Uterine and placental specific localization of AQP2 and AQP8 is related with changes of serum progesterone levels in pregnant queens.

Aquaporins play vital roles in reproductive physiology. This study evaluates the expression and localization dynamics of AQP1, AQP2, AQP3 and AQP8 in the endometrium and placental transference zone during pregnancy in queens by means of immunohistochemistry and Western blot. Animals were distributed into six groups: non-pregnant queens with low levels of serum progesterone (P4), non-pregnant animals with high P4 levels, and queens at 30, 40, 50 and 60 days of pregnancy. All AQPs were present in glandular and luminal epithelia and myometrium. AQP1 was also present in the endometrial endothelia. AQP2, AQP3 and AQP8 were found in trophoblast. In endometrial samples with P4 above 2 ng/mL, AQP2 and AQP8 were distributed across plasma membrane and cytoplasm, whereas progesterone levels under 1 ng/mL kept both AQPs confined to the plasma membrane. Western blot showed no significant changes in AQPs expression among the stages. In conclusion, our results indicate that the distribution of AQP2 and AQP8 in the queen reproductive tract is related to P4 levels.

Placental and uterine expression of GLUT3, but not GLUT1, is related with serum progesterone levels during the first stages of pregnancy in queens.

The present study investigated the expression of GLUT1 and GLUT3 in the uterus and placental transference zone of non-pregnant and pregnant queens throughout different pregnancy ages, using immunohistochemistry and immunoblotting techniques. Both GLUT1 and GLUT3 were expressed in both uterine glandular and luminal epithelia and myometrium in pregnant and non-pregnant queens. While endometrial endothelia showed expression of GLUT1 in both pregnant and non-pregnant queens, GLUT3 was only expressed in the pregnant counterparts. Regarding placental structures, GLUT3 was present in cytotrophoblasts, syncytiotrophoblasts and chorionic endothelia and GLUT1 showed a similar location but was absent in cytotrophoblasts. The presence of GLUT1 (55 kDa) and GLUT3 (60 kDa) was confirmed in both uterine and placental tissues through immunoblotting. When the expression of both GLUT1 and GLUT3 were analysed as a whole in the total of the pregnancy period, no significant differences in the relative content of both GLUTs were observed between pregnant and non-pregnant queens. However, when GLUTs expression was analysed in a time-period basis and related with progesterone levels, results were different. Thus, whereas the relative content of GLUT1 showed no correlation with serum progesterone levels, a significant (P < 0.05) and negative correlation was found between the relative GLUT3-content in the uterus on days 30 and 40 of pregnancy as well as in the placental transference zone on day 30 and serum progesterone levels. In summary, our results indicate that whereas GLUT1 could be considered as a basal, constant sugar intake system for the whole of pregnancy in queens, GLUT3 is specially required for optimizing glucose uptake during the first half of pregnancy in this species through a progesterone-related mechanism.

Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes.

This study examines whether incorporating cholesterol-loaded methyl-ß-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage.

Functionalization of 3D scaffolds with protein-releasing biomaterials for intracellular delivery.

Appropriate combinations of mechanical and biological stimuli are required to promote proper colonization of substrate materials in regenerative medicine. In this context, 3D scaffolds formed by compatible and biodegradable materials are under continuous development in an attempt to mimic the extracellular environment of mammalian cells. We have here explored how novel 3D porous scaffolds constructed by polylactic acid, polycaprolactone or chitosan can be decorated with bacterial inclusion bodies, submicron protein particles formed by releasable functional proteins. A simple dipping-based decoration method tested here specifically favors the penetration of the functional particles deeper than 300µm from the materials' surface. The functionalized surfaces support the intracellular delivery of biologically active proteins to up to more than 80% of the colonizing cells, a process that is slightly influenced by the chemical nature of the scaffold. The combination of 3D soft scaffolds and protein-based sustained release systems (Bioscaffolds) offers promise in the fabrication of bio-inspired hybrid matrices for multifactorial control of cell proliferation in tissue engineering under complex architectonic setting-ups.