Jacky Cosson

Sperm motility in fishes. (II) Effects of ions and osmolality: a review.

The spermatozoa of most fish species are immotile in the testis and seminal plasma. Therefore, motility is induced after the spermatozoa are released into the aqueous environment during natural reproduction or into the diluent during artificial reproduction. There are clear relationships between seminal plasma composition and osmolality and the duration of fish sperm motility. Various parameters such as ion concentrations (K+, Na+, and Ca2+), osmotic pressure, pH, temperature and dilution rate affect motility. In the present paper, we review the roles of these ions on sperm motility in Salmonidae, Cyprinidae, Acipenseridae and marine fishes, and their relationship with seminal plasma composition.

Biology of sperm and artificial reproduction in carp

Although the common carp, Cyprinus carpio, has been cultivated for several thousand years and is produced in large quantities, research on reproduction has been very limited. Traditionally, spawning occurred naturally in situ in rearing ponds. In slightly improved methods large breeding ponds stocked with brood fish were devoted to reproduction with fry collection in autumn, or in small spawning ponds with collection of larvae a few days after hatching. Controlled reproduction in hatcheries started only in the 1950s. This paper reviews some of the basic work on carp sperm and describes the technologies of artificial reproduction. The sperm is of a primitive type with uncondensed chromatin and a small midpiece. As in most teleost fish, the sperm is immotile in the male genital tract and in the semen and is activated after release into fresh water. The initiation of motility is due to the decrease in osmotic pressure. The structure of the flagellum is rapidly disorganized in fresh water and sperm stop moving after 30 s. When dilution occurs in a 50 mM NaCl solution, the osmotic change is sufficient to initiate motility, but the flagellum is not disorganized and swimming lasts a few minutes. Motility depends mainly on endogenous ATP stores, about 12 nmol108 spermatozoa, and stops when 50–80% of the ATP is exhausted by hydrolysis. The procedure of artificial insemination includes collection of gametes (from “hypophysised” males and females), mixing in an extender (45 mM NaCl, 5 mM KCl, Tris 2.5 mM, glycine 19 mM, pH 8) in a ratio 1 litre of eggs to 1 litre of diluent and 1 ml of semen. Sperm motility can be triggered during collection by contamination of the semen with urine; this could interfere with fertilization and, if present, urine should be discarded by pouring out the top part of the tube. The sticky layer of the egg is removed by adding a “dissolving solution” (20 g urea/1 + 4g NaCl/1 of water) or milk (diluted 15 in water) to the fertilized egg and stirring. One hour later the swollen eggs are transferred to incubators (usually Zug bottles, sometimes 200 litre circular jars).

Sperm motility in fishes. I. Effects of temperature and pH: a review

Sperm motility is a key factor in allowing us to determine semen quality and fertilizing capacity. Motility in semen is mainly controlled by K+ in salmonids, and probably also in sturgeons, and by osmotic pressure in other freshwater and seawater fish species, but other factors, such as concentration of surrounding metabolites and ions (Ca2+, Mg2+, etc.), pH and temperature also influence motility characteristics. In the present study, we have mainly reviewed and summarized the effects of temperature and pH on the motility of spermatozoa in three fish species: salmonids, cyprinids and sturgeons. Data in the literature show that motility, fertilizing ability and velocity of spermatozoa, as well as the duration of the motility period, depend on the temperature of the assay medium and also of that of the brood fish holding tank. In contrast, the pH of the swimming medium, and thus the intracellular pH of spermatozoa, has less influence on sperm motility parameters in cyprinids, salmonids and sturgeons.

The Ionic and Osmotic Factors Controlling Motility of Fish Spermatozoa

This review presents actual knowledge about energetic, ionic, osmotic and gaseous control of fish sperm motility and its duration. Right after they are activated, fish spermatozoa of most species swim for a short period of time, in the range of one to several minutes. What determines the activation process? Is it due to the new ionic, gaseous and/or osmotic environment? Why is the duration of motility so short? Is it resulting from a fast exhaustion of energy stores (ATP, ADP, AMP, PCr) combined with the above-mentioned ionic/osmotic stress leading to morphological alterations? The motility criteria (flagellar beat frequency, head displacement velocity, flagellar waves morphology, etc.) used to characterize fish sperm movement and sperm flagella will be described. Most parameters change very rapidly during the brief motility period of fish sperm. Then will be considered the main environmental factors, ionic and/or osmotic signals, responsible of the activation of fish sperm motility. Then the metabolic compounds involved in cell energetics will be considered as their concentrations also rapidly change during the motility phase. An additional feature will then be discussed concerning the mechanisms by which fish sperm cell can be revived for a second motility round at the end of the first motility period. A model is proposed to explain how external osmolarity can control internal ionic composition, the latter being the key factor controlling flagellar activity.

Frenetic activation of fish spermatozoa flagella entails short-term motility, portending their precocious decadence.

In most species, fish spermatozoa activate their motility on contact with the external medium (sea or fresh water depending of their reproductive habitat). Their flagella immediately develop waves propagated at high beat frequency (up to 70 beats s(-1)), which propel these sperm cells at high velocity (6-10 mm min(-1)), but for a quite short period of time, usually limited to minutes. Their specific inability to restore their energy content (mostly adenosine triphosphate) fast enough relatively to their high rate of energy consumption by flagellar contributes mainly to the activity arrest of motility, as the spermatozoa need to rely on early accumulated energy prior to activation. This review of the published data explains the present understanding of physico-chemical mechanisms by which flagellar motility is activated (mostly through osmotic and ionic regulation) and then propels sperm cells at speed. It aims also to describe the gradual arrest of their motility much of which occurs within a few minutes.

Nucleotide content, oxydative phosphorylation, morphology, and fertilizing capacity of turbot (Psetta maxima) spermatozoa during the motility period

The interdependence between motility, respiration, ATP production, and utilization was investigated in intact spermatozoa of turbot (Psetta maxima), a marine teleost. When spermatozoa were diluted in a hyperosmotic medium (>300 mOsmol/kg), they immediately became motile, and the intracellular concentration of ATP as well as the adenylate energy charge ratio dropped concomitant with the straight‐line velocity. The ADP and AMP levels increased from 1.4 to 8.0 nmole/108 cells and from 0.6 to 6.0 nmole/108 cells, respectively. Moreover, 31P‐NMR spectra recorded prior to the swimming phase revealed the presence of phosphomonoesters (PMEs) and phosphodiesters (PDEs), intracellular inorganic phosphate (Pi), and phosphocreatine (PCr). At the end of the motility period, PCr, PDE, and PME decreased, while the Pi level increased markedly. Following initiation of motility, O2 consumption of spermatozoa increased from 34.9 to 124.8 O2 nmole/109 spermatozoa/min. FCCP, an uncoupler of oxydative phosphorylation, did not significantly affect the respiratory rate of motile spermatozoa. Ouabain, a specific inhibitor of (Na+/K+)/ATPase, slightly decreased the respiration rate of motile spermatozoa, indicating that the major part of ATP catabolism was linked to dynein ATPase. Inhibitors of the respiratory chain (KCN, NaN3, NaHCO3–, oligomycin) reduced sperm respiration, percentage of motile cells, velocity, and adenylate contents. Following the reactivation of motility of demembranated spermatozoa, KCN, NaN3, NaHCO3– altered the flagellar beat frequency, demonstrating that these respiratory inhibitors possess action sites other than mitochondria. Mitochondrial oxydative phosphorylation is highly requested to produce energy required during motion. Nevertheless it is insufficient to maintain endogenous ATP stores. A second phase of motility was induced by a transfer of exhausted spermatozoa into an ionic medium of low osmolality (200 mOsmol/kg) for 30 min. Spermatozoa, once reactivated in AM, recovered 55% of initial motility and 31% of initial fertilization rate. In hypo‐osmotic medium, mitochondrial oxydative phosphorylation also induced ATP regeneration. Following activation of movement, several morphological changes were observed in the mitochondria and the midpiece. Mol. Reprod. Dev. 53:230–243, 1999.

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa.

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.

Artificial insemination in turbot (Scophthalmus maximus): determination of the optimal sperm to egg ratio and time of gamete contact

Abstract In high-quality batches of eggs (mean egg viability rate 89.6%), fertilization success (FS = number of 4-cell-stage eggs/number of viable eggs) was maximal when the ratio of spermatozoa to egg was above 6 × 10 3 . For lower ratios, FS decreased and became highly variable. In lower-quality batches of eggs (mean egg viability rates 72.0 and 75.9%), FS was variable even for the highest sperm to egg ratios. For the ratio of 1.5 × 10 3 spermatozoa per egg, the occurrence of maximum FS in relation to increasing contact time between gametes was scattered and mainly recorded after 2 and 3 min. For the ratio of 6 × 10 3 spermatozoa per egg, maximum FS was mainly observed after a contact of 1 min between gametes. In commercial production, a minimum ratio of 6 × 10 3 spermatozoa per egg and a contact time between gametes of 3 min is recommended for the artificial insemination of turbot eggs.

Initiation of carp spermatozoa motility and early ATP reduction after milt contamination by urine

Abstract Fish sperm collected by stripping males is frequently contaminated by urine. In this study, carp milt mixed with urine (0.5–7.5% of volume) was studied in order to evaluate the changes of some motility parameters (percentage of motile spermatozoa, velocity and beat frequency) and the ATP content of spermatozoa. In the absence of urine contamination, spermatozoa had an ATP content in the range of 8–9 nmol/10 8 spermatozoa, an initial velocity of 100–160 μ m s −1 and a flagellar beat frequency around 30–50 Hz, 10 s after a 1/2000 dilution in an activating medium (45 mM NaCl, 5 mM KCl, 30 mM Tris–HCl, pH 8.0, osmolality −1 ). In contrast, when milt was contaminated with 7.5% of urine for 1 h, the ATP content was 4–5 nmol/10 8 spermatozoa and most spermatozoa had low initial velocity (30–100 μ m s −1 ) and flagellar beat frequency (10–30 Hz). It appears that the low osmolality of urine was responsible for the degradation in the quality of carp spermatozoa by an early activation in the collecting tube which induced an early reduction of the intracellular ATP store. From a practical point of view, milt contamination by urine during stripping can be avoided by first pressing the abdomen before sampling and then collecting the remaining urine by means of a catheter introduced into the urinary bladder.

Influence of external pH on ciliary beat frequency in human bronchi and bronchioles

Ciliated respiratory epithelial cells have to tolerate variations in local pH caused by the respiratory cycle and potential ventilation-perfusion mismatches. We showed previously that peripheral bronchiolar cilia beat at a lower frequency than bronchial cilia, and have now investigated whether they show differences in tolerance to changes in pH. Using the image analysis system applied in the previous study, we compared variations in the ciliary beat frequencies (CBF) of bronchi and bronchioles sampled from human lung resections at various pH in vitro. Application of nonparametric tests (the variance of samples was not similar) indicated that CBF was not significantly modified when pH was varied between 7.5 and 10.5 for bronchi, and between 5.5 and 10.5 for bronchioles. Reversible and significantly lower CBF were observed below pH 7.0 for bronchi and below pH 5.0 for bronchioles. Extreme pH values such as 11.0 or 3.0 were lethal within a few minutes. Thus, respiratory ciliary beating is able to tolerate external pH variations between 3.5 and 10.5 without permanent impairment. In addition we found that alkaline pH values are more favourable than acidic ones and that bronchiolar ciliated cells are more tolerant to acidic pH than bronchial cells.