B Hoffmann

Justus-Liebig-Universität Gießen, Gieben, Hesse, Germany

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Publications (84)136.07 Total impact

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    ABSTRACT: Luteal development is regulated by many locally produced mediators, e.g., prostaglandins and angiogenic factors. However, the role and function of vasoactive factors in the canine corpus luteum (CL) remain largely unknown. Consequently, expression of the endothelin (ET) receptors-A and -B (ETA and ETB,, revealing vasoconstriction and vasodilator properties, respectively), the ET-converting enzyme (ECE-1) and ET-1, -2 and -3, was investigated in CL from non-pregnant dogs (days 5, 15, 25, 35, 45 and 65 post-ovulation), and at selected stages of pregnancy (pre-implantation, post-implantation, mid-gestation), and during normal and antigestagen-induced prepartum luteolysis/abortion. The interrelationship between PGE2 and the ET system was investigated in PGE2-treated canine primary lutein cells from early CL. ET-1 did not change significantly over time; ET-2, ECE-1 and ETB were elevated in early CL and were downregulated towards the mid/late-luteal phase. The prepartum increase of ET-2 was significant. ET-3 increased gradually, and was highest in late CL and/or at prepartum luteolysis. ETA remained constant until the late CL phase and increased only during prepartum luteolysis. ET-1 was localized to the luteal cells, and ET-2, ET-3 and ETA to vascular endothelium. ECE-1 and ETB were detected at both locations. Except for upregulated ET-1 and lack of effect on ET-2, antigestagen applied to mid-pregnant dogs evoked similar changes to those observed during normal luteolysis. PGE2 upregulated ETB in treated cells; ETA and ET-1 remained unaffected, and ET-2 decreased. A modulatory role of the ETs in canine CL, possibly in association with other factors (e.g., PGE2 and progesterone receptor), is strongly indicated.
    Reproduction 08/2015; DOI:10.1530/REP-15-0256
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    Jl Riveros · G Schuler · B Urquieta · B Hoffmann · C Bonacic
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    ABSTRACT: This study evaluated ovarian activity in late gestation and post-partum in guanacos in captivity. Follicular dynamics was monitored every second day from 40 days before and other 40 after delivery by transrectal sonography and by plasma steroids concentrations. Seven out of eight (87.5%) of gestating females presented ovarian follicular activity under progesterone levels >3 nmol/l with maximum follicular size of 8.42 ± 0.83 mm from days 23 to 1 before delivery. After delivery, all females have follicular wave development from day 0 to 38, with larger follicular size and longer follicular wave phases and interwave interval when compared with pre-partum data. During post-partum period, there was a close relationship between follicle size and estradiol-17β concentration, with r = 0.69 at the beginning of growth phase and r = 0.86 in association with the largest dominant follicle. Plasma estradiol-17β concentration varied from 11.92 to 198.55 pmol/l. Plasma estrone sulfate, free estrone and progesterone returned to baseline concentrations during peripartal period and remained basal thereafter. The results described follicular activity during late gestation and early post-partum period. These findings provide relevant information to understand physiological changes occurring during this reproductive key period in seasonal breeders with long gestation duration as New and Old World camelids. © 2014 Blackwell Verlag GmbH.
    Reproduction in Domestic Animals 12/2014; 50(1). DOI:10.1111/rda.12462 · 1.18 Impact Factor
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    ABSTRACT: The mechanisms governing corpus luteum (CL) function in domestic dogs remain not fully elucidated. The upregulated expression of cyclooxygenase 2 and prostaglandin (PG) E2 synthase (PGES) at the beginning of the canine luteal phase indicated their luteotrophic roles, and the steroidogenic activity of PGE2 in the early canine CL has been confirmed in vitro. Recently, by applying a cyclooxygenase 2 (COX2)-specific inhibitor (firocoxib [Previcox]; Merial) from the day of ovulation until the midluteal phase, the luteotrophic effects of PGs have been shown in vivo. This is a follow-up study investigating the underlying endocrine mechanisms associated with the firocoxib-mediated effects on the canine CL. Experimental groups were formed with ovariohysterectomies performed on Days 5, 10, 20, or 30 of firocoxib treatments (10 mg/kg bw/24h; TGs = treated groups). Untreated dogs served as controls. A decrease of steroidogenic acute regulatory (STAR) protein expression was observed in TGs. The expression of PGE2 synthase was significantly suppressed in TGs 5 and 10, and both PGE2 and PGF2α levels were decreased in luteal homogenates, particularly from CL in TG 5. Similarly, expression of the prolactin receptor (PRLR) was diminished in TGs 5 and 20. The expression of PGE2 receptors PTGER2 (EP2) and PTGER4 (EP4), the PG- transporter (PGT) , and 15-hydroxy PG dehydrogenase (HPGD) was not affected in TGs. Our results substantiate a direct luteotrophic role of PGs in the early canine CL, i.e., by upregulating the steroidogenic machinery. Additionally, the possibility of an indirect effect on PRL function arises from the increased prolactin receptor expression in response to PGE2 treatment in canine lutein cells observed in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.
    Theriogenology 12/2014; 83(6). DOI:10.1016/j.theriogenology.2014.12.006 · 1.85 Impact Factor
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    ABSTRACT: The prepartum output of PGF2alpha in the bitch is associated with increased placental PGE2-synthase (PTGES)-mRNA levels. Contrasting with this is a decreased expression of PGF2alpha-synthase (PGFS/AKR1C3) in utero-placental compartments during prepartum luteolysis, suggesting an involvement of alternative synthetic pathways in PGF2alpha synthesis, e.g., conversion of PGE2 to PGF2alpha. However, since the expression and possible functions of the respective PTGES protein remained unknown, no further conclusion could be drawn. Therefore, a canine-specific PTGES antibody was generated and used to investigate the expression, cellular localization and biochemical activities of canine utero-placental PTGES throughout pregnancy and at prepartum luteolysis. Additionally, the biochemical activities of these tissues involved in the conversion of PGE2 to PGF2alpha were investigated. The endometrial PTGES was localized in the uterine surface epithelium pre-implantation, and in superficial and deep uterine glands, endothelial cells and myometrium throughout pregnancy and at parturition. Placental signals were mostly in the trophoblast. The biochemical properties of recombinant PTGES protein were confirmed. Additionally, expression of two PGE2-receptors, PTGER2/EP2 and PTGER4/EP4, revealed their decreasing expression during luteolysis. In contrast, the utero-placental expression of prostaglandin transporter (PGT) was strongly elevated prior to parturition. These localization patterns resembled that of PTGES. The increased expression of PTGES and PGT at parturition, together with the accompanying decreased levels of PGE2-receptors, and the capability of canine uterine and placental homogenates to take part in the conversion of PGE2 to PGF2alpha, as found in this study, suggest that PGE2 could be utilized locally as a substrate for prepartum PGF2alpha synthesis in the dog.
    Biology of Reproduction 10/2014; 91(6). DOI:10.1095/biolreprod.114.122929 · 3.45 Impact Factor
  • M. P. Kowalewski · E. Kautz · E. Hoegger · B. Hoffmann · A. Boos
    18th Annual Conference of the; 09/2014
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    ABSTRACT: In boars high concentrations of estrone sulfate (E1S) are found which are produced in the testicular-epididymal compartment (TEC). However, the exact site of E1S production and the underlying synthetic pathway are still unknown. Previous studies identified the Leydig cells as the only cell type of the TEC exhibiting significant aromatase expression. However, the estrogen specific sulfotransferase SULT1E1-mRNA and estrogen sulfotransferase activity were virtually absent in the testis but readily detectable in the epididymis. In order to locate E1S production measurements were performed in blood samples taken from arterial (CA) and venous blood vessels (CV) of the testicular capsule. Concentrations in CV were significantly higher compared to CA (geometric mean/deviation factor: 56.3/3.3 vs. 3.9/4.4 ng/ml; p = 0.002; n = 4) clearly pointing to the testis as the major source of E1S in TEC. Consistently, E1S tissue concentrations were 16.8 ± 8.1 ng/g wet tissue in testis but below 1 ng/g in epididymal head, body and tail (n = 3). In immunostained sections from epididymis, the most intense signals for SULT1E1 were found in cytoplasmic protrusions of the epithelial cells, which were obviously released into the lumen of the epididymal duct (epididymosomes). In connection with our previous observations the results suggest that in porcine Leydig cells sulfonated precursors may be used for the synthesis of E1S. This hypothesis is further corroborated by the high correlations found between the secretory profiles of pregnenolone sulfate, dehydroepiandrosterone sulfate and E1S measured by liquid chromatography–tandem mass spectrometry. With support from the DFG research group 1369 Sulfonated Steroids in Reproduction.
    Reproductive Biology; 08/2014
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    ABSTRACT: The aim of this study was to test for the postulated luteotropic effect of prostaglandin E2 during early diestrus in the dog in an in vivo study. This study was performed on 30 bitches which were randomly assigned to a treatment group (TG) and a control group. Starting on the day of ovulation (Day 0), dogs of the TG were treated for 5, 10, 20, or 30 days with 10 mg firocoxib/kg body weight per day (Previcox, a selective PTGS2 inhibitor) and ovariohysterectomized for collection of corpora lutea on the last day of treatment. Similarly, dogs of the control group were ovariohysterectomized on Days 0, 5, 10, 20, and 30. Blood samples for progesterone measurement were collected every second day; additionally, the area of luteal cell nuclei and the expression of 3β-hydroxysteroid-dehydrogenase at the mRNA and the protein levels were assessed. Mean P4 concentrations were lower in TGs; however, a significant difference was only observed on Day 10. This observation is in line with the finding that treatment with firocoxib reduced expression of 3β-hydroxysteroid-dehydrogenase mRNA and protein (P < 0.05) and the area of luteal cell nuclei (P < 0.05). The results of this study further point to the postulated luteotropic function of prostaglandin E2.
    Theriogenology 07/2014; 82(6). DOI:10.1016/j.theriogenology.2014.07.005 · 1.85 Impact Factor
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    ABSTRACT: Sulfated steroids have been traditionally regarded as inactive metabolites. However, they may also serve as precursors for the production of active free steroids in target cells. We use the boar as a model to study the metabolism, transport and function of steroid sulfates due to their high production in the porcine testicular-epididymal compartment, of which the role is unknown. To characterize the secretion of free and sulfated steroids, plasma samples were collected from six postpubertal boars over six hours every 20 minutes from the jugular vein. Long-term secretion profiles were also established in seven boars stimulated with hCG. To directly characterize the testicular output, samples were collected from superficial testicular arterial and venous blood vessels. Testosterone, androstenedione and sulfated pregnenolone, dehydroepiandrosterone, estrone and estradiol-17β were determined by liquid chromatography-tandem mass spectrometry. Free estrone and estradiol-17ß were measured by radioimmunoassay. Irrespective of a high variability between individuals, the results suggest that 1) all steroids assessed are primarily produced in the testis, 2) they exhibit similar profiles pointing to a pulsatile secretion with low frequency (3-5 pulses/day), 3) after synthesis at least a major proportion is immediately released into peripheral circulation. The fact that all steroid sulfates assessed are original testicular products and the high correlations to one another suggest their role as being intermediates of testicular steroidogenesis rather than inactivated end products. Moreover, a substantial use of sulfated steroids in porcine testicular steroidogenesis would assign a crucial regulatory role to steroid sulfatase which is highly expressed in Leydig cells.
    Reproduction (Cambridge, England) 06/2014; DOI:10.1530/REP-14-0193 · 3.26 Impact Factor
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    ABSTRACT: BACKGROUND: 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 PGF2alpha 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. METHODS: Several key factors involved in the production and/or actions of PGs were studied: cyclooxygenase 2 (COX2, PTGS2), PGF2alpha-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. RESULTS: 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. CONCLUSIONS: 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.
    Reproductive Biology and Endocrinology 05/2014; 12(1):46. DOI:10.1186/1477-7827-12-46 · 2.41 Impact Factor
  • Klymiuk M.C · Hoffmann B · Bingsohn L · Dezhkam Y · Schuler G
    57th Symposium fur Deutsche Gesellschaft für Endokrinologie, Dresden, Germany; 03/2014
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    ABSTRACT: In adult boars high levels of various sulfated steroids have been determined with maximum concentrations in the systemic circulation up to 150 ng/ml for dehydroepiandrosterone sulfate (DHEAS), 45 ng/ml for estrone sulfate (E1S) and 20 ng/ml for pregnenolone sulfate (P5S). Measurements in the testicular circulation clearly indicate that they mainly originate from the testis. In order to obtain further information on their formation, the expression of sulfotransferases (SULTs) considered relevant for the sulfation of estrogens (SULT1E1), DHEA (SULT2A1) and of P5 and cholesterol (SULT2B1) was investigated on the mRNA (SULTs 1E1, 2A1 and 2B1) and the protein level (SULTs 1E1 and 2B1) in the porcine testis and epididymis applying real-time RT-PCR, western blot and immunohistochemistry (IHC). Moreover, sulfation of E1, DHEA and P5 was assessed in an in vitro assay using cytosolic fractions in the presence of 3′-phosphoadenosine-5′-phosphosulfate. Relative gene expression (RGE) levels of SULT1E1 were high in the epididymal head (EH), decreased gradually in the epididymal body (EB) and tail (ET) but were at the limit of detection in the testis. Corresponding results were obtained from IHC and determinations of enzyme activity. RGE levels for SULT2B1 were minimal in the testis and EH, increased gradually in EB and were maximal in the hind part of ET. In the epididymis this expression pattern was consistent with sulfation of P5 and with cytosolic immunostaining in epithelial cells. However, in the EH epithelium and in various cell types of the testis distinct nuclear signals occurred in the absence of sulfation of P5. RGE levels of SULT2A1-mRNA were high in the testis and variable in the epididymis. However, sulfation of DHEA was virtually undetectable. The release of high amounts of steroid sulfates from the porcine testis in the virtual absence of relevant sulfotransferase activities remains unclear but might be explained by the extensive utilization of sulfated precursors.
    Experimental and Clinical Endocrinology & Diabetes; 03/2014
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    MC Klymiuk · B Hoffmann · L Bingsohn · Y Dezhkam · G Schuler
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    ABSTRACT: 3ß-hydroxysteroid dehydrogenase (3ß-HSD) is a steroidogenic key enzyme catalyzing the conversion of 3ß-hydroxy-5-ene- into 3-keto-4-ene steroids. In pigs so far only one isoenzyme (HSD3B1) has been identified. During investigations into the steroidogenic capacity of the adult porcine epididymis using real-time RT-PCR a surprisingly high expression of HSD3B1- mRNA was found in 6 of 7 animals. Relative gene expression (RGE) levels (geometric mean [dispersion factor]) increased continuously from 6.7 [6.4] in the proximal part of the epididymal head to 143.8 [8.5] in the hind part of the tail, while the RGE levels determined in the testes were only 4.8 [11.7]. In comparison, measurement in adrenal tissue from an adult boar yielded an RGE level of 1494.6. To test for 3ß-HSD activity, the conversion of pregnenolone into progesterone was assessed in an in vitro assay in the presence of an NAD+-regenerating system using microsomal fractions prepared from the testis and the hind part of the epididymal tail as the source of the enzyme (n=5 animals). With 32.9 [2.77] resp. 14.6 [1.65] ng/mg protein/min formation of progesterone in the epididymis vs. testis was much closer than was expected from the mRNA data. This enigmatic observation might be explained by the expression of an enzyme with 3ß-HSD activity in the testis different from HSD3B1. The physiological substrate and the biological role of HSD3B1 highly expressed in the porcine epididymal tail is currently unclear, especially in the light of a high pregnenolone sulforansferase activity and SULT2B1 expression observed in this tissue in a concomitant study.
    47. Jahrestagung Physiologie und Pathologie derFortpflanzung 2014, Giessen; 02/2014
  • B Hoffmann · A W Bernhardt · K Failing · G Schuler
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    ABSTRACT: Objectives: To gain further data on the hormonal control of pregnancy in the donkey and to obtain reference values for hormonal pregnancy testing. Material and methods: Blood samples were collected at monthly intervals from 23 donkey mares with normal singleton pregnancies. Further samples were obtained from six mares displaying pregnancies with clinical disorders. Progesterone (P4), total estrone (TE), free (E) and conjugated estrone (ES) were determined using radioimmunoassay. Results: Mean duration of pregnancy was 372 ± 16 days. It was longer (p < 0.05) in large (375.9 ± 5.7 days) and standard (385.8 ± 20.7 days) donkeys than in miniature donkeys (357.4 ± 5.7 days) and negatively correlated to the age of the mare (p = 0.043). P4-concentrations varied between 12-35 ng/ml during weeks 2-5 of pregnancy and increased thereafter in eight jennies concomitant with the formation of the secondary corpora lutea (sCL), reaching values of 40-110 ng/ml during weeks 12-17. The decrease observed thereafter resulted in concentrations between 5-16 ng/ml until week 46, followed by a slight increase in most of the mares prior to parturition. Concentrations of TE remained < 1 ng/ml until week 6. They increased thereafter to 600-2700 ng/ml during midpregnancy and displayed a decrease to 1-20 ng/ml during the last 2 weeks of pregnancy. The course of E and ES was correlated (p < 0.0001) and E concentrations were up to 1000 times lower than those of ES. The course of hormone concentrations did not provide any clear indications in relation to the observed clinical disorders. Conclusion: The course of P4-concentrations resembles largely the situation in the horse. In contrast to the horse, the course of ES does not show an increase concomitant with the formation of the sCL. Breed-specific effects became apparent regarding pregnancy duration. Clinical relevance: Hormonal pregnancy diagnostic in the jenny could be put on a solid basis with TE values > 5 ng/ml being indicative for pregnancy. At present, monitoring of P4 and estrone during pregnancy does not allow the prediction of clinical disorders.
    Tierärztliche Praxis. Ausgabe G, Grosstiere/Nutztiere 02/2014; 42(1):32-9. · 0.47 Impact Factor
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    ABSTRACT: The canine corpus luteum (CL) functions as a source of progesterone (P4) and 17β-estradiol (E2); however, the transport of energy substrates to maintain its high hormonal output has not yet been characterised. The present study involved the localisation and temporal distribution of the facilitative glucose transporter 1 and the quantification of the corresponding protein (GLUT1) and gene (SLC2A1) expression. Some GLUT1/SLC2A1 regulatory proteins, such as HIF1A (hypoxia inducible factor 1 alpha) and FGF2 (fibroblast growth factor 2), and mRNAs, such as HIF1A, FGF2, and VEGFA (vascular endothelial growth factor), and VEGFA receptors 1 and 2 (FLT1 and KDR) were also analysed from days 10 to 70 after ovulation. Additionally, plasma P4 and E2 levels were assessed via chemiluminescence. Moreover, the canine KDR sequence has been cloned; thereby, enabling subsequent semi-quantitative PCR (qPCR) analysis. Our results demonstrate time-dependent variations in the expression profile of SLC2A1 during dioestrus, which were accompanied by highly correlated changes (0.84 < r < 0.98; P <0.03) in the gene expression of HIF1A, VEGF, and FLT1 as well as in P4 plasma concentrations. FGF2 mRNA correlated with E2 plasma concentrations (r = 0.61; P = 0.01). Our data reveal that glucose transporter is regulated throughout the CL life span and suggest that CL depends upon the sensing of hypoxia and the status of luteal vascularisation. Moreover, time-dependent expression of GLUT1/SLC2A1 may lie underneath increased metabolic and energetic requirements for sustaining P4 production.
    Reproduction 10/2013; DOI:10.1530/REP-13-0398
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    ABSTRACT: Testicular function in the dog was downregulated using two different GnRH agonist implants, with adult and juvenile testes serving as controls. Treatment resulted in an increased % of the interstitial area and decreased area of Leydig cell nuclei. Expression of Steroidogenic acute regulatory protein (StAR) and the steroidogenic enzymes cytochrome P450 side-chain cleavage enzyme (P450scc) and cytochrome P450 17α-hydroxylase- 17, 20-lyase (P450c17) in Leydig cells was blocked on the mRNA- and protein level, showing no differences between the two agonists. Staining for androgen receptor (AR) by IHC was positive in Sertoli-, Leydig- and peritubular cells and some spermatogonia with ISH confirming expression in Sertoli cells. Expression on the AR-mRNA was not affected, however, translation was blocked (reduced percentage of AR-positive Sertoli cells), with the number of nuclei in basal position being decreased. In the juvenile testes mRNA expression of StAR, P450scc and P450c17 was higher compared to the other groups but distinctly lower for the AR. On the protein level expression was at the limit of detection for StAR; AR positive Sertoli cells were not detected.Our observations show that the downregulated testis is different from the juvenile one rather resembling the testicular status in seasonal breeders out of season.
    Reproduction 09/2013; 146(6). DOI:10.1530/REP-13-0195
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    ABSTRACT: There is no distinct explanation of the mechanism for the prepartal prostaglandin F2alpha (PGF2alpha) increase in pregnant dogs. While 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-prostaglandin 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 (CL) throughout pregnancy and at prepartum luteolysis. PGFS (AKR1C3) expression was weak or absent in luteal samples. Uterine PGFS (AKR1C3) was up-regulated post-implantation 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 post-implantation, lower at mid-gestation, 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 co-localization of HPGD with the PGFS (AKR1C3) expression suggests a modulatory role for the HPGD as a "gate keeper" of the supply of prostaglandin in the pregnant canine uterus.
    Biology of Reproduction 05/2013; 89(1). DOI:10.1095/biolreprod.113.109918 · 3.45 Impact Factor
  • P Khatri · B Hoffmann · G Schuler
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    ABSTRACT: OBJECTIVES: In cattle no biological role has been definitely identified for placental estrogens and progesterone. However, in the bovine trophoblast androgens may also be produced and have local effects. Thus, the aims of this study were to identify androgen receptor (AR) expressing cells and to monitor testosterone tissue concentrations in bovine placentomes throughout gestation. METHODS: Placental AR expression was characterized at the mRNA and protein level applying conventional and real-time RT-qPCR, western blot and immunohistochemistry. Testosterone was measured by radioimmunoassay. RESULTS: AR-mRNA was qualitatively detected from day 50 of gestation until term. Mean relative gene expression levels were constant between day 100 and late gestation. A slight non-significant increase was observed in the prepartal period. With immunohistochemistry distinct nuclear signals were predominantly observed in invasive trophoblast giant cells (TGC) from day 80 until term. In mature TGC of the trophoblast, immature TGC and uninucleated trophoblast cells, stromal cells of the chorionic villi, caruncular epithelial and stromal cells immunoreactive score values were low at early and midgestation but increased significantly (p < 0.01) during late gestation and remained high until parturition. With western blot in placentomal tissue a specific band of approximately 110 kDa was detected as it was the case in epididymis used as a positive control. Testosterone concentrations increased from 0.70 ± 0.29 pmol/g wet tissue between days 60-220 to 4.22 ± 1.29 pmol/g during late gestation (p < 0.001). DISCUSSION: The results are consistent with androgens as active products of bovine placental steroidogenesis. The substantial up-regulation of AR expression during TGC differentiation suggests that androgens may be related to this process.
    Placenta 03/2013; 34(5). DOI:10.1016/j.placenta.2013.01.018 · 3.29 Impact Factor
  • H. Körber · A. Wehrend · B. Hoffmann · S. Goericke-Pesch
    Reproductive biology 02/2013; 13:30–31. DOI:10.1016/j.repbio.2013.01.081 · 1.05 Impact Factor
  • M Gentil · B Hoffmann · A Spang · K Failing · S Goericke-Pesch
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    ABSTRACT: To date, no details are available concerning the restart of steroidogenesis following the downregulation of testicular endocrine and germinative function by gonadotrophin-releasing hormone (GnRH)-agonist implants. This restart was assessed by determining the expression of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage enzyme (P450scc) and cytochrome P450 17α-hydroxylase,17,20-lyase (P450c17). The re-establishment of steroidogenesis was initiated by the removal of the GnRH-agonist implant (18.5 mg azagly nafarelin, Gonazon) at 5 months after treatment. Testes were removed at 3-week intervals (weeks 0-24) and four groups were formed according to the stage of spermatogenesis as revealed by the most developed germ cells observed (developmental group [DG] spermatocytes to DG elongated spermatids). Five dogs served as untreated controls. Positive immunostaining for StAR, P450scc and P450c17 was restricted to Leydig cells. Western blot indicated the specifity of the respective antibodies with hints of a expression of canine-specific P450scc and P450c17 proteins. A significant effect of group was observed for a percentage of the immunopositive area (PIA) as an indicator of active Leydig cells for StAR (P<0.05), P450scc (P<0.001) and P450c17 (P<0.001), with PIA being lowest for the DG spermatocytes. With regard to the strength of the immunopositive signal, a significant effect of group was found for P450scc (P<0.01) and P450c17 (P<0.05), with the lowest intensity being observed in DG spermatocytes. At the mRNA level, the upregulation from DG spermatocytes to DG round spermatids was clearly evident but was only significant for P450scc (P<0.05). Thus, downregulation affects the whole cascade of steroidogenesis, whereas withdrawal of inhibition results in a rapid restart, in part indicating a rebound phenomenon.
    Cell and Tissue Research 10/2012; 350(3). DOI:10.1007/s00441-012-1506-5 · 3.33 Impact Factor