Differential cellular localization of galectin-1 and galectin-3 in the regressing corpus luteum of mice and their possible contribution to luteal cell elimination.
ABSTRACT: Galectin-1 and galectin-3, beta-galactoside-binding lectins, are predominantly expressed in the regressing corpus luteum (CL) of mouse ovary. This study revealed the expression patterns and cellular localizations of galectins during CL formation and regression by ISH and IHC. Galectin-1 mRNA expression temporarily increased in active CL, preceding the expression of progesterone degradation enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which represents functional luteolysis. The expressions of both galectin-1 and galectin-3 remarkably increased in the structurally regressing CL, which vigorously expressed 20alpha-HSD and contained abundant apoptotic luteal cells. Ultrastructurally, galectin-1- and galectin-3-immunoreactive cells were identified as fibroblasts and infiltrating macrophages, respectively. In addition, some populations of luteal cells themselves expressed galectin-3 in regressing CL and formed unique demarcation membranes in the cytoplasm, showing a non-typical apoptotic feature. Ovary of adult mice with repeated estrus cycles contained CL of three different generations. Among them, the old CL formed during previous estrus cycles consisted of galectin-3-positive luteal cells. The galectin-3-positive old CL was resistant to apoptosis and seemed to be eliminated by a mechanism different from apoptosis. The stage- and cell-specific expression of galectin in CL suggests its differential contribution to luteolysis, and this expression may be mediated by major regulatory molecules of CL function, prolactin and/or prostaglandin F2alpha.
Project description:In mammals, the corpus luteum (CL) is an essential endocrine gland for the establishment and maintenance of pregnancy. If pregnancy is not established, the CL regresses and disappears rapidly from the ovary. A possible explanation for the rapid disappearance of the CL is that luteal cells are transported from the ovary via lymphatic vessels. Here, we report the presence of cells positive for 3?-hydroxysteroid dehydrogenase (3?-HSD), an enzyme involved in progesterone synthesis, in the lumen of lymphatic vessels at the regressing luteal stage and in the lymphatic fluid collected from the ovarian pedicle ipsilateral to the regressing CL. The 3?-HSD positive cells were alive and contained lipid droplets. The 3?-HSD positive cells in the lymphatic fluid were most abundant at days 22-24 after ovulation. These findings show that live steroidogenic cells are in the lymphatic vessels drained from the CL. The outflow of steroidogenic cells starts at the regressing luteal stage and continues after next ovulation. The overall findings suggest that the complete disappearance of the CL during luteolysis is involved in the outflow of luteal cells from the CL via ovarian lymphatic vessels.
Project description:BACKGROUND: During female reproductive cycles, a rapid fall in circulating progesterone (P4) levels is one of the earliest events that occur during induced luteolysis in mammals. In rodents, it is well recognized that during luteolysis, P4 is catabolized to its inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP) by the action of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) enzyme and involves transcription factor, Nur77. Studies have been carried out to examine expression of 20alpha-HSD and its activity in the corpus luteum (CL) of buffalo cow. METHODS: The expression of 20alpha-HSD across different bovine tissues along with CL was examined by qPCR analysis. Circulating P4 levels were monitored before and during PGF2alpha treatment. Expression of 20alpha-HSD and Nur77 mRNA was determined in CL at different time points post PGF2alpha treatment in buffalo cows. The chromatographic separation of P4 and its metabolite, 20alpha-OHP, in rat and buffalo cow serum samples were performed on reverse phase HPLC system. To further support the findings, 20alpha-HSD enzyme activity was quantitated in cytosolic fraction of CL of both rat and buffalo cow. RESULTS: Circulating P4 concentration declined rapidly in response to PGF2alpha treatment. HPLC analysis of serum samples did not reveal changes in circulating 20alpha-OHP levels in buffalo cows but serum from pseudo pregnant rats receiving PGF2alpha treatment showed an increased 20alpha-OHP level at 24 h post treatment with accompanying decrease in P4 concentration. qPCR expression of 20alpha-HSD in CL from control and PGF2alpha-treated buffalo cows showed higher expression at 3 and 18 h post treatment, but its specific activity was not altered at different time points post PGF2alpha treatment. The Nur77 expression increased several fold 3 h post PGF2alpha treatment similar to the increased expression observed in the PGF2alpha-treated pseudo pregnant rats which perhaps suggest initiation of activation of apoptotic pathways in response to PGF2alpha treatment. CONCLUSIONS: The results taken together suggest that synthesis of P4 appears to be primarily affected by PGF2alpha treatment in buffalo cows in contrast to increased metabolism of P4 in rodents.
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:The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone into its inactive form, 20alpha-hydroxyprogesterone. To gain information about the exact sites of 20alpha-HSD mRNA expression, we performed in situ hybridization using a (35)S-labeled cRNA probe in tissues of adult mice of both sexes. 20alpha-HSD mRNA was expressed in both male and female gonads. In the ovary, high expression was found in luteal cells of corpora lutea, while much lower expression could be detected in granulosa cells of growing follicles. In the testis, a specific hybridization signal was detected only in Leydig cells. In the female reproductive tract, 20alpha-HSD mRNA was found in the epithelial cells of the uterine cervix. In the adrenal cortex, only the zona reticularis exhibited specific radiolabeling, the expression being very high in the female and very low in the male. In the skin, specific labeling was restricted to sebaceous glands, the hybridization signal being much higher in the female than in the male. In the liver, 20alpha-HSD mRNA was found in hepatocytes, with a higher degree of expression in the female. In the kidney, specific labeling was observed in the epithelial cells of distal convoluted tubules, the signal being also much more striking in the female than in the male. In non-reproductive tissues, it clearly appears that the expression of 20alpha-HSD mRNA is higher in the female than in the male, suggesting that 20alpha-HSD may play an important role in reducing the intracellular concentration of progesterone originating from the circulation at a much higher level in the female.
Project description:The mechanisms regulating the function and regression of the corpus luteum (CL) have not yet been elucidated in detail. The regressed CL of cows was previously reported to be filled with unusual vessels like arteriovenous anastomosis (AVA); however how these vessels are being established during luteolysis remains unknown.The bovine CL at different luteal stages and regressing bovine CL induced by prostaglandin F2? (PGF) were histologically analyzed using light and electron microscopic levels. The changes in mRNA expression of genes encoding ?-smooth muscle actin (SMA; Acta2) and transforming growth factor ?1 (Tgfb1) in luteal tissues were analyzed by quantitative RT-PCR.AVA-like vessels appeared in the regressed CL with a diameter less than 1.5 cm in which no functional luteal cells and macrophages were observed. Epithelioid cells in the AVA-like vessel wall were immunoreactive for SMA, and the lumen of the vessels were narrow. Immunoreaction for SMA was found in the tunica media of typical arteries and arterioles, and pericytes around capillary vessel. Cells with elongated cytoplasmic processes -resident fibroblasts expressing vimentin- distributed in the CL parenchyma without any association with blood vessels are also immunoreactive for SMA, and accumulated around arteries and arterioles during the late-luteal stage. In the regressed CL, walls of arteries and arterioles consisted of more than two layers of epithelioid cells positive for both SMA and desmin, suggesting that they are myofibroblasts transformed from fibroblasts. The percentage of the area positive for SMA and the mRNA expression of Acta2 were significantly increased in the regressed CL; however, they did not alter when a luteolytic dose of PGF was injected in vivo and collected within 24 h after the injection. On the other hand, Tgfb1, a known regulator for myofibroblast transformation, was significantly increased in PGF-induced regressing CL as well as in the CL during the late-luteal stage.SMA-positive myofibroblasts accumulates around the arteries and arterioles to form AVA-like vessels during luteolysis in cows. PGF indirectly regulates myofibroblast transformation through enhancing the expression of TGF?1. These peculiar AVA-like vessels may be involved in the regulation of blood flow in the bovine CL during luteolysis.
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:Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3alpha-HSD catalyzes the inactivation of androgens in the prostate by converting 5alpha-dihydrotestosterone to 3alpha-androstanediol, where excess 5alpha-dihydrotestosterone is implicated in prostate disease. By contrast, 20alpha-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20alpha-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3alpha-HSDs and 20alpha-HSDs belong to the aldo-keto reductase superfamily and share 67% amino acid sequence identity yet show positional and stereospecificity for the formation of secondary alcohols on opposite ends of steroid hormone substrates. The crystal structure of 3alpha-HSD indicates that the mature steroid binding pocket consists of 10 residues located on five loops, including loop A and the mobile loops B and C. 3alpha-HSD was converted to 20alpha-HSD by replacing these loops with those found in 20alpha-HSD. However, when pocket residues in 3alpha-HSD were mutated to those found in 20alpha-HSD altered specificity was not achieved. Replacement of loop A created a 17beta-HSD activity that was absent in either 3alpha- or 20alpha-HSD. Once loops A and C were replaced, the chimera had both 3alpha- and 20alpha-HSD activity. When loops A, B, and C were substituted, 3alpha-HSD was converted to a stereospecific 20alpha-HSD with a resultant shift in k(cat)/K(m) for the desired reaction of 2 x 10(11). This study represents an example where sex hormone specificity can be changed at the enzyme level.
Project description:To explore chorionic gonadotrophin (CG)-regulated gene expression in the primate corpus luteum (CL), adult female rhesus macaques were treated in a model of simulated early pregnancy (SEP). Total RNA was isolated from individual CL after specific intervals of exposure (1, 3, 6 and 9 days) to recombinant hCG in vivo and hybridized to Affymetrix™ GeneChip Rhesus Macaque Genome Arrays. The mRNA levels of 1192 transcripts changed ?2-fold [one-way ANOVA, false discovery rate (FDR) correction; P< 0.05] during SEP when compared with Day 10 untreated controls. Real-time PCR validation indicated that 15 of 17 genes matched in expression pattern between PCR and microarray. Protein levels of three genes identified as CG-sensitive, CYP19A1 (aromatase), PGRMC1 (progestin-binding protein) and STAR (steroidogenic acute regulatory protein) were quantified by western blot analysis. To further analyze global changes in gene expression induced by CG exposure, luteal gene expression was compared between SEP (rescued) and regressing CL, utilizing previously banked GeneChip data from the luteal phase of the menstrual cycle. Expression patterns and mRNA levels were analyzed between time-matched intervals. Transcripts for 7677 mRNAs differed in expression patterns ?2-fold (one-way ANOVA, FDR correction; P< 0.05) between the hCG-exposed (SEP) CL and regressing CL. Regressed CL (at menses) were most unlike all other CL. Pathway analysis of significantly affected transcripts was performed; the pathway most impacted by CG exposure was steroid biosynthesis. Further comparisons of the genome-wide changes in luteal gene expression during CG rescue and luteolysis in the natural menstrual cycle should identify additional key regulatory pathways promoting primate fertility.
Project description:We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2? (PGF), interferon ? (IFNG) and tumor necrosis factor ? (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8-12 after ovulation) were cultured with PGF (0.01, 0.1, 1 ?M), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 ?M PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One ?M PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.
Project description:In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols.