Research Article J Forensic Sci & Criminal Inves Volume 15 Issue 1 - February 2021 Copyright © All rights are reserved by Hao PANG DOI: 10.19080/JFSCI.2021.15.555905 Recombinant Human Zona Pellucida 2 Protein was not Detected to Bind to the Non-Viable Spermatozoa in Vitro Xinrui Yang1,2, Zhixin Huo1,3 and Hao Pang1* 1Department of Forensic Genetics and Biology, China Medical University, China 2School of Forensic Medicine, Wannan Medical College, China 3Department of Forensic Medicine, Inner Mongolia Medical University, China Submission: January 21, 2021; Published: February 08, 2021 *Corresponding author: Hao PANG, School of Forensic Medicine, Department of Forensic Genetics and Biology, China Medical University, Shenyang 110122, PR China Abstract secondary zona ligand during fertilization to be related to acrosome exocytosis and block to polyspermy. Various studies have been performed Zona pellucida (ZP) binds to spermatozoa is the first step for sperm-egg interaction. ZP2, one of four human ZPs, is considered as a employing either purified native or recombinant proteins to gain insight into the role of ZP2 with the capacitated spermatozoa. In the current study recombinant human ZP2 (rhZP2) was first produced by overexpression in HEK293 cell, and the overexpressed rhZP2 was next purified. Presence of the rhZP2 was identified by Western blotting analysis. The effect of the rhZP2 binding to non-viable spermatozoa was assessed by indirect immunofluorescence and ELISA. After the non-viable spermatozoa were incubated with the rhZP2 in vitro, the interactions reflected by their dependent fluorescent labeling were not observed under microscope. The rhZP2 present in resultant supernatant was not significantly reduced by ELISA analysis after the rhZP2 was reacted with spermatozoa. Further, there was no relation found between spermatozoon number or incubation time and the amount of rhZP2 bound to the non-viable spermatozoa. The present results provide experimental evidence that the rhZP2 has not shown a character binding to non-viable spermatozoa in vitro, suggesting that the capacitated spermatozoa have still been preferentially employed in fertilization. An insufficiency of direct hit interaction, as a ligand vs an acceptor, between the rhZP2 and non-viable spermatozoaKeywords: implies that the rhZP2 has not applicability in separation of spermatozoa from the mixtures with vaginal fluid in forensic cases. Recombinant, ZP2, Purified rhZP2, Capacitation, Non-viable spermatozoa, Binding Introduction is effused into the extracellular matrix. A transmembrane region Zona pellucida (ZP) is a kind of glycoprotein matrix secreted of ZP2 protein distributes on the carboxyl terminal side and its by oocytes and granulosa cells that is essential for mammalian neighbored amino acid sequence can be recognized by furin. ZP2 fertilization. In human, the extracellularly glycoproteinaceous has been implicated as a secondary spermatozoon ligand that binds spermatozoon only after the induction of the spermatozoa coat is composed of four ZPs, ZP1-4. Reproductive studies recognition of the spermatozoa to the oocyte and subsequent prove that ZP glycoproteins have a major part to play in specific undergo a proteolytic cleavage. Spermatozoon binding to ZP2 is acrosome reaction of spermatozoa. In addition, ZPs can also acrosome reaction and is modified by the acrosome reaction to necessary for successful mammalian fertilization. Further studies on the molecular basis of gamete recognition have indicated that polyspermy and protect the embryo development [1]. Human prevent the formation of non-viable polypoid embryos against the N terminus of ZP2 is a key target region for spermatozoon ZP2 glycoprotein is one member of the ZP glycoprotein complex. ZP2 produced in baculovirus can in vitro bind to their cognate binding. Recombinant amino acid residues 39-154 of human Its 745 amino acid encoded by the Zp2 gene on chromosome 16 contain all structural domains of ZP protein family [2]. The data for the role of ZP2 in mediating spermatozoon binding to signal peptide, the remaining polypeptide as a functional protein spermatozoon [3]. These results not only cumulate experimental 34 amino acids of N-terminal are first cleaved and secreted as a J Forensic Sci & Criminal Inves 15(1): JFSCI.MS.ID.555905 (2021) 001 Journal of Forensic Sciences & Criminal Investigation the ZP and but also support human sperm-egg recognition with expression vector, which contains an additional C-terminal dependentIn mammalian, on ZP2 involvement spermatozoon [4]. capacitation leads to its 6-histidine (6×His) tag (Produced by Taihe Biotechnology, movement hyperactivated and prepares the spermatozoon to Beijing, China). The recombinant vector and a negative control go through acrosome reaction. At molecular level, cholesterol were transfected into the human embryonic kidney cell line 293 depletion from the spermatozoon plasma membrane associated (HEK293) cells. After the HEK293 cells were harvested, and finely crushed, the resultant lysates were stored at -80℃ until used eitherDetection for identification of recombinant or protein proteins purification by western of the rhZP2. blotting with spermatozoon capacitation, which quickens membrane hyperpolarizes the spermatozoon plasma membrane [6]. During fluidity, modulates intracellular ion concentrations [5], and mammalian fertilization, spermatozoa must pass through the female reproductive tract to capacitate or in vitro may be The cell lysates were tested for the presence of the rhZP2 proteins. The samples were mixed with a reducing sample capacitated spermatozoon is able to bind to the ZP. In contrast buffer, boiled for five minutes. Proteins were separated by SDS- capacitated by incubation with chemical analog, and then the polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.45mm nitrocellulose (Merck Millipore Bioscience). Membranes to the sperm-egg binding investigations, which the capacitated were blocked with 5% skim milk in Tris buffered saline containing spermatozoa were overwhelmingly employed to explore its 0.05% Tween-20 (TBST) for 1h at room temperature. The anti-His interactions with ZP glycoproteins during fertilization in humans, hamster gametes in vitro monoclonal antibody (1:5000, TransGen Biotech, Beijing, China) the incapacitated and non-viable ones were rarely exploited. With and anti-ZP2 polyclonal antibody (1:5000, Invitrogen, Carlsbad, , it was founded that an incapacitated California) were diluted in TBST as the primary antibody, spermatozoon would bind specifically to ZP of the same species. spermatozoon indicates that maturation and capacitation of respectively. Second antibody, goat anti-mouse IgG-HRP or goat The examination from the binding of ZP3 to incapacitated mouse spermatozoon are prerequisites for spermatozoon to expose its anti-rabbit IgG-HRP, was used to incubate the corresponding membrane. After washing, the immunoreactive proteins were Beijing, China). recognition in vitro revealed that uncapacitated spermatozoa visualized with enhanced chemiluminescence (TransGen Biotech, ZP3 binding sites [7]. The results from human spermatozoa-ZP Purification of rhZP2 protein the capacitated ones [8,9]. In human, primary spermatozoon had an obviously lower ZP-binding capacity when compared with The expressed rhZP2 protein was with affinity purified receptors interacting with ZP3 and ZP4 located in the surface using the 6×His tag binding capacity of the Ni-NTA agarose resin of the intact spermatozoon acrosome, whereas secondary the acrosome reaction [9,10]. Most models of gamete recognition (Merck Millipore Bioscience). The cell lysate was mixed with spermatozoon receptors binding to ZP2 were only exposed after preequilibrated Ni-NTA resin and incubated overnight. After a spermatozoon surface receptor [11,12]. Although the ligand extensive washing with the buffer containing 10mmol/L Tris-HCl suppose that a single ligand in the extracellular ZPs interacts with (pH 8.0), 0.3mol/L NaCl, and 5mmol/L imidazole, the resins were signaling cascades resulting in diverse cellular responses, it may be resuspended in the washing buffer. The rhZP2 was eluted off by can bind a cell surface receptor and trigger relative downstream a series of imidazole solutions with different concentrations. The receptor is experimentally evaluated at the protein level in vitro. eluted solutions, which the rhZP2 showed higher concentration a showing rationality that the interaction between the ligand and In addition, except for the potential application in genesiology, characterized by Western blot, were together dialyzed against 10mmol/L Tris-HCl (pH 8.0) and 0.3mol/L NaCl buffer. The in vitro collected solution through dialysis was condensed in Ultrafree-15 it is a very meaningful exploration for ZPs-spermatozoa binding Centrifugal Filter Units (MW 10kDa cut off, Millipore, MA, USA). to separate spermatozoa from the mixtures with vaginal The above finalized rhZP2 was in quality and quantity assessed fluidA in biologically forensic cases. active synthetic ZP, including recombinant bySpermatozoon Western blots. samples preparation human ZP proteins (rhZPs), would be distinguishingly useful as ligands for investigating spermatozoon-ZP recognitions. Thus, Freshly ejaculated semen was obtained by masturbation spermatozoon binding capacity to rhZP2 in vitro. the purpose of this study was to evaluate the non-viable human from three healthy normozoospermic donors after 3 to 4 days Materials and Methods of abstinence, following the clearance