GP130, the shared receptor for the LIF/IL6 cytokine family in the mouse,is not required for early germ cell differentiation, but is required cell-autonomously in oocytes for ovulation

Abstract

GP130 is the shared receptor for members of the IL6 family of cytokines. Members of this family have been shown to enhance the survival of migratory (E10.5) or postmigratory (E12.5) murine primordial germ cells (PGCs) in culture; however, it is uncertain what role these cytokines play during PGC development in vivo. We have examined PGC numbers in E13.5 GP130-deficient mouse embryos and found that males exhibited a slight decrease in PGC numbers; females were normal. Also, we used the Cre-loxP system to inactive GP130 specifically in germ cells and found that this resulted in a fertility defect in females. These animals were found to have a slight reduction in the number of primary follicles and a major defect in ovulation. This data suggests that GP130 is required in female germ cells for their normal function, but is dispensable in male germ cells. These cytokines share common receptors and common signal transduction machinery, and this might explain the relatively mild phenotypes resulting from inactivation of a single family member or a single high affinity receptor. Ablation of the common receptor (GP130), or a common cytoplasmic component (STAT3), should affect all IL6 family members and result in stronger phenotypes. This appears to be the case. STAT3-knockout animals die by E7.5 (Takeda et al., 1997), and GP130-null animals die of cardiac and hematological disorders by E15.5 (Yoshida et al., 1996), or at birth (Kawasaki et al., 1997), depending on the genetic background. Intriguingly, GP130-null animals have been reported to have fewer numbers of PGCs (T. Taga, unpublished).In order to clarify the role of the IL6 family in PGC development, we examined PGC numbers in GP130-deficient males and females. In addition, we have used the Cre-loxP system to generate germ-cell-specific ablations of GP130. Surprisingly, our data demonstrate that GP130-mediated signaling is not required for the early stages of PGC development, but reveal a novel role for GP130-mediated signaling late in oogenesis. MATERIALS AND METHODS Mouse strains and genotypingThe TNAP-Cre, GP130Flox and Oct4∆PE:GFP lines have been described previously (Anderson et al., 1999; Betz et al., 1998;Lomeli et al., 2000). For breed tests, CD1 males and females were purchased from Charles River. Genotyping of animals was performed by PCR. DNA was isolated from tail snips (adults) or from heads (embryos) using the Wizard Genomic DNA purification kit (Promega). PCR was performed on 1-100 ng of genomic DNA using RedMix Plus (PGC Scientific) or Platinum PCR Supermix (Invitrogen) as a source of Taq, buffer and dNTPs. Final primer concentrations were 0.4 µM. PCR consisted of: an initial denaturing step of 5 minutes at 95°C; followed by 5 cycles of 30 seconds at 95°C, 1 minute at 65°C and 30 seconds at 72°C; followed by 35 cycles of 30 seconds at 95°C, 1 minute at 60°C and 30 seconds at 72°C; followed by a 10 minute extension step at 72°C. Primers for genotyping the Oct4∆PE:GFP strain are described by Anderson et al. (Anderson et al., 1999). Primers for the TN...

FGF9 promotes survival of germ cells in the fetal testis

“…ablation of GP130 led to reduced gonocyte numbers in 13.5 dpc XY animals (Molyneaux et al, 2003). However, the basis for this effect was unclear, and, interestingly, these GP130-null males are fertile.…”

The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo

“…In previous work, using a living marker of germ cells, we have shown that germ cells leave the hindgut between E9.0 and E9.5, and migrate laterally towards each gonad (Molyneaux et al, 2001;Molyneaux et al, 2003). By E10.5, the embryos has grown significantly in size, and a mesentery has formed that displaces the hindgut from the dorsal body wall, and increases the distance of migration.…”

“…PGC isolation and reverse transcription was performed as previously described (Molyneaux et al, 2003). PGC and somatic cDNAs were diluted 1:10 and 1 µl used for PCR (25 µl).…”

Germ cell pluripotency, premature differentiation and susceptibility to testicular teratomas in mice

“…FACS with the Oct4::GFP transgene has been previously described (Heaney et al, 2009;Molyneaux et al, 2003). Briefly, gonads were digested in 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) for 15 minutes at 37°C.…”