Zona pellucida (ZP) plays a key role in the recognition, combination and penetration of sperms and oocytes, the prevention of multiple impregnation, the protection of embryos, and so on. The paper reviews the constitution, molecular structure, biological function and application of zona pellucida in male fertilization.
Acrosome Reaction ; physiology ; Animals ; Female ; Fertility ; physiology ; Humans ; Male ; Mice ; Molecular Structure ; Zona Pellucida ; chemistry ; physiology

Human sperm-egg recognition, adhesion and fusion are key steps in the whole reproductive process. Some abnormalities in human gamete interaction have been shown to be due to defects in the sperm, others attributed to defects in the zona pellucida (ZP) and the egg plasma membrane. This paper reviews the molecular basis and molecular mechanisms of human sperm-egg interaction. More and more advances in the studies of these aspects are shown to be of significant value to the diagnosis and treatment of infertility as well as to the development of assisted reproductive techniques.
Acrosome Reaction ; physiology ; Female ; Humans ; Infertility, Male ; physiopathology ; Male ; Sperm-Ovum Interactions ; physiology ; Zona Pellucida

Human zona pellucida (hZP) plays a critical role in the recognition, binding of sperms and oocytes, induction of acrosomal exocytosis, and avoidance of polyspermy. Human ZP is composed of four glycoproteins designated as hZP1, hZP2, hZP3 and hZP4. This paper reviews the actions of native hZP or recombinant hZP on acrosomal exocytosis and sperm-ZP binding.
Acrosome ; physiology ; Egg Proteins ; physiology ; Humans ; Male ; Membrane Glycoproteins ; physiology ; Receptors, Cell Surface ; physiology ; Spermatozoa ; physiology ; Zona Pellucida Glycoproteins

The purpose was to optimize the vitrification for porcine embryos cryopreservation. Blastocyst/Morula (5-6th day-embryos) were collected from superovulated Bama mini-pigs (sows/gilts). We compared different cryopreservation methods, cryopreservation tools, thining of zona pellucida (ZP) and recipient breeds on the efficiency of porcine embryo cryopreservation. The results showed that: in embryo survival rate and blastocyst cell number, there were no significant differences between cryopreservation method I [embryos were vitrified by two step method with open pulled straw (OPS) and glass micropipette (GMP) in solution 1 (TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3 min, and solution 2 (TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4 mol/L SUC) for 1 min, stored in liquid nitrogen] and method II[Blastocysts were cultured for 25 min in NCSU23 + 7.5 microg/mL cytochalasin B, centrifuged at approximately 13 000 xg for 12-13 min, and recovered back into pNCSU23. They were then equilibrated for 5 min in 2 mol/L ethylene glycol in pNCSU23, washed quickly in the vitrification medium, 8 mol/L ethylene glycol, 7% polyvinylpyrrolidone (PVP) in pNCSU23, loaded into OPS/GMP, and plunged into liquid nitrogen]. GMP vitrification method was more suitable and efficient than OPS method (P < 0.05) in embryo survival rate (83.8% vs 77.6%) and blastocyst cell number (53.1 vs 47.5) after thawing. Thining of ZP did not increase the survival rate, but significantly improved blastocyst cell number in the survival blastcysts (60.1 and 46, P < 0.01). Local pig breeds (Fengjing sows) were more suitable as recipients for embryo transfer of vitrified/warmed blastcysts, which can improve pregnant rate and embryo efficiency.
Animals ; Blastomeres ; cytology ; Cryopreservation ; methods ; veterinary ; Embryo Transfer ; veterinary ; Embryo, Mammalian ; Swine ; Swine, Miniature ; Vitrification ; Zona Pellucida ; physiology

Assisted hatching (AH), which is known to improve the hatching potential of mammalian embryos, has been used to increase the pregnancy rate in in vitro fertilization cycles. However, the effect of AH on a trypsin-like protease, which is known to be associated with the hatching process, has not been studied. In this study, we evaluate whether the intactness of zona pellucida affects the secretion of a trypsin-like protease from mouse blastocyst. Four- to 8-cell stage mouse embryos were collected at 66- to 68 hr after hCG injection and divided into 3 groups according to the manipulation of zona pellucida. The groups are no treatment (control), drilling of zona pellucida (ZD) and thinning of zona pellucida (ZT). The activity of a trypsin-like protease, blastocyst development and hatching rate were compared among the three groups at 110 and 135 hr after hCG injection, respectively. The protease activity and blastocyst development were not significantly different among control, ZD and ZT groups at 110 and 135 hr after hCG injection, respectively. However, the hatching rate of ZD and ZT groups was significantly higher than that of control group at each time, respectively (p>0.001). Even in the zona pellucida removed embryos, the protease activity did not differ from the control group. In conclusion, the secretion of a trypsin-like protease from mouse blastocyst does not seem to be affected by the intactness of zona pellucida.
Animal ; Blastocyst/secretion ; Blastocyst/enzymology* ; Female ; Fertilization in Vitro/methods ; Gonadotropins, Chorionic/pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Pregnancy ; Serine Endopeptidases/secretion ; Serine Endopeptidases/metabolism* ; Zona Pellucida/physiology* ; Zona Pellucida/drug effects

AIMTo investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa.

METHODSThe effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA)/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline (CTC) assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa.

RESULTSImmunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively.

CONCLUSIONOur results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.

Acrosome Reaction ; drug effects ; physiology ; Animals ; Cattle ; Enzyme Inhibitors ; pharmacology ; Genistein ; pharmacology ; Male ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Sperm Capacitation ; drug effects ; physiology ; Zona Pellucida ; drug effects ; physiology

OBJECTIVE: To determine the sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction in patients with unexplained infertility, and to discuss the relationship between ZP-induced acrosome reaction and fertilization rate. METHODS: We compared the fertilization rate and good embryo rate in patients with unexplained infertility after fertilization in 2 ways. Based on the causes of infertility, patients were divided into an unexplained infertility group (Group A) and a pure female tubal factor group (Group B). Oocytes which were obtained by super ovulation from 25 patients with unexplained infertility were randomly divided into 2 groups with conventional in vitro fertilization (IVF) (Group A1) and intracytoplasmic sperm injection (ICSI) fertilization (Group A2). The pure female tubal factor group (Group B) had conventional IVF. We conducted sperm-ZP binding and ZP-induced acrosome reaction experiments with 2 groups of men's sperms separately. We compared the number of sperm-egg binding and ZP-induced acrosome reaction rate and discussed the relationship between the ZP-induced acrosome reaction and fertilization rate, and also the fertilization rate, good embryo rate and pregnancy rate in patients with unexplained infertility after fertilization in 2 ways. RESULTS: The average number of sperm-egg binding (78.29 ± 16.31) and the ZP-induced acrosome reaction rate (55.87 ± 27.69) % in Group A were lower than those of Group B [94.63 ± 6.72, (82.53 ± 17.99)%]. The difference between the average number of sperm-egg binding and the ZP-induced acrosome reaction was significant (P <0.01). The fertilization rate of Group A1 was significantly lower than that of Group B and Group A2 (P <0.01). But there was no significant difference in the good embryo rate among the 3 groups. There was no significant difference between Group A2 and B in fertilization rate and good embryo rate (P <0.05). There was no significant difference in pregnancy rate between Group A and B (P <0.05). Fertilization rate and the rate of acrosome reaction had marked positive correlation with statistical significance (r =0.932, P <0.01). CONCLUSION ZP binding and ZP-induced acrosome reaction are very important experiments in sperm function test for patients with unexplained infertility. It can not only effectively avoid no embryo transferring due to complete failure of fertilization but also get a desirable outcome of pregnancy using half-ICSI fertilization in patients with unexplained infertility.
Acrosome Reaction ; Embryo Transfer ; Female ; Fertilization in Vitro ; Humans ; Infertility ; etiology ; therapy ; Male ; Oocytes ; physiology ; Ovulation Induction ; Sperm Injections, Intracytoplasmic ; Sperm-Ovum Interactions ; physiology ; Treatment Outcome ; Zona Pellucida ; physiology

The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed.
Acrosome Reaction ; Aneuploidy ; Chromatin ; DNA Damage ; Fertility* ; Humans ; Infertility ; Infertility, Male ; Male* ; Physical Examination ; Physiology ; Reactive Oxygen Species ; Semen Analysis ; Sperm Injections, Intracytoplasmic ; Sperm Motility ; Spermatozoa ; Zona Pellucida

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER^® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER^® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER^® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.
Acrosome Reaction/physiology* ; Calcimycin/pharmacology* ; Calcium/pharmacology* ; Calcium Ionophores/pharmacology* ; Drug Delivery Systems ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/metabolism* ; Male ; Progesterone/pharmacology* ; Spermatozoa/metabolism* ; Zona Pellucida/metabolism*

AIMTo detect the expression of hepatitis B virus (HBV) genes (HB S and C genes) in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization (IVF) technique.

METHODSHuman sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction (PCR) was used to detect HB S and pre-Core/Core (pre-C/C) coding genes both in one- and two-cell embryos. Reverse transcription-PCR (RT-PCR) analysis was used to study the expression of the two genes. Fluorescence in situ hybridization (FISH) analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome.

RESULTSBoth HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences.

CONCLUSIONSperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Animals ; Blastula ; virology ; Cricetinae ; DNA Primers ; Female ; Fertilization in Vitro ; Gene Expression Regulation, Viral ; Genome, Viral ; Hepatitis B virus ; genetics ; Humans ; Male ; Mesocricetus ; Oocytes ; physiology ; Ovum ; virology ; Polymerase Chain Reaction ; Semen ; virology ; Spermatozoa ; virology ; Transfection ; Virus Replication ; Zona Pellucida ; physiology