Orientador: Gilson Hélio Toniollo / Coorientador: Silvia Regina Rogatto / Banca: Paola Castro Moraes / Banca: Luís Gustavo Gosuen Gonçalves Dias / Banca: Carolina Franchi João / Banca: Fabiana Ferreira de Souza / Resumo: O processo degenerativo progressivo em consequência a resposta exagerada do endométrio à exposição crônica de progesterona e estrógeno, endógeno ou exógeno, resulta em hiperplasia endometrial cística (HEC) na maioria das cadelas idosas, e consequente degeneração tecidual, distensão glandular e acúmulo de secreções, que pode resultar em contaminação bacteriana, inflamação supurativa e degenerativa do endométrio, com acúmulo de exsudato nas glândulas endometriais e lúmen uterino, bacteremia, afecção hepatorenal e toxemia. A análise oligoarrays tem sido aplicada com sucesso no estudo da expressão gênica do endométrio; o único tecido com capacidade dinâmica de sofrer remodelação em resposta a variações cíclicas de hormônios esteróides e fatores locais autócrinos e parácrinos. Objetivou-se com este estudo identificar os genes diferencialmente expressos responsáveis pela proliferação e hiperplasia no endométrio de fêmeas caninas acometidas por HEC, mucometra e piometra, comparado com o tecido endometrial normal de fêmeas caninas na fase de diestro. Foi utilizado teste estatístico SAM (Significance Analysis Of Microarray), do programa TMeV v.4.5, por meio de teste estatístico paramétrico (teste t), p<0,05 e ajuste pelo teste de Bonferroni, considerando apenas os conjuntos de genes que com False Discovery Rate iguais ou inferiores a zero, e fold-change > ou < que 3,0. O grupo HEC apresentou 33 genes com expressão aumentada (upregulated) e 45 genes com expressão diminuída (downregulated) em relação ao grupo diestro, com 15 moléculas envolvidas no desenvolvimento, crescimento e proliferação celular e morfologia tumoral. O grupo mucometra apresentou 189 genes upregulated e 150 genes downregulated em relação ao grupo diestro, sendo 26 moléculas associadas ao desevolvimento embrionário e 21 moléculas envolvidas com movimentação celular, desenvolvimento e funcionamento do sistema ... / Abstract: Hyperplasic and cystic alteration of the canine endometrium compromise reproduction and fertility and may lead to a degenerative inflammation and infection of endometrium. The progressive process usually proposed as the initiating lesion, is mediated by progesterone and potentially aggravated by estrogens. A separate process caused by local uterine irritation to trophoblastic reaction and bacterial proliferation has been recently proposed as an alternate mechanism leading to pyometra. In order to identify molecular similarity and potential targets for therapeutic intervention and biomarkers for endometrium proliferative conditions in humans and dogs, gene expression profiles were performed in fifteen endometrial biopsy samples from female dogs during luteal phase (diestrus), fifteen affected by endometrial cystic hyperplasia, fifteen by mucometra and fifteen by pyometra, and processed on Affymetrix Canine 1.0 ST array. The transcriptome analysis revealed expression of 115, 23 and 284 significantly genes (p<0,05) altered in the hyperplasic compared with normal endometrium, hyperplasic and aseptic secretory compared with hyperplasic endometrium, and infected compared to hyperplasic and aseptic secretory endometrium, respectively. Gene ontology enrichment analysis revealed genes associated with the cell development, growth, proliferation, function and maintenance, and cell-to-cell signaling and interaction were altered in the hyperplasic and proliferative lesion, and also in aseptic secretory endometrium, such as elevated expression of CYR61, EGR1, FOS, GALNT14, IGKC, SLC47A2, IGFBP3, and low expression of ESR2, SOCS3 and MCOLN3. The proliferative to secretory transition revealed genes associated with immune cell trafficking, and cell-mediated immune response, such as FOSB, MAL, CCL4 and SLPI. The infected endometrium due to bacterial infection revealed elevated expression of chemokines (CCL2, CCL3, CXCS), cytokines (IL8, IL6), proteases ... / Doutor

Orientador: Cezinande de Meira / Banca: Sony Dimas Bicudo / Banca: João Carlos Pinheiro Ferreira / Banca: Lucianao Andrade de Silva / Banca: Rubens Paes de Almeida / Resumo: O objetivo do presente estudo foi avaliar a relação entre a intensidade de alterações degenerativas endometriais e da idade com a hemodinâmica uterina durante o período posterior à infusão de sêmen (Experimento 1) e durante a fase inicial da gestação (Experimento 2). Vascularidade, contratilidade e índices Doppler do útero foram mensurados durante as 12 primeiras horas pós-inseminação artificial (IA) no experimento1. Não foi observado (P>0,1) efeito da posição do folículo pré-ovularório sobre a hemodinâmica e contratilidade uterina. Um aumento na vascularidade foi detectado (P<0,001) em éguas velhas e com degeneração endometrial moderada (GII) e intensa (GIII) nas duas primeiras horas pós-IA. Índices de pulsatilidade (PI) e resistência (RI) uterina mantiveram-se constante durante as 12 horas pós-IA em éguas dos grupos GII, jovens e velhas (P>0,05). Um aumento na atividade contrátil do útero foi detectado (P<0,0001) nas horas 1 e 2, independente do grupo experimental (P>0,05). No experimento 2, os parâmetros citados acima, além de características luteais (área e vascularidade) e diâmetro da vesícula embrionária foram avaliados diariamente em éguas gestantes e não gestantes. Aumento e diminuição, respectivamente, na vascularidade e índices Doppler uterinos foram detectados (P<0,05) em D4 (D0=dia da ovulação) em éguas gestantes e não gestantes. Um aumento progressivo na perfusão sanguínea com pronunciada vascularidade no corno ipsi-lateral ao embrião foi observado em éguas gestantes a partir de D12 (P<0,001). Efeito da idade e da intensidade das alterações degenerativas endometriais sobre a vascularidade e índices Doppler do útero gravídico foram detectados (P<0,01). A contratilidade uterina foi máxima (P<0,001) entre D10 e D17 da gestação independente da classificação histológica do... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The overall purpose of the present study was to evaluate the relationship between endometrial degenerative changes and age on the uterine hemodynamics of mares during the post-breeding (Experiment 1) and early gestation phases (Experiment 2). Vascularity, contractility and Doppler indices from the uterus were measured hourly during the first 12 hours post-artificial insemination (AI) in experiment 1. Hemodynamics and contractility of the uterus did not change (P>0.1) by pre-ovulatory follicle position. Increased uterine vascularity was observed (P<0,001) on hours 1 and 2 in older and moderate (GII) and intense (GIII) endometrial degeneration mares. Pulsatility and resistance indices (PI and RI, respectively) did not change (P>0.05) through the experiment 1 age groups (younger and older mares) mares with moderate endometrial degeneration. Independently of the age and endometrial degenerative changes, increased uterine contractility was observed (P<0,0001) in the first two hours post-AI. In experiment 2, the same end points were measured every day. Additionally, luteal vascularity and embryonic vesicle diameter were evaluated. On D4 (D0 = day of ovulation), increased vascularity and decreased Doppler indices were detected (P <0.05) in pregnant and nonpregnant mares. A progressive increase on the uterine vascular perfusion and greater vascularity in the horn with embryo were observed in pregnant mares from D12 (P <0.001). Effect of age and intensity of endometrial degenerative changes on the vascularity and Doppler indices of the gravidic uterus were detected (P<0.01). Uterine contractility was greater (P<0.001) between days 10 and 17 of pregnancy regardless of the uterine histological classification and age of mares (P> 0.05). Mean luteal vascularity and plasmatic... (Complete abstract click electronic access below) / Doutor

Nas éguas cíclicas, a luteólise ocorre entre os dias 14 e 16 após ovulação, pela ação da PGF2&#940 endometrial. Entretanto, durante a gestação, a luteólise deve ser bloqueada, ao mesmo passo que a ação da PGE2 deve ser estimulada. Ambos hormônios possuem baixa difusão pela membrana plasmática, sendo necessária a presença da proteína transportadora de prostaglandina (PGT) para o influxo e efluxo destes hormônios nas células. Os objetivos deste experimento foram identificar e relacionar o RNAm da PGT no endométrio de éguas cíclica e gestante aos 14 dias (experimento 1) e avaliar o perfil do RNAm para PGT no endométrio eqüino em final de diestro sob efeito de secreção embrionária (experimento 2). Para o experimento 1, um ciclo estral de 11 éguas foi acompanhado. Seis éguas não foram inseminadas e somente detectado o tempo de ovulação e cinco foram inseminadas. Biópsias endometriais foram realizadas quando detectado folículo pré-ovulatório (&#8805;35mm de diâmetro) e edema endometrial (E0; n=6), sete (E7; n=6) e quatorze (E14; n=6) dias após ovulação nas fêmeas cíclicas e aos quatorze dias de gestação (EG; n=4) nas fêmeas gestantes. No experimento 2, 5 embriões eqüinos de 13,5 dias de idade foram coletados, cultivados por 24 horas em ambiente com temperatura e CO2 controlados e o meio condicionado embrionário (MCEE) gerado foi armazenado a -80&ordm;C. Em seguida, amostras endometriais de sete éguas cíclicas aos 14 dias pós ovulação foram coletadas por biópsia uterina e cultivadas por 24 horas, em ambiente com temperatura e CO2 controlados, na presença do MCEE. O RNA total foi extraído de todas as amostras endometriais e amplificado pela reação em cadeia da polimerase em tempo real (RT-PCR), de um passo. A abundância relativa média dos transcritos foi submetida a análise de variância e as médias foram separadas pelo teste LSD (P<0,05). No experimento 1, o RNAm da PGT em tecido equino foi identificado, de maneira que as quantidades relativas deste gene foram similares entre E0, E7, E14 e EG. No experimento 2, o MCEE não modificou a quantidade de RNAm para PGT no endométrio em fase final do diestro. / In cyclic mares, luteolysis occurs between the 14th and 16th days after ovulation, due to endometrial PGF2&#940 However, in pregnant mares luteolysis must be blocked, whereas the PGE2 action must be stimulated. Both hormones have low diffusion through the plasma membrane, wherein the Prostaglandin Transporter Protein (PGT) is needed to influx and efflux of these hormones in the cells. The objectives of this experiment are to identify and to relate with the mRNA to PGT in the endometrium of cyclic and pregnant mares (experiment 1) and to evaluate the mRNA profile to PGT in equine endometrium at end of diestrous, under embryonic secretion effect (experiment 2). In the experiment 1, one estrous cycle of 11 mares (5 to 12 years old) was examined. Six mares were not inseminated and only the time of ovulation was recorded, and five mares were inseminated. Endometrial biopsies were performed when pre-ovulatory follicles (diameter &#8805; 35mm) and endometrial edema were detected (E0; n=6), seven (E7; n=6) and fourteen days (E14; n=6) after ovulation in cyclic mares, and fourteen days after ovulation in pregnant mares (EG; n=4). In the experiment 2, five embryos of 13,5 days of age were collected, cultured during 24 hours in controlled temperature and CO2 and the embrionic conditioned medium (ECM) was stored at -80&ordm;C. After that, endometrium samples of 7 mares at fourteen days after ovulation were collected by uterine biopsy and they were cultured during 24 hours, in controlled temperature and CO2, with ECM. Total RNA was extracted and submitted to amplification by one step real-time polymerase chain reaction (RT-PCR). The abundance relative average of trancripts was submitted to variance analysis and averages were separated by LSD test (P<0,05). In the experiment 1 the mRNA to equine PGT was identified so that the relative quantities of this gene were equal among E0, E7, E14 e EG. In the experiment 2, the ECM did not modify the mRNA quantity to PGT in the endometrium at end of diestrous.

Abstract Apoptosis is a genetically programmed mechanism for a multicellular organism to remove cells that are unnecessary, or potentially harmful. The female reproductive system is characterised by a high rate of cellular proliferation. At the same time, apoptosis is also abundant during the normal physiological function of the ovary and endometrium. More than half of the 7 million oocytes that are produced during human ovarian development are deleted before birth and only about 400 oocytes reach the stage of ovulation during the female fertile lifespan. The fate of the non-ovulatory follicles is atresia, occurring through the mechanism of apoptosis. The endometrium goes through radical renewal processes during each menstrual cycle. Apoptosis has been suggested to participate in the regulation of endometrial cellular homeostasis. Errors in this mechanism can result in endometrial diseases such as hyperplasia and cancer. In this work, apoptosis and its regulation were studied in the human fetal and adult ovary, normal endometrium and endometrial pathologies. In fetal ovaries, apoptosis was already abundantly present in oocytes at 13 weeks of gestation. The maximum rate of apoptosis was seen between the 14th and 20th weeks, after which apoptosis decreased towards term. Ovarian Bcl-2 expression was detected in early fetal life during weeks 13 and 14. Bax expression was observed throughout the studied period, from week 13 to 40. The expression of transcription factor GATA-4, which is linked to follicular survival, was localised to the granulosa cells and was high in early fetal life and decreased somewhat towards term. In adult life apoptosis was located in the granulosa cells of the growing follicles. In ovarian biopsies from women homozygous for the inactivating C566T mutation of the FSH receptor, apoptosis or GATA-4 expression was not detected. During corpus luteum regression a peak in apoptosis was detected 10 - 12 days after the LH surge, and was preceded by an increase in 17HSD type 1 and TNF-α expression. During normal menstrual cycles, the highest rate of apoptosis was observed in the menstrual endometrium. This increase in apoptosis was preceded by a decreased Bcl-2/Bax ratio. In endometrial hyperplasia, the rate of apoptosis was similar to that seen during normal proliferation of the endometrium, but an apparent increase was observed in grade II endometrial carcinoma. In grade III carcinoma, the rate of apoptosis was lower than in grade II carcinoma but higher than in hyperplasia. These results indicate that apoptosis is the mechanism behind the substantial oocyte demise during ovarian development. During adult life, apoptosis was mainly localised to the granulosa cells of the growing follicles which do not reach the stage of a dominant follicle. In ovaries where FSH action is abolished, folliculogenesis was impaired and ovarian apoptosis was negligible. Apoptosis is also the underlying mechanism of corpus luteum regression. In the endometrium, apoptosis has a role in rejuvenating the endometrium for growth during the next endometrial cycle and in regulating cellular homeostasis.

Neste estudo caracterizamos a expressão de Mif nas células trofoblásticas (CT), placentárias e decidua (D) na gestação em camundongos.A imunolocalização foi realizada nos dias de gestação(dg) 7,5, 10,5, 13,5 e 17,5.Com cones ectoplacentários e placentas fetais (PL) realizou-se ensaios de Western blotting e qRT-PCR nos mesmos dg.D foram submetidas a qPCR.Aos 7,5 dg as CT gigantes e D apresentaram imunomarcação.Dos 10,5 ao 17,5 dg o Mif concentrou-se nas CTG e no espongiotrofoblasto.Na D, a imunomarcação foi menor que aos 7,5 dg.A expressão protéica na PL aumentou do 7,5 dg para o 10,5 dg (p=0,005) e para o 13,5 dg (p=0.03).A maior expressão gênica foi em 10,5 dg e diferente em 13,5 dg (p=0,048) e 17,5 dg (p=0,009).Na D, a maior expressão gênica foi em 7,5 dg e diferente dos 10,5 (0,012) e 13,5 (0,032) dg.O aumento da expressão de Mif na PL coincide com a organização em quatro camadas e com o início da circulação fetal.Esta distribuição temporal e tecido-específica sugere o MIF como modulador no início da placentação ou na sua adaptação ao ambiente uterino / The goal of this study was to characterize Mif expression by trophoblast (TC),fetal placenta (FP) and decidua (D) during mouse pregnancy.Mif was immunolocalized at TC and D on gestation days (gd) 7.5, 10.5, 13.5 and 17.5.Ectoplacental cones (EC) and FP were used for Western blotting and qPCR.D were also used for qRT-PCR.On gd 7.5,DC and TC giant (TGC) showed strong reactivity.On gds 10.517.5,were concentrated in TGC and spongiotrophoblast cells. D reactivity was weaker than 7.5 gd.Protein expression at FP increased from gd 7.5 to 10.5 (p=0.005) and to 13.5 (p=0.03).Higher mRNA expression was found on gd 10.5 and was different from gds 13.5 (p=0.048) and 17.5 (p=0.009).At D on gd 7.5 was greater than those on gds 10.5 (0,012) and 13.5 (0,032).The up-regulation of Mif coincides with the stage that placenta assumes its four-layered organization and the fetal blood circulation begins,This temporal tissue-specific distribution and expression data suggests that Mif may play a modulator role in the onset of placentation or in it adaptation to the uterine environment

Orientador: Claudia Maria Bertan Membrive / Resumo: Em bovinos, o 17β-estradiol (17β-E2) estimula a expressão de receptores de estradiol (ER) e ocitocina (OXTR) no endométrio. A ativação de OXTR induz a ativação de uma complexa cascata que resulta na síntese de PGF2α. A hipótese é que o tratamento com 17β-E2, 15 dias após o estro (D15), modula a expressão gênica das proteínas quinase (PKC) e fosfolipase A2 (PLA2), ambas envolvidas na síntese de PGF2α e dependentes de cálcio. Objetivou-se neste estudo determinar os efeitos do 17β-E2 na abundância de trancritos (PKCα, PKCβ, PLA2G4, AKR1B1, AKR1C4 e PTGS2) diretamente envolvidos na síntese de PGF2α. Novilhas (N=50), não prenhes, cíclicas, foram sincronizadas pela inserção de um dispositivo de liberação intravaginal contendo 0,558g de progesterona (P4), pela administração de 1 mg de benzoato de estradiol e 0,075 mg de D-Cloprostenol, ambos intramuscular (IM). Após 6 dias, foi injetado 0,075 mg de D-Cloprostenol, IM. Após 48 horas, o dispositivo contendo P4 foi removido e 0,150 mg de D-Cloprostenol foi administrado IM. Nesta ocasião, um adesivo foi inserido na base da cauda para a identificação dos estros (Boviflag Red Estrus Detector - ABS Pecplan) e observações de estro foram realizadas nos próximos 4 dias. Participaram do experimento somente novilhas identificadas em estro (D0 = dia do estro) e que ovularam (N=46). Entre D14 e D23, a área do corpo lúteo (CL; cm2), fluxo sanguíneo (%) e as concentrações plasmáticas de progesterona (P4) foram avaliadas diariamente. No D15, as novi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In cattle, 17β-estradiol (17β-E2) stimulates expression of estradiol (ER) and oxytocin receptor (OXTR) in the endometrium. The activation of OXTR leads to the induction of a complex cascade of molecular activation resulting in PGF2α synthesis. The hypothesis was that 17β-E2 treatment on day 15 (D15) after estrus modulates the gene expression of protein kinase (PKC) and phospholipase A2 (PLA2), both directly involved in the synthesis of PGF2α and calcium dependent. The aim of this study was to determine the effects of 17β-E2 on the abundance of key transcripts (PKCα, PKCβ, PLA2G4, AKR1B1, AKR1C4 and PTGS2) involved in PGF2α signaling and synthesis. Nelore heifers (N=50), don't pregnant, cyclic were synchronized by insertion an intravaginal release device containing 0.558g of progesterone (P4), and by the administration of 1 mg of estradiol benzoate and 0.075 mg de D-Cloprostenol, both intramuscularly (IM). After 6 days, 0.075 mg D-Cloprostenol was injected, IM. After 48 hours the P4 device was removed and 0.150 mg D-Cloprostenol was administered IM. On this occasion, an adhesive was inserted at the base of the tail for the identification of estrus (Boviflag Red Estrus Detector - ABS Pecplan) and estrous observation were made in the next 4 days. Participated in the experiment only the heifers identified in estrus (D0 = day of estrus) that ovulated (N=46). Between D14 and D23, corpus luteum (CL) area (cm2), blood flow (%), and progesterone (P4) plasmatic concentrations were eval... (Complete abstract click electronic access below) / Mestre

The final, definitive version of this paper has been published in Acta radiologica by SAGE Publications Ltd, All rights reserved.Final publication is available at http://acr.sagepub.com/ / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19582号 / 医博第4089号 / 新制||医||1014(附属図書館) / 32618 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 戸井 雅和, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L-arginine is one of the most versatile amino acids due to the fact that it serves as a precursor for many molecules which have important roles in bodily functions including mammalian reproduction. The current studies sought to further examine the role that L-arginine has in mammalian reproduction utilizing both in vivo and in vitro approaches. In the first study, a novel bioluminescent murine pregnancy model was developed to monitor VEGFR2 transcription activity non-invasively in the fetoplacental unit. Secondly, the effect that dietary L-arginine supplementation has during mouse gestation was examined. L-arginine supplementation increased weight gain during the latter third of gestation, total litter size, number of implantation sites, and litter birth weight. Additionally, L-arginine supplementation increased VEGFR2 transcription activity in the fetoplacental unit which may create a more favorable environment for fetal survival. Moreover, the increased number of implantation sites observed suggests an effect of L-arginine at the level of the endometrium. To this end, the effect that L-arginine has on apoptosis and cell proliferation in an established endometrial cell line was examined. The addition of L-arginine at physiological (200 micromolar) and supra-physiological (800 micromolar) concentrations increased cell proliferation , and this effect was achieved through biosynthesis of polyamines and nitric oxide. L-arginine also decreased the proportion of cells that were experiencing mitochondrial mediated apoptosis, and it was observed that this decrease in mitochondrial mediated apoptosis was concurrent with increased phosphorylation of BAD protein, which induces apoptosis when not phosphorylated. The final study examined the ability of porcine uterine epithelial (PUE) cells to synthesize L-arginine from L-citrulline. L-citrulline was able to support PUE cell proliferation in the absence of L-arginine. Additionally, ASS-1 and ASL, L-arginine synthesizing enzymes, were expressed in PUE cells and were regulated by the presence of L-arginine and L-citrulline, respectively. This data would support the hypothesis that PUE cells may be able to convert L-citrulline to L-arginine. Together, the current findings along with the plethora of relevant literature provide further evidence for the role of L-arginine in mammalian reproduction and allow for new questions to be investigated regarding this particular amino acid’s role in mammalian reproduction.

The uterine endometrium undergoes cyclical phases of cell proliferation, cell differentiation, and menstruation under the influence of the ovarian hormones, estrogen and progesterone. Since the data necessary to create a classical kinetic model of these signaling pathways is lacking, we used a Boolean network approach that includes the influences of various growth factors and the differential expression of their receptors under the influence of estrogen and progesterone. Results show a gain in endometrial tissue and loss of tissue during menstruation that mirrors what can be expected over the course of a normal menstrual cycle in women, where the endometrium typically reaches a thickness of approximately 10 mm. We utilized an existing model of the normal menstrual cycle that was used to predict hormonal changes following administration of GnRH analogues. We adapted this model to provide the hormonal and ovarian compartments that would interact with our model of the endometrial cycle.

Interest in women's reproductive variation within the subfield of Physical Anthropology known as Human Reproductive Ecology is dominated by energetic models for fecundity that disregard genetic variation as a potential cause of differences in reproduction. Further, a strong correlation between ovarian and uterine markers of fecundity is assumed, although this assumption is not supported by the available data. A polymorphism in the progesterone receptor gene, called PROGINS, shows diminished progesterone response in vitro and is associated with a number of uterine disorders in women. To elucidate the discrepancy between ovarian and uterine markers of fecundity, carriers of the PROGINS variant were compared to non-carriers with regard to endometrial thickness and menstrual cycle characteristics. Gene-environment interactions between PROGINS and life history, lifestyle factors, progesterone levels, anthropometric measures, and physical activity were also considered. The PROGINS polymorphism was found to impact both luteal phase length and menses duration, as well as to modify endometrial sensitivity to life history factors, progesterone levels, anthropometric measures, and physical activity. These results support the notion that PROGINS diminishes progesterone response, and indicate that the polymorphism also alters endometrial sensitivity to acute and chronic energetic stress. The findings of this study indicate that Human Reproductive Ecologists must consider genetically-based variation in sensitivity to energetic stress in future adaptive models of women's reproduction. / Anthropology