Supplemental Figure 1. The parthenogenetic activation of oocytes recovered from oviducts of gcnrg1 KO mice and wild type mice.

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1 % (a) MII AII Pronucleus (b) 3 F-Actin / Tubulin / DAPI 1 WT KO Supplemental Figure 1. The parthenogenetic activation of oocytes recovered from oviducts of gcnrg1 KO mice and wild type mice. (a) Triple staining (acetylated tublin, actin and nuclei) of oocytes recovered form oviducts at 16, or 24 h after hcg in gcnrg1ko mice. (b) The percentage of pronuclear formation in oocytes recovered from oviduct of ecghcg primed female mice at 24 h after hcg injection.

2 tublin / DAPI A B C Supplemental Figure 2. oocytes recovered form oviducts at 24 h after hcg in gcnrg1ko mice mated with wild type mice Dual staining (acetylated tublin and nuclei) of oocytes recovered from oviducts at 24 h after hcg in gcnrg1ko mice mated with wild type mice was done to assess fertilization. White arrows represented pronuclei and blue arrows showed sperm tail.

3 % (blastocyst stage / 2 cell embryo) P<.5 WT KO WT KO WT KO WT KO 14h 16h 18h h Supplemental Figure 3. The developmental ability to blastocyst stgae of oocytes recovered from WT or gcnrg1ko mice. The oocytes were recovered from oviduct of ecg-hcg primed female mice at different time periods, 12, 14, 16, 18, or h after hcg injection, and then fertilized in vitro. The 2 cell stage embryos were further cultured for 3 days.

4 firefly/renilla firefly/renilla firefly/renilla Ptgs2 (CREB) control Forskolin Forskolin+PMA Areg (CREB) control Forskolin Forskolin+PMA Ereg (SP-1) control Forskolin Forskolin+PMA Supplemental Figure 4. The promoter activity of genes that are expressed at elevated levels in granulosa cells of gcnrg1ko mice was analyzed in gcnrg1ko granulosa cells by transfection of promoter-luciferase reporter constructs of each gene. The Ptgs2-, Areg-, Ereg-luciferase constructs were transfected into cultured gcnrg1ko granulosa cells and then treated with forskolin and/or PMA. Firefly luciferase activities were normalized by Renilla luciferase activities. Values represent the mean +/- SEM of 3 replicates.

5 Number of oocytes in oviduct WT KO WT KO WT KO WT KO WT KO WT KO WT KO 12h 14h 16h 18h h 22h 24h Supplemental Figure 5. The kinetic changes of number of ovulated oocytes in gcnrg1 KO mice and wild type mice. The COCs were recovered from oviduct of ecg-hcg primed female mice at different time periods, 12, 14, 16, 18,, or 24 h after hcg injection, and then counted number of oocytes.

6 Supplemental Table1. List of primers employed for RT-PCR and the expected size gene Forward Primer Reverse Primer Size anneling temperature Areg 5' -TTACTTTGGCGAACGGTGTG -3' 5' -TGTGCAGTCCCGTTTTCTTG -3' Btc 5' -CACAGCACGGTTGATGGACT -3' 5' -CCGAGAGAAGTGGGTTTTCG -3' Cyp11a1 5' -GGGAGACATGGCCAAGATGG -3' 5' -CAGCCAAAGCCCAAGTACCG -3' Ereg 5' -ACACTGGTCTGCGATGTGAG -3' 5' -TCCGTAACTTGATGGCACTG -3' Has2 5' -AGACATTCAATGGGGGTTGG -3' 5' -CCACACAAAGCATGGCAAGT -3' Hsd3b 5' -TGGGGAGAGAAGTCCATTCA -3' 5' -GGAGCCCCCATTCCTTACTA -3' L19 5' -GGCATAGGGAAGAGGAAGG -3' 5' -GGATGTGCTCCATGAGGATGC -3' Nrg1 5 -AGAACCGGCTGTCTGCTTTT -3 5 TAGAGCTCCTCCGCTTCCAT Pgr 5' -AGGTCTACCCGCCATACCTT -3' 5' -GTTATGCTGCCCTTCCATTG -3' Ptgs2 5' -TGTACAAGCAGTGGCAAAGG -3' 5' -GCTGTGGATCTTGCACATTG -3' Ptx3 5' -TGGCTGAGACCTCGGATGAC -3' 5' -GCGAGTTCTCCAGCATGATGA -3' Sfrp4 5' -GAAGTGCCCCCCAAAAAGTCA -3' 5' -CCACAAGCCTTCCCTCTTGT -3' Snap25 5' -GAGATGCAGAGGAGGGCTGAC -3' 5' -GCTGGCCACTACTCCATCCTG -3' StAR 5' -GCAGCAGGCAACCTGGTG -3' 5' -TGATTGTCTTCGGCAGCC -3' Tnfaip6 5' -GTCTGTGCTGCTGGGTGGAT -3' 5' -CGTACTTGAGCCGGATGTGC -3'

7 Supplemental Table2. List of for westernblotting or immunohistochemistry (IHC) Protein target NRG1 EGF domain of NRG1 StAR ERK1/2 Antigen sequence (if known) A synthetic peptide derived from human NRG1. A synthetic peptide (PNEFTGDR) corresponding EGF domain of NRG1 Epitope corresponding to amino acids representing full length StAR of human origin a synthetic peptide corresponding to the sequence of p42 MAP Kinase. Name of Anti-NRG1 ab5314 Anti-EGF domain of NRG1 1 StAR (FL-285): sc-2586 p44/42 MAPK (Erk1/2) (L34F12) Mouse mab Manufacturer,catalog #,and/or name of individual providing the Abcam Cambridge, MA ab5314 Santa Cruz Biotecnology Santa Cruz,CA,USA sc-2586 K19 Cell Signaling Technology, Inc Danvers, MA 4696 Species raised in; monoclonal or polyclonal Ditution used rabbit polyclonal 1 in 1 rabbit polyclonal Mouse monoclonal in 5 for both WB and IF 1 in 5 for IHC 1 in 1 Phosphorylated ERK1/2 phosphorylated CREB connexin43 a synthetic phosphopeptide corresponding to residues surrounding Thr2/Tyr4 of human p44 MAP kinase. Synthetic phosphopeptide corresponding to residues surrounding Ser133 of human CREB Synthetic peptide corresponding to residues of human connexin 43. Synthetic phosphopeptide phosphorylated corresponding to residues Ser 368 connexin43 surrounding Ser368 of human connexin 43 acetyl tubulin βactin Epitope located on the α3 isoform of Chlamydomonas axonemal α-tubulin,1 within four residues of Lys-4 when this amino acid is acetylated.2 Synthetic peptide, Ac-Asp-AspAsp- Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly- Ser-Gly-Lys, conjugated to KLH Phospho-p44/42 MAPK (Erk1/2) (Thr2/Tyr4) (G11) Rabbit mab Phospho-CREB (Ser133) (87G3) Rabbit mab Connexin 43 Antibody Phospho-Connexin 43 (Ser368) Antibody Monoclonal Anti-Tubulin, Acetylated produced in mouse Monoclonal Anti-β-Actin produced in mouse Cell Signaling Technology, Inc Danvers, MA 4376 Cell Signaling Technology, Inc Danvers, MA 9198 Cell Signaling Technology, Inc Danvers, MA 3512 Cell Signaling Technology, Inc Danvers, MA 3511 Sigma St. Louis, MO T M476 Sigma St. Louis, MO A K485 Rabbit monoclonal rabbit monoclonal rabbit polyclonal rabbit polyclonal mouse monoclonal mouse monoclonal 1 in 1 1 in 1 1 in 1 1 in 1 1 in 1 in 1

8 Supplemental Table3. The fertilization ability of oocytes recovered from WT or gcnrg1ko mice at different time point after hcg injection. hr genotype number of Normal Abnormal fertilization (%) Parthenogenesis Nonfertilization (%) Other (%)# oocyte fertilization (%) Total (%) 1-pro (%) 3-pro (%) (%) WT (82) 1 (1) 1 (1) () 2(2) 3 (4) 9 (11) KO (87) 6 (7) 1 (1) 5 (6) 7 (8) () 7 (8) WT (83) 3 (5) () 3 (5) 4 (6) () 4 (6) KO (77) 6 (9) 1 (2) 5 (8) 2 (3) 5 (8) 2 (3) WT (79) 5 (6) () 5 (6) 5 (6) 1 (1) 6 (7) KO 9 29 (32)* 14 (16)* 4 (4) 1 (11) 12 (13) 9 (1) 26 (29) WT 7 5 (71) 6 (9) () 6 (9) 4 (6) () 1 (14) KO (23)* 19 (26)* 12 (16) 7 (9) 4 (5) 22 (3) 12 (16) #; Some oocytes contained 2 or three metaphase plate.

9 Supplemental Table4.1. The fertilization status of putative eggs recovered from WT or gcnrg1ko mice mated with WT male mice at different time point after hcg injection KO Plug formation observed Number of Normal Abnormal fertilization (%) Parthenogenesis Other Non- (hr after hcg injection) oocyte fertilization(%) Total (%) 1-pro (%) 3-pro (%) (%) (%) fertilization(%) (91) 2 (9) 1 (5) 1 (5) () () () (88) 1 (3) 1 (3) () 1 (3) () 2 (6) (81) 4 (13) () 4 (13) 1 (3) () 1 (3) (74) 1 (4) () 1 (4) 1 (4) () 4 (17) (77) 5 (19) 1 (4) 4 (15) 1 (4) () () (72) 1 (23) 1 (2) 9 (21) () 1 (2) 1 (2) (79) 1 (5) () 1 (5) 1 (5) () 2 (11) (75) 2 (8) () 2 (8) () () 4 (17) (78) 3 (17) 2 (11) 1 (6) () () 1 (6) (74) 1 (5) 1 (5) () 1 (5) () 3 (16) (74) 5 (19) 2 (7) 3 (11) 2 (7) () () (69) 5 (31) 1 (6) 4 (25) () () () (65) 4 (15) 1 (4) 3 (12) 1 (4) 3 (12) 1 (4) (48) 4 (17) 4 (17) () 6 (26) 1 (4) 1 (4) (63) 1 (31) 1 (3) 9 (28) 2 (6) () () (55) 14 (37) 4 (11) 1 (26) 2 (5) () 1 (3) (41) 12 (41) 3 (1) 9 (31) 3 (1) 1 (3) 1 (3) (37) 14 (52) 4 (15) 1 (37) 1 (4) () 2 (7)

10 Supplemental Table4.2. The fertilization status of putative eggs recovered from WT or gcnrg1ko mice mated with WT male mice at different time point after hcg injection Plug formation observed (hr after hcg injection) Number of oocyte Normal fertilization(%) Abnormal fertilization (%) Parthenogenesis (%) Other (%) Nonfertilization(%) WT Total (%) 1-pro (%) 3-pro (%) (96) 1 (4) () 1 (4) () () () (94) () () () 1 (3) () 1 (3) (87) 1 (4) 1 (4) () () () 2 (9) (93) () () () 1 (7) () () (9) () () () () () 2 (1) (86) () () () 1 (7) () 1 (7) (9) () () () 1 (5) () 1 (5) (88) 1 (4) () 1 (4) () () 2 (8) (86) () () () () () 3 (14) (94) () () () () () 1 (6) (93) () () () () () 1 (7) (82) 1 (5) 1 (5) () 1 (5) () 2 (9) (8) 2 (1) 1 (5) 1 (5) () () 2 (1) (9) () () () () 1 (5) 1 (5) (82) 1 (5) 1 (5) () () 1 (5) 2 (9) (83) 1 (4) () 1 (4) 1 (4) () 2 (8) (84) () () () 2 (11) () 1 (5) (85) 2 (1) () 2 (1) () () 1 (5) (81) 1 (5) () 1 (5) 1 (5) () 2 (1) (72) 1 (6) 1 (6) () () 2 (11) 2 (11) (75) () () () 1 (4) 1 (4) 4 (17) (77) () () () () () 3 (23)

11 Supplemental Table5. The fertilization ability of oocytes matured in vitro with AREG and/or NRG1 for 16 or 18 h number of normal Abnormal fertilization(%) Parthenogenesis Other Nongenotype hr treatment oocyte fertilization (%) Total (%) 1-pro (%) 3-pro (%) (%) (%) fertilization(%) AREG (71)a () () () 3 (8) () 8 (21) 16 AREG+NRG (7) () () () () () 8 (29) WT AREG 36 8 (22)a,b 11 (31) 9 (25) 2 (6) 5 (14) () 12 (33) 18 AREG+NRG (76)b () () () 1 (3) 1 (3) 6 (17) AREG 33 7 (21)c 15 (45) 4 (12) 11 (33) 3 (9) 1 (3) 7 (21) KO 18 AREG+NRG1 15 (75)c 2 (1) 1 (5) 1 (5) () () 3 (15)