Tgfβ Signaling in Male Germ Cells Regulates Gonocyte Quiescence and Fertility in Mice

Developmental Biology 342 (2010) 74–84

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Developmental Biology

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TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

Stéphanie G. Moreno a,d,e,⁎, Myriam Attali a,d,e, Isabelle Allemand b,d,e, Sébastien Messiaen a,d,e, Pierre Fouchet b,d,e, Hervé Cofﬁgny a,d,e, Paul-Henri Romeo c,d,e, René Habert a,d,e a Laboratoire de Développement des Gonades, CEA, DSV-iRCM-SCSR, Fontenay-aux-Roses, F-92265, France b Laboratoire de Gamétogenèse, Apoptose et Génotoxicité, CEA, DSV-iRCM-SCSR, Fontenay-aux-Roses, F-92265, France c Laboratoire de recherche sur la Réparation et la Transcription dans les cellules Souches, CEA, DSV-iRCM-SCSR, Fontenay-aux-Roses, F-92265, France d INSERM, U967, Fontenay-aux-Roses, F-92265, France e Université Paris Diderot - Paris 7, Fontenay-aux-Roses, F-92265, France article info abstract

Article history: During testis development, proliferation and death of gonocytes are highly regulated to establish a standard Received for publication 3 December 2009 population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Revised 16 March 2010 Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions Accepted 17 March 2010 during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases Available online 24 March 2010 gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but Keywords: TGFbeta the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with Conditional knockout time. Using an organ culture system mimicking in vivo development, we show higher proportions of Germ line proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal Quiescence quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, Apoptosis isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence. Fertility These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis. © 2010 Elsevier Inc. All rights reserved.

Introduction (Vergouwen et al., 1991)(Nagano et al., 2000). Nevertheless, the mechanisms regulating the establishment of the quiescence of germ During testicular development, the germ lineage passes through cells and resumption of their cell cycle at birth are not yet clear. cycles of proliferation and apoptosis. In the mouse, after sexual Many pathways have been explored and some results suggest differentiation occurring at 11.5 days post-conception (dpc), fetal that genes such as Pten, P27, P15 or P53 could be involved in the male germ cells (named gonocytes) actively proliferate until 16.5 dpc. germ lineage regulation (Beumer et al., 1999)(Moreno et al., 2001) Between 16.5 and 19.5 dpc, gonocytes are quiescent, and after birth (Moreno et al., 2002)(Kimura et al., 2003)(Western et al., 2008). they resume mitosis. In parallel, numerous gonocytes during both the Because the establishment of normal spermatogenesis is a funda- fetal and neonatal phases of proliferation will die by apoptosis, mental process, there probably exists a redundancy of several path- whereas none of them will die during the quiescent phase (Olaso and ways to preserve germ cell integrity in case of a defect in the main Habert, 2000). These stages are essential for the establishment of a regulation system. normal spermatogenesis. After birth, the gonocyte population gives One of the pathways known to be involved in cell regulation is the rise to type A spermatogonia, which will differentiate during Transforming Growth Factor beta (TGFbeta) signaling. TGFbeta spermatogenesis to produce sperm (de Rooij and Grootegoed, 1998). (Moses et al., 1981)(Roberts et al., 1981) belongs to a superfamily This fetal and neonatal period of testis development has been of genes including also the bone morphogenetic proteins (BMPs), the largely studied in rodents, especially in the rat (Beaumont and Mandl, anti-Müllerian hormone (AMH), the growth and differentiation factor 1963)(Hilscher et al., 1974)(Boulogne et al., 1999) and mouse (GDF), the distantly related glial cell line-derived neurotropic factor (GDNF), the activins / inhibins (Kingsley, 1994), and Nodal (Schier, 2003). TGFbeta signaling controls numerous cellular processes including cell proliferation, differentiation and apoptosis, both during ⁎ Corresponding author. DSV/iRCM/SCSR, CEA – 18, route du Panorama, BP6, F- 92265 Fontenay-aux-Roses cedex, France. Fax: +33 1 46 54 99 06. embryogenesis and adulthood. Three isoforms of TGFbeta have E-mail address: [email protected] (S.G. Moreno). been currently described in mammals: TGFbeta1, beta2 and beta3.

They initiate signaling via the transmembrane type I (TβRI) and background, were mated with TNAP-Cre mice (kindly provided by A. type II (TβRII) receptors serine/threonine kinase on the cell surface Nagy, Samuel Lunenfeld Research Institute, Canada), maintained on a (Derynck, 1994)(Kingsley, 1994). The signal is then transmitted to hybrid 129 Sv/C57BL/6 background. Male TβRII+/flox/TNAP-Cre mice the Smad proteins (Attisano and Wrana, 2002). A third type of were then crossed with female TβRIIflox/flox mice, as ectopic recom- TGFbeta receptor (TβRIII), also called betaglycan, has been described bination may be avoided when the TNAP-Cre locus is transmitted from as an accessory receptor, and is known to facilitate the binding of the male (Lomeli et al., 2000)(Suzuki et al., 2001). TGFbeta (especially TGFbeta2) to TβRII (Lopez-Casillas et al., 1993). TβRII was identified for the first time in 1992 (Lin et al., 1992) and Genotyping appears to be the most important signaling receptor (Wrana, 1998). TGFbeta ligands must bind to TβRII before TβRI is recruited. Once Animals were genotyped by PCR analysis of genomic DNA from the holo-complex is formed, the kinase domain from TβRII trans- tail biopsy. To facilitate genotyping of mice, primer pairs were de- phosphorylates the GS domain of TβRI, which in turn phosphorylates signed for detection of both wild-type (WT) and floxed (FL) alleles Smad proteins (Lin et al., 2006). (8w-a/LA-LoxP: 5’-TAAACAAGGTCCGGAGCCCA-3’ /5’-ACTTCTG- Expression of TGFbeta isoforms and receptors has been described CAAGAGGTCCCCT-3’), or for deleted (DEL) allele (8w-a/mSAr: 5’- in the rat during testis development and it was shown that targets of TAAACAAGGTCCGGAGCCCA-3’ /5’-AGAGTGAAGCCGTGGTAGGT- TGFbeta signaling seem to be Leydig cells and gonocytes (Teerds and GAGCTTG-3’)(Chytil et al., 2002). The Cre-specificprimerpairwas Dorrington, 1993)(Gautier et al., 1994)(Olaso and Habert, 2000). as follows: CreForw / CreRev: 5’-GATGCAACGAGTGATGAGGTTCGC- Additionally to immunohistochemical studies, functional assays 3’ /5’-ACCCTGATCCTGGCAATTTCGGC-3’. performed in vitro demonstrated a potential effect of TGFbeta on steroidogenesis, cord formation and gonocyte behavior (Gautier et al., Immunohistochemistry 1997)(Olaso et al., 1998)(Cupp et al., 1999). Other in vitro data showed an anti-proliferative effect of TGFbeta1 on mouse primordial Testes were fixed overnight at 4 °C with Bouin's fluid (2% (w/v) germ cells (PGCs) (Godin and Wylie, 1991)(Richards et al., 1999) and picric acid, 7% (v/v) formaldehyde, 3.8% (v/v) acetic acid), dehy- an apoptotic role of TGFbeta on rat fetal and neonatal gonocytes during drated and embedded in paraffin. the proliferative phases of testis development (Olaso et al., 1998). Sections were rehydrated and boiled twice for 5 min in a 10 mM Nevertheless, a physiological role of TGFbeta signaling in gonocyte sodium-citrate solution for antigen retrieval (Shi et al., 1991). development has never been demonstrated in vivo. The best way to Endogenous peroxidase activity was blocked with 0.3% (v/v) H2O2 establish such a physiological role for TGFbeta signaling would be the in methanol, and non-specific binding sites were blocked by use of transgenic models. incubating the sections with 5% (w/v) bovine serum albumin in Models of mice lacking TGFbeta isoforms or receptors have been PBS. Subsequently, the slides were incubated overnight at 4 °C with generated (Dickson et al., 1995)(Oshima et al., 1996)(Sanford et al., the following primary antibodies: anti-TGFβ1 (1:50; sc-146, Santa- 1997)(Nguyen and Pollard, 2000)(Larsson et al., 2001)(Stenvers Cruz Biotechnology Inc., Santa Cruz, CA); anti-TGFβ2 (1:50; # 905- et al., 2003)(Memon et al., 2008). To avoid a probable redundancy 582, Assay Designs); anti-TGFβ3 (1:200; sc-82, SantaCruz Biotech- between isoforms, the best way is to disrupt one of the receptors. It is nology Inc.); anti-TβRI (1:50; sc-398, SantaCruz Biotechnology Inc.); known that each type II receptor is specific for its subfamily, unlike anti-TβRII (1:50; sc-220, SantaCruz Biotechnology Inc.), anti-TNAP type I receptors. This means that deletion of TβRII would disturb the (1:50; sc-23430, Santa Cruz Biotechnology Inc.), and anti-PLZF (1:50; TGFbeta signaling only, and not the signaling of the other family sc-22839, Santa Cruz Biotechnology Inc.). After washing in PBS, slides members. Null mutation of TβRII in all cells from early embryonic were incubated at room temperature with a secondary biotinylated stages leads to growth retardation and severe anemia between 10.5 antibody (ABC peroxidase staining kit; Vector Laboratories Inc., and 12.5 dpc (Oshima et al., 1996). In order to overcome this limita- Burlingame, CA) diluted 1:200. The avidin-biotin complex reaction tion, we undertook to generate TβRII conditional knock-out mice was performed according to the manufacturer's protocol. To visualize (condKO), via the Cre/Lox system, to target the disruption specifically bound antibodies, sections were covered with 3, 3’-diaminobenzidine in germ cells. (DAB; substrate kit for peroxidase, Vector Laboratories Inc.). Negative With this model, we demonstrated that TGFbeta signaling in control sections were treated as described above, except that primary germ cells is physiologically involved in the regulation of male antibody was omitted during the procedure and replaced by 5% gametogenesis as a direct anti-proliferative and anti-apoptotic factor. bovine serum albumin in PBS. This study also showed that TGFbeta might regulate gonocyte quiescence phase and is fundamental in the initiation and mainte- Organotypic cultures nance of adult spermatogenesis. Mouse fetal testes were cultured on Millicell CM (Fischer Scientific Materials and methods Labosi, Elancourt, France) filters (pore size 0.4 mm) as previously described (Livera et al., 2006). Testes from embryos were collected at Mice 13.5 dpc and the mesonephros was removed. Briefly, for each animal, each testis was placed on a filter floating on medium in tissue-culture Allanimalstudieswereconductedinaccordancewiththe dishes and incubated at 37 °C in a humidified atmosphere containing guidelines for the care and use of laboratory animals of the French 95% air and 5% CO2. The culture medium used was a mix of Dulbecco's Ministry of Agriculture. Animals were housed under controlled modified Eagle's medium (DMEM) and Nutrient Mixture F-12 Ham's photoperiod conditions (lights on 08.00-20.00) and were supplied (HAM'sF12) (1:1) (Gibco, Cergy Pontoise, France). No serum, hormone with commercial feed and tap water ad libitum. Males were caged or growth factor was added to the medium. Testes were cultured for one with females overnight and the day after mating was counted as day (corresponding to 14.5 dpc in vivo), four days (corresponding to 0.5 day post-conception (dpc). Birth occurs at 19.5 dpc, which was 17.5 dpc in vivo), six days (corresponding to 0 dpp in vivo), or seven days counted as 0 day post-partum (dpp). (corresponding to 1 dpp in vivo). The generation of the TβRIIflox/flox mice has been previously To measure testicular responsiveness to TGFβ, tissue-culture dishes described (Chytil et al., 2002). had been previously siliconed with Sigmacote (Sigma-Aldrich Lyon, To knock out TβRII in the PGCs, these floxed mice (kindly provided France) to avoid TGFβ binding to the plastic dishes. One testis was by H. Moses, Vanderbilt University, USA), maintained on a C57BL/6 cultured for 24 hours in a control medium and the contra-lateral testis 76 S.G. Moreno et al. / Developmental Biology 342 (2010) 74–84 was cultured in a medium containing 5 ng/ml porcine TGFβ2(R&D Dickinson). Elongated spermatids were excluded to avoid any Systems Europe, France). contamination of the germinal side population (SP).

Proliferation and quiescence study Statistics

In vivo: pregnant females at 13.5 dpc were injected intra- All values are means±SEM. The significance of the differences peritoneally with 5-bromo–2’-deoxyuridine (BrdU, Sigma-Aldrich, between mean values was evaluated by the Student t-test (Instat3, France) at a dose of 50 mg per kilogram body-weight (Merlet et al., GraphPad software). A pb0.05 was considered significant and 2007). Fetuses were sacrificed 3 hours later and testes were fixed illustrated with one asterisk. overnight at 4 °C in a 4% (w/v) paraformaldehyde-PBS solution. In vitro: testes were incubated at the end of the culture period for 3 Results hours, with 10 µg/ml BrdU added to the culture medium. Then, testes were fixed overnight at 4 °C with a 4% (w/v) paraformaldehyde-PBS Expression of TGFbeta isoforms and receptors in mouse testis during solution. development Incorporated BrdU was revealed by immunohistochemistry with a BrdU/AMH (Anti-Müllerian Hormone) double-staining, as previously Expression of the TGFbeta isoforms and receptors type I and type II described (Merlet et al., 2007). Briefly, the AMH staining will color was studied by immunohistochemistry during testis development purple the cytoplasm of all the Sertoli cells, making easier the (Fig. 1 and S1). Whereas the receptors were both expressed throughout recognition of germ cells, which keep a colorless cytoplasm. testis development in germ and Leydig cells (Figs. 1A, B and S1), the The phosphorylated form of the histone H3 (P-H3) was detected three TGFbeta isoforms were expressed differently during development. by immunohistochemistry (phospho-H3 #9701, 1:100, Cell Signaling TGFbeta1 could not be detected in testis (Figs. 1D, G and J) and TGFbeta3 Technology). was expressed in both gonocytes and Leydig cells at all studied stages of The proportion of quiescent germ cells was quantified after Ki67 testis development (Figs. 1F, I and L). TGFbeta2 was the only TGFbeta staining (anti-Ki67 antibody, # 550609, 1:50, BD Pharmingen). Ki67- isoform expressed differently according to the gonocyte proliferation negative gonocytes were considered quiescent. status. During fetal and neonatal phases of gonocyte proliferation, The percentage of proliferating or quiescent germ cells was TGFbeta2 was detected in Leydig cells and only in some gonocytes determined in fetuses and newborns by counting at least 1000 cells (Figs. 1E and K), whereas during the quiescent phase it is expressed in and in adults by counting cells in 100 round tubule sections. Leydig cells and all gonocytes (Fig. 1H). The proportion of Ki67-negative gonocytes (G0-phase) was determined at 13.5 dpc, 16.5 dpc and 2 dpp, Apoptosis detection and compared to the proportion of TGFbeta2-positive gonocytes (Fig. 1M): no significant difference was observed at the three stages. Apoptosis was detected by immunostaining for the cleaved- These results suggested that the TGFbeta signaling pathway might be caspase3 and by using the ApopTag In Situ Apoptosis Detection Kit involved in the control of gonocyte proliferation. (ISEL method; S7100-KIT, MP Biomedicals, Illkirch, France), as previously described (Duan et al., 2003). TGFbeta signaling pathway regulates both gonocyte proliferation and death in vitro Testicular single cell suspension Wild-type testes were explanted at 13.5 dpc and cultured for 24 h Cells were isolated from 4-month-old male mice as previously with or without TGFbeta2. TGFbeta2 treatment resulted in a three- described (Lassalle et al., 2004). Briefly, the seminiferous tubules were fold decrease of gonocyte proliferation, measured by BrdU/AMH enzymatically dissociated for 20 min at 32 °C with collagenase type I double-staining (Figs. 2A, B and C), and in a four-fold decrease of (Invitrogen) at 100 U/ml in Hanks’ balanced salt solution (HBSS) dying gonocytes, measured using cleaved caspase 3/AMH double- supplemented with 20 mM HEPES pH 7.2, 1.2 mM MgSO47H2O, staining (Figs. 2D, E and F) or an ISEL assay (data not shown). These 1.3 mM CaCl22H2O, 6.6 mM sodium pyruvate (Invitrogen), 0.05% results indicated that in vitro the TGFbeta signaling pathway regulated lactate (Sigma-Aldrich, France). To discard interstitial cells, the both proliferation and death of gonocytes and prompted us to study suspension was filtered through a 40 µm nylon mesh and the tubules the role of TGFbeta during testis development. were recovered on the filter to be incubated in a Cell Dissociation Buffer (Invitrogen) for 25 min at 32 °C. The resulting cell suspension Generation and characterization of the TβRII -/flox/TNAP-Cre mice was filtered through a 40 µm nylon mesh to remove cell clumps. After an HBSS wash, the cell pellet was resuspended in the incubation To suppress specifically in germ cells the TGFbeta signaling buffer (HBSS supplemented with 20 mM HEPES pH 7.2, 1.2 mM pathway activated by TGFbeta but not by the other members of the

MgSO47H2O, 1.3 mM CaCl22H2O, 6.6 mM sodium pyruvate, 0.05% TGFbeta superfamily, we crossed mice harboring a floxed TGFbeta lactate, 2 mM L-glutamine and 1% decomplemented fetal calf serum) receptor type II (TβRII) gene and TNAP-Cre mice that have been shown and further incubated at 32 °C. Cell concentrations were estimated to express the Cre recombinase in germ cells during testis develop- using Trypan Blue (N95% viable cells). ment. After mating TβRII+/flox/TNAP-Cre and TβRIIflox/flox mice, we obtained 170 newborns, but only 4% of TβRII -/flox/TNAP-Cre mice Flow cytometry analysis (condKO) instead of the 25% expected for Mendelian transmission. This abnormal percentage indicated that the majority of condKO As previously described (Barroca et al., 2009), 106 cells were animals unexpectedly died in utero in spite of the germ cell-targeted stained with Hoechst 33342 (5 µg in 1 ml incubation buffer) for deletion of TβRII. To determine when lethality occurs, female mice 50 min at 32 °C. Cells were then labeled for 20 min at 4 °C with were sacrificed at different stages of pregnancy. At 14.5 dpc, from a monoclonal antibodies or corresponding isotypes (1 µg per 106 cells): total of 37 embryos, 24 were alive, 3 had no beating heart and 10 were anti-c-kit-APC and anti-α6 integrin-PE (BD Pharmingen). After aborted embryos. Genotyping of the 24 living embryos showed that washing, cells were resuspended in incubation buffer with 2 µg/ml only one was a condKO. The 3 embryos without beating heart were all propidium iodide (to exclude dead cells) and maintained at room condKO, indicating that the lethality occurred around this stage of temperature before analysis with a LSR II flow cytometer (Becton development. This was confirmed by genotyping 214 embryos at 13.5 S.G. Moreno et al. / Developmental Biology 342 (2010) 74–84 77

Fig. 1. Expression of TGFbeta receptors type I (A) and type II (B) and isoforms TGFbeta1 (D, G, J), TGFbeta2 (E, H, K) and TGFbeta3 (F, I, L) during mouse testis development. Immunostaining for both receptors was performed at 18.5 dpc (A-B) and for the different isoforms at 13.5 dpc (D-F) during the fetal phase of germinal proliferation, at 16.5 dpc (G-I) during the quiescent phase of germ cells and at 2 dpp (J-L) during the neonatal phase of germinal proliferation. A negative control is illustrated in C. Proportions of Ki67-negative gonocytes and TGFbeta2-positive gonocytes were quantiﬁed and compared at 13.5 dpc, 16.5 dpc and 2 dpp (M). Black arrow: positive germ cell; white arrow: negative germ cell; lc: Leydig cells. Bars represent 10 µm. dpc from 42 litters. 15.9% of embryos were living condKO indicating The TGFbeta signaling pathway regulates gonocyte proliferation during that death of condKO occurs between 13.5 dpc and 14.5 dpc. At 13.5 development dpc, the condKO embryos displayed 3 distinct phenotypes: a normal one (N) (37.5% of total condKO)(Fig. S2B) quite similar to a WT As condKO embryos were alive at 13.5 dpc, we analyzed the embryo (Fig. S2A), an intermediate one (I) (14.6% of total condKO) deletion of TβRII in their germ cells by immunohistochemistry and (Fig. S2C) and a severe one (S) (47.9% of total condKO)(Fig. S2D). showed that the TβRII was no longer expressed in the condKO germ Compared with 13.5 day-old WT embryos, the (I) and (S) condKO cells (data not shown). Proliferation of condKO and control littermate embryos had the same size but looked anemic. The most severely gonocytes was compared by BrdU incorporation in vivo at 13.5 dpc disturbed phenotype (S) showed brain bleeding and a small and 1 dpp (fetal and neonatal proliferation phases) and in vitro after hemorrhagic liver. TNAP immunohistochemical staining on sagittal testis explantation at 13.5 dpc followed by 1 day, 4 days, 6 days and sections of entire 10.5 dpc embryos and in 13.5 dpc placental sections 7 days of organotypic culture (Fig. 3A). In vivo at 13.5 dpc and in vitro showed that TNAP was not speciﬁc to germ cells, but was also strongly after 1 day of culture, the percentage of gonocytes that incorporate expressed in cardiac cells and in placenta (Fig. S3), which both BrdU was almost doubled in condKO compared with control. After strongly expressed TβRII (data not shown). This indicated that the 4 days of culture (which corresponds in vivo to the quiescent phase), TNAP-Cre-mediated deletion of TβRII might also occur in the heart quiescence was observed both in control and condKO testes. After 6 and placenta additionally to germ cells, and could explain the and 7 days of culture (corresponding in vivo to the neonatal prolif- observed embryonic lethality. eration phase), a two-fold higher proportion of condKO gonocytes had 78 S.G. Moreno et al. / Developmental Biology 342 (2010) 74–84

Fig. 2. TGFbeta2 decreased gonocyte proliferation and apoptosis in vitro. The proliferation level of germ cells (BrdU incorporation) was measured in wild-type (WT) testes, explanted at 13.5 dpc and cultured for 24 h with or without 5 ng/ml TGFbeta2 (A). BrdU/AMH double-staining was used to count the proliferating germ cells in control (B) and TGFbeta2- treated (C) testes. Nuclei of cells positive for BrdU are stained brown, and the cytoplasm of Sertoli cells, positive for AMH, is colored purple. Counterstaining of all the nuclei was performed with hematoxylin (blue). Proliferating germ cells have a brown nucleus and white cytoplasm (black arrowhead), and non-proliferating germ cells have a blue nucleus and white cytoplasm (white arrowhead). Bars represent 10 µm. The apoptotic level of gonocytes was also measured in WT testes, explanted at 13.5 dpc and cultured for 24 h with or without 5 ng/ml TGFbeta2 (D). Cleaved caspase 3/AMH double-staining was used to count the apoptotic germ cells in control (E) and TGFbeta2-treated (F) testes. Apoptotic gonocytes are stained brown (arrows). Bar represents 10 µm. Significance is illustrated with an asterisk. resumed the cell cycle. This increased level of cycling gonocytes was signaling pathway negatively regulated both proliferation and also found in vivo in one condKO male that was alive after birth. apoptosis of gonocytes during development. Then, we compared the proportion of gonocytes in the different phases of the cell cycle in condKO and control littermate mice at 13.5 dpc Phenotype of adult TβRII -/flox/TNAP-Cre mouse spermatogenesis and found, in condKO, a decreased number of gonocytes in G0 (Fig. 3Ba; Bb-Bc), an increased number of gonocytes in S-phase (Fig. 3Ba; Bd-Be) In spite of the early death of most of the TβRII conditional KO mice, and a reduced number of gonocytes in G2/M (Fig. 3Ba; Bf-Bg). two males survived until adulthood. By mating them with wild-type Finally, we studied in vitro the impact of a TGFbeta2-treatment on female mice, fertility of one male was studied between the age of 3 gonocyte quiescence. Gonocytes from WT testes explanted every day and 4 months, and fertility of the second one was studied from the age from 13.5 dpc to 1 dpp and cultured with or without TGFbeta2 for 24 h of 3 months onwards (Fig. 6A). Whereas the mean number of pups were counted using Ki67/AMH double-staining (Fig. 4). In control per litter remained fairly constant for wild-type males aged from 3 to testes, the “quiescent phase” (100% of gonocytes are Ki67-negative) 9 months, this number showed a progressive age-related decrease for occurred between 15.5 dpc +24 h and 17.5 dpc +24 h. After TGFbeta2 the older condKO male, since it became totally infertile after 8 months. treatment, proportion of Ki67-negative gonocytes reached 98% at 14.5 The efficiency of the TβRII deletion in germ cells was demonstrated by dpc +24 h and 99.6% at 18.5 dpc +24 h, indicating that more (i) immunohistochemical study of TβRII on condKO testis that showed gonocytes have entered the “quiescence phase” one day before and that expression was absent specifically in germ cells, whereas the one day after the classical period. Leydig cells still express the receptor (Fig. S4), and (ii) by genotyping These results suggested that TGFbeta negatively regulates fetal and the condKO offspring which was 100% heterozygous. neonatal gonocyte proliferation. Inactivation of TGFbeta signaling did These two condKO males were sacrificed at 4 or 9 months to not completely suppress the period during which 100% of gonocytes characterize their testis abnormalities. Histological study showed an are quiescent, but increased the gonocyte proliferation before and abnormal morphology of the 4 month-old condKO testis with some after this period. In contrast, addition of exogenous TGFbeta2 on WT tubules being completely depleted in germ cells and some others testes induced the quiescence of more than 98% of gonocytes, both being totally disorganized (Fig. 6B). Additionally, the number of one day before and one day after the classical quiescence phase. TUNEL-positive germ cells was increased in condKO (data not shown). This phenotype became extremely severe at 9 months with no normal The TGFbeta signaling pathway regulates gonocyte death during tubules in condKO testis. No spermatozoa could be detected and the development interstitial tissue was more abundant compared with WT. In order to determine the step of impairment, we performed a flow As the number of gonocytes is strongly dependent on proliferation cytometry analysis in one WT and one condKO adults at the age of but also on cell death during testis development we studied the role of 4 months. We analyzed the population of germinal stem cells and the TGFbeta signaling pathway in the death of gonocytes. At 13.5 dpc, spermatogonial progenitors, which was shown to harbor the side the percentage of apoptotic gonocytes was increased two-fold in population (SP) phenotype, based on the Hoechst 33342 efflux condKO mice compared with control littermates (Fig. 5A). This (Lassalle et al., 2004)(Falciatori et al., 2004). CondKO displays a difference could not be evidenced after 4 days of culture, i.e. during smaller SP than WT (9.2% versus 13.4%) (Figs. 7A and C). Additionally, the gonocyte quiescent phase, but was observed again after 6 and on the gated SP, the α6-integrin and c-Kit labeling showed a slight + -/low 7 days of culture i.e. when gonocytes re-enter the cycle. In vivo,at1 reduction of the SP/α6 /c-Kit population (As to Aal spermatogonia dpp, we also found a three-fold increase of apoptotic cells in condKO corresponding to stem cells and immature progenitors) in condKO gonocytes. A similar result was also obtained in vivo at 13.5 dpc using compared with WT (15.1% versus 17.6%) (Figs. 7B and D). The reduced + -/low the ISEL method (Fig. 5B). These results seemed specific to the role of SP population combined with the reduced SP/α6 /c-Kit sub- the TGFbeta in gonocytes and not due to side effects related to the population suggested a 41%-reduced population of germ stem cells condKO phenotype, as we found similar percentages of both apoptotic and immature progenitors in the 4 month-old condKO. and proliferating germ cells in N-condKO and S-condKO testes at 13.5 In agreement with the FACS results, immunostaining for PLZF, a dpc (Fig. S5). Altogether, these results indicated that the TGFbeta specific marker of stem/immature progenitor cells (c-Kit-/low S.G. Moreno et al. / Developmental Biology 342 (2010) 74–84 79

Fig. 3. Inactivation of the TGFbeta pathway increases the proportion of gonocytes in S-phase and decreases the proportion of gonocytes in G0 and G2/M. (A) The proliferation level of gonocytes was measured in condKO and littermate control testes, by determining the proportion of cells that incorporated BrdU. This study was performed in vivo at 13.5 dpc and 1 dpp, and after culture for 1, 4, 6 or 7 days. The number of cases is noted in brackets. Significance is illustrated with an asterisk. (Ba) The proportion of gonocytes in G0, S, and G2/M was determined in condKO and littermate control testes at 13.5 dpc. The G0-phase was measured after Ki67/AMH double-staining (Bb-Bc): gonocytes in G0 are Ki67-negative (arrowhead); The S-phase was measured after BrdU/AMH double-staining (Bd-Be); The G2/M-phases were measured after P-H3/AMH double-staining (Bf-Bg). Significance is illustrated with an asterisk. population) (Costoya et al., 2004) showed that the number of PLZF- of this family, particularly BMPs (Ross et al., 2007; Zhao et al., 1996; positive cells was reduced by 35% in the condKO compared with the Zhao et al., 1998), in the initiation and maintenance of male and WT (Fig. 7E). Finally, we performed PLZF/Ki67 double-staining to female gametogenesis. The present study demonstrates that TGFbeta study the proportion of cycling stem/ immature progenitor cells. is physiologically involved in the development of mouse germ cells, Whereas around 30% of stem/immature progenitor cells were cycling particularly during their quiescent period, and in the maintenance of in WT testes, only 5 to 10% of these cells were cycling in condKO adult spermatogenesis. The Cre/Lox system allowed us to demon- (Figs. 7F–G). strate that TGFbeta acts directly on gonocytes, in accordance with the All these data suggest that normal adult spermatogenesis is immunolocalization of TGFbeta isoforms and receptors. dependent on functional TGFbeta signaling in developing germ cells. The TNAP-Cre model induces a specific gene deletion in the germ line as early as the PGC stage as described by Lomeli et al. on a 129/SvJ Discussion background (Lomeli et al., 2000). Several teams have already used it successfully (Kimura et al., 2003)(Kaneda et al., 2004)(Kehler et al., During fetal and neonatal life, development of gonocytes is critical 2004). Although it is known that TNAP expression is not restricted to for the establishment of adult spermatogenesis. Involvement of the the PGCs and gonocytes (MacGregor et al., 1995), all these conditional TGFbeta superfamily during gametogenesis has been largely investi- knock-out models survived until adulthood. gated in drosophila (Kawase et al., 2004)(Li et al., 2007) and rodents. In our model, the ectopic excision of the TβRII gene led to Numerous studies have revealed the critical role of several members embryonic lethality of the majority of condKO mice. We observed that 80 S.G. Moreno et al. / Developmental Biology 342 (2010) 74–84

Fig. 4. TGFbeta2 can increase the duration of the germinal quiescence. The proportion of quiescent gonocytes (Ki67-negative) was determined in WT testes, explanted from 13.5 dpc to 1 dpp, and cultured for 24 h with or without TGFbeta2 (5 ng/ml). The number of cases is noted in brackets. Signiﬁcance is illustrated with an asterisk.

TNAP is importantly expressed in fetal cardiac cells, in placental cells In addition, our data suggest that TGFbeta regulates also the and in the umbilical cord. As the cardiac and placental tissues express gonocyte quiescence. Gonocytes do not enter synchronously into the TGFbeta isoforms and receptors (Roelen et al., 1994), the TβRII quiescence, but the “quiescent phase” is defined as the time period deletion in these tissues is probably responsible for the mouse during which 100% of gonocytes are quiescent. In our WT mice, 100% lethality. Thus, our study demonstrates the limits of the TNAP-Cre of gonocytes are quiescent between 16.5 and 18.5 dpc, but at 13.5 dpc, model. 30% of them are already in G0. When TβRII is disrupted in germ cells, TGFbeta is known to negatively regulate cell proliferation in the “quiescent phase” is not completely abolished, but only 20% of different systems. It acts through different signaling pathways gonocytes are quiescent at 13.5 dpc, and the proportion of gonocytes including the cyclin inhibitor p27. Recently, Western et al (Western in S-phase is at least two-fold higher before and after the “quiescent et al., 2008) have reported that gonocyte mitotic arrest is probably phase”. More precisely, the proportion of proliferating gonocytes at mediated by preventing pRb phosphorylation through the activation 13.5 dpc+6d in condKO is equivalent to that observed in WT one day of P27. However, the factors involved upstream of this signaling later. These observations may simply reflect the regulating role of cascade have not been elucidated yet. In mouse, in vitro studies have TGFbeta on both fetal and neonatal proliferation of gonocytes outside described an anti-proliferative effect of TGFbeta on PGCs during their the “quiescent phase”. Nevertheless, these results may be consistent migration to the gonads between 8.5 and 11.5 dpc (Godin and Wylie, with several other hypotheses involving directly TGFbeta in the 1991)(Richards et al., 1999). In this paper, we showed, both in vitro control of the “quiescence phase”, by regulating, (i) either the and in vivo, that TGFbeta2 also inhibits gonocyte proliferation at 13.5 individual gonocyte quiescence, (ii) or all the phases of the gonocyte dpc and later on. In vitro, addition of exogenous TGFbeta2 led to a cell cycle. A combination of these two possibilities is also probable. significant decrease of the level of BrdU-positive gonocytes, associated The fact that the “quiescence phase” is not completely aborted in with a significant increase of the quiescent gonocyte proportion. By condKO mice might be due, either to a redundancy between TGFbeta contrast, the in vivo germ-specific disruption of the TGFbeta signaling and one or several members of its superfamily such as, for example, pathway increased the level of cycling gonocytes and revealed a direct activin, or to a redundancy with another signaling pathway. Activin is anti-proliferative effect of TGFbeta on germ cells. expressed in rat gonocytes during their quiescence (Meehan et al.,

Fig. 5. Inactivation of the TGFbeta pathway increases the level of apoptotic gonocytes. The level of apoptotic gonocytes was measured in condKO and littermate control mice, by determining the proportion of cleaved caspase3-positive cells. This study was performed in vivo at 13.5 dpc and 1 dpp, and after organotypic culture for 4, 6 or 7 days (A). The ISEL method was used at 13.5 dpc in vivo to conﬁrm the results obtained with the cleaved caspase3 staining (B). The number of cases is noted in brackets. Signiﬁcance is illustrated with an asterisk. S.G. Moreno et al. / Developmental Biology 342 (2010) 74–84 81

Fig. 6. Spermatogenesis is impaired in adult condKO mice. (A) After breeding with WT females, fertility of WT and condKO males was studied between 3 and 9 months of age. The number of litters is noted in brackets. (B) Histology of testes was studied after hematoxylin/eosin staining, in 4 and 9 month-old condKO mice and compared with WT adult mice.

2000) and TGFbeta is able to bind the activin receptor type II (ActRIIA/ the opposite role of TGFbeta on gonocyte development. The present IIB) (Itman et al., 2006). Thus, in the absence of TβRII in germ cells, study suggests that the main regulation of mouse gonocytes by TGFbeta could bind ActRIIA/IIB and induce a cell cycle arrest by TGFbeta is mediated by TGFbeta2, which acts as an autocrine factor. activating the activin signaling pathway. A second hypothesis is that By contrast, previous data suggest that gonocyte regulation in the rat TGFbeta and Activin regulate the quiescence phase together. is mediated by TGFbeta1, acting as a paracrine factor produced by the Even if a quiescent period still exists in the condKO mice, the Leydig cells (Olaso et al., 1998). However, due to the Cre ectopic reduction of its duration could probably be involved in the action, we cannot completely exclude that TGFbeta signaling has been impairment of adult spermatogenesis. Kimura et al (2003) suggested inactivated in other cells or tissues regulating the testis function. In that quiescence was a critical event in the establishment of male germ the gonad, TβRII inactivation is likely only germ-speciﬁc as TNAP, cell commitment. They reported that gonocytes lacking Pten do not which targets the Cre excision, is not expressed in somatic cells enter quiescence, leading to bilateral testicular teratoma in adult. In (MacGregor et al., 1995; Urven et al., 1993). Nevertheless, a TβRII our model, we can suppose that the absence of TGFbeta signaling in inactivation in the pituitary axis cannot be eliminated as no data have gonocytes prevents the activation of the Pten/Akt pathway, leading to been published about an eventual expression of TNAP in this tissue. higher germinal proliferation. This would be in agreement with A correlation between the TGFbeta pathway inactivation in PGCs ﬁndings, which have demonstrated the involvement of the Pten/Akt and the impairment of adult spermatogenesis could be established. pathway in a TGFbeta-induced inhibition of lung endothelial cell The condKO males that survived until adulthood were primarily proliferation (Xing et al., 2008). fertile, but the older male, which was studied during 9 months, Additionally to an anti-proliferating effect, we showed here that became sterile from the age of 8 months. Histological analysis of testes TGFbeta has an anti-apoptotic effect on gonocytes. TGFbeta has been showed some depleted seminiferous tubules in the young adult and a previously described as an inducer of gonocyte apoptosis in fetal rat totally disorganized testis with atrophic tubules and no sperm in the testis in vitro (Olaso et al., 1998). In contrast, here we demonstrate, older one. both in vitro and in vivo, that TGFbeta is a negative regulator of In condKO testes, some undifferentiated spermatogonia (SP/α6+/ gonocyte apoptosis in mouse. This difference between two rodent c-Kit-/low) remain, but their number was reduced by around 35% species is intriguing and exhibits the complexity of the intracellular compared with WT testes. Moreover, the majority of these remaining regulation of gonocyte apoptosis. We show here in mouse that undifferentiated cells were quiescent (90-95% versus 70% in WT). TGFbeta2 is expressed in all the gonocytes during their quiescent Despite the presence of undifferentiated spermatogonia in tubules, period. On the contrary, it is not expressed in rat quiescent gonocytes. these seemed unable to recover, since spermatogenesis is impaired. This species difference in TGFbeta expression pattern correlates with This means that, either it doesn't remain stem cells among these 82 S.G. Moreno et al. / Developmental Biology 342 (2010) 74–84

Fig. 7. The number of spermatogonial stem/immature progenitor cells and the proportion of cycling PLZF-positive cells are reduced in condKO mice. Using a Ho staining, the SP was analyzed by flow cytometry analysis in one 4-month-old WT (A) and one 4-month-old condKO (C) mice during the same experiment. After gating the SP, the α6-integrin+/c-Kit-/low and α6-integrin+/c-Kit+ populations were compared in WT (B) and condKO (D) animals. The number of PLZF-positive cells per 100 round tubule sections was counted in adult WT and condKO mice after immunostaining (E). The proportion of PLZF+/Ki67+ cells was measured in WT and condKO testes, after double-immunostaining (F-G). PLZF+ cells appeared in red (arrowheads), Ki67+ cells in green and PLZF+/Ki67+ cells in yellow (arrow). undifferentiated spermatogonia, or these are not functional. Stem cell similar phenotype of transitory fertility has been observed in mice exhaustion could be explained differently: either (i) by a reduced pool overexpressing the activin βA subunit (Tanimoto et al., 1999). In of stem cells in the young male, or (ii) by a stem cell hyper- addition, it has been reported that a decrease of the activin level is proliferation during their asymmetric division, associated with a necessary to the gonocyte-spermatogonia transition (Meehan et al., defect in their self-renewal. 2000). Consequently, TGFbeta ligands, interacting with the activin Our first hypothesis is that TGFbeta is involved in adult receptor, could induce the activin signaling pathway and mimic an spermatogenesis with an essential role in the maintenance of stem overexpression of activin leading to a defect in the gonocyte- cell capacity for self-renewal. Inactivation of the TGFbeta signaling spermatogonia transition and finally to a lower pool of stem cells. resulted in an early testis senescence due to a rapid stem cell Thirdly, we can hypothesize that inactivation of TGFbeta signaling exhaustion. This is in agreement with findings about other members could result in a hyper-proliferation of the differentiated progenitors of the TGFbeta family (such as Nodal or BMP4), which have been (A1 to B spermatogonia) in adult, leading to a rapid loss of these cells reported to play important roles in the maintenance of self-renewal and to an early sterility. and pluripotency of mouse ES cells (Watabe and Miyazono, 2009)(Qi In conclusion, our present results demonstrate a direct and et al., 2004) or in germ stem cells (He et al., 2009). physiological role of the TGFbeta signaling pathway in male germ cell A second hypothesis for this early sterility is that a defect in the development. We show that TGFbeta2 is an autocrine negative regulator gonocyte-spermatogonia transition during neonatal life has led partly of the proliferation and apoptosis of fetal and neonatal germ cells and to a reduced pool of stem cells in the young condKO adult. A quite that the inactivation of the TGFbeta pathway does not completely S.G. Moreno et al. / Developmental Biology 342 (2010) 74–84 83 prevent, but does reduce the gonocyte quiescent phase. Then, germ cell Kawase, E., Wong, M.D., Ding, B.C., Xie, T., 2004. Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the regulation mediated by TGFbeta during testis development seems to be Drosophila testis. Development 131, 1365–1375. important to initiate and maintain a normal adult spermatogenesis. Kehler, J., Tolkunova, E., Koschorz, B., Pesce, M., Gentile, L., Boiani, M., Lomeli, H., Nagy, A., McLaughlin, K.J., Scholer, H.R., Tomilin, A., 2004. 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