Brucellosis, caused by Gram-negative bacteria of the genus <i>Brucella</i>, is a zoonotic disease with global impacts on human, livestock, and wildlife health. Around 500,000 cases of human brucellosis are reported by the World Health Organization annually. Even though brucellosis has been eradicated from domestic livestock in the United States of America, the causative bacterial pathogen is still present in elk, bison, and feral swine. With the growth of free-range farming, domestic livestock and wildlife populations come into close contact, spreading the disease. Feral swine interactions are of particular concern. They carry a number of zoonotic diseases including brucellosis. As there is no commercial vaccination protocol to prevent brucellosis in wildlife and swine, interactions with these populations are especially dangerous for public health. Feral swine population is increasing nationwide even with the current population control practices. There is an urgent need for efficient control of feral swine and preventing the spread of brucellosis. To aid in the prevention of the spread of feral swine across the USA, immunocontraceptives have been employed. Over the years several candidates have been tested, but the search for the perfect vaccine is still ongoing. The monumental task includes reversibly preventing one of life’s most basic necessities, reproduction, through an oral route with no effect on non-target species. One way that science is tackling both of these threats at once is through dual-purpose vaccines. Dual-purpose vaccines produce an immune response that targets two different pathogens, or in this case a pathogen and reproductive hormones. In the effort to produce this vaccine, more knowledge was needed in regards to <i>B. neotomae</i>. This dissertation showed that it has the ability to survive in a variety of cells from different species, in a similar manner to known virulent <i>Brucella</i> species. This is of concern when using <i>B. neotomae</i> as a vaccine, but it has also been shown that attenuated <i>B. neotomae</i> can provide protection against virulent <i>B. suis, B. abortus</i>, and <i>B. melitensis</i> challenge. This is a major finding in the effort towards a universal brucellosis vaccine. After genetic manipulation, cell culture assays, and mouse trials, several leucine auxotrophic <i>B. neotomae</i> and <i>B. abortus</i> strains show promise in the effort towards a dual-purpose vaccine. Strains of <i>B. neotomae</i> <i>ΔwboA ΔleuB</i> pNS4-trcD-fliC-Gonadotropins were discontinued in this effort towards a brucellosis immunocontraceptive dual-purpose vaccine due to lethality issues in mice. These stability and lethality issues are still under investigation. Instead, a proven stable strain of <i>B. abortus</i> RB51 (a USDA approved cattle vaccine) was used to investigate its effects on fertility in mice when expressing fliC-Gonadotropins. Strains <i>B. abortus</i> RB51 ΔleuB pNS4-trcD-fliC-porcineFSHβ (RB51LFSHβ) and <i>B. abortus</i> RB51 ΔleuB pNS4-trcD-fliC-GnRH (RB51LGnRH) confer reduced fertility characteristics in both male and female purpose bred mice. Strain RB51 ΔleuB has also been shown to protect against virulent <i>B. abortus</i> challenge in the literature. These findings warrant further investigation to determine the efficacy of these vaccine strains in swine as an oral vaccine. Ultimately, their ability to prevent brucellosis, while causing immunocontraception needs to be determined in feral swine. / Ph.D. / While brucellosis has been eradicated from domestic livestock in the United States, the causative agent is still present in wildlife like elk, bison, and feral swine. The interactions between these infected wildlife populations with domestic livestock and human populations pose a great health risk. Many tools are employed to mitigate these interactions including vaccination programs and population management. In particular, the feral swine population has proven difficult to control. It has quadrupled in the past ten years and continues to expand nationwide, making their population control an important national objective. Furthermore, feral swine are known carriers of zoonotic diseases, including hemorraghic colitis, leptospirosis, trichinosis, swine influenza, and brucellosis. Many cases of these diseases in humans have been traced back to interactions with feral swine. The current population control practices have failed to minimize the $1.5 billion of damage they cause to the agricultural industry per year. Thus, there is a need to effectively control the feral swine population and prevent the spread of zoonotic diseases like brucellosis. Rough leucine auxotrophic strains of <i>Brucella</i> expressing gonadotropin releasing hormone (GnRH) or porcine follicle stimulating hormone beta subunit (FSH) conjugated to <i>Salmonella</i> fliC show promise. They have been shown to provide protection from virulent Brucella strain challenge and reduce fertility characteristics in mice. Their effectiveness as an immunocontraceptive for feral swine management, while reducing the spread of brucellosis needs to be tested in swine. These vaccine strains [<i>B. abortus</i> RB51 ΔleuB pNS4-trcD-fliC-porcineFSHβ (RB51LFSHβ), <i>B. abortus</i> RB51 ΔleuB pNS4-trcD-fliC-GnRH (RB51LGnRH) and <i>B. neotomae ΔwboA ΔleuB</i> pNS4-trcD-fliC-GnRH (BNWLGnRH)] could pave the way for effective novel immunocontraceptive tools to be used in wildlife management.

Osteoporosis which is frequently referred to as a pediatric disease with geriatric consequences (Golden, 2000) can result from a lack of optimal bone accrual during the development (NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy, 2001). Pubertal timing is a key factor that contributes to optimal bone accrual and strength (Bonjour et al., 1994; 21 Warren et al., 2002). Bone mass doubles during the onset of puberty and young adulthood (Katzman et al., 1991) with more than 90% of peak bone mass being accrued at the end of second decade of life (Schneider & Wade, 2000). The rate of periosteal expansion is elevated during the pubertal period (Specker et al., 1987; Bradney et al., 2000) and this expansion parallels longitudinal growth (Parfitt, 1994). Irrespective of other changes, periosteal expansion lowers fracture risk by improving the strength of long bones by increasing the moment of inertia (Orwoll, 2003). Therefore, a delay in puberty may actually increase the time available for periosteal development and positively affect bone strength. Previous animal studies have shown decreases in strength, endocortical bone formation and increases in periosteal bone formation with delayed puberty. Clinical studies report negative effects of delayed puberty on bone mass accrual suggesting that delayed puberty is a multifactorial problem affecting bone strength development. The purpose of this study was to determine the effect of delayed puberty on mechanical strength and endocortical bone marrow cells in two models: female rats treated with gonadatropin releasing hormone antagonists (GnRH-a) and energy restriction (30%). Thirty-two female Sprague Dawley rats (21 to 22 days-of-age) were received from (Charles Rivers Laboratories, Wilmington, MA, USA) and housed individually at the Temple University Central Animal Facility (Temple University Weiss Hall). Animals were randomly assigned to one of three groups; control (n=10), GnRH-a (n=10) and energy restriction (ER) (n=12). The GnRH-a group was injected with gonadotropin releasing antagonist injections (GnRH-a) (Antide, Bachem, Torrance, Ca. USA) at a dose of 2.5 mg/kg/BW. The ER group received a 30% energy restricted diet (0pen Source diet (D07100606)(Research Diets, New Brunswick, NJ). All animals were sacrificed on Day 51. One way analysis of variance testing (ANOVA) with a significance level of 0.05 was used to assess group differences. Following the two protocols the uterine weight in the GnRH-a group was 80.6% lower than control; no change in the ER group. Ovarian weight was significantly lower in the GnRH-a group (83.3%) and in the ER group (33.3%) as compared to controls. A 22.7% lower muscle weight was found in the ER group but was equal to control and GnRH-a when normalized by body weight (BW). The retro-peritoneal fat pad weight was significantly decreased by 64.95% in the ER group as compared to controls. Energy restriction did not result in any deficit in bone strength when normalized by body weight however the GnRH-a group had a 26.2% lower bone strength compared to control. Histomorphometric changes were not significantly different between groups, but the ratio for periosteal versus endocortical bone formation rates for the control group was 1.38, GnRH-a was significantly higher with a ratio of 5.54 and for ER was 3.02 indicating that periosteal BFR are almost twice endocortical BFR in the experimental groups. There was a significant decrease in the trabecular percent bone volume (BV/TV) of the lumbar vertebra in the GnRH-a group (20.2%) compared to control. However BV/TV was significantly higher in the ER (18.4%) compared to the control group. Proliferation was suppressed to 59.6% of control in the GnRH-a group but only 85.5% of control in the ER group. The alkaline phosphatase activity was 31.2% lower in the GnRH-a group and 63.9% lower in the ER group. The relative quantification (RQ) of RUNX2 gene expression was lowest in control followed by GnRH-a and highest in ER group although no statistical significance was observed between any groups. Thus our data infers that 30% energy restriction does not negatively impact bone health. Thirty percent food restriction with no deficits in micronutrients or hormone suppression may just suppress growth as indicated by the maintenance of bone strength per body weight and equivalent muscle mass per body weight in the ER group compared to control. The GnRH-a injections resulted in decreased bone strength and trabecular bone volume. Female Athlete Triad or Anorexia Nervosa are the two clinical conditions hypothesized to result from a combination of ER and estrogen deficient environment. Studies replacing estrogen in hypothalamic amenorrhea or IGF-1 in anorexia alone have failed to improve bone mineral density (BMD), but a combination of IGF-1 and estrogen has been successful in improving BMD. This suggests that estrogen dependant and independent mechanisms work in combination to protect bone. Our study investigated both mechanisms separately and indicates that ER at 30% may be protective for bone health. Since estrogen deficiency may be the extreme end of the spectrum affecting trabecular bone, treatment therapies may have to be based on age, magnitude and severity of energy restriction and presence or absence of menstrual status. / Kinesiology

Die Überlebensrate von Patienten mit duktalem Adenokarzinom des Pankreas ist sowohl in primär resektablen als auch im lokal fortgeschrittenen und metastasierten Stadium kurz. Das duktale Adenokarzinom des Pankreas breitet sich rasch aus und wird aufgrund fehlender Frühsymptome oft erst im fortgeschrittenen Stadium diagnostiziert. Gegenwärtig fehlen spezifische Tumormarker, die eine frühzeitige Diagnose erlauben würden. Aufgrund zahlreicher Mechanismen der primären und sekundären Chemoresistenz ist das Pankreaskarzinom verhältnismäßig resistent gegenüber konventioneller systemisch verabreichter Chemotherapie, antikörperbasierten sowie niedermolekularen Therapiestrategien, Enzyminhibitoren, Bestrahlung und chirurgischer Therapie. Ein vielversprechender Angriffspunkt zur zielgerichteten Therapie des Pankreaskarzinoms eröffnet die tumorspezifische Expression des GnRH-I Rezeptors. In dieser Arbeit konnte der GnRH-IRezeptor durch RT-PCR und immunhistochemisch in 32,5% der duktalen Adenokarzinome des Pankreas nachgewiesen werden. Es wurde gezeigt, dass die Behandlung mit dem Hybridwirksoff AESZ-108 (AN-152), einem zytotoxischen GnRH-Analogon in vitro und in vivo Apoptose in den GnRH-IRezeptor- positiven Pankreaskarzinomzelllinien induziert. Apoptose wurde durch den intrinsischen Signalweg über den Zusammenbruch des mitochondrialen Membranpotentials vermittelt und führte zu DNA-Fragmentierung des Nukleus wie fluoreszenzmikroskopisch gezeigt werden konnte. Das zytotoxische GnRH-Analogon AESZ-108 (AN-152) führt in vivo zu einer signifikanten Inhibition des Tumorwachstums im Vergleich zur Therapie mit dem Anthrazyklin Doxorubicin, welches zu keiner signifikanten Inhibition des Pankreaskarzinomwachstums führt. Die rezeptorvermittelte Aufnahme ermöglicht eine selektive Therapie. Nach rezeptorvermittelter Endozytose wird das an die D-Lys6-Seitenkette gebundene Doxorubicin spezifisch im Nukleus der rezeptorpositiven Karzinomzellen freigesetzt. Die Ergebnisse dieser Arbeiten zeigen, dass AESZ-108 (AN-152) ein geeigneter Ansatz zur selektiven Chemotherapie GnRH-I-Rezeptor positiver humaner duktaler Adenokazinome des Pankreas ist.

Doctor of Philosophy / Department of Animal Sciences and Industry / Jeffrey S. Stevenson / Four experiments were conducted in beef cows to determine factors that increased the probability of pregnancy per AI when cows are inseminated by appointment. Cows in all experiments were inseminated after a 7-d CO-Synch + CIDR program (100 μg GnRH [2 mL Factrel, Pfizer Animal Health, Whitehouse Station, NJ] 7 d before 25 mg PGF₂[subscript]α [d 0; 5 mL Lutalyse; Pfizer Animal Health]). Experiment 1 compared 1 vs. 2 inseminations and GnRH injection times at 60 and 75 h after the CO-Synch + CIDR program. Delaying AI until 75 h, according to interpretation of estrus-detection patches, for cows not in estrus by 60 h after CIDR insert removal increased (P < 0.05) pregnancy risk (PR) compared with cows not in estrus and inseminated at 60 h (51.4 vs. 41.7%), respectively. The necessity of GnRH injection concurrent with AI was tested in experiment 2. Cows displaying estrus by 65 h that were injected with GnRH had similar PR to cows in estrus and not treated with GnRH (61.9 vs. 60.4%), respectively. Cows in experiment 2 that did not display estrus, but were treated with a GnRH injection at 65 h and then inseminated at 84 h after CIDR insert removal had increased PR compared with similar cows not treated with GnRH (33.4 vs. 15.0%; P < 0.01), respectively. Experiments 3 and 4 were observational studies conducted to determine if blood metabolites glucose and beta-hydroxy butyrate (BHB experiment 3), or physical body and blood metabolites, (glucose, BHB, non-esterified fatty acids [NEFA], blood urea nitrogen [BUN], body weight, rump fat [RF], or BCS; experiment 4) were indicative of future reproductive success in suckled beef cows enrolled in a timed AI program. In experiment 3, plasma glucose concentration 10 d before AI was lesser (P = 0.01; 52.2 vs. 56.9 mg/dL) and serum BHB concentration was lesser (P < 0.01) in cows that became pregnant 35 d after timed AI than for cows that did not become pregnant (600 vs. 690 μM), respectively. Experiment 4 identified relationships between indicators and reproductive success including the finding that serum NEFA concentration 2 to 4 wk before AI is negatively correlated (P < 0.05) with PR to AI.

INTRODUÇÃO: Aventa-se a hipótese de quepacientes com endometriose poderiam apresentar risco elevado para doenças cardiovasculares. Existe, porém, controvérsia quanto ao perfil lipídico observado nessas pacientes. OBJETIVOS: avaliar os marcadores de risco cardiovascular associados à endometriose, comparando-se o efeito sobre eles de dois diferentes tratamentos para esta doença: Sistema Intra-uterino liberador de levonorgestrel (SIU-LNG) e o análogo do GnRH na forma de depósito (aGnRH). Marcadores inflamatórios, parâmetros clínicos e avaliação lipídica foram utilizados como marcadores de risco cardiovascular. MATERIAL E MÉTODOS: Quarenta pacientes entre 18 e 40 anos, com diagnóstico laparoscópico de endometriose, foram randomizadas para receber tratamento com SIU-LNG (n=22) e com o aGnRH (n=18), durante 6 meses. Foram avaliados o índice de massa corporal, a freqüência cardíaca, as pressões arteriais sistólica e diastólica, além dos seguintes parâmetros laboratoriais: lipidograma (colesterol total (CT), HDL-colesterol (HDL-C), LDL-colesterol (LDL-C), triglicérides (TGL)), interleucina-6 (IL-6), proteína C reativa (PCR), homocisteína, molécula de adesão decélula vascular (VCAM), fator de necrose tumoral ?(TNF-?) e contagem de leucócitos (LCT), sendo realizados no início e após seis meses de tratamento. RESULTADOS: no grupo do SIU-LNG, houve redução dos níveis do VCAM (92,8 ± 4,2ng/mL para 91,2 ± 2,7ng/mL, p=0,04), PCR (0,38 ± 0,30mg/dL para 0,28 ± 0,21mg/dL, p=0,03), CT (247,0 ± 85,0 mg/dL para 180,0 ± 31,0 mg/dL, p=0,0002), TGL (118,0 ± 76,0 mg/dL para 86,5 ± 41,5 mg/dL,p=0,003), LDL-C (160,5 + 66,0mg/dL para 114,5 + 25,5mg/dL, p=0,0005) e HDL-C (63,0 + 20,5mg/dL para 48,5 + 10,5mg/Dl, p=0,002). No grupo do aGnRH, houve aumento da homocisteína (11,5 + 2,9 µmol/L para 13,0 + 2,7µmol/L, p=0,04) e diminuição dos níveis de IL-6 (4,3 + 3,9pg/mL para 2,3 + 0,8pg/mL, p=0,005), VCAM (94,0 + 3,8ng/mL para 92,0 + 1,6ng/mL, p=0,03) e LCT (7330 + 2554 para 6350 + 1778, p=0,01). Esse estudo mostra que alguns marcadores de risco cardiovascular são influenciados por ambos aGnRH e SIU-LNG, mas esse último reduz mais os níveis lipídicos e pode ter efeitos mais favoráveis em longo prazo. / The aim of this prospective and controlled study was to evaluate the cardiovascular risk markers associated with endometriosis and the influence of the levonorgestrel intra-uterine system (LNG-IUS) compared with the GnRH analogue (GnRHa) leuprolide acetate on these risks after six monthsof treatment. Methods: This was a randomized, prospective, open clinical study, with44 patients with laparoscopically and histologically confirmed endometriosis. Patients were randomized into two groups: LNG-IUS group, 22 patients submittedto LNG-IUS insertion, and GnRHa group, 22 patients who received a monthly GnRHa injection for six months. Body mass index, systolic and diastolic arterial blood pressure, heart rate and laboratory cardiovascular risk markers such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein (CRP), homocysteine (HMC), lipid profile, total leucocytes, and vascular cell adhesion molecule (VCAM) were measured before and six months after treatment. Results: in the LNG-IUS group, there was reduction of the levels of VCAM(92.8 +4.2 to 91.2+2.7 ng/mL, p = 0.04), CRP (0.38+0.30 to 0.28+0.21 mg/dL, p = 0.03), total cholesterol (247.0+85.0 to 180.0+31.0 mg/dL, p = 0.0002), triglycerides (118.0+ 76.0 to 86.5+41.5 mg/dL, p = 0.003), LDL (160.5+66.0 to 114.5+25.5 mg/dL, p = 0.0005) and HDL (63.0+20.5 to 48.5+10.5 mg/dL, p = 0.002). The GnRHa group showed an increase of HMC levels (11.5+2.9 to 13.0+2.7 µmol/L, p = 0.04) and a reduction of IL-6 levels (4.3+3.9 to 2.3+0.8 pg/mL, p = 0.005), VCAM (94.0+3.8 to 92.0+1.6 ng/mL, p = 0.03) and total leucocytes (7330+2554 to 6350+1778, p = 0.01). Conclusions: This study shows that some cardiovascular risk markers are influenced by both GnRHa and LNG-IUS,but the latter had a greater positive impact on the lipid profile, which could lead to a favorable effect during long-term treatment.

The wave-like developmental pattern of follicles ≥1 mm in temporal relationship with follicle stimulating hormone (FSH) and the existence of 2- and 3-waves of follicular development during an interovulatory interval (IOI) have been clearly defined in cattle. However, information about the developmental pattern of antral follicles <1 mm and the repeatability of the wave pattern (2- or 3-wave IOI) is lacking. Using approaches such as immunization against GnRH (to suppress circulating concentrations of FSH) and histomorphometric study of ovarian tissues collected from cyclic heifers on different days after ovulation, the developmental pattern of antral follicles <1 mm and the role of FSH in their development were studied in heifers. Ultrasonographically acquired follicular data were used to determine the repeatability of 2- and 3-wave patterns and the effect of season on the wave patterns. The ovulatory follicle in 3-wave IOI is exposed to a shorter term high-progesterone environment than that of 2-wave IOI, and it has been argued that the less-aged ovulatory follicle of 3-wave IOI yields a more fertile oocyte than the 2-wave IOI. The developmental competence of oocytes in preovulatory follicles of 2- versus 3-wave IOI was compared using in vivo environments created to mimic short-term low- and high-progesterone environments similar to 2- and 3-wave IOI, respectively. The developmental competence of oocytes in persistent dominant-type follicles was also determined.<p>The vaccination against GnRH attenuated FSH surges but did not suppress the basal circulating concentrations of FSH. The attenuation of FSH surges suppressed the wave-like emergence of follicles ≥4 mm but not of the antral follicles <4 mm. The study revealed an inverse relationship between the mean and peak circulating concentrations of FSH and the number of follicles recruited into ≥1 mm size category. Histomorphometric study revealed that antral follicles <1 mm developed in a wave-like fashion in response to a rise in the circulating concentrations of FSH. After treatment with exogenous FSH, the growth rate of follicles in GnRH-immunized heifers was similar to controls. <p>The duration of IOI was predictive of the wave pattern (i.e., 2- or 3-wave IOI), and the pattern was repeatable within individuals throughout the year. The dominant follicle of Wave 1 in 2-wave IOI had a longer duration of dominance than in 3-wave IOI. Hence, the dominant follicle of Wave 1 may have a primary role in the regulation of 2- and 3-wave patterns. Greater attrition of follicles in 3-wave IOI, due to the emergence of an extra wave compared to 2-wave IOI, may contribute to earlier follicular depletion and onset of reproductive senescence in heifers with primarily a 3-wave pattern. The fertilization capacity of oocytes that were exposed to the short-term low-progesterone environment (i.e., similar to the early growing phase of the ovulatory follicle of 3-wave IOI) was increased, but the developmental competence post-fertilization was not different from oocytes that were exposed to a short-term high-progesterone environment (i.e., similar to the early growing phase of preovulatory follicle of 2-wave IOI). Multiple follicles developed under the prolonged-low progesterone environment, but failed to ovulate.

The wave-like developmental pattern of follicles ≥1 mm in temporal relationship with follicle stimulating hormone (FSH) and the existence of 2- and 3-waves of follicular development during an interovulatory interval (IOI) have been clearly defined in cattle. However, information about the developmental pattern of antral follicles <1 mm and the repeatability of the wave pattern (2- or 3-wave IOI) is lacking. Using approaches such as immunization against GnRH (to suppress circulating concentrations of FSH) and histomorphometric study of ovarian tissues collected from cyclic heifers on different days after ovulation, the developmental pattern of antral follicles <1 mm and the role of FSH in their development were studied in heifers. Ultrasonographically acquired follicular data were used to determine the repeatability of 2- and 3-wave patterns and the effect of season on the wave patterns. The ovulatory follicle in 3-wave IOI is exposed to a shorter term high-progesterone environment than that of 2-wave IOI, and it has been argued that the less-aged ovulatory follicle of 3-wave IOI yields a more fertile oocyte than the 2-wave IOI. The developmental competence of oocytes in preovulatory follicles of 2- versus 3-wave IOI was compared using in vivo environments created to mimic short-term low- and high-progesterone environments similar to 2- and 3-wave IOI, respectively. The developmental competence of oocytes in persistent dominant-type follicles was also determined.<p>The vaccination against GnRH attenuated FSH surges but did not suppress the basal circulating concentrations of FSH. The attenuation of FSH surges suppressed the wave-like emergence of follicles ≥4 mm but not of the antral follicles <4 mm. The study revealed an inverse relationship between the mean and peak circulating concentrations of FSH and the number of follicles recruited into ≥1 mm size category. Histomorphometric study revealed that antral follicles <1 mm developed in a wave-like fashion in response to a rise in the circulating concentrations of FSH. After treatment with exogenous FSH, the growth rate of follicles in GnRH-immunized heifers was similar to controls. <p>The duration of IOI was predictive of the wave pattern (i.e., 2- or 3-wave IOI), and the pattern was repeatable within individuals throughout the year. The dominant follicle of Wave 1 in 2-wave IOI had a longer duration of dominance than in 3-wave IOI. Hence, the dominant follicle of Wave 1 may have a primary role in the regulation of 2- and 3-wave patterns. Greater attrition of follicles in 3-wave IOI, due to the emergence of an extra wave compared to 2-wave IOI, may contribute to earlier follicular depletion and onset of reproductive senescence in heifers with primarily a 3-wave pattern. The fertilization capacity of oocytes that were exposed to the short-term low-progesterone environment (i.e., similar to the early growing phase of the ovulatory follicle of 3-wave IOI) was increased, but the developmental competence post-fertilization was not different from oocytes that were exposed to a short-term high-progesterone environment (i.e., similar to the early growing phase of preovulatory follicle of 2-wave IOI). Multiple follicles developed under the prolonged-low progesterone environment, but failed to ovulate.

Female reproductive senescence is widespread among mammalian species, but menopause is limited to species with menstrual cycles. While hormonal changes at menopause have profound impacts in the lives of women at middle age, the complex mechanisms underlying this process remain obscure. All three levels of the hypothalamic-pituitary-gonadal (HPG) axis are involved in reproductive aging, and evidence highlights a critical role for the dysregulation of gonadotropin-releasing hormone (GnRH) neurons, the hypothalamic cells that drive reproductive function. To investigate neuroendocrine mechanisms that may initiate and perpetuate reproductive decline at each step in the transition to acyclicity, I utilized an ovarian-intact middle-aged female rat model of natural reproductive senescence. These studies focused on three hypothalamic nuclei that are known to control GnRH activity: the anteroventral periventricular nucleus (AVPV), the site of positive hormone feedback onto GnRH neurons; the arcuate nucleus (ARC), the site of negative feedback; and the median eminence (ME), the site of GnRH release, with the following specific aims: 1) Characterize neuroendocrine gene and protein expression in female rats throughout the natural transition to acyclicity; 2) Determine the effects of chronic N-methyl-D-asparate receptor subunit 2b (NMDAR-NR2b) inhibition in acyclic females; and 3) Examine neuroendocrine gene expression during premature reproductive senescence after perturbation of the HPG axis. The results of these studies identified novel molecular and cellular changes with age and reproductive cycle status in the ARC and ME, two regions that are underappreciated for their roles in reproductive senescence. Surprisingly, few molecular targets were identified in the AVPV, a region that is much better-studied in this context. In the ME and ARC, I found changes in transcription factors and evidence of altered hormone feedback via changes in sex steroid hormone receptors and enzyme expression with reproductive aging. I also discovered decreased expression of genes for the excitatory neuropeptides, kisspeptin and neurokinin B, as well as decreased percentage of kisspeptin immunoreactive cells and their co-expression with estrogen receptor alpha in the ARC. And finally, in the ME, neurotrophic factor expression was changed with age, and the presence and phosphorylation state of the NR2b subunit of the NMDA receptor contributes to a greater inhibitory tone with acyclicity. Together these studies have identified novel pathways, especially in the ARC and ME, that are related to reproductive decline. Furthermore, changes in the hypothalamic neural and glial network of neurotransmitters, neuropeptides, hormone receptors and other transcription factors are likely contributing to altered responses to hormonal feedback and decreased excitatory drive for GnRH release. / text

Hermaphroditism, associated with territoriality and dominance behavior, is common in the marine environment. Male sex-specific coloration patterns and behavior are particularly evident in species where males are territorial and guard harems of females such as wrasses and groupers. Protogynous hermaphrodites that change sex from female to male are good models to study sexual behavior and related changes in the brain due to their abilities to reorganize their sexual phenotype as adults. Two hormones produced in the brain and implicated in the process of sex-specific behavior and reproductive development are arginine vasotocin (AVT) and gonadotropin releasing hormone (GnRH). While a wealth of data exists regarding these hormone systems separately, little is known about linkage between these two systems. Especially there is no data tracking these two systems together in any protogynous fish. This study was conducted to test the hypothesis that coordinated interactions between AVT and GnRH facilitate the process of behavioral and gonadal sex change in the rock hind Epinephelus adscensionis. Four topics were addressed to investigate the relationship between behavior and reproduction: i) rock hind sex change, sexual characteristics and conditions causing sex change to occur in captivity were detailed as a basis for examining the AVT system and GnRH during this process, ii) the distribution of a vasotocin V1a type receptor identified in rock hind brain was examined for the first time in a fish species using a custom designed antibody then the receptor protein was co-localized with GnRH producing cells within the brain to confirm that a pathway exists for AVT action on GnRH, iii) levels of AVT, AVT receptors, and GnRH messenger RNA (mRNA) were compared between male and female rock hind phenotypes, and iv) female rock hind at early stages of sex change were compared for brain mRNA expression of AVT, AVT receptors, and GnRH to determine the order of hormonal change during the process of sexual inversion in this species. This study provides a better understanding of the relationship between sex-specific behavior and reproductive development via AVT and GnRH systems that are conserved in all vertebrates. / text

Das Ziel der vorliegenden Arbeit war es, die brunststimulierende Wirkung des synthetisch hergestellten Gonadorelin[5-His, 6-Asp, 7-Trp, 8-Lys] (International Nonproprietary Name: Peforelin), das in dem Präparat Maprelin® XP10 enthalten ist, bei abgesetzten pluriparen Sauen zu prüfen. Im ersten Versuchsabschnitt wurde die zweckmäßige Dosierung ermittelt. Im zweiten Versuchsabschnitt wurde die Wirkung von Maprelin® XP10 mit der einer eCG- und einer Placebobehandlung verglichen. Die Bedingungen waren für die Versuchstiere in beiden Versuchsabschnitten homogen (etwa vierter Wurf, Säugezeit vier Wochen, Brunstkontrolle zweimal täglich in Anwesenheit eines geschlechtsaktiven Ebers, zweimal täglich sonographische Ovaruntersuchung, duldungsorientierte Besamung einmal täglich). In der Dosisfindungsstudie, in die 88 Tiere einbezogen wurden, erwies sich die Dosierung von 150 µg Peforelin, 24 Stunden nach dem Absetzen appliziert, als zweckmäßig zur Brunststimulation. Andere getestete Varianten (100 µg 24 Stunden, 150 µg 0 Stunden, 150 µg 48 Stunden, 187,5 µg 24 Stunden, 255 µg 24 Stunden nach dem Absetzen) waren hierzu weniger geeignet. In der Untersuchung zur klinischen Wirksamkeit wurden die 313 einbezogenen Tiere in drei Gruppen aufgeteilt und erhielten 24 Stunden nach dem Absetzen pro Tier 150 µg Peforelin (Gruppe I), 800 IE eCG (Gruppe II) oder 2 ml physiologische NaCl-Lösung als Placebo (Gruppe III). Zur Befunderhebung an den Ovarien wurden die Tiere zweimal täglich sonographisch untersucht. Die Östrusrate nach der Peforelin-Behandlung war derjenigen nach eCG-Injektion gleichwertig (95,1 bzw. 96,3 %), beide waren der Placebobehandlung signifikant überlegen (80,6 %). In die weiteren Auswertungen wurden nur Tiere mit Brunstbeginn bis zum siebten Tag nach dem Absetzen einbezogen. Das Absetz-Östrus-Intervall betrug 100,5, 94,2 bzw. 104,1 Stunden in den Gruppen I, II bzw. III. In der Brunstdauer und dem Intervall vom Östrusbeginn bis zur Ovulation unterschieden sich die drei Gruppen nicht. Die durchschnittliche Follikelgröße war in allen drei Gruppen zum Zeitpunkt des Absetzens 4 mm und zum Zeitpunkt der ersten Duldung 6 mm. Die Ovulationen fanden sowohl nach eCG als auch nach Maprelin® XP10 zwischen dem Mittag des sechsten (13.00 h) und der Nacht des siebten Tages (1.00 h) nach dem Absetzen statt. Bei der sonographischen Trächtigkeitsuntersuchung in der vierten Woche post inseminationem waren 100,0, 99,0 bzw. 97,6 % in den Gruppen I, II bzw. III positiv. Die Trächtigkeitsrate betrug 96,9, 97,1 bzw. 91,6 % in den Gruppen I, II bzw. III. Die Abferkelrate ergab 92,2, 93,4 und 73,8 % in den Gruppen I, II und III. Das Abferkelergebnis war in allen drei Gruppen gleich (11,7, 12,0 bzw. 11,6 insgesamt geborene Ferkel in den Gruppen I, II bzw. III). Es wurde eine negative Korrelation zwischen der Dauer des Absetz-Östrus-Intervalls und der Brunstdauer bzw. dem Intervall vom Östrusbeginn bis zur Ovulation nachgewiesen. Darüber hinaus korrelierten die Brunstdauer und das Intervall Östrusbeginn bis Ovulation positiv miteinander. In allen drei Gruppen stand die Follikelgröße bei der ersten Duldung in positiver Korrelation mit der Länge des Absetz-Östrus-Intervalls. Nach längerer Säugezeit kamen die Sauen tendenziell und in der Gruppe I signifikant früher in die Brunst als nach kürzerer Laktation. Weitere überprüfte potentielle Einflussfaktoren hatten weder auf den Brunsteintritt noch auf die erzielten Wurfgrößen oder andere Parameter Auswirkungen. Mit der vorliegenden Untersuchung wurde erstmals die Wirksamkeit des synthetisch hergestellten Peforelin zur Brunststimulation bei abgesetzten pluriparen Sauen nachgewiesen. Inwieweit das Präparat für diese Indikation auch bei primiparen Sauen oder Jungsauen wirksam eingesetzt werden kann, bleibt weiteren klinischen Prüfungen vorbehalten.