Transcriptome analysis reveals differences in mechanisms regulating cessation of luteal function in pregnant and non-pregnant dogs.
ABSTRACT: 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.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.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.
Project description: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, and following antigestagen (Aglepristone)- treatment. The analysis of transcriptomes presented herein supports the hypothesis describing luteal regression in non-pregnant dogs as a degenerative process devoid of the acute luteolytic principle and without an acute involvement of the immune system observed prepartum. The contribution of the immune system seems, however, to be critical in the PGF2alpha-mediated active prepartum luteolysis, which appears to be an acute immune process. The antigestagen-medited effects point towards the withdrawal of the luteotropic function of P4 with lesser involvement of immune system than during natural luteolysis. In summary, a deeper insights have been obtained into possible endocrine, paracrine and autocrine mechanisms governing the luteal life span in the domestic dog during pregnancy and in non-pregnant cycles. Overall design: Corpora lutea (CL) from clinically healthy, cross-breed bitches (aged 2-8 years) were used representing the following experimental groups: (Group-1) mid-pregnancy (days 35-40); (Group-2) active prepartum luteolysis; (Group-3) antigestagen-treated mid-gestation group (days 40-45); (Group-4) non-pregnant bitches at late CL regression (day 65 after ovulation). In all dogs the time of ovulation was determined by measurements of P4 (> 5ng/ml) and by vaginal histology. Pregnant dogs were mated 2 days after ovulation (Day 0). To determine active prepartum luteolysis (Group-2), P4 concentrations in peripheral blood plasma were measured at 6 h intervals beginning on day 58 of pregnancy; when P4 levels in 3 consecutive measurements decreased below 2-3 ng/ml, the tissue material was collected. In Group 3, prepartum luteolysis/abortion was induced with the antigestagen aglepristone (Alizine; 10mg/Kg bw, 2x/24 h apart) and the tissues were collected 24h after the second application. All dogs underwent routine ovariohysterectomy. For RNA preservation, immediately after surgery the CL tissue were trimmed of surrounding ovarian tissue, washed with phosphate-buffered saline (PBS) and placed in RNAlater® (Ambion Biotechnologie GmbH, Wiesbaden, Germany) for 24 h at +4°C, and then stored at -80°C until use.
Project description:BACKGROUND: Endocrine mechanisms governing canine reproductive function remain still obscure. Progesterone (P4) of luteal origin is required for maintenance of pregnancy. Corpora lutea (CL) are gonadotrop-independent during the first third of dioestrus; afterwards prolactin (PRL) is the primary luteotropic factor. Interestingly, the increasing PRL levels are accompanied by decreasing P4 concentrations, thus luteal regression/luteolysis occurs in spite of an increased availability of gonadotropic support. PRL acts through its receptor (PRLr), the expression of which has not yet been thoroughly investigated at the molecular and cellular level in the dog. METHODS: The expression of PRLr was assessed in CL of non-pregnant dogs during the course of dioestrus (days 5, 15, 25, 35, 45, 65 post ovulation; p.o.) as well as in CL, the utero/placental compartments (Ut/Pl) and interplacental free polar zones (interplacental sites) from pregnant dogs during the pre-implantation, post-implantation and mid-gestation period of pregnancy and during the normal and antigestagen-induced luteolysis. Expression of PRLr was tested by Real Time PCR, immunohistochemistry and in situ hybridization. RESULTS: In non-pregnant CL the PRLr expression was significantly upregulated at day 15 p.o. and decreased significantly afterwards, towards the end of dioestrus. CL of pregnancy showed elevated PRLr expression until mid gestation while prepartal downregulation was observed. Interestingly, placental but not interplacental expression of PRLr was strongly time-related; a significant upregulation was observed towards mid-gestation. Within the CL PRLr was localized to the luteal cells; in the Ut/Pl it was localized to the fetal trophoblast and epithelial cells of glandular chambers. Moreover, in mid-pregnant animals treated with an antigestagen, both the luteal and placental, but not the uterine PRLr were significantly downregulated. CONCLUSIONS: The data presented suggest that the luteal provision of P4 in both pregnant and non-pregnant dogs may be regulated at the PRLr level. Furthermore, a role of PRL not only in maintaining the canine CL function but also in regulating the placental function is strongly suggested. A possible functional interrelationship between luteal P4 and placental and luteal PRLr expression also with respect to the prepartal luteolysis is implied.
Project description:Next-generation sequencing (RNA-Seq) was performed on canine placenta Placental samples collected at natural prepartum luteolysis were compared with those samples obtained from dogs at mid-pregnancy stage. Furthermore, in order to better understand the involvement of P4 and PGR-dependent downstream regulatory mechanisms during initiation of parturition in the dog, samples derived from dogs in which prepartum luteolysis/abortion was induced with the antigestagen aglepristone at mid-pregnancy, were included. With this approach the aim was to acquire new information that could be translated to clinical and breeding practice for more accurate patient management. The sample set derived from antigestagen-treated dogs was compared with mid-pregnant non-treated group that served as non-treated control. Finally, to identify differences in molecular events occurring in the placenta prior to natural parturition and/or abortion, placental transcriptomes of both groups were compared. Overall design: Placentae from nine (n=9) clinically healthy, cross-breed bitches (aged 2-8 years) were included in this study. Animals were assigned to following experimental groups: 1) mid-gestation (days 35-40 of pregnancy; n=3); 2) natural prepartum luteolysis (n=3); 3) antigestagen-induced luteolysis (n=3). Dogs were mated 2 days after ovulation, which was determined by vaginal cytology and measurements of serum P4 concentrations (> 5ng/ml). Day of mating represented Day 0 of gestation. The time of natural prepartum luteolysis (Group 2) was ascertained by measurements of serum P4 beginning on day 58 of pregnancy, in 6h intervals. Under physiological conditions, parturition in dogs occurs 12-42h following the luteolytic P4 drop. Thus, when P4 levels dropped below 3ng/ml in three consecutive measurements indicating its prepartum decrease, the surgery was performed and the tissue samples were collected. Additionally, abortion was induced in bitches at mid-pregnancy (Group 3) using P4-receptor (PGR) blocker, aglepristone (Alizine®, Virbac, Bad Oldesloe, Germany) in dosage of 10 mg/kg bw, 2 times in 24h interval. The surgery and tissue collections were done 24h after the second treatment. All dogs used for the study were subjected to routine ovariohysterectomy. Following surgery, placentae were separated from uteri, rinsed with phosphate-buffered saline (PBS) and immersed in RNAlater® for 24h at +4°C. After 24h, tissue material was stored at -80°C until RNA isolation.
Project description:By acting through its receptors (RXFP1, RXFP2), relaxin (RLN) exerts species-specific effects during pregnancy; possible luteotropic effects through stimulation of prolactin (PRL) release have been suggested. In the domestic dog (Canis lupus familiaris) serum PRL increases in pregnant bitches shortly after RLN appears in the circulation, and a possible functional relationship between the RLN and the PRL systems in regulating progesterone secretion has been implied. Therefore, here (Study 1) the luteal expression and localization of the RLN system was investigated by immunohistochemistry using custom-made antibodies and semi-quantitative PCR, at selected time points during gestation: pre-implantation (d. 8-12), post-implantation (d. 18-25), mid-gestation (d. 35-40) and at normal and antigestagen-induced luteolysis. Further, (Study 2) hypophyseal expression of the RLN system and its spatial association with PRL was assessed. Luteal expression of RLN, but not of its receptors, was time-dependent: it increased significantly following implantation towards mid-gestation and decreased at prepartum. Antigestagen treatment resulted in downregulation of RLN and RXFP2. Whereas RLN was localized in steroidogenic cells, RXFP1 and RXFP2 also stained strongly in macrophages and vascular endothelial cells. The RLN system was detected in the canine adenohypophysis and was co-localized with PRL in hypophyseal lactotrophs. The intraluteal RLN seems to be involved in regulating the canine corpus luteum (CL) in a time-dependent manner. The presence of RLN family members in the adenohypophysis implies their possible involvement in regulating the availability of PRL and other pituitary hormones.
Project description:Utero-placental (Ut-Pl) angiogenesis and blood flow are fundamental for successful outcome of pregnancy. They are controlled by numerous vasodilator and vasoconstrictor systems such as endothelins (EDNs) and the renin angiotensin system. Dogs possess an invasive type of placentation, classified as endotheliochorial. Despite increasing knowledge regarding canine Ut-Pl function, little information exists on uterine and placental vascular activity during initiation, maintenance and termination of pregnancy in this species. The current study investigated expression of EDNs and their receptors (EDNRA and EDNRB) in the pre-implantation uterus and Ut-Pl compartments during gestation and at normal parturition, as well as in mid-pregnant dogs treated with the antigestagen aglepristone. The Ut-Pl mRNA expression of EDN1 and EDNRA was constant until mid-gestation and increased significantly during prepartum luteolysis. In contrast, EDN2 was highest pre-implantation and decreased following placentation, remaining low thereafter. Expression of the EDN-activating enzyme ECE1 and mRNA of EDNRB increased towards mid-gestation and was further elevated at prepartum luteolysis. Antigestagen treatment resulted in increased levels of EDN1 and EDNRA. At the cellular level, the uterine expression of EDN1, ECE1 and EDNRB was found predominantly in the endometrial surface and glandular epithelial cells; uterine signals for EDNRA were weak. In Ut-Pl all targets were mainly localized in the placenta fetalis, with syncytiotrophoblast staining stronger for ECE1 and EDNRB. In contrast, EDNRA stained strongly at the base of the placental labyrinth. Expression and localization of EDNs (EDN1, -2), EDN receptors and ECE1 in the placenta fetalis suggests their involvement in the trophoblast invasion and proliferation.
Project description:Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20?-hydroxysteroid dehydrogenase (20?-HSD) catabolizes P4 into its biologically inactive form, 20?-hydroxyprogesterone (20?-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20?-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3?-hydroxysteroid dehydrogenase (3?-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3?-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20?-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.
Project description:BACKGROUND: In higher primates, although LH/CG play a critical role in the control of corpus luteum (CL) function, the direct effects of progesterone (P4) in the maintenance of CL structure and function are unclear. Several experiments were conducted in the bonnet monkey to examine direct effects of P4 on gene expression changes in the CL, during induced luteolysis and the late luteal phase of natural cycles. METHODS: To identify differentially expressed genes encoding PR, PR binding factors, cofactors and PR downstream signaling target genes, the genome-wide analysis data generated in CL of monkeys after LH/P4 depletion and LH replacement were mined and validated by real-time RT-PCR analysis. Initially, expression of these P4 related genes were determined in CL during different stages of luteal phase. The recently reported model system of induced luteolysis, yet capable of responsive to tropic support, afforded an ideal situation to examine direct effects of P4 on structure and function of CL. For this purpose, P4 was infused via ALZET pumps into monkeys 24 h after LH/P4 depletion to maintain mid luteal phase circulating P4 concentration (P4 replacement). In another experiment, exogenous P4 was supplemented during late luteal phase to mimic early pregnancy. RESULTS: Based on the published microarray data, 45 genes were identified to be commonly regulated by LH and P4. From these 19 genes belonging to PR signaling were selected to determine their expression in LH/P4 depletion and P4 replacement experiments. These 19 genes when analyzed revealed 8 genes to be directly responsive to P4, whereas the other genes to be regulated by both LH and P4. Progesterone supplementation for 24 h during the late luteal phase also showed changes in expression of 17 out of 19 genes examined. CONCLUSION: These results taken together suggest that P4 regulates, directly or indirectly, expression of a number of genes involved in the CL structure and function.
Project description:In the non-pregnant dog, ovarian cyclicity is independent of a uterine luteolysin. This is in contrast to pregnant animals where a prepartum increase of luteolytic PGF2? occurs, apparently originating in the pregnant uterus. Recently, the placenta as a source of prepartum prostaglandins (PGs) was investigated, indicating fetal trophoblast cells as the likely main source. However, the possible contribution of uterine interplacental tissues to the production of these hormones has not yet been thoroughly examined in the dog.Several key factors involved in the production and/or actions of PGs were studied: cyclooxygenase 2 (COX2, PTGS2), PGF2?-synthase (PGFS/AKR1C3), PGE2-synthase (PGES), and the respective receptors FP (PTGFR), EP2 (PTGER2) and EP4 (PGTER4), 15-hydroxyprostaglandin dehydrogenase (HPGD), PG-transporter (PGT, SLCO2A1) and progesterone receptor. Their expression and localization patterns were assessed by Real Time PCR and immunohistology in the interplacental uterine sites from pregnant dogs during the pre-implantation period (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and during antigestagen-induced luteolysis/abortion.Whereas only low COX2 expression was observed in uterine samples at all the selected time points, expression of PGFS/AKR1C3 strongly increased post-implantation. A gradual increase in PGES-mRNA expression was noted towards mid-gestation. FP-mRNA expression decreased significantly with the progression of pregnancy until mid-gestation. This was associated with clearly detectable expression of HPGD, which did not change significantly over time. The expression of FP and EP2-mRNA decreased significantly over time while EP4-mRNA expression remained unaffected. The antigestagen-treatment led to a significant increase in expression of COX2, PGES, EP2 and PGT (SLCO2A1) mRNA. COX2 was localized predominantly in the myometrium. The expression of PGFS/AKR1C3, which was unchanged, was localized mostly to the surface luminal epithelium. The expression of EP4, PGT and HPGH did not change during treatment, they were co-localized with PGES and EP2 in all uterine compartments.The data clearly demonstrate the basic capability of the canine pregnant uterus to produce and respond to PGs and suggests their functions both as local regulatory factors involved in the establishment and maintenance of pregnancy, as well as potential contributors to the process of parturition, supporting the myometrial contractility associated with fetal expulsion.
Project description:Prostaglandin F2alpha (PGF) causes luteolysis of the pig corpus luteum (CL) only after Day 12 of the estrous cycle. Recent evidence indicates that progesterone (P4) may protect the CL from cell death. The present study tested the hypothesis that acute inhibition of P4 by treatment with epostane (EPO; 3betaHSD inhibitor) in CL lacking luteolytic capacity (Day 9 CL) will allow PGF to induce responses associated with luteolysis. Multiple PGF-induced responses were evaluated, including genes involved in production of PGF and estradiol-17beta, apoptosis (caspase 3), and transcription (FOSB). These responses are associated with PGF-induced luteolysis and do not normally occur in CL lacking luteolytic capacity. Animals on Day 7 after estrus were divided into four groups: 1) control (C), 2) PGF, 3) EPO, and 4) PGF plus EPO (PGF+EPO). Treatment with EPO (10 mg/kg) or vehicle was given every 12 h for 36 h. Treatment with PGF (25 mg) or vehicle was given at 38 h, and CL were collected from all animals at 48 h. Some CL from each animal were frozen in liquid nitrogen for mRNA and protein analysis. Remaining CL were incubated in media for 2 h for determination of P4 and PGF production. EPO dramatically decreased production of P4 by luteal tissue (ng/mg tissue) by 90% and 95% in EPO and PGF+EPO groups, respectively, compared to C (P < 0.01). Low production of PGF by luteal tissue was found in C, PGF, and EPO groups; however, treatment with PGF+EPO dramatically increased (782%) luteal PGF production. Similar to intraluteal PGF production, increased mRNA for cyclooxygenase 2 (PTGS2) and phospholipase A2 (group IB; PLA2G1B) was found in the PGF+EPO, but not in the EPO or PGF, group. Aromatase (CYP19A1) mRNA was not induced by PGF or EPO; however, PGF+EPO caused a more than 40-fold increase in CYP19A1 mRNA (P < 0.01). CASP3 mRNA was increased (P < 0.01) by EPO (3.4-fold) and by PGF (2.7-fold) but was most dramatically increased by PGF+EPO (5.3-fold), whereas caspase activity was only increased by PGF (1.5-fold) or PGF+EPO (2.2-fold). Thus, these data support the hypothesis that elimination of the protective effect of intraluteal P4 does not directly cause luteolysis of the early CL but allows PGF to induce luteolytic responses in CL lacking luteolytic capacity.
Project description:Luteolysis of the corpus luteum (CL) during nonfertile cycles involves a cessation of progesterone (P4) synthesis (functional regression) and subsequent structural remodeling. The molecular processes responsible for initiation of luteal regression in the primate CL are poorly defined. Therefore, a genomic approach was used to systematically identify differentially expressed genes in the rhesus macaque CL during spontaneous luteolysis. CL were collected before [d 10-11 after LH surge, mid-late (ML) stage] or during (d 14-16, late stage) functional regression. Based on P4 levels, late-stage CL were subdivided into functional-late (serum P4 > 1.5 ng/ml) and functionally regressed late (FRL) (serum P4 < 0.5 ng/ml) groups (n = 4 CL per group). Total RNA was isolated, labeled, and hybridized to Affymetrix genome microarrays that contain elements representing the entire rhesus macaque transcriptome. With the ML stage serving as the baseline, there were 681 differentially expressed transcripts (>2-fold change; P < 0.05) that could be categorized into three primary patterns of expression: 1) increasing from ML through FRL; 2) decreasing from ML through FRL; and 3) increasing ML to functional late, followed by a decrease in FRL. Ontology analysis revealed potential mechanisms and pathways associated with functional and/or structural regression of the macaque CL. Quantitative real-time PCR was used to validate microarray expression patterns of 13 genes with the results being consistent between the two methodologies. Protein levels were found to parallel mRNA profiles in four of five differentially expressed genes analyzed by Western blot. Thus, this database will facilitate the identification of mechanisms involved in primate luteal regression.