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Relationship: 2527

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

increased atRA concentration leads to premature meiosis, male germ cells

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

increased atRA concentration

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

premature meiosis, male germ cells

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Increased (ectopic) concentration of all-trans retinoic acid (ATRA) if fetal testis leading to reduced sperm count, males	adjacent	Moderate	Low	Terje Svingen (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
mouse	Mus musculus	High	NCBI
rat	Rattus norvegicus	Moderate	NCBI
human	Homo sapiens	Low	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Male	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Fetal	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Germs cells undergo meiotic cell division to produce haploid sperm or eggs from diploid gonocytes or oocytes, respectively. Meiotic entry occurs during fetal life in germ cells of ovaries but postnatally in germ cells of testes (Spiller et al, 2017). During late fetal life, germ cells in testes remain in a state of mitotic quiescence and it isn’t until prior to puberty that they initiate meiotic entry.

All-trans retinoic acid (atRA) is suggested to induce meiosis in both males and females at the appropriate life stages. atRA prompts germ cells of the developing ovary to initiate first round of meiosis during fetal life, whereas male germ cells in the developing testis is prevented from entering meiosis until puberty by the effective breakdown of atRA by CYP26 enzymes. In the absence of atRA, fetal male germ cells enter cell cycle quiescence (Spiller et al, 2017). If, however, atRA is ectopically expressed in the fetal testis, premature meiotic initiation is induced in fetal male germ cells, which ultimately disrupts gonocyte development.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

The majority of evidence for this KER is derived from rodent studies. Gonad explants are predominantly used for evidence supporting this KER in humans.

The overall strength of evidence will be evaluated upon further studies of relevant literature included from the literature search.

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

In mammalian germ cells, expression of the pre-meiotic marker Stimulated by retinoic acid gene 8 (Stra8) is a critical factor for meiotic onset and progression (Baltus et al, 2006; Bowles et al, 2006; Feng et al, 2021; Koubova et al, 2014; Koubova et al, 2006). In male mice lacking Stra8, the germ cells fail to initiate meiosis at puberty (Anderson et al, 2008), which has also been shown to be the case in the fetal female (Baltus et al, 2006) underlining that Stra8 seems to be required for correct meiotic initiation in both sexes. Though the mechanistic details of the spatiotemporal regulation of Stra8 are yet unclear, robust evidence suggests that atRA is a main regulator of Stra8 expression (Griswold et al, 2012). Germ cells from embryos from pregnant vitamin A (the precursor of atRA) deficient rats fail to upregulate Stra8 and enter meiosis (Li & Clagett-Dame, 2009) and several functional atRA response elements have been identified within the Stra8 promoter (Feng et al, 2021).

In the fetal male, somatic expression of the cytochrome P450 (CYP) enzyme CYP26B1 ensures atRA degradation, thus avoiding initiation of meiosis (Bowles et al, 2018; Bowles et al, 2006; Koubova et al, 2006; Li et al, 2009; MacLean et al, 2007). However, if atRA is not cleared effectively, male germ cells will initiate expression of Stra8, and aberrantly enter meiosis (Bowles et al, 2006; Koubova et al, 2006).

Genetic deletion of Cyp26b1 in mice increases endogenous atRA levels in the fetal testis and the germ cells abnormally start to express Stra8 and enter meiosis (Bowles et al, 2006; MacLean et al, 2007). Exogenous atRA can stimulate male germ cells to induce Stra8 expression and meiosis in mouse testis explants (Bowles et al, 2006; Koubova et al, 2006; Trautmann et al, 2008).

This effect seems to be conserved in humans, though limited studies are available. A key difference, however, has been the observation that CYP26B1 is expressed at equal or even higher levels in the fetal ovary than in the fetal human testis (Childs et al, 2011; Jørgensen et al, 2012). This is in contrast to the sexually dimorphic expression patterns observed in fetal rodents. Lastly, in accordance with the above mentioned rodent studies, single cell suspension cultures of human fetal testes showed increased STRA8 expression in response to exogenous atRA, however, no effect was observed on the gene expression of meiosis markers SYCP3 and DMC1 (Childs et al, 2011).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Animal models

Model	Relevant observations	Reference
Cyp26b1 knockout mice	Stra8 and Sycp3 are abnormally expressed in the embryonic testes of Cyp26b1 knockout mice, and this is followed by a loss of male germ cells	(MacLean et al, 2007)
Cyp26b1 knockout mice	Stra8 and Sycp3 are abnormally expressed in the embryonic testes of Cyp26b1 knockout mice	(Bowles et al, 2006)

In vitro/ex vivo

Study type	Species	Compound	Effect Dose	Duration	Results	Reference
Embryonic stem cells	Mouse	atRA	100 nM	4-10 d	Activates meiosis-related gene network	(Aoki & Takada, 2012)
Fetal testes in culture	Mouse	atRA	1 µM	48 h or 72 h	Ectopic expression of meiotic markers (Stra8, Sycp3, Dmc1) and decrease in pluripotency marker Oct4	(Bowles et al, 2006)
Fetal testes in culture	Mouse	Ketoconazole (Cyp26 inhibitor)	40 µM	48 h or 72 h	Ectopic expression of meiotic markers (Stra8, Sycp3, Dmc1) and decrease in pluripotency marker Oct4	(Bowles et al, 2006)
Fetal testes in culture	Mouse	BMS-194753 (RAR-α agonist)	0.5 µM	2 d	Ectopic Stra8 expression	(Koubova et al, 2006)
Fetal testes in culture	Mouse	BMS-213309 (RAR-β agonist)	0.5 µM	2 d	Ectopic Stra8 expression	(Koubova et al, 2006)
Fetal testes in culture	Mouse	BMS-270394 (RAR-γ agonist)	0.5 µM	2 d	Ectopic Stra8 expression	(Koubova et al, 2006)
Fetal testes in culture	Mouse	atRA	0.7 µM	2 d	Ectopic Stra8 expression	(Koubova et al, 2006)
Fetal testes in culture	Mouse	Ketoconazole (Cyp26 inhibitor)	0.7 µM	2 d	Ectopic Stra8 expression	(Koubova et al, 2006)
Fetal testes in culture	Mouse	R115866 (Cyp26 inhibitor)	0.7 µM	2 d	Ectopic Stra8 expression	(Koubova et al, 2006)
Fetal testes in culture	Rats	atRA	1 µM and 0.031 µM	2 or 3 d	Increased apoptosis of germ cells, reduction in total number of germ cells	(Livera et al, 2000a)
Culture of fetal testis single cell suspension	Human	atRA	1 µM	24 h	Increased STRA8 expression, but had no effect on DMC1 or SYCP3 expression	(Childs et al, 2011)

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Mouse deletion model for the atRA synthesis enzymes Aldh1a1, Aldh1a2 and Aldh1a3 showed decreased expression of Stra8 in double (Aldh1a2/3) and triple (Aldh1a1/2/3) knockouts, although ultimately germ cells were observed undergoing meiosis in these ovaries, suggesting redundant role for atRA (Chassot et al, 2020; Kumar et al, 2011). Similarly, transgenic mice lacking the three atRA nuclear receptors (RAR-a, -b, -g) showed reduced levels of Stra8, although ultimately germ cells were observed undergoing meiosis and were capable of producing live offspring (Vernet et al, 2020). Whether or not these models led to impaired fertility (such as sub-fertility) has not been elucidated and the size of their oocyte pools were not determined.

Gain of function mouse ovary models for CYP26A1 and CYP26B1 shows that CYP26B1 can prevent oocytes from entering meiosis (as in, failure to induce Stra8 expression), whereas CYP26A1 does not have the same effect despite being a potent atRA degrading enzyme. This suggests that factor(s) in addition to atRA are required for meiosis induction (Bellutti et al, 2019).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

There is a very limited quantitative understanding of this KER other than the knowledge that when germ cells, from either testis or ovary, are cultured in the presence of atRA, at concentrations as low as 10nM, Stra8 expression is rapidly induced (Spiller & Bowles, 2019).

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

In vitro and ex vivo, it has been conclusively shown that low levels of exogenous atRA can induce germ cells to enter meiosis in mice (Bowles et al, 2006) and rats (Livera et al, 2000b) and, similarly, that it is necessary to achieve meiosis in in-vitro-derived oocytes via PGCLCs (Miyauchi et al, 2017). Yet, its exact role in vivo is debated.

Whilst the relative levels of endogenous atRA produced by the ovary (for any species) remains unknown, similarly, the quantitative relationship between atRA levels and induction of meiosis also remains unclear. As such, the quantitative understanding of how much atRA needs to be reduced to prevent germ cells from entering meiosis in vivo is rated low.

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help