De-Shien Jong

Kisspeptin acts as an upstream regulator of the hypothalamus–pituitary–gonad axis, which is one of the main regulatory systems for mammalian reproduction. Kiss1 and its receptor Kiss1r (also known as G protein-coupled receptor 54 (Gpr54)) are expressed in various organs, but their functions are not well understood. The purpose of this study was to investigate the expression profiles and functions of kisspeptin and KISS1R in the reproductive tissues of imprinting control region mice. To identify the expression pattern and location of kisspeptin and KISS1R in gonads, testes and ovarian tissues were examined by immunohistochemical or immunofluorescent staining. Kisspeptin and KISS1R were expressed primarily in Leydig cells and seminiferous tubules respectively. KISS1R was specifically localized in the acrosomal region of spermatids and mature spermatozoa. Kisspeptin, but not KISS1R, was expressed in the cumulus–oocyte complex and oviductal epithelium of ovarian and oviductal tissues. The sperm intracellular calcium concentrations significantly increased in response to treatment with kisspeptin 10 in Fluo-4-loaded sperm. The IVF rates decreased after treatment of sperm with the kisspeptin antagonist peptide 234. These results suggest that kisspeptin and KISS1R might be involved in the fertilization process in the female reproductive tract. In summary, this study indicates that kisspeptin and KISS1R are expressed in female and male gametes, respectively, and in mouse reproductive tissues. These data strongly suggest that the kisspeptin system could regulate mammalian fertilization and reproduction.

A major reason for infertility is due to male factors, including the quality of spermatozoa, which is a primary factor and often difficult to assess, particularly the total sperm concentration and its motile percentage. This work presents a simple microfluidic device to assess sperm quality by quantifying both total and motile sperm counts. The key design feature of the microfluidic device is two channels separated by a permeative phase-guide structure, where one channel is filled with raw semen and the other with pure buffer. The semen sample was allowed to reach equilibrium in both chambers, whereas non-motile sperms remained in the original channel, and roughly half of the motile sperms would swim across the phase-guide barrier into the buffer channel. Sperms in each channel agglomerated into pellets after centrifugation, with the corresponding area representing total and motile sperm concentrations. Total sperm concentration up to 10⁸ sperms per ml and motile percentage in the range of 10–70% were tested, encompassing the cutoff value of 40% stated by World Health Organization standards. Results from patient samples show compact and robust pellets after centrifugation. Comparison of total sperm concentration between the microfluidic device and the Makler chamber reveal they agree within 5% and show strong correlation, with a coefficient of determination of R² = 0.97. Motile sperm count between the microfluidic device and the Makler chamber agrees within 5%, with a coefficient of determination of R² = 0.84. Comparison of results from the Makler Chamber, sperm quality analyzer, and the microfluidic device revealed that results from the microfluidic device agree well with the Makler chamber. The sperm microfluidic chip analyzes both total and motile sperm concentrations in one spin, is accurate and easy to use, and should enable sperm quality analysis with ease.

We present a microfluidic device to generate either statically spatial or dynamically temporal logarithmic concentrations. The temporal logarithmic concentration generator was also integrated with planar patch-clamp chips for dose-response assays on ion channels. Proposed serial dilution principle controls the flow pattern at each branching point via designing the flow resistance of microchannels. Simple and linear ratios of the flow resistance results in desired logarithmic concentration at outlets, where the concentrations can be dynamically altered by different combination of valve actuations, were demonstrated. Single-cell pharmacology on ion channels was implemented by sequentially applying logarithmic drug concentrations to patched cells. Inhibitory activity of potassium channels of human embryonic kidney cells was examined by tetraethylammonium solutions. Resulted IC₅₀ and Hill slope reveal excellent agreement with assays from manually prepared drug concentrations showing the practicability and preciseness of the present approach. Applications include cellular analysis under various drugs and/or logarithmic concentrations at the single-cell level.

We present a dielectrophoresis (DEP)-based cellular microarray chip for cell-based anticancer drug screening in perfusion microenvironments. Human breast cancer cells, MCF7, were seeded into the chip and patterned via DEP forces onto the planar interdigitated ring electrode (PIRE) arrays. Roughly, only one third of the cell amount was required for the chip compared to that for a 96-well plate control. Drug concentrations (cisplatin or docetaxel) were stably generated by functional integration of a concentration gradient generator (CGG) and an anti-crosstalk valve (ACV) to treat cells for 24 hours. Cell viability was quantified using a dual staining method. Results of cell patterning show substantial uniformity of patterned cells (92 ± 5 cells per PIRE). Furthermore, after 24 hour drug perfusion, no statistical significance in dose–responses between the chip and the 96-well plate controls was found. The IC₅₀ value from the chip also concurred with the values from the literature. Moreover, the perfusion culture exhibited reproducibility of drug responses of cells on different PIREs in the same chamber. The chip would enable applications where cells are of limited supply, and supplement microfluidic perfusion cultures for clinical practices.

Planar patch clamp has revolutionized characterization of ion channel behavior in drug discovery primarily via advancement in high throughput. Lab use of planar technology, however, addresses different requirements and suffers from inflexibility to enable wide range of interrogation via a single cell. This work presents integration of planar patch clamp with microfluidics, achieving multiple solution exchanges for tailor‐specific measurement and allowing rapid replacement of the cell‐contacting aperture. Studies via endogenously expressed ion channels in HEK 293T cells were commenced to characterize the device. Results reveal the microfluidic concentration generator produces distinct solution/drug combination/concentrations on‐demand. Volume‐regulated chloride channel and voltage‐gated potassium channels in HEK 293T cells immersed in generated solutions under various osmolarities or drug concentrations show unique channel signature under specific condition. Excitation and blockage of ion channels in a single cell was demonstrated via serial solution exchange. Robustness of the reversible bonding and ease of glass substrate replacement were proven via repeated usage of the integrated device. The present approach reveals the capability and flexibility of integrated microfluidic planar patch‐clamp system for ion channel assays. Biotechnol. Bioeng. 2011; 108:1395–1403.

Planar patch-clamp has revolutionized ion-channel measurement by eliminating laborious manipulation from the traditional micropipette approach and enabling high throughput. However, low yield in gigaseal formation and/or relatively high cost due to microfabricated processes are two main drawbacks. This paper presents patch clamping on glass substrate—an economical solution without sacrificing gigaseal yield rate. Two-stage CO₂ laser drilling methodology was used to generate an hourglass, funnel-like aperture of a specified diameter with smooth and debris-free surfaces on 150 µm borosilicate cover glass. For 1–3 µm apertures as patch-clamp chips, seal resistance was tested on human embryonic kidney, Chinese hamster ovary, and Jurkat T lymphoma cells with a gigaseal success rate of 62.5%, 43.6% and 66.7% respectively. Results also demonstrated both whole-cell and single channel recording on endogenously expressed ion channels to confirm the capability of different patch configurations.

We report production of a polyclonal antibody against the StAR (steroidogenic acute regulatory) protein of steroidogenic cells and immunohistochemistry (IHC) staining of bovine adrenal gland tissue available in paraffin block. The epitope-specific polyclonal antibody was produced in a rabbit immunized against a synthetic 26 amino acid peptide (82AMQRALGILKDQEGWKKESRANGDE107) derived from the coding sequences reported for the bovine StAR gene (Gene Bank Accession No. Q28918). Western blots were developed using the StAR-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The antiserum was found to be highly specific for StAR, which exhibited an estimated molecular weight of about 30 KDa for all species analyzed. Finally, the peptide antiserum was successfully employed to localize StAR protein by immunohistochemical staining of thin sections prepared from bovine adrenal gland tissue. This study is the first to report a polyclonal peptide antiserum that apparently recognizes native StAR protein, regardless of the species of origin. The successful production of the antibody has provided a useful tool for studying regulation of StAR protein.

We present a fast and easy fabrication method to create an hourglass-shaped aperture for cellular electrophysiology. A series of short pulses of CO 2 laser were applied to drill on borosilicate cover glass. Special reflow phenomenon of melted glass occurred at the rear of the surface resulted in the unique tailored-contour aperture. Smooth and debris-free surface of the aperture reveals the capability of planar patch-clamp application. In seal resistance characterization, calculated seal resistance was 1.2MΩ before and 862MΩ after cell attached to a 4μm aperture. These values are promising and they are most likely due to freshly activated contact surface during seal formation. This efficient and economic approach could be extended to array configuration and integrate with microfluidic system.