Olga Bondarenko

In vitro sperm maturation in sterlet, Acipenser ruthenus.

The aim of the study was to examine sperm maturation in sturgeon and to establish the localization of the maturation. We demonstrated that sperm maturation occurs in sturgeon outside the testes via dilution of sperm by urine. The process involves the participation of high molecular weight (>10kDa) substances and calcium ions.

Volume changes during the motility period of fish spermatozoa: Interspecies differences

The aim of this study was to describe spermatozoa volume changes during the motility period of fish species with either osmotic (common carp Cyprinus carpio) or with ionic (sterlet Acipenseri ruthenus and brook trout Salvelinus fontinalis) modes of motility activation. Nephelometry, light microscopy, and spermatocrit methods were used for quantitative assessment of cell volume changes in media of different osmolalities. Significant correlation (R(2) = 0.7341; P < 0.001) between parameter of volume changes measured using nephelometry and light microscopy methods confirmed nephelometry as a sufficiently sensitive method to detect changes of spermatozoa volume. The spermatocrit alteration method resulted in a large proportion of damaged and potentially immotile spermatozoa in media of osmolality less than 150 mOsm/kg in carp and osmolalities from 10 to 300 mOsm/kg in sterlet and brook trout. Therefore, this method is not reliable for assessing spermatozoa swelling in hypotonic solutions, because the integrity of the cells is not fully preserved. Increase in carp spermatozoa (osmotic activation mode) volume occurred during the motility period in hypotonic conditions, but no indications of volume changes were found in sterlet and brook trout spermatozoa (ionic activation mode) associated with environmental osmolality alteration. Accordingly, we conclude that sperm volume changes are differentially involved in the motility activation process. Species-specific differences in spermatozoa volume changes as a response to a hypotonic environment during the motility period are discussed in relation to their potent physiological role.

Hypotonic treatment prior to freezing improves cryoresistance of common carp (Cyprinus carpio L.) spermatozoa.

Post-thaw motility rate, curvilinear velocity (VCL), and fertilizing ability of carp spermatozoa can be improved by short-term treatment with moderately hypotonic media prior to freezing. Before cryopreservation, carp sperm samples were treated with NaCl solutions of differing osmolalities, ranging from 100 to 300mOsmkg(-1) for 10s, after which final osmolality was adjusted to 300mOsmkg(-1). The resulting sperm suspension was diluted 1:1 with cryoprotective medium and frozen using conventional techniques. Control samples were treated in the same way, without the pre-dilution step. Post-thaw motility rate in samples pretreated with 200mOsmkg(1) NaCl was significantly higher (44±10%) than in controls (21±15%) and samples pretreated with 100mOsmkg(-1) (25±15%) and 300mOsmkg(-1) (25±12%) NaCl. Significantly higher mean VCL were observed in samples pretreated with 100, 150, and 200mOsmkg(-1) (119±24, 118±22, and 115±32μms(-1), respectively) compared to controls (92±27μms(-1)). Fertilization rate of frozen-thawed sperm treated with 200mOsmkg(-1) solution of 2M NaCl was significantly higher (25±18%) than that of sperm treated with 300mOsmkg(-1) NaCl solutions (12±7%) and the control (9±6%).

Motility of sturgeon spermatozoa can sustain successive activations episodes.

Here we report for the first time the possibility of sequential sperm motility activation in sturgeon (sterlet, Acipenser ruthenus), a fish with external fertilization, through changes either in osmolality (global solute concentration) or in the Ca(2+) concentration of the medium surrounding the spermatozoa. Sperm motility was initiated in any of three solutions containing buffer and sucrose at 80, or 40 or 10mM (called S80, S40, S10, respectively); S80 is hypertonic relative to sterlet seminal fluid, while S40 is isotonic and S10 is hypotonic. After cessation of sperm movement at the end of this first motility period, a second and then a third, subsequent motile phase were observed. The second motility period was induced at cessation of motility in S80 by imposing a two-fold decrease in osmolality. After arrest of motility in this half-diluted S80, a third motility period could be initiated by addition of CaCl2 to 1mM final concentration. At the end of a first motility period in either S40 or S10, subsequent motility re-activation episodes were achieved only by addition of 1mM CaCl2. Depending on conditions in which sperm samples were activated, significant differences in curvilinear velocity, percent motile spermatozoa, motility duration time, and specific external features of spermatozoa flagella were observed. Altogether, these observations on the ability of sturgeon spermatozoa to sustain sequential activation episodes by experimental adjustment of their environmental conditions represent a potent model for deeper investigations on the sperm motility activation mechanisms.

Calcium ion supplementation increases brook trout Salvelinus fontinalis spermatozoa activation at the end of the spawning season.

The role of environmental ion composition and osmolality in calcium ion (Ca(2+) ) signalling of spermatozoa activation over the course of the spawning period of brook trout Salvelinus fontinalis was investigated. Motility at the end of spawning was low (mean ± s.d. of 5 ± 2% motile spermatozoa with curvilinear velocity of 25 ± 8 µm s(-1) ). Addition of 10 mM Ca(2+) to the activation medium resulted in values similar to those recorded during the middle of the spawning period (mean ± s.d. of 95 ± 6% motile spermatozoa with curvilinear velocity of 130 ± 15 µm s(-1) ).

Sperm maturation in sturgeon (Actinopterygii, Acipenseriformes): A review

The morphology of the reproductive system of acipenseriform fishes is quite different from that of teleostean species, but an associated unique physiological difference in male sturgeons was not discovered until recently; sperm of sturgeons passes through the kidneys then via Wolffian ducts into the environment rather that emptying directly through seminal ducts. The mixing of sperm with excretory products has been found to be a requisite for the capacity to be activated (maturation step) instead of being deleterious. In the current review we summarize results of studies performed in our laboratory on physiological processes involved in sturgeon sperm maturation, namely changes in: 1) ionic environment; 2) sensitivity of spermatozoa to calcium ions (Ca2+); 3) antioxidant enzymes and proteolytic activities; and 4) content in macroergic phosphates arising during this maturation process. We also discuss taxa-specific aspects of sturgeon sperm maturation in relation to hormonal regulation of spermiation, and the unusual features of sturgeon sperm maturation relative to using testicular sturgeon sperm in aquaculture.