Unveiling the role of protein kinase A (PKA) activity in bovine oviductal epithelial cells: implications on apoptotic signaling pathways during the estrous cycle.

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70 kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Metabolic stressful environment drives epigenetic modifications in oviduct epithelial cells.

The oviduct provides a suitable microenvironment from the gametes' final maturation until initial embryo development. Dynamic functional changes are observed in the oviduct cells, mainly controlled by steroid hormones and well-orchestrated during the estrous cycle. However, based on the roles played by the oviduct, additional layers of complexity might be present in its regulatory process. There is a cellular process that includes metabolic adaptation that can guide molecular modifications. This process is known as metaboloepigenetics. Therefore, we aimed to better understand how this crosstalk occurs in oviductal epithelial cells (OEC). Due to limited in situ access to the oviduct, we used the primary in vitro cell culture as a culture model and glucose as a metabolic disturbed factor. For that, cells derived from the oviductal epithelial layer were collected from cows at either follicular or luteal stages (n = 4 animals per group). They were cultured on a monolayer culture system under normoglycemic (2.7 mM glucose) or hyperglycemic conditions (27 mM glucose). On day five of culture, attached cells were submitted to analysis of mitochondrial metabolism (mitochondrial membrane potential - MMP) and epigenetics markers (5- methylcytosine - 5 mC and histone 3 lysine 9 acetylation - H3K9ac). Moreover, the culture media were submitted to the metabolites analysis profile by Raman spectrometry. Data were analyzed considering the effect of glucose level (normoglycemic vs. hyperglycemic), stages when OEC were harvested (follicular vs. luteal), and their interaction (glucose level * cycle stage) by two-way ANOVA. As a result, the high glucose level decreased the H3K9ac and MMP levels but did not affect the 5 mC. Regardless of the metabolic profile of the culture media, the glucose level was the only factor that changed the Raman shifts abundance. Although this present study evaluated oviductal epithelial cells after being submitted to an in vitro monolayer culture system, which is known to lead to cell dedifferentiation, yet, these results provide evidence of a relationship between epigenetic reprogramming and energy metabolism under these cell culture conditions. In conclusion, the levels of metabolites in culture media may be crucial for cellular function and differentiation, meaning that it should be considered in studies culturing oviductal cells.

The interaction between the environment and embryo development in assisted reproduction.

It can be assumed that the natural processes of selection and developmental condition in the animal provide the best prerequisites for embryogenesis resulting in pregnancy and subsequent birth of a healthy neonate. In contrast, circumventing the natural selection mechanisms and all developmental conditions in a healthy animal harbors the risk of counteracting, preventing or reducing the formation of embryos or substantially restricting their genesis. Considering these facts, it seems to be obvious that assisted reproductive techniques focusing on early embryonic stages serve an expanded and unselected germ cell pool of oocytes and sperm cells, and include the culture of embryos outside their natural habitat during and after fertilization for manipulation and diagnostic purposes, and for storage. A significant influence on the early embryonic development is seen in the extracorporeal culture of bovine embryos (in vitro) or stress on the animal organism (in vivo). The in vitro production per se and metabolic as well as endocrine changes in the natural environment of embryos represent adequate models and serve for a better understanding. The purpose of this review is to give a brief presentation of recent techniques aimed at focusing more on the complex processes in the Fallopian tube to contrast in vivo and in vitro prerequisites and abnormalities in early embryonic development and serve to identify potential new ways to make the use of ARTs more feasible.

Importance of preovulatory estradiol on uterine receptivity and luteal function.

Animals that exhibited estrus had greater pregnancy success compared to animals that did not exhibit estrus before fixed-time AI (FTAI). Estradiol is synthesized in bovine ovarian follicles under gonadotropin regulation and can directly and indirectly regulate the uterine receptivity and luteal function. Estradiol concentrations at FTAI impacted oviductal gene expression and has been reported to play an important role in establishing the timing of uterine receptivity. These changes have been reported to impact uterine pH and sperm transport to the site of fertilization. After fertilization, preovulatory estradiol has been reported to improve embryo survival likely by mediating changes in uterine blood flow, endometrial thickness and changes in histotroph. Cows with greater estradiol concentrations at the time of GnRH-induced ovulation also had a larger dominant follicle size and greater circulating progesterone concentrations on day 7. Therefore, it is impossible to accurately determine the individual benefit of greater estradiol concentrations prior to ovulation and greater progesterone concentrations following ovulation to pregnancy establishment, as these two measurements are confounded. Research has indicated an importance in the occurrence and timing of increasing preovulatory concentrations of estradiol, but increasing estradiol concentrations by supplementation may not be sufficient to increase fertility. Increased production of estradiol by the preovulatory follicle may be required to enhance fertility through the regulation of sperm transport, fertilization, oviductal secretions, the uterine environment, and embryo survival.

Histomorphometric comparison of left and right oviduct structure from alpaca (Vicugna pacos).

Alpacas are species of induced ovulation and with foetal development only in the left uterine horn (98%). The histoarchitecture of the oviductal regions determine a spatio-temporal interaction between the gametes/embryos and the oviduct. This study compares the morphometric changes of the left and right oviducts in alpaca during the follicular phase. Five oviducts (n = 05), from adult alpacas with dominant follicle in the right ovary were recovered, dissected, and processed by histological technique with H&E and PAS stain for measurement of morphometric parameters and cell characteristics, respectively. Also, a 3D image reconstruction was performed (by reconstruct software). Resin moulds (polyurethane PU4ii) were applied for visualization of oviductal lumen. The multivariable data of parameters were analysed with ANOVA and principal component analysis (PCA). The histomorphometric parameters of left and right oviducts did not show statistically significant differences (p ≥ 0.05), although PCA showed morphometric differences between regions of the oviduct. No differences were observed between the 3D reconstruction of the left and right oviducts, as well as in the luminal spaces examined in the resin moulds. In conclusion, the histomorphometry of the oviduct is not affected by its location on either the left or right side; therefore, it cannot explain why 98% of foetuses implant in the left uterus.

A review of the reproductive system in anuran amphibians.

Reproductive biology is an important topic that is well explored in many vertebrates, but information about frogs' reproductive mechanisms could be improved. Therefore, this review aims to provide organized and specific information on frog reproduction. First, we developed schemes that illustrate the general information regarding reproductive biological mechanisms in frogs in a specific way. Then, we described the physiological, histological, and morphological mechanisms of each organ of the reproductive system of male and female frogs. Finally, this manuscript may contribute to a broader understanding of anuran reproductive biology. Since, understanding frogs' reproductive system permits one to make a comparison with reproduction with other anurans.

An assessment of responses to egg production and liver health of Japanese quails subjected to different levels of metabolizable energy.

OBJECTIVE: Current quail production is configured as an economic activity in scale. Advancements in quail nutrition have been limited to areas such as breeding and, automation of facilities and ambience. The objective of this study was to evaluate the performance responses, liver and oviduct morphometry, and liver histology of Japanese laying quails subjected to different levels of nitrogen-corrected apparent metabolizable energy (MEn). METHODS: A completely random design was used that consisted of nine levels of MEn, six replicates, and five hens per cage with a total of 270 quails. The experimental period lasted for 10 weeks. The variables of performance were subjected to analysis of variance and then regression analysis using the broken-line model. The morphometric and histological variables were subjected to multivariate exploratory techniques. RESULTS: The MEn levels influenced the responses to zootechnical performance. The brokenline model estimated the maximum responses for feed intake, egg production, egg weight, and egg mass as 3,040, 2,820, 1,802, and 2,960 kcal of MEn per kg of diet, respectively. Multivariate analysis revealed that the occurrence of hepatic steatosis and increased levels of Kupffer cells were not related to MEn levels. CONCLUSION: The level of 2,960 kcal/kg of MEn meets performance variable requirements without compromising hepatic physiology.

Importance of preovulatory estradiol on uterine receptivity and luteal function

Animals that exhibited estrus had greater pregnancy success compared to animals that did not exhibit estrus before fixed-time AI (FTAI). Estradiol is synthesized in bovine ovarian follicles under gonadotropin regulation and can directly and indirectly regulate the uterine receptivity and luteal function. Estradiol concentrations at FTAI impacted oviductal gene expression and has been reported to play an important role in establishing the timing of uterine receptivity. These changes have been reported to impact uterine pH and sperm transport to the site of fertilization. After fertilization, preovulatory estradiol has been reported to improve embryo survival likely by mediating changes in uterine blood flow, endometrial thickness and changes in histotroph. Cows with greater estradiol concentrations at the time of GnRH-induced ovulation also had a larger dominant follicle size and greater circulating progesterone concentrations on day 7. Therefore, it is impossible to accurately determine the individual benefit of greater estradiol concentrations prior to ovulation and greater progesterone concentrations following ovulation to pregnancy establishment, as these two measurements are confounded. Research has indicated an importance in the occurrence and timing of increasing preovulatory concentrations of estradiol, but increasing estradiol concentrations by supplementation may not be sufficient to increase fertility. Increased production of estradiol by the preovulatory follicle may be required to enhance fertility through the regulation of sperm transport, fertilization, oviductal secretions, the uterine environment, and embryo survival.(AU)

The interaction between the environment and embryo development in assisted reproduction

It can be assumed that the natural processes of selection and developmental condition in the animal provide the best prerequisites for embryogenesis resulting in pregnancy and subsequent birth of a healthy neonate. In contrast, circumventing the natural selection mechanisms and all developmental conditions in a healthy animal harbors the risk of counteracting, preventing or reducing the formation of embryos or substantially restricting their genesis. Considering these facts, it seems to be obvious that assisted reproductive techniques focusing on early embryonic stages serve an expanded and unselected germ cell pool of oocytes and sperm cells, and include the culture of embryos outside their natural habitat during and after fertilization for manipulation and diagnostic purposes, and for storage. A significant influence on the early embryonic development is seen in the extracorporeal culture of bovine embryos (in vitro) or stress on the animal organism (in vivo). The in vitro production per se and metabolic as well as endocrine changes in the natural environment of embryos represent adequate models and serve for a better understanding. The purpose of this review is to give a brief presentation of recent techniques aimed at focusing more on the complex processes in the Fallopian tube to contrast in vivo and in vitro prerequisites and abnormalities in early embryonic development and serve to identify potential new ways to make the use of ARTs more feasible.(AU)

Effect of estrous cycle phases on gene expression in bovine oviduct epithelial cells.

Background and Aim: The oviduct environment is of particular importance because it is the site of fertilization and early embryo development. The oviduct, as a component of the reproductive system, responds to ovarian hormone (estradiol [E2] and progesterone [P4]) stimuli depending on the estrous cycle phase. This study aimed to elucidate the effect of estrous cycle phases (follicular and early and late luteal phases) on gene expression patterns in bovine oviduct epithelial cells (BOECs). Materials and Methods: Oviducts were obtained from healthy slaughterhouse animals, corresponding to ipsilateral ovaries with dominant follicles or corpus luteum during early and late luteal phases. BOECs were recovered from the isthmus (IST) and ampulla (AMP), and the expression patterns of genes related to cytokinesis and mitosis mechanisms (rho-associated coiled-coil containing protein kinase and cellular communication network factor 2 [CCN2]), growth factors (insulin-like growth factor-binding protein 3, epidermal growth factor receptor [EGFR], vascular endothelial growth factor A, and EGFR), antioxidant mechanisms (glutathione peroxidase 4 [GPX4]), apoptosis (B-cell lymphoma 2), complement component (C3), energy metabolism (aldose reductase gene family 1-member b1 [AKRIB1] and solute carrier family 2), hormone receptors (estrogen receptor 1 and luteinizing hormone/choriogonadotropin receptor), and specific glycoproteins (oviductal glycoprotein 1) were analyzed. Results: High P4 levels (late luteal phase) affected the expression of important genes related to antioxidant mechanisms (GPX4), energy metabolism (AKRIB1), growth factors (IGBP3 and EGFR), and cell growth regulation (CCN2) in the AMP. Low P4 levels (early luteal phase) affected the expression of AKR1B1, IGBP3, and CCN2. In addition, estrogen likely had an effect on OVPGP expression in the cattle oviduct. Conclusion: Differential gene expression patterns of BOECs in the AMP during the luteal phase (antioxidant mechanisms, energy metabolism, growth factors, and immunological regulators) and in the IST during the follicular phase (glycoproteins) may influence their renewal and population proportions, modulating the oviduct environment as well as gamete and embryo physiology.

Histological changes and transglutaminase 2 expression in the oviduct of advanced pregnant cows.

The oviduct is a dynamic organ that has not been assigned specific functions during advanced pregnancy. However, since changes in the oviductal epithelium during the estrous cycle are attributed mainly to variations in estradiol (E2) levels, and E2 levels increase along pregnancy, we hypothesized that advanced pregnant cows should present changes in the oviductal epithelium. In advanced pregnant cows, the oviducts showed higher leaf-like folds and lower mucosa width and epithelium height than those of cycling animals. Also, PAS-positive apical protrusions and TUNEL-positive extruded cytoplasmic material were observed in advanced pregnant cows. Oviductal fluid from advanced pregnant cows showed lower protein concentration than that from cycling cows. Transglutaminase 2 (TG2) was detected exclusively in oviductal fluid of pregnant cows but not in cells from any stage, whereas its mRNA was detected in different amounts in cells from all stages. This protein was identified by LC/MS-MS and its identity was corroborated by Western blot. The observations in histology of the epithelium and the presence of TG2 in oviductal fluid correlate with high levels of E2 in serum. In conclusion, important histological changes in the oviductal epithelium and secretion of TG2 to the oviductal fluid appear to be triggered by the high E2 levels exclusive of advanced pregnancy.

A stallion spermatozoon's journey through the mare's genital tract: In vivo and in vitro aspects of sperm capacitation.

Conventional in vitro fertilization is not efficacious when working with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, these gametes fail to initiate the acrosome reaction in the vicinity of the oocyte and cannot, therefore, penetrate into the perivitelline space. Failure of sperm penetration most likely relates to the absence of optimized in vitro fertilization media containing molecules essential to support stallion sperm capacitation. In vivo, the female reproductive tract, especially the oviductal lumen, provides an environmental milieu that appropriately regulates interactions between the gametes and promotes fertilization. Identifying these 'fertilization supporting factors' would be a great contribution for development of equine in vitro fertilization media. In this review, a description of the current understanding of the interactions stallion spermatozoa undergo during passage through the female genital tract, and related specific molecular changes that occur at the sperm plasma membrane is provided. Understanding these molecular changes may hold essential clues to achieving successful in vitro fertilization with equine gametes.

Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we established an in vitro OEC culture model to select a sperm population with potential fertilizing capacity in this species. We demonstrated E-cadherin and cytokeratin expression in cultures of OECs obtained. When sperm-OEC cocultures were performed, the attached spermatozoa were motile and presented an intact acrosome, suggesting a selection by the oviductal model. When co-cultures were incubated in capacitating conditions a greater number of alive (p < 0.05), capacitated (p < 0.05), with progressive motility (p < 0.05) and with the intact acrosome sperm population was observed (p < 0.05) suggesting that the sperm population released from OECs in vitro presents potential fertilizing capacity. Improvements in handling and selection of cryopreserved sperm would improve efficiencies in ARTs allowing the use of a population of higher-quality sperm.

A long-term in vitro culture of bovine epithelial cells on collagen rafts.

In the present work, we established and characterized a 3D functional polarized primary bovine oviduct epithelial cells (BOECs) culture on free-floating type I collagen hydrogels (rafts) at an air-liquid interface (ALI). Intercellular junctions, ultrastructural cellular morphology and the expression of the OVGP1 closely recapitulated those of the in vivo epithelium lining. These morphological and physiological epithelial cell features were maintained under standard DMEM/F12 with 10% foetal bovine serum culture medium for at least 28 days of ALI culture. The versatility of the BOECs raft cultures should allow testing of toxicity compounds, in vitro evaluation of physiological or pathological oviductal states, and the study of epithelial-mesenchymal interactions that are critical for the maintenance of oviductal homeostasis.

In vitro sperm storage with poultry oviductal secretions.

In the hen oviduct, tubules have been identified that preserve the sperm, maintaining viability for up to 15 weeks. This study aimed to evaluate the physiological status of rooster sperm when preserved in vitro with uterus vaginal junction secretions (UVJS). Males and females of the Rhode Island breed were used. Sperm aliquots were prepared using Lake extender and Lake extender with UVJS (10.00%, 30.00%, 60.00%, and 90.00%). Subsequently, a basic sperm evaluation was performed and sperm physiological status was determined through the presence and distribution of Ca2+ and its acrosomal reaction capability via perivitelline layer (PVL) co-incubation. It was observed that motility was decreased in sperm preserved with UVJS at 6 and 24 hr) compared to 40 min and fresh semen. The sperm decapacitation percentage was increased when preserved with UVJS at 40 min, 6 and 24 hr compared to fresh semen. The acrosomal reaction was increased in sperm co-incubated with PVL, even when preserved with UVJS. It was concluded that UVJS induced physiological changes in sperm by inducing a decapacitation process, which increased sperm viability when preserved in vitro.

Effect of mating on mRNA and protein expression of beta nerve growth factor and its receptor, TrKA, in the oviduct of llama (Lama glama).

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of ß-nerve growth factor (ß-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. ß-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both ß-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative ß-NGF abundance was higher in the UTJ of copulated females (p < .05). ß-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the ß-NGF/TrKA system and that an increase in ß-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas.

Histopathological characterization of the reproductive organs of heifers experimentally infected with Campylobacter fetus venerealis / Caracterización histopatológica de los órganos reproductivos de vaquillonas experimentalmente infectadas con Campylobacter fetus venerealis / Caracterização histopatológica em órgãos reprodutores de novilhas infetadas no modo experimental com Campylobacter fetus venerealis

Abstract Background: Bovine campylobacteriosis is a venereal disease due to infection with Campylobacter fetus venerealis. It causes mainly reproductive failures that lead to considerable economic losses. Objective: To perform a histopathological description of the mucosa from reproductive organs of heifers experimentally infected with Campylobacter fetus venerealis. Methods: Twelve 15-18-months-old Aberdeen Angus heifers were treated for estrous synchronization and exposed to natural breeding. They were then randomly divided into two groups: group A (n=9) was inoculated with C. fetus venerealis; group B (n=3, control) was inoculated with a placebo. Ultrasonography was performed at days 29, 38, and 42 post-breeding, and plasmatic progesterone levels were quantified using ELISA to confirm pregnancies. Animals in group A with plasma progesterone levels below 1 ng/mL and/or diagnosed as non-pregnant were further divided into three subgroups: A1 (n=4), euthanized at day 30 post-breeding; A2 (n=3), euthanized at day 40 post-breeding and A3 (n=2), euthanized at day 55 post-breeding. Heifers from group B, all diagnosed as pregnant, were euthanized each at day 30, 40, and 55 days post-breeding as well. Histological sections from every group were taken from oviducts, uterus, and vagina. Results: Lymphocytic inflammation was the most common lesion in all infected heifers. Trophoblast cells were found in the non-pregnant heifers euthanized at days 40, and 55 post-breeding. The inflammatory process with the presence of lymphoid cells probably altered the balance in the activity of maternal lymphoid cells, as well as gene expression of the trophoblast, finally affecting the embryo survival. Conclusion: This work contributes to the understanding of the histopathological process involved in post-mating infection of Campylobacter fetus bovine.

Resumen Antecedentes: La campilobacteriosis bovina es una enfermedad venérea causada por el Campylobacter fetus venerealis, que produce principalmente fallas reproductivas ocasionando grandes pérdidas económicas Objetivo: Describir las características histopatológicas de la mucosa de órganos reproductores de vaquillonas infectadas experimentalmente con Campylobacter fetus venerealis. Métodos: Doce vaquillonas Aberdeen Angus (15 a 18 meses de edad) con celo sincronizado, recibieron servicio natural, e inmediatamente se dividieron aleatoriamente en dos grupos: A (n=9), inoculadas con Campylobacter fetus venerealis; B (n=3; control), inoculadas con placebo. El diagnóstico de preñez se realizó por ultrasonografía a los 29, 38 y 42 días post-servicio; los niveles plasmáticos de progesterona fueron determinados por ELISA. Las vaquillonas del grupo A con niveles de progesterona plasmáticos menores a 1 ng/mL y/o diagnosticadas no preñadas, fueron consideradas para eutanasia y divididas en tres subgrupos: A1-eutanasia día 30 (n=4); A2-día 40 (n=3); y A3-día 55 (n=2) post-servicio. Las vaquillonas del grupo B, diagnosticadas preñadas, fueron eutanasiadas a los 30, 40 y 55 días. Se tomaron muestras de oviductos, útero y vagina. Resultados: Se observó inflamación linfocitaria en la totalidad de muestras del grupo A. Células trofoblásticas fueron encontradas en muestras correspondientes a los grupos A2 y A3. Probablemente, el proceso inflamatorio alteró el equilibrio de las células linfoides maternas y la expresión génica del trofoblasto, afectando la supervivencia embrionaria. Conclusión: Este trabajo contribuye a la comprensión del proceso histopatológico involucrado en la infección poscoital por Campylobacter fetus bovino.

Resumo Antecedentes: A campilobacteriose bovina é uma doença venérea originada pelo Campylobacter fetus venerealis, quem produz principalmente falha reprodutiva e porém grandes perdas económicas. Objetivo: Descrever as características histopatológicas da mucosa dos órgãos reprodutores de novilhas infetadas no modo experimental com Campylobacter fetus venerealis. Métodos: Doze novilhas Aberdeen Angus de 15 até 18 meses com cio sincronizado, receberam serviço natural. Logo após, foram aleatóreamente separados em grupos: A (n=9) inoculados com Campylobacter fetus venerealis e grupo B (n=3; controle) inoculadas com um placebo. O diagnóstico da gestação foi realizado por ultrasom nos dias 29, 38 y 42 pós-serviço. Os níveis plasmáticos da progesterona foram determinados por ELISA. As novilhas do grupo A, com níveis plasmáticos de progesterona menores a 1 ng/mL e/ou diagnosticadas não grávidas, foram consideradas para eutanásia e foram divididas em três subgrupos: A1-eutanásia aos 30 dias pós- serviço (n=4); A2-dia 40 (n=3); A3-dia 55 (n=2). Foram realizada eutanásia ás novilhas do grupo B diagnosticadas prenhadas, aos 30, 40 e 55 dias e a amostragem de ovidutos, útero e vagina. Resultados: A presença de inflamação linfocitária foi observada na totalidade das amostras do grupo A. Foram achadas células trofoblásticas nas amostras correspondente aos grupos A2 e A3. Provavelmente, pelo processo inflamatório tenha sido alterado o equilíbrio das células linfoides maternas, assim também como a expressão gênica do trofoblasto, afetando a supervivência embrionária. Conclusão: Este trabalho contribue á compreensão do processo histopatologico na infecção com Campylobacter fetus bovino pós-acasalamento.

Impact of extracellular folic acid levels on oviductal gene expression.

Folate plays a specific role as methyl donor for nucleotide synthesis and genomic methylation patterns, which in turn are important epigenetic determinants in gene expression. Previous studies have revealed the presence of folate in bovine oviductal fluid as well as the existence of a fine-tuned regulation of the gene expression of folate receptors and transporters in bovine oviduct epithelial cells (BOECs). However, the functional implications of folate in the oviduct remain unknown. The present study aimed to assess the effect of folic acid (FA) on expression levels of selected genes that potentially respond to the folate status in in vitro BOECs. To obtain an insight into the optimization of a culture system for assays, gene expression of folate receptors and transporters was compared between BOECs grown in monolayers and in suspension. The results showed that BOECs from isthmus and ampulla in suspension culture better preserved the region-dependent gene expression profile than in monolayers. Subsequently, BOECs from both anatomical regions were separately cultured in suspension for 24 h assaying different FA concentrations: I) TCM-199 (control); II) TCM-199 + 1 µM FA (similar to the oviduct concentration); III) TCM-199 + 10 µM FA and IV) TCM-199 + 100 µM FA. Expression analysis of genes related to important cellular processes including folate transport, DNA methylation, cell-cell interaction, antioxidant activity and signaling pathways was performed in BOECs using RT-qPCR. Our data demonstrated that addition of 1 µM FA did not affect mRNA levels of most genes analyzed. In contrast, BOECs cultured with 10 µM FA exhibited increased mRNA expression levels of genes involved in folate intake, DNA methylation and antioxidant protection. It is worth noting that at 100 µM FA, transcriptional response in BOECs mainly resulted in decreased mRNA levels of the majority of the genes assayed. Interestingly, cytotoxicity analysis showed a similar LDH activity in the culture media of the experimental groups, indicating that cell integrity was not affected by the FA concentrations assayed. In conclusion, our findings suggest that folate can affect BOECs, promoting changes in gene activity in a framework of functional readjustments in response to environmental conditions.

Local influence of the corpus luteum on the ipsilateral oviduct and early embryo development in the ewe.

The objective of this study was to evaluate the local effect of the corpus luteum (CL) on ipsilateral oviduct-uterus functionality and early embryo development in ewes. A total of 499 embryos were transferred on Day 1 after in vitro fertilization into the ipsilateral (n = 250) and contralateral oviducts (n = 249) of 13 ewes on Day 1 after ovulation (18-20 embryos per oviduct). On Day 6, their reproductive tracts were collected and their uterine horns were flushed for embryo recovery. More recovered embryos, a higher proportion of blastocysts, and more viable embryos were collected when the embryos were transferred into the ipsilateral oviducts (P < 0.05). In addition, almost five times higher P4 concentrations and significantly lower E2 concentrations, with higher P4:E2 ratio, were found in the ipsilateral than contralateral oviductal tissue (P < 0.05). Furthermore, a higher concentration of adiponectin was found in the ipsilateral uterine tissue macerates than in the contralateral side to the CL. The ipsilateral oviductal tissue had a lower expression of PGR and IGFBP5, but the transcript expression of ADIPOR1 was higher in the ipsilateral oviductal tissue. In the uterus, the mRNA expression of ESR1, IGFBP3, IGFBP5, and LEPR was higher or tended to be higher in the ipsilateral than contralateral uterine tissue. Uterine flushing fluid collected from the ipsilateral uterine horn had lower insulin concentrations than the contralateral horn, while no differences were found in the P4 and E2 concentrations. In conclusion, on Day 6 post-ovulation, P4 was elevated in the ipsilateral oviductal tissue, embryo development was advanced, and differential gene expression of PGR, ESR1, IGFBP3, IGFBP5, LEPR, and ADIPOR1 in the oviductal or uterine tissue was found between the ipsilateral and contralateral side. This study demonstrates local regulation of the ovary on the ipsilateral oviduct/uterine horn in the ewe.