The Expression Of CRISP1 In Human Semen And Its Effect On IVF Fertilization Outcomes

Objective:To detect the localization of cysteine-rich secretory protein 1（CRISP1）on human sperm surface and its expression in semen of normal semen and asthenospermia,oligospermia,oligoasthenospermia patients,to explore the change of localization of CRISP1 during fertilization and the relationship between the expression level of human CRISP1 and semen quality;Study the expression level of CRISP1 on the surface of capacitated sperm of IVF patients with different fertilization outcomes,to investigate the effect of human CRISP1 expression level on IVF fertilization outcome.Methods:Prepare the sperm that before capacitation,after capacitation and after acrosome reaction,the localization of CRISP1 on the sperm surface during fertilization was detected by indirect immunofluorescence assay.The semen of male patients who underwent semen analysis at the Reproductive Medical Center of Renmin Hospital of Wuhan University from 2016 to 2018 was collected.According to the standardized analysis results of the fifth edition of the World Health Organization Laboratory Manual for Human Semen Examination and Handling,the patients were divided into normal semen group,asthenospermia group,oligospermia group and oligoasthenospermia group.The expression of CRISP1 in semen was detected by PCR and Western Blot.To compare the difference of CRISP1 expression in semen of different quality,and to explore the relationship between CRISP1 expression and semen quality;To collect the semen of male patients who underwent IVF-ET fertilization treatment at the Reproductive Medical Center of Renmin Hospital of Wuhan University from 2016 to 2018,excluding the influence of oocyte quality and semen quality,and to divide them into normal fertilization group and fertilization disorder group according to the results of short-term fertilization.The expression level of CRISP1 on the surface of capacitated sperm was detected by PCR and Western Blot,to compare the difference of the expression of CRISP1 on the surface of capacitated sperm in patients with different IVF fertilization outcomes,and to analyse the effect of CRISP1 expression on the IVF fertilization outcomes.Results:1.The results of indirect immunofluorescence showed that CRISP1 was distributed on the whole sperm surface before capacitation.After sperm capacitation,the fluorescence signal of CRISP1 in sperm head is reduced.After acrosome reaction,the CRISP1 fluorescence signal disappeared in the sperm tail,and the fluorescence signals were concentrated in the equatorial segment of the sperm head and the front end of the equatorial segment.2.The expression level of m RNA and protein of CRISP1 was detected in normal semen,asthenospermia semen,oligozoospermia semen and oligoasthenospermia semen.There was no significant difference in CRISP1 m RNA levels among the four groups.But the expression level of CRISP1 protein in different quality semen were significantly different.CRISP1 expression level in asthenospermia and oligoasthenospermia patients was lower than that in normal semen patients,and CRISP1 expression level in oligoasthenospermia patients was also lower than that in oligozoospermia patients;CRISP1 expression level in oligozoospermia patients was not different from that in normal semen patients,and there was also no difference in CRISP1 expression between patients with oligospermia and oligozoospermia.3.Among the patients who underwent IVF short-term fertilization,the level of CRISP1 on the surface of capacitated sperm in the fertilization disorder group was significantly lower than that in normal fertilization group.Conclusion:1.The location of CRISP1 changes during fertilization,and it initially demonstrated its participation behavioral in the sperm-egg interaction process,which laid the foundation for further revealing the mechanism of CRISP1 involvement in fertilization.2.The expression of human CRISP1 is not related to sperm concentration,but significantly lower than the normal level when PR is abnormal,suggesting that CRISP1 may regulate the important Ca²⁺channel Cat Sper on sperm membrane through the CRD domain,thus enhancing the progressive motility of the sperm before capacitation,but the specific molecular mechanism needs further research.3.The level of CRISP1 on the surface of capacitated sperm in IVF fertilization disorder patients was significantly decreased.The protein level of CRISP1 in capacitated sperm may affect the fertilization process of sperm in IVF.The defect of CRISP1 in capacitated sperm may lead to the failure of IVF fertilization,which further illustrates the important influence of CRISP1 tightly bound to sperm surface on the process of sperm-egg interaction.4.CRISP1 which is tightly bound to sperm surface,plays an important role in the process of fertilization.It can participate in the interaction between sperm and egg through migration on sperm surface and affect the IVF fertilization outcome.The level of CRISP1 on the surface of capacitated sperm may be a predictor of IVF fertilization outcomes and help clinical selection of ART therapy.However,the predictive value of CRISP1 on the surface of capacitated sperm for fertilization outcomes still needs a lot of research.