- Email: [email protected]
Mammary secretion of oestrogens in the cow
Domestic Animal Endocrinology 23 (2002) 125–137
Mammary secretion of oestrogens in the cow T. Janowski∗ , S. Zdu´nczyk, J. Małecki-Tepicht, W. Bara´nski, A. Ra´s Department of Obstetrics and Pathology of Reproduction, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Abstract Two experiments in vivo and one experiment in vitro were conduced to examine the mechanisms involved, which lead to mammary secretion of oestrogens and its importance for milk production and udder health in cows. In experiment 1 in six cows of the White–Black breed on day 268 of pregnancy catheters were inserted into uterine vein of pregnant horn, the abdominal aorta and the caudal superficial epigastric (milk) vein. Blood samples for estimation of oestrone, oestrone sulphate, oestradiol-17␣ and -17 by RIA were obtained daily from day 7 pre-partum until day 1 post-partum. Only the concentration of oestradiol-17 was statistically higher (P ≤ 0.01) in mammary venous plasma than in aortal and uterine plasma. In experiment 2 forty late-pregnant cows were divided into two groups according to their milk production in the previous lactation: group 1 (n = 20) high-yielding cows (>6500 kg milk per lactation), and group 2 (n = 20) low-yielding cows (<3700 kg milk per lactation). Blood samples for measurement of oestradiol-17 by RIA were collected from milk and tail veins every fourth day during a period from day 20 prior to parturition to day 4 post-partum. The concentration of oestradiol-17 was significantly higher (P ≤ 0.01) in the milk vein than in the peripheral plasma from day 12 pre-partum to parturition. In high-yielding cows the level of oestradiol-17 in mammary venous blood was significantly higher (P ≤ 0.01) than in low-yielding cows. In six cows with pathological udder oedema ante-partum the concentration of oestradiol-17 in milk vein was significantly higher (P ≤ 0.05) than in control cows. There were no statistically significant differences in the levels of oestradiol-17 in cows with clinical mastitis (n = 10) during 2 weeks after parturition and without it (P ≥ 0.05). In an in vitro experiment, homogenates of mammary tissue collected on day 7 pre-partum from two cows were incubated with 3 H-androstendione. After incubation the samples were extracted and 3 H-oestradiol-17 was separated by HPLC. 3 H-oestradiol-17 was formed in a total yield of 37%. These results indicate that oestrone, oestrone sulphate and oestradiol-17␣ are not secreted by bovine mammary gland. Furthermore, the secretion of oestradiol-17 starts about day 12 pre-partum and is associated with milk yield and udder oedema. Preliminary in vitro study suggests the synthesis of oestradiol-17 by mammary tissue. © 2002 Elsevier Science Inc. All rights reserved.
∗
Corresponding author. Tel.: +48-89-233-497; fax: +48-89-233-440. E-mail address: [email protected] (T. Janowski).
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 5 1 - 0
126
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
1. Introduction The development of mammary gland and lactation are well known hormonal regulated processes and the bovine udder is a target organ for many hormones produced outside this organ. It is generally agreed that prolactin, growth hormone, insuline and steroids are the main hormones involved in this regulation [1–4]. The role of oestrogens in mammo- and lactogenesis has been well established; however, the mode of action of these hormones on the mammary gland is still discussed. It has been suggested that oestrogens are acting either directly via their specific receptor or indirectly via local or systemic production of growth factors like IGF-1, EGF or TGF␣ [5–8]. Previous studies have shown that the bovine mammary gland secretes oestradiol-17 and prostaglandin F2 ␣ into the mammary venous blood [9,10]. However, these studies were limited to the last week of pregnancy. The secretion of other oestrogen fractions by bovine mammary gland has been not investigated. The finding that the mammary gland itself is the source of the oestradiol-17 suggests biological importance of this phenomenon for mammo- and lactogenesis. The high level of peripheral plasma oestrogens seems to increase the occurrence of udder oedema and mastitis [11–14]. However, there are no studies about the relationship between mammary gland secretion of this hormone and milk production as well as udder health. The mechanisms involved, which lead to the secretion of oestradiol-17 by mammary gland, is also not known; however, conversion of unconjugated oestrogens, hydrolysis of conjugated oestrogens or synthesis de novo of oestradiol-17 by mammary tissues have been suggested [15–17]. In the view of these facts, the main aims of this study were: 1. to identify the oestrogen fractions secreted by mammary gland; 2. to establish the oestradiol-17 level in the mammary venous drainage during 3 weeks prior to parturition; 3. to compare mammary secretion of this hormone in cows with different milk yield as wells as to determine the relationship between release of oestradiol-17 and health status of the udder; 4. to investigate in vitro conversion of androstendione into oestradiol-17 by mammary tissue. 2. Materials and methods 2.1. In vivo 2.1.1. Experiment 1 In six cows of the White–Black breed on day 268 of pregnancy catheters were inserted into uterine vein of pregnant horn, the abdominal aorta and the caudal superficial epigastric (milk) vein as described previously [10]. Heparinised blood samples for estimation of oestrone, oestrone sulphate and oestradiol-17␣ and -17 were obtained daily from day 7 pre-partum until day 1 post-partum. After centrifugation the plasma was stored at −20◦ C until analysis.
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
127
2.1.2. Experiment 2 Forty late-pregnant dairy cows of the White–Black breed were used. The animals were divided into two groups according to their milk production in the previous lactation: group 1 (n = 20), high-yielding cows (>6500 kg milk per lactation period) and group 2 (n = 20), low-yielding cows (<3700 kg milk per lactation period). Blood samples for measurement of oestradiol-17 were collected from the milk vein and tail vein into heparinised evacuated tubes every fourth day during a period from day 20 prior to parturition to day 4 post-partum. After centrifugation the plasma was stored at −20◦ C until analysis. Additionally, the clinical observation of mammary gland pre-partum as well as the clinical, cytological and bacteriological control of the health status of the udder during 2 weeks post-partum were carried out. 2.1.3. Hormone assay Oestradiol-17, oestrone and oestrone sulphate values were determined by radioimmunoassay according methods described by Hoffmann [18], oestradiol-17␣ as described by Möstl et al. [19]. Antiserum against estradiol-17 showed the following cross-reactivites: oestradiol-17 100%, oestrone 0.58%, oestradiol-17␣ 0.04%, androstendione and testosterone lower than 0.01%. Cross-reactivites of the antiserum against oestrone were as follows: oestrone 100%, oestradiol-17 1.94%, oestradiol-17␣ 1.02%, androstendione, progesterone and testosterone lower than 0.05%. Intra- and inter-assay coefficients of variation and sensitivity of the assays were 6.0, 9.2% and 74 pmol for oestradiol-17, and 8.2, 15.8% and 92.5 pmol/L for oestrone, respectively. Antiserum against oestradiol-17␣ showed the following cross-reactions: oestradiol-17␣ 100%, oestradiol-17 0.4%, oestrone 0.2% and androstendione lower than 0.1%. The intra- and inter-assay coefficients of variation of the assay for oestradiol-17␣ were 8.5 and 14.2%, respectively. The sensitivity of the assay was 58.7 pg/mL. 2.1.4. Statistical analysis The results are expressed as arithmetical means±SEM. The differences in hormone concentrations were analysed by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test (GraphPad, San Diego, CA, USA). 2.2. In vitro 2.2.1. Sample collection and preparation Samples of mammary gland tissue were collected from two cows 7 days prior parturition as described by Knight et al. [20]. The tissue was cut into small pieces and washed with Ringer solution. After the final wash, the tissue was homogenised in Ringer-HEPES-buffer (4 mL/g of tissue). 2.2.2. Incubation An amount of 0.2 mL homogenate (=50 mg wet tissue) was incubated with 100 L 3 Handrostendione (ca. 50,000 cpm) in 1 mL incubation medium (Ringer-HEPES-buffer with NADPH-regenerating system) in 15 mL glass tubes [21]. The incubation was carried out in quadruplets in shaking water bath at 37◦ C. Incubation periods were 1, 5, 30 and 60 min.
128
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
Fig. 1. Standard chromatogram: separation of tritiated oestrone (E1), oestradiol-17 (E2) and androstendione (A) on reversed phase HPLC.
2.2.3. Extraction and HPLC The samples were extracted three times with diethyl ether. Extracts were dried, dissolved in 35% methanol and submitted for clean up procedure on Bakerbond spe® C18 cartridges (J.T. Baker Inc., Philipsburg, NJ, USA). Following elution with 95% methanol and drying, the samples were dissolved in 0.22 mL 40% methanol, and 0.1 mL was separated by HPLC (Fig. 1). The column-system consisted of a pre-column (Zorbax, 4.6 mm×12.5 mm, 5 m) and a RP 18-column (Zorbax, 4.6 mm × 250 mm, 5 m; DuPont Co., Wilmington, DE, USA). The mobile phase was a mixture of methanol and water (6:4, v/v) and the flow rate was 0.5 mL/min. Radioactivity of fractions containing oestradiol-17 was measured with a liquid scintillation counter and calculated as the percentage of total radioactivity.
3. Results 3.1. In vivo 3.1.1. Experiment 1 The concentration of oestradiol-17 was statistically higher (P ≤ 0.01) in mammary venous plasma than in aortal and uterine plasma (Fig. 2). In contrast to this hormone, higher or
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
129
Fig. 2. The concentrations (means ± SEM) of oestradiol-17 (a) and oestradiol-17␣ (b) in abdominal aorta, caudal superficial epigastric and uterine veins in cows. ∗∗ P ≤ 0.01 compared with the corresponding values in abdominal aorta and uterine vein.
comparable concentrations of other measured oestrogens were observed in uterine and peripheral blood compared to those in mammary venous blood (Figs. 2 and 3). 3.1.2. Experiment 2 The concentration of oestradiol-17 was significantly higher (P ≤ 0.01) in the milk vein than in the peripheral plasma from day 12 pre-partum to parturition. During this period,
130
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
Fig. 3. The concentrations (means ± SEM) of oestrone sulphate (a) and oestrone (b) in abdominal aorta, caudal superficial epigastric and uterine veins in cows. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01 compared with the corresponding values in abdominal aorta and caudal superficial epigastric vein.
the levels of this hormone increased in mammary as well in peripheral circulation, reaching maximum at parturition and decreasing thereafter (Fig. 4). In high-yielding cows, the level of oestradiol-17 in mammary venous blood was significantly higher (P ≤ 0.01) than in low-yielding cows. This difference was not observed in peripheral blood (Fig. 5).
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
131
Fig. 4. The concentrations (means ± SEM) of oestradiol-17 in mammary (- - -) and peripheral (—) venous plasma in cows during the peri-partum period. ∗ P ≤ 0.05 compared with the corresponding values in peripheral plasma.
In six cows, pathological oedematic swelling of the udder was observed ante-partum. In this group of cows, the concentration of oestradiol-17 was significantly higher (P ≤ 0.05) than in cows without this phenomenon, but this difference was noted only in mammary venous blood (Fig. 6). Although the levels of oestradiol-17 in cows with clinical mastitis (n = 10) during 2 weeks after parturition were higher in both mammary and tail veins, the differences were not statistically significant (P ≥ 0.05; Fig. 7). 3.2. In vitro After incubation of mammary tissue homogenates from prepartal cows with 3 H-androstendione, 3 H-oestradiol-17 was formed in a total yield of 37%. The formation of 3 H-oestradiol17 reached maximum after 30 min. (Fig. 8).
4. Discussion The information about the local mammary secretion of oestrogens is until now limited. In earlier studies of Maule Walker et al. [9] and Janowski et al. [10] about three times higher concentration of oestradiol-17 in mammary gland venous blood than in peripheral circulation has been shown. The new aspect of the present study was the comparison of venous mammary and uterine blood concentration of other main oestrogen fractions like oestrone, oestrone sulphate and oestradiol-17␣ in late pregnancy (experiment 1). It has been well documented that the placenta
132
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
Fig. 5. The concentrations (means ± SEM) of oestradiol-17 in the milk (a) and tail (b) veins of high- (- - -) and low-yielding (—) cows during the peri-partum period. ∗ P ≤ 0.05 compared with the corresponding values in low-yielding cows.
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
133
Fig. 6. The concentrations (means ± SEM) of oestradiol-17 in the milk (a) and tail (b) veins of cows with udder oedema (—) and without it (- - -) during the peri-partum period. ∗ P ≤ 0.05 compared with the corresponding values in animals without udder oedema.
is the major source of oestrogens around parturition [21–23]; on the contrary, the mammary secretion of these hormones into venous drainage has not been investigated. The results of the experiment 1 indicate that in contrast to oestradiol-17, other oestrogens are not secreted by the mammary gland prior to parturition.
134
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
Fig. 7. The concentrations (means ± SEM) of oestradiol-17 in the milk (a) and tail (b) veins of cows with mastitis (—) and without it (- - -) during the peri-partum period.
In the second experiment, the levels of oestradiol-17 in mammary and peripheral plasma showed a similar pattern, with concentrations rising for around day 12 pre-partum. These results agree with the period in which oestradiol-17 was present in peripheral plasma before parturition [23]. However, the concentration of this hormone in mammary venous drainage was
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
135
Fig. 8. Conversion of 3 H-androstendione into 3 H-oestradiol-17 by homogenates of bovine mammary tissue in relation to time of incubation; values expressed as percent of total activity collected on HPLC.
significantly higher (P ≤ 0.01) than in peripheral circulation. The above described profiles further point to the mammary gland as the site of extra secretion of oestradiol-17 ante-partum and additionally determine the start of this release, which was unknown till now. Biologically, oestradiol-17 is the most active oestrogen and is essential for development of mammary gland in late pregnancy and lactation [2–4]. It is possible that this hormone produced in the udder operates locally in mammary tissue and is involved in control of these processes pre-partum. No investigations of the relationship between mammary oestradiol-17 concentration and milk yield have been conduced until now. In the present study, the levels of oestradiol-17 in the milk vein was significantly higher (P ≤ 0.05) in high-yielding cows compared with low-yielding cows. This difference was not observed for the peripheral blood. These results indicate that oestradiol-17 may have a local effect on mammary gland function. Erb et al. [24] reported the positive correlation for oestradiol-17 pre-partum and milk production during the first 60 days of lactation. This result supports our finding suggesting that the mammary secretion of oestradiol-17 seems to be associated with milk yield. In contrast, we couldn’t find any relationship between concentration of mammary oestradiol17 and clinical mastitis during 2 weeks post-partum. The influence of oestrogens on immunological status and defence mechanisms of the udder is not clear. In general, oestrogens
136
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
are known as hormones stimulating defence processes of different organs [25,26]; on the other hand, it has been documented that under influence of these hormones the incidence of mastitis increases [13,14]. In addition, the ethiopathogenesis of this disease is very complex with involvement of many other factors. On the basis of our study, due to the small number of animals, it is difficult to evaluate this problem properly. In our in vivo study, we have observed relationship between the higher level of mammary oestradiol-17 and the degree of pre-partal mammary oedema. Oedematic swelling of the udder around the time of parturition is normally a physiological process, but in some cases it is so severe that may cause milking difficulties [11]. This kind of oedema we noted in cows with higher level of mammary oestradiol-17. The general mechanism of oedema formation is complex, but changes in the blood flow and capillary permeability are involved in this process. Oestrogens are well known as factor increasing the permeability of blood vessels and causing the softening of the birth canal and mammary gland pre-partum [27]. Malven et al. [12] could also observe the relationship between the concentration of oestrogens and incidence of severe udder oedema, whereas Kellermann and Wendt [28] did not confirm this observation. The mechanisms responsible for the mammary production and secretion of oestradiol-17 are not exactly known. Thus, conversion of other oestrogens could be also involved in secretion of oestradiol-17 by udder [15]. Aromatase activity in the goat mammary gland was detected by Peaker and Taylor [16]. Our data are consistent with those of Prandi and Gaiani [17], who found that oestradiol-17 was synthesised from androgens by mammary gland in the goat. In summary, the results obtained indicate that oestrone, oestrone sulphate and oestradiol-17␣ are not secreted by bovine mammary gland. Furthermore, the secretion of oestradiol-17 starts about day 12 pre-partum and is associated with milk yield and udder oedema. Preliminary in vitro study suggests the synthesis of oestradiol-17 by mammary tissue. Acknowledgments We thank Prof. Dr. B. Hoffmann for the gift of antibodies against oestrone and oestradiol-17 and Prof. Dr. E. Möstl for antibodies against oestradiol-17␣. References [1] Collier RJ, McGrath MF, Bayatt JC, Zurfluh LL. Regulation of bovine mammary growth by peptide hormones: involvement of receptors, growth factors and binding proteins. Livest Prod Sci 1993;35:21–33. [2] Cowie AT, Forsyth IA, Hart IC. Hormonal control of lactation. New York: Springer, 1980. [3] Delouis CJ, Dijane J, Houdebine LM, Terqui M. Relation between hormones and mammary gland function. J Dairy Sci 1980;63:1492–513. [4] Schams D. Endokrinologie. der Laktation. In: Döcke F, editor. Veterinärmedizinische endokrinologie. Jena-Stuttgart: Fischer Verlag, 1994. p. 571–93. [5] Haslam SZ. Local versus systemically mediated effects of estrogen on normal mammary epithelial cell DNA synthesis. Endocrinology 1988;122:860–7. [6] Houdebine LM, Dijane J, Dusanter-Fourt I, Martel P, Kelly PA, Devinoy E, Servely JL. The mechanism of action of the hormone controlling the mammary gland activity. J Dairy Sci 1985;68:489–500. [7] Plaut K. Role of epidermal growth factor and transforming growth factors in mammary development and lactation. J Dairy Sci 1993;76:1526–38.
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
137
[8] Ruan W, Catanese V, Wieczorek M, Feldman M, Kleinberg DL. Estradiol enhances the stimulatory effect of insulin-like growth factor-1 (IGF-1) on mammary development and growth hormone-induced IGF-1 messenger ribonucleic acid. Endocrinology 1995;136:1296–302. [9] Maule Walker FM, Davis AJ, Fleet JR. Endocrine activity of the mammary gland: oestrogen and prostaglandin secretion of the cow and sheep mammary gland during lactogenesis. Br Vet J 1983;139:171–7. [10] Janowski T, Zdu´nczyk S, Ra´s A, Okrasa S. Mammary secretion of oestrogens and prostaglandin F2 ␣ in cows near parturition. Anim Reprod Sci 1988;17:297–302. [11] Al-Ani FK, Vebster JGE. Udder oedema: an update review. Vet Bull 1986;56:763–9. [12] Malven PV, Erb RE, D’Amico MF, Stewart TS, Chew BP. Factors associated with edema of the mammary gland in primigravid dairy heifers. J Dairy Sci 1983;66:246–52. [13] Saad AM, Aström G. Effects of exogenous estrogen administration to bovine ovariectomized cows on the blood and milk-leucocyte counts and -neutrophil phagocytosis measured by flow cytometry. J Vet Med B 1988;35:654–63. [14] Zdu´nczyk S, Malecki-Tepicht J, Janowski T. Untersuchungen zum Einfluss von exogenem Estron auf die Eutergesundheit bei Kühen. Tierärztl Umschau 2001;56:463–70. [15] Challis JRG, Linzell JL. Oestrone metabolisms in pregnant and lactating goats. J Endocrinol 1973;57:451–7. [16] Peaker M, Taylor E. Oestrogen production by the goat mammary gland: transient aromatase activity during late pregnancy. J Endocrinol 1990;125:1–3. [17] Prandi A, Gaiani R. In vivo and in vitro studies of the steroidogenic activity of the goat mammary gland in late pregnancy. Atti Della Societa Italiana Delle Scinze Veterinarie 1984;38:194–7. [18] Hoffmann B. Bestimmung von Steroidhormonen beim weiblichen Rind. Entwicklung von Meßverfahren und physiologische Daten. Berlin und Hamburg: Verlag Paul Parey, 1977. [19] Möstl E, Choi KS, Wurm W, Ismail N, Bamberg E. Pregnancy diagnosis in cows and heifers by determination of oestradiol-17␣ in faeces. Br Vet J 1984;140:287–91. [20] Knight CH, Hillerton JE, Teverson RM, Winter A. Biopsy of the bovine mammary gland. Br Vet J 1992;148:129–32. [21] 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. [22] Hoffmann B, Wagner WC, Gimenez T. Free and conjugated steroids in maternal and fetal plasma in the cow near term. Biol Reprod 1976;15:126–33. [23] 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. [24] Erb RE, Chew BP, Malven PV, D’Amico MF, Zamet CN, Colenbrander VF. Variables associated with peripartum traits in dairy cows. VII. Hormones, calf traits and subsequent milk yields. Theriogenology 1980;51:143–52. [25] Wissendorf G, Scheibl P, Zerbe H. Einfluß von Östrogenen auf das Immunsystem mit Berücksichtigung der bovinen Retentio secundinarum. Dtsch tierärztl Wschr 1988;105:32–4. [26] Roth JA, Kaeberle ML, Appel LH, Nachreiner RF. Association of increased estradiol and progesterone values with altered bovine polymorphonuclear leucocyte function. Am J Vet Res 1981;44:247–53. [27] Döcke F. Wirkung der Keimdrüsenhormone. In: Döcke F, editor. Veterinärmedizinische Endokrinologie. Jena-Stuttgart: Fischer Verlag, 1994. p. 418–44. [28] Kellermann F, Wendt K. Untersuchungen zu Ursachen und Folgen des physiologischen Euterödems. Mh Vet-Med 1988;43:746–9.
Mammary secretion of oestrogens in the cow T. Janowski∗ , S. Zdu´nczyk, J. Małecki-Tepicht, W. Bara´nski, A. Ra´s Department of Obstetrics and Pathology of Reproduction, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Abstract Two experiments in vivo and one experiment in vitro were conduced to examine the mechanisms involved, which lead to mammary secretion of oestrogens and its importance for milk production and udder health in cows. In experiment 1 in six cows of the White–Black breed on day 268 of pregnancy catheters were inserted into uterine vein of pregnant horn, the abdominal aorta and the caudal superficial epigastric (milk) vein. Blood samples for estimation of oestrone, oestrone sulphate, oestradiol-17␣ and -17 by RIA were obtained daily from day 7 pre-partum until day 1 post-partum. Only the concentration of oestradiol-17 was statistically higher (P ≤ 0.01) in mammary venous plasma than in aortal and uterine plasma. In experiment 2 forty late-pregnant cows were divided into two groups according to their milk production in the previous lactation: group 1 (n = 20) high-yielding cows (>6500 kg milk per lactation), and group 2 (n = 20) low-yielding cows (<3700 kg milk per lactation). Blood samples for measurement of oestradiol-17 by RIA were collected from milk and tail veins every fourth day during a period from day 20 prior to parturition to day 4 post-partum. The concentration of oestradiol-17 was significantly higher (P ≤ 0.01) in the milk vein than in the peripheral plasma from day 12 pre-partum to parturition. In high-yielding cows the level of oestradiol-17 in mammary venous blood was significantly higher (P ≤ 0.01) than in low-yielding cows. In six cows with pathological udder oedema ante-partum the concentration of oestradiol-17 in milk vein was significantly higher (P ≤ 0.05) than in control cows. There were no statistically significant differences in the levels of oestradiol-17 in cows with clinical mastitis (n = 10) during 2 weeks after parturition and without it (P ≥ 0.05). In an in vitro experiment, homogenates of mammary tissue collected on day 7 pre-partum from two cows were incubated with 3 H-androstendione. After incubation the samples were extracted and 3 H-oestradiol-17 was separated by HPLC. 3 H-oestradiol-17 was formed in a total yield of 37%. These results indicate that oestrone, oestrone sulphate and oestradiol-17␣ are not secreted by bovine mammary gland. Furthermore, the secretion of oestradiol-17 starts about day 12 pre-partum and is associated with milk yield and udder oedema. Preliminary in vitro study suggests the synthesis of oestradiol-17 by mammary tissue. © 2002 Elsevier Science Inc. All rights reserved.
∗
Corresponding author. Tel.: +48-89-233-497; fax: +48-89-233-440. E-mail address: [email protected] (T. Janowski).
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 5 1 - 0
126
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
1. Introduction The development of mammary gland and lactation are well known hormonal regulated processes and the bovine udder is a target organ for many hormones produced outside this organ. It is generally agreed that prolactin, growth hormone, insuline and steroids are the main hormones involved in this regulation [1–4]. The role of oestrogens in mammo- and lactogenesis has been well established; however, the mode of action of these hormones on the mammary gland is still discussed. It has been suggested that oestrogens are acting either directly via their specific receptor or indirectly via local or systemic production of growth factors like IGF-1, EGF or TGF␣ [5–8]. Previous studies have shown that the bovine mammary gland secretes oestradiol-17 and prostaglandin F2 ␣ into the mammary venous blood [9,10]. However, these studies were limited to the last week of pregnancy. The secretion of other oestrogen fractions by bovine mammary gland has been not investigated. The finding that the mammary gland itself is the source of the oestradiol-17 suggests biological importance of this phenomenon for mammo- and lactogenesis. The high level of peripheral plasma oestrogens seems to increase the occurrence of udder oedema and mastitis [11–14]. However, there are no studies about the relationship between mammary gland secretion of this hormone and milk production as well as udder health. The mechanisms involved, which lead to the secretion of oestradiol-17 by mammary gland, is also not known; however, conversion of unconjugated oestrogens, hydrolysis of conjugated oestrogens or synthesis de novo of oestradiol-17 by mammary tissues have been suggested [15–17]. In the view of these facts, the main aims of this study were: 1. to identify the oestrogen fractions secreted by mammary gland; 2. to establish the oestradiol-17 level in the mammary venous drainage during 3 weeks prior to parturition; 3. to compare mammary secretion of this hormone in cows with different milk yield as wells as to determine the relationship between release of oestradiol-17 and health status of the udder; 4. to investigate in vitro conversion of androstendione into oestradiol-17 by mammary tissue. 2. Materials and methods 2.1. In vivo 2.1.1. Experiment 1 In six cows of the White–Black breed on day 268 of pregnancy catheters were inserted into uterine vein of pregnant horn, the abdominal aorta and the caudal superficial epigastric (milk) vein as described previously [10]. Heparinised blood samples for estimation of oestrone, oestrone sulphate and oestradiol-17␣ and -17 were obtained daily from day 7 pre-partum until day 1 post-partum. After centrifugation the plasma was stored at −20◦ C until analysis.
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
127
2.1.2. Experiment 2 Forty late-pregnant dairy cows of the White–Black breed were used. The animals were divided into two groups according to their milk production in the previous lactation: group 1 (n = 20), high-yielding cows (>6500 kg milk per lactation period) and group 2 (n = 20), low-yielding cows (<3700 kg milk per lactation period). Blood samples for measurement of oestradiol-17 were collected from the milk vein and tail vein into heparinised evacuated tubes every fourth day during a period from day 20 prior to parturition to day 4 post-partum. After centrifugation the plasma was stored at −20◦ C until analysis. Additionally, the clinical observation of mammary gland pre-partum as well as the clinical, cytological and bacteriological control of the health status of the udder during 2 weeks post-partum were carried out. 2.1.3. Hormone assay Oestradiol-17, oestrone and oestrone sulphate values were determined by radioimmunoassay according methods described by Hoffmann [18], oestradiol-17␣ as described by Möstl et al. [19]. Antiserum against estradiol-17 showed the following cross-reactivites: oestradiol-17 100%, oestrone 0.58%, oestradiol-17␣ 0.04%, androstendione and testosterone lower than 0.01%. Cross-reactivites of the antiserum against oestrone were as follows: oestrone 100%, oestradiol-17 1.94%, oestradiol-17␣ 1.02%, androstendione, progesterone and testosterone lower than 0.05%. Intra- and inter-assay coefficients of variation and sensitivity of the assays were 6.0, 9.2% and 74 pmol for oestradiol-17, and 8.2, 15.8% and 92.5 pmol/L for oestrone, respectively. Antiserum against oestradiol-17␣ showed the following cross-reactions: oestradiol-17␣ 100%, oestradiol-17 0.4%, oestrone 0.2% and androstendione lower than 0.1%. The intra- and inter-assay coefficients of variation of the assay for oestradiol-17␣ were 8.5 and 14.2%, respectively. The sensitivity of the assay was 58.7 pg/mL. 2.1.4. Statistical analysis The results are expressed as arithmetical means±SEM. The differences in hormone concentrations were analysed by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test (GraphPad, San Diego, CA, USA). 2.2. In vitro 2.2.1. Sample collection and preparation Samples of mammary gland tissue were collected from two cows 7 days prior parturition as described by Knight et al. [20]. The tissue was cut into small pieces and washed with Ringer solution. After the final wash, the tissue was homogenised in Ringer-HEPES-buffer (4 mL/g of tissue). 2.2.2. Incubation An amount of 0.2 mL homogenate (=50 mg wet tissue) was incubated with 100 L 3 Handrostendione (ca. 50,000 cpm) in 1 mL incubation medium (Ringer-HEPES-buffer with NADPH-regenerating system) in 15 mL glass tubes [21]. The incubation was carried out in quadruplets in shaking water bath at 37◦ C. Incubation periods were 1, 5, 30 and 60 min.
128
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
Fig. 1. Standard chromatogram: separation of tritiated oestrone (E1), oestradiol-17 (E2) and androstendione (A) on reversed phase HPLC.
2.2.3. Extraction and HPLC The samples were extracted three times with diethyl ether. Extracts were dried, dissolved in 35% methanol and submitted for clean up procedure on Bakerbond spe® C18 cartridges (J.T. Baker Inc., Philipsburg, NJ, USA). Following elution with 95% methanol and drying, the samples were dissolved in 0.22 mL 40% methanol, and 0.1 mL was separated by HPLC (Fig. 1). The column-system consisted of a pre-column (Zorbax, 4.6 mm×12.5 mm, 5 m) and a RP 18-column (Zorbax, 4.6 mm × 250 mm, 5 m; DuPont Co., Wilmington, DE, USA). The mobile phase was a mixture of methanol and water (6:4, v/v) and the flow rate was 0.5 mL/min. Radioactivity of fractions containing oestradiol-17 was measured with a liquid scintillation counter and calculated as the percentage of total radioactivity.
3. Results 3.1. In vivo 3.1.1. Experiment 1 The concentration of oestradiol-17 was statistically higher (P ≤ 0.01) in mammary venous plasma than in aortal and uterine plasma (Fig. 2). In contrast to this hormone, higher or
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
129
Fig. 2. The concentrations (means ± SEM) of oestradiol-17 (a) and oestradiol-17␣ (b) in abdominal aorta, caudal superficial epigastric and uterine veins in cows. ∗∗ P ≤ 0.01 compared with the corresponding values in abdominal aorta and uterine vein.
comparable concentrations of other measured oestrogens were observed in uterine and peripheral blood compared to those in mammary venous blood (Figs. 2 and 3). 3.1.2. Experiment 2 The concentration of oestradiol-17 was significantly higher (P ≤ 0.01) in the milk vein than in the peripheral plasma from day 12 pre-partum to parturition. During this period,
130
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
Fig. 3. The concentrations (means ± SEM) of oestrone sulphate (a) and oestrone (b) in abdominal aorta, caudal superficial epigastric and uterine veins in cows. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01 compared with the corresponding values in abdominal aorta and caudal superficial epigastric vein.
the levels of this hormone increased in mammary as well in peripheral circulation, reaching maximum at parturition and decreasing thereafter (Fig. 4). In high-yielding cows, the level of oestradiol-17 in mammary venous blood was significantly higher (P ≤ 0.01) than in low-yielding cows. This difference was not observed in peripheral blood (Fig. 5).
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
131
Fig. 4. The concentrations (means ± SEM) of oestradiol-17 in mammary (- - -) and peripheral (—) venous plasma in cows during the peri-partum period. ∗ P ≤ 0.05 compared with the corresponding values in peripheral plasma.
In six cows, pathological oedematic swelling of the udder was observed ante-partum. In this group of cows, the concentration of oestradiol-17 was significantly higher (P ≤ 0.05) than in cows without this phenomenon, but this difference was noted only in mammary venous blood (Fig. 6). Although the levels of oestradiol-17 in cows with clinical mastitis (n = 10) during 2 weeks after parturition were higher in both mammary and tail veins, the differences were not statistically significant (P ≥ 0.05; Fig. 7). 3.2. In vitro After incubation of mammary tissue homogenates from prepartal cows with 3 H-androstendione, 3 H-oestradiol-17 was formed in a total yield of 37%. The formation of 3 H-oestradiol17 reached maximum after 30 min. (Fig. 8).
4. Discussion The information about the local mammary secretion of oestrogens is until now limited. In earlier studies of Maule Walker et al. [9] and Janowski et al. [10] about three times higher concentration of oestradiol-17 in mammary gland venous blood than in peripheral circulation has been shown. The new aspect of the present study was the comparison of venous mammary and uterine blood concentration of other main oestrogen fractions like oestrone, oestrone sulphate and oestradiol-17␣ in late pregnancy (experiment 1). It has been well documented that the placenta
132
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
Fig. 5. The concentrations (means ± SEM) of oestradiol-17 in the milk (a) and tail (b) veins of high- (- - -) and low-yielding (—) cows during the peri-partum period. ∗ P ≤ 0.05 compared with the corresponding values in low-yielding cows.
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
133
Fig. 6. The concentrations (means ± SEM) of oestradiol-17 in the milk (a) and tail (b) veins of cows with udder oedema (—) and without it (- - -) during the peri-partum period. ∗ P ≤ 0.05 compared with the corresponding values in animals without udder oedema.
is the major source of oestrogens around parturition [21–23]; on the contrary, the mammary secretion of these hormones into venous drainage has not been investigated. The results of the experiment 1 indicate that in contrast to oestradiol-17, other oestrogens are not secreted by the mammary gland prior to parturition.
134
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
Fig. 7. The concentrations (means ± SEM) of oestradiol-17 in the milk (a) and tail (b) veins of cows with mastitis (—) and without it (- - -) during the peri-partum period.
In the second experiment, the levels of oestradiol-17 in mammary and peripheral plasma showed a similar pattern, with concentrations rising for around day 12 pre-partum. These results agree with the period in which oestradiol-17 was present in peripheral plasma before parturition [23]. However, the concentration of this hormone in mammary venous drainage was
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
135
Fig. 8. Conversion of 3 H-androstendione into 3 H-oestradiol-17 by homogenates of bovine mammary tissue in relation to time of incubation; values expressed as percent of total activity collected on HPLC.
significantly higher (P ≤ 0.01) than in peripheral circulation. The above described profiles further point to the mammary gland as the site of extra secretion of oestradiol-17 ante-partum and additionally determine the start of this release, which was unknown till now. Biologically, oestradiol-17 is the most active oestrogen and is essential for development of mammary gland in late pregnancy and lactation [2–4]. It is possible that this hormone produced in the udder operates locally in mammary tissue and is involved in control of these processes pre-partum. No investigations of the relationship between mammary oestradiol-17 concentration and milk yield have been conduced until now. In the present study, the levels of oestradiol-17 in the milk vein was significantly higher (P ≤ 0.05) in high-yielding cows compared with low-yielding cows. This difference was not observed for the peripheral blood. These results indicate that oestradiol-17 may have a local effect on mammary gland function. Erb et al. [24] reported the positive correlation for oestradiol-17 pre-partum and milk production during the first 60 days of lactation. This result supports our finding suggesting that the mammary secretion of oestradiol-17 seems to be associated with milk yield. In contrast, we couldn’t find any relationship between concentration of mammary oestradiol17 and clinical mastitis during 2 weeks post-partum. The influence of oestrogens on immunological status and defence mechanisms of the udder is not clear. In general, oestrogens
136
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
are known as hormones stimulating defence processes of different organs [25,26]; on the other hand, it has been documented that under influence of these hormones the incidence of mastitis increases [13,14]. In addition, the ethiopathogenesis of this disease is very complex with involvement of many other factors. On the basis of our study, due to the small number of animals, it is difficult to evaluate this problem properly. In our in vivo study, we have observed relationship between the higher level of mammary oestradiol-17 and the degree of pre-partal mammary oedema. Oedematic swelling of the udder around the time of parturition is normally a physiological process, but in some cases it is so severe that may cause milking difficulties [11]. This kind of oedema we noted in cows with higher level of mammary oestradiol-17. The general mechanism of oedema formation is complex, but changes in the blood flow and capillary permeability are involved in this process. Oestrogens are well known as factor increasing the permeability of blood vessels and causing the softening of the birth canal and mammary gland pre-partum [27]. Malven et al. [12] could also observe the relationship between the concentration of oestrogens and incidence of severe udder oedema, whereas Kellermann and Wendt [28] did not confirm this observation. The mechanisms responsible for the mammary production and secretion of oestradiol-17 are not exactly known. Thus, conversion of other oestrogens could be also involved in secretion of oestradiol-17 by udder [15]. Aromatase activity in the goat mammary gland was detected by Peaker and Taylor [16]. Our data are consistent with those of Prandi and Gaiani [17], who found that oestradiol-17 was synthesised from androgens by mammary gland in the goat. In summary, the results obtained indicate that oestrone, oestrone sulphate and oestradiol-17␣ are not secreted by bovine mammary gland. Furthermore, the secretion of oestradiol-17 starts about day 12 pre-partum and is associated with milk yield and udder oedema. Preliminary in vitro study suggests the synthesis of oestradiol-17 by mammary tissue. Acknowledgments We thank Prof. Dr. B. Hoffmann for the gift of antibodies against oestrone and oestradiol-17 and Prof. Dr. E. Möstl for antibodies against oestradiol-17␣. References [1] Collier RJ, McGrath MF, Bayatt JC, Zurfluh LL. Regulation of bovine mammary growth by peptide hormones: involvement of receptors, growth factors and binding proteins. Livest Prod Sci 1993;35:21–33. [2] Cowie AT, Forsyth IA, Hart IC. Hormonal control of lactation. New York: Springer, 1980. [3] Delouis CJ, Dijane J, Houdebine LM, Terqui M. Relation between hormones and mammary gland function. J Dairy Sci 1980;63:1492–513. [4] Schams D. Endokrinologie. der Laktation. In: Döcke F, editor. Veterinärmedizinische endokrinologie. Jena-Stuttgart: Fischer Verlag, 1994. p. 571–93. [5] Haslam SZ. Local versus systemically mediated effects of estrogen on normal mammary epithelial cell DNA synthesis. Endocrinology 1988;122:860–7. [6] Houdebine LM, Dijane J, Dusanter-Fourt I, Martel P, Kelly PA, Devinoy E, Servely JL. The mechanism of action of the hormone controlling the mammary gland activity. J Dairy Sci 1985;68:489–500. [7] Plaut K. Role of epidermal growth factor and transforming growth factors in mammary development and lactation. J Dairy Sci 1993;76:1526–38.
T. Janowski et al. / Domestic Animal Endocrinology 23 (2002) 125–137
137
[8] Ruan W, Catanese V, Wieczorek M, Feldman M, Kleinberg DL. Estradiol enhances the stimulatory effect of insulin-like growth factor-1 (IGF-1) on mammary development and growth hormone-induced IGF-1 messenger ribonucleic acid. Endocrinology 1995;136:1296–302. [9] Maule Walker FM, Davis AJ, Fleet JR. Endocrine activity of the mammary gland: oestrogen and prostaglandin secretion of the cow and sheep mammary gland during lactogenesis. Br Vet J 1983;139:171–7. [10] Janowski T, Zdu´nczyk S, Ra´s A, Okrasa S. Mammary secretion of oestrogens and prostaglandin F2 ␣ in cows near parturition. Anim Reprod Sci 1988;17:297–302. [11] Al-Ani FK, Vebster JGE. Udder oedema: an update review. Vet Bull 1986;56:763–9. [12] Malven PV, Erb RE, D’Amico MF, Stewart TS, Chew BP. Factors associated with edema of the mammary gland in primigravid dairy heifers. J Dairy Sci 1983;66:246–52. [13] Saad AM, Aström G. Effects of exogenous estrogen administration to bovine ovariectomized cows on the blood and milk-leucocyte counts and -neutrophil phagocytosis measured by flow cytometry. J Vet Med B 1988;35:654–63. [14] Zdu´nczyk S, Malecki-Tepicht J, Janowski T. Untersuchungen zum Einfluss von exogenem Estron auf die Eutergesundheit bei Kühen. Tierärztl Umschau 2001;56:463–70. [15] Challis JRG, Linzell JL. Oestrone metabolisms in pregnant and lactating goats. J Endocrinol 1973;57:451–7. [16] Peaker M, Taylor E. Oestrogen production by the goat mammary gland: transient aromatase activity during late pregnancy. J Endocrinol 1990;125:1–3. [17] Prandi A, Gaiani R. In vivo and in vitro studies of the steroidogenic activity of the goat mammary gland in late pregnancy. Atti Della Societa Italiana Delle Scinze Veterinarie 1984;38:194–7. [18] Hoffmann B. Bestimmung von Steroidhormonen beim weiblichen Rind. Entwicklung von Meßverfahren und physiologische Daten. Berlin und Hamburg: Verlag Paul Parey, 1977. [19] Möstl E, Choi KS, Wurm W, Ismail N, Bamberg E. Pregnancy diagnosis in cows and heifers by determination of oestradiol-17␣ in faeces. Br Vet J 1984;140:287–91. [20] Knight CH, Hillerton JE, Teverson RM, Winter A. Biopsy of the bovine mammary gland. Br Vet J 1992;148:129–32. [21] 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. [22] Hoffmann B, Wagner WC, Gimenez T. Free and conjugated steroids in maternal and fetal plasma in the cow near term. Biol Reprod 1976;15:126–33. [23] 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. [24] Erb RE, Chew BP, Malven PV, D’Amico MF, Zamet CN, Colenbrander VF. Variables associated with peripartum traits in dairy cows. VII. Hormones, calf traits and subsequent milk yields. Theriogenology 1980;51:143–52. [25] Wissendorf G, Scheibl P, Zerbe H. Einfluß von Östrogenen auf das Immunsystem mit Berücksichtigung der bovinen Retentio secundinarum. Dtsch tierärztl Wschr 1988;105:32–4. [26] Roth JA, Kaeberle ML, Appel LH, Nachreiner RF. Association of increased estradiol and progesterone values with altered bovine polymorphonuclear leucocyte function. Am J Vet Res 1981;44:247–53. [27] Döcke F. Wirkung der Keimdrüsenhormone. In: Döcke F, editor. Veterinärmedizinische Endokrinologie. Jena-Stuttgart: Fischer Verlag, 1994. p. 418–44. [28] Kellermann F, Wendt K. Untersuchungen zu Ursachen und Folgen des physiologischen Euterödems. Mh Vet-Med 1988;43:746–9.