The interaction between tumor cells and extracellular matrix (ECM) proteins influences cell migration and the invasive behavior of cancer cells. In this study, we provide experimental evidence that collagen I and fibronectin affect ovarian cancer cell migration. In vitro wound healing assays and transwell migration assays were used to measure both total wound healing and directionality of individually migrating OV2008 and C13 ovarian cancer cells on glass, collagen I and fibronectin. Involvement of p21-activated kinase 2 (Pak2) in the motility of these cell lines was investigated using a chemical inhibitor as well as siRNA transfection. Culturing ovarian cancer cells on collagen type I (COLL) increased the migratory ability of OV2008 and C13 cells by increasing the directional migration of cells. In contrast, fibronectin (FN) decreased the migratory ability of OV2008 cells by reducing their ability to migrate directionally. When both cell lines are cultured on COLL and treated with increasing concentrations of a PAK inhibitor (IPA-3), there is a dose-dependent response such that there is a decrease in migration with an increase in inhibitor concentration. Further experiments utilizing PAK2 knockdown via siRNA transfection demonstrated significantly reduced migration of OV2008 cells on COLL as compared to those receiving control siRNA. In conclusion, our results indicate that FN and COLL affect the motility of the selected ovarian cancer cells lines and the effect of COLL is likely mediated, at least in part, by PAK2. A better understanding of the molecular events that contribute to tumor invasion and metastasis is crucial for developing novel treatment strategies to improve the long-term survival of women with ovarian cancer. As PAK2 is involved in motility, it should be further explored as a pro-metastatic gene in ovarian cancer.

Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28-day-old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14-day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells--RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross-hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3' region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT+) and RT- reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre- and postmeiotic germ cells and in somatic cells of the testes.

Cases of gonadal dysgenesis, both Turner syndrome and mixed, were studied with Y centromeric and short-arm probes. The Y-centromeric alphoid repeat clone, Y97, allowed sensitive detection of Y-chromosomal material in marker chromosomes or mosaics by in situ analysis or Southern hybridization with purified DNA. The Y short-arm probe, p75/79, allowed detection of sequences normally associated with proximal Yp by Southern analysis. The presence of DNA fragments characteristic of Yp correlates well with partial male sexual differentiation in the cases of mixed gonadal dysgenesis. Thus, the combined use of molecular and cytogenetic techniques has proven to be a powerful approach to the analysis of chromosomal sex disorders.

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Testicular and Leydig cell function were studied in four inbred strains of mice. Significant strain-related differences were found in the number of LH receptors and in the production of testosterone in vitro in response to increasing concentrations of human CG (hCG) by both decapsulated testes and isolated Leydig cells. Maximal testicular testosterone production was similar in the C57BL/10J and C57BL/6J strains and considerably less in the DBA/2J (DBA) and C3H/HeJ strains, which were similar. However, the number of testicular LH receptors was less in C57BL/6J mice than in all three other strains. The pattern of maximal testosterone production by isolated Leydig cells among the four strains was similar to that observed for whole testes, whereas the number of LH receptors per 10(6) Leydig cells was least for Leydig cells from the DBA strain. When the number of Leydig cells per testis was estimated by dividing the number of testicular LH receptors by the number of LH receptors in 10(6) Leydig cells, it was apparent that testes from DBA mice contain approximately twice as many Leydig cells as those from the other strains. Differences in maximal testicular testosterone production appear to be reflected in differences in maximal testosterone production by isolated Leydig cells from each strain. However, there were no differences in testicular sensitivity to hCG but there were differences in Leydig cell sensitivity to hCG among the four strains. Furthermore, the existence of strain-related differences in well defined functional characteristics of Leydig cells offers the opportunity to study the genetic as well as physiological mechanisms involved in the regulation of Leydig cell function in normal individuals.