ABSTRACT
Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50-60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G(1)/G(0) and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.
ABSTRACT
The roles of recombinant human inhibin A (rh-inhibin A) and rh-activin A in regulating the pituitary and ovary of the adult female rat were examined. Serum and pituitary FSH and LH and serum estradiol and progesterone concentrations were evaluated at 1, 2, and 6-12 h after sc hormone administration on metestrus and on proestrus. A second study examined the effect of the hormones 24 h after injection at 1000 h on each day of the cycle. Rh-inhibin A inhibited FSH secretion 60 min after injection on proestrus but did not alter serum FSH concentration on metestrus. FSH remained low for the 12 h examined during the evening of proestrus and on the morning of estrus. Serum LH concentration, pituitary FSH content, and pituitary LH content were not significantly changed under any experimental condition. Rh-inhibin A did not regulate estradiol concentration on metestrus or on proestrus; however, it did cause a rise in serum estradiol in animals treated on metestrus and diestrus and examined 24 h later. This suggests that inhibin may participate in regulating follicular maturation in a subset of developing follicles. Last, after rh-inhibin A treatment, the duration of the proestrus progesterone surge was shortened. Serum FSH concentration rose by 6 h after rh-activin A administration on proestrus and both FSH and LH rose by 6 h after hormone administration on metestrus. Rh-activin A significantly increased serum estradiol through 6 h of treatment on proestrus. Progesterone levels were significantly greater in animals treated on metestrus and killed 24 h later. The increased length of the midcycle progesterone surge may be the result of increased LH on metestrus. These studies suggest that rh-inhibin A and rh-activin A may regulate ovarian and pituitary function in a cycle dependent manner. Specifically, rh-inhibin A can acutely regulate FSH and progesterone on proestrus and estradiol during follicular development. Rh-activin A acutely regulates FSH on both proestrus and metestrus and LH on metestrus. Whether circulating endogenous inhibin or activin participate physiologically in these functions is under investigation.
