Reconstitution of Caruncle Placenta through the 20α-HSD/Casp-3 Apoptotic Pathway during Early Pregnancy in Bovines.

Trophoblast cells of endometrium during bovine pregnancy with different characteristics undergo dynamic changes during uterine remodeling, which can be observed as continuous changes, as P4 secreted by the mother is replaced by placental hormones. In this context, the present study analyzed tissues' morphological changes through uterine apoptosis during early pregnancy. In addition, the expression pattern associated with apoptosis genes and 20α-HSD was determined in the endometrium and caruncle tissues. The localization of 20α-HSD, VEGF, Casp3, and mTOR protein was also determined in endometrium and caruncle during early pregnancy. From around 30 days, caruncle trophoblast cells with very high invasiveness expanded the villus section as the gestation period progressed. The surrounding cells detached and reorganized into new cells. In addition, an analysis of the effect of apoptosis on cell reorganization in the caruncle revealed that the expression of 20α-HSD/Casp-3 signals in the villus section gradually increased from 30 to 90 days. However, on the 30th day, glandular epithelial cells occurred sporadically in the trophoblast cell section. Moreover, the apoptosis of trophoblast cells increased at 90 days. Taken together, the results of the present study show that changes in the uterus during early pregnancy promote changes during later pregnancy by inducing the reorganization through the stimulation of 20α-HSD and Casp-3, promoting uterine and caruncle tissues, unlike cell development mediated by hormone signaling.

Steroid Metabolism in Children and Adolescents With Obesity and Insulin Resistance: Altered SRD5A and 20α/20ßHSD Activity.

Alterations in glucocorticoid metabolism may contribute to the development of obesity and insulin resistance (IR). Obesity in turn affects the androgen balance. The peripheral metabolism of steroids is equally an important determinant of their bioavailability and activity. The aim of this study was to evaluate steroid metabolism in obese children and to define which enzyme alterations are associated with IR. Clinical characteristics and anthropometric measurements were determined in 122 obese children and adolescents (72 girls, 50 boys) aged 8 - 18 years. 26 of them (21.3%) were diagnosed with IR (13 boys, 13 girls). Routine laboratory tests were performed and 24h urinary steroid excretion profiles were analyzed by gas chromatography/mass spectrometry. Positive relationship between 5α-reductase (SRD5A) activity and IR was found. According to the androsterone to etiocholanolone (An/Et) ratio the activity of SRD5A was significantly increased in obese children with IR, but the difference remained insignificant once the 5α-dihydrotestosterone to testosterone (5αDHT/T) ratio was considered. Furthermore, this relationship persisted in boys but was not observed in girls. The activity of 20α-hydroxysteroid dehydrogenase (20αHSD) and 20ß-hydroxysteroid dehydrogenase (20ßHSD) was reduced only in obese girls with IR. Conclude, in the context of obese children and adolescents with IR, we surmise that increased SRD5A represents a compensatory mechanism to reduce local glucocorticoid availability. This phenomenon is probably different in the liver (restriction) and in the adipose tissue (expected increase in activity). We show significant changes in 20αHSD and 20ßHSD activity in obese girls with IR, but it is difficult to clearly determine whether the activity of these enzymes is an indicator of the function in their ovaries or adrenal glands.

Pro-inflammatory signals induce 20α-HSD expression in myometrial cells: A key mechanism for local progesterone withdrawal.

Metabolism of progesterone (P4) by the enzyme 20α hydroxysteroid dehydrogenase (20α-HSD) in myometrial cells is postulated to be a mechanism for P4 withdrawal, which occurs concomitant to uterine inflammation (physiologic or infection-induced) and associated activation of transcription factors: NF-кB and AP-1, common to term and preterm labour. We found that 20α-HSD protein is significantly increased in human myometrium during term labour, and in mouse uterus during term and preterm labour. Treatment of human myometrial cells with the pro-inflammatory mediators, lipopolysaccharide (LPS, mimicking infection) and 12-O-tetradecanoylphorbol-13-acetate (TPA, mimicking inflammation), induced 20α-HSD gene expression and increased 20α-HSD protein abundance. LPS treatment decreased P4 release into the culture medium and resulted in up-regulation of GJA1 in the hTERT-HM cells. The NF-кB /AP-1 transcription factors mediated effects of LPS and TPA on 20α-HSD gene transcription. Both pro-inflammatory stimuli induced 20α-HSD promoter activity in LPS/TPA-treated cells which was significantly attenuated by inhibition of NF-кB (JSH: 20 µM) or AP-1 signalling (T5224: 10 µM). Deletion of NF-кB consensus sites abrogated LPS-mediated promoter induction, while removal of AP-1 sites reversed the TPA-mediated induction of 20α-HSD promoter. We conclude that inflammatory stimuli (physiologic or pathologic) that activate NF-кB or AP-1 induce 20α-HSD transcription and subsequent local P4 withdrawal resulting in up-regulation of GJA1 and activation of myometrium that precedes labour.

Neuromodulatory effect of oestradiol in the metabolism of ovarian progesterone and oestradiol during dioestrus II: participation of the superior mesenteric ganglion.

The aims of the present study were to determine: (1) whether oestradiol (E2) in the superior mesenteric ganglion (SMG) modifies the release of ovarian progesterone (P4), androstenedione (A2) and E2, the activity and gene expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-HSD and the expression of P450 aromatase (Cyp19a1) and (2) whether any such modifications are related to changes in ovarian nitric oxide (NO) and noradrenaline (NA) levels during dioestrus II. Using an ex vivo SMG-ovarian nervous plexus-ovary system, ovarian P4 release was measured following the addition E2 plus tamoxifen (Txf) (10-6M) to the ganglion, whereas A2, E2, NA and NO were measured following the addition of E2 alone. Steroids were measured by radioimmunoassay, NA concentrations were determined by HPLC and gene expression was evaluated using reverse transcription-polymerase chain reaction. Oestradiol in the ganglion decreased ovarian P4, E2 and NA release, as well as 3ß-HSD activity, but increased the release of A2 and nitrites, as well as the 20α-HSD expression and its activity. No changes were observed in Cyp19a1 gene expression. The addition of E2 plus Txf to the ganglion reversed the effects of E2 alone. The action of oestradiol in SMG favours the beginning of functional luteolysis, due to an increase in NO release and a decrease in NA in the ovary. These results may help elucidate the role of E2 in hormone-dependent pathologies in women.

Low Prolactin and High 20-α-Hydroxysteroid Dehydrogenase Levels Contribute to Lower Progesterone Levels in HIV-Infected Pregnant Women Exposed to Protease Inhibitor-Based Combination Antiretroviral Therapy.

BACKGROUND: It has been reported that pregnant women receiving protease inhibitor (PI)-based combination antiretroviral therapy (cART) have lower levels of progesterone, which put them at risk of adverse birth outcomes, such as low birth weight. We sought to understand the mechanisms involved in this decline in progesterone level. METHODS: We assessed plasma levels of progesterone, prolactin, and lipids and placental expression of genes involved in progesterone metabolism in 42 human immunodeficiency virus (HIV)-infected and 31 HIV-uninfected pregnant women. In vitro studies and a mouse pregnancy model were used to delineate the effect of HIV from that of PI-based cART on progesterone metabolism. RESULTS: HIV-infected pregnant women receiving PI-based cART showed a reduction in plasma progesterone levels (P= .026) and an elevation in placental expression of the progesterone inactivating enzyme 20-α-hydroxysteroid dehydrogenase (20α-HSD; median, 2.5 arbitrary units [AU]; interquartile range [IQR], 1.00-4.10 AU), compared with controls (median, 0.89 AU; IQR, 0.66-1.26 AU;P= .002). Prolactin, a key regulator of 20α-HSD, was lower (P= .012) in HIV-infected pregnant women. We observed similar data in pregnant mice exposed to PI-based cART. In vitro inhibition of 20α-HSD activity in trophoblast cells reversed PI-based cART-induced decreases in progesterone levels. CONCLUSIONS: Our data suggest that the decrease in progesterone levels observed in HIV-infected pregnant women exposed to PI-based cART is caused, at least in part, by an increase in placental expression of 20α-HSD, which may be due to lower prolactin levels observed in these women.

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.

C21-steroids inactivation and glucocorticoid synthesis in the developing lung.

Glucocorticoids (GCs) are important regulators of lung development. The genes normally involved in GC synthesis in adrenals are co-expressed with 20α-hydroxysteroid dehydrogenase (20α-HSD) in the developing lung. In this study, C21-steroid metabolism was investigated in fetal and postnatal mouse lungs. Incubation of [(3)H]-progesterone with lung explant cultures of different perinatal developmental time points revealed two different (antenatal vs. postnatal) complex metabolization patterns. Progesterone inactivation was predominant. 20αOH-derivatives were more abundant after birth and some metabolites were 5α-reduced. Using [(3)H]-progesterone as substrate, corticosterone synthesis was only observed in a fraction of lung explants from gestation day (GD) 15.5. Neither aldosterone synthase nor P450c17 activity was observed. With epithelial-enriched primary cell cultures, deoxycorticosterone synthesis from [(3)H]-progesterone was observed. With lung explants incubated with [(3)H]-corticosterone as substrate, [(3)H]-4-pregnen-21-ol-3,11,20-trione (11-dehydrocorticosterone), the product of 11ß-HSD2, accumulated in higher proportion on GD 15.5 than at later developmental time points. The temporal correlation observed between levels of progesterone inactivation by 20α-HSD (higher after birth) and the sensitivity of lung development to GCs suggests a role for 20α-HSD in the modulation of GR occupancy through the control of 21-hydroxylase substrate and product levels. In conclusion, the developing lung is characterized by effective inactivation of c21-steroids by 20α-HSD. The formation of active GCs from the "adrenal"-like pathway was observed with some lung explants and primary epithelial cell cultures. Coexistence of this GC synthesis pathway with 20α-HSD activity strongly suggests local regulation of GC action and is compatible with intracrine/paracrine actions of GC.

Expression and localization of 20α-hydroxysteroid dehydrogenase (20α-HSD) in porcine reproductive tissues during pregnancy.

The aims of this study were to determine the specific site of 20α-HSD expression in the reproductive tissues on day 30 of pregnancy and during pre-parturition. 20α-HSD mRNA was demonstrated to have the highest expression in the placenta on day 30 of pregnancy and in the ovary during pre-parturition. Weak mRNA expression was observed in the uterus and ovary on day 30 of pregnancy. However, the mRNA was not expressed in the oviduct on day 30 of pregnancy. The mRNA was also specifically detected in the placenta on day 30 of pregnancy by northern blot analysis. Western blot analysis indicated that the expression pattern of the 20α-HSD protein in the reproductive tissues was similar to that of 20α-HSD mRNA. Immunohistochemical analysis also revealed that the pig 20α-HSD protein was localized in the trophoblast villus in the placenta on day 30 of pregnancy. It was highly expressed in the glandular epithelial cells of the endometrium and the luminal epithelial cells of the uterus. The 20α-HSD protein was highly localized in the large luteal cells of the ovary on day 30 of pregnancy and during pre-parturition. Taken together, our study demonstrated that the pig 20α-HSD mRNA and protein are mainly localized in the trophoblast villus in the placenta on day 30 of pregnancy. The expression of the protein is also localized in the large luteal cells of the ovary. In addition, the protein is highly expressed in the glandular epithelial cells of the endometrium and the luminal epithelial cells of the uterus.

Ontogeny of adrenal-like glucocorticoid synthesis pathway and of 20α-hydroxysteroid dehydrogenase in the mouse lung.

BACKGROUND: Glucocorticoids exert recognized positive effects on lung development. The genes involved in the classical pathway of glucocorticoid synthesis normally occurring in adrenals were found to be expressed on gestation day (GD) 15.5 in the developing mouse lung. Recently, expression of two of these genes was also detected on GD 17.5 suggesting a more complex temporal regulation than previously expected. Here, we deepen the knowledge on expression of "adrenal" glucocorticoid synthesis genes in the mouse lung during the perinatal period and we also study expression of the gene encoding for the steroid inactivating enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). RESULTS: We performed an ontogenic study of P450scc, 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase 1 (3ß-HSD1), 21-hydroxylase, 11ß-hydroxylase, 11ß-HSD1, and 11ß-HSD2 expression up to post natal day (PN) 15. The substrate (progesterone) and the product (deoxycorticosterone) of 21-hydroxylase are substrates of 20α-HSD, thus 20α-HSD (Akr1c18) gene expression was investigated. In lung samples collected between GD 15.5 and PN 15, 11ß-hydroxylase was only detected on GD 15.5. In contrast, all the other tested genes were expressed throughout the analyzed period with different temporal expression patterns. P450scc, 21-hydroxylase, 20α-HSD and 11ß-HSD2 mRNA levels increased after birth with different patterns including an increase from PN 3 with a possible sex difference for 21-hydroxylase mRNA. Also, the 21-hydroxylase protein was observed by Western blot in perinatal lungs with higher levels after birth. CONCLUSION: Progesterone is present at high levels during gestation and the product of 21-hydroxylase, deoxycorticosterone, can bind the glucocorticoid receptor with an affinity close to that of corticosterone. Detection of 21-hydroxylase at the protein level during antenatal lung development is the first evidence that the adrenal-like glucocorticoid synthesis pathway detected during lung development has the machinery to produce glucocorticoids in the fetal lung. Glucocorticoids from lung 21-hydroxylase appear to modulate lung ontogenesis through paracrine/intracrine actions.

Ovarian LGR5 is critical for successful pregnancy.

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is expressed in many organs, including female reproductive organs, and is a stem cell marker in the stomach and intestinal epithelium, hair follicles, and ovarian surface epithelium. Despite ongoing studies, the definitive physiological functions of Lgr5 remain unclear. We utilized mice with conditional deletion of Lgr5 (Lgr5(d/d)) in the female reproductive organs by progesterone receptor-Cre (Pgr(Cre)) to determine Lgr5's functions during pregnancy. Only 30% of plugged Lgr5(d/d) females delivered live pups, and their litter sizes were lower. We found that pregnancy failure in Lgr5(d/d) females was due to insufficient ovarian progesterone (P4) secretion that compromised decidualization, terminating pregnancy. The drop in P4 levels was reflected in elevated levels of P4-metabolizing enzyme 20α-hydroxysteroid dehydrogenase in corpora lutea (CL) inactivated of Lgr5. Of interest, P4 supplementation rescued decidualization failure and supported pregnancy to full term in Lgr5(d/d) females. These results provide strong evidence that Lgr5 is critical to normal CL function, unveiling a new role of LGR5 in the ovary.

Substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase.

In this study, we examined the substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase (AKR1C5), which plays a role in the termination of pregnancy by progesterone inactivation. AKR1C5 moderately reduced the 3-keto group of only 5α-dihydrosteroids with 17ß- or 20α/ß-hydroxy group among 3-ketosteroids. In contrast, the enzyme reversibly and efficiently catalyzed the reduction of various 17- and 20-ketosteroids, including estrogen precursors (dehydroepiandrosterone, estrone and 5α-androstan-3ß-ol-17-one) and tocolytic 5ß-pregnane-3,20-dione. In addition to the progesterone inactivation, the formation of estrogens and metabolism of the tocolytic steroid by AKR1C5 may be related to its role in rabbit parturition. AKR1C5 also reduced various non-steroidal carbonyl compounds, including isatin, an antagonist of the C-type natriuretic peptide receptor, and 4-oxo-2-nonenal, suggesting its roles in controlling the bioactive isatin and detoxification of cytotoxic aldehydes. AKR1C5 was potently and competitively inhibited by flavonoids such as kaempferol and quercetin, suggesting that its activity is affected by ingested flavonoids.

Involvement of the ganglion cholinergic receptors in gonadotropin-releasing hormone, catecholamines, and progesterone release in the rat ovary.

OBJECTIVE: To investigate whether cholinergic ganglionic stimulus modifies the release of gonadotropin-releasing hormone (GnRH), catecholamines, and progesterone at the ovarian level. DESIGN: Animal study. SETTING: University animal laboratory. ANIMAL(S): Six to eight virgin adult Holtzman rats. INTERVENTION(S): Superior mesenteric ganglion-ovarian nerve plexus-ovary system removed and placed in one cuvette with two compartments, with acetylcholine added to the ganglion in the experimental group. MAIN OUTCOME MEASURE(S): Measurement of ovarian liquid obtained from catecholamines by high-performance liquid chromatography; measurement of progesterone (P(4)), GnRH, and luteinizing hormone (LH) by radioimmunoassay; and measurement of gene expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD) by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULT(S): The study focused on the estrus and diestrus II (DII) stages. On the estrus days, the release of GnRH, NA, and 20α-HSD increased, while P(4) and 3ß-HSD decreased. On the DII days, GnRH, P(4), and 3ß-HSD increased, while 20α-HSD and NA decreased. The ovarian liquid with GnRH showed biologic activity, namely, an increase in LH release during the DII stage and a decrease during the estrus stage. CONCLUSION(S): Neural stimulus from the superior mesenteric ganglion influences the release of NA, adrenaline, and GnRH. We also have demonstrated that these neurotransmitters participate in the atretogenic processes of the ovary, thus providing evidence of the necessity of the sympathetic neural pathway.

Protective effect of oestradiol in the coeliac ganglion against ovarian apoptotic mechanism on dioestrus.

The aims of this work were to investigate if oestradiol 10(-8)M in the incubation media of either the ovary alone (OV) or the ganglion compartment of an ex vivo coeliac ganglion-superior ovarian nerve-ovary system (a) modifies the release of ovarian progesterone (P4) and oestradiol (E2) on dioestrus II, and (b) modifies the ovarian gene expression of 3ß-HSD and 20α-HSD enzymes and markers of apoptosis. The concentration of ovarian P4 release was measured in both experimental schemes, and ovarian P4 and E2 in the ex vivo system by RIA at different times. The expression of 3ß-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and antiapoptotic bcl-2 and proapoptotic bax by RT-PCR were determined. E2 added in the coeliac ganglion caused an increase in the ovarian release of the P4, E2 and 3ß-HSD, while in the ovary incubation alone it decreased P4 and 3ß-HSD but increased and 20α-HSD and bax/bcl-2 ratio. It is concluded that through a direct effect on the ovary, E2 promotes luteal regression in DII rats, but the addition of E2 in the coeliac ganglion does not have the same effect. The peripheral nervous system, through the superior ovarian nerve, has a protective effect against the apoptotic mechanism on DII.

MicroRNA-200a serves a key role in the decline of progesterone receptor function leading to term and preterm labor.

During pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) activity, but labor is facilitated by a series of events that impair PR function. Previously, we discovered that miR-200 family members serve as progesterone (P(4))-modulated activators of contraction-associated genes in the pregnant uterus. In this study, we identified a unique role for miR-200a to enhance the local metabolism of P(4) in myometrium and, thus, decrease PR function during the progression toward labor. miR-200a exerts this action by direct repression of STAT5b, a transcriptional repressor of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). We observed that miR-200a expression increased and STAT5b expression coordinately decreased in myometrium of mice as they progressed to labor and in laboring myometrium from pregnant women. These changes were associated with a dramatic increase in expression and activity of 20α-HSD in laboring myometrium from mouse and human. Notably, overexpression of miR-200a in cultured human myometrial cells (hTERT-HM) suppressed STAT5b and increased 20α-HSD mRNA levels. In uterine tissues of ovariectomized mice injected with P(4), miR-200 expression was significantly decreased, STAT5b expression was up-regulated, and 20α-HSD mRNA was decreased, but in 15 d postcoitum pregnant mice injected with the PR antagonist RU486, preterm labor was associated with increased miR-200a, decreased STAT5b, and enhanced 20α-HSD expression. Taken together, these findings implicate miR-200a as an important regulator of increased local P(4) metabolism in the pregnant uterus near term and provide insight into the importance of miR-200s in the decline in PR function leading to labor.

Estradiol promotes luteal regression through a direct effect on the ovary and an indirect effect from the celiac ganglion via the superior ovarian nerve.

There is evidence suggesting that estradiol (E(2)) regulates the physiology of the ovary and the sympathetic neurons associated with the reproductive function. The objective of this study was to investigate the effect of E(2) on the function of late pregnant rat ovaries, acting either directly on the ovarian tissue or indirectly via the superior ovarian nerve (SON) from the celiac ganglion (CG). We used in vitro ovary (OV) or ex vivo CG-SON-OV incubation systems from day 21 pregnant rats. Various concentrations of E(2 )were added to the incubation media of either the OV alone or the ganglion compartment of the CG-SON-OV system. In both experimental schemes, we measured the concentration of progesterone in the OV incubation media by radioimmunoassay at different times. Luteal messenger RNA (mRNA) expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD) enzymes, respectively, involved in progesterone synthesis and catabolism, and of antiapoptotic B-cell lymphoma 2 (Bcl-2) and proapoptotic Bcl-2-associated X protein (Bax), were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) at the end of the incubation period. Estradiol added directly to the OV incubation or to the CG of the CG-SON-OV system caused a decline in the concentration of progesterone accumulated in the incubation media. In addition, E(2), when added to the OV incubation, decreased the expression of 3ß-HSD and the ratio of Bcl-2/Bax. We conclude that through a direct effect on the OV, E(2) favors luteal regression at the end of pregnancy in rats, in association with neural modulation from the CG via the SON.

Expression of aldo-keto reductase family 1 member C1 (AKR1C1) gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy.

BACKGROUND: The aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPH-dependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) is a member of AKR family. The aims of this study were to determine its expression in the ovary and uterus endometrium during the estrous cycle and pregnancy. METHODS: Rapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine 20 alpha-HSD cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20 alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary. RESULTS: The porcine 20 alpha-HSD cDNA is 957 bp in length and encodes a protein of 319 amino acids. The cloned cDNA was virtually the same as the porcine AKR1C1 gene (337 amino acids) reported recently, and only differed in the C-terminal region (the AKR1C1 gene has a longer C-terminal region than our sequence). The 20 alpha-HSD gene (from now on referred to as AKR1C1) cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of AKR1C1 genes from other animals. Porcine AKR1C1 mRNA was expressed on day 5, 10, 12, 15 of the cycle and 0-60 of pregnancy in the ovary. The mRNA was also specifically detected in the uterine endometrium on day 30 of pregnancy. Western blot analysis indicated that the pattern of AKR1C1 protein in the ovary during the estrous cycle and uterus during early pregnancy was similar to that of AKR1C1 mRNA expression. The recombinant protein produced in CHO cells was detected at approximately 37 kDa. Immunohistochemical analysis also revealed that pig AKR1C1 protein was localized in the large luteal cells in the early stages of the estrous cycle and before parturition. CONCLUSIONS: Our study demonstrated that AKR1C1 mRNA and protein are coordinately expressed in the luteal cell of ovary throughout the estrous cycle and in the uterus on day 30 of pregnancy. Thus, the porcine AKR1C1 gene might control important mechanisms during the estrous cycle.

Molecular characterization of bovine placental and ovarian 20α-hydroxysteroid dehydrogenase.

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme plays a critical role in the regulation of luteal function in female mammals. In this study, we conducted the characterization and functional analyses of bovine 20α-HSD from placental and ovarian tissues. The nucleotide sequence of bovine 20α-HSD showed significant homology to that of goats (96%), humans (84%), rabbits (83%), and mice (81%). The mRNA levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. Northern blot analysis revealed a 1.2  kb mRNA in the bovine placental and ovarian tissues. An antibody specific to bovine 20α-HSD was generated in a rabbit immunized with the purified, recombinant protein. Recombinant 20α-HSD protein produced in mammalian cells had a molecular weight of ∼37  kDa. Bacterially expressed bovine 20α-HSD protein showed enzymatic activity. The expression pattern of the 20α-HSD protein in the pre-parturition placenta and the CL1 stage of the estrous cycle was similar to the level of 20α-HSD mRNA expression. Immunohistochemical analysis also revealed that bovine 20α-HSD protein was intensively localized in the large luteal cells during the late estrous cycle.

Expression and localization of the 20α-hydroxysteroid dehydrogenase (HSD) enzyme in the reproductive tissues of the cynomolgus monkey Macaca fascicularis.

This study was conducted to characterize and functionally analyze the monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) in the ovary, placenta, and oviduct. We focused on 20α-HSD mRNA expression and protein localization in monkey reproductive tissues and the molecular characterization of the promoter region. Reverse transcription-polymerase chain reaction (RT-PCR) monkey 20α-HSD mRNA was more strongly detected in the ovary at pre-ovulation than in the placenta and oviduct at pre-parturition. The mRNA was approximately 1.2kb in size and the expression was high in the ovary, which was the same as the RT-PCR result. We also produced His tagged 20α-HSD proteins by using an Escherichia coli expression system. In a western blot for the 20α-HSD protein, only 1 band of approximately 37-kDa was detected in the ovary, oviduct tissue, and recombinant protein produced in the Chinese hamster ovary (CHO) cell line. However, in the placenta, additional 2 bands (35 and 39 kDa) were detected. Immunohistochemical analyses suggested that the monkey 20α-HSD protein was localized mainly in the syncytiotrophoblast of the placenta and the isthmus cells of the oviduct. According to promoter analyses with the enhanced green fluorescent protein (EGFP) gene, the monkey 20α-HSD promoter was efficiently expressed in the CHO-K1 cell line; however, the promoter was not expressed in bovine fetal fibroblast (bFF) cell. Taken together, our study showed that the 20α-HSD mRNA and protein are coordinately expressed in the ovary at pre-ovulation and in the placenta and oviduct at pre-parturition. Therefore, monkey 20α-HSD in the placenta, ovary and oviduct plays an important role in the estrous cycle, pregnancy, and parturition.

Biotechnological production of 20-alpha-dihydrodydrogesterone at pilot scale.

The human sex hormone progesterone plays an essential and complex role in a number of physiological processes. Progesterone deficiency is associated with menstrual disorders and infertility as well as premature birth and abortion. For progesterone replacement therapy, the synthetic progestogen dydrogesterone is commonly used. In the body, this drug is metabolized to 20α-dihydrodydrogesterone (20α-DHD), which also shows extensive pharmacological effects and hence could act as a therapeutic agent itself. In this study, we describe an efficient biotechnological production procedure for 20α-DHD that employs the stereo- and regioselective reduction of dydrogesterone in a whole-cell biotransformation process based on recombinant fission yeast cells expressing the human enzyme AKR1C1 (20α-hydroxysteroid dehydrogenase, 20α-HSD). In a fed-batch fermentation at pilot scale (70 L) with a genetically improved production strain and under optimized reaction conditions, an average 20α-DHD production rate of 190 µM day(-1) was determined for a total biotransformation time of 136 h. Combined with an effective and reliable downstream processing, a continuous production rate of 12.3 ± 1.4 g 20α-DHD per week and fermenter was achieved. We thus established an AKR-dependent whole-cell biotransformation process that can also be used for the production of other AKR1C1 substrates (as exemplarily shown by the production of 20α-dihydroprogesterone in gram scale) and is in principle suited for the production of further human AKR metabolites at industrial scale.

Differential stimulation pathways of progesterone secretion from newly formed corpora lutea in rats treated with ethylene glycol monomethyl ether, sulpiride, or atrazine.

Ethylene glycol monomethyl ether (EGME), sulpiride, and atrazine are known ovarian toxicants, which increase progesterone (P4) secretion and induce luteal cell hypertrophy following repeated administration. The aim of this study was to define the pathways by which these compounds exerted their effects on the ovary and hypothalamic-pituitary-gonadal (HPG) axis. In the ovary, changes in the steroidogenic activity of new and old corpora lutea (CL) were addressed. EGME (300 mg/kg), sulpiride (100 mg/kg), or atrazine (300 mg/kg) were orally given daily for four times from proestrus to diestrus in normal cycling rats. Treatment with all chemicals significantly increased serum P4 levels, and EGME as well as sulpiride induced increases in prolactin (PRL) levels. In new CL, at both the gene and the protein levels, all three chemicals upregulated the following steroidogenic factors: scavenger receptor class B type I, steroidogenic acute regulatory protein, P450 cholesterol side-chain cleavage, and 3ß-hydroxysteroid dehydrogenase (HSD) and downregulated the luteolytic gene, 20α-HSD. Coadministration of EGME and bromocriptine, a D2 agonist, completely inhibited PRL but not P4 secretion. Additionally, steroidogenic factor expression levels were upregulated, and 20α-HSD level was downregulated in new CL. These results suggest that EGME both directly and indirectly stimulates P4 production in luteal cells, whereas sulpiride elevates P4 through activation of PRL secretion in the pituitary. Atrazine may directly activate new CL by stimulating steroidogenic factor expressions. The present study suggests that multiple pathways mediate the effects of EGME, sulpiride, and atrazine on the HPG axis and luteal P4 production in female rats in vivo.