Estradiol-17ß Regulates Expression of Luteal DNA Methyltransferases and Genes Involved in the Porcine Corpus Luteum Function In Vivo.

The corpus luteum (CL) is a temporary endocrine gland vital for pregnancy establishment and maintenance. Estradiol-17ß (E2) is the major embryonic signal in pigs supporting the CL's function. The mechanisms of the luteoprotective action of E2 are still unclear. The present study aimed to determine the effect of E2 on luteal expression of factors involved in CL function. An in vivo model of intrauterine E2 infusions was applied. Gilts on day 12 of pregnancy and the estrous cycle were used as referential groups. Concentrations of E2 and progesterone were elevated in CLs of gilts receiving E2 infusions, compared to placebo-treated gilts. Estradiol-17ß stimulated luteal expression of DNA-methyltransferase 1 (DNMT1), but decreased expression of DNMT3B gene and protein, as well as DNMT3A protein. Similar results for DNMT3A and 3B were observed in CLs on day 12 of pregnancy compared to day 12 of the estrous cycle. Intrauterine infusions of E2 altered luteal expression of the genes involved in CL function: PTGFR, PTGES, STAR, HSD17B1, CYP19A1, and PGRMC1. Our findings indicate a role for E2 in expression regulation of factors related to CL function and a novel potential for E2 to regulate DNA methylation as putative physiological mechanisms controlling luteal gene expression.

Prolactin modulates luteal regression from the coeliac ganglion via the superior ovarian nerve in the late-pregnant rat.

There is considerable evidence of the neuroendocrine control involved in luteal regression in the rat. In addition, circulating prolactin (PRL), which increases during the night before parturition, may gain access to the coeliac ganglion (CG), indirectly impacting the physiology of the ovary because of the known connection between the CG and the ovary via the superior ovarian nerve (SON). In this work we investigated in the CG-SON-ovary system and whether PRL added to the CG has an impact, indirectly via the SON, on luteal regression on Day 21 of pregnancy. The system was incubated without (control) or with PRL added to the CG. We measured the ovarian release of progesterone (P), oestradiol and prostaglandin F2 alpha (PGF2α) by radioimmunoassay, and nitrites (NO) by the Griess method. Luteal mRNA expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 20α-HSD, aromatase, inducible nitric oxide synthase (iNOS) and apoptosis regulatory factors was analysed by reverse transcription-polymerase chain reaction. P release, the expression of Bcl-2 and the Bcl-2:Bax ratio was lower than control preparations, while the expression of 20α-HSD and the release of NO and PGF2α were higher in the experimental group. In conclusion, PRL acts at the CG and, by a neural pathway, modulates luteal function at the end of pregnancy.

Expression of aldo-keto reductase 1C23 in the equine corpus luteum in different luteal phases.

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. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-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 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3ß-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-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.

Biosynthesis and degradation of canine placental prostaglandins: prepartum changes in expression and function of prostaglandin F2α-synthase (PGFS, AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD).

There is no distinct explanation of the mechanism for the prepartal prostaglandin F2alpha (PGF2alpha) increase in pregnant dogs. Although the PGF2alpha-synthase (PGFS [AKR1C3]) mRNA expression and localization profiles have been previously investigated in canine utero/placental compartments, the availability and biochemical activity of the PGFS (AKR1C3) protein remain unknown. In order to better understand the regulation of canine uterine PGF2alpha availability and eventual prepartum release in luteolytic amounts in dogs, canine-specific PGFS (AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD) antibodies were generated and used to characterize the expression, cellular localization, and biochemical properties of PGFS (AKR1C3) and HPGD in the utero/placental compartments and corpus luteum throughout pregnancy and at prepartum luteolysis. PGFS (AKR1C3) expression was weak or absent in luteal samples. Uterine PGFS (AKR1C3) was up-regulated postimplantation and declined prepartum. The utero/placental expression of PGFS (AKR1C3) was identified in the superficial uterine glands throughout gestation and in the trophoblast cells within the feto-maternal contact zone during placentation, suggesting a possible role for PGFS (AKR1C3) in the trophoblast invasion. Utero-placental HPGD was up-regulated until postimplantation, lower at midgestation, and greatly suppressed at prepartum. Expression was routinely identified in the endometrial surface and glandular epithelia, and positive signals were also observed in the trophoblast cells at the feto-maternal contact zone. The biochemical activity of recombinant PGFS (AKR1C3) and HPGD was confirmed after its expression in a heterologous system. The colocalization of HPGD with PGFS (AKR1C3) expression suggests a modulatory role for HPGD as a gatekeeper of the supply of prostaglandin in the pregnant canine uterus.

Evidence for a luteotropic role of peroxisome proliferator-activated receptor gamma: expression and in vitro effects on enzymatic and hormonal activities in corpora lutea of pseudopregnant rabbits.

The expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and its role in corpora lutea (CL) function were studied in pseudopregnant rabbits. Corpora lutea were collected at an early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. Immunohistochemistry found evidence for the presence of PPARgamma in the perinuclear cytoplasm and nucleus of all the luteal cells; immunoreactivity decreased from the early to the late stage, with immunonegativity of the nuclei of late stage CL. PPARgamma mRNA transcript was expressed in all the luteal stages with the lowest level in the late stage. In CL cultured in vitro, the PPARgamma agonist (15-deoxy delta12,14 prostaglandin J2 [15d-PGJ2], 200 nM) increased and the antagonist (T0070907, 50 nM) decreased progesterone secretion at early and midluteal stages, whereas 15d-PGJ2 reduced and T0070907 increased PGF2alpha at the same stages. Prostaglandin-endoperoxide synthase 2 (PTGS2) activity was reduced by 15d-PGJ2 and increased by T0070907 in CL of early and midluteal stages. Conversely, 15d-PGJ2 increased and T0070907 reduced 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in early and midluteal stage CL. PGE2 in vitro secretion as well as PTGS1 and 20alpha-HSD enzymatic activities were not affected by 15d-PGJ2 and T0070907 in any CL types. These results indicate that PPARgamma plays a luteotropic role in pseudopregnant rabbits, through PTGS2 down-regulation and 3beta-HSD up-regulation, with a consequent PGF2alpha decrease and progesterone increase.

Role of tumor necrosis factor-α, interferon-γ and Fas-ligand on in vitro nitric oxide activity in the corpus luteum.

Normal reproductive function involves the expression of inflammatory mediators. Regarding the corpus luteum (CL), cytokines promote the cross-talk between immune, vascular and steroidogenic cells, among others. Moreover, TNF, IFNG and FASL were shown to regulate equine CL establishment and regression. We hypothesized that cytokines action on equine CL may be mediated by nitric oxide (NO), through the regulation of endothelial NO synthase (eNOS) expression. TNF increased eNOS mRNA level and NO metabolite (nitrite) production during CL growth. Cytokines combined action (TNF+IFNG+FASL) promoted eNOS protein upregulation in mid-CL and nitrite production in mid and late-CL. However, in late-CL, TNF alone decreased nitrite secretion. These results indicate that in equine CL, cytokines TNF, IFNG and FASL regulate NO activity, via eNOS expression modulation.

Analysis of 20alpha-hydroxysteroid dehydrogenase expression in the corpus luteum of the buffalo cow: effect of prostaglandin F2-alpha treatment on circulating 20alpha-hydroxyprogesterone levels.

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.

P450-aromatase mRNA is expressed in the corpus luteum (CL) of the non-pregnant sheep and goat: the expression of the enzyme is present throughout pregnancy in the goat CL.

In most mammals, the corpus luteum (CL) and placenta are the major sources of progesterone. The goat pregnancy depends on the presence of CL after mid-gestation, while sheep pregnancy does not. The expression and distribution of P450-aromatase (P450-Aro) mRNA throughout gestation has not been investigated in the goat CL and partially in the sheep CL. The present research was designed to characterize the expression of P450-Aro mRNA in small ruminant CL with emphasis in the goat. For this purpose, ovaries from Criollo goats and Pelibuey sheeps were analysed using in situ reverse transcription-polymerase chain reaction (RT-PCR) for the histological detection of P450-Aro transcripts. In addition, P450-Aro expression was determined by in vitro RT-PCR. In situ RT-PCR studies showed that the goat and sheep CL were rich in cells positive for P450-Aro mRNA. We have also found in vitro RT-PCR expression of P450-Aro mRNA in goat CL at 1, 3 and 4 months of gestation. This study shows that the goat CL expresses P450-Aro mRNA along gestation, suggesting that this structure is capable to produce oestrogens up to the end of gestation.

Immunopresence and functional activity of prostaglandin-endoperoxide synthases and nitric oxide synthases in bovine corpora lutea during diestrus.

The aim of this study was to evaluate the occurrence and the activity of prostaglandin-endoperoxide synthase 1 (PTGS1), PTGS2, and endothelial, neuronal, and inducible nitric oxide synthase (e-, n-, and iNOS) in early, mid, late, and regressive corpora lutea (CL) of bovines during diestrus. PTGS1 immunoreactivity was localised mainly in the cytoplasm of small luteal cells, whereas PTGS2 was detected in the cytoplasm of large luteal cells during early, mid, and late stages. The immunoexpression of all NOS isoforms was observed in the nuclei of luteal cells in the CL stages examined. PTGS1 enzyme activity was higher in late CL and lower in regressive ones; PTGS2 increased from early to late CL and lowered in regressive ones. Constitutive NOS enzymatic activity (eNOS plus nNOS) was higher in late CL and lower in regressive ones; iNOS was lower in regressive CL. These results support the idea that PTGSs and NOSs regulate the bovine CL life span mainly during the transition from the luteotrophic to the luteolytic phase.

Roles of antioxidant enzymes in corpus luteum rescue from reactive oxygen species-induced oxidative stress.

Progesterone produced by the corpus luteum (CL) regulates the synthesis of various endometrial proteins required for embryonic implantation and development. Compromised CL progesterone production is a potential risk factor for prenatal development. Reactive oxygen species (ROS) play diverse roles in mammalian reproductive biology. ROS-induced oxidative damage and subsequent adverse developmental outcomes constitute important issues in reproductive medicine. The CL is considered to be highly exposed to locally produced ROS due to its high blood vasculature and steroidogenic activity. ROS-induced apoptotic cell death is involved in the mechanisms of CL regression that occurs at the end of the non-fertile cycle. Luteal ROS production and propagation depend upon several regulating factors, including luteal antioxidants, steroid hormones and cytokines, and their crosstalk. However, it is unknown which of these factors have the greatest contribution to the maintenance of CL integrity and function during the oestrous/menstrual cycle. There is evidence to suggest that antioxidants play important roles in CL rescue from luteolysis when pregnancy ensues. As luteal phase defect impacts fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of ROS-scavenging antioxidant enzymes in the control of mammalian CL function and integrity. The corpus luteum (CL) is a transient endocrine organ that develops after ovulation from the ovulated follicle during each reproductive cycle. The main function of the CL is the production and secretion of progesterone which is necessary for embryonic implantation and development. Compromised CL progesterone production is a potential risk factor for prenatal development and pregnancy outcomes. Reactive oxygen species (ROS), which are natural by-products of cellular respiration and metabolism, play diverse roles in mammalian reproductive biology. ROS-induced oxidative damage and subsequent development of adverse pregnancy outcomes constitute important issues in reproductive medicine. Before the end of the first trimester, a high rate of human and animal conceptions end in spontaneous abortion and most of these losses occur at the time of implantation in association with ROS-induced oxidative damage. Every cell in the body is normally able to defend itself against the oxidative damage caused by the ROS. The cellular antioxidant enzymes constitute the first line of defence against the toxic effects of ROS. The CL is considered to be highly exposed to locally produced ROS due to its high blood vasculature and metabolic activity. There is now evidence to suggest that cellular antioxidants play important roles in CL rescue from regression when pregnancy ensues. As defective CL function impacts fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of antioxidant enzymes in the control of mammalian CL function and integrity.

The bovine genome contains three differentially methylated paralogous copies of the P450c17 encoding gene (CYP17A1).

CYP17A1 encodes the key enzyme of androgen biosynthesis, P450c17. The gene is expressed in a number of steroidogenic tissues among them testis, ovary, placenta and adrenal gland. The proper analysis of CYP17A1 expression and of epigenetic parameters however, is hampered by the presence of more than one copy of the gene within the bovine genome. Therefore, as a prerequisite for future studies we characterized these copies and analyzed their promoter methylation and expression profiles in different tissues. DNA methylation levels were determined by bisulfite modification, amplification, cloning and sequencing. Transcription was analyzed by RT-PCR. From bovine genomic DNA three different CYP17A1 promoter sequences could be amplified with a sequence similarity of 94.8%, 95.6% and 98.7%. Based on these sequences we could reconstruct, by in silico analysis, the promoter regions and eight potentially coding exons of two loci, CYP17A1a and CYP17A1b, and the promoter region and truncated first exon of a third locus, CYP17A1x. By using locus-specific primers, only transcripts of CYP17A1a, but not of CYP17A1b could be detected in testis, epididymis, theca, corpus luteum, placental cotyledons, adrenal gland and preoptic brain area. Methylation analysis revealed that only the CYP17A1a promoter was hypo-methylated in the tested P450c17 active tissues, whereas both other copies showed higher levels of methylation. From these data we conclude that the bovine genome contains three paralogous copies of the CYP17A1 gene, of which two (CYP17A1b and CYP17A1x) might be silenced by epigenetic modification (promoter methylation).

A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes prostaglandin2α-induced corpus luteum regression.

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2α induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2α but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knock-in mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.

The antioxidative enzyme SOD2 is important for physiological persistence of corpora lutea in lynxes.

Corpora lutea (CL) are transient endocrine glands supporting pregnancy by progesterone production. They develop at the site of ovulation from the remaining follicle, are highly metabolically active and undergo distinct, transformative processes during their lifetime. In contrast to other species, CL of lynxes do not regress at the end of cycle, but remain functionally active (persist) for years. Reactive oxygen species (ROS) and anti-oxidative enzymes are described to be important for the functionality of CL. We examined ten anti-oxidative enzymes in fresh and persistent CL of lynxes as well as in domestic cat CL of different luteal stages. The gene expression profiles, especially those of SOD1 and SOD2, showed some remarkable differences between CL stages during non-pregnant and pregnant cycles of domestic cats and between fresh and persistent CL of lynxes. Lynx gene expression profiles of SODs were confirmed by western blot analysis, immunohistochemistry and activity assays. SOD2 was characterized by a conspicuous high expression and enzyme activity exclusively in persistent CL. We suggest that SOD2 is required to detoxify potential elevated superoxide anion levels by producing H2O2 in the physiologically persistent CL. This product might also act as a signaling molecule, securing the CL from apoptosis and insuring long-term luteal cell survival.

N-Acetylglucosamine 6-O-sulfotransferase-2 as a tumor marker for uterine cervical and corpus cancer.

N-Acetylglucosamine 6-O-sulfotransferase-2 (GlcNAc6ST2) is ectopically expressed in ovarian mucinous and clear cell adenocarcinoma [Kanoh et al., Glycoconj J 23:453-460, 2006]. Here we studied whether GlcNAc6ST2 protein can be detected in sera from patients with gynecological cancers and could serve as a tumor marker. First, we created a monoclonal antibody and polyclonal antiserum against GlcNAc6ST2. These antibodies were specific for GlcNAc6ST2, as shown by Western blot analysis and immunoprecipitation. Using these antibodies, we constructed a sandwich ELISA method for detecting GlcNAc6ST2 in the serum. GlcNAc6ST2 provided lower positive rates for ovarian cancer than CA125, but higher positive rates for uterine cervical and corpus cancer than SCC antigens and CA125, respectively. A significantly higher percentage of stage I uterine cervical and corpus cancers were positive for GlcNAc6ST2 than for SCC antigens and CA125, respectively. GlcNAc6ST2 could therefore be a good serological marker for detecting early-stage uterine cervical and corpus cancers.

Expression and localization of the serine proteases high-temperature requirement factor A1, serine protease 23, and serine protease 35 in the mouse ovary.

Proteolytic degradation of extracellular matrix components has been suggested to play an essential role in the occurrence of ovulation. Recent studies in our laboratory have indicated that the plasminogen activator and matrix metalloproteinase systems, which were previously believed to be crucial for ovulation, are not required in this process. In this study we have used a microarray approach to identify new proteases that are involved in ovulation. We found three serine proteases that were relatively highly expressed during ovulation: high-temperature requirement factor A1 (HtrA1), which was not regulated much during ovulation; serine protease 23 (PRSS23), which was down-regulated by gonadotropins; and serine protease 35 (PRSS35), which was up-regulated by gonadotropins. We have further investigated the expression patterns of these proteases during gonadotropin-induced ovulation in immature mice and in the corpus luteum (CL) of pseudopregnant mice. We found that HtrA1 was highly expressed in granulosa cells throughout follicular development and ovulation, as well as in the forming and regressing CL. PRSS23 was highly expressed in atretic follicles, and it was expressed in the ovarian stroma and theca tissues just before ovulation. PRSS35 was expressed in the theca layers of developing follicles. It was also highly induced in granulosa cells of preovulatory follicles. PRSS35 was also expressed in the forming and regressing CL. These data suggest that HtrA1 and PRSS35 may be involved in ovulation and CL formation and regression, and that PRSS23 may play a role in follicular atresia.

Presence of arylsulfatase A and sulfogalactosylglycerolipid in mouse ovaries: localization to the corpus luteum.

Arylsulfatase A (AS-A) is a lysosomal enzyme, which catalyzes the desulfation of certain sulfogalactolipids, including sulfogalactosylglycerolipid (SGG), a molecule implicated in cell adhesion. In this report, immunocytochemistry revealed the selective presence of AS-A in the corpus luteum of mouse ovaries. Immunoblotting indicated that mouse corpus luteum AS-A had a molecular mass of 66 kDa, similar to AS-A of other tissues. Corpus luteum AS-A was active, capable of desulfating the artificial substrate, p-nitrocatechol sulfate, at the optimum pH of five. To understand further the role of AS-A in female reproduction, levels of AS-A were determined during corpus luteum development in pseudopregnant mice and during luteolysis after cessation of pseudopregnancy. Immunocytochemistry, immunoblotting and desulfation activity showed that AS-A expression was evident at the onset of pseudopregnancy in the newly formed corpora lutea, and its level increased steadily during gland development. The increase in the expression and activity of AS-A continued throughout luteolysis after the decrease in serum progesterone levels. We also observed the selective presence of SGG on the luteal cell surface in developed corpora lutea, as shown by immunofluorescence of mouse ovary sections as well as high-performance thin-layer chromatography of lipids isolated from mouse and pig corpora lutea. The identity of the "SGG" band on the thin layer silica plate was further validated by electrospray ionization mass spectrometry. Significantly, SGG disappeared in regressing corpora lutea. Therefore, lysosomal AS-A may be involved in cell-surface remodeling during luteolysis by desulfating SGG after its endocytosis and targeting to the lysosome.

Monoamine oxidase A is highly expressed by the human corpus luteum of pregnancy.

To investigate the physiological characteristics of the corpus luteum (CL) of pregnancy, we raised a mAb, human corpus luteum (HCL)-4, against human luteal cells obtained from CL of pregnancy. The affinity-purified antigen from human CL of pregnancy or placenta using HCL-4 was a 61 kDa protein. The partial amino acid sequence of the antigenic protein was identical to that of human monoamine oxidase A (MAOA, EC1.4.3.4). MAOA has been shown to catabolize catecholamines that were reported to regulate luteal function in CL and vasoconstriction in various organs. Immunohistochemistry using HCL-4 mAb showed that MAOA was intensely expressed on large luteal cells and moderately expressed on small luteal cells in the CL of pregnancy. In the CL of menstrual cycle, MAOA was weakly detected on large luteal cells but not detected at all on small luteal cells. Western blotting analysis confirmed the high expression of MAOA in CL of pregnancy. Northern blot analysis also showed the expression of MAOA mRNA in human CL, and showed that its expression was higher in CL of pregnancy than in CL of menstrual cycle. The increased expression of MAOA in the CL of pregnancy suggests the contribution of MAOA to the function of the CL of pregnancy.

Prostaglandin F2alpha increases endothelial nitric oxide synthase in the periphery of the bovine corpus luteum: the possible regulation of blood flow at an early stage of luteolysis.

Prostaglandin F(2)(alpha) (PGF(2)(alpha)) released from the uterus causes alterations in luteal blood flow, reduces progesterone secretion, and induces luteolysis in the bovine corpus luteum (CL). We have recently discovered that luteal blood flow in the periphery of the mature CL acutely increases coincidently with pulsatile increases in a metabolite of PGF(2)(alpha) (PGFM). In this study, we characterized changes in regional luteal blood flow together with regional alterations in endothelial nitric oxide synthase (eNOS) expression during spontaneous luteolysis and in response to PGF(2)(alpha). Smooth muscle actin-positive blood vessels larger than 20 microm were observed mainly in the periphery of mature CL. PGF(2)(alpha) receptor was localized to luteal cells and large blood vessels in the periphery of mid-CL. PGF(2)(alpha) acutely stimulated eNOS expression in the periphery but not in the center of mature CL. Injection of the NO donor S-nitroso-N-acetylpenicillamine into CL induced an acute increase in luteal blood flow and shortened the estrous cycle. In contrast, injection of the NOS inhibitor l-NAME into CL completely suppressed the acute increase in luteal blood flow induced by PGF(2)(alpha) and delayed the onset of luteolysis. In conclusion, PGF(2)(alpha) has a site-restricted action depending on not only luteal phase but also the region in the CL. PGF(2)(alpha) stimulates eNOS expression, vasodilation of blood vessels, and increased luteal blood flow in periphery of mature CL. Furthermore, the increased blood flow is mediated by NO, suggesting that the acute increase in peripheral blood flow to CL is one of the first physiological indicators of NO action in response to PGF(2)(alpha).

Generation and utilization of P450 cholesterol side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase antibodies for universal detection.

The biosynthesis of steroids from steroidogenic cells are catalyzed by the two major enzymes, P450 side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). This article describes the construction of two novel polyclonal antibodies against conserved recombinant protein and the validation of these antibodies on fixed tissue sections of bovine corpus luteum. The polyclonal antibodies were used successfully in Western blots and specifically reacted with P450scc and 3beta-HSD protein in bovine luteal cell extracts. Thus, P450scc and 3beta-HSD are two specific polyclonal antibodies that are integral products in the investigation of the biological function and regulatory mechanism involved in steroidogenesis.

Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo.

Nitric oxide is reportedly involved in the regulation of several ovarian processes, yet the isoforms of nitric oxide synthase (NOS) expressed in the ovary are unknown. Our purpose was to identify and localize NOS isoenzymes in the rat ovary and to examine++ if mRNA expression of NOS isoenzymes change after gonadotropin stimulation. Using reverse transcriptase-PCR, we demonstrated that inducible (iNOS) and endothelial (eNOS), but not neuronal, NOS mRNAs are expressed in the ovary. In a gonadotropin-stimulated rat model, unstimulated ovaries had the highest levels of iNOS mRNA as quantified by ribonuclease protection assay. After gonadotropin injection, iNOS mRNA declined to undetectable levels in ovaries containing ovulatory follicles before increasing slighty in ovaries containing copora lutea. In situ hybridization studies localized iNOS to granulosa cells of secondary follicles and small antral follicles. Western blots of unstimulated ovaries demonstrated iNOS protein. In contrast to iNOS, eNOS mRNA levels, determined by quantitative PCR, increased after gonadotropin stimulation and peaked in ovaries containing ovulatory follicles before declining in the luteal phase. eNOS protein was localized to blood vessels in the ovary by immunohistochemistry. We conclude that two isoforms of NOS are expressed in the ovary and the mRNA levels for these isozymes are differentially regulated.