Unexpected Role of α-Fetoprotein in Spermatogenesis

BACKGROUND: Heat shock severely affects sperm production (spermatogenesis) and results in a rapid loss of haploid germ cells, or in other words, sperm formation (spermiogenesis) is inhibited. However, the mechanisms behind the effects of heat shock on spermatogenesis are obscure. METHODOLOGY/PRINCIPAL FINDINGS: To identify the inhibitory factor of spermiogenesis, experimental cryptorchid (EC) mice were used in this study. Here we show that α-fetoprotein (AFP) is specifically expressed in the testes of EC mice by proteome analysis. AFP was also specifically localized spermatocytes by immunohistochemical analysis and was secreted into the circulation system of EC mice by immunoblot analysis. Since spermatogenesis of an advanced mammal cannot be reproduced with in vitro, we performed the microinjection of AFP into the seminiferous tubules of normal mice to determine whether AFP inhibits spermiogenesis in vivo. AFP was directly responsible for the block in spermiogenesis of normal mice. To investigate whether AFP inhibits cell differentiation in other models, using EC mice we performed a partial hepatectomy (PH) that triggers a rapid regenerative response in the remnant liver tissue. We also found that liver regeneration is inhibited in EC mice with PH. The result suggests that AFP released into the blood of EC mice regulates liver regeneration by inhibiting the cell division of hepatocytes. CONCLUSIONS/SIGNIFICANCE: AFP is a well-known cancer-specific marker, but AFP has no known function in healthy human beings. Our findings indicate that AFP expressed under EC conditions plays a role as a regulatory factor in spermatogenesis and in hepatic generation

Localization of AFP in EC testicular cells and microinjection of human AFP into the seminiferous tubules of normal mice.

<p>Immunohistochemical staining of EC 14 Ds testis (A). Conventional HE stained paraffin sections of EC 14 Ds testis (B). Basal aspect of the seminiferous epithelium of EC 14 Ds mice (C). Microinjection of AFP into the seminiferous tubules through the efferent ducts of normal 49 Ds male mice (D). Fixed testes five days after the microinjection (E). AFP microinjected testis (F). BSA microinjected testis (G). White arrows indicate AFP positive cells (A), and black arrows indicate spermatocytes (B). Scale bar, 50 µm. In the pachytene stage of the EC 14 Ds mice spermatocytes, synaptonemal complexes were clearly seen (C, arrows). Scale bar, 2 µm. SPC, spermatocyte; S, Sertoli cells; L, lumen of seminiferous tubule, Li, lipid droplet. Blue staining of the seminiferous tubules right under the tunica albuginea reflects the success of the microinjection (D). Scale bar, 1 mm. Five days after the microinjection, fixed testes containing faint blue staining of the seminiferous tubles right under the tunica albuginea (E). Scale bar, 1 mm. Semithin sections were stained with 1% toluidine blue dye (F and G). No evidence of spermiogenesis is observed in the shrunken seminiferous tubule of AFP microinjected testis (asterisk in F), but normal appearing spermatogenesis is observed in the BSA microinjected testis (G). Scale bar, 50 µm. TA, tunica albuginea.</p

Testis specific expression of AFP in EC mice.

<p>SDS-PAGE and Western blotting of multiple tissues reacts using an anti-mice AFP antibody (A). Dot blotting of EC mice sera after different days of cryptorchidism (B). The AFP antibody reacts with two bands (about 72 and 26 kDa) in the testis (A, lane 8, arrows), and no signals are seen in the other tissues. Asterisks indicate the two bands which correspond to the positive bands of Western blot. All of the EC mice sera react positively, but the 35 Ds control sera are negative (C in B). N, negative control, 0.1 mg/ml BSA; P, positive control, 0.1 mg/ml synthetic peptide.</p

Partial hepatectomy on control and EC mice.

<p>Remnant liver of control mice 35 days after PH (A). Remnant liver of EC mice 35 days after PH (B). Scale bar, 1 cm. Volume of remnant liver in control and EC mice 21 and 42 days after PH (C). Dot blotting of control and EC mice sera 21 and 42 days after PH reacted using an anti-mice AFP antibody (D). Quantification of the remnant liver volume in EC mice 42 days after PH shows a statistically significant low value (C, *<i>p</i> = 0.05 by unpaired <i>t</i> test). Data are presented as mean ± SD. EC mice sera reacted positively, but the control sera are negative (D). N, negative control, 0.1 mg/ml BSA; P, positive control, pregnant mice sera.</p

Proteome analysis of EC testes.

<p>Silver-stained 2-D gels of testis proteins from EC 14 Ds and normal 20 Ds mice (A). Full-length amino acid sequence of mice AFP (B). Homology of mouse and human AFP (C). Spots I (about 72 kDa) and II (about 26 kDa) are detected in testis proteins from EC 14 Ds (A, white circles). Internal N-terminal amino acid sequence of spot I is highlighted in green and red. Internal N-terminal amino acid sequence of spot II is highlighted in red. The homology of mouse and human AFP is 65% and the matching amino acid residues are shown in yellow. Metal binding site (22; black circle), glycosylation site (251; asterisk) and disulfide bonds (99–114, 113–124, 148–193, 192–201, 224–270, 269–277, 289–303, 384–393, 416–462, 461–472, 485–501, 500–511, 538–583, and 582–591) were completely conserved (C).</p

The Detergent-Soluble Cytoplasmic Pool of Survivin Suppresses Anoikis and Its Expression Is Associated with Metastatic Disease of Human Colon Cancer

<div><p>Survivin is a component of the chromosomal passenger complex (CPC) that is essential for accurate chromosome segregation. Interfering with the function of Survivin in mitosis leads to chromosome segregation errors and defective cytokinesis. Survivin contains a Baculovirus IAP Repeat (BIR) and therefore was originally classified as inhibitor of apopotosis protein (IAP), yet its role in apoptosis after cellular stress remains largely unknown. We demonstrate here, that Survivin predominantly suppresses anoikis, a form of programmed cell death induced by loss of cellular adhesion to extracellular matrix. Interestingly, cells ectopically overexpressing EGFP-Survivin showed after loss of cell-matrix-interaction a decreased expression of IκB-α. Subsequent subcellular protein fractionation and immunoprecipitation experiments revealed that XIAP interacts with detergent-soluble Survivin which is known to cooperatively activate NF-κB signaling. Examination of the expression levels of detergent soluble Survivin in colorectal cancer cell lines and in colorectal cancerous tissues revealed that detergent soluble cytoplasmic Survivin levels correlated inversely with anoikis susceptibility in colorectal cancer. Therefore, the detergent soluble cytoplasmic Survivin might be a promising predictive biomarker for lymph node and distant metastases of colorectal cancer. We conclude that an anti-apoptotic function of detergent-soluble Survivin in interphase cells experiencing anoikis is mediated at least via XIAP/IκB-α/NF-κB signaling.</p> </div