New insights in the immuno-endocrine regulation of equine reproduction : in vitro studies on luteal and endometrial function

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e BiomédicasCoordination of reproductive events in the ovary and uterus demands the action of diverse factors as steroid hormones, eicosanoids, growth factors or cytokines on the regulating of processes such as angiogenesis, cell growth and differentiation, and apoptosis. Thus, the objectives of this study were to evaluate the influence of cytokines tumor necrosis factor α (TNF), interferon gamma (IFNG) and Fas Ligand (FASL) on the regulation of secretory function, angiogenesis, cell viability and apoptosis in the equine: (i) corpus luteum (CL) during luteal establishment and functional and structural regression; and (ii) endometrium during follicular phase (FP) and mid luteal phase (MLP), at cell, gene and molecular levels. All studied cytokine ligands and receptors were expressed in the equine CL, throughout the luteal phase, and in the endometrium, throughout the estrous cycle. During CL growth, TNF was shown to stimulate in vitro P4 and PGE2, to inhibit PGF2α secretion and to increase VEGF expression and angiogenic factors production. Thus, among all cytokines studied, TNF might give a luteotrophic contribution for CL establishment. Conversely, during CL regression, all cytokines alone reduced P4 and PGE2 secretion, while both FASL and TNF stimulated PGF2α secretion. TNF and IFNG reduced angiogenic factors secretion and FASL decreased VEGF expression. Cytokine association (TNF+IFNG+FASL) effectively promoted apoptosis and reduced luteal cell viability. Besides, they stimulated PGF2α and inhibited P4, PGE2 secretion and angiogenesis. In conclusion, cytokines interaction appears to coordinate functional and structural luteolysis in the mare. Concerning TNF role on endometrial cells, it can be concluded that during MLP, this cytokine stimulated PGE2 secretion, promoted angiogenic activity and NO secretion and increased endometrial cells viability. The interaction between TNF, oxytocin and steroid hormones was shown to be determinant for physiologic regulation of equine endometrium.RESUMO - Novas perspectivas na regulação imuno-endócrina da reprodução equina: estudos in vitro da função lútea e endometrial. - A coordenação da função reprodutora no ovário e no útero requer a participação de diversos factores como hormonas esteróides, eicosanóides, factores de crescimento ou citocinas, responsáveis por regular processos biológicos como a angiogénese, o crescimento e diferenciação celular e a apoptose. Assim, o presente estudo teve como objectivo avaliar a influência das citocinas factor de necrose tumoral α (FNT), interferão gama (IFNG) e Fas Ligando (FASL) na regulação da função secretora, angiogénese, viabilidade celular e apoptose: (i) no corpo lúteo (CL) durante o seu estabelecimento e regressão funcional e estrutural; e (ii) no endométrio durante as fases folicular (FF) e lútea média (FLM), a nível celular, molecular e genético na égua. A expressão de todos os ligandos e receptores das citocinas estudadas foi confirmada no CL, ao longo da fase lútea, e no endométrio, ao longo do ciclo éstrico. Durante o crescimento do CL, FNT estimulou a produção in vitro de P4 e PGE2, inibiu a secreção de PGF2α e aumentou a expressão de VEGF e a produção de factores angiogénicos. Desta forma, de todas as citocinas estudadas, FNT poderá contribuir como factor luteotrófico para o estabelecimento do CL. Contrariamente, aquando da regressão lútea, todas as citocinas individualmente reduziram a secreção de P4 e PGE2, enquanto FASL e FNT estimularam a secreção de PGF2α. FNT e IFNG inibiram a secreção de factores angiogénicos e FASL diminuiu a expressão de VEGF. A associação de citocinas FNT+IFNG+FASL promoveu de forma efectiva a apoptose e a redução da viabilidade das células lúteas. Além disso, estimulou a secreção de PGF2α e diminuiu as de P4 e PGE2, bem como inibiu a angiogénese. Concluindo, a interacção entre as várias citocinas parece coordenar a regressão funcional e estrutural do CL na égua. Considerando o papel do TNF nas células endometriais, conclui-se que, durante a FLM, este factor estimulou a secreção de PGE2, promoveu a produção de factores angiogénicos e de NO e aumentou a viabilidade das células endometriais. A interacção entre o FNT, a ocitocina e as hormonas esteróides provou ser determinante para a regulação fisiológica do endométrio equino

Cellular mechanisms of luteal regression in the bovine corpus luteum (CL)

The corpus luteum (CL) is a transient endocrine gland that produces progesterone (P4) for the establishment and maintenance of pregnancy. In absence of pregnancy, timely regression of the CL is essential for normal ovarian cyclicity. Several factors are known to participate in luteal regression. In this study, two factors, PGF2 alpha and endothelin (ET-1) are being studied. Protein kinase C (PKC) and calcium are the two main intracellular mediators of PGF2 alpha. The role of ET-1 in the regulation of luteal regression is unclear. The early CL is insensitive to the luteolytic actions of PGF2 alpha and the cellular mechanism(s) involved in this process are poorly understood. This study investigates: (1) the array of PKC isozyme expression as a function of development in the bovine CL and the ability of PGF2 alpha and ET-1 to activate the PKC isozymes in the early (day-4) and late (day-10) luteal phase, (2) the physiological role of the luteal PKC isozymes on PGF2 alpha-induced rise in intracellular calcium concentration and luteinizing hormone (LH) stimulated P4 accumulation at the mid luteal phase and (3) the cellular source of the luteal PKC isozymes. PKC alpha, beta I, beta II, epsilon and meu were observed to be expressed in the bovine CL with beta II and epsilon being differentially expressed as a function of development. In day-10 CL PGF2 alpha and ET-1 were able to activate PKC alpha, beta I and epsilon. More importantly, PKC epsilon was found to be involved in the regulation of PGF2 alpha induced rise in intracellular calcium concentration and antagonized the inhibitory effect of PGF2 alpha and ET-1 on LH-stimulated P4 accumulation in cultures of day-10 luteal steroidogenic cells (SC). PKC epsilon was found exclusively expressed in SC. In contrast, PKC alpha, beta I and beta II were expressed in both SC and endothelial cells (EC), with SC expressing higher amounts than EC. In this study we have proposed that the differential expression and activation of PKC epsilon as a function of development may be one of several factors responsible for the insensitivity of the early CL. Expression of PKC epsilon in the mid luteal phase shifts the PGF2 alpha induced rise in intracellular calcium concentration from a P4 favorable to a P4 inhibitory condition. Based on these observations it is hypothesized that the insensitivity of the early CL towards the luteolytic actions of PGF2 alpha may be due to differences in the intracellular mediators with respect to luteal development

Mechanisms Behind the Luteoprotective Effects of Fish Meal Supplementation in Bovine: Cytokine Sensitivity and Luteal Blood Flow

Early pregnancy loss continues to be problematic in the cattle industry. A large proportion of the pregnancy loss occurs during the maternal recognition of pregnancy (MRP) phase of gestation. During the MRP, a developing conceptus must secrete sufficient interferon tau to inhibit release of the uterine- derived and luteolytic mediator, prostaglandin F2 alpha (PGF2). If successful, the progesterone- secreting corpus luteum (CL) will be maintained, which is necessary for establishment and maintenance of pregnancy. Pregnancy loss at the point of MRP may be due to a slow developing conceptus that does not effectively block PGF2. Therefore, reducing the influence of PGF2 on the CL may lengthen the window of time to establish pregnancy. Fish meal (FM) supplementation, rich in the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), serves as a luteoprotective agent following PGF2 exposure; an effect we repeated here. However, the mechanistic actions of FM supplementation are not yet apparent. Here, we postulated two mechanisms by which FM supplementation (and components) reduces rates of luteal regression: 1) EPA and DHA protect cells against the cytotoxic effects of TNF and IFN and (or) 2) FM supplementation maintains luteal blood flow in functional CL 48 hrs following exposure to PGF2. We found that EPA and DHA did not protect cells from the cytotoxic effects of TNF and IFN exposure, although omega-3 fatty acids altered transcriptional regulation of necroptotic cell death in cells of an early and middle-stage CL. Moreover, we demonstrated that cells of a middle stage CL are less sensitive to the cytotoxic effects of TNF and IFN, which may be due to reduced expression of TNFRI. Exposure to TNF and IFN increased expression of TNF and NFkB, and reduced expression of steroidogenic genes STARD1 and CYP11A1. With respect to our second postulated mechanism, we found no change in luteal blood flow 48 hrs following PGF2 exposures, as detected with power Doppler ultrasonography. However, we did find that FM-PGF2 infused cows had a significant reduction in luteal hyperemia when compared to the control supplementation, indicating FM supplementation may be altering the acute blood flow response. In conclusion, the differing micro-environments of each luteal stage pre-conditions cellular sensitivity to TNF and IFN exposure. Moreover, FM supplementation may maintain functional CL by blunting the acute changes in luteal blood flow during luteal regression

An Examination of Preimplantation Embryos and Endometrium Developed During Induced Aluteal Cycles in the Mare

Changes to the embryonic environment that occur within the first few days of development can have significant effects on subsequent fetal development. The following studies investigate the effects of progesterone-deprivation during the early preimplantation period in the mare. The first study described embryo morphology (diameter, stage, and quality grade) collected from mares with induced aluteal cycles. The second study evaluated the effect of induced aluteal cycles on the future fertility of embryo collections as a potential method for cycle manipulation of embryo donor mares. The third study characterized embryonic and endometrial transcript expression of progesterone-mediated transcripts as well as those related to normal embryo growth and pregnancy establishment during induced aluteal cycles. Finally, the fourth study evaluated the potential for the successful establishment of pregnancy when preimplantation embryos were deprived of progesterone. Embryos developed in a progesterone-deprived environment were developmentally delayed by all parameters evaluated. A significant effect was seen on the transcript expression of embryos and endometrium collected from progesterone-deprived cycles in the mare. Embryos deprived of progesterone during the early preimplantation period demonstrated delayed embryonic vesicle growth compared to control cycles. No effect was seen on the future fertility of embryo collections after utilizing progesterone-deprived cycles. The results of these studies elucidate the significance of adequate progesterone levels beginning immediately post-ovulation in the mare

Mare endometrium : physiological and pathological involvement of hormones and neutrophil extracellular traps

Tese de Doutoramento em Ciências Veterinárias, Especialidade de Ciências Biológicas e Biomédicas.Two reproductive topics in mares were addressed in this thesis. The aims of the studies were to evaluate: (i) the effect of chronic oxytocin administration to mid-luteal phase mares on luteal maintenance and its cellular and molecular mechanisms at endometrial level; (ii) the capacity of equine neutrophils to produce neutrophil extracellular traps (NETs) in vitro when stimulated with bacteria obtained from mares with endometritis, and to determine if NETs release also occurred in vivo in mares with endometritis; (iii) the in vitro effects of some NETs components on mare endometrial fibrogenic capacity and to determine if they could depend on endometrial inflammatory lesions or estrous cycle phases; and (iv) the involvement of PGF2α and PGE2 pathways in collagen deposition on mare endometrium, challenged with NETs proteases. In the first study, luteal maintenance occurred in 67% of oxytocin treated mares, which may be related to oxytocin and progesterone (PGR) receptors spatial expression in endometrium. Reduction of endometrial estrogen receptor 2 (ESR2) may be responsible for the maintenance of PGR in luminal and glandular epithelium and may attenuate ESR1 endometrial transcriptional activity. Equine neutrophils were able to release NETs in the presence of bacteria that cause mare endometritis, and might be a complementary mechanism to fight endometritis. By in vitro studies with NETs proteases, increased collagen type I (COL1) production characteristic of fibrosis was observed, although endometrial response to each NETs protease depended on estrous cycle and/or endometrial category. Also, NETs proteases were linked to fibrogenesis, by increased synthesis of PGF2a and/or PGF2a receptor transcripts and impaired PGE2 or PGE2 receptor 2 transcripts associated to increased COL1. These effects were influenced by endometrium type and estrous cycle phases. Injury induced-changes on PG mediators by NETs components may instigate PGF2α or PGE2 vias, as additional pathways in mare endometrial fibrogenesis.RESUMO - Endométrio da égua: envolvimento fisiológico e patológico de hormonas e de redes extracelulares de neutrófilos - Nesta tese foram abordados dois temas reprodutivos em éguas. Os objectivos destes estudos consistiram na avaliação: (i) da administração crónica de ocitocina na manutenção do corpo lúteo em éguas na fase lútea média, e os mecanismos celulares e moleculares no endométrio; (ii) da capacidade dos neutrófilos equinos para produzirem redes extracelulares de neutrófilos (NETs) in vitro e in vivo quando estimulados com bactérias de éguas com endometrite; (iii) dos efeitos in vitro de componentes das NETs na fibrogénese do endométrio equino e sua associação com lesões inflamatórias no endométrio ou com o ciclo éstrico; e (iv) do envolvimento das vias da PGF2α e PGE2 na deposição de colagénio no endométrio da égua, incubado com proteases das NETs. No primeiro estudo, o prolongamento da função lútea em 67% das éguas tratadas com ocitocina, parece estar relacionado com alteração da expressão espacial dos receptores de ocitocina e progesterona (PGR) no endométrio. A diminuição do receptor 2 dos estrogénios (ESR2) pode ser responsável pela manutenção do PGR no epitélio luminal e glandular do endométrio e atenuar a transcrição do ESR1. Os neutrófilos equinos libertaram NETs na presença de bactérias causadoras de endometrite podendo ser um mecanismo complementar no combate à endometrite. As proteases das NETs in vitro aumentaram a produção de colagénio do tipo I (COL1), característico de fibrose, no endométrio, embora a resposta a cada protease dependesse do ciclo éstrico e/ou da categoria do endométrio. O aumento do COL1 associado ao incremento de PGF2α e/ou da transcrição do seu receptor e à diminuição da PGE2 e/ou do seu receptor 2, parecem implicar as prostaglandinas e as proteases das NETs na fibrogénese no endométrio equino. Alterações nos mediadores das prostaglandinas, induzidas pelos componentes das NETs, podem instigar as vias da PGF2a ou da PGE2, como mecanismos adicionais de fibrose do endométrio da égua.N/

Use of steroid hormones or GnRH to synchronize and resynchronize follicular wave emergence, estrus, and ovulation in cattle

A series of experiments were designed to study alternative estrus synchronization and resynchronization protocols to facilitate the use of artificial insemination in cattle. Studies were conducted to study the effects of estradiol cypionate (ECP) on follicular dynamics, time of ovulation, and pregnancy rate to timed-AI (TAI) in CIDR-based protocols. Although administration of 1 mg ECP did not result in synchronous follicular wave emergence, a dose of 0.5 mg ECP synchronized LH release and ovulation. Administration of ECP 24 h after CIDR removal resulted in acceptable pregnancy rate. However, treatment with ECP at CIDR removal resulted in acceptable pregnancy rate only if follicular wave emergence was synchronized with estradiol-17â (E-17â). The efficacy of two estradiol preparations (5 mg of E-17â or estadiol valerate; EV) and reduced doses of EV on CL and ovarian follicular dynamics and superovulatory response were examined. When doses of 5 mg were compared, EV treatment resulted in a more variable interval to follicular wave emergence and a lower superovulatory response than E-17â. However, EV at a dose of 1 or 2 mg was efficacious in synchronizing follicle wave emergence in CIDR-treated cattle.Pregnancy rates were compared following TAI in cattle given a new or previously used CIDR and injections of estradiol, with or without progesterone, to synchronize follicular wave emergence. Pregnancy rate following TAI did not differ between cattle treated with a new or once-used CIDR, but pregnancy rate was lower in cattle treated with one or two twice-used CIDR. The addition of an injection of progesterone to the estradiol treatment at CIDR insertion did not enhance pregnancy rate. The efficacy of progestins (used CIDR and MGA), and E-17â, ECP, GnRH, or progesterone treatment for resynchronization of estrus in cattle not pregnant following TAI were investigated. Progestin treatment resulted in the majority of nonpregnant heifers detected in estrus over a 4-d interval. Conception rates were higher in heifers resynchronized with a once-used CIDR than with MGA. GnRH at CIDR insertion synchronized follicular wave emergence in cows, but did not increase conception rate in heifers. E-17â at CIDR insertion (1.5 mg) and removal (0.5 mg) resulted in decreased pregnancy rate following TAI. In summary, protocols described in this thesis resulted in acceptable pregnancy rates following TAI and resynchronization of previously inseminated heifers with progestins resulted in variable estrus and pregnancy rates

All in good time : dynamics of the bovine estrous cycle investigated with a mathematical model

Bovine fertility is subject of extensive research in animal sciences, especially since a decline in dairy cow fertility has been observed during the last decades. One factor is reduced expression of estrous behavior. Fertility is a complex process, regulated by interactions between brain and reproductive organs. The objective of this thesis was to improve insight in the regulation of dairy cow fertility by developing and using a mechanistic mathematical model of the bovine estrous cycle. The model that was developed describes the dynamics of the bovine estrous cycle on individual cow level. It simulates follicle and CL development and the periodic changes in hormone levels that control these processes by a set of linked differential equations. The model captures a number of key physiological processes of the bovine estrous cycle, and serves as a starting point for further simulation studies, model validation, and extended models. The model was used to find candidate mechanisms that regulate follicular development. A normal estrous cycle contains 2 or 3 waves of follicular development, but why some cycles consist of 3 and others of 2 waves is unknown. Results showed that variation of (combinations of) model parameters regulating follicle growth rate or time point of CL regression can change the model output from 3 to 2 waves of follicular growth in a cycle. Several factors may perturb the regular oscillatory behavior of a normal estrous cycle. Such perturbations are likely the effect of simultaneous changes in multiple parameters. It was investigated how multiple parameter perturbation changes the behavior of the estrous cycle model, so as to identify biological mechanisms that could play a role in the development of cystic ovaries, a common reason for reproductive failure in dairy cows. Simulation results indicated that CL functioning, luteolytic signals, and GnRH synthesis are likely involved in the development of cystic ovaries. Empirical data of individual cows was used to identify mechanisms that explain individual differences in cycle characteristics by fitting the model to the data. Finding specific parameter configurations for individual cows shows the capability of the model to simulate ‘real’ data. Certain combinations of estimated parameter values induced a clear qualitative shift in model behavior (e.g. a different number of follicular waves), suggesting possible routes how environmental or genetic influences could affect estrous cycle characteristics. Experimental data to verify simulation results are not always available, but hypotheses based on the model predictions could be investigated in future animal experimen</p

Modelling physiological reproductive inflammatory networks in vivo.

The immune and reproductive systems have long been known to be inextricably linked, with components of immune pathways, particularly cytokines, mediating processes such as ovarian/menstrual cyclicity, endometrial remodelling, mating-induced immunomodulation, implantation, pregnancy, parturition and lactation. The nature of this involvement has often been investigated at the level of single mediators, with little consideration of the fact that cytokines are increasingly understood to function as complex networks. This study aimed to characterise inflammatory networks using both traditional and novel machine-learning Bayesian network-based methods in the context of keystone aspects of reproduction, viz., in the endometrial response to seminal plasma, cytokine:hormone interactions during lactation, and oocyte maturation following controlled ovarian hyperstimulation. ‘Traditional’ pathway analyses used to examine the murine endometrial response to seminal plasma revealed previously unidentified mediators and showed compartmentalised epithelium/stroma-specific responses. However, they proved ineffective in describing novel cytokine interactions. This led to the development a highly effective novel Bayesian network-based approach to explore cytokine:hormone networks during murine lactation. This revealed that prolactin, a putative potent immunomodulator, was far less influential than expected in vivo. The method also identified previously unknown cytokine interactions and described features such as synergy and antagonism. Further refinement of these network analyses as modified variational Bayesian state space models enabled the display of core, conserved subnetworks (communities) of human follicular fluid cytokines whose interactions varied with oocyte maturity. Moreover, these cytokine signatures also allowed the prediction of an oocytes’ fertilisability potential, with potential attendant benefits to assisted conception. This thesis represents the first endeavour to model inflammatory networks in vivo in any setting to date. It has revealed their central role, functional conservation and key features of cytokine interactions across a spectrum of reproductive processes. Further development of this methodology appears set to offer invaluable new insights into the complex immune signalling that underpins reproductive biology

Characterization of the Immune Response to Lipopolysaccharide in Early Pregnant Ewes as a Model to Study Bacterial Infection Induced Embryonic Loss

An immunological balance has to be established during pregnancy that protects the mother yet tolerates the semi-allogenic fetus. To understand the innate immune response during bacterial infections that may cause early embryonic loss, a lipopolysaccharide (LPS) treated sheep model was used. Two objectives of this study were to examine if omega-3 PUFAs in the form of supplementary whole flaxseed could reduce the inflammatory response to an LPS challenge and to examine if there is a differential immune response to LPS in Suffolk and Dorset ewes. A total of 3 experiments were conducted; two investigating the effect of supplement and one investigating the effect of breed. Estrus was synchronized by CIDR insertion for 5 days, followed by 20 mg of PGF 2alpha at CIDR withdrawal. Early pregnant ewes received via the jugular vein, either phosphate buffered saline (PBS) (3 ml) or LPS (2.5 mug/kg). Blood was collected via jugular venipuncture at hour: 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 9, 12, and 24. Whole blood samples were used to determine white blood cell counts (WBCs) before centrifugation to collect white blood cells for RNA extraction. Rectal temperature and change in behavior/physical (lethargy, coughing, nasal discharge, absence of eating) appearance were recorded hourly. Real-time PCR was performed for expression of cytokines (CXCL8, IL6, TNFalpha, IFNgamma, IL-10, and TGFbeta), receptors (TLR4, MRC1), enzymes (COX2, SOD2), transcription factors (NF-kappaB, PPARgamma, and Foxp3) and complement component 3. In all experiments, temperature increased in response to LPS, peaking at hour 4 before returning to normal by hours 6-9; WBCs dropped by hour 1 before returning to normal by hours 6-9. In trial 1 of the supplement study (flaxseed versus a control supplement) (Dorset ewes flaxseed + LPS n=3; flaxseed + PBS n=3; control + LPS n=5; control + PBS n=5), LPS increased haptoglobin and cortisol levels and affected gene transcription of CXCL8, IFN?, TLR4, MRC1, SOD2, Foxp3 (by hour), and C3.There was a diet effect with regard to cortisol and gene expression of CXCL8, and TLR4. There was a diet x LPS interaction with regard to temperature, WBCs (by hour), haptoglobin, serum amyloid A and gene expression of CXCL8, IL-6, and TLR4. In trial 2 of the supplement study (Dorset ewes flaxseed + LPS n=11, flaxseed + PBS n=10, control + LPS n=11, control + PBS n=10), LPS increased haptoglobin and cortisol and affected gene expression of CXCL8, TLR4, MRC1, SOD2, PPARgamma, Foxp3, and C3. There was a diet effect on cortisol and gene expression of C3.There was a diet x LPS interaction with regard to temperature and cortisol. In the breed effects study (Dorset + LPS n=11; Dorset + PBS n=10; Suffolk + LPS n=16; Suffolk + PBS n=16), LPS increased cortisol and affected gene transcription of CXCL8, TLR4, MRC1, SOD2, PPARgamma, and C3. There was an effect of breed on temperature; haptoglobin, serum amyloid A (by hour), and cortisol levels; and gene transcription of IL-6, IFNgamma, IL-10, TLR4, COX2, SOD2, PPARgamma, Foxp3, and C3. There was a breed x LPS interaction on change in temperature from hour 0, the frequency of behavior/physical changes; haptoglobin, serum amyloid A (by hour), and cortisol; and gene transcription of IL-6 and C3. Pregnancy status was assessed at 25 dpc with transrectal ultrasound and progesterone was measured in plasma samples. In trial 1 of the supplement study, flaxseed increased progesterone but it did not differ between groups in trial 2. There were no differences in progesterone between the breeds tested. The number of ewes that lambed in each treatment group was not different in any of the experiments. In summary, acute infections may cause embryonic loss by shifting the environment to be pro-inflammatory. There was no clear benefit of supplementary o-3 PUFAs in reducing the inflammatory response. Suffolk ewes had an elevated inflammatory response to LPS compared to Dorset ewes and may be more susceptible to embryonic loss in response to infection