Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-05-02T17:48:14Z No. of bitstreams: 1 2012 - Joaquim Esquerdo Ferreira.pdf: 1044844 bytes, checksum: 50873f4b4aaa018dbcef3230dfd6b272 (MD5) / Made available in DSpace on 2017-05-02T17:48:14Z (GMT). No. of bitstreams: 1 2012 - Joaquim Esquerdo Ferreira.pdf: 1044844 bytes, checksum: 50873f4b4aaa018dbcef3230dfd6b272 (MD5) Previous issue date: 2012-03-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Although the bovine embryo transfer is a technique widely used around the world, the variability of donor response to superovulatory treatment is still an important limitation. The objective of this study was to evaluate the effect of the synchronization method of follicular wave on superovulatory response of bovine embryo donors. For this, seven Girolando cows and five Girolando heifers previously selected (depending on the reproductive history, sanitary conditions, nutritional and reproductive tract without anatomical abnormalities) were randomly assigned to three treatments according to the synchronization of follicular wave before the superovulation: i) synchronization with GnRH ii) synchronization with progestagen implant and application of estradiol benzoate iii) without synchronization (based on estrus detection - Control Group). After submitted to the treatment for synchronization of follicular wave, the donors were superovulated with eight decreasing FSH doses, applied twice daily by deep intramuscular injection (IM) with intervals of 12 hours for each application, with a total dose of 133 mg for each animal. The first insemination was conducted 12 hours after application of ovulation inducer (GnRH) and the second insemination conducted 12 hours after the first. The superovulatory response was measured with an ultrasound device (MINDRAY D2200 VET) to quantify the number of corpus luteum present in the ovaries on the day of embryo flushing and also assessed the number and quality of embryos recovered. It was observed that animal category and crossbred influenced the superovulatory response (p <0.05). The heifers showed a higher response to superovulatory treatment (100% of response in all treatments) when compared to cows had showed 85.7%, 57.1% and 57.1% of superovulatory response to estrus based group, GnRH and P4+EB, respectively. The genetic group (crossbred) also showed influence on superovulatory response (p <0.05) since the 3/8 animals responded better than 3/4animals. The treatments for follicular wave emergence synchronization did not show differences for superovulatory response (p>0.05), recovered structures and viable structures. So, we conclude that the treatments for follicular wave synchronization with GnRH or P4+BE can be used in Girolando donors with same efficiency of estrus based group. Besides, we conclude also that Girolando heifers have better superovulatory response than cows as well as 3/8 crossbred donors. / Embora a transfer?ncia de embri?es bovinos seja uma t?cnica amplamente empregada em todo o mundo, a variabilidade da resposta de doadoras ao tratamento superovulat?rio ainda ? uma importante limita??o. O objetivo deste trabalho foi avaliar o efeito do m?todo de sincroniza??o da onda folicular na resposta superovulat?ria de doadoras de embri?o Girolando. Para tanto, sete vacas e cinco novilhas, previamente selecionadas (em fun??o do hist?rico reprodutivo, condi??es sanit?ria, nutricional e trato reprodutivo sem anormalidades anat?micas) foram aleatoriamente distribu?das em tr?s tratamentos de acordo com o m?todo de sincroniza??o da onda folicular previamente ? superovula??o: i) sincroniza??o com GnRH; ii) sincroniza??o com implante de progest?geno e aplica??o de Benzoato de Estradiol; iii) sem sincroniza??o (observa??o do cio base - Grupo Controle). Depois de submetidas ao tratamento para sincroniza??o da onda folicular, as doadoras foram superovuladas com oito subdoses decrescentes de FSH, aplicadas duas vezes ao dia, por via intramuscular profunda (IM), com intervalos de 12 horas a cada aplica??o, somando 133 mg por animal. A primeira insemina??o foi realizada 12 horas ap?s a aplica??o do indutor da ovula??o (GnRH) e a segunda insemina??o realizada 12 horas ap?s a primeira. A resposta superovulat?ria foi avaliada com auxilio de um aparelho de ultrassom (MINDRAY D2200 VET) para quantificar o n?mero de corpos l?teos presentes nos ov?rios no dia da coleta assim como pelo n?mero e qualidade dos embri?es recuperados. Foi observado que a categoria animal e o grau de sangue influenciaram a resposta superovulat?ria (p<0.05). As novilhas apresentaram uma maior resposta ao tratamento superovulat?rio (100% de resposta em todos os tratamentos) quando comparadas ?s vacas que apresentaram 85,7%, 57,1% e 57,1% de resposta superovulat?ria para os grupos cio base, GnRH e P4+BE, respectivamente. O grupo gen?tico (grau de sangue) tamb?m mostrou influ?ncia na resposta superovulat?ria (p<0.05) sendo que os animais 3/8 responderam melhor ao tratamento superovulat?rio do que os animais 3/4. Os tratamentos de sincroniza??o da emerg?ncia da onda folicular n?o apresentaram diferen?as na resposta superovulat?ria (p>0.05), nas estruturas recuperadas e nas estruturas vi?veis. Desta forma, conclui-se que os tratamentos de sincroniza??o da onda folicular com GnRH ou com P4+BE podem ser utilizados em doadoras Girolando com a mesma efici?ncia do cio base. Al?m disso, conclui-se tamb?m que novilhas Girolando respondem melhor ao tratamento superovulat?rio do que vacas assim como as doadoras de grau de sangue 3/8 quando comparadas as 3/4.

Background Ovarian follicular fluid serves as the microenvironment for a maturing oocyte prior to ovulation. Previous studies have shown that gilts fed a high fibre (HF) diet before ovulation have improved fertility compared to gilts fed a control (C) diet, including a higher proportion of metaphase II oocytes following in vitro maturation (IVM). Hypothesis The molecular composition of porcine follicular fluid (pFF) was altered by the diet and that these alterations conferred the fertility benefits. Aims The aim of this study was to compare the protein composition of pFF from pigs fed a control diet with pFF of pigs fed a high fibre diet, to identify whether a high fibre diet fed to pigs during their oestrous cycle altered the composition of pFF. Additionally, the pFF of fertile animals was compared with the pFF of non-fertile animals to identify whether pFF composition was associated with fertility; fertile animals produced an embryo following in vitro fertilisation (IVF). Differences in the molecular composition were to be used to ascertain the potential underlying mechanism(s) involved in dietary induced improvements to oocyte maturation. Results The protein composition of pooled pFF from 12 HF-pigs and 12 C-pigs was compared by liquid chromatography tandem mass spectrometry (LC-MS/MS). Additionally, within each dietary group, the composition of pooled pFF from pigs whose oocytes produced blastocysts following in vitro fertilisation (C-Bl and HF-Bl) was compared with pFF from pigs whose oocytes did not produce blastocysts (C-No and HF-No respectively; n=6 per group). These proteomic analyses identified differentially expressed proteins, associated with several canonical pathways including acute phase response signalling, complement system and LXR/RXR activation, as determined by Ingenuity Pathway Analysis. Quantitative western blots revealed the differential expression of candidates associated with these canonical pathways. Plasminogen expression was lower (P≤0.05) in pFF of HF-pigs compared to pFF of C-pigs. In pFF from C-Bl gilts, apolipoprotein A4 (P≤0.01) and apolipoprotein M (P≤0.05) expression were higher compared to pFF from C-No gilts. Plasmin expression was lower (P≤0.05) in pFF from HF-Bl gilts compared to pFF from C-Bl gilts. Due to the interest in the differentially expressed apolipoproteins (involved in cholesterol and lipid efflux), a targeted metabolomic analysis was carried out to measure the concentration of nine fatty acids (FAs) in pFF of individual pigs in C-No, C-Bl, HF-No, HF-Bl groups (n=6 per group); adrenic, arachadonic, arachidic, dihomo- γ-linolenic, docosapentaenoic, erucic, linoleic, palmitoleic and oleic acids were measured by LC-MS/MS. The analysis revealed the lower concentration of linoleic acid (LA, p≤0.05) and higher concentration of erucic acid (P≤0.05) in HF-pFF compared to C-pFF. Following the results of the targeted metabolomic analysis, cumulus-oocytecomplexes (COCs) were matured in TCM 199 medium supplemented with 0 (No-LA), 50, 100 or 200 μM LA for 44 hours (n = 320 per treatment). COC diameters were measured and the COCs were categorised into "full", "partial" or "no" expansion. COCs were denuded, fixed and stained to determine their stage of maturation. IVM with 200 μM LA resulted in the reduced diameter of COCs (p≤0.01), fewer COCs with full cumulus expansion (p≤0.05) and fewer metaphase II oocytes (p≤0.05). Discussion Plasminogen is the precursor to plasmin, a proteolytic enzyme involved in weakening the follicular wall prior to ovulation. The lower expression of plasminogen and plasmin in pFF of high fibre pigs implies a delay in the accumulation of the inflammatory proteins required for ovulation. The delay in ovulation can result in the lengthening of the oocyte maturation process, leading to more mature oocytes, as observed in the previous studies. A disruption in the expression of apolipoproteins may also occur in high fibre-fed pigs. The increase in apolipoproteins associated with blastocyst development was only observed with pFF of control pigs but not high fibre pigs. An alteration in lipid homeostasis in the high fibre pigs could potentially affect oocyte energy consumption. LA concentration was also lower in pFF of high fibre pigs. LA is an essential fatty acid, indicating that the difference in concentration is directly from the diet. The lower levels of LA can potentially be beneficial to oocyte maturation, which is substantiated by the negative effects of a high LA concentration on IVM of abattoir derived oocytes.

Les cellules T folliculaires helper (Tfh) jouent un rôle central dans la mise en place de réponses humorales efficaces. En effet, les Tfh participent à la sélection des lymphocytes B permettant le développement de lymphocytes B mémoires et d’anticorps de haute affinité. Les Tfh représentent ainsi une cible prometteuse pour la mise en place de nouvelles stratégies thérapeutiques, notamment pour augmenter l’efficacité de la vaccination. Ainsi, il apparaît crucial de mieux comprendre les étapes menant à leur développement, en particulier chez l’Homme. L’initiation de la polarisation Tfh se déroule dans les organes lymphoïdes secondaires et met en jeu les cellules myéloïdes mononucléaires (MMC). Les MMC présentes dans les organes lymphoïdes comprennent les macrophages résidents et trois sous populations de DC résidentes : les cDC1 (CD141+), les cDC2 (CD1c+) et les pDC. Nous nous sommes plus particulièrement intéressés au rôle respectif des sous populations de MMC humaines dans l’induction de la polarisation Tfh. Ainsi, les travaux effectués au cours de ma thèse avaient pour objectifs dans un premier temps d’analyser la capacité des différentes populations de MMC à induire la polarisation Tfh, afin de mettre en évidence de potentielles spécialisations fonctionnelles. Dans un second temps, nous nous sommes concentrés sur les mécanismes moléculaires impliqués dans l’induction par les MMC de la polarisation Tfh. Nous avons montré une spécialisation fonctionnelle des cDC2 et des macrophages des amygdales pour la polarisation Tfh. Toutefois, des différences ont été observées entre les cDC2 et macrophages, puisque les macrophages induisent la sécrétion par les lymphocytes T d’une grande quantité de CXCL13 par rapport au cDC2, qui sont plus efficaces pour induire la production d’IL21. Nous avons pu également montrer que les cDC2 et macrophages sécrétaient des cytokines précédemment identifiées comme ayant un rôle dans l’induction des Tfh telles que IL12p70, ActivinA et TGFβ. Afin de confirmer le rôle de ces cytokines dans la polarisation induite par les MMC d’amygdales, nous avons utilisé des anticorps bloquants dans nos expériences de polarisation T helper. Ainsi, nous avons confirmé le rôle de l’IL12p70, de l’Activin A et du TGFβ dans l’induction des Tfh humains. Nos résultats suggèrent également un rôle de l’Activin A et de TGFβ dans l’induction de la sécrétion de CXCL13, alors que l’IL12p70 serait impliqué dans l’induction de la sécrétion d’IL21. Nos résultats suggèrent aussi l’existence de deux sous populations de Tfh caractérisées soit par l’expression d’IL21 soit par l’expression de CXCL13. Les travaux réalisés au cours de ma thèse enrichissent ainsi les connaissances sur la spécialisation fonctionnelle des sous populations de DC et des macrophages humains, et apportent de nouveaux éléments pour la compréhension de la différenciation des Tfh humains. / T follicular helper cells (Tfh) play a key role in the establishment of efficient humoral responses. Indeed, Tfh are involved in B lymphocyte selection allowing the development of high affinity memory B cells and antibodies. Tfh are promising targets for new therapeutic strategies, especially to increase the effectiveness of vaccination. Thus, it is crucial to better understand the stages leading to their development, especially in human. Initiation of Tfh polarisation occurs in secondary lymphoid organs and involves mononuclear myeloid cells (MMC). MMC from secondary lymphoid organs include resident macrophages and three subsets of resident Dendritic Cells (DC): cDC1 (CD141+), cDC2 (CD1c+) and pDC. We were particularly interested in human MMC subsets respective roles in the induction of Tfh polarisation. Thus, the work carried out during my thesis aimed first at analysing the ability of different populations of MMC to induce Tfh polarisation, in order to highlight potential functional specialisations. In a second step, we focused on the molecular mechanisms involved in Tfh polarisation by MMC. We have shown a functional specialisation of cDC2 and tonsillar macrophages for Tfh polarisation. However, differences have been observed between cDC2 and macrophages, since macrophages induce secretion by T cells of a large amount of CXCL13 compared to cDC2, which are more effective in inducing IL21 production. We have also been able to show that cDC2 and macrophages secreted cytokines previously shown to play a role in Tfh induction such as IL12p70, ActivinA and TGFβ. In order to confirm the role of these cytokines in Tfh polarisation induced by tonsil MMCs, we used blocking antibodies in our T helper polarisation experiments. Thereby, we confirmed the role of IL12p70, Activin A and TGFβ in the induction of human Tfh. Our results also suggest a role for Activin A and TGFβ in inducing secretion of CXCL13, whereas IL12p70 would be involved in the induction of IL21 secretion. Besides, our results suggest the existence of two Tfh subsets characterised by expression of either IL21 or CXCL13. The work performed during my thesis broadens the knowledge on the functional specialisation of human DC subsets and macrophages, and provides new insight into the differentiation of human Tfh.

<p>Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. </p><p>In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis.</p><p>In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.</p>

Le tropisme cellulaire des prions infectieux diffère selon lespèce animale, celui-ci est corrélé à la souche infectieuse et à des facteurs spécifiques de lhôte. Par exemple, certains prions infectieux sont lymphotropiques, notamment en cas de scrapie chez les moutons et de variant de la maladie de Creutzfeldt-Jakob (vMCJ) chez lhomme. Par opposition, certains prions se caractérisent par un neurotropisme comme observé chez des patients Creutzfeldt-Jakob atteints de la forme sporadique ou chez des bovins atteints dencéphalopathies spongiformes bovines (ESB). Lhypothèse de notre travail repose sur les observations suivantes : dans le cas du variant de la maladie de Creutzfeldt-Jakob et des encéphalopathies spongiformes bovines, lagent responsable est identique, la voie dinoculation et les lésions neurologiques le sont également, seul le tropisme de cette souche pour les organes lymphoïdes diffère. En effet, les amygdales, la rate et lappendice sont infectieux chez lhomme. Par contre, linfectiosité est surtout confinée au niveau du système nerveux chez le bovin. Lors dune inoculation expérimentale par voie orale de lagent responsable de lESB chez les bovins, les plaques de Peyer iléales sont les seuls tissus lymphoïdes infectieux. Notre hypothèse de travail est que des propriétés de lhôte interviennent dans le tropisme de lagent infectieux. Deux axes de recherche ont été envisagés afin de vérifier cette hypothèse :  Lanalyse de la distribution des fibres nerveuses au sein des tissus lymphoïdes associés aux muqueuses (MALT) des espèces bovine et humaine  Létude de lexpression de PrPc et de ses isoformes au sein des tissus lymphoïdes et nerveux des espèces bovine et humaine. Pour atteindre au mieux nos objectifs, il nous manquait un outil essentiel permettant la caractérisation spécifique des FDC bovines. En effet, aucun marqueur spécifique de ces cellules nétait commercialisé. Nous avons donc produit, en collaboration avec le Centre dEconomie Rural de Marloie, un anticorps monoclonal spécifiquement dirigé contre les cellules folliculaires dendritiques (FDC) bovines. Cet anticorps nous a permis détudier la distribution des FDC au sein des organes lymphoïdes bovins. Une attention particulière a été portée aux FDC isolées à partir des plaques de Peyer jéjunales (PPJ) et iléales (PPI). Lapparente différence dinfectivité de ces tissus lymphoïdes chez des bovins atteints expérimentalement dESB nous a conduit à comparer les capacités fonctionnelles des FDC isolées à partir de PPJ et de PPI. Ces observations sont décrites et discutées dans le chapitre 1. Dans le chapitre 2, nous avons établi une cartographie des fibres nerveuses au sein des amygdales, des plaques de Peyer iléales et jéjunales bovines de plusieurs catégories dâge et ensuite comparé ce pattern dinnervation à celui des amygdales humaines; ceci permettra de pister les voies potentielles de neuro-invasion. Une attention particulière a été portée à linterface cellules folliculaires dendritiques fibres nerveuses. En effet, les FDC matures jouent un rôle prépondérant dans la pathogenèse des maladies à prion puisquen leur absence, une infection périphérique na pas lieu. De plus, la proximité entre fibres nerveuses et FDC est un paramètre intervenant dans la neuro-invasion; nous avons dès lors aussi analysé les contacts entre les FDC et les éléments nerveux. Lexpression de la PrPc est une condition sine qua non pour la formation de PrPres. Cette protéine cellulaire sert probablement de récepteur pour son homologue infectieux mais sert surtout de substrat pour lamplification de PrPres ; toute modification au niveau de sa synthèse pourrait entraîner un changement de la cinétique dinfection et pourrait expliquer lapparente absence dinfectivité constatée au niveau du système immunitaire chez les bovins. Lexpression tissulaire et cellulaire spécifique disoformes de la PrPc représente un facteur de lhôte potentiellement capable dinfluencer le tropisme cellulaire de lagent infectieux chez lhumain et le bovin. Cette expression a été étudiée dans les systèmes MALT bovins et humains. Pour affiner notre étude, nous avons analysé, par des techniques de western-blotting, le glycopattern de la PrPc ainsi que lexpression de ses formes tronquées dans les tissus lymphoïdes humains et bovins mais également dans des populations cellulaires spécifiques, les lymphocytes et les FDC. Afin de vérifier si les isoformes de PrPc sont spécifiques aux tissus lymphoïdes, nous avons effectué une étude comparative du pattern de glycosylation et du ratio des formes clivées de PrPc, exprimés au sein de différentes régions du système nerveux central bovin et humain. Les résultats de ces travaux sont repris dans le chapitre 3.

Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis. In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.

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.

Transrectal ovarian ultrasonography and hormone measurements were used to study ovarian antral follicular dynamics and development of luteal structures during the middle portion of the breeding season in non-prolific cross-bred Western white-faced ewes and prolific Finn sheep. Studies were also done on ovarian activity in Western white-faced ewes during the transition to seasonal anoestrus and at the onset of the breeding season. Lastly, two experiments were carried out to examine ovulatory responses and subsequent luteal function in Western white-faced ewes treated with luteolysin (PgF 2á) and progestogen (medroxyprogesterone acetate-MAP) during the luteal phase of the oestrous cycle and after ovulation induction with gonadotrophin-releasing hormone (GnRH) in mid-anoestrus. The results of the present experiments showed that the growth of ovine antral follicles reaching ovulatory sizes of >=5 mm in diameter occurred in a wave-like pattern throughout the oestrous cycle in both breeds of sheep under study. There were typically 3 or 4 waves of follicle production throughout the 17-day interovulatory period. Ovarian follicular emergence, or beginning of growth from the pool of 3-mm follicles, appeared to be primarily controlled by changes in circulating concentrations of follicle-stimulating hormone (FSH). In cyclic ewes, the largest ovarian follicles acquired the ability to secrete oestradiol from the day of emergence and a peak of oestradiol secretion occurred about the time they reached their maximum diameter. The high ovulation rate in prolific Finn sheep appeared to be achieved mainly by the ovulation of follicles emerging in the last two waves of the interovulatory interval. Interestingly, prolific Finn ewes produced more but smaller corpora lutea (CL) and had lower serum concentrations of progesterone during the luteal phase of the oestrous cycle as compared to non-prolific Western white-faced ewes. During the transition into seasonal anoestrus in Western white-faced ewes, FSH secretion resembled that during the breeding season but the pattern of emergence of sequential follicular waves was dissociated from FSH and oestradiol secretion. Prior to the first ovulation of the breeding season, there was a distinct elevation in circulating concentrations of progesterone produced by luteinized unovulated follicles and/or interstitial tissue of unknown origin. This increase in serum levels of progesterone, heralding the resumption of ovulatory cycles, did not alter the rhythmic pattern FSH secretion or follicular wave emergence. Treatment of non-prolific Western white-faced ewes with PgF2á and MAP applied late in the oestrous cycle changed follicular dynamics and increased ovulation rate to resemble that in prolific Finn sheep. Effects of MAP on the recruitment and growth of ovulatory follicles in Western white-faced ewes did not have a clear gonadotrophic dependancy, suggesting a possible local regulation of ovarian activity by progestins in ewes. Following the induction of ovulation with GnRH in anoestrous Western white-faced ewes, an array of ovarian responses were detected with ultrasonography, including failure of ovulation of large antral follicles, normal (fall-lifespan) and short-lived CL post-ovulation, and luteinized cystic-like follicles. The normal luteinization of ovulated follicles appeared to be related to the amplitude of episodic elevations in daily serum FSH concentrations before induction of ovulation and characteristics of the preovulatory LH surge.

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.

Background: Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods: We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results: High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers. Conclusions: Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.