The Exploratory Study Of The Potential Of CatSper As Target For Male Infertility Screening And Immunocontraception

The recent cloning and characterization of CatSper family is a matter of significance to the study of male reproduction and infertility:①CatSper family is the first calcium ion channel required for sperm hyperactivation and male fertility. CatSper deficient mice are infertile as a result of an impairment of sperm mobility and an inability to fertilize intact oocytes;②The vital role of mice CatSper family in male fertility and the restricted localization of CatSper family to the mice testis and mature sperm suggest that CatSper family could be a potential target for male infertility screening and treatment, and it might be an excellent target for contraception. Herein, this thesis reports some elementary results about these potentials of CatSper family in two parts.(PARTⅠ) Expression of CatSper family transcripts in the mouse testis during post-natal development and human ejaculated spermatozoa: relationship to sperm motility(EXPERIMENTⅠ) Expression of CatSper family transcripts in the mouse testis during postnatal developmentBackground:CatSper is a unique sperm cation channel-like protein family exclusively expressed in the testis and plays important roles in sperm functions. The four members of this family were assumed to form a functional hetero-tetrameric channel in sperm, but more recently, the distinct localization patterns between CatSper1/2 and CatSper3/4 and the interactions of other auxiliary proteins, such as the voltage-gated T type calcium channel Cav3.3, CatSperβwith CatSper1 and CatSper2 blurred the relationships between these four members. Aim: To investigate the expression patterns of 4 CatSper mRNAs during postnatal mice testis development so as to understand the relationship between CatSper family members and the further study of their precise roles.Methods: The temporal expression of CatSper1-4 mRNAs in the mice testis during postnatal development through to adulthood (at day 8, 11, 15, 18, 21, 25, 28, 35, 42, 56, and 120 postpartum) were investigated by real time RT-PCR, using SYBR GREENⅠfluorescence detection of amplified products.β-Actin was used in parallel for each run as internal control. A four-step experimental run protocol was used and the high temperature fluorescence measurement point was set at the end of the fourth segment to melt the unspecific PCR products below the chosen temperature and ensure accurate quantification of the desired product.Results: The expressions of CatSper family members were developmentally regulated during the mouse sexual maturation, but the initiation and the expression pattern of these four members were considerably different.Very low levels of CatSper1 mRNA were detected in the mouse testes on day 18 and 21 (after meiosis). The significant upregulations of its mRNA expression were observed at day 25, 28 and 42(compared with their former time point, P<0.001, P<0.005, P<0.05 respectively)Then, CatSper1 mRNA increased gradually from day 42 onward, such that the amount of CatSper1 mRNA was maxmal in adult testis stage.A weak expression of CatSper2 mRNA was found as early as day 8 (before meiosis). With further development, the sharp upregulations of CatSper2 mRNA occurred at day 18 and 35 (compared with their former time point, P<0.001 both), and then the expression of CatSper2 mRNA decreased to adult levels, thus during testis postnatal development CatSper2 mRNA peaks around day 35.CatSper3 and CatSper4 mRNAs have similar expression profiles during mouse postnatal development, initiated on day 15 (after meiosis), increased significantly at 21, 25 and 35 (compared with their former time point, P<0.005, P<0.05, P<0.001 respectively), and then increased gradually as the animals aged till maximal levels in the adult testis.Conclusion: The differences between the temporal expression profiles of the CatSper transcripts in postnatal mouse testis development suggest different regulations to their transcriptions, and shed lights on the possibility of forming heteromeric channels among these four CatSper family members. It is possible that CatSper1 and CatSper2 fulfill their functions at different stages during testis development, through binding to Cav3.3 or CatSperβ. As to the possibility of CatSper3 to form heteromeric channel with CatSper4. It was postulated that the two proteins may form heterodimer.(EXPERIMENTⅡ) Expression of CatSper family transcripts in human spermatozoa and their relationships to sperm motilityBackground: Sperm ion channels are difficult to study by using conventional electrophysiological methods, due to their smaller size, complex geometry and motile nature. During the last decade, investigations have revealed the presence of a complex population of mRNA, including transcripts of some ion channels, in mature spermatozoa of mammals. The mRNA of ion channels might be a feasible target to study ion channels in spermatozoa. Furthermore, although the origin and the role of the mRNAs found in the human mature spermatozoa are yet to be discussed, the most common idea is to consider that these transcripts represent remnants of stored mRNA from genes activated during spermatogenesis. The study of mRNAs in spermatozoa could reflect past events of spermatogenesis and/or spermiogenesis, and could be used as a tool for study and a clinical assay to provide a panoramic view of testis gene expression that can be difficult to achieve from a testicular biopsy.Aim: Aimed to provide a tool to study CatSpers in sperm in which relative fewer methods could be applied, the presence of four CatSper mRNAs in human ejaculated spermatozoa was examined and the levels of their mRNAs in the high and low motile spermatozoa were compared. Methods: Human semen samples were obtained from healthy donors who met all the World Health Organization criteria for normozoospermia. To rule out the possibility of contamination by residual cells, the five samples used for the study of the presence of CatSper mRNAs in spermatozoa were individually purified by two sequential centrifugations through 40:80 discontinuous gradients of Percoll. C-Kit, a positive marker for testicular germ cells, was included to control the quality of sperm purification as well as high and low motile spermatozoa isolation described below. The 80% fraction of the second centrifugation through the Percoll gradient was pooled for RNA extraction after a microscopic examination and a hypotonic treatment. Non-quantitative RT–PCR was performed to identify the presence of transcripts of CatSper1-4 in the human ejaculated spermatozoa.High and low motile sperm fractions were fractioned on the four-layer Percoll gradients with the following densities: 95, 76, 57 and 47.5%. high and low motile spermatozoa were collected respectively from the 95% layer and from the interface 76~57%. The purity of these two fractions was confirmed as described above. The motility of was examined by computer-assisted semen assessment. Sperm of the high motile fractions showing motility <90% and sperm of the low motile fraction showing motility >30% were excluded. Sperm fraction was stained by an equal volume of 0.4% eosin solution to determine the viability. Relative quantitative RT-PCR was performed to determine the mRNA levels of the high and low motile spermatozoa, by takingβ-Actin as internal control, and the four-step experimental run protocol was used.Results: Round cells and cytoplasmic droplets were absent in the purified spermatozoa as well as the high and low motile sperm fractions isolated by Percoll gradients. Moreover, the CatSper family members are testified to express solely in testis, and the absent of C-Kit mRNA discards the possible contamination of testicular germ cells.The transcripts of CatSper2 and CatSper3 were found in purified spermatozoa from all 5 semen samples by RT-PCR, and CatSper1 mRNA was detected in 2 samples, while there was no expression of CatSper4 mRNA in these purified spermatozoa.The quantification of CatSper2 and CatSper3 mRNA in the high and low motile sperm fractions isolated from 10 semen samples individually was performed by real time RT-PCR. The threshold cycle ofβ-Actin varied from 14.23 to 15.16 and mRNA levels ofβ-Actin in the high and low motile sperm fractions were similar (P=0.932). CatSper2 and CatSper3 transcript amounts varied in different samples with normal distributions. In every semen sample and in 9 of 10 semen samples, higher CatSper2 and CatSper3 transcript amounts normalized byβ-Actin in high motile sperm fractions than in low motile sperm fractions were observed respectively. Only in 1 sample, the ratio of CatSper3 mRNA toβ-Actin mRNA was 0.88 in low motile sperm fraction and was higher than 0.82 in high motile sperm fraction. The ratios of both CatSper2 and CatSper3 mRNA toβ-Actin mRNA were statistically different between high motile sperm and low motile fraction (P=0.008, P=0.004 respectively). The percentage of live cells was not different between the high motile spermatozoa and low motile spermatozoa (P=0.121).Conclusion: CatSper1~3 transcripts were identified to be present in human ejaculated spermatozoa and may be good tools for the further study. The significant higher expressions of CatSper2 and CatSper3 mRNA levels observed in the high motile spermatozoa than in the low motile fraction also suggest that CatSper2 and CatSper3 transcripts in human ejaculated spermatozoa be potential targets for male infertility screening. (PARTⅡ) The exploratory study of the immunocontraceptive potential of CatSper1(EXPERIMENTⅠ) The expression and significance of CatSper1 in human testis and ejaculated spermatozoaBackground:As the first member of the family, mice CatSper1 was cloned in 2001. In mice, CatSper1 protein expressed exclusively in the testis and not in other tissues. In mice sperm, CatSper1 is localized primarily in the tail's principal piece and is required for hyperactivated sperm mobility. Moreover, CatSper1 is essential for male fertility. Based on its restricted expression pattern and important role in sperm mobility and male fertility, CatSper1 is predicted to be a potential target for male infertility screening and an ideal target for contraception. However, little is known about the expression and function of human CatSper1.Aim: To evaluate the expression and distribution of CatSper1 protein in human testis and human ejaculated spermatozoa.Methods: Human ejaculated sperm from normozoospermic donors and liquid nitrogen frozen human testis were used for the study of CatSper1 protein expression and distribution by Western blot and immunohistochemistry, respectively.Results: The CatSper1 protein (about 80 kD) was detected both in human testis and ejaculated spermatozoa. In human testis, abundant CatSper1 protein was detected in the membrane of spermatid. In human ejaculated spermatozoa, CatSper1 protein was localized in the principal piece of the sperm tail.Conclusion: Consistent with the postmeiotic expression in mouse testis, CatSper1 protein expressed in the membrane of spermatid. In ejaculated spermatozoa, CatSper1 protein was highly localized to the principal piece of the sperm tail. The same expression and localization of CatSper1 in human as in mice indicate the similar role of CatSper1 in human male fertility.(EXPERIMENTⅡ) Inhibition of human sperm function and mouse fertilization by a polyclonal antibody against CatSper1: the contraceptive potential of its transmembrane domains and pore regionBackground:CatSper1 was implicated as a potential target for contraception, but there is no report about this potential of CatSper1 and its antagonist. Because CatSper1 is specifically localized on the membrane of spermatozoa, it may be an ideal sperm specific antigen for immunocontraception. Although there is no ion channel that has been testified to be target for immunocontraception, some autoimmune diseases caused by antoantibodies to ion channel implied that it is possible to block the function of ion channel by the antibody.Aim: To estimate the contraceptive potential of the transmembrane domains and pore region of CatSper1, the influence of anti CatSper1 IgG targeting these regions upon human sperm functions and mouse sperm in vitro fertilization were evaluated. On the other hand, we aimed to evaluate the function of human CatSper1 indirectly.Methods: Spermatozoa from normozoospermic donors (n=9) were individually processed using a swim-up procedure and were then incubated with anti CatSper1 IgG at a final concentration of 50μg/ml. Sperm agglutination was observed by microscopic examination. Sperm motility and hyperactivited sperm motility were determined by computer-assisted semen assessment. Acrosome reaction was evaluated by a coomassie brilliant blue procedure. Mouse sperm form cauda epididymidis were incubated with anti CatSper1 IgG at a final concentration of 50μg/ml for 90 min, before to fertilize eggs. The fertilization rates were compared between the mouse sperm pretreated with anti CatSper1 IgG and the control.Results: The anti CatSper1 IgG specifically bound with the principal piece of human and mouse sperm tail. The influence of anti CatSper1 IgG (final concentration of 50μg/ml) on the human sperm functions: 1) After 2 h incubation with anti CatSper1 IgG, no sperm agglutination was observed under microscop.2) Significant changes in the percentage of progressively motile sperm were observed after 1, 2 and 4 h incubation with anti CatSper1 IgG(P<0.005). Moreover, under the influence of anti CatSper1 IgG(P<0.01, P<0.005), similar changes of the percentage of fast progressively motile sperm were observed. The decline of progressive motility caused by anti CatSper1 IgG was mainly ascribed to the changes of fast progressive motility.3) After 4 h incubation, no effect of anti CatSper1 IgG on human sperm acrosome reaction was observed.4) Significant inhibition of the hyperactivited sperm motility was observed after 5 h incubation with anti CatSper1 IgG.The incubation of mouse sperm with anti CatSper1 IgG significantly reduced the fertilization rates (45.8% vs. 80.4%, P<0.05).Conclusion: It is possible and effective to block the function of CatSper1 by applying the antibody targeting its transmembrane domains and pore region. our results suggest that human CatSper1 could be a possible target for immunocontraception. The antibody may be a tool to study function of ion channel in sperm in which relative fewer methods could be applied.