1

2 2 2

male anim_als 3

(a) General 3

(b) ^Antler_s of deer 3

(c) Gonads and acce�sory sex glands 4

(d) Male libido 5

(e) ^Semen production 6

(4) Hormonal changes 7

(a) Pituitary and hypothalamic hormone

levels 7

(b) Plasma hormone levels 8 (5) Male sexual cycles

(6) Male-female reproductive seasonality inter­

relationships (7) ^Summary

10

10 11 2. Factors involved in the seasonality of repro.-:::ction 11

(1) Light

(?.) ^Temp^erature (3) Nutrition (4) ^Olfaction

(a) General

(b) Photoperiodicity of reproduction in m.rymmals

(5) Other factors

3. Neuroendocrinolop,y of repronuction in m^ale mammals (1) ^General

11 11

16 22 25 26 28 29 29

(2) Hyuothalamus 29

(3) Hypothalamic regions involved in gonadotrophin

release 30

(4) Extr8hypothalaruic areas involved in gonadotrophin release

(5) Mechanisms of GnRH release (6) Negative feedback of steroids (7) Si_tes of steroid feedback

30 31 32 32

PAGE (8) Hypoth�lamic sites o f steroid feedback 33 (9) Role of steroids in hypothalamic differentiation 33

(10) Sy nthetic GnRH 34

(11) Actions of GnRIT 35

(12) GonAdotrophins

(13) ^Androgens

(�) General (^b) ^{FSH anrl LH} (^c) ^Prolactin

4. The role of the pineal gland ⁱn ^mammalian repror3uctio n

36 36 36 37 3 8

39

(1) Ge neral 39

(2) Ef fects o f pinealectomy 39

(3) Pineal indoleamines 40

(4) Pⁱn^{eal peptides} 41

(5) Gonadal regulation o f pineal function 42

(6) Tra nsport of _pin�al principles 42

(7) Afferent nervous pathways 43

(8) Role of the pineal .gland in the seasonality o f

reproductio n 44

(9) Pⁱn^eal function a fter puberty 45 (10) Pineal gland and ol factory function

5. The purpose of th8 present study CHAPTER _II: HATERIALS AND HETHODS

45 46

· 1. Animals 47

2 . Animal management procedures

(l) On i)asture (2) ^Indoors

3 . Surgical tech niques

(^l) General surgical procedure (2) Ol factory bulbectomy

(3) Cranial cervical ganglionectomy

(4) Ol factory bulbectomy/cranial cervical ganglio nectomy

(5) Pinealectomy (6) Sham-pinealectomy 4. Semen collection

5. Semen appraisal

(1 ) Ej_ac ulate volume

47 47 47 48 48 49 50

50 50 55 55 55 55

(2) Motility and percentage motile spermatozoa (3) Per^centages of unstained and morphologically

normal �permatozoa

(4) Concentration of so^ermat^{ozoa per ml semen and}

ix

PAGE 56

56

number of spermatozoa per ejaculate 56 (5) Conc�^ntratⁱon of fructose in semen and s^{em inal}

plasm?, ann total ejaculate fru^ctos^{e content 56} 6. Blood collection

7. Autopsy procedure

(1) Histological procedures

61 62 62 (2) Seminal ve�iculer fructose content and

concentration 63

64 (3) Epididymal sperm�.^toz^{oal re}s^erves

(4) Hydroxyindole-0-methyl transferase a�say 65

8. Hormone assays (1) ^Reagents (2) LH assay

(^a) Discuseion of HIOMT assay metho d 66

(^a) Radioiodination o f ovine-LH with 125r

(b) Preparation of precipitating antisera

(c) Radioimmunoassay pr^ocedure (d) Validation of ovi^ne-LH radio-

67 67 68

68 69 69

immunoassay 70

(3) Prolactin assay 73

(^a) Labellⁱⁿg o f ovine prolactin

with 125r ₇₃

(b) _Prep^aration o f prolactin antiserum 7 5 (c) Radioimmunoassay procedure 7 5 (d) _Valid^ation of ovine prolactin

assay 76

(4) Testosterone assays 76

76 84 87 (^a) Protein-binding assay

(b) Radioim�unoas�ay _I (c) _Radioimm^unoa�s^ay II

(5) ^Cortⁱs^ol ^assay

9. Experimental design and analysi^s (1) A nalyse� o f variance

(2) Missing data

(3) Trancform�tions

(4) Computations

90 91 91 94 94 95

CHAPTER I II: SEASONALITY OF SEMEN PRODUCTION AND PLASMA HORl'lONE L"SVELS IN NE'.V _ZEALAND RONNEY,

MERINO AND POLLED _DORSET RANS AT PASTURE 1. Introduction

2� Materials and methods

(1) Experimental procedure

PAGE

96 96 96

(2) Statistical analyses 97

(^a) Semen data 97

(b) LH and testosterone data 97

(^c) ^{Prolactin data 97}

(^d) Rationale for division o f study period into seasons

(^e) Breed contrasts 3. Results

(1) Semen data

(2) Plasma hormone data (a) LH

(b) Testosterone (c) Prolactin (3) Autopl'ly data

(4) Meteorological data 4 .. Discussion

(^l) ^{Seasonal changes in semen} production (2) ^{Seasonal changes} in plasma hormone levels ..

(a) ^{LH e.nd} testor;terone (b) Prolactin

( 3) ^{Autopsy data}

(4) General discussion

CHAPTER IV� SEASONALITY OF SEMEN PRODUCTION AND PLASMA HORMONE _LEVELS IN RAMS WITH MO D IFIED OLFAC TORY AND PINEAL FUNCTION

l. Introduction

2. Materials and methods

(1) Animals and management procedure

(2) Data collection (3) Statistical analyses 3 .. Results

(1) Semen data

(2) Plasma hormone levels (^a) ^LH (3) Autopsy data

(b) TeE"tosterone (^c) ^Prolactin

97 102 102 102 113 113 113 113 121 121 126 126 130 130 131 133 133

139 140 140 141 141 142 142 152 153 153 153 161

4. Discus sion

(1) Semen data

(2) LH and testosterone

(3) _Prolactin (4) Autopsy data

(5) General discussion

CHAPTER V: EFFECTS OF DIFFERENT LIGHTING REGIMES ON SEMEN PRODUCTION AND PLA₃MA HORMONE LEVELS IN RAMS

1. Introduction

2. Materials and methods

(1) Animals and experimental procedure

(2) Statistical analyses

3. Results

(1) Semen data

(a) Semen data

(b) LH _and tectosterone (c) Prol9.ctin

(d) Treatment contrasts

(2) LH and tP-stoderone (3) Prolactin

4. Discussion

CHAPTER VI: EFFECTS OF PINEALECTOMY ON SEMEN PRODUCTION AND PLASMA HORMONE LEVELS IN RAMS SUBJECTED

TO CONTRASTING LIGHTING REGIMES

. 1. Introduction

2. Materials and methods

(1) Animals and experimenta l proce dure

(2) Statistical analyses

3. Results

(1) Semen data

(a) Semen and hormone data

(b) Treatment contrasts

(2) Plasma hormone data (a) LH

( 3) Autopsy da.ta

4. Discussion

(b) Testosterone (c) Prolactin

(1) Semen production

xi

PAGE

166 166 167 168 170 172

174 175 175 175 175 180 180 180 180 180 189 189 197

203 203 203 204 204 209 209 209 221 221 225 225 233 236 236

(2) Pla<ma hormone levels (a ) ^LH (3) Autopsy data

(b) Testosterone (c) Prolactin (4) Gener�l discussion

CHAPTER VII: PERIPHERAL PLASMA HORHON� LEVELS RECORDED FROM PIN�ALECTOMIZED OR SH�M-OPERATED RAMS SUBJECTED TO CONTRASTING LIGHTING REGIMES 1.

2.

3.

4.

Introduction

Materials and methods

(1) ^{Animals and} experimental (2) Hormone assay procedure (3) Statistical

Results Discussion

(1) ^LH

(2) Testosterone (3) Prolactin (4) Cortisol

analyses

(5) General discussion

procedure

CHAPTER VIII: GENERAL DISCUSSION AND CONCLUSIONS

PAGE 237 237 239 240 240 241

245 246 246 247 247 248 270 270 273 274 278 279

281 1. Sea�onality of reproduction in grazing rams 282 2. Effects of lighting regimes on reproduction in rams 287 3. Reproductive functions of prolactin in rams 292 4. Pineal gland function and reproduction in rams 294 5. Possible applications of the present findings 298

REFERENCES 301

xiii

LIST OF FIGURES

FIGURE PAG_E

2.1 Head of r3m clipued _and mRrked to show approximate

line of skin incision 52

2. 2 Skull expoeed sho�ing cent�ring hole for trephine 5 2 2.3 Re�ov�l o f circular plate of bone from the skull

after trephining 53

2.4 Flap of dura mRter reflected to expose the occipital

lobe of the left cerebr�l cortex 53

2. 5 Pineal gland just visible rostral to the rostral

cerebral colliculus 54

2.6 Removal of pineal gland with Allis forceps 54 2. 7 Flow diagram of AutoAnalyzer assay for seminal

fructose 58

2. 8 Standard curve for AutoAnalyzer seminal fructose

assay 59

2.9 Composite standard curve for ovine LH radioimmunoassay 71 2.10 Composite standard curve for ovine prolactin radio­

immunoassay .

2.11 Cross-reaction of anterior pituitary hormones in

77

the ovine prolactin radioimmunoassay 78 2.1 2 Composite standard curve for testosterone protein-

binding assay 82

2. 13 Composite standard curve for testosterone radio­

immunoassay I

2.1 4 Comp6site standard curve for testosterone radio­

immunoassay II

3. 1 Diagram to show how the time-course of Experiments 3 and 4 was subdivided into sampling periods and seasons for data analyses

3.2 Seasonal veriations in numbers of spermatozoa/ejaculate in semen collected from N.Z. Romney, Merino and Polled

86

Dorset rams, between March 1 972 and June 1 973 1 10 3. 3 Seasonal variations in seminal plasma fructose con­

centrations in semen collected from N.Z. Romney, Merino and Polled Dorset rams, between March 1 972

and June 1973 1 1 1

FIGURE

3.4 Seasonal v�riation� in plasma LH concentrations recorded from N.Z. Romney, Merino and Polled Dorset

PAGE

rams, between March 1972 an d June 1973 116 3.5 Seasonal variations in plasma testosterone con­

centrations recorded from _N.Z. Romney, Merino an d

Polled Dorset rams, between March 1972 and June 1973 117 3.6 Seasonal variations in plasma prolactin concentrations

recor ded from N.Z. Romney, Merino an d Polle d Dorset

rams, between March 1972 an d June 1973 120 3.7 Monthly variations in daily photoperiod, temperature

an d total rainfall, during the time-course of Ex-

periments 3 and 4 125

3.8 Interrelationships between seasonal variations in plasma LH and testosterone, an d in seminal plasma fructose concentrations, for N.Z. Romney, Merino an d

Polle d Dorset r ams 134

4.1 Seasonal variations in numbers of spermatozoa/ejaculate in semen collected f rom Controls, BulbX, GanglionX an d BulbX/GanglionX rams, between March 1972 an d June

1973 150

4.2 Seasonal variations in seminal plasma fructose con­

centrations in semen collected from Controls, BulbX, GunglionX and BulbX/GanglionX rams, between March 1972

an d June 1973 151

4.3 Seasonal variations in plasma LH concentrations re­

cor de d from Controls, BulbX, GanglionX and BulbX/

GanglionX rams, between March 1972 and June 1973 156 4.4 Seasonal variations in pla sma testosterone con­

centrations recorde d from Controls, BulbX, GanglionX and BulbX/GanglionX rams, between March 1972 and

June 1973 157

4.5 Seasonal variations in pla sma prolactin c-oncentrations recorded from Controls, BulbX, GanglionX and BulbX/

G anglionX rams, between March 1972 and June 1973 160 4.6 Mean hydroxyindole-0-methyl transferase activity in

pineal glan ds from Controls, BulbX, GanglionX and

BulbX/GanglionX rams 165

5.1 Diagram showing the cyclic changes in daily photo­

period for the lighting regimes use d in Experiment 5, and the sub division of the time-course of the ex-

periment for data analyses 176

FIGURE

5.2 Monthly variations in seminal plasma fructose con­

centrations in semen collected from rams subjected to

XV

PAGE

Normal, Even or Reversed liGhting regimes 188 5.3 Monthly variations in plasma LH concentrations re­

corded from rams subjected to Normal, Even or Reversed

lighting regimes 192

5. 4 Monthly variations in plasma testosterone concentrations recorded from rams subjected to Normal, Even or Re-

versed lightine regimes 193

5. 5 Monthly variations in plasma prolactin concentrations recorded from rams subjected to Normal, Even or Re-

versed liehting regimes ^· 196

6.1 Midsagittal section of brain from sham-operated ram

showing intact pineal gland 205

6. 2 Midsaeittal section of brain from pinealectomized ram

showing remnant of pineal stalk 205

6.3 Diagram showing the cyclic changes in daily photoperiod for the lighting regimes used in Experiment 6, and the subdivision of the time-course of the experiment for data analyses

6.4 Monthly variation� in numbers of spermatozoa/ejaculate in semen collected from sham-operated and pineal-

206

ectomized rams subjected to the Normal lighting regime 217 6.5 Monthly variations in numbers of spermatozoa/ejaculate

in semen collected from sham-operated and pineal­

ectomized rams subjected to the Reversed lighting regime

·6.6 Monthly variations in seminal plasma fructose con­

centrations in semen collected from sham-operated and pinealectomized rams subjected to the Normal lighting regime

6 .7 Monthly variations in seminal plasma fructose con­

centrations in semen collected from sham-operated and pinealectomized rams subjected to the Reversed light­

ing regime

6. 8 Fortnightly variations in plasma LH concentrations recorded from sham-operated and pinealectomized rams

218

219

220

subjected to the Normal lighting regime 223 6 .9 Fortnichtly variations in plasma LH concentrations re­

corded from sham-operated and pinealectomized rams

subjected to the Reversed lighting regime 224 6.10 Fortnightly variation� in plasma testosterone con­

centrationG recorded from sham-operated and pineal­

ectomized rams subjected to the Normal lighting regime 227

FIGURE _PAGE 6.11 Fortnightly variations in plasma testosterone con­

centr�tions recorded from sham-operated and pineal­

ectomized rams subjected to the Reversed lighting regime

6.12 Fortnightly variations in plasma prolactin con-

228

centrations recorded from sham-operated and pineal­

ectomized rams subjected to the Normel lighting regime 2 30 6.13 Fortnightly variations in plasma prolactin con­

centrations recorded from sham-operated and pineal­

ectomized rams subjected to the Reversed lighting regime

7.1 LH secretion patterns recorded from plasma samples collected from four sham-operated rams , sampled at

2 31

20 minute intervals for 26 hours 249

7.2 LH secretion patterns recorded from plasma samples collected from four pinealectomized rams, sampled at

20 minute intervals for 26 hours 2 50

7.3 26-Hour cumulative LH levels showing pooled cumulative regression lines

7.4 Testosterone secretion patterns recorded from plasma samples collected f rom four sham-operated rams ,

2 51

sampled at 20 minute intervals for 26 hours 2 53 7. 5 Testosterone secretion patterns recorded from plasma

samples collected from four pinealectomized rams ,

sampled at 20 minute intervals for 26 hours 2 54 7.6 Prolactin secretion patterns recorded from plasma sam­

ples collected from four sham-operated rams , sampled

at 20 minute intervals for 26 hours 2 5 5

7.7 Prolactin secretion patterns recorded from plasma sam­

ples collected from four pinealectomized rams , sampled

at 20 minute intervals for 26 hours. 2 56 7. 8 Cortisol secretion patterns recorded from plasma sam­

ples collected from four sham-operated rams , sampled

at 20 minute intervals for 26 hours 2 57 7. 9 Cortisol secretion patterns recorded from plasma sam­

ples collected from four pinealectomized rams , sampled

at 20 minute intervals for 26 hours 2 58 7.10 LH levels in sham-operated and pinealectomized rams

recorded from plasma samples collected at 30 minute

intervals for a four hour period on March 5 , 197 5 261

F IGURE

7.1 1 Prolactin levels in sham-operated and pinealectomized rams recorded from plasma samples collected at 30 minute intervals for a four hour period on March 5,

xvii

PAGE

1 975 26 2

7.1 2 LH levels in sham-operated and pinealectomized rams recorded from plasma samples collected at 30 minute

intervals for a six hour period on May 20, 1975 26 6 7.1 3 Prolactin levels in sham-operated and pinealectomized

rams recorded from plasma samples collected at 30

minute intervals for a six hour period on May 20, 1 975 26 7