Background: The progesterone metabolite allopregnanolone is involved in several clinical conditions in women, e.g. premenstrual dysphoric disorder. It is a very potent GABA-steroid with GABA-A receptor effects similar to other GABA-agonists, e.g. benzodiazepines, and it causes sedation. An objective way to examine effects on the GABA-A receptor in humans is to measure saccadic eye velocity (SEV), which is reduced by GABA-agonists, e.g. allopregnanolone. Animal studies suggest that allopregnanolone is involved in the regulation of gonadotropin secretion via the GABA-A receptor, but this has not been studied in humans. Polycystic ovary syndrome (PCOS) is the most common endocrine disturbance among women of fertile age (5–10%), characterized by polycystic ovaries, menstrual dysfunction, hyperandrogenity, and 50% have obesity. Studies have shown higher allopregnanolone levels in overweight people. PCOS women have increased levels of androstanediol, an androgen metabolite which is an GABA-A receptor agonist. Tolerance often occurs when persons are exposed to high levels of GABAergic modulators. It has not been studied whether GABA-A receptor sensitivity in PCOS women is changed. Another progesterone metabolite, isoallopregnanolone, is the stereoisomere of allopregnanolone but has not been shown to have any GABA-A receptor effect of its own. Instead it has often been used to control steroid specificity to allopregnanolone. Aims: To compare the effects of allopregnanolone and isoallopregnanolone on gonadotropin secretion. To compare allopregnanolone levels, GABA-A receptor sensitivity to allopregnanolone and effects on gonadotropin secretion in both cycle phases and PCOS conditions. To examine pharmacokinetics and pharmacodynamic properties for isoallopregnanolone. Method: In the follicular phase healthy women were examined for the effect of allopregnanolone or isoallopregnanolone on gonadotropin secretion. PCOS women and healthy women in both cycle phases were given allopregnanolone and the differences in effects on SEV were examined, as well as changes in serum levels of gonadotropins and allopregnanolone at baseline and during the test day. Pharmacokinetics and GABA-A receptor sensitivity using SEV were explored for isoallopregnanolone in healthy women. Results: Allopregnanolone decreases gonadotropin serum levels in healthy controls in both cycle phases, but has no effect on gonadotropin secretion in women with PCOS. PCOS women have higher baseline serum levels of allopregnanolone than follicular phase controls, but lower levels than luteal phase controls. PCOS women show greater reduction in SEV to allopregnanolone than controls. Isoallopregnanolone has no effect on gonadotropin secretion. There is an effect of isoallopregnanolone on SEV, explained by a metabolism of isoallopregnanolone into allopregnanolone. Conclusion: There are significant differences in the GABA-A receptor response to a GABA-steroid in different endocrine conditions in women of fertile age examined with saccadic eye velocity. The GABA-steroid allopregnanolone decreases gonadotropin serum levels in healthy women but not in PCOS women. The lack of effect on gonadotropins by isoallopregnanolone suggests an involvement of the GABA-A receptor.

Thesis (M.Sc. (Biochemistry))--Mahidol University, 1984.

Thesis (M.Sc (Biochemistry))--Mahidol University, 1982.

Thesis (Ph. D.)--University of Wisconsin, 1973. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-08-14Bitstream added on 2014-06-13T19:35:12Z : No. of bitstreams: 1 cipriano_rs_me_araca.pdf: 1046361 bytes, checksum: 779be3d9a25d3b3245c78549a32c9bf2 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Avaliou-se a secreção de LHe FSH de plasmático em novilhasdurantea exposição à diferentes concentrações de progesterona(P4) e após a administração deGnRH ou benzoatodeestradiol (BE). Novenovilhas Nelore, pré-sincronizadas com PgF2a, emintervalo de 12 dias, foram submetidas a 6protocolos comrepetições casualizadas. Os 3 grupos de P4foram: CL (Corpo Lúteo), IMPL+CL (DIB® ecorpo lúteo) e IMPL(DIB®), eapós a remoçãodaP4 estimuladascom GnRH ou BE. Durante a P4, foicoletado sangue a cada12h e nos dias 3, 4 e 5 acada 15 min durante 6 h em 1 animalde cada grupo, depois da retiradados implantes e/ou aplicação de PgF2a, coletas foramrealizadas a cada 3hpor 24 h (BE) ou a cadahora por10h (GnRH) para quantificação de LH e FSH. O exame ultrasonográfico foi realizado a cada 12 he após o términodascoletas de sangue às 24h e 48h. Às 12 h após colocação do implante, o grupo IMPL+CLapresentou menorconcentração de LHque o grupo IMPL,após 36 h os grupos IMPL+C e IMPL apresentaram menorconcentração de LH que o grupo CL e às 60 h o grupo IMPL apresentou menor secreção que o grupo CL. Após 24 h da colocação dos implantes, o grupo IMPL apresentou maior secreção de FSH que os demais grupos, e após 48 e 60 h o grupo IMPL+CL apresentou maior secreção de FSH que o grupo CL. No grupo IMPL, a amplitude máxima do pico de LH foi maior após o GnRH quando comparado com o BE. No grupo CL as novilhas apresentaram menor número de folículos ovarianos e maior diâmetro do maior folículo em relação aos grupos IMPL+CL e IMPL. Quando foi aplicado BE, o grupo IMPL+CL apresentou menor taxa de ovulação com 24 h que os grupos CL e IMPL. Os protocolos de sincronização da ovulação empregados foram... / The LH and FSH secretion and follicle profile was evaluated during expositionto different progesterone (P4) concentration and afterGnRH or estradiol benzoate (BE) administration inNelore heifers. Nine heifers were pré-sinchronized with PGF2a (two injection with 12 days interval) received 6 treatments randomly repeated . There were 3 P4 groups: CL (corpus luteum), IMPL+CL (DIB®and CL) and IMPL (DIB®)that were stimulatedwith GnRH or BE after P4 removal. During P4 blood samples were collected every12 hand on days 3, 4and 5 every 15 minfor 6 hfrom 1 animal per group, after P4 device removal, every 3 hfor 24 h (BE)orevery hour for 10 h(GnRH), for LH and FSH quantification. Ultrasound examination was realizedevery 12 h until the end of blood samples, andthereafter 24 and 48 h. At 12hafter implant insertion the IMPL+CLgroup presented lower LH concentration than IMPL group, after 36 hthe animals with implant presented lower LH concentration than CL group, and after 60h the IMPL group had lower LH secretionthan CL one. On IMPL group,themaximum LH peak amplitude was higher in animalsthat received GnRH comparedto BE. Group with CL presented fewer follicles and higher largest follicle diameterthan IMPL+CLand IMPL. When BE was injected the IMPL+CL group presented lower ovulation rateat 24 h than CL and IMPLgroups. The ovulation synchronization protocols used wereefficient in promoting a preovulatory LH peak in Neloreheifers, independentlyof an associated CL or not to progesterone device. The GnRH treatment induced a higher LH peak amplitude and with an higer efficiency in stimulating ovulation in 24h than BE treatment, in animals with higher progesterone...(Complete abstract, click electronic access below)

Made available in DSpace on 2014-10-09T12:52:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:55Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:26:59Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:49Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:52:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:55Z (GMT). No. of bitstreams: 0 / Um método de cromatografia líquida de alta eficiência por fase reversa (RP-HPLC) para análise qualitativa e quantitativa do hormônio folículo estimulante humano íntegro (hFSH), foi estabelecido e validado quanto à exatidão, precisão e sensibilidade. O FSH humano é um hormônio glicoprotéico dimérico largamente utilizado em medicina reprodutiva tanto para diagnóstico quanto para terapia. A metodologia desenvolvida preserva a integridade da proteína, permitindo a análise da forma heterodimérica intacta, e não somente de suas subunidades, como é normalmente obtida na maioria das condições geralmente empregadas. Esta técnica foi também utilizada para a comparação da hidrofobicidade relativa de preparações de hFSH hipofisária, urinária e derivadas de células de ovário de hamster chinês (CHO) bem como de outros dois hormônios glicoprotéicos, sintetizados na hipófise anterior: hormônio humano estimulante da tireóide (hTSH) e hormônio luteinizante humano (hLH). O menos hidrofóbico dos três hormônios analisados foi o hFSH, seguido do hTSH e do hLH. Uma diferença significativa (p<0,005) foi observada entre o tempo de retenção (tR) das preparações hipofisária e recombinante de hFSH, refletindo diferenças estruturais nas suas cadeias de carboidratos. Duas isoformas principais foram detectadas no hFSH urinário, incluindo uma forma que foi significativamente diferente (p<0,005) das preparações hipofisária e recombinante. Foram demonstradas linearidade da curva dose-resposta (r=0,9965, n=15) para esta metodologia de RP-HPLC, bem como uma precisão inter-ensaio, cujo coeficiente de variação é menor que 4%, para a quantificação de diferentes preparações de hFSH e uma sensibilidade da ordem de 40 ng. Foram também analisados o comportamento cromatográfico e a hidrofobicidade relativa das subunidades individuais das preparações recombinantes e hipofisária de hFSH. Além disso, a exata massa molecular das subunidades individuais de hFSH e do heterodímero foram simultaneamente determinadas por espectrometria de massa MALDI-TOF. A presente metodologia representa, em nossa opinião, uma ferramenta essencial para a caracterização e controle de qualidade deste hormônio, que ainda não consta das principais farmacopéias. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:26:59Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:49Z (GMT). No. of bitstreams: 0 / Neste trabalho foram desenvolvidas condições específicas de RP-HPLC para análise de preparações recombinantes e naturais de hLH, de hCG, e de suas subunidades. O hLH e o hCG heterodimérico e suas subunidades e migraram com tempos de retenção (tR) significativamente diferentes, na seguinte ordem de hidrofobicidade crescente: -hCG < -hLH < hCG < hLH < -hCG < -hLH. Nestas condições, onze preparações foram estudadas: o Padrão Internacional recombinante hLH-WHO 96/602, uma preparação comercial recombinante, duas preparações hipofisárias altamente purificadas de hLH, uma preparação recombinante e duas preparações urinárias de hCG e quatro produtos urinários heterogêneos, contendo hLH + hFSH. Todas as preparações de hLH mostraram um tempo de retenção similar para o pico principal (tR = 38,35 ± 0,42 min; DPR = 1,1 %; n = 4 preparações), enquanto o pico principal do hCG migrou cerca de 4% mais rápido, quando comparado a este valor médio. Picos de hLH, hFSH e hCG foram também identificados nas preparações urinárias heterogêneas. O método foi validado para as sete preparações homogêneas, sendo a exatidão, precisão e sensibilidade calculadas com base na curva dose-resposta, altamente linear (r=0,99998; p<0,0001; n=20). A quantificação de diferentes gonadotrofinas nas preparações heterogêneas foi também realizada, embora com claras limitações de exatidão. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Corpus luteum (CL) dysfunction has been implicated among various factors predisposing early embryonic mortality in cattle. Two experiments were conducted to evaluate the efficacy of using human chorionic gonadotropin (hCG) given either at the time of breeding (day 0) , day 7 or 14 post breeding, in reducing that component of early embryonic mortality caused by CL dysfunction. The aims of experiment 1 were to investigate the effectiveness of using hCG, in inducing the development of accessory CL, their formation and growth, and the effect of such treatments on the function of both the induced and spontaneous CL. Thirty-four lactating Holstein cows were randomly assigned to one of four treatments. A single intramuscular injection of 1000 IU of hCG was given either at the time of breeding (day 0, n=8), day 7 (n=9) or 14 (n=9) post breeding or no hCG given (control, n=8). A real-time ultrasound machine was used to study follicular dynamics and CL growth. The CL and antral follicle diameter was determined using a built-in system of calibrated callipers. Ultrasound scanning was carried out on days 7, 9, 11, 14, 16, 18, 21, 28, 35 and 42 post breeding. Blood and milk samples, for progesterone (P₄) determination using radioimmunoassay, were collected on days coincident with ultrasonography. Diameter of the CL is presented as the sum of the diameter of all luteal tissue in each animal. Differences in CL diameter, milk and plasma P₄ were analyzed using the General Linear Models Procedures while pregnancy data were analyzed using Chi-Square analysis in Statistical Analysis Systems (SAS, version 6.3). Based on the day 7 ultrasound scanning, the incidence of twin ovulations was higher among cows treated on day 0 (3/8) compared to control cows (1/8) and day 7 (1/9). Accessory CL were detected in 7/9 of the day 7-treated cows compared to 4/9 among the day-14 treated cows. Least squares means (LSMeans) for CL diameter were significantly higher (P<0.001) among cows treated with hCG compared to control cows starting at day 7 continually until day 42. Plasma P₄ profiles were significantly higher (P<0.05), at days 18, 35 and 42, in cows treated on day 7 or 14 compared to control cows. The first detectable differences (P<0.05) between hCG treated and control cows, in milk P₄ occurred at day 21 and persisted until day 42. Pregnancy rates were highest among cows treated with hCG on day 7 where 6 of the 9 cows were diagnosed pregnant. Corresponding pregnancy rates for day 0, 14 or control cows, were 4/8, 5/9 and 3/8, respectively. In the second experiment, two trials were conducted at two different farms to investigate the efficacy of using hCG to increase milk P₄ and pregnancy rates. In trial one, 79 lactating Holstein cows were exposed to the treatment protocol described in experiment 1. In addition to the milk sample collection schedule given in experiment 1, a sample was collected on day 0. Milk samples were stored at 4°C and later transported to the UBC laboratories for P₄ analysis. LSMeans for milk P₄ concentrations were different only at days 16 and 18 post breeding. Pregnancy rates were improved (P<0.01) by hCG treatments. The respective pregnancy rates for cows receiving hCG on day 0 (n=20), 7 (n=20), 14 (n=20) or control (n=19) were 25, 35, 35 and 21 %. In the second trial, 121 lactating Holstein cows were randomly assigned to treatments as described earlier. Weekly milk samples were collected from each animal and assayed for P₄ as described above. LSMeans for milk P₄ were significantly different (P<0.05) among groups starting at day 14 until day 42 post breeding. hCG increased pregnancy rates over control cows. The pregnancy rates for cows treated on day 0, 7, 14 and control were 31, 50, 41 and 26 %, respectively. In conclusion, this study revealed that treatment with hCG induced accessory CL development, increased P₄ production and improved pregnancy rates. It is evident, too, that treatment with hCG on day 7 post breeding may have greater potential for improving pregnancy rates not only in dairy and beef cattle but equally beneficial to the embryo transfer programmes. Increased pregnancy rates confirm the hypothesis that CL dysfunction does cost the livestock industry appreciable losses in embryos. / Land and Food Systems, Faculty of / Graduate