An in vitro culture system for studying vitellogenin uptake into ovarian follicles of the rainbow trout, Salmo gairdneri

Abstract

The development of a short-term culture system to study vitellogenin (VTG) sequestration in vitro into vitellogenic follicles of the rainbow trout, Salmo gairdneri, is described.The outer epithelial cell layer of the trout follicle restricted VTG uptake considerably and its removal was a prerequisite to study VTG uptake in vitro. Vitellogenic follicles divested of the surface epithelium sequestered VTG at rates of up to 170 ngmm2 follicle surface-1.h-1(ng.mm-2.h-1). Uptake of VTG over an 8 h period of incubation was linear.Increasing the titre of VTG in the culture medium induced greater rates of sequestration in a dose-dependent manner; follicles cultured in medium containing 45 mg VTG-ml-sequestered 170 * 9ng.mm-2.h-1 whereas those follicles cultured in medium containing 2 mg.ml-l only incorporated 6 % 0.5 ng-mm-2.h-1.Uptake of VTG was temperature dependent with higher temperatures inducing higher levels of incorporation. Sequestration of VTG persisted at temperatures below 5"C, showing that the uptake mechanisms are adapted to low temperatures in this species.Size of follicles also had a considerable bearing on the rate of VTG sequestration; follicles of less than 1 mm diameter (pre-vitellogenic follicles) did not sequester VTG in amounts that were detectable. All follicles measuring 1 mm or greater in diameter sequestered VTG. As the follicles increased in size between approximately 1 and 3 mm in diameter there was an increase in both the total amount of VTG sequestered and the rate of uptake of VTG relative to surface area. Limited data suggested that in follicles closely approaching ovulation the rate of VTG sequestration was considerably reduced.Female rainbow trout expend considerable energy on reproduction. Each year the ovaries grow from less than 1% of the body weight up to 20% just prior to ovulation, when between 2,000 and 3,000 eggs per kg body weight are released into the body cavity. At ovulation the oocytes attain a diameter of approximately 5 mm, constituting a volume of 250 x lo6 greater than oogonia (Sumpter, '84). This enormous growth of the oocytes occurs predominantly during the phase of development termed vitellogenesis, when large amounts of an extra-ovarian protein called vitellogenin (VTG) are sequestered from the blood. Despite this, however, there has been little work conducted on the mechanisms controlling VTG sequestration. Studies of this nature, however, are difficult to conduct in vivo for they are prone to difficulties of interpretation because the involvement of a parameter may be either direct or indi- rect (in the latter case being relayed by other physiological and endocrinological processes). The availability of an in vitro system for culturing trout follicles, on the other hand, would allow such studies on VTG sequestration to be conducted, for they do not incur such problems of interpretation. Furthermore, tissues in culture are provided with a "controlled" environment, unlike in vivo where the environment is dynamic.Twenty years ago such an in vitro system was...

Oocyte growth and development in teleosts

“…Preliminary evidence suggests that in teleosts, as in mammals, growth factors play an integral role in oocyte growth. For example, insulin has been shown to stimulate VTG uptake in rainbow trout follicles (Tyler et al, 1987;Shibata et al, 1993), an effect that may well be mediated through the IGF receptor, rather than the receptor for insulin itself. Indeed, there is considerable structural homology between the insulin receptor and IGF-I receptor (Scavo, 1991).…”

Identification and Transcriptional Modulation of the Largemouth Bass, Micropterus salmoides, Vitellogenin Receptor During Oocyte Development by Insulin and Sex Steroids1

“…Furthermore, the existence of a new system of multiple Vtgrs that disparately bind functionally different types of Vtgs in white perch has been suggested [16]. Short-term in vitro studies have reported successful incorporation of radiolabeled Vtg into fish oocytes [17,18]. Several publications have explored the signals controlling Vtg uptake and suggest control by various hormones including gonadotropin I [19], insulin (INS) and thyroxine [20], a pituitary glycoprotein fraction from salmon [21], 11-ketotestosterone (11-KT), and INS-like growth factor hormone I (IGF-I) [22].…”

Molecular endocrinology of oocyte growth and maturation in fish

“…In vitro culture systems have been developed for studies on VT sequestration into vitellogenic follicles of rainbow trout (Oncorhynchus mykiss) and F. heteroclitus, and have been used to demonstrate receptor-mediated VT incorporation by these oocytes (Tyler et al 1990;Kanungo et al 1990;Tyler 1991). The binding for VT was characterized in isolated oocyte membranes of the tilapia, Oreochromis niloticus (Chan et al 1991).…”