Characterization and regulation of a mRNA encoding the prostaglandin F2alpha receptor in the rat ovary.

The recent cloning of several cDNAs encoding prostaglandin (PG) receptors has paved the way for a more detailed investigation of the postulated regulatory role of prostaglandins in corpus luteum function. We have utilized the reverse transcription-polymerase chain reaction (RT-PCR) to isolate a mRNA encoding the ovarian PGF(2alpha) (FP) receptor, using oligonucleotides based on the recently cloned mouse cDNA as primers. The 5'-untranslated region of the rat ovarian mRNA was isolated following 5'-RACE (rapid amplification of cDNA ends). The isolated 1526 base-pair sequence, which spans the entire open reading frame, was found 100% identical in the protein coding region to a similar sequence isolated from a rat astrocyte cDNA library, but different in the first 32 nucleotides of the 5'-untranslated region, possibly due to tissue-specific splicing heterogeneity. Using ribonuclease protection assay, a quantitative analysis of FP receptor mRNA levels was performed in corpora lutea excised from adult pseudopregnant rats (Day 8) at different timepoints (0.5-48 h) following the in vivo s.c. regimen of a luteolytic dose of the FP receptor agonist cloprostenol (5 microg). Already 3 h after cloprostenol injection, FP receptor mRNA levels exhibited a pronounced increase to values 4.0-fold higher (P < 0.01) than before injection. At 7 h through 24 h, the amount of luteal FP receptor mRNA decreased, approaching preinjection levels, whereafter they were again 3.0-fold higher (P < 0.01) at 48 h than before injection. We conclude that following homologous stimulation of the FP receptor, abundance of this mRNA is tissue-specifically regulated in a dynamic pattern, suggestive of an important role for FP receptor-mediated action on gene expression during the demise of corpus luteum function.

Rat luteinizing hormone receptor messenger ribonucleic acid expression and luteolysis: inhibition by prostaglandin F2 alpha.

To elucidate the mechanisms by which prostaglandin F2 alpha (PGF2 alpha) permanently inhibits LH-dependent steroidogenesis during luteolysis, we investigated the effect on luteal LH receptor mRNA levels of the stable PGF2 alpha analogue cloprostenol injected into adult pseudopregnant rats on different days during the luteal period. After treatment, LH receptor mRNA expression was determined by RNase protection assay. Twelve hours after cloprostenol injection on Day 8 of pseudopregnancy, the luteal LH receptor mRNA levels were drastically reduced (0.95 +/- 0.18 fmol mRNA/microgram DNA, p < 0.01) as compared with those in untreated controls (12.3 +/- 1.3 fmol mRNA/microgram DNA) or in corresponding controls given an injection of saline (8.8 +/- 0.7 fmol mRNA/microgram DNA) (n = 6-8 per group). At 24 h the levels rose to 4.3 +/- 0.8 fmol mRNA/ microgram DNA but were still significantly decreased compared to control values. Forty-eight hours after cloprostenol injection, the luteal LH receptor mRNA levels were not significantly different from control levels; but if the rats received an injection every twelfth hour, levels were significantly decreased compared to those in controls. When PGF2 alpha was injected, LH receptor mRNA levels were reduced in the same manner as seen after cloprostenol injection. LH receptor mRNA of young corpora lutea (CL) (Day 3) was more resistant to down-regulation by cloprostenol than that of CL of the mid (Day 8)-or late (Day 11) luteal phase. On the eighth day of pseudopregnancy, serum progesterone levels were decreased at 0.5 h after cloprostenol injection and fell further at 3 h; serum 20 alpha-dihydroprogesterone levels were first increased at 7 h after cloprostenol injection. We conclude that luteal LH receptor mRNA expression is under direct regulatory control by PGF2 alpha in a both time-and dose-dependent manner and thereby may decisively contribute to the inhibition of LH responsiveness during luteolysis.

Prolactin stimulates the expression of luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acid in the rat corpus luteum and rescues early pregnancy from bromocriptine-induced abortion.

Timed pseudopregnancy (psp) and pregnancy were induced in adult female rats by mating with infertile and fertile males, respectively. Corpora lutea (CL) and the residual parts of the ovaries were isolated and analyzed for luteinizing hormone/chorionic gonadotropin (LH/CG) receptor mRNA by Northern blot and solution hybridization analyses. Several LH/CG receptor mRNA transcripts were detected that could code for an intact functional receptor (6.8, 4.4, and 2.6 kb) as well as several smaller truncated transcripts. LH/CG receptor mRNA abundance in CL varied dramatically during both psp and pregnancy, with peak levels seen during the period of maximal progestational activity (Days 5-10 of psp and Days 7-14 of pregnancy). During the period of functional luteolysis, LH/CG receptor mRNA abundance decreased to low levels. The changes in LH/CG receptor expression could be explained by hormonal regulation. Bromocriptine treatment inhibited pituitary prolactin secretion. This treatment had a potent luteolytic effect by decreasing the levels of LH/CG receptor mRNA and plasma progesterone during early pregnancy, resulting in embryonal resorption in pregnant rats. Exogenous prolactin acted as a anti-luteolysin to reverse these effects by restoring LH/CG receptor mRNA abundance either by increasing gene expression or by stabilizing mRNA transcripts from degradation in young CL.

Luteolysis induced by a prostaglandin F2alpha analogue occurs independently of prolactin in the rat.

The hypothesis that prolactin exerts a stimulatory dominance over the luteolytic effect of prostaglandin (PG) F2alpha on corpus luteum maintenance and progesterone production was experimentally tested. A dose-dependent effect of the stable PGF2alpha analogue cloprostenol (dose range 200 ng(-5) microg) was found 12 h after s.c. injection, in Day 9 adult pseudopregnant rats: 1) LH receptor mRNA levels, as measured by RNase protection assay, were dramatically decreased (by 67%) by a single s.c. dose of 200 ng cloprostenol; and 2) serum progesterone levels were significantly (p < 0.05) decreased (by 43%) whereas 20alpha-dihydroprogesterone significantly (p < 0.05) increased (by 80%) initially at a 0.5-microg dose of cloprostenol. To study the integrated response to prolactin and PGF2alpha, we investigated the effect of cloprostenol treatment in sterile-mated female rats with or without circulating prolactin. Prolactin secretion was inhibited by s.c. injection of bromocriptine (1 mg) in the morning of the ninth day of pseudopregnancy. A group of rats was left prolactin-depleted; in another group prolactin was reintroduced by adding 8 IU ovine prolactin. It was found that after injection of 0.5 microg cloprostenol the LH receptor mRNA levels and the serum progesterone/20alpha-dihydroprogesterone ratio were not significantly different whether the rats had circulating endogenous/exogenous prolactin or were prolactin-depleted. Therefore, although prolactin exerts a stimulatory influence on both progesterone production and corpus luteum LH receptor gene expression, the conclusion is reached that prolactin alone cannot antagonize the luteolytic effect of PGF2alpha.

Increased LH receptor mRNA and extended corpus luteum function induced by prolactin and indomethacin treatment in vivo in hysterectomized pseudopregnant rats.

To assess the effects of prostaglandins and prolactin on corpus luteum function and regression, sterile-mated adult pseudopregnant rats hysterectomized on day 5 after mating were injected with indomethacin or prolactin. Daily samples of blood were collected via the tail, from day 12 to day 21, and assayed for serum concentrations of progesterone, 20 alpha-dihydroprogesterone and LH, whereafter corpora lutea and the remainder of ovaries were separated and the tissue content of PGF2 alpha, PGE2 and LH receptor mRNA were measured. Injections of prolactin (8 iu) s.c. or a low dose of indomethacin (200 micrograms kg-1) s.c. were administered twice a day, beginning on day 13 after mating. Both indomethacin and prolactin significantly increased serum progesterone concentrations (P < 0.05; n = 8), and extended the period of functional corpora lutea when compared with controls. Indomethacin, but not prolactin, lowered the concentration of serum 20 alpha-dihydroprogesterone. In the corpora lutea of indomethacin-treated animals, collected on day 21, both prostaglandins measured were reduced in concentration by 50% or more, compared with controls (P < 0.05; n = 8), whereas prolactin had no effect. Both prolactin and indomethacin treatment caused a substantial (tenfold) increase in the concentration of LH receptor mRNA, confined solely to the luteal compartment. These findings in vivo provide further evidence for a luteolytic role of locally synthesized prostaglandins in the rat ovary. Furthermore, prolactin can sustain corpus luteum function by exerting a luteotrophic effect during the late luteal phase, as judged by the stimulation of progesterone synthesis and the expression of LH receptors.

Bacterial contamination and sperm recovery after semen preparation by density gradient centrifugation using silane-coated silica particles at different g forces.

The effects of density gradient centrifugation through silane-coated silica particles (PureSperm) using 100, 200, 300 and 500 g on bacterial contamination of sperm samples and recovery of motile spermatozoa from sperm samples were investigated with conventional culturing techniques and microscopic visual assessment. The recovery of motile spermatozoa was variable and was not improved using 500 g compared to the recommended 300 g. The bacterial contamination was highly decreased by gradient centrifugation through PureSperm and was almost abolished when strict aseptic techniques were used, with changes to sterile Pasteur pipettes and tubes prior to washing procedures.