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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00650

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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

Division of Developmental Biology, Children's Hospital, Cincinnati, OH 45229, USA

View larger version (64K): [in a new window]   Fig. 1. GP130 is expressed in PGCs throughout early development. (A) RT-PCR showing expression of GP130 and STAT3 in PGCs at E10.5 and E12.5. LIFR message is present at E12.5. The PGC marker gene Kit is expressed at both stages, and the somatic marker Kitl (St) is absent. `+' indicates RT+ and `-' indicates RT-controls. (B) GP130 protein is present in extracts prepared from E14.5 testes (T) and ovaries (O). The equivalent of 1/3 of a gonad was loaded per lane. Blotting was performed using the M20 antibody. (C,D) Whole mounts stained with goat anti-GP130 (gGP130; red). (C) Control (E10.5; no primary antibody). PGCs express GFP (green) driven by the Oct4PE promoter. (D) GP130 is expressed on the cell surface of PGCs at E10.5. Cell surface expression is evident on somatic as well as germ cells. (E,F) 12 mm frozen sections from E15.5 embryos stained with gGP130. GP130 is expressed in the ovary (E) and testis (F) at E15.5. Cell surface staining is much stronger in the somatic cells than in the PGCs. Scale bars: 20 µm.

View larger version (180K): [in a new window]   Fig. 2. GP130 is expressed in the adult ovary and testis. (A) Control. (B) Primordial follicle stained with gGP130. Staining is weak and diffuse in the oocyte. (C) Growing follicle stained with gGP130. Cell surface staining is evident at the surface of the oocyte, and in the thecal and granulosa cells of the follicle. (D) Seminiferous tubule stained with naïve goat IgG. Staining at the periphery of the tubule and in Leydig cells is nonspecific. (E) Seminiferous tubule stained with gGP130. This tubule contains elongating spermatids but no mature sperm. Cell surface staining is most prominent in the primary spermatocytes, which also exhibit a strong perinuclear spot of GP130 staining. (F) Seminiferous tubule stained with gGP130. This tubule contains mature sperm. GP130 is strongly expressed on the surface of all spermatocytes and spermatids. (G,H) The M20 antibody also stains growing oocytes (G) and primary spermatocytes (H); however, staining is more diffuse. Scale bars: 20 µm for A,D,E,G,H; 10 µm for B,C,F.

View larger version (86K): [in a new window]   Fig. 3. GP130-deficient embryos have almost normal numbers of PGCs. PGCs express the gfp marker (green), and GP130 staining (red) was performed using the gGP130 antibody. Staining and counts were performed on gonads dissected at E13.5. (A-D) GP130 expression in a / ovary (A), a wild-type ovary (B), a / testis (C), and a wild-type testis (D). Both male and female / gonads exhibit reduced staining. (E) Germ cell counts in /, /+ and +/+ littermates. PGCs were counted in individual testes or ovaries as described (Materials and Methods). The males exhibit a slight reduction in PGC numbers, which is statistically significant in the case of the / (-/-) littermates (Student's t-test, P<0.02). Error bars show standard deviations. Scale bars: 20 µm.

View larger version (45K): [in a new window]   Fig. 4. Germ cell specific ablation of GP130 causes a breeding defect in females. (A) Breeding performance of GCKO females and their wild-type or heterozygous littermates. Four-month-old females were bred three times and the number of pups in each litter were recorded. GCKO females (n=8) produced statistically fewer pups in their second and third litters (Student's t-test) as compared with their littermates (n=8). Error bars show standard deviations. (B-E) Cre-mediated excision of GP130 results in accumulation of a secreted form of the protein. GP130 staining (red) was performed using the ggp130 antibody. (B) Staining in an antral-stage oocyte from a GP130Flox/GP130 Cre+ (GCKO) animal. The surface of the oocyte can be seen in the light transmitted image (right). GP130 staining fills the zona. (C) Staining in an antral-stage oocyte from a wild-type female. (D) Staining in an antral-stage oocyte from a GP130+/GP130 Cre-female. (E) Summary of staining data from six GCKO ovaries and six heterozygous sibling controls. Staining intensity was quantified by summing the pixel intensities within the secreted ring of GP130. Three oocytes per ovary were quantified and their staining intensity was averaged. Error bars represent standard deviations. Scale bars: 20 µm.

View larger version (52K): [in a new window]   Fig. 5. Ovaries from GCKO females exhibit no gross morphological defects. (A,B) Hematoxylin and Eosin stained sections from a GCKO ovary (A) and a GP130Flox/GP130+ Cre+ sibling (B). Follicles at various stages are evident. Primordial (p), primary (pr), growing (g), atretic (a) and antral (an) stage follicles are indicated. (C) GCKO females (n=6) have normal numbers of total follicles relative to their siblings (n=5). Follicles were counted in every fifth section. (D) Counted follicles were staged based on morphology. GCKO females have a slight, but statistically significant reduction in the percentage of follicles in the primary stage (Student's t-test, P<0.03). Error bars show standard deviations. Scale bars: 30 µm.

View larger version (80K): [in a new window]   Fig. 6. (A) Eggs ovulated from a GCKO female after one day in culture. (B) Eggs ovulated from a GP130Flox/ Cre-sibling control. (C) Summary of ovulation data. Typically, GCKO females release fewer eggs than their siblings, and few of those eggs, apart from those released from GCKO 5, cleave in culture.