Human liver glycerol-3-phosphate dehydrogenase (hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzymesubstrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E•NAD, and ternary E•NAD•DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain, and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains was likewise examined by double mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

Successful fertilization in mammals requires spermatozoa to efficiently traverse the female reproductive tract to meet the egg. This process naturally selects high quality sperm cells for fertilization, but when artificial reproductive technologies are used such as in vitro fertilization, intracytoplasmic sperm injection, or intrauterine insemination, other methods of sperm selection are required. Currently, technology enables sperm sorting based on motility, maturity as defined by zeta potential or hyaluronic acid binding site expression, absence of apoptotic factors, appropriate morphology, and even sex. This review summarizes current knowledge on all known methods of sperm cell sorting, compares their efficiency, and discusses the advantages and limitations of each technique. Scope for further refinement and improvement of current methods are discussed as is the potential to utilize a variety of materials to innovate new methods of sperm separation.

Motility is an indicator of sperm cell viability due to higher probability in swimming through the female reproductive tract and undergo fertilization with the egg cell. Centrifugation method is a technique to process high volume semen and isolate motile sperm cells but decreases the biochemical integrity of spermatozoa due to the contact with reactive oxygen species (ROS) from dead cells released during centrifugation. This study uses solution electrospun poly(ε-caprolactone) membranes as an alternative in isolating motile spermatozoa by utilizing a rationally designed 3D printed module set up, providing the same benefits as commercially available techniques with minimal processing time, and bypassing the centrifugation step to provide higher quality sperm cells. The membranes, with nominal pore size distributions ranging from 5 to 6 µm are highly porous structures suitable for establishing baseline data for sperm cell sorting by motility. The proposed method allows for isolation of motile sperm cells with 74% purity, while decreasing the processing time by 98% when compared to centrifugation techniques. This novel approach provides a facile method for isolating motile spermatozoa directly from frozen semen samples without any pretreatments and is easily scalable for small and medium scale farms as well as larger industries.