Comparison of two methods to obtain hemizonae pellucidae for sperm function tests

Sánchez, Raúl; Finkenzeller, Caroline; Schill, W.‐B.; Miska, W.

doi:10.1093/oxfordjournals.humrep.a135825

Cited by 13 publications

(10 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Prior to each assay, salt-stored oocytes were desalted by washing with BWW containing 0.3% BSA. Oocytes were bisected into equal halves under a stereomicroscope [24].…”

Section: Hemizona Assaymentioning

confidence: 99%

Nitric Oxide Synthase Inhibition in Human Sperm Affects Sperm-Oocyte Fusion but Not Zona Pellucida Binding1

Francavilla

Santucci

Macerola

et al. 2000

Biology of Reproduction

View full text Add to dashboard Cite

There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). N(G)-nitro-L-arginine methyl ester (L-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. L-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by L-arginine in a dose-dependent manner. L-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 microM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to L-NAME following capacitation in both versions of HEPT. On the contrary, L-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.

show abstract

“…Prior to each assay, salt-stored oocytes were desalted by washing with BWW containing 0.3% BSA. Oocytes were bisected into equal halves under a stereomicroscope [24].…”

Section: Hemizona Assaymentioning

confidence: 99%

Nitric Oxide Synthase Inhibition in Human Sperm Affects Sperm-Oocyte Fusion but Not Zona Pellucida Binding1

Francavilla

Santucci

Macerola

et al. 2000

Biology of Reproduction

View full text Add to dashboard Cite

show abstract

“…Originally developed to predict the fertilization potential of human spermatozoa in IVF settings (Franken et al ., 1989, 1993), its application today has extended to different fields as reproductive physiology (Francavilla et al ., 2000; Miranda et al ., 2000) and immunocontraception (Govind et al ., 2000). Matching hemizonae are usually generated using micromanipulation (Burkman et al ., 1988) or handcutting (Sánchez et al ., 1995). In order to simplify the technically demanding cutting procedures, the laser‐bisecting method presented in this paper was developed.…”

Section: Discussionmentioning

confidence: 99%

“…Its application is extremely easy, even the inexperienced will be able to perform an exact oocyte bisection within a few seconds with the laser method. In contrast, the handcutting method (Sánchez et al ., 1995) and the conventional micromanipulator method (Burkman et al ., 1988) require higher technical skills and experience in the use of the micromanipulator. Furthermore, there is no need for specially bended holding pipettes and microscalpels.…”

Section: Discussionmentioning

confidence: 99%

“…The zona halves are conventionally generated using a micromanipulator, working with a holding pipette and a microscalpel (Burkman et al ., 1988). An alternative manual bisection technique was introduced in 1995 (Sánchez et al ., 1995). In 1999 we presented first data on using a 1.48‐μm diode laser for bisecting oocytes into two equal hemizonae (Schöpper et al ., 1999).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Application of a 1.48‐μm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay

et al. 2002

View full text Add to dashboard Cite

Laser systems are very promising new technical tools in assisted reproduction. It was investigated if laser radiation can replace the mechanical cutting procedure via micromanipulator in the hemizona assay (HZA), a commonly used bioassay to determine the sperm-zona pellucida binding capacity. An oocyte was bisected precisely into two identical hemizonae with approximately 20 laser pulses (pulse length 30 msec) using a 1.48-microm diode laser. Compared with the conventional method using microscalpels for zona bisection, laser treated hemizonae showed equivalent sperm-binding and within the two groups there was no detectable difference between matching hemizonae in their capacity for tight sperm-binding. To evaluate whether laser radiation affects the outcome of the HZA when effects of certain substances are investigated, the spermatozoa were preincubated with human follicular fluid (hFF), which inhibits the binding of spermatozoa to zona pellucida in vitro. Supplementation with follicular fluid exerted an inhibitory effect in both groups. The hemizona index (HZI) showed no statistical differences between the two methods. Therefore, the 1.48-microm diode laser is a suitable new instrument for generating equally sized hemizonae. There is no use for holding pipettes and microscalpels, on the contrary, for performing the HZA the laser is a precise, very quick and easy to use new working tool.

show abstract

“…Hemizona binding of human spermatozoa to laser-bisected and manually bisected hemizonae was essentially performed as described by Sanchez et al (1995) and following the recommendations by Yao et al (1996). Motile spermatozoa were prepared by standard procedurc:.…”

Section: Methodsmentioning

confidence: 99%

A new method to produce equally sized hemizonae pellucidae for the hemizona assay

2009

View full text Add to dashboard Cite

Summary. The hemizona assay (HZA) is a valuable tool to study the binding potential and interaction of spermatozoa with the zona pellucida. Its accuracy strongly depends on the use of equally sized hemizonae. Usually, manipulator‐guided microblades are used for cutting the zona pellucida. Recently, lasers were introduced for precise local thermolysis of the zona. The use of a 1.48 μm diode laser for the generation of hemizonae from human oocytes was investigated. This laser allowed drilling of equally sized hemizonae which were used for hemizona binding assays. It is concluded that the 1.48 μm diode laser system can be applied for the production of hemizonae. The method is easy to perform and offers a fast and efficient access to hemizonae of identical size.

show abstract

Comparison of two methods to obtain hemizonae pellucidae for sperm function tests

Cited by 13 publications

References 0 publications

Nitric Oxide Synthase Inhibition in Human Sperm Affects Sperm-Oocyte Fusion but Not Zona Pellucida Binding1

Nitric Oxide Synthase Inhibition in Human Sperm Affects Sperm-Oocyte Fusion but Not Zona Pellucida Binding1

Application of a 1.48‐μm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay

A new method to produce equally sized hemizonae pellucidae for the hemizona assay

Contact Info

Product

Resources

About