Mammalian Fluid Research Articles

In vitro approach points to a chemotactic effect of melatonin on ram spermatozoa

Sperm orientation mechanisms, such as chemotaxis, are essential for the sperm to reach the oocyte and fertilize it. Melatonin is secreted by the cumulus cells and is also present in the follicular fluid in mammals. The presence of membrane receptors for melatonin in ram spermatozoa, and its proven involvement in the sperm functionality, may suggest a possible role in the guided movement towards the oocyte. Hence, the objective of the present work is to study the in vitro potential chemotactic action of melatonin on ram spermatozoa, analysing the influence of the season (breeding and non-breeding) and the sperm capacitation state. The first experimental approach consisted in the inclusion of melatonin in the upper layer of a swim-up selection method. During the non-breeding season, the presence of melatonin at 100 pM and 1 μM concentrations significantly increased the cell recovery rate, and induced changes in the sperm location of the MT2 melatonin receptor, compared with the standard swim-up. Moreover, the selected sperm population with 100 pM melatonin presented a higher percentage of capacitated spermatozoa. The greater recovery rate obtained with melatonin could be due to the stimulation of sperm movement in random directions, i.e., a chemokinetic effect, or due to a guided movement (chemotaxis) towards the gradient of the melatonin. To elucidate this issue, together with the study of the influence of the sperm capacitation status, we performed a second experimental approach which consisted in the use of chemotaxis chambers and an open-source software (Open-CASA) that analyses the sperm trajectories towards the hormone gradient and calculates a chemotaxis index (SL index). There was a significant difference between the SL index in the presence of 1 μM melatonin and the control without hormone. This effect was only observed in capacitated spermatozoa with cAMP-elevating agents (Cap-CK samples) obtained during the non-breeding season. These results would point to an in vitro chemotactic effect of melatonin on ram spermatozoa, although chemokinesis cannot be ruled out. Nonetheless, the inclusion of this hormone in the swim-up procedure could enhance the sperm recovery rate.

Analysis of post-translational modifications in soluble proteins involved in chemical communication from mammals and insects.

Different soluble, small polypeptide molecules isolated from biological tissues/body fluids of mammals and insects have been reported to bind odorant compounds and pheromones, and have been accordingly named odorant-binding proteins, pheromone-binding proteins and chemosensory proteins. Mammal and insect odorant-binding and pheromone-binding proteins show evident differences in their primary structure; in insects, the same condition occurs for odorant- and pheromone-binding proteins, which belong to the same protein family, and chemosensory proteins. In order to understand their molecular characteristics, these proteins have been subjected to an extensive analysis for their post-translational modifications through mass spectrometry approaches. Depending on proteins, they have been characterized as having specific disulfide bridge arrangements, which were consistent among molecular sequence homologs. Only selected odorant-binding proteins from mammals have been demonstrated bearing N-linked glycan structures and other modifications. In this review article, we discuss the application of mass spectrometry procedures for the analysis of post-translational modifications in odorant-binding proteins, pheromone-binding proteins and chemosensory proteins. Different example proteins are reported, and protocols are presented for obtaining definitive information in this context.

Gene expression to contaminated or not contaminated Triatoma dimidiata faeces with Trypanosoma cruzi in vivo and in vitro assay.

Triatoma dimidiata is considered one of the most important vectors in the spread of Chagas disease in Mesoamerica. Hematophagous insects may deposit faeces contaminated or not with Trypanosoma cruzi on the surface of the skin during the feeding process. In endemic areas humans can be in contact several times to this fluid before get infection. Due to limited work on the of immune response to insect's fluids in mammals. In this work, we explored the early response of chemokine and cytokines of skin tissue at the inoculation site against faeces from T. dimidiata contaminated or not with T. cruzi in murine model, also we explored the in vitro response of epitheloid cells line "HeLa cells" to this fluid. Mice were exposed intradermically to faeces free of metacyclic trypomastigotes, oneor multiple times along the experiment before challenged with faeces contaminated with metacyclic trypomastigotes, mimicking natural conditions. In in vitro assay with HeLa cells only one exposure was carried out. It was observed that mice multiexposed to faeces, induced the strong response of TNF-α and CXCL3 expression at the inoculation site; but faeces contaminated with metacyclic trypomastigotes lower the response of TNF-α, and CXCL3. In in vitro assay with HeLa cell line, the faeces induced a strong response of CXCL8 and IL-6, but the presence of faeces contaminated with metacyclic trypomastigotes lower the response of CXCL8. In contrast, to in vivo model null response of TNF α and IL-1-α was observed in HeLa cells. In conclusion, Triatoma dimidiata faeces induces at the inoculation site chemokines that mediate preferentially chemotaxis of neutrophils and macrophages but the presence of metacyclic trypomastigotes moderate their induction. Epithelial cells act as efficient sentinels producing chemokines that may recruit neutrophils and monocytes.

Composing the Early Embryonic Microenvironment: Physiology and Regulation of Oviductal Secretions.

The oviductal fluid is the first environment experienced by mammalian embryos at the very beginning of life. However, it has long been believed that the oviductal environment was not essential for proper embryonic development. Successful establishment of in vitro embryo production techniques (which completely bypass the oviduct) have reinforced this idea. Yet, it became evident that in vitro produced embryos differ markedly from their in vivo counterparts, and these differences are associated with lower pregnancy outcomes and more health issues after birth. Nowadays, researchers consider the oviduct as the most suitable microenvironment for early embryonic development and a substantial effort is made to understand its dynamic, species-specific functions. In this review, we touch on the origin and molecular components of the oviductal fluid in mammals, where recent progress has been made thanks to the wider use of mass spectrometry techniques. Some of the factors and processes known to regulate oviductal secretions, including the embryo itself, as well as ovulation, insemination, endogenous and exogenous hormones, and metabolic and heat stress, are summarized. Special emphasis is laid on farm animals because, owing to the availability of sample material and the economic importance of fertility in livestock husbandry, a large part of the work on this topic has been carried out in domestic animals used for dairy and/or meat production.

Profile of bile acid subspecies is similar in blood and follicular fluid of cattle.

The composition of follicular fluid (FF) has an impact on the developmental capacity of the oocyte and the resulting embryo. FF is composed of blood plasma constituents which cross the blood follicular barrier and the secretory components of granulosa and theca cells. Moreover, it has been shown recently that follicular cells have the ability to synthesize bile acids (BAs). BAs are present in several fluids of mammals especially in bile, blood and urine. FF is an essential impacting factor on the oocyte quality and therefore resulting embryos. To achieve a better understanding of this subject, the presence and concentration of BAs were measured in fluid collected from bovine follicles, categorized according to their size, throughout two entire oestrus cycles and compared to those in blood and urine. The body fluids were collected during the same examination procedure and in total samples from four heifers were obtained. A broad spectrum of 11 BA derivatives was measured applying liquid chromatography–tandem mass spectrometry (LC‐MS/MS). The simultaneous and direct quantification of BAs in different body fluids of cattle are reported. Within the follicular fluid, blood and urine, cholic acid and glycocholic acid are the dominant BA subspecies irrespective of the oestrus cycle stage. Moreover, BA concentrations in blood compared to those in the FF were similar. For the first time these results clearly highlight the presence of different BA subspecies in FF, blood and urine during the oestrus cycle in cattle.

Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes.

Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media.

High Potassium Concentration during Culturing of Early Mammalian Embryos: Normal or Extreme Situation?

Analysis of the element composition of oviduct and uterine fluid in mammals showed high potassium concentrations in the early embryo microenvironment in vivo. The results of early embryogenesis of mammals in vitro in the presence of high potassium concentrations are discussed. The data are summarized in accordance with the conditions of experimentally modeled pre-implantation development. Comparative assessment of the quality of embryo development until the blastocyst stage in vitro proved the embryos more successfully developed at potassium concentrations close to those registered in the oviductal fluid.

Regulation and roles of the hyaluronan system in mammalian reproduction.

Hyaluronan (HA) is a non-sulphated glycosaminoglycan polymer naturally occurring in many tissues and fluids of mammals, including the reproductive system. Its biosynthesis by HA synthase (HAS1-3) and catabolism by hyaluronidases (HYALs) are affected by ovarian steroid hormones. Depending upon its molecular size, HA functions both as a structural component of tissues in the form of high-molecular-weight HA or as a signalling molecule in the form of small HA molecules or HA fragments with effects mediated through interaction with its specific cell-membrane receptors. HA is produced by oocytes and embryos and in various segments of the reproductive system. This review provides information about the expression and function of members of the HA system, including HAS, HYALs and HA receptors. We examine their role in various processes from folliculogenesis through oocyte maturation, fertilisation and early embryo development, to pregnancy and cervical dilation, as well as its application in assisted reproduction technologies. Particular emphasis has been placed upon the role of the HA system in pre-implantation embryo development and embryo implantation, for which we propose a hypothetical sequential model.

Seminal Fluid Signalling in the Female Reproductive Tract: Implications for Reproductive Success and Offspring Health.

Carriage of sperm is not the only function of seminal fluid in mammals. Studies in mice show that at conception, seminal fluid interacts with the female reproductive tract to induce responses which influence whether or not pregnancy will occur, and to set in train effects that help shape subsequent fetal development. In particular, seminal fluid initiates female immune adaptation processes required to tolerate male transplantation antigens present in seminal fluid and inherited by the conceptus. A tolerogenic immune environment to facilitate pregnancy depends on regulatory T cells (Treg cells), which recognise male antigens and function to suppress inflammation and immune rejection responses. The female response to seminal fluid stimulates the generation of Treg cells that protect the conceptus from inflammatory damage, to support implantation and placental development. Seminal fluid also elicits molecular and cellular changes in the oviduct and endometrium that directly promote embryo development and implantation competence. The plasma fraction of seminal fluid plays a key role in this process with soluble factors, including TGFB, prostaglandin-E, and TLR4 ligands, demonstrated to contribute to the peri-conception immune environment. Recent studies show that conception in the absence of seminal plasma in mice impairs embryo development and alters fetal development to impact the phenotype of offspring, with adverse effects on adult metabolic function particularly in males. This review summarises our current understanding of the molecular responses to seminal fluid and how this contributes to the establishment of pregnancy, generation of an immune-regulatory environment and programming long-term offspring health.

Responsiveness of Human Neonates to the Odor of 5α-Androst-16-en-3-one: A Behavioral Paradox?

The odorous steroid 5α-androst-16-en-3-one (AND) occurs in numerous biological fluids in mammals, including man, where it is believed to play a chemocommunicative role. As AND was recently detected in milk and amniotic fluid, sensitivity and hedonic responses to this substance were assessed in human neonates. To this aim, respiration and facial expressions were recorded in 3-day-old newborns in response to aqueous solutions of AND, ranging from 500ng/mL to 0.5 fg/mL. Although analyses of respiratory rate did not lead to clear-cut results, the newborns changed their facial expressions at concentrations not detected by adults in a triangle test. Newborns displayed negative facial actions of longer duration to AND relative to an odorless control. Thus, AND may be considered to be offensive to newborns, which is a counterintuitive outcome as they are exposed to this compound in the womb (and it should therefore be familiar), in milk, and on the mother's skin surface (and it should therefore be conditioned as positive). Multiple reasons for this perceptual-behavioral paradox are discussed.

Phytate levels in biological fluids of mammals

Phytate levels in biological fluids of mammals

Antisecretory factor as a potential health-promoting molecule in man and animals

Antisecretory factor (AF) is a protein secreted in plasma and other tissue fluids in mammals with proven antisecretory and anti-inflammatory activity; its immunohistological distribution suggests a role in the immune system. The expression level and the distribution of AF protein are altered during an immunological response. Exposure to bacterial toxins induces secretion of AF in plasma, probably reflecting a natural defence mechanism to agents causing diarrhoea, thereby contributing to a favourable clinical outcome and disease termination. An increase of AF levels in plasma by dietary means, such as specially processed cereals (SPC), has been demonstrated in human subjects and animals. Administration of SPC to patients affected by inflammatory bowel disease, gastroenteritis and Ménière's disease relieved symptoms and improved quality of life. A recent study showed the positive effect of SPC diet supplementation on prevention of the effects of exposure to low levels of blast overpressure in rats, reducing the extent of intracranial pressure increase and cognitive function impairment. AF-rich egg yolk powder improved health status in children suffering acute and chronic diarrhoea, reducing the frequency and increasing the consistency of stools. This kind of functional food could be used for prophylaxis in populations exposed to a high risk of morbidity and mortality caused by diarrhoea and as a complementary therapy in patients affected by chronic intestinal inflammatory disease to improve well-being. In pig husbandry AF-inducing diets, owing to their antisecretory activity and anti-inflammatory action, are a suitable option as an alternative to antibiotic growth promoters to counteract post-weaning diarrhoea.

Early embryonic brain development in rats requires the trophic influence of cerebrospinal fluid

Cerebrospinal fluid has shown itself to be an essential brain component during development. This is particularly evident at the earliest stages of development where a lot of research, performed mainly in chick embryos, supports the evidence that cerebrospinal fluid is involved in different mechanisms controlling brain growth and morphogenesis, by exerting a trophic effect on neuroepithelial precursor cells (NPC) involved in controlling the behaviour of these cells. Despite it being known that cerebrospinal fluid in mammals is directly involved in corticogenesis at fetal stages, the influence of cerebrospinal fluid on the activity of NPC at the earliest stages of brain development has not been demonstrated. Here, using “in vitro” organotypic cultures of rat embryo brain neuroepithelium in order to expose NPC to or deprive them of cerebrospinal fluid, we show that the neuroepithelium needs the trophic influence of cerebrospinal fluid to undergo normal rates of cell survival, replication and neurogenesis, suggesting that NPC are not self-sufficient to induce their normal activity. This data shows that cerebrospinal fluid is an essential component in chick and rat early brain development, suggesting that its influence could be constant in higher vertebrates.

D-amino acid analysis.

AbstractIn nature D-amino acids occur rarely as compared with their enantiomers. Their presence has been observed in several tissues and fluids of mammals (1), molluscs (2), invertebrates (3) and plants (4), as well as in microbial fermented foods as result of bacterial metabolism (5). Gram-positive and -negative bacteria contain D-amino acids as fundamental components of cell-wall peptidoglycans (6). Similarly, they have been found inserted in polypeptide chains of several antibiotics synthesized by microbial multienzymatic complexes (7) or of bioactive peptides of ribosomal origin isolated from various vertebrates (8). In the latter case, their presence, which is essential to exert the specific biological activity, is the result of a post-translational modification occurring on larger precursors encoded by mRNAs where the base triplet coding for the L-amino acid is present at the position occupied by the corresponding D-enantiomer in the mature product (9). Furthermore, in proteins the presence of small quantities of D-amino acids is considered to be the result of a racemization process related to molecular aging (10). Recently, the occurrence of D-serine in mammalian brain has been described and its role as neuromodulator has been proposed; subsequently, serine racemase, a glial pyridoxal phosphate-dependent enzyme synthesizing D-serine, has been cloned and expressed (11). Thus, D-amino acids appear to be related to many important biological processes, but the poor diffusion in protein chemistry laboratories of methodologies and instruments dedicated to D-amino acid detection makes their occurrence in nature still underestimated. KeywordsImportant Biological ProcessSerine RacemaseAmide MoietySpecific Biological ActivityAmino Acid EnantiomerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Anti-Inflammatory Effects of Mutant Forms of Secretory Leukocyte Protease Inhibitor

The secretory leukocyte protease inhibitor (SLPI) is found in a variety of secreted fluids in mammals and is a known inhibitor of serine proteases. Wild-type (WT) SLPI has recently been shown to block nuclear factor kappaB (NF-kappaB) activation in rat lungs and to interfere with the ensuing inflammatory response and recruitment of neutrophils after an intrapulmonary deposition of IgG immune complexes. In this study, WT SLPI and SLPI mutants with various degrees of protease-inhibitory capacity (for trypsin, chymotrypsin, and elastase) were evaluated for their ability to suppress the lung-vascular leak, neutrophil accumulation, and NF-kappaB activation in the lung inflammatory model. The SLPI mutant with Gly(72) (replacing Leu(72) ) lost its ability to block in vivo activation of NF-kappaB, as well as its ability to suppress the lung vascular leak and neutrophil recruitment. The Phe(72) and Gly(20) mutants were as effective as the WT SLPI in suppressing NF-kappaB activation and neutrophil recruitment. The Lys(72) mutant had the most suppressive effects of the lung vascular leak and for neutrophil recruitment into the lung. The in vivo suppressive effects of SLPI mutants on lung vascular permeability, neutrophil recruitment, and NF-kappaB activation appear to be most closely related to their trypsin-inhibiting activity. These data suggest that the suppressive effects of SLPI on the intrapulmonary activation of NF-kappaB and neutrophil recruitment into the lung may be linked to their antiprotease activity, directed, perhaps, at the intracellular proteases.

Distribution of free and conjugated dopamine in rats and mice

Free and conjugated dopamine occurs in many tissues and fluids of mammals. We have used reversed phase liquid chromatography with electrochemical detection in sequence with an alumina adsorption procedure to separate and then detect amounts of free and conjugated dopamine in the major visceral organs and brain of the rat and mouse. We report those results here. Values for free and conjugated dopamine are given for all tissues studied. Conjugated DA was found in the rat kidney, liver and adrenal gland and in the kidney of the mouse. Those tissues which have a significant amount of conjugated dopamine will be studied further for the presence of dopamine sulfate.

III.—On the Source of the Amniotic and Allantoic Fluids in Mammals

In all vertebrates, as the embryo develops at one pole of the egg, it sinks inwards till it becomes completely enclosed in a hood or sac filled with fluid, the amnion. When the sac or true amnion has completely closed over the embryo, it becomes separated from the outer layer, which remains in contact with the covering of the egg as the false or chorionic amnion.