Yuli Zhao, Yu Zhang, Ying Zhong, Jing Guo, Mengyue Lu, Lang Gui,＊,Mingyou Li,＊

aInternational Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai, 201306, China

bKey Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, 201306, China

Keywords:

ABSTRACT

1.Introduction

The teleost fish,

Oryzias celebensis

has an XX-XY sex-determination(SD) system and is evolutionarily close to

Oryzias latipes

(Myosho,Takehana, Hamaguchi, & Sakaizumi, 2015; Zhu, Gui, Zhu, Li, & Li,2018). In

O. latipes

, a testis determining genes

DMY/Dmrt1bY

has been identified and is expressed throughout testicular differentiation in somatic cells of primordial gonads surrounding germ cells (Kobayashi et al., 2004). However, the

O. latipes

testis determining gene(

DMY/Dmrt1bY

) is absent in

O. celebensis

although their developmental process and morphology is similar (Zhu et al., 2018). Considering that they have a common gene and the same XY SD system (Myosho et al.,2015), this makes it easy to compare their sex-related factors to track the evolution of SD genes. Furthermore, understanding the genotypic sex determination (GSD) of

O. celebensis

may provide insights into the evolution of sex determination and differentiation among teleost fish,thus opening-up new horizons for management of breeding and reproduction of fish of commercial interest (Li et al., 2018; Xiong, Wang, Gui,& Mei, 2019).There are two types of genes involved in sex differentiation in mammals, those that are involved in ovarian differentiation, such as

foxl2

,

cyp19a

,

star

etc (Cao et al., 2012; He, Wang, Wu, Zhu, & Yang,2018), and those involved in testis differentiation, such as

sox9

,

dmrt1

,

amh

,

cyp11b

etc (Diaz & Piferrer, 2015; Shen & Wang, 2014). Among these genes,

sox9

and

foxl2

are two early gonadal factors related to male or female sex differentiation (Hersmus et al., 2008; Huang, Ye, & Chen,2017). Interestingly, studies in mice and chickens have found antagonistic effects between

foxl2

and

sox9

, while knockdown of

foxl2

expression can ectopically activate

sox9

in the female gonads (Major,Ayers, Chue, Roeszler, & Smith, 2019; Wilhelm et al., 2009).

Foxl2

is a member of the forkhead family of transcription factors and is important in the development and maintenance of female sexualcharacteristics from mammals to teleost (Fan et al., 2019; Li et al., 2014;Schmidt et al., 2004; Valenzuela, 2008). Moreover, it has been reported that a 17 bp deletion at nucleotides 1092–1108 in

foxl2

result in an autosomal dominant disorder blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) in humans, which is associated with premature ovarian failure (Yamada et al., 2001). During the development of the ovary,

foxl2

is mainly expressed in the granulosa cells and a few theca cells (Herpin et al., 2013), which are involved in the production of estrogen to maintain ovarian differentiation (Pannetier et al., 2006).Remarkably, a recent study in

O. latipes

revealed that Foxl2 protein expression began in germ line stem cells and persisted during meiosis until early oogenesis (Herpin et al., 2013). Furthermore, studies in Nile tilapia and mice reported that loss of

foxl2

resulted in female-to-male sex reversal (Auguste et al., 2011; Zhang et al., 2017). Collectively, all the above reports indicate that

foxl2

is involved in the differentiation of ovarian granulosa cells and maintain ovarian development.

Table 1 Sequences of primers used in the present study.

Due to the teleost specific genome duplication, two

sox9

genes named

sox9a

and

sox9b

have also been reported in

O. latipes

(Klüver,Kondo, Herpin, Mitani, & Schartl, 2005), zebrafish (van Boxtel et al.,2010), and rainbow trout (Takamatsu et al., 1997). The copy,

sox9a

is not expressed in the gonadal supporting cells necessary for sex determination, but is expressed in the oocytes of the adult ovary (Yokoi et al.,2002). In contrast,

sox9b

initiates expression in gonadal precursor cells,which develop into supporting cells and promote cellular association(Nakamura et al., 2012). In addition, the transcription factor

sox9b

is an intermediate downstream target of the Sex-determining Region on the Y Chromosome (SRY) and is absolutely required for testis development(Wei et al., 2019). In

Oryzias luzonensis

,

sox9b

mRNA is in the somatic cells surrounding the spermatogonia (Nakamoto et al., 2009). Consistent with previous research, the

O. latipes sox9b

transcript is also detected in the somatic cells of the developing gonads of both sexes (Nakamoto,Suzuki, Matsuda, Nagahama, & Shibata, 2005). Subsequent studies demonstrated that

sox9b

is positive in the testis somatic cells, the Sertoli cells (Nakamura et al., 2008). Considering that

sox9b

is present in many species, its function has been explored in zebrafish where it was shown the absence of

sox9b

results in craniofacial malformations and organ defects, as reported in humans and mice (Hofsteen, Plavicki, Johnson,Peterson, & Heideman, 2013; Plavicki et al., 2014). At present,

sox9b

has been cloned in a variety of teleost fish, but its expression pattern in gonads is controversial.The present study, investigates whether

foxl2

and

sox9b

are involved in ovarian and testicular development and differentiation in

O. celebensis

as has been reported in other vertebrates. The cDNA of

Ocfoxl2

and

Ocsox9b

were cloned and their expression patterns in gonads analyzed by RT-PCR and

in situ

hybridization (ISH). To identify their differential expression, the co-localization of

Ocfoxl2

and

Ocsox9b

with the germ cell marker gene

Ocdnd

was also analyzed.

2.Materials and methods

2.1.Fish and embryos

All animal experiments were carried out in accordance with the guidance of the Committee for Laboratory Animal Research at Shanghai Ocean University.

O. celebensis

was maintained at 26C under an artificial photoperiod of 14 h light and 10 h darkness, embryo developmental stage was determined as described previously (Iwamatsu, 2004).

2.2.RNA isolation and gene cloning

Total RNA was extracted from seven adult tissues of

O. celebensis

(eye, brain, kidney, liver, gut, ovary and testis) and different embryo stages (3, 10, 13, 17, 30, 37 and 40) using TRIzol reagent (Invitrogen,Carlsbad, CA). The RNA quality and quantity were detected by Nano-Drop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA),and the integrity was checked by 1% agarose gel stained with ethidium bromide. A Super SMART™ PCR cDNA Synthesis Kit (Clontech, USA)was used for cDNA synthesis from 1 μg of total RNA. To amplify a partial cDNA fragment of

Ocfoxl2

and

Ocsox9b

, degenerate primers were designed based on the amino acid sequence alignment of the genes from different species. Then 5and 3RACE was used to obtain the full-length cDNA sequence of

foxl2

and

sox9b

. Primer sequences used for gene cloning are listed in Table 1.

2.3.Semi-quantitative RT-PCR and sequence analysis

Ocfoxl2

and

Ocsox9b

transcript distribution was analyzed by semiquantitative RT-PCR using the primers indicated in Table 1, with the expression of β-actin as the internal control (Vandesompele et al., 2002).The reaction mixture contained 1 μL of cDNA template (100 ng), 0.5 μL of dNTPs (10 mM), 0.5 μL of EX-taq enzyme (5 U/μL) (TaKaRa, Shiga,Japan), 2.5 μL of 10×EX-taq buffer, and 0.5 μL of the forward and reverse primers (10 mM), and deionized water was added to give a final reaction volume of 25 μL. The thermocycle was as follows: 95C for 10 s,annealing at 58C for 10 s and extension at 72C for 1 min 10 s, 35 cycles were performed. The PCR products were detected on a 1%agarose gel stained with ethidium bromide and visualized and quantified on a bio-imaging system (Bio-Rad, Hercules, CA, US).

A phylogenetic tree was constructed using MEGA 7 program with the neighbor-joining (NJ) and using the deduced protein sequence of Foxl2 and Sox9b (Zhu et al., 2018). Protein alignment was performed with Vector NTI Advance® 11.5 software (Thermo Fisher, US).

2.4.RNA in situ hybridization

Sections for

in situ

hybridization (SISH) and fluorescence ISH (FISH)were performed as described previously with minor modifications (Li et al., 2011; Yuan, Chen, Zhu, Yuan, & Li, 2018). For production of the probe the CDS of

Ocfoxl2

,

Ocsox9b

and

Ocdnd

were ligated into pGEM-T vector and were sequenced. The plasmid was linearized with a suitable restriction enzyme for the synthesis of the antisense and sense probes from the T7 or SP6 promoter using a DIG or FITC RNA Labelling Kit(Roche, Basel, Switzerland). Detection of riboprobe annealing to tissue sections was established by staining with BCIP/NBT for ISH and FISH was performed using the (TSA™) Plus Fluorescence Systems and following the manufacturers manual (Life Technologies, Carlsbad, CA).DAPI was used to stain the nucleus of cells and the slides were mounted using Gold anti-fade reagent (Invitrogen, Carlsbad, CA).

2.5.Microscopy

Fig.1.Nucleotide and amino acid Sequences of Foxl2 and Sox9b. The upper line indicates the nucleotide sequence and the lower line the amino acid sequence. The start codon ATG is in bold; the stop codon is indicated by an asterisk. (A) The regions of fork head (FH) superfamily (foxl2) is shown in a solid-line box. (B) The HMG box and C-terminal transactivation domain are boxed.

Microscopy was performed as previously described (Sun, Gui, Liu,Hong, & Li, 2019). In brief, observation and photography were performed using a Leica TCS SP8 Laser Scanning Confocal Microscope(Leica, Germany) and a Nikon Ds-Ri2 camera (Nikon, Tokyo, Japan).

3.Results

3.1.Cloning and characterization of Ocfoxl2 and Ocsox9b gene

The CDS of

Ocfoxl2

gene is 915 bp and encoded a protein of 304 amino acids (GenBank accession no. MN864781) (Fig.1A). Multiple amino acid sequence alignments of Foxl2 orthologs indicated that the OcFoxl2 is most similar to the previously cloned

O. latipes

Foxl2(98.3%), and 84.4%, 83.7%, 82.0%, 74.2% and 73.6%, identical to carp,zebrafish, African clawed frog, human and mouse, respectively(Fig.S1A). The forkhead domain and the C-terminal region were highly conserved among different species (Fig.1A). Analysis of the phylogenetic tree indicated that fish and mammalian Foxl2 proteins cluster in two distinct branches (Fig.2A).The CDS of

Ocsox9b

gene is 1431 bp (GenBank accession no.MN864782) and encodes a protein of 476 amino acids that contains the characteristic high-mobility-group box (HMG box) DNA binding domain(Fig.1B). Multiple amino acid sequence alignments indicated that

O.

celebensis

Sox9b is 98.2%,82.8%, 82.8% and 72.7% identical to

O. latipes

, common carp, torafugu and zebrafish, respectively (Fig.S1B).Phylogenetic tree analysis revealed that OcSox9

b

is located on the same branch as

O. latipes

Sox9b (Fig.2B).

Fig.2.Neighbor-joining phylogenetic tree of Foxl2 and sox9b proteins constructed using MEGA 7. (A) The phylogenetic tree of Foxl2 was constructed using 13 different species. (B) The phylogenetic tree of Sox9b was constructed using 10 different species.

3.2.Gonad and embryonic expression of Ocfoxl2 and Ocsox9b mRNA

RT-PCR was used to analyze the mRNA expression of

Ocfoxl2

and

Ocsox9b

. In adult tissues,

Ocfoxl2

transcript was detected in the eye and ovary, but was absent in the other tissues examined (Fig.3A). In contrast, the

Ocsox9b

transcript was present in brain, eye, liver, gut and testis (Fig.3A). In the different developmental stages of the embryo,

Ocfoxl2

transcript was found at a low level in blastula and increased significantly in subsequent stages and reached its highest level in 7 day embryos (Fig.3B). While

Ocsox9b

transcript persisted and gradually increased throughout embryogenesis (Fig.3B).

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3.3.Expression of Ocfoxl2 and Ocsox9b mRNA in adult gonad by ISH

To further elucidate the cellular expression of

Ocfoxl2

and

Ocsox9b

in the ovary and testis, of

O. celebensis

, ISH was performed. In the adult ovary,

Ocfoxl2

mRNA was localized in follicular cells of the previtellogenic and vitellogenic follicles and gradually decreased during follicular development (Fig.3C and D). In the adult testis,

Ocsox9b

mRNA was detected in the Sertoli cells that surround the spermatogonia, as well as in the spermatids and sperm (Fig.3E, F, G). The sense probes gave no signal in the ovary (Fig.S2A) or testis (Fig.S2B) and confirmed the specificity of the

Ocfoxl2

and

Ocsox9b

ISH.

3.4.OcFoxl2 and Ocsox9b co-localized with Ocdnd in adult gonads

To accurately confirm the cellular distribution of

Ocfoxl2

and

Ocsox9b

in adult gonads, the co-localization of

foxl2

and

sox9b

with the germ cells marker

dnd

were performed using dual color fluorescent ISH(FISH). Based on the results of previous studies,

Ocdnd

mRNA is known to be specifically expressed in gonadal germ cells (Zhu et al., 2018). In the ovary, the

Ocdnd

signal was relatively weak in stage I oocytes and became most abundant in stage II and III oocytes (Fig.4A, C, D). At higher magnification, the

Ocdnd

transcript was also detected in oogonia(Fig.4E, G). The

Ocfoxl2

signal was persistent throughout oogenesis but in gradually decreasing levels (Fig.4B, D), which was similar to the expression pattern of

Ocdnd

during oogenesis (Fig.4A, C, D). At higher magnifications the

Ocfoxl2

transcript was observed to be localized in the oogonia and the granulosa cells and a low intensity signal was also detected in the theca cells (Fig.4F and G).In the testis, the

Ocdnd

transcript is most abundant in spermatogonia and lower level was observed in spermatocytes and the signal was lost in post meiotic spermatids and sperm (Fig.5B, C, F). In contrast,

Ocsox9b

transcript was abundant in the somatic cells surrounding spermatogonia(Fig.5A, D, E, G). Furthermore, the signal of

Ocsox9b

was also detected in spermatids and sperm (Fig.5A, D, H). In summary, the results reveal two populations of

sox9b

-positive cells in later stages of development that are either Sertoli cells or germ cells.

4.Discussion

To investigate whether

foxl2

and

sox9b

are involved in gonad differentiation and development in

O. celebensis

as in other fish, we isolated the CDS of

Ocfoxl2

and

Ocsox9b

and analyzed their expression patterns in gonads by RT-PCR and ISH. Some differences in the expression patterns of

foxl2

and

sox9b

were found between

O. celebensis

and

O. latipes

.For example,

sox9b

transcript is detected in the adult ovary of

O. latipes

(Nakamura et al., 2012), but it was not detected in

O. celebensis

, in our study indicating that the spatiotemporal regulation of this gene has changed during the divergence process of different fish lineages. It is worth noting that

Ocfoxl2

and

Ocsox9b

mRNA were not expressed simultaneously in the testis or ovary, which provides indirect support for their gonad-biased expression in the testis or ovary, respectively.Multiple sequence alignments and phylogenetic tree analysis revealed that the amino acid sequence of

Ocfoxl2

is similar to other fishes, with no proline or glycine repeats or polyalanine bundles,although the characteristic forkhead domain was conserved (Hu, Guo,Gao, Tang, & Li, 2014). It has been proposed that the forkhead domain is responsible for protein import from the nucleus, and it is involved in various cellular processes, such as cell proliferation, differentiation,migration and DNA damage response (Zhu, 2016). The HMG box in

Ocsox9b

is highly conserved and has more than 98% amino acid homology with the protein in

O. latipes

, so we speculate that the gene function of

sox9b

may be similar among species of the same genus.Moreover, it has been reported that

sox9

may activate transcription of downstream target genes through its HMG box (Yokoi et al., 2002).Given the highly conserved structure of these two genes, we speculate that

Ocfoxl2

and

Ocsox9b

are probably involved in

O. celebensis

gonad differentiation and development.The expression of

Ocfoxl2

and

Ocsox9b

were first analyzed in adult organs and embryo. We found that the

Ocfoxl2

mRNA signal was present in the eye and ovary of adult individuals, which is similar to what has been reported in

O. latipes

(Nakamoto, Matsuda, Wang, Nagahama, &Shibata, 2006),

Oryzias luzonensis

(Nakamoto et al., 2009), zebrafish(Yang, Wang, Li, Zhou, & Gui, 2017) and Nile tilapia (

Oreochromis niloticus

) (Zhang et al., 2017). In addition, expression of

Ocfoxl2

was also detected during embryogenesis. In mice,

foxl2

is expressed in cranial neural crest cells and cranial mesodermal cells and contribute to early eyelid development and eyelid extension (Heude et al., 2015). Interestingly,

foxl2

has also been found during fetal eyelid development in the dogfish and goat (Cocquet et al., 2002; Wotton, French, & Shimeld,2007), and the eyelids are structures characteristic of gnathostomes,suggesting that

foxl2

may affect the morphology of vertebrate during evolution. These data indicate that

foxl2

may play a vital role in the organ formation and embryo development.

Fig.3.RNA expression of Ocfoxl2 and Ocsox9b. RT-PCR analysis of Ocfoxl2 and Ocsox9b in adult organs (A) and developing embryos (B). (C and D) Adult ovarian cryosections were hybridized to antisense Ocfoxl2 probe and visualized by chromogenic staining. (D) Higher power magnification of the framed area in (C). (E–G) Adult testicular cryosections were hybridized to the antisense Ocsox9b probe and visualized by chromogenic staining. (F and G) Higher power magnification of the framed area in(E). I–V, stages of oocytes; gc, granulosa cells; tc, theca cells. sg, spermatogonia; sc,spermatocytes; sm, sperm; st, spermatids; se,Sertoli cell. Scale bars, 25 μm.

In adult tissues, the

Ocsox9b

transcript was enriched in the testis and detectable in other tissues such as brain, eye, liver, and gut. The testis expression of

Ocsox9b

is identical to that of

O. latipes sox9b

, however,

Ocsox9b

is not expressed in the ovary, which is different from

O. latipes

and zebrafish (Chiang et al., 2001; Klüver et al., 2005). These results indicate that

Ocsox9b

may be involved in the growth and development of these organs. Consistent with the results of whole mount

in situ

hybridization of

sox9b

in

O. latipes

embryos (Klüver et al., 2005),

Ocsox9b

transcript persisted during embryogenesis. Therefore,

sox9b

may play an important role in the regulation of embryonic development and it has been documented in zebrafish that

sox9b

is involved in the formation of various cartilage tissues (Chiang et al., 2001). Further research will be necessary to elucidate the regulatory mechanisms of

foxl2

and

sox9b

in gonadal development.

Foxl2

is the earliest known gene to exhibit a sexually-dimorphic expression pattern in ovarian somatic cells and is involved in the lineage of female support cells, the differentiation of granulosa cells, and the formation or maintenance of ovarian follicles (Jiao, Ke, Qin, & Chen,2018; Nakamoto et al., 2006; Pannetier et al., 2006). In the present study, the co-localization of

Ocfoxl2

and

Ocdnd

revealed that

Ocfoxl2

is expressed in the granulosa cells and theca cells and persisted throughout oogenesis including in the oogonia. Interestingly,

Ocfoxl2

initiated expression in the oogonia entering meiosis and increased significantly in the early meiotic phase as shown in oocytes at stages I and II.

Foxl2

expression in oocytes has been reported in only a few species, such as hens (

Gallus gallus

) (Qin et al., 2015), human (Ernst, Franks, Hardy,Villesen, & Lykke-Hartmann, 2018) and Japanese grenadier anchovy(

Coilia nasus

) (Fang et al., 2019). In the mouse the deficiency of

foxl2

leads to subsequent up-regulation of

sox9

(Sekido & Lovell-Badge,2008), and leads to trans-differentiation of the ovarian granulosa and theca cells into testis specific Sertoli cells and Leydig cells, respectively(Uhlenhaut et al., 2009). Based on this we speculate that

foxl2

is crucial for the maintenance of granulosa and theca cell identity. In addition,

foxl2

may be involved in ovarian development of

O. celebensis

by regulating oogenesis and oocyte differentiation, as shown by the functional studies of

foxl2

knockout in zebrafish and tilapia (Yang et al., 2017;Zhang et al., 2017).

O. latipes sox9b

has been proposed to act by promoting cellular association, which is indispensable for the proliferation and survival of germ cells, rather than directly regulating the testicular identification and differentiation (Nakamura et al., 2012). In addition, studies in zebrafish have found that

sox9b

is crucial for the development of the hepatopancreatic ducts and the regeneration of pancreatic endocrine cells (Manfroid et al., 2012). The present study reveals

Ocsox9b

is expressed by Sertoli cells and it is assumed to be involved in the function of Sertoli cells throughout testicular development. Surprisingly, the

Ocsox9b

signal was also detected in spermatids and sperm, suggested that

Ocsox9b

may also be involved in the spermatogenesis of

O. celebensis

, but the exact regulatory mechanism is unclear. Considering the tissue expression pattern and cellular localization of

Ocsox9b

, we propose that it plays an important role in male gonadal development of

O. celebensis

.

Fig.4.Expression of Ocfoxl2 and Ocdnd mRNA in the ovary. Adult ovarian cryosections were hybridized to the antisense probe, the signal was visualized by fluorescence staining. (A and B) The Ocdnd is stained in red and the Ocfoxl2 is stained in green by FISH. Nuclei are stained in blue with DAPI. (A–D) Merges of Ocdnd with DAPI, Ocfoxl2 with DAPI, Ocfoxl2 with Ocdnd, Ocfoxl2 with Ocdnd and DAPI. (E–G) Higher power magnifications of the framed areas in (A, B, D). I–V, stages of oocytes; og, oogonia; gc, granulosa cells; tc, theca cells. Scale bars, 25 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

In conclusion,

foxl2

and

sox9b

were identified in

O. celebensis

, and their cellular localization in adult gonads was elucidated. The results reveal that

foxl2

may play a critical role in the differentiation of

O. celebensis

ovary, and its function may be evolutionarily conserved among vertebrates, while

sox9b

may play an important role in testicular development and spermatogenesis of

O. celebensis

testis. Since some commercially important fish have individual differences between males and females, or differences in disease resistance, the present study not only provides impetus for further research on the molecular antagonism that determines the testicular and ovarian pathways in

O. celebensis

, but also provides reference value for the selection of male and female breeding varieties.

Authorship Contribution

L. G and M. Y. L conceive and design the experiments. Y. L. Z. and Y.Z. performed the experiments. Y. L. Z., Y. Z., L. G and M. Y. L analyzed the data. J. G. and M. Y. L contributed reagents and materials. Y. L. Z., Y.Z., L. G and M. Y. L wrote and corrected the manuscript.

Fig.5.Expression of Ocsox9b and Ocdnd mRNA in the testis. Adult testicular cryosections were hybridized to the antisense probe, the signals were visualized by fluorescence staining. (A and B) Ocsox9b is stained in red and the Ocdnd is stained in green by FISH. Nuclei are stained in blue with DAPI. (A–D) Merges of Ocsox9b with DAPI, Ocdnd with DAPI, Ocsox9b with Ocdnd, Ocsox9b with Ocdnd and DAPI. (E–H) Higher power magnifications of the framed areas in (A, B, D). sg, spermatogonia; sc, spermatocytes; sm, sperm; st, spermatids; se, Sertoli cell. Scale bars, 25 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgement

This work was supported by National Key R&D Program of China(2018YFD0901205) and National Natural Science Foundation of China(31672700, 31372520).

Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.aaf.2020.06.009.