Failure of LH and/or prolactin to prevent PGF 2alpha-induced luteolysis of ovine corpora lutea

Infusions of phosphate buffered saline, LH (4 microgram/min or 14 microgram/min), prolactin (42 microgram/min) or LH (4 microgram/min) plus prolactin (42 microgram/min) for 12 hr did not prevent luteolysis following intramuscular injections of prostaglandin F2alpha-tham salt two and six hr after beginning the infusion. Likewise, these treatments did not delay luteolysis since a similar rate of decline in peripheral plasma progesterone occurred in all groups. It was concluded that elevation of serum concentrations of LH and prolactin to high levels had no effect on PGF2alpha-induced luteolysis on day 8 following induced ovulation.     

Changes in refractoriness of rabbit corpora lutea to a prostaglandin F2 alpha analogue, alfaprostol, during pseudopregnancy

Seventeen care providers were interviewed about their caring experiences on a hospital psychiatric ward. The interviews focused on the meaning of their work, including the care they provide and the nature of the patient as a person. The study was guided by a phenomenological hermeneutic perspective inspired by Ricoeur (1976). The analysis focused on context and form. Three themes illuminate the meaning of care provided. These themes are as follows; being in the midst of human storage, moving towards a human care of relations, and struggling with 'the old' and 'the new'. Experiencing work as being in the midst of a human storage reflects the historical and human situation of warehousing psychiatric patients. The care providers are experiencing a shift in their view of the patient and the meaning of their work, towards a more human care of relations. For these care providers, there is a struggle between the past, the present and the future. This struggle between 'the old' and 'the new' conveys a struggle between doing as a nurse, which dominates the past, and relating, which is, or needs to be, the current and future focus in psychiatric care. The shift in view distinguished itself by the care providers viewing the patient as being vulnerable and having problems with relations. The results have been interpreted and discussed in the light of a previously published interview study with the patients, carried out at the same time on the same ward. Attending to ingrained attitudes of the past and their influence on new approaches to care is essential to understanding not only changes in ways of doing nursing tasks, but also ways of relating.     

Polyubiquitin up-regulation in corpora lutea of prostaglandin-treated ewes

Genes that encode mRNAs for ubiquitin are activated by cells in metabolic distress. Cytosolic proteins that consequently become conjugated to ubiquitin are targeted for degradation. We hypothesized that ubiquitin mediates the endocrine demise of the corpus luteum induced by prostaglandin (PG) F2alpha. Indeed, polyubiquitin gene expression increased abruptly (within 2 h) in luteal tissues of ewes treated with PGF2alpha--before the precipitous decline in glandular progesterone accumulation indicative of functional luteolysis. A corresponding elevation in ubiquitin immunostaining was localized to large (PG-sensitive) luteal cells. It is suggested that luteal progesterone biosynthesis is disrupted by ubiquitination of steroidogenic regulatory proteins--perhaps those involved in the mechanics of mitochondrial delivery and side-chain cleavage of cholesterol.     

Inhibition by tocopherol of prostaglandin-induced apoptosis in ovine corpora lutea

Accumulation of toxic oxidants within corpora lutea is a prelude of apoptotic cell death. Vitamin E (alpha-tocopherol) is a biological antioxidant that protects cells from the inductive effects of reactive oxygen on DNA damage and nuclear/cytoplasmic condensation that dictate apoptosis. Ewes were challenged with a luteolytic dose of PGF2 alpha on d 10 of the estrous cycle. The acute decline in circulatory progesterone indicative of the onset of functional luteolysis was not affected by systemic administration of alpha-tocopherol; however, corpora lutea consequently (beyond 24 h) rebounded from the steroidogenic insult. Luteal tissues obtained at 24 h after PGF2 alpha revealed that internucleosomal DNA fragmentation and cellular collapse were inhibited by alpha-tocopherol. These observations indicate that regressive corpora lutea can be spared from terminal involution by diminishing the apoptotic influence of luteolytic hormone with an antioxidant.     

Relationship between membrane potential and progesterone release in ovine corpora lutea

When slices of ovine luteal tissue were perfused with medium containing luteinizing hormone (LH), the output of progesterone was increased significantly (P less than 0.01) in eleven of twelve experiments. However, addition of LH to the medium did not influence the luteal cell membrane potential. The addition of 47 mM potassium to the medium resulted in increased progesterone output (P less than 0.01) and depolarization of the luteal cell membrane within 2 min. Progesterone output decreased to approximate pretreatment levels within 2 min of the return to normal potassium levels in the perfusion medium. High levels of potassium further increased the output of progesterone from tissue stimulated with LH. Perfusion of the slices with sodium-free medium also resulted in increased (P less than 0.01) progesterone output within 2 min, which returned to pretreatment levels within 2 min after normal sodium levels were restored to the medium. Perfusion of the slices with sodium-free medium did not influence the membrane potential. Perfusion of the tissue with LH, 47 mM potassium, or sodium-free medium had no effect on progesterone output if the medium was calcium-free and/or contained 2 mM EGTA. These data suggested that the calcium ion plays an important role in mediating the steroidogenic response of ovine luteal tissue to LH. A second series of experiments was designed to ascertain if luteal cells were coupled electrically. Sixty-six pairs of luteal cells separated by 150-300 mum were penetrated with electrodes and the membrane potential of both cells was studied. One cell of each pair was hyperpolarized by passage of 0.4 nA current into the cell, but in no case was there an effect on the membrane potential of the other penetrated cell. Likewise, when five cells were injected iontophoretically with Procion Yellow there was no evidence of diffusion of the dye to adjacent cells. There was no evidence obtained in this study which suggested that ovine luteal cells were coupled electrically.     

Effect of prostaglandin F2 alpha treatment before norgestomet and estradiol valerate treatment on regression, formation, and function of corpora lutea in beef heifers

Despite the presence of several human disease genes on chromosome 11q13, few of them have been molecularly cloned. Here, we report the construction of a contig map encompassing 11q13.1-q13.3 using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The contig map comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and 1 YAC clone and spans a 3-Mb region from D11S480 to D11S913. The map encompasses all the candidate loci of Bardet-Biedle syndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5), one-third of the distal region for hereditary paraganglioma 2 (PGL2), and one-third of the central region for insulin-dependent diabetes mellitus 4 (IDDM4). In the process of map construction, 61 new sequence-tagged site (STS) markers were developed from the Not I linking clones and the termini of clone inserts. We have also mapped 30 ESTs on this map. This contig map will facilitate the isolation of polymorphic markers for a more refined analysis of the disease gene region and identification of candidate genes by direct cDNA selection, as well as prediction of gene function from sequence information of these bacterial clones.     

Identification and distribution of tumor necrosis factor alpha receptors in pig corpora lutea

This study examined whether the pig CL contains specific tumor necrosis factor alpha (TNF alpha) receptors and compared the binding affinities and capacities of small and large cell membranes. Aliquots of membranes, isolated from intact or dispersed luteal tissue, were homogenized, and membrane protein content was quantified. Luteal membranes were assayed for specific TNF alpha binding by displacement analysis, with use of [125I]TNF alpha and varying concentrations of unlabeled TNF alpha. Preliminary experiments demonstrated that TNF alpha binding was maximal after incubation at 22 degrees C for 180 min. In addition, [125I]TNF alpha binding was displaced by TNF alpha, but not by other cytokines. Small cell membranes contained a TNF alpha binding site with an affinity (Kd = 11.6-19 nM) different (p < 0.05) from that of the binding site on large cell membranes (Kd = 56.2-99.6 nM). TNF alpha binding capacities were similar in small and large cell membranes. These data demonstrate that pig CL contain specific, saturable TNF alpha binding sites. The higher-affinity binding sites were localized in the small cell population, which contains predominantly endothelial cells and small luteal cells, suggesting that TNF alpha acts primarily on one or both of these cell types within the CL.     

Effect of corpora lutea grafted under renal capsule on pregnancy in the rabbit

Implantation of the blastocyst and maintenance of pregnancy were examined by autografting corpora lutea under renal capsule in rabbits. Implantation of the blastocyst did not occur when the corpora lutea were autografted because of small amounts of lutein tissue, low secretory activity of lutein cells, and the period required for functional activity of grafts. Autografts of 4-day-old corpora lutea were capable of maintaining pregnancy after 15 days post coitum, when follicles and interstitial tissue remained intact. Pregnancy was maintained in a limited number of fetuses when 6-day-old corpora lutea were autografted in ovariectomized estradiol-treated does.     

Progesterone levels in amniotic fluid and maternal plasma in prostaglandin F2alpha-induced midtrimester abortion

Progesterone concentrations in amniotic fluid and maternal plasma were determined in 11 midtrimester pregnant patients following the intraamniotic administration of prostaglandin F2alpha. Samples were obtained at 3-hour intervals until abortion or spontaneous rupture of membranes occurred or fetal heart tones disappeared. In amniotic fluid, the mean progesterone concentrations increased throughout the sampling period. The plasma progesterone levels declined by about one-third of basal values in the first 3 hours after prostaglandin administration. The paradoxic increase in amniotic fluid progesterone is probably secondary to alterations in uterine blood flow and intrauterine pressure.     

Epidermal growth factor receptors in human corpora lutea during the menstrual cycle and pregnancy

We have demonstrated the presence of epidermal growth factor (EGF) and its receptors in human non-gestational corpora lutea. To determine further the characteristics of EGF receptor binding, we examined 30 human corpora lutea throughout the luteal phase and during pregnancy. Scatchard plots of EGF binding in 29 of the 30 corpora lutea were curvilinear, suggesting negative co-operativity. The mean +/- SE of the association constant Ka was (0.9 +/- 0.2) x 10(9) l/mol, the dissociation constant Kd was (2.2 +/- 0.3) x 10(-9) mol/l and the number of binding sites (Rt) was (15.8 +/- 2.1) x 10(-19) mol/micrograms protein for non-gestational corpora lutea. The Kd increased significantly in late pregnancies compared to early pregnancies (P = < 0.005), while Rt was significantly higher in term pregnancies than in either early pregnancy (P < 0.01) or the menstrual cycle (P < 0.001). Corpora lutea atretica (n = 2) and ovarian stroma (n = 6) did not show any EGF binding activity. Our findings demonstrate the presence of specific EGF receptors in human corpora lutea of both the menstrual cycle and pregnancy. The changes in EGF binding parameters in early pregnancy suggest that there may be a relationship between the role of EGF and ovarian steroidogenesis.     

Synergistic effects of prostaglandin F2alpha and tumor necrosis factor to induce luteolysis in the pig

Telomerase activity was detected in germ cells, stem cells and cancer cells. In tumors of the ovary, an organ that contains germ cells, the authors examined availability to detect telomerase activity. Telomerase activity of malignant tumors was extremely high compared with that of normal ovaries and benign tumors. Strength and frequency of telomerase activity in malignant tumors was significant different from that in benign tumors. Telomere length tended to be smaller for malignant tumors of advanced stage, but no significant relationship between telomere length and telomerase activity and tumor stage could be recognized. Telomerase activity may be a useful marker for the diagnosis of ovarian tumors.     

Altering conception of dairy cattle by gonadotropin-releasing hormone preceding luteolysis induced by prostaglandin F2 alpha

The objective of these experiments was to determine the effect on fertility of GnRH when used in conjunction with one or two injections of PGF2alpha. In Experiment 1, GnRH was administered 7 d before the second of two injections of PGF2alpha (14 d apart). The control group received two injections of PGF2alpha without GnRH. Conception was reduced from 63.5% for 74 controls to 48.7% for the 79 heifers and cows that had been treated with GnRH, but estrus detection and pregnancy rates were similar. In Experiment 2, 85 heifers and cows received GnRH at a random stage of the estrous cycle, followed in 7 d by PGF2alpha. Thirty to 32 h after PGF2alpha, a second dose of GnRH was given to induce ovulation of the preovulatory follicle, followed by one fixed-time insemination 18 to 19 h later (treatment designated as GnRH, PGF2alpha, and GnRH). Controls (n = 85) were given PGF2alpha and inseminated at estrus. Although conception rate was not different, one fixed-time insemination after the GnRH, PGF2alpha, and GnRH treatment tended (35.3%) to reduce fertility compared with effects of the control (47.1%). It is unclear how an injection of GnRH during the intervening week between two injections of PGF2alpha reduced fertility in Experiment 1. However, in Experiment 2, when GnRH was given 7 d before one injection of PGF2alpha and when ovulation was induced with a second GnRH injection, one fixed-time insemination seemed to produce acceptable fertility in dairy cows but probably less than that when inseminations were based on detected estrus.     

Effect of endothelin-1 on bovine luteal cell function: role in prostaglandin F2alpha-induced antisteroidogenic action

Endothelin-1 (ET-1) a vasoactive peptide, is synthesized and secreted by endothelial cells. In the bovine corpus luteum (CL), endothelial cells constitute a major proportion (53.5%) of total CL cells. This study was designed to examine the effects of ET-1 on bovine luteal cell functions and its involvement in the action of PGF2alpha. To better define the cells implicated in this process, we used CL slices, whole CL-derived cells, and steroidogenic large (LLC) and small (SLC) luteal-like cells. High affinity binding sites for ET-1 (K(d), approximately 0.3 x 10(-9)) were present in both steroidogenic luteal cells. The binding affinity of ET-1 was 3 orders of magnitude higher than that of ET-3, and a selective ETA receptor antagonist (BQ123) competed similarly to ET-1, suggesting the presence of ETA receptors. The lack of effect of ET-3 on CL-derived cells further supported this conclusion. Both basal progesterone secretion and bovine LH (5 ng/ml)-stimulated progesterone secretion from CL-derived cells were significantly inhibited by ET-1 in a dose-dependent manner, whereas preincubation of these cells with ETA receptor antagonist prevented the inhibitory effect of added ET-1. Incubation of LLC with 10(-8) M ET-1 inhibited their progesterone secretion (114.8 vs. 176.7 ng/10(5) cells-20 h; P < 0.05). On the other hand, ET-1 did not affect progesterone production from SLC despite the presence of ET-binding sites. PGF2alpha only inhibited LH-stimulated progesterone secretion by luteal slices. This antisteroidogenic effect of PGF2alpha could be prevented by the addition of a selective ETA receptor antagonist. Luteal tissue and microvascular endothelial cells isolated from bovine CL produced ET-1; in contrast, the peptide was undetectable in the culture medium or in cell extracts of either LLC or SLC. These data support the concept that ET-1 may play a paracrine regulatory role in bovine luteal function and propose a novel role for this peptide in mediating PGF2alpha-induced luteal regression.     

Regulation of mRNA encoding low density lipoprotein receptor and high density lipoprotein-binding protein in ovine corpora lutea

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.     

Prostaglandin F2alpha as a mediator of pulmonary changes during platelet aggregation

Increases in pulmonary arterial pressure, tracheal insufflation pressure, and blood levels of the prostaglandin F2alpha metabolite, 15-keto-13, 14-dihydro F2alpha, were observed after protamine chloride or thrombin-induced platelet aggregation and release reaction in dogs. These effects were largely eliminated after administration of acetylsalicylic acid, an inhibitor of prostaglandin synthesis. The platelet aggregation was not noticeably affected. It is suggested that release of prostaglandin F2alpha from platelets is an important factor for the pulmonary changes during induced platelet aggregation. The necessity of measuring metabolite levels, rather than prostaglandin F2alpha levels, in blood during in vivo conditions is demonstrated.     

Demonstration of luteotrophic responses of human recombinant gamma interferon in porcine corpora lutea using an in-vivo microdialysis system

Conceptuses from several mammalian species prior to implantation secrete proteins belonging to the family of interferons. The main species of interferons known to be secreted by the pig blastocyst is interferon gamma (IFNgamma), the precise role of which is unclear. We decided to explore its effects on corpus luteum (CL) function using the novel microdialysis technique in vivo. Six cycling miniature pigs were monitored for estrus by daily plasma progesterone analysis and visual symptoms. On day nine of the cycle (day zero being the day of ovulation) the animals underwent surgery, and microdialysis tubing (vitafiber, Amicon U.S.A, cut off mol. wt. 1 million) were implanted in 17 corpora lutea. The inlets and outlets of all tubings were exteriorized and the entry and exit points of tubings in the CLs sealed with tissue glue. The afferent extension tubings were connected to a fraction collector and the system was continuously flushed with Ringer at a flow rate of 2.4 ml/h. After an initial flushout phase of 8 h, fractions were collected every half hour over 3 days. On days 10, 11 and 12 post estrus 12 CLs were stimulated for 4 h with 10(-7) M, 2 x 10(7) M and 4 x 10(-7) M human recombinant IFNgamma (Pharma Biotechnologie) respectively. Simultaneously, fractions were also collected from the remaining five unstimulated corpora lutea which served as controls. Progesterone concentrations in the dialysates were estimated by a sensitive enzymeimmunoassay (EIA). A significant increase (P < 0.01) in progesterone release was observed in all 3 days following stimulation. The progesterone increase was more marked on the first day of stimulation (1 x 10[-7] M) with the hormone levels rising further even after the end of stimulation. The overall increase in progesterone concentration was 2-fold on day 10 in comparison to 15-30% on subsequent days even though IFN concentrations for stimulation were 2- and 4-fold higher. In the unstimulated CLs, a gradual decline (P < 0.01) in progesterone levels were observed over days. In conclusion, these data provide evidence that the early conceptus signals its presence by way of IFNgamma to maintain the CL in pigs.     

Identification of matrix metalloproteinases and metalloproteinase inhibitors in bovine corpora lutea and their variation during the estrous cycle

Corpora lutea (CL) were collected from cattle to study key physiologic events in angiogenesis. Our objective was to evaluate the activity of matrix metalloproteinases (MMP) and endogenous inhibitors. Corpora lutea were collected 2, 4, 6, 8, 10, 12, 14, and 16 d (n = 3/d) after estrus was first detected. In zymograms, a band of protein migrating at a relative molecular mass (M(r)) of 98 kDa was increased early in the cycle; a M(r) = 88 kDa band was detectable on all days. The molecular weights of these proteins are consistent with the MMP-9 family members. In all samples, a band of enzyme activity was detected at M(r) = 62 kDa, and another band of lesser density was detected at M(r) = 60 kDa. The molecular weights of these proteins are consistent with the MMP-2 family members. An immunoreactive band, detected in all samples with equal density, migrated between M(r) = 27 and 29 kDa, as did the tissue inhibitor of metalloproteinase (TIMP-1) standard. A second band, which was less dense in samples from d 2 through 6, migrated at M(r) = 19 kDa, as did the TIMP-2 standard. A third band was detected in all samples; it migrated at M(r) = 35 kDa, as did the cartilage-derived inhibitor (CDI) standard, and was less dense in d 8 and d 12 through 16 samples. In summary, MMP (gelatinases) and MMP inhibitors are present in developing luteal tissue, and the M(r) = 98 kDa enzyme, CDI, and TIMP-2 varied during the estrous cycle.     

Expression of progesterone receptor isoforms in corpora lutea of human subjects: correlation with serum oestrogen and progesterone concentrations

To understand the biological significance of progesterone receptor forms A (PR-A) and B (PR-B) in human corpus luteum, the expression of mRNA and serum steroid hormone concentrations were determined simultaneously in the luteal stages. The expression of PR-A mRNA predominated over PR-B mRNA in all samples analysed. Total PR (PR-AB) and PR-B mRNA concentrations at the late secretory phase were significantly (P < 0.01) lower than those at the early and mid secretory phases of the menstrual cycle. The ratio of PR-B to PR-AB mRNA concentration showed no significant change during the secretory phase. In the early and mid secretory phases, there was a negative correlation between PR-B mRNA concentration and serum progesterone concentration, and between the ratio of PR-B to PR-AB mRNA concentrations and serum progesterone concentration (P < 0.01). These findings suggest that human corpus luteum might intracellularly synthesize PR-A and PR-B, and thus be involved in the steroid functional regulation of the corpus luteum, especially at the early and mid secretory phases, and that progesterone might regulate the synthesis of PR-A and PR-B.     

Tyrosine kinase inhibitors suppress prostaglandin F2alpha-induced phosphoinositide hydrolysis, Ca2+ elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.