Kenji Oka

Hypoxia Attenuates the Expression of E-Cadherin via Up-Regulation of SNAIL in Ovarian Carcinoma Cells

Since ovarian carcinoma cells detach from the primary lesion and metastasize via peritoneal dissemination, we hypothesized that these cells are exposed to hypoxia, which may affect cell attachment and invasiveness. To address this hypothesis, we first examined in vivo the immunohistochemical expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and its topological correlation with E-cadherin expression in ovarian carcinomas. We then examined in vitro the effect of hypoxia on the mRNA and protein expressions of E-cadherin using two ovarian cancer cell lines, SKOV3 and OVCAR3, and normal ovarian surface epithelial (OSE) cells. In addition, hypoxia-induced change in the expression of SNAIL, a transcriptional factor repressing E-cadherin expression, was also analyzed. Finally, we examined the facilitation of invasiveness of ovarian cancer cells under hypoxia using Matrigel invasion assay. Immunohistochemically, nuclear localization of HIF-1alpha was observed in 32 of the 76 (42%) carcinomas studied, and showed a topological correlation with loss of E-cadherin expression. Northern blotting, real-time PCR and Western blotting demonstrated that E-cadherin expression was remarkably decreased under hypoxia in both SKOV3 and OVCAR3 cells, but not in normal OSE cells. mRNA expression of SNAIL was increased under hypoxia in both ovarian cancer cell lines. Invasion assay revealed that hypoxia increases the invasiveness of ovarian cancer cells. Accordingly, the present study demonstrated that hypoxia induces down-regulation of E-cadherin in ovarian carcinoma cells, via up-regulation of the transcriptional repressor SNAIL. These findings suggest that hypoxia plays an important role in the change in intercellular attachment, which may be involved in the initiation of tumor progression of ovarian cancer cells.

Expression of steroid receptor coactivators and corepressors in human endometrial hyperplasia and carcinoma with relevance to steroid receptors and Ki‐67 expression

To examine the steroid hormone dependent growth mechanism of human endometrial hyperplasia and carcinoma, expression levels of steroid receptor cofactors, such as coactivators (steroid receptor coactivator 1 [SRC‐1] and p300/cyclic AMP‐response element‐binding protein (p300/CBP]) and corepressors (nuclear receptor corepressor [NCoR] and silencing mediator for retinoid and thyroid‐hormone receptors [SMRT]), were investigated.

A comparative study of pre-operative procedures to assess cervical invasion by endometrial carcinoma

Objective To compare the accuracy of different diagnostic procedures currently used to assess cervical involvement in endometrial carcinoma.

Immunohistochemical detection of steroid receptor cofactors in ovarian endometriosis: involvement of down-regulated SRC-1 expression in the limited growth activity of the endometriotic epithelium

To study the steroid hormone-induced growth mechanisms of endometriosis, the immunohistochemical expression of steroid hormone receptor cofactors was investigated in 37 cases of endometriotic epithelia and was compared with that of eutopic endometria of identical patients. The expression of steroid receptor coactivators (p300/CBP and SRC-1) and corepressors (NCoR and SMRT) was examined in relation to the estrogen receptor (ER), the progesterone receptor (PR), and Ki-67. Results of immunostaining were indicated as a “positivity index” (PI, full score; 100). The expression of ER and PR in endometriotic epithelia largely resembled that in eutopic endometria, however, the expression of Ki-67 in the proliferative phase (PI 13.8 ± 2.4, mean ± SD) was significantly lower than that in eutopic endometria (32.6 ± 10.6). The expression of SRC-1 in eutopic endometria was increased in the proliferative phase (56.5 ± 16.8) and decreased in the secretory phase (14.8 ± 6.9). In endometriosis, however, the PI for SRC-1 did not show apparent cyclic changes during the menstrual cycle. Moreover, the expression of SRC-1 in endometriotic epithelia in the proliferative phase was significantly lower than that in eutopic endometria. These findings suggested the reduced proliferative activity in endometriotic epithelia to be related to the reduced expression of SRC-1.

Evaluation of sperm head shape at high magnification revealed correlation of sperm DNA fragmentation with aberrant head ellipticity and angularity

OBJECTIVE To test for an association between DNA fragmentation and head shape at high magnification in fresh motile spermatozoa. DESIGN Observational study. SETTING Academic tertiary care center. PATIENT(S) A total of 60 men in our assisted reproductive program. INTERVENTION(S) Quantifying sperm head shape using elliptic Fourier analysis, and detecting DNA fragmentation by use of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. MAIN OUTCOME MEASURE(S) Correlation between percentage of spermatozoa with abnormal head shape and percentage of DNA fragmentation. RESULT(S) Elliptic Fourier analysis decomposed sperm head shapes into four quantitative parameters: ellipticity, anteroposterior (AP) symmetry, lateral symmetry, and angularity. The DNA fragmentation was statistically significantly correlated with abnormal angularity, and moderately with abnormal ellipticity but not with abnormal AP symmetry or lateral symmetry. Forward stepwise multiple logistic regression analysis revealed a statistically significantly higher percentage of DNA fragmentation in spermatozoa with abnormal ellipticity and abnormal angularity than in spermatozoa with normal-shaped head (6.1% and 5.4% vs. 2.8%). Spermatozoa with large nuclear vacuoles also correlated with sperm DNA fragmentation, and had a statistically significantly higher percentage of DNA fragmentation (4.7%). CONCLUSION(S) Among the morphologic features of the sperm head, abnormal ellipticity, angularity, and large nuclear vacuoles are associated with DNA fragmentation.

Morphological alterations in protamine-deficient spermatozoa

STUDY QUESTION How are protamine deficiencies associated with sperm head morphology in subfertile men? SUMMARY ANSWER The prevalence of morphological variations and large nuclear vacuoles was slightly higher in protamine-deficient spermatozoa than in non-deficient spermatozoa. WHAT IS KNOWN ALREADY A protamine deficiency was previously reported to be associated with an abnormal sperm morphology; however, how they are related to each other remains unclear. This is further confounded by a number of protamine-deficient spermatozoa having a normal head morphology. STUDY DESIGN, SIZE, DURATION This is a cross-sectional study, including 36 men diagnosed with male factor infertility or participating in an assisted reproduction program. To assess sperm head morphology, this study analyzed 2400 spermatozoa with a protamine deficiency and 2400 spermatozoa with a normal protamine status. An additional 21 men were analyzed to examine DNA fragmentation and its relationship with protamine deficiencies and sperm head morphologies. PARTICIPANTS/MATERIALS, SETTING, METHODS The morphology of the sperm head was evaluated based on its shape, size and nuclear vacuoles at a magnification of >6000×. Using elliptic Fourier analysis, the shape was summarized into four numeric variables. The protamine status was evaluated with chromomycin A3 (CMA3). Sperm head size, vacuoles and shape were compared between protamine-deficient and non-deficient spermatozoa. DNA fragmentation was evaluated with the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay. The percentages of protamine-deficient spermatozoa and DNA fragmentation were compared between spermatozoa with morphologically normal heads and those with abnormal heads. MAIN RESULTS AND THE ROLE OF CHANCE Variations in head size (P < 0.0001) and shape (P < 0.0001) were significantly higher, with narrower (P < 0.001), more fan-shaped (P < 0.01) and more square-shaped forms (P < 0.001) in protamine-deficient spermatozoa than in non-deficient spermatozoa; however, the distribution of morphological variations markedly overlapped. Protamine deficiencies were more frequently observed in spermatozoa with large nuclear vacuoles than in those without them (32.0 ± 3.1 versus 39.4 ± 2.9%, P < 0.001). The percentage of protamine-deficient spermatozoa was significantly lower in spermatozoa with a normal head morphology than in those with an abnormal head morphology (25.4 ± 2.6 versus 38.0 ± 2.5%, P < 0.001). The percentage of DNA fragmentation was significantly higher in protamine-deficient spermatozoa than in non-deficient spermatozoa (11.3 ± 2.1 versus 1.6 ± 0.6%, P < 0.001), and was lower in spermatozoa with a normal head morphology than in those with an abnormal head morphology (2.6 ± 0.7 versus 6.4 ± 0.2%, P < 0.001). LIMITATIONS, REASONS FOR CAUTION We were unable to discriminate the kind of protamines or quantify the extent of the protamine deficiency in spermatozoa using the CMA3 staining method. WIDER IMPLICATIONS OF THE FINDINGS This study provided a novel insight into how abnormal protamination affects sperm head morphology as well as the relationship between sperm head morphology and its own molecular integrity. Our results will contribute to a deeper understanding of the benefits and limitations of the morphological selection of spermatozoa for ICSI. STUDY FUNDING/COMPETING INTERESTS This study was supported by a JSPS Grant-in-Aid for the Encouragement of Scientists (25931009, 26931010). All authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A.

FGF2 Has Distinct Molecular Functions from GDNF in the Mouse Germline Niche

Summary Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1+ subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche.