Identifying disruptors of male germ cell development by small molecule screening in ex vivo gonad cultures

Background Germ cell development involves formation of the spermatogenic or oogenic lineages from the bipotential primordial germ cells. Signaling mechanisms in the fetal testis and ovary determine whether germ cells enter the male or female developmental pathway, respectively. These signaling processes underpin an important phase of germ cell development, disruption of which can lead to failed germ cell function resulting in infertility or the formation of germ cell tumours. Findings In this study we have developed a small molecule screening protocol combined with flow cytometry to identify signaling pathways that direct male-specific development of germ cells. Here we provide a detailed method for this screening protocol, which we have used to identify signaling pathways important for male germ cell development. Conclusion This method will be of particular use in screening inhibitors of signaling pathways, endocrine disruptors or other chemicals for their ability to disrupt testis and germ cell development, thereby providing insight into testicular dysgenesis and factors underlying poor male reproductive health.


Background
Spermatogenesis and oogenesis are founded on the development of the male and female germ cell lineages in the developing fetus. In mammals, primordial germ cells are specified from the pluripotent epiblast in the pregastrulation embryo [1]. The primordial germ cells migrate through the embryo and populate the developing gonads at approximately embryonic day (E) 10.5. At this stage, germ cells express key regulators of pluripotency and can readily establish pluripotent embryonic germ cell lines in vitro, reflecting their ability to retain germ line totipotency [2][3][4][5]. After entering the developing testes or ovary the germ cells differentiate down the spermatogenic or oogenic pathways in response to their respective environments [6][7][8][9]. The molecular pathways directing male and female germ line development are poorly understood, even though these processes are crucial for later fertility and for preventing germ cell tumours.
Testis development is initiated with the expression of Sex Region Y chromosome (Sry), which activates Sry box gene 9 (Sox9), thereby promoting specification and proliferation of Sertoli cells and a cascade of events culminating in testis differentiation [10][11][12]. Sertoli cells proliferate and form testis cords, which enclose the fetal germ cells and define the interstitial space in which Leydig cell differentiation occurs [10,13]. These somatic cell types promote testis development and differentiation of the male germ line.
At the onset of testis or ovary development the germ cells can enter the male or female developmental pathway, regardless of whether they are genetically male (XY) or female (XX). The earliest indication of male and female germ cell development is their entry into mitotic arrest or meiosis, which occurs from E12.5 and E13.5, respectively [8]. Experimental evidence collected over the last three decades demonstrate that male and female germ cells are responsive to signals from the gonadal environment and this signaling leads to sex specification by E12.5 and E13.5, respectively [6,8,9,14].
The signaling mechanisms leading to male germ cell development and regulating testis development are not fully understood. However, germ cell mitotic arrest occurs in 48 hours from approximately E12.5, depending on the mouse strain [15,16]. Mitotic arrest of male germ cells involves activation of a number of G1-S phase check-point controlling proteins, including Retinblastoma and p27 KIP1 [15,17]. Male germ cell differentiation also involves the repression of the core pluripotency genes Oct4, Sox2 and Nanog, which are maintained in germ cell derived teratomas [4,5,[18][19][20].
We have previously analysed male germ cell development using flow cytometry [15,16,21]. In this study we aimed to develop an ex-vivo screening protocol for identifying signaling pathways involved in male germ cell development with the expectation that disrupting these signaling processes would block male germ cell mitotic arrest and differentiation, without causing sex-reversal. We isolated E12.5 fetal testes after male sex determination had occurred and testis cords had formed, but before germ cells had entered mitotic arrest [15,16]. These fetal testes were cultured with a range of specific small molecule chemical inhibitors and germ cell mitotic arrest was monitored using a flow cytometric assay. Here we provide a detailed account of this protocol and its application in screening small molecule inhibitors for their ability to disrupt mouse fetal germ cell or gonad development. This system provides an effective medium throughput, ex-vivo model for identifying small molecules or chemicals, such as endocrine disruptors, that inhibit germ cell mitotic arrest, reflecting compromised differentiation of the fetal germ cells, and potentially gonad development.

Screening germ cell development by flow cytometric analysis
Initially in a series of single pass experiments (n=1, 4 gonads per culture) we screened 18 small molecule inhibitors for their ability to disrupt male germ cell development in fetal mouse testes. Testes were isolated from E12.5 Oct4-GFP positive mouse fetuses and cultured for 72 hours in the presence of inhibitor or vehicle control (e.g. dimethyl sulphoxide; DMSO) using a culture system we have previously validated [22]. In this screen the inhibitors used targeted a range of common signaling pathways, including those mediated by (or through) platelet derived growth factor (PDGF), glycogen synthase kinase 3 beta (GSK3β), mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), janus kinase (JAK), vascular endothelial growth factor (VEGF) and ACTIVIN/NODAL/TGFβ. At the end of the culture period the gonads were labeled for two hours with 5-ethynyl-2′deoxyuridine (EdU), which marks cells progressing through S-phase. After 72 hours of culture we examined gonadal phenotype in whole mount to identify overall effects of the inhibitors on gonad development. Testis cord morphology was viewed under phase contrast microscopy, while germ cells were viewed in-situ using GFP fluorescence, which is driven by germ cell specific expression of the Oct4-GFP transgene ( Figure 1A). The gonads were then dissociated and subject to flow cytometric assessment of germ cell mitotic arrest, used in this context as a marker of male germ cell differentiation ( Figure 1B) [15,16].
Flow cytometric analysis of the gonads treated in these cultures revealed that the two most effective inhibitors of male germ cell mitotic arrest were SB431542 and PD0325901, which inhibit the ACTIVIN/TGFβ/NODAL receptors ALK4, ALK5 and ALK7, and MEK1/2 signaling, respectively. Treatment with SB431542 or PD0325901 resulted in 14.5% and 13.5% of the germ cell population incorporating 5′-ethynyl-2′-deoxyuridine (EdU) (i.e. transiting S-phase) compared with 3.2% in DMSO (control) treated gonads. Detailed analyses of the impacts of SB431542 on male germ cell development has been reported in detail elsewhere [22] (D. Miles, S. Wakeling and P. Western unpublished data). Here we provide a step-by-step protocol for screening similar chemical inhibitors and other reagents for their ability to disrupt male germ cell development.

Protocol
This protocol is based on Click-iT Edu chemistry (Invitrogen). Germ cells are separated from gonadal somatic cells using an antibody specific for Mouse Vasa Homologue (MVH) (AbCam Ab13840), detected using an alexa fluor donkey-anti-rabbit 488 nm secondary antibody, but both the primary and secondary antibody can be varied depending on your target cell (eg anti-SOX9 can be used to isolate Sertoli cells and anti-FOXL2 or anti-GATA4 can be used for female somatic cells), flow cytometer and preference for secondary detection. This protocol is written for analysis of germ cells in the fetal testis, but can also be applied to fetal ovary.  (Figure 2A). Display cells from P1 in a new scatter plot of PI width (linear) vs PI area (linear) ( Figure 2B). 3.2: Using P1 as the parent population, establish a gate (P2) around the single cells as shown in Figure 2B. Set PI intensity so that the G1 population is located over 50 to100 and G2/M population is located over 100 to 200 fluorescence units ( Figure 2B). This value is not critical, as long as sufficient separation is achieved using PI to model cell cycle in a third party software such as ModFit or FlowJo. 3.3: Showing only the P2 events and using P2 as the parent population, establish a scatter plot for alexa-flour 488 nm (log scale) vs PI (PE-Cy5) area (linear scale). Set 488 nm laser power so that the 488 nm negative somatic cells are below just 1000. 3.4: Change to control sample 2 (Table 1). You should see MVH positive germ cells in the 1000-10,000 range. Expect the staining intensity for MVH to be a little higher in males than females and higher at E15.5 than at E12.5.
In an untreated E12.5 samples cultured for 72 hours most of the MVH positive cells should be located over the G1 PI value (ie over 50-100) with few scattered over S-G2/M (100-200) ( Figure 2C).   Figure 2E). P6 should be essentially identical to the somatic cell P5 gate, but specific for the somatic cells. These are the somatic cells passing through S-phase in the two-hour culture period during which the gonads were exposed to EdU. 3.8: Using control sample 1 (Table 1) [22].

Discussion
Over recent decades male reproductive health has declined and it has been suggested that various chemicals or environmental toxins may be responsible through their ability to disrupt testis and germ-line development [23]. This is manifest in increased incidence of pluripotent germ cell tumours, which have their origin in poorly differentiated fetal germ cells. Male germ cell differentiation is initiated and directed by unknown signaling processes within the fetal testis, which results in strict regulation of germ cell proliferation and the repression of germ cell pluripotency. Although these signaling processes are poorly understood, disruption of male germ cell development at this stage can lead to pluripotent germ cell tumours [19,20].
A tractable and sensitive model that facilitates rapid screening of signaling inhibitors, environmental toxins (e.g. endocrine disruptors) or other reagents that result in compromised testis and/or germ cell development would be of significant benefit in male reproductive biology. We have combined our existing flow cytometric method with gonad organ culture to develop a drug screening protocol that facilitates identification of key signaling pathways in testis and male germ cell development. Our model provides a means to rapidly screen drugs that affect the early processes in testis and male germ cell development and allow the analysis of perturbed germ cell differentiation in a compromised testicular environment. By using E12.5 rather than E11.5 testes this protocol provides a method for perturbing germ cell development after somatic sex determination, reducing the complicating factors introduced by sex-reversal. The flow cytometric assay allows robust detection of small changes in cell proliferation in small amounts of tissue (2-4 gonads are sufficient). As quality antibodies suitable for flow cytometry are commercially available and/or existing transgenic mouse models provide access to various testis specific cell types (i.e. germ cells, Sertoli cells, endothelial cells, Leydig cells) [24], this approach is applicable to screening many inhibitors, endocrine disruptors or other reagents that might impact testis or germ cell development.
To test the veracity of the screening approach we initially screened 20 inhibitors of common signaling pathways for their ability to disrupt male germ cell development in the early testis, soon after male sex determination. We identified an important role for ACTIVIN/NODAL/TGFβ signaling pathways in male germ cell development, which we analyzed in detail in a separate study [22]. We also identify negative affects on mitotic arrest of fetal male germ cells using the MEK1/2 (MAPK) inhibitor germ cell PD0325901 (S. Wakeling, D. Miles and P. Western, unpublished data) and are now performing a more detailed analysis of this pathway. Following identification of drugs or chemicals that exhibit activity in this screening protocol, further detailed analyses, including immunofluorescence, qRTPCR and cell biological assays can be performed to establish the mechanisms through which the inhibitor is active. Given appropriate antibodies for flow cytometry, a similar approach could be applied to other stages of gonad development and may allow identification of drugs, chemicals or toxins leading to reduced fertility and germ cell function. We have applied this screening approach to ex-vivo gonad cultures in mice because germ cell development is best characterized in this species and ex-vivo screening facilitates a higher throughput system. However, the same approach can be applied to mice using in-vivo treatment, or a similar approach could be used in other species.
We have developed a robust protocol facilitating medium throughput screening of chemical inhibitors that can be used to identify signaling pathways involved in male germ cell development. This model provides an accessible system in which environmental and developmental processes influencing testis and male germ cell development can be manipulated and will provide important insights into the processes underlying testicular dysgenesis and the early stages of germ cell tumour formation.