Yolk proteolysis in rainbow trout oocytes after serum‐free culture: Evidence for a novel biochemical mechanism of atresia in oviparous vertebrates

Abstract

Our recent studies show little evidence for increased granulosa cell apoptosis during atresia in teleost follicles, in direct contrast to the mammalian model. Histological evidence suggests that atresia in many oviparous vertebrates involves proteolytic degradation of the energy-rich yolk storage proteins within the oocyte. This study tests the hypothesis that physiological conditions that promote atresia (hormone withdrawal) lead to increased lysosomal protease activity in rainbow trout oocytes. We subjected rainbow trout ovarian follicles to conditions that promote atresia (serum-free culture) for up to 72 hr, and measured the activity of lysosomal proteases using routine enzymatic assays. Furthermore, we used high performance liquid chromatography to quantify the increase in free amino acids resulting from proteolysis of yolk proteins. Concomitantly, we evaluated the extent of follicular apoptosis during prolonged serum-free culture, using caspase-3-like activity and DNA fragmentation as indicators of apoptosis. Our results show a significant, time-dependent increase in cathepsin L-like, but not cathepsin D-like, activity levels during culture in serum-free medium; increased cathepsin L-like activity is confirmed by a significant increase in oocyte free amino acid content after 72 hr culture. In contrast, we detected only a transient increase in apoptosis during prolonged serum-free culture, as revealed through both radioactive 3'end-labeling of oligonucleosomal DNA fragments, and caspase-3-like activity. The results of this study provide the first evidence for a novel mechanism of follicular atresia in teleosts involving cathepsin-mediated yolk proteolysis.

Relationship among follicular apoptosis, integrin β1 and collagen type IV during early ovarian regression in the teleost Prochilodus argenteus after induced spawning

“…In P. argenteus, the immunoreactivity for caspase 3 has been identified mainly in the follicular cells of postovulatory follicles. Caspase-3-like activity has also been detected in cultured atretic follicles of rainbow trout (Wood and Van Der Kraak 2003) and gonadal differentiation of the gilthead seabream, Sparus aurata (Soverchia et al 2007), but not at the cellular level. Studies with zebrafish indicate a surprising similarity with mammalian Bcl-2 family members, which control the intrinsic apoptotic pathway .…”

Thirty years of using the postovulatory follicles method: Overview, problems and alternatives

“…Indeed, in a recent study Santos et al (2005) showed that the apoptotic index (per cent of apoptotic cells) of the granulosa increased in POFs of L. taeniatus soon after spawning suggesting that it could be used in validating and improving POF staging keys. The apoptosis may also be used to discriminate late stages of POFs from atretic follilces (Wood and Van der Kraak, 2003).…”

Microarray gene expression profiles from mature gonad tissues of Atlantic bluefin tuna, Thunnus thynnus in the Gulf of Mexico

“…Cathepsin proteases have been reported to be responsible for yolk protein hydrolysis in teleosts [42-45]. Significant sequence similarities with cathepsin coding genes of 15 T. thynnus transcripts that were differentially expressed in the maturing ovarian tissue of T. thynnus in this investigation indicate a similar putative function for these ESTs (Table 1).…”