The bovine placenta; a source and target of steroid hormones: observations during the second half of gestation

Domestic Animal Endocrinology 23 (2002) 309–320

The bovine placenta; a source and target of steroid hormones: observations during the second half of gestation B. Hoffmann, G. Schuler∗ Klinik für Geburtshilfe, Gynäkologie und Andrologie der Groß- und Kleintiere mit Tierärztlicher Ambulanz, Justus-Liebig-Universität, D-35392 Giessen, Germany

Abstract Apart from estrone-3-sulfate (E1S) the bovine placenta produces progesterone (P4), though the corpus luteum is the major source of P4 responsible for maintaining pregnancy. So far the biological function of placental steroids in cattle is largely unknown. However, since the local availability of free estrone (E1) in the placenta seems to be controlled by sulfatase and sulfotranferase, the hypothesis was developed that placental estrogens and P4 might act as local regulatory factors. To test for such a function placentomes from 150, 220, 240, 270 days (D) pregnant and parturient cows were screened immunohistochemically for progesterone and estrogen receptors (PR, ER). PR were found at all stages in the caruncle in stromal cells and capillary pericytes but only at parturition in arterial walls. Percentage of PR-positive caruncular stromal cells (CSC) increased (P < 0.05) from 51.8 ± 2.6% at D150 to 58.9 ± 1.8% at parturition. ER were detected in CSC, caruncular epithelial (CE) cells and in caruncular capillary pericytes. Mean percentage of ER-positive CSC decreased from 39.0 ± 5.9% in pregnant cows to 17.5 ± 8.3% at parturition (P < 0.05). In CE all cells exhibited positive signals with the exception of those immediately surrounding large primary chorionic villi. Proliferation was assessed immunohistochemically by determining the percentage of Ki67-antigen positive cells. Highest values (P < 0.001) were obtained for CE (58.0–68.3%), followed by the trophoblast (23.3–25.4%), CSC (10.6–45.3%) and the stroma of the chorionic villi (2.9–10.5%). A transient depression of proliferation in CSC between D150–270 (P < 0.05) paralleled local estrogen tissue concentrations. The results suggest that placental estrogens and P4 are important factors controlling caruncular growth, differentiation and function. © 2002 Elsevier Science Inc. All rights reserved.

∗

Corresponding author. E-mail address: [email protected] (G. Schuler).

0739-7240/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 0 2 ) 0 0 1 6 6 - 2

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1. Introduction As in many mammalian species, also in cattle the placenta produces estrogens and progesterone (P4). Estrone-3-sulfate (E1S) is the major placental estrogen. Local estrogen production has been detected as early as on day 33 of gestation; however, in peripheral maternal blood E1S concentrations only start to increase between days 70 and 100 to reach a plateau of about 15–30 nmol/L between days 265 and parturition. Estrone (E1) follows a basically similar pattern but on a 10–15-fold lower level until the last 2 weeks of gestation, when it starts to increase disproportionally to E1S reaching almost equimolar concentrations immediately prior to parturition [1,2]. Concerning the biological role of placental estrogens in the bovine species, so far no function and target organ have been identified in the period between the onset of their production and late gestation. Largely based on clinical observations and only immediately prior to birth, parturition-related functions such as stimulation of myometrial activity, placental maturation and softening of the birth canal have been suggested [3]. In comparison to ovarian estrogens of which the biological roles have been widely elucidated, placental estrogen production in cattle exhibits some particular features. (1) With the exception of the immediate prepartal phase, the main product is E1S which is not active at the nuclear estrogen receptors [4]. After hydrolysis, the biological activity of the resulting E1 is clearly lower than that of estradiol-17␤ [5] which is the main product of ovarian estrogen synthesis. (2) Plasma concentrations of placental estrogens by far exceed those of ovarian origin and may continuously exert their activity over a period of several months. (3) Any biological activity of placental estrogens must be expressed in the presence of high P4 levels. Thus on a molecular level the processes induced by placental estrogens could be partially different from the function of ovarian estrogens. Also placental P4 synthesis in cattle has not yet been fully understood. In contrast to other species also exhibiting placental progestagen production like the sheep and the horse, where the placenta adopts the role as the main source of progestagens after a species specific phase of pregnancy maintenance by luteal P4 [6], in cattle the placenta only contributes temporarily and to a minor extent to maternal systemic P4 levels since the corpus luteum is the main source of P4 throughout gestation [7–10]. Thus, the question for the biological need of an apparently subordinate accessory P4 production in bovine gestation arises. As for placental estrogens also for the extra produced placental P4 in cattle so far no specific target organs or target cells have been identified. In view of this statement it must, however, be considered that apart from acting as classical endocrine hormones, sex steroids may also function as auto or paracrine regulators, as has been demonstrated for testosterone within the testis [11] or as is indicated for P4 and estradiol-17␤ by the presence of progesterone receptors (PR) [12] and estrogen receptors (ER) [13,14] in the ovary. Similarly also placental steroids could exert their actions in close proximity to the site of their production which is the trophoblast. With the present paper it is attempted to put into perspective recent data on the local expression of PR [15] and ER [16,17] and on cellular dynamics [18,19] in the bovine placenta with placental steroidogenesis in cattle [20,21] to forward a concept of a putative role of placental steroids as local regulators of placental growth, differentiation and functions.

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2. In vitro investigations on bovine placental steroidogenesis In previous studies [22–26] cotyledonary tissue was identified as the main source of placental estrogens. However, in spite of many investigations, a complete picture of the underlying steroidogenic pathways was only obtained by Schuler et al. [20]; in the in vitro investigations performed pregnenolone (P5) was incubated with cotyledonary homogenates from 220 and 270 days pregnant and from parturient cows (n = 3 per group) and the formation of E1, androstenedione, dehydroepiandrosterone (DHEA) and 17␣-OH-P5 (collected as one fraction), 17␣-OH-P4 and P4 was monitored by submitting sample extracts to HPLC-separation. 3 H-P5 was rapidly metabolized (Fig. 1) with P4 clearly being the main metabolite on days 220 and 270 (42±3.2%, respectively, 53.8±2.2% of 3 H-activity recovered in the HPLC-fraction corresponding to P4), whereas at parturition the 3 H-activity according to P4 was only at 1.6 ± 0.8%. E1 was the only estrogen detected; formation was minimal on day 220 (2.3 ± 0.9%) and day 270 (3.7±1.0%), whereas it was the main product of placental steroidogenesis (50.8±9.2%) at parturition. Parallel incubations with 3 H-P4 (Fig. 2) showed that cotyledonary metabolism of P4 was low on days 220 and 270 (71.3 ± 4.0%, respectively, 84.8 ± 1.3% unmetabolized after 5 min of incubation) with 17␣-OH-P4 being the main metabolite (17.2 ± 3.5%, respectively, 8.0 ± 1.5%); no significant formation of E1 could be detected. At parturition, however, 3 H-P4 was rapidly converted (12.2±5.8% unmetabolized after 5 min of incubation) with 17␣-OH-P4 again being the main metabolite (70.3 ± 3.8%); the formation of E1 (4.6 ± 1.1%) was just above background level (2.6 ± 0.4%). These results show that (1) the bovine placenta contains all enzymes to autonomously produce estrogens from P5, (2) the 5-pathway is the main route of bovine placental estrogen synthesis as the conversion of P4 into androstenedione is almost completely blocked after 17␣-hydroxylation, (3) the increased risk of abortion in late gestation after ablation of luteal function is not a result of a decrease in placental P4 synthesis, (4) the prepartal collapse of placental P4 synthesis and the concomitant increase of free estrogens are not due to a direct conversion of P4 into E1 but obviously mainly results from an increased

Fig. 1. Conversion of 1.74 pmol 3 H-pregnenolone (P5) by 50 mg homogenized cotyledonary tissue from 220 and 270 days pregnant and from parturient cows (n = 3 per group) after 5 min of incubation. Results are presented as percent 3 H-activity measured in HPLC-fractions corresponding to the substrate P5 and the metabolites estrone (E1), androstenedione (A), dehydroepiandrosterone plus 17␣-hydroxyprogesterone (DHEA/HP5), 17␣-hydroxyprogesterone (HP4) and progesterone (P4).

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Fig. 2. Conversion of 0.37 pmol 3 H-progesterone (P4) by 50 mg homogenized cotyledonary tissue from 220 and 270 days pregnant and from parturient cows (n = 3 per group) after 5 min of incubation. Results are presented as percent 3 H-activity measured in HPLC-fractions corresponding to the substrate P4 and the metabolites estrone (E1), androstenedione (A) and 17␣-hydroxyprogesterone (HP4).

influx of P5 into the 5-pathway, (5) bovine placental estrogen synthesis occurs at a relatively constant basal level until the immediate prepartal period, when a sudden increase is observed, and (6) The mechanism underlying the prepartal switch in bovine placental steroidogenesis is a distinct up-regulation of 17␣-hydroxylase-C17,20-lyase (P450c17␣) activity. These data are consistent with the in vivo observations of an immediate prepartal increase of E1 [1], however, they provide no evidence of the in vivo observation clearly suggesting that E1S is the major placental estrogen [26]. To clarify this discrepancy, further in vitro studies were performed to test for the activity of steroid sulfatase (STS) and estrogen sulfotransferase (OST) in cotyledonary and caruncular tissue homogenates [21]. Following incubation with 3 H-E1S the rapid formation of 3 H-E1 indicated high STS activities in both tissues. Apparently STS activity completely overrode OST activities, as OST activity only became detectable after competitive inhibition of STS activity by the addition of 4-nitrophenyl sulfate to the incubation medium. These observations may explain why preceding in vitro studies on placental steroidogenesis [20] only yielded free E1. Investigations performed in 150, 220, 240 and 270 days pregnant and parturient animals (n = 3 per group) showed moderately but significantly higher OST activities in the cotyledons than in the caruncles, whereas STS activities were clearly higher in the maternal part of the placentome (Fig. 3). No influence of the observational group was found with OST, whereas STS activity was significantly lower in parturient compared to pregnant animals. These observations suggest that the marked prepartal increase of free estrogens results from a dramatic increase of the de novo synthesis of E1 and not from an enhanced hydrolysis of conjugated estrogens accumulated in the feto-placental compartment as has been suggested by Janszen et al. [27]. However, in spite of the scarce data on the cellular distribution and expression of STS and OST, the established co-localization of these two enzymes catalyzing opposite reactions leading to the inactivation of free estrogens and activation of conjugated estrogens might represent a mechanism controlling the local availability of E1 within the placentome; a similar mechanism was postulated for human breast cancer tissue [28]. In addition, this observation provides a first subtle hint towards a function of placental estrogens as local para/autocrine factors.

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Fig. 3. (A) Sulfoconjugation of 0.6 pmol 3 H-estrone and (B) hydrolysis of 0.6 pmol 3 H-estrone-3-sulfate by 50 mg homogenized caruncular and cotyledonary tissue from 150, 220, 240 and 270 days pregnant and from parturient cows (n = 3 per group) after 5 min of incubation.

3. Expression of estrogen receptors and progesterone receptors in the bovine placenta The concept of placental steroids as local regulatory factors implies the presence of the respective receptors. Thus, immunohistochemical studies on the expression of PR [15] and ER [16,17] were performed to identify putative target cells of placental steroids within the placentome. For the immunolocalization of PR, the primary antibody 10A9 directed against the highly conserved C-terminus of the PR-molecule was used. Using this antibody, PR were detected at all stages under investigation in a proportion of cells of the caruncular stroma (Fig. 4). Positive reactions were primarily found in the nuclei of the fibrocyte-like cells of the maternal caruncular septa. Moreover, some vascular pericytes, especially of capillaries located in the free margin of superficial maternal septa, exhibited also specific nuclear staining. From day 150 to 270 the staining patterns and intensity were very uniform and no alterations in relation to the stage of gestation could be observed. A representative micrograph from a 220 days pregnant cow is shown in Fig. 4A. In placentomes from parturient cows, few positive nuclear reactions were additionally found in the walls of small caruncular arteries. In these animals a higher and more homogenous nuclear staining in caruncular stromal cells (CSC) was observed (Fig. 4B) than in the pregnant animals. No positive reaction could be identified in any other cell type of the caruncle or in the fetal part of the placentome.

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Fig. 4. Immunolocalization of progesterone receptors in placentomes from a 220 days pregnant (A) and a parturient cow (B). Positive reactions are exclusively found in a proportion of stromal cells of maternal septa surrounding cross-sectioned chorionic villi (CV). Magnifications: A: 320×; B: 160×.

Fig. 5. Staining patterns of the monoclonal antibodies 1D5 (A) and AER311 (B)—both directed against estrogen receptor ␣ (ER␣)—in an identical placentome specimen from a 150 days pregnant cow. With 1D5 recognizing the N-terminus of ER␣, positive staining is exclusively found in a proportion of stromal cells of maternal septa surrounding cross-sectioned chorionic villi (CV). With the C-terminally directed AER311, additional signals are present in all caruncular epithelial cells. Magnifications: A: 340×; B: 300×.

Fig. 6. Proliferative activity in placentomes from a 150 days (A) and a 270 days (B) pregnant cow as indicated by the immunohistochemical detection of the proliferation marker Ki67-antigen. At both stages of gestation the vast majority of positive reactions was located in the caruncular epithelium. Magnifications: A: 200×; B: 320×.

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Table 1 Percentage (X¯ ± SD) of estrogen receptor (ER) and progesterone receptor (PR) positive caruncular stromal cells (CSC) in bovine placentomesa Observational group (OG); n = 3 animals

PR-positive CSC (%)

ER-positive CSC (%)

Day 150 Day 220 Day 240 Day 270 Parturition

51.8 ± 2.6 51.5 ± 2.2 53.9 ± 1.9 56.2 ± 5.7 58.9 ± 1.8

34.0 ± 5.26 41.9 ± 3.19 40.4 ± 5.56 39.6 ± 5.92 17.5 ± 8.28

P(OG)

0.047

0.011

a

Values are based on the evaluation of three tissue sections of one placentome per animal with a minimum total number of 600 cells classified per section.

The percentage of PR-positive CSC rose slightly from 51.8 ± 2.6% on day 150 to 58.9 ± 1.8% at parturition (Table 1). Analysis of covariance revealed a linear trend between days 150 and 270 (P < 0.05). For the immunolocalization of ER four different monoclonal antibodies specific for the ER␣ were applied: 1D5 [29], AER311 [30], AER314 [30] and HT277 [31,32]. Two of these antibodies, 1D5 and AER314 recognized epitopes located within the N-terminus of the ER-molecule, whereas AER311 and HT277 were directed towards the C-terminus. In calf endometrium used as a positive control, these antibodies concordantly gave positive nuclear signals in a proportion of the stromal cells while all luminal and glandular epithelial cells showed a positive cytoplasmic and nuclear staining. In contrast to the uniform staining pattern in calf endometrium, these antibodies produced different staining patterns in the placentomes, obviously depending on the localization of the respective epitope in the C- or the N-terminus of the ER-molecule. Both type antibodies identified ER in a proportion the caruncular stroma cells (Fig. 5). As was the case with PR, the majority of positive reactions was located in the nuclei of the fibrocyte-like cells; sporadically also vascular pericytes, especially of capillaries located in the free margin of superficial maternal septa, exhibited a specific nuclear staining. Using clone 1D5, the mean percentage of ER-positive CSC was 39.0 ± 5.9% in pregnant cows and decreased to 17.5 ± 8.3% at parturition (P < 0.05; Table 1). However, in the caruncular epithelium only AER311 (Fig. 5B) and HT277, antibodies recognizing the C-terminus of the ER, gave positive nuclear and cytoplasmic signals which were found in all caruncular epithelial (CE) cells with the exception of those facing the chorionic plate or immediately surrounding large primary chorionic villi. No positive reactions could be identified in the fetal part of the placentome. Possible reasons for the diverging staining patterns may be differential masking of the respective epitopes induced by ligand binding or interaction of the ER with other regulatory proteins or DNA, differential breakages of fixer-induced cross-linkages by microwave irradiation [31] which was used for antigen retrieval, N-terminal hyperphosphorylation of ER [33], or the differential recognition of a N-terminally truncated [34] or exon-deleted variants [35]. These results strongly suggest that ER are expressed in caruncular stromal and epithelial cells. This conclusion is substantiated by the detection of ER␣-specific mRNA by means of RT-PCR in the bovine placentome [17].

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Table 2 Percentage (X¯ ± SD) of Ki67-antigen positive cells in various cell types of the bovine placentome at different stages of gestation and at parturitiona Observational group (OG); n = 3 animals

Caruncular stroma

Caruncular epithelium

Stroma of chorionic villi

Trophoblast

Day 150 Day 220 Day 240 Day 270 Parturition P(OG)

30.9 ± 1.2 10.6 ± 3.4 20.2 ± 5.7 27.6 ±15.6 45.3 ± 5.5 0.016

67.2 ± 4.5 58.0 ± 6.9 63.7 ± 6.8 68.3 ± 5.7 – 0.379

10.5 ± 1.7 6.1 ± 2.0 7.9 ± 1.1 2.9 ± 0.4 – 0.005

24.9 ± 1.7 25.4 ± 4.7 23.3 ± 3.4 25.1 ± 5.1 – 0.953

a For each cell type, values are based on the evaluation of three tissue sections of one placentome per animal with a minimum total number of 600 classified cells per section and cell type.

4. Proliferative activity in different cell types of the placentome In an attempt to identify functions which may be under the control of placental steroids, cellular dynamics in the bovine placentome were characterized [18,19] in order to test for correlations to the expression of ER and PR and the local availability of the respective ligands. Immunohistochemical detection of the proliferation marker Ki67-antigen was used to characterize proliferative activity [19]. The percentage of positive cells was registered in four different cell types of the placentome, the caruncular epithelium, the caruncular stroma, the stroma of the chorionic villi and the trophoblast. Proliferative activity followed very different patterns in the cell categories under investigation (P < 0.0001; see Fig. 6 and Table 2). The lowest percentage of Ki67-antigen positive cells (%Ki67+ ) was found in the stroma of the chorionic villi. It decreased linearly from 10.5 ± 1.7 on day 150 to 2.9 ± 0.4 on day 270 (P < 0.005). In the trophoblast %Ki67+ exhibited only slight variations between 23.3 ± 3.4 on day 240 and 25.4±4.7 on day 220. In the caruncular stroma %Ki67+ decreased (P < 0.05) from 30.9±1.2 on day 150 to 10.6 ± 3.4 on day 220 to increase thereafter to 45.3 ± 5.5 at parturition. With %Ki67+ ranging between 58.0 ± 6.9 on day 220 and 68.3 ± 5.7 on day 270, proliferation in caruncular epithelium by far exceeded that in other cell types; it followed a similar pattern as in caruncular stroma. However, influence of experimental group was not significant. In parturient animals, proliferation was only assessed in the caruncular stroma as identification of other cell types was uncertain due to the characteristic prepartal changes in placentomal architecture [36,37]. However, apart from numerous positive signals in the caruncular stroma, positive reactions were also found in remnants of the caruncular epithelium and in some uninucleated trophoblast cells, whereas in the stroma of chorionic villi positive staining was virtually absent. 5. Evidence for a role of placental steroids as local regulators of growth, differentiation and functions in the bovine placenta The identification of ER and PR in caruncular tissue in the immediate proximity to the site (trophoblast) of estrogen and P4 production suggests that both type steroids also exert local,

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paracrine activities in respect to control of caruncular growth and function. Concerning placental estrogens, this conclusion is supported by the observation that the proliferative activity in the ER-expressing stroma exactly parallels local E1 tissue concentrations [38,39] with a transient decrease between days 150 and 270, followed by an increase towards parturition. Particular target cells seem to be those of the caruncular epithelium with an almost excessive rate of proliferation. Proliferation of these cells seems to be largely independent from general caruncular growth as it continues undiminishedly until late gestation (Fig. 6B) when placentomal growth is minimal [40]; further on it clearly exceeds proliferation of its fetal counterpart, the trophoblast, where additionally to growth and continuous tissue remodelling proliferation also reflects the continuous replacement of degenerating trophoblast giant cells [41]. The high turn over of CE cells may occur in order to avoid the formation of a high, multi-layered epithelium. However, also another observation should be considered. As observed by light microscopy degenerating CE cells are either phagocytosed by the trophoblast or perish as components of the short-living feto-maternal hybrid cells [42,43]. Hence the hypothesis is put forward that the phagocytosis of apoptotic cell detritus in the trophoblast which was observed throughout the second half of gestation [18] may be an important source of nutrients in addition to those delivered via diffusion. Thus, similar to the production of the histiogenic “uterine milk” [44] by endometrial glands in early gestation bovine placentomes may provide nutrients to the fetus, and placental estrogens may be involved in the stimulation and maintenance of this process. A still open question concerns the production of E1 by the placenta versus estradiol-17␤ by the ovary. In comparison to estradiol-17␤, E1 is commonly regarded as a weakly to moderately effective estrogen [5], and sulfoconjugated estrogens are not active at the nuclear ER [4]. These observations in conjunction with the expression of STS and OTS at the placental level suggest a complex but subtle regulatory mechanism, where in addition to estrogen synthesis the availability of free E1 is regulated by both enzymes on a cellular level. Sulfoconjugation in ER-positive target cells could serve to limit the actions of placental E1, thereby avoiding undesired local or systemic actions. Moreover, E1 could be the predominant placental estrogen not to induce the full spectrum of estrogenic actions but only a limited one, meeting the needs of pregnancy in terms of an endogenous SERM (selective estrogen receptor modulator [45]). As for E1 there are still many questions concerning the biological role of placental P4 in cattle. Certainly, the need for an additional endocrine source to the corpus luteum is rather unlikely since the bovine placenta only contributes to a minor extent to the peripheral maternal plasma levels which are rather constant throughout bovine gestation [46]. However, the close proximity between the P4 producing trophoblast and the putative target cells in the caruncular stroma suggest that the PR-expressing cells of the caruncles are under the control of placental rather than luteal P4. This concept is supported by the fact that—despite its negligible contribution to systemic maternal levels—the onset of placental P4 production is associated with a considerable increase in local tissue concentrations [39,47]. Thus, whereas some actions of P4 like the closure of the cervix and the quiescence of the myometrium may occur at comparatively low systemic plasma levels, other functions, especially at the feto-maternal interface, may depend on clearly higher concentrations. Interestingly, in the pregnant ovine uterus specific functions of high P4 levels have been identified in the regulation of local immunotolerance [48]. A possible importance of locally available P4 is also stressed by the continuous increase of PR concentrations in the caruncular stroma until parturition. Hence not

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only the withdrawal of luteal but also of placental P4 immediately prior to parturition might be an important biological phenomenon. The expression of PR in caruncular capillary pericytes detected from day 150 to parturition and in the walls of small caruncular arteries in parturient animals suggests that placental P4 may also be involved in the local regulation of caruncular angiogenesis and/or blood flow. In conclusion, investigations on placental steroidogenesis, ER and PR expression, local tissue steroid concentrations and cellular dynamic clearly point to a role of placental steroids as local regulators of placental growth, differentiation and functions in cattle. However, further research is needed to identify the underlying processes on a molecular level.

Acknowledgments The authors acknowledge the assistance of Prof. R. Leiser, Drs. Karl Klisch and Christiane Pfarrer, Institut für Veterinär-Anatomie, -Histologie und -Embryologie, Justus-LiebigUniversität Giessen, with the preparation of tissue samples. We highly appreciate the financial support by the German Research Foundation (DFG grant SCHU 1195/1-1), the Ewald and Hilde Berge-Foundation and the Kogge-Foundation. References [1] Hoffmann B, Goes de Pinho T, Schuler G. Determination of free and conjugated oestrogens in peripheral blood plasma, feces and urine of cattle throughout pregnancy. Exp Clin Endocrinol Diabetes 1997;105:296–303. [2] Eley RM, Thatcher WW, Bazer FW. Hormonal and physical changes associated with bovine conceptus development. J Reprod Fertil 1979;55:181–90. [3] Birgel EH, Zerbe H, Grunert E. Untersuchungen über Zusammenhänge zwischen Anzeichen der nahenden Abkalbung und Steroidhormonprofilen. Prakt Tierarzt 1996;77:627–30. [4] Hähnel R, Twaddle E, Ratajczak T. The specificity of the estrogen receptor of human uterus. J Steroid Biochem 1973;4:21–31. [5] Hoffmann B, Karg H. Metabolic fate of anabolic agents in treated animals and residue levels in their meat. In: Lu FC, Rendel J, editors. Anabolic agents. Stuttgart: Georg Thieme Publishers, 1976. p. 181–91. [6] Hoffmann B. Gravidität, Geburt, Puerperium. In: Döcke F, editor. Veterinärmedizinische Endokrinologie. 3rd ed. Stuttgart: Gustav Fischer Verlag, 1994. p. 509–41. [7] Chew B, Erb RE, Fessler JF, Callahan CJ, Malven PV. Effects of ovariectomy during pregnancy and of prematurely induced parturition on progesterone estrogens, and calving traits. J Dairy Sci 1979;62:557–66. [8] Day AM. Cloprostenol for termination of pregnancy in cattle. N Z Vet J 1977;25:139–44. [9] Estergreen VL, Frost OL, Gomes WR, Erb RE, Bullard JF. Effect of ovariectomy on pregnancy maintenance and parturition in dairy cows. J Dairy Sci 1967;50:1293–5. [10] Johnson WH, Manns JG, Adams WM, Mapletoft RJ. Termination of pregnancy with cloprostenol and dexamethasone in intact or ovariectomized cows. Can Vet J 1981;22:288–90. [11] Zirkin BR. Spermatogenesis: its regulation by testosterone and FSH. Semin Cell Dev Biol 1998;9:417–21. [12] Slomczynska M, Krok M, Pierscinski A. Localization of the progesterone receptor in the porcine ovary. Acta Histochem 2000;102:183–91. [13] Slomczynska M, Wozniak J. Differential distribution of estrogen receptor beta and estrogen receptor alpha in the porcine ovary. Exp Clin Endocrinol Diabetes 2001;109:238–44. [14] Rosenfeld CS, Roberts RM, Lubahn DB. Estrogen receptor- and aromatase-deficient mice provide insight into the roles of estrogen within the ovary and uterus. Mol Reprod Dev 2001;59:336–46.

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[15] Schuler G, Wirth C, Klisch K, Pfarrer C, Leiser R, Hoffmann B. Immunolocalization of progesterone receptors in bovine placentomes throughout mid and late gestation and at parturition. Biol Reprod 1999;61:797–801. [16] Schuler G, Wirth C, Hoffmann B, Klisch K, Leiser R, Pfarrer C. Placental steroids in cattle: evidence for a role as local regulators of placental growth and differentiation. Biol Reprod 1999;60(Suppl 1):126. [17] Schuler G, Wirth C, Teichmann U, Failing K, Leiser R, Thole H, Hoffmann B. Occurrence of estrogen receptor ␣ in bovine placentomes throughout mid and late gestation and at parturition. Biol Reprod (in press). [18] Pfarrer C, Wirth C, Schuler G, Klisch K, Leiser R, Hoffmann B. Frequency, ultrastructural features, and relevance of apoptosis in the bovine placenta. Biol Reprod 1999;60(Suppl 1):222. [19] Schuler G, Wirth C, Klisch K, Failing K, Hoffmann B. Characterization of proliferative activity in bovine placentomes between day 150 and parturition by quantitative immunohistochemical detection of Ki67-antigen. Reprod Dom Anim 2000;35:157–62. [20] Schuler G, Hartung F, Hoffmann B. Investigations on the use of C-21-steroids as precursors for placental oestrogen synthesis in the cow. Exp Clin Endocrinol 1994;102:169–74. [21] Hoffmann B, Falter K, Vielemeier A, Failing K, Schuler G. Investigations on the activity of bovine placental oestrogen sulfotransferase and sulfatase from midgestation to parturition. Exp Clin Endocrinol Diabetes 2001;109:294–301. [22] Evans G, Wagner WC. In vitro oestrogen synthesis by bovine placenta during pregnancy and induced parturition. Acta Endocr 1981;98:119–25. [23] Larson K, Wagner WC, Sachs N. Oestrogen synthesis by bovine foetal placenta at normal parturition. Acta Endocr 1981;98:112–8. [24] Gross TS, Williams WF. In vitro steroid synthesis by the placenta of cows in late gestation and at parturition. J Reprod Fertil 1988;83:565–73. [25] Hoedemaker M, Weston PG, Wagner WC. Influence of cortisol and different steroidogenic pathways on estrogen synthesis by the bovine placenta. Am J Vet Res 1990;51:1012–5. [26] Hoffmann B. Untersuchungen zur Steroidhormonsynthese in der Plazenta des Rindes. Wien Tierärztl Mschr 1983;70:224–8. [27] Janszen BPM, Bevers MM, Van Tol HTM, Dieleman SJ, Van der Weijden GC, Taverne MAM. Oestrogen sulphatase activity in endometrium and foetal membranes of late gestational and parturient cows. Anim Reprod Sci 1995;37:251–6. [28] Pasqualini JR, Chetrite GS. Estrone sulfatase versus estrone sulfotransferase in human breast cancer: potential clinical applications. J Steroid Biochem Mol Biol 1999;69:287–92. [29] Al Saati T, Clamens S, Cohen-Knafo E, Faye JC, Prats H, Coindre JM, Wafflart J, Caverivière P, Bayard F, Delsol G. Production of monoclonal antibodies to human estrogen receptor protein (ER) using recombinant ER (RER). Int J Cancer 1993;55:651–4. [30] Abbondanza C, de Falco A, Nigro V, Medici N, Armatta I, Molinari AM, Moncharmont B, Puca GA. Characterization and epitope mapping of monoclonal antibodies to estradiol receptor. Steroids 1993;58:4– 12. [31] Sierralta WD, Thole HH. Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies. Histochem Cell Biol 1996;105:357–63. [32] Qualmann B, Kessels MM, Thole HH, Sierralta WD. A hormone pulse induces transient changes in the subcellular distribution and leads to a lysosomal accumulation of the estradiol receptor ␣ in target tissues. Eur J Cell Biol 2000;79:383–93. [33] Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS. Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity. J Biol Chem 1994;269:4458–66. [34] Ogle TF, George P. Regulation of the estrogen receptor in the decidua basalis of the pregnant rat. Biol Reprod 1995;53:65–77. [35] Pfeffer U, Fecarotta E, Arena G, Forlani A, Vidali G. Alternative splicing of the estrogen receptor primary transcript normally occurs in estrogen receptor positive tissues and cell lines. J Steroid Biochem Mol Biol 1996;56:99–105.

320

B. Hoffmann, G. Schuler / Domestic Animal Endocrinology 23 (2002) 309–320

[36] Woicke J, Schoon H-A, Heuwieser W, Schulz L-C, Grunert E. Morphologische und funktionelle Aspekte plazentarer Reifungsmechanismen beim Rind. J Vet Med A 1986;33:660–7. [37] Schoon H-A. Lungen- und Plazentareifung beim Rind in der Endphase der Gravidität. Hannover: Habilitation, 1989. [38] Tsumagari S, Kamata J, Takagi K, Tanemura K, Yosai A, Takeshi M. Aromatase activity and oestrogen concentrations in bovine cotyledons and caruncles during gestation and parturition. J Reprod Fertil 1993;98:631–6. [39] Schuler G. Plazentare Steroide beim Rind. Biosynthese und Beziehungen zu Wachstum und Differenzierung der Plazentome. Giessen: Habilitation, 2000. [40] Björckman NH. Morphological and histochemical studies on the bovine placentome. Acta Anat 1954;22(Suppl):1–99. [41] Wooding FBP, Wathes DC. Binucleate cell migration in the bovine placentome. J Reprod Fertil 1980;59: 425–30. [42] Wooding FB, Beckers JF. Trinucleate cells and the ultrastructural localisation of bovine placental lactogen. Cell Tissue Res 1987;247:667–73. [43] Wooding FB. Current topic: the synepitheliochorial placenta of ruminants: binucleate cell fusions and hormone production. Placenta 1992;13:101–13. [44] Leiser R, Wille KH. Cytochemical establishment of acid phosphatase in the bovine endometrium and trophoblast during implantation. Anat Embryol (Berl) 1975;148:159–73. [45] Dutertre M, Smith CL. Molecular mechanisms of selective estrogen receptor modulator (SERM) action. J Pharmacol Exp Ther 2000;295:431–7. [46] Schams D, Hoffmann B, Fischer S, März E, Karg H. Simultaneous determination of LH and progesterone in peripheral bovine blood during pregnancy, normal and corticoid-induced parturition and the post-partum period. J Reprod Fertil 1972;29:37–48. [47] Tsumagari S, Kamata J, Takagi K, Tanemura K, Yosai A, Takeshi M. 3␤-Hydroxysteroid dehydrogenase activity and gestagen concentrations in bovine cotyledons and caruncles during gestation and parturition. J Reprod Fertil 1994;102:35–9. [48] Hansen PJ. Regulation of uterine immune function by progesterone: lessons from the sheep. J Reprod Immunol 1998;40:63–79.