In an attempt to establish a defined model system for studies aimed at elucidating the mechanism of PGF2α action, we examined the effects of medium supplementation with soluble hydroxycholesterol analogues, alone and in combination with ovine luteinizing hormone (oLH) in the presence and absence of PGF2α' on progesterone secretion by mixed ovine luteal cells in vitro. In short-term cultures (2-6 h), supplementary 22R-hydroxycholesterol (22R-OHC; 0.16–20 μg ml−1) increased (P < 0.05) progesterone production in a dosedependent manner, whereas similar concentrations of 22S-hydroxycholesterol (22S-OHC) and 25-hydroxycholesterol (25-OHC) had little effect. In incubations of ≤24 h duration, 22R-OHC (1 μg ml−1) dramatically increased progesterone secretion, whereas oLH (100 ng ml−1) in the presence or absence of PGF2α (250 ng ml−1) had no consistent effects, alone or in combination with 22R-OHC. In contrast, 22R-OHC (1 μg ml−1) alone had no effect in long-term incubations (72–192 h), nor did treatment with oLH (100 ng ml−1) in the presence or absence of PGF2α (250 ng ml−1) in the absence of 22R-OHC. Together, however, 22R-OHC and oLH stimulated (P < 0.05) progesterone secretion, a synergistic effect consistently inhibited (P < 0.05) by PGF2α' Equimolar (2.5 μmol l−1) concentrations of 22R-OHC and homologous serum low- or high-density lipoprotein cholesterol exhibited comparable capacities to maintain progesterone secretion in long-term cultures (24–168 h), with and without gonadotrophin (oLH or human chorionic gonadotrophin, 100 ng ml−1) stimulation. These data establish 22R-OHC as an effective steroidogenic substrate for progesterone biosynthesis in ovine luteal cells in vitro, exhibit its capacity (when combined with gonadotrophin) to extend the useful life of cultured ovine luteal cells in a manner similar to serum lipoproteins and demonstrate the ability to reproduce PGF2α-induced luteolytic actions in a defined model system in vitro.
T. A. Fitz, D. F. Contois, M. M. Marr, C. E. Rexroad and M. A. Fritz
M. C. Schiewe, T. A. Fitz, J. L. Brown, L. D. Stuart and D. E. Wildt
Summary. Ewes were treated with exogenous follicle-stimulating hormone (FSH) and oestrus was synchronized using either a dual prostaglandin F-2α (PGF-2α) injection regimen or pessaries impregnated with medroxy progesterone acetate (MAP). Natural cycling ewes served as controls. After oestrus or AI (Day 0), corpora lutea (CL) were enucleated surgically from the left and right ovaries on Days 3 and 6, respectively. The incidence of premature luteolysis was related (P < 0·05) to PGF-2α treatment and occurred in 7 of 8 ewes compared with 0 of 4 controls and 1 of 8 MAP-exposed females. Sheep with regressing CL had lower circulating and intraluteal progesterone concentrations and fewer total and small dissociated luteal cells on Day 3 than gonadotrophin-treated counterparts with normal CL. Progesterone concentration in the serum and luteal tissue was higher (P < 0·05) in gonadotrophin-treated ewes with normal CL than in the controls; but luteinizing hormone (LH) receptors/cell were not different on Days 3 and 6. There were no apparent differences in the temporal patterns of circulating oestradiol-17β, FSH and LH. High progesterone in gonadotrophin-treated ewes with normal CL coincided with an increase in total luteal mass and numbers of cells, which were primarily reflected in more small luteal cells than in control ewes. Gonadotrophin-treated ewes with regressing CL on Day 3 tended (P < 0·10) to have fewer small luteal cells and fewer (P < 0·05) low-affinity PGF-2α binding sites than sheep with normal CL. By Day 6, luteal integrity and cell viability was absent in ewes with prematurely regressed CL. These data demonstrate that (i) the incidence of premature luteal regression is highly correlated with the use of PGF-2α; (ii) this abnormal luteal tissue is functionally competent for 2–3 days after ovulation, but deteriorates rapidly thereafter and (iii) luteal-dysfunctioning ewes experience a reduction in numbers of small luteal cells without a significant change in luteal mass by Day 3 and, overall, have fewer low-affinity PGF-2α binding sites.
Keywords: sheep; superovulation; premature luteal regression; prostaglandin F-2α; receptor