Complementary effects of propranolol and nonoxynol-9 upon human sperm motility.

The inhibitory effects of nonoxynol-9, DL- and D-propranolol upon human sperm motility were determined in vitro. All three compounds were capable of causing complete cessation of sperm movement. However, greater efficacy was achieved using combinations of nonoxynol-9 and propranolol, suggesting a complementary interaction between these compounds. Investigations of the mechanism of action of propranolol revealed that an influx of calcium accompanied the loss of motility. However, since incubation in the absence of calcium enhanced the spermicidal effects of this compound, it was concluded that this calcium influx did not constitute the primary means by which motility was disrupted. Low doses of propranolol, which did not affect motility, were found to inhibit the capacity of human spermatozoa for sperm-oocyte fusion.

PIP: The inhibitory effects of nonoxynol-9, DL-, and D-propranolol upon human sperm motility were determined in vitro. Semen samples were obtained from a panel of over 50 donors exhibiting normal semen profiles. At the higher concentration of 500 mcM, DL-propranolol caused a significant (p 0.01) suppression of motility within 15 minutes of addition. At a dose of 50 mcg/ml, nonoxynol-9 caused a significant (p 0.01) reduction of motility within 30 minutes of addition. At a concentration of 500 mcg/ml, nonoxynol-9 completely abolished all sperm movement within 1 minute of addition. In contrast, D-propranolol at a concentration of 5 mcM caused a significant (p 0.01) reduction in percentage motility over an incubation period of 120 minutes. A similar response profile to that expressed in the presence of 5 mcM D-propranolol was found for 50 mcM of this compound. In view of the apparent complementary effects between DL-propranolol and nonoxynol-9 in the suppression of sperm movement, a study was undertaken of the interaction between nonoxynol-9 and D-propranolol in the disruption of sperm motility. When added at a concentration of 300 mcg/ml, nonoxynol-9 had a slight, insignificant suppressive effect on seminal sperm motility within 20 seconds of drug addition. D-propranolol at a dose of 850 mcM significantly (p 0.01) inhibited sperm movement within 20 seconds, although approximately 20% of cells were still motile after this time. However, when both drugs were added together at these concentrations, motility was reduced almost to zero within this short incubation period. To investigate the mechanism of action of DL-propranolol, intracellular calcium levels were analyzed using the fluorescent probe Quin-2. The suppression of motility caused by a concentration of 500 mcM of this compound was associated with a concomitant increase in the free calcium content of these spermatozoa. At a dose of 100 mcM, this reagent significantly (p 0.05) inhibited the ability to penetrate hamster oocytes, while having no significant inhibitory effect on sperm motility.

Generation of reactive oxygen species, lipid peroxidation, and human sperm function.

Recent studies have demonstrated that human spermatozoa are capable of generating reactive oxygen species and that this activity is significantly accelerated in cases of defective sperm function. In view of the pivotal role played by lipid peroxidation in mediating free radical damage to cells, we have examined the relationships between reactive oxygen species production, lipid peroxidation, and the functional competence of human spermatozoa. Using malondialdehyde production in the presence of ferrous ion promoter as an index of lipid peroxidation, we have shown that lipid peroxidation is significantly accelerated in populations of defective spermatozoa exhibiting high levels of reactive oxygen species production or in normal cells stimulated to produce oxygen radicals by the ionophore, A23187. The functional consequences of lipid peroxidation included a dose-dependent reduction in the ability of human spermatozoa to exhibit sperm oocyte-fusion, which could be reversed by the inclusion of a chain-breaking antioxidant, alpha-tocopherol. Low levels of lipid peroxidation also had a slight enhancing effect on the generation of reactive oxygen species in response to ionophore, without influencing the steady-state activity. At higher levels of lipid peroxidation, both the basal level of reactive oxygen species production and the response to A23187 were significantly diminished. In contrast, lipid peroxidation had a highly significant, enhancing effect on the ability of human spermatozoa to bind to both homologous and heterologous zonae pellucidae via mechanisms that could again be reversed by alpha-tocopherol. These results are consistent with a causative role for lipid peroxidation in the etiology of defective sperm function and also suggest a possible physiological role for the reactive oxygen species generated by human spermatozoa in mediating sperm-zona interaction.

Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia.

The ability of human spermatozoa to exhibit sperm-oocyte fusion in response to the ionophore, A23187, was examined in relation to the capacity of these cells to generate reactive oxygen species. In 70 fertile control donors, there was an overwhelming pattern of high levels of sperm-oocyte fusion associated with low levels of reactive oxygen species production. By contrast, 88% of the 74 oligozoospermic patients exhibited less than 25% oocyte penetration in response to A23187 and 58% exhibited no penetration whatsoever. Of the 40 oligozoospermic patients who failed to respond to A23187, nine had low levels of reactive oxygen species production in association with impaired liquefaction of seminal plasma. Of the remainder, 17 (55%) exhibited defective sperm function together with elevated production of reactive oxygen species. These observations, which are the first to describe a biochemical defect in the spermatozoa of oligozoospermic patients, may carry significant implications for the etiology and treatment of this condition.

Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques.

The mechanisms responsible for mediating the influence of sperm preparation protocols on human sperm function have been investigated. Techniques that involved the separation of motile spermatozoa prior to centrifugation were found to yield sperm suspensions of highest quality. If the spermatozoa were centrifuged prior to isolation of the motile cells, sperm function was impaired. The detrimental effects of centrifugation were associated with a sudden burst of reactive oxygen species production by a discrete subpopulation of cells (characterized by significantly diminished motility and fertilizing capacity) that could be separated from normal functional spermatozoa on Percoll gradients. If unfractionated sperm suspensions were subjected to centrifugation, the reactive oxygen species generated by this subpopulation impaired the functional competence of normal spermatozoa in the same suspension. Assessment of the ability of the antioxidants, butylated hydroxytoluene, and vitamin E, to curtail the peroxidative damage inflicted by such cells in response to centrifugation revealed a significant improvement of sperm function in the presence of vitamin E.

Analysis of calmodulin acceptor proteins and the influence of calmodulin antagonists on human spermatozoa.

The possible role of calmodulin in regulating a number of calcium-dependent functions exhibited by human spermatozoa was investigated by using the antagonists trifluoperazine and calmidazolium. At high doses both antagonists inhibited the motility of human spermatozoa and induced a concomitant rise in [Ca2+]i and a decline in cAMP. Lower doses of these antagonists, particularly calmidazolium, suppressed the ability of human spermatozoa to generate reactive oxygen species and exhibit sperm-oocyte fusion, without influencing [Ca2+]i, cAMP, or motility. This inhibition of sperm-oocyte fusion was effective even if the spermatozoa were subsequently exposed to A23187, suggesting that calmodulin may regulate this aspect of human sperm function at a point downstream from calcium influx. Both radiolabelling and affinity chromatography techniques were used to detect a number of calcium-dependent and calcium-independent calmodulin acceptor proteins in the human spermatozoon. The major calcium-dependent acceptor proteins exhibited Mr values of 32,000 and 22,000-27,000, respectively, and did not appear to be associated with the sperm plasma membrane.

Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.

Addition of the divalent cation ionophore, A23187, to washed populations of human spermatozoa resulted in a sudden burst of production of reactive oxygen species which peaked within 3-5 min. This activity was dependent upon the presence of calcium in the external medium and was unaffected by the mitochondrial inhibitors, oligomycin, antimycin and rotenone. Studies with scavengers of reactive oxygen species revealed that, while reagents directed against singlet oxygen and the hydroxyl radical were without effect, cytochrome C reduced the response to A23187 by about 50%, suggesting that the superoxide anion radical is a major product of the activated human spermatozoon. The clinical implications of these studies stem from the considerable variation observed between individuals in the levels of reactive oxygen species produced by the spermatozoa. This variability was shown to be inversely related to the ability of the spermatozoa to exhibit sperm-oocyte fusion on exposure to A23187; defective samples exhibited a basal level of reactive oxygen species production which was 40 times that observed with normal functional cells.

Relative ability of modified versions of the hamster oocyte penetration test, incorporating hyperosmotic medium or the ionophore A23187, to predict IVF outcome.

This study was designed to assess the relationship between IVF outcome and the results obtained with two modified versions of the zona-free hamster oocyte penetration test in which the spermatozoa were pre-incubated with either hyperosmotic medium or the divalent cation ionophore A23187. When the former system was used, a poor correlation with IVF outcome was observed. Samples screened prior to IVF exhibited a 60% false negative rate (failed penetration test, successful IVF), while for those assessed concurrently with IVF, the equivalent figure was 85.7%. Addition of A23187 optimized the penetration system giving higher levels of sperm--oocyte fusion and a more accurate prediction of the capacity of the spermatozoa to fertilize human ova in vitro. With this system the false negative rate was 4.3% for screened samples and 0% for those assessed simultaneously with IVF. These results suggest that the A23187-enhanced system may be of value as a screening criterion for IVF.

Contribution of defective sperm function to infertility.

The nature and significance of some biologically orientated tests of human sperm function is discussed by the Authors. In particular tests for motility and the fertilizing capacity of spermatozoa are examined, motility tests and the zona-free hamster oocyte penetration test are particularly criticized. The Authors affirm that hamster oocyte tests carried out in the presence of A 23187 have shown to correlate with fertility in vivo and may be of critical value in decreasing the incidence of false negative scores. The data furnished by this test will provide a logical basis for discovering the aetiology of male infertility as well as devising techniques to both detect and treat these conditions.