Thesis (Ph. D.)--Lehigh University, 1996. / Includes vita. Includes bibliographical references (leaves 78-90).

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 126-145). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 126-145). Also available on the Internet.

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vi, 84 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 76-83).

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

AN ABSTRACT OF THE DISSERTATION OF LAN HAI, for the Doctor of Philosophy degree in Molecular Cellular and Systemic Physiology, presented on 11th December, 2015 at Southern Illinois University Carbondale. TITLE: EFFECT OF CONSTITUTIVELY ACTIVATED LUTEINIZING HORMONE RECEPTOR ON THE MOUSE REPRODUCTIVE SYSTEM MAJOR PROFESSOR: Dr. Prema Narayan The luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is crucial for fertility, and genetic mutations in LHCGR cause adverse effects in reproductive development. Among the activating mutations identified in LHCGR, replacement of aspartic acid 578 by glycine (D578G) is the most common inherited mutation. Boys with this mutation undergo puberty by 2-4 years, caused by elevated testosterone in the context of prepubertal luteinizing hormone levels and present with Leydig cell hyperplasia. Clinically, these symptoms are associated with familial male-limited precocious puberty (FMPP). Our lab has published a mouse model for FMPP (KiLHRD582G) with D582G mutation equivalent to D578G in human LHCGR. We have previously demonstrated that KiLHRD582G male mice exhibited precocious puberty, Leydig cell hyperplasia and elevated testosterone and was a good model for FMPP. However, unlike women with the D578G mutation who show no abnormal phenotype, our studies revealed that female KiLHRD582G mice were infertile. KiLHRD582G female mice exhibit precocious puberty and irregular estrous cyclicity. A temporal study from 2-24 weeks of age indicated elevated steroid levels and upregulation of steroidogenic acute regulatory protein as well as several steroidogenic enzyme genes. Ovaries of KiLHRD582G mice exhibited significant pathology with the development of large hemorrhagic cysts as early as 3 weeks of age, extensive stromal cell hyperplasia with luteinization, numerous atretic follicles and granulosa cell tumors. Anovulation could not be rescued by exogenous gonadotropins. The body weights of the KiLHRD582G mice was higher that wild type counterparts, but there were no differences in the body fat composition. Hyperandrogenism and polycystic ovary phenotype was not accompanied by impaired glucose tolerance. Blocking the androgen action and estrogen synthesis indicated that reproductive phenotype was primarily due to excess estradiol. These studies demonstrate that activating LHCGR mutations do not produce the same phenotype in humans and mice and clearly illustrates species differences in the expression and regulation of LHCGR in the ovary. As we use male KiLHRD582G mice as breeders, we observed that the KiLHRD582G mice became progressive infertile, and only 8% of KiLHRD582G were fertile at 6 months of age despite normal sperm production. The infertile KiLHRD582G males were not able to form copulatory plugs in WT females, and mating studies suggested that the KiLHRD582G males were not capable of mating and/or ejaculating. Sexual behavioral testing revealed that the infertile KiLHRD582G males were capable of mounting the receptive WT females but were unable to achieve ejaculation indicating a problem with erectile and/or ejaculatory function. To address the reason for the ejaculatory dysfunction, we performed histopathological analysis of the accessory glands and penis. Hematoxylin and eosin staining showed that the normal columnar epithelium was replaced by pseudostratified columnar epithelium in the ampulla and several aggregates of chondrocyte metaplasia were apparent in the penile body of KiLHRD582G male mice. A temporal study indicated the histopathological changes in ampulla and penile body initiated at 7-8 and 12 weeks of age, respectively. Immunohistochemistry indicated that the chondrocytes stained positive for collagen type II, SOX9 and androgen receptor in the nucleus and for LHCGR in the cytoplasm. Penile fibrosis is a major cause of erectile dysfunction and is characterized by an increased collagen/smooth muscle ratio. However, our Image J analysis, hydroxyproline assay and western blot showed that KiLHRD582G penile body exhibited reduced levels of smooth muscle actin but similar total collagen content compared to WT mice. Thus, penile fibrosis was not responsible for the progressive infertility of adult KiLHRD582G mice. We also observed Leydig cell adenoma and disruption of spermatogenesis at 1 year of age. Our results suggest FMPP patients may be susceptible to infertility and testicular tumors later in their life and a follow-up study of FMPP patients is recommended.

Two forms of gonadotropin-releasing hormone (GnRH) were extracted from brain-pituitary tissues of two species of Thai catfish, Clarias inacrocephalus and C. batrachus. The peptides were detected using high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA), The amino acid sequences of both forms were determined using Edman degradation. One form of GnRH in the brain-pituitary tissues of the Thai catfish was novel, whereas the second form of GnRH was identical to chicken GnRH-XI. The presence of the N-terminal pGlu residue in both peptides was established by digestion with pyroglutamyl aminopeptidase. In addition, catfish GnRH-I was studied by mass spectrometry. The localization of these two peptides was determined to be in the discrete brain areas and in the pituitary of female and male catfish, C. macrocaphalus, using heterologous and homologous radioimmunoassays. Initially a heterologous RIA was used with mammalian GnRH as iodinated tracer and standard, and an antiserum made against salmon GnRH. Catfish GnRH-I (novel form) was found in most areas of the female and male brain with the highest content and concentration in the female pituitary and in the male hypothalamus,, Catfish GnRH-II (chicken GnRH-II) was found with the highest content in the female Cerebellum and highest concentration in the pituitary, catfish GnRH-II (chicken GnRH-II) was found with the highest content and concentration for males in the same area, hypothalamus. Additionally, a homologous RIA was used with catfish GnRH-II (chicken GnRH-II) as iodinated tracer and standard, and an antiserum prepared against chicken GnRH-II. Catfish GnRH-II was detected with the highest content and concentration in the cerebellum of both sexes. These values are higher than the results obtained in the heterologous assay. The location of catfish GnRH-I suggests that it plays a role in regulating the release of gonadotropin from the pituitary since the high content and concentration of this immunoreactive GnRH are detected in the hypothalamus and pituitary gland. In contrast, catfish GnRH-II may act as a neurotransmitter in the catfish brain, in particular in tha cerebellum where a high content and concentration of immunoreactive GnRH are detected. Physiological in vivo studies indicate that catfish GnRH-II is more effective than catfish GnRH-I and other forms of GnRH such as mammalian and dogfish GnRH for induction of ovulation in catfish, C. macrocephalus. Eight GnRH analogs had varying potencies for the induction of ovulation, but the most effective forms were two forms of catfish GnRH-II (chicken GnRH-II) modified in positions six and ten. In vitro studies found that catfish GnRH-I not only causes the release of gonadotropin but also the release of growth hormone in a dose-dependent manner. The primary structures of the two catfish GnRH peptides are important for understanding the evolution of this family peptide. The novel catfish GnRH shows that only positions 5, 7 and 8 vary in the GnRH molecule in jawed vertebrates, whereas catfish GnRH-II provides direct evidence that the structure of this GnRH is conserved in teleosts. / Graduate

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The exact mechanisms controlling uterine secretion of prostaglandin F₂α (PGF₂α) are not known. This study (Experiments 1, 2 and 3) was conducted to evaluate the relationships of progesterone and estrogen to changes in 13,14-dihydro-15-keto-prostaglandin F₂α (PGFM) and PGF₂α in ewes. Experiment 1 was designed to determine whether a radioimmunoassay (RIA) for progesterone would detect pessary-released 6α-methyl-17α-hydroxy-progesterone (MPA; n=3) and oral 17α-acetoxy-6-methyl-16-methylene-4, 6-pregnadiene-3, 20 dione (MGA; n=3) in blood plasma of ovariectomized ewes. Neither progestogen treatment interfered with the RIA. Experiment 2 was conducted to answer the question: Do MPA-containing pessaries delay luteolysis in intact ewes? Ewes were treated with MPA containing (n=10) or blank pessaries (controls; n=8) from d 7 and until d 18 of the estrous cycle for control and until d 22 for MPA-treated ewes; d 0 was the day of estrus. Blood samples were collected from the jugular vein throughout the experiment. Pessaries containing MPA did not affect the timing of luteolysis (d 15.4 ± .2), but they prolonged (P<.O5) the interestrous interval (17.5 d for control vs 24.1 d for MPA-treated ewes). Experiment 3 was designed to study the relationships among progesterone, estrogen, PGFM and PGF₂α in ewes. Ewes were treated with MPA-containing (n=7; 60 mg), progesterone-containing (n=8i 45 mg) or blank pessaries (n=8) from d 7 until d 20 of the estrous cycle. From d 14 and continuing until 24 h after estrus, jugular and vena caval blood samples were collected during two sampling periods daily. Plasma was assayed for progesterone, estrogen, PGFM and PGF₂α. Treatment did not affect the profiles of change in concentration of progesterone, PGFM and jugular PGF₂α, but treatment affected (P < .05) estrogen and vena caval PGF₂α profiles. Overall, treatment affected (P < .05) the mean concentrations of estrogen, progesterone, PGFM and PGF₂α. sampling site (jugular vs. vena cava) affected (P < .0001) the mean concentration of progesterone, estrogen and PGF₂α, but site did not affect PGFM concentrations. Hormonal relationships associated with changes in release of PGF₂α were evaluated. Estrogen seemed to be the primary hormone controlling PGF₂α release. In conclusion, MPA treatment did not delay the timing of luteolysis, but it increased the interestrous interval. Of the compounds measured, estrogen accounted for the greatest proportion of the variation in PGF₂α release in ewes around the time of luteolysis. / Master of Science