No description available.

No description available.

Prostaglandin (PG) F₂α, is luteolytic in ewes, but the exact mechanism for this effect is not clear. 6α-Methyl-17α hydroxyprogesterone acetate (MPA) reduces uterine secretion of PGF₂α, but it does not interfere with luteolysis. The mechanism by which MPA suppresses PGF₂α and permits luteal regression remains to be determined. Three experiments were conducted to determine whether MPA reduces PGF₂α in uteroovarian blood, causes the release of PGF₂α before d 14 of the estrous cycle, or increases sensitivity of corpora lutea (CL) to exogenous PGF₂α. In Exp. 1 and 2, blank (control) or MPA-impregnated pessaries were inserted six days after estrus (i.e., d 6). Jugular, vena caval, and(or) uteroovarian blood samples were collected frequently (i.e., at .5 h intervals during twice daily collection periods of 2 h each) before and after luteolysis. The MPA reduced (P < .05) jugular, vena caval, and uteroovarian concentrations of PGF₂α, but MPA did not affect the interval from estrus to the first day of luteolysis. Average PGF₂α concentrations in uteroovarian and vena caval blood differed (P < .05) but the profiles did not. In Exp. 3, blank (control), MPA-impregnated, or no (untreated) pessaries were inserted on d 7. A minimal luteolytic dose of PGF₂α (Lutalyse ®; 4 mg/58 kg of body weight) was administered on d 8 or 12, blood samples were collected and the CL were collected 48 h after PGF₂α treatment. The MPA increased the sensitivity of CL to exogenous PGF₂α. Progesterone and CL weights were less (P < .05) in MPA-treated ewes than in control and untreated ewes given PGF₂α on d-8. The MPA-treated ewes given PGF₂α on d-12 had smaller CL than did ewes in the other two groups, but MPA did not affect progesterone concentrations. In summary, MPA reduces the ability of the uterus to secrete PGF 7a and increases the sensitivity of CL to a minimal luteolytic dose of PGF₂α. This information should improve our ability to control the estrous cycle ewes. / Master of Science

The corpus luteum (CL) is a transient endocrine structure formed from the remnants of an ovulated follicle with the primary purpose of producing progesterone (P4), a hormone vital for the establishment and maintenance of pregnancy. The precise regulation of CL function is essential for normal reproductive cycles and maintenance of early pregnancy. In mammals, the pituitary hormones prolactin (PRL) and luteinizing hormone (LH) function as luteotrophic factors during pregnancy, and these two hormones form a functional luteotrophic complex to control CL function in rodents. The mechanistic underpinnings of the luteotrophic actions of PRL and LH, as well as the interplay between the two hormones are poorly understood, and has been the focus of the current investigation. There are several limitations involved in studying luteal function under cell culture conditions. Hence, in vivo animal models employing dopaminergic receptor agonist, 2-Bromo-α-Ergocryptine Mesylate (CB-154; inhibits pituitary PRL secretion) and GnRH receptor antagonist, cetrorelix (CET; inhibits pituitary LH secretion) have been standardized for purposes of examining the roles of PRL and LH in the regulation of CL structure and function in rats. Administration of CB-154 or CET to pregnant rats caused inhibition of CL function and concomitant loss of conceptuses. The CB-154 treatment induced loss of implants was determined to be the result of inhibition of luteal function, rather than the non-specific effects of CB-154 or requirement of PRL for uterine maintenance of implants. To understand how PRL and LH regulate luteal function, targets of PRL and LH in the rat CL needs to be established; however, this has not been well defined by previous studies. The present study observed that CB-154 induced inhibition of luteal function was gradual in its onset; hence, transcriptional changes of genes involved in steroid genesis were examined. mRNA expression of genes involved in P4 production were found to be down regulated, while 20α-hydroxysteroid dehydrogenate (20α-HSD), a P4 catabolizing enzyme was unregulated by CB-154 treatment. CET treatment also had a similar effect on mRNA expression of steroidogenic genes. Interestingly, mRNA expression of the steroidogenic acute regulatory protein (StAR), a key regulator of steroid genesis was not regulated by CB-154 or CET treatment. The luteolytic factor PGF2α also inhibited CL function in pregnant rats but did not down regulate mRNA expression of StAR. However, examination of phospho-StAR (Ser-195), the activated form of StAR, during CET and PGF2α-induced luteolysis suggested that regulation of StAR in the CL of pregnant rats might primarily be at the level of phosphorylation. PRL has been implicated in maintaining luteal expression of LH/choriogonadotrophin receptor (LH/CGR), the cognate receptor for LH. Hence, the luteotrophic actions of PRL may be indirect, by way of regulating LH signalling. Hence, the importance of the LH/CGR pathway and its regulation were examined. LH/CGR mRNA expression was found to correlate with CL function, with CET and CB-154 treatments resulting in down regulation of LH/CGR mRNA expression. Further, CB-154 treatment down regulated LH/CGR pre-mRNA levels, suggesting a role for PRL in the regulation of LH/CGR transcription. mRNA expression of LRH-1, a constitutively active transcription factor previously reported to be important in CL function was down regulated by both CB-154 and CET treatments and hence correlated with LH/CGR mRNA expression. Further, luciferase assays in HeLA cells transiently expressing LRH-1 suggests its involvement in activating the LH/CGR promoter. Estrogen receptor (ER)-α and ER-β also appear to correlate with LH/CGR expression and may play a role along with LRH-1 in the regulation of LH/CGR mRNA expression in the CL of pregnant rats. To examine mechanisms by which PRL may regulate its downstream targets, pathways employed by PRL in the CL of pregnant rats were analysed. The Akt pathway including downstream targets were down regulated by CB-154 treatment. The pathway was found to be regulated at the level of Akt1 mRNA expression. Hence, actions of PRL may regulate the survival of CL. This study has also made observations of LH playing a similar role in survival of the CL. The results of these studies taken together, shed light on the regulation of CL structure and function by PRL and LH, and provide molecular evidence for the two hormones having similar downstream targets and functioning as a luteotrophic complex in pregnant rats, which could only mean a robust interaction between the signalling pathways employed by the two hormones.

Iberische und Eurasische Luchse zeigen einen saisonalen Monoöstrus. Nach der Ovulation findet man frisch gebildete (freshCL) und physiologisch persistierende Gelbkörper (corpora lutea, perCL). Funktionelle perCL verhindern eine Ovulation außerhalb der Zuchtsaison durch konstant erhöhte Progesteron-(P4)-Plasmawerte. Hauskatzen zeigen einen saisonalen Polyöstrus. Nach der Ovulation werden CL gebildet, deren Lebensspanne in Abhängigkeit von einer Trächtigkeit durch unterschiedliche P4-Plasmaprofile charakterisiert ist. Ziel der Dissertation war es, die Synthese und Rezeption von Steroiden und Prostaglandinen (PG) in CL von Feliden zu untersuchen, um potentiell luteotrophe und luteolytische Faktoren zu identifizieren. Während der Gelbkörperphase trächtiger und nicht-trächtiger Katzen weisen CL gleicher Histomorphologie, unabhängig vom Vorhandensein einer Trächtigkeit, ähnliche steroidogene Kapazitäten auf. Die Abnahme der CL-Funktion spiegelt sich im graduellen Verlust der Steroidbiogenese wider. Bei Luchsen ist die steroidogene Kapazität der perCL im Proöstrus herabgesetzt, aber im Metöstrus wieder verstärkt. Die steroidogene Kapazität ist demnach mit verschiedenen CL-Stadien und dem Reproduktionszyklus assoziiert. Die Synthese und Rezeption von PGE2 erfolgen bei Katze und Luchs unabhängig vom CL-Stadium und dem Reproduktionszyklus. Hohe Werte an luteotrophem PGE2 in perCL könnten für die funktionelle und strukturelle CL-Persistenz beim Luchs verantwortlich sein. Der feline CL ist zur Bindung von luteolytischem PGF2alpha fähig, jedoch ist die Kapazität zur Synthese begrenzt. Feliden weisen keine PGF2alpha-assoziierte luteale Regression in Abwesenheit einer Trächtigkeit auf. Allerdings wurden Höchstwerte an PGF2alpha in der Plazenta, wie auch im Plasma (PGFM), im letzten Trächtigkeitstrimester der Katze gemessen. Folglich ist die feline Plazenta zur Synthese von luteolytischem PGF2alpha fähig, welches die CL-Regression und Geburt am Ende der Trächtigkeit ermöglicht. / Iberian and Eurasian lynxes exhibit a seasonal monooestrus. After ovulation, freshly formed (freshCL) coexist with physiologically persistent luteal bodies (corpora lutea, perCL). Functional perCL prevent ovulation outside the breeding season through constantly elevated plasma progesterone (P4) levels. Domestic cats show a seasonal polyoestrus. After ovulation, CL are built with lifespans being characterised by different plasma P4 profiles dependent on pregnancy. The aim of the dissertation was to characterise the synthesis and reception of steroids and prostaglandins (PGs) in CL of felids to identify potential luteotrophic and luteolytic factors. During the luteal lifespan of pregnant and non-pregnant cats, CL of equal histomorphology exhibit similar steroidogenic capacities, irrespectively of an ongoing pregnancy. The functional demise of CL mirrors the gradual loss of steroid biogenesis. In lynxes, the steroidogenic capacity of perCL is limited at prooestrus, but is enhanced again during metoestrus. The steroidogenic capacity is thus associated with different CL stages and the reproductive cycle. The synthesis and reception of PGE2 in cat and lynx is independent on the CL stage and reproductive cycle. High levels of luteotrophic PGE2 in perCL might be responsible for the functional and structural CL persistence in lynxes. The feline CL is capable of binding luteolytic PGF2alpha; however, the capacity to synthesise PGF2alpha is limited. Felids show no PGF2alpha-associated luteal regression in the absence of pregnancy. Interestingly, peak levels of PGF2alpha in the placenta, as well as in plasma (PGFM), were measured during the last trimester of pregnancy in the cat. Therefore, the feline placenta is capable of synthesising luteolytic PGF2alpha, which enables CL regression and parturition at the end of pregnancy.

Corpus luteum (CL), a transient endocrine structure formed from the ruptured ovarian follicle after ovulation, secretes progesterone (P4) that is essential for establishment and maintenance of pregnancy in mammals. The biosynthesis and secretion of P4 from CL depends, in general, on trophic hormones of the anterior pituitary gland and on hormones or factors originating from ovary, uterus, embryo and placenta. The structure and function of CL tissue is regulated by intricate interplay between two types of factors, namely, the luteotrophic factors, which stimulate CL growth and function, i.e., P4 secretion, and the luteolytic factors, which inhibit CL function and lead to luteal regression. In monoovulatory species such as higher primates and bovines, a striking diversity in the regulation of CL function exists not only between species, but also within the species during different stages of the luteal phase. In higher primates, unlike other species, one of the important characteristics of CL regulation is that, during non-fertile cycle, circulating LH appears to be the sole trophic factor responsible for maintenance of its function, and during fertile cycle, chorionic gonadotropin (CG), an LH analogue, originating from placenta maintains CL function. In higher primates, the role/involvement of luteolytic factors during luteolysis remains elusive. On the other hand, in the bovine species, the role/involvement of luteolytic factor, prostaglandin (PG) F2α during luteolysis is well established. It should be pointed out that in both the species, the mechanism of luteolysis is still poorly understood and the work presented in this thesis attempts to address these lacunae. Further, in bovines, studies have been carried out to examine potential trophic factor(s) responsible for the maintenance of CL function. Chapter I provides an extensive review of literature on CL structure and function with emphasis on factors that influence its growth, development, function and demise in primates and bovines. In Chapter II, employing bonnet monkey (Macaca radiata) as the representative animal model for higher primates, various studies have been conducted to examine the role of molecular modulators involved in regulation of CL function, particularly during spontaneous luteolysis. Although, it is well established that LH is essential for the maintenance of CL function in higher primates, the mechanism(s) responsible for the decline in serum P4 levels at the end of non-fertile cycles, without a concomitant change in circulating LH milieu, remains to be addressed. Several experiments have been conducted to examine the component(s) of luteotrophic (LH/CG) signaling that is/are modulated during luteolysis in the bonnet monkey CL. To understand the relative lack of responsiveness of CL to the circulating LH during the late luteal phase, LH/CG receptor (R) dynamics (expression of LH/CGR and its various transcript variants) was examined throughout the luteal phase and during different functional states of the monkey CL. The results indicated presence of LH/CGR mRNA, its transcript variants and functional LH/CGR protein in the monkey CL on day 1 of menses. Moreover, the functionality of receptors was tested by confirming the biological response of the CL to bolus administration of exogenous LH preparations, which eventually suggested factor(s) downstream of LH/CGR activation to account for the decline in CL function observed during non-fertile cycle. Studies have been conducted to identify molecular modulators that would selectively exploit intraluteal processes to regulate trophic signaling pathways that are critical to the control of luteal function. Immunoblot and qPCR analyses were carried out to examine presence and activation of Src family of kinases (SFKs) and cAMP-phosphodiesterases (PDEs) during various functional states of CL. The results revealed an increased activation of Src (phosphorylated at Tyr 416) during spontaneous and PGF2α/CET-induced luteolysis that may participate in the regulation of cAMP levels in part by increasing the cAMP-PDE activity observed during spontaneous luteolysis. This observation raised the question on the possible mechanism by which CG, an analog of pituitary LH, rescues CL function during early pregnancy. Thus, subsequent experiments involving LH/hCG administration in CET-treated animals as well as simulated early pregnancy animal model were conducted and the results revealed that, a bolus of LH/hCG decreased Src activation and cAMP-PDE activity accompanying a momentous increase in cAMP levels in both these models that further led to a concomitant increase in P4 secretion. Although the mechanisms of action of LH/CG involve modulation of a number of signaling pathways in the CL, by far, the results from various experiments suggested that it leads to activation of Src kinase and cAMP-PDE, thus causing inhibition of various elements of the primary signaling cascade- AC/cAMP/PKA/CREB during spontaneous luteolysis. One of the consequences of activation of Src kinase and cAMP-PDE was the regulation of expression of genes associated with steroidogenesis and it was observed that expression of SR-B1, a membrane receptor associated with trafficking of HDL-CE into the luteal cells, was lower in the regressed CL. The results taken together suggest that the decrease in responsiveness of CL to LH milieu during non-fertile cycles is not associated with changes in LH/CGR dynamics, but, is instead coupled to the activation of Src kinase and cAMP-PDE, inhibition of molecules downstream of LH signaling, and a decrease in the SR-B1 expression that regulates cholesterol economy of the luteal cell, and in turn, P4 secretion. The control of primate CL function appears to be dominated by the luteotrophic factors (LH/CG) over the luteolytic factors, since the process of luteal regression was overcome by administration of LH/CG. Further, in the primate CL, the molecular modulators of LH/CG signaling (Src kinase and PDE) are maintained in the repressed state by the luteotrophic factor LH/CG for maximum steroidogenic function. In contrast, in non-primate species, without invoking a role for the luteotrophic factor, essentially the synthesis and secretion of luteolytic factor, PGF2α, from the uterus is kept in check during pregnancy by the trophoblast derived IFN- and thus allowing CL to continue to function that is essential for maintenance of pregnancy. In the bovine species, the mechanism of PGF2α-induced luteolysis that involves a change in expression of genes associated with various processes of cellular function is poorly understood. Experiments were conducted utilizing buffalo cows (Bubalus bubalis) as a model system, to determine temporal changes in the global gene expression profile of the CL in response to PGF2α treatment. For this purpose, CL tissues were collected on day 11 of estrous cycle without treatment (designated as 0 h) and at 3, 6 and 18 h post PGF2α treatment for various analyses. Global changes in gene expression pattern in the CL were investigated employing Affymetrix GeneChip bovine genome array and the results are presented in Chapter III. The hybridization intensity values obtained by microarray analysis were subjected to R/Bioconductor tool. Following the application of highly stringent statistical filters to eliminate false positives, a set of differentially expressed genes were identified. The differentially expressed genes were further classified based on a fold change cut-off filter of ≥2, and the analysis revealed 127 genes to be differentially expressed within 3 h of PGF2α administration, of these 64 and 63 genes were up-regulated and down-regulated, respectively. Analysis of microarray data at 6 h post PGF2α administration revealed 774 genes to be differentially expressed, of which 544 genes were up-regulated, while 230 genes were down-regulated. The microarray analysis performed on CL tissues collected at 18 h post PGF2α administration showed that out of the total 939 differentially expressed genes, 571 genes were up-regulated, while 368 genes were down-regulated. Analysis of the ontology report for the biological processes category showed that initially in response to PGF2α administration, genes regulating steroidogenesis, cell survival and transcription were differentially regulated in the CL, but at later time points, differential expression of genes involved in apoptosis, PGF2α metabolism, tissue remodeling and angiogenesis was observed. Further, involvement of molecules downstream of LH/IGF-1 activation was investigated and the results obtained indicated that PGF2α interfered with the LH/IGF-1 signaling since the expression of LH/CGR, GHR and pAkt were down-regulated following PGF2αadministration. Furthermore, the functional luteolysis observed post PGF2αadministration appeared to be due to an interruption in cholesterol trafficking to inner mitochondrial membrane, since StAR expression was inhibited. The results obtained also demonstrated that the expression of AGTR1, VEGFR2 and R3 were down-regulated following PGF 2α administration. Further, the data obtained also suggested modulation of expression of pro- and anti-angiogenic factors upon PGF2α-treatment indicative of an involvement of other autocrine or paracrine factor(s) in the regression of bovine CL. This was an interesting finding as it suggests a novel and potential functional relationship between angiogenesis and the luteolytic response of CL to PGF2α administration. In bovines, despite extensive research being carried out to examine factors involved in the regulation of development and function of the CL, the trophic factor(s) required for maintenance of CL function, especially, P4 biosynthesis and secretion are not well characterized. It was hypothesized that the function of the CL during its finite lifespan must be responsive to LH as well as to various growth factors. Thus, experiments were conducted to examine the effects of increased LH and GH/IGF-I on the maintenance of CL function during mid luteal phase and post PGF2α administration and the results of these studies are presented in Chapter IV. To elucidate the role of LH as a trophic factor in the regulation of CL function, effects of increased endogenous LH through GnRH administration and exogenous hCG injections were examined. The results indicated an absence of noticeable effect of various hCG/GnRH treatments on circulating P4 levels. On the other hand, administration of GH resulted in increased serum IGF-1 and P4 levels. It was further observed that the administration of a combination of hCG and GH increased serum P4 levels better than treatment with GH alone. Further experiments were carried out to examine the complex reciprocal relationship between LH/GH and PGF2α on expression of genes involved in the regulation of luteal structure and function. In buffalo cows, administration of exogenous hCG and/or GH following inhibition of CL function by PGF2α administration did not prevent the PGF2α-induced decline in serum P4 levels, but PGF2-mediated decrease in expression of LH/CGR and GHR genes was prevented upon GH administration. However, the decrease in StAR expression was not restored by hCG and GH treatments, thereby indicating that PGF2 action was not prevented by hCG and/or GH treatments. Taken together, the results of studies carried out in buffalo cows employing various experimental model systems suggest essential role for LH and GH/IGF-1, however, these factors were unable to reverse PGF2α-induced luteolysis. Further, our crucial findings of the effects of increased endogenous LH and IGF-1, in addition to their relationship with luteolytic agents such as PGF2α will open new avenues for studying the mechanisms involved in the regulation of structural and functional properties of the buffalo CL. It is well known that a large number of buffalo cows experience loss of pregnancy and infertility due to inadequate luteal function and/or failure of timely insemination. Results from our studies suggest that the incorporation of PGF2α and hCG or GH/IGF-1 protocols in buffalo cows to be beneficial for improving their breeding efficiency as these protocols are likely to increase luteal function with defined luteolysis. To summarize, the results of studies described in the present thesis provide new insights into the physiological and molecular mechanisms involved in the regulation of CL function during luteolysis in the monoovulatory species. The results suggest that the maintenance of CL function appears to be dependent on both luteotrophic and luteolytic factors, but with a varied degree of dominance between the two species examined. Further, the results indicate that while the luteotrophic factors (LH/CG) dominate the CL regulation in primates, the regulation of CL function in bovines is dominated by the actions of luteolytic factor (PGF2α). In monoovulatory species, the luteotrophic and luteolytic factors following binding to their specific plasma membrane receptors on the luteal cells, would counteract each other and modulate activation of various downstream signaling molecules subsequently leading to regulation of gene expression and P4 secretion (Fig.5.1). LH: luteinizing hormone; CG: chorionic gonadotropin; LH/CGR: LH/CG receptor; Gαs: stimulatory α-subunit of trimeric G-protein; AC: adenylate cyclase; cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; p: phosphorylation: CREB: cAMP response element binding protein; SR-B1: scavenger receptor class B, type I; SF-1: steroidogenic factor 1; LRH-1: liver receptor homologue 1; P4; progesterone; Src; sarcoma; PDE4D: cAMP phosphodiesterase 4D; StAR, steroidogenic acute regulatory protein; PGF2α: prostaglandin F2α; PTGFR: PGF2α receptor; PLC: phospholipase C; CYP19A1: cytochrome P450 aromatase; PTGR1: Prostaglandin reductase 1; AREG: Amphiregulin; RTK: receptor tyrosine kinase; Akt: protein kinase B; FKHR: forkhead transcription factor; DAPL1: death associated protein like 1; ARG2: Arginase, type II Growth factor LH/CGR RR AC Gαs ? Gα TT P? Gα K PKP src cAMP ? P Akt PDE4D P PFKHR FKHR CREB P LRH-1CREB P SF-1 Genes associated with Genes associated with apoptosis ? CYP19A1, apoptosis SR-B1 PTGR1 DAPL1 SF-1, LRH-1 AREG ARG 2 P4 biosynthesis Apoptosis? P4 biosynthesis Apoptosis MONKEY BUFFALO COW Shown here is the diagram depicting intracellular signaling pathways regulated by luteotrophic factor (LH) and luteolytic factor (PGF2α) and their cross talk to counteract changes in the expressions of genes associated with the biosynthesis and secretion of P4 and apoptosis in the CL. In primates, LH/CG activates a multitude of intracellular signaling cascades, primarily Gαs/AC/cAMP/PKA/CREB leading to changes in gene expression. LH during early and mid luteal phase and CG during pregnancy maintain the activation of Src and PDE in an inhibitory state. However, during the late luteal phase of non-fertile cycle, results in present study suggests that activated Src levels and PDE activity increase, with accompanying decrease in cAMP and pCREB levels leading to concomitant decrease in SR-B1 expression, and in turn, P4 secretion. Surprisingly, regulation of apoptotic gene expression and CL regression are still unclear. In bovines, PGF2α of uterine origin mediates changes in luteal gene expression and results in decreased P4 secretion, principally by reduction in StAR level. The present study suggests that during luteolysis PGF2α affects the genes regulated by LH, by interfering with LH (and perhaps IGF-1) signaling leading to alteration in the expression of genes crucial for CL structure and function. (Pl refer the abstract file for figures)

Glicocorticoides (GCs) modulam a reprodução, interferindo na produção de hormônios gonadais, estradiol (E2) e progesterona (P4). Concomitantemente, E2 e P4 também influenciam a liberação de cortisol. Além disso, em altas concentrações os GCs interferem na via de sinalização da insulina prejudicando o funcionamento dos órgãos, incluindo os do sistema reprodutivo bem como a produção de P4 pelo corpo lúteo (CL). O CL utiliza glicose para manter sua atividade, e o cortisol interfere na via de sinalização da glicose em diversos tecidos, porém ainda não existem estudos quanto à sua atividade no CL canino, tampouco quanto à presença de genes relacionados à via de sinalização dos GCs neste órgão. Os objetivos deste estudo foram caracterizar a expressão dos genes relacionados à via de sinalização dos GCs e da insulina em CL de cadelas cíclicas e identificar expressão diferencial destes genes entre os dias 20 e 60 do diestro, que correspondem à fase final de crescimento do CL e à fase de regressão luteínica, respectivamente. Ainda, avaliar as concentrações basais de cortisol salivar (CS) e metabólitos fecais de glicocorticoides (MGFs) em diferentes dias do diestro (10, 20, 30, 40, 50 e 60) de cadelas cíclicas, comparando com os níveis de E2 e P4 já publicados; correlacionar os valores obtidos nas duas técnicas; e estabelecer um intervalo de valores basais de CS e MFGs para o diestro. Para esta pesquisa foram selecionadas 28 cadelas cíclicas saudáveis; foi realizado o sequenciamento de nova geração (RNA-Seq) dos CLs coletados de 18 animais em dias específicos do diestro (dia 10, 20, 30, 40, 50 e 60 pós-ovulação); das outras 10 cadelas foram coletadas amostras de fezes e saliva, durante todo o diestro, e realizada mensuração da concentração de MFGs e CS. Nos resultados de RNA-Seq foi identificada a expressão dos genes NR3C1, HSD11B1, HSD11B2, SLC2A4, INSR e IRS1. Os genes NR3C1 (p&gt;0,3; FDRCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,6) e SLC2A4 (pCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,02; FDRCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,1) não apresentaram expressão diferencial no diestro. Os genes HSD11B1 (p=0,0001; FDR=0,0021), HSD11B2 (p=0,013; FDR=0,06), INSR (p=0,002; FDR=0,01) mostraram maior expressão no dia 20 do diestro, quando comparado ao dia 60. O gene IRS1 (p=0,0006; FDR= 0,006) estava mais expresso no dia 60 do diestro em relação do dia 20. As dosagens hormonais demonstraram que no dia 10 do diestro (CS=0,0656 &#177; 0,0237&micro;g/dL e MGFs=110,41 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 46,51pg/ng) as duas mensurações apresentaram concentrações menores que nos demais dias do diestro (CS=0,1027 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,0496&micro;g/dL e MFGs=220,22 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 183,74pg/ng). A concentração média de CS=0,0972&micro;g/dL (0,011-0,246&micro;g/dL) e MFGs=189,875pg/ng (9pg/ng-1067,2pg/ng) foi definida para o diestro canino. Estes resultados sugerem que o CL canino também é influenciado pelo cortisol circulante da mesma forma que outros tecidos já estudados, podendo interferir na via de sinalização da insulina e, consequentemente, prejudicar seu funcionamento e o sucesso reprodutivo. Além disso, em relação aos resultados das dosagens hormonais, os níveis baixos de CS e MFGs no dia 10 do diestro corroboram com os achados em literatura e com a queda de E2 neste mesmo período, o que sugere uma associação entre a produção de ambos os hormônios. / Glucocorticoids (GCs) modulate reproduction by interfering in the gonadal hormones production, estradiol (E2) and progesterone (P4). Likewise, E2 and P4 influence release of cortisol. Furthermore, high concentrations of GCs may harm the functioning of organs, including reproductive organs and progesterone (P4) production by the corpus luteum (CL). CL utilizes glucose to maintain its own activity and cortisol interferes with the glucose signaling pathway in several tissues. Studies characterizing cortisol activity and expression of genes related to GC signaling pathways in canine CL are still scarce. The aims of this study were to characterize the expression of genes related to GCs and insulin signaling pathways in cyclic canine CL and to identify differential expression of these genes between day 20 and day 60 of diestrus which correspond, respectively to luteal final growth and regression phases. Moreover to evaluate the basal concentrations of salivary cortisol (SC) and fecal glucocorticoid metabolites (FGMs) on different days of diestrus (10, 20, 30, 40, 50 e 60) in cyclic bitches, moreover to compare E2 and P4 levels already published. As well as to correlate the values of SC and FGMs, and also to establish an interval of basal concentrations of SC and FGMs for diestrus. For this research 28 healthy cyclic bitches were selected; RNA-Seq of the corpora lutea from 18 animals on specific days of diestrus (day 10, 20, 30, 40, 50, 60) was made. Saliva and fecal samples were collected from the other 10 bitches during diestrus phase. Enzyme immunoassay was made to measure the concentrations of SC and FGMs. The RNA-Seq results identified the expression of the genes NR3C1, HSD11B1, HSD11B2, SLC2A4, INSR e IRS1. No difference on the expression of NR3C1 (p&gt;0,3; FDR&gt;0,6) and SLC2A4 (p&gt;0,02; FDR&gt;0,1) was observed during diestrus. Nevertheless HSD11B1 (p=0,0001; FDR=0,0021), HSD11B2 (p=0,013; FDR=0,06), INSR (p=0,002; FDR=0,01) showed a higher expression on day 20 of diestrus and the gene IRS1 (p=0,0006; FDR= 0,006) presented higher expression on day 60 of diestrus. Results related to hormonal evatuations showed lower SC and FGMs concentrations on day 10th (SC=0,0656 &#177; 0,0237&micro;g/dL and FGMs=110,41 &#177; 46,51pg/ng) than on the other days of diestrus (SC=0,1027 &#177; 0,0496 &micro;g/dL e FGMs=220,22 &#177; 183,74 pg/ng). Average concentrations of SC=0,0972&micro;g/dL (0,011-0,246&micro;g/dL) and FGMs=189,875pg/ng (9pg/ng-1067,2pg/ng) were defined for canine diestrus. These data suggest that canine CL is influenced by cortisol similarly to other tissues in which cortisol may interfere in the insulin signaling pathway and, consequently, its function. Besides that, lower levels of SC and FGMs on day 10th of the diestrus corroborate with the literature data and with the decrease in E2 production at the same period. This data suggest that the production of both hormones are associated.

Orientador: Sony Dimas Bicudo / Resumo: O objetivo do presente trabalho foi avaliar os corpos lúteos (CL) formados em decorrência da aspiração folicular guiada por laparoscopia (LOPU) na espécie ovina, quanto ao desenvolvimento morfológico, funcionalidade e potencial de desenvolvimento gestacional. Foram realizados dois experimentos, em ambos o estro das ovelhas foi sincronizado utilizando dispositivo intravaginal de acetato de medroxiprogesterona por 14 dias, aplicação de 140 μg cloprostenol no quarto dia e retirada do dispositivo no décimo quarto dia com concomitante administração de gonadotrofina coriônica equina (eCG). No Experimento I, 48 horas após a remoção do dispositivo realizou-se a LOPU dos folículos ovarinos em seis ovelhas (Grupo LOPU – LG), ou a LOPU associada com reposição de células em sete animais (Grupo reposição – RG) em cinco animais a ovulação foi espontânea (Grupo controle – CG). Foi realizada avaliação ultrassonográfica ovariana dos animais diariamente até o momento de uma nova ovulação e coleta de sangue para dosagem de progesterona (P4) plasmática a cada 48 horas até o decimo quinto dia. Os animais do LG (1,7±0,5) e RG (1,4±0,5) formaram mais CLs do que os animais do CG (1,0±0,0). A área lútea individual dos CLs foi menor no LG (0,69±0,30 cm2), em comparação ao RG (0,79±0,25 cm2) e CG (0,85±0,32 cm2). A área lútea total foi semelhante entre o LG (1,2±0,4 cm2) e RG (1,1±0,5 cm2), sendo ambas maiores que a do CG (0,9±0,3 cm2). A concentração de P4 plasmática não diferiu entre os grupos, fican... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of the present study was to evaluate the corpus luteum (CL) formed as a result of laparoscopic ovum pick-up (LOPU) in the ovine, regarding the morphological development, functionality and potential of gestational development. Two experiments were carried out, in both of them, the oestrous of the ewes was synchronized using a 14-day intravaginal device of medroxyprogesterone acetate, 140 μg cloprostenol on the fourth day and withdrawal of the device on the fourteenth day with concomitant administration of equine chorionic gonadotrophin (eCG). In Experiment I, 48 hours after the removal of the device, was realized a LOPU of the ovarian follicles in six sheep (LOPU Group - LG), or LOPU associated with cell replacement in seven animals (Replacement Group - RG) was performed in five animals a ovulation was spontaneous (Control Group - CG). Ultrasonographic evaluation of the animals was performed daily until the time of ovulation and blood collection for progesterone plasma concentration (P4) every 48 hours until the 15th day. LG animals (1.7 ± 0.5) and RG (1.4 ± 0.5) formed more CLs than CG animals (1.0 ± 0.0). The individual luteal area of CLs was lower in LG (0.69 ± 0.30 cm2), compared to RG (0.79 ± 0.25 cm2) and CG (0.85 ± 0.32 cm2). The total luteal area was similar between LG (1.2 ± 0.4 cm2) and RG (1.1 ± 0.5 cm2), both larger than CG (0.9 ± 0.3 cm2). Plasma P4 concentration did not differ between groups, being above 2 ng / mL in the static phase of CLs development. I... (Complete abstract click electronic access below) / Doutor

O presente estudo visou avaliar o efeito das características morfológicas e da dinâmica vascular do folículo e corpo lúteo sobre a fertilidade de vacas de corte submetidas à protocolo de sincronização de estro e ovulação. Para isso o estudo foi dividido em três experimentos sendo: No primeiro experimento foi avaliado, no momento da transferência de embriões (TE), a vascularização do corpo lúteo e sua relação com as taxas de prenhez em vacas multíparas Nelore utilizadas como receptoras de embriões. No segundo experimento analisou-se a área e vascularização do folículo no momento da inseminação artificial em vacas, novilhas e primíparas Nelore e vacas Tabapuã multíparas submetidas a protocolo de sincronização de estro e ovulação. No dia 11 após as inseminações foram avaliados a área, diâmetro e vascularização do corpo lúteo e no dia 30 realizou-se o diagnóstico gestacional dos animais. No terceiro experimento foram utilizadas vacas multíparas Nelore e cruzadas submetidas ao mesmo protocolo de sincronização de estro e ovulação do experimento 2. Neste experimento foi analisada a área dos folículos no momento da inseminação artificial e aplicados 400 UI de eCG 14 dias após, quando então avaliou-se o tamanho do corpo lúteo e dos folículos existentes. No dia 30 após a IA foi realizado o diagnóstico gestacional e análise da vascularização do corpo lúteo permitindo avaliar a relação da aplicação do eCG com o aumento da vascularização e taxas de prenhez. No presente estudo foi encontrada uma provável correlação positiva em relação à vascularização e tamanho do folículo pré-ovulatório com o tamanho e vascularização do corpo lúteo, porém apenas nos experimentos 1 e 3 houve um aumento nas taxas de prenhez conforme o aumento na vascularização. / The present study evaluated the effect of the morphological characteristics and the vascular dynamics of the follicle and corpus luteum in the fertility of beef cows submitted to a protocol of estrus synchronization and ovulation. Three experiments were done. In the first experiment, the corpus luteum vascular perfusion and its relationship with the pregnancy rates of Nelore recipient cows after embryo transfer (ET) were evaluated. In the second experiment, the area and vascular perfusion of the follicle at the time of artificial insemination (AI), the area, diameter and vascular perfusion of the corpus luteum on Day 11 after ovulation of Nelore cows submitted to a protocol of estrus synchronization and ovulation were evaluated. In the third experiment, Nelore cows were submitted to the same protocol of estrus synchronization and ovulation used in experiment 2 and the area of the follicle was measured at the time of the AI. After 14 days of the AI, 400 UI de eCG was given to the cows and the size of the corpus Luteum and the largest follicle were evaluated. On Day 30 after AI the size and vascular perfusion of the corpus luteum was evaluated. The pregnancy diagnosis was done on Day 30 after ovulation for all the three experiments. In the present study, it was found a positive correlation between the vascular perfusion and the size of the pre-ovulatory follicle, and the size and vascular perfusion of the corpus luteum. However, only in the experiments 1 and 3 the pregnancy rates increased with the increase in the corpus luteum vascular perfusion.

Biotécnicas aplicadas à reprodução animal são empregadas com o intuito de melhorar qualitativa e quantitativamente os rebanhos. A superovulação assim como a estimulação do folículo dominante utilizando-se doses suprafisiológicas de eCG (gonadotrofina coriônica eqüina) são empregadas em bovinos e bubalinos, mas nem sempre alcançam os resultados esperados. Entretanto, foram relatadas alterações morfofuncionais em corpos lúteos (CL) de animais submetidos a estes tratamentos, o que implica, necessariamente, em levantamento de hipóteses relativas ao(s) mecanismo(s) pelo(s) qual(is) as gonadotrofinas exógenas alteram as funções celulares nos folículos e corpos lúteos resultantes. Com o intuito de melhor compreensão do papel exercido pelo eCG sobre a célula luteínica bovina, 16 vacas foram sincronizadas e submetidas (grupos tratados) ou não (grupo controle) ao tratamento com eCG. O tratamento foi realizado previamente (superovulação) ou posteriormente (estimulação do folículo dominante) ao desvio folicular. No dia 6 após a ovulação, estes animais foram abatidos e os CL coletados para realização de análises histológicas e ultra-estruturais, assim como o sangue coletado para dosagem de progesterona. Para comparação dos dados obtidos entre os diferentes grupos utilizou-se o programa GraphPrism e as médias foram consideradas diferentes quando p < 0.05. Em relação à densidade de mitocôndrias e densidade numérica das mitocôndrias elipsóides não houve diferença significativa entre os grupos estudados. Quando se calculou o volume total das mitocôndrias no CL bovino, houve diferença significativa do grupo superovulado em relação aos outros grupos. Já para as mitocôndrias esferóides observou-se diferença significativa do grupo estimulado comparado aos grupos controle e superovulado. À imunofluorescência os animais do grupo superovulado apresentaram visualmente uma marcação mais fluorescente intensa para mitocôndrias quando comparados aos grupos controle e estimulado; e o grupo estimulado apresentou uma marcação mais intensa em relação ao grupo controle. Houve diferença significativa entre os três grupos em relação à quantidade das células luteínicas grandes. Quando se corrigiu o número de células luteínicas grandes pelo volume do CL, os mesmos resultados foram encontrados. Quanto às medidas do maior diâmetro das células luteínicas grandes houve uma diferença significativa quando comparado o grupo estimulado com os demais. Aspectos qualitativos da microvascularização do CL em animais controle e superovulados revelam vascularização mais abundante no grupo tratado. Estes dados apontam para modificações morfológicas da célula luteínica e do CL bovino direcionadas à maior produção hormonal após tratamento com eCG e indicam que doses diferentes aplicadas em momentos diferentes do ciclo estral levam à respostas diversas da célula luteínica e do próprio CL. / Biotechnologies applied to animal reproduction are employed in order to improve livestock quality and quantity. Superovulation and the stimulation of the dominant follicle using supraphysiological doses of eCG (equine chorionic gonadotropin) are used in cattle and buffaloes, but not always achieve the expected results. However, morphofunctional changes in corpora lutea (CL) derived from animals submitted to these treatments were reported, which necessarily implies a survey of hypotheses regarding the mechanism(s) by which exogenous gonadotrophins result in altered cellular functions in the follicles and corpora lutea. In order to better understand the role played by eCG on bovine luteal cells, 16 cows were synchronized and submitted (treated groups) or not (control group) to treatment with eCG. The treatment was performed previously (superovulation) or after (stimulation of the dominant follicle) follicular deviation. At 6 days after ovulation, animals were slaughtered and the CL collected for histological and ultrastructural analysis, as well as blood for determining plasmaprogesterone.For statistical analysis the program Graph Prism was used and mean considered significant different when p < 0.05. In relation to mitochondria number and volume density there were no differences among the studied groups. Values of mitochondria total volume in CL were different if stimulated was compared to the other groups. Imunofluoresce for mitochondria showed a more intensive signal for animals from the superovulated group, followed by animals belonging to the stimulated group. The number of large luteal cells was different among all groups. Even though this number was corrected by CL volume, the same results were found. The biggest diameter of large luteal cells was compared among the groups and stimulated group showed higher values. Qualitative aspects of CL microvascularization in control and superovulated animals reveal more abundant vascularization in treated group. These data point towards morphological modification of bovine CL and luteal cell directed to increased hormonal production after eCG treatment. Moreover, results indicate that different eCG dosis applied in different time points of estrous cycle lead to different responses from CL and luteal cells.