This AOP is licensed under a Creative Commons Attribution 4.0 International License.
Inhibition of ALDH1A (RALDH) causing reduced all-trans retinoic acid levels leading to impaired fertility, females
Point of Contact
- Terje Svingen
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite||Under Development||1.97||Included in OECD Work Plan|
This AOP was last modified on January 13, 2022 17:17
|Inhibition of ALDH1A (RALDH)||November 11, 2021 14:48|
|Decreased all-trans retinoic acid (atRA) concentration||February 27, 2022 10:55|
|Disrupted meiotic initiation of fetal oogonia of the ovary||February 27, 2022 10:59|
|Reduced size of the ovarian follicle pool||November 11, 2021 14:57|
|impaired, Fertility||December 02, 2016 09:21|
|irregularities, ovarian cycle||November 29, 2016 19:09|
|ALDH1A (RALDH), inhibition leads to decreased, atRA concentration||November 11, 2021 15:32|
|decreased, atRA concentration leads to Oocyte meiosis, disrupted||February 27, 2022 10:51|
|Oocyte meiosis, disrupted leads to Ovarian follicle pool, reduced||November 11, 2021 15:44|
|Ovarian follicle pool, reduced leads to irregularities, ovarian cycle||November 12, 2021 08:36|
|irregularities, ovarian cycle leads to impaired, Fertility||December 03, 2016 16:37|
This AOP links inhibition of ALDH1A during fetal life with female infertility in adulthood. A key step in this AOP is a reduction in all-trans retinoic acid (atRA) locally in the fetal ovary, which prevents resident germ cells (oocytes) from entering meiosis. Evidence for this AOP, especially upstream events, draws heavily from mouse studies, both genetic models and from exposure studies (including explanted ovaries). Human evidence is also available, especially for downstream events where the oocyte pool/ovarian reserve is known to directly impact on fertility. In reproductive toxicity (animal studies and human epidemiology) fertility is an apical endpoint of high importance and has strong utility for chemical safety assessments. Infertility can be caused by many, and varied, factors, but this AOP focusses on linking perturbed meiosis through disrupted atRA signaling during development, thus supporting the use of data from in silico and in vitro measurements for interference with nuclear receptor activity (RAR/RXR) and atRA synthesis/expression to infer potential to cause in vivo effects.
In mammals, the primordial germ cells are initially ‘bipotential’. They will develop into either oocytes or gonocytes in ovaries or testis, respectively, depending on cues from the somatic environment. Germ cells in the developing testis will enter a quiescent state and reactivate at the onset of puberty. In contrast, germ cells in the developing ovary will enter meiosis (prophase I) during fetal life. A key signaling event for this sexual dimorphic germ cell programming is retinoid signaling, with all-trans retinoic acid (atRA) acting as a meiosis-inducing factor (Spiller & Bowles, 2019).
The source of atRA during ovary development differs to some degree between species. In mice, the adjacent mesonephros, which expresses two enzymes necessary for the final step in atRA production, ALDH1A2 and ALDH1A3, is likely the main source of atRA at early developmental stages (Bowles et al, 2018; Bowles et al, 2006; Koubova et al, 2006; Niederreither et al, 1999). There is also the capacity for atRA to be produced within the ovary itself, due to local expression of the atRA-synthesizing enzyme ALDH1A1 (Bowles et al, 2016; Mu et al, 2013).
In humans, ALDH1A enzymes (ALDH1A, -1B and -1C) are expressed in both testes and ovaries of the developing fetus, which suggest a capacity for de novo synthesis of atRA (Childs et al, 2011; Jørgensen & Rajpert-De Meyts, 2014; le Bouffant et al, 2010), as is also the case in rabbits (Díaz-Hernández et al, 2019). One team studying human fetal ovaries reported a peak of ALDH1A1 expression at the onset of meiosis (le Bouffant et al, 2010), suggesting that meiotic onset in the human ovary depends on provision of atRA at the correct time. There seems to be conservation from rodent to human in terms of the requirement for atRA to induce the pre-meiotic factor STRA8. However, in mice atRA is produced by adjacent tissue and is present at high concentrations in the ovaries, whereas in human ovaries RA is present at only low levels and is then actively produced to induce meiosis in the ovary (Spiller & Bowles, 2019).
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||1880||Inhibition of ALDH1A (RALDH)||ALDH1A (RALDH), inhibition|
|2||KE||1881||Decreased all-trans retinoic acid (atRA) concentration||decreased, atRA concentration|
|3||KE||1882||Disrupted meiotic initiation of fetal oogonia of the ovary||Oocyte meiosis, disrupted|
|4||KE||1883||Reduced size of the ovarian follicle pool||Ovarian follicle pool, reduced|
|5||KE||405||irregularities, ovarian cycle||irregularities, ovarian cycle|
|6||AO||406||impaired, Fertility||impaired, Fertility|
Relationships Between Two Key Events (Including MIEs and AOs)
|ALDH1A (RALDH), inhibition leads to decreased, atRA concentration||adjacent||High||Moderate|
|decreased, atRA concentration leads to Oocyte meiosis, disrupted||adjacent||High||Low|
|Oocyte meiosis, disrupted leads to Ovarian follicle pool, reduced||adjacent||Moderate||Moderate|
|Ovarian follicle pool, reduced leads to irregularities, ovarian cycle||adjacent||Moderate||Low|
|irregularities, ovarian cycle leads to impaired, Fertility||adjacent||High||Low|
Life Stage Applicability
|During development and at adulthood||Moderate|
Overall Assessment of the AOP
The majority of evidence supporting this AOP is derived from mouse studies, both in vitro (fetal ovary cultures) and in vivo (incl. genetic mouse models). There is also evidence from humans (in vitro ovary cultures), yet it is also recognized that there are some differences between mice and humans with regard to atRA synthesis, expression and potential role in meiotic initiation. Notably, an important link, yet not described as a separate key event, is the role for Stra8 in meiotic initiation alongside the established role for atRA to control Stra8 expression via RAR/RXR.
The evidence linking MIE with KE1 is considered as strong and regarded as canonical knowledge. Likewise, evidence for the downstream key events linking reduced oocyte pool/ovarian reserve with reduced fertility is very strong and regarded as canonical knowledge. The weak link in the overall AOP is the connection between reduced atRA levels and fertility via loss of oocytes during development. To strengthen this link, more evidence must be obtained; nevertheless, the remaining links are very strong and can be used to assess the impact of chemical stressors on female fertility. Yet, caution should be exercised with directly linking inhibition of ALDH1A2 with reduced fertility.
Domain of Applicability
- Sex: This AOP applies to females. atRA is also involved in meiosis of testicular gonocytes, but this occurs postnatally. In the female ovaries, atRA induces meiosis of oocytes during gestation, thus the spatiotemporal expression of atRA in the ovaries are tightly controlled. Finally, as this AOP is concerned with establishing the ovarian reserve/follicle pool through mechanisms that are unique to ovaries, restricting the AOP to female only is appropriate.
- Life stages: This AOP spans the period from mid- to late-gestation in mammals, all the way to adulthood where fertility is manifested. The upstream event pertains to fetal/neonatal life stages, whereas the downstream events pertain to adult reproductive life stages.
- Taxonomy: Strongest evidence for the role of atRA in regulating oocyte entry into meiosis stems from moue studies, so the taxonomic applicability is strongest for this animal model. Evidence also exists for the same modes of action being relevant across mammalian species, including human (Kalampokas et al, 2014), albeit the evidence for taxonomic applicability is still weaker.
Essentiality of the Key Events
The critical first stage of this AOP is the lack of atRA in ovaries at the stage where oocytes need to enter meiosis during gestation. Failure to enter meiosis at the correct time during development is detrimental to oocyte development and ultimately this will compromise the follicle pool in adulthood; a non-renewable source for producing viable eggs for fertilization. However, reduced atRA is not defined as the MIE in this AOP, as atRA synthesis by the action of ALDH1A enzymes is required, and this synthesis step is a potential vulnerable point for chemical perturbation.
- MIE (Inhibition, ALDH1A): atRA is synthesized from dietary vitamin A in a two-step enzymatic process, where ALDH1A is responsible for the second, irreversible oxidation of retinal to atRA (Chatzi et al, 2013; Shannon et al, 2017).
- KE1 (Decreased atRA): Although ALDH1A enzymes can make different retinoid metabolites (9-cis, 13-cis, all-trans), atRA is considered the only active metabolite in mammals (Krężel et al, 2019). atRA acts locally in tissues as a paracrine, short-range, signaling molecule (Teletin et al, 2017). In the mouse, atRA is synthesized in the adjacent mesonephros and diffuses into the gonad proper to establish local concentrations required for organogenesis (Chassot et al, 2020; Kumar et al, 2011), including prompting oocytes to enter meiosis (Bowles et al, 2006; Koubova et al, 2006). In humans, atRA appears to be synthesized locally in the ovary. This KE is essential for this AOP as represents the essential link between ALDH1A inhibition by chemicals with reduced fertility in adult females.
- KE2 (disrupted meiosis): The downstream KE of this AOP, reduced follicle pool, can arise from multiple events, of which disrupted meiosis is only one. Nevertheless, disrupted meiosis is essential for this AOP in that it is the clear rational link between inhibited retinoid signaling and reduced fertility via diminished ovarian reserve. For meiosis to initiate in the mouse, oocytes need to express Stra8 (Baltus et al, 2006), a factor that is regulated by atRA via retinoid receptors RAR/RXR (Bowles et al, 2016; Bowles et al, 2006; Feng et al, 2021; Koubova et al, 2006). Although the role for atRA for initiating meiosis in humans is still under some debate, the role for Stra8 appears essential also for human oocytes to initiate meiosis (Childs et al, 2011).
- KE3 (reduced ovarian reserve): In mammals, it is broadly accepted that females are born with a set number of follicles, termed the ovarian reserve, which is dependent on proper development, including meiotic initiation, during fetal life (Grive & Freiman, 2015). A large number of oocytes are lost during ovarian/oocyte development to ensure a quality ovarian reserve. However, a minimum amount of oocytes are required to establish and maintain adult reproductive function. Therefore this KE represents an essential step in the AOP, since falling below a critical number of follicles will lead to disrupted ovary function and irregular cyclicity (as normally occurs during menopause).
- KE4 (irregular ovarian cycle): Female fertility depends on the ovarian cycle to produce competent follicles for ovulation and fertilization. Thus, this KE is an essential step in determining fertility status.
- AO impaired fertility, female: Fertility represents the capability to reproduce and as such is the essential AO of this AOP. It is measurable both at the individual and population level. Although the AO of this AOP (ID 406) describe impaired fertility independent of sex, the AOP is specific to females as it involves oocyte/ovary development and function.
Biological Plausibility, coherence, and consistency of the experimental evidence
The role for ALDH1A2 in the synthesis of atRA is well established as an essential component of regulating regional expression of retinoid species during development. It is also well established that atRA is an inducer of meiosis in germ cells in mice; however, there is some debate about the essentiality of atRA in this process in human fetal ovaries. The requirement for oocytes to enter the first phase of meiosis during fetal development is also well established, hence the biological plausibility linking meiotic failure with loss of oocytes at later developmental stages is strong.
Although non-meiotic oocytes can survive in germ cell nests and during nest breakdown, they will ultimately be eliminated from the oocyte pool of competent follicles. There is therefore a direct link between meiotic entry and fertility during adulthood. Thus, this AOP provides a plausible chain of events linking reduced atRA during fetal life with reduced ovarian reserve and fertility during reproductive age. The strength of the downstream KEs and KER – reduced ovarian reserve and reduced fertility – is very well documented and thus the biological plausibility is very strong. Evidence for a direct link between the AO and perturbed atRA synthesis, or reduced atRA levels, during early development comes mainly from mouse studies; yet the relationship is regarded biologically plausible also in humans, but with weight of evidence not being as strong.
Concordance of dose-response relationships
The quantitative understanding of dose-response relationships in this AOP is limited. 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. Nevertheless, it is has been conclusively shown that low levels of exogenous atRA can induce mouse and rat germ cells to enter meiosis both in vitro and ex vivo (Bowles et al, 2006; Livera et al, 2000). Likewise, atRA is necessary to achieve meiosis in in vitro-derived oocytes via PGCLCs (Miyauchi et al, 2017).
Temporal concordance among the key events and the adverse outcome
This AOP bridges two different life stages: fetal/perinatal and adult/reproductive age. The adverse outcome is the result of perturbation taking place during early stages of ovary development. In mice, rats and humans, the oocytes must enter meiosis prophase in order to establish the follicle pool/ovarian reserve postnatally. Thus, the AOP focusses on chemical perturbations during fetal life, which occurs around E13-E16 in mice and E15-E18 is rats, or first trimester in humans (Peters, 1970), but the adverse outcome does not manifest until adulthood.
There is strong temporal concordance between the various key events, from inhibition of ALDH1A2 (RALDH2) that leads to reduced atRA synthesis. In turn, atRA must be present in the fetal ovaries at the time when oocytes are supposed to enter meiosis mid-gestation in mice (or first trimester in human). With a significant reduction in available atRA the oocytes will not enter meiosis, ultimately leading to the downstream key event of loss of oocytes beyond what is normal. The number of oocytes, or the oocyte pool/ovarian reserve, in turn will affect ovary function and fertility at reproductive stages, hence the temporal sequence of events is rational based on the biological process.
Strength, consistency, and specificity of association of adverse effect and initiating event
In mice, there is strong evidence to support the view that atRA is an inducer of meiosis in germ cells, with consistent results from in vitro (PGCLCs), ex vivo (ovary cultures) and in vivo studies as listed under KE 2477. There is strong evidence showing the importance of RA for female fertility, but this relates to many aspects of reproductive development and function from fetal life to adulthood, including maintaining pregnancy (Clagett-Dame & Knutson, 2011). Thus, it can be difficult to distill exactly how atRA-controlled meiotic entry of oocytes directly link to reduced fertility. Nevertheless, a direct relationship is strongly supported by the fact that Stra8-depleted mice are infertile with small ovaries lacking oocytes (Baltus et al, 2006) and that Stra8 induction in germ cells is controlled by atRA in mice, rats and humans (Bowles et al, 2006; Childs et al, 2011; Koubova et al, 2006; Livera et al, 2000). Furthermore, vitamin A-deficient (VAD) mice display delayed or failed meiotic entry of fetal oocytes depending on level of Vitamin A deficiency (Li & Clagett-Dame, 2009).
Uncertainties, inconsistencies and data gaps
In mice, there is strong evidence to support the view that atRA is important for initiating meiosis in germ cells (Bowles et al, 2016; Spiller et al, 2017; Teletin et al, 2017). Some studies suggest that atAR is not critical but important for meiotic entry under normal physiological conditions by evidencing meiosis in Aldh1a1, Aldh1a2 and Aldh1a3 ablated mice, individually and in tandem (Bellutti et al, 2019; Chassot et al, 2020; Kumar et al, 2011); however, additional studies have shown redundant roles between all three Aldha isoforms which can compensate for deletion of one or two (Bowles et al, 2016). More specifically, both double (Aldh1a2/3) and triple (Aldh1a1/2/3) knockout mouse models display reduced Stra8 expression in oocytes, yet oocytes eventually go through meiosis, which could suggest a redundant role for atRA for meiosis in the ovaries (Chassot et al, 2020; Kumar et al, 2011). A similar phenotype with reduced Stra8 expression but eventual meiotic initiation is seen for deletion of atRA receptors RAR-α, -β, -γ) in mice (Vernet et al, 2020). But, although RAR knockouts were also capable of producing offspring, it remains unclear if any of the above-mentioned mouse models display impaired fertility or whether the size of their oocyte pools are affected.
This AOP is still largely qualitative, as the quantitative understanding between chemical potency and perturbation of KEs are insufficient. This relates to the dose-response relationship between concentrations of atRA in the ovary relative to meiotic initiation of oocytes. It also relates to the relationship between number of lost oocytes during development relative to the oocyte pool/ovarian reserve, as there naturally is a large loss of oocytes during development.
Considerations for Potential Applications of the AOP (optional)
Baltus AE, Menke DB, Hu YC, Goodheart ML, Carpenter AE, de Rooij DG, Page DC (2006) In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication. Nat Genet 38: 1430-1434
Bellutti L, Abby E, Tourpin S, Messiaen S, Moison D, Trautmann E, Guerquin MJ, Rouiller-Fabre V, Habert R, Livera G (2019) Divergent Roles of CYP26B1 and Endogenous Retinoic Acid in Mouse Fetal Gonads. Biomolecules 9: 536
Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson MJ, Rossant J, Hamada H, Koopman P (2006) Retinoid signaling determines germ cell fate in mice. Science 312: 596-600
Chassot AA, Le Rolle M, Jolivet G, Stevant I, Guigonis JM, Da Silva F, Nef S, Pailhoux E, Schedl A, Ghyselinck NB, Chaboissier MC (2020) Retinoic acid synthesis by ALDH1A proteins is dispensable for meiosis initiation in the mouse fetal ovary. Sci Adv 6: eaaz1261
Feng CW, Burnet G, Spiller CM, Cheung FKM, Chawengsaksophak K, Koopman P, Bowles J (2021) Identification of regulatory elements required for Stra8 expression in fetal ovarian germ cells of the mouse. Development 148: dev194977
le Bouffant R, Guerquin MJ, Duquenne C, Frydman N, Coffigny H, Rouiller-Fabre V, Frydman R, Habert R, Livera G (2010) Meiosis initiation in the human ovary requires intrinsic retinoic acid synthesis. Hum Reprod 25: 2579-2590
Miyauchi H, Ohta H, Nagaoka S, Nakaki F, Sasaki K, Hayashi K, Yabuta Y, Nakamura T, Yamamoto T, Saitou M (2017) Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice. EMBO J 36: 3100-3119
Vernet N, Condrea D, Mayere C, Féret B, Klopfenstein M, Magnant W, Alunni V, Teletin M, Souali-Crespo S, Nef S, Mark M, Ghyselinck NB (2020) Meiosis occurs normally in the fetal ovary of mice lacking all retinoic acid receptors. Sci Adv 6: eaaz1139