Sperm Binding to the Zona Pellucida, Hyaluronic Acid Binding Assay, and PICSI

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilisation. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 μg/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2–3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomologus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.

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Chromomycin A3 staining, sperm chromatin structure assay and hyaluronic acid binding assay as predictors for assisted reproductive outcome

Reproductive BioMedicine Online ◽

10.1016/j.rbmo.2009.07.002 ◽

2009 ◽

Vol 19 (5) ◽

pp. 671-684 ◽

Cited By ~ 49

Author(s):

Martine Nijs ◽

Eva Creemers ◽

Annemie Cox ◽

Kim Franssen ◽

Mia Janssen ◽

...

Keyword(s):

Hyaluronic Acid ◽

Chromatin Structure ◽

Binding Assay ◽

Sperm Chromatin ◽

Reproductive Outcome ◽

Chromomycin A3 ◽

Sperm Chromatin Structure Assay ◽

Hyaluronic Acid Binding

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119 VITRIFICATION OF IMMATURE FELINE OOCYTES: EFFECTS ON ZONA PELLUCIDA ULTRASTRUCTURE AND DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab119 ◽

2010 ◽

Vol 22 (1) ◽

pp. 218

Author(s):

T. Tharasanit ◽

T. Sananmuang ◽

S. Manee-In ◽

A. Adirekthaworn ◽

C. Lohachit ◽

...

Keyword(s):

Zona Pellucida ◽

Binding Assay ◽

Research Unit ◽

Developmental Competence ◽

Domestic Cats ◽

Sperm Binding ◽

Genetic Potential ◽

Maturation Rate ◽

Senior Research

Oocyte cryopreservation has been used to restore fertility problems in women undergoing oocyte-toxic chemotherapy and also to salvage the genetic potential of endangered/valuable species. In domestic cats, knowledge regarding the cryopreservation technique of feline oocytes is rather limited. We examined the effect of OPS vitrification on zona pellucida ultrastructure and also the developmental competence of immature cat oocytes. Immature cat oocytes were vitrified using different CPA and CPA exposure techniques: (1) 2-step DMSO, n = 71; (2) 4-step DMSO, n = 78; (3) 2-step EG, n = 73; (4) 4-step EG, n = 117; (5) 2-step EG + DMSO, n = 67 and (6) 4-step EG + DMSO, n = 88. A combination of EG and DMSO or EG alone using 4-step equilibration technique yielded the highest maturation rates (28/88: 31.8% and 44/117: 37.6%, respectively). This was, however, significantly lower than nonvitrified controls (36/59, 61% maturation rate). We further examined the effect of OPS vitrification on ultrastructure of the zona pellucida using scanning electron microscopy and also to test the quality of the zona pellucida using sperm binding assay. Our modified OPS protocols did not significantly affect the appearance of zona pellucida of vitrified oocytes and also the number of tightly bound sperm as compared with controls (80.8 ± 63.8 v. 81.6 ± 70.4, respectively). Vitrification of immature oocytes significantly reduced the number of oocytes reaching cleavage and blastocyst stages compared with nonvitrified controls (24.8 and 7.5% v. 62.5 and 30.8% for cleavage and blastocyst formation rates, respectively). It is concluded that modification of OPS vitrification can be successfully applied for immature cat oocytes, in terms of zona pellucida ultrastructure, ability to bind sperm, and also their developmental competence. This study was financially supported by MRG4980108, CHE-TRF Senior Research Scholars RTA-5080010, The Thailand Research Fund, and The Research Unit of Reproductive and Biological Science, Chulalongkorn University.

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Relationship between hyaluronic acid binding assay and outcome in ART: a pilot study

Andrologia ◽

10.1111/j.1439-0272.2009.00992.x ◽

2010 ◽

Vol 42 (5) ◽

pp. 291-296 ◽

Cited By ~ 26

Author(s):

M. Nijs ◽

E. Creemers ◽

A. Cox ◽

M. Janssen ◽

E. Vanheusden ◽

...

Keyword(s):

Hyaluronic Acid ◽

Pilot Study ◽

Binding Assay ◽

Hyaluronic Acid Binding

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Zona pellucida-binding protein 2 (ZPBP2) and several proteins containing BX7B motifs in human sperm may have hyaluronic acid binding or recognition properties

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gax053 ◽

2017 ◽

Vol 23 (12) ◽

pp. 803-816 ◽

Cited By ~ 6

Author(s):

F Torabi ◽

O A Bogle ◽

J M Estanyol ◽

R Oliva ◽

D Miller

Keyword(s):

Hyaluronic Acid ◽

Zona Pellucida ◽

Binding Protein ◽

Human Sperm ◽

Hyaluronic Acid Binding

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Hyaluronic Acid Binding Assay Is Highly Sensitive to Select Human Spermatozoa with Good Progressive Motility, Morphology, and Nuclear Maturity

Gynecologic and Obstetric Investigation ◽

10.1159/000439530 ◽

2015 ◽

Vol 81 (3) ◽

pp. 244-250 ◽

Cited By ~ 9

Author(s):

Leila Rashki Ghaleno ◽

Mojtaba Rezazadeh Valojerdi ◽

Mohammad Chehrazi ◽

Fazel Sahraneshin Samani ◽

Reza Salman Yazdi

Keyword(s):

Hyaluronic Acid ◽

Binding Assay ◽

Human Spermatozoa ◽

Progressive Motility ◽

Highly Sensitive ◽

Hyaluronic Acid Binding

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Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes

Reproduction ◽

10.1530/reprod/119.1.77 ◽

2000 ◽

pp. 77-83 ◽

Cited By ~ 15

Author(s):

B Strom Holst ◽

B Larsson ◽

C Linde-Forsberg ◽

H Rodriguez-Martinez

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Zona Pellucida ◽

Binding Assay ◽

Binding Capacity ◽

Storage Conditions ◽

Deep Freezing ◽

Sperm Binding ◽

Significant Difference ◽

Scanning Electron

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P </= 0.05) irrespective of washing procedure and there was no significant difference between the two methods of storage. Scanning electron microscopy revealed that the zona pellucida of fresh oocytes was sponge-like in appearance with fenestrations surrounded by a meshwork of filaments. In stored oocytes, the meshwork tended to be wider, with larger fenestrations. The thickness of the zona pellucida was 0.3-1.0 microm in salt-stored oocytes and 6-8 microm in oocytes from frozen ovaries, whereas it was 3-6 microm in fresh oocytes. Changes in morphology that occur under the various storage conditions tested might have contributed to the reduced sperm binding capacity of the stored oocytes. Although stored canine oocytes do not bind as many spermatozoa as do fresh oocytes, the former can be used in a zona pellucida binding assay, and the deep freezing of the ovaries appears to be a better storage method than storing oocytes in salt solution.

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