John A. McCracken

Hormone receptor control of pulsatile secretion of PGF2α from the ovine uterus during luteolysis and its abrogation in early pregnancy

Abstract Studies on the control of PGF2α secretion by hormone receptors in the ovine uterus based on in vivo infusion experiments in the autotransplanted uterus are summarized. In addition, direct measurement of hormone receptor dynamics for oestradiol-17β, progesterone and oxytocin in the ovine uterus under experimental conditions are reported. Investigation of the antiluteolytic effect of early pregnancy in sheep was approached in two ways: (a) studies on the antiprostaglandin secreting effect of the embryo and (b) studies on a putative luteoprotective effect of the embryo. In the former study, the measurement of PGF2α in samples of uterine venous plasma collected hourly from 5 sheep on days 12 to 17 postmating failed to reveal the presence of any major peaks of PGF2α secretion. In the latter approach, several putative luteoprotective substances were examined for their ability to block the luteolytic effect of PGF2α given as 5 separate hour-long pulses over 25 h into the arterial supply of the autotransplanted ovary. These substances included: day 14–15 uterine venous plasma from pregnant sheep, PGE2, ovine prolactin, ovine LH, ovine placental lactogen and porcine relaxin. In no case was a convincing luteoprotective effect demonstrated suggesting that the antiprostaglandin secreting effect of the embryo may be the more important of these two antiluteolytic mechanisms. Experiments were performed to investigate circulating levels of endogenous oxytocin by monitoring intramyometrial pressure on a continuous basis in conscious sheep. These studies revealed the presence of hour-long periods of intense uterine activity at intervals of 5.5 to 6.5 hours on days 14 to 16 in the cyclic animal, but not in the early pregnant animal. Based on these results and on recent reports on the production of oxytocin by the corpus luteum, a hypothesis is presented to explain the hormone receptor control of episodic pulsatile secretion of PGF2α in the cyclic sheep, including a possible mechanism for its abrogation during early pregnancy.

Hormonal and related factors affecting the release of prostaglandin F2α from the uterus

Abstract While evidence is accumulating that prostaglandin F 2α (PGF 2α ) is the uterine luteolytic factor in several sub-primate species, factors controlling the release of PGF 2α from the uterus are not fully documented. The present study utilizes two models consisting of either the in situ or the autotransplanted uterus of the sheep. Four factors affecting the release of PGF 2α from the uterus are described, (a) Ovarian steroid hormones : Spontaneous peaks of estradiol-17β (E 2 -17β) occur throughout the estrous cycle but it is not until the time of corpus luteum regression that peaks of PGF 2α release from the uterus become associated with peaks of E 2 -17β secretion. When physiological amounts of E 2 -17β are infused into the arterial supply of the autotransplanted uterus, PGF 2α , is released only late in the luteal phase suggesting that a priming effect of progesterone may also be necessary for PGF 2α synthesis, (b) Oxytocin : When physiological amounts of oxytocin are infused into the arterial supply of the in situ uterus, uterine tonus and amplitude of contractions increase immediately and are associated with a simultaneous increase in PGF 2α release, an effect which varied with the steroid status of the animal. Indo-methacin inhibits the oxytocin-induced release of PGF 2α but not uterine contractions even although infusions of PGF 2α alone mimic this effect of oxytocin. It is possible that PGF 2α released during oxytocin action may have other important physiological functions such as altering uterine blood flow or changing cervical tone. (c) Mechanical stimulation : When the in situ uterus is mechanically stimulated by massaging it for 10 min, a rapid and sustained release of PGF 2α occurs only very early and very late in the cycle suggesting a dependence of this stimulus on the steroid hormone status of the animal. Peripheral blood levels of oxytocin were found to be elevated during massage at certain stages of the cycle, suggesting that at least some of the massage-induced release of PGF 2α may be mediated through oxytocin action on the uterus, (d) Bacterial endotoxin : Endotoxin has long been known to cause abortion in women as well as in lower species. Recent studies in pregnant mice have implicated uterine PGF 2α as the mediator of the abortifacient action of endotoxin. Minute quantities of endotoxin injected into the arterial supply of the uterus produce a marked and immediate increase in PGF 2α release, an effect which is abolished by Indomethacin. These studies at the organ level confirm and extend the evidence that the abortifacient action of endotoxin is mediated via the release of PGF 2α from the uterus. In conclusion the response of the uterus to a variety of physiological and pathological stimuli appears to center invariably on the release of PGF 2α (and possibly other PGs) and evidence exists that this event is wholly or partially dependent on the endogenous steroid status of the animal.

Corpus luteum regression induced by ultra-low pulses of prostaglandin F2α

In view of the pulsatile nature of PGF2 alpha secretion from the ovine uterus at the time of luteolysis, experiments were designed to examine the effect of pulsed infusions of PGF2 alpha on luteal function and to re-examine the minimal effective levels of PGF2 alpha required to induce luteolysis. To mimic physiological conditions, hour-long infusions of PGF2 alpha in increasing concentrations were given either 4 times in 19 h or 5 times in 25 h into the arterial supply of the autotransplanted ovary in conscious sheep on day 12 of an induced cycle. Blood flow and progesterone secretion rate from the ovary were used to monitor directly the luteolytic effect of administered PGF2 alpha. The concentration of LH in peripheral plasma was measured throughout each infusion experiment and the presence of a preovulatory peak of LH was used as an indicator of the permanence of luteal regression. Four pulses of PGF2 alpha in 19 h caused complete corpus luteum regression in only 1 of 4 animals whereas the addition of a fifth pulse (5 pulses in 25 h) caused permanent regression in 4 out of 4 animals. Infusion of 5 hour-long pulses of saline or PGF2 alpha at a rate less than 0.04 microgram/h did not induce permanent suppression of progesterone secretion. The average total effective dose of PGF2 alpha required to induce luteal regression when given as 5 pulses was 1/40th of the amount currently regarded as the minimal effective one when given by constant infusion into the ovarian artery. In another series of experiments the luteolytic effect of a single hour-long pulse of 0.1 microgram/h PGF2 alpha given daily for either 3 or 4 days was investigated. A significant fall (ANOVA, F0.01) in progesterone secretion rate, which reached a nadir at 5.3 +/- 2.2 h (means +/- S.D., n = 15), was followed by a recovery of progesterone secretion rate. Permanent luteal regression did not occur with this protracted regimen, suggesting that a relatively short pulse frequency of PGF2 alpha over a minimal period of 24 h is a necessary condition for physiological regression of the corpus luteum in sheep.

Transplantation of the uterus in sheep: Methodology and early reperfusion events

Aim:  Uterine transplantation is developing into a clinical treatment for uterine factor infertility. An animal model with a similar uterus size and vessels to humans and with pregnancy extending over several months would be beneficial for research on uterine transplantation. The aim of this study was to develop and evaluate autotransplantation of the sheep uterus to an orthotopic position in the pelvis.

The evolution of titer and specificity of aldosterone binding antibodies in hyperimmunized sheep

Abstract Two sheep were immunized with an aldosterone-21-hemisuccinate bovine serum albumin complex. Four sheep were immunized to an aldosterone-18,21-dihemisuccinate bovine serum albumin complex. All sheep produced anti-aldosterone antibodies in high titer. The development of antibody titers and cross reactions with related steroids were studied for up to 264 days. Three of the six animals died during this period. There was a large between animal variation in both cross reaction and titer of the antibodies produced. Cross reactions tended to decrease, whereas titers tended to increase with protracted immunization. No definite relation was seen between maximum titer and minimum cross reaction.

Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal

We previously showed that progesterone rapidly down regulates nuclear estrogen receptor (Re) in the estrogen-primed rodent uterus. We have now extended these studies to test the response of the Re system in sheep uterus to progesterone withdrawal. Since the estrogen-Re complex is believed to regulate hormone-dependent gene expression, it was of interest to determine whether withdrawal of progesterone under constant estrogen stimulation would lead to the recovery of nuclear Re levels and estrogen action, i.e. oxytocin receptor (ROT) synthesis. Ovariectomized ewes were primed with estradiol-17 beta and serum steroid levels were maintained by constant infusion of estradiol (0.5 microgram/h) and progesterone (500 micrograms/h) for 5 days. The animals were anesthetized with fluothane/O2, and uterine samples were excised 1 h before and 3, 6 and 12 h after progesterone withdrawal. Estradiol infusion was continued during the experiment in order to maintain estrogen levels at a steady state (14 pg/ml plasma). Re, ROT and progesterone receptor (Rp) were measured in endometrium and myometrium using standard 3H-hormone binding assays. Following progesterone withdrawal, the nuclear Re concentration increased in both uterine compartments, and the nuclear Re level was correlated significantly with the ROT concentration in the membrane fraction of both uterine tissues (endometrium, r = 0.79; myometrium, r = 0.86). Although cytosol Re rose between 6 and 12 h in the endometrium, cytosol Re levels remained unchanged in myometrium. Cytosol Rp appeared to increase in endometrium but not in myometrium. Uterine tissue sampled from a control animal before stopping the progesterone infusion revealed that the observed changes in receptor concentration following progesterone withdrawal were not due to regional differences in receptor levels. These results demonstrate that the recovery of nuclear Re in the ovine endometrium and myometrium following progesterone withdrawal represents a selective effect on Re retention in the nucleus rather than on cytosol Re availability or Re activation which was controlled by constant estrogen infusion. Thus, these results are consistent with the hypothesis that progesterone induces an Re regulatory factor which acts to down regulate nuclear Re, and that the activity of this factor diminishes rapidly after progesterone withdrawal.

Immunogold cytochemistry of cytochromes P-450 in porcine adrenal cortex

SummaryIn order to study the distribution of mitochondrial cytochromes P-450 in porcine adrenal glands, the glands of anesthetized pigs were fixed in situ. Polyclonal antibodies against two cytochromes P-450, i.e., C27 sidechain cleavage enzyme and 11 beta-hydroxylase, were used to study the distribution of these enzymes in cryosections of the adrenal cortex. Ultrathin cryosections were evaluated by both protein-A/gold/silver immunocytochemistry and immunielectron microscopy using double labeling with protein-A/colloidal-gold. At light microscopy, the two cytochrome P-450 enzymes were found to be broadly distributed in both the fasciculata and glomerulosa zones of the adrenal cortex. Quantitative immunoelectron microscopy revealed that both enzymes were localized only in mitochondria, in which they were present on the inner aspects of the inner mitochondrial membrane. Both cytochromes P-450 were demonstrable in all of the mitochondria examined, and statistical evaluation of the ratios of the two enzymes present in individual mitochondria yielded a normal distribution curve. Since no evidence was found for the preferential localization of either enzyme in a special population of mitochiondria, we conclude that all mitochondria of the adrenal cortex contain both enzymes. We discuss implications of these findings with respect to the regulation of steroidogenesis.

Intraluteal Prostaglandin Biosynthesis and Signaling Are Selectively Directed Towards PGF2alpha During Luteolysis but Towards PGE2 During the Establishment of Pregnancy in Sheep

ABSTRACT In ruminants, endometrial prostalgandin (PG) F2alpha causes functional luteolysis, whereas luteal synthesis of PGF2alpha is required for structural luteolysis. PGE2 is considered to be a luteoprotective mediator. Molecular aspects of luteal PGF2alpha and PGE2 biosynthesis and signaling during the estrous cycle and establishment of pregnancy are largely unknown. The objectives of the present study were 1) to determine the regulation of proteins involved in PGF2alpha and PGE2 biosynthesis, catabolism, transport and signaling in the corpus luteum (CL); 2) to investigate the transport of interferon tau (IFNT), PGF2alpha, and PGE2 from the uterus to the ovary through the vascular utero-ovarian plexus (UOP); and 3) to compare the intraluteal production of PGF2alpha and PGE2 on Days 12, 14, and 16 of the estrous cycle and pregnancy in sheep. Our results indicate that luteal PG biosynthesis is selectively directed towards PGF2alpha at the time of luteolysis and towards PGE2 during the establishment of pregnancy. Moreover, the ability of the CL of early pregnancy to resist luteolysis is due to increased intraluteal biosynthesis of PGE2 and PGE2 receptor (PTGER) 2 (also known as EP2)- and PTGER4 (also known as EP4)-mediated signaling. We also found that IFNT protein is not transported through the UOP from the uterus to the ovary; in contrast, a large proportion of endometrial PGE2 is transported from the uterus to the ovary through the UOP. These results indicate that endometrial PGE2 stimulated by pregnancy is transported locally to the ovary, which increases luteal PGE2 biosynthesis and hence activates luteal PTGER2 and PTGER4 signaling, thus protecting the CL during the establishment of pregnancy in sheep.

The structure of steroids and their diffusion through blood vessel walls in a counter-current system

Several substances including prostaglandin F2 alpha, progesterone and 85-krypton have been shown to be transferred from the venous side to the arterial side of the circulation in the ovarian vascular pedicle. Experiments were therefore carried out to study the transfer of three pairs of steroids (progesterone and 20 alpha-dihydroprogesterone, C-21; androstenedione and testosterone, C-19; and estrone and estradiol-17 beta, C-18) in which each member of a pair differed by one hydroxyl group. Each pair of steroids, one labeled with 3H and the other with 14C, were infused in sequence for 30 minutes into a side branch of an ovarian vein near the hilus of the ovary with a rest period of 90 minutes between infusions. An increase in radioactivity in ovarian arterial plasma compared to the radioactivity in an equal volume of aortic plasma sampled simultaneously was used as the index for a direct transfer of steroids from the ovarian vein to the adjacent ovarian artery. All six steroids showed such a transfer which began 3 to 6 minutes after the start of each infusion and decreased rapidly after the infusion was stopped. The results of this study also showed that a larger quantity of the less polar (ketonic) form of each steroid pair examined was transferred than its hydroxyl counterpart.

In vivo desensitization of a high affinity PGF2α receptor in the ovine corpus luteum

Abstract The corpus luteum (CL) of the sheep exhibits a differential sensitivity to PGF2α in vivo in terms of an increase in oxytocin (OT) secretion and a decrease in progesterone secretion, pointing to the presence in vivo of both high and low affinity receptors for PGF2α. The presence of the high affinity PGF2α receptor was assessed by monitoring the secretion rate of OT from the ovine CL in response to subluteolytic infusions of PGF2α. Rapid desensitization to PGF2α occurred after only one hour of infusion, while a minimum rest period of six hours was required to restore sensitivity. The possibility that these findings could be explained by the depletion and resynthesis of OT was excluded by demonstrating an increase in OT secretion rate with supra-physiological levels of PGF2α two hours after desensitization. Collectively, these results indicate the presence of a high affinity receptor for PGF2α in the ovine CL which exhibits desensitization and recovery in vivo . The temporal nature of the desensitization and recovery of the high affinity PGF2α receptor controlling luteal OT secretion may contribute to the pulsatile nature of PGF2α release from the ovine uterus.