In vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor β2 (ERβ2) during embryogenesis of medaka, and here we investigated the functional importance of ERβ2 in female gonad development and maintenance using a transgenerational ERβ2-knockdown (ERβ2-KD) line and ERβ2-null mutants. We found that ERβ2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERβ2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERβ2 increased intracellular Ca 2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.

This study was conducted to determine the specific roles of ERβ2 in early germ cell development and its consequences on sexual identity. In brief, we have developed transgenic ERβ2 knockdown (ERβ2-KD) and knockout (ERβ2-KO) medaka lines and assessed the ERβ2-mediated effects on sex differentiation/maintenance and sex reversal. Furthermore, we have ascertained the direct involvement of ERβ2 in SDF1/CXCR4-mediated chemotactic PGC migration, and calcium homeostasis-related germ cell survival and death, which, in some cases, eventually affects the seeding population of germ cells in the gonadal anlagen and disrupts normal sexual development.

Estrogen is known to affect the transcriptional profiles of several major sex-related genes (e.g., DMRT1, GSDF, Aromatase, RSPO1), germ cell proliferation characteristics, and sexual identity in medaka (). Earlier we found that, during early sex differentiation, ERβ2 predominantly expresses in the germ cells of embryonic female gonad, while estrogen treatment specifically induces ERβ2 expression in XY male fish, and thereby speculated the ERβ2 association in germ cell maintenance ().

Differential expression of three estrogen receptor subtype mRNAs in gonads and liver from embryos to adults of the medaka, Oryzias latipes.

Differential expression of three estrogen receptor subtype mRNAs in gonads and liver from embryos to adults of the medaka, Oryzias latipes.

Germ and somatic cells are two essential variables in gonadal differentiation and sexual identity in mouse, chicken, and fish (). In vertebrates, primordial germ cells (PGCs) arise at a distant site, divide, migrate through the gut mesentery and bloodstream, and arrive at and colonize at the gonadal primordium during the bipotential stage (). During early embryonic development, PGCs migrate to the newly formed gonadal anlagen with the help of germ-soma chemotaxis. Notably, cellular chemotaxis is affected by external steroids (), ERs (), and calcium homeostasis (). More or less at the same time, the somatic cells undergo a series of genetic sex- or environmental-dependent modifications and, along with germ cell, decide the gonadal sexuality (). Furthermore, migrational disruption and depletion of PGC number causes suboptimal PGC settlement in the gonadal primordium and, in turn, blocks female fate and sometimes triggers male development (). In medaka, an important model species for gonadal sex determination and differentiation studies (), the early gonadal settlement of PGCs is regulated by SDF1/CXCR4-mediated chemotactic migration (). Later, during medaka sex differentiation, both germ and somatic cells co-operatively regulate the gonadal development in an estrogen-dependent manner () and help the PGCs to undergo a proliferative mitosis and meiosis in females, while restricting proliferation in males.

Sex steroids and their receptors harmoniously maintain the reproductive physiology, and any disruption in sexual development leads to a huge impact on individual or species physiology (). The action of the female hormone, estrogen, is mostly mediated by estrogen receptors (ERs), whose action on sterility, infertility, or subfertility and sexual behavior have been thoroughly investigated using selective ER modulators, ERα- and ERβ-knockout (KO) mice (), and ERα-, ERβ1-, and ERβ2-KO zebrafish (). Although ERβs have recently gained popularity in vertebrate folliculogenesis and infertility studies (), their involvement in early gonadal sex differentiation and maintenance requires further investigation.

Despite significant effects on PGC maintenance and initiation of primary gonadal sex reversal, when analyzed at adult stages approximately 25%–47% of fish possessed male-like secondary sexual characteristics (fan-like anal fin and forked dorsal fin; Figures 5 E–5G ), 14%–30% of ERβ2-KD-XX and ERβ2-XX fish showed complete testis ( Figures 5 C and 5H–5I; Table 1 ), and another 10%–17% had testis-ova ( Figure 5 D and Table 1 ). ISH and qPCR analysis of XX testis showed an inverse expression pattern between male- and female-responsive genes, except for RSPO1 and β-catenin ( Figure S6 ), a phenomenon similar to early-stage embryos. However, only 25% (3/12) of adult ERβ2-KD Ffish had integration of KD cassette and were able to produce viable progeny. The Fand subsequent progenies generated from the three Fphenotypic males were used for all our experiments. The XX fertile male production increased in later generations ( Table S2 ) when the ERβ2-KD-XX males were mated with XX normal females, further cementing the significance of ERβ2 in gonadal sex development and maintenance.

Randomly selected fish (from the aforementioned pool of screened fish) were examined for secondary sexual characteristics and tested with mature normal fish of opposite sex. A, B, and C, respectively denote male-like, female-like, and no sexual behavior.

c Randomly selected fish (from the aforementioned pool of screened fish) were examined for secondary sexual characteristics and tested with mature normal fish of opposite sex. A, B, and C, respectively denote male-like, female-like, and no sexual behavior.

Four different populations of fish, generated from four different parents, were mixed and sampled randomly for histological analysis to ascertain the gonadal sexual status and sex reversal. All the examined fish were screened with DMY-specific genomic PCR and, whenever necessary, followed by ERβ2-mutation-specific PCR and sequencing.

a Four different populations of fish, generated from four different parents, were mixed and sampled randomly for histological analysis to ascertain the gonadal sexual status and sex reversal. All the examined fish were screened with DMY-specific genomic PCR and, whenever necessary, followed by ERβ2-mutation-specific PCR and sequencing.

(A–D) Histological analysis of control-XX (A), control-XY (B), and ERβ2-KD-XX fish showed evidence that knockdown of ERβ2 results in partial (D) to complete (C) testis formation in XX adults. Inset: representative low-magnification photomicrograph of testicular (blue boundary) and ovarian (red boundary) prevalent gonadal areas.

ERs, SDF1a, and CXCR4b are largely associated with calcium signaling, and the latter two are known to cause cell death through the Ca-signaling pathway (). Moreover, our microarray analysis revealed that several Ca-signaling-related genes, e.g., plasma membrane CaATPase (PMCA) 1b, and plasma membrane Na/Caexchanger (NCX) 1, and Calmodulin (CaM) (), were significantly altered in the ERβ2-KD-XX fish. To prove our hypothesis that calcium is indispensable for ERβ2-associated cell death, we incubated the OLVAS-eGFP-ERβ2-KD embryo (from 1-cell stage) in CaCl(2 mM) solution and checked the PGC migration at stage 33. Unexpectedly, the migration remained identical to that of their ERβ2-KD control counterpart ( Figure 4 A ). Although NCX1 and PMCA1b transcription were significantly reduced in ERβ2-KD-XX, CaM showed a pattern completely opposite to that of both control-XX and ERβ2-KD-SDF1/CXCR4b-overexpressed (OV)-XX (at stage 33; Figure 4 C). These opposite transcriptional alteration patterns are probably related to their differential role in calcium transport, i.e., Caextrusion (NCX1 and PMCA1b;) and influx inhibition (CaM;). This, along with calcium homeostasis-related gene transcriptions in ERβ2XX ( Figure 4 C), implies that ERβ2 reduction overloads the germ cells with Caby suppressing the transcriptions of Caoutflux-related genes and triggers a secondary negative regulatory mechanism, via CaM (), to eventually control the exponential overload. In a subsequent experiment, we reduced the intracellular Caof ERβ2-KD fish by BAPTA_AM, and recorded a substantial decrease in CaM level, thus validating the germ cell Caoverloading theory. On the contrary, extracellular Cachelation by EGTA did not bring about any significant changes. Further in vitro analysis showed that E/ERE-responsive ERβ2 transcription was accelerated by Ca, unaffected by intracellular Cachelation, and reduced by extracellular Cachelation ( Figure 4 B), thereby highlighting the importance of Cainflux-outflux ratio in ERβ2 transcription management ( Figure S6 ). To solve the puzzle of whether addition of excessive Cabalances the intracellular and extracellular calcium ion concentration, and further reduces the cell death in mismigrated cells, we sorted three different groups (control-, ERβ2-KD-XX-, and CaCl-treated ERβ2-KD-XX) of stage-33 OLVAS-eGFP embryonic single-cell suspension, stained with Alexa 488-GFP and Alexa 546-LC3 conjugates, and collected three populations of cells, namely, L (LC3 positive), D (LC3 and eGFP positive), and G (eGFP positive). Morphologically, D- and G-cell populations were similar to PGCs, while L had more likeness with the somatic cells. Moreover, ERβ2-KD-S33-XX fish showed lowest G-cell portion and highest number of D-type cells ( Figures 4 D–4F). The significantly diminished D population after calcium addition ( Figure 4 F) further confirmed that cell death was indeed reduced by calcium. qPCR analysis of each cell population depicted a substantially elevated CaM level in D population than G-cell fraction, while PMCA1b transcription was further suppressed in LC3-positive germ cells ( Figure 4 G). Our data suggest that cellular-level calcium ion threshold is critical for cell survivability (). Longer incubation with BAPTA_AM, and not with EGTA, increased the ERβ2-KD-XX gonad size and germ cell numbers, further emphasizing that intracellular Caoverload is crucial for increased PGC degeneration and further germ cell loss ( Figures 4 H–4J). As mentioned earlier, ERβ2-KD donor cells, upon being transplanted into control-XX embryos, showed that the majority of donor cells mismigrated and became apoptotic, while the host germ cells did not show such a phenomenon ( Figure S5 ). Most likely, in the ERβ2-reduced situation, when Caimbalance was irreparable, the defective cells underwent programmed cell death. Thus, these findings validate the importance of ERβ2 in germ cell migration and maintenance.

PGC migration was analyzed using confocal microscopy. In graphs, data are plotted as means ± SEM; different letters denote significant differences at p < 0.05. BAPTA_AM and EGTA were used as intracellular and extracellular Ca 2+ chelator, respectively. Control and OLVAS-eGFP-ERβ2-KD fish lines were used for the experiments. Scale bars, 20 μm.

(H–J) ERβ2-KD-XX or control-XX embryos were incubated in BAPTA_AM (10 pM) and EGTA (5 pM) solution until hatching, and gonadal histology was performed at 12 dah (n = 9) to visualize the germ cell proliferation status.

(G) The mRNA transcription profile of PMCA1b and CaM were analyzed using qPCR of populations D and G cells to discern the difference between LC3-positive and -negative PGCs.

(C) qPCR analysis (n = 10 individuals/group) of several calcium transport-related genes showed significant alteration in both ERβ2-KD (upper panel) and ERβ2 −/− (lower panel) fish than their control, SDF1a/CXCR4b-overexpressed, or intra-/extracellular Ca 2+ -reduced counterparts.

(B) In vitro ERE-dependent ERβ2 transcriptional analysis (using HEK-293 cells, and various combination of 2 mM CaCl 2 , 5 pM EGTA, 10 pM BAPTA-AM, 1 ng/mL 17β-estradiol, and 100 ng of ERβ2 overexpression plasmid) demonstrated that cellular calcium concentration significantly alters the E 2 -responsive ERβ2 transcription.

To interrogate the ERβ2 involvement in PGC migration, we transplanted the NANOS-dsRED-ERβ2-KD-positive PGCs to the OLVAS-eGFP host and found that only dsRed-positive cells were prone to mismigration and degradation ( Figure S5 ). Germ cell mismigration is associated with the imbalance of SDF1 and associated CXCR4 machinery, and both overexpression and knockdown of SDF1 or knockdown of CXCR4 triggers PGC mismigration in fish and frog (). Similarly, in the present study, co-injection of SDF1a-Cyan and CXCR4b-mCherry mRNAs in 1-cell-stage control and OLVAS-eGFP-ERβ2-KD cross hybrids helped to resume proper migration ( Figures 3 H and 3I) and further increased germ cell population in the gonad, which could not be achieved with singular injection of either mRNAs ( Figures 3 I and S5 Table S1 ). These results, along with the ChIP data, clearly indicate that ERβ2 is essential for SDF1a- and CXCR4b-responsive chemotactic migration () of PGCs and their settlement in gonadal anlagen. However, the germ cell population in the co-injected fish was half that of the normal gonad at 10 dah and also devoid of proper meiotic initiation, thus implying that PGC migration is independent of PGC maintenance and meiosis. The increased WT1b activity in ERβ2-KD embryonic gonads at later stages might be a possible factor in the germ cell survivability ().

Control-XX fish possessed clustered germ cells, while ERβ2-KD-, ERβ2-, and AI-treated XX embryos demonstrated disorganized settlement of germ cells in gonadal primordium ( Figures 3 D–3F and S5 ). These observations emphasize that the ERβ2-deprived situation created by knockdown mimics the estrogen-reduced situation and becomes critical for maintaining embryonic estrogen action and associated sexual development. In this regard, zygotic estrogen synthesis and actions were found to be critical for embryonic gonadal sexuality (). Contrastingly, CYP19a1-null mutant medaka did not produce primary sex reversal (sex reversal from early stages), or any germ cell reduction. This highlights that, probably, brain type CYP19b isoform, which also expresses in the gonad during early development, is critical for early estrogen synthesis in medaka. Moreover, our finding suggests that CYP19b actions are impaired in ERβ2-KD embryos ( Figure S4 ), further illuminating the importance of estrogen in early gonadal development. Interestingly, the total OLVAS expressions remained unaffected at stages 22 and 33 of ERβ2-KD-XX embryos and significant suppression was noticed at 4 daf, at which point the mislocalized cells became autophagic ( Figure 3 G). This autophagy induction might be associated with the disturbance in E/ER associated ion homeostasis ().

Chromatin immunoprecipitation (ChIP) analysis further confirmed that ERβ2 directly influences the transcription of SDF1, CXCR4, and WT1b ( Figure 3 B), and thus illuminated the relevance of E/ERβ2 in germ cell migration. Our qPCR and WISH ( Figures 3 C and S5 ) results showed a significant decrease in SDF1a and CXCR4b, and elevation in WT1b expression in the ERβ2-KD group compared with their respective controls. The transcriptional difference among control and ERβ2-KD-XX fish became wider at stage 33 than stage 22 ( Figure 3 C). Similarly, morpholino knockdown of CXCR4b induces PGC mismigration and slowly causes germ cell reduction (), suggesting that a continuous ERβ2-dependent regulation is in place to strictly regulate the germ cell settlement and maintenance. Given the fact that SDF1 and CXCR4 expresses in somatic and germ cells, respectively, and play significant roles in PGC maintenance in fish (), it is probably likely that ERβ2 simultaneously manipulates both somatic and germ cells and controls PGC maintenance.

Our previous data, along with results from tet-ERβ2-KD and ERβ2 expression analysis ( Figures 2 and S3 ) experiments, suggest that the action of ERβ2 is predominant during 0–7 daf (). This window represents two different major gonadal development-related phenomena, i.e., proliferative mitosis and initiation of meiosis, which in turn determine the phenotypic sex of hatchlings. Total germ cell number in a developing gonad decides the sexual identity (). Reports also confirm that the abnormal cell migration is closely related to estrogen and ERs (). Our present data suggest that ERβ2 is essential for germ cell population maintenance, and we found severely mismigrated PGCs in the ERβ2-KD embryos (discussed later). To prove the importance of ERβ2 in PGC migration, firstly, we performed a comprehensive microarray analysis using stage-18 (before the onset of PGC migration) and stage-22 (just after the initiation of PGC migration) control and ERβ2-KD-XX embryos and identified several germ cell migration- and survival-related candidate genes (SDF1a, CXCR4b, and WT1b). Later, we checked the ERβ2-responsive and E-dependent promoter activity of these candidates by measuring the luciferase activity, using the HEK-293 cell line. We observed an increased SDF1a and CXCR4b activity, but reduced WT1b expression, upon Eaddition ( Figure 3 A ), highlighting the importance of these genes in estrogenic medaka germ cell maintenance ().

qPCR analyses were performed using pooled samples, n = 10 individuals/pool. In graphs, data are plotted as means ± SEM; different letters and asterisks () denote significant differences at p < 0.05. The probable gonadal primordia and mismigrating cells are, respectively, marked with white dotted lines and white arrowheads. See also Figure S5 and Table S1

(H and I) Co-injection of SDF1a and CXCR4b mRNAs (H), but not a singular injection of either SDF1a or CXCR4b (I), rescued the PGC mismigration. The percentile ratio between total and mismigrating germ cells were subgrouped into normal (0%–5%), mildly abnormal (>5%–15%), moderately abnormal (>15%–35%), and severely abnormal (>35%), and plotted as percent occurrence at group level, to ascertain the rescue effect.

(D–F) The estrogen/ERβ2-responsive mismigration of PGCs to the gonadal primordium was also confirmed by live confocal z-stage imaging of OLVAS-eGFP-control-XX (D), OLVAS-eGFP-XX-AI (E), and OLVAS-eGFP-ERβ2-KD-XX (F) embryos (generated by crossing ERβ2-KD-XX F 3 males with OLVAS-GFP-XX females at stage 33).

(B and C) ChIP assay demonstrated direct relation between SDF1a, CXCR4b, WT1b, and ERβ2 (C) (n = 3). Comparative qPCR analysis of stage 22 (migrating PGC) and stage 33 (PGC settled in the gonadal primordium) of ERβ2-KD-XX depicted significant downregulation of SDF1a and CXCR4b at both stages while WT1b showed upregulation at stage 33 (B) (n = 10).

Differential expression of three estrogen receptor subtype mRNAs in gonads and liver from embryos to adults of the medaka, Oryzias latipes.

Earlier, we reported that ERβ2 expression peaks at 7 days after fertilization (daf) in the germ cells of XX medaka (). This implies that ERβ2 is likely to play a critical role in germ cell maintenance and gonadal development of medaka. To test the hypothesis, we conducted tetracycline (tet)-responsive conditional knockdown of ERβ2, induced at different time points during embryogenesis (knockdown efficiency: 65%–92%, depending on stages), and observed a clear decrease in both mitotic and meiotic proliferation ( Figures 2 A and 2B) as well as OLVAS expression in 0–7 daf, which gradually reduced in later stage groups of tet-ERβ2-KD-XX embryos and became non-significant from control female by 10 daf ( Figures 2 C–2G). Meanwhile, ethanol-treated tet-ERβ2-KD-XX embryos had similar germ cell numbers as the control ethanol-/doxycycline-treated embryos. These data were further confirmed by both qPCR and whole-mount ISH (WISH) of ERβ2 at 12 dah and 0 dah, respectively ( Figures 2 H–2K). Notably, ERβ2 and aromatase co-localizes in female medaka brain to regulate estrogen synthesis (), thereby validating the fact that ERβ2 is critical for embryonic gonadal development. Although slightly different, it was observed that zebrafish ERα is essential for embryonic PGC mismigration, and the governance of PGC migration is transferred to ERβ2 in enhanced estrogen condition (). Additionally, recent investigation using zebrafish CRISPR knockout showed that ERβ2 null mutants have relatively slower pace of female gonadal development and male-biased sex ratio than their control counterparts (). All these data highlight that a delicate ER synergy, which might be species specific in some instances or related to intricate organismal-sexual development, is in place to control gonadal development.

(H–K) At 12 dah, ERβ2 expression was measured using qPCR (n = 6) and plotted against average total germ cell numbers of respective treatment groups. In (H), the various treatment groups are marked as 1 (5 dah), 2 (6 dah), 3 (7 dah), 4 (8 dah), 5 (10 dah), and 6 (12 dah). WISH analysis using ERβ2 at 0 dah further corroborated the sex reversal (I–K). WISH gonadal positions and representative ERβ2 signal are, respectively, marked with black dotted boundary and red arrows.

(C–G) Histologically, restricted germ cell proliferation was evident in 7-daf groups (C) but germ cell proliferation in 10-daf (D) and 12-daf (E) groups depicted a control-XX-like pattern (F). qPCR analysis (n = 6) of OLVAS gene (G) substantiated the histological observations and suggested XX male development. Representative oogonia (Og), oocyte (Oc), spermatogonia (Sg), and spermatocytes (Sc) are marked with black arrows.

(B) At 12 dah, the total germ cell numbers of XX fish were counted and average numbers (n = 18) were plotted against starting days of doxycycline induction to postulate the gonadal sex.

(A) ERβ2-KD tet-on plasmid-injected embryos were treated with doxycycline from either 5, 6, 7, 8, 10, or 12 daf. Tetracycline knockdown effects at representative time points were evaluated by qPCR analysis of ERβ2 gene at 20 dah and the data were plotted as percentile reduction against control-XX medaka.

Use of antagonists and morpholinos in loss-of-function analyses: estrogen receptor ESR2a mediates the effects of 17alpha-ethinylestradiol on primordial germ cell distribution in zebrafish.

Differential expression of three estrogen receptor subtype mRNAs in gonads and liver from embryos to adults of the medaka, Oryzias latipes.

Our recent data highlight that RSPO1, an estrogen-responsive gene, regulates the GSDF expression (). The unchanged RSPO1 expression in the ERβ2-KD XX embryos suggests some unknown intricate connections between RSPO1, ERβ2, and GSDF in medaka gonad. Although we observed a male-specific gene expression pattern in ERβ2-KD-XX and ERβ2-XX fish, similar alteration in DMRT1, GSDF, SPO11, and FOXL2 expressions were also noticed upon androgen/aromatase inhibitor (AI) treatment (). This indicates that ERβ2 action is associated with either direct blockage of estrogen action or indirect induction of androgen activity/production (). To confirm this, we treated the control and ERβ2-KD-XX fish with estrogen and ERβ agonist (WAY20070). Although estrogen failed, WAY20070 helped to regain both mitotic and meiotic germ cell proliferation of ERβ2-KD fish to some extent ( Figure S2 ). This insinuates that in a receptor-reduced situation, estrogen fails to form receptor-ligand complex and further fails to regulate the ERE-responsive transcription of downstream genes. The mild rescuing effect associated with WAY20070 might be related to simultaneous triggering of both ERβ1- and ERβ2-mediated pathways (as discussed above), and/or indirect activation of certain mitosis- and meiosis-related genes, which could further influence germ cell proliferation. Thus, to verify the specificity of ERβ2 knockdown, we injected synthetic (gene sequence was modified to avoid knockdown) ERβ2-eGFP mRNA into 1- to 2-cell stages of ERβ2-KD-XX For control embryos. eGFP mRNA was injected into ERβ2-KD-XX embryos, which served as control. Interestingly, we observed nearly 100% rescuing effect in ERβ2-overexpressed ERβ2-KD-XX embryos, while no significant influence was noticed in the gonadal development of control groups ( Figures 1 L and S2 ). The reoccurrence of gonadal femininity after ERβ2 overexpression was further confirmed by ISH ( Figure S2 ).

To determine the importance of ERβ2 in estrogen-dependent sex differentiation of medaka, we knocked down the ERβ2 expression ( Figure S2 ) in 1- to -2 cell stage embryo, using a pre-established transgenerational knockdown technology (). We simultaneously scouted the medaka tilling mutant library and generated ERβ2-KO line ( Figures S2 and S3 ). ERβ2 reduction resulted in an average decrease of ERβ2 transcript of 67% (66%–80% in females [ Figure 1 A ] and 41%–69% in males). Interestingly, the germ cell number and corresponding OLVAS (vasa homolog and medaka germ cell marker) expression ( Figures 1 B and S3 ) showed a remarkable direct correlation with ERβ2 mRNA expression (CR XX = 0.89, CR XY = 0.91, and CR ERβ2-KD-XX = 0.9; p < 0.01). Live confocal imaging and subsequent qPCR analysis of OLVAS-eGFP-ERβ2-KD and control OLVAS-eGFP embryos ( Figures 1 C–1E), and in situ hybridization (ISH) ( Figures 1 F–1K) demonstrated that ERβ2 reduction not only increased the GSDF abundance in gonadal primordium but also significantly suppressed the mitotic and meiotic germ cell count. Notably, at 10 dah, in control female gonad germ cells undergo rapid mitotic and meiotic proliferation ( Figure 1 C), while males possess mitotically quiescent sporadically distributed germ cells in the gonad ( Figure 1 D), resulting in differences among germ cell numbers of both sexes. The ERβ2-KD-XX and ERβ2XX fish gonads showed more similarity toward males than females ( Figure 1 E) and also harbored fewer germ cells in the gonad ( Figure 1 L). Several authors have suggested that GSDF negatively regulates the initiation of meiosis in medaka (), probably by influencing the germ-somatic cell interaction. In silico analysis depicted several half-ERE in the GSDF promoter sequence, making it a potential target for ERs, and in vitro analysis confirmed the ERβ2-responsive GSDF promoter activity ( Figure S4 ). qPCR analysis demonstrated a significant increase in DMRT1 and GSDF expression, and simultaneous reduction in several ovarian responsive genes, i.e., SPO11 and FOXL2 ( Figure 1 A) in ERβ2-KD-XX fish. Additionally, we observed sex-biased, but relatively ubiquitous, ERβ2 expression in germ and various somatic cells during PGC migration and gonadogenesis ( Figure S3 ), which suggests that the action of ERβ2 might be associated with GSDF during gonadal sex differentiation. However, interaction with other ovary-responsive GSDF-linked genes cannot be ruled out.

(F–L) In situ hybridization analysis using GSDF (F–H) and ERβ2 (I–K) confirmed the gonadal masculinity. (L) Furthermore, different embryonic treatments, i.e., 17β-estradiol (E 2 , 1 ng/L), ERβ agonist (WAY20070, 1 nM), ERβ antagonist (Cyclofenil, 10 nM), and ERβ2 overexpression, were performed using ERβ2-KD-XX and control-XX embryos to rescue the masculine effect of ERβ2-KD. Ethanol (EtOH)-treated samples were used as vehicle control. In graphs, data are plotted as means ± SEM; different letters denote significant differences at p < 0.01. In (L), letters in lower case (a–f) and upper case (A–D), respectively, indicate significant differences among mitotic and meiotic cell population (black, continuous error bars) and total cell population (red, continuous inverted error bars) at p < 0.01. Red arrows indicate candidate mitotic cells while black arrowheads denote the cells undergoing first meiosis. Black dotted lines mark the gonadal boundary. n = 10 fish used for each experiment per group.

(C–E) Proliferative mitosis and meiosis was evident in control-XX fish (C), while control-XY (D) and ERβ2-KD-XX (E) fish demonstrated male-type gonadal development, characterized by mitotic and meiotic blockage.

(B) Germ cell numbers showed a strong correlation with ERβ2 expression. Chronologically, gonadal germ cell population was determined by confocal imaging, ERβ2 concentration was measured by qPCR in the same OLVAS-eGFP-ERβ2-KD embryos, and later general linear modeling was used for statistical analysis (n = 10 individual samples/group). The individual gonads that housed the meiotic cells are marked with black arrows.

To ascertain the role of ERβs in gonadal development in vivo, we treated fertilized embryos with an ERβ-specific agonist (WAY20070, 1 nM), antagonist (cyclofenil, 10 nM), or ethanol (vehicle control) for a period of 18 days (until 10 days after hatching [dah]). Histological analysis depicted a significant reduction in gonad size and germ cell numbers in both agonist- and antagonist-treated fish ( Figure S1 ). Although meiotic cell numbers were slightly higher in agonist-treated fish, the mitotic proliferation reduced drastically in the ERβ agonist-treated than in the ERβ antagonist-treated XX fish. Previously, based on E-dependent ERβ1 and ERβ2 expression profiles in medaka embryos, we hypothesized that both these ERβ subtypes work, respectively, on “cessation of male germ cell proliferation” and “mitotic burst in female.” Thus, the present agonist and antagonist (both in vivo and in vitro; Figure S1 ) treatments further corroborate that ERβ1 and ERβ2 has an antagonistic role in medaka (). Even though histologically no significant phenotypic changes were observed, ERβ agonist selectively reduced the male-dominated genes, i.e., SOX9a2 and GSDF, in the XY fish, while the female-dominated genes (FOXL2 and CYP19a1) were found to decrease in XX females upon antagonist treatment ( Figure S1 ). Interestingly, the correlation (p < 0.05) between histological and transcriptional changes was much higher for ERβ2 (correlation coefficient [CR] = 0.78, n = 15) than ERβ1 (CR = 0.27, n = 15), thus highlighting the importance of ERβ2 in early medaka gonadogenesis (). This finding is supported by our previous report wherein ERβ2 showed female-dominated expression in the early sex determination period ().

Differential expression of three estrogen receptor subtype mRNAs in gonads and liver from embryos to adults of the medaka, Oryzias latipes.

Differential expression of three estrogen receptor subtype mRNAs in gonads and liver from embryos to adults of the medaka, Oryzias latipes.

Differential expression of three estrogen receptor subtype mRNAs in gonads and liver from embryos to adults of the medaka, Oryzias latipes.

Discussion

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Zhang Y.G. Rspo1-activated signaling molecules are sufficient to induce ovarian differentiation in XY medaka (Oryzias latipes). 2 /ER actions are critical for embryonic gonadal development. In vertebrates, ERs and their ligands play several important and conserved physiological functions. Furthermore, maternal estrogen is known to have a critical role in early development and differentiation (). Numerous reports suggest that estrogen is synthesized in the mammalian fetus, especially in the brain, and helps in the smooth execution of ER-regulated estrogenic functions (). Recently, we reported that estrogen is indeed synthesized in the developing medaka embryos (), interestingly at the same time as zygotic ER and brain type aromatase (CYP19b) transcriptional onset (), thus emphasizing that fetal estrogenic concentration and actions are instrumental in gonadal sex differentiation in medaka (). In this work, we found that the ER-agonist, ER-antagonist, ER-KD, and ER-null mutation affect the estrogenic actions in medaka from very early stages of embryonic development, thus establishing the fact that E/ER actions are critical for embryonic gonadal development.

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Valenzuela N. Ecotoxicological perspectives of sex determination. Conversely,found that null mutation of CYP19a1 gene does not affect the early gonadal development. In medaka, despite the rapid reduction in maternal estrogen from yolk stores, it is probable that traces of remaining estrogen or other estrogen by-products might be enough to drive the normal gonadal development in CYP19a1medaka. Moreover, chronologically, first zygotic expression of CYP19b (2 daf) and ERβ2 (2 daf) precedes the CYP19a1 expression (5–10 dah), and ERβ2 knockdown reduces the CYP19b expression in both gonad and brain. It is possible that during early development estrogen synthesis is mainly regulated by CYP19b or some other estrogen-like machinery (). Notably, it has been repeatedly shown that environmental estrogenic chemicals can mimic endogenous estrogen's action and alter sexual development in various organisms, suggesting that estrogen actions are vital for sexual differentiation (). Further investigations to decipher the sources of endogenous estrogenic components are necessary to completely understand the estrogenic role in early gonadal development.

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et al. Biallelic and monoallelic ESR2 variants associated with 46, XY disorders of sex development. In an exemplary study, using morpholino knockdown and cell transplantation,demonstrated that the threshold number of PGC is required to retain ovarian stability in zebrafish and is instrumental for testicular differentiation. In our ERβ2-KD fish, PGC mismigration and cell death likely significantly reduce the initial PGC number in the embryonic gonad and mimic a situation similar to male-type gonad in medaka. This gonadal state is further aggravated by the reduced estrogen signaling-associated transcription of male-biased genes. However, in this regard the indirect involvement of the interactively modulated AR pathway () cannot be overlooked. Since ERβ2 actions are critical throughout embryogenesis, it is possible that elimination of ERβ2 affects several other important pathways, and in turn orchestrates the sex reversal. Despite a significant increase in male-biased gene transcription and germ cell reduction, both initiated by ERβ2 reduction, the percentages of adult sex reversal were somewhat less than expected. This suggests that some unknown mechanism, probably variable non-genomic action or substitution effect or a threshold of gene(s)/pathway(s), are in action, which needs further investigation. In contrast to previous reports on the absence of sex-reversal phenotype in ER-KO mice (), it was recently found that biallelic/monoallelic mutation of ERβ causes sex reversal in humans (). This difference has been suggestively attributed to the non-genomic ER action, i.e., MAPK signaling (). Moreover, the intensity of sex reversal in humans is significantly related to zygosity, further supporting the idea that a threshold limit might be important for functional manifestation of ERβ2 actions in medaka. Nevertheless, irrespective of different non-genomic interaction or fish-specific genome duplication, it seems that the estrogen/ER pathway directly controls the reproductive fitness in both human and medaka. Hence, this study will help us to rethink the estrogen/ER involvement in successful reproduction and reproductive disorder management.

In conclusion, our data suggest that ERβ2 has multi-point regulation, starting from regulation of chemotactic germ cell migration, calcium homeostasis, and cell sustenance, to controlling meiotic initiation, which eventually influences sexual development. Our data also highlight the importance of ERβ2 in estrogen transmission to gonad to maintain the gonadal sexuality. Thus, this study might be a key to comprehending the diverse estrogen-reproduction relationship in vertebrates.