Temporal changes in the corpus luteum during early pregnancy reveal regulation of pathways that enhance steroidogenesis and suppress luteolytic mechanisms†.

Although rescue of the corpus luteum (CL) is required for pregnancy, luteal function during maternal recognition of pregnancy remains largely unexplored. CL were collected from pregnant cattle on days 14, 17, 20, and 23, to encompass the maternal recognition of pregnancy period. Next-generation sequencing was used to profile mRNA abundance during this time, while tandem mass spectrometry and nanostring technology were used to profile proteins and miRNA, respectively. A total of 1157 mRNA were differentially abundant, while 27 miRNA changed, and 29 proteins tended to change. mRNA that increased were regulators of interferon signaling and DNA repair, while those that decreased were associated with luteolytic processes, such as calcium signaling and matrix metallopeptidase (MMP) signaling, indicating inhibition of these processes. One of these, MMP12, was regulated by prostaglandin F2A in vitro. mRNA that were maximally abundant on day 20 were primarily associated with immune processes. Two of these, C-C motif chemokine ligand 1 and NFKB inhibitor alpha, were regulated by interferon tau in vitro. MiRNA that increased were predicted to inhibit phosphatidylinositol signaling, while those that decreased may be negative regulators of steroidogenesis. One protein that was greater on day 20 than on day 14 was aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which synthesizes retinoic acid. Pharmacological inhibition of this enzyme, or of retinoic acid receptor signaling, led to suppression of progesterone production in vitro. Overall, these data indicate that there are changes in the CL of pregnancy that are important for continued luteal function.

Differential fractal dimension is associated with extracellular matrix remodeling in developing bovine corpus luteum.

To assist in evaluating and quantifying tissue changes, fractal dimension (FD) is a useful method for assessing the organization in an image from fractals that describes the amount of space and the self-similarity of the structure, once FD detects subtle morphological changes and performs functional quantitative measures. Here, we hypothesized that fractal analysis may be different in functional and regressing bovine corpus luteum (CL) and may be correlated with differential expression of genes involved in extracellular matrix remodeling. CL presents two developmental stages, the functional and regressing CL, according to progesterone levels and morphology. First, we found a lower FD in functional CL using HE staining and picrosirius red approach. Additionally, we found a great amount of total collagen in regressing CL. Regarding gene expression, we showed an up regulation of COL1A1, COL1A2, MMP2, and MMP14 and a down regulation of TIMP1 and TIMP2 in regressing CL compared to the functional one. Thus, we concluded that differential FD observed during luteal regression is an effective method to evaluate the tissue changes observed during luteal development in cattle and is related to differential quantity of genes involved in extracellular matrix remodeling.

Gene expression profiling of the canine placenta during normal and antigestagen-induced luteolysis.

The domestic dog is the only domestic animal species that does not produce steroids in the placenta and instead relies on luteal steroids throughout pregnancy. Nevertheless, the canine placenta is highly responsive to steroids, and withdrawal of progesterone (P4) affects the feto-maternal unit, initializing the parturition cascade. Similar effects can be observed during antigestagen-induced abortion. Here, aiming to provide new insights into mechanisms involved in the termination of canine pregnancy, next generation sequencing (NGS, RNA-seq) was applied. Placental transcriptomes derived from natural prepartum and antigestagen-induced abortions were analyzed and compared with fully developed mid-gestation placentas. The contrast "prepartum luteolysis over mid-gestation" revealed 1973 differentially expressed genes (DEG). Terms associated with apoptosis, impairment of vascular function and activation of signaling of several cytokines (e.g., IL-8, IL-3, TGF-ß) were overrepresented at natural luteolysis. When compared with mid-term, antigestagen treatment revealed 135 highly regulated DEG that were involved in the induced luteolysis and showed similar associations with functional terms and expression patterns as during natural luteolysis. The contrast "antigestagen-induced luteolysis over prepartum luteolysis" revealed that, although similar changes occur in both conditions, they are more pronounced during natural prepartum. Among P4-regulated DEG were those related to immune system and cortisol metabolism. It appears that, besides inducing placental PGF2α output, both natural and induced P4 withdrawal is associated with disruption of the feto-maternal interface, leading to impaired vascular functions, apoptosis and controlled modulation of the immune response. The time-related maturation of the feto-maternal interface needs to be considered because it may be clinically relevant.

Circadian rhythms of factors involved in luteal regression are modified in p55 tumour necrosis factor receptor (TNFRp55)-deficient mice.

The rhythm of factors involved in luteal regression is crucial in determining the physiological duration of the oestrous cycle. Given the role of tumour necrosis factor (TNF)-α in luteal function and circadian regulation and that most of the effects of TNF-α are mediated by p55 TNF receptor (TNFRp55), the aims of the present study were to analyse the following during the luteal regression phase in the ovary of mice: (1) whether the pattern of expression of progesterone (P4) and the enzymes involved in the synthesis and degradation of P4 is circadian and endogenous (the rhythm persists in constant conditions, (i.e., constant darkness) with a period of about 24 hours); (2) circadian oscillations in clock gene expression; (3) whether there are daily variations in the expression of key genes involved in apoptosis and antioxidant mechanisms; and (4) the consequences of TNFRp55 deficiency. P4 was found to oscillate circadianally following endogenous rhythms of clock factors. Of note, TNFRp55 deficiency modified the circadian oscillation in P4 concentrations and its enzymes involved in the synthesis and degradation of P4, probably as a consequence of changes in the circadian oscillations of brain and muscle ARNT-Like protein 1 (Bmal1) and Cryptochrome 1 (Cry1). Furthermore, TNFRp55 deficiency modified the circadian rhythms of apoptosis genes, as well as antioxidant enzymes and peroxidation levels in the ovary in dioestrus. The findings of the present study strengthen the hypothesis that dysregulation of TNF-α signalling may be a potential cause for altered circadian and menstrual cycling in some gynaecological diseases.

Transcriptome analysis reveals differences in mechanisms regulating cessation of luteal function in pregnant and non-pregnant dogs.

BACKGROUND: In the domestic dog, corpora lutea (CL) are the only source of progesterone (P4), both in pregnant and non-pregnant cycles because there is no placental steroidogenesis. The absence of an endogenous luteolysin in absence of pregnancy results in long-lasting physiological pseudopregnancy, strongly contrasting with the acute luteolysis observed prepartum. The underlying biological mechanisms and the involvement of P4 signalling remain, however, not fully understood. Therefore, here, next-generation sequencing (RNA-Seq) was performed on CL from the late luteal phase and compared with normally luteolyzing CL collected at the prepartum P4 decrease. RESULTS: The contrast "luteal regression over luteolysis" yielded 1595 differentially expressed genes (DEG). The CL in late luteal regression were predominantly associated with functional terms linked to extracellular matrix (p = 5.52e-05). Other terms related to transcriptional activity (p = 2.45e-04), and steroid hormone signalling (p = 2.29e-04), which were more highly represented in late regression than during luteolysis. The prepartum luteolysis was associated with immune inflammatory responses (p = 2.87e-14), including acute-phase reaction (p = 4.10e-06). Immune system-related events were also more highly represented in CL derived from normal luteolysis (p = 7.02e-04), compared with those from dogs in which luteolysis was induced with an antigestagen (1480 DEG in total). Additionally, the withdrawal of P4 at mid-gestation resulted in 92 DEG; over-represented terms enriched in antigestagen-treated dogs were related to the inflammatory response (p = 0.005) or response to IL1 (p = 7.29e-05). Terms related to proliferation, e.g., centrosome organization (p = 0.002) and steroid metabolic processes (p = 0.001), prevailed at mid-gestation. Thereby, our results revealed the nature of luteotropic effects of P4 within canine CL. It appears that, even though they result in diminished steroidogenic output, the effect of antigestagens is more related to the withdrawal of P4 support than to the PGF2alpha-related inflammatory reaction observed at physiological parturition. CONCLUSIONS: We report the differential gene expression associated with maintenance and cessation of luteal function in pregnant and non-pregnant dogs. Based on the differentially expressed genes, we indicate functional pathways and gene networks that are potentially involved in the underlying endocrine and molecular mechanisms. This study establishes future research directions that may be helpful in understanding some of the clinical conditions, such as luteal insufficiency, associated with negative pregnancy outcome in dogs.

Loss of luteotropic prostaglandin E plays an important role in the regulation of luteolysis in women.

STUDY QUESTION: Do intraluteal prostaglandins (PG) contribute to luteal regulation in women? SUMMARY ANSWER: Prostaglandin E (PGE), which is produced in human granulosa-lutein cells stimulated with luteotropic hCG, exerts similar luteotropic effects to hCG, and the expression of PG synthetic and metabolic enzymes in the human CL is driven toward less PGE but more prostaglandin F (PGF) during luteolysis. WHAT IS KNOWN ALREADY: Uterine PGF is a major luteolysin in many non-primate species but not in women. Increases in the PGF synthase, aldo-ketoreductase family one member C3 (AKR1C3), have been observed in the CL of marmoset monkeys during luteolysis. PGE prevents spontaneous or induced luteolysis in domestic animals. STUDY DESIGN, SIZE, DURATION: Human CL tissues staged as the early-luteal (n = 6), mid-luteal (n = 6), late-luteal (n = 5) and menstrual (n = 3) phases were obtained at the time of hysterectomy for benign gynecological conditions. Luteinized granulosa cells (LGCs) were purified from follicular fluids obtained from patients undergoing assisted conception. PARTICIPANTS/MATERIALS, SETTING, METHODS: Upon collection, one half of the CL was snap-frozen and the other was fixed with formalin and processed for immunohistochemical analysis of a PGE synthase (PTGES). Quantitative RT-PCR was employed to examine changes in the mRNA abundance of PG synthetic and metabolic enzymes, steroidogenic enzymes, and luteolytic molecules in the staged human CL and in human LGCs in vitro treated with hCG, PGE and PGF. A PGE withdrawal experiment was also conducted in order to reveal the effects of the loss of PGE in LGCs. Progesterone concentrations in the culture medium were measured. MAIN RESULTS AND THE ROLE OF CHANCE: The key enzyme for PGE synthesis, PTGES mRNA was abundant in the functional CL during the mid-luteal phase (P < 0.01), while mRNA abundance for genes involved in PGF synthesis (AKR1B1 and AKR1C1-3) increased in the CL during the late-luteal phase and menstruation (P < 0.05-0.001). PTGES mRNA expression positively correlated with that of 3ß-hydroxysteroid dehydrogenase (HSD3B1; r = 0.7836, P < 0.001), while AKR1C3 expression inversely correlated with that of HSD3B1 (r = -0.7514, P = 0.0012) and PTGES (r = -0.6923, P = 0.0042). PGE exerted similar effects to hCG-promoting genes, such as steroidogenic acute regulatory protein (STAR) and HSD3B1, to produce progesterone and luteotropic PGE, suppress PGF synthetic enzymes and down-regulate luteolytic molecules such as ßA- and ßB-inhibin subunits (INHBA and INHBB) and bone morphogenetic proteins (BMP2, BMP4 and BMP6). PGE withdrawal resulted in reductions in the enzymes that produce progesterone (STAR; P < 0.001) and PGE (PTGES; P < 0.001), and the capacity to produce PGE decreased, while the capacity to produce PGF increased during the culture. The addition of PGF did not recapitulate the luteolytic effects of PGE withdrawal. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Changes in mRNA expression of PG synthetic and metabolic enzymes may not represent actual increases in PGF during luteolysis in the CL. The effects of PGF on luteal cells currently remain unclear and the mechanisms responsible for decreases in the synthesis of PGE in vitro and at luteolysis have not been elucidated in detail. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained strongly support a luteotropic function of PGE in regulation of the human CL. They suggest that the main PG produced in human luteal tissue changes from PGE to PGF during the maturation and regression of the CL, and the loss of PGE is more important than the effects of PGF during luteolysis in women. This may be accompanied by reduced effects of LH/hCG in luteal cells, particularly decreased activation of cAMP/protein kinase A; however, the underlying mechanisms remain unknown. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported by the Cunningham Trust to WCD, a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science and the Suntory Foundation for Life Sciences to J.N.-K.; W.C.D. is supported by an MRC Centre Grant G1002033 and a Scottish Senior Clinical Fellowship. The authors have nothing to disclose.

Interferon-tau and fertility in ruminants.

Establishment of pregnancy in domestic ruminants includes pregnancy recognition signalling by the conceptus, implantation and placentation. Despite the high fertilisation success rate in ruminants, a significant amount of embryo loss occurs, primarily during early gestation. Interferon-tau (IFNT), a type I interferon that is exclusively secreted by the cells of the trophectoderm of the ruminant conceptus, has been recognised as the primary agent for maternal recognition of pregnancy in ruminants. It produces its antiluteolytic effect on the corpus luteum by inhibiting the expression of oxytocin receptors in the uterine epithelial cells, which prevents pulsatile, luteolytic secretion of prostaglandin F2α by the uterine endometrium. While the importance of IFNT in maternal recognition of pregnancy and prevention of luteolysis in ruminants is unequivocal, important questions, for example, relating to the threshold level of IFNT required for pregnancy maintenance, remain unanswered. This paper reviews data linking IFNT with measures of fertility in ruminants.

Interferon-tau promotes luteal endothelial cell survival and inhibits specific luteolytic genes in bovine corpus luteum.

Interferon-tau (IFNT), a maternal recognition of pregnancy (MRP) signals in domestic ruminants, suppresses the release of luteolytic pulses of uterine prostaglandin F2a (PGF2a), thus extending the corpus luteum (CL) life span. We hypothesized that IFNT also exerts anti-luteolytic actions in bovine CL. To examine the direct effects of IFNT on bovine CL, luteal slices and enriched luteal endothelial cells (LECs) were utilized. We found that recombinant ovine IFNT (roIFNT) markedly elevates interferon-associated genes (STAT1, STAT2 and IRF9) and interferon-stimulated genes (ISGs: MX2, ISG15 and OAS1Y) in both models. Furthermore, IFNT time-dependently induced STAT1 phosphorylation in LECs without affecting total STAT1. roIFNT-stimulated viable LECs numbers and the knockdown of protein inhibitor of activated STAT1 (PIAS1) abolished this effect, suggesting that PIAS1 may mediate the proliferative effect of IFNT. IFNT significantly downregulated luteolytic genes such as TGFB1, thrombospondin-1 (THBS1), endothelin-1 (EDN1) and serpin family E member-1 (SERPINE1) in LECs. However, less robust effects were observed in luteal slices. Moreover, PGF2a alone induced THBS1, SERPINE1 and EDN1 mRNA in CL slices whereas in the presence of IFNT, THBS1 and SERPINE1 stimulation was abolished. Collectively, these results indicate that IFNT acts via STAT1- IRF9-dependent and independent pathways and affects diverse luteal functions. Most interestingly, this study suggests the existence of an anti-luteolytic effect of IFNT in bovine CL, namely, inhibiting key PGF2a-induced luteolytic genes. The proliferative effect of IFNT may constitute an additional mechanism that promotes luteal cell survival, thus, extending the luteal life span during early pregnancy in cows.

Expressions of lipoprotein receptors and cholesterol efflux regulatory proteins during luteolysis in bovine corpus luteum.

The corpus luteum (CL) synthesises and secretes progesterone (P4), which is essential for the establishment and maintenance of pregnancy in mammals. P4 is synthesised from cholesterol. Cholesterol is internalised by low-density lipoprotein receptor (LDLR) and/or scavenger receptor B1 (SR-BI), and is effluxed by ATP-binding cassette (ABC) transporter A1 (ABCA1) and G1 (ABCG1). To test the hypothesis that lipoprotein receptors and ABC transporters are involved in functional luteolysis, we examined the expression of LDLR, SR-BI, ABCA1 and ABCG1 in bovine CL during the luteal stages and after injection of prostaglandin (PG) F2α on Day 10 after ovulation. Expression of LDLR and SR-BI mRNA and protein was lower in the regressed luteal than late luteal stage. Injection of cows with a PGF2α did not affect LDLR mRNA and protein levels in the CL. Although expression of SR-BI mRNA did not change, SR-BI protein expression decreased 12 and 24h after PGF2α injection. The overall findings of the present study suggest that the decreased expression of SR-BI induced by PGF2α is one of the factors responsible for the continuous decrease in P4 production during functional luteolysis.

Toll-like receptor and related cytokine mRNA expression in bovine corpora lutea during the oestrous cycle and pregnancy.

Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll-like receptors (TLR) mediate innate immune mechanisms via the production of pro-inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30-50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll-like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN-G), and interleukin (IL)-12, mRNA expressions were greatest in regressing CL compared with earlier stages (p < .05), whereas no change was observed for IL-6 mRNA expression. Cytokine mRNA expression in cultured luteal cells was not altered by LPS. Based on these data, one or more of the TLRs found within the CL may play a role in luteolysis, perhaps via pro-inflammatory cytokine mRNA expression.

Steroid hormones, prostanoids, and angiogenic systems during rescue of the corpus luteum in pigs.

In order to characterize the transition of the corpora lutea (CL) from acquisition of luteolytic sensitivity to rescue of luteal function: i) the expression of 38 factors associated with steroids, prostanoids, and angiogenic systems and ii) concentrations of the main hormones responsible for maintenance of CL function in cyclic and pregnant pigs were examined. Additionally, the effect of prostaglandin (PG) E2 and F2 α on luteal function during the estrous cycle and pregnancy was evaluated in vitro. Significantly up-regulated gene expression was revealed in CL collected on day 14 of the estrous cycle (CYP19A1, ESR2, PTGS2, HIF1A, and EDN1) and on days 12-14 of pregnancy (SCARB1, PGRMC1, STAR, HSD3B1, NR5A1, PTGFR, PTGER4, and VEGFA). Elevated concentrations of estradiol-17ß and PGE2 occurred in CL on days 12 and 14 of pregnancy respectively, while an increased intraluteal PGF2 α content was noted on day 14 of the estrous cycle. Both PGs increased the synthesis of progesterone by cultured luteal slices obtained on day 14 of pregnancy, in contrast to the action of PGF2 α on the corresponding day of the estrous cycle. PGE2 stimulated cAMP production via PTGER2 and PTGER4, while PGF2 α elevated the content of CREB in cultured luteal slices from CL of pregnant pigs. In silico analysis showed that infiltration of lymphocytes and apoptosis of microvascular endothelium were activated in CL on day 12 of the estrous cycle vs pregnancy. Summarizing, an abundance of E2 and PGE2 during pregnancy regulates specific pathways responsible for steroidogenesis, the prostanoid signaling system and angiogenesis during rescue from luteolysis in porcine CL.

Regulated C-C motif ligand 2 (CCL2) in luteal cells contributes to macrophage infiltration into the human corpus luteum during luteolysis.

Intense macrophage infiltration is observed during luteolysis in various animals including women; however, we still do not know how macrophage infiltration into the human corpus luteum (CL) during luteolysis is regulated. In this study, we examined the expression, localization and regulation of an important chemokine for the recruitment of monocyte/macrophage lineages, C-C motif ligand 2 (CCL2), in the human CL across the luteal phase and in cultured human luteinized granulosa cells (LGCs), with special reference to the number of infiltrating macrophages and luteal cell function. CCL2 mRNA increased in the non-functional regressing CL during menstruation (P < 0.01), corresponding to an elevated mRNA expression of a macrophage-derived cytokine, tumor necrosis factor (TNF), and an increased number of infiltrating macrophages positively stained with a macrophage marker, CD68. CCL2 protein was immunohistochemically localized to the cytoplasm of granulosa-lutein and theca-lutein cells, and CCL2 mRNA was significantly reduced by hCG both in vivo (P < 0.05) and in vitro (P < 0.01). CCL2 was also down-regulated by luteotrophic prostaglandin (PG) E (P < 0.0001), but up-regulated by luteolytic PGF (P < 0.05) in vitro. Administration of TNF significantly enhanced the CCL2 mRNA expression in cultured LGCs (P < 0.01). A greater abundance of infiltrating macrophages were found around granulosa-lutein cells lacking 3ß-HSD or PGE synthase (PGES) immunostaining. CCL2 mRNA expression was negatively correlated with both HSD3B1 and PGES, suggesting that locally produced progesterone and PGE suppress macrophage infiltration into the CL. Taken together, the infiltration of macrophages in the human CL is regulated by endocrine and paracrine molecules via regulation of the CCL2 expression in luteal cells.

Progesterone, estradiol, arachidonic acid, oxytocin, forskolin and cAMP influence on aquaporin 1 and 5 expression in porcine uterine explants during the mid-luteal phase of the estrous cycle and luteolysis: an in vitro study.

BACKGROUND: The cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression. METHODS: Uterine tissues were collected on Days 10-12 and 14-16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry. RESULTS: The results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P4, E2, AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus. CONCLUSIONS: This study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs.

Transcriptomic analysis of the myometrium during peri-implantation period and luteolysis--the study on the pig model.

In pigs, implantation begins with the attachment of embryos to the endometrium. As the process is regulated by the expression of numerous genes, endometrial transcriptomic profiles have been extensively studied in early gravid pigs. However, the myometrium, a secretory tissue, should not be neglected, as it can also participate in the regulation of implantation in early pregnant pigs. To clarify this issue, the transcriptomic profile of the porcine myometrium during the peri-implantation period (i.e. on days 15 to 16 of pregnancy) was compared with the profile observed during luteolysis (i.e. on days 15 to 16 of the oestrous cycle) with an Agilent's Porcine (V2) Two-Colour Gene Expression Microarray 4 × 44 (Agilent, USA). Analysis of the microarray data revealed that of 526 unique, accurately annotated genes, the expression of 271 unique genes was upregulated, while the expression of 255 genes was downregulated in pregnant versus cyclic myometrium. The in-depth data analysis revealed differential expression of genes encoding for factors involved in immunomodulation, tissue growth and differentiation, and prostaglandin and steroid biosynthesis and action. Moreover, the comparison of the obtained data on the myometrial transcriptome with our previously published results on the endometrial transcriptome allowed us to determine substantial differences in the regulatory function of both tissues. The new insights into the function of the myometrium of early pregnant pigs obtained here are in agreement with our previous results that suggest that this tissue plays an important role in providing optimal conditions for developing embryos. Therefore, the importance of the myometrium as an active embryo signal-responsive tissue during early pregnancy cannot be underestimated.

Canine placental prostaglandin E2 synthase: expression, localization, and biological functions in providing substrates for prepartum PGF2alpha synthesis.

The prepartum output of PGF2alpha in the bitch is associated with increased placental PGE2-synthase (PTGES) mRNA levels. Contrasting with this is a decreased expression of PGF2alpha-synthase (PGFS/AKR1C3) in uteroplacental compartments during prepartum luteolysis, suggesting an involvement of alternative synthetic pathways in PGF2alpha synthesis, for example, conversion of PGE2 to PGF2alpha. However, because the expression and possible functions of the respective PTGES proteins remained unknown, no further conclusion could be drawn. Therefore, a canine-specific PTGES antibody was generated and used to investigate the expression, cellular localization, and biochemical activities of canine uteroplacental PTGES throughout pregnancy and at prepartum luteolysis. Additionally, the biochemical activities of these tissues involved in the conversion of PGE2 to PGF2alpha were investigated. The endometrial PTGES was localized in the uterine surface epithelium at preimplantation and in superficial and deep uterine glands, endothelial cells, and myometrium throughout pregnancy and at parturition. Placental signals were mostly in the trophoblast. The biochemical properties of recombinant PTGES protein were confirmed. Additionally, expression of two PGE2-receptors, PTGER2/EP2 and PTGER4/EP4, revealed their decreasing expression during luteolysis. In contrast, the uteroplacental expression of prostaglandin transporter (PGT) was strongly elevated prior to parturition. These localization patterns resembled that of PTGES. The increased expression of PTGES and PGT at parturition, together with the accompanying decreased levels of PGE2-receptors and the capability of canine uterine and placental homogenates to take part in the conversion of PGE2 to PGF2alpha, as found in this study, suggest that PGE2 could be used locally as a substrate for prepartum PGF2alpha synthesis in the dog.

Unfolding protein response signaling is involved in development, maintenance, and regression of the corpus luteum during the bovine estrous cycle.

The corpus luteum (CL) is a transient endocrine organ. Development, maintenance, and regression of CL are effectively controlled by dynamic changes in gene expression. However, it is unknown what types of gene are affected during the CL life span of the estrous cycle in bovine. Here, we determined whether unfolded protein response (UPR) signaling via eIF2α/ATF4/GADD34, p90ATF6/p50ATF6, and IRE1/XBP1, which is a cellular stress response associated with the endoplasmic reticulum (ER), is involved in the bovine CL life span. Our results indicated that expression of Grp78/Bip, the master UPR regulator, was increased during the maintenance stage and rapidly decreased at the regression stage. Additionally, UPR signaling pathways genes were found to be involved in luteal phase progression during the estrous cycle. Our findings suggested that Grp78/Bip, ATF6, and XBP1 act as ER chaperones for initiating CL development and maintaining the CL. In addition, we investigated whether ER stress-mediated apoptosis is occurred through three UPR signaling pathways in CL regression stage. Interestingly, pIRE1 and CHOP were found to be involved in both the adaptive response and ER stress-mediated apoptosis. During the CL regression stage, increased expression of pJNK and CHOP, two components of ER stress-mediated apoptotic cascades, occurred before increased level of cleaved caspase 3 were observed. The present investigation was performed to identify a functional link between UPR signaling and CL life span during the bovine estrous cycle. Taken together, results from this study demonstrated that UPR protein/gene expression levels were different at various stages of the bovine CL life span. Variations in the expression of these protein/genes may play important roles in luteal stage progression during the estrous cycle.

Expression and regulation of secreted phosphoprotein 1 in the bovine corpus luteum and effects on T lymphocyte chemotaxis.

Secreted phosphoprotein 1 (SPP1) in the bovine corpus luteum (CL) regulates cell function during the transitional periods of luteinization and luteal regression. The objectives were to i) characterize SPP1 expression in the CL throughout the estrous cycle, ii) determine factors that regulate SPP1 expression in luteal cells, and iii) examine the role of SPP1 in lymphocyte chemotaxis, proliferation, and function. SPP1 mRNA was greater in fully functional (d10) CL and late cycle (d18) CL compared with developing (d4) CL. Additionally, SPP1 mRNA increased within 1 h and remained elevated 4 and 8 h following induction of luteolysis with prostaglandin (PG)F2α. Expression of the SPP1 receptor, ß3 integrin, was not different throughout the estrous cycle but decreased following induction of luteolysis. Expression of CD44 increased during the estrous cycle but did not change during luteal regression. In cultured luteal cells, SPP1 mRNA was upregulated by PGF2α and/or tumor necrosis factor α. Western blots revealed the presence of both full-length SPP1 and multiple cleavage products in cultured luteal cells and luteal tissue. Depletion of endogenous SPP1 did not hinder luteal cell-induced lymphocyte proliferation or lymphocyte phenotype but did inhibit lymphocyte migration toward luteal cells. Based on these data, it is concluded that SPP1 is initially activated to establish and maintain cellular interactions between steroidogenic and nonsteroidogenic cells during the development of the CL. Upon induction of luteolysis, SPP1 serves as a signaling molecule to recruit or activate immune cells to facilitate luteal regression and tissue degradation.

Patterns of gene expression in the bovine corpus luteum following repeated intrauterine infusions of low doses of prostaglandin F2alpha.

Natural luteolysis involves multiple pulses of prostaglandin F2alpha (PGF) released by the nonpregnant uterus. This study investigated expression of 18 genes from five distinct pathways, following multiple low-dose pulses of PGF. Cows on Day 9 of the estrous cycle received four intrauterine infusions of 0.25 ml of phosphate-buffered saline (PBS) or PGF (0.5 mg of PGF in 0.25 ml of PBS) at 6-h intervals. A luteal biopsy sample was collected 30 min after each PBS or PGF infusion. There were four treatment groups: Control (n = 5; 4 PBS infusions), 4XPGF (4 PGF infusions; n = 5), 2XPGF-non-regressed (2 PGF infusions; n = 5; PGF-PBS-PGF-PBS; no regression after treatments), and 2XPGF-regressed (PGF-PBS-PGF-PBS; regression after treatments; n = 5). As expected, the first PGF pulse increased mRNA for the immediate early genes JUN, FOS, NR4A1, and EGR1 but unexpectedly also increased mRNA for steroidogenic (STAR) and angiogenic (VEGFA) pathways. The second PGF pulse induced immediate early genes and genes related to immune system activation (IL1B, FAS, FASLG, IL8). However, mRNA for VEGFA and STAR were decreased by the second PGF infusion. After the third and fourth PGF pulses, a distinctly luteolytic pattern of gene expression was evident, with inhibition of steroidogenic and angiogenic pathways, whereas, there was induction of pathways for immune system activation and production of PGF. The pattern of PGF-induced gene expression was similar in corpus luteum not destined for luteolysis (2X-non-regressed) after the first PGF pulse but was very distinct after the second PGF pulse. Thus, although the initial PGF pulse induced mRNA for many pathways, the second and later pulses of PGF appear to have set the distinct pattern of gene expression that result in luteolysis.

Roles of prostaglandin F2alpha and hydrogen peroxide in the regulation of Copper/Zinc superoxide dismutase in bovine corpus luteum and luteal endothelial cells.

BACKGROUND: Prostaglandin F2alpha (PGF) induces luteolysis in cow by inducing a rapid reduction in progesterone production (functional luteolysis) followed by tissue degeneration (structural luteolysis). However the mechanisms of action of PGF remain unclear. Reactive oxygen species (ROS) play important roles in regulating the luteolytic action of PGF. The local concentration of ROS is controlled by superoxide dismutase (SOD), the main enzyme involved in the control of intraluteal ROS. Thus SOD seems to be involved in luteolysis process induced by PGF in cow. METHODS: To determine the dynamic relationship between PGF and ROS in bovine corpus luteum (CL) during luteolysis, we determined the time-dependent change of Copper/Zinc SOD (SOD1) in CL tissues after PGF treatment in vivo. We also investigated whether PGF and hydrogen peroxide (H2O2) modulates SOD1 expression and SOD activity in cultured bovine luteal endothelial cells (LECs) in vitro. RESULTS: Following administration of a luteolytic dose of PGF analogue (0 h) to cows at the mid-luteal stage, the expression of SOD1 mRNA and protein, and total SOD activity in CL tissues increased between 0.5 and 2 h, but fell below the initial (0 h) level at 24 h post-treatment. In cultured LECs, the expression of SOD1 mRNA was stimulated by PGF (1-10 microM) and H2O2 (10-100 microM) at 2 h (P<0.05). PGF and H2O2 increased SOD1 protein expression and total SOD activity at 2 h (P<0.05), whereas PGF and H2O2 inhibited SOD1 protein expressions and total SOD activity at 24 h (P<0.05). In addition, H2O2 stimulated PGF biosynthesis at 2 and 24 h in bovine LECs. Overall results indicate that, SOD is regulated by PGF and ROS in bovine LECs. SOD may play a role in controlling intraluteal PGF and ROS action during functional and structural luteolysis in cows.

In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) prevent luteolysis in cows. II: mRNA for PGF2α, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue.

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-17ß 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.