Article Nuclear and Membrane Receptors for Sex Steroids Are Involved in the Regulation of Delta/Serrate/LAG-2 Proteins in Rodent Sertoli Cells Sylwia Lustofn 1,†, Alicja Kami´nska1,†, Ma gorzata Brzoskwinia 1, Joanna Cyran 1 , Ma gorzata Kotula-Balak 2 , Barbara Bili´nska1 and Anna Hejmej 1,* 1 Department of EndocrinologyInstitute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University in Krakow, 30-387 Krakow, Poland; s.lustofn@doctoral.uj.edu.pl (S.L.); ala.kaminska@uj.edu.pl (A.K.); m.brzoskwinia@doctoral.uj.edu.pl (M.B.); joanna.cyran@doctoral.uj.edu.pl (J.C.); barbara.bilinska@uj.edu.pl (B.B.) 2 Departmentof Anatomy andPreclinical Sciences, University CentreofVeterinary Medicine JU-UA, University of Agriculture in Krakow, 30-059 Krakow, Poland; malgorzata.kotula-balak@urk.edu.pl * Correspondence: anna.hejmej@uj.edu.pl † These authors contributed equally to this work. Abstract: Delta/Serrate/LAG-2 (DSL) proteins, which serve as ligands for Notch receptors, mediate direct cell–cell interactions involved in the determination of cell fate and functioning. The present study aimed to explore the role of androgens and estrogens, and their receptors in the regulation of DSLproteinsin Sertoli cells.To this end, primary rat Sertoli cells and TM4 Sertoli cell line were treated with either testosterone or 17ß-estradioland antagonistsoftheirreceptors.To confrmtherole of particular receptors, knockdown experiments were performed. mRNA and protein expressions of Citation: Lustofn,S.;Kami´nska,A.; Jagged1(JAG1), Delta-like1(DLL1),and Delta-like4(DLL4)were analyzedusingRT-qPCR,Western Brzoskwinia,M.;Cyran,J.; blot,and immunofuorescence.Testosterone causeddownregulationofJAG1andDLL1expression, Kotula-Balak,M.;Bili´nska,B.;Hejmej, actingthrough membraneandrogenreceptorZRT-and Irt-likeprotein9(ZIP9)or nuclearandro- A. Nuclear and Membrane Receptors gen receptor (AR), respectively. DLL4 was stimulated by testosterone in the manner independent for Sex Steroids Are Involved in the of AR and ZIP9 in Sertoli cells. The expression of all studied DSL proteins was upregulated by Regulation of Delta/Serrate/LAG-2 17ß-estradiol. Estrogen action on JAG1 and DLL1 was mediated chiefy via estrogen receptor . Proteins in Rodent Sertoli Cells. Int. J. (ER.), while DLL4 was controlled via estrogen receptor ß (ERß)and membrane G-protein-coupled Mol. Sci. 2022, 23, 2284. https:// estrogenreceptor (GPER).To summarize, the co-operationof nuclear and membranereceptors for doi.org/10.3390/ijms23042284 sex steroids controlsDSLproteinsin Sertoli cells, contributingto balancedNotch signaling activityin Academic Editor: Jerome seminiferous epithelium. F. Strauss III Keywords: androgen receptors; estrogen receptors; DSL proteins; Notch signaling; Sertoli cells Received: 23 January 2022 Accepted: 15 February 2022 Published: 18 February 2022 Publisher’s Note: MDPI stays neutral withregardtojurisdictional claimsin published maps and institutional affl­iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license(https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction Aseries of studiesperformed at the beginning of the 21st century and subsequent research clearly demonstrated that proper androgen/estrogen balance is fundamental for normal male sexual development and function in humans and animals[1–6]. This balance is governed primarilybyaromatase, which catalyzestheirreversible conversionofandrogenic steroids (testosterone and androstenedione), produced by testicular interstitium, into the estrogens (estradiol and estrone), as well as by the expression of nuclear androgen (AR) and estrogen receptors (ER. and ERß), determining cell response to these hormones[7,8]. Duetoadiscoveryof membrane androgenand estrogenreceptors(ZRT-and Irt-likeprotein 9, ZIP9, and membrane G-protein-coupled estrogen receptor, GPER) and their localization in the male gonad, it has become increasingly apparent that the testicular androgen– estrogen systemis more complex than initially thought[9–13].Variationsin the balanceof sex steroidsor their actionarerelatedto testicular disordersand infertility [14–16]andare Int. J. Mol. Sci. 2022, 23, 2284. https://doi.org/10.3390/ijms23042284 https://www.mdpi.com/journal/ijms alsoa featureofageing[17].Therefore,it appears importantto understandtherolethatthe components of this system play in the regulation of testicular cell function. Sertoli cells constitute the somatic component of the seminiferous epithelium, which controls male germ cell development by providing structural, nutritional, and regulatory support for spermatogenic cells. Androgens are major regulators of postnatal Sertoli cell physiology and, indirectly, spermatogenesis.Testosterone actionis indispensable for Sertoli cell maturation, blood–testis barrier formation and maintenance, germ cell meiosis, their differentiation, andreleasefrom seminiferous epithelium[18]. Although theroleof estrogens in the seminiferous epithelium is less clear-cut, these hormones are considered important modulators of Sertoli cell proliferation, differentiation, survival, and energy metabolism[19,20]. Rodent postnatal Sertoli cellsshowadynamic patternoftheexpression of nuclear and membrane androgen and estrogenreceptors, thereby enablingaco-ordinated response to hormonal stimulation[12,19,21,22]. Delta/Serrate/LAG-2(DSL)proteinsaretypeItransmembraneproteins characterized by the presence of an extracellular N-terminal DSL motif and epidermal growth factor (EGF)-like repeats. They serve as ligands for the receptors of Notch family (Notch 1– 4) to mediate direct cell–cellinteractions involved in the determination of cell fate and functioning. In seminiferous epithelium, the proper activity of the Notch pathway is crucial forthe balance between spermatogoniaproliferationanddifferentiation.It controlsgerm cell fate and survival throughout the spermatogenesis and regulates the expression of tight junction proteins[19,23–26]. In mammals, fve canonical DSL proteins were identifed. They are classifed on the basis of the presence (Jagged/Serrate; JAG1 and JAG2) or absence (Delta-like; DLL1, DLL3, and DLL4) of a cysteine-rich domain. JAG1, JAG2, and DLL4 possess additional Delta and OSM-11-like (DOS) domain[27]. Upon ligand–receptor binding, the ligand undergoes endocytosis into the signal-sending cell. This createsa force that causesa conformational change and promotes receptor activation (called trans-activation). Then Notch receptor undergoestwoproteolytic cleavages.Asaresult,the Notch intracellular domainisreleased and translocatesintothe nucleustoengagein transcriptionregulation[28].DSLligands co-expressedinthe samecellwith Notchreceptorsareableto inhibitthe activationofthe receptors(cis-inhibition), thereby suppressing the intracellular signal. Recently, the process of cis-activation wasalso describedin diverse cell types, extending the rangeof possible modesof Notch signaling[29]. Both trans-and cis-interactions are highly sensitive to the relative levelsof ligandsandreceptors,anda switch betweentwocell states, signal-sending and signal-receiving states,maybe generated[30].Of note,rodent Sertoli cells express Notch receptors and DSL ligands, and thus may be considered as both signal-receiving and signal-sending cells[31–33]. Although the expression levelsof Notch ligandsin Sertoli cellsarelowerin comparisontogermcells[24],the biological signifcanceofDSLproteins in Sertoli cells has already beenreported[32]. Moreover, thepresenceof Notchrecep­ tors in germ cells suggests that Sertoli-cell-derived DSL proteins are implicated in Notch signaling in germ cells, which is important for normal spermatogenesis [31,34–36]. The pre­cise control of Notch ligand expression may be, therefore, crucial for the maintenance of seminiferous epithelium homeostasis. Nevertheless, to date, little is known about factors and mechanisms controlling DSL proteins in Sertoli cells. Our previous study revealed that anti-androgen exposure and androgen withdrawalresultedin disturbed expression of DLL1, DLL4, and JAG1 in rat testis in vivo [33], but detailed mechanisms were not determined.Veryrecently, peroxisomeproliferator-activatedreceptor . was also identifed as aregulator of Dll4 mRNA expressionin boar postnatal testis[37]. In the present study, we aimed to explore the role of testicular sex steroids and their receptors in the regulation of DSL protein expression in rodent Sertoli cells. Primary rat Sertoli cells (PSC) and TM4 Sertoli cell line were treated with either testosterone or 17ß­estradiol and pharmacological inhibitors of classical (nuclear) or nonclassical (membrane) androgenandestrogenreceptors.In addition,to unveilthepreciseroleofeachreceptor, the silencing of genes that encode the receptors was performed in TM4 cells. 2. Results 2.1. The RoleofTestosteronein the Controlof DLL1, DLL4, and JAG1in Sertoli Cells All studied DSL proteins (DLL1, DLL4, and JAG1), as well as AR and ZIP9, were ex­pressedinbothPSCandTM4 Sertolicells(Figure 1,Figure 2andFigureS1).Asdemon-stratedbyRT-qPCR,Western blot, and immunofuorescence analyses, mRNA andprotein expression of JAG1 decreased following testosterone exposure(p < 0.01; p < 0.001) in both PSC (Figure 1a,b,g) and TM4 cells (Figure 2a,b,g). Exposure to hydroxyfutamide (HF; AR antagonist) had no effect on testosterone-stimulated JAG1 expression, whereas bicalutamide (Bic; AR and ZIP9 antagonist) abrogated the effect of testosterone on JAG1. This suggests the role ofmembrane androgen receptor ZIP9 in the control of JAG1 ex­pression.To further confrm the mechanisms involvedin JAG1regulationby testosterone, AR and ZIP9 were knocked down in Sertoli cells. Since transfection effciency in primary Sertoli cells is low ([38]; our unpublished observations), we used TM4 cell line for these experiments. Following knockdownof ZIP9in TM4 cells, testosterone did notreduce JAG1 expression(p<0.01;p<0.001). In contrast, AR knockdown was ineffective in blocking the actionof testosterone (Figure 2h,i). The sameeffects were also clearly demonstrated using immunofuorescence analysis (Figure 2n). These observations confrmed that mainly ZIP9 is involved in the regulation of JAG1 by testosterone. Testosterone reduced the expression of mRNA and protein expression of DLL1 (p < 0.05; p < 0.01), both in PSC (Figure 1c,d) and TM4 cells (Figure 2c,d). Treatment with HF or Bic abolished the effect of testosterone on DLL1 expression. These fndings were confrmedby theresultsof immunofuorescence analysis, showing decreased signal intensity in the cells treated with testosterone alone, but not in the cells incubated with testosteroneandHForBic,whencomparedtothecontrolcells(Figures 1gand2g). Silenc­ ing experiments demonstratedthat only AR knockdown abolished the effect of testosterone (p< 0.001) on DLL1 expressionin TM4 cells (Figure 2j,k,n), which corroboratesresults obtainedfromexperiments with pharmacological inhibitors. Theseresults suggest that the ARis involvedin theregulationof DLL1 expression. In contrast to the effect on DLL1 and JAG1, testosterone enhanced the expression of DLL4 mRNA and protein(p< 0.01; p< 0.001) in both PSC (Figure 1e,f,g) and TM4 cells (Figure 2e,f,g).Testosterone-induced expressionof DLL4 was inhibited neitherby HF nor Bic. Likewise, increased DLL4 expression in TM4 cells treated with testosterone (p < 0.01;p<0.001)persisted following the AR or ZIP9 knockdown (Figure2l,m). Im­munofuorescence analysis confrmedWestern blot data (Figure 2n). Based on these fnd­ ingsDLL4regulationbytestosterone seemstobe independentoftheARandZIP9 activation in Sertoli cells. 2.2. The Role of 17ß-Estradiol in the Control of DLL1, DLL4, and JAG1 in Sertoli Cells We confrmed that ER., ERß, and GPER proteins are expressed both in PSC and TM4 cells. The level of ER. protein in PSC is lower than in TM4 cells, whereas protein expression levels of ERß and GPER arecomparable between both cellular models (FigureS1). Exposure of PSC and TM4 to 17ß-estradiolresultedin the marked increasein the expressionof DLL1, DLL4, and JAG1 mRNA andprotein(p<0.05;p<0.01;p<0.001) as detectedbyRT-qPCR andWesternblot,respectively(Figures 3and4).Inthe caseof immunofuorescence analysis, a morepronouncedeffect was foundin TM4 cells thanin PSC (Figures 3gand4g,n). Figure 1. Theeffectofandrogenreceptor antagonistsonmRNAandproteinexpressionof Jag1/JAG1, Dll1/DLL1, and Dll4/DLL4 in primary rat Sertoli cells. Cells were treated with 10-8Mtestosterone (T), 10-4Mhydroxyfutamide (HF),HF+T, 10-6Mbicalutamide (Bic), Bic+T, or vehicle (C) for 24 h.(a,c,e)Relative expressionof mRNAs (RQ) was determined usingreal-timeRT-PCR analysis. The expression values of the individual genes were normalized to the mean expression of the reference genes.(b,d,f)Western blot detection of the proteins. The relative level of studied protein was normalized to ß-actin. The protein levels within the control group were arbitrarily set at 1. The histograms are the quantitative representation of data (mean ± SD) of three independent ex­periments, each in triplicate. Signifcant differences from control values are denoted as * p< 0.05, ** p<0.01, and ***p<0.001.(g)Immunofuorescence analysis of JAG1, DLL1, and DLL4 expression. Scale bar = 10 µm. Figure 2. Theeffectof androgenreceptor antagonists or androgenreceptor silencing on mRNA and protein expression of Jag1/JAG1, Dll1/DLL1, and Dll4/DLL4in TM4 Sertoli cell line.(a–g)Cells were treated with 10-8Mtestosterone (T), 10-4Mhydroxyfutamide (HF), HF+T, 10-6Mbica­lutamide (Bic), Bic+T, or vehicle (C) for 24 h.(h–n)Cells were treated with transfection reagent alone (C), transfectionreagent+5 × 10-8Mnon-targeting siRNA (negative control, NT), transfection reagent +5 × 10-8 M AR siRNA (AR-Kd), or ZIP9 siRNA (ZIP9-Kd). After 24 h, 10-8MT or vehicle was added to the culture.(a,c,e,h,j,l)Relative expression of mRNAs (RQ) was determined usingreal-timeRT-PCR analysis. The expression valuesof the individual genes were normalized tothe mean expressionofthereference genes.(b,d,f,i,k,m)Western blot detection of the proteins. The relative level of studied protein was normalized to ß-actin. The protein levels within the control groupwere arbitrarilysetat1.The histogramsarethe quantitativerepresentationofdata(mean ± SD) of three independent experiments, each in triplicate. Signifcant differences from control values are denoted as * p<0.05, **p<0.01, and ***p<0.001.(g,n)Immunofuorescence analysis of JAG1, DLL1, and DLL4 expression. Scale bar = 10 µm. The 17ß-estradiol-stimulated increase in JAG1 expression was blocked by ICI 182,780 (ER./ß antagonist)(p < 0.01), but not by G15 (GPER antagonist), as detected by RT-qPCR,Western blot, and immunofuorescence analyses (Figure 3a,b,g and Figure 4a,b,g). This indicates that primarily nuclear ERs are involved in the regulation of JAG1 in Sertoli cells. Knockdown experiments demonstrated that ER. silencing completely blocked estradiol-induced upregulation of JAG1 mRNA and protein expression(p<0.001) in TM4 cells. ERß silencingreducedtheeffectof estradiolonJAG1(p< 0.05). GPER silencing reduced this effect at mRNA level(p< 0.01), but not at the protein level (Figure 4h,i,n). JAG1 regulation by 17ß-estradiol in Sertoli cells is, therefore, mediated via the ER. and ERß, but ER. hasa prevailing role. ICI 182,780 abolished the effect of 17ß-estradiol on the expression of DLL1 mRNA and protein(p<0.01;p<0.001), whereas G15 was ineffective in blocking 17ß-estradiol action in PSC (Figure 3c,d) and TM4 cells (Figure 4c,d). Immunofuorescence analysis confrmed Westernblotdata(Figures3gand4g). Theseresults suggestacontributionof nuclearERsto theregulationof DLL1in Sertoli cells. Knockdown experimentsrevealed thatan increasein Dll1 mRNA expressioninresponseto17ß-estradiol was blocked by ER. or ERß silencing (p<0.001), while GPER silencing was ineffcacious (Figure4j).Protein expressionof DLL1, however, was suppressed only by ER. silencing(p<0.01) (Figure4k,n). Thus,in TM4 cells, ER. seems to play a dominant role in the control of DLL1. Neither ICI 182,780 nor G15 infuenced 17ß-estradiol-stimulated mRNA expression of Dll4 in PSC (Figure 3e) and TM4 cells (Figure 4e); however, a decreasein DLL4protein expression was found when compared to 17ß-estradiol-stimulated cells(p<0.05) (Figures3f and4f). Moreover, a decreased immunofuorescence signal was observed after exposureof PSCandTM4 cellstoICI 182,780orG15 (Figures 3gand4g), which impliesthe involvement of both nuclear and membrane estrogenreceptors. In TM4 cells, estrogen-stimulated mRNA and protein expression of DLL4 was abolished by ERß knockdown(p<0.05), while ER. silencing had some effect only on Dll4 mRNA expression(p< 0.05). GPER knockdown inhibited the effect of 17ß-estradiol on DLL4 protein expression(p< 0.01), but not on mRNA expression (Figure 4l,m). Immunofuorescence analysis confrmedWestern blot data (Figure 4n).Taken together,ERß and GPER seem to mediate estrogen action on DLL4 protein expression. Figure 3. The effect of estrogen receptor antagonists on mRNA and protein expression of Jag1/JAG1, Dll1/DLL1, and Dll4/DLL4 in primary rat Sertoli cells. Cells were treatedwith 10-9M17ß-estradiol (E), 10-6MICI 182,780 (ICI), ICI+E,10-8MG15,G15+E,or vehicle(C)for24h.(a,c,e)Relative expression of mRNAs (RQ) was determined using real-time RT-PCR analysis. The expression values of the individual genes were normalized to the mean expression of the reference genes. (b,d,f)Western blot detection of the proteins. The relative level of studied protein was normalized to ß-actin. The protein levels within the control group were arbitrarily set at 1. The histograms are the quantitativerepresentationof data (mean ± SD) of three independent experiments, each in triplicate. Signifcant differences from control values are denoted as * p< 0.05, ** p< 0.01, and *** p< 0.001. (g)Immunofuorescence analysis of JAG1, DLL1, and DLL4 expression. Scale bar =10 µm. Figure 4. The effect of estrogen receptor antagonists or estrogen receptor silencing on mRNA and protein expression of Jag1/JAG1, Dll1/DLL1, and Dll4/DLL4 in TM4 Sertoli cell line.(a–g)Cells were treated with 10-9M17ß-estradiol (E), 10-6MICI 182,780 (ICI), ICI+E,10-8MG15,G15+E, or vehicle (C) for 24 h.(h–n)Cells were treated with transfection reagent alone (C), transfection reagent+5 × 10-8Mnon-targeting siRNA (negative control, NT), transfectionreagent+5× 10-8M ER. siRNA (ER. -Kd), ERß siRNA (ERß -Kd), or GPER siRNA (GPER-Kd). After 24 h, 17ß-estradiol or vehicle was added to the culture.(a,c,e,h,j,l)Relative expression of mRNAs (RQ) was determined usingreal-timeRT-PCR analysis. The expression valuesof the individual genes were normalized tothe mean expressionofthereference genes.(b,d,f,i,k,m)Western blot detection of the proteins. The relative level of studied protein was normalized to ß-actin. The protein levels within the control groupwerearbitrarilysetat1.The histogramsarethe quantitativerepresentationofdata(mean ± SD) of three independent experiments, eachin triplicate. Signifcantdifferencesfrom control values are denoted as * p<0.05, **p<0.01, and ***p<0.001.(g,n)Immunofuorescence analysis of JAG1, DLL1, and DLL4 expression. Scale bar = 10 µm. 3. Discussion Inthepresentstudy,wehave demonstratedtheroleofsexsteroidsandtheirrespective receptors in the control of DSL proteins in rodent Sertoli cells. Our fndings provide evidence that androgens and estrogens have an opposite effect on the expression of DLL1 and JAG1 proteins in Sertoli cells: testosterone downregulates their expression, whereas estradiol exertsa stimulatoryeffect. Our previous study showed increased expression of JAG1 in the testes of pubertal rats after androgen signaling disruption in vivo [33], suggesting an inhibitory effect of androgensonJAG1. However,inthatstudy,testicularcelltypesthatrespondedtoandrogen signaling disruption with changes in JAG1 expression were not identifed. The results presented hereinrevealed thattestosterone suppresses JAG1in Sertoli cells and thiseffect appearedtobe mediatedbyZIP9.Inagreement,Okadaetal.[39]showedthatcAMP,a second messenger involved, i.a.,in testosterone/ZIP9 signal transduction[10], decreased Jag1 mRNA levelin isolated mouse Sertoli cells. The limitedroleoftheARintheregulation of JAG1 was also described earlier in DU145 and PC3 prostate cancer cells, in which AR overexpressionhad almostnoeffectonthe levelofJAG1[40]. JAG1is knownto undergo ectodomain shedding, whichisa post-translational event independentof the expression level of mRNA[41], and the ability of Sertoli cells to release JAG1 was documented previously[32]. Martinetal.[42]foundthat syntheticandrogenR1881,anagonistofthe AR, increased JAG1 level in prostate cancer cells LNCaP conditioned medium. It cannot, therefore, be excluded that, although Jag1 gene expression in Sertoli cells is controlled primarilyby ZIP9, testosterone action viaAR modulates thereleaseof JAG1 ectodomain. This issue remains to be elucidated. In contrast to the effect observed in Sertoli cells, elevated testosterone induced JAG1 expression in the interstitial tissue of murine immature testis, as well asin the activated macrophages[43,44], indicating clearly context-dependent regulation of this protein. Inagreementwiththeresultsofourprevious invivo study, whichrevealedupregu­lated mRNA and protein expression of DLL1 in the testes of rats in response to futamide (antiandrogen) or testosterone deprivation[33], herein,itis found that DLL1 expression in Sertoli cells is negatively regulated by testosterone. Based on data from antagonist exposures and knockdown experiments, we demonstrated that this regulation is medi­ated through the AR, while ZIP9 is not involved. According to our knowledge, these are, to date, the frst reports on the role of androgen–AR system in the control of DLL1 expres­sion in mammals. In the only earlier published paper, no effect of testosterone on Dll1 in the murine gubernaculum was demonstrated[45]. Wehaverecentlyreportedthat testosteroneincreasesthe activityofthe Notch pathway in rat and mouse Sertoli cells, upregulating the expression of Notch1 intracellular domain and the effector genes Hes1 and Hey1 [46]. In light of these data, the results of the present study suggest that enhanced Notch pathway activity following testosterone exposure may be, at least to some extent, associated with reduced expression of DLL1 or JAG1 proteins, which potentially prevents cis-inhibitory interactions within Sertoli cells. Moreover, our earlier study showed that JAG1 and DLL1 are involved in the control of androgen recep­tor expression in Sertoli cells. Exposure of the cells to immobilized recombinant JAG1 inhibited ZIP9 mRNA and protein expression, whereas immobilized DLL1 reduced the AR expression[12]. Theseresults, together with ourpresent fndings, implya feedback regulatory mechanism in which androgen receptors are involved in the maintenance of proper expression level of DSL proteins in Sertoli cells. In contrasttoJAG1andDLL1,DLL4was positivelyregulatedbytestosterone.Thestim­ulatoryeffectof testosterone on DLL4in Sertoli cells supports theresultsof our abovemen­tioned study in which androgen withdrawal in vivo caused decreased expression of DLL4 in pubertal rat testis[33]. Although theprevious study alsorevealed markedly decreased immunostaining for DLL4 in Sertoli cells of futamide-treated rats, herein, neither the exposure to AR antagonists nor AR knockdown abrogated testosterone-stimulated DLL4 expression. One possible explanation for this discrepancy couldbe thepresenceof other AR-expressing cells in the testis of rats used in the former study. It is likely that changes in DLL4 expression in Sertoli cells observed following futamide treatment in vivo could be mediated by an indirect mechanism (e.g., paracrine), not by AR blockade in Sertoli cells. Notably, specifc ablation of AR expression in peritubular myoid cells disturbed Sertoli cell functions and altered gene expressionin Sertoli cells[47]. Moreover,in mice lacking testicular AR specifcally in the Leydig cells, dysfunction of seminiferous epithelium was reported[48]. Thus, the effect of testosterone on DLL4 expression in Sertoli cells seems to be independent of Sertoli-cell-expressed AR. Furthermore, ZIP9 silencing was also ineffective in preventing the effect of testosterone, indicating the lack of ZIP9 involvement in DLL4 control in Sertoli cells. Further studies are required to elucidate a mechanism of DLL4 regulation by testosterone in Sertoli cells. It cannot be ruledout, however, that this effect is mediatedby otherreceptors, suchasG-protein-coupledreceptor classCgroup6memberA (GPRC6A)or transientreceptor potential cation channel subfamilyMmember8(TRPM8), which are localizedinrodent Sertoli cells and androgens are among their ligands[49–52]. The opposite effects of testosterone on different DSL proteins are likely related to the fact that different DSL ligands may trigger different responses of the cell. Recently, Nandagopaletal.[53]proposedthe mechanismthatleadstoeitherpromotionor inhibition of somite myogenesis, depending on the activating ligand, DLL1 or DLL4. Moreover, the results of our previous study demonstrated that different DSL proteins, DLL1 and JAG1, negativelyregulatetheexpressionofdifferentandrogen-dependent junctionproteins in Sertoli cells, claudin 11 or claudin 5, respectively, whereas DLL4 has no effect on their expression[12]. Summing up, androgens exert diverse effects on the expression of DSL proteins in Sertoli cells, which may contribute to the complex regulation of Notch pathway activity in rodent seminiferous epithelium. Ourresults demonstrated that17ß-estradiol stimulates the expression of DLL1, DLL4, and JAG1 in both PSC and TM4 cell line. The effect of estradiol on DLL1 and JAG1 expressionwasclearly abolishedbyICI182,780, indicatingthe involvementof nuclearERs. We found that estradiol upregulated the expression of JAG1 in Sertoli cells acting through both ER. and ERß, but ER. seemstoplaythemainroleinthisregulation. Earlier studies on human breast cancer cell line MCF-7 and endometrial stromal cells demonstrated that promoter of JAG1 contains estrogen-responsive elements. Moreover, luciferase reporter analysisrevealed that estrogen stimulated the expressionof JAG1 viaER., which is bound to estrogen-responsive elementin the JAG1promoter[54,55]. Interestingly, knockdown of JAG1 in MCF-7 cells resulted in the loss of ER. expression[56], indicatinga mutual relationship between these proteins. In the present study, we showed that 17ß-estradiol enhanced the expression of DLL1 acting chiefy through ER.. IncreasedDLL1 protein expression following estrogen treat­ment was also foundpreviouslyinbreast cancer cells[57]. The authorsreported that ER. knockdownledtoasignifcantdecreaseinDLL1protein levelbutnotmRNA level. In contrast, downregulationof both transcriptandprotein wasrevealedinTM4 Sertoli cells following ER. silencing, indicating that the loss of ER. mayreduce DLL1 level not onlyby enhancingprotein ubiquitination and degradation asreportedpreviously[56], but alsoby the effect on Dll1 mRNA expression. It should be mentioned that the regulation of Dll1 by estradiol clearly depends on the tissue or cell type. In the uterus, estradiol upregulated Dll1 mRNA; in the fallopian tube, estrogen acting through ERß ledto thereductionin DLL1protein, whereas,in human umbilical vein endothelial cells, it had no effect on DLL1 protein expression[58–60]. Although the effect of ERß on estrogen-stimulated expression of JAG1 and DLL1 was less evident in TM4 cells, it cannot be ruled out that, in PSC (that have lower ER. expression), contribution of ERß in theregulationof theseproteinsis also important. Herein, we found estrogen-dependent upregulation of DLL4 in Sertoli cells. Increased mRNA expression of Dll4 was also detected in human endothelial cells after 17ß-estradiol exposure, whereas downregulation of Dll14 was observed in the vagina and uterus[58,59]. Direct exposuretoestrogenic compound bisphenolAenhancedtheexpressionofDLL4in rat testis explants[61]. Our data provided evidence that both ERß and GPER are involved in estrogenic stimulation of DLL4 protein in Sertoli cells. The role of GPER in the control ofDSLproteinshasnotbeenreportedyet; however,Pupoetal.[62]demonstratedthat estrogen/GPER signaling induced activation of Notch1 and Notch target protein HES1 in breast cancer cells. Of note, GPER silencing in TM4 cells produced signifcant change in DLL4 expression only at the protein level, which suggests that activity of nonclassical estrogen signaling may be involved in the post-translational regulation of DLL4. Recently, theroleofGPER independentof transcriptional activation wasreportedin theregulation of endothelial glucose transporter1[63]. Taken together, although DSL protein expression in Sertoli cells appears to be con­trolled mainly via nuclear ERs, our fndings provided evidence that GPER also contributes to this regulation. Of note, the mechanisms of cross-talk between nuclear and membrane estrogen receptors and their downstream pathways were described[64,65]. Potential involvement of such interactions in the regulation of DSL proteins needs further research. Based on the available data, the importance of the proper expression levels of DSL proteins in Sertoli cells for the control of germ cell differentiation and survival may be considered. Studies on human testicular samples, whichrevealeda common expressionof Notch2and Notch4receptorsin seminomaandcarcinomainsitu, raisedthe possibilitythat Notch signalingplaysarolein controlling the mitotic/meiotic switchin primordialgerm cells. The authors proposed that dysfunction of this mechanism could result in abnormal chromosomal segregation and the generation of aneuploid cells—precursors for further developmentto cancer cells[66].Inthis context,it cannotbe excludedthatdisruption of other members of the Notch signaling pathway, such as DSL ligands in Sertoli cells, could also lead to abnormal germ cell development and tumor promotion. The signifcance ofproper Notch pathway activityin germ cells was also indicatedby Okudaetal.[34], who found that aberrant activation of Notch pathway in spermatogonial stem cells (evoked by a deletion of Nkapl, germ-cell-specifc transcriptional suppressor of Notch signaling) caused enhanced germ cell apoptosis and affected several transcriptional factors associated with early germ cell differentiation. In addition, it was demonstrated that exposure of adult rats to the inhibitor of canonical Notch signaling (DAPT) affected the expression of Notch components, including the loss of DLL4 in Sertoli cells at stages VI to IX of seminiferous epithelium cycle. This was accompaniedby abnormal morphologyof germ cells,a failure of cell division, disturbed spermatid elongation and itsprematurerelease, andby increased apoptosis of zygotene spermatocytes and germ cells undergoing the last steps of meiotic division[25]. Notably, meiosis completion, as well as proper spermatid adhesion and release from seminiferous epithelium, are also dependent on androgen action in Sertoli cells[18].Thus,itmaybe hypothesizedthatthecontrolofDSLligandsinSertolicellsby sexsteroids contributestotheeffectsofthese hormonesonmalegermcell maintenance and the course of spermatogenesis. Finally, even thoughDSLproteinsare usually describedas ligandsfor Notchreceptors, they can also interact with other signaling pathways through their intracellular domains. DSLproteins undergoproteolytic cleavageby gamma-secretase, which generates intracel­lular domain with nuclear localization[67]. Intracellular domainof DLL1 was shownto mediate transforming growth factor-ß/activin signaling through binding to Smad2/3 in mouseneuralstemcells[68].In addition, intracellulardomainofDLL1maydownregu­ late Notch receptor activity, disrupting the formation ofthe Notch intracellular domain andrecombination signal bindingprotein for immunoglobulin kappaJ region (RBP-Jk) complex[69]. Recently, JAG1 intracellular domain was identifed asa negativeregulator of Leydig cell steroidogenesis via Nur77-dependent mechanism[70]. The functionof in­ tracellular domains of DSL ligands in Sertoli cells has not been determined to date, but it becomes increasingly apparent that the signifcance of DSL proteins in these cells may extend well beyond theirrole as ligands for Notchreceptors. To summarize, data presented herein highlight a substantial role of the co-operation of classical and nonclassical signaling pathways triggered by androgens and estrogens in maintainingtheproperexpressionofDSLproteinsinSertolicells(Figure 5).Thus,adelicate balance between testicular androgens and estrogens and their nuclear and membrane receptors existing in seminiferous epithelium seems to be important for appropriate Notch signaling activity in the testis, and, thereby, for seminiferous epithelium homeostasis and spermatogenesis. Figure 5. Aschematic model of the regulation of DSL proteins by sex steroids and their receptors in rodent Sertoli cells. AR—nuclear androgen receptor; DLL1—Delta-like 1; DLL4—Delta-like 4; ER.—estrogenreceptor .;ERß—estrogen receptor ß;GPER -Gprotein-coupled estrogenreceptor; JAG1—Jagged1; ZIP9—ZRT-and Irt-like protein 9. 4. Materials and Methods 4.1. Cell Cultures andTreatments Primary cultures of Sertoli cells (PSC) were isolated from the testes of 20-day-old rats usingenzymatic digestionand washing, accordingtopreviously publishedprotocol[71,72]. Briefy, PSC were seeded at 0.5 × 106 cells/cm2 in 6-well plates or on coverslips and incubated in Dulbecco’s modifed Eagle medium (DMEM) supplemented with growth factors and an antibioticina humidifed atmosphereof 95% air and5%CO2(vol/vol)at 35 .C.At48hafter plating, culturesweretreatedwitha hypotonicbuffer(20mMTrispH 7.4 at 22 .C) tolyse contaminating germ cells and obtain PSC with 98% purity. Cells were treated with: (i) 10-8Mtestosterone (Cat NO. 86500; Sigma-Aldrich, St. Louis, MO, USA), anti-androgens 10-4Mhydroxyfutamide (HF; Cat NO. H4166; Sigma-Aldrich, St. Louis, MO, USA), or 10-6Mbicalutamide (Bic) (Cat NO. B9061; Sigma-Aldrich, St. Louis, MO, USA), alone or with the addition of 10-8Mtestosterone for 24 h; (ii) 10-9M17ß-estradiol (Cat NO. E2758; Sigma-Aldrich, St. Louis, MO, USA), 10-6MER./ß antagonist ICI 182,780 (ICI, Cat NO. 5.31042; Sigma-Aldrich, St. Louis, MO, USA), or 10-8Mselective GPER antagonist G15 (Cat NO. 3678,Tocris Bioscience, Bristol, UK), alone or with the addition of 10-9M17ß-estradiol. All compounds were dissolvedin dimethyl sulfoxide (DMSO) before addition to the culture medium to obtain the above fnal concentrations. Control cells were incubated in the presence of the vehicle only (0.01% DMSO). Murine Sertoli cell line TM4 (Cat NO. CRL-1715;ATCC, Manassas,VA, USA) was cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scien­tifc, Rocheford, IL, USA) at 37 .Cin 5% CO2. Properties of TM4 cell line were evaluated as describedpreviously[12]. Beforethe experiments, cells were serum starvedfor24h. The same cell concentration (0.5 × 106/cm2)was used in all experimental groups. In the frst experiment, the cells cultured in plates or on the coverslips were treated with steroid hormones or steroid hormone receptor antagonists according to the protocol described aboveforPSC.Inthe second experiment,TM4 cells were seededat0.1 × 105 cells/cm2 in 6-well plates or on coverslips and transfected with Silencer Select siRNAs (AR-specifc siRNA assay ID: s62547; ZIP9-specifc siRNA assay ID: s116149; ER.-specifc siRNA assay ID: s65686; ERß-specifc siRNA assay ID: s65689; GPER-specifc siRNA assay ID: s94713, Thermo Fisher Scientifc, Rocheford, IL, USA) using Lipofectamine RNAiMAXTransfection Reagent (Thermo Fisher Scientifc, Rocheford, IL, USA) in serum-free Opti-MEM (Cat NO. 11058021; LifeTechnologies, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Negative control cells were treated with transfection reagent alone or trans­fectionreagent plus Silencer Select Negative Control No.1(nontargeting siRNA; Cat NO. 4404020; Thermo Fisher Scientifc, Rocheford, IL, USA). Silencer Select GAPDH Positive Control siRNA (Cat NO. 4390849; Thermo Fisher Scientifc, Rocheford, IL, USA) was used for positive control.Transfectioneffciencies were determined withWestern blot analysis basedontherelativeexpression levelsofthereceptorproteinsin transfectedcellpopula­tions vs. control cultures.Transfectioneffciencies were:87 ± 2% for AR siRNA, 73 ± 5% for ZIP9 siRNA, 76% ± 9% for ER. siRNA, 80% ± 1% for ERß siRNA, and 68% ± 9% for GPER siRNA. After 24 h, cells were washed to remove silencing duplexes and transfec­tion medium. Cells were treated with testosterone, 17ß-estradiol, or a vehicle for 24h as described above. 4.2. RNA Isolation, ReverseTranscription, and QuantitativeRT-PCR(RT-qPCR) Total RNA was extracted with TRIzol® reagent (Cat NO. 15596026; LifeTechnolo­gies, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Residual DNA was removed with TURBO DNA-free Kit (Cat NO. AM1907; Ambion, Austin, TX, USA). The yield and quality of the RNA were evaluated by checking the A260:A280 ratio (NanoDrop ND2000 Spectrophotometer, Thermo Scientifc, Rocheford, IL, USA) and by electrophoresis. High-Capacity cDNA ReverseTranscription Kit (Cat NO. 4368814; Ap­plied Biosystems, Carlsbad, CA, USA) was used to generate cDNA. For each RNA sample, reactionsinthe absenceofRTwererunto appraise genomicDNA contamination.RT-qPCR analyses were performed with the 10 ng cDNA templates, 0.5 µMprimers (Institute of Biochemistryand Biophysics, Polish Academyof Sciences,Table 1),andSYBRGreen master mix(CatNO. 4309155; Applied Biosystems, Carlsbad,CA,USA)ina fnal volumeof10 µL with the StepOne Real-timePCR system (Applied Biosystems, Carlsbad, CA,USA). PCR conditions: 55 .Cfor2min,94.Cfor10 min, followedby denaturation temperature95.C for15sand annealing temperaturefor60sto determinethecyclethreshold(Ct)for quanti­tative measurement. Amplifcationeffciency was between 97% and 104%. Meltingcurve analysis and agarose gel electrophoresis were used to confrm amplifcation specifcity. Negative controlreactions correspondingtoRT reaction withoutthereverse transcriptase enzyme anda blank sample were carried out. Thereference gene candidates were tested on experimental and control samples. The Microsoft Excel-based application NormFinder was used to analyze the expression stability of commonly used reference genes. Based on these analyses, housekeeping genes for normalizing RNA expression were selected: Rn18s, B2m, Actb, Rpl13a, Hprt1, and Gapdh. mRNA expressions were normalized to the mean expressionofthereference genes(relative quantifcation,RQ =1) withthe useofthe2-..Ct method[73]. 4.3.Western Blot Analysis Lysates were obtainedby cell sonifcation withTris/EDTAbuffer(50mMTris,1mM EDTA, pH 7.5) containing protease inhibitors (Cat NO. P8340; Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined with DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) using BSA as a standard. Proteins were resolved by SDS-PAGE underreducing conditions, transferredto polyvinylidene difuoride membranes (Sigma-Aldrich, St. Louis, MO, USA), and detected by immunoblotting as previously reported in detail[60]. Primary antibodies used in the analyses were: anti-JAG1 (1:3000; Cat NO.PA5–72843, Thermo Fisher Scientifc, Rocheford, IL, USA), anti-DLL1 (1:1000; Cat NO. SAB2100593, Sigma-Aldrich, St. Louis, MO, USA), and anti-DLL4 (1:2000; Cat NO. AB7280, Abcam, Cambrige, UK). Secondary horseradish peroxidase-conjugated antibody (1:3000; Cat NO. 31460, Thermo Fisher Scientifc, Rocheford, IL, USA), followed by enhanced chemiluminescence, was used to detect targeted protein bands. Bands were visualized with a ChemiDocTM XRS+ System (Bio–Rad Labs., Mchen, Germany). The molecular weightsoftargetedproteins were assessedby referenceto standardproteins (PageRuler Prestained Protein Ladder, Thermo Fisher Scientifc, Rocheford, IL, USA). All immunoblots were stripped(25mM glycine-HCl,1%(w/v)sodium dodecyl sulfate, pH 2.1 for 30 min) and reprobed with an antibody against ß-actin (1:3000; Cat NO. A2228, Sigma-Aldrich, St. Louis,MO,USA), which servedastheprotein loading control, followedby secondary horseradish peroxidase-conjugated antibody (1:3000, Cat NO. 1706516, Bio-Rad Labs., Mchen, Germany). Relative intensitiesofprotein bands werequantifedbythe ImageLab software (Bio-Rad Labs., Mchen, Germany). Table 1.Sequencesof forwardandreverse primers. Gene Forward Sequence Reverse Sequence Product Size (bp) Annealing Temp (.C) Mouse Actb AAGAGCTATGAGCTGCCTGA TACGGATGTCAACGTCACAC 160 58 B2m GGCCTGTATGCTATCCAGAA GAAAGACCAGTCCTTGCTGA 198 58 Dll1 TCAGATAACCCTGACGGAGGC AGGTAAGAGTTGCCGAGGTCC 185 56 Dll4 GCTGGAAGTGGATTGTGG CTTGTCGCTGTGAGGATAC 405 51 Gapdh CTGGAGAAACCTGCCAAGTA TGTTGCTGTAGCCGTATTCA 223 58 Hprt1 GCTGACCTGCTGGATTACAT TTGGGGCTGTACTGCTTAAC 242 58 Jag1 AACTGGTACCGGTGCGAA TGATGCAAGATCTCCCTGAAAC 216 54 Rn18s CTCTGGTTGCTCTGTGCAGT GGCTCCTTGTAGGGGTTCTC 455 52 Rpl13a ATGACAAGAAAAAGCGGATG CTTTTCTGCCTGTTTCCGTA 215 58 Rat Actb CACACTGTGCCCATCTATGA CCGATAGTGATGACCTGACG 272 58 B2m TGCTACGTGTCTCAGTTCCA GCTCCTTCAGAGTGACGTGT 196 58 Dll1 TCAGATAACCCTGACGGAGGC AGGTAAGAGTTGCCGAGGTCC 185 56 Dll4 GCTGGAAGTGGATTGTGG CTTGTCGCTGTGAGGATAC 405 51 Gapdh AGACAGCCGCATCTTCTTGT CTTGCCGTGGGTAGAGTCAT 207 58 Hprt1 GACTTTGCTTTCCTTGGTCA AGTCAAGGGCATATCCAACA 152 58 Jag1 AACTGGTACCGGTGCGAA TGATGCAAGATCTCCCTGAAAC 216 54 Rn18s GCCGCGGTAATTCCAGCTCCA CCCGCCCGCTCCCAAGATC 320 61 Rpl13a GTGAGGGCATCAACATTTCT CATCCGCTTTTTCTTGTCAT 242 58 4.4. Immunofuorescence Immunofuorescence was performed on PSC and TM4 cells seeded on coverslips. The cells were washed with phosphate-buffered saline (PBS), fxed with cold methanol– acetone, and immunostained as described previously[74]. Analysis was performed with the corresponding primary antibodies listed in Section 4.3. (anti-JAG1, dilution 1:100; anti-DLL1, dilution 1:50; anti-DLL4, dilution 1:50) and Cy3-conjugated goat anti-Rabbit IgG (1:200; Cat NO. A10520; Thermo Fischer Scientifc, Rocheford, IL, USA) secondary antibody. Images were captured with epifuorescence microscope Nikon Eclipse Ni (Nikon InstechCo.,Tokyo,Japan).Nofuorescencewas observedinthenegativecontrols,where the respective primary antibodies were omitted (not shown). 4.5. Statistical Analysis Each data point was a mean ± SDof theresultsfrom three independent experiments. Normality and the homogeneity of variance were tested with Shapiro–WilkW-test and Levene’s test, respectively. Statistical signifcance was assessed using one-way ANOVA, followedbyTukey’s post hoc comparison test. Statistical analyses were performed on raw data using Statistica 10 software (StatSoft Inc.,Tulsa, OK, USA). Data were considered statistically signifcant at * p<0.05, **p<0.01, ***p<0.001. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23042284/s1. Author Contributions: Conceptualization, A.K., S.L. and A.H.; methodology, A.K., S.L., M.K.-B. and A.H.; formal analysis, A.K., S.L., B.B. and A.H.; investigation, A.K., S.L., J.C. and M.B.; data curation, A.K., S.L. and A.H, writing—original draft preparation, A.H.; writing—review and editing, A.K., S.L., J.C., M.B., M.K-B., B.B. and A.H.; visualization, A.K., S.L. and A.H.; supervision, A.H. and B.B.; project administration, A.H. and S.L.; funding acquisition, A.H. and S.L. All authors have read and agreed to the published version of the manuscript. Funding: Thisresearch was fundedbyNational Science Centre(Poland), grant 2017/25/B/NZ4/01037 (OPUS13) andbyJagiellonian University, Facultyof Biology, grant N18/MNW/000022. The open­access publication of this article was funded by the program “Excellence Initiative—Research Univer­sity” at the Jagiellonian University in Krakow, Poland. Institutional Review Board Statement: Sacrifcing rats for testis collection and Sertoli cell isolation was performed in accordance with Polish regulations (Act of 15 January 2015 on the protection of animals used for scientifc and educational purposes; Journal of Laws of 2015, item 266). DataAvailability Statement: The data presented in this study are available on request from the corresponding author. Conficts of Interest: The authors declare no confict of interest. References 1. McKinnell, C.; Atanassova, N.;Williams, K.; Fisher, J.S.;Walker, M.;Turner, K.J.; Saunders,T.K.; Sharpe,R.M. 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