G. Ferreira-Dias

Regulation of Luteal Function and Corpus Luteum Regression in Cows: Hormonal Control, Immune Mechanisms and Intercellular Communication

The main function of the corpus luteum (CL) is production of progesterone (P4). Adequate luteal function to secrete P4 is crucial for determining the physiological duration of the oestrous cycle and for achieving a successful pregnancy. The bovine CL grows very fast and regresses within a few days at luteolysis. Mechanisms controlling development and secretory function of the bovine CL may involve many factors that are produced both within and outside the CL. Some of these regulators seem to be prostaglandins (PGs), oxytocin, growth and adrenergic factors. Moreover, there is evidence that P4 acts within the CL as an autocrine or paracrine regulator. Each of these factors may act on the CL independently or may modify the actions of others. Although uterine PGF(2 alpha) is known to be a principal luteolytic factor, its direct action on the CL is mediated by local factors: cytokines, endothelin-1, nitric oxide. The changes in ovarian blood flow have also been suggested to have some role in regulation of CL development, maintenance and regression.

Cytokines and Angiogenesis in the Corpus Luteum

In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum (CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant from the balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase. Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.

Gene transcription of TLR2, TLR4, LPS ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia–pyometra complex

Escherichia coli (E. coli) is the most frequent bacterium isolated in cases of cystic endometrial hyperplasia-pyometra complex, the most frequent endometrial disorder in the bitch. Toll-like receptors (TLRs) play an essential role in the innate immune system. The aim of this study was to compare transcription of genes encoding TLR2, TLR4 and LPS ligands (CD14, MD-2, LBP), prostaglandin synthesis enzymes (COX1, COX2, PGES1 and PGFS), and to compare COX1 and COX2 protein expression and PGE(2) and PGF(2alpha) endometrial content in the endometrium of canine diestrous uteri with (n=7) or without (n=7) pyometra. All cases of pyometra were hyperplastic and E. coli was the only isolated bacteria, while diestrous normal uteri did not present signs of cystic endometrial hyperplasia and were negative for bacteriology. Except for COX1, transcription of all genes was significantly higher in pyometra than in normal endometria. COX1 protein was observed in both normal and pyometra uteri, but COX2 protein was only present in pyometra cases. Endometrial PGE(2) and PGF(2alpha) content were significantly higher in pyometra than in normal diestrous endometria. In conclusion, data obtained in this study provides evidence that pyometra-isolated E. coli induces the up-regulation of TLR2 and TLR4 genes in the canine diestrous endometrium. This up-regulation, which is probably the result of the stimulation by LPS and lipoprotein E. coli constituents, leads to the endometrial up-regulation of PG synthesis genes. This, in turn, results in a higher endometrial concentration of PGE(2) and PGF(2alpha), which may further regulate the local inflammatory response.

Actions of a nitric oxide donor on prostaglandin production and angiogenic activity in the equine endometrium

Nitric oxide (NO) plays an important role in prostaglandin secretion and angiogenesis in the reproductive system. In the present study, the roles of the NO donor spermine NONOate and tumour necrosis factor-alpha (TNF; as a positive control) in prostaglandin production and angiogenic activity of equine endometria during the oestrous cycle were evaluated. In addition, the correlation between NO production and the expression of key prostaglandin synthase proteins was determined. The protein expression of prostaglandin F synthase (PGFS) increased in early and mid-luteal stages, whereas that of prostaglandin E synthase (PGES) was increased in the early luteal stage. The in vitro release of NO was highest after ovulation. There was a high correlation between NO production and PGES expression, as well as NO release and PGFS expression. There were no differences detected in prostaglandin H synthase 2 (PTGS-2) throughout the oestrous cycle and there was no correlation between PTGS-2 expression and NO. In TNF- or spermine-treated endometria, the expression of prostaglandin (PG) E(2) increased in the early and mid-luteal phases, whereas that of PGF(2alpha) increased in the follicular and late luteal phases. Bovine aortic endothelial cell (BAEC) proliferation was stimulated in TNF-treated follicular-phase endometria. However, in spermine-treated endometria, NO delivered from its donor had no effect, or even an inhibitory effect, on BAEC proliferation. In conclusion, despite no change in PTGS-2 expression throughout the oestrous cycle in equine endometrial tissue, there were changes observed in the expression of PGES and PGFS, as well as in the production of PGE(2) and PGF(2alpha). In the mare, NO is involved in the secretory function of the endometrium, modulating PGE(2) and PGF(2alpha) production. Even though TNF caused an increase in the production of angiogenic factors and prostaglandins, its complex action in mare uterus should be elucidated.

Is FAS/Fas Ligand System Involved in Equine Corpus Luteum Functional Regression?

Proapoptotic factor Fas ligand (FASL) and its cell surface receptor FAS are tumor necrosis factor superfamily members that trigger apoptosis in different cell types. However, their influence on luteal steroidogenesis is not clearly understood. The aim of the present work was to determine (i) the presence of the cytokine FASL and its receptor FAS in the mares corpus luteum (CL) throughout the luteal phase, as well as (ii) the influence of FASL alone, or together with the cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), on equine luteal cell production of luteotrophic and luteolytic factors, cell viability, and apoptosis. FASL and FAS protein expression and mRNA transcription were evaluated in different luteal stages of the equine CL by Western blotting and real-time PCR assays, respectively. Protein expression and FASL mRNA transcription increased in the late CL. Also, FAS and FASL proteins were present in large steroidogenic and endothelial CL cells throughout the luteal phase, as demonstrated by immunohistochemistry. Equine luteal cells isolated from midluteal phase CL were stimulated without (control) or with exogenous cytokines: FASL (10 ng/ml); TNF+IFNG (10 ng/ml each; positive control) or FASL+TNF+IFNG (10 ng/ml each). FASL clearly inhibited in vitro progesterone and prostaglandin E2 (PGE2) production by equine luteal cells but increased prostaglandin F2alpha (PGF2alpha). Furthermore, FASL effect on equine luteal cell viability depended on the presence of cytokines TNF and IFNG. In conclusion, this study shows the presence of FASL and FAS in the equine CL and suggests their importance in functional luteolysis.

Equine luteal function regulation may depend on the interaction between cytokines and vascular endothelial growth factor: an in vitro study.

ABSTRACT We hypothesized that cytokines influence luteal angiogenesis in mares, while angiogenic factors themselves can also regulate luteal secretory capacity. Therefore, the purpose of this study was to evaluate the role of cytokines—tumor necrosis factor alpha (TNF), interferon gamma (IFNG) and Fas ligand (FASL)—on in vitro modulation of angiogenic activity and mRNA level of vascular endothelial growth factor A (VEGF), its receptor VEGFR2, thrombospondin 1 (TSP1), and its receptor CD36 in equine corpus luteum (CL) throughout the luteal phase. After treatment, VEGF protein expression was determined in midluteal phase (mid) CL cells. The role of VEGF on regulation of luteal secretory capacity was assessed by progesterone (P4) and prostaglandin E2 (PGE2) production and by mRNA levels for steroidogenic enzymes 3-beta-hydroxysteroid dehydrogenase (3betaHSD) and PGE synthase (PGES). In early CL cells, TNF increased angiogenic activity (bovine aortic endothelial cell viability) and VEGF and VEGFR2 mRNA levels and decreased CD36 (real-time PCR relative quantification). In mid-CL cells, TNF increased VEGF mRNA and protein expression (Western blot analysis) and reduced CD36 mRNA levels, while FASL and TNF+IFNG+FASL decreased VEGF protein expression. In late CL cells, TNF and TNF+IFNG+FASL reduced VEGFR2 mRNA, but TNF+IFNG+FASL increased TSP1 and CD36 mRNA. VEGF treatment increased mRNA levels of 3betaHSD and PGES and secretion of P4 and PGE2. In conclusion, these findings suggest a novel auto/paracrine action of cytokines, specifically TNF, on the up-regulation of VEGF for angiogenesis stimulation in equine early CL, while at luteolysis, cytokines down-regulated angiogenesis. Additionally, VEGF stimulated P4 and PGE2 production, which may be crucial for CL establishment.

Caspase-3-mediated apoptosis and cell proliferation in the equine endometrium during the oestrous cycle

Cell proliferation and apoptosis are hormone-dependent physiological processes involved in endometrial growth and regression. The aims of the present study were: (1) to evaluate endometrial cell proliferation using proliferating cell nuclear antigen (PCNA) expression; (2) to evaluate the induction of endometrial cell death by the expression of active caspase-3 and the apoptotic phenotype visualised by DNA fragmentation; and (3) to relate these observations to endometrial tissue dynamics in the equine endometrium throughout the oestrous cycle. Endometria were assigned to follicular and luteal phases based on ovarian structures and plasma progesterone. Cell proliferation and active caspase-3-mediated apoptosis were expressed in both phases of the oestrous cycle. In the luteal phase, PCNA expression was higher than in the follicular phase. Highest PCNA activity was noted in the luminal and glandular structures. Active caspase-3 staining was increased in luminal epithelium and deep glandular cells during the luteal phase. However, in the follicular phase, stromal cells showed greater active caspase-3 expression. Only a few apoptotic endometrial cells were detected by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) and these cells were mostly present in luminal and glandular structures. A simultaneous increase in DNA, cell proliferation and protein synthesis was observed in the endometrium during the mid-luteal phase. This suggests that cell hyperplasia occurs at the time the histotroph is needed for eventual embryo nourishment.

Oestrous cycle-related changes in production of Toll-like receptors and prostaglandins in the canine endometrium

The objectives of this study were to evaluate the following events in the canine endometrium over the course of the oestrous cycle: (i) the transcriptional profiles of genes encoding the Toll-like receptors (TLR1-TLR7 and TLR9); (ii) the transcription and protein expression levels of TLR2 and TLR4; (iii) the gene transcription profile of prostaglandin synthesis enzymes (PTGS2, PGES and PGFS); (iv) the response pattern of PGF(2α) and PGE(2) following exposure of endometrial explants to LPS and LTA. TLR1-TLR7 and TLR9 genes were transcribed in the endometrium of bitches throughout the oestrous cycle, which indicates that TLR-mediated immune surveillance is an important component of the defence mechanisms within the uterus. Canine endometrial mRNA and protein expression of TLR2 and TLR4 was up-regulated at the late dioestrus and anoestrus and was the lowest in the follicular phase and early dioestrus. The decreased mRNA and protein levels observed at early dioestrus may favour implantation, but may also be linked to the high prevalence of pyometra at this stage of the oestrous cycle. After LPS and LTA stimulation, endometrial explants produced more PGF(2α) than PGE(2), which may be related to the early demise of the corpus luteum observed in vivo in canine pyometra cases. Overall, these results indicate that TLRs are involved in the activation of the inflammatory response associated with pyometra in the bitch. TLRs may therefore be therapeutic targets for the control of uterine bacterial infections in the bitch and potentially in other species.

Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.

Progesterone receptors and proliferating cell nuclear antigen expression in equine luteal tissue

Steroid hormones act via specific receptors, and these play an important physiological role in the ovary. The objective of this study was to evaluate the cellular distribution of progesterone receptors and their staining intensity in different equine luteal structures during the breeding season, as well as their relationship to luteal cell composition, cell proliferation pattern and plasma progesterone (P4) concentration. There was an increase in proliferating cell nuclear antigen (PCNA) expression in large luteal cells from the corpus hemorrhagicum (CH) to mid-luteal phase, followed by a decrease toward the late luteal stage. In the CH, the number of large luteal cells was lower than in other structures. Only large luteal cells showed positive staining for P(4) receptors. An increase in staining intensity for P(4) receptors was observed between CH and mid-phase corpus luteum, and CH and late-phase corpus luteum. Synthesis of P(4) started at a very early stage of the luteal structure and was accompanied by an increase in P(4) receptors and PCNA expression, and proliferation of large luteal cells, until mid-luteal phase. These data suggest that large luteal cells might play an important role in the regulation or synthesis of P(4) in equine luteal structures.