Aging influences steroid hormone release by mink ovaries and their response to leptin and IGF-I.

The aim of our study was to understand whether ovarian steroid hormones, and their response to the metabolic hormones leptin and IGF-I leptin, could be involved in the control of mink reproductive aging via changes in basal release of ovarian progesterone and estradiol. For this purpose, we compared the release of progesterone and estradiol by ovarian fragments isolated from young (yearlings) and old (3-5 years of age) minks cultured with and without leptin and IGF-I (0, 1, 10 or 100 ng/ml). We observed that isolated ovaries of older animals produced less progesterone but not less estradiol than the ovaries of young animals. Leptin addition stimulated estradiol release by the ovarian tissue of young animals but inhibited it in older females. Leptin did not influence progesterone output by the ovaries of either young or older animals. IGF-I inhibited estradiol output in young but not old animals, whereas progesterone release was inhibited by IGF-I irrespective of the animal age. Our observations demonstrate the involvement of both leptin and IGF-I in the control of mink ovarian steroid hormones release. Furthermore, our findings suggest that reproductive aging in minks can be due to (a) reduction in basal progesterone release and (b) alterations in the response of estradiol but not of progesterone to leptin and IGF-I.

Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos.

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19-20 h after hCG injection. About 5 microg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3' flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

Effect of ghrelin on activities of some lysosomal hydrolases in rabbits.

OBJECTIVES: Observing the changes of activity of some lysosomal enzymes in blood serum of female rabbits subjected to injection of 10 microg of ghrelin/kg of body weight. METHODS: In the blood serum the activity of cathepsins D and L, alanine aminopeptidase, acid phosphatase, lysosomal lipase and lysosomal esterase was determined. RESULTS: As a result of ghrelin injection the activity of all the enzymes examined in blood serum increased markedly. CONCLUSION: Changes of lysosomal enzymes activities in the blood serum caused by the effects of ghrelin should be regarded as the response of the lysosomal system.