Sperm Binding Of Zona Pellucida Research Articles

169 Use of stored zonae pellucidae from young and old mares to study sperm–oocyte binding

Natural and standard IVF requires sperm binding to the zona pellucida (ZP); however, difficulties in obtaining equine ZP limit research in this area. In a previous study, an in vitro assay was used to assess ZP sperm binding for oocytes collected from old and young mares’ dominant follicles, with similar numbers of sperm bound per ZP (means of 45.5 and 58.1 sperm per ZP; P=0.12) (Lupole et al. 2010 Anim. Reprod. Sci. 121, 252-253). We hypothesised that ZP from uncleaved equine oocytes after intracytoplasmic sperm injection (ICSI) could be used to study ZP sperm binding. For this study, oocytes from mares >20 years (old, n=13 oocytes) and 4 to 12 years (young, n=16 oocytes) were collected by transvaginal, ultrasound-guided aspirations and injected with a single sperm. Only oocytes that failed to cleave into at least 2 cells within 2 days after ICSI were used. The oocytes were stored in hyperosmotic salt solution at 4°C for 2 years. The sperm binding assay was performed similar to the previous study (Lupole et al. 2010). Oocytes were removed from salt solution and placed into TALP and incubated for 1h at 38.5°C in 6% CO2 and air. Following incubation, oocytes were moved to a 45-µL drop of TALP. Frozen-thawed sperm (100,000 total sperm) from one stallion of good fertility were added and incubated for 2h. Oocytes were then washed through 4 drops of TALP using a pipette tip with a 175-µm inner diameter to remove loosely bound sperm. Oocytes and tightly bound sperm were stained in 4’6-diamidino-2-phenyindole (DAPI; 10µg mL−1) for 15min. The oocytes were secured on a glass slide under a coverslip that was supported by a mix of paraffin and petroleum jelly and viewed at 400× magnification using an epifluorescence microscope (Nikon Eclipse E800, Nikon, Tokyo, Japan). The number of tightly bound sperm was recorded. Sperm counts for age groups were compared using Student’s t-test, with data presented as mean±s.e.m. Sperm binding was not different between groups (59.2±16.9 for old mares and 36.8±10.6 for young mares; P=0.25). As previously observed with oocytes that had not been injected with sperm and minimal time in salt storage, we found no significant effect of mare age on sperm binding to the ZP. In addition, numbers of sperm bound to the ZP were similar between projects. Our findings suggest that equine oocytes that fail to cleave after ICSI could provide a valuable source of ZP for research. Further work is ongoing to determine the effect of time that oocytes are stored in salt solution on sperm binding.

Traditional intracytoplasmic sperm injection provides equivalent outcomes compared with human zona pellucida-bound selected sperm injection

The capability of human zona pellucida (ZP) to bind selectively to normal functional sperm with normal chromatin has been reported widely in the literature. The aim of this study was to evaluate whether ZP-binding sperm selection may represent a method to retrieve superior spermatozoa for intracytoplasmic sperm injection (ICSI). Patients were divided into two groups: a ZP-ICSI and a conventional ICSI group. In the ZP-ICSI group, spermatozoa for injection were selected after ZP-sperm incubation and spermatozoa that were tightly bound to the ZP were used for ICSI (ZP-ICSI). Clinical outcomes of ZP-ICSI were compared with the outcomes of traditional scientist-selected sperm injection (conventional ICSI). Results did not show any significant difference in fertilization, pregnancy, implantation and take-home-baby rates between conventional ICSI and ZP-ICSI. However, when data relative to patients who received ZP-ICSI were analyzed, an interesting result was observed: higher sperm concentration and morphology correlated with higher ZP-sperm binding. Additionally, patients with higher ZP-sperm binding seem to have improved pregnancy and take-home-baby rates. In conclusion, this study shows that ZP-ICSI is not a superior method compared with conventional ICSI. However, clinical ICSI outcomes were apparently improved in the presence of good ZP-sperm binding. We therefore speculate that sperm competence to ICSI could be reduced when the sperm's ability to bind the ZP is impaired.

Fertility prediction in stallions

Abstract The prediction of a stallion's fertility is one of the main unresolved problems in horse breeding. Despite many studies of the relationship between seminal characteristics and fertility, we are not able to evaluate with certainty the breeding capacity of a stallion. Stallion selection and fertility prediction are routinely based on the analysis of seminal and behavioural parameters. Basal blood hormone concentrations and their modification after different stimulation are also used for diagnosis. Although the methods developed in some laboratories may explain established subfertility or infertility, mainly detected by abnormal seminal characteristics and/or pregnancy rate, they cannot yet be used for general infertility detection. Such tests are based on spermatozoal morphology, e.g. chromatin and membrane integrity analyses, and spermatozoal functionality, e.g. motility evaluation, zona pellucida-sperm binding assay, and on seminal plasma composition. At this stage we have no idea of the frequency of the abnormalities demonstrated by these methods nor any idea of their correlation with subfertility or infertility.

Involvement of N-linked carbohydrate chains of pig zona pellucida in sperm-egg binding.

The sperm receptor activity of pig zona pellucida has been previously shown to exist in one of the components, pig zona protein 3 alpha (PZP3 alpha), that can be purified after the removal of sialylated and/or sulfated N-acetylpoly(lactosamine) by digestion with endo-beta-galactosidase. In this study, we examined whether N-linked or O-linked carbohydrate chains are involved in the sperm receptor activity of pig zona pellucida. The elimination of N-linked carbohydrate chains from endo-beta-galactosidase-digested PZP3 alpha by digestion with N-glycanase markedly reduced its inhibitory effect on sperm-egg binding in an in vitro competition assay, whereas the elimination of O-linked carbohydrate chains by alkali treatment hardly reduced the inhibitory effect. These results indicate that N-linked carbohydrate chains of PZP3 alpha play a major role in mediating the sperm binding of zona pellucida in pig.