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In vivo intra-luteal implants of prostaglandin (PG) E-1 or E-2 (PGE(1), PGE(2)) prevent luteolysis in cows. I. Luteal weight, circulating progesterone, mRNA for luteal luteinizing hormone (LH) receptor, and occupied and unoccupied luteal receptors for LH
Abstract
Previously, it was reported that chronic intra-uterine infusion of PGE1 or PGE2 every four hours inhibited luteolysis in ewes. However, estradiol-17β or PGE2 given intra-uterine every 8 h did not inhibit luteolysis in heifers, but infusion of estradiol + PGE2 inhibited luteolysis in heifers. The objective of this experiment was to determine whether and how intra-luteal implants containing PGE1 or PGE2 prevent luteolysis in Angus or Brahman cows. On day-13 post-estrus, Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE1, or PGE2 and corpora lutea were retrieved on day-19. Coccygeal blood was collected daily for analysis for progesterone. Breed did not influence the effect of PGE1 or PGE2 on luteal mRNA for LH receptors or unoccupied or occupied luteal LH receptors did not differ (P > 0.05) so the data were pooled. Luteal weights of Vehicle-treated Angus or Brahman cows from days-13–19 were lower (P < 0.05) than those treated with intra-luteal implants containing PGE1 or PGE2. Day-13 Angus luteal weights were heavier (P < 0.05) than Vehicle-treated Angus cows on day-19 and luteal weights of day-13 corpora lutea were similar (P > 0.05) to Angus cows on day-19 treated with intra-luteal implants containing PGE1 or PGE2.
... Other pregnancy-associated factors, such as PGE 2 , could induce processes that are important for maintenance of CL in pregnancy (Lee, et al. 2012a, Weems, et al. 2011, Weems, et al. 2012. PGE 2 has long been suggested as a potent luteotrophic factor that can induce P 4 production (Miyamoto, et al. 1993, Shelton, et al. 1990), furthermore, intra-luteal implants containing PGE 2 were shown to maintained luteal weights and circulating P 4 on days 14-19 in cows (Weems, Fig. 4. Simplified model describing three potential pathways involved in CL maintenance by interferon tau (IFNT). ...
... For the list of references supporting these actions, please refer to the text. et al. 2011, Weems, et al. 2012. Arosh et al. reported major changes in PGE 2 and PGF2α synthesis and in their receptors induced by intrauterine IFNT infusion in the endometrium, myometrium, and CL of cattle (Arosh, et al. 2004b). ...
... Reviewing the findings published thus far support the notion that three potential pathways (Fig. 4) may be involved in luteal maintenance by IFNT: First, the classical mechanism whereby IFNT changes uterine gene expression, resulting in reduced pulses of PGF2α and thus prevents luteolysis (Dorniak, et al. 2013, Gifford, et al. 2007, Knickerbocker, et al. 1986, Spencer, et al. 2007, Thatcher, et al. 2001. Second, IFNT decreases uterine PTGFS; INFT also increases the transported of PGE 2 from the uterus to the ovary through the UOP, thus inducing luteal PGE 2 , which maintains luteal function (Arosh, et al. 2004b, Asselin, et al. 1997, Lee, et al. 2012a, Wasielak, et al. 2009, Weems, et al. 2011, Weems, et al. 2012. Third, recent evidence indicates that the endocrine mode of IFNT action, by interacting directly with the CL, protects the gland from undergoing luteolysis by a mechanism involving ISGs and survival genes (Antoniazzi, et al. 2013, Bott, et al. 2010, Gifford, et al. 2007, Hansen, et al. 2010, Oliveira, et al. 2008. ...
... In addition, a single im injection of PGE 1 increased progesterone for the duration of the 72-hour sampling period in cows [39]. Preventing a loss of luteal LH unoccupied and occupied receptors is the common mechanism for PGE 1 or PGE 2 to prevent spontaneous or induced luteolysis [1,17,18,36,37]. The common mechanism whereby PGE 1 or PGE 2 inhibits spontaneous or induced premature luteolysis is similar, namely by preventing a loss of luteal mRNA for LH receptors and luteal unoccupied and occupied LH receptors in ewes [36] and cows [37]. ...
... Preventing a loss of luteal LH unoccupied and occupied receptors is the common mechanism for PGE 1 or PGE 2 to prevent spontaneous or induced luteolysis [1,17,18,36,37]. The common mechanism whereby PGE 1 or PGE 2 inhibits spontaneous or induced premature luteolysis is similar, namely by preventing a loss of luteal mRNA for LH receptors and luteal unoccupied and occupied LH receptors in ewes [36] and cows [37]. In ewes given progesterone early in the estrous cycle, the levels of luteal unoccupied and occupied receptors for LH were also greater in PGE 1 or PGE 1 þ progesterone-treated ewes than in any other treatment group. ...
... In ewes given progesterone early in the estrous cycle, the levels of luteal unoccupied and occupied receptors for LH were also greater in PGE 1 or PGE 1 þ progesterone-treated ewes than in any other treatment group. Similar effects of both PGE 1 or PGE 2 preventing luteolysis were also reported in Angus and Brahman cows with silastic implants in the CL containing PGE 1 or PGE 2 on Days 13 to 19, which prevented luteolysis by preventing the loss of luteal mRNA for LH receptors and luteal unoccupied and occupied receptors for LH [37]. Thus, the common antiluteolytic mechanism of PGE 1 or PGE 2 is to prevent loss of luteal mRNA for LH receptors and luteal LH unoccupied and occupied receptors in ewes [36] and cows [37]. ...
The objective of this study was to determine whether prostaglandin E1 (PGE1) or prostaglandin E2 (PGE2) prevents premature luteolysis in ewes when progesterone is given during the first 6 days of the estrous cycle. Progesterone (3 mg in oil, im) given twice daily from Days 1 to 6 (estrus = Day 0) in ewes decreased (P < 0.05) luteal weights on Day 10 postestrus. Plasma progesterone concentrations differed (P < 0.05) among the treatment groups; toward the end of the experimental period, concentrations in jugular venous blood decreased (P < 0.05) compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE1 or PGE1 + progesterone were greater (P < 0.05) than in vehicle controls or in ewes receiving PGE2 or PGE2 or PGE2 + progesterone. Chronic intrauterine treatment with PGE1 or PGE2 prevented (P < 0.05) decreases in plasma progesterone concentrations, luteal weights, and the proportion of luteal unoccupied and occupied LH receptors on Day 10 postestrus in ewes given exogenous progesterone, but did not affect (P > 0.05) concentrations of PGF2α in inferior vena cava blood. Progesterone given on Days 1 to 6 in ewes advanced (P < 0.05) increases in PGF2α in inferior vena cava blood. We concluded that PGE1 or PGE2 prevented progesterone-induced premature luteolysis by suppressing loss of luteal LH receptors (both unoccupied and occupied).
... These data indicate differences in PGE 1 and PGE 2 actions to prevent luteolysis in ewes. In addition, intra-luteal implants of PGE 1 or PGE 2 on days 13-19 prevented the decline in circulating progesterone, luteal mRNA for LH receptors, and unoccupied and occupied receptors for LH in Angus or Brahman cows [25]. ...
... Prior to surgery, the flank of cows was clipped, washed with soap and water, scrubbed with Betadine (Purdue Frederick, Stamford, CT), and given Lidocaine (Hospira Inc., Lake Forest, I) was administered subcutaneously into the flank as a local anesthesia. Ten cm 3 of penicillin were given around the incision and intramuscularly (Bimeda Inc., Le Sueur, MN) after surgery [25]. ...
... intra-luteal implant weighs more than 500 mg, circulating progesterone will decline to less than 1 ng/ml within 24 h [25]. Implants with PGE 1 or PGE 2 secreted an average of 5.1 and 4.7 ng/4 h, respectively, in vitro. ...
Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every 4h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in cows, but infusion of estradiol+PGE(2) inhibited luteolysis. In contrast, intra-luteal implants containing PGE(1) or PGE(2) in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE(1) or PGE(2) alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE(1) and PGE(2) up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE(1) and PGE(2) down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE(1) or PGE(2) up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE(1) and PGE(2) support their agonistic effects. We conclude that both PGE(1) and PGE(2) may prevent luteolysis by altering expression of mRNA for prostanoid receptors, which is correlated with changes in luteal mRNA for LH receptors reported previously in these same cows to prevent luteolysis.
... More recently, evidence that IFNT also exits the uterus and can act directly on peripheral tissues, including the CL, has been reported [6,7]. However, there is also evidence from both early research and from recent research that IFNT alters PGE secretion by the endometrium [8][9][10][11], and this may also be a key factor in maintenance of the CL during early pregnancy, by acting as a luteoprotective agent [8,9,12,13]. ...
... Similar to the local effects of PGF 2α during CL regression, the protective effect of the embryo appears to be mediated by local pathways, as evidenced by elegant vascular anastomoses experiments showing that the uterine venous effluent from the gravid uterine horn contains a small molecule that is transferred to the ipsilateral ovarian artery and this blocks the normal luteolytic process [44,45]. A number of reports indicate that this locally active, luteoprotective factor is likely to be PGE [8,9,12,13,46]. During pregnancy, the bovine or ovine uterus produces much greater amounts of PGE 2 than during a similar time period in nonpregnant animals [47,48]. ...
In ruminants, uterine pulses of prostaglandin (PG) F2α characterize luteolysis, while increased PGE2/PGE1 distinguish early pregnancy. This study evaluated intrauterine (IU) infusions of PGF2α and PGE1 pulses on CL function and gene expression. Cows on day 10 of estrous cycle received 4 IU infusions (every 6h; n = 5/treatment) of saline, PGE1 (2 mg PGE1), PGF2α (0.25 mg PGF2α), or PGE1 + PGF2α. A luteal biopsy was collected at 30 min after third infusion for determination of gene expression by RNA-Seq. As expected, IU pulses of PGF2α decreased (P < 0.01) P4 luteal volume. However, there were no differences in circulating P4 or luteal volume between Saline, PGE1, and PGE1 + PGF2α indicating inhibition of PGF2α -induced luteolysis by IU pulses of PGE1. After third pulse of PGF2α, luteal expression of 955 genes were altered (FDR < 0.01), representing both typical and novel luteolytic transcriptomic changes. Surprisingly, after third pulse of PGE1 or PGE1 + PGF2α there were no significant changes in luteal gene expression (FDR > 0.10) compared to Saline cows. Increased circulating PGFM (after PGF2α and PGE1 + PGF2α) and PGEM (after PGE1 and PGE1 + PGF2α) demonstrated that PGF2α and PGE1 are entering bloodstream after IU infusions. Thus, IU pulses of PGF2α and PGE1 allow determination of changes in luteal gene expression that could be relevant to understanding luteolysis and pregnancy. Unexpectedly, by third pulse of PGE1 there is complete blockade of either PGF2α transport to the CL or PGF2α action by PGE1 resulting in complete inhibition of transcriptomic changes following IU PGF2α pulses.
... Much of this resistance to PGF action is ascribed to actions of prostaglandin E2 and E1 (termed PGE in this proposal) coming from the pregnant uterus. Indeed, PGE can block natural or PGF-induced luteolysis either in vivo or in vitro (Michael et al., 1993;Miyamoto et al., 1993;Fortier et al., 2004;Weems et al., 2011). In addition, recent studies showed that endocrine delivery of recombinant ovine IFNT, via uterine or jugular vein, protected the ovine CL from the luteolytic actions of PGF, maintaining intraluteal and circulating P4 and CL volume (Antoniazzi et al., 2013). ...
... First, the classical mechanism is that IFNT changes uterine gene expression resulting in reduced pulses of PGF and thus lack of luteolysis (Thatcher et al., 1984;Knickerbocker et al., 1986;Danet-Desnoyers et al., 1994;Spencer et al., 2007a;Dorniak et al., 2013). Second, IFNT increases uterine production of PGEs (PGE1 and PGE2) and PGE blocks the action of PGF at the CL, maintaining CL function (Ottobre et al., 1984;Silvia et al., 1984a, b;Wiltbank and Ottobre, 2003;Krishnaswamy et al., 2009;Weems et al., 2011Weems et al., , 2012Lee et al., 2012). Third, recent convincing evidence demonstrates that IFNT exits the uterine lumen and interacts directly with the CL and may directly block PGF action at the CL (Gifford et al., 2007;Oliveira et al., 2008;Bott et al., 2010;Hansen et al., 2010;Antoniazzi et al., 2013). ...
In ruminants, there are specific times during the estrous cycle or pregnancy when the corpus luteum (CL) may undergo regression. This review has attempted to summarize the physiological and cellular mechanisms involved in CL regression or maintenance during four distinct periods. The first period is near day 7 when animals that are ovulating after a period of low circulating progesterone (P4), such as first pubertal ovulation or first postpartum ovulation, are at risk of having a premature increase in Prostaglandin F2α (PGF) secreted from the uterus resulting in early CL regression and a short estrous cycle. The second period is when normal luteolysis occurs at day 18-25 of the cycle or when the CL is rescued by interferon-tau secreted by the elongating embryo. The uterine mechanisms that determine the timing of this luteolysis or the prevention of luteolysis have been generally defined. Induction and activation of endometrial E2 receptors result in induction of endometrial oxytocin receptors that can now be activated by normal pulses of oxytocin. Of particular importance is the observation that the primary mechanisms are only activated through local (ipsilateral) and not a systemic route due to transfer of PGF from the uterine vein to the ovarian artery. In addition at the CL level, studies are providing definition to the cellular and molecular mechanisms that are activated in response to uterine PGF pulses or pregnancy. The third period that is discussed occurs in the second month of pregnancy (day 28-60) when undefined mechanisms result in CL maintenance of an ipsilateral CL but regression of a contralateral (opposite side from pregnancy) CL. The final period that is discussed is regression of the CL just prior to parturition. Although, cortisol from the fetus appears to be the primary initiator of luteolysis, PGF seems to be the final signal that causes regression of the CL. Thus, in all four periods, regression of the CL is likely to be caused by the direct actions of PGF that is secreted from the uterus. The uterine mechanisms that result in secretion of PGF seem to be normally inhibited during the early luteal phase, making short luteal phases not a normal event, and are altered during early pregnancy (day 18-25) resulting in prevention of luteolysis. During much of pregnancy, the mechanisms that cause PGF secretion from the uterus in response to oxytocin are intact but luteolysis does not normally occur, perhaps due to lack of efficient utero-ovarian transfer of PGF.
... PGE2 released from the uterus and conceptus are involved in the control of porcine CL lifespan by antagonizing PGF2a in a luteotropic or antiluteolytic manner (Christenson et al., 1994;Waclawik and Ziecik, 2007). In cows, luteoprotective PGE2 induced the mRNA expression of LH receptors and prevented the decrease in unoccupied or occupied luteal receptors for LH (Weems et al., 2011). ...
BACKGROUND
Obesity has now been recognized as a high-risk factor for reproductive health. Although remarkable advancements have been made in ART, a considerable number of infertile obese women still suffer from serial implantation failure, despite the high quality of embryos transferred. Although obesity has long been known to exert various deleterious effects on female fertility, the underlying mechanisms, especially the roles of lipid metabolism in endometrial receptivity, remain largely elusive.
OBJECTIVE AND RATIONALE
This review summarizes current evidence on the impacts of several major lipids and lipid-derived mediators on the embryonic implantation process. Emerging methods for evaluating endometrial receptivity, for example transcriptomic and lipidomic analysis, are also discussed.
SEARCH METHODS
The PubMed and Embase databases were searched using the following keywords: (lipid or fatty acid or prostaglandin or phospholipid or sphingolipid or endocannabinoid or lysophosphatidic acid or cholesterol or progesterone or estrogen or transcriptomic or lipidomic or obesity or dyslipidemia or polycystic ovary syndrome) AND (endometrial receptivity or uterine receptivity or embryo implantation or assisted reproductive technology or in vitro fertilization or embryo transfer). A comprehensive literature search was performed on the roles of lipid-related metabolic pathways in embryo implantation published between January 1970 and March 2022. Only studies with original data and reviews published in English were included in this review. Additional information was obtained from references cited in the articles resulting from the literature search.
OUTCOMES
Recent studies have shown that a fatty acids-related pro-inflammatory response in the embryo-endometrium boundary facilitates pregnancy via mediation of prostaglandin signaling. Phospholipid-derived mediators, for example endocannabinoids, lysophosphatidic acid and sphingosine-1-phosphate, are associated with endometrial receptivity, embryo spacing and decidualization based on evidence from both animal and human studies. Progesterone and estrogen are two cholesterol-derived steroid hormones that synergistically mediate the structural and functional alterations in the uterus ready for blastocyst implantation. Variations in serum cholesterol profiles throughout the menstrual cycle imply a demand for steroidogenesis at the time of window of implantation (WOI). Since 2002, endometrial transcriptomic analysis has been serving as a diagnostic tool for WOI dating. Numerous genes that govern lipid homeostasis have been identified and, based on specific alterations of lipidomic signatures differentially expressed in WOI, lipidomic analysis of endometrial fluid provides a possibility for non-invasive diagnosis of lipids alterations during the WOI.
WIDER IMPLICATIONS
Given that lipid metabolic dysregulation potentially plays a role in infertility, a better understanding of lipid metabolism could have significant clinical implications for the diagnosis and treatment of female reproductive disorders.
... Downregulation of LH/CG receptors in the CL during spontaneous as well as PGF-induced luteolysis has been reported in cattle [73][74][75]. Moreover, a reduced receptor mRNA expression preceded a reduction in luteal LH/CG receptor during PGF-induced luteolysis [76]. ...
By virtue of the secretion of progesterone (P4), corpus luteum (CL) is important not only for normal cyclicity but also for conception and continuation of pregnancy in female mammals. Luteolysis (also called luteal regression) is defined as loss of the capacity to synthesize and secrete P4 followed by the demise of the CL. There is strong evidence that sequential pulses of prostaglandin F2α (PGF) secreted from the uterus near the end of luteal phase induces luteolysis in farm animals. Loss of luteal sensitivity to luteinizing hormone (LH) at the end of menstrual cycle has been reported to be critical for initiation of luteolysis in primates, however this has not been investigated in farm animals. A closer observation of the published real-time profiles of circulating hormones (P4, LH, and PGF) and their inter-relationships around the time of the beginning of spontaneous luteolysis in cattle revealed- 1) A natural pulse of PGF causes a transient P4 suppression lasting a couple of hours followed by a rebound in P4 concentration, 2) The P4 secretions that occur in response to LH pulses before the beginning of luteolysis (i.e., preluteolysis) either fail or do so to a lesser extent during luteolysis indicating a loss of sensitivity to LH, and 3) The loss of sensitivity coincides with the beginning of luteolysis (i.e., transition), and apparently luteolysis does not initiate until there is loss of sensitivity to LH. The CL is sensitive to LH during preluteolysis, and the LH-stimulated P4-dependent and/or independent local survival mechanisms maintain the steroidogenic capability and viability of the CL until the very end of preluteolysis. Luteolysis does not appear to initiate with the PGF pulse(s) that occur during this period. With the loss of sensitivity to LH at the transition, however, a progressive decline in P4 begins initiating luteolysis. Also, the survival mechanisms become compromised making the CL less viable. The uterine PGF pulses that occur after the beginning of luteolysis induces increase in the local luteolytic factors, which contribute to further luteolysis, more importantly, structural luteolysis with ultimate demise of the CL. Therefore, I hypothesize that the loss of luteal sensitivity to LH underlies luteolysis in cattle. The hypothesis not only unifies the basic mechanism of luteolysis in a farm animal and primates but also provides a perspective to view luteolysis as a process rather than a factor-mediated event. A novel unified working model for luteolysis in a farm animal and primates is described. A better understanding of the luteal physiology including how responsiveness to LH diminishes in aging CL would help in the development of novel strategies in modulating CL structure-function to improve and/or control fertility in humans as well as in animals.
... Some eicosanoids are important for the regression of the CL such as PGF 2a [16], which allows for the non-pregnant female to begin another follicular phase of the estrous cycle. Other eicosanoids are important for the maintenance of the CL such as PGE 2 , which prolongs its lifespan and stimulates P 4 , especially around maternal recognition of pregnancy [17]. The insufficient profiling of oxylipins within the CL of cattle, has created a significant gap in knowledge because of the known importance of eicosanoid metabolism during maternal recognition of pregnancy. ...
The objective was to determine the effects of pregnancy status on oxylipin profiles and eicosanoid metabolizing enzymes and in corpora lutea (CL) or endometrial (caruncle; CAR and intercaruncle; IC) tissues. Angus crossed cattle were synchronized with the CO-Synch protocol and artificially inseminated (AI). Sixteen days after AI, cattle were euthanized, and reproductive tracts collected from 6 non-pregnant and 6 pregnant cows. Oxylipin profiles and concentrations of progesterone (P4) were obtained from CL tissues. The activity of cytochrome P450 1A (CYP1A) and UDP-glucuronosyltransferase (UGT) enzymes were determined using specific luminogenic substrates. Data were analyzed using the MIXED procedure of SAS, and the model included pregnancy status. Corpora lutea of pregnant cattle contained greater (P < 0.05) concentrations of 9,10-DiHODE, 15,16-DiHODE, and 9,10-DiHOME. These oxylipins have been observed to increase cellular proliferation and vasodilation. Activity of CYP1A in the CL and UGT in CAR and IC was not different (P > 0.05) between pregnant and non-pregnant cattle. In the CL, activity of UGT was decreased (P < 0.05) in pregnant vs. non-pregnant cattle. The decrease in CL UGT activity during pregnancy indicates alterations in local hormone metabolism, while no differences in CL weight nor amount of P4 in CL were different between pregnant and non-pregnant cattle. Moreover, the increase in specific concentrations of oxylipins in the CL may indicate a novel pathway of steroid and eicosanoid metabolism during maternal recognition of pregnancy.
... A number of reports indicate that the locallyactive, luteoprotective factor is likely to be PGE2 or PGE1 (PGE) secreted from the endometrium in response to pregnancy or IFNT (Huie et al., 1981;Arosh et al., 2004Arosh et al., ,2016Weems et al., 2011). One type of evidence is that during pregnancy, the bovine or ovine uterus produces much greater amounts of PGE than during a similar time period in non-pregnant animals (Danet-Desnoyers et al., 1995;Arnold et al., 2000;Lee et al., 2012). ...
Maintenance of the corpus luteum (CL) during pregnancy is essential for continuing the elevated circulating progesterone (P4) that is required to maintain pregnancy. The mechanisms that protect the CL during early pregnancy when the non-pregnant animal would typically undergo CL regression have been extensively investigated. It is clear uterine prostaglandin F2α (PGF) causes regression of the CL in non-pregnant ruminants and that maintenance of the CL during early pregnancy is dependent upon secretion of interferon-tau (IFNT) from the elongating embryo. A number of specific mechanisms appear to be activated by IFNT. Most studies indicate that there is an inhibition of oxytocin-induced secretion of uterine PGF. There is also evidence for increased resistance to PGF action, perhaps due to secretion of PGE2 and PGE1 or direct endocrine actions of circulating IFNT. These mechanisms occur concurrently and each may help to maintain the CL during the first month of pregnancy. However, during the second month of pregnancy, IFNT is no longer secreted by the embryo. Attachment of the embryo to the uterus and subsequent placentome development have been linked to silencing of expression from the IFNT gene. In addition, there is some evidence that oxytocin responsiveness of the uterus returns during the second month of pregnancy leading to substantial basal secretion of PGF and perhaps PGF pulses. There is also no evidence that the CL during the second month of pregnancy is resistant to the actions of PGF as observed during the first month. Thus, this manuscript attempts to compare the mechanisms that maintain the CL during the first and second months of pregnancy in ruminants and provides a new, speculative, physiological model for maintenance of the CL during month two of pregnancy that is distinct from the previously-described mechanisms that maintain the CL during the first month of pregnancy.
... The luteotro- press.endocrine.org/journal/endo phic role of PGE 2 in the mare is not as well established as in other species, such as human (38), cow (39) or dog (40). Nevertheless, our data are in line with previous reports, because the in vitro treatment of equine CL cells with PGE 2 promoted mRNA transcription of StAR ( Figure 6E). ...
In the present work, we investigated the role of Nodal, an embryonic morphogen from TGFβ-superfamily, in corpus luteum (CL) secretory activity using cells isolated from equine CL as a model. Expression pattern of Nodal and its receptors ACVR2B, Alk7 and Alk4, as well as Nodal physiological role, demonstrate the involvement of this pathway in functional luteolysis. Nodal and its receptors were immune-localized in small and large luteal cells and endothelial cells, except ACVR2B, which was not detected in the endothelium. Nodal mRNA in situ hybridization confirmed its transcription in steroidogenic and endothelial cells. Expression analysis of the aforementioned factors evidenced that Nodal and Alk7 proteins peaked at the Mid-CL (p<0.01), the time of luteolysis initiation, while Alk4 and ACVR2B proteins increased from Mid- to Late CL (p<0.05). Nodal treatment of luteal cells decreased progesterone (P4) and prostaglandin (PG) E2 concentrations in culture media (p<0.05), as well as mRNA and protein of secretory enzymes StAR, CYP11A1, cPGES and mPGES1 (p<0.05). Conversely, PGF2α secretion and gene expression of PTGS2 and PGFS were increased after Nodal treatment (p<0.05). Mid-CL cells cultured with PGF2α had increased Nodal protein expression (p<0.05) and Smad3 phosphorylation (p<0.05). Finally, the supportive interaction between Nodal and PGF2α on luteolysis was shown to its greatest extent because both factors together more significantly inhibited P4 (p<0.05) and promoted PGF2α (p<0.05) synthesis than Nodal or PGF2α alone. Our results neatly pinpoint the sites of action of the Nodal signaling pathway towards functional luteolysis in the mare.
... Progesterone (P 4 ) is the main steroid hormone synthesized by the corpus luteum (CL), and plays a major role in preparing the uterus for pregnancy by promoting and controlling the secretion of uterine proteins and growth factors, the growth rate of embryos and the embryonic secretion of interferon-tau (Spencer & Bazer, 1995;Weems et al., 2011). If the secretion of interferon-tau by the embryo is not delivered appropriately, endometrial prostaglandin F2α is released, inducing luteolysis, which ends in embryo death (Bonnin et al., 1999). ...
Thirty-two ewes were used to study how the ovarian response to a superovulatory treatment determines quality of oocytes recovered from ovaries after embryo collection, and their developmental capacity after in vitro maturation (IVM) and fertilization (IVF). Ewes were superovulated, and seven days after oestrus, embryos were collected and ewes divided into three groups: (+ +), n = 19, ewes responding to the treatment with embryos collected after flushing; (+), n = 8, ewes responding, but only oocytes were found; and (- -), n = 5, ewes not responding to the treatment and no embryos collected. Ovaries were recovered and oocytes collected from the three groups. A significant effect of the response to the treatment was observed for oocyte quality, so that (- -) ewes presented the higher number of oocytes per ewe (p < 0.001). Total number of oocytes selected for IVM and IVF was significantly higher in the same group, in comparison with (+ +) and (+ -) (p < 0.001). Group (+ -) ewes presented the lowest maturation (p < 0.001), fertilization (p < 0.05) and cleavage rates (p < 0.001). In conclusion, the ovarian response to a superovulatory treatment determines the number and quality of the oocytes recovered 7 days after the oestrus induced by the hormonal treatment. In vitro techniques could be an important tool to increase embryo production by particular ewes when they are not able to produce a significant amount of in vivo embryos.
... Progesterone (P 4 ) is the main steroid hormone synthesized by the corpus luteum (CL), and plays a major role in preparing the uterus for pregnancy by promoting and controlling the secretion of uterine proteins and growth factors, the growth rate of embryos and the embryonic secretion of interferon-tau (Spencer & Bazer, 1995;Weems et al., 2011). If the secretion of interferon-tau by the embryo is not delivered appropriately, endometrial prostaglandin F2α is released, inducing luteolysis, which ends in embryo death (Bonnin et al., 1999). ...
The effect of undernutrition on gene expression of progesterone and oestrogen receptors (PGR and ESR1), and insulin-like growth factors 1 and 2 (IGF1 and IGF2) in the uterus and oviducts of ewes on day 5 after oestrus was investigated. The effect of the side of the uterus/oviduct regarding the ovary bearing a corpus luteum (CL) (ipsi vs. contralateral) was also analyzed. Fourteen oestrous synchronized ewes were fed either 1.5 (C, n =7) or 0.5 (L, n = 7) times their maintenance requirements from the onset of the hormonal treatment (day 14), till slaughter on day 5 postoestrus. Oviducts and samples of uterus were collected and their gene expression studied by real time RT-PCR. Undernourished ewes had greater PGR expression in the oviduct than control ewes, but lower expression of IGF1 in uterus and of IGF2 in oviducts. The ipsilateral oviduct presented lower expression of PGR, ESR1 and IGF2 mRNA than the contralateral one, but this did not occur in the uterus. In conclusion, there is an effect of undernutrition on gene expression that is transcript and organ dependent (uterus/oviduct). This work reports for the first time that growth factors and sex steroid receptor expression on day 5 after oestrus vary depending on the side of the CL-bearing ovary and the region of the reproductive tract.
... Their observations of 88 to 90 days ovine corpora lutea of pregnancy led to the conclusion that these corpora lutea produce their own luteotropin which is PGE2. In the cow intraluteal implants containing PGE2 prevented or inhibited luteolysis when implanted on Day 13 post-estrus, with the effect apparently based on the prevention of the loss of luteal LH receptors, as delineated from their expression on the mRNA level [27]. In the dog, the present study has provided further evidence that prostaglandins, and presumably mostly PGE2, seem to act luteotropically in the early phase of diestrus in the dog. ...
The aim of this study was to test for the postulated luteotropic effect of prostaglandin E2 during early diestrus in the dog in an in vivo study. This study was performed on 30 bitches which were randomly assigned to a treatment group (TG) and a control group. Starting on the day of ovulation (Day 0), dogs of the TG were treated for 5, 10, 20, or 30 days with 10 mg firocoxib/kg body weight per day (Previcox, a selective PTGS2 inhibitor) and ovariohysterectomized for collection of corpora lutea on the last day of treatment. Similarly, dogs of the control group were ovariohysterectomized on Days 0, 5, 10, 20, and 30. Blood samples for progesterone measurement were collected every second day; additionally, the area of luteal cell nuclei and the expression of 3β-hydroxysteroid-dehydrogenase at the mRNA and the protein levels were assessed. Mean P4 concentrations were lower in TGs; however, a significant difference was only observed on Day 10. This observation is in line with the finding that treatment with firocoxib reduced expression of 3β-hydroxysteroid-dehydrogenase mRNA and protein (P < 0.05) and the area of luteal cell nuclei (P < 0.05). The results of this study further point to the postulated luteotropic function of prostaglandin E2.
... If this reflects a biological difference and effects on STAR turnover as well as synthesis is not known. In cattle, PGE 2 has been demonstrated to up-regulate expression of LH receptor (Weems et al. 2011). Whether or not the survival of our primary cell culture would enable one to investigate such an effect in vitro merits investigation. ...
The luteal phase in dogs is governed by many, poorly-understood regulatory mechanisms. Functioning of the CL is unaffected by hysterectomy. Recently, the role of prostaglandins in regulating canine CL function was addressed suggesting a luteotropic effect of PGE2 during the early luteal phase. However, compelling functional evidence was lacking. The potential of PGE2 to stimulate steroidogenesis was tested in canine primary luteal cells isolated from developing CL of non-pregnant dogs. In addition, the luteal expression of prostaglandin transporter (PGT) and steroidogenic acute regulatory protein (STAR) was demonstrated and characterized in CL from non-pregnant bitches during the course of dioestrus as well as from pregnant animals during the pre-implantation, post-implantation and mid-gestation periods of pregnancy and during luteolysis; the luteal expression of PGE2-receptors (EP2 and EP4) has been investigated at the protein level throughout pregnancy. Our findings show that PGE2 is an activator of STAR expression in canine luteal cells from early luteal phase, significantly upregulating STAR promoter activity and protein expression resulting in increased steroidogenesis. The 3βHSD- and P450scc-expression remained unaffected by PGE2 treatment. The expression of PGT was confirmed in CL both during pregnancy and dioestrus and generally localized to the luteal cells. After initial upregulation during the earlier stages of the CL-phase, its expression declined towards the luteal regression. Together with the demonstration of EP2 and EP4 throughout pregnancy, and the decline in EP2 at prepartum, our findings further support our hypothesis that intra-luteal PGE2 may play an important role in regulating progesterone secretion in the canine CL.
Reproducția asistată (așa cum este denumită în termenii de specialitate
în limba engleză) a ajuns să reprezinte, atât prin trecerea timpului cât și
datorită minții iscoditoare a omului, o multitudine de tehnici care au drept scop
suprem crearea unor noi vieți. Contribuția omului este covârșitoare și
copleșitoare dacă este să ne amintim că în secolul trecut au fost puse bazele
însămânțărilor artificiale, ale embriotransferului, IVF, ICSI, clonării. În trecutul
recent, pe baza principiilor elaborate pentru aceste tehnici în conjugare cu
tehnicile de biologie moleculară au fost posibile progrese inimaginabile precum
generarea celulelor stem pluripotente induse (IPSC) sau mult mai recenta
descriere a tehnicii CRISPR-Cas9, recompensată cu Premiul Nobel în anul
2020 în domeniul chimiei, tehnică ce permite „editarea genetică”, pusă la punct
în cele mai mici detalii de către Jennifer Doudna și Emmanuelle Charpentier.
În această lucrare am inventariat tehnicile mai sus amintite în ordinea
creșterii complexității lor fără a ne manifesta un partizanat fățiș pentru vreuna
dintre acestea. Informațiile sunt la un nivel superior ceea ce poate îngreuna în
cazul unor capitole lectura acestora. În ideea înlesnirii receptării informației am
considerat util să preluăm din anumite lucrări scheme sau poze, cu
menționarea sursei de proveniență, iar la altele să ne aducem contribuția
proprie pentru o și mai bună descriere a detaliilor. Suntem încrezători că tot
ceea ce a fost cuprins în această lucrare va fi util cititorilor (mai mult sau mai
puțin avizați) constituindu-se pentru toți în mănunchiuri de informație care să
poată sta la baza altor demersuri de natură științifică și practică. În mod firesc,
toți cei care găsesc elemente mai puțin reprezentate sau explicate,
raționamente oprite pe parcursul lor sau împărtășesc alte opinii sunt invitați să
ne convoace la dialog pentru ca toți cei interesați să avem de câștigat.
In previous work, an EP2 prostanoid receptor (EP2R) agonist in vivo increased mRNA expression of luteal LH receptors (LHR), unoccupied and occupied luteal; LHR, and circulating progesterone, while an EP3R or FPR agonist decreased; mRNA expression of luteal LHR, unoccupied and occupied luteal LHR, and; circulating progesterone. An EP4R and lysophosphatidic acid (LPA) LPA2R and LPA3R agonists were reported to inhibit luteal function and sirtuins have been proposed to increase prostaglandin synthesis. The objectives were to determine; whether an EP4R, LPA2R, or LPA3R agonist affect ovine luteal function in vivo or; in vitro. In addition, whether sirtuin (SIRT)-1, 2, or 3; LPA2R or LPA3R; or EP1R, EP2R, EP3R, or EP4R agonists affect caruncular endometrial PGF2α or PGE (PGE1+PGE2) secretion in vitro. Day-10 nonpregnant ewes received a single injection of Vehicle (N = 5); an LPA2R (N = 5); LPA3R (N = 6); or EP4R (N = 5) agonist given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary to determine effects on circulating progesterone, mRNA expression of luteal LHR, and luteal unoccupied and occupied LHR. In addition, agonists for LPA2R, LPA3R, EP1R, EP2R, EP3R, or EP4R or SIRT-1, SIRT-2, or SIRT-3 activators were incubated with caruncular endometrial slices in vitro to determine their effect on carunclar endometrial PGF2α, or PGE secretion. LPA2R, LPA3R, or an EP4R agonist in vivo did not affect (P ≥ 0.05) luteal weight, circulating progesterone, or occupied luteal LHR. However, an LPA2R or EP4R agonist, but; not LPA3R agonist, in vivo increased (P ≤ 0.05) mRNA expression of luteal LHR. An; LPA2R, LPA3R, or EP4R agonist increased (P ≤ 0.05) luteal unoccupied LHR, but; not occupied LHR. An LPA2R, LPA3R, or an EP4R agonist did not affect (P ≥ 0.05); luteal progesterone secretion in vitro. An LPA2R or LPA3R agonist did not affect (P ≥ 0.05) luteal PGF2α, or PGE secretion in vitro. However, an EP4R agonist tended to decrease (P < 0.066) luteal PGF2α secretion and increased (P ≤ 0.05) luteal PGE; secretion in vitro. EP1R, EP2R, EP3R, or an EP4R agonist did not affect (P ≥ 0.05); caruncular endometrial PGF2α secretion in vitro. However, EP1R, EP3R, or an EP4R agonist increased caruncular endometrial PGE secretion in vitro, while two different EP2R agonists did not affect (P ≥ 0.05) caruncular endometrial PGE; secretion. A SIRT-1 activator, but not SIRT-2 or SIRT-3 activators, increased (P ≤ 0.05) caruncular endometrial PGE secretion, while sirtuin 1, 2, or 3 activators did not affect (P ≥ 0.05) caruncular endometrial PGF2α secretion. In conclusion, receptors for EP4, LPA2, and LPA3 do not appear to be involved; in luteolysis, but EP4R and LPA2R might participate in preventing luteolysis by maintaining luteal mRNA expression for LHR and preventing loss of unoccupied luteal LHR. In addition, SIRT-1, EP1R, EP3R, and EP4R might be involved in; regulating caruncular endometrial PGE secretion, but not PGF2α secretion.
Cyclic phosphatidic acids (cPAs) are naturally occurring, very active signaling molecules, which are involved in several pathological states, such as cancer, diabetes, or obesity. As molecules of highly lipidic character found in the circulatory system, cPAs are bound and transported by the main extracellular lipid binding protein - serum albumin.
Here, we present the detailed interactions between human serum albumin (HSA) and equine serum albumin (ESA) with a derivative of cPA, 1-O-myristoyl-sn-glycerol-2,3-cyclic phosphorodithioate (Myr-2S-cPA). Initial selection of the ligand used for the structural study was made by the analysis of the therapeutically promising properties of the sulfur containing analogues of cPA in respect to the unmodified lysophospholipids. Substitution of one or two non-bridging oxygen atoms in the phosphate group with one or two sulfur atoms increases the cytotoxic effect of cPAs up to 60% on the human prostate cancer cells. Myr-2S-cPA reduces cancer cell viability in a dose-dependent manner, with IC50 value of 29.0 μM after 24h incubation, which is almost 30% lower than IC50 of single substituted phosphorothioate cPA.
Although, the structural homology between HSA and ESA is big, their crystal complexes with Myr-2S-cPA demonstrate significantly different mode of binding of this lysophospholipid analogue. HSA binds three molecules of Myr-2S-cPA, while ESA only one. Moreover, none of the identified Myr-2S-cPA binding sites overlap in both albumins.
Reproduction in the ewe is dominated by two distinct rhythms: a 16-17-day estrous cycle and an annual cycle of fertility. Events of the estrous cycle are controlled by the interactions of several hormones (gonadotropin-releasing hormone from the hypothalamus; luteinizing hormone and follicle-stimulating hormone from the pituitary; estradiol, inhibin, and progesterone from the ovary; and prostaglandin F2α from the uterus), while seasonal reproductive patterns are determined by melatonin from the pineal gland. In this chapter, we will first describe the behavioral and hormonal patterns during the cycle, and then consider the mechanisms controlling estrous behavior and the secretion of each hormone. We will next synthesize these components to provide an integrated model for the ovarian cycle. Finally, we will discuss the mechanisms underlying the annual breeding and anestrous seasons, and how pheromones and social cues act in anestrus to re-initiate fertility. Throughout, we will emphasize new data obtained in the last decade, while integrating this information with established concepts described in the previous version of this chapter.
Previously, it was reported that intraluteal implants containing PGE1 or PGE2 in Angus or Brahman cows prevented luteolysis by preventing loss of mRNA expression for luteal LH receptors and luteal unoccupied and occupied LH receptors. In addition, intraluteal implants containing PGE1 or PGE2 upregulated mRNA expression for FP prostanoid receptors and downregulated mRNA expression for EP2 and EP4 prostanoid receptors. Luteal weight during the estrous cycle of Brahman cows were reported to be lighter than in Angus cows, but not during pregnancy. The objective of this experiment was to determine whether intraluteal implants containing PGE1 or PGE2 alter VEGF, FGF2, ANG-1, and ANG-2 protein in Brahman or Angus cows. On Day-13 of the estrous cycle Angus cows received no intraluteal implant and corpora lutea were retrieved or Angus and Brahman cows received intraluteal silastic implants containing Vehicle, PGE1, or PGE2 on Day-13 and corpora lutea were retrieved on Day-19. Corpora lutea slices were analyzed for VEGF, FGF-2, ANG-1, and ANG-2 angiogenic proteins via westernblot. Day-13 Angus cow luteal tissue served as preluteolytic controls. Data for VEGF were not affected (P>0.05) by day, breed or treatment. PGE1 or PGE2 increased (P<0.05) FGF-2 in luteal tissue of Angus cows compared to Day-13 and Day-19 Angus controls, but decreased (P<0.05) FGF-2 in luteal tissue of Brahman cows when compared to Day-13 or Day-19 Angus controls. There was no effect (P>0.05) of PGE1 or PGE2 on ANG-1 in Angus luteal tissue when compared to day-13 or Day-19 controls, but ANG-1 was decreased (P<0.05) by PGE1 or PGE2 in Brahman cows when compared to Day-19 Brahman controls. ANG-2 was increased (P<0.05) on Day-19 in Angus Vehicle controls when compared to Day-13 Angus controls, which was prevented (P<0.05) by PGE1, but not by PGE2 in Angus cows. There was no effect (P>0.05) of PGE1 or PGE2 on ANG-2 in Brahman cows. PGE1 or PGE2 may alter cow luteal FGF-2, ANG-1, or ANG-2, but not VEGF to prevent luteolysis; however, species or breed differences may exist.
The objective was to determine whether PGE1 or PGE2 prevents a premature luteolysis when oxytocin is given on Days 1-6 of the ovine estrous cycle. Oxytocin given into the jugular vein every 8 h on Days 1-6 postestrus in ewes decreased (P<0.05) luteal weights on Day-8 postestrus. Plasma progesterone differed (P<0.05) amongst the treatment groups; towards the end of the experimental period concentrations of circulating progesterone in the oxytocin only treatment group decreased (P<0.05) when compared to the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE1 or PGE1+oxytocin were greater (P<0.05) than in Vehicle controls or in ewes receiving PGE2 or PGE2+oxytocin and was greater (P<0.05) in all treatment groups receiving PGE1 or PGE2 than in ewes treated only with oxytocin. Chronic intrauterine treatment with PGE1 or PGE2 also prevented (P<0.05) oxytocin decreases in luteal unoccupied and occupied LH receptors on Day-8 postestrus. Oxytocin given alone on Days 1-6 postestrus in ewes advanced (P<0.05) increases in PGF2α in inferior vena cava or uterine venous blood. PGE1 or PGE2 given alone did not affect (P>0.05) concentrations of PGF2α in inferior vena cava and uterine venous blood when compared to Vehicle controls or oxytocin–induced PGF2α increases (P<0.05) in inferior vena cava or uterine venous blood. We concluded that PGE1 or PGE2 prevented oxytocin-induced premature luteolysis by preventing a loss of luteal unoccupied and occupied LH receptors.
The bovine corpus luteum (CL) is hypothesized to utilize a local auto-amplification system for prostaglandin (PG) F2α production. The objective of the present study was to determine if such a PGF2α auto-amplification system exists in the bovine CL, and if so, which factors regulate it. PGF2α significantly stimulated intra-luteal PGF2α production in all luteal phases, but did not affect PGE2 production. The stimulatory effect of exogenous PGF2α on CL PGF2α production was lower at the early luteal phase. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthase (PTGS), significantly suppressed the PGF2α-stimulated PGF2α production by luteal tissue, indicating that the PGF2α in the medium was of luteal origin. Consistent with these secreted-PGF2α profiles, PGF2α receptor (PTGFR) protein expression was higher during the mid and late luteal phases than at early and developing luteal phases. Treatment of cultured bovine luteal cells obtained from the mid luteal phase with PGF2α (1 µM) significantly increased the expressions of PTGS2, PGF synthase (PGFS), and carbonyl reductase 1 (CBR1) at 24 h post-treatment. Together, these results suggest the presence of a local auto-amplification system for PGF2α mediated by PTGS2, PGFS, and CBR1 in the bovine CL, which may play an important role in luteolysis. Mol. Reprod. Dev. © 2014 Wiley Periodicals, Inc.
Functional differences between the corpus luteum (CL) of pregnancy and CL of the cycle in cows were examined. Messenger RNA and protein levels of prostaglandin (PG) E synthase (PGES), PGF2α receptor (PGFR), tumor necrosis factor-α (TNF) and Fas were found to be higher in the CL of pregnancy than in CL of the cycle. Oxytocin (OT) mRNA and protein levels were lower in the CL of pregnancy. Messenger RNA levels of progesterone receptor (PR), luteinizing hormone receptor (LHR), PGE2 receptor (PGER), PGF synthase (PGFS), TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) did not differ between the cycle and pregnancy. PGE2 and PGF2α production by cultured bovine endometrial tissues was decreased by a supernatant derived from the homogenized CL of pregnancy but not by that of the CL of the cycle, suggesting that specific substances in the CL of pregnancy affect endometrial PG production in cows. Collectively, PGES, PGFR, TNF, Fas or OT may contribute to differences between the CL of pregnancy and CL of the estrous cycle in cows.
Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1–6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful
in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1−/− mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in
Lpar1−/− mice but not in Lpar2−/− and Lpar3−/− animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific
transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type
osteoclasts treated with Ki16425, mature Lpar1−/− osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally,
LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely,
systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy
revealed that Debio0719 reduced the retention of CX3CR1-EGFP+ osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that
LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss.
Lysophospholipids are bioactive molecules that are implicated in the control of fundamental biological processes such as proliferation, differentiation, survival and motility in different cell types. Here we review the role of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in the regulation of skeletal muscle biology. Indeed, a wealth of experimental data indicate that these molecules are crucial players in the skeletal muscle regeneration process, acting by controllers of activation, proliferation and differentiation not only of muscle-resident satellite cells but also of mesenchymal progenitors that originate outside the skeletal muscle. Moreover, S1P and LPA are clearly involved in the regulation of skeletal muscle metabolism, muscle adaptation to different physiological needs and resistance to muscle fatigue. Notably, studies accomplished so far, have highlighted the complexity of S1P and LPA signaling in skeletal muscle cells that appears to be further complicated by their close dependence on functional cross-talks with growth factors, hormones and cytokines. Our increasing understanding of bioactive lipid signaling can individuate novel molecular targets aimed at enhancing skeletal muscle regeneration and reducing the fibrotic process that impairs full functional recovery of the tissue during aging, after a trauma or skeletal muscle diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
LPA and LPA(1) have been shown to increase osteoblastic proliferation and differentiation as well as activation of osteoclasts. Cell and animal model studies have suggested that LPA is produced by bone cells and bone tissues. We obtained data from invalidated mice which support the hypothesis that LPA(1) is involved in bone development by promoting osteogenesis. LPA(1)-invalidated mice demonstrate growth and sternal and costal abnormalities, which highlights the specific roles of LPA(1) during bone development. Microcomputed tomography and histological analysis demonstrate osteoporosis in the trabecular and cortical bone of LPA(1)-invalidated mice. Moreover, bone marrow mesenchymal progenitors from these mice displayed decreased osteoblastic differentiation. Infrared analysis did not indicate osteomalacia in the bone tissue of LPA(1)-invalidated mice. LPA(1) displays opposite effects to LPA(4) on the related G proteins G(i) and G(s), responsible for decrease and increase of the cAMP level respectively, which itself is essential to the control of osteoblastic differentiation. The opposite effects of LPA(1) and LPA(4) during osteoblastic differentiation support the possibility that new pharmacological agents derived from the LPA pathways could be found and used in clinical practice to positively influence bone formation and treat osteoporosis. The paracrine effect of LPA is potentially modulated by its concentration in bone tissues, which may result from various intracellular and extracellular pathways. The relevance of LPA(1) in bone remodeling, as a receptor able to influence both osteoblast and osteoclast activity, still deserves further clarification. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Bone is a common metastatic site for solid cancers. Bone homeostasis is tightly regulated by intimate cross-talks between osteoblast (bone forming cells) and osteoclasts (bone resorbing cells). Once in the bone microenvironment, metastatic cells do not alter bone directly but instead perturb the physiological balance of the bone remodeling process controlled by bone cells. Tumor cells produce growth factors and cytokines stimulating either osteoclast activity leading to osteolytic lesions or osteoblast function resulting in osteoblastic metastases. Growth factors, released from the resorbed bone matrix or throughout osteoblastic bone formation, sustain tumor growth. Therefore, bone metastases are the sites of vicious cycles wherein tumor growth and bone metabolism sustain each other. Lysophosphatidic acid (LPA) promotes the growth of primary tumors and metastatic dissemination of cancer cells. We have shown that by acting on cancer cells via the contribution of blood platelets and the LPA-producing enzyme Autotaxin (ATX), LPA promotes the progression of osteolytic bone metastases in animal models. In the light of recent reports it would appear that the role of LPA in the context of bone metastases is complex involving multiple sources of lipid combined with direct and indirect effects on target cells. This review will present our current knowledge on the LPA/ATX axis involvement in osteolytic and osteoblastic skeletal metastases and will discuss the potential activity of LPA upstream and downstream metastasis seeding of cancer cells to bone as well as its implication in cancer induced bone pain. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
The simplest signalling lipid Lysophosphatidic acid (LPA) elicits pleiotropic actions upon most mammalian cell types. Although LPA has an established role in many biological processes, particularly wound healing and cancer, the function of LPA for human osteoblast (hOB) biology is still unravelling. Early studies, identified in this review, gave a reliable indication that LPA, via binding to one of several transmembrane receptors, stimulated multiple intracellular signalling networks coupled to changes in cell growth, fibronectin binding, maturation and survival. The majority of studies exploring the actions of LPA on hOB responses have done so using the lipid in isolation. Our own research has focussed on the co-operation of LPA with the active vitamin D3 metabolite, 1α25,dihydroxycholecalciferol (calcitriol), in light of a serendipitous discovery that calcitriol, in a serum-free culture setting, was unable to promote hOB maturation. We subsequently learnt that the serum-borne factor co-operating with calcitriol to enhance hOB differentiation was LPA bound to the albumin fraction of whole serum. Recent studies from our laboratory have identified that LPA and calcitriol are a potent pairing for securing hOB formation from their stem cell progeny. Greater understanding of the ability of LPA to influence, for example, hOB growth, maturation and survival could be advantageous in developing novel strategies aimed at improving skeletal tissue repair and regeneration. Herein this review provides an insight into the diversity of studies exploring the actions of a small lipid on a major cell type key to bone tissue health and homeostasis. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Lysophosphatidic acid (LPA) is the simplest signalling lipid eliciting pleiotropic actions upon most mammalian cell types. Although LPA has an established role in many biological processes, particularly wound healing and cancer, the participation of LPA in skeletal biology is just beginning to emerge. Early studies, identified in this review, gave a solid indication that LPA, via binding to one of several cell surface receptors, activated multiple intracellular systems culminating in altered cell morphology, growth, motility and survival. More recently the ablation of murine LPA1 and 4 receptors implies that this lipid has a role in skeletal development and post natal bone accrual. Greater understanding of the ability of LPA to influence, for example, osteoblast growth, maturation and survival could be advantageous in developing novel strategies aimed at improving skeletal tissue repair and regeneration. Herein this review provides an insight into the diversity of studies exploring the actions of a small lipid on those major cell types key to skeletal tissue health and homeostasis.
A number of morphological and biochemical changes occur as the cells of the recently ovulated follicle luteinize and develop into a functional CL. There are two distinct steroidogenic luteal cell types that appear to differentiate from thecal and granulosal cells in the follicle. The control of progesterone secretion is quite different in the two cell types. Prostaglandin F2 alpha (PGF2 alpha) is the primary luteolytic hormone in most mammals. PGF2 alpha appears to exert its antisteroidogenic actions via activation of the protein kinase C system, while its cytotoxic effects appear to be mediated via a dramatic increase in intracellular levels of free calcium. The mechanisms involved in maternal recognition of pregnancy are very diverse between species and may involve direct luteotropic stimulation of the CL, reduced uterine secretion of PGF2 alpha, and/or inhibition of actions of PGF2 alpha at the level of the CL.
Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection
and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative
expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene.
Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and
the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the
calculation of relative expression ratio allow only for the determination of a single transcription difference between one
control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which
compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target
genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample
and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance
by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression
in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded
at http://www.wzw.tum.de/gene-quantification/.
Cholesterol provided by low- or high-density lipoprotein is the precursor for biosynthesis of progesterone. Once inside the cell, cholesterol can be used for steroidogenesis or esterified with long-chain fatty acids and stored as cholesterol esters in lipid droplets. When it is needed for steroidogenesis, free cholesterol is transported to the mitochondrion via a mechanism that involves cytoskeletal elements and sterol carrier proteins. Cytochrome P450 cholesterol side chain cleavage enzyme complex converts the cholesterol to pregnenolone, which is then converted to progesterone by 3beta-hydroxysteroid dehydrogenase/Delta(5),Delta(4) isomerase in the smooth endoplasmic reticulum. Transport of cholesterol from the cytoplasm to the inner mitochondrial membrane is both the rate-limiting step in progesterone biosynthesis and the step most acutely influenced by second messengers. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptors (PBR) are involved in this transport. StAR may bind cholesterol in the cytosol and transport it to the mitochondrial membrane where PBR is involved in transport from the outer to the inner mitochondrial membrane. Phosphorylation of StAR by protein kinase A (PKA) stimulates cholesterol transport, whereas phosphorylation by PKC may inhibit this process. Endozepine, the natural ligand for PBR, also appears to be involved in regulation of the rate of cholesterol transport to the inner mitochondrial membrane and to play a role in the stimulatory effects of PKA on steroidogenesis. Increased concentrations of endozepine were detected in large luteal cells, and may explain the increased progesterone secretion from this type of cell. Fluorescence energy transfer procedures indicate that StAR associates with PBR in mitochondrial membranes. A model is presented for the proposed interactions of StAR, PBR and endozepine in the transport of cholesterol from the outer to the inner mitochondrial membrane.
The second messengers mediating hormonal regulation of the corpus luteum are incompletely defined, particularly for the primary luteolytic hormone prostaglandin F2 alpha (PGF2 alpha). In this study, hormonally induced changes in free intracellular calcium concentrations were measured in individual small and large ovine luteal cells by using computer-assisted microscopic imaging of fura-2 fluorescence. This technique could readily detect transient increases in free calcium concentrations within both small and large luteal cells after treatment with 1 microM of the calcium ionophore, A23187. Treatment with PGF2 alpha (1 microM) caused a dramatic increase in free calcium concentrations in large (before = 73 +/- 2 nM; 2 min after PGF2 alpha = 370 +/- 21 nM; n = 33 cells) but not in small (before = 66 +/- 4 nM; 2 min after PGF2 alpha = 69 +/- 8 nM; n = 12 cells) luteal cells. The magnitude and timing of the calcium response was dose- and time-dependent. The PGF2 alpha-induced increase in free intracellular calcium is probably due to influx of extracellular calcium, since inclusion of inorganic calcium channel blockers (100 microM manganese or cobalt) attenuated the response to PGF2 alpha and removal of extracellular calcium eliminated the response. In contrast to PGF2 alpha, luteinizing hormone (LH) (100 ng/ml) caused no change in intracellular levels of free calcium in small or large luteal cells, even though this dose of LH stimulated (p less than 0.01) progesterone production by small luteal cells. Therefore, alterations in free calcium concentrations could be the intracellular second message mediating the luteolytic action of PGF2 alpha in the large ovine luteal cell.
The effect of pregnancy on concentrations of prostaglandins E2, F2 alpha and 6-keto-prostaglandin F1 alpha (PGE2, PGF2 alpha and 6-keto-PGF1 alpha) in utero-ovarian venous plasma was examined in ewes on Days 10 through 14 after estrus, an interval which includes the critical period for maternal recognition of pregnancy. The utero-ovarian vein ipsilateral to a corpus luteum was catheterized on Day 9 or 10 in 6 pregnant and 8 nonpregnant ewes. Five blood samples were collected at 30-min intervals for 2 h beginning at 0500 and 1700 h daily. Sampling began at 0500 h on the day after catheterization. The mean and variance within each 2-h collection period were calculated for each ewe. The natural logarithm of the variance in each collection period (ln variance) was used as an estimate of the fluctuations in secretory activity by the endometrial-conceptus complex. Patterns of the mean concentrations of PGE2 were different between pregnant and nonpregnant ewes (P less than 0.01); PGE2 being higher in the pregnant ewes beginning on Day 13. There was a trend for the patterns of ln variance in PGE2 to differ (P less than 0.1) with pregnancy status over the entire period; ln variance was greater in pregnant ewes beginning on Day 13. The patterns of the mean concentrations and ln variances for PGF2 alpha and 6-keto-PGF1 alpha did not differ between pregnant and nonpregnant ewes. There were significant increases in both of these prostaglandins over time, independent of pregnancy status (P less than 0.01). The association of higher concentrations of PGE2 in utero-ovarian venous plasma with early pregnancy is consistent with the hypothesis that PGE2, originating from the uterus and/or conceptus, is one factor involved in maintenance of the corpus luteum of pregnancy.
To determine the temporal expression of vascular growth factors during the lifespan of the primate corpus luteum, experiments were designed to detect mRNA for vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1 and Ang-2 and to localize protein expression for VEGF in macaque luteal tissue during the menstrual cycle. Corpora lutea (n 3–5/stage) were collected during the early (3–5 days post-luteinizing hormone surge), mid- (6–8 days), mid-late (10–12 days), and late (14–16 days) luteal phase and at menstruation (17–18 days). Reverse transcription-polymerase chain reaction products equated to cDNA for VEGF, Ang-1 and Ang-2 in all corpora lutea. VEGF mRNA levels increased (P < 0.05) from early to mid-luteal phase and declined in the late luteal phase and at menstruation. Immunostaining for VEGF was detected in the cytoplasm of steroidogenic luteal cells, with the most intense staining in the early luteal phase. Ang-1 and Ang-2 mRNA expression was low in the early to mid-luteal phase but increased (P < 0.05) at late luteal phase before declining at menstruation. These data suggest transcriptional control of VEGF, Ang-1 and Ang-2, as well as post-transcriptional control of VEGF, in macaque corpus luteum. Dynamic expression of angiogenic/angiostatic factors appears critical for development, maintenance and regression of the luteal microvasculature during the menstrual cycle.
Injection of antiserum to bovine LH into hysterectomized heifers caused significant decreases in corpus luteum weight, total progesterone, total progestins and 20β-hydroxypregn-4-en-3-one (20-β-ol) concentration but had no effect on progesterone concentration. Gonadotropin secreting cells in the anterior pituitaries of these heifers were markedly degranulated and pituitary LH contents lowered. Thyroid epithelial cell heights were lowered suggesting that the antiserum also contained anti-TSH. The administration of antibovine LH to cyclic heifers from Day 2 to Day 6 of the cycle caused a significant decrease in corpus luteum weight and total progesterone content, but only a slight decrease in progesterone concentration. Histological signs of regression were clearly evident in the corpora lutea of the hysterectomized anti-LH treated heifers, but little difference could be noted between the corpora lutea of control and treated cyclic animals.
Injection of 50 ml of antibovine LH daily from Day 2 to Day 6 of the cycle increased the interval between corpus luteum removal at Day 11 and return to estrus. Injections of 50 ml of antibovine LH daily from Day 12 to Day 16 and from Day 16 to Day 20 of the cycle increased the lengths of the treatment cycles to 28.5 and 36.7 days, respectively. The first post-treatment cycle in both groups was significantly shortened. The increase in cycle length was primarily due to a failure of follicles to develop to mature size, rather than to prolongation of the life span of the corpora lutea. The results of these experiments indicated that LH is required for follicular development and maturation and for the induction of estrus, as well as for corpus luteum maintenance.
Data from several studies are presented which suggest that blood flow to the corpus luteum may be important in the regulation of this gland. Blood flow to the luteal ovary increases from less than 1 ml/mm to 3 to 7 ml/min as the corpus luteum develops and is maintained. During regression, blood flow to the luteal ovary declines sharply. Data obtained with radioactive microspheres indicates that the changes in blood flow to the luteal ovary can be attributed to changes in flow to the corpus luteum, which receives the majority of the blood. In addition, the entrapment of 15 µ microspheres was less than that for 50 µ microspheres during the late luteal phase of the cycle suggesting that arteriole-venule shunting of blood occurs. As a result of the decline in total ovarian blood flow and the shunting of blood within the corpus luteum, there appears to be a severe restriction in the quantity of blood available to the luteal cell during regression. Morphological data obtained during this period indicate that there is even a decrease in the relative volume of the capillary network within the corpus luteum.
Treatment of ewes with a combination of CB-154 and anti-prolactin serum did not influence either blood flow to the luteal ovary or systemic levels of progesterone. It was demonstrated in this study that CB-154 treatment was followed by a dramatic decline in serum levels of prolactin. In addition, an excess of antibodies against prolactin was demonstrated in sera collected from treated ewes. It was also demonstrated that the antibodies in serum were capable of binding prolactin. Treatment of ewes during mid-cycle with anti-LH serum resulted in a rapid decline in blood flow to the luteal ovary and in circulating levels of progesterone. Infusion of exogenous LH resulted in a dramatic increase in serum levels of progesterone associated with a less dramatic increase in blood flow to the luteal ovary.
When ewes were administered PGF2α or analogs of PGF2α serum levels of progesterone and blood flow to the luteal ovary declined to basal levels within 6 h. The hemodynamic changes associated with luteal regression in PGF2α-treated ewes were similar to those observed in cycling ewes.
Finally, regional blood flow within the corpus luteum was studied using 15 µ radioactive microspheres. When small pieces of tissue were taken following infusions of HCG-1 2 5 I and 8 5Sr microspheres into the ovarian artery the cpm/mg tissue of the two isotopes were highly correlated. These data suggest that the specific binding of HCG to luteal cells is influenced by the flow of blood to these cells. From these studies it appears that blood flow may be an important factor in regulating the activity of the gonadotropic hormones at the luteal cell level. It further appears that a secondary mechanism of action of LH may be to increase blood flow to the corpus luteum.
To determine if the synthesis and/or secretion of prostaglandins PGF2α and PGE2 differed during the late luteal phase of the cycle and early pregnancy, a combination of in vivo and in vitro parameters were studied. Synthesis of PGF2α and PGE2 by ovine endometrial tissue in vitro was studied on Days 13, 15 and 17 of the estrous cycle and pregnancy. The prostaglandins were quantified by radioimmunoassay. There was no difference in the quantity of PGF2α synthesized by tissue collected on Day 13 of either the cycle or pregnancy. Synthesis of PGF2α was greater on Day 15 (P<0.01) and tended to be greater on Day 17 (P<0.10) of pregnancy than on Day 15 or 17 of the estrous cycle. Synthesis of PGE2 was also significantly greater on Days 15 (P<.001) and 17 (P<0.05) of pregnancy than on the same days of the estrous cycle. Synthesis rates of both PGs in vitro tended to decrease from Days 13-17 in tissue collected from cycling ewes, while increasing from Days 13-15, then falling again on Day 17 of pregnancy. Concentrations of PGF2α and PGE2 in endometrial tissue followed the same pattern as in vitro secretion, suggesting that in vitro secretion accurately reflects the in vivo capacity of endometrial tissue to synthesize PGs. Concentrations of PGF2α in uteroovarian venous serum were greater on Day 13 (P<0.05) and not different on Days 15 and 17 of pregnancy compared with the same days of the estrous cycle. However, concentrations of PGE2 in uteroovarian venous serum were greater on Days 15 (P<0.01) and 17 (P<0.05) of pregnancy than on the same days of the estrous cycle. Prostaglandin F2α was present in uterine flushings in extremely small quantities (0.3-0.6 ng/ml) and PGE2 was nondetectable (<10 pg/ml) on Days 13, 15 and 17 of the estrous cycle. In contrast, concentrations of PGF2α and PGE2 were relatively high on all days of pregnancy examined and on Day 15 were at least 50 times greater than on Day 15 of the estrous cycle.
These data indicate that the capacity of endometrial tissue to secrete PGF2α is increased during early pregnancy. The finding that PGE2 is also secreted at a greater rate during early pregnancy lends support to the hypothesis that PGE2 may be the factor responsible for maintenance of the corpus luteum of early pregnancy.
A procedure which utilized elutriation was developed which permitted the separation of two populations of luteal cells from enzymatically dispersed corpora lutea taken from superovulated ewes on the 10-12th day of the estrous cycle. One population consisted of cells which measured 23-35 pm in diameter, while the other consisted of cells with a diameter of 12-22 pm. Preparations of both cell types were analyzed for rate of secretion of progesterone, responsiveness to secretagogues, and number of specific binding sites for luteinizing hormone/human chorionic gonadotropin (LH/hCG), and prostaglandins E2 and F2a (PGE2 and PGF2a). Without stimulation, the secretion of progesterone by large cells was approximately 20-fold greater than for an equal number of small luteal cells. However, the ability of large luteal cells to respond to LH (100 ng/mI) or N6,02-dibutyryladenosine 3’: 5’-cyclic monophosphoric acid (dbcAMP, 10 mM) was minimal and approximately 10% of that obtained with small luteal cells. Marked differences were also observed between the two cell types with respect to the number of specific binding sites for LH/hCG, PGE2 and PGF2 a. The large cells had very few receptors for LH/hCG compared to small cells (3074 receptors/large cell vs. 33,260/small cell during the breeding season). The majority of receptors for prostaglandins was found in large cells (68,143 sites of PGF2a and 10,955 sites for PGE2/large cells; 2115 sites for PGF2a and 904 sites for PGE2/small cell). These data suggest that functional differences exist between the large and small steroidogenic cells in the corpus luteum of the ewe. The large luteal cells appear to secrete most of the progesterone produced by the corpus luteum, however, this secretion appears to be independent of LH and dbcAMP. In contrast, the small luteal cells secrete minimal quantities of progesterone in the unstimnulated state butresponddramatically to LH or dbcAMP.
Experiment I was designed to determine the optimal dose of phorbol 12-myristate 13-acetate (PMA) that inhibited progesterone production when infused into the ovarian artery. The most efficacious dose of PMA was 2 mol. Experiment II was designed to determine whether activation of protein kinase C (PKC) inhibited progesterone production without initiating luteolysis. Ewes received ovarian arterial infusions of 4a-phorbol (2 tLmol, n = 4), PMA (2 mol, n = 8), or prostaglandin F2 (PGFa; 1 mol, n = 5). Concentrations of progesterone in serum decreased by 3 h in PMA-treated ewes and by 5 h in PGFatreated ewes (p < 0.05). By 48 h, serum levels of progesterone in PMA-treated ewes had returned to control values; but in PGF, 0-treated ewes they remained low for the duration of the experiment. Luteal weights and progesterone contents at 48 h were similar in 4a-phorbol- and PMA-treated ewes but were decreased in PGF2,-treated ewes (p < 0.05). Experiment III was designed to determine whether PGF,, or PKC activation induced oligonucleosome formation or influenced mRNA levels for cytochrome P450,. or 3-hydroxysteroid dehydrogenase/A 5 -A 4 isomerase (3[-HSD). Ewes received treatments as in experiment II, and CL were collected at 3, 12, or 24 h (n = 3-4 per group). Luteal weights were decreased (p < 0.05) and oligonucleosome formation was increased (p < 0.05) in PGF,,-treated ewes compared to controls or to PMA-treated ewes by 12 h. Concentrations of mRNA encoding for cytochrome P450,, were reduced (p < 0.05) at 3 and 12 h after the PMA infusion compared to the value in 4-phorbol-treated controls, but were not different in PGF,2-treated ewes compared to controls. Infusion of PMA or PGF,, decreased concentrations of mRNA encoding 3-HSD at 3 and 12 h (p < 0.05), and these values remained low at 24 h in the PGF,,-treated ewes. In the PMA-treated ewes, levels of mRNA encoding 3-HSD were intermediate between those in control and PGF,.-treated ewes by 24 h. Thus, PKC activation decreased progesterone production without initiating luteolysis. Treatment with PGF,. increased oligonucleosome formation while PKC activation did not, and PGF2, decreased levels of mRNA for 3-HSD, probably through activation of PKC.
High-affinity LH/hCG binding sites have been characterized in porcine, lepine, and murine uteri. In the present study, LH/hCG binding sites were characterized in bovine endometrium. Radioreceptor assays were performed with membrane homogenates of endometrial tissues and analyzed for binding site specificity and capacity. There was little competition for receptor occupancy between hCG and ovine FSH (5%) or ovine prolactin (< 0.1%), but there was a 20% cross-reaction with eCG. There was no affinity for LH/hCG in crude membrane preparations of kidney, skeletal muscle, or vascular tissues. Concentrations of endometrial LH/hCG binding sites were determined during the bovine estrous cycle. LH/hCG receptors were found in cell preparations from Days 2-4 and 15-17 of the cycle, but not in preparations from the other stages of the cycle tested (Days 8-12, pre- and post-estrus, and ovulation). The concentration of uterine LH/hCG receptor varied during the estrous cycle, with higher values at Days 15-17 (3.1 fmol/mg protein) and lower values at Days 2-4 (1.2 fmol/mg protein). However, the binding capacity of hCG by luteal cells (9.7 fmol/mg protein) was 3-fold higher (p < 0.01) than that by endometrial tissue on any day studied. No differences in affinity constant (Ka) were seen between endometrial LH/hCG receptors (either) from Days 2-4 or 15-17) and mid-cycle luteal cells (0.60 x 10(11) M-1). Using Western blot analysis, we determined the expression of cyclooxygenase (COX) during the estrous cycle of the cow. It was found that the signal for COX was strongest at 15-17 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Plasma membrane receptors for prostaglandins (PG) F 2α and E 2 were quantified in ovine corpora lutea obtained from non-pregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF 2α were 7- to 10-fold greater than those for PGE 2 . In pregnant ewes the concentration of receptors for PGF 2α was highest on Day 10 (35.4 ± 2.8 fmol/mg) and lowest on Day 25 (22.3 ± 2.5 fmol/mg)
The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.
Preliminary characterization indicated the presence of separate prostaglandin (PG)E1 and (PG)F2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10−9M and 2.1 × 10−8M for PGE1 and PGF2α, respectively. Competition of several natural prostaglandins for the PGE1 and PGF2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit 3H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE1 resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE1 treatment compared to pretreatment values. In so far as data obtained on PGF2α relative binding affinity to the bovine CL can be compared to data obtained independently on PGF2α induced luteolysis in the bovine, PGF2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE1 relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.
Procedure is given for analysis of data from studies with time-profiles of information, some of which are incomplete, because data are missing randomly across all periods or because some animals have truncated profiles. For investigations with relatively few animals, the procedure is more powerful than alternative methods that utilize multivariate theory or one or more summary functions for the profiles of individual animals (to eliminate the time factor from the analysis of variance). The procedure also permits answering of highly specific questions related to time, which cannot be done directly with alternative methods. For the standard split-plot model for repeated measurements, the procedure relies on estimation of two basic parameters: 1. Variation among animals treated alike (with the effect of correlation removed), and 2. Average inter-period correlation. An example is given, which involves effects of different doses of a drug presumed to be detrimental to rate of growth.
Analyse wiederholter Messungen bei Tieren mit unvollständigen Zeitprofilen
Die Notwendigkeit hierfür entsteht, weil bei einigen Tieren Beobachtungen in allen Perioden entweder zufällig fehlen oder in anderen Fällen die Beobachtungsperioden abgeschnitten sind. Bei Untersuchung mit relativ wenigen Tieren ist die Methode mächtiger als alternative Methoden, welche Multivariable Theorie oder eine oder mehrere Summenfunktionen für die Profile individueller Tiere verwenden (zur Elimination des Zeitfaktors in der Varianzanalyse). Die Methode erlaubt die Beantwortung relativ spezieller Fragen in bezug auf Zeit, die nicht direkt mit alternativen Methoden behandelt werden können. Für die Standard split-plot Modelle wiederholter Messungen beruht sie auf Schätzung zweier Größen:
Es wird ein Beispiel gegeben, welches die Wirkung unterschiedlicher Drogendosen auf Wachstumsverschlechterung widerspiegelt.
Secretion of progesterone in vitro by mature day 8 ovine corpora lutea (CL) of the estrous cycle was increased linearly by ovine LH (1, 10 and 100 ng/ml) or prostaglandin E 2 (PGE 2 ) 1, 10 and 100 ng/ml) in a dose dependent manner (P ≤ 0.05). Progesterone secretion in vitro by 88–90 day ovine CL of pregnancy was not affected P ≥ 0.05 by LH (1, 10 and 100 ng/ml) while prostaglandin E 1 (PGE 1 ) 1, 10 and 100 ng/ml) increased (P ≤ 0.05) secretion of progesterone in a dose dependent manner and PGE 2 (1, 10 and 100 ng/ml) increased (P ≤ 0.05) secretion of progesterone only at the 100 ng/ml dose. Day 8 ovine CL of the estrous cycle did not secrete (P ≥ 0.05) detectable quantities of prostaglandin F 2 α (PGF 2 α) or prostaglandin E (PGE) while 88–90 day ovine CL of pregnancy secrete PGE (P ≤ 0.05) but not PGF 2 α (P ≥ 0.05). Regulation of PGE secretion by 88–90 day ovine CL of pregnancy may be via pregnancy specific protein B (PSPB), which increased (P ≤ 0.05) PGE and progesterone but not PGF 2 α (P ≥ 0.05) secretion. Secretion of progesterone by CL of 88–90 days of pregnancy was not affected by IGF 1 , IGF 2 , PAF-16, PAF-18, oxytocin, PGI 2 , PGD 2 or leukotriene C 4 (P ≥ 0.05). It is concluded that PGE 1 or PGE 2 but not LH regulates secretion of progesterone in vitro by 88–90 day ovine CL of pregnancy. In addition, it is concluded that 88–90 day ovine CL of pregnancy secretes it's own luteotropin, which is PGE. Secretion of PGE by ovine CL of pregnancy may be regulated by PSPB.
One objective of this experiment was to evaluate our hypotheses that estradiol-17β regulates secretion of pregnancy specific protein B (PSPB) and that secretion of progesterone during pregnancy is regulated by a prostanoid by examining the effects of prostaglandin F2α (PGF2α), a luteolyic agent; indomethacin, a prostanoid synthesis inhibitor; tamoxifen, an estrogen receptor antagonist; estradiol 17-β; and interaction of these factors on the incidence of abortion and progesterone and PSPB secretion. Another objective was to determine if there is a luteal source of PSPB. Weights of corpora lutea were decreased (P ≤ 0.05) by PGF2α, indomethacin, PGF2α + tamoxifen, PGF2α + indomethacin, and PGF2α + estradiol-17β but not (P ≥ 0.05) by tamoxifen or estradiol-17β alone. No ewe treated with PGF2α alone aborted (P ≥ 0.05). Forty percent of ewes treated with PGF2α + estradiol-17β aborted (P ≤ 0.05), but ewes were not aborted by any other treatment within the 72-h sampling period. Profiles of progesterone in jugular venous blood differed (P ≤ 0.05) among control, indomethacin-, tamoxifen-, and PGF2α + indomethacin-treated ewes. Progesterone in jugular venous blood of control ewes decreased (P ≤ 0.05) by 24 h, followed by a quadratic increase (P ≤ 0.05) from 24 to 62 h. Progesterone in jugular venous blood of indomethacin-, PGF2α-, PGF2α + tamoxifen-, PGF2α + indomethacin-, PGF2α + estradiol-17β-, and tamoxifen-treated ewes was reduced (P ≤ 0.05) by 18 h and did not vary (P ≥ 0.05) for the remainder of the 72-h sampling period. Progesterone in vena cava and in uterine venous blood was reduced (P ≤ 0.05) at 72 h in PGF2α-, indomethacin-, tamoxifen-, PGF2α + indomethacin-, PGF2α + tamoxifen-, and PGF2α + estradiol-17β-treated ewes. Weights of placentomes did not differ among treatment groups (P ≥ 0.05). Profiles of PSPB in inferior vena cava blood differed (P ≤ 0.05) among control, estradiol-17β-, indomethacin-, tamoxifen-, PGF2α + indomethacin-, and PGF2α + tamoxifen-treated 88- to 90-day pregnant ewes. Concentrations of PSPB in inferior vena cava blood were increased (P ≤ 0.05) in indomethacin-, estradiol-17β-, tamoxifen-, PGF2α + tamoxifen-, and PGF2α + indomethacin-treated 88- to 90-day pregnant ewes within 6 h and did not vary (P ≥ 0.05) for the remainder of the 72-h sampling period. Concentrations of PSPB in uterine venous blood of indomethacin-, tamoxifen-, PGF2α + tamoxifen-, and PGF2α + indomethacin-treated ewes were greater (P ≤ 0.05) at 72 h than at 0 h. PSPB in ovarian venous blood did not differ (P ≥ 0.05) adjacent or opposite to the ovary with the corpus luteum. It is concluded from these data that estrogen regulates placental secretion of PSPB and that a prostanoid, presumably prostaglandin E, regulates placental secretion of progesterone during 88–90 days of gestation in sheep and that there is no luteal source of PSPB.
Holstein and Guernsey bulls of different nonreturn rates in artificial insemination have been compared to determine their fertilization rates and to estimate their embryonic death rates. The 64 bulls for which fertilization tests were made on first service heifers had an average nonreturn rate of 65.1%, a fertilization rate of 85.9%), and an embryonic death date, estimated as the per cent of fertilized ova that died, of 21.0%. When the bulls were divided according to nonreturn rates into a high half (67-79%) and a low half (40-66%), they had fertilization rates of 100% and 71.9% (P < 0.01) and embryonic death estimates of 25.5% and 14.9% for the high and the low fertility bulls, respectively.
The differences were not significant between the fertilization rates of the Holsteins (89.2%) and the Guernseys (81.5%). The nonreturn rates of 65.8% and 64.0% were similar.
It appears from this study that approximately 40% of all potential young in dairy cattle are lost by 60-90 days after breeding. Their loss may be estimated as 3% due to genital abnormalities of the cow, 9% to defective ova, 12% to failure of fertilization resulting from undetermined causes, and 16% to death of the embryo, all calculated as percentages of the potential young producible by a female group.
Evidence that the pattern of secretion of prostaglandin E2 (PGE2) by the uterus: conceptus unit is consistent with that compound playing a role in the local antiluteolytic effect of the embryo is reviewed briefly. Utero-ovarian venous concentrations of PGE2 increased on day 13 in pregnant compared to non-pregnant ewes, both absolutely and in relation to prostaglandin F2α (PGF2α).The roles of oestrogen and progesterone in regulation of utero-ovarian venous concentrations of PGF2α were examined during two intervals of the oestrous cycle and on day 13 of the cycle or pregnancy. The first interval (Experiment l, days 10 to 14) was chosen to study the initial rises of PGF2α that precede luteolysis. At laparotomy on day 9, a catheter was inserted into a utero-ovarian vein. Eight ewes received no further treatment. At surgery, 12 ewes were given 10 mg of progesterone (i.m.) along with an intravaginal pessary containing 30 mg flurogestone acetate and were either ovariectomized (n=5) or lutectomized (n=7). Five ewes were ovariectomized only. The second interval (Experiment 2, days 14 to 17) was chosen to study the maximal rises of PGF2α associated with the completion of luteal regression and the preovulatory increase of oestrogen. Ewes were laparotomized and catheterized on day 14 and either received no further treatment (n=5), or were lutectomized (n=6), ovariectomized (n=5) or ovariectomized and implanted with oestradiol-17β (n=5). Five utero- ovarian venous samples were collected, every 30 min for 2 hours beginning at 0600 and 1800 on each day in each experiment, and assayed for PGF2α. In a third experiment, 15 bred and 12 nonbred ewes were laparotomized and catheterized on day 12. One third of each type remained intact, while two thirds were ovariectomized and half of those received 10 mg of progesterone (s.c.) and an intravaginal sponge containing 30 mg of progesterone on day 12. Utero-ovarian samples were collected at 15-minute intervals for 3 hours on day 13.Neither the average day of occurrence of the first peak of PGF2α (control, 12.1; lutectomy plus progesterone, 11.6; ovariectomy plus progesterone, 12.0; ovariectomy, 12.2) nor the mean number of peaks detected in each ewe during the sampling periods on days 10 to 14 (2.8, 3.7, 3.2 and 3.4, respectively) differed among groups in Experiment 1. The mean number of peaks of PGF2α detected in each ewe during days 14 to 17 did not differ among groups in Experiment 2. Neither of the patterns of concentrations of PGF2α studied (mean within sampling period over time and natural logarithm of the variance within sampling period over time) differed among groups in either experiment. Concentrations of PGF2α increased after ovariectomy in Experiment 3 and were restored to values similar to intact ewes by replacement therapy with progesterone. These data do not support the idea that increases in oestrogen are involved in regulating patterns of uterine secretion of PGF2α during the ovine oestrous cycle or the initiation of maternal recognition of pregnancy. In addition, progesterone may not be necessary after day 9 for initial rises of PGF2α to occur on days 12 and 13, but does appear to be necessary to sustain normal patterns of secretion of PGF2α beyond day 14.
This study examined the effects of tumour necrosis factor-α (TNFα) on basal and stimulated progesterone secretion, as well as prostaglandin F 2α production, by small, large and mixed porcine luteal cells and assessed the action of TNFα in the presence and absence of indomethacin. Corpora lutea were isolated from gilts on days 8–9 of the oestrous cycle and enzymatically dissociated. Luteal cells were either subjected to elutriation to isolate small and large cells or were separated from erythrocytes by a polysucrose gradient to serve as the mixed luteal cell group. Then 24-well culture plates were seeded with 150 000 small, 30 000 large and mixed (30 000 large+100 000-250 000 small) luteal cells suspended in 1 ml medium 199 media supplemented with 5 μg insulin/ml, 40 ng cortisol/ml and 50 μg low-density lipoproteins/ml. Cells were cultured for up to 24 h in a humidified incubator at 37 °C with 5% CO 2 in air. TNFα time- and dose-dependently suppressed ( P <0·05) LH-induced, but not basal, progesterone secretion by small luteal cells. Moreover, TNFα inhibited ( P <0·05) forskolin-mediated, but not cyclic AMP-mediated, progesterone secretion by small luteal cells. The LH-stimulated progesterone secretion by small luteal cells was not affected by TNFα in the presence of indomethacin. Progesterone secretion by large and mixed luteal cells was not affected by TNFα. Prostaglandin F 2α production by small and mixed, but not large, luteal cells was enhanced ( P <0·05) by TNFα. These data demonstrated that TNFα acts primarily on a target cell(s) in the small cell fraction, and the cytokine-induced inhibition of LH-stimulated progesterone secretion by small cells was mediated by prostaglandin F 2α and involved a site(s) proximal, but not distal, to cyclic AMP generation.
Journal of Endocrinology (1994) 143 , 75–83
Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that attracts monocytes and macrophages. It is known that macrophages accumulate in the corpus luteum (CL) during luteal regression in many species. In this study, we investigated the regulation of MCP-1 mRNA in ovine and bovine CL during prostaglandin (PG) F2α-induced luteolysis, after LH treatment, or after pharmacologic activation of the protein kinase (PK) A or PKC intracellular effector systems. In experiment 1, ewes on day 11 or 12 of the estrous cycle were infused with saline or PGF2α. PGF2α increased MCP-1 mRNA at 1 and 4 h after treatment. MCP-1 mRNA returned to basal level at 12 h and increased again at 24 h post treatment. In experiment 2, ewes received saline, PGF2α, phorbol 12-myristate 13-acetate (PMA), luteinizing hormone (LH), or forskolin infusion and CL were collected at 0 (untreated), 4, 12, or 24 h after infusion. Similar to experiment 1, PGF2α induced MCP-1 mRNA at 4 and 24 h post treatment. PMA increased mRNA for MCP-1 at 4, 12, and 24 h. Treatment with LH or forskolin transiently decreased MCP-1 mRNA expression. In experiment 3, cows were treated with a luteolytic dose (25 mg) of PGF2α on day 4 or day 11 of estrous cycle and expression of MCP-1 mRNA was quantified. Steady-state concentrations of mRNA for MCP-1 were induced by PGF2α treatment only in mid-cycle CL but not in early CL. In summary, administration of PGF2α or activation of PKC induced MCP-1 mRNA expression. Expression of MCP-1 may be important for stimulating immune processes during luteal regression.
One of the postulated main luteolytic actions of prostaglandin (PG) F2α is to decrease ovarian blood flow. However, before Day 5 of the normal cycle, the corpus luteum (CL) is refractory to the luteolytic action of PGF2α. Therefore, we aimed to determine in detail the real-time changes in intraluteal blood flow after PGF2α injection at the early and middle stages of the estrous cycle in the cow. Normally cycling cows at Day 4 (early CL, n = 5) or Days 10–12 (mid CL, n = 5) of the estrous cycle (estrus = Day 0) were examined by transrectal color and pulsed Doppler ultrasonography to determine the blood flow area, the time-averaged maximum velocity (TAMXV), and the volume of the CL after an i.m. injection of a PGF2α analogue. Ultrasonographic examinations were carried out just before PG injection (0 h) and then at 0.5, 1, 2, 4, 8, 12, 24, and 48 h after the injection. Blood samples were collected at each of these times for progesterone (P) determination. The ratio of the colored area to a sectional plane at the maximum diameter of the CL was used as a quantitative index of the changes in blood flow within the luteal tissue. Blood flow within the midcycle CL initially increased (P < 0.05) at 0.5–2 h, decreased at 4 h to the same levels observed at 0 h, and then further decreased to a lower level from 8 h (P < 0.05) to 48 h (P < 0.001). Plasma P concentrations decreased (P < 0.05) from 4.7 ± 0.5 ng/ml (0 h) to 0.6 ± 0.2 ng/ml (24 h). The TAMXV and CL volume decreased at 8 h (P < 0.05) and further decreased (P < 0.001) from 12 to 24 h after PG injection, indicating structural luteolysis. These changes were not detected in the early CL, in which luteolysis did not occur. In the early CL, the blood flow gradually increased in parallel with the CL volume, plasma P concentration, and TAMXV from Day 4 to Day 6. The present results indicate that PGF2α induces an acute blood flow increase followed by a decrease in the midcycle CL but not in the early CL. This transitory increase may trigger the luteolytic cascade. The lack of intraluteal vascular response to PG injection in the early CL appears to be directly correlated with the ability to be resistant to PG.
Insulin-like growth factor-I (IGF-I) is produced within the porcine corpus luteum (CL) and is thought to play an autocrine/paracrine role in CL development/function during the early luteal phase. This study examines the hypotheses that the luteolytic actions of prostaglandin F2α (PGF2α) during the early luteal phase may involve either a decrease in IGF-I or IGF receptor (IGF-IR), or an increase in IGF binding protein (IGFBP)-3, expression, any of which could interfere with the luteotropic actions of IGF-I in this tissue. Cycling gilts were treated twice daily with PGF2α (or saline) on Days 5–9 of the cycle to induce premature luteolysis. CL were collected on Days 6–9, and RNA, protein, or progesterone was extracted. By slot blot analysis, steady-state levels of IGF-I and IGFBP-3 mRNA were not different in PGF2α-treated vs. control animals; however, IGF-IR mRNA was increased in treated animals on Day 9. No changes in IGF-I content (ng/CL measured by RIA) were observed with respect to treatment. According to ligand blot analysis, the levels of IGFBP-3 increased on Day 6 and decreased on Days 8–9, while IGFBP-2 was higher on Days 6–7 and decreased on Day 9 in treated animals. IGF-IR levels, determined from Western blots, were higher on Day 7 (P < 0.05) and lower on Day 9 in PGF2α-treated animals vs. control animals (P < 0.05). In conclusion, PGF2α-induced premature luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. However, since IGFBP-2 and -3 protein levels increased early in the treatment period (Days 6–7), it is possible that they may mediate the luteolytic actions of PGF2α by sequestering IGF-I and preventing its interaction with the IGF-IR.
The objective of this study was to investigate tumor necrosis factor a (TNF-a) expression, the presence of functional TNF-a receptors, and expression of TNF receptor type I (TNF-RI) mRNA in the bovine corpus luteum (CL) during different stages of the estrous cycle. Reverse transcription (RT)-polymerase chain reaction (PCR) showed no difference in TNF-a mRNA expression during the estrous cycle. Concentrations of TNF-a in the CL tissue increased significantly from the mid to the late luteal stage and decreased thereafter (P , 0.05). An RT-PCR analysis showed higher levels of TNF-RI mRNA in CL of Days 3‐7 than of other stages (P , 0.05). 125 I-TNF-a binding to the membranes of bovine CL was maximal after incubation at 388C for 48 h. The binding was much greater for TNF-a than for related peptides. A Scatchard analysis revealed the presence of a high-affinity binding site in the CL membranes collected at each phase of the estrous cycle (dissociation constant: 3.60 6 0.58‐5.79 6 0.19 nM). In contrast to TNF-RI mRNA expression, the levels of receptor protein were similar at each stage of the estrous cycle. When cultured cells of all luteal stages were exposed to TNF-a (1‐100 ng/ml), TNF-a stimulated prostaglandin F2a and prostaglandin E2 secretion by the cells in a dose-dependent fashion (P , 0.01), especially during the early luteal phase, although it did not affect progesterone secretion. These results indicate the local production of TNF-a and the presence of functional TNF-RI in bovine CL throughout the estrous cycle, and suggest that TNFa plays some roles in regulating bovine CL function throughout the estrous cycle.
There is sufficient evidence to prove that tumor necrosis factor α (TNFα) modulates bovine corpus luteum (CL) function. Our previous study demonstrated that functional TNFα receptors are present on luteal cells in bovine CL throughout the estrous cycle. The purpose of the present study was to identify the presence of functional TNFα receptors on the microvascular endothelial cells derived from developing bovine CL. TNFα receptors were analyzed by a radioreceptor assay using ¹²⁵I-labeled TNFα on two types of cultured endothelial cells. One has a cobblestone appearance (CS cells), and the other has a tube-like structure (TS cells). ¹²⁵I-Labeled TNFα binding was maximal after incubation for 30 h at 37°C, and the specificity of binding was confirmed. A Scatchard analysis showed the presence of two binding sites (high- and low-affinity) for TNFα receptors on both CS and TS cells. The dissociation constant (Kd) values and concentrations of the high-affinity binding sites for TNF receptors were similar for CS and TS cells. However, Kd values and concentrations of the low-affinity binding sites in CS cells were significantly higher than those in TS cells (P < 0.05 or lower). The expression of TNF receptor type 1 (TNF-RI) mRNA was determined in both cell types. Furthermore, TNFα significantly stimulated prostaglandin E2 and endothelin-1 secretion by both CS and TS cells (P < 0.05 or lower). These results indicate the presence of two types of TNF receptors and the expression of TNF-RI mRNA in the endothelial cells derived from bovine CL, and suggest that TNFα plays two or more roles in regulating the secretory function of the endothelial cells.
There is ample evidence that prostaglandin F2alpha (PGF2alpha) is a luteolytic substance in sows, however, there is also some evidence that it may stimulate progesterone (P4) secretion in young corpora lutea (CL). In vitro studies also suggested that tumor necrosis factor alpha (TNF) is inhibitory to luteal cell P4 and estradiol-17beta (E2) release. Since E2 is a strong luteotropic substance in porcine CL, we studied the effects of intraluteal application of PGF2alpha and TNF alone and in combination on the secretion of P4 and E2 in freely moving sows. Furthermore, the effects of intraluteal infusion of E2 and its stereoisomer, estradiol-17alpha, on luteal function, were also determined. Microdialysis systems were implanted into CL at Day 10 of the estrous cycle. After a 24-h recovery period, PGF2alpha (10(-6) M) or E2 (10(-6) M) was applied daily for 6 h into the CL. PGF2alpha caused a stimulation of E2 and P4, and E2 also stimulated P4 secretion at Days 11 and 12, but the stimulatory effect of both substances diminished as the CL approached luteolysis. Intraluteal TNF application resulted in a transient increase of P4 secretion, which was followed by a dramatic reduction of P4 release. When TNF-pretreated CL were exposed to PGF2alpha at Day 11 of the estrous cycle, the prostaglandin was no longer able to stimulate but rather inhibited E2 and P4 secretion. Intraluteal application of estradiol-17alpha had no effect on P4 secretion. These results are suggestive that the PGF2alpha-induced E2 secretion in young and middle-aged CL is stimulatory to P4 secretion. Under the influence of macrophage-derived TNF production, E2 secretion is inhibited, and thereby PGF2alpha and TNF cause functional luteolysis.
An experiment was conducted to determine if injections of prostaglandin (PG) F2α influenced the number of receptors for LH in ovine luteal tissue and to compare the time course of change in the numbers of LH receptors and progesterone secretion. Ewes on Day 9 of the estrous cycle were given 2 injections of PGF2α-tham salt (6.7 mg, i.m.) at 4 h intervals. Corpora lutea were removed from each of 6 ewes at 2.5, 7.5, and 22.5 h after the initial injection of PGF2α and from 6 control ewes on Day 10 of the cycle. Jugular blood samples for analysis of progesterone were collected at 20 min intervals for 1 h prior to treatment and at 0.5, 1 or 2 h intervals in ewes in which corpora lutea were collected at 2.5, 7.5 and 22.5 h after injection of PGF2α, respectively. Luteal weight, luteal progesterone concentrations and total number of luteal LH receptors and the number of receptors occupied by endogenous LH were determined.
Serum concentrations of progesterone were not decreased at 2.5 h after the initial injection of PGF2α, but had decreased by 7.5 and 22.5 h (P<0.01). There was no change in luteal weight, luteal concentration of progesterone, total number of LH receptors or the number of receptors occupied by LH at 2.5 or 7.5 h when compared to luteal tissue from control ewes. However, each of these parameters had decreased by 22.5 h. The affinity of the receptor for LH did not differ at any of the times studied. These data indicate that progesterone secretion by the corpus luteum after PGF2α administration decreased well before (7.5 h) decreases in the number of occupied or unoccupied receptors for LH could be detected (22.5 h).
Fifty-six ewes were used to study the effects of PGE1 or PGE2 plus PGF2α given into the perivascular space of the ovarian vascular pedicle on luteal function of nonbread ewes. All ewes receiving PGF2α had reduced levels of plasma progesterone and unoccupied receptors for LH at 24 hr after treatment regardless even if they received PGE1 or PGE2 concomitantly. Levels of plasma progesterone in ewes receiving only PGF2α were reduced further at 48 hour. Plasma progesterone and unoccupied receptors for LH of ewes receiving PGE2 + PGF2α were maintained at 48 hr at levels seen at 24 hr after treatment, while progesterone in ewes receiving PGE1 + PGF2α at 48 hr returned to levels seen in controls at 48 hr and unoccupied receptors for LH were three fold greater then controls.
The role of tumor necrosis factor α (TNFα) and its type I receptor (TNFRI) in structural luteolysis was investigated. A semiquatitative reverse-transcription polymerase chain reaction (RT-PCR) was used to characterize the pattern of TNFRI mRNA expression within the corpus luteum (CL) throughout the estrous cycle and its cellular distribution. Increase in TNFRI mRNA levels was recorded both in regressed luteal tissue and in CL of cows injected with prostaglandin F2α. All three major cell types composing the CL, steroidogenic (large and small) and endothelial cells expressed the TNFRI gene. A densitometric analysis of TNFRI mRNA expression revealed that resident endothelial cells had significantly higher levels of TNFRI mRNA than steroidogenic luteal cells. The physiological effects associated with TNFRI expression were investigated in the various luteal cell types. TNFα-induced programmed cell death (PCD) in dose- and time-dependent manners of cultured luteal endothelial cells (LECs) but not of in vitro luteinized steroidogenic cells. Several lines of evidence are provided to show that progesterone regulates luteal cell survival: 1) CL and LECs express progesterone receptor mRNA, 2) physiological levels of the steroid abolished TNFα-induced PCD of LECs, and 3) progesterone-producing cells are protected from PCD. In conclusion, this study suggests that TNFα-induced PCD during structural luteolysis is mediated by TNFRI, primarily affects endothelial cells, and that the decline in progesterone, preceding structural luteolysis, is a prerequisite for the initiation of apoptosis in endothelial cells.
In experiments and we have previously shown that PGF2α directly antagonized the action of gonadotrophins on the corpus luteum. To determine if this action of PGF2α may occur as a consequence of an induced loss of gonadotrophin receptors, binding of hCG to rat luteal tissue was measured following PGF2α treatment . In immature rats which were treated with exogenous gonadotrophin to luteinize the gonads, PGF2α produced a marked and highly significant decrease in circulating progesterone when administered 24 hours before sacrifice. Although the affinity constant (Ka; 1.2-2 × 1010 L/M) of the luteal receptor to hCG was not affected, PGF2α treatment produced a marked fall in the binding capacity of the luteal tissue to hCG. This response was absent, however, when PGF2α was incubated directly with luteal receptor or administered during early pseudopregnancy when corpora lutea are more resistant to luteolysis. Experiments are in progress to determine if the decrease in capacity of luteal receptors to bind hCG is the mechanism or a consequence of luteolysis produced by PGF2α.
Prostaglandins (PGs) are believed to play important roles in the establishment of pregnancy. Glandular and stromal cells were isolated from pig endometrium on days 11 through 19 of pregnancy and cultured in the presence of estradiol-17β (E2) and progesterone (P4) to determine the effect of day pregnancy and steroids on the secretion of PGE and PGF2α. Estradiol at concentrations between .01 and 1 μM did not affect PGE and PGF2α secretion into the medium by glandular and stromal cells. Progesterone (.1 μM) suppressed (P < .001) PGE and PGF2α production from both cell types. Glandular cell secreted more (P < .01) PGF2α than PGE, whereas stromal cells collected on days 11, 12, 13, and 19 secreted more (P < .05) PGE than PGF2α. Stromal cells isolated from tissues collected on day 13 of pregnancy produced PGs with higher (P < .01) PGE:PGF2α ratio than those from tissues harvested on other days of pregnancy. Glandular cells isolated from tissues collected on days 13 and 19 and stromal cells isolated from tissue collected on day 13 of pregnancy secreted more (P < .05) PGE and PGF2α than cells isolated on other days of pregnancy. We conclude that: 1) P4 has a suppressing effect on PG secretion; 2) endometrial glandular and stromal cells each produce a unique profile of PGs; and 3) endometrial cells harvested on different days of pregnancy secrete different amounts of PGE and PGF2α.
Periodic collections of uterine venous blood were obtained from three nonmated, three pregnant and two mated but nonpregnant ewes in which uterine veins were cannulated with polyvinyl tubing on day 11 postestrus. Frequent sampling was achieved in three of these ewes with additional cannulae in the ovarian veins. Blood samples were collected at 3-hr intervals from 0600 on day 12 to 1800 on day 13 and then at 6-hr intervals through day 15. On day 13, three additional samples at 30-min intervals were collected between 1400 and 1530. Prostaglandins F (PGF) in plasma were quantified by radioimmunoassay.
PGF2α has been postulated to be the uterine luteolytic factor. If this is true, then one might expect the levels of PGF2α to be lower during early pregnancy. However, in the present study levels of PGF2α were found to be significantly higher in both content (ng) and concentration (ng/gm) in the endometrium of day-13 pregnant ewes (2375, 152 respectively) compared to day-13 nonpregnant animals (1478, 89 respectively). These results imply that the luteotropic effects of the embryo must occur by some mechanism other than inhibition of production or release of the uterine luteolytic factor.