Gene expression and functional abnormalities in xx/sry leydig cells

ABSTRACT The _SRY_ gene induces testis development even in XX individuals. However, XX/_Sry_ testes fail to produce mature sperm, due to the absence of Y chromosome carrying genes essential

for spermatogenesis. XX/_Sry_ Sertoli cells show abnormalities in the production of lactate and cholesterol required for germ cell development. Leydig cells are essential for male functions

through testosterone production. However, whether XX/_Sry_ adult Leydig cells (XX/_Sry_ ALCs) function normally remains unclear. In this study, the transcriptomes from XY and XX/_Sry_ ALCs

demonstrated that immediate early and cholesterogenic gene expressions differed between these cells. Interestingly, cholesterogenic genes were upregulated in XX/_Sry_ ALCs, although

downregulated in XX/_Sry_ Sertoli cells. Among the steroidogenic enzymes, CYP17A1 mediates steroid 17α-hydroxylation and 17,20-lyase reaction, necessary for testosterone production. In

XX/_Sry_ ALCs, the latter reaction was selectively decreased. The defects in XX/_Sry_ ALCs, together with those in the germ and Sertoli cells, might explain the infertility of XX/_Sry_

testes. SIMILAR CONTENT BEING VIEWED BY OTHERS IMPAIRED KETOGENESIS IN LEYDIG CELLS DRIVES TESTICULAR AGING Article Open access 07 May 2025 DAPL1 IS A NOVEL REGULATOR OF TESTOSTERONE

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OF TIBETAN SHEEP Article Open access 22 October 2024 INTRODUCTION It has been established that the _SRY_ (sex-determining region on the Y chromosome) gene is responsible for the

differentiation of the testes in mammals1,2. Indeed, injection of the _Sry_ gene into fertilized XX mouse eggs leads to testis development in XX fetuses. However, XX mice carrying the _Sry_

transgene (XX/_Sry_ mice) suffer from spermatogenic failure3,4. Although the developmental defects of germ cells have been thought to be caused by the lack of Y-chromosome genes essential

for spermatogenesis5, the reason for this infertility in XX/_Sry_ mice is still under discussion. In fact, our previous study identified disfunction of XX/_Sry_ Sertoli cells6. In general,

Sertoli cells support the differentiation of germ cells by providing them with nutrients including lactate7 and cholesterol8. XX/_Sry_ Sertoli cells were found to synthesize these substances

less than XY Sertoli cells, due to lower expression levels of the genes required for their synthesis6. In addition to Sertoli cells, testes contain Leydig cells, which are developmentally

divided into two types, fetal-type (FLCs) and adult-type (ALCs). During the fetal stage, FLCs emerge within the interstitial space of the fetal testes and increase in number during embryonic

development. After birth, FLCs are gradually substituted with ALCs9. Finally, in the adult stage, the testicular interstitial space is predominantly occupied by ALCs, although a small

population of FLCs remains9,10,11. With respect to the Leydig cells in XX/_Sry_ testes, it remains largely unclear whether the ALCs in XX/_Sry_ mice exhibit functions equivalent to XY ALCs.

In general, ALCs are characterized by the functional capacity to produce testosterone. Four enzymes have been implicated in the synthesis of testosterone from cholesterol: cytochrome P450

family members cholesterol side-chain cleavage enzyme (CYP11A1) and 17α-hydroxylase/17,20-lyase (CYP17A1); 3β-hydroxysteroid dehydrogenase (HSD3B1 and HSD3B6); and 17β-hydroxysteroid

dehydrogenase (HSD17B3)12,13. Of these enzymes, CYP17A1 uniquely mediates two distinct reactions: 17α-hydroxylation and C17,20-cleavage of steroids14. Both reactions are successively

mediated by CYP17A1 in the Leydig cells of all mammalian species. Ad4BP/SF-1 (adrenal-4 binding protein/steroidogenic factor 1/NR5A115) was initially identified as a nuclear receptor-type

transcription factor that regulates the gene transcription of _CYP11A1_ and _CYP11B1_ (steroid 11β-hydroxylase)16,17,18. Thereafter, many studies have investigated whether other

steroidogenic genes are also regulated by Ad4BP/SF-1. These studies identified _HSD3B2_19,20, _CYP17A1_21,22,23, and _CYP19A1_24 as target genes of this factor. Thus, it has been widely

accepted that Ad4BP/SF-1 plays a central role in the regulation of steroidogenic genes25,26. All steroid hormones are synthesized from cholesterol. In addition to special usage for

steroidogenesis, cholesterol is known to be an essential component of various cellular membranes27. In accordance with this broad range of requirements for cholesterol, cholesterogenic genes

are expressed in a variety of cell types. Extensive investigation of cholesterogenic gene regulation in the liver has led to the identification of sterol regulatory element binding protein

2 (SREBP2, encoded by _SREBF2_) as the key transcription factor regulating all cholesterogenic genes28. In addition to this key molecule, Ad4BP/SF-1 has recently been shown to be involved in

cholesterogenic gene regulation in steroidogenic cells29. In this study, we investigated whether XX/_Sry_ ALCs are functionally different from XY ALCs. Comparison of the transcriptomes

obtained from these two types of cells revealed that the expression of immediate early genes and cholesterogenic genes was altered in the XX/_Sry_ ALCs. In addition, we found that the

17,20-lyase reaction mediated by CYP17A1 was specifically affected in XX/_Sry_ ALCs. RESULTS INCREASE OF ALCS IN XX/SRY TESTES It was previously believed that FLCs are completely replaced by

ALCs after birth. However, our previous studies have demonstrated that FLCs persist in adult mouse testes11,30. Therefore, to selectively investigate ALCs, we established a mouse line

carrying _Ad4BP-BAC-EGFP_ and _mFLE-mCherry_ as transgenes. In the mouse testes, FLCs were labeled with both EGFP and mCherry, whereas ALCs were labeled with EGFP alone. This double

transgenic mouse line thus enabled us to isolate ALCs and FLCs with no mutual contamination. We transferred these two transgenes into XY and XX/_Sry_ mice to obtain XY and XX/_Sry_ ALCs as

EGFP single-positive cells. As shown in Fig. 1a, we found both EGFP single-positive and EGFP/mCherry double-positive Leydig cells in both XY and XX/_Sry_ testes. HSD3B6, an ALC marker, was

colocalized with the EGFP in the single-positive Leydig cells, indicating that these cells were ALCs. Fluorescence-activated cell sorting (FACS) of the testicular cells enabled us to isolate

two distinct Leydig cell populations, EGFP single-positive ALCs and EGFP/mCherry double-positive FLCs, from both XY and XX/_Sry_ testes (Fig. 1b). Since the XX/_Sry_ adult testes were

hypoplastic and lacked all germ cells (Supplemental Fig. 1), the total number of cells in a single XX/_Sry_ testis was substantially lower than that in a single XY testis. Surprisingly,

however, the number of ALCs in the XX/_Sry_ testis was close to double that in the XY testis (Fig. 1b). The purity of the ALC fraction prepared by FACS was examined using qRT-PCR for

testicular cell marker genes. _EGFP_ and _Hsd3b1_ were highly enriched in ALCs from both XY and XX/_Sry_ testes, whereas _mCherry_ was barely detectable in either group (Fig. 1c). Germ cell

markers _Ddx4_ and _Prm2_ were undetectable in the ALCs, as was Sertoli cell marker _Sox9_. These results indicate that the ALC fraction used in this study was unlikely to have been

contaminated with FLCs, germ cells, or Sertoli cells. DIFFERENTIAL GENE EXPRESSION BETWEEN XY AND XX/SRY ALCS To investigate whether XX/_Sry_ ALCs differ from XY ALCs, transcriptomes were

obtained from three biologically independent sets of ALC samples each from XY and XX/_Sry_ testes. Considering the high unique mapping rate of the sequence reads (approximately 90%) and the

high reproducibility between the biological triplicates (correlation coefficient >  = 0.992; Supplemental Fig. 2a and 2b), the quality of the transcriptome datasets was considered

sufficient for further examination. Comparison of the transcriptomes revealed that the expression levels of 302 and 285 genes were more than 1.5-fold higher and lower, respectively, in the

XX/_Sry_ ALCs compared to the XY ALCs (Fig. 2a, Supplemental Tables 1 and 2). These differentially expressed genes were subjected to GO pathway analysis to investigate which biological

processes are associated with the genes up- and downregulated in the XX/_Sry_ ALCs. As listed in Fig. 2b (left panel), ‘sterol biosynthetic process’, ‘cholesterol biosynthetic process’,

‘steroid biosynthetic process’, and ‘steroid metabolic process’ were strongly related to the genes upregulated in the XX/_Sry_ ALCs. Of these genes, the ones commonly associated with these

processes were predominantly cholesterogenic. The next most strongly represented process was ‘lipid metabolic process’. Although the gene list for this process includes cholesterogenic

genes, it also includes genes specifically required for lipid synthesis. In accordance with the sharing of cholesterogenic genes, REVIGO plot analysis suggested that these processes

involving cholesterogenesis seemed to form a cluster at the top left (right panel in Fig. 2b). Multiple terms related to blood vessels were listed, and these formed another cluster (Fig. 

2b). This suggests that although we could not find any clear defect, the blood vessels of the XX/_Sry_ testes may be affected by the differential expression of these genes. In addition to

the genes included in the terms above, we noticed that the expression of extracellular matrix genes (such as those associated with several types of collagen, laminin, and biglycan) was

higher in the XX/_Sry_ ALCs, suggesting that the extracellular matrix surrounding XX/_Sry_ ALCs is different from that surrounding XY ALCs. A few biological processes were related to the

genes downregulated in the XX/_Sry_ ALCs, and their _p_-values were relatively large compared with those related to the upregulated genes (left panel in Fig. 2c). REVIGO plot analysis

suggested that these biological terms were not closely related (right panel in Fig. 2c). Although any close correlations between the listed terms and Leydig cell functions were unlikely, we

noticed that the term ‘response to cytokine’ includes the _Fos_, _Junb_, and _Jund_ genes. These gene products, leucine zipper-type transcription factors, have been studied extensively and

found to be activated in response to a variety of stimuli, such as serum, growth factors, and cytokines31. Since these genes have been classified into immediate early genes, we examined

whether the expression levels of other genes in this group were affected in XX/_Sry_ ALCs. Interestingly, many other immediate early genes, such as _Atf3_, _Egr1_, and _Myc_, were also

downregulated in XX/_Sry_ ALCs compared to XY ALCs (Fig. 2d). CHOLESTEROGENIC GENE EXPRESSION INCREASED IN XX/SRY ALCS Since cholesterogenic pathway is involved in the biological functions

activated in the XX/_Sry_ ALCs, we examined the expression of cholesterogenic genes in the XY and XX/_Sry_ ALCs. The transcriptome data indicated that almost all the cholesterogenic genes

were upregulated more than 1.5-fold in the XX/_Sry_ ALCs (Fig. 3a). This increased expression was confirmed by qRT-PCR (Fig. 3b). Numerous studies have demonstrated that SREBP2, encoded by

_Srebf2_, plays a crucial role in cholesterogenic gene regulation28. In fact, it has been demonstrated that SREBP2 accumulates in the regions upstream of cholesterogenic genes32. In

addition, we recently demonstrated that Ad4BP/SF-1 also accumulates at cholesterogenic gene loci in steroidogenic cells, including ALCs29. Therefore, we expected that at least one of these

two transcription factors would also be upregulated in the XX/_Sry_ ALCs. Although the expression of _Ad4BP/SF-1_ was unaltered in these cells, _Srebf2_ expression was slightly higher in the

XX/_Sry_ ALCs. This altered expression of _Srebf2_ could be responsible, at least in part, for the observed enhanced expression of cholesterogenic genes in the XX/_Sry_ ALCs. DIFFERENTIAL

EFFECTS ON GENE EXPRESSION BETWEEN XX/SRY ALCS AND SERTOLI CELLS We previously compared gene expression between XY and XX/_Sry_ Sertoli cells and found that cholesterogenic genes were

downregulated in the latter6. Accordingly, the present study showed that cholesterogenic gene expression was affected in opposite ways between the XX/_Sry_ ALCs and Sertoli cells. We

graphically compared whole-gene expression changes in the two types of cells. Fold changes in gene expression levels (XX/_Sry_ vs. XY) for Sertoli cells were plotted on the vertical axis and

for ALCs on the horizontal axis (Fig. 4). If genes were up- or downregulated in both types of XX/_Sry_ cells, they would be lie on or near the red broken line in Fig. 4a. However, there was

no particular pattern of distribution along this line. Instead, a considerable number of genes were aligned along the lines x = 0 or y = 0, suggesting that the alteration of gene expression

was probably cell-type specific. Cholesterogenic genes are indicated with red dots in the plot shown in Fig. 4b. As expected, many of these genes are localized in the lower right quadrant,

which is consistent with our finding that cholesterogenic genes were upregulated in the XX/_Sry_ ALCs but downregulated in the XX/_Sry_ Sertoli cells. As mentioned above, immediate early

genes were downregulated in the XX/_Sry_ ALCs. However, no biased expression of this group was detected in the XX/_Sry_ Sertoli cells. Consistent with this, the immediate early genes are

distributed within the left half of the plot area (Fig. 4c). ALTERED EXPRESSION OF GENES NORMALLY ENRICHED IN ALCS It has been established that the expression levels of _Insl3_,

_Ad4BP/SF-1_, and _Lhcgr_ (Luteinizing hormone/choriogonadotropin receptor) are enriched in ALCs33,34. In addition, we previously found several candidate genes that are probably enriched in

ALCs by comparing the transcriptomes of ALCs and FLCs30. In the present study, we examined the expression of these genes via in situ hybridization. As shown in Fig. 5a, _Agt_

(angiotensinogen) was expressed in ALCs but not in Sertoli or germ cells in adult testes. Enriched expression in ALCs has previously been observed for _Hmgcs2_

(3-hydroxy-3-methylglutaryl-CoA synthase 2)35,36, _Lcn2_ (lipocalin-2)37, and _Sepp1_ (selenoprotein P, plasma, 1)38. A high level of _Ptgds_ (prostaglandin D2 synthase) expression was

detected in ALCs, although the expression was also detected in Sertoli cells from some, but not all, testicular tubules39. Interestingly, the transcriptomes obtained in the present study

revealed that many of these genes were downregulated in the XX/_Sry_ ALCs (Fig. 5b). STEROIDOGENESIS POSSIBLY AFFECTED BY DECREASED STEROID 17,20-LYASE ACTIVITY We previously demonstrated

that the amount of testosterone synthesized in XX/_Sry_ testes at postnatal day 21 was smaller than in XY testes6. We therefore extracted the expression data for steroidogenic genes from our

transcriptome datasets. The expression of _Star_, _Cyp11a1_, _Cyp17a1_, and _Hsd17b3_ was decreased to approximately 70% of XY ALC levels in the XX/_Sry_ ALCs (Fig. 6a). Similar expression

profiles for these genes were obtained using qRT-PCR (Fig. 6b). The expression of _Ad4BP/SF-1_, a key regulator of steroidogenic gene expression, was not significantly affected in the

XX/_Sry_ ALCs, while that of _Lhcgr_ was more than doubled. To examine whether these changes affected steroidogenesis, the quantities of steroidal molecules were determined for both XY and

XX/_Sry_ testes. Testosterone synthesis from cholesterol is mediated by multiple enzymes (Fig. 7a). As indicated in Fig. 7b, the quantities of P5 (pregnenolone), P4 (progesterone), 17αOH-P5

(17α-hydroxypregnenolone), and 17αOH-P4 (17α-hydroxyprogesterone) in the XX/_Sry_ testes were greater than those in the XY testes. Interestingly, however, the quantities of DHEA

(dehydroepiandrosterone), A-dione (androstenedione), A-diol (androstenediol), and T (testosterone) in the XX/_Sry_ testes were smaller than those in the XY testes. Based on these steroid

quantities, the enzymatic activities were evaluated by calculating metabolic ratios. While 17α-hydroxylation, 3β-dehydrogenation, and 17β-hydroxylation were not significantly altered, the

17,20-lyase reaction was substantially reduced in the XX/_Sry_ testes (Fig. 7c). Interestingly, 17α-hydroxylation and 17,20-lyase reaction are mediated by a single enzyme, CYP17A1. Electrons

from NADPH/NADH required for these reactions are transferred to CYP17A1 from POR (P450 oxidoreductase) and/or CYB5A (cytochrome b5a). The expression of _Cyb5a_ was increased in the XX/_Sry_

ALCs (Fig. 7d), but that of _Por_ was not significantly altered. DISCUSSION In the present study, we aimed to determine whether XX/_Sry_ ALCs are functionally equivalent to XY ALCs. To

investigate it, transcriptomes obtained from XY and XX/_Sry_ ALCs were compared. As the consequence, the expression of 302 and 285 genes was found to be up- and downregulated, respectively,

in the XX/_Sry_ ALCs compared to XY ALCs. These gene sets suggested that several biological activities and processes are affected in XX/_Sry_ ALCs. There are potential reasons why the number

of ALCs was increased in XX/_Sry_ testes. LH has been established to be one of the key molecules for differentiation of ALCs. In fact, ALCs were decreased in the testis of _Lhcgr_ KO

mice40. Moreover, transgenic overexpression of human chorionic gonadotropin (_HCG_), which potentially binds and activates LHCGR, resulted in an increase of ALCs41,42. Based on these

findings, the increase of ALCs in the XX/_Sry_ testes might be due to the increased expression of _Lhcgr_. In addition to the endocrine factor above, there are several paracrine factors

regulating differentiation of ALCs. Desert hedgehog (DHH), secreted by Sertoli cells, stimulates proliferation of stem Leydig cells and their differentiation into ALCs43. However, our

previous study showed that the expression of _Dhh_ was not altered in the XX/_Sry_ Sertoli cells compared to XY cells6 (Supplemental Fig. 3a). Likewise, the expression of the hedgehog

signaling components such as _Ptch1/2_ and _Smo_ was not affected in the XX/_Sry_ ALCs (Supplemental Fig. 3b). PDGF is another factor to activate proliferation of stem Leydig cells43.

Although it has been unclear which cells synthesize PDGFs in adult testes, the expression of _Pdgfc_ was increased in the XX/_Sry_ Sertoli cells. Interestingly, the expression of the

receptor gene, _Pdgfra_, was increased in the XX/_Sry_ ALCs. Taken together, it was suggested that the increase of ALCs in the XX/_Sry_ testes might be attributable to the augmentation of

PDGF together with LH signaling. ALCs actively synthesize testosterone through abundant expression of steroidogenic genes. Our transcriptomic analysis revealed that the expression of all

steroidogenic genes except _Hsd3b1_ and _Hsd3b6_ was lower in the XX/_Sry_ ALCs compared to XY ALCs. Similarly, we found that the expression of genes normally enriched in ALCs was

suppressed, suggesting that the characteristic features of ALCs were affected in the XX/_Sry_ ALCs. With respect to the reason for the suppressed expression of these genes, it is interesting

to note the downregulation of immediate early genes, whose expression is activated by multiple stimuli31, in the XX/_Sry_ ALCs. Indeed, the immediate early genes such as _Fos_, _Jun_,

_Junb_, and _Jund_ (AP1 family members) are activated in ALCs by hCG44. It could therefore be assumed that the gene products above activate cellular functions by enhancing the transcription

of certain sets of target genes. In fact, steroidogenic gene transcription is regulated by the AP1 family members45,46. In addition to the steroidogenic genes, it has been demonstrated that

_LCN2_ displaying ALC-enriched expression is regulated by EGR147. In the present study, we demonstrated that the expression levels of immediate early genes were decreased in the XX/_Sry_

ALCs. The decreased expression of steroidogenic and ALC-enriched genes might therefore be caused by the downregulation of immediate early genes. Based on this scenario, the concentration of

LH secreted by the pituitary and the expression of its receptor, LHCGR, in ALCs should be considered. Our previous study showed that the serum LH concentration in the XX/_Sry_ mice was

comparable to that of the XY mice6, but the present study showed that the expression of _Lhcgr_ was higher in the XX/_Sry_ ALCs than in the XY ALCs. Therefore, the XX/_Sry_ ALCs might

receive more effectively the LH signal than the XY ALCs. If it is the case, gene transcription downstream of LH signal such as _Fos_ and _Jun_ could be activated. Nevertheless, the

expression of the immediate early genes was found to be downregulated. Therefore, this inconsistent outcome suggests that intracellular signal transduction might be abnormally regulated in

XX/_Sry_ ALCs, although it remains unknown which components and/or steps may be affected. Many transcription factors have been shown to regulate steroidogenic genes. Our transcriptome

datasets revealed that the expression of _Cebpb_ (_C/EBPβ_) and _Fos_ was decreased less than 0.67-fold while that of _Nr3c1_ was increased more than 1.5-fold in the XX/_Sry_ ALCs

(Supplemental Fig. 4). C/EBPβ and FOS were reported to regulate positively mouse _Star_ and human _CYP11A1_ genes46,48,49. Therefore, the decreased expression of _Cyp11a1_ and _Star_ genes

might be due to the downregulated expression of _Cebpb_ and _Fos_ in the XX/_Sry_ ALCs. NR3C1 (GR) was reported to act as a suppressor of mouse _Star_ gene transcription50. Thus, the

upregulated expression of _Nr3c1_ might be responsible for the decreased expression of _Star_ gene in the XX/_Sry_ ALCs. Steroidogenesis from cholesterol takes place via multiple enzymatic

reactions. Based on our analyses of the metabolic ratios, we realized that the 17,20-lyase reaction mediated by CYP17A1 was selectively affected in the XX/_Sry_ ALCs. CYP17A1 catalyzes two

reactions: 17α-hydroxylation and 17,20-lyase reaction14. In many mammalian species, cortisol (glucocorticoid) is synthesized in the zona fasciculata of the adrenal cortex, while testosterone

is synthesized in ALCs. In the former process, CYP17A1 mediates only 17α-hydroxylation, whereas in the latter process it mediates both 17α-hydroxylation and 17,20-lyase reaction. Many

studies have been performed to improve our understanding of the mechanism for selective regulation of these two reactions by CYP17A151. Some of them have focused on the two components, POR

and CYB5A, which transport electrons to CYP17A1. One study reported that POR preferentially activates the 17,20-lyase reaction52, while another reported that CYB5A is responsible for this

activation53. Concordantly, a KO study has shown that _Cyb5a_ is necessary for 17,20-lyase activity in ALCs54. Unexpectedly, however, the expression of _Por_ and _Cyb5a_ was not decreased in

the XX/_Sry_ ALCs_._ Another possible regulatory mechanism of the two reactions, phosphorylation of CYP17A1 by cAMP-dependent protein kinase, p38α, and an unknown kinase activated under

serum-free condition has been shown to selectively increase 17,20-lyase activity55,56,57. Unfortunately, however, our preliminary study failed to detect the phosphorylated CYP17A1 in the XY

and XX/_Sry_ testes. Although we could not unveil the mechanism for the selective regulation of the CYP17A1-mediated reactions, our study revealed that XX/_Sry_ ALCs could be an excellent

cellular tool for future investigation of it. In our previous study, we examined histone modifications and showed that accumulation of H3K4me3 around the upstream regions of cholesterogenic

genes was reduced in XX/_Sry_ Sertoli cells6. Considering that H3K4me3 is a mark for an active promoter, we concluded that this reduction may have led to the decreased expression of

cholesterogenic genes in the XX/_Sry_ Sertoli cells. Interestingly, our present study demonstrated that immediate early genes and cholesterogenic genes were differentially altered in

XX/_Sry_ ALCs and XX/_Sry_ Sertoli cells. Comparison of whole-genome histone modifications could contribute to a deeper understanding of the mechanisms underlying cell-type-specific

alteration of gene expression in XX/_Sry_ mice. MATERIALS AND METHODS ANIMALS Wild-type XY C57BL/6 and XX sex-reversed transgenic mice carrying the _Hsp-Sry_ transgene were used58. The

presence of the transgene and genetic sex were confirmed via PCR with primers for _Hsp-Sry_ and SX59 (Supplemental Table 3). SX is a single set of primers to amplify _Xlr_ and _Sly_ on the

X- and Y-chromosome, respectively, giving distinct banding patterns after electrophoresis. We also used _Ad4BP-BAC-EGFP_ mice and _mFLE-mCherry_ mice60, in which Leydig cells and FLCs are

labeled with EGFP and mCherry, respectively. _Sry_ transgenic mice were crossed with _Ad4BP-BAC-EGFP;mFLE-mCherry_ mice to obtain EGFP single-positive ALCs from the testes of XX/_Sry_ mice.

All protocols for the animal experiments were approved by the Animal Care and Use Committee of Kyushu University. All experiments were performed in accordance with the institutional

guidelines. CELL COUNTING AND SORTING Testes were collected from eight-week-old _Ad4BP-BAC-EGFP;mFLE-mCherry_ double-transgenic mice and dispersed with collagenase30. Total numbers of cells

from XY and XX/_Sry_ testes were counted using a Countess II FL (Thermo Fisher Scientific, Waltham, MA, USA). The dispersed cells were subjected to FACS using a BD FACS Aria SORP (BD

Biosciences, San Jose, CA, USA) and FACS Diva software (BD Biosciences) to sort the cells into two populations based on EGFP and mCherry fluorescence (ALCs: EGFP single-positive; FLCs:

EGFP/mCherry double-positive). 1,000,000 cells were analyzed to obtain the percentages of ALCs and FLCs, which were converted to the absolute numbers per testis by multiplying the total

numbers of testicular cells. The EGFP single-positive ALCs were purified by performing two FACS cycles. IMMUNOFLUORESCENCE ANALYSES Eight-week-old mice were perfused with 4% paraformaldehyde

(PFA) and their testes were collected and then immersed in 4% PFA at 4 °C for 48 h. The samples were subsequently cryoprotected in 20% sucrose at 4 °C and embedded in OCT Compound (Sakura

Finetek, Torrance, CA, USA). Immunofluorescence analyses were performed as described previously11. A rabbit antibody against HSD3B661 (1:500), a chicken antibody against EGFP (ab13970,

1:1000; Abcam, Cambridge, UK), and a mouse antibody against mCherry (ab125096, 1:200; Abcam) were used as the primary antibodies. Alexa Fluor 488-labeled goat anti-chicken IgY antibody

(ab150169, 1:500; Abcam), Alexa Fluor 555-labeled goat anti-mouse IgG antibody (A28180, 1:500; Life Technologies, Carlsbad, CA, USA), and Alexa Fluor 647-labeled goat anti-rabbit IgG

antibody (A27040, 1:500; Life Technologies) were used as the secondary antibodies. Nuclei were stained with DAPI (4′6-diamidino-2-phenylindole; Sigma–Aldrich, St. Louis, MO, USA).

Immunofluorescence was observed under a BZ-X700 microscope (Keyence, Osaka, Japan). IN SITU HYBRIDIZATION AND IMMUNOHISTOCHEMISTRY In situ hybridization was performed as previously

described62. RIKEN FANTOM cDNA clones for _Agt_ (angiotensinogen, A730059G17), _Hmgcs2_ (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2, 1300002P16), _Ptgds_ (prostaglandin D2 synthase,

2010004I02), _Lcn2_ (lipocalin-2, 2G530015N18), and _Sepp1_ (selenoprotein P, plasma, 1; I920052L16) were purchased (DNAFORM, Yokohama, Japan). Digoxigenin-labeled riboprobes for these genes

were used (Roche, Basel, Switzerland). QRT-PCR qRT-PCR was performed as previously described63 and conducted following the MIQE guidelines64. In brief, total RNA was isolated from the

sorted cells or tissues using RNeasy Micro Kit or RNeasy Mini Kit (Qiagen, Hilden, Germany) and 50 ng of total RNA was reverse-transcribed to cDNA using random hexamers and M-MLV Reverse

Transcriptase (Thermo Fisher Scientific). RNA integrity numbers (RINs) of all samples were confirmed to be higher than 7.5 using a Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

qRT-PCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the SYBR Select Master Mix (Applied Biosystems, Foster City, CA, USA). Gene expression

was determined using the standard curve method. The correlation coefficients (R2) for the standard curves were higher than 0.99. Gene expression levels were normalized to those of _Actb_

(β-actin). The primers used for the PCR are listed in Supplemental Table 3. MRNA SEQUENCING mRNA sequencing was performed as described previously30. Briefly, poly(A) RNA content was isolated

from total RNA (10 ng per sample) prepared from sorted XY and XX/_Sry_ ALCs using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA). Sequence

libraries were constructed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) and NEBNext Multiplex Oligo for Illumina (Dual Index Primers Set 1; New

England Biolabs). cDNA libraries were sequenced using a NovaSeq 6000 (51-bp pair-end; Illumina, San Diego, CA, USA). DATA PROCESSING The FastQ files were mapped using STAR software65

(version 2.7.0a; standard option) to the mouse reference genome (UCSC mm10) and the genome annotation (modified to integrate the EGFP and mCherry transgenes) was downloaded from the UCSC

Genome Browser. Bam files were generated using SamTools66 (version 0.3.3). Quality control, mapping, read count, and CPM (counts per million mapped reads) were computed using featureCounts67

(version 1.6.4; option ‘-O -p’), edgeR68 (version 3.20.9), and an in-house pipeline. MicroRNA and small nucleolar RNA genes were excluded from the analyses. Gene expression data are

presented as CPM. Mean values for biological replicates (n = 3) were calculated, and genes with CPM values < 20 in both XY and XX/_Sry_ ALCs were removed. Differentially expressed genes

were identified based on fold change and subjected to Gene Ontology (GO) analyses using DAVID. The significantly enriched biological process GO terms with _p_-values < 0.001 were

visualized in two-dimensional plots using REVIGO69. Fold changes in gene expression levels (XX/_Sry_ vs. XY) for Sertoli cells were also calculated using the transcriptome data in our

previous study6 (accession number: DRA004090). When comparing whole gene expression changes in the two types of cells, a pseudo-count of 10 was added to the CPM values before the fold

changes were calculated. MEASUREMENT OF INTRATESTICULAR SEX STEROIDS Testes obtained from eight-week-old XY and XX/_Sry_ mice were lyophilized using a Vacuum Centrifugal Evaporator

(CVE-2000; EYELA, Tokyo, Japan) and stored at − 80 °C until later use. Gas chromatography–mass spectrometry steroid profiling was performed using a Shimadzu GC 2010 Plus gas chromatograph

coupled to a triple-quadrupole GCMS-TQ8050 (Shimadzu Corporation, Kyoto, Japan) as previously described70. Quantitative results were based on absolute quantities of steroid molecules per

testis, and their metabolic ratios were also calculated to express their corresponding enzymatic activities. STATISTICAL ANALYSIS At least three biologically independent samples were used in

all experiments. Data are presented as mean ± SEM. Differences between XY and XX/_Sry_ cells or testes were examined using two-tailed Student’s _t_-tests or Mann–Whitney _U_ tests, and

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  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP17H06427

(K.-i.M.), JP16H05142 (K.-i.M.), JP16K08593 (T.B.), and JP19J12133 (S.Y.), Takeda Science Foundation (T.B.), and The Shin-Nihon Foundation of Advanced Medical Research (T.B.). This work was

technically supported by the Research Support Center, Graduate School of Medical Sciences, Kyushu University. We are profoundly thankful to Prof. Mikita Suyama (Kyushu University, Japan) for

technical support in the NGS data processing, and Profs Hitoshi Okamura and Masao Doi (Kyoto University, Japan) for kindly providing the HSD3B6 antibody. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Systems Life Sciences, Graduate School of Systems Life Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan Shogo Yanai, Takashi

Baba, Kai Inui, Fumiya Takahashi, Yasuyuki Ohkawa & Ken-ichirou Morohashi * Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1,

Higashi-ku, Fukuoka, 812-8582, Japan Takashi Baba, Kanako Miyabayashi, Miki Inoue & Ken-ichirou Morohashi * Molecular Recognition Research Center, Korea Institute of Science and

Technology, Seoul, 02792, Korea Soyun Han & Man Ho Choi * Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan Yoshiakira Kanai *

Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan Yasuyuki Ohkawa * AMED-CREST, Japan Agency for

Medical Research and Development, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan Yasuyuki Ohkawa Authors * Shogo Yanai View author publications You can also search for this author

inPubMed Google Scholar * Takashi Baba View author publications You can also search for this author inPubMed Google Scholar * Kai Inui View author publications You can also search for this

author inPubMed Google Scholar * Kanako Miyabayashi View author publications You can also search for this author inPubMed Google Scholar * Soyun Han View author publications You can also

search for this author inPubMed Google Scholar * Miki Inoue View author publications You can also search for this author inPubMed Google Scholar * Fumiya Takahashi View author publications

You can also search for this author inPubMed Google Scholar * Yoshiakira Kanai View author publications You can also search for this author inPubMed Google Scholar * Yasuyuki Ohkawa View

author publications You can also search for this author inPubMed Google Scholar * Man Ho Choi View author publications You can also search for this author inPubMed Google Scholar *

Ken-ichirou Morohashi View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.Y., T.B. and K.-i.M. conceived and designed the experimental

approach and performed experiments. S.Y. and K.-i.M. prepared the manuscript. K.I. contributed to the computational analyses for mRNA-seq. M.I. and K.Mi. performed in situ hybridization.

S.H. and M.H.C. measured intratesticular sex steroids. F.T. constructed the mRNA-seq libraries. Y.K. provided the transgenic mice. Y.O. performed deep sequencing of the mRNA-seq libraries.

CORRESPONDING AUTHOR Correspondence to Ken-ichirou Morohashi. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE

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