Osmotically induced cell swelling triggers a chain of events leading to a net loss of major cell ions and water, resulting in cell volume recovery, a process known as regulatory volume decrease (RVD). In many cell types, there is an evidence that the cytoskeleton may play a role in the initial sensing and transduction of the signal of volume change. In this study, we tested the hypothesis that an intact microfilament and microtubule network is required for volume response and RVD in boar sperm before and after capacitation treatment and whether addition of cytochalasin D and colchicine to the capacitation medium would affect volumetric behaviour. Capacitation is a series of cellular and molecular alterations that enable the spermatozoon to fertilize an oocyte. Cell volume measurements of washed sperm suspensions were performed electronically in Hepes-buffered saline solutions of 300 and 180 mosmol/kg. After exposure to hypoosmotic conditions, boar sperm showed initial swelling (up to 150% of initial volume within 5 min), which was subsequently partially reversed (to about 120–130% after 20 min). Treatment with cytochalasin D led to reduced initial swelling (1 μmol/l) and loss of RVD in washed sperm (1–10 μmol/l) and at the beginning of incubation under capacitating conditions (5 μmol/l). Short treatment with 500 μmol/l colchicine affected the volume regulatory ability in sperm under capacitating conditions but not in washed sperm. No significant differences in cell volume response were observed after subsequent addition of cytochalasin D and colchicine to the suspensions of sperm incubated for 3 h under capacitating conditions. However, the incubation under capacitating conditions in the presence of cytochalasin D led to improved volume regulation at the end of the incubation period (23%). The microfilament network appears to be important for volume regulation in washed boar spermatozoa while intact microtubules do not seem to be necessary for osmotically induced RVD. The changes in cytoskeleton microfilament organization during capacitation, possibly affecting the osmotically induced volume response, appear to occur at the later stages of capacitation, whereas changes in microtubules, related to volume regulatory ability, may be programmed within the first stages of capacitation.
A M Petrunkina, M Hebel, D Waberski, K F Weitze and E Töpfer-Petersen
AM Petrunkina, RA Harrison, M Hebel, KF Weitze and E Topfer-Petersen
The ability to reverse swelling caused by hypo-osmotic stress is an important cell function; in spermatozoa, it is likely to be of consequence during ejaculation and also during the thawing process that terminates cryopreservation. In this study, the time course of boar and bull sperm volume changes after exposure to hypo-osmotic conditions at 39 degrees C was recorded. Cell volume measurements of washed sperm suspensions were performed electronically in Hepes-buffered saline solutions of 300 and 180 mosmol kg(-1) containing 2.5 mmol K(+) l(-1). Treatment with quinine in the presence or absence of the potassium ionophore valinomycin was used to determine whether potassium channels were involved in the reversal of swelling. After exposure to hypo-osmotic conditions, both bull and boar spermatozoa showed initial swelling (up to 200% and 140% of initial volume, respectively, within 5 min), which was subsequently partially reversed (to about 150% and 120%, respectively, after 20 min). Incubation with quinine led to an increase in swelling in both species. However, bull sperm volume was already maximal (up to 294%) after 30 s and declined thereafter, whereas boar sperm volume increased slowly to a maximum of about 220% after 20 min. Valinomycin treatment caused quinine-induced swelling in bull spermatozoa to decrease rapidly to control (no quinine, no valinomycin) values, whereas in quinine-treated boar spermatozoa it had an opposite, enhancing effect. Interpreting these results in the light of data from studies by others on a variety of cell types, it is proposed that swelling-activated potassium channels are involved in regulatory volume decrease in both species of spermatozoa, but that boar spermatozoa may contain fewer swelling-activated chloride channels than do bull spermatozoa.