Luteolytic Factor Research Articles

Expression and regulation of plasminogen activators, plasminogen activator inhibitor type-1, and steroidogenic acute regulatory protein in the rhesus monkey corpus luteum.

The corpus luteum (CL) is a transient endocrine organ that secretes progesterone to support early pregnancy. Using primate materials obtained from rhesus monkeys, we have in this study investigated the expression and regulation of the plasminogen activators (PAs) and PA inhibitor type 1 (PAI-1) during CL development and regression. Adult (5-7 yr old) female rhesus monkeys were treated with pregnant mare serum gonadotropin/human chorionic gonadotropin to induce ovulation and follicular luteinization. At various luteal developmental stages, CL or whole ovaries were obtained for preparing luteal cells, Northern blot, in situ hybridization, and immunohistochemistry. We demonstrated that luteal cells from the rhesus monkey were able to produce both tissue type PA (tPA) and urokinase type PA, as well as the physiological PAI-1. During luteal development in the monkey, urokinase type PA was the major PA species taking part in the active angiogenesis and tissue remodeling processes in the forming CL. However, the mRNA as well as the enzymatic activity levels of tPA increased dramatically in monkey CL with the advent of luteolysis. This change of tPA levels was in a temporal coordination with the regulation of PAI-1 expression, resulting in an increased tPA activity at the initiation of luteolysis. Therefore, we suggest that tPA might be a luteolytic factor to the monkey CL. A PAI-1 modulated tPA activity might be important for the initiation of luteolysis in the monkey. In addition, we have also demonstrated that the expression of steroidogenic acute regulatory protein in the monkey CL was in accordance with the changes of progesterone production, suggesting that steroidogenic acute regulatory protein expression may be considered as a reliable marker for CL function in primates.

Enhancer regions of ovine interferon-τ gene that confer PMA response or cell type specific transcription

Interferon-τ (IFNτ), produced by the trophectoderm of peri-implantation conceptuses in ruminant ungulates, attenuates the uterine production of a luteolytic factor, prostaglandin F 2α, resulting in the maintenance of corpus luteum function. However, molecular mechanisms regulating the temporal/spatial expression of IFNτ gene are not clearly understood. The 5′-upstream region of the sheep IFNτ (oIFNτ) gene was examined for its transcriptional regulation in two different cell types; JEG3 cells supported the transactivation of oIFNτ-reporter construct, but HeLa cells did not. In a heterologous SV40 enhancer–oIFNτ promoter or oIFNτ enhancer–SV40 promoter systems, elements required for such cell specific transactivation were localized between −654 and −555 bases, the enhancer, but not the basal promoter region of the oIFNτ gene. In these combinations, high degrees of transactivation were observed in JEG3 cells and the activity was further enhanced by the addition of phorbol 12-myristate 13-acetate (PMA), while those responses were absent in HeLa cells. To identify nucleotide sequences responsible for cell specific expression, transient transfection studies with sequential point mutations in the enhancer elements were executed. Transactivation of oIFNτ enhancer-reporter constructs was primarily regulated by three regions containing AP-1 site, GATA like sequence and site(s) unidentified. In gel mobility shift assays (GMSAs), the AP-1 site located in the enhancer region was recognized by nuclear extracts from both cell types. However, one of the GMSA probes containing GATA-like sequence exhibited different DNA-protein complex patterns in JEG3 and HeLa cells. Observations, in which the same upstream sequence behaved differently due possibly to kinds of nuclear factors available in these cell lines, suggest that such a sequence may be involved in cell specific transactivation of the oIFNτ gene. Furthermore, the same enhancer sequences were also recognized by nuclear extracts from sheep trophoblasts, suggesting that the enhancer sequences between −654 and −555 bases of oIFNτ gene may be functioning in vivo.

Evidence for nitric oxide acting as a luteolytic factor in the human corpus luteum.

The aims of the present study were to characterize the expression and cellular localization of isoforms of nitric oxide synthase (NOS) in the human corpus luteum (CL) and to determine the effects of nitric oxide (NO) on CL steroidogenesis. Immunoblotting analyses revealed that endothelial NOS (eNOS) is the most abundant isoform in human CL with highest values during the late luteal phase. Immunoreactive eNOS was localized predominantely in the theca lutein layer, being particularly abundant in endothelial cells, but with positive staining also in some steroidogenic cells. Immunoreactive inducible NOS (iNOS) was also detected, but to lesser degree, and did not display apparent phase-specific changes. The effect of NO on CL steroid synthesis was examined using human chorionic gonadotrophin (HCG)-stimulated dispersed CL cells cultured in vitro. Progesterone production was significantly decreased (P < 0.05) by the NO donor spermine NONOate (10(-5) mol/l) in cells of the late, but not mid-, luteal phase. To investigate a potential link between NO and the local prostaglandins (PG), concentrations of PGF(2alpha) and PGE(2) were measured in culture medium. NO significantly increased (P < 0.05) concentrations of both PGF(2alpha) and PGE(2) during the late luteal phase. It is concluded that NO may be luteolytic in the human CL of menstruation.

Detection and regulation of the messenger for a putative bovine endometrial 9-keto-prostaglandin E(2) reductase: effect of oxytocin and interferon-tau.

During reproductive processes, prostaglandin (PG) E(2) (PGE(2)) and PGF(2alpha) play important roles in which they often exert opposite effects. At the time of recognition of pregnancy in vivo, PGF(2alpha) is recognized as the luteolytic factor in ruminants and in most species including human, whereas PGE(2) may exert a luteoprotective action. We have previously demonstrated that recombinant interferon-tau (rIFN-tau), the embryonic signal responsible for recognition of pregnancy in ruminants, stimulated in vitro the production of PGE(2) and prostaglandin-endoperoxide synthase 2 (Ptgs2; also called cyclooxygenase-2) gene expression in both epithelial and stromal endometrial cells. Since PGE(2) is the major prostaglandin produced by stromal cells, the effect on Ptgs2 could explain the increase in PGE(2) output. At high concentrations, however, recombinant ovine (ro) IFN-tau acts on epithelial cells by changing the primary PG produced from PGF(2alpha) to PGE(2). This change in the primary PG produced could be explained by a decrease in PGF synthase (PGFS) activity or an increase in PGE synthase activity, or by modulation of a putative PGE(2)-9-ketoreductase, which converts PGE(2) into PGF(2alpha). Therefore, we have investigated the regulation of the mRNAs for PGFS and PGE(2)-9-ketoreductase (9K-PGR), two enzymes that lead to the production of PGF(2alpha). Others have described 9K-PGR activity in uterus, ovaries, kidney, and liver of different species and have established that this enzyme could possess both 9K-PGR and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity. Some have concluded that 9K-PGR and 20alpha-HSD are identical enzymes. Using primers sequences chosen from homologous nucleotide sequences of published rabbit 20alpha-HSD/9K-PGR and rat 20alpha-HSD cDNAs, a 317-base pair (bp) fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned, and sequenced. Homologies of 83% and 78% were found with rabbit and rat 20alpha-HSD, respectively. The presence of 20alpha-HSD/9K-PGR and prostaglandin F synthase (PGFS) mRNA expression was studied semiquantitatively in cultured epithelial cells using RT-PCR. Stimulation of cells with roIFN-t resulted in a biphasic response, an inhibition of PGF(2alpha) production at low dose (1 ng/ml) and a stimulation of PGE(2) at high dose (10 microg/ml). The increase of PGE(2) was accompanied by reduced 9K-PGR and PGFS mRNA gene expression. The effect of oxytocin (OT) was also studied, and the presence of OT had no effect on either 9K-PGR or PGFS gene expression. The 20alpha-HSD/9K-PGR transcript was also detected in other bovine tissues at different intensity (liver > kidney > testis > ovaries). We believe that the 9K-PGR and PGFS can be key enzymes in the regulation of specific PGs in the endometrium during the periimplantation period.

Angiotensin II interacts with prostaglandin F2alpha and endothelin-1 as a local luteolytic factor in the bovine corpus luteum in vitro.

Recent findings suggest that the ovarian renin-angiotensin system may regulate ovarian function through the paracrine/autocrine actions of angiotensin II (Ang II). In this study, we have examined and characterized the local effects of Ang II as a luteolytic factor and its interaction with prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1) in the bovine corpus luteum (CL) of the mid-luteal phase, by using an in vitro microdialysis system (MDS). Ang II was detected in the MDS perfusate (4 pg/ml), and infusion of PGF2alpha (10(-6) M) for 2 h increased the Ang II release by 50-100% during the following experimental period, in addition to its stimulation of ET-1 release. Two 2-h infusions of Ang II (10(-7)-10(-5) M) separated by a 2-h interval induced a dose- and time-dependent decrease of progesterone (P4) release by 41-66%. When the luteal explants were pre-perfused with PGF2alpha (10(-6) M) for 2 h, two consecutive perfusions of Ang II (10(-6) M) at a 2-h interval rapidly reduced the P4 release (by 50%). This reduction occurred 6 h earlier than those of infusions of PGF2alpha or Ang II alone. The simultaneous infusion of either 1) Ang II (10(-6) M) with PGF2alpha (10(-6) M), 2) ET-1 (10(-7) M) with PGF2alpha, or 3) Ang II + ET-1 with PGF2alpha (10(-6) M) for 2 h also induced a rapid and pronounced (60%) decrease in P4 release. Perfusion with the Ang II antagonist blocked the P4-suppressing activity of Ang II alone or PGF2alpha + Ang II infusion. Ang II stimulated the release of ET-1 and oxytocin during infusion but inhibited them after infusion. These results show that Ang II is released in the bovine midcycle CL in vitro, and this peptide, either alone or together with PGF2alpha, can suppress the release of P4. As PGF2alpha directly stimulated Ang II release, Ang II may influence the critical period for starting the cascade of functional luteolysis in vivo and might lead to structural luteolysis with ET-1 as a major vasoconstrictor. The overall results suggest that Ang II may have an important role at luteolysis in the bovine CL.

Hormonal and non-hormonal agents at implantation as targets for contraception.

The processes leading to implantation and the establishment of pregnancy involve hormonal and non-hormonal agents that offer opportunities as targets for contraception. Hormonal agents include progesterone, luteolytic factors (prostaglandin F2 alpha) and embryonic signals (chorionic gonadotrophin, oestradiol-17 beta, interferon-tau) responsible for maintaining the corpus luteum. Non-hormonal agents include surface antigens (attachment and adhesion molecules), vasoactive agents, tissue-remodelling enzymes (matrix metalloproteinases) and inhibitors (TIMPs), growth factors (epidermal growth factor and insulin-like growth factor families) and cytokines (such as leukaemia inhibitory factor, colony-stimulating factor-1, interleukin-1 (IL-1) and IL-6) associated with the pre-attachment period and the apposition, adhesion and invasion of the blastocyst. This review describes some of the hormonal and non-hormonal agents present at the time of implantation that may be exploited as targets for contraception in feral species. Particular attention is paid to the mouse as an experimental model and potential target species. The considerable species differences which exist in the models of implantation and placentation and the way in which the female 'recognizes' the presence of a viable conceptus offer a means of conferring species specificity on potential contraceptive targets for feral species.

Influence of Supernatants from Rabbit Luteal Tissue Cultures on Blastogenesis of Peripheral Blood Lymphocytes.

To evaluate the correlation between luteolysis and T lymphocytes (T-cells) found in the corpus luteum (CL), peripheral blood lymphocyte (PBL) blastogenesis to mitogens tested for the presense of luteolytic factor(s) in the supernatant culture medium of luteal tissues from various stages of pseudopregnancy (PSP). PBL blastogenesis was measured by 3H-thymidine uptake. The culture medium of luteal tissue from Day 15 PSP caused a significantly high 3H-thymidine uptake in the Con A stimulation group of the PBL blastogenesis test. The present finding suggests the presence of some factors which activate in the regressive CL.

Phospholipase A2 activity in endometrium from early pregnant and non-pregnant ewes

In the ewe, synthesis of the luteolytic factor, prostaglandin F 2α, increases from day 13 to the end of the estrous cycle. Availability of free arachidonic acid is usually the rate-limiting step in prostaglandin biosynthesis. Phospholipase A 2 (PLA 2) may be the key enzyme for the hydrolysis of arachidonic acid from membrane-bound phospholipids. To investigate uterine PLA 2 activity during the estrous cycle and early pregnancy, we monitored the release of [ 14C]oleic acid from the substrate 1-palmitoyl-2-[ 14C]oleoyl-phosphorylcholine by homogenates and cytosolic fractions of endometrium from ewes on days 12, 14 and 16 of the estrous cycle or pregnancy. We observed that PLA 2 activity dropped by 58% (p<0.02) in day-16 pregnant endometrium compared to day-16 non-pregnant endometrium. We then investigated whether the reduced PLA 2 activity was due to induction of a specific inhibitor. The PLA 2-inhibitor activity was determined by monitoring the inhibition of release of [ 14C]oleic acid from the radioactive substrate by porcine pancreatic PLA 2. Inhibition by endometrial homogenates of pregnant animals of the control enzyme activity was 27% and only 14% by cyclic ones. Inhibition was dose-dependent and was as high as 53% (p<0.01) with 1 mg protein from pregnant endometrial homogenates. Endometrial PLA 2 behaved as a Michaëlian enzyme in the endometrium of day-16 cyclic ewes (Km = 79.4 μmol/l). Furthermore, the inhibitory activity from pregnant endometrium had characteristics of competitive inhibition. Our results suggest that inhibition of endometrial PLA 2 activity could occur in early pregnant ewes.

2 Prostaglandins in the ovary and fallopian tube

More than 20 years following the recognition of a possible role for eicosanoids in ovarian function a physiological role for prostaglandins and/or leukotrienes in human ovulation, corpus luteum function and tubal motility remains to be demonstrated. With respect to ovarian function, the well-characterized preovulatory rise in eicosanoid production in animal species and humans, in conjunction with the large body of experimental evidence employing inhibitors of prostaglandin synthesis and replacement of individual prostaglandins, has provided strong evidence for a role in follicular rupture independent of other LH-mediated ovulatory events. The possible mechanism of prostaglandin-induced follicle rupture may involve stimulation of proteolytic activity via substances such as plasmin and PA; however, this is controversial. A role for prostaglandins in ovarian luteal function is well established in laboratory animals and large ruminant species, where PGF2 alpha derived from the uterus has been demonstrated to be the luteolytic factor. In humans, luteal function may be influenced by local intraovarian eicosanoid production, which has been suggested to involve the paracrine interaction of local ovarian hormones such as oxytocin, noradrenaline, insulin and IGFs, to name but a few. Several lines of evidence have also implicated prostaglandins as an aetiological factor in ovarian pathological states such as seen in the OHSS. However, the bulk of clinical experimental evidence to date has failed to support this contention. Prostaglandin production has likewise been well characterized in the fallopian tube in both humans and animal species. Whereas a role for prostaglandins in tubal transport has been demonstrated with animal species such as the rabbit, several studies have failed to define a similar function in humans. More recently, direct injections of prostaglandin analogues into the fallopian tube and the corpus luteum have been shown to be efficacious as a treatment for ectopic pregnancy. Whether the primary mechanism of action involves effects on tubal musculature or corpus luteum function, or is simply a local vascular effect, remains to be demonstrated. Therefore, although the physiological role for eicosanoids in ovarian and tubal function remains unclear, particularly in the human, an increasing body of recent evidence has suggested an important paracrine function for this class of cellular mediators whose interaction with other more recently characterized local ovarian factors has only begun to be recognized.

In Vivo Levels of Prostaglandin F2α, E2 and Prostacyclin in the Corpus Luteum of Pregnant and Pseudopregnant Rats1

Prostaglandin F2 alpha (PGF2 alpha) is a well-known luteolytic factor in the rat corpus luteum. To investigate a possible luteal origin of PGF2 alpha, measurements of this prostaglandin were performed in different luteal tissues in vivo. Prostaglandin E2 (PGE2) and the stable metabolite of prostacyclin, 6-keto-PGF1 alpha, were assayed simultaneously. Corpora lutea of different ages from 57 pregnant and pseudopregnant rats (mated with sterile males) were rapidly excised, dissected in 0 degree C indomethacin solution, homogenized, and extracted for prostaglandins with solid-phase extraction cartridges. Prostaglandins were determined by radioimmunoassay. Plasma levels of progesterone and 20 alpha-dihydroprogesterone were also monitored. In the adult pseudopregnant rat model, luteolysis occurs at Day 13 +/- 1, and maximal levels of all three prostaglandins were detected on Day 13 of pseudopregnancy: 0.40 +/- 0.02, 2.6 +/- 0.29, and 1.76 +/- 0.24 pmol/mg protein (mean +/- SEM, n=7) for PGF2 alpha, PGE2, and 6-keto-PGF1 alpha respectively. In pregnant rats, on the corresponding day, levels were considerably lower: 0.15 +/- 0.02, 0.90 +/- 0.13, and 0.50 +/- 0.06 pmol/mg protein (mean +/- SEM, n=9, p less than 0.0001), respectively. Luteal levels in pregnant rats showed a continuous decline on Days 13 and 19 for all prostaglandins measured, whereas in pseudopregnant rats an increment of PGF2 alpha was noted between Days 7 and 13 and remained high on Day 19. PGE2 closely followed levels of PGF2 alpha, but at a 5- to 10-fold higher level. The coefficient of correlation between PGF2 alpha and PGE2 in the luteal compartment of both models was 0.87 (p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Initiation of parturition and lactation in the sow: effects of delaying parturition with medroxyprogesterone acetate

The synthetic progestagen, medroxyprogesterone acetate (MPA), was administered to sows in late pregnancy with the objective of slightly delaying the time of farrowing and thereby providing more marked associations between hormonal changes and the termination of pregnancy, and the initiation of farrowing and lactation in this species. MPA was administered orally (140 mg, twice daily) to eight sows in late pregnancy on days 112, 113 and 114 of gestation. Parturition was then induced to occur on day 116 by injecting 200 micrograms cloprostenol i.m. on day 115 of gestation. The peripartum changes in the plasma concentrations of progesterone, cortisol, oestradiol-17 beta, relaxin, prolactin, lactose and 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) were measured in these sows together with a group of untreated sows. The gestational length for the MPA-treated sows (116.3 +/- 0.3 days, mean +/- S.E.M.) was significantly (P less than 0.01) greater compared with the untreated sows (114.9 +/- 0.3 days). Plasma progesterone declined earlier (P less than 0.05) with respect to the time of parturition in the treated sows compared with the untreated group. With respect to the timing of parturition, the time at which maximal concentrations of relaxin were attained and the timing of the subsequent decline were earlier in the MPA-treated sows. In both groups of sows, the concentration of relaxin increased before the decline in plasma progesterone. In the untreated sows, the concentration of PGFM increased either slightly before or at the same time as the decline in plasma progesterone, whereas in sows treated with MPA, progesterone concentrations began to decline before any significant increase in the plasma concentration of PGFM. The profiles of cortisol, oestradiol-17 beta and PGFM were similar in both groups of sows. In both groups of sows, the timing of the initial increase in the concentration of plasma prolactin coincided with a similar rise in plasma lactose (P less than 0.01). Plasma progesterone either declined earlier or at the same time as the rise in plasma lactose (P less than 0.01) in the treated group of sows only. We conclude that since the prepartum changes in the concentration of progesterone and relaxin occurred before significant changes in the concentration of PGFM in the MPA-treated sows, the nature of the luteolytic factor and the mechanism by which it exerts its action remains obscure. The higher concentration of lactose in the mammary secretion at birth in the MPA-treated sows compared with the untreated group suggested that lactogenesis was initiated earlier with respect to parturition following MPA treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on the prostaglandin E-2-9-ketoreductase mediated production of prostaglandin F-2 alpha and its metabolism in cultured rabbit luteal cells.

Luteal cells were isolated from pseudopregnant rabbits on days 3, 6, 9, 12 and 15 post ovulation. Prostaglandin concentration and the activities of the enzymes prostaglandin E-2-9-ketoreductase and prostaglandin-15-hydroxydehydrogenase were determined. Luteal cells from day 7 and 12 of pseudopregnancy were maintained in culture for 24 h and then exposed to a mixture of [1-14C]PGE2 (70 mumol/l) in the presence or absence of estradiol-17 beta. After a 24 h incubation period, the culture medium was adjusted to pH 3.5, immediately extracted and analysed for PG. Cultured luteal cells were able to convert exogenously applied PGE2 to PGF2 alpha and to metabolize both PGs. Primary PGs as well as their metabolites 15-keto PGE2, 15-keto PGF2 alpha, 13,14-dihydro-15-keto PGE2, and 13,14 dihydro-15-keto PGF2 alpha were detected in the culture medium and in the cells. The addition of estradiol-17 beta together with PGE2 caused a significant reduction in the PGE2-9-ketoreductase mediated PGF2 alpha synthesis, whereas the metabolism of PGE2 remained unchanged. This inhibitory effect of estradiol-17 beta was dose-dependent on day 12 of pseudopregnancy. The results demonstrate that isolated rabbit luteal cells are able to synthesize and metabolize the luteolytic factor PGF2 alpha. Whether the inhibitory effect of estradiol-17 beta may have any physiological relevance has to be examined in further studies.

Prostaglandins and progesterone secretion in the in vitro perfused pseudopregnant rabbit ovary.

The relationship between progesterone and prostaglandin (PG) secretion in the pseudopregnant rabbit corpus luteum was investigated using isolated in vitro perfused ovaries. Progesterone and PG secretion were measured on days 1, 11, and 18 of hCG-induced pseudopregnancy. The mean progesterone secretion increased significantly from days 1 to 11, and then decreased significantly by day 18. PG secretion was inversely correlated with progesterone secretion, suggesting that PG might inhibit progesterone secretion. To test this hypothesis, indomethacin, an inhibitor of PG secretion, was administered to intact rabbits from days 11-18 of pseudopregnancy and/or on day 18 ovaries were perfused in vitro with indomethacin. Indomethacin administered in vivo, in vitro, or both in vivo and in vitro significantly reduced PG secretion compared to that in controls, but did not affect progesterone secretion. In addition, perfusion of ovaries in vitro with PGF2 alpha did not alter progesterone secretion on either day 11 or day 18. Thus, although there is an inverse relationship between progesterone and PG secretion during pseudopregnancy, PGF2 alpha alone had no effect on progesterone secretion. These results question the hypothesis that PGF2 alpha alone is the luteolytic factor.

Luteotropic and luteolytic factors regulating human corpus luteum function.

Annals of the New York Academy of SciencesVolume 541, Issue 1 p. 485-497 Luteotropic and Luteolytic Factors Regulating Human Corpus Luteum Function a L. HAMBERGER, L. HAMBERGER Departments of Obstetrics and GynecologySearch for more papers by this authorM. HAHLIN, M. HAHLIN Departments of Obstetrics and GynecologySearch for more papers by this authorT. HILLENSJÖ, T. HILLENSJÖ Departments of Obstetrics and GynecologySearch for more papers by this authorC. JOHANSON, C. JOHANSON Physiology University of Göteborg Göteborg, SwedenSearch for more papers by this authorA. SJÖGREN, A. SJÖGREN Departments of Obstetrics and GynecologySearch for more papers by this author L. HAMBERGER, L. HAMBERGER Departments of Obstetrics and GynecologySearch for more papers by this authorM. HAHLIN, M. HAHLIN Departments of Obstetrics and GynecologySearch for more papers by this authorT. HILLENSJÖ, T. HILLENSJÖ Departments of Obstetrics and GynecologySearch for more papers by this authorC. JOHANSON, C. JOHANSON Physiology University of Göteborg Göteborg, SwedenSearch for more papers by this authorA. SJÖGREN, A. SJÖGREN Departments of Obstetrics and GynecologySearch for more papers by this author First published: October 1988 https://doi.org/10.1111/j.1749-6632.1988.tb22285.xCitations: 4 a This work was supported by Grant No. 2873 from the Swedish Medical Research Council. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume541, Issue1In Vitro Fertilization and Other Assisted ReproductionOctober 1988Pages 485-497 RelatedInformation

Effect of photoperiod, injection of pentobarbitone sodium or lesion of the suprachiasmatic nucleus on pre-partum decrease of blood progesterone concentrations or time of birth in the rat.

In Sprague-Dawley rats kept under 14L:10D (lights on 05:00-19:00 h), parturition occurred during the light phase on Day 23, and the pre-partum decrease in progesterone concentrations was observed between 07:00 and 15:00 h during the light period on Day 22. When the rats were transferred to reversed light-dark regimen (lights on 17:00-07:00 h) on Day 7, the progesterone decrease and parturition still occurred during the light period on Day 21 and 22-23, respectively. However, when rats were kept in constant darkness from Day 7, parturition occurred independently of the time of day between Day 22 and 24. A gradual decline of progesterone concentrations was randomly observed in individual rats. In Wistar rats kept under the usual light-dark regimen, parturitions were biphasic, occurring during the light periods on Day 22 and 23. The progesterone decrease occurred at the usual time even when the lighting regimen was changed only on the day of the expected progesterone decrease. However, treatment with pentobarbitone sodium at 15:00, 19:00 or 21:00 h, but not at 12:00 or 23:00 h, on Day 21 resulted in a delay of progesterone decrease and of parturition. Complete lesion of the suprachiasmatic nucleus on Day 13 or 14 led to advancement and random distribution of the time of birth. These results suggest that the time of parturition and of pre-partum progesterone decrease may be closely associated with an endogenous circadian system, and a luteolytic factor involving the nervous system may be present during a limited period before parturition.

Luteolytic influence of intrauterine dead embryos in the early pregnant rat.

The present investigation was an endeavour to study if annihilation of embryos in the uterus of the rat before the establishment of placental luteotropic functions has any influence on corpus luteal function, and, if there is any, whether it is local or systemic. The responsibility of pregnancy maintenance was imposed on a single ovary by performing unilateral ovariectomy after implantation (on Day 5 postcoitum). The implantation sites in one uterine horn, either ipsilateral or contralateral to the remaining ovary, were selectively destroyed by injecting 0.1 ml of sterile normal saline to that particular horn only, and the peripheral progesterone level and viability of the embryos in the untreated horn, which depended on the functions of the remaining ovary, were examined. Selective killing of embryos in the uterine horn of the ovariectomized side did not exert any influence on the fetal viability in the untreated horn ( nonovariectomized side) and the peripheral progesterone level also remained statistically unaffected. On the contrary, induction of fetal resorptions in the uterine horn of the intact side produced a significant fall in the peripheral level of progesterone and induced resorption of embryos of the ovariectomized side also. The latter could significantly be prevented by simultaneous administration of exogenous progesterone, indicating luteolysis as the major, if not sole, factor responsible for fetal resorption in the untreated horn. The luteolytic effect was attributed neither to saline itself, nor to the distension of the uterine horn caused by saline injection. Luteolytic factors from the dead embryo-bearing horn which act locally on the adjacent ovary only, are discussed.

Biochemical aspects of conceptus--endometrial interactions.

Mammalian conceptuses must provide a chemical signal to the maternal system to insure maintenance of corpus luteum (CL) function and of progesterone production and continuation of uterine endometrial secretory activity. These events insure that the developing conceptus is provided with appropriate nutrients, regulatory enzymes and endocrine state to allow successful establishment and maintenance of pregnancy. Pig blastocysts begin to produce estrogens by Day 11 of pregnancy, which prevents secretion of the uterine luteolytic factor (PGF2 alpha) in an endocrine direction, but allows secretion in an exocrine direction, i.e., into the uterine lumen. Therefore, CL are "protected." Blastocyst estrogens also trigger secretion of a group of proteins, including uteroferrin, an iron transport protein, and a family of protease inhibitors whose biosynthesis within the uterine glandular epithelium is under the control of progesterone. Estrogen also appears to promote accumulation of glucose and fructose within the uterine lumen. A complex in vivo "culture medium" is thereby established to promote conceptus development. Pig blastocysts do not undergo invasive implantation within the uterine lumen although they produce the protease, plasminogen activator. Invasion may be prevented by endometrial secretion of progesterone-induced protease inhibitors which are produced in large amounts. In addition to estrogens of conceptus origin, calcium and prostaglandins PGF2 alpha and E2 may affect the uterine vasculature, water and electrolyte transport, capillary permeability, conceptus steroid production, and related events during pregnancy. The blastocysts of the large domestic animals also secrete proteins which include a large glycoprotein (Mr approximately 600,000) and a small acidic protein (Mr approximately 17,000). The latter has been purified from sheep and named ovine trophoblast protein I. These proteins may play unique roles in early pregnancy with respect to establishment and maintenance of pregnancy in the ewe, sow, mare, and cow.

Luteotropic and antiluteolytic activities of the rabbit conceptus.

We have previously confirmed that maternal recognition of pregnancy in the rabbit occurs between Days 10 and 18 following mating. However, the mechanisms by which the conceptuses affect continued maternal corpus luteum progesterone secretion have not been completely defined. To determine whether the conceptuses develop a gonadotropin-like activity as a function of gestation time, preimplantation embryos collected on Day 6 post coitum (p.c.), early postimplantation conceptuses obtained on Day 10 p.c., and medium from preincubations of fetal placentas obtained on Days 18 and 26 p.c. were tested for their ability to increase testosterone production by dispersed rat testicular interstitial cells. A very low gonadotropin-like activity was evident in post-implantation conceptuses; however, as gonadotropin-like activity in maternal peripheral serum was greater than that observed in uterine venous serum, the significance of a direct gonadotropic signal from the conceptus remains unclear. Furthermore, in an additional bioassay for gonadotropin-like activity, rabbit fetal placentas obtained on Days 17-20 p.c. failed to stimulate ovarian growth and ovulation in immature rats. These data suggest that a direct luteotropic stimulus from the conceptus may not be a significant factor in continued corpus luteum function during pregnancy. Other investigators have reported that the rabbit conceptus plays a role in blocking a uterine induced luteolysis at or near the time of maternal pregnancy recognition. Following hysterectomy of pregnant and pseudopregnant rabbits on Days 6 or 10 p.c., the decline in serum progesterone levels was gradual, but the progesterone levels were significantly different from those of intact pregnant does as early as Days 16-18 p.c. In contrast, after hysterectomy on either Day 18 or 26 of pregnancy, serum progesterone concentrations declined abruptly. These data indicate that removal of a uterine luteolytic factor may only partially sustain corpus luteum function for a brief period of time following pregnancy recognition. An additional antiluteolytic role of the conceptus, in blocking a possible extrauterine prostaglandin mediated luteolysis was examined by treating pregnant rabbits hysterectomized on Day 18 with indomethacm, a potent inhibitor of prostaglandin synthesis. This treatment, initiated immediately postsurgery, failed to prevent the rapid decline in serum progesterone levels following hysterectomy, suggesting that in the rabbit prostaglandins may not be involved as extrauterine luteolytic factors.

Regulation of cholesterol acquisition and utilization in the corpus luteum.

In this report we have focused on two aspects of control of corpus luteum function; the regulation of lipoprotein uptake and the modulation of cholesterol side chain cleavage through alterations in the phospholipid milieu. Until recently, a role of gonadotropins in the acquisition of lipoprotein-carried cholesterol had not been generally appreciated. However, it is likely that the regulation expression of cell surface lipoprotein receptors will prove to be an important function of tropic hormones, particularly LH, since luteal cells generate progestins at a substantial rate and must, therefore, have an equally substantial supply of cholesterol. Factors other than gonadotropins may have a function in control of lipoprotein receptors and it would not be surprising if luteolytic factors, such as prostaglandin F2 alpha, are found to diminish lipoprotein-uptake as part of their action in reducing steroidogenesis. The control of mitochondrial pregnenolone synthesis by gonadotropin-induced alterations in phospholipid composition of mitochondrial membranes represents a novel concept. Whether certain polar lipids will emerge with the status of intracellular messengers remains to be seen. In any event, our observations clearly demonstrate the possibility for such a mechanism of control.

Prolongation of the oestrous cycle in cows and ewes after passive immunization with PGF antibodies.

Three cows and 2 sheep were passively immunized against prostaglandin (PG) F on Day 16 and Days 13-15 of the oestrous cycle respectively. The PGF antiplasma was raised in ovariectomized ewes against a PGF-2 alpha-bovine serum albumin complex and showed 100%, 12.5%, 0.3%, less than 0.05% and less than 0.01% cross-reactivity with PGF-2 alpha, PGE-2, PGA-2, PGB-2 and arachidonic acid, respectively. Control animals were given an equivalent amount of ovariectomized ewe plasma. In all passively immunized animals there was evidence of a persistent corpus luteum as indicated by plasma progesterone concentration and the failure of the animals to return to oestrus until at least 29 days after treatment. These data are consistent with previous proposals that PGF-2 alpha is the uterine luteolytic factor in sheep and cattle.