Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model.

Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the 'Non-cultured control'. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early antral stage while Fshr was only affected in the former compared to the non-cultured control. Pre-incubating follicles in 350 mOsm medium for 24 h enhanced (P < 0.05) Star and Aqp7 while decreasing (P < 0.05) Aqp1 expression compared to the control in secondary follicles, but not in the early antral stage. In summary, short-term hypertonic exposure promoted cat follicle development in vitro (including the meiotic competence of the enclosed oocyte) possibly through a mechanism that does not involve water transport genes.

Expression pattern of matrix metalloproteinases changes during folliculogenesis in the cat ovary.

Matrix metalloproteinase (MMP) has been implicated as having roles in ovarian folliculogenesis. Here, we determined the expression pattern of six MMPs (MMP1, MMP2, MMP3, MMP7, MMP9 and MMP13) and their endogenous tissue inhibitor, TIMP1, during cat follicle growth. Different developmental stage follicles were mechanically isolated and gene expression analysed by real-time qPCR while MMP1, 2, 9 and 13 localization was determined by immunohistochemistry. With the exception of MMP13, the amount of MMP mRNA was lowest in primordial follicles and increased thereafter. Peak levels were detected in early antral follicles for MMP1 (72.2-fold increase above primordial follicle amount), MMP2 (10-fold), MMP3 (57-fold) and MMP9 (2.8-fold). MMP7 transcripts increased 2-fold by the primary follicle stage and then plateaued. MMP13 mRNA peaked in primary follicles (2.5-fold) and was lower in more advanced counterparts. TIMP1 sharply increased (6-fold) in secondary follicles and gradually declined in the later stages. MMP1 and MMP9 expression were expressed in the granulosa cells of all follicle stages. MMP2 was immunoreactive in early and antral follicles, especially at granulosa cells adjacent to the antral cavity. By contrast, the MMP13 was weakly detected in primary follicles onward. In summary, there are distinctive and consistent changes in MMPs and TIMP1 expression during follicle development, suggesting that these enzymes play one or more roles in cat folliculogenesis. In particular, high mRNA and protein expression levels of MMP1 and MMP2, especially at the antral stage, indicate that these enzymes likely are involved in antrum formation and expansion.

Effect of κ-opioid receptor agonist on the growth of non-small cell lung cancer (NSCLC) cells.

BACKGROUND: It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial. METHODS: The effects of κ-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth. RESULTS: Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated-glycogen synthase kinase 3ß (p-GSK3ß). CONCLUSION: The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3ß.