Ana Lía Errecalde

The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and early stage embryos.

Hyaluronic acid (HA) is a high molecular weight polysaccharide found in the extracellular matrix of most animal tissues, that exerts a profound influence on cell behavior. HA is one of the most abundant glycosaminoglycans (GAGs) in the uterine, oviductal and follicular fluids in mouse, pig, human and cattle. CD44, the principal cell membrane receptor for HA, is expressed from the 1- to 8-cell stage in human embryos, during post-implantation mouse embryogenesis and on the surface of differentiated embryonic stem cells. In the present study, we have analyzed by immunofluorescence, whether CD44 is present in bovine oocytes, fertilized oocytes and early stage embryos. Bovine cumulus-oocyte complexes (COCs) were aspirated from follicles (2-5mm) and were selected for IVM and incubated for 24h. Oocytes showing an expanded cumulus (generally 90-95%) were used for IVF. Fertilized oocytes were separated for immunofluorescence assay after 16h of sperm incubation in order to fix the eggs at the pronuclear stage. The embryos were cultured for 8 days and the different stages of development for immunofluorescence assay were separated every 24h of culture. The CD44 receptor was detected at every observation time examined. Fluorescence-tagged HA for the internalization assay was prepared by mixing fluorescein amine, Isomer I and 1mg of HA from umbilical cord. Fluorescence-tagged HA was internalized in 2-, 4-, 8- and 16-cell-stage embryos, morulae and blastocysts. CD44 is expressed on the surface and in the cytoplasm of bovine oocytes and embryos in different stages of development.

A therapy-grade protocol for differentiation of pluripotent stem cells into mesenchymal stem cells using platelet lysate as supplement

IntroductionMesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes.MethodsIn this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement.ResultsWe showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum.ConclusionsThis protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.

Pesticide-induced decrease in rat testicular steroidogenesis is differentially prevented by lipoate and tocopherol

We have previously demonstrated that the sub-chronic administration of low doses of Toc or α-Toc, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5 weeks) provoked severe oxidative stress (OS) in testicles. These effects were also reflected in plasma. Lipoic acid (LA) and α-tocopherol are considered as antioxidants due to their ability to neutralize reactive oxygenated species (ROS) and reset endogenous antioxidant levels. To investigate the possible protective effect on reproductive function, LA and Toc (i.p. 25, 50 and 100mg/kg) were administered simultaneously with the pesticide mixture (PM) for 5 weeks. Both drugs prevented OS and the damage to proteins and lipids caused by PM in a dose-dependent manner. The PM-induced increase levels of prostaglandins E2 and F2α was completely restored by LA but not by Toc. Similarly, only LA was able to restore the inhibition of testosterone production, the decrease of 3β- and 17β-hydroxysteroid dehydrogenases activities, and the elevation of gonatropins (FSH and LH) levels produced by PM. Furthermore, LA was more efficient than Toc in normalizing the histological alterations produced by PM administration, suggesting that pesticides act though other mechanisms that generate oxidative stress. In our experimental model LA displayed a higher protective role against pesticide-induced damage than that observed by Toc administration. Our results suggest that LA administration is a promising therapeutic strategy for coping with disorders suspected to be caused by OS generators - such as pesticides - in male reproductive system.

Influence of manganese on apoptosis and glutathione content of cumulus cells during in vitro maturation in bovine oocytes.

We have investigated the effect of different Mn concentrations on (1) DNA integrity of cumulus cells by olive tail moment (OTM); (2) cumulus cells apoptosis by Annexin V staining assay; (3) intracellular total glutathione (GSH‐GSSG) content; and (4) oocyte nuclear maturation and embryo cleavage after in vitro fertilisation (IVF). For this purpose, 0 (control), 2 (Mn1), 5 (Mn2) and 6u2009ng/mL (Mn3) Mn concentrations were added to IVM medium. Comet assay analysed by OTM was significantly higher in cumulus cells arising from COCs matured without Mn (control, Pu2009<u20090.01) respect to cumulus cells obtained from COCs matured with Mn (control: 5.18u2009±u20092.3; Mn1: 2.93u2009±u20092.2; Mn2: 2.63u2009±u20092.4; Mn3: 2.92u2009±u20092.4). The frequency of apoptotic cells was higher in the control group (control: 6.63u2009±u20090.59; Mn1: 5.05u2009±u20090.5; Mn2: 4.61u2009±u20090.49; Mn3: 3.33u2009±u20090.42). Intracellular concentration of GSH‐GSSG increased in oocytes and cumulus cells matured in the presence of Mn (Pu2009<u20090.01). There were no differences in percentages of nuclear maturation when Mn was added to IVM medium at any concentration, but at 6u2009ng/mL Mn a higher cleavage rate was observed respect to the control group (Pu2009<u20090.05). In conclusion, deficiency in Mn concentration during in vitro maturation increased the damage in the DNA molecule and the frequency of apoptotic cumulus cells. However, the addition of an adequate Mn concentration (6u2009ng/mL Mn) to IVM medium improved the health of cumulus‐oocyte complexes and produced more cleaved embryos 48u2009h after IVF.

Changes in VEGF expression and DNA synthesis in hepatocytes from hepatectomized and tumour-bearing mice

Transplanted tumours could modify the intensity and temporal distribution of the cellular proliferation in normal cell populations, and partial hepatectomy alters the serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. The following experiments were designed in order to study the SI (S‐phase index) and VEGF (vascular endothelial growth factor) expression in regenerating liver (after partial hepatectomy) of adult male mice bearing a hepatocellular carcinoma, throughout one complete circadian cycle. We used adult male C3H/S‐strain mice. After an appropriate period of synchronization, the C3H/S‐histocompatible ES2a hepatocellular carcinoma was grafted into the subcutaneous tissue of each animals flank. To determine the index of SI and VEGF expression of hepatocytes, we used immunohistochemistry. The animals were divided into two experimental groups: Group I, control, hepatectomized animals; Group II, hepatectomized tumour‐bearing animals. The statistical analysis of SI and VEGF expression was performed using Anova and Tukey as a postcomparison test. The results show that in the second group, the curve of SI changes the time points for maximum and minimum activity, and the peak of VEGF expression appears before the first group. In conclusion, in the hepatectomized mice, the increases of hepatic proliferation, measured by the SI index, may produce a rise in VEGF expression with the object of generating a vascular network for hepatic regeneration. Lastly, as we have mentioned, in hepatectomized and tumour‐bearing mice, the peak of VEGF expression appears before the one of DNA synthesis.

Vascular endothelial growth factor expression along a circadian time span in intact adult mice liver

In the present study we analyzed VEGF-A expression in adult male and female C3H/S mice along a circadian time span. The animals were sacrificed at 00:00, 04:00, 08:00, 12:00, 16:00 and 20:00 h. Samples of the liver from each animal were processed for immunohistochemistry. The VEGF expression index was calculated as positives immunostaining hepatocytes x 100 and analyzed by ANOVA, and the Student–Newman–Keuls Multiple Comparison Test. The results show that in both male and female mice there were significant differences between the values corresponding to all the time points studied. In male mice the values of VEGF expression of 20:00 h are significantly higher than all the others analyzed while in females the value of the 20:00 h was significantly different from the value of the 04:00 h. In conclusion, the hepatocytes of the perivenular areas in the liver of intact adult male and female mice expressed VEGF with a defined circadian rhythm.

Circadian rhythm of VEGF expression in the liver of hepatectomized tumor bearing mice

In this study we analyzed VEGF-C expression in regenerating liver (after partial hepactectomy) of ES2 hepatocellular carcinoma bearing mice, throughout one complete circadian cycle. The animals were sacrificed every 4 h throughout one complete circadian cycle from 26 to 50 h post-hepatectomy. Tumor samples were processed for immunohistochemistry. The expression of VEGF was assessed according to the percentage of immunoreactive cells in a total of 1000 cells (quantitative analysis). The results show that controls and tumor bearing mice have statistical differences at all analysed time points, but the maximun value of VEGF expression of treated animals is significantly higher (p < 0.01) than the control group. We can conclude that when a partial hepatectomy is made with the purpose of eradicating a hepatic tumor, the presence of possible metastasis could release factors related with cellular proliferation that could increase the possibilities of tumoral recidives.

Changes in DNA synthesis circadian rhythms in a hepatocellular carcinoma after hepatectomy

The effect of partial hepatectomy on the DNA synthesis rhythms of ES12a hepatocellular carcinoma grafted into C3H/S male mice was studied and compared with tumor growth in unoperated animal controls. The subtotal hepatectomy also changes the concentration of substances related to cellular proliferation to generate a compensatory hyperplasia in the liver. Thus, we analyzed the interaction between liver regeneration after hepatectomy and the DNA synthesis activity of the tumor. The animals were sacrificed every 4 h throughout one complete circadian cycle. All experimental groups of animals received an intraperitoneal dose of 50 mg of 5-bromodeoxiuridine per kg of body weight before being killed. Tumor samples were processed for histology and immunohistochemistry. DNA synthesis indices were expressed as labeled nuclei per 1000 nuclei. The results show that the ES12a tumor displays the DNA synthesis activity rhythm which, as in normal hepatocytes, passes through a peak during the nocturnal phase, and the surgery alters the temporal structure of the DNA synthesis indices. This fact should be considered in both neoplastic drug treatments and in growth related experiments.

VEGF EXPRESSION IN HEPATECTOMIZED TUMOR-BEARING MICE

The experiments were designed in order to study the VEGF expression in intact (group I), hepatectomized (group II), and hepatectomized-tumor bearing mice (group III) throughout one complete circadian time span. Adult male mice were used for the VEGF expression study. The statistical analysis was performed using analysis of variance (ANOVA). The results showed statistical differences in the VEGF expression between groups I and II, but the most significant differences were found between groups I and III. In conclusion, these expressions have a circadian rhythm in all groups; moreover, in group III, this expression was higher and appeared before than in the others.

DNA synthesis in tongue keratinocytes of hepatectomized and tumor-bearing mice

Tongue keratinocytes have high S‐phase and mitotic indices with evident circadian variation. Transplanted tumors modify the intensity and temporal structure of the S‐phase index in cell populations in tumor‐bearing animals; also, partial hepatectomy changes the concentrations of substances involved in cellular proliferation, leading to compensatory liver hyperplasia. The aim of our study was to analyze the interaction between tumor growth and the liver regeneration that follows partial hepatectomy, and the effects of both these processes on lingual keratinocytes. We used 380 adult male mice divided into six groups: tumor‐free and tumor‐bearing mice without surgery, with sham hepatectomy, and with partial hepatectomy. Each group was divided into six subgroups, which were killed at 4‐h intervals until a circadian cycle was completed (from 26 until 50 h post‐surgery in the operated animals). Each animal was injected with 5‐bromodeoxyuridine (50 mg/kg) 1 h before it was killed, and tongue samples were obtained and processed for histology. The sections were placed on silanized slides and incubated with the primary antibody Bu 20a (1/100 dilution). The reaction was developed using diaminobenzidine and staining was detected visually. SIs were measured as the number of labeled nuclei per thousand cells. The mean ± S.E. of each group was calculated. Differences among experimental groups were analyzed by ANOVA and the Student—Newman—Keuls Multiple Comparisons Test. The results show that the presence of a tumor alters the normal circadian curve of SI in lingual keratinocytes, irrespective of whether the mice underwent surgery. This finding has to be considered in drug treatments for neoplasms and in experiments related to growth.