'ZP domain' of human zona pellucida glycoprotein-1 binds to human spermatozoa and induces acrosomal exocytosis

<p>Abstract</p> <p>Background</p> <p>The human egg coat, zona pellucida (ZP), is composed of four glycoproteins designated as zona pellucida glycoprotein-1 (ZP1), -2 (ZP2), -3 (ZP3) and -4 (ZP4) respectively. The zona proteins possess the archetypal 'ZP domain', a signature domain comprised of approximately 260 amino acid (aa) residues. In the present manuscript, attempts have been made to delineate the functional significance of the 'ZP domain' module of human ZP1, corresponding to 273-551 aa fragment of human ZP1.</p> <p>Methods</p> <p>Baculovirus-expressed, nickel-nitrilotriacetic acid affinity chromatography purified 'ZP domain' of human ZP1 was employed to assess its capability to bind and subsequently induce acrosomal exocytosis in capacitated human spermatozoa using tetramethyl rhodamine isothiocyanate conjugated Pisum sativum Agglutinin in absence or presence of various pharmacological inhibitors. Binding characteristics of ZP1 'ZP domain' were assessed employing fluorescein isothiocyanate (FITC) labelled recombinant protein.</p> <p>Results</p> <p>SDS-PAGE and immunoblot characterization of the purified recombinant protein (both from cell lysate as well as culture supernatant) revealed a doublet ranging from ~35-40 kDa. FITC- labelled 'ZP domain' of ZP1 binds primarily to the acrosomal cap of the capacitated human spermatozoa. A dose dependent increase in acrosomal exocytosis was observed when capacitated sperm were incubated with recombinant 'ZP domain' of human ZP1. The acrosome reaction mediated by recombinant protein was independent of Gi protein-coupled receptor pathway, required extra cellular calcium and involved both T- and L-type voltage operated calcium channels.</p> <p>Conclusions</p> <p>Results described in the present study suggest that the 'ZP domain' module of human ZP1 has functional activity and may have a role during fertilization in humans.</p

Nonendoscopic endonasal dacryocystorhinostomy: Outcome in 134 eyes

Aims: To evaluate the outcome of nonendoscopic endonasal dacryocystorhinostomy (NEN-DCR) in patients with nasolacrimal duct obstruction (NLDO) in India. Methods: Retrospective case series of NEN-DCR between July 2012 and October 2014. All patients had follow-up >3 months. Success was defined anatomically as patency on irrigation and functionally as relief from epiphora. Statistical Analysis Used: Fischer's exact test and Chi-square test. Results: A total of 122 patients (134 eyes; 81 female; mean age 37 ± 18 years) were included. Indications were primary acquired NLDO in 92 (68%) eyes of adults (>18 years), NLDO in children (<18 years) in 22 eyes (16%), acute dacryocystitis in 13 eyes, failed prior DCR in six eyes, and secondary acquired NLDO in one eye. Mean duration of surgery was 36 min (range: 16–92). At a median follow-up of 6 months (range: 3–15), 86% eyes had functional success and 85% had anatomical success. Revision NEN-DCR was successful in 13/16 eyes. All patients with acute dacryocystitis were completely symptom-free at final visit. In children, (17/22) 77% achieved functional success after primary NEN-DCR which improved to 100% after one revision. Tube-related epiphora and granuloma in ten eyes resolved after removal. Conclusion: NEN-DCR gives good outcome in primary NLDO and is also effective in those with acute dacryocystitis and in children with NLDO. The technique obviates the need for an endoscope and has an acceptable safety profile and thus may be particularly suited for the developing nations

Bonnet monkey (Macaca radiata) ovaries, like human oocytes, express four zona pellucida glycoproteins

Recent studies document that the zona pellucida matrix of human oocytes is composed of four glycoproteins designated as ZP1, ZP2, ZP3, and ZP4 instead of three as proposed in mouse model. In the present study, investigations were carried out to find the presence of the fourth ZP glycoprotein in the ovaries of non-human primates namely bonnet monkey (Macaca radiata). Employing total RNA isolated from bonnet monkey ovaries, the complementary deoxyribonucleic acid (cDNA) encoding bonnet monkey ZP1 (up to furin cleavage site) was successfully amplified by reverse transcribed polymerase chain reaction (RT-PCR). The deduced amino acid (aa) sequence of bonnet monkey ZP1 revealed 96.0% identity with human ZP1. The 21 cysteine residues present in bonnet monkey ZP1 were conserved in human, mouse, rat, quail, and chicken. Simultaneously, polyclonal antibodies were generated in mice against synthetic peptides corresponding to human ZP1 (P1, 137-150 aa; P2, 223-235 aa; P3, 237-251 aa; P4, 413-432 aa; and P5, 433-451 aa). Employing anti-peptide antibodies that were devoid of cross-reactivity as determined by ELISA with human/bonnet monkey recombinant ZP2, ZP3, and ZP4, the presence of ZP1 in the ovaries of bonnet monkey and human oocytes was demonstrated by indirect immunofluorescence. The antibodies against peptides P3 and P4 reacted only with the ZP of bonnet monkey ovaries and not other ovarian associated cells. The data presented in this manuscript provide evidence, for the first time, that the bonnet monkey ZP matrix is composed of four glycoproteins. Mol. Reprod. Dev. 75: 156-166, 2008

Transconjunctival dacryocystorhinostomy: An aesthetic approach

Purpose: To report the anatomical and cosmetic outcome of transconjunctival dacryocystorhinostomy (TDCR) in an Asian Indian population. Methods: TDCR was initially performed in cadaver eyes followed by patients with primary acquired nasolacrimal duct obstruction (NLDO). This was a prospective noncomparative case series of all consecutive TDCRs performed between April 2013 and June 2015. Outcome measures were anatomical patency, epiphora, presence of diplopia, aesthetic outcome, and health status. Results: A total of 17 (18 eyes) patients with a mean age 43.9 ± 11.8 years (range, 32–75) were included in the study. Eight were males, and one patient underwent TDCR in both eyes. TDCR was successfully performed in 15/18 (82%) eyes under local anesthesia. Procedure converted to transcutaneous external DCR in two and dacryocystectomy in one patient each. Mean duration of surgery was 52.6 (range, 29–110) min. Anatomical patency and relief from epiphora was achieved in all (15/15) eyes after TDCR at a median follow-up of 15.5 months. At final follow-up, objective assessment of the cosmetic outcome graded the surgical scar at the lateral canthus as invisible in all except one and conjunctival fornix as visible only after eyelid eversion in all patients. Disturbance of the medial fat pad was not seen in any patient. A questionnaire-based health status evaluation showed marked improvement in anxiety/depression before and after TDCR with an overall well-being score of 88 on a scale of 0–100 (worst–best) after TDCR. Conclusions: TDCR offers a promising aesthetic approach in patients with primary acquired NLDO and gives excellent functional and cosmetic outcome

Human zona pellucida glycoproteins: functional relevance during fertilization

The zona pellucida (ZP), a glycoproteinaceous matrix surrounding the mammalian oocyte plays an important role in species-specific sperm-egg binding, induction of acrosome reaction in the ZP-bound spermatozoa, avoidance of polyspermy and protection of the embryo prior to implantation. In contrast to mouse, human ZP matrix is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4 (Zp4 pseudogene in mouse). Recent studies employing recombinant and immunoaffinity purified human zona proteins revealed that in addition to ZP3, capacitated acrosome-intact spermatozoa also bind ZP4. Human ZP2 primarily binds to the acrosome-reacted spermatozoa, supporting its role as secondary sperm receptor, as delineated in the murine model. For binding of human zona proteins to spermatozoa, glycosylation is not critical. Both human ZP3 and ZP4 induce dose-dependant acrosomal exocytosis in capacitated sperm. In contrast to the murine model, N-linked glycosylation is more critical for the human ZP3/ZP4 mediated induction of acrosomal exocytosis. Subtle differences in the downstream signaling events associated with ZP3 vs. ZP4 mediated induction of acrosomal exocytosis have been observed. To conclude, in humans, ZP3 and ZP4 are involved in binding of the spermatozoa to the egg and subsequent induction of acrosome reaction. The contribution, if any, of human ZP glycoprotein-1 (ZP1) during these stages of fertilization remains to be elucidated

Primary nonendoscopic endonasal versus external dacryocystorhinostomy in nasolacrimal duct obstruction in children

Purpose: The aim is to compare the outcome of nonendoscopic endonasal dacryocystorhinostomy (NEN DCR) with external DCR (EXT-DCR) in the treatment of nasolacrimal duct obstruction (NLDO) in children. Methods: A retrospective, comparative chart analysis of all consecutive children <16 years after EXT-DCR or NEN-DCR between June 2012 and February 2016. Results: Seventy-one children (79 eyes) underwent DCR in the study, of which 37 children (40 eyes) underwent EXT-DCR and 34 (39 eyes) NEN-DCR. Mean age of both groups (8.7 vs. 7.7 years) was comparable. Etiologically, persistent congenital NLDO was the most common indication (50% vs. 72%), followed by acquired and secondary NLDO. Mean duration was shorter for NEN-DCR (47 vs. 37 min; P = 0.0021). Mitomycin C 0.04% was used more often in NEN-DCR (10% vs. 56.41%). Success after primary EXT-DCR was 100% as compared to 75% for primary NEN-DCR at median follow-up of 12 and 16 months respectively. At revision, the main cause of failure was granuloma (60%). After revision, all eyes were symptom-free at a median follow-up of 9.5 months. Conclusion: Primary NEN-DCR has a poorer outcome than EXT-DCR in the treatment of NLDO in children and is more likely to need a revision procedure

DNA vaccine encoding chimeric protein encompassing epitopes of human ZP3 and ZP4: immunogenicity and characterization of antibodies

Immunization with zona pellucida (ZP) glycoproteins leads to curtailment of fertility often associated with ovarian dysfunction. To avoid ovarian dysfunction, synthetic peptides corresponding to ZP glycoproteins have been proposed as candidate immunogens. In the present study, plasmid DNA encoding a human ZP glycoprotein-3 (ZP3) epitope corresponding to amino acid (aa) residues 334-343 and a human ZP glycoprotein-4 (ZP4) epitope corresponding to aa residues 251-273 separated by a triglycine spacer was constructed using the mammalian expression vector, VR1020. The plasmid DNA construct expressed both human ZP3 and ZP4 epitopes, as revealed by transient transfection of COS-1 (African green monkey, kidney) mammalian cells. Active immunization of female BALB/cJ mice with the DNA vaccine led to generation of antibodies reactive with baculovirus-expressed recombinant human ZP3, ZP4 and ZP3<SUB>(334-343aa)</SUB>-GGG-ZP4<SUB>(251-273aa)</SUB> synthetic peptide in an ELISA as well as T cell responses. Antibodies generated by the DNA vaccine also recognized native ZP. The immune sera significantly inhibited (p/&lt;/0.005) the binding of FITC-labeled ZP3 to capacitated human sperm, whereas no inhibition in the binding of FITC-labeled ZP4 was observed. However, a significant decrease in acrosomal exocytosis mediated by both recombinant human ZP3 (p/&lt;/0.005) and ZP4 (p/&lt;/0.005) was observed in presence of the immune sera. These studies demonstrate that a DNA vaccine can be designed to elicit antibodies against small epitopes of ZP

In humans, zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis

Background: It has been suggested that the zona pellucida (ZP) may mediate species-specific fertilization. In human the ZP is composed of four glycoproteins: ZP1, ZP2, ZP3 and ZP4. In the present study, the expression profile of ZP1 in human oocytes and ovaries, and its role during fertilization, is presented. Methods: Human ZP1 (amino acid residues 26-551) was cloned and expressed in both non-glycosylated and glycosylated forms and its ability to bind to the capacitated human spermatozoa and to induce acrosomal exocytosis was studied. Monoclonal antibodies (MAbs), specific for human ZP1 and devoid of reactivity with ZP2, ZP3 and ZP4 were generated and used to localize native ZP1 in oocytes and ovarian tissues. Results: The MAbs generated against ZP1 recognized specifically the zona m-atrix of secondary and antral follicles, ovulated oocytes, atretic follicles and degenerating intravascular oocytes, but failed to react with the Fallopian tube, endometrium, ectocervix and kidney. Escherichia coli and baculovirus-expressed recombinant human ZP1 revealed bands of ~75 and ~85 kDa, respectively, in western blot. Lectin binding studies revealed the presence of both N- and O-linked glycosylation in baculovirus-expressed ZP1. Fluorescein isothiocyanate-labelled E. coli- and baculovirus-expressed recombinant ZP1 bound to the anterior head of capacitated spermatozoa, however, only baculovirus-expressed ZP1 induced acrosomal exocytosis in capacitated sperm suggesting the importance of glycosylation in mediating the acrosome reaction. The human ZP1-mediated acrosome reaction involved the activation of both T- and L-type voltage-operated calcium channels, but does not activate the Gi-coupled receptor pathway. Inhibition of protein kinase A and C significantly also reduced the ZP1-mediated induction of the acrosome reaction. Conclusion: These studies revealed for the first time that in humans ZP1, in addition to ZP3 and ZP4, binds to capacitated spermatozoa and induces acrosomal exocytosis