DEVELOPMENTAL CHANGES IN GONADOTROPltf RELEASING HORMONE NEURONS IN THE BRAIN OF THE FEMALE RABBIT (oryctolagus cuniculus)

Transcription

1 DEVELOPMENTAL CHANGES IN GONADOTROPltf RELEASING HORMONE NEURONS IN THE BRAIN OF THE FEMALE RABBIT (oryctolagus cuniculus) By WARREN G. FOSTER, B.Sc., M.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfilment of the Requirements for the Degree Doctor of Philosophy McMaster University, (c) Copyright by Warren George Foster, November, 1990

2 DEVELOPMENTAL CHANGES IN THE GnRH NEURON o

3 DOCTOR OF PHILOSOPHY (1990) Hamilton, Ontario McMASTER UNIVERSITY (Health Sciences) TITLE: Developmental Changes in Gonadotropin Releasing Hormone Neurons in the Brain of the Female Rabbit (Oryctolagus cuniculus) AUTHOR: Warren George Foster, B.Sc. (Upiversity of Guelph) SUPERVISOR: M.Sc. (University of Guelph) Professor E.V. YoungLai NUMBER OF PAGES: xv, 178 ii

4 ABSTRACT of the GnRH This thesis examined developmental changes in morphology neuron in the female rabbit brain during sexual maturation. In the adult virgin rabbit approximately 1,000 GnRH cells were counted in half the hypothalamus. Two morphologically distinct populations of GnRH neurons were found. Fusiform cells with relatively smooth contours (smooth) accounted for 34% of the total. (rough) represented 64% Cells with irregular contours of the immunoreactive cells counted. In a subsequent experiment GnRH cell types were quantified in rabbits treated with Tamoxifen (TAM, lg ~g/kg/day), vehicle, and pregnant mare serum (PHS, 50 IU on postnatal days (PND) 25 and 28). Sexual maturity was considered achieved when rabbits attained a body weight of 3.0 kg. The proportion of rough cells increased while the smooth cells decreased with sexual maturation. This change was advanced by PHS treatment and prevented by TAM treatment compared to controls. Sexual maturity was advanced by PMS treatment (92 PND) versus controls (108 PND) and delayed by TAM treatment (128 PND). Mean plasma estradiol was significantly (P= 0.01) elevated in PHS rabbits versus controls between PND 25 and 34 and again at PND 75 (p= 0.05). Since the total number of immunoreactive cells remained.-.-::~. constant, it is concluded that smooth cells are transformed to rough cells. In ano~her experiment chronic ovariectomy did not change the total number of GnRH cells counted when compared to

5 .' iv sham operated rabbits. However, the developmental shift of smooth cells to rough cells was prevented (p<o.05) in ovariectomized rabbits. These results suggest that the developmental change in GnRH cell morphology is functionally related to puberty onset. Moreover, estradiol seems to induce these changes through indirect mechanisms. It is proposed that estradiol augments the growth neural imputs to GnRH cellls.

6 ACKNOWLEDGEMENTS There are many people who were instrumental in making my experience at McMaster enjoyable and successful as well as contributing to the preparation of this thesis. First and foremost I would like to thank my supervisor Dr. E. V. YoungLai for his time and friendship over the preceding four years. Dr. YoungLai has provided me with the opportunity and freedom to pursue my thesis and non-thesis research interests in a relatively independent manner, which is most appreciated. Dr. YoungLai ' s support and friendly discussions are gratefully acknowledged. Dr. J. F. Jarrell, my cosupervisor is also deserving of many thanks for his insights into the work described in this thesis. I would also like to thank the other members of my supervisory committee, Drs. G. Brown and A. Ball for their time and help. I am especially indebted to Dr. Ball for his instruction in immunohistochemical techniques and critical evaluation of my work. I would also like to pay special thank you's to Dr. J. D. Booth, Roberta Petitti, Lisa Deys, Nicolle Thompson, Cindy Todoroff, Joanne Gunby, Jan Yeo, Ann Schwidder, Derick Waters, and Avril McMahon for the friendship that has been extended to me whiie at McMaster. The many varied discussions with Dr. Booth have been most enjoyable and have served to broaden my perspectives and understanding v

7 vi of computers, endocrinology, and medicine. Jan Yeo's technical skills of ovariectomy and radioimmunoassay are greatly appreciated. 1 gratefully acknowledge the agencies ',Ilhich have provided me with the following scholarships: ontario Graduate Scholarship, Medical Research Council Studen~ship and McMaster University Scholarship. The financial support from scholarships has been very helpful in the pursuit of my training. Lastly, I thank my wife Helen for her support and patience during my graduate study. For her understanding Helen deserves more of a thank you than can be expressed here.

8 TABLE OF CONTENTS ACKNOWLEDGEMENTS <. v LIST OF FIGtJR.ES "... xi LIST OF TABLES LIST OF ABBREVIATIONS xiii xiv CHAPTER I INTRODUCTION... 1 CHAPTER II NEUROENDOCRINOLOGY OF PUBERTY IN THE FEMALE RABBIT: A REVIEW '.' Introduction. Puberty In The Rabbit Physical Signs Of Puberty circulating Gonadotropins Pituitary Responsiveness to Exogenous GnRH Ontogeny Of GnRH Receptors Pulsatile Secretion Of Gonadotropins Pulsatile GnRH Secreti~n Ontogeny Of Ovarian Estrogens In the Rabbit Thg Gonadostat Theory Synaptogenesis Theory Hypothalamic Localization Of GnRH Radioimmunoassay Immunohistochemical In situ Hybridization.. Regulation of GnRH Secretion Catecholamines.... i) Morphological studies.. ii) Functional Studies Opioid Peptides..... i) Morphological Studies. ii) Functional Studies Gonadal steroids... i) Morphological Studies... ii) Functional Studies Summary Hypothesis. Experimental Questions <;' 57 vii

9 viii CHAPTER III MATERIAl,S AND METHODS Animals & '" Experimental Design Experiment I Localization Of Hypothalamic Nuclei Immunohistochemistry a Paraffin Technique b Thick sections c Comparison Of Thick sections Technique With Vibratome And Cryostat Methods Controls '" '" Quantification Of Immunoreactive GnRH. Neurons CHAPTER IV Topography Of GnRH Ueural Elements In The Hypothalamus Experi~ent II '" Des~gn '" a Evaluation Of Estradiol Effects On Puberty And GnRH. Cytoarchitecture b Effect Of Ovariectomy On GnRH cytoarchitecture GnRH Immunohistochemistry Radioimmunoassays Follicular Morphometry Data Analysis Experiment III... RESULTS EXPERIMENT I Immunohistochemistry Paraffin Technique Thick sections a Specificity of Antisera b Immunoreactive GnRH Cells. 80 4,1.1.3 comparison of Thick vs. Vibratome and Cryostat Techniques Topography of Immunoreactive Neural Elements EXPERIMENT II Immunoreactive Cell Counts Plasma and Ovarian Estradiol Measurements specificity of Estradiol Antiserum

10 ix Developmental Chanes in Mean Plasma Estradiol _ Developmental Changes in Ovarian Estradiol Developmental Changes in Plasma Gonadotropins Plasma FSH Plasma LH FSH/LH Ratio Physical Measurements Body Weights Ovarian Weights Uterine and Pituitary Weights Mating Response Follicle Morphometry Ovariectomy Experiment Plasma Gonadotropins FSH/LH Ratio Immunoreactive Cell counts EXPERIMENT III CHAPTER V DISCUSSION Introduction Immunohistochemical Quantification, Morphological Topography of GnRH Cells in the Brain of the Adult Rabbit specificity of GnRH Antisera Quantification of GnRH Cells S~2.3. Topography of GnRH Neural Elements Developmental Changes in GnRH Neurons Immunoreactive Cell Counts Developmental Changes in Plasma Estradiol and Gonadotropins Relationship Between Body Weight and Sexual Maturation in the Rabbit Ovariectomy Impairs Developmental. Changes in GnRH Neurons Summary of Developmental Changes in the GnRH Neuron 141 Chronic Treatment with Tamoxifen Appears to Prevent the Development of Estrogen Negative Feedback Proposed stages of Sexual Development in the Rabbit Summary Conclusions 150

11 x REFERENCES.~. 151 ::,,~,

12 LIST OF FIGURES Figure Hypothalamic-pituitary-ovarian axis 3 2. Ontogeny of Serum Gonadotropins in the Female Rabbit Amino Acid Sequence of GnRH Models of Hypothalamic Neurocircuitry Ventral View of Rabbit Hypothalamus Immunoreactive GnRH containing Cells of the Preoptic Area GnRH cells clustered in the retrochiasmatic area MUltiple GnRH cell types seen in the retrochiasmatic area A smooth bipolar GnRH cell and 11. process Rough GnRH cells with multiple thickened protuberances , 13a and 13b. Rough GnRH cells with spiked contours and 15. Camera lucida drawings of GnRH neural elements in the rabbit hypothalamus (Fig. 14). Hypothalamic nuclei (Fig. 15) and 17. GnRH fibers in fr~ntal section Immunoreactive fiber at ependymal surface Beaded swellings on GnRH-IR processes to 23. Developmental changes in the number of rough and smooth GnRH cells Specificity of Estradiol Antisera Developmental changes in the mean plasma estradiol levels xi

13 xii Figure 26. Developmental changes in ovarian estradiol content Developmental changes in mean plasma FSH levels Developmental changes in mean plasma LH levels Graph of body weight changes during development Cell counts in ovariectomized rabbits propos7d d 7 velopmental changes in hypothalamic neuroc1rcu1try 142

14 LIST OF TABLES TABLE I Summary of Reported Outcome Measures of Sexual Development in the Female Rabbit II Summary of Experimental Design III IV Comparison of Cell Counts Performed on Thick vs. Vibratome Sections Developmental Changes in FSH/LH Ratio of Treated vs. Control Rabbits V Mean Rate of Weight Gain VI Developmental Changes in Body, Pituitary, Adrenal, uterine and Ovarian Weights VII Mating Response VIII IX X Developmental Changes in Follicular Maturation 109 Developmental Changes in the FSH/LH Ratio of Ovariectomized Rabbits Compared to Controls 111 Effect of Tamoxifen Citrate on LH and FSH secretion in the Adult Female Rabbit. 114 " xiii

15 LIST OF ABBREVIATIONS USED ARCN CNS DA DAB E EB EOP FSH GABA GH GHRH GnRH GnRH-IR h hpg IGF-I i.v. LH MBH ME min mrna mths NAL NE arcuate nucleus central nervous system dopamine diaminobenzidine epinephrine estradiol benzoate endogenous opioid peptides follicle stimulating hormone gamma-amino-butyric acid growth hormone growth hormone releasing hormone gonadotropin releasing hormone gonadotropin releasing hormone immunoreactivity hour hypogonadal insulin like growth factor I intravenous luteinizing hormone medial basal hypothalamus median eminence minute messenger ribonucleic acid months naloxone norepinephrine xiv

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205