RESUMEN

Three experiments were conducted to examine the regulation of steady-state concentrations of mRNA encoding ovine low density lipoprotein receptor (LDL-R) and high density lipoprotein-binding protein (HBP) in corpora lutea. In Experiment 1, corpora lutea were collected from ewes on Days 3, 6, 9, 12 and 15 (Day 0, oestrus) of the oestrous cycle. Enriched preparations of small and large steroidogenic luteal cells were also obtained on Days 6, 9, 12 and 15 of the oestrous cycle. In Experiment 2, 16 ewes were hypophysectomized on Day 5 of the oestrous cycle and received saline, luteinizing hormone (LH), growth hormone (GH) or a combination of LH+GH until collection of luteal tissue on Day 12 of the oestrous cycle. Corpora lutea were also collected from pituitary-intact control ewes on Day 5 and Day 12 of the oestrous cycle. In Experiment 3, 13 ewes on Day 11 or Day 12 of the oestrous cycle were administered prostaglandin F2 alpha (PGF2 alpha) and corpora lutea were collected 4 h, 12 h and 24 h later. Corpora lutea were also collected from 4 non-injected and 4 saline-injected (at 24 h) ewes. Results demonstrated that concentrations of mRNA encoding LDL-R did not differ throughout the oestrous cycle. Luteal tissue collected on Day 3 of the oestrous cycle had higher concentrations of mRNA encoding HBP than luteal tissue collected on any other day of the oestrous cycle. Hypophysectomy increased concentrations of mRNA encoding LDL-R but had no effect on concentrations of mRNA encoding HBP. Twelve hours following PGF2 alpha injection concentrations of mRNA encoding LDL-R were decreased but concentrations of mRNA encoding HBP were increased. Concentrations of both LDL-R and HBP mRNA were decreased 24 h following injection of PGF2 alpha. Thus, long-term positive and acute negative regulation of progesterone secretion from the corpus luteum by luteotrophic and luteolytic hormones was not mediated by changes in steady-state concentrations of mRNA encoding LDL-R or HBP.

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A partial cDNA was used to measure steady-state concentrations of mRNA encoding the receptor for luteinizing hormone (LH) in ovine corpora lutea. In experiment 1, luteal tissue and purified preparations of small and large steroidogenic luteal cells (n=4 per day) were obtained on days 3 (tissue only), 6, 9, 12 and 15 of the estrous cycle (estrus=day 0). Steady-state concentrations (fmoles receptor mRNA/µg poly(A)(+) RNA) and total quantities of mRNA (fmoles/corpus luteum) encoding the receptor for LH in luteal tissue increased (P<0.05) from day 3 to days 9 and 12 of the cycle; values on days 6 and 15 were intermediate. Small luteal cells contained at least four-fold greater (P<0.001) concentrations of mRNA encoding the receptor for LH than large luteal cells on days 6, 9, 12 and 15 of the cycle. In experiment 2, ewes on days 11 or 12 of the cycle received an infusion of either 1 µmol prostaglandin F(2α) (PGF(2α)) or saline into the ovarian artery. Luteal tissue was collected 1 (n=6), 4 (n=5), 12 (n=5) or 24 (n=5) h following PGF(2α) infusion, and 0 (no infusion;n=3), 12 (n=3) or 24 (n=4) h following saline administration. Concentrations of progesterone in sera decreased (P<0.05) within 12 h and remained low, whereas luteal weight and concentrations of progesterone in luteal tissue did not decrease (P<0.05) until 24 h after PGF(2α) treatment. Steady-state concentrations of mRNA encoding the receptor for LH were reduced (P<0.05) within 4 h of PGF(2α) infusion, and continued to decrease at 12 and 24 h post treatment. Calculated amounts of mRNA encoding the receptor for LH per corpus luteum were reduced (P<0.05) at 12 h after the PGF(2α) treatment and were 10% (P<0.05) of the values in saline-treated ewes at 24 h post-treatment. The increase during the estrous cycle in steady-state concentrations of mRNA encoding the receptor for LH appears to occur prior to the previously observed increase in number of receptors for LH. Following PGF(2α)-induced luteal regression, concentrations of mRNA encoding LH receptor decreased prior to the previously reported decrease in LH binding. Thus, changes in the number of receptors for LH in ovine luteal tissue during luteal development and luteolysis appears to be preceded by corresponding changes in mRNA encoding this receptor.

RESUMEN

To test the hypothesis that growth hormone (GH) as well as luteinizing hormone (LH) is required for normal luteal growth and function, 16 western range ewes were hypophysectomized (HPX) on day 5 of the estrous cycle. Ewes were randomly assigned to receive saline (S), LH, GH, or LH + GH (n=4 per group) from the time of HPX until collection of corpora lutea 7 days after HPX (day 12). Corpora lutea were also collected from pituitary-intact ewes on days 5 (day 5 control,n=4) and 12 (day 12 control,n=4) of the estrous cycle. To assess luteal function, concentrations of progesterone in sera, luteal weights and luteal concentrations of mRNA encoding cytochrome P450 side-chain cleavage enzyme (P450(scc)) and 3ß-hydroxysteroid dehydrogenase/Δ5,Δ4 isomerase (3ß-HSD) were determined. Concentrations of progesterone in sera and luteal weights increased between days 5 and 12 of the estrous cycle in control ewes, but not in HPX + S ewes. In HPX ewes treated with LH, concentrations of progesterone in sera and luteal mRNA for P450(scc) and 3ß-HSD increased but luteal weights were unaffected. Treatment with GH increased luteal weight and luteal concentrations of mRNA encoding P450(scc) but did not increase concentrations of mRNA encoding 3ß-HSD compared to HPX + S ewes. Concentrations of progesterone in sera of GH-treated, HPX ewes were similar to those of day 12 control ewes but not significantly different from those in HPX + S ewes. Treatment of HPX ewes with LH + GH increased all parameters of luteal function measured to values similar to those in day 12 controls. In conclusion, both GH and LH are necessary for normal luteal development in the ewe.

RESUMEN

The objective of these experiments was to determine the pattern of mRNA expression for cytochrome P450 side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5,delta 4 isomerase (3 beta-HSD) during luteinization of the follicle and in ovine luteal tissue on Days 3, 6, 9, 12, and 15 of the estrous cycle. Mean concentration of mRNA for P450scc was not different in follicles collected 4 or 24 h after the onset of estrus but increased (p < 0.05) 3-fold by 48 h (corpus hemorrhagicum). With the methods used, mRNA for 3 beta-HSD was not detected until after ovulation and formation of the corpus hemorrhagicum (48 h after onset of estrus). In luteal tissue, mean concentration of mRNA for P450scc increased from Days 3 to 9 (p < 0.05) and had decreased (p < 0.05) by Day 15. Mean concentration of mRNA for P450scc was higher (p < 0.05) in small luteal cells on Day 9 than on Day 15, with values on Days 6 and 12 being intermediate. In large luteal cells, mean concentrations of P450scc mRNA increased (p < 0.05) between Days 6 and 12 and then decreased (p < 0.05) on Day 15. Mean concentration of mRNA for 3 beta-HSD was not different (p = 0.33) in luteal tissue on any day examined. In small luteal cells, mean concentrations of mRNA for 3 beta-HSD decreased between Days 6 and 15 (p < 0.05) while in large luteal cells, mean concentrations decreased (p < 0.05) between Days 12 and 15.(ABSTRACT TRUNCATED AT 250 WORDS)

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The binding and degradation of 125I-hIGF-I by isolated sheep hepatocytes have been examined. Hepatocytes were isolated by collagenase perfusion of 32-55 kg wether lambs and were incubated at 20 or 37 C at pH 7.4 in a 95% O2/5% CO2 atmosphere. Maximal binding was obtained at 60 min and declined slightly over the following 60-min period at both 20 and 37 C. Degradation of 125I-hIGF-I by the hepatocytes was minimal with 10-12% degradation over a 120-min period at 37 C. The lysosomal inhibitors chloroquine (0.2 mM), leupeptin and ammonium chloride had no significant effects on 125I-hIGF-I degradation or binding. At 20 C (60-min incubation), half maximal inhibition of 125I-hIGF-I binding was obtained with 8.4 +/- 1.1 nM hIGF-II, 16 +/- 2.4 nM hIGF-I, 36 +/- 6.2 nM oIGF-II, and 60 +/- 5.9 nM oIGF-I. Ovine insulin (0.01-10 uM) had no effect on 125I-hIGF-I binding. These observations suggest that IGF-I binds to the type II IGF receptor. The low molecular weight sheep serum IGF binding protein inhibited binding of 125I-hIGF-I to hepatocytes in a dose-dependent manner with half maximal inhibition occurring at 16.5 micrograms/ml, but did not affect IGF-I degradation. The current studies show that IGF-I interacts with ruminant hepatocytes via type II IGF receptors. The liver is not a major site of IGF-I degradation and the observed degradation is nonlysosomal and independent of receptor interaction.

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This study examines the binding and degradation of IGF-II by the ovine liver. Binding and degradation of 125I-IGF-II to isolated hepatocytes was time, temperature and cell number dependent. Ovine and human IGF-II were 2-5 times more effective in inhibiting 125I-hIGF-II binding than were the IGF-I preparations. Insulin did not affect binding. Autoradiographs of 125I-hIGF-II affinity cross-linked to hepatocytes showed a major band of molecular weight 271,000 under reduced conditions. This band was eliminated by 100 nM hIGF-II or oIGF-II but not by excess hIGF-I, oIGF-I or insulin. The internalization of IGF-II was examined by treating the cells with trypsin or sodium acetate to remove surface-bound IGF-II. Both treatments showed that 20-25% of 125I-hIGF-II was internalized. Mannose-6-phosphate at 1, 2 and 4 mM enhanced the binding of 125I-hIGF-II to hepatocytes 3.5, 12.8 and 16.4%, respectively. The lysosomal inhibitors ammonium chloride, chloroquine and leupeptin had no effect on 125I-hIGF-II degradation or cell-associated radioactivity indicating a nonlysosomal pathway of degradation for 125I-hIGF-II in the ovine hepatocyte. The low molecular weight sheep serum IGF binding protein inhibited binding of 125I-hIGF-II in a dose-dependent manner but had no effect on degradation, which suggests that degradation of 125I-hIGF-II is independent of receptor interaction. These studies demonstrate that IGF-II binds to specific high affinity sites in sheep hepatocytes which display the characteristics of type II IGF receptors. A significant fraction of the receptor bound IGF-II is internalized but not degraded by these cells, which suggests that the biological actions of IGF-II may be exerted by an intracellular pathway in sheep hepatocytes.

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