An evolutionary conserved interaction between the Gcm transcription factor and the SF1 nuclear receptor in the female reproductive system

NR5A1 is essential for the development and for the function of steroid producing glands of the reproductive system. Moreover, its misregulation is associated with endometriosis, which is the first cause of infertility in women. Hr39, the Drosophila ortholog of NR5A1, is expressed and required in the secretory cells of the spermatheca, the female exocrine gland that ensures fertility by secreting substances that attract and capacitate the spermatozoids. We here identify a direct regulator of Hr39 in the spermatheca: the Gcm transcription factor. Furthermore, lack of Gcm prevents the production of the secretory cells and leads to female sterility in Drosophila. Hr39 regulation by Gcm seems conserved in mammals and involves the modification of the DNA methylation profile of mNr5a1. This study identifies a new molecular pathway in female reproductive system development and suggests a role for hGCM in the progression of reproductive tract diseases in humans.


Fertility and egg laying assays
For fertility assays, three 1-day-old virgins of a given genotype were crossed with one 1-day-old male Oregon-R on standard medium at 25˚C. The cross was flipped every three days for twelve days. The progeny issued from the four bottles is counted at the adult stage. Each cross was replicated at least ten times and paired with a control (Oregon-R females). The average number of progeny per female of the ten replicates and standard error of the mean are represented in Figure 1a. For the egg laying assays, five 1-day-old females of the indicated genotypes were crossed with ten 1-day-old males Oregon-R for 3 days, then the flies were transferred to a cage to count the number of eggs laid over 48 hrs. The number of eggs was then reported to the number of females and the number of days in Figure 2i. The p-values were estimated after variance analysis using bilateral student test with equal variance (ns for not significant; "*" for p-value < 0.05, 0.01 <; "**" for p-value < 0.01, 0.001 <; "***" for p-value < 0.001)).

Secretory cell and basal cell counts
The spermatheca were dissected and labelled with anti-Hnt antibody and DAPI as described above. For each spermatheca, the Hnt/DAPI positive cells (secretory cells) were counted from the stack of six focal plans taken at 3µm interval in the middle of the spermatheca (the plan giving the largest cross-section of the spermatheca). This was repeated in at least six independent spermathecae for each genotype. The average number of secretory cells and the standard error of the mean are represented in Figure 2h and Figure 4h. The p-values were estimated after variance analysis using bilateral student test with equal variance (ns for not significant; "*" for p-value <0.05, 0.01<, "**" for p-value <0.01, 0.001<, "***" for p-value < 0.001).

qPCR and luciferase assay in S2 cells
The transfection of S2 cells, the quantitative PCR (qPCR) and the luciferase assay were performed as described in Cattenoz et al. 10 . For the qPCR, 6 million S2 cells were plated per well in 6-well plates in 1.5 mL of Schneider medium complemented with 10% Fetal Calf Serum (FCS) and 0.5% penicillin and 0.5% streptomycin (PS). Cells were transfected 12 hrs after plating using the Effectene transfection reagent (Qiagen) using 2 µg of pPac-gal4 vector and 1 µg of pUAS-GFP for the negative control (ppacEmpty) and 2 µg of pPac-gcm 11 and 1 µg of 4.3kb repo-GFP (repoGFP) 12 for the gcm GOF assays (ppacGcm). After 48 hrs of transfection, the cells were sorted on a BD FACSAria according to GFP expression to obtain more than 80% of transfected cells in the sample. The RNA was then extracted using TRI reagent (Sigma), 1 µg of RNA per sample was DNAse treated with RNAse free DNAse 1 (Thermo Fisher) and reverse transcribed with Superscript II (Invitrogen). Quantitative PCR (qPCR) assays were performed on a lightcycler LC480 (Roche) with SYBR master (Roche) on the equivalent of 5 ng of reverse transcribed RNA with the primer pairs targeting Hr39, hnt, Gapdh1 and Act5c listed below. Each PCR was carried out in triplicates on at least three biological replicates. The quantity of each transcript was normalized to the quantity of Gapdh1 and Act5c. The p-values were measured comparing the control with the transfected cells using student test, the bars represent the standard error of the mean.
For the luciferase assay, WT and mutant reporters were built for each GBS at Hr39 and hnt loci. Sense and antisense oligonucleotides covering the GBS in each gene were synthesized using flanking restriction sites for KpnI at the 5' extremity and NheI at the 3' extremity. Each pair of oligonucleotides was designed with the WT GBS and with a mutated GBS that is not bound by Gcm (mutated for nucleotides 2, 3, 6 and/or 7: list below, the restriction sites are indicated in capital letters). For each WT and mutant GBS, 2 μg of annealed oligonucleotide were digested with 20 U of KpnI (NEB # R3142S) and 20 U of NheI (NEB # R3131S) in Cutsmart buffer (NEB # B7204S) for 1 h 30 min at 37ºC. The digested double stranded probes were then cleaned and ligated in pGL4.23 (Promega #E841A) (ratio plasmid:probe = 1:6). Transfections of Drosophila S2 cells were carried out in 12-well plates using Effectene transfection reagent (Qiagen #301427) according to manufacturer's instructions. Cells were transfected with 0.5 μg pPac-lacZ, 0.5 μg pGL4.23 carrying the indicated GBS, 0.5 μg pPac-gcm 11 or 0.5 μg pPac 13 . 48 hrs after transfection, cells were collected, washed once in cold PBS and resuspended in 100 μL of lysis buffer (25 mM Tris-phosphate pH7.8, 2 mM EDTA, 1 mM DTT, 10% glycerol, 1% Triton X-100). The suspensions were frozen / thawed four times in liquid nitrogen and centrifuged 30 min at 4ºC at 13000 g. The Luciferase and βgal activities were measured in triplicates for each sample. For βgal measurements, 20 μL of lysate were mixed with 50 μL of β-galactosidase assay buffer (60 mM Na2PO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 50 mM β-mercaptoethanol) and 20 μL ONPG (4mg / mL) and incubated at 37ºC for 20 min. The reaction was stopped by adding 50 μL 1M Na2CO3 and the DO at 415 nm was measured. For Luciferase activity, 10 μL of protein lysate were analysed on an opaque 96-well plate (Packard instrument # 6005290) with a Berthold Microluminat LB96P Luminometer by injecting 50 μL of luciferase buffer (20 mM Tris-phosphate pH 7.8, 1 mM MgCl2, 2.5 mM MgSO4, 0.1 mM EDTA, 0.5 mM ATP, 0.5 mM luciferine, 0.3 mM coenzyme A, 30 mM DTT). For both βgal and Luciferase assays, background levels were estimated using lysate from not transfected S2 cells. The relative Luciferase activities were calculated as follow: first the background was subtracted from each value, then the average values of the technical triplicate were calculated. From there, the Luciferase activity of each sample was normalized to the βgal activity (Luciferase activity / βgal activity) to correct for transfection efficiency variability and the ratio (Luciferase with Gcm / Luciferase without Gcm) was calculated. For each WT and mutant GBS, biological triplicates were carried out.
In situ hybridisation and RNA extraction from mouse uterus RNA in situ hybridisation with digoxigenin-labelled probes for mGcm2 transcripts was performed as described in Vernet et al. 14 with slight modifications. Cryosections (10 µm sections) of mouse (C57BL/6) uterus were labelled with sense or anti-sense probes targeting mGcm2 (only the anti-sense probe is shown). The probes were synthesized from the clone 40054293 inserted into pCR-BluntII-TOPO using the Ribo-probe in vitro transcription system (Promega).
To assess mGcm1, mGcm2 and mNr5a1 levels of expression in uterus (Figure 5f), the RNA was extracted from the uterus of C57BL/6 using TRI reagent (Sigma) and the qPCR were carried out as described below for mammalian cells. The levels were estimated from 3 different animals.

Transfection and qPCR in mammalian cells
HeLa cells were plated in 6-well plates, 400,000 cells per well, in 1.6 mL of DMEM medium complemented with 5% FCS and gentamycin. Cells were transfected 12 hrs after plating using Effectene transfection reagent (Qiagen). Briefly, 1 µg of pCIG vector, 1 µg of pCIG vector expressing mGCM1 (pCIG-mGcm1) 15 or 1 µg of pCIG vector expressing mGCM2 (pCIG-mGcm2) were mixed with 100 µL of EC buffer and 8 µL of enhancer, incubated 5 min at room temperature, then 10 µL of Effectene were added and the mix was incubated at room temperature for 20 min. 200 µL of DMEM medium + 5% FCS + gentamycin were added to the mix before spreading it on the cells. 48 hrs after transfection, the RNA was extracted using TRI reagent (Sigma).
MEF cells were plated in 6-well plates, 400,000 cells per well, in 1.6 mL of DMEM medium (4.5g/L glucose) complemented with 10% FCS, 1% sodium pyruvate and 0.5% penicillin and 0.5% streptomycin. Cells were transfected 12 hrs after plating using Effectene transfection reagent (Qiagen). Briefly, 1 µg of pCIG vector, 1 µg of pCIG vector expressing mGCM1 (pCIG-mGcm1) 15 or 1 µg of pCIG vector expressing mGCM2 (pCIG-mGcm2) were mixed with 100 µL of EC buffer and 8 µL of enhancer, incubated 5 min at room temperature, then 10 µL of Effectene were added and the mix was incubated at room temperature for 20 min. 200 µL of DMEM medium + 5% FCS + gentamycin were added to the mix before spreading it on the cells. After 48 hrs of transfection, the cells were sorted on a BD FACSAria according to GFP expression (the pCIG vectors express GFP constitutively) to obtain more than 80% of transfected cells in the sample. The RNA was then extracted using TRI reagent (Sigma).
Reverse transcription and qPCR were carried out as described for the S2 cells with the primer pairs listed below. The quantity of each transcript was normalized to the quantity of the housekeeping genes Glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and Actin Beta (ActnB).

Bisulfite sequencing in MEF cells
MEF cells transfected and sorted as described above were used to analyse the methylation profile of mNr5a1 locus. After sorting, the cells were incubated 1.5 hrs at 37°C in 20 mM EDTA, 10 mM Tris pH 8.0, 200 mM NaCl, 0.2% Triton X-100 and 100 mg/mL proteinase K and centrifuged at room temperature for 5 min at 14000 rpm. The DNA was precipitated from the supernatant by adding 1 vol. of isopropanol and 1/20 vol. of 4M NaCl, incubating the sample overnight at -20°C and centrifugation at 4°C for 25 min at 14000 rpm. The DNA pellet was suspended in demineralized water and treated with RNAse A for 1 hr at 37°C. Then 500 ng of DNA was digested with BamH1 restriction enzyme and converted with bisulfite using EZ DNA methylation Direct Kit (ZYMO #D5020) according to the manufacturer instruction. The loci of interest were then amplified by PCR using the ZymoTaq DNA polymerase (ZYMO #E2001) and the primers indicated below. The PCR products were cloned in pGEM-T Easy vector and sequenced by Sanger sequencing (GATC Biotech). At least 10 clones were sequenced per condition. The p-values were estimated after variance analysis using bilateral student test for paired samples (ns for not significant; "*" for p-value <0.05, 0.01<, "**" for p-value <0.01, 0.001<, "***" for p-value < 0.001).