by Poon Chuen Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 161-176). / Abstract --- p.i / Acknowledgements --- p.vi / Abbreviations --- p.viii / Introduction --- p.1 / Review of the literature --- p.2 / Incidence and historical background --- p.3 / Theories on etiology of emesis/hyperemesis gavidarum --- p.5 / Mechanism of vomiting --- p.7 / Temporal association between hCG and emesis/hyperemesis gravidarum --- p.9 / Thyroid function and emesis/hyperemesis gravidarum --- p.13 / Steroid hormone and emesis/hyperemesis gravidarum --- p.15 / Hyperemesis caused by corpus luteum in right ovary --- p.15 / "Formulation of a new hypothesis for emesis/hyperemesis gravidarum : “hCG stimulated electrolyte and fluid secretion in the UGIT, leading to pregnancy associated vomiting 226}0ح" --- p.16 / Is hCG a humoural secretory agent ？ --- p.18 / Can hCG stimulate the secretion of fluid and electrolytes from the GIT ？ --- p.18 / Are LH-CGR present in the GIT ？ --- p.20 / Can electrolyte and fluid secretion in the UGIT lead to vomiting during pregnancy ？ --- p.24 / HCG and regulation of electrolyte and fluid movement --- p.24 / Gut- gonad axis --- p.25 / Objectives --- p.27 / Section 1 hCG and in vitro secretion of anions from cells with functional LH-CGR - A cell model --- p.28 / Introduction --- p.28 / Materials and Methods / Materials --- p.30 / Methods / Chapter 1. --- Test for the presence of LH-CGR in human gastrointestinal cell lines --- p.31 / Chapter 2. --- Establishment of cells expressing functional receptor for hCG --- p.35 / Chapter 3. --- Effect of hCG on [125I] iodide efflux from COS-1 cells transiently expressing MC1+2 LH-CG/TSH chimera receptor --- p.38 / Chapter 4. --- Anion secretion from MLTC-1 cells with wild type LH-CGR --- p.38 / Chapter 5. --- Statistics --- p.41 / Results --- p.42 / Discussion --- p.55 / Section 2 Presence of LH-CGR in the upper gastrointestinal tract of rat --- p.61 / Introduction --- p.61 / Materials and Methods / Materials --- p.62 / Methods / Chapter 1. --- Screening for hCG binding sites along the rat GIT --- p.63 / Chapter 2. --- Detection of Triton X-100 soluble plasma membrane hCG binding protein along the rat GIT --- p.64 / Chapter 3. --- Measurement of affinity of the hCG binding proteins by Scatchard analysis --- p.66 / Chapter 4. --- Determination of the isoelectric points for the duodenal hCG binding protein and ovarian LH-CGR --- p.67 / Chapter 5. --- Developement of antibodies to LH-CGR --- p.69 / Chapter 6. --- Western blotting to determine the molecular weight of the LH-CGR --- p.78 / Chapter 7. --- Immunohistochemical localization of LH-CGR in the UGIT --- p.80 / Chapter 8. --- Statistics --- p.81 / Results --- p.82 / Discussion --- p.109 / Section 3 hCG and in vivo secretion of electrolytes from pancreas / duodenal region in rats --- p.119 / Introduction --- p.119 / Materials and Methods / Materials --- p.121 / Methods --- p.121 / Chapter 1. --- The effect of hCG on the secretion of electrolytes into bile and pancreatic/duodenal effluent --- p.121 / Chapter 2. --- Effect of ovariectomy on pancreatic content and electrolyte levels in blood --- p.125 / Chapter 3. --- Statistics --- p.128 / Results --- p.129 / Discussion --- p.151 / General discussion --- p.155 / Reference --- p.161 / Original data --- p.176

Biologically active peptides are a class of molecules which can, in general, be grouped into families of structurally related moieties which have a wide somatic distribution and which subserve multiple physiological roles. Thus, the releasing factors of the hypothalamus or their structural analogues may carry out multiple endocrine, paracrine, or neurotransmitter functions outside the hypothalamus. This thesis describes the distribution and biosynthesis of Luteinizing Hormone-Releasing Hormone (LH-RH) and structurally related peptides in mammalian tissues and tumours. Two strategies were employed in this investigation: (a) Immunological screening of tissues for suitable sources of LH-RH and the partial characterization of the LH-RH immunoreactive peptides of selected tissues; (b) Complementary oligonucleotide screening of mRNA from certain tissues, cDNA and genomic libraries as well as genomic DNA restriction endonuclease fragments to establish the nature of the LH-RH precursor and related molecules. Acetic acid extracts of nineteen rat tissues were assayed in serial dilution for LH-RH immunoreactivity (LH-RH-IR). An antibody directed to the middle of the LH-RH sequence was used in these screening radioimmunoassays. LH-RH-IR was found in extrahypothalamic brain areas, in gastroenteropancreatic tissues, in the retina, the submandibular gland, the thyroid and the testes. In view of a putative role of endogeonous LH-RH-like immunoreactive peptides in intra-testicular regulation, the molecular nature of the LH-RH-like material detected in rat testes was investigated. Acetic acid extracts of adult rat testes were partially purified by LH-RH-immunoaffinity chromatography. On Sephadex G-100 this material separated into four major peaks of >100K, ~32K, ~5K and <4K daltons. The <4K peak of LH-RH-IR eluted differently on Sephadex G-25 and analytical reverse phase HPLC than did synthetic hypothalamic LH-RH decapeptide. Antibodies directed at the C-terminus of LH-RH gave higher estimates of LH-RH-IR for all the chromatographically separated testicular polypeptides than did N-terminally and middle directed antisera. The small testicular LH-RH-like peptides displaced radiolabelled LH-RH agonist from rat pituitary membranes more effectively than did the larger immunoreactive molecules. Monkey, pig and dog testes as well as dog Sertoli cell tumours were extracted with acetic acid and shown to contain LH-RH immunoreactive material similar to that of the rat. Human seminal plasma was also investigated as a source of LH-RH-IR. When pooled seminal plasma from azoospermic males was fractionated by gel filtration some fractions had significant amounts of the rat testicular type of LH-RH.

Changes in circulating progesterone (P4) and estradiol (E2) during proestrus produce dynamic changes in endometrial function and pituitary release of gonadotropins. Independent and combined effects of P4 and E2 on endometrium and pituitary were evaluated. In Exp.1, an exogenous hormone model of proestrus was created by removal of CL and follicles >=5 mm followed by gradual removal of intravaginal P4 implants during 18h and treatment with increasing doses of estradiol benzoate during 48h to mimic proestrus using HighE2 (n=9) or LowE2 (n=9). Decreased P4, increased E2, and increased endometrial area (EA) simulated proestrus values in HighE2 cows and this was used subsequently. Experiment 2 used a 2x2 factorial design with: HighE2&LowP4 (n=11); HighE2&HighP4 (n=11); LowE2&HighP4 (n=11); LowE2&LowP4 (n=10). At 48h, GnRH-induced LH and FSH release was determined. Variables were analyzed using PROC MIXED of SAS and AUC calculated with MESS. The EA increased dramatically during 48h only in HighE2&LowP4 cows. For FSH, HighE2 cows had greater AUC and FSH peak after GnRH than LowE2 groups, with mild negative effects of HighP4. For LH, concentration at peak and AUC were 2-fold greater in HighE2 compared to LowE2 groups, with HighP4 reducing in a similar proportion compared to LowP4 groups. Thus, maximal changes in uterus and pituitary during proestrus depend on both LowP4 and HighE2 but E2 and P4 have different actions. Changes in endometrium depend on LowP4 and HighE2, whereas GnRH-induced FSH secretion primarily depends on HighE2, and GnRH-induced LH secretion is independently increased by HighE2 or reduced by HighP4. / As mudanças na progesterona (P4) e estradiol (E2) circulantes durante o proestro produzem alterações dinâmicas na função endometrial e na liberação das gonadotrofinas pela adenohipófise. Efeitos independentes e combinados da P4 e do E2 no endométrio e adenohipófise foram avaliados. No Exp.1, foi elaborado um modelo de proestro usando apenas hormônios exógenos a partir da remoção do CL e folículos >= 5 mm seguido por remoção gradual de dois dispositivos intravaginais contendo P4 durante 18 h e tratamento com doses crescentes de benzoato de estradiol (BE) durante 48 h para mimetizar o proestro, usando AltoE2 (n = 9) ou BaixoE2 (n = 9). Redução da P4, aumento do E2, e aumento da área endometrial (AE) simularam valores similares ao proestro em vacas do grupo AltoE2, o qual foi usado subsequentemente. No Exp. 2 foi realizado um arranjo fatorial 2x2 contendo: AltoE2&BaixaP4 (n = 11); AltoE2&AltaP4 (n = 11); BaixoE2&AltaP4 (n = 11); BaixoE2&BaixaP4 (n = 10). Após 48 h do início dos tratamentos com BE, foi avaliada a liberação de LH e FSH após desafio com GnRH. As variáveis foram analisadas usando o PROC MIXED do SAS e área sob a curva (AUC) foi calculada com MESS. A AE aumentou durante 18 h apenas nas vacas HighE2&LowP4. Para FSH, animais do AltoE2 apresentaram maior AUC e FSH no pico após GnRH comparados aos grupos BaixoE2, com efeitos negativos discretos da AltaP4. Para LH, concentração ao pico e AUC foram 2 vezes maiores nas vacas de AltoE2 comparados a BaixoE2, com AltaP4 reduzindo essas variáveis em uma proporção similar quando comparado aos grupos de BaixaP4. Assim, máximas mudanças no útero e na adenohipófise durante o proestro dependem de ambos, BaixaP4 e AltoE2, mas E2 e P4 apresentam diferentes ações. Alterações no endométrio dependem de BaixaP4 e AltoE2, enquanto que a liberação de FSH induzida por GnRH depende primariamente de AltoE2, e a liberação de LH induzida por GnRH é independentemente aumentada pelo AltoE2 ou reduzida pela AltaP4.

We hypothesized that marked reductions in secretion of luteinizing hormone (LH) during transitional and anovulatory periods can be accounted for by similar reductions in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. Catheters were inserted surgically into the intercavernous sinus (ICS) of seven non-pregnant mares via the superficial facial vein during the ovulatory season (August 12-23), fall transition (November 15-30), the anovulatory season (January 19 - February 1) and spring transition (March 24 - May 12). Catheter placement was confirmed and standardized in each mare by lateral radiography. Ovarian status was monitored throughout the study by transrectal ultrasonography and serum concentrations of progesterone. During the breeding season, ICS blood samples were collected at 5-min intervals for 8 h when the dominant follicle reached approximately 35 mm and estrous behavior was observed. All mares ovulated within 5 d after sampling, except one mare who ovulated < 24 h before sampling. During the fall, mares were anovulatory (n = 5) or had a final ovulation within 5 d following intensive sampling (n = 2). Winter anovulation sampling was performed when all mares were anovulatory. During spring transition, each mare was sampled just before the second ovulation of the season. Similar to the ovulatory season, mares were sampled when the dominant, preovulatory follicle reached approximately 35 mm and estrous behavior was observed. Mean concentrations of LH were markedly higher (P < 0.01) during the breeding season than during all other seasons. Lower mean concentrations of LH in the fall transition, winter anovulation and spring transition sampling periods occurred coincident with a similar reduction (P < 0.01) in amplitude of LH pulses. Unexpectedly, neither the frequency (pulse/8 h) of LH pulses, frequency and amplitude of GnRH pulses, nor mean concentrations of GnRH differed among seasons. In addition, there were no differences observed due to season in mean concentrations of FSH or amplitude of FSH pulses. However, a small but significant (P < 0.05) reduction in the frequency of FSH pulses was observed during fall transition compared to all other seasons. In summary, contrary to accepted dogma, these results indicate that the photoperiodic initiation of seasonal anovulation in the mare is mediated at the level of the anterior pituitary, and appears to occur through a dampening of gonadotroph responsiveness to an unchanging pattern and magnitude of GnRH secretion.

We hypothesized that marked reductions in secretion of luteinizing hormone (LH) during transitional and anovulatory periods can be accounted for by similar reductions in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. Catheters were inserted surgically into the intercavernous sinus (ICS) of seven non-pregnant mares via the superficial facial vein during the ovulatory season (August 12-23), fall transition (November 15-30), the anovulatory season (January 19 - February 1) and spring transition (March 24 - May 12). Catheter placement was confirmed and standardized in each mare by lateral radiography. Ovarian status was monitored throughout the study by transrectal ultrasonography and serum concentrations of progesterone. During the breeding season, ICS blood samples were collected at 5-min intervals for 8 h when the dominant follicle reached approximately 35 mm and estrous behavior was observed. All mares ovulated within 5 d after sampling, except one mare who ovulated < 24 h before sampling. During the fall, mares were anovulatory (n = 5) or had a final ovulation within 5 d following intensive sampling (n = 2). Winter anovulation sampling was performed when all mares were anovulatory. During spring transition, each mare was sampled just before the second ovulation of the season. Similar to the ovulatory season, mares were sampled when the dominant, preovulatory follicle reached approximately 35 mm and estrous behavior was observed. Mean concentrations of LH were markedly higher (P < 0.01) during the breeding season than during all other seasons. Lower mean concentrations of LH in the fall transition, winter anovulation and spring transition sampling periods occurred coincident with a similar reduction (P < 0.01) in amplitude of LH pulses. Unexpectedly, neither the frequency (pulse/8 h) of LH pulses, frequency and amplitude of GnRH pulses, nor mean concentrations of GnRH differed among seasons. In addition, there were no differences observed due to season in mean concentrations of FSH or amplitude of FSH pulses. However, a small but significant (P < 0.05) reduction in the frequency of FSH pulses was observed during fall transition compared to all other seasons. In summary, contrary to accepted dogma, these results indicate that the photoperiodic initiation of seasonal anovulation in the mare is mediated at the level of the anterior pituitary, and appears to occur through a dampening of gonadotroph responsiveness to an unchanging pattern and magnitude of GnRH secretion.

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An excitatory role for norepinephrine (NE) in the regulation of luteinizing hormone (LH) release was first suggested when it was demonstrated that noradrenergic receptor antagonists were able to block ovulation. More recently it has been proposed that NE has both an excitatory role and an inhibitory role in the neuroendocrine regulation of LH release. The excitatory effects may be mediated by alpha-adrenergic receptors and the inhibitory effects may be mediated via beta-adrenergic receptors. These experiments were performed to better understand the role of NE, the receptor type through which NE exerts its effects, and the role of the two major NE pathways in the brain, on LH secretion in the rat. To further understand the role of NE in pulsatile LH release, NE or one of its agonists was infused into the third ventricle of ovariectomlzed rats pretreated with an adrenergic antagonist. In the second set of experiments ascending noradrenergic pathways were electrically stimulated to determine their effect on pulsatile LH release. These experiments demonstrated that the inhibitory effect of NE on pulsatile LH release is blocked when alpha-1- or alpha-2- receptors are blocked but not when beta-receptors are blocked. Electrical stimulation experiments in unprimed ovariectomlzed rats demonstrated that activation of the dorsal noradrenergic tract (DNT) but not the ventral noradrenergic tract (VNT) inhibited pulsatile LH release. Another series of experiments were performed to determine the role NE in the regulation of LH release in the steroid-primed ovariectomlzed rat. These experiments demonstrated that activation of alpha- or beta-adrenergic receptors inhibited the LH surge when adrenergic agonists are infused during the rising phase of the surge. In a similar manner electrical stimulation of either the DNT or VNT inhibited LH release if stimulation occured during the rising phase of the surge. The inhibitory effects of the DNT appear to be via activation of alpha-adrenergic receptors since inhibition was prevented by an alpha-adrenergic antagonist. Under a variety of steroidal conditions and stimulation parameters, activation of the DNT or VNT did not enhance LH release. The lone exception to this was stimulation of the VNT in anaesthetized, steroid-primed ovariectomized rats pretreated with an alpha-adrenergic antagonist. In this case stimulation of the VNT did enhance LH release over non-stimulated and electrically stimulated, saline-treated controls. These results suggest that LH release is enhanced by stimulation of the VNT only when alpha-adrenergic receptors are blocked. In conclusion, it is evident from these studies that activation of alpha-adrenergic receptors either by intraventricular infusion of NE or alpha-agonists, as well as electrical stimulation of noradrenergic tracts inhibits LH secretion. This suggests that the inhibitory effects of NE may be more of a factor in the regulation of LH release than has been previously proposed. In conclusion, NE, in addition to its well established excitatory role, may also have an important inhibitory role in the regulation of LH release. It appears that both inhibitory and excitatory effects of NE on LH release may be mediated by both alpha- and beta-receptors. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

No description available.

The physiological mechanisms through which nutrition mediates its effects in controlling reproduction are not well characterized. Both neural and endocrine components have been implicated in the communication of nutritional status to the central nervous system. Leptin, a hormone synthesized and secreted mainly by adipocytes, is heavily involved in this communication network. The objectives of studies reported herein were 1) to determine the effects of short-term restriction of nutrients on circulating leptin, leptin gene expression in adipose tissue, and leptin receptor (LR) gene expression in the adenohypophysis of ovariectomized cows; and 2) to investigate the responsiveness of the hypothalamic-adenohypophyseal (AP) axis of fasted and non-fasted cattle to leptin. Studies demonstrated that circulating concentrations of leptin and leptin gene expression in subcutaneous adipose tissue are decreased by fasting. Although 2 to 3 days of fasting did not affect patterns of release of luteinizing hormone (LH), cerebroventricular infusions of leptin increased mean circulating concentrations of LH in fasted, but not normal-fed cows, without affecting frequency or amplitude of pulses of LH. In vitro studies were conducted to determine whether the in vivo effects of leptin could be accounted for at the hypothalamic and/or AP levels. Leptin did not affect the release of gonadotropin-releasing hormone (GnRH) from hypothalamic-infundibular explants from either normal-fed or fasted cattle. Moreover, leptin did not affect the basal release of LH from bovine AP cells or AP explants from normal-fed cows. However, leptin induced a higher basal release of LH from AP explants of fasted cows and increased GnRH-stimulated release of LH from AP explants of normal-fed cows. Results demonstrate that leptin acts directly at the AP level to modulate the secretion of LH, and its effects are dependent upon nutritional status. Cellular mechanisms associated with the increased responsiveness of gonadotropes to leptin in fasted cows were investigated. Expression of LR and suppressor of cytokine signaling-3 (SOCS-3) in the adenohypophysis did not account for the increased responsiveness of fasted cows to leptin. Therefore, although leptin clearly stimulates the hypothalamic-gonadotropic axis in nutrient-restricted cattle, it is unclear why cattle maintained under neutral or positive energy balance are resistant to leptin.

In Experiment 1, 12 cyclic mares were assigned randomly to one of two groups (n = 6/group): 1) Control, saline; and 2) GnRH, 100 mu g/h. Between 3 and 6 d after ovulation (Day 0), Alzet osmotic minipumps (Model 2ML1) containing saline or GnRH were placed subcutaneous and connected to a jugular infusion catheter. Five-min samples were collected from the intercavernous sinus (ICS) of 10/12 mares (5/group) during 8 h on Day 4, followed by an additional 6-h intensive sampling period 36 h after induced luteal regression (Day 6). Treatment with GnRH markedly increased (P < 0.01) secretion of LH during both luteal and follicular phases. During the luteal phase, treatment with GnRH eliminated the very large, intermittent secretory episodes of LH characteristic of controls and produced frequent episodes of LH release of short duration. In Experiment 2, 12 anovulatory mares and 3 mares with some residual follicular activity (n = 15) were used during the fall (December 5 to 20) and winter (February 15 to 29) seasons. Mares were assigned randomly to: 1) Control, 2) GnRH-20; continuous infusion of GnRH at 20 mu g/h, or 3) GnRH-100; continuous infusion of GnRH at 100 mu g/h. Treatments were administered subcutaneously for 14 d using Alzet minipumps. Both the 20- and 100-mu g/h treatments increased (P less than 0.01) mean circulating concentrations of LH compared to controls before the winter solstice, but mares did not respond to the GnRH- 20 dose after the winter solstice. GnRH-100 caused a seasonally-independent increase (P less than 0.0001) in follicle size and ovulation frequency compared to controls The equine gonadotrope responded to continuous administration of high-dose GnRH during both ovulatory and anovulatory seasons, but was less responsive late compared to early in the anovulatory season.