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Transcriptome profiling reveals male- and female-specific gene expression pattern and novel gene candidates for the control of sex determination and gonad development in Xenopus laevis

  • Rafal P. PiprekEmail author
  • Milena Damulewicz
  • Jean-Pierre Tassan
  • Malgorzata Kloc
  • Jacek Z. Kubiak
Open Access
Original Article
  • 156 Downloads

Abstract

Xenopus laevis is an amphibian (frog) species widely used in developmental biology and genetics. To unravel the molecular machinery regulating sex differentiation of Xenopus gonads, we analyzed for the first time the transcriptome of developing amphibian gonads covering sex determination period. We applied microarray at four developmental stages: (i) NF50 (undifferentiated gonad during sex determination), (ii) NF53 (the onset of sexual differentiation of the gonads), (iii) NF56 (sexual differentiation of the gonads), and (iv) NF62 (developmental progression of differentiated gonads). Our analysis showed that during the NF50, the genetic female (ZW) gonads expressed more sex-specific genes than genetic male (ZZ) gonads, which suggests that a robust genetic program is realized during female sex determination in Xenopus. However, a contrasting expression pattern was observed at later stages (NF56 and NF62), when the ZW gonads expressed less sex-specific genes than ZZ gonads, i.e., more genes may be involved in further development of the male gonads (ZZ). We identified sexual dimorphism in the expression of several functional groups of genes, including signaling factors, proteases, protease inhibitors, transcription factors, extracellular matrix components, extracellular matrix enzymes, cell adhesion molecules, and epithelium-specific intermediate filaments. In addition, our analysis detected a sexually dimorphic expression of many uncharacterized genes of unknown function, which should be studied further to reveal their identity and if/how they regulate gonad development, sex determination, and sexual differentiation. Comparison between genes sex-specifically expressed in developing gonads of Xenopus and available transcriptome data from zebrafish, two reptile species, chicken, and mouse revealed significant differences in the genetic control of sex determination and gonad development. This shows that the genetic control of gonad development is evolutionarily malleable.

Keywords

Testis Ovary Sex determination Gonad development Xenopus Transcriptome 

Introduction

Xenopus laevis is a good model to study molecular mechanisms of gonad development because the structural changes in developing gonads and the master gene determining sex, the W-linked DM-domain gene (dm-w), are well known. The dm-w is located on W chromosome and thus is present only in the genetic females (ZW) (Yoshimoto et al. 2008). At the earliest stage of gonad development, the gonads are undifferentiated and bipotential. The expression of dm-w triggers ovary development, while its absence promotes testis development. It is believed that the DM-W protein blocks the DMRT1 (doublesex and mab-3-related transcription factor 1) involved in male sex determination (Yoshimoto et al. 2010). In addition to the dm-w, many other genes, which act independently or downstream of dm-w, are involved in the development of bipotential gonads into the ovaries or the testes (Piprek et al. 2016). However, the expression and role of many genes involved in gonadal development is still vague. At the initial stage of gonadogenesis (NF50, Nieuwkoop-Faber stage 50), the gonads consist of the gonadal cortex and the medulla. The gonadal cortex contains coelomic epithelium and the germ cells, which adhere to the interior face of the epithelium. The medulla is sterile and contains medullar cells only (Piprek et al. 2016, 2017). At this stage, the sex-determining genes (dm-w and dmrt1) are expressed in the somatic cells of the gonads. In the absence of dm-w, i.e., in the differentiating testis (ZZ), around stage NF53, the cortex and medulla fuse. Subsequently, around stage NF56, the germ cells become enclosed by the somatic cells, which results in the formation of testis cords (Piprek et al. 2017). The typical structure of the testis, i.e., fully differentiated testis cords separated by the interstitium, is established at stage NF62. In contrast, in differentiating ovaries, which express dm-w, the germ cells remain in the cortical position, and at stage NF56, the ovarian cavity forms inside the gonad. Around NF62, the ovaries are fully differentiated, with the oocytes located in the cortex (Piprek et al. 2017; Yoshimoto et al. 2008). This divergent development of the female and male gonads has to be controlled by differential gene expression. A global analysis of Xenopus gonad transcriptome, which we performed in this study, is the step in obtaining a broad database of gene expression pattern in developing male and female Xenopus gonads.

Among vertebrates, the transcriptome of developing gonads has been studied in the mouse (Beverdam and Koopman 2006; Chen et al. 2012; Gong et al. 2013; Jameson et al. 2012; Nef et al. 2005; Small et al. 2005), chicken (Ayers et al. 2015; Scheider et al. 2014), slider Trachemys scripta (Czerwinski et al. 2016), Alligator mississippiensis (Yatsu et al. 2016), and in several teleost fish species (Bar et al. 2016; Lin et al. 2017; Sreenivasan et al. 2008; Sun et al. 2018; Xu et al. 2016). These studies provided valuable insights into the genes involved in gonad development and identified new sex-determining gene candidates.

Among anurans, a transcriptome analysis was performed only in Silurana (Xenopus) tropicalis and only on already sexually differentiated gonads (from stage NF58) (Haselman et al. 2015). Thus, the genes expressed before and during the sexual differentiation of amphibian gonads are still unknown. The aim of our study was to examine the transcriptome of developing Xenopus gonads from the earliest stage of gonad development. We studied the gene expression pattern in four different stages of gonad development: the undifferentiated gonad during the period of sex determination (NF50), gonads at the onset of sexual differentiation (NF53), the differentiating gonads (NF56), and during the developmental progression of differentiated gonads (NF62) (Fig. 1).
Fig. 1

Structural changes in developing gonads. a, b At stage NF50, there is no difference in the gonad structure between genetic sexes (ZW and ZZ). Such undifferentiated gonads (arrows) are composed of the somatic cells of coelomic epithelium (ce) covering the gonad, and germ cells (g) located inside; the germ cells are attached to the coelomic epithelium. The somatic cells gather in the gonad center forming gonadal medulla (m). At stage NF53, the first sexual differences appear in the gonad structure; in the differentiating ovaries (c, ZW), the germ cells remain in the peripheral position forming the ovarian cortex, whereas the centrally located medulla remains sterile. In the ZZ (male) gonads at the onset of sexual differentiation (d, the onset of the testis differentiation), the germ cells (g) detach from the coelomic epithelium and move towards the gonad center (medulla, m). At stage NF56, the differentiating ovaries (e) becomes compartmentalized into cortex and medulla; all germ cells (g) are located in the cortex and are attached to the coelomic epithelium; an ovarian cavity forms in the medulla (asterisk). In the differentiating testes (f), the germ cells (g) are dispersed and the cortex and medulla are absent. At stage NF62, the ovaries (g) contain large ovarian cavity (asterisk); the ovarian cortex contains meiotic cells (o). In the testes (h), the germ cells (g) are located within the testis cords (encircled). Scale bar, 25 μm

Results and discussion

Sex-specific changes in the level of gene expression

In developing Xenopus laevis gonads (stages NF50, NF53, NF56, and NF62 combined), we detected the expression of 63,084 transcripts in total. We found that while the expression level of the majority of genes was similar between stages and between male and female gonads, a subpopulation of genes showed distinct changes in the expression level between stages and sexes, which suggested that they may play a role in sex determination and/or sexual differentiation (Figs. 2A, B and 3, Tables 1 and 2).
Fig. 2

Diagram of changes in the number of genes upregulated and downregulated (≥ 2-fold change) between different stages in ZW gonads (a) and ZZ gonads (b)

Fig. 3

Diagram of changes in the number of genes with higher expression in ZW or ZZ gonads (≥ 2-fold change)

Table 1

Number of genes with up- and downregulated (≥ 2-fold change) expression in ZW and ZZ gonads

Compared stages

ZW (females)

ZZ (males)

Upregulated

Downregulated

Upregulated

Downregulated

NF53 vs. NF50

376

1078

659

436

NF56 vs. NF53

143

128

340

340

NF62 vs. NF56

918

1834

334

831

Table 2

Number of genes with up- and downregulated (≥ 2-fold change) expression in ZW versus ZZ gonads

ZW vs. ZZ compared at stages

Upregulated in ZW

Downregulated in ZW

NF50

820

372

NF53

193

890

NF56

75

346

NF62

594

2630

Analysis of gene expression level in the gonads showed that in the genetic females (ZW), the gonads at the onset of sexual differentiation (NF53) had 376 genes with upregulated expression and 1078 genes with downregulated expression in comparison to the undifferentiated gonad during sex determination period (NF50) (Fig. 2, Table 1). In the differentiating ovaries (NF56), only 143 genes were upregulated and 128 genes were downregulated in comparison to NF53 (Table 1). In differentiated ovaries (NF62), there were 918 genes with upregulated expression and 1834 genes with downregulated expression in comparison to NF56 (Table 1).

The genetic male (ZZ) gonads at the onset of sexual differentiation (NF53) had 659 genes with upregulated expression and 436 genes with downregulated expression in comparison to NF50 stage (Fig. 2, Table 1). In differentiating testes (NF56), 340 genes were up-, and 340 downregulated in comparison to NF53 stage. The differentiated testes at stage NF62 had 334 genes with upregulated expression and 831 genes with downregulated expression in comparison to NF56 stage.

Altogether, these data indicate that in both sexes, the transcriptional regulation is more robust during early gonadal development, i.e., at the onset of sexual differentiation of the gonad (NF50-NF53) and in the already differentiated gonads NF56-NF62 than in the differentiating gonads (NF53-NF56).

The comparison of gene expression level in between ZW and ZZ gonads showed significant differences between the sexes and revealed sexually dimorphic pattern of gene expression. At the initial phase of gonad development, i.e., in the undifferentiated gonads during sex determination (NF50), there were 1192 genes (i.e., 3.4%) with sexually dimorphic expression (≥ 2-fold change). Eight hundred twenty genes showed higher expression in ZW (genetic females), and only 372 showed higher expression in ZZ (genetic males) gonads (Fig. 3, Table 2). This indicates that female sex determination in Xenopus involves a robust transcriptional regulation. In contrast, in mice, during the sex determination period (between embryonic day E10.5 and E12.5), a higher number of genes were upregulated in the XY (genetic males) than in the XX (genetic females) gonads (Nef et al. 2005), which suggested that programs of sex determination may be diverse among vertebrates.

Our analysis showed that at NF53, i.e., at the beginning of sexual differentiation of Xenopus gonads, 1083 genes (i.e., 3%) showed sexually dimorphic expression (≥ 2-fold change), which was slightly lower number than at NF50 (during sex determination). One hundred ninety-three genes showed higher expression in ZW gonads, and 890 in ZZ gonads (Fig. 3, Table 2). Thus, at the onset of sexual differentiation, more genes were specifically expressed in ZZ (male) gonads than in ZW (female) gonads in Xenopus, which was opposite to the mouse, where more genes were specifically expressed in XX (female) than XY (male) gonads at the beginning of sexual differentiation (E13.5) (Nef et al. 2005). This again indicates differences in the molecular programs of gonad development among vertebrates.

At NF56, i.e., in the differentiating gonads, only 421 genes (i.e., 1.2%) showed sexually dimorphic expression (≥ 2-fold change). This stage showed the lowest percentage of genes with sexually dimorphic expression among all stages. Seventy-five genes had higher expression in ZW, and 346 in ZZ gonads (Fig. 3, Table 2). Thus, more genes were highly expressed in ZZ gonads (differentiating testes) than in ZW (differentiating ovaries). We previously showed that the testis differentiation in Xenopus is a complex process during which the basement membranes between gonadal cortex and medulla disintegrate, the cortex and medulla fuse, and the germ cells and somatic cells gather to form the testis cords (Piprek et al. 2017). This sequence of profound structural changes certainly requires an involvement of a number of different genes, which is reflected in the high number of genes expressed in ZZ gonads at this stage.

At stage NF62, the sexual dimorphism of gene expression is the most pronounced. At this stage, 3224 genes (i.e., 5%) showed sexually dimorphic expression (≥ 2-fold change). However, only 594 genes showed higher expression in ZW (ovaries), and as many as 2630 in ZZ (testes) gonads. This is the stage when the gonads of both sexes are already differentiated and fully prepared to perform their sex-specific functions, and therefore the sexual dimorphism is evident not only at structural but also at molecular level.

The expression of genes during different stages of ovary development

We found that in ZW gonads at stage NF53, in comparison to stage NF50, 376 genes had upregulated expression. The list of genes is presented in Suppl. Table 1, and chosen genes are presented in Table 3. Functional analysis grouped these genes in several distinct categories shown in Table 4. Among the upregulated genes, monoacylglycerol O-acyltransferase 2 gene 1 (mogat2.1) is involved in synthesis of diacylglycerol (DAG) that acts as a messenger lipid in cell signaling (Toker 2005); retinol-binding protein 2 (rbp2) is involved in retinoic acid regulation; extracellular proteins: collagen 2 and collagen 9, cysteine protease cathepsin K, epithelium-specific intermediate filaments: keratin 14 and keratin 19, estrogen receptor 1 (esr1), and synuclein gamma. At this early stage, the germ and somatic cells proliferate, and somatic cells start gathering in the gonad center forming medulla (Fig. 1A, C). Collagens accumulate between the gonad cortex and medulla (Piprek et al. 2017). Importantly, around stage NF50, a sex determination period takes place and gene expression analysis suggest that DAG, retinol, and estradiol may be involved in Xenopus sex determination.
Table 3

Chosen genes up- and downregulated in ZW (genetic females) gonads at NF53 in relation to NF50 stage

Probe name

Gene symbol

Gene name

Log FC

Genes upregulated (higher expression at NF53 than at NF50)

  A_10_P009259

mogat2.1

Monoacylglycerol O-acyltransferase 2.1

6.53907

  A_10_P079665

rbp2

Retinol-binding protein 2

5.67257

  A_10_P002950

col9a1

Collagen, type IX, alpha 1

4.86313

  A_10_P005551

srpx2

Sushi repeat–containing protein, X2

4.74263

  A_10_P000515

bcan

Brevican

4.008

  A_10_P136703

krt14

Keratin 14

3.56258

  A_10_P007276

aldh3b1

Aldehyde dehydrogenase 3 B1

3.5464

  A_10_P143593

ctsh

Cathepsin H

3.39144

  A_10_P004976

matn4

Matrilin 4

3.3843

  A_10_P027124

col2a1b

Collagen, type II, alpha 1

3.12339

  A_10_P002931

matn2

Matrilin 2

3.10895

  A_10_P041821

sncg-b

Synuclein, gamma b

2.79753

  A_10_P032181

sncg-a

Synuclein, gamma a

2.7756

  A_10_P046256

ctsk

Cathepsin K

2.75751

  A_10_P165493

krt19

Keratin 19

2.48345

  A_10_P006607

col9a3

Collagen, type IX, alpha 3

2.41836

  A_10_P033056

esr1-a

Estrogen receptor 1

2.36005

  A_10_P224323

racgap1

Rac GTPase activating protein 1

2.29739

  A_10_P036156

dcn

Decorin

2.25563

  A_10_P065984

itga11

Integrin, alpha 11

2.17377

Genes downregulated (higher expression at NF50 than at NF53)

  A_10_P174228

chrd

Chordin

11.53231

  A_10_P030946

rbp4

Retinol-binding protein 4

6.862097

  A_10_P056207

vtn

Vitronectin

6.558013

  A_10_P075910

serpini2

Serpin peptidase inhibitor, clade I .2

5.739304

  A_10_P008816

serpina3

Serpin peptidase inhibitor, clade A .3

4.968027

  A_10_P065884

wnt10b

Wingless-type MMTV integration site 10B

4.090623

  A_10_P002182

serpinc1

Serpin peptidase inhibitor, clade C .1

3.044408

  A_10_P009298

igf3

Insulin-like growth factor 3

3.030882

  A_10_P043816

dmrt2

Doublesex and mab-3 related transcription factor 2

2.872563

  A_10_P178123

mafb

v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B

2.66641

Table 4

Number of genes assigned to functional groups up- and downregulated in ZW (genetic female) gonads

Functional gene groups

ZW (genetic females)

NF53 vs. NF50

NF56 vs. NF53

NF62 vs. NF56

Up

Down

Up

Down

Up

Down

Signaling factors

20

61

7

8

103

Calcium-binding proteins

6

3

Iron-binding proteins

4

Monooxygenases

4

11

Oxidoreductases

5

11

22

Sushi domain–containing proteins

2

Metalloproteinases

3

8

Intermediate filaments

3

EGF-like domain–containing proteins

3

ECM-receptor interaction pathway

3

Progesterone-mediated oocyte maturation pathway

4

11

Proteases

12

18

Hydrolases

27

33

Disulfide bond–containing proteins

5

45

Extracellular matrix components

5

Markers of epithelial differentiation

2

Meiosis regulation factors

8

RNA-binding proteins

15

Phosphoproteins

16

Proteins involved in development

22

Proteins involved in oogenesis

3

Cytoplasmic proteins

35

Cytoskeletal proteins

12

Proteins involved in differentiation

9

Nuclear proteins

45

Transcriptional repressors

8

DNA-binding proteins

3

Oocyte meiosis

10

p53 signaling

6

Basal transcription factors

4

Proteins involved in DNA Replication

4

Proteins involved in the formation of dorso-ventral axis

3

Secreted proteins

23

Transport proteins

36

We also found that in ZW gonads at stage NF53, there were 1078 genes with a downregulated expression in comparison to stage NF50. All these genes are listed in Suppl. Table 2, and chosen genes are presented in Table 3. Functional analysis grouped these genes in four categories shown in Table 4. Among these downregulated genes, there were signaling protein chordin (chrd), retinol-binding protein (rbp4), several protease inhibitors serpins, signaling proteins wnt10b and igf3 (insulin-like growth factor 3), transcription factors dmrt2, and mafb (Table 3).

In developing ZW gonad at stage NF56, in comparison to stage NF53, there were 143 genes with upregulated expression (Suppl. Table 3, and chosen genes are presented in Table 5). Functional analysis grouped these genes in three categories shown in Table 4. One of important genes upregulated in this period is a neurotrophin receptor a-1 (p75NTRa) (Table 2); its role in gonad development has never been studied; however, its upregulation suggests that neurotrophins (ligands of this receptor) can play a role in ovarian differentiation. We also found that in ZW gonad at stage NF56, in comparison to stage NF53, there were 128 genes with downregulated expression (Suppl. Table 4, and chosen genes are presented in Table 5). Functional analysis grouped these genes in several categories shown in Table 4. At NF56 stage, more genes responsible for reorganization of extracellular matrix and epithelial differentiation in ZW gonads are expressed than at stage NF53. Between stages NF53 and NF56, the medulla cells disperse, which results in the formation of the cavity in the ovary center (Fig. 1E). The mechanism of this event is not known and would be interesting to study how the neurotrophins, extracellular matrix, and epithelial differentiation are involved in this process.
Table 5

Chosen genes up- and downregulated in ZW (genetic females) gonads at NF56 in relation to NF53 stage

Probe name

Gene symbol

Gene name

Log FC

Genes upregulated (higher expression at NF56 than at NF53)

  A_10_P259017

sag

Arrestin

3.6903

  A_10_P000364

p75NTRa

p75 neurotrophin receptor a-1

3.24871

Genes downregulated (higher expression at NF53 than at NF56)

  A_10_P000515

bcan

Brevican

3.073221

  A_10_P136703

krt14

Keratin 14

2.897822

  A_10_P004976

matn4

Matrilin 4

2.768856

  A_10_P002950

col9a1

Collagen, type IX, alpha 1

2.713584

  A_10_P140568

krt5.6

Keratin 5, gene 6

2.618006

  A_10_P006607

col9a3

Collagen, type IX, alpha 3

2.601338

  A_10_P084685

krt14

Keratin 14

2.530431

  A_10_P038721

col2a1b

Collagen, type II, alpha 1

2.509659

  A_10_P032181

sncg-a

Synuclein, gamma a

2.497221

In developing ZW gonad at stage NF62, in comparison to stage NF56, there were 918 genes with upregulated expression (Suppl. Table 5, and chosen genes are presented in Table 6). Functional analysis grouped these genes in the many categories (Table 4). Among known genes upregulated in the ovaries at stage NF62 are genes involved in meiosis and oocyte development, such as poly(A)-binding protein, oocyte-specific pou5f3.3, zygote arrest 1, zona pellucid proteins (zp2, zpd, zpy1), sycp3 (synaptonemal complex protein 3),and lhx8 (LIM homeobox 8). This reflects the onset of meiosis at stage NF62 and appearance of first oocytes (Fig. 1G). Also, more genes involved in the regulation of development, such as genes encoding the following: vegt protein, growth differentiation factor (gdf1), foxh1, foxr1, wnt11b, ddx25, and the survivin which prevents apoptosis, were upregulated at stage NF62 than at stage NF56.
Table 6

Chosen genes up- and downregulated in ZW (genetic females) gonads at NF62 in relation to NF56 stage

Probe name

Gene symbol

Gene name

Log FC

Genes upregulated (higher expression at NF62 than at NF56)

  A_10_P000661

spdyc-b

Speedy/RINGO cell cycle regulator C

5.91483

  A_10_P041271

pabpn1l-a

Poly(A) binding protein, nuclear 1-like

5.78779

  A_10_P078660

rnf138

Ring finger protein 138

5.43076

  A_10_P004355

pou5f3.3

POU class 5 homeobox 3, gene 3

4.82381

  A_10_P002029

zar1

Zygote arrest 1

4.7962

  A_10_P038461

LOC398389

Survivin

4.75826

  A_10_P027361

vegt-a

vegt protein

4.68137

  A_10_P007276

aldh3b1

Aldehyde dehydrogenase 3 family, B1

4.65557

  A_10_P032511

cldn6.1

Claudin 6, gene 1

4.50308

  A_10_P162298

zp2

Zona pellucida glycoprotein 2

4.43055

  A_10_P009533

gdf1

Growth differentiation factor 1

4.40831

  A_10_P002027

velo1

velo1 protein

4.36483

  A_10_P027280

zpd

Zona pellucida protein D

4.2713

  A_10_P205908

foxh1

Forkhead box H1

4.2256

  A_10_P031016

foxr1

Forkhead box R1

4.10517

  A_10_P008731

wnt11b

Wingless-type MMTV integration site family, member 11B

4.0833

  A_10_P033516

zpy1

Zona pellucida protein Y1

4.00754

  A_10_P117061

ddx25

DEAD box helicase 25

3.89223

  A_10_P040816

sycp3

Synaptonemal complex protein 3

3.70889

  A_10_P071715

lhx8

LIM homeobox 8

3.56271

  A_10_P056732

dppa2

Developmental pluripotency-assoc 2

3.51303

  A_10_P027350

adam21

ADAM metallopeptidase domain 21

2.89064

Genes downregulated (higher expression at NF56 than at NF62)

  A_10_P047196

LOC100037217

Uncharacterized LOC100037217

6.582348

  A_10_P180718

hrg

Histidine-rich glycoprotein

6.249551

  A_10_P004053

rbp4

Retinol-binding protein 4

5.794043

  A_10_P034336

serpina1

Serpin peptidase inhibitor, A1

5.541168

  A_10_P006319

sag

Arrestin

5.153979

  A_10_P075910

serpini2

Serpin peptidase inhibitor, I2

4.285183

  A_10_P030976

LOC398504

Villin-1-like

3.897723

  A_10_P068493

fetub

Fetuin B

3.871496

  A_10_P110124

krt12

Keratin 12

3.5294

  A_10_P006916

emx1.2

Empty spiracles homeobox 1, gene 2

3.507484

  A_10_P002103

mmp7

Matrix metallopeptidase 7

3.50358

  A_10_P153143

igf3

Insulin-like growth factor 3

3.452683

  A_10_P003788

igfbp1-a

Insulin-like growth factor–binding 1

3.06992

  A_10_P005507

ctsl

Cathepsin L

2.882569

  A_10_P137683

gata2

GATA binding protein 2

2.5687

  A_10_P053899

cdh26

Cadherin 26

2.529154

  A_10_P126889

rdh16

Retinol dehydrogenase 16 (all-trans)

2.355785

  A_10_P174228

chrd

Chordin

2.347432

  A_10_P007857

timp2

TIMP metallopeptidase inhibitor 2

2.066979

In developing ZW gonad at stage NF62, in comparison to stage NF56, there were 1834 genes with downregulated expression (Suppl. Table 6, and chosen genes are presented in Table 6). Functional analysis grouped these genes into several categories (Table 4). Also, many (24) pathways were downregulated, including metabolic pathways, steroid hormone biosynthesis, retinol metabolism, PPAR signaling pathway, and adipocytokine signaling pathway (Table 4). Among known genes downregulated in the ovaries at stage NF62 are the following genes: retinol-binding protein 4 (rbp4), rdh16 (retinol dehydrogenase 16), several serpins, emx1.2 (empty spiracles homeobox 1), igf3 (insulin-like growth factor 3), igfbp1-a (insulin-like growth factor–binding protein 1), gata2 (GATA binding protein 2), and chordin. This indicates that retinol pathway and insulin-like growth factor pathway are downregulated at a later stage of ovarian development (NF62), and that these two pathways may be important for earlier stages of ovarian development. The PPAR signaling pathway and adipocytokine signaling pathway are involved in fat tissue differentiation (Ogunyemi et al. 2013) and are probably important for the development of corpora adiposa (fat tissue) at the anterior edges of the developing gonads at stages before NF62. Thus, after the fat tissue had been formed, these pathways are downregulated at stage NF62.

Another interesting gene expressed at the onset of gonadogenesis (NF50), showing upregulation at NF53 and downregulated at NF62 is chordin (chrd). Several studies showed that this gene is crucial for early organogenesis (dorsalization, gastrulation, and head development (Pappano et al. 1998; Bachiller et al. 2000), but its role in gonad development is unknown. Overall, our gene expression analysis showed that the later development of the ovary (NF62) is a very transcriptionally active period (many genes become upregulated and downregulated between NF56 and NF62), which may be related to the initialization of meiosis and oocyte formation during this developmental period.

The expression of genes during different stages of testis development

Our analysis showed that in the genetic male (ZZ) gonads at stage NF53, i.e., at the beginning of sexual differentiation, there were 659 genes with upregulated expression in comparison to the stage NF50 gonad (Suppl. Table 7, and chosen genes are presented in Table 7). Functional analysis grouped these genes into several categories (Table 8). There were the following genes with known function: igf3 (insulin-like growth factor 3), rbp4 (retinol-binding protein 4), vtn (vitronectin), several serpins, esr2 (estrogen receptor 2), several components of extracellular matrix (collagen 9, matrilin 2), and extracellular matrix (timp3) enzymes. A role of these genes in the early phase of ZZ gonad development is not known, and it would be interesting to study if retinol and/or igf3 are involved in male sex determination in Xenopus. Upregulation of PPAR and adipocytokine signaling pathways, characteristic for fat tissue, possibly reflects the onset of the development of the fat bodies at the anterior edge of the gonad.
Table 7

Chosen genes up- and downregulated in ZZ (genetic males) gonads at NF53 in relation to NF50 stage

Probe name

Gene symbol

Gene name

Log FC

Genes upregulated (higher expression at NF53 than at NF50)

  A_10_P030946

rbp4

Retinol-binding protein 4

4.97523

  A_10_P056207

vtn

Vitronectin

4.51992

  A_10_P075910

serpini2

Serpin peptidase inhibitor, clade I. 2

4.4381

  A_10_P041856

igf3

Insulin-like growth factor 3

4.34284

  A_10_P003882

timp3

TIMP metallopeptidase inhibitor 3

2.37097

  A_10_P007964

serpinf2

Serpin peptidase inhibitor, F2

2.32024

  A_10_P030126

esr2

Estrogen receptor 2 (ER beta)

2.28938

  A_10_P058537

col9a1-b

Collagen, type IX, alpha 1

2.24147

  A_10_P048579

ocln-b

Occludin

2.22878

  A_10_P002931

matn2

Matrilin 2

2.06945

Genes downregulated (higher expression at NF50 than at NF53)

  A_10_P017957

ocm

Oncomodulin

6.204741

  A_10_P140568

krt5.6

Keratin 5, gene 6

4.866387

  A_10_P138508

krt15

Keratin 15

4.154655

  A_10_P126949

mmp1

Matrix metallopeptidase 1

2.745253

  A_10_P008082

fgfbp1

Fibroblast growth factor–binding 1

2.66819

  A_10_P203798

lum

Lumican

2.460273

  A_10_P222743

isyna1-b

Inositol-3-phosphate synthase 1

2.399986

  A_10_P002391

capn8-a

Calpain 8

2.388139

  A_10_P040276

wnt7b

Wingless-type MMTV integration site family, member 7B

2.038541

Table 8

Number of genes assigned to functional groups up- and downregulated in ZZ (genetic male) gonads

Functional gene groups

ZZ (genetic males)

NF53 vs NF50

NF56 vs NF53

NF62 vs NF56

Up

Down

Up

Down

Up

Down

Signaling factors

48

13

43

17

Calcium-binding proteins

5

Metal-binding proteins

30

21

Monooxygenases

3

8

Oxidoreductases

9

8

5

14

Metalloproteinases

4

3

EGF-like domain–containing proteins

4

Proteases

13

14

9

Hydrolases

20

25

12

Disulfide bond–containing proteins

35

31

13

Secreted proteins

12

Transport proteins

3

Steroid hormone synthesis pathway

4

2

Insulin signaling pathway

4

7

PPAR signaling pathway

4

3

Adipocytokine signaling pathway

5

6

Mitochondrial proteins

7

Ion transport

5

Terpenoid backbone biosynthesis pathway

3

3

ER protein processing pathway

5

Receptors

6

Metabolic pathway

23

8

FoxO signaling pathway

7

45

Cell membrane proteins

48

Intercellular transport

21

Retinol metabolism

5

Our analysis also showed that in the genetic male (ZZ) gonads at stage NF53, there were 436 genes with downregulated expression (Suppl. Table 8, and chosen genes are presented in Table 7). Functional analysis grouped these genes in the categories shown in Table 8.

Comparison of gene expression level in the ZZ gonads between stage NF56 and NF53 showed that at stage NF56, there were 340 genes with upregulated expression (Suppl. Table 9, and chosen genes are presented in Table 9). Functional analysis grouped these genes in categories shown in Table 8. Some of these upregulated genes are rbp2 (retinol-binding protein 2), receptor of prostaglandin E (ptger3), stromal cell-derived factor 2-like 1 (sdf2l1), and neurotrophin receptor (p75NTRa). Further, studies are necessary to establish what is the exact role of the prostaglandin E, retinol, and neurotrophins in testis differentiation. Importantly, around NF53-NF56, the cortex and medulla fuse in differentiating testes, and the germ cells lose their connection with the superficial coelomic epithelium and disperse in the whole testis (Fig. 1F). There were also 340 genes downregulated at stage NF56 ZZ gonad in comparison to stage NF53 gonad (Suppl. Table 10, and chosen genes are presented in Table 9). Functional analysis grouped these genes into several categories (Table 8).
Table 9

Chosen genes up- and downregulated in ZZ (genetic males) gonads at NF56 in relation to NF53 stage

Probe name

Gene symbol

Gene name

Log FC

Genes upregulated (higher expression at NF56 than at NF53)

  A_10_P079665

rbp2

Retinol-binding protein 2, cellular

6.44222

  A_10_P043951

ptger3

Prostaglandin E receptor 3

2.75218

  A_10_P036706

sdf2l1

Stromal cell-derived factor 2-like 1

2.32111

  A_10_P000364

p75NTRa

p75 neurotrophin receptor a-1

2.02373

Genes downregulated (higher expression at NF53 than at NF56)

  A_10_P036346

LOC100189571

Uncharacterized LOC100189571

8.899836

  A_10_P102465

rbp4

Retinol-binding protein 4

7.963364

  A_10_P056207

vtn

Vitronectin

7.657184

  A_10_P027027

ptx

Pentraxin

7.367071

  A_10_P041856

igf3

Insulin-like growth factor 3

3.440657

  A_10_P002182

serpinc1

Serpin peptidase inhibitor C1

2.826371

  A_10_P094993

krt12

Keratin 12

2.032246

Comparison of gene expression level in the ZZ gonads between stages NF62 and NF56 showed that at stage NF62 gonad, there were 334 genes with the upregulated expression (Suppl. Table 11, and chosen genes are presented in Table 10). Functional analysis grouped these genes into several categories (Table 8). Around stage NF56-NF62, cells group into the testis cords (Fig. 1H). Genes involved in this process are not known, and presumably, the genes upregulated at this stage may be responsible for the formation of testis cords. There were also 831 genes downregulated in ZZ gonad at stage NF62 in comparison to stage NF56 (Suppl. Table 12, and chosen genes are presented in Table 10, and the gene categories, which were analyzed functionally are shown in Table 8).
Table 10

Chosen genes up- and downregulated in ZZ (genetic males) gonads at NF62 in relation to NF56 stage

Probe name

Gene symbol

Gene name

Log FC

Genes upregulated (higher expression at NF62 than at NF56)

  A_10_P049320

prss1

Protease, serine, 1

6.7897

  A_10_P045961

prss3

Protease, serine, 3

6.64558

  A_10_P259137

tfip11

Tuftelin-interacting protein 11

6.14112

  A_10_P027545

mmp11

Matrix metallopeptidase 11

3.31378

  A_10_P027246

klf9-a

Kruppel-like factor 9

2.7011

  A_10_P203798

lum

Lumican

2.10476

Genes downregulated (higher expression at NF56 than at NF62)

  A_10_P032408

ocm.2

Oncomodulin

7.542298

  A_10_P004053

rbp4

Retinol-binding protein 4

5.656075

  A_10_P000084

krt5.5

Keratin 5, gene 5

4.410092

  A_10_P003972

mmp28-b

Matrix metallopeptidase 28

3.191715

  A_10_P044151

fgfr4-b

Fibroblast growth factor receptor 4

3.091348

  A_10_P002657

isyna1-a

Inositol-3-phosphate synthase 1

3.030967

  A_10_P094993

krt12

Keratin 12

2.838533

Genes with sexual dimorphism of expression in ZW and ZZ gonads in different developmental stages

The master sex-determining gene in Xenopus the dm-w was discovered in 2008 (Yoshimoto et al. 2008), but the molecular machinery of sex determination is certainly very complex and contains many other genes. We previously published the expression profile of known genes involved in sex determination and sexual differentiation in the Xenopus gonads (Piprek et al. 2018). We showed that the gata4, sox9, dmrt1, amh, fgf9, ptgds, pdgf, fshr, and cyp17a1 had upregulated expression in testes, while dm-w, fst, foxl2, and cyp19a1 had upregulated expression in the ovary (Piprek et al. 2018).

Here, we compared gene expression level between ZW and ZZ gonads at different stages of gonad development. These analyses showed that at stage NF50 (undifferentiated gonads during sex determination period), there were 820 genes with upregulated expression in ZW gonad (Suppl. Table 13, and chosen genes are shown in Table 12). Functional analysis grouped these genes into several categories (Table 11). Many genes upregulated in this period are uncharacterized. Among known genes upregulated in ZW gonad at stage NF50 is chordin (chrd). Chordin is a secreted protein responsible for several developmental processes such as dorsalization, head development, and gastrulation (Sasai et al. 1994; Pappano et al. 1998; Bachiller et al. 2000); our study indicates that it may play a crucial role in female sex determination (Table 12, Suppl. Table 13). Other genes upregulated in ZW gonad at NF50 are two protease inhibitors, serpin A3 and serpin I2, extracellular glycoprotein vitronectin, metalloproteinases mmp7 and adam27, retinol-binding protein rbp4, signaling molecules wnt10b, wnt11b, and igf3, helicase ddx25, and transcription factors foxa2 and lhx8. A role of these factors in sex determination in Xenopus is unknown and requires further study.
Table 11

Number of genes assigned to functional groups expressed at higher level in ZW and ZZ gonads

Functional gene groups

NF50

NF53

NF56

NF62

ZW

ZZ

ZW

ZZ

ZW

ZZ

ZW

ZZ

Signaling factors

64

18

50

18

73

Calcium-binding proteins

3

Metal-binding proteins

28

26

Metalloproteinases

7

Progesterone-mediated oocyte maturation pathway

8

Proteases

20

9

Hydrolases

28

21

25

Disulfide bond–containing proteins

42

34

10

6

52

Extracellular matrix components

3

Markers of epithelial differentiation

2

Meiosis regulation factors

4

Oocyte meiosis

7

RNA-binding proteins

11

Phosphoproteins

11

Proteins involved in development

18

19

Cytoplasmic proteins

30

Cytoskeletal proteins

10

Nuclear proteins

35

p53 signaling

6

Secreted proteins

15

7

14

6

19

Transport proteins

5

Metabolic pathway

14

33

Intermediate filaments

3

Mitochondrial proteins

5

Insulin signaling pathway

7

Steroid hormone synthesis

3

3

Adipocytokine signaling pathway

4

FoxO signaling pathway

8

Cell membrane proteins

5

63

Cell junction proteins

4

Ion channel proteins

4

Cell division proteins

10

Mitotic proteins

6

Wnt signaling pathway

5

Table 12

Chosen genes upregulated in ZW (genetic females) in relation to ZZ (genetic males) gonads at NF50 stage [higher gene expression level in ZW than in ZZ gonads]

Probe name

Gene symbol

Gene name

Log FC

A_10_P174228

chrd

Chordin

11.30213

A_10_P007346

MGC85508

MGC85508 protein

8.151194

A_10_P008816

serpina3

Serpin peptidase inhibitor, clade A3

6.774417

A_10_P075910

serpini2

Serpin peptidase inhibitor, clade I2

6.378762

A_10_P233398

vtn

Vitronectin

5.368433

A_10_P187778

wnt11b

Wingless-type MMTV integration site family, member 11B

5.00604

A_10_P004053

rbp4

Retinol-binding protein 4, plasma

4.876474

A_10_P065884

wnt10b

Wingless-type MMTV integration site family, member 10B

4.20504

A_10_P027350

adam21

ADAM metallopeptidase domain 21

3.851196

A_10_P009298

igf3

Insulin-like growth factor 3

3.848738

A_10_P202038

MGC69070

Matrix metalloproteinase 7

3.690095

A_10_P006376

anxa13

Annexin A13

3.483353

A_10_P003549

MGC69070

Matrix metalloproteinase 7

3.459862

A_10_P000388

ddx25

DEAD box helicase 25

3.239557

A_10_P082395

foxa2

Forkhead box A2

3.049031

A_10_P003648

lhx8

LIM homeobox 8

2.965778

There were 372 genes with higher expression level in the ZZ (genetic males) gonads at stage NF50 (Suppl. Table 14, and chosen genes are shown in Table 13, and the functional groups are shown in Table 11). Among these upregulated genes are known genes such as epithelium markers keratin 5, 12, and 14, coiled-coil domain containing 50 (ccdc50) that acts as an effector in EGF signaling and negative regulator of NF-kB factor (Tsukiyama et al. 2012), signaling molecules: wnt3a, wnt7b, growth differentiation factor 3 (gdf3), fibroblast growth factor–binding protein 1 (fgfbp1), proteases cathepsin K and H, extracellular matrix molecules lumican, collagen IX and I, and decorin. A role of these genes in male sex determination and early testis development remains unknown.
Table 13

Chosen genes downregulated in ZW in relation to ZZ gonads at NF50 stage [higher gene expression level in ZZ than in ZW gonads]

Probe name

Gene symbol

Gene name

Log FC

A_10_P136703

krt14

Keratin 14

7.50258

A_10_P183185

ccdc50

Coiled-coil domain containing 50

6.57626

A_10_P140568

krt5.6

Keratin 5, gene 6

5.84154

A_10_P003366

lum

Lumican

5.75697

A_10_P193923

krt14

Keratin 14

5.1494

A_10_P008082

fgfbp1

Fibroblast growth factor–binding protein 1

3.66899

A_10_P002950

col9a1

Collagen, type IX, alpha 1

3.07046

A_10_P046256

ctsk

Cathepsin K

2.60816

A_10_P036156

dcn

Decorin

2.60212

A_10_P244713

col1a1

Collagen, type I, alpha 1

2.56712

A_10_P040276

wnt7b

Wingless-type MMTV integration site family, member 7B

2.3506

A_10_P026995

wnt3a

Wingless-type MMTV integration site family, member 3A

2.25544

A_10_P094993

krt12

Keratin 12

2.23416

A_10_P000272

gdf3

Growth differentiation factor 3

2.07545

A_10_P046876

ctsh

Cathepsin H

2.01352

There are 193 genes with a higher expression in ZW (genetic females) gonad at stage NF53 (the onset of sexual differentiation of gonads) (Suppl. Table 15, and chosen genes are shown in Table 14). Functional analysis did not link these genes to any specific pathway. Among these upregulated genes, there are the following known genes: retinol-binding protein 2 (rbp2), protease calpain 8, synuclein gamma with unknown function, cell adhesion gene claudin 6, metalloproteinases mmp1 and adam21, and galectin-la involved in cell adhesion and signaling.
Table 14

Chosen genes upregulated in ZW in relation to ZZ gonads at NF53 stage [higher gene expression level in ZW than in ZZ gonads]

Probe name

Gene symbol

Gene name

Log FC

A_10_P079665

rbp2

Retinol-binding protein 2

5.229889

A_10_P032636

LOC100101274

Uncharacterized LOC100101274

3.804135

A_10_P062524

lgalsia-a

Galectin-Ia

2.939513

A_10_P008579

krt5.2

Keratin 5, gene 2

2.846329

A_10_P057292

sncg-a

Synuclein, gamma

2.52171

A_10_P002391

capn8-a

Calpain 8

2.404349

A_10_P032511

cldn6.1

Claudin 6, gene 1

2.207111

A_10_P027350

adam21

ADAM metallopeptidase domain 21

2.177794

A_10_P126949

mmp1

Matrix metallopeptidase 1

2.07391

There were 890 genes with higher expression in ZZ (genetic males) gonad at stage NF53 (the onset of sexual differentiation of gonads) (Suppl. Table 16, and chosen genes are shown in Table 15). Functional analysis grouped these genes into several categories (Table 11). The upregulated known genes are coiled-coil domain containing 50 (ccdc50), retinol-binding protein 4 (rbp4), signaling molecules igf1 and igf3, estrogen receptor 2 (esr2), transcription factors, Kruppel-like factor 9 (klf9), Kruppel-like factor 15 (klf15), and foxo1 (forkhead box O1), enzyme glycerophosphodiester phosphodiesterase 1 (gde1) responsible for synthesis of signaling molecule lysophosphatidic acid (LPA), cell adhesion proteins gap junction protein alpha 3 (gja3), occluding (ocln), and extracellular matrix component vitronectin (vtn).
Table 15

Chosen genes downregulated in ZW in relation to ZZ gonads at NF53 stage [higher gene expression level in ZZ than in ZW gonads]

Probe name

Gene symbol

Gene name

Log FC

A_10_P183185

ccdc50

Coiled-coil domain containing 50

7.79896

A_10_P009082

gde1

Glycerophosphodiester phosphodiesterase 1

6.52222

A_10_P030946

rbp4

Retinol-binding protein 4, plasma

5.8968

A_10_P233398

vtn

Vitronectin

5.85368

A_10_P009298

igf3

Insulin-like growth factor 3

3.50579

A_10_P002488

gja3

Gap junction protein, alpha 3, 46 kDa

3.2409

A_10_P001965

klf15

Kruppel-like factor 15

2.9746

A_10_P027246

klf9-a

Kruppel-like factor 9

2.74415

A_10_P030126

esr2

Estrogen receptor 2 (ER beta)

2.42444

A_10_P027093

igf1

Insulin-like growth factor 1

2.40431

A_10_P000763

foxo1

Forkhead box O1

2.14949

A_10_P048579

ocln-b

Occludin

2.12035

There were 75 genes with higher expression in ZW (genetic females) gonad at stage NF56 (Suppl. Table 17, and chosen genes are shown in Table 16, and the functional groups are shown in Table 11). Among known genes are retinoic binding protein 4 and vitronectin.
Table 16

Chosen genes upregulated in ZW versus ZZ gonads at NF56 stage [higher gene expression level in ZW than in ZZ gonads]

Probe name

Gene symbol

Gene name

Log FC

A_10_P036346

LOC100189571

Uncharacterized LOC100189571

5.456322

A_10_P056207

vtn

Vitronectin

4.026518

A_10_P030946

rbp4

Retinol-binding protein 4, plasma

3.555976

There were 346 genes with higher expression in ZZ (genetic males) gonad at stage NF56 (Suppl. Table 18, and chosen genes are shown in Table 17, and the functional groups are shown in Table 11). Among known genes are keratin 14 and 15, cell molecule gap junction protein, alpha (gja3), endophilin B2 (sh3glb2) and coiled-coil domain containing 50 (ccdc50).
Table 17

Chosen genes downregulated in ZW in relation to ZZ gonads at NF56 stage [higher gene expression level in ZZ than in ZW gonads]

Probe name

Gene symbol

Gene name

Log FC

A_10_P084685

krt14

Keratin 14

3.53568

A_10_P171263

sh3glb2

SH3-domain GRB2-like endophilin B2

3.50581

A_10_P183185

ccdc50

Coiled-coil domain containing 50

3.3565

A_10_P138508

krt15

Keratin 15

3.13432

A_10_P002488

gja3

Gap junction protein, alpha 3, 46 kDa

2.0898

There were 594 genes with higher expression in ZW (genetic females) gonad at stage NF62 (Suppl. Table 19, and chosen genes are shown in Table 18, and the functional groups are shown in Table 11). Many genes expressed at this stage such as zona pellucida glycoprotein 4 (zp4) and zona pellucida C glycoprotein (xlzpc) are involved in ovarian follicles and oocytes formation and development. Other genes with upregulated expression at this stage were enzyme arachidonate 12-lipoxygenase 12R type (alox12b) responsible for metabolism of a signal compound—arachidonic acid (ARA), signaling factors such as growth differentiation factor 1 (gdf1), Wnt11b, cell adhesion molecules claudin 6 and connexin 38, transcription factors foxr1 and foxh1, and survivin—an inhibitor of apoptosis.
Table 18

Chosen genes upregulated in ZW versus ZZ gonads at NF62 stage [higher gene expression level in ZW than in ZZ gonads]

Probe name

Gene symbol

Gene name

Log FC

A_10_P009488

alox12b

Arachidonate 12-lipoxygenase, 12R

5.326002

A_10_P031553

zp4-a

Zona pellucida glycoprotein 4

4.350343

A_10_P032511

cldn6.1

Claudin 6, gene 1

4.081705

A_10_P038461

LOC398389

Survivin

3.781997

A_10_P034497

kpna2

Importin alpha 1b

3.634424

A_10_P048511

foxh1

Forkhead box H1

3.486778

A_10_P009533

gdf1

Growth differentiation factor 1

3.42356

A_10_P031016

foxr1

Forkhead box R1

3.378015

A_10_P005051

xlzpc

Zona pellucida C glycoprotein

2.893829

A_10_P205908

foxh1

Forkhead box H1

2.859015

A_10_P004066

LOC397866

Connexin 38

2.845325

A_10_P008731

wnt11b

Wingless-type MMTV integration site family, member 11B

2.404891

There were 2630 genes with upregulated expression in ZZ (genetic males) gonad at stage NF62 (Suppl. Table 20, and chosen genes are shown in Table 19). Functional analysis grouped these into many categories (Table 11). Among known genes with upregulated expression were factors involved in signaling and signaling pathways: igf1, desert hedgehog (dhh), sonic hedgehog (shh), indian hedgehog (ihh), wnt3a, wnt8b, wnt7b, Janus kinase 2 (jak2), frizzled receptor 4 and 10 (fzd4, fzd10), cellular retinoic acid–binding protein 2 (crabp2), SMAD family member 4 (smad4); proteases: serine protease 3 (prss3), cathepsin H (ctsh), peptidase inhibitor—serpini2; transcription factors: LIM homeobox 1 (lhx1), homeobox a9, d10, and d13 (hoxa9, hoxd10, hoxd13), foxf1, foxa2, gata2; extracellular matrix components: collagen III (col3a1), collagen I (col1a1), fibrillin 3 (fbn3); extracellular matrix enzymes: mmp2, mmp16, cell adhesion molecule 3 (cadm3); and intermediate filaments: keratin 15 and nestin (nst).
Table 19

Chosen genes downregulated in ZW in relation to ZZ gonads at NF62 stage [higher gene expression level in ZZ than in ZW gonads]

Probe name

Gene symbol

Gene name

Log FC

A_10_P077615

MGC116439

Uncharacterized protein MGC116439

8.36828

A_10_P045961

prss3

Protease, serine, 3

7.894

A_10_P075910

serpini2

Serpin peptidase inhibitor, clade I2

4.04603

A_10_P143593

ctsh

Cathepsin H

3.91699

A_10_P041916

smad4.1

SMAD family member 4, gene 1

3.54869

A_10_P186858

lhx1

LIM homeobox 1

3.43296

A_10_P067362

igf1

Insulin-like growth factor 1

3.21852

A_10_P037301

dhh-b

Desert hedgehog

3.07896

A_10_P004008

hoxd10

Homeobox D10

2.92652

A_10_P036201

krt15

Keratin 15

2.86458

A_10_P027055

shh

Sonic hedgehog

2.84344

A_10_P047936

hoxd13

Homeobox D13

2.7812

A_10_P026995

wnt3a

Wingless-type MMTV integration site family, member 3A

2.78056

A_10_P002038

mmp16

Matrix metallopeptidase 16

2.77842

A_10_P137013

col3a1

Collagen, type III, alpha 1

2.75957

A_10_P143748

crabp2

Cellular retinoic acid–binding protein 2

2.74791

A_10_P116556

wnt8b

Wingless-type MMTV integration site family, member 8B

2.72865

A_10_P139638

nes

Nestin

2.71329

A_10_P000674

foxf1-a

Forkhead box F1

2.69515

A_10_P232633

fbn3

Fibrillin 3

2.64504

A_10_P002666

cadm3

Cell adhesion molecule 3

2.53779

A_10_P040276

wnt7b

Wingless-type MMTV integration site family, member 7B

2.52685

A_10_P016774

foxa2

Forkhead box A2

2.48915

A_10_P050489

jak2

Janus kinase 2

2.48864

A_10_P000087

fzd10-a

Frizzled class receptor 10

2.46006

A_10_P267657

col1a1

Collagen, type I, alpha 1

2.43227

A_10_P162773

gata2

GATA binding protein 2

2.41128

A_10_P141938

hoxa9

Homeobox A9

2.3827

A_10_P000694

fzd4

Frizzled class receptor 4

2.3253

A_10_P027230

ihh

Indian hedgehog

2.11478

A_10_P164973

mmp2

Matrix metallopeptidase 2

2.05927

Genes identified here that showed sexual dimorphism of expression can be categorized into several functional groups: (1) signaling molecules: chordin (upregulated in ♀), wnt3a (upregulated in ♂), wnt7b (♂), wnt8b (♂), wnt10b (♀), wnt11b (♀), igf1 (♂), igf3 (♀ and ♂), gdf1 (♀), gdf3 (♂), ccdc50 (effector in EGF pathway) (♂), including hedgehog factors (♂): dhh, shh, ihh; (2) retinoic binding proteins: rbp2 (♀), rbp4 (♀ and ♂); (3) enzymes involved in signaling: enzyme glycerophosphodiester phosphodiesterase 1 (gde1) responsible for synthesis of signaling molecule lysophosphatidic acid (LPA) (♂), enzyme arachidonate 12-lipoxygenase 12R type (alox12b) responsible for metabolism of a signal compound—arachidonic acid (♀); (4) receptors of wnt signaling: fzd4 (♂), fzd10 (♂); (5) proteases: cathepsin H (♂), cathepsin K (♂), calpain 8 (♀); (6) protease inhibitors: serpin A3 (♀), serpin I2 (♀ and ♂); (7) transcription factors: foxa2 (♀), foxf1 (♂), foxh1 (♀), foxo1 (♂), foxr1 (♀), lhx1 (♂), lhx8 (♀), gata2 (♂), Kruppel-like factor 9 (klf9) (♂), Kruppel-like factor 15 (klf15) (♂); (8) helicase: ddx25 (♀); (9) cell adhesion molecules: occludin (♂), claudin 6 (♀),galectin-a (♀); (10) extracellular matrix components (mainly in ♂): collagens 1,3,9 (♂), vitronectin (♂), decorin (♂), lumican (♂), fibrillin 3 (♂); (11) extracellular matrix enzymes: mmp1 (♀), mmp2 (♂), mmp7 (♀), mmp16 (♂), adam21 (♀), adam27 (♀); (12) oocyte-specific proteins (♀): zp4, xlzpc; (13) epithelium-specific intermediate filaments (♂): keratins 5, 12, 14, 15.

The changes in the level of the expression of several genes listed above indicate that EGF signaling and lysophosphatidic acid (LPA) signaling may be involved in testis differentiation, arachidonic acid signaling may be involved in ovarian differentiation, while the wnt signaling, insulin-like growth factor signaling, and retinol signaling may be involved in gonad development in both sexes.

Interestingly, from the moment of sexual differentiation (after stage NF53), the genes encoding cytoplasmic and nuclear proteins are upregulated in ZW gonads (developing ovaries), while the genes encoding cell membrane proteins are upregulated in ZZ gonads (developing testes) (Fig. 4). The same trend was noted during gonad development in Silurana tropicalis (Haselman et al. 2015). This indicates that there are important molecular differences between developing ovaries and testes.
Fig. 4

Subcellular distribution of gene products (obtained from the Ingenuity Pathway Analysis)

Comparison of sex-specifically expressed genes in developing gonads of Xenopus and other vertebrates

We compared Xenopus microarray data to the published microarray data of developing gonads in other vertebrates: mouse (Jameson et al. 2012), chicken (Ayers et al. 2015), a red-eared slider Trachemys scripta (Czerwinski et al. 2016), American alligator (Yatsu et al. 2016)—both species with temperature-dependent sex determination, and zebrafish (Sreenivasan et al. 2008). The comparison is shown in Tables 20, 21, and 22.
Table 20

Comparison of sex-specifically expressed genes in developing gonads of Xenopus and mouse

Gene

Xenopus laevis (this paper)

Mouse (Jameson et al. 2012)

Wnt3, Wnt7, Wnt8, Wnt10, Wnt11, chordin

Sexual dimorphism

No sexual dimorphism

Igf1

Higher in ZZ

Higher in XX

Gdf1

Higher in ZW

Not expressed

Igf3, Gdf3

Higher in ZZ

Not expressed

Ccdc50

Higher in ZZ

No sexual dimorphism

Dhh, Shh, Ihh

Higher in ZZ

Only Dhh expressed

Rbp

rbp2 higher in ZW and rbp4 in ZZ and ZW

Rbp1 (in XX) and Rbp4 (in XY)

Gde1

Higher in ZZ

No sexual dimorphism

Alox12b

Higher in ZW

Not expressed

serpins

Several expressed

Not expressed

Cathepsin H (ctsh)

ctsh higher in ZZ

Only Ctsh higher in XY

Foxo1

Higher in ZZ

Higher in XY

Lhx1

Higher in ZZ

Higher in XY

Col9

Higher in ZZ

Higher in XY

MMP2

Higher in ZZ

Higher in XY

calpain 8 (Capn8)

Higher in ZW

Not expressed

Table 21

Comparison of sex-specifically expressed genes in developing gonads of Xenopus and chicken

Gene

Xenopus laevis (this paper)

Chicken (Ayers et al. 2015)

calpain 5 (Capn5)

No sexual dimorphism

Higher in ZW

gpr56

Not expressed

Higher in ZW

fgfr3

Not expressed

Higher in ZW

Table 22

Comparison of sex-specifically expressed genes in developing gonads of Xenopus and red-eared slider (Trachemys scripta), American alligator, and zebrafish

Gene

Xenopus laevis (this paper)

Red-eared slider (Czerwinski et al. 2016)

American alligator (Yatsu et al. 2016)

Zebrafish (Sreenivasan et al. 2008)

fdxr2

Slightly higher in ZZ

Higher at male-producing temperature

hspb6

Slightly higher in ZZ

Higher at female-producing temperature

twist1

Slightly higher in ZZ

Higher at female-producing temperature

nov, pcsk6

No sexual dimorphism

Higher at male-producing temperature

vwa2, rbm20

Not expressed

Higher at male-producing temperature

frank1, avil

Not expressed

Higher at female-producing temperature

kdm6b

Not expressed

Higher at male-producing temperature

wnt11

Higher in ZW

Higher at male-producing temperature

Estrogen receptor 2

esr2

Higher in ZZ

Higher in testes

The transcriptome of developing mouse gonad did not show the expression of Wnt3, Wnt7, Wnt8, Wnt10, Wnt11, and chordin (Jameson et al. 2012), which were expressed in Xenopus developing gonads. The Igf1 was expressed in XX (genetic females) mouse gonads at a higher level than in XY gonads (Jameson et al. 2012); however, in Xenopus, this gene was expressed in ZZ developing gonads (genetic males). In mouse, in contrast to Xenopus (data presented in this study), the developing gonads did not express the Igf3, Gdf1, and Gdf3 (Jameson et al. 2012). The Ccdc50 was expressed in the developing mouse gonads but did not show sexual dimorphism of expression (Jameson et al. 2012). In Xenopus, this gene had an upregulated expression in ZZ gonads. Among hedgehog growth factors, in developing mouse gonads, only the dhh was expressed (Jameson et al. 2012). In Xenopus, gonads dhh and also shh and ihh were expressed. In mice, the Rbp1 (in XX) and Rbp4 (in XY gonads) were expressed (Jameson et al. 2012). In Xenopus, the rbp2 was expressed in ZW and rbp4 in ZZ and ZW gonads. Gde1 gene was expressed in developing mouse gonads; however, it did not show sexual dimorphism of expression (Jameson et al. 2012) In Xenopus, this gene had an upregulated expression in ZZ gonads. Alox12b gene was not expressed in the developing mouse gonads (Jameson et al. 2012) but was upregulated in Xenopus ZW gonads. A subpopulation of fzd receptors was expressed in the developing mouse gonads. In Xenopus, fzd4 and fzd10 had an upregulated expression in developing ZZ gonads. The calpain 8 (Capn8) was not expressed in developing mouse gonads (Jameson et al. 2012) but was upregulated in Xenopus ZW gonads. The serpins were not expressed in developing mouse gonad (Jameson et al. 2012), but they were expressed in Xenopus developing gonads. In developing mouse gonads, several cathepsins (Cts) were expressed; however, only cathepsin H (ctsh) was upregulated in XY gonads (Jameson et al. 2012), and this gene was also upregulated in ZZ Xenopus gonads. Among forkhead box factors, only Foxo1 was expressed in XY developing mouse gonads (Jameson et al. 2012) and in ZZ Xenopus gonads. Similarly, Lhx1 was expressed in XY developing mouse gonads (Jameson et al. 2012) and ZZ Xenopus gonads. Considering proteins of extracellular matrix, only collagen 9 and metalloproteinase Mmp2 were expressed in a similar manner in XY developing mouse gonads (Jameson et al. 2012) and ZZ Xenopus gonads.

Analysis of transcriptome of developing chicken gonads showed that calpain 5 (Capn5), Gpr56, and Fgfr3 were upregulated in ZW (female) gonads, which suggested that they may be involved in sexual differentiation (Ayers et al. 2015). Calpain 5 was expressed in developing Xenopus gonads, but not in a sex dimorphic manner. We showed the upregulation of calpain 8 in ZW (females) Xenopus gonads, which suggests a role of this group of proteases in sexual differentiation of vertebrate gonads. However, calpain 5 or 8 was not expressed in developing mouse gonads (Jameson et al. 2012). Gpr56 was upregulated in XY mouse and ZW chicken gonads (Ayers et al. 2015; Jameson et al. 2012), but it was not expressed in Xenopus developing gonads. Fgfr3 showed sexual dimorphism of expression in developing chicken gonads (upregulated in ZW) (Ayers et al. 2015) and was also expressed, equally in both sexes, in mouse (Jameson et al. 2012) and Xenopus gonads.

Analysis of transcriptome of a red-eared slider (T. scripta) developing gonads showed that Vwa2, Fdxr, Nov, Kdm6b, Rbm20, and Pcsk6 were upregulated in the male-producing temperature, while Fank1, Avil, Twist1, and Hspb6 were upregulated in the female-producing temperature (Czerwinski et al. 2016). Fdxr2 and Hspb6 were also upregulated in ZW (male) developing gonads of Xenopus, but the sexual dimorphism in the level of expression was not statistically significant. Twist1 gene was slightly upregulated in ZZ gonads of Xenopus, but the sexual dimorphism in the level of expression was also not significant. We detected the expression of Nov and Pcsk6 in Xenopus gonads but these genes did not show a sexual dimorphism of expression. Among Kdms genes, we detected only the expression of kdm6a but it did not show sexual dimorphism. We did not detect the expression of Vwa2, Rbm20, Frank1, or Avil in developing Xenopus gonads.

In American alligator, the expression of Wnt11 was shown at male-producing temperature, which induces the development of the testes (Yatsu et al. 2016). We detected the expression of this gene in ZW (female) developing gonads in Xenopus. Analysis of transcriptome of zebrafish developing gonads showed that the estrogen receptor 2 (esr2) was upregulated in developing testes (Sreenivasan et al. 2008). The ZZ developing Xenopus gonads also upregulated the expression of this gene.

This comparison indicates that there is a profound difference in the pattern of gene expression and sexual dimorphism of gene expression between Xenopus and other vertebrates. Only few genes indicated above show a similar pattern of expression between Xenopus and other vertebrates. This shows how complex and fast-evolving is a molecular regulation of gonad development.

Conclusion

In this study, we revealed genes representing many functional groups, which showed sexual dimorphism of expression in developing Xenopus gonads. Some of these genes are probably involved in sex determination and sexual differentiation of the gonads. We also detected a sexual dimorphism of expression of many uncharacterized and unnamed genes. These genes should be characterized and studied further to discover if they are involved in sex determination and sexual differentiation. Comparative analysis of genes expressed in developing gonads of different classes of vertebrates showed striking inter-specific differences. Only few genes showed similarities of expression pattern between the species. This indicates how little we know and how complex, diversified, and evolutionary malleable are molecular mechanisms driving gonad development in vertebrates.

Material and methods

Animals

Tadpoles of the African clawed frog (Xenopus laevis Daudin, 1802) were raised in 10-L aquaria (30 tadpoles per 10 L) at 22 °C, fed daily with powder food Sera Micron (Sera), and staged according to Nieuwkoop and Faber (1956). The tadpoles at four stages (NF50, NF53, NF56, and NF62) were anesthetized with 0.1% MS222 solution, and the gonads were manually dissected under the dissecting microscope. All individuals used in the experiments were handled according to Polish legal regulations concerning the scientific procedures on animals (Dz. U. nr 33, poz. 289, 2005) and with the permission from the First Local Commission for Ethics in Experiments on Animals.

Sex determination by PCR

The genetic sex of each tadpole was determined using PCR detection of female-specific dm-w gene. DNA was isolated from tadpole tails using NucleoSpin Tissue Kit (Macherey-Nagel, 740952.240C). The dm-w gene (W-linked female-specific marker) and dmrt1 gene (positive control) were used to determine ZZ or ZW status of tested animals. PCR was performed as previously described (Yoshimoto et al. 2008). Following pairs of primers were used: for dm-w, 5′-CCACACCCAGCTCATGTAAAG-3′ and 5′-GGGCAGAGTCACATATACTG-3′, and for dmrt1, 5′-AACAGGAGCCCAATTCTGAG-3′ and 5′-AACTGCTTGACCTCTAATGC-3′.

Histological analysis

Bouin’s solution-fixed and paraffin-embedded samples were sectioned at 4 μm. Sections were deparafinated, rehydrated, and stained with hematoxylin and picroaniline according to Debreuill’s procedure (Piprek et al. 2012). Sections were viewed under the Nikon Eclipse E600 microscope.

RNA isolation

Total RNA was isolated using Trizol and purified with Direct-zol RNA kit according to the manufacturer’s protocol (Zymo Research, R2061). The total RNA was quantified using NanoDrop 2000, and RIN (RNA Integrity Number) was assessed with Bioanalyzer 2100. All samples used in the study had RIN above 8. In order to obtain a sufficient amount of RNA, the samples from 10 individuals were pooled in each experiment as previously described (Piprek et al. 2018). Total RNA in RNase-free water was frozen at − 80 °C until further use.

Microarray analysis

Microarray analysis was performed as previously described (Piprek et al. 2018). Total RNA was labeled with fluorescent dyes using Agilent One-Color Quick Amp Labeling Protocol. RNA isolated from ZW gonads were labeled with Cy3, and RNA from ZZ gonads with Cy5. Fluorescently labeled RNA samples were mixed with Agilent Hi-RPM Hybridization Buffer, and hybridized at 65 °C for 17 h in HybArray12 hybridization station (Perkin Elmer). RNA from ZW and ZZ were mixed together and hybridized to the same chip. The RNA isolated from the gonads in different stages of development was labeled with the same fluorochrome (either Cy3 or Cy5) and hybridized individually to the separate chips. Samples were washed in Gene Expression Wash Buffer 1 (6X SSPE, 0.005% N-lauroylsarcosine; at RT) and Gene Expression Wash Buffer (0.06X SSPE, 0.005% N-lauroylsarcosine; at RT) for 1 min each and immersed in a solution of acetonitrile. Air-dried slides (custom-commercial Agilent-070330 X. laevis Microarray slides) were scanned in the Agilent Technologies G2505C Microarray Scanner at a 5-μm resolution. The microarray experiment was repeated three times.

Data processing

Data processing was performed as previously described (Piprek et al. 2018). TIF files obtained in microarray scanner were processed using Agilent Feature Extraction software version 10.5.1.1. Control and non-uniform features were removed; remaining values for each unique probe sequence were averaged. Log base 2 intensities were median centered between arrays. Differential gene expression was filtered using a statistical significance threshold (FDR < 0.05) and a fold change threshold (2-fold). The data were published in Gene Expression Omnibus (accession number GSE105103). Functional analysis and gene ontology were carried out using DAVID 6.8 (https://david.ncifcrf.gov/tools.jsp) and IPA (Ingenuity Pathway Analysis, Qiagen). First, we compared the level of gene expression between gonads in different stages of development within each sex. The gene expression level at each stage of gonad development was compared to the gene expression level at the previous developmental stage, i.e., the stage NF53 was compared to the stage NF50, the stage NF56 was compared to the stage NF53, and the stage NF62 was compared to the stage NF56. In each comparison, the level of gene expression in the younger stage of gonad development was arbitrarily designated as the reference level of expression. The results of these analyses gave us an overview of the pattern of gene expression in consecutive stages of gonad development. Subsequently, we compared the level of gene expression between genetic female (ZW) versus male (ZZ) gonads at each studied developmental stage.

Notes

Funding information

The study was conducted within the project financed by the National Science Centre assigned on the basis of the decision number DEC-2013/11/D/NZ3/00184.

Compliance with ethical standards

All individuals used in the experiments were handled according to Polish legal regulations concerning the scientific procedures on animals (Dz. U. nr 33, poz. 289, 2005) and with the permission from the First Local Commission for Ethics in Experiments on Animals.

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References

  1. Ayers KL, Lambeth LS, Davidson NM, Sinclair AH, Oshlack A, Smith CA (2015) Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq. BMC Genomics 16:704CrossRefGoogle Scholar
  2. Bachiller D, Klingensmith J, Kemp C, Belo JA, Anderson RM, May SR, McMahon JA, McMahon AP, Harland RM, Rossant J, De Robertis EM (2000) The organizer factors Chordin and Noggin are required for mouse forebrain development. Nature 403:658–661CrossRefGoogle Scholar
  3. Bar I, Cummins S, Elizur A (2016) Transcriptome analysis reveals differentially expressed genes associated with germ cell and gonad development in the Southern bluefin tuna (Thunnus maccoyii). BMC Genomics 17:217CrossRefGoogle Scholar
  4. Beverdam A, Koopman P (2006) Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes. Hum Mol Genet 15:417–431CrossRefGoogle Scholar
  5. Chen H, Palmer JS, Thiagarajan RD, Dinger ME, Lesieur E, Chiu H, Schulz A, Spiller C, Grimmond SM, Little MH, Koopman P, Wilhelm D (2012) Identification of novel markers of mouse fetal ovary development. PLoS One 7:e41683CrossRefGoogle Scholar
  6. Czerwinski M, Natarajan A, Barske L, Looger LL, Capel B (2016) A timecourse analysis of systemic and gonadal effects of temperature on sexual development of the red-eared slider turtle Trachemys scripta elegans. Dev Biol 420:166–177CrossRefGoogle Scholar
  7. Gong W, Pan L, Lin Q, Zhou Y, Xin C, Yu X, Cui P, Hu S, Yu J (2013) Transcriptome profiling of the developing postnatal mouse testis using next-generation sequencing. Sci China Life Sci 56:1–12CrossRefGoogle Scholar
  8. Haselman JT, Olmstead AW, Degitz SJ (2015) Global gene expression during early differentiation of Xenopus (Silurana) tropicalis gonad tissues. Gen Comp Endocrinol 214:103–113CrossRefGoogle Scholar
  9. Jameson SA, Natarajan A, Cool J, DeFalco T, Maatouk DM, Mork L, Munger SC, Capel B (2012) Temporal transcriptional profiling of somatic and germ cells reveals biased lineage priming of sexual fate in the fetal mouse gonad. PLoS Genet 8(3):e1002575CrossRefGoogle Scholar
  10. Lin R, Wang L, Zhao Y, Gao J, Chen Z (2017) Gonad transcriptome of discus fish (Symphysodon haraldi) and discovery of sex-related genes. Aquac Res 48:5993–6000CrossRefGoogle Scholar
  11. Nef S, Schaad O, Stallings NR, Cederroth CR, Pitetti JL, Schaer G, Malki S, Dubois-Dauphin M, Boizet-Bonhoure B, Descombes P, Parker KL, Vassalli JD (2005) Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development. Dev Biol 287:361–377CrossRefGoogle Scholar
  12. Nieuwkoop PD, Faber J (1956) Normal tables of Xenopus laevis (Daudin), 1st edn. North-Holland, AmsterdamGoogle Scholar
  13. Ogunyemi D, Xu J, Mahesan AM, Rad S, Kim E, Yano J, Alexander C, Rotter JI, Chen YD (2013) Differentially expressed genes in adipocytokine signaling pathway of adipose tissue in pregnancy. J Diabetes Mellitus 3:86–95CrossRefGoogle Scholar
  14. Pappano WN, Scott IC, Clark TG, Eddy RL, Shows TB, Greenspan DS (1998) Coding sequence and expression patterns of mouse chordin and mapping of the cognate mouse chrd and human CHRD genes. Genomics 52:236–239CrossRefGoogle Scholar
  15. Piprek RP, Pecio A, Kubiak JZ, Szymura JM (2012) Differential effects of busulfan on gonadal development in five divergent anuran species. Reprod Toxicol 34(3):393–401CrossRefGoogle Scholar
  16. Piprek RP, Kloc M, Kubiak JZ (2016) Early development of the gonads: origin and differentiation of the somatic cells of the genital ridges. Results Probl Cell Differ 58:1–22CrossRefGoogle Scholar
  17. Piprek RP, Kloc M, Tassan JP, Kubiak JZ (2017) Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix. Dev Biol 432:298–310CrossRefGoogle Scholar
  18. Piprek RP, Damulewicz M, Kloc M, Kubiak JZ (2018) Transcriptome analysis identifies genes involved in sex determination and development of Xenopus laevis gonads. Differentiation 100:46–56CrossRefGoogle Scholar
  19. Sasai Y, Lu B, Steinbeisser H, Geissert D, Gont LK, De Robertis EM (1994) Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79:779–790CrossRefGoogle Scholar
  20. Scheider J, Afonso-Grunz F, Hoffmeier K, Horres R, Groher F, Rycak L, Oehlmann J, Winter P (2014) Gene expression of chicken gonads is sex- and side-specific. Sex Dev 8:178–191CrossRefGoogle Scholar
  21. Small CL, Shima JE, Uzumcu M, Skinner MK, Griswold MD (2005) Profiling gene expression during the differentiation and development of the murine embryonic gonad. Biol Reprod 72:492–501CrossRefGoogle Scholar
  22. Sreenivasan R, Cai M, Bartfai R, Wang X, Christoffels A, Orban L (2008) Transcriptomic analyses reveal novel genes with sexually dimorphic expression in the zebrafish gonad and brain. PLoS One 3:e1791CrossRefGoogle Scholar
  23. Sun LX, Teng J, Zhao Y, Li N, Wang H, Ji XS (2018) Gonad transcriptome analysis of high-temperature-treated females and high-temperature-induced sex-reversed neomales in nile tilapia. Int J Mol Sci 19(3):E689CrossRefGoogle Scholar
  24. Toker A (2005) The biology and biochemistry of diacylglycerol signalling. EMBO Rep 6(4):310–314CrossRefGoogle Scholar
  25. Tsukiyama T, Matsuda-Tsukiyama M, Bohgaki M, Terai S, Tanaka S, Hatakeyama S (2012) Ymer acts as a multifunctional regulator in nuclear factor-κB and Fas signaling pathways. Mol Med 18:587–597CrossRefGoogle Scholar
  26. Xu D, Shen KN, Fan Z, Huang W, You F, Lou B, Hsiao CD (2016) The testis and ovary transcriptomes of the rock bream (Oplegnathus fasciatus): a bony fish with a unique neo Y chromosome. Genom Data 7:210–213CrossRefGoogle Scholar
  27. Yatsu R, Miyagawa S, Kohno S, Parrott BB, Yamaguchi K, Ogino Y, Miyakawa H, Lowers RH, Shigenobu S, Guillette LJ Jr, Iguchi T (2016) RNA-seq analysis of the gonadal transcriptome during Alligator mississippiensis temperature-dependent sex determination and differentiation. BMC Genomics 17:77CrossRefGoogle Scholar
  28. Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehara C, Matsuda Y, Takamatsu N, Shiba T, Ito M (2008) A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis. Proc Natl Acad Sci U S A 105:2469–2474CrossRefGoogle Scholar
  29. Yoshimoto S, Ikeda N, Izutsu Y, Shiba T, Takamatsu N, Ito M (2010) Opposite roles of DMRT1 and its W-linked paralogue, DM-W, in sexual dimorphism of Xenopus laevis: implications of a ZZ/ZW-type sex-determining system. Development 137:2519–2526CrossRefGoogle Scholar

Copyright information

© The Author(s) 2019

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Department of Comparative Anatomy, Institute of Zoology and Biomedical ResearchJagiellonian UniversityKrakowPoland
  2. 2.Department of Cell Biology and Imaging, Institute of Zoology and Biomedical ResearchJagiellonian UniversityKrakowPoland
  3. 3.Univ Rennes, UMR 6290, Cell Cycle Group, Faculty of MedicineInstitute of Genetics and Development of RennesRennesFrance
  4. 4.The Houston Methodist Research InstituteHoustonUSA
  5. 5.Department of SurgeryThe Houston Methodist HospitalHoustonUSA
  6. 6.MD Anderson Cancer CenterUniversity of TexasHoustonUSA
  7. 7.Laboratory of Regenerative Medicine and Cell BiologyMilitary Institute of Hygiene and Epidemiology (WIHE)WarsawPoland

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