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doi: 10.1242/10.1242/dev.00584

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Pin1 regulates the timing of mammalian primordial germ cell proliferation

Fawn W. Atchison1, Blanche Capel2 and Anthony R. Means1,*

1 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA



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  		Fig. 1. Postnatal Pin1-/- males and females have fewer germ cells. (A,C,E,G) Wild-type; (B,D,F,H) Pin1-/-. (A,B) Adult testis section shows that Pin1-/- testis has mostly empty seminiferous tubules containing only Sertoli cells and no germ cells. (C,D) Adult ovary section reveals that few ovarian follicles are seen in Pin1-/- females. (E,F) Newborn testis section shows that Pin1-/- testis has many fewer gonocytes, identified by the GCNA1 antigen (brown), within properly formed testis cords, while many gonocytes populate the wild-type testis. (G,H) Numerous oocytes, highlighted by the blue circles, are in a newborn wild-type ovary. The newborn Pin1-/- ovary has few oocytes. (A,B) Periodic acid-Schiff histochemistry. (C,D,G,H) Hematoxylin and Eosin. (E,F) Immunohistochemistry with antibodies to GCNA1.

 


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  		Fig. 2. Primordial germ cells express the Pin1 protein. (A) Immunohistochemistry using anti-Pin1 antibodies reveals high Pin1 protein expression in postnatal day 1 (P1) gonocytes (arrows). (B) No Pin1 protein is detected in Pin1-/- gonocytes (arrow). (C,E) Immunofluorescence shows intense Pin1 protein expression (green) in 13.5 dpc male XY (C) and female XX (E) PGCs, identified as large round cells by their surface antigen PECAM (red, round cells). (D,F) Negative control using pre-absorbed anti-Pin1 antibodies with Pin1 proteins shows the lack of green Pin1 staining, demonstrating the specificity of the anti-Pin1 antibody. (G) Immunostaining for Pin1 in 9.5 dpc embryo section reveals Pin1 expression (green) in the embryo. (H) Alkaline phosphatase detection of PGCs (brown, arrowhead) in the adjacent serial-section shows the location of PGCs in the hindgut (hg) at 9.5 dpc. Pin1 protein (green) is expressed in the PGC containing regions of the hindgut in G. (I) Negative control using pre-absorbed anti-Pin1 antibodies shows the lack of green Pin1 staining. (G,I) Red, DAPI stained nuclei. s, somite.

 


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  		Fig. 3. PGC number is progressively impaired in Pin1-/- embryos. (A,C,E,G) Wild-type. (B,D,F,H) Pin1-/-. (A,B) Light micrograph images of 8.5 dpc wild-type (A) and Pin1-/- (B) wholemount embryos after alkaline phosphatase assays show PGCs (arrow) are properly allocated at the base of the allantois. (C,D) Light micrograph images of 9.5 dpc wild-type (C) and Pin1-/- (D) wholemount embryos after alkaline phosphatase assays show PGCs (arrows) migrating through the hindgut. The Pin1-/- embryo has fewer PGCs. (E-H) Confocal images of 13.5 dpc wild-type (E,G) and Pin1-/- (F,H) male XY (E,F) and female XX (G,H) gonads after whole-mount immunohistochemistry. PGCs (red, round cells, white arrows) are identified as large round cells by their surface antigen PECAM. Testis cords are identified by laminin at the basement membrane as green crescents (white arrowheads). The Pin1-deficient gonads have few PGCs, but normal cords are formed in the male gonad. (I) PGC number in male XY (left, blue) and female XX (right, pink) whole embryos (8.5 and 9.5 dpc) and comparable gonadal sections (11.5-13.5 dpc) at different stages: wild-type, solid; Pin1-/-, hatched. Values represent mean±s.e.m. *Statistically significant (ANOVA): not significant at 8.5 dpc; P<0.0005 at 9.5 dpc; P<0.0001 at 11.5-13.5 dpc. Subtests at each age were legitimatised by the genotype x age interaction, P<0.0001.

 


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  		Fig. 4. Pin1-/- PGCs do not undergo cell cycle arrest and apoptosis. (A,C,E) Wild type; (B,D,F) Pin1-/-. (A-F) Confocal images of 12.5 dpc male (XY) gonads after wholemount immunohistochemistry. (A,B) Nearly all PGCs (green, round cells) are positive for the Ki67 antigen (red, arrowheads) in wild-type (A) and Pin1-mutant (B) gonads, indicating that PGCs are actively cycling in the presence and absence of Pin1. (C,D) Phosphohistone H3 (green, arrows) identifies PGCs (red, round cells) in mitosis in wild-type (C) and Pin1-/- (D) gonads. No accumulation of phosphohistone H3-positive cells is seen in Pin1-/- gonads, indicating that Pin1-/- PGCs are not arrested in mitosis. (E,F) Wild-type (E) and Pin1-mutant (F) PGCs (green, round cells) are not positive for the apoptosis marker LysoTracker (red), indicating that Pin1-/- PGCs do not undergo increased apoptosis.

 


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  		Fig. 5. Decreased proliferation in Pin1-/- PGCs. (A-C) Male XY; (D-F) female XX. (A,B,D,E) Confocal images of 12.5 dpc male (A,B) and female (D,E) gonads after wholemount immunohistochemistry. (A,D) Many PGCs (green, round cells) incorporated BrdU (red, solid arrows) in wild-type gonads. (B,E) Few Pin1-/- PGCs were labeled with BrdU (red, solid arrows) in the same period, while many Pin1-/- PGCs did not incorporate BrdU (broken arrows). (A,B) Pin1-/- Sertoli cells (arrowheads) appeared to be labeled with BrdU similar to wild type. (C,F) Bar graphs of BrdU labeling indices of wild-type (solid) and Pin1-/- (hatched) male XY (blue) and female XX (pink) PGCs. Values represent mean±s.e.m. *Statistically significant using unpaired Student's t-test, P<0.001.

 


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  		Fig. 6. Illustrative model of cell cycle progression and proliferation in PGCs. Cycling Pin1-/- PGCs (bottom) have a lower BrdU labeling index (red), but normal Ki67, phosphohistone H3 and apoptosis marker profiles. This indicates that Pin1-/- PGCs have a prolonged cell cycle because of defective cell cycle progression (larger, hatched circle) rather than cell cycle arrest and apoptosis. The net effect of decreased proliferation is fewer cell divisions (represented by arrows) in Pin1-deficient PGCs in the same time period, resulting in fewer PGCs at the end of the proliferative phase on 13.5 dpc in the absence of Pin1.

 

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