Aquaporins 8 and 9 as Possible Markers for Adult Murine Lacrimal Gland Cells

Aquaporins (AQPs) are proteins that selectively transport water across the cell membrane. Although AQPs play important roles in secretion in the lacrimal gland, the expression and localization of AQPs have not been clarified yet. In the current study, we investigated the expression pattern of AQP family members in the murine lacrimal gland during development. Lacrimal gland tissues were harvested from E13.5 and E17.5 murine embryos and from mice 8 weeks of age (adults). Corneal and conjunctival tissues from the latter served as controls. Total RNA was isolated and analyzed for the expression of AQP family members using qPCR. The localization of AQPs in the adult lacrimal gland in adult murine lacrimal glands was also analyzed. Expression of Aqp8 and Aqp9 mRNAs was detected in the adult lacrimal gland but not in the cornea, conjunctiva, or fetal lacrimal gland. AQP8 and AQP9 and α-SMA partially colocalized around the basal regions of the acinar unit. The levels of Aqp3 mRNAs and protein were much lower in the adult lacrimal gland but were readily detected in the adult cornea and conjunctiva. Our study suggests that AQP8 and AQP9 may serve as markers for adult murine lacrimal gland, ductal, and myoepithelial cells.

Effect of N-acetyl cysteine on the quality of blastocyst formation rate using cultured vitrified murine embryos

Vitrification is the best method for embryo cryopreservation although it increases endogenous reactive oxygen species (ROS) production. N-acetylcysteine (NAC) a free radical scavenger may be used for reducing ROS toxic effects. The aim of the present study is to investigate potential beneficial effects of NAC on the developmental embryo competence applying different culture conditions in vitrified-warmed 2-cell embryos derived in vivo or in vitro. Thus, 2-cell embryos were vitrified or cultured fresh in presence or absence of 1 mM of NAC during: a) the entire embryo culture, b) for 24 hours with NAC at days 1.5 (G1) or 2.5 (G2) and returned to basal embryo culture (KSOM) or c) cultured in the presence of NAC for 12 hours at day 3.5 (G3). Despite NAC addition to fresh or vitrified embryos produced in vivo or by IVF, blastocyst rates remained unchanged. In vitrified-warmed IU or IVF-derived embryos, total cell number varied when NAC was added at day 1.5 although differences were not significant (60.1 ± 1.9 vs. 59.4 ± 1.3 for IU G1 and control respectively; and 59.3 ± 1.6 and 52.6 ± 3.0 IVF G1 and control respectively; mean cell number ± SEM, p > 0.05). It seems that the embryo culture medium supplementation with 1 mM of NAC in the first day after vitrification of development improves blastocyst quality of murine embryos and does not exert any beneficial effect at oyher culture points.

Unveiling the Potential of Droplet Generation, Sorting, Expansion, and Restoration in Microfluidic Biochips

Microfluidic biochip techniques are prominently replacing conventional biochemical analyzers by the integration of all functions necessary for biochemical analysis using microfluidics. The microfluidics of droplets offer exquisite control over the size of microliter samples to satisfy the requirements of embryo culture, which might involve a size ranging from picoliter to nanoliter. Polydimethylsiloxane (PDMS) is the mainstream material for the fabrication of microfluidic devices due to its excellent biocompatibility and simplicity of fabrication. Herein, we developed a microfluidic biomedical chip on a PDMS substrate that integrated four key functions—generation of a droplet of an emulsion, sorting, expansion and restoration, which were employed in a mouse embryo system to assess reproductive medicine. The main channel of the designed chip had width of 1200 μm and height of 500 μm. The designed microfluidic chips possessed six sections—cleaved into three inlets and three outlets—to study the key functions with five-day embryo culture. The control part of the experiment was conducted with polystyrene (PS) beads (100 μm), the same size as the murine embryos, for the purpose of testing. The outcomes of our work illustrate that the rate of success of the static droplet culture group (87.5%) is only slightly less than that of a conventional group (95%). It clearly demonstrates that a droplet-based microfluidic system can produce a droplet in a volume range from picoliter to nanoliter.

Mitochondrial function in vitrified versus slow-frozen murine embryos

The effects of vitrification and slow-freezing on mitochondrial functions of in vitro produced murine embryos at various developmental stages were investigated using the Confocal Laser Scanning Microscope (CLSM). Oocytes were obtained from superovulated females, fertilized with sperm and cultured. Resulting 2-, 4- and 8-cell embryos were collected and cryopreserved by vitrification and slow-freezing. Mitochondria were stained with MitoTracker Red (CMXRos). Images were viewed by CLSM and analyzed using QWin SoftwareV.3. Fluorescent intensities were used to indicate viability. Results showed that mitochondrial fluorescence intensities of cryopreserved embryos were significantly lower as compared to non-cryopreserved embryos (p<0.01). Vitrification was found to be superior to slow-freezing at all developmental stages, based on mitochondrial function.