Sphingosine-1-phosphate, regulated by FSH and VEGF, stimulates granulosa cell proliferation.

Sphingosine-1-phosphate (S1P) is a bioactive polar sphingolipid which stimulates proliferation, growth and survival in various cell types. In the ovary S1P has been shown protect the granulosa cells and oocytes from insults such as oxidative stress and radiotherapy, and S1P concentrations are greater in healthy than atretic large follicles. Hence, we postulate that S1P is fundamental in follicle development and that it is activated in ovarian granulosa cells in response to FSH and VEGF. To test this hypothesis we set out: i) to evaluate the effect of FSH and VEGF on S1P synthesis in cultured bovine granulosa cells and ii) to analyse the effect of S1P on proliferation and survival of bovine granulosa cells in vitro. Seventy five thousand bovine granulosa cells from healthy medium-sized (4-7mm) follicles were cultured in 96-well plates in McCoy's 5a medium containing 10ng/mL of insulin and 1ng/mL of LR-IGF-I at 37°C in a 5% CO2/air atmosphere at 37°C. Granulosa cell production of S1P was tested in response to treatment with FSH (0, 0.1, 1 and 10ng/mL) and VEGF (0, 0.01, 0.1, 1, 10 and 100ng/mL) and measured by HPLC. Granulosa cells produced S1P at 48 and 96h, with the maximum production observed with 1ng/mL of FSH. Likewise, 0.01ng/mL of VEGF stimulated S1P production at 48, but not 96h of culture. Further, the granulosa cell expression of sphingosine kinase-1 (SK1), responsible for S1P synthesis, was demonstrated by Western blot after 48h of culture. FSH increased the expression of phosphorylated SK1 (P<0.05) and the addition of a SK1 inhibitor reduced the constitutive and FSH-stimulated S1P synthesis (P<0.05). Sphingosine-1-phosphate had a biphasic effect on granulosa cell number after culture. At low concentration S1P (0.1µM) increased granulosa cell number after 48h of culture (P<0.05) and the proportion of cells in the G2 and M phase of the cell cycle (P<0.05), whereas higher concentrations decreased cell number (10µM; P<0.05) by an increase (P<0.05) in the proportion of cells in apoptosis (hypodiploid cells). In addition, treatment with SK-178 suppressed the FSH- and VEGF-stimulated rise of the granulosa cells number (P<0.05). Interestingly, the effect of 0.1µM S1P on granulosa cell number and their proportion in G2/M phases is similar to that observed with 1ng/mL FSH. The results of this study are the first to demonstrate sphingosine-1-phosphate (S1P) synthesis in granulosa cells under the control of FSH and VEGF. The later achieved through the regulation of sphingosine kinase 1 expression. This S1P augments the proportion of cells in the G2/M phase of the cell cycle that translates in increased granulosa cell proliferation.

P450-aromatase mRNA is expressed in the corpus luteum (CL) of the non-pregnant sheep and goat: the expression of the enzyme is present throughout pregnancy in the goat CL.

In most mammals, the corpus luteum (CL) and placenta are the major sources of progesterone. The goat pregnancy depends on the presence of CL after mid-gestation, while sheep pregnancy does not. The expression and distribution of P450-aromatase (P450-Aro) mRNA throughout gestation has not been investigated in the goat CL and partially in the sheep CL. The present research was designed to characterize the expression of P450-Aro mRNA in small ruminant CL with emphasis in the goat. For this purpose, ovaries from Criollo goats and Pelibuey sheeps were analysed using in situ reverse transcription-polymerase chain reaction (RT-PCR) for the histological detection of P450-Aro transcripts. In addition, P450-Aro expression was determined by in vitro RT-PCR. In situ RT-PCR studies showed that the goat and sheep CL were rich in cells positive for P450-Aro mRNA. We have also found in vitro RT-PCR expression of P450-Aro mRNA in goat CL at 1, 3 and 4 months of gestation. This study shows that the goat CL expresses P450-Aro mRNA along gestation, suggesting that this structure is capable to produce oestrogens up to the end of gestation.

Expression of P450-aromatase in the goat placenta throughout pregnancy.

The enzyme P450-aromatase (P450-Aro) is essential for the conversion of androgens to estrogens. The objective was to study the expression and distribution of P450-Aro in goat placentae throughout pregnancy. For this purpose, we used reverse transcription-polymerase chain reaction (RT-PCR) with primers derived from the ovarian cDNA sequence found by our group. The expression of P450-Aro mRNA was first detected by in vitro RT-PCR in cotyledons at 4 months and was still present at term. Based on in situ RT-PCR, cotyledon microvilli expressed P450-Aro mRNA early in pregnancy; the signal was detected in the syncytiotrophoblast and in non-fused cytotrophoblasts inside the microvilli, but was scarce in the interstitial cells of the villous core. In the last 2 months of pregnancy (including at term), the expression of P450-Aro mRNA was still detected in the syncytiotrophoblast. However, P450-Aro was never detected in the caruncule (regardless of stage of pregnancy). In conclusion, P450-Aro was expressed in the goat placenta microvilli starting early in pregnancy; the expression and distribution of the enzyme increased throughout pregnancy and was still present at term.

[Comparative efficiency of 2 products for cleaning the instrumented dentin wall]. / Eficacia comparativa de dos productos para limpieza de la pared dentinaria instrumentada.

Two dental cleansing products, Rc-Prep and Largal Ultra, were subjected to a comparative study, evaluating their efficacy in vitro on 15 recently-extracted dentary units, through optic microscopy applied on the dentine wall of the instrumented root canal. Both materials were applied on separate hemisections of the canal during 15 minutes intervals, with two applications on each canal. Rc-Prep was observed to have a slight, not significant advantage over Largal-Ultra in its cleansing effect over the dentine smear, although the compact, granular and amorphous layer of dentine smear over the root canal wall, blocking the entry to dentine channels, persisted after use of both products. In view of conditions observed in the dentine walls, the authors assume that adhesion and adaptation of obturating materials over these structures is exceedingly difficult. Although variability was not considered as is usual in clinical studies, in vitro evaluation as observed in this study allows a more accurate comparative analysis, since it was performed on one individual tooth, with analogous instrumentation and on dentary tissue with similar characteristics.