Movement Of Human Sperm And Their Impregnating Ability Biology Essay

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NR2b receptor was found in our previous study to highly express in human sperm. This study aimed to evaluate potential pharmacological efficacy of NR2b-targeting reagents on sperm by using a sperm-activity assay and a sperm-cervical mucus-contact test. This study showed that the direction and speed of sperm movement was obviously disturbed and the ability of sperm to pass through uterus mucilage was apparently inhibited by MK-801. It is concluded that MK-801 may inhibited sperm movement via NR2B antagonism. This study indicate that NR2B-targeting compounds such as MK-801 would be potential to be used clinically for decreasing the probability of human pregnancy, thereby meeting a possible contraceptive purpose.

Glutamate receptors are classified into ionotropic and metabotropic receptors according to their pharmacological and molecular biological evidence. The ion-type receptors are subdivided into N-methyl-D-aspartate (NMDA), kainate (KA), and α-Amino-3-hydroxy-5-methylisoxazole-4-propionicacid (AMPA) receptors [Hollmann M 1994, Nakannishi S 1992], each of which is capable of coupling ion channels directly and thereby known to possess a ligand-gating property. Until now, it is confirmed that the sodium ion-dependent glutamate receptor includes GLAST,GLT1,EAAC1,EAAT4 and EAAT5 subtypes.[Pines G 1992, Kanai Y 1992, Storck T 1992, Fairman WA 1995, Arriza JL 1997] Moreover, these ion channels can be further divided into various subtypes such as NR1 and NR2A-D receptors (these five for NMDA receptors), GluR1-4 receptors (for AMPA receptors), and GluR5-7, KA1, and KA2 receptors (for KA receptors) [Hollmann M 1994, Kanai Y 1992], according to their homology sequence and ligand affinity. As to NMDA receptors, there exist two major subunits expressed at high abundance in tissues, i.e. NR1 and NR2; wherein, NR2 family (NR2A, NR2B, NR2C, and NR2D subunit) has recently received much attention in the field of pharmacology. These NR2 subunits express alternatively at different developmental stages, wherein the expression of NR2B is proved to be the highest within the bodies of grown-up animals and human [Storo M 2001]. Our previous study showed that NR2B highly expressed in the body and tail of mouse and human sperm [Hu JH 2004]. However, its function remained to be unclear. This study aimed to determine whether a NR2B-targeting compound could affect sperm activity or not. A known glutamate receptor antagonist MK801 was used to treat human sperm, and a seminal fluid analyzer was applied to monitor the change of sperm movement after the drug intervention. Also, a cervix-mucilage interaction experiment was utilized simultaneously to explore the influence of MK801 on the fertilizing capacity of sperm.

2. Materials and Methods

2.1. Detection of sperm activity

MK801 was bought from Sigma Company. Human sperm were obtained from several 25-40-year-old men with marriage and child-rearing history. All sperm samples were obtained via masturbation. On experimentation, the sperm were suspended with BWW fluid in a water bath at 37°C after liquation, and then adjusted to 6-107/ml. The specimen were then treated with MK-801 at different concentrations, and then incubated in a water bath at 37°C for 30 minutes. Finally, the treated specimens were subject to an activity measurement done by using a US Hamilton-IVOS sperm analyzer (computer assisted sperm analysis system, CASA). The process was repeated 3 times.

2.2. Sperm-cervical mucus contact (SCMC) test

Sample cervical mucus was prepared by using an absorption method. The cervical-mucus preparation was conducted 1-2 days prior to ovulation. The standards of sample collection were: 1) that suppliers must be healthy women at age of 25-40 years old and with a history of marriage and child rearing; 2) according to the Moghissi scoring system, the cervical mucus score should be more than 10 points. Sperm-specimen preparation and treatment were done as mentioned above. A sperm-cervical mucus-contacting test was initiated by addition of the treated sperm to cervical mucus. The experiment was repeated 3 times.

2.3. Statistical analysis

Data are expressed as mean ± standard deviation for continuous variables. The one-way analysis of variance (ANOVA) was used to compare measurement data. All p values <0.05 were considered to be statistically significant. Statistical analysis was performed using the statistical software Origin 7.0 (OriginLab, Northampton, MA).

3. Results

3.1. Effect of MK-801 on human sperm activity

As shown in Figure 1, straight-line velocity (VSL) and average path velocity (VAP) of sperm movement were significantly decreased after treatment of sperm with 100 and 500 μm MK-801, with p value less than 0.05 as compared to control group, which displayed that MK801 can interfere with the direction and speed of sperm movement.

3.2. Effect of MK-801 on sperm penetration of cervical mucus

After the MK-801-treated sperm and the cervical mucus were mixed together, passage of sperm through cervical mucus was analyzed by a SCMC test. As shown in Figure 2, sperm groups pre-incubated with concentrations of MK-801 showed lower levels of SCMC values relative to sperm control group (each p<0.05 for 100, 200, and 300 μm treatment group, respectively), which illustrated that MK-801 can inhibit the ability of sperm to pass through cervical mucus.

4. Discussion

It is reported that γ-aminobutyric acid (GABA) system exists on the rats and mouse testicle and sperm, and has a vital impact on maturation of sperm [Akinci MK 1999, He XB 2001, Hu JH 2000, Ma YH 2000]. This study is an extension of our previous investigation that disclosed that NR2b highly expressed in the body and tail of mice and human sperm [Hu JH 2004]. This high expression provides an indication that NR2B inhibitors may affect the biological properties of human sperm. When applying MK-801, a potent selective, non-competitive antagonist of NMDA receptors, to interfere with human sperm, it was shown that the straight-line velocity (VSL) and average path velocity (VAP) of sperm movement decreased significantly in the groups with 100 and 500 μm of the testing drug, suggesting that MK-801 could affect sperm -ovum interactions. To test this hypothesis, an in vitro SCMC test was conducted upon the sperm-cervical mucus system. Obviously, MK-801, in a concentration-dependent manner, strongly inhibited the sperm penetration capacity at the contact interface between sperm and cervical mucus. These findings pharmacologically confirm the hypothesis of NR2B-targeting activity in sperm. Recently, several external contraceptives such as alfenoxynol and benzene alcohol in different formulations are extensively used in clinic. Generally speaking, the currently used contraceptives or compounds with clinical potential mainly act by killing sperm. In this study, the glutamate receptor NR2B that exists in human sperm was adopted as a therapy target, and its potent selective antagonist MK-801 was used showing inhibitory actions not only on human sperm movement but also on the capacity of sperm penetration through cervical mucus. NMDA receptor antagonists such as MK-801 are thereby suggested to be potential to be applied for contraception.

In summary, NMDA receptor antagonist MK801 not only reduces the linear activity of human sperm but also inhibits sperm penetration through cervical mucus. Results from this study will provide a new idea for solving the problems of human reproduction and contraception.

Figure 1 Effect of MK801 on the line moving ability of human sperm

A: Action of MK801 on VSL. B: action of MK801 on VAP. Both VSL and VAP were monitored by CASA. *: p<0.05.

Figure 2 Effect of MK801 on the ability of human sperm to pass through cervical mucus

MK-801, at differing indicated concentrations, was used to insult sperm followed by SCMC test. *, **, and ***: p<0.05, 0.01, and 0.001, respectively.