The in vitro effect of benzo[a]pyrene on human sperm hyperactivation and acrosome reaction.

OBJECTIVE: To evaluate the in vitro effect of benzo[a]pyrene on sperm hyperactivation and acrosome status in normozoospermic semen samples of nonsmokers analyzed by computer-assisted semen analysis (CASA). DESIGN: Experimental in vitro study. SETTING: Andrology laboratory. PATIENT(S): Thirteen proven fertile, normozoospermic, and nonsmoking men. INTERVENTION(S): Spermatozoa were washed free of seminal plasma and were treated with different concentrations of benzo[a]pyrene and compared with controls treated with medium alone. The benzo[a]pyrene concentrations were: 100, 50, 25, and 12.5 microg/mL. MAIN OUTCOME MEASURE(S): Effect of varying concentrations of benzo[a]pyrene on sperm hyperactivation and acrosomal reaction. RESULT(S): A statistically significant increase in sperm hyperactivation was observed in presence of benzo[a]pyrene at concentrations of >or=50 microg/mL. The result of the acrosome halo test showed that concentrations of benzo[a]pyrene >or=25 microg/mL statistically significantly decreased the percentage of halo formation, indicating an inappropriate (false) acrosome reaction. CONCLUSION(S): Benzo[a]pyrene statistically significantly affected sperm functional competence as evidenced by increased hyperactivation as well as premature acrosomal reaction.

Semen quality and age-specific changes: a study between two decades on 3,729 male partners of couples with normal sperm count and attending an andrology laboratory for infertility-related problems in an Indian city.

OBJECTIVE: To compare the semen quality and age-specific changes in men between the 1980s and 2000s. DESIGN: Prospective study. SETTING: Andrology laboratory, University of Calcutta, India. PATIENT(S): A semen sample was obtained from 3729 men presenting for infertility problems in two distinct decades, that is, between 1981-85 and 2000-2006. INTERVENTION(S): Subjects with sperm count >20 x 10(6)/mL without any extreme pathological disorders were selected. Samples having a major liquefaction problem were excluded. MAIN OUTCOME MEASURE(S): A standard World Health Organization procedure for semen analysis was performed that included assessment of volume, sperm concentration, and percentage motility. The motility parameters were further classified into forward progressive motility and nonprogressive motility. RESULT(S): The present large-scale study confirms a significant decline in the sperm motility parameters and seminal volume in the present decade. However, no change in overall sperm concentration was noted. A decline was seen in sperm motility with increasing age in both decades. CONCLUSION(S): There are significant changes in sperm motility and volume between the two decades, and the age-related changes in semen parameters are also different in the two decades.

Expression of metallothionein-1 (MT-1) mRNA in the rat testes and liver after cadmium injection.

Metallothioneins (MTs) belong to the family of stress proteins that are present in the majority of living organisms. The MTs play an important task in detoxifying heavy metals. The mammalian scrotal testis is known to be susceptible to cadmium (Cd) exposure. The present work focuses on the MT-1 isoform and aims to ascertain and confirm previous findings to answer whether rodent testes indeed contain MT-1 mRNA, whether its level is increased with Cd injection in liver and testes, and lastly what is the relative difference in the expression of MT-1 mRNA in liver and testes both with and without Cd injection. Adult male Wistar rats weighing 270-290 g received a subcutaneous injection of 4.0 mumol Cd/kg and were sacrificed by cervical dislocation 6 h later. RNA was isolated from testes as well as the liver. There were 2 replicates per treatment for RNA analyses. MT-1 mRNA levels were determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis and then assessed by densitometry scanning. The results of RT-PCR clearly demonstrated that the rodent testes express MT-1 mRNA. The densitometry data shows that the expression of MT-1 mRNA increases with Cd treatment in testes. The relative level of MT1-mRNA is greater in the control-liver than in the control-testes. However, upon Cd injection, the level of testes MT-1 mRNA increases 2.16 fold. These results suggest that the testes respond to Cd for at least 6 h post injection through a transcriptional mechanism.

Emerging technologies for the molecular study of infertility, and potential clinical applications.

The techniques currently used to treat infertility cases are quite limited in their capabilities, due to an incomplete understanding of the molecular activities of germ cells. Fortunately, several technologies are presently being researched that should aid the understanding of the various molecular causes of germ cell pathologies. This review discusses microarray technology, proteomics, metabolic profiling, the PolScope, atomic force microscopy and microfluidics. These technologies have all seen success in preliminary studies, and promise directly or indirectly to improve the low success rates of IVF and other related therapies. However, their widespread use in laboratories and clinics may not be seen until preliminary studies confirming their safety and effectiveness are published, and until standardized protocols for their utilization are established.

Does human sperm nuclear DNA integrity affect embryo quality?

Abnormalities in the male genome are a clear potential reason for post fertilization failure. Male infertility may arise due to high levels of loosely packaged chromatin and damaged DNA. The achievement of a correct chromatin packaging level is essential for successful fertilization. The chromatin contained in the nuclei of mammalian spermatozoa is an extremely compact and stable structure. The reports on mammalian spermatozoa indicate that available volume is insufficient to contain sperm chromatin packed in nucleosome like structure and thus is organized in a special way. Different unique properties of sperm DNA like high degree of inertness and stability, absence of transcription, replacement of somatic histone by protamine etc have made the study of sperm chromatin more interesting. Increased levels of sperm nuclear DNA damage exist in infertile men with abnormal sperm parameters (i.e. concentration, motility and morphology), and various assay techniques have been developed to evaluate sperm chromatin maturity/DNA integrity. These assays are based on the facts that defects in chromatin structure have been shown to lead to increased DNA instability and sensitivity to denaturing stress. DNA integrity in the sperm is essential for the accurate and successful transmission of genetic information. Importance of sperm DNA has also become more obvious in the context of assisted reproductive techniques. While recent advances in assisted reproductive technologies have made possible and practical for many infertile men to become father, the risk of transmission of genetic mutation to the offspring, however, still remains. Further research is necessary to devise techniques for identification and selection of sperm with undamaged DNA for ICSI or to remove sperm with damaged DNA from the semen sample to improve the pregnancy outcome in ICSI.

Acrosin activity as a potential marker for sperm membrane characteristics in unexplained male infertility.

OBJECTIVE: To evaluate sperm membrane system integrity in unexplained infertile male subjects with three consecutive conception failures on IUI even though semen clinical parameters were normal. DESIGN: Prospective study. SETTING: Medical biotechnology laboratory, School of Medical Science and Technology IIT Kharagpur, India. PATIENT(S): Twenty-nine patients with unexplained infertility, 17 normal proven-fertile healthy donors, and 21 infertile males with low motility but with other semen parameters remaining normal. INTERVENTION(S): Semen samples were collected from unexplained infertile patients as well as from healthy fertile donors after abstinence of 3-5 days and were analyzed according to World Health Organization guidelines. MAIN OUTCOME MEASURE(S): Release of 5'-nucleotidase (plasma membrane marker), lactate dehydrogenase (mitochondrial marker) and free acrosin, proacrosin, and total acrosin (acrosomal membrane marker). RESULT(S): Plasma membrane integrity and respiratory activity of sperm cells were comparable in all three groups. The proacrosin-acrosin system was adversely affected in unexplained infertile subjects despite high sperm motility. CONCLUSION(S): Total acrosin activity may be considered as a sensitive biochemical marker for clinical evaluation of unexplained infertility in males.

Assessment of sperm functional competence and sperm-egg interaction.

A precise understanding in the functional competence of mammalian sperm is essential to generate clinical advances for the treatment of infertility and novel contraceptive strategies. The fundamental knowledge on the controlling parameters for spermatozoal activation process will help in the identifying the causes in fertilization failure due to male factor as well as in developing male contraceptive methodologies. The defects in the sperm-egg interaction seem to be one of the controlling mechanisms, however, none of the presently available methods for the evaluation of the fertilizing ability of sperm precisely indicates the reason for the failure or the success of sperm entry into egg. Adequate number of motile spermatozoa with normal morphology and timely occurrence of acrosome reaction are presumably the major prerequisites for the penetration through the egg investments. The present communication briefly reviews some of the main features of mammalian sperm which control the success or the failure of fertilization and existing clinical methods indicating the lack of fundamental knowledge on the sub-cellular and molecular aspects of this unique and species-specific cell-cell interaction.

Sperm immobilization activity of Allium sativum L. and other plant extracts.

AIM: To identify possible spermicidal agents through screening a number of edible medicinal plants with antimicrobial activity. METHODS: Initial screening was made on the basis of ram cauda epididymal sperm immobilization immediately after addition of extracts. The most potent extract was selected and was evaluated on both ram and human spermatozoa. To unravel its mode of action several sperm functional tests were carried out, namely viability of cells, hypo-osmotic swelling test for membrane integrity and assays of membrane-bound enzyme 5'-nucleotidase and acrosomal marker enzyme acrosin. RESULTS: The crude aqueous extract of the bulb of Allium sativum L. showed the most promising results by instant immobilization of the ram epididymal sperm at 0.25 g/mL and human ejaculated sperm at 0.5 g/mL. Sperm immobilizing effects were irreversible and the factor of the extract responsible for immobilization was thermostable up to 90 deg. On boiling at 100 deg for 10 minutes, this activity was markedly reduced. Moreover, this extract was able to cause aggregation of ram sperms into small clusters after 30 minutes of incubation at 37 deg. However this property was not found in human spermatozoa. More than 50 % reduction in sperm viability and hypo-osmotic swelling occurred in treated sperm as compared with the controls, indicating the possibility of plasma membrane disintegration which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase and acrosomal acrosin. CONCLUSION: The crude aqueous extract of A. sativum bulb possesses spermicidal activity in vitro.