Oxidative potential of two Brazilian endophytic fungi from Handroanthus impetiginosus towards progesterone.

Biotransformation has been successfully employed to conduct uncommon reactions, which would hardly be carried out by chemical synthesis. A wide diversity of compounds may be metabolized by fungi, leading to chemical derivatives through selective reactions that work under ecofriendly conditions. Endophytic fungi live inside vegetal tissues without causing damage to the host plant, making available unique enzymes for interesting chemical derivatization. Biotransformation of steroids by endophytic fungi may provide new derivatives as these microorganisms came from uncommon and underexplored habitats. In this study, endophytic strains isolated from Handroanthus impetiginosus leaves were assayed for biotransformation of progesterone, and its derivatives were identified through GC-EI-MS analysis. The endophyte Talaromyces sp. H4 was capable of transforming the steroidal nucleus selectively into four products through selective ene-reduction of the C4-C5 double bond and C-17 oxidation. The best conversion rate of progesterone (>90 %) was reached with Penicillium citrinum H7 endophytic strain that transformed the substrate into one derivative. The results highlight endophytic fungi's potential to obtain new and interesting steroidal derivatizations.

Towards the Importance of a Roller Mill in Compounding Practice: An Experimental Approach.

There are a substantial amount of suppliers for roller mills in the market, but there is a lack of scientific evidence of a roller mill's capacity to improve particle size reduction/distribution or homogenization. In this concise paper, we evaluate the use a roller mill in the final steps of compounding semisolid dosage forms. We performed three simple tests to verify these claims: 1) particle size evaluation through dynamic light scattering and scanning electron microscopy techniques, 2) content uniformity through high-performance liquid chromatography technique, and 3) cross contamination through a cleaning validation method. Dynamic light scattering and scanning electron microscopy techniques of benzoyl peroxide 5% (gel) and testosterone 1% (cream) showed a significant reduction on particle diameter. Content uniformity testing of creams containing progesterone 5%, estradiol 0.1%, and estriol 0.4% showed better homogeneity when using the roller mill. Finally, the proposed cleaning procedure decreased the presence of the compounded preparation to a "none-detection" level after the procedure. This suggests that the roller mill used does, in fact, play a role in the final aspect and quality of pharmaceutical semisolid dosage forms.

Allopregnanolone and Progesterone in Experimental Neuropathic Pain: Former and New Insights with a Translational Perspective.

In the last decades, an active and stimulating area of research has been devoted to explore the role of neuroactive steroids in pain modulation. Despite challenges, these studies have clearly contributed to unravel the multiple and complex actions and potential mechanisms underlying steroid effects in several experimental conditions that mimic human chronic pain states. Based on the available data, this review focuses mainly on progesterone and its reduced derivative allopregnanolone (also called 3α,5α-tetrahydroprogesterone) which have been shown to prevent or even reverse the complex maladaptive changes and pain behaviors that arise in the nervous system after injury or disease. Because the characterization of new related molecules with improved specificity and enhanced pharmacological profiles may represent a crucial step to develop more efficient steroid-based therapies, we have also discussed the potential of novel synthetic analogs of allopregnanolone as valuable molecules for the treatment of neuropathic pain.

Changes in mucins and matrix metalloproteases in the endometrium of early pregnant alpacas (Vicugna pacos).

South American Camelids (SAC) have unique reproductive features, one of which is that 98% of the pregnancies develop in the left uterine horn. Furthermore, early pregnancy is an uncharacterized process in these species, especially in regard to the ultrastructural, biochemical and genetic changes that the uterine epithelial surface undergoes to allow embryo implantation. The present study describes the uterine horn luminal surface and the characteristics of the mucinous glycocalyx in non-pregnant and early pregnant (15 days) female alpacas. In addition, the relative abundance of Mucin 1 and 16 genes (MUC1 and MUC16) was determined, as well as the relative mRNA abundance of matrix metalloproteinases (MMPs) that could be involved in MUC shedding during early pregnancy. Noticeable changes were detected in the uterine luminal epithelium and glycocalyx of pregnant alpacas in comparison to non-pregnant ones, as well as presence of MUCs and MMPs in the endometrial environment. The decrease in glycocalyx staining and in the relative abundance of MUC 1 and MUC 16 transcripts in pregnant females would allow embryo attachment to the luminal epithelium and its subsequent implantation, as has been described in other mammals. These results suggest a crucial role of MUC1 and MUC16 and a possible role of MMPs in successful embryo implantation and survival in alpacas.

Nonenzymatic electrochemical sensor based on imidazole-functionalized graphene oxide for progesterone detection.

The modification of electrode surfaces has been the target of study for many researchers in order to improve the analytical performance of electrochemical sensors. Herein, the use of an imidazole-functionalized graphene oxide (GO-IMZ) as an artificial enzymatic active site for voltammetric determination of progesterone (P4) is described for the first time. The morphology and electrochemical performance of electrode modified with GO-IMZ were characterized by scanning electron microscopy and cyclic voltammetry, respectively. Under optimized conditions, the proposed sensor showed a synergistic effect of the GO sheets and the imidazole groups anchored on its backbone, which promoted a significant enhancement on electrochemical reduction of P4. Figures of merits such as linear dynamic response for P4 concentration ranging from 0.22 to 14.0 µmol L-1, limit of detection of 68 nmol L-1 and limit of quantification and 210 nmol L-1 were found. In addition, presented a higher sensitivity, 426 nA L µmol-1, when compared to the unmodified electrode. Overall, the proposed device showed to be a promising platform for a simple, rapid, and direct analysis of progesterone.

Effect of particle size, polydispersity and polymer degradation on progesterone release from PLGA microparticles: Experimental and mathematical modeling.

Poly(lactic-co-glycolic acid) (PLGA) microparticles containing progesterone were prepared by the solvent extraction/evaporation and microfluidic techniques. Microparticles were characterized by their size distribution, encapsulation efficiency, morphology and thermal properties. The effect of particle size, polydispersity and polymer degradation on the in vitro release of the hormone was studied. A triphasic release profile was observed for larger microparticles, while smaller microspheres showed a biphasic release profile. This behavior is related to the fact that complete drug release was achieved in a few days for smaller microparticles, during which polymer degradation effects are still negligible. A mathematical model was developed that predicts the progesterone release profiles from different-sized PLGA microspheres. The model takes into account both the dissolution and diffusion of the drug in the polymeric matrix as well as the autocatalytic effect of polymer degradation. The model was adjusted and validated with novel experimental data. Simulation results are in very good agreement with experimental results.

21-Hydroxy-6,19-epoxyprogesterone: A Promising Therapeutic Agent and a Molecular Tool for Deciphering Glucocorticoid Action.

Glucocorticoids are steroid hormones that exert most of their effects through their binding to the glucocorticoid receptor (GR), a ligand regulated transcription factor. Although glucocorticoids are widely used in the clinic, their usage in chronic therapies provokes severe adverse reactions. In the quest for safer glucocorticoids a dissociated model was established that proposes a disconnection between GR activated pathways responsible of desired pharmacological effects and pathways involved in adverse GR reactions. Under this model, a myriad of steroidal and non-steroidal compounds has been characterized, with most of them still producing side effects. X-ray crystallographic studies followed by molecular dynamics analysis led research to insights on the receptor Ligand Binding Domain (LBD), which undergoes specific ligand dependent conformational changes that influence receptor activities. In this sense, the flexibility of the ligand structure would contribute to the final GR outcome. Here, we review different data of 21-hydroxy-6,19-epoxyprogesterone (21OH-6,19OP), a rigid steroid with potential pharmaceutical interest due to its anti-inflammatory and immunosuppressive activities, lacking several GR adverse reactions. The rigid structure endows this compound with an enhanced selectivity towards GR. Molecular characterization of the GR/21OH-6,19OP complex revealed specific intermediate conformations adopted by the receptor that would explain the influence on GR dimerization and the recruitment of a specific set of GR transcription modulators. We summarize recent data that will contribute to understand the complexity of glucocorticoid response.

Distribution and concentration of maternal progesterone in the yolk of Greater Rhea eggs (Rhea americana).

Progesterone is the most concentrated maternal yolk steroid characterized to date in birds; however, no information about it is available in ratite eggs. We collected freshly laid eggs from zoo-housed Greater Rhea females (Rhea americana) bred under similar rearing conditions during two breeding seasons to characterize concentration and distribution of maternal yolk progesterone. After high-performance liquid chromatography analysis, yolk hormone was measured using a commercial electrochemiluminescence immunoassay. Progesterone concentrations were found to vary significantly among the yolk layers, supporting a follicular origin for this steroid in Greater Rhea eggs. Additionally, highly similar mean absolute yolk progesterone concentrations were detected between 2013 and 2015 breeding seasons (1,332.98 ± 82.59 and 1,313.59 ± 85.19 ng/g, respectively). These values are also comparable to those found in some domestic carinate species. Findings suggest that at population level, when rearing conditions are similar, mean absolute yolk maternal progesterone concentrations also appear bounded. Future research on the factors and mechanisms that regulate progesterone deposition in Greater Rhea eggs is needed to better understand whether its levels depend on different rearing conditions.

Modeling of Progesterone Release from Poly(3-Hydroxybutyrate) (PHB) Membranes.

Poly(3-hydroxybutyrate) (PHB) biodegradable polymeric membranes were evaluated as platform for progesterone (Prg)-controlled release. In the design of new drug delivery systems, it is important to understand the mass transport mechanism involved, as well as predict the process kinetics. Drug release experiments were conducted and the experimental results were evaluated using engineering approaches that were extrapolated to the pharmaceutical field by our research group. Membranes were loaded with different Prg concentrations and characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). SEM images showed that membranes have a dense structure before and after the progesterone addition. DSC and FTIR allowed determining the influence of the therapeutic agent in the membrane properties. The in vitro experiments were performed using two different techniques: (A) returning the sample to the receptor solution (constant volume of the delivery medium) and (B) extracting total volume of the receptor solution. In this work, we present a simple and accurate "lumped" second-order kinetic model. This lumped model considers the different mass transport steps involved in drug release systems. The model fits very well the experimental data using any of the two experimental procedures, in the range 0 ≤ t ≤ ∞ or 0 ≤ M t ≤ M ∞. The drug release analysis using our proposed approaches is relevant for establishing in vitro-in vivo correlations in future tests in animals.

In vitro evaluation of suspoemulsions for in situ-forming polymeric microspheres and controlled release of progesterone.

One possibility to obtain a higher dose of drug in a lower formulation volume can be by using of saturated quantity of drug in one of the phases of an emulsion. These formulations are called suspoemulsions (S/O/W). When a hydrophobic polymer is added to the organic phase of suspoemulsions, these formulations can be used to entrap the drug inside microspheres after in situ precipitation of the polymer-drug-excipients mix. In this work, performance and stability of progesterone suspensions in triacetin as organic phase of suspoemulsions were evaluated. These formulations were compared with O/W emulsions. Mathematical models were used to study in vitro release profiles. The results confirmed that S/O/W systems could be an attractive alternative to O/W formulations for the entrapment of progesterone inside poly(d,l-lactide-co-glycolide) microspheres. Diffusive-based models fit the in vitro release of progesterone from in situ-formed microspheres. For longer release periods, a time-dependent diffusion coefficient was successfully estimated.

Crystallization of progesterone polymorphs using polymer-induced heteronucleation (PIHn) method.

Progesterone is a natural hormone steroid used in humans for several treatments and in livestock for artificial insemination, which exhibits two polymorphic forms at ambient conditions: form 1 and form 2. Form 2 is metastable and more soluble than form 1; however, it is not suitable to use as powder raw material because it transforms into form 1 by the effects of grinding. A polymorphic screening of progesterone based on polymer-induced heteronucleation method was performed as an alternative to prepare the metastable form. Polyvinyl alcohol, hydroxypropyl methylcellulose (HPMC), dextran, gelatin, polyisoprene (PI) and acrylonitrile-butadiene (NBR) copolymer were used. Crystals were prepared from 0.5, 10 and 40 mg/mL solutions in acetone at room temperature by solvent evaporation. The samples were characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), scanning electron microcopy and attenuated total reflectance infrared Fourier transform spectroscopy. Form 1 was nucleated from 40 mg/mL solutions on the six polymers and from 10 mg/mL solutions on PI and NBR. The mixture of form 1 and form 2 was obtained from 10 mg/mL solution on HPMC, dextran and gelatin and from 0.5 mg/mL solution crystallizations. Therefore, the polymeric devices, which crystallized the metastable and more soluble polymorph (2) of progesterone, would be a promissory alternative for the pharmaceutical applications.

Dissolution properties, solid-state transformation and polymorphic crystallization: progesterone case study.

Progesterone is a natural steroid hormone and a poor soluble drug which presents two polymorphs (forms 1 and 2). Different methods to obtain form 2 were tested and a complete solid-state characterization of both polymorphs (forms 1 and 2) was conducted. X-ray powder diffraction, hot stage microscopy, Fourier transform infrared, dispersive Raman, (13)C solid-state nuclear magnetic resonance spectroscopy, thermal analysis, scanning electron microscopy techniques and intrinsic dissolution rates (IDR) were applied to investigate physical-chemical and dissolution properties of these two polymorphs. Form 2 was obtained from diluted solutions and from melting after cooling at room temperature. Form 1 was obtained from concentrated solutions and, a mixture of both polymorphs was crystallized from intermediate solutions. The crystal habit was not a distinctive characteristic of each polymorph. The effect of mechanical stress was evaluated in the metastable polymorph (form 2). We observed that grinding form 2 produced seeds of form 1 that induced the transformation of form 2 into form 1 at high temperature. The polymorphic quantification from XRD patterns of ground samples were carried out by the Rietveld method. After grinding and at room temperature conditions (∼25 °C), it was observed the transformation of 17% of form 2 into form 1 in 10 days.

A polymorphism in the insulin-like growth factor 1 gene is associated with postpartum resumption of ovarian cyclicity in Holstein-Friesian cows under grazing conditions.

BACKGROUND: Insulin-like growth factor 1 (IGF-1) gene is considered as a promising candidate for the identification of polymorphisms affecting cattle performance. The objectives of the current study were to determine the association of the single nucleotide polymorphism (SNP) IGF-1/SnaBI with fertility, milk production and body condition traits in Holstein-Friesian dairy cows under grazing conditions. METHODS: Seventy multiparous cows from a commercial herd were genotyped for the SNP IGF-1/SnaBI. Fertility measures evaluated were: interval to commencement of luteal activity (CLA), calving to first service (CFS) and calving to conception (CC) intervals. Milk production and body condition score were also evaluated. The study period extended from 3 wk before calving to the fourth month of lactation. RESULTS AND DISCUSSION: Frequencies of the SNP IGF-1/SnaBI alleles A and B were 0.59 and 0.41, respectively. Genotype frequencies were 0.31, 0.54 and 0.14 for AA, AB and BB, respectively. Cows with the AA genotype presented an early CLA and were more likely to resume ovarian cyclicity in the early postpartum than AB and BB ones. No effect of the SNP IGF-1/SnaBI genotype was evidenced on body condition change over the experimental period, suggesting that energy balance is not responsible for the outcome of postpartum ovarian resumption in this study. Traditional fertility measures were not affected by the SNP IGF-1/SnaBI. CONCLUSION: To our knowledge this is the first report describing an association of the SNP IGF-1/SnaBI with an endocrine fertility measure like CLA in cattle. Results herein remark the important role of the IGF-1gene in the fertility of dairy cows on early lactation and make the SNP IGF-1/SnaBI an interesting candidate marker for genetic improvement of fertility in dairy cattle.

Non-genomic, direct modulatory effect of 17ß-estradiol, progesterone and their synthetic derivatives on the activity of human erythrocyte CuZn superoxide dismutase.

This study aimed to evaluate whether natural or synthetic steroid hormones could directly modulate the activity of the different superoxide dismutase (SOD) isoforms found in human blood fractions without changing enzyme expression. Enzyme samples of human erythrocytes, the human platelet-rich plasma fraction (PRP) or isolated CuZnSOD, which was purified from human erythrocytes were pre-incubated with natural steroids (17ß-estradiol 17-acetate and progesterone) and their synthetic derivatives (ß-estradiol 3-benzoate and medroxyprogesterone 17-acetate). Then, CuZn and MnSOD activities were measured using the xanthine/xanthine oxidase/nitroblue tetrazolium method. Hormones had no effect on MnSOD activity from the PRP, but we show for the first time that natural and synthetic steroid hormones have a direct, bell-shaped effect on the activity of CuZnSOD from both male and female human erythrocytes. Low (physiological) hormone concentrations caused a dose-dependent increase in enzyme activity, which disappeared at higher hormone concentrations. In addition, the combination of synthetic and natural estrogens and progestins had a synergistic stimulatory effect on the activity of CuZnSOD from human erythrocytes. The molecular interaction between CuZnSOD and steroid hormones was preliminarily studied. Natural hormones did not change the electrophoretic mobility of SOD under denaturing conditions, but they did increase the absorption spectra of SOD in the 230-290 nm range. These data suggest that hormone-mediated modulation of CuZnSOD is related to subtle changes in protein conformation, possibly related to Trp and Phe residues. We propose that this effect may account for the physiological regulation of enzyme activity during conditions where steroid hormones undergo alterations as the ovulatory cycle.

Morphology study of progesterone polymorphs prepared by polymer-induced heteronucleation (PIHn).

In this article, morphology of progesterone polymorphs prepared by polymer-induced heteronucleation (PIHn) technique was studied. Hydroxypropyl methylcellulose(HPMC), such as dextran T-500 and gelatin G-9382, polyisoprene (PI), and acrylonitrile/butadiene copolymer (NBR) were used as substrates. The crystallizations were performed by solvent evaporation at room temperature from 0.5, 10, and 40 mg/ml solutions in chloroform and acetone. Progesterone polymorphs were identified by X-ray diffraction. Differential scanning calorimetry and total attenuated reflectance infrared spectroscopy were used as complementary techniques in the identification. Depending on the polymeric matrix and the concentration used, form 1, form 2, or mixture of both polymorphs were obtained. Scanning electron microscopy pictures evidenced difference in morphology and in homogeneity of the two progesterone polymorphs. These polymorphs prepared by PIHn, did not present a distinctive morphology that allows identifying polymorph by its crystal habit. Hence, polymeric matrix induced the crystallization, affecting polymorphism and morphology.

Bimodal binding and free energy of the progesterone receptor in the induction of female sexual receptivity by progesterone and synthetic progestins.

Synthetic progestins (SPs) are used for regulation of fertility, contraception and hormone replacement therapy. The acetylated medroxyprogesterone (MPA), megestrol (MGA) and chlormadinone (CLA) are related to progesterone (P). Other SPs are 19-nortestosterone derivatives such as: norethisterone (NET), norethynodrel (NED) or the 13-ethyl gonane, levonorgestrel (LNG). We studied MPA, NET, NED and LNG in a dose-response manner to induce sexual receptivity in rats. Results showed that MPA, NET and NED act as partial agonists, with similar or lower potency than P. However, LNG is a full agonist. Additionally, the molecules of MPA, MGA, CLA, NET, NED, LNG, and P, were submitted to computer calculations at ab initio quantum mechanics theory, to obtain their electronic structure and molecular properties. The aim was to correlate their behavioral effect with their physicochemical properties. In addition, the crystals of P, NET and LNG bound to the progesterone receptor (PR) were studied. The PR crystallizes as a dimer forming two monomers (mA and mB), in which Gln725 interacts in either of two possible ways with the C3-carbonyl pharmacophore of progestins. P binds differentially to both PR monomers, while NET binds exclusively as mA and LNG binds only as mB in both monomers with no difference. Energetically, binding of LNG and P to mB, is more favorable than that of NET and P to mA. Consequently, this bimodal mechanism increases the action possibilities of SPs on biological systems. Interestingly, progestin potency depends mostly on local molecular structure and electronic features, prevailing over total molecular properties.

Development of poly(lactic acid) nanostructured membranes for the controlled delivery of progesterone to livestock animals.

Solution blow spinning (SBS) is a novel technology feasible to produce nanostructured polymeric membranes loaded with active agents. In the present study, nanofibrous mats of poly(lactic acid) (PLA) loaded with progesterone (P4) were produced by SBS at different P4 concentrations. The spun membranes were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR). The in vitro releasing of P4 was evaluated using high-performance liquid chromatography (HPLC). Interactions between progesterone and PLA were confirmed by rheological measurements of the PLA/P4 solutions and in the spun mats by microscopy (SEM), thermal (DSC) and spectral (FTIR) analyses. SEM micrographs provided evidences of a smooth and homogeneous structure for nanostructured membranes without progesterone crystals on fiber surface. FTIR spectroscopy indicated miscibility and interaction between the ester of PLA and the ketone groups of the P4 in the nanofibers. X-ray analysis indicated that the size of PLA crystallites increased with progesterone content. Finally, by in vitro release experiments it was possible to observe that the progesterone releasing follows nearly first-order kinetics, probably due to the diffusion of hormone into PLA nanofibers.

Relationships between growth of the preovulatory follicle and gestation success in lactating dairy cow

This report summartizes three studies conducted with lactating dairy cws aiming to increase pregnancy rates to fixes time artificial insemination (TAI) protocols. Experiment I was designed to determine if changing the the timing of PGF2α treatment during an E2/P4 based program would affect fertility to TAI or fixedtime embryo transfer (TET). In experiment 2, pregnancy rates to AI were compared following synchronized ovulation using two protocols that have been developed to reduce the period between follicular wave emergence and TAI. The Ovsynch type protocol utilizes GnRH to synchronize the follicular wave by inducing ovulati on of a dominant follicle at the beginning of the protocol, and to synchronize ovulation at the end of the protocol allowing TAI. In contrast, E2/P4 based protocols utilize E2 products in the presence of P4 to induce atresia of antral follicles and synchro nize emergence of a new follicular wave. At the end of E2/P4 based protocol another E2 treatment in the absence of P4 is used to induce LH release and synchronize ovulation and allow TAI. Experiment 3 was designed to determine whether increasing the length time interval with reduced circulating P4 (proestrus) would increase fertility in a TAI program that utilized E2 and P4 to synchronize ovulation of cycling, lactating dairy cows. The overall conclusions are that circulating concentrations of progesterone and estradiol prior to and circulating concentrations of progesterone following ovulation can affect fertility in cattle. In addition, small increases in P4 concentrations near the time of AI, due to lack of complete CL regression, result in reductions in fertility. Earlier treatment with PGF2α should allow greater time for CL regression, an increase in estradiol and subsequent reductions in circulating P4 that could be critical for fertility. Optimization of follicle size in TAI programs is clearly an intr icate balance between oocyte quality, adequate circulating E2 near AI, and adequate circulating P4 after AI.

Relationships between growth of the preovulatory follicle and gestation success in lactating dairy cow

This report summartizes three studies conducted with lactating dairy cws aiming to increase pregnancy rates to fixes time artificial insemination (TAI) protocols. Experiment I was designed to determine if changing the the timing of PGF2α treatment during an E2/P4 based program would affect fertility to TAI or fixedtime embryo transfer (TET). In experiment 2, pregnancy rates to AI were compared following synchronized ovulation using two protocols that have been developed to reduce the period between follicular wave emergence and TAI. The Ovsynch type protocol utilizes GnRH to synchronize the follicular wave by inducing ovulati on of a dominant follicle at the beginning of the protocol, and to synchronize ovulation at the end of the protocol allowing TAI. In contrast, E2/P4 based protocols utilize E2 products in the presence of P4 to induce atresia of antral follicles and synchro nize emergence of a new follicular wave. At the end of E2/P4 based protocol another E2 treatment in the absence of P4 is used to induce LH release and synchronize ovulation and allow TAI. Experiment 3 was designed to determine whether increasing the length time interval with reduced circulating P4 (proestrus) would increase fertility in a TAI program that utilized E2 and P4 to synchronize ovulation of cycling, lactating dairy cows. The overall conclusions are that circulating concentrations of progesterone and estradiol prior to and circulating concentrations of progesterone following ovulation can affect fertility in cattle. In addition, small increases in P4 concentrations near the time of AI, due to lack of complete CL regression, result in reductions in fertility. Earlier treatment with PGF2α should allow greater time for CL regression, an increase in estradiol and subsequent reductions in circulating P4 that could be critical for fertility. Optimization of follicle size in TAI programs is clearly an intr icate balance between oocyte quality, adequate circulating E2 near AI, and adequate circulating P4 after AI.(AU)

Avaliação da influência da expressão da indoleamina 2,3-dioxigenase no ciclo celular de células placentárias e embrionárias murinas e de ratas em cultivo celular, frente à ação de hormônios e citocinas / Evaluation of the influence of the expression of indoleamine 2,3-dioxygenase on the cell cycle of murine and rat embryonic cells and placental cells in culture supplemented with hormones and cytokines

A indoleamina 2,3-dioxigenase (IDO) desempenha um papel importante na tolerância materno-fetal devido á sua ação de catabolizar o triptofano e, consequentemente, impedir a proliferação de linfócitos T, que necessitam desse aminoácido para se manter. Hormônios da reprodução também participam do processo de sobrevivência do feto alogênico, como a progesterona que bloqueia o estímulo mitogênico da proliferação de células T, modula a produção de anticorpos, favorece a produção de IL-10, etc. e o estradiol, que pode modular o perfil imune Th1 ou Th2 na gestação, dependendo de sua concentração. Contudo, a existência ou não de correlação da ação da IDO com esses hormônios ou vice-versa ainda encontra-se pouco evidenciada. Desta forma este trabalho verificou a influência da expressão da IDO e às ações de hormônios da reprodução e citocinas no ciclo celular de células oriundas de fetos e placenta de gestação a termo de fêmeas de ratas e camundongos em cultivo. (...)As células que foram suplementadas com 1- metil DL - triptofano + triptofano mantiveram o mesmo comportamento no ciclo celular , se mantendo em fase G1 nos tempos de 4, 24 e 48 horas grupos prenhes e não prenhes de ratas e nos tempos de 4 e 24 horas nos grupos de camundongos fêmeas prenhes e não prenhes, estando com seu DNA fragmentado e em fase de síntese, respectivamente, no tempo de 48 horas. A suplementação dos diversos fatores aos cultivos celulares permitiu observar alterações na dinâmica do ciclo celular, representada principalmente por um aumento de células em fase G1 nos grupos de células placentárias e embrionárias que receberam progesterona, estradiol, interferon γ e triptofano e apresentaram um significativo aumento da expressão de IDO, e que permite inferir que provavelmente a síntese de RNAm esteja correlacionada à produção da IDO

The indoleamine 2,3-dioxygenase (IDO) plays an important role in maternal-fetal tolerance due to its capacity to catabolize tryptophan and thereby preventing the proliferation of T lymphocytes, necessary for their maintenance. Reproductive hormones are also involved in the process of survival of semi-allogeneic fetus, as progesterone that blocks mitogenic stimulation of T cell proliferation, modulates antibodies production, promotes production of IL-10, etc. and estradiol, that can modulate the Th1 or Th2 immune profile during pregnancy, depending on its concentration. However, there is very few evidences regarding a possible correlation between IDO expression and these hormones; thus this study examined the influence of the expression of IDO and the actions of reproductive hormones and cytokines in the cell cycle of cells derived from fetuses and placenta at term gestation of female rats and mice in culture. (…) In the group of pregnant rats a predominance of cells in the G1 phase was observed in periods of 4 and 24 hours, and 48 hours, where the cells undergone DNA fragmentation. In pregnant and non-pregnant rats the cells supplemented with 1 - methyl - DL - tryptophan + tryptophan remained at G1 phase in periods of 4, 24 and 48 hours and 4 and 24 hours for cells from pregnant and non-pregnant mice , that show ragmented DNA followed by synthesis at 48 hours period. Supplementation of the various factors to cell cultures allowed to observe dynamic changes in the cell cycle, represented primarily by an increase of cells in G1 phase in groups of embryonic and placental cells that received progesterone, estradiol, interferon γ and tryptophan and showed a significant increase in the expression of IDO, that allows us to infer that the mRNA synthesis is probably correlated to the production of IDO