Effect of a low water salinity on oocyte maturation, oocyte hydration, ovulation and egg quality in rainbow trout.

Water salinity is an important environmental factor known to have detrimental effects on salmonid reproduction, mostly when migrating female broodfish are held in sea water. In contrast, data obtained in freshwater are scarce and the impact of low water salinity during reproduction in freshwater is currently unknown. For this reason, and because ion and water fluxes are critical for the final steps of the female gamete formation, including oocyte hydration and ovulation, the aim of the present study was to investigate the impact of low salinity water on final oocyte maturation, ovulation and, ultimately, on egg quality, using rainbow trout as a physiological model and relevant aquaculture species. Fish from the same commercial strain were raised throughout their lifecycle either in a site characterized by low concentrations of Na+, K+, and Cl- ions in the water or in a closely located control site exhibiting standard salinity levels. Egg quality and duration of final oocyte maturation were investigated using innovative phenotyping tools such as automatic assessment of egg viability using the VisEgg system and non-invasive echograph-based monitoring of final oocyte maturation duration, respectively. Oocyte hydration during final oocyte maturation and after ovulation was also investigated. Finally, molecular phenotyping was performed using real-time PCR-based monitoring of several key players of final oocyte maturation and ovulation associated with ion and water transport, inflammation, proteolytic activity, and coagulation. Oocyte hydration and gene expression data were analyzed in the light of the duration of final oocyte maturation. Here we show that low water salinity negatively influences final oocyte maturation, ovulation and, ultimately, egg quality. Low water salinity triggers delayed ovulation and lower oocyte viability. When investigating the impact of low water salinity on final oocyte maturation duration, individuals presenting the most severe phenotypes exhibited impaired oocyte hydration and abnormally reduced gene expression levels of several key players of the ovulatory process. While the under expression of water (e.g., aquaporins) and ion (e.g., solute carriers) transporters is consistent with impaired oocyte hydration, our observations also indicate that the entire ovulatory gene expression program is disrupted. Our results raise the question of the mechanisms underlying the negative influence of low salinity water on the dynamics of the preovulatory phase, on the control of the oocyte homeostasis, including hydration, and on the overall success of the maturation-ovulation process.

Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by an H+-ATPase inhibitor: effects on the hydration mechanism

In teleost oocytes, yolk proteins (YPs) derived from the yolk precursors vitellogenins are partially cleaved into free amino acids and small peptides during meiotic maturation before ovulation. This process increases the osmotic pressure of the oocyte that drives its hydration, which is essential for the production of buoyant eggs by marine teleosts (pelagophil species). However, this mechanism also occurs in marine species that produce benthic eggs (benthophil), such as the killifish ( Fundulus heteroclitus), in which oocyte hydration is driven by K+. Both in pelagophil and benthophil teleosts, the enzymatic machinery underlying the maturation-associated proteolysis of YPs is poorly understood. In this study, lysosomal cysteine proteinases potentially involved in YP processing, cathepsins L, B, and F (CatL, CatB, and CatF, respectively), were immunolocalized in acidic yolk globules of vitellogenic oocytes from the killifish. During oocyte maturation in vitro induced with the maturation-inducing steroid (MIS), CatF disappeared from yolk organelles and CatL became inactivated, whereas CatB proenzyme was processed into active enzyme. Consequently, CatB enzyme activity and hydrolysis of major YPs were enhanced. Follicle-enclosed oocytes incubated with the MIS in the presence of bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, underwent maturation in vitro, but acidification of yolk globules, activation of CatB, and proteolysis of YPs were prevented. In addition, MIS plus bafilomycin A1-treated oocytes accumulated less K+ than those stimulated with MIS alone; hence, oocyte hydration was reduced. These results suggest that CatB is the major protease involved in yolk processing during the maturation of killifish oocytes, whose activation requires acidic conditions maintained by a vacuolar-type H+-ATPase. Also, the data indicate a link between ion translocation and YP proteolysis, suggesting that both events may be equally important physiological mechanisms for oocyte hydration in benthophil teleosts.

In vivo oocyte hydration in Atlantic halibut (Hippoglossus hippoglossus); proteolytic liberation of free amino acids, and ion transport, are driving forces for osmotic water influx

SUMMARY The in vivo swelling and hydration of maturing oocytes of Atlantic halibut Hippoglossus hippoglossus were studied in order to characterise the osmotic mechanism underlying oocyte hydration in oviparous marine teleosts that spawn pelagic eggs. Sequential biopsies from two females, spanning four hydration cycles, were examined by osmometry, solute analysis and electrophoresis of dissected hydrating oocytes and ovulated eggs. The hydration cycle of the biopsied halibuts lasted 33–54 h. The majority of ovarian oocytes existed in a pre-hydrated condition (individual wet mass approx. 3.7 mg, diameter approx. 1.87 mm, 63 % H2O) with easily visible, non-coalesced, yolk platelets. Group-synchronous batches of the pre-hydrated oocytes increased in individual wet mass, diameter and water content to reach the ovulated egg stage of approximately 15 mg, 3.0 mm and 90 % H2O, respectively. The yolk osmolality of the hydrating oocytes was transiently hyperosmotic to the ovarian fluid (range 305–350 mOsmol l–1) with a peak osmolality of about 450 mOsmol l–1 in oocytes of 6–8 mg individual wet mass. The transient hyperosmolality was well accounted for by the increase in oocyte content of free amino acids (FAAs; approx. 2300 nmol oocyte–1), K+ (approx. 750 nmol oocyte–1), Cl– (approx. 900 nmol oocyte–1), total ammonium (approx. 300 nmol oocyte–1) and inorganic phosphate (Pi; approx. 200 nmol oocyte–1) when relating to the increase in cellular water. The oocyte content of Na+ did not increase during the hydration phase. Extensive proteolysis of yolk proteins, in particular a 110 kDa protein, correlated with the increase in the FAA pool, although the latter increased by approx. 20 % more than could be accounted for by the decrease in the oocyte protein content. Both indispensable and dispensable amino acids increased in the FAA pool, and particularly serine, alanine, leucine, lysine, glutamine and glutamate. Taurine content remained stable at approx. 70 nmol oocyte–1 during oocyte hydration. The results show that final hydration of Atlantic halibut oocytes is caused by an osmotic water uptake in which FAAs, derived mainly from the hydrolysis of a 110 kDa yolk protein, contribute approximately 50 % of the yolk osmolality and ions (Cl–, K+, Pi, NH4+) make up the balance.