Histological architecture and endocrine profile of Pik3cb^K805R/K805R mice. (A) Histological architecture of Pik3cb^WT/WT (WT) and Pik3cb^K805R/K805R (KR) mice (hematoxylin-eosin staining). Scale bar, 50 μm. (B) Plasma testosterone levels (ng/ml) of adult WT and KR mice (n = 9 WT, 5 KR), measured by ELISA. The difference is not statistically significant. (C) Plasma FSH levels (ng/ml) of adult WT and KR mice (WT, n = 9; KR, n = 5), measured by IRMA. *p < 0.05, KR versus WT.

Loss of spermatogones in adult Pik3cb^K805R/K805R testes. Representative immunostaining for Tra98 (marking undifferentiated spermatogones), PLZF (marking SSCs), and Gata-4 (L, Leydig cells; S, Sertoli cells) of testicular sections obtained from postnatal days 5 and 10 (P5 and P10) and week 16 (W16) Pik3cb^WT/WT (left) and Pik3cb^K805R/K805R mice (right). Testes were fixed in Carnoy and PFA, and immunostaining was obtained with a secondary antibody linked to HRP (appearing in brown and red), whereas counterstaining was performed with hematoxylin. Black arrows, SSCs. Scale bar, 50 μm.

p110β activates Akt downstream of the c-Kit receptor in spermatogones (A) Analysis of c-Kit–positive cells in Pik3cb^WT/WT and Pik3cb^K805R/K805R adult testis. Testes were fixed with 4% PFA (top) or Carnoy (bottom), and sections were stained with an antibody specific for the c-Kit receptor (marking Leydig cells and spermatogones). Scale bar, 50 μm. (B) Analysis of Akt and Erk phosphorylation after treatment with TGX221. Isolated spermatogones were treated with 200 nM TGX221 and stimulated with 100 ng/ml SCF. A representative Western blot is presented. (C) Bars represent the quantification of Akt phosphorylation on Ser⁴⁷³ (n = 6; **p < 0.01).

The absence of p110β activity reduces the survival of the spermatogenic lineage. (A) Number of actively proliferating PCNA and cyclin D1–positive cells in testicular sections of Pik3cb^WT/WT (WT) and Pik3cb^K805R/K805R (KR) testes obtained from postnatal day 10 (P10) and adult (W8) mice. Representative immunostaining for PCNA and cyclin D1 of a WT and a KR section are presented. PCNA and cyclin D1–positive cells appear in brown and red, respectively, whereas counterstaining was performed with hematoxylin. PCNA-positive cells were counted for each tubule in different sections. Data are presented as average ± SEM (n = 5 animals per genotype); ***p < 0.001 KR versus WT. (B) Number of TUNEL-positive cells in tubules from WT and KR mice. Representative micrographs are shown. TUNEL staining appears as a red fluorescence, whereas nuclei were stained with DAPI (blue). The data (number of TUNEL-positive cells/microscopic field) are reported as average ± SEM (n = 5 animals per genotype); *p < 0.05 KR versus WT. Scale bar, 50 μm.

The absence of p110β activity causes severe testicular hypotrophy, defective fertility, and fecundity. (A) Number of litters from mutant mice. Mutant and control mice (n = 6) were mated with receptive female for 6 mo, and the number of litters produced was counted. (B) Number of spermatozoa obtained from the flushing of the deferens ducts of wild-type (WT) and Pik3cb^K805R/K805R (KR) mice (n = 5). Spermatozoa were counted as the number of cells per microscopic field. Data (number of cells/microscopic field) are reported as average ± SEM; *p < 0.05, KR versus WT. (C) Average weight of testes from Pik3cb^WT/WT (WT) and Pik3cb^K805R/K805R (KR) mice (n = 10). A representative image of testes dissected from a WT and a KR mouse is presented. The data (mg/testis) is reported as average ± SEM; ***p < 0.001, KR versus WT. (D) Representative fluorescent immunostaining of testicular sections obtained from Pik3cb^WT/WT and Pik3cb^K805R/K805R mice. p110β was detected with a specific mAb (green), whereas nuclei were stained in red with DAPI. Scale bar, 50 μm.

The absence of p110β activity reduces the survival but does not affect the proliferation of SSCs. (A) Inhibition of cell proliferation of SSCs by a specific p110β or p110α inhibitor: TGX221 (200 nM) or PIK75 (25 nM), respectively. Cells were treated with TGX221 or PIK75 for 72 h in the presence or absence of GDNF, and the relative growth was calculated in comparison to day 0. (B) Proliferation analysis. SSCs were treated with TGX221 and PIK75 for 72 h in the presence of GDNF, fixed with 5% PFA, and stained for Ki67, a positive marker for cell proliferation. The percentage of Ki67-positive cells was measured in more than 10 microscope fields. (C) Analysis of cell cycle markers. Cells were treated with TGX221 or PIK75 for 72 h in the presence or absence of GDNF, and the expression of cyclin D1 was analyzed by Western blot. (D) Analysis of Akt phosphorylation in response to GDNF after treatment with TGX221 or PIK75. SSCs were treated for 1 h with inhibitors and stimulated with 10 ng/ml GDNF. Left, a representative Western blot is presented. Right, bars represent the quantification of phosphorylated Akt on Ser⁴⁷³ (n = 6; **p < 0.001). (E) Analysis of apoptosis after treatment with TGX221 or PIK75. After 72 h of treatment, the percentage of apoptotic cells was quantified by TUNEL assay.