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An in vitro ovarian explant culture system to examine sex change in a hermaphroditic fish.

ABSTRACT: Many teleost fishes undergo natural sex change, and elucidating the physiological and molecular controls of this process offers unique opportunities not only to develop methods of controlling sex in aquaculture settings, but to better understand vertebrate sexual development more broadly. Induction of sex change in some sequentially hermaphroditic or gonochoristic fish can be achieved in vivo through social manipulation, inhibition of aromatase activity, or steroid treatment. However, the induction of sex change in vitro has been largely unexplored. In this study, we established an in vitro culture system for ovarian explants in serum-free medium for a model sequential hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). This culture technique enabled evaluating the effect of various treatments with 17?-estradiol (E2), 11-ketotestosterone (11KT) or cortisol (CORT) on spotty wrasse ovarian architecture for 21 days. A quantitative approach to measuring the degree of ovarian atresia within histological images was also developed, using pixel-based machine learning software. Ovarian atresia likely due to culture was observed across all treatments including no-hormone controls, but was minimised with treatment of at least 10 ng/mL E2. Neither 11KT nor CORT administration induced proliferation of spermatogonia (i.e., sex change) in the cultured ovaries indicating culture beyond 21 days may be needed to induce sex change in vitro. The in vitro gonadal culture and analysis systems established here enable future studies investigating the paracrine role of sex steroids, glucocorticoids and a variety of other factors during gonadal sex change in fish.

SUBMITTER: Goikoetxea A

PROVIDER: S-EPMC7666549 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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An in vitro ovarian explant culture system to examine sex change in a hermaphroditic fish.

Goikoetxea Alexander A Damsteegt Erin L EL Todd Erica V EV McNaughton Andrew A Gemmell Neil J NJ Lokman P Mark PM

PeerJ 20201111

Many teleost fishes undergo natural sex change, and elucidating the physiological and molecular controls of this process offers unique opportunities not only to develop methods of controlling sex in aquaculture settings, but to better understand vertebrate sexual development more broadly. Induction of sex change in some sequentially hermaphroditic or gonochoristic fish can be achieved in vivo through social manipulation, inhibition of aromatase activity, or steroid treatment. However, the induct ...[more]

PMID: 33240644

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Project description:Can cultured follicles model the ovarian cycle, including follicular- and luteal-phase hormone synthesis patterns and ovulation?Under gonadotrophin stimulation, murine follicles grown in an encapsulated three-dimensional system ovulate in vitro and murine and human follicle hormone synthesis mimics follicular and luteal phases expected in vivo.Studies of the human ovary and follicle function are limited by the availability of human tissue and lack of in vitro models. We developed an encapsulated in vitro follicle growth (eIVFG) culture system, which preserves 3D follicular structure. Thus far, the alginate system has supported the culture of follicles from mice, dog, rhesus macaque, baboon and human. These studies have shown that cultured follicles synthesize steroid hormones similar to those observed during the follicular phase in vivo.Cultured murine follicles were treated with human chorionic gonadotrophin (hCG) and epidermal growth factor (EGF) and either assayed for luteinization or removed from alginate beads and assayed for ovulation. Human follicles were also cultured, treated with follicle-stimulating hormone (FSH), hCG and EGF to mimic gonadotrophin changes throughout the ovarian cycle, and culture medium was assayed for hormone production.Murine and human follicles were cultured in alginate hydrogel and hormone production [17?-estradiol, progesterone, inhibin A, inhibin B, activin A and anti-Müllerian hormone (AMH)] was quantified in medium by enzyme-linked immuno assay (ELISA). Human ovarian tissue was acquired from females between 6 and 34 years of age with a cancer diagnosis. These participants were undergoing ovarian tissue cryopreservation at National Physicians Cooperative sites as part of the Oncofertility Consortium.When grown in this system, 96% of mouse follicles ovulated in response to hCG and released meiotically competent eggs. Ovulated follicles recapitulated transcriptional, morphologic and hormone synthesis patterns post-luteinizing hormone (LH/hCG). In addition to rodent follicles, individual human follicles secreted steroid and peptide hormones that mimicked the patterns of serum hormones observed during the menstrual cycle.This was a descriptive study of an in vitro model of ovulation and the ovarian hormone cycle. The ovulation studies were limited to murine tissue and further studies are needed to optimize conditions using other species.The eIVFG system reliably phenocopies the in vivo ovarian cycle and provides a new tool to study human follicle biology and the influence of cycling female hormones on other tissue systems in vitro.This work was supported by NIH U54 HD041857, NIH U54 HD076188, NIH UH2 E5022920, NIH UH3 TR001207 and F30 AG040916 (R.M.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Project description:?Many angiosperms are hermaphroditic and produce bisexual flowers in which male (pollen export) and female (stigma receptivity) functions are separated temporally. This sequential hermaphroditism may be associated with variation in flower size, color, or pattern, all of which may influence pollinator attraction. In this study, we describe variation in these traits across discrete functional sex stages within and between 225 greenhouse-grown individuals of Clarkia unguiculata (Onagraceae). In addition, to identify the effects of floral phenotype on pollinator attraction in this species, we examine the effects of these floral traits on pollen receipt in ~180 individuals in an experimental field array.?Petal area, ultraviolet (UV)-absorbing nectar guide area, and blue and green mean petal reflectance differ significantly across the functional sex stages of C. unguiculata. Male- and female-phase flowers display significantly different pollinator attraction traits. Petal and UV nectar guide area increase as flowers progress from male phase to female phase, while blue reflectance and green reflectance peak during anther maturation.?In field arrays of C. unguiculata, female-phase flowers with large UV nectar guides receive more pollen than those with small nectar guides, and female-phase flowers with high mean blue reflectance values are more likely to receive pollen than those with low blue reflectance. Female-phase flowers with green mean reflectance values that differ most from background foliage also receive more pollen than those that are more similar to foliage. These findings indicate that components of flower color and pattern influence pollen receipt, independent of other plant attributes that may covary with floral traits. We discuss these results in the context of hypotheses that have been proposed to explain sex-specific floral attraction traits, and we suggest future research that could improve our understanding of sexual dimorphism in sequentially hermaphroditic species and the evolution of features that promote outcrossing.

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An in vitro ovarian explant culture system to examine sex change in a hermaphroditic fish.

Publications

An in vitro ovarian explant culture system to examine sex change in a hermaphroditic fish.

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