Sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle of Sprague-Dawley rats ranging in age from 4 months to 28 months were studied and compared. A marked decline, with age, was observed in the amount of (total) SR proteins isolated per gram of muscle tissue used. This decline is in line with the known loss of muscle fiber mass and size with advancing age; however, whether the magnitudes of these two effects are indeed identical, remains to be studied. In contrast, no analogous age-related change was detected in the amount of SR protein per unit mass of rat cardiac muscle. The calcium contents, per mg protein, in SR vesicles isolated from rats of all age groups studied did not differ significantly, and represented only a small fraction of the total capacity of the vesicles for this cation. This capacity was found to decline at old age and this effect, combined with the age-related decrease in the concentration of SR proteins in the tissue, indicate a significant decline in calcium sequestration ability in old muscle. Both basal (Ca2+ independent) and calcium stimulated ATPase activities were found not to be affected by age. In contrast, the efficiency of Ca2+ transport across the SR membrane, as reflected by the number of calcium ions pumped into the vesicles per ATP cleaved, declined from a value of 0.37 at 3-4 months to 0.15 at 24 months. This change may represent an age-related reduction in the fraction of coupled SR vesicles, possibly due to alterations in the membrane. SR vesicle preparations from both young and old rats displayed strongly biphasic inactivation kinetics when incubated at 37 degrees C. This may reflect the heterogeneity of muscles in the tissue used, or be due to the presence of a mixture of coupled and uncoupled vesicles in the SR preparations. The rate of the first step in the ATPase inactivation, in which about 75% of the activity is lost, was found to be affected by age, the old SR vesicles being markedly more labile than their young counterparts. In contrast, no difference was detected between the inactivation kinetics of young and old ATPase proteins dissolved in Triton X-100 and the inactivation was monophasic down to less than 6% of the original activity. These results indicate that the age-related modifications in the stability of the SR calcium pump system involve the membrane but not the ATPase protein. The inactivation of the SR ATPase is believed to proceed via dissociation of the dimeric enzyme to (unstable) subunits. It is therefore likely that changes in the SR membrane in old muscle render the ATPase more dissociable.

The effects of estradiol on luteal estrogen receptor and steroidogenesis were examined on Days 9 through 11 of pseudopregnancy. In normal pseudopregnant rabbits, unoccupied cytoplasmic and total nuclear estrogen receptor were 2.6 +/- 0.4 fmol/microgram DNA and 0.4 +/- 0.1 fmol/microgram DNA, respectively, on Day 10 of pseudopregnancy. An i.v. injection of 4 micrograms of estradiol caused the translocation of cytoplasmic receptor and a 6.6-fold increment in total nuclear receptor within 15 min, which was followed by rapid processing of the nuclear receptor. Both cytoplasmic and nuclear estrogen receptor returned to normal values within 24 h, and during this period, serum progesterone did not change significantly. Withdrawal of an estradiol implant from animals on Day 9 of pseudopregnancy initiated a marked decline in serum progesterone within 24 h. Following an injection of saline or of 4 micrograms estradiol on Day 10, unoccupied cytoplasmic estrogen receptor in saline- and estradiol-injected rabbits was 1.0 +/- 0.1 fmol/microgram DNA and 1.9 +/- 0.1 fmol/microgram DNA, respectively, on Day 11 of pseudopregnancy. Associated with the increase in cytoplasmic receptor there was an increase in serum progesterone (8.2 +/- 1.5 ng/ml), in contrast to saline-injected animals in which serum progesterone continued to decline (1.6 +/- 0.4 ng/ml). Despite the significant differences in cytoplasmic receptor and in progesterone synthesis, total nuclear estrogen receptor was not different in these animals. These data suggest that in corpora lutea already secreting progesterone at high rates during midpseudopregnancy, the rapid translocation of available estrogen receptor does not cause further stimulation of progesterone synthesis. However, if steroidogenesis is first reduced experimentally, then an injection of 4 micrograms of estradiol can readily stimulate progesterone synthesis, and this stimulation is associated with an increase in cytoplasmic receptor.

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.

On Day 10 of pseudopregnancy, rabbits were given an i.v. injection of hCG (10-20 i.u.) that was sufficient to cause new ovulations and the loss of follicular oestradiol secretion. There was an immediate 3-4-fold rise in serum progesterone which returned to near prestimulation values (approximately 27 ng/ml) within 12 h in the presence of an implant containing oestradiol-17 beta. In the absence of oestradiol, serum progesterone continued to decline to reach low values (approximately 4 ng/ml) within 24 h and the original corpora lutea subsequently regressed. The administration of oestradiol 24 h after injection of hCG, when progesterone secretion was low, arrested any further decline in progesterone and then restored serum progesterone to normal values. This steroidogenic effect of oestradiol in vivo was a function of enhanced luteal steroidogenesis; corpora lutea removed and incubated for 12 h produced progesterone at high, linear rates, whereas the corpora lutea from animals that did not receive oestradiol produced low or insignificant quantities of progesterone in vitro. We conclude that hCG at these doses is compatible with continued responsiveness of the corpora lutea to oestrogen and that hCG produces its luteolytic effect primarily by ovulating follicles, thus stopping the secretion of the luteotrophic hormone, oestradiol.

The requirement of the pituitary gland and the role of 17 beta-estradiol in the early development of the corpus luteum was investigated in rabbits hypophysectomized the day after sterile mating (day 1). Serum progesterone in hypophysectomized rabbits was normal for 2 days after hypophysectomy. Luteal tissue from hypophysectomized and sham-hypophysectomized rabbits had similar wet weight (4.0 +/- 0.4 vs. 5.3 +/- 0.2 mg/corpus luteum) and similar concentrations of available cytoplasmic estrogen receptor (1.2 +/- 0.2 vs. 1.5 +/- 0.3 fmol/micrograms DNA) and luteinizing hormone (LH) receptor (4.0 +/- 0.2 vs. 6.1 +/- 1.4 fmol/micrograms DNA) on day 4 of pseudopregnancy. Serum progesterone in hypophysectomized rabbits began to decline on day 4 and was undetectable by day 6. Estrogen receptor and luteal weight in hypophysectomized animals also declined after day 4 to low values by day 6, and serum estradiol was undetectable. However, if estradiol was administered by Silastic capsule implanted subcutaneously at the time of hypophysectomy or 3 days after hypophysectomy, serum progesterone, luteal weight, estrogen receptor, and LH receptor were maintained on day 6 of pseudopregnancy. These results indicate that after a preovulatory gonadotropin surge, the function of newly formed corpora lutea is normal for 3-4 days in the absence of pituitary hormones. However, by 4 days after ovulation, estradiol is required to sustain the structural and functional integrity of corpora lutea.