Use of an organotypic mammalian in vitro follicle growth assay to facilitate female reproductive toxicity screening

Xu, Yuanming; Duncan, Francesca E.; Xu, Min; Woodruff, Teresa K.

doi:10.1071/rd14375

Cited by 17 publications

(7 citation statements)

References 57 publications

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“…The primary functions of the follicle are to synthesize and secrete steroid hormones to maintain the ovarian cyclicity and to support oocyte maturation for fertilization and pregnancy. In vitro follicle cultures provide a dynamic method for evaluating the ovarian toxicity of both pharmaceutical drugs and environmental chemicals and efforts in harmonizing patients dosing with in vivo animal data and in vitro models are critically important (Ahn et al, 2013;Mahalingam et al, 2016;Patel et al, 2016;Xu et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

Doxorubicin Has Dose-Dependent Toxicity on Mouse Ovarian Follicle Development, Hormone Secretion, and Oocyte Maturation

Xiao

Zhang

Liu

et al. 2017

Toxicological Sciences

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Doxorubicin (DOX), one of the most commonly used anticancer medications, has been reported to affect fertility by damaging ovarian follicles; however, the dose-dependent toxicity of DOX on the dynamic follicle development and oocyte maturation has not been well-defined. Our objective is to determine the effects of human-relevant exposure levels of DOX on follicular functions across developmental time. In vitro cultured multilayered secondary mouse follicles were treated with DOX at 0, 2, 20, 100, and 200 nM for 24 h, and follicle development, hormone secretion, and oocyte maturation were analyzed. DOX caused dose-dependent toxicity on follicle growth, survival, and secretion of 17β-estradiol (E2). At 200 nM, DOX induced DNA damage and apoptosis in follicle somatic cells first and then in oocytes, which was correlated with the uptake of DOX first to the somatic cells followed by germ cells. Follicles treated with DOX at 0, 2, and 20 nM showed similar oocyte metaphase II (MII) percentages after in vitro oocyte maturation; however, 20 nM DOX significantly increased the number of MII oocytes with abnormal spindle morphology and chromosome misalignment. In an effort to harmonize the in vitro study to in vivo treatment, dose-dependent toxicity on oocyte meiotic maturation was found in 16-day-old CD-1 mice treated with DOX at 0, 0.4, 2, and 10 mg/kg, consistent with the in vitro oocyte maturation outcomes. Our study demonstrates that DOX has dose-dependent toxicity on ovarian follicle development, hormone secretion, and oocyte maturation, which are three key factors to support the female reproductive and endocrine functions.

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Section: Discussionmentioning

confidence: 99%

Doxorubicin Has Dose-Dependent Toxicity on Mouse Ovarian Follicle Development, Hormone Secretion, and Oocyte Maturation

Xiao

Zhang

Liu

et al. 2017

Toxicological Sciences

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“…The ovarian follicle provides a unique organotypic system in which to investigate the impact of FSH glycoforms without any systemic side effects of the body or of surrounding ovarian cells (Xu et al 2016). In the follicle, FSH drives granulosa cell proliferation and production of estrogen, which in turn promotes follicle growth, follicle and oocyte maturation, and ovulation (Hunzicker-Dunn & Maizels 2006).…”

Section: Introductionmentioning

confidence: 99%

Recombinant FSH glycoforms are bioactive in mouse preantral ovarian follicles

et al. 2019

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Female reproductive aging is characterized by a rise in follicle-stimulating hormone (FSH) levels during peri-menopause. N-linked glycans are co-translationally attached to the Asn7 and Asn24 residues on the FSHβ subunit. Differences in the number of N-glycans on the FSHβ subunit result in distinct glycoforms: hypo-glycosylated (FSH21/18, glycans absent on either Asn24 or Asn7, respectively) or fully-glycosylated (FSH24, glycans present on both Asn7 and Asn24). The relative abundance of FSH glycoforms changes with advanced reproductive age, shifting from predominantly FSH21/18 in younger women to FSH24 in older women. Previous in vitro studies in granulosa cell lines and in vivo studies using Fshb-null mice showed these glycoforms elicit differential bioactivities. However, the direct effects of FSH glycoforms on the mouse ovarian follicle have not yet been determined. In this study, we isolated secondary follicles from pre-pubertal mice and treated them with 20- or 100 ng/mL purified recombinant FSH glycoforms for 1 h or 18–20 h. Analysis of phosphorylated PKA substrates showed that glycoforms were bioactive in follicles following 1-h treatment, although differential bioactivity was only observed with the 100 ng/mL dose. Treatment of follicles with 100 ng/mL of each glycoform also induced distinct expression patterns of FSH-responsive genes as assessed by qPCR, consistent with differential function. Our results, therefore, indicate that FSH glycoforms are bioactive in isolated murine follicles.

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“…reproductive toxicity and bioactivity (27,31,45,46), and as a means for nonhormonal contraceptive discovery (38). The following protocol focuses on standard eIVFG methods, but various modifications in culture conditions, alginate composition, and uses for eIVFG are well documented in the literature (2-34, 38, 46-49).…”

Section: Introductionmentioning

confidence: 99%

Recapitulating folliculogenesis and oogenesis outside the body: encapsulated in vitro follicle growth

Converse

Zaniker

Amargant

et al. 2022

Biology of Reproduction

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Folliculogenesis is a tightly coordinated process essential for generating a fertilization-competent gamete while also producing gonadal hormones that sustain endocrine function. In vitro follicle growth systems have been critical to our understanding of key events in folliculogenesis, such as gonadotropin-independent and -dependent growth, steroid hormone production, and oocyte growth and maturation (cytoplasmic and meiotic). Although there are several successful follicle culture strategies, the following protocol details an encapsulated in vitro follicle growth (eIVFG) system for use with mouse ovarian follicles. eIVFG is performed with alginate hydrogels, which are biologically inert, maintain cell-to-cell interactions between granulosa cells and the oocyte, and preserve follicle architecture as found in the ovary. The system supports follicle growth, development, and differentiation from the early primary follicle to the antral follicle stage. Moreover, post-folliculogenesis events including meiotic maturation, ovulation, and luteinization are also supported. Importantly, the culture of secondary follicles has successfully resulted in viable pups after blastocyst transfer. This alginate-based eIVFG system is versatile and has broad applications as a tool for interrogating the fundamental biology of the ovarian follicle in a controlled manner, a screening platform for toxicity and bioactivity, and a potential fertility preservation method for endangered species as well as humans.

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Use of an organotypic mammalian in vitro follicle growth assay to facilitate female reproductive toxicity screening

Cited by 17 publications

References 57 publications

Doxorubicin Has Dose-Dependent Toxicity on Mouse Ovarian Follicle Development, Hormone Secretion, and Oocyte Maturation

Doxorubicin Has Dose-Dependent Toxicity on Mouse Ovarian Follicle Development, Hormone Secretion, and Oocyte Maturation

Recombinant FSH glycoforms are bioactive in mouse preantral ovarian follicles

Recapitulating folliculogenesis and oogenesis outside the body: encapsulated in vitro follicle growth

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