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Relationship: 2937

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

Impaired, Spermatogenesis leads to Decreased, Viable Offspring

Upstream event

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

Impaired, Spermatogenesis

Downstream event

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

Decreased, Viable Offspring

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
PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-Ketotestosterone	adjacent	Moderate	Low	Jennifer Olker (send email)	Open for citation & comment
Essential element imbalance leads to reproductive failure via oxidative stress	adjacent			Travis Karschnik (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 Do not cite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
teleost fish	teleost fish	High	NCBI
Murinae gen. sp.	Murinae gen. sp.	High	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
Adult, reproductively mature	High

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

Spermatogenesis is a multiphase process of cellular transformation that produces mature male gametes known as sperm for sexual reproduction. The process of spermatogenesis can be broken down into 3 phases: the mitotic proliferation of spermatogonia, meiosis, and post meiotic differentiation (spermiogenesis) (Boulanger et al., 2015). Male fertility is dependent on the quantity as well as the proper cellular morphology of the sperm formed in the testes. The fusion of sperm and oocytes is the key step for the beginning of life known as fertilization. Oocyte fertilization and the production of viable offspring from sexual reproduction are dependent on spermatogenesis and sufficient quantity and quality of sperm. When the impairment of spermatogenesis occurs, it can result in impaired reproduction with a decrease in viable offspring.

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

The majority of papers used in evidence supporting the key event relationship were found through AbstractSifter, a Microsoft Excel-based application that extracts papers from PubMed. AbstractSifter ranks abstracts based on their relevance through key search and filter terms. An initial set of papers was identified through Google Scholar with search terms “Impaired spermatogenesis male infertility” and “Impaired spermatogenesis male infertility in fish”; these papers helped identify search and filter terms used in Abstract Sifter. In AbstractSifter, searches were done to curate a subset of 40 papers using search terms “spermatogenesis AND fish” and “spermatogenesis AND zebrafish”. This initial set of papers was filtered with the terms “male, infertility, and reduced”, “male, infertility, and impaired”, and “male and infertil”. The first 2 filter set of words were used for the spermatogenesis and fish search which yielded 9 and 11 papers respectively. The last set of filter terms was used for the spermatogenesis and zebrafish search which yielded a respective 25 papers. Additional sources used towards the weight of evidence were provided through expert knowledge and found through sources in papers initially curated in the AbstractSifter search.

Evidence Supporting this KER

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

Table 1A - Concordance table [authors A-N] (full table as PDF)

Species	Experimental design	Evidence of Impaired Spermatogenesis (IS)	Evidence of Viable Offspring, Decreased (VOD)	IS observed?	VOD observed?	Citation	Notes
Zebrafish (Danio rerio)	Two generation exposure to 1nM BPA	Significant decrease in sperm density of F1 and F2 males compared to control Decreased sperm quality as measured by motility, velocity, ATP content and lipid peroxidation in F1 and F2 males	Delayed hatching at 48hpf and increased malformation and mortality were observed in the offspring from BPA- exposed F2; paternal-specific resulting from BPA-exposed males No significant difference in egg production and fertilization of F1 and F2 females	Yes	No: F1 and F2 Yes: offspring of F2	Chen et al., 2015	Female-biased sex ratio observed in both F1 and F2 adults
Tilapia (Oreochromis niloticus)	CRISPR/Cas9 mediated mutation of eEF1A1b; F1 sampled at 90, 120, 150 and 180 days after hatch	Significant downregulation of key genes involving spermatogenesis Spermatogenesis arrested; reduced number of spermatogonia and spermatocytes Altered morphology Delayed spermatogenesis Reduced motility	Reduced in vitro fertilization rate (5% vs 80% in WT) due to abnormal spermiogenesis	Yes	Yes	Chen et al., 2017	eEF1A1b - elongation factor
Zebrafish (Danio rerio)	Adult males exposed to two concentrations of bis-(2-ethylexhyl) phthalate (DEHP; 0.2 or 20 μg/L) for three weeks; 25 ng ethynylestradiol positive control	Areas of spermatogonial and spermatid cysts were larger in fish exposed to 20 μg/L DEHP compared with controls Testicular area of spermatocyte cysts was lower in males exposed to 0.2 μg/L DEHP Testicular area occupied by spermatocytes was reduced in fish exposed to DEHP compared to controls, with a concomitant increase in the area occupied by spermatogonia	Significant decrease in embryo production (up to 90%) observed in males treated with DEHP (0.2 and 20 μg/L) Hatch rate of embryos significantly lower in DEHP-exposed males	Yes	Yes	Corradetti et al., 2013	Reproductive performance evaluated with untreated females in clean water
Zebrafish (Danio rerio)	Targeted genetic disruption of tdrd12 through TALEN techniques	Reduced expression of germ cell markers vasa, dnd, piwil1 and amh in mutants Deformed and apoptotic spermatogonia at 35 dpf found in mutants Lack of spermatozoa at adult stage	Infertile under standard breeding despite being able to induce female egg laying (0% fertilization)	Yes	Yes	Dai et al., 2017	Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and Piwi-interacting RNA (piRNA) pathway
Zebrafish (Danio rerio)	Fish were exposed from 2 to 60 days post-hatch (dph) to nonylphenol (NP; 10, 30, or 100 μg/L nominal) or ethinylestradiol (EE2; 1, 10, or 100 ng/l nominal); reared until adulthood (120 dph) for breeding studies	Majority of fish exposed to 10 ng/l EE lacked differentiated gonadal tissue (undeveloped gonads) at 60 dph One fish at NP-30 μg/l and two fish at NP-100 μg/l were observed to have ovatestes at 60 dph	Zebrafish exposed to 10 ng/l of EE exhibited a significant reduction in the percent of viable eggs (clear vs opaque) Significant decrease in hatch and swim-up success observed with EE2 and 100 μg NP/L	Yes	Yes	Hill and Janz, 2003	Due to high mortality in the 100 ng/l EE group, insufficient fish were available for analyses
Roach (Rutilus rutilus)	Mature adult roach collected from both reference and river (effluent contaminated) sites during two consecutive spawning seasons; artificially induced to spawn in laboratory	Volume of milt released from spermiating male fish significantly lower in the intersex fish than in the reference males Most fish that did not spermiate had testes that were clearly immature	Fertilization rate significantly reduced when sperm from intersex males used to fertilize eggs collected from females Both proportion of fertilized embryos reaching eyed stage and hatching success decreased with increased feminization	Yes	Yes	Jobling et al., 2002	Embryo viability was determined after 24 h (fertilization success), at eyed stage and at swim-up stage (hatching success)
Japanese medaka (Oryzias latipes)	Adult medaka exposed for 21 days to 29.3, 55.7, 116, 227, and 463 ng/L 17β-estradiol (E2)	In males exposed to 463 ng/l, a few oocytes were observed in testis, and testicular tissue almost completely replaced by connective tissue Accompanied by presence of macroscopic atrophy and degenerated spermatozoa and spermatocytes suggest a lack of spermatogenesis	Total number of egg spawned and fertility significantly reduced at 463 ng/l E2 compared to the control	Yes	Yes	Kang et al., 2002
Zebrafish (Danio rerio)	Founder fish with originally mlh1 mutation was crossed out twice to WT fish of the TL line from which the founder was generated	Significant decrease in weight of spermatids and spermatozoa); some spermatozoa were visible in testes of all mutant fish Increased number and proportion of spermatogenic stages prior to spermatids compared to WT Increase in apoptotic cells	Reduced fertilization rates under standard breeding conditions (0.4%) Eggs fertilized from mutant sperm were malformed and and aneuploid	Yes	Yes	Leal et al., 2008	Mlh1 is a member of DNA mismatch repair machinery and essential for stabilization of crossovers during first meiotic division
Zebrafish (Danio rerio)	3-month-old male fish exposed to 10 ug/L of DEHP for 3 months	No effect	No effect	No	No	Ma et al., 2018	Semi-static exposure; half water renewed daily and whole water renewed weekly; exposed males mated with WT females
	3-month-old male fish exposed to 30 ug/L of DEHP for 3 months	No effect	Concentration-dependent decrease in fertilization rate	No	No
	3-month-old male fish exposed to 100 ug/L of DEHP for 3 months	Percent of spermatocytes increased significantly by 27.4% Significant decrease of 32.2% in spermatids	Significant decrease in fertilization rate by 22% compared to the control	Yes	Yes
Zebrafish (Danio rerio)	Multi-generational study to 0.5, 5 and 50 ng/L ethynylestradiol (EE2) or 5 ng/L 17β-estradiol (E2)	None of the F₁ males exposed to 5 ng/L EE2 had normal testes; 43% had gonads not fully differentiated	Time-related decrease in egg production and egg viability 14 hpf in F₀ generation at 50 ng/L EE2 and no survival of F₁ 100 hpf; no eggs produced after 10 d exposure Exposure to 5 ng/L EE2 in the F₁ caused a 56% reduction in fecundity and no survival past 14 hpf Proportion of nonviable eggs significantly higher for all treatments compared to control	Yes	Yes	Nash et al., 2004

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

Spermatogenesis is one of the most conserved biological processes from Drosophila to humans (Wu et al., 2016). The process itself is well understood and gametes produced from spermatogenesis are required for sexual reproduction.

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

Dose concordance

When exposed to 50 mg DEHP kg-1 via intraperitoneal injection for 10 days, zebrafish experienced a reduction in the proportion of spermatozoa present compared to the control group. However, at this exposure concentration there was no effect on evidence for decrease in viable offspring. Whereas when exposed to 5000 mg of DEHP kg-1, there was a significantly lower proportion of spermatozoa and a significant decrease in fertilization success (Uren-Webster et al., 2010).
When exposed to DEHP for 3 months, zebrafish had a significant decrease in spermatids and increase in spermatocytes at the highest exposure concentration (100 ug/L) and no effect at the lowest exposure concentration (10 ug/L) (Ma et al. 2018)

Table 1B - Concordance table [authors O-Z] (full table as PDF)

Species	Experimental design	Evidence of Impaired Spermatogenesis (IS)	Evidence of Viable Offspring, Decreased (VOD)	IS observed?	VOD observed?	Citation	Notes
Zebrafish (Danio rerio)	Targeted genetic disruption of fdx1b using a TALEN approach	Reduced sperm count compared to control (p=0.0097%) Promale sox9a downregulated Spermatogenic genes igf3 and insl3 downregulated	Infertile under standard breeding despite being able to cause spawning of eggs (0% fertilization)	Yes	Yes	Oakes et al., 2019	fdx1b is an electron- providing cofactor for steroidogenic cytochrome P450
Zebrafish (Danio rerio)	ENU mutagenesis screen to find mutations that lead to defects in gonadogenesis 3 mutants focused on (its, isa, imo)	Post meiotic germ cells absent at 3 months age (found aberrant germ cells instead) Only spermatogonia and primary spermatocytes were present; no spermatids or sperm observed	Decreased fertilization rates in cells from mutant testes (<2% vs 41.9-65.8 in WT) Only 1 mutant embryo survived at 1 dpf compared to nearly 100% in WT	Yes	Yes	Saito et al., 2011	ENU= N‐ethyl‐N‐nitrosourea
Zebrafish (Danio rerio)	hsf5 mutants obtained by CRISPR/Cas9 technology targeting exon2	Loss of spermatozoa along with increase in primary spermatocytes compared to WT Decrease in sperm count and sperm motility Altered morphology (microtubule arrangement, flagellar axoneme, sperm heads)	No viable offspring when mutants were crossed with any types of females Lethality of embryos via in vitro fertilization with WT females (before 1 dpf)	Yes	Yes	Saju et al., 2018	Heat shock protein 5
Medaka (Oryzias latipes)	Mature fish exposed to 32.6, 63.9, 116, 261, and 488 ng ethinylestradiol (EE2)/L for 21 d under ﬂow-through conditions	Testicular tissue composed of abnormally developed connective tissue, with only a few sper-matozoa and spermatocytes compared to control	Significant decrease in fecundity observed at 448 ng/L	Yes	Yes	Seki et al., 2002
Zebrafish (Danio rerio)	ar mutant line generated using TALENs	Upregulation of amh and gsdf Downregulation igf3 Reduced number of sperm Reduction in number of germ cells observed in AR mutant fish Increased proportion of pre-spermatids sperm cells Small amount of mature spermatozoon still present in mutants	Reduced in vitro fertilization rate ≤ 20% with WT female	Yes	Yes	Tang et al., 2018	Androgen receptor
Mice	mPCI deficient mice	Morphologically abnormal sperm (lacked tails and were degenerated) Reduced motility (12.5%) compared to control (51.5%) Apoptotic spermatocytes likely due to destruction of Sertoli cells	Reduced in vivo fertilization rate (0.5%) vs control (94%) with WT females	Yes	Yes	Uhrin et al., 2000	PCI - present in seminal plasma; inhibitor of activated protein C and a variety of proteases
Zebrafish (Danio rerio)	Adult males exposed to 0.5 mg DEHP kg-1 (body weight) for 10 days via intraperitoneal injection	No effect	No effect	No	No	Uren-Webster et al., 2010	DEHP is phthalate which is a plasticizer in many mass-produced products
	Adult males exposed to 50 mg DEHP kg-1 for 10 days via intraperitoneal injection	Significantly lower proportion of spermatozoa and a significantly greater proportion of spermatocytes	No effect	Yes	No
	Adult males exposed to 5000 mg DEHP kg-1 for 10 days via intraperitoneal injection	Significantly lower proportion of spermatozoa and a significantly greater proportion of spermatocytes	Significant decrease in fertilization success of males, especially during the second 5-day period of exposure	Yes	Yes
Mice (C57BL/6)	BRD7-deficient mice	Irregular head shape Deformed acrosome Post-meiotic development of elongating spermatids disrupted Abnormal morphology and degeneration of spermatids Increased proportion of abnormal spermatids Downregulation of various spermatogenic markers	WT female mice coupled with homozygous mutant males did not produce any pups	Yes	Yes	Wang et al., 2016	BRD7 is a bromodomain gene that inhibits cell growth and cell cycle progression and is a co-factor for p53 BRD7 has high expression in mice testes
Zebrafish (Danio rerio)	mettl3 mutant fish generated using TALENs	Significantly increased proportions of spermatogonia (24.4% vs 7.5% in WT) and spermatocytes (56.1% vs 26.7% in WT) Significantly decreased proportion of spermatozoa (10.4% vs 50.1% in WT) Very little or no mature sperm Sperm motility significantly reduced (average path velocity, curvilinear velocity, and straight-line velocity)	Decreased fertilization rate (48.8.% vs 91.4% in WT) 8.1% of mutant male x WT female spawned successfully vs 94.4% in WT	Yes	Yes	Xia et al., 2018	MEttl3 - multicomponent methyltransferase complex
Zebrafish (Danio rerio)	CRISPR/Cas9 gene targeting of E2f5	Reduced number of spermatozoa compared to WT Increased % of spermatocytes at leptotene and zygotene stages compared to WT Suggests arrest of spermatogenesis at zygotene stage; later stages rarely observed Increased germ cell apoptosis	Decreased fertilization rates (3% vs 94% in WT) under standard breeding conditions	Yes	Yes	Xie et al., 2020	E2f5 is a transcriptional repressor during cell-cycle progression
Marine medaka (Oryzias melastigma)	0.1 mg/L of DEHP for 6 months from larval stage	Contained mostly spermatocytes (Sp) and spermatids (Sd) with few spermatozoa especially in this treatment	Significant decrease in fecundity compared to control (21.78 vs 29.89 eggs/f/d) Significant decrease in fertilization success (84.12 vs 94.21%)	Yes	Yes	Ye et al., 2014	DEHP - phthalate MEHP - active metabolite of DEHP; fertilization success defined as proportion of fertilized eggs
	0.5 mg/L of DEHP for 6 months from larval stage	Contained mostly Sp and Sd with few spermatozoa	Significant decrease in fecundity compared to control (20.44 vs 29.89 eggs/f/d) Significant decrease in fertilization success (81.61 vs 94.21%)	Yes	Yes
	0.1 mg/L of MEHP for 6 months from larval stage	Contained mostly Sp and Sd with few spermatozoa	Significant decrease in fertilization success vs control (87.46% vs 94.21%)	Yes	Yes
	0.5 mg/L of MEHP for 6 months from larval stage	Contained mostly Sp and Sd with few spermatozoa	Significant decrease in fertilization success vs control (82.16% vs 94.21%)	Yes	Yes

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

When exposed to 10 and 100 ng/L of EE2 for 62 days leading to spawning, rainbow trout exhibited an increase in sperm density, concentration, and spermatocrit and decrease in GSI but overall there were no significant changes to spermatogenesis. Despite this, there was a decrease in viability of embryos (Schultz et al., 2003).
Two-generation zebrafish study with 1 nM bisphenol A (BPA) showed a significant decrease in sperm density along with decreased sperm quality, however, no significant different in egg fertilization (Chen et al., 2015).
There are multiple other factors involved in producing viable offspring, including but not limited to oocyte maturation and ovulation, development including successful organogenesis, and adequate nutrition.

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

Response-response Relationship

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

Empirical response-response data is very limited; thus, the response-response relationship has not yet been evaluated.

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

The duration of spermatogenesis in humans is reported to be 74 days (Griswold, M.D, 2016). Consequently, effects on spermatogenesis may not manifest as observable impacts on fertility until perhaps 74 days after impacts on spermatogenesis began. This may vary depending on the stage(s) of spermatogenesis that are impacted by the stressor.
The duration of the meiotic and spermiogenic phases in zebrafish is reported to be 6 days which means there could be a delay of at least 6 days before signs of impaired fertility and downstream effects may be detected (Leal et al., 2009).

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

Feedforward/feedback loops haven’t been evaluated yet. However, given that that oocyte fertilization and production of viable offspring are external to the male it seems unlikely there would feedback that impacts spermatogenesis.

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

Taxonomic Applicability: Spermatogenesis is one of the most conserved biological processes from Drosophila to humans (Wu et al., 2016). As a result, animals who utilize sexual reproduction as their way to produce offspring are heavily reliant on spermatogenesis being effective and normal. There are studies on reproduction and spermatogenesis across a multitude of taxa.

Sex Applicability: Spermatogenesis is a male-specific process (Schulz et al., 2010, Tang et al., 2018, Wu et al., 2015 ). Thus, the present relationship is only relevant for males.

Life Stage Applicability: Spermatogenesis and reproduction are only relevant for sexually-mature adults.

References

List of the literature that was cited for this KER description. More help

Boulanger, G., Cibois, M., Viet, J., Fostier, A., Deschamps, S., Pastezeur, S., Massart, C., Gschloessl, B., Gautier-Courteille, C., & Paillard, L. (2015). Hypogonadism Associated with Cyp19a1 (Aromatase) Posttranscriptional Upregulation in Celf1 Knockout Mice. Molecular and cellular biology, 35(18), 3244–3253. https://doi.org/10.1128/MCB.00074-15

Chen, J., Jiang, D., Tan, D., Fan, Z., Wei, Y., Li, M., & Wang, D. (2017). Heterozygous mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in male tilapia, Oreochromis niloticus. Scientific reports, 7, 43733. https://doi.org/10.1038/srep43733

Chen, J., Xiao, Y., Gai, Z., Li, R., Zhu, Z., Bai, C., Tanguay, R. L., Xu, X., Huang, C., & Dong, Q. (2015). Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects. Aquatic toxicology (Amsterdam, Netherlands), 169, 204–214. https://doi.org/10.1016/j.aquatox.2015.10.020

Chen, J., Xiao, Y., Gai, Z., Li, R., Zhu, Z., Bai, C., Tanguay, R. L., Xu, X., Huang, C., & Dong, Q. (2015). Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects. Aquatic toxicology (Amsterdam, Netherlands), 169, 204–214. https://doi.org/10.1016/j.aquatox.2015.10.020

Corradetti, B., Stronati, A., Tosti, L., Manicardi, G., Carnevali, O., and Bizzaro, D. (2013). Bis-(2-ethylexhyl) phthalate impairs spermatogenesis in zebrafish (Danio rerio). Reprod Biol. 13(3):195-202.

Dai, X., Shu, Y., Lou, Q., Tian, Q., Zhai, G., Song, J., Lu, S., Yu, H., He, J., & Yin, Z. (2017). Tdrd12 Is Essential for Germ Cell Development and Maintenance in Zebrafish. International journal of molecular sciences, 18(6), 1127. https://doi.org/10.3390/ijms18061127

Griswold M. D. (2016). Spermatogenesis: The Commitment to Meiosis. Physiological reviews, 96(1), 1–17. https://doi.org/10.1152/physrev.00013.2015

Hill, R.L Jr and Janz, D.M. (2003). Developmental estrogenic exposure in zebrafish (Danio rerio): I. Effects on sex ratio breeding success. Aquat Toxicol. 63(4):417-429.

Jobling, S., Coey, S., Whitmore, J.G., Kime, D.E., Van Look, K.J.W., McAllister, B.G., Beresford, N., Henshaw, A.C., Brighty, G., Tyler, C.R., and Sumpter, J.P. (2002). Wild intersex roach (Rutilus rutilus) have reduced fertility. Biol Reprod. 67(2):515–524.

Kang, I.J., Yokota, H., Oshima, Y., Tsuruda, Y., Yamaguchi, T., Maeda, M., Imada, N., Tadokoro, H., and Honjo, T. (2002). Effect of 17β-estradiol on the reproduction of Japanese medaka (Oryzias latipes). Chemosphere 47(1): 71-80,

Leal, M. C., Cardoso, E. R., Nóbrega, R. H., Batlouni, S. R., Bogerd, J., França, L. R., & Schulz, R. W. (2009). Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations. Biology of reproduction, 81(1), 177–187. https://doi.org/10.1095/biolreprod.109.076299

Leal, M. C., Feitsma, H., Cuppen, E., França, L. R., & Schulz, R. W. (2008). Completion of meiosis in male zebrafish (Danio rerio) despite lack of DNA mismatch repair gene mlh1. Cell and tissue research, 332(1), 133–139. https://doi.org/10.1007/s00441-007-0550-z

Ma, Yan-Bo, Jia, Pan-Pan, Junaid, Muhammad, Yang, Li, Lu, Chun-Jiao, & Pei, De-Sheng. (2018). Reproductive effects linked to DNA methylation in male zebrafish chronically exposed to environmentally relevant concentrations of di-(2-ethylhexyl) phthalate. Environmental Pollution (1987), 237, 1050-1061.

Nash, J.P, Kime, D.E., Van der Ven, Leo T.M., Wester, P.W., Brion, F., Maack, G., Stahlschmidt-Allner, P., and Tyler, C.R., (2004). Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish. Environ Health Perspect 112(17):1725-1733.

Oakes, J. A., Li, N., Wistow, B., Griffin, A., Barnard, L., Storbeck, K. H., Cunliffe, V. T., & Krone, N. P. (2019). Ferredoxin 1b Deficiency Leads to Testis Disorganization, Impaired Spermatogenesis, and Feminization in Zebrafish. Endocrinology, 160(10), 2401–2416. https://doi.org/10.1210/en.2019-00068

Saito, K., Siegfried, K. R., Nüsslein-Volhard, C., & Sakai, N. (2011). Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants. Developmental dynamics : an official publication of the American Association of Anatomists, 240(7), 1779–1792. https://doi.org/10.1002/dvdy.22661

Saju, J. M., Hossain, M. S., Liew, W. C., Pradhan, A., Thevasagayam, N. M., Tan, L., Anand, A., Olsson, P. E., & Orbán, L. (2018). Heat Shock Factor 5 Is Essential for Spermatogenesis in Zebrafish. Cell reports, 25(12), 3252–3261.e4. https://doi.org/10.1016/j.celrep.2018.11.090

Schultz, I. R., Skillman, A., Nicolas, J. M., Cyr, D. G., & Nagler, J. J. (2003). Short-term exposure to 17 alpha-ethynylestradiol decreases the fertility of sexually maturing male rainbow trout (Oncorhynchus mykiss). Environmental toxicology and chemistry, 22(6), 1272–1280.

Schulz, R. W., de França, L. R., Lareyre, J. J., Le Gac, F., Chiarini-Garcia, H., Nobrega, R. H., & Miura, T. (2010). Spermatogenesis in fish. General and comparative endocrinology, 165(3), 390–411. https://doi.org/10.1016/j.ygcen.2009.02.013

Seki, M., Yokota, H., Matsubara, H., Tsuruda, Y., Maeda, M., Tadokoro, H. and Kobayashi, K. (2002). Effect of ethinylestradiol on the reproduction and induction of vitellogenin and testis-ova in medaka (Oryzias latipes). Environ. Toxicol. Chem. 21(8):1692-1698.

Tang, H., Chen, Y., Wang, L., Yin, Y., Li, G., Guo, Y., Liu, Y., Lin, H., Cheng, C., & Liu, X. (2018). Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor. Biology of reproduction, 98(2), 227–238. https://doi.org/10.1093/biolre/iox165

Tang, H., Chen, Y., Wang, L., Yin, Y., Li, G., Guo, Y., Liu, Y., Lin, H., Cheng, C., & Liu, X. (2018). Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor. Biology of reproduction, 98(2), 227–238. https://doi.org/10.1093/biolre/iox165

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