- Email: [email protected]
Activity of steroid sulphatase and estrogen sulphotransferase in the boar epididymis during the postpubertal period
reproductive biology 12 (2012) 374–378
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/repbio
Original research article
Activity of steroid sulphatase and estrogen sulphotransferase in the boar epididymis during the postpubertal period Sławomir Zdun´czyk *, Tomasz Janowski, Andrzej Ras´, Wojciech Baran´ski Department of Obstetrics and Pathology of Reproduction, Faculty of Veterinary Medicine, University of Warmia and Mazury, Olsztyn, Poland
article info
abstract
Article history:
The activities of steroid sulphatase (StS) and estrogen sulphotransferase (EST) were deter-
Received 26 July 2011
mined in the epididymis of 18 boars. The animals were divided into three groups (n = 6)
Accepted 30 March 2012
according to age (8, 12 and 16 months). The boars were anesthetized and castrated. The tissue samples of different epididymal parts (caput, corpus and cauda) were taken and
Keywords:
homogenized. Activities of StS and EST were assessed using 3H-estrone sulphate (3H-E1S)
Boar
and free 3H-oestrone (3H-E1) as substrates, respectively. The substrate conversion rates after
Epididymis
60 min of incubation were 51.25% for 3H-E1S and 45.65% for 3H-E1. The activities of both
Steroid sulphatase
enzymes were significantly higher in the caput epididymis compared to the cauda epididy-
Estrogen sulphotransferase
mis ( p < 0.05). A significant age-dependent increase of StS and EST activities ( p < 0.05) was observed. These results suggest that the availability of estrogens in the boar epididymis may be locally controlled also by StS and EST. The age-dependent increase of StS and EST activities may be related to the process of ‘‘biochemical maturation’’ of the reproductive system during the postpubertal period. # 2012 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
1.
Introduction
The spermatozoa become motile and gain the fertilizing ability during epididymal storage and transit. The boar testes produce large amounts of estrogens, which also may be found in semen. The main estrogen secreted by testis is estrone, predominantly in its sulphoconjugated form [1–4]. Estrogens play an important role in the regulation of development and function of the reproductive tract in the boar, for review see [5,6]. Estrogens in mice are involved in
reabsorption of epididymal fluid, a process essential for the maintenance of normal male fertility [7,8]. In estrogen receptor alpha knockout (aERKO) mice and mice treated with estrogen receptor (ER) antagonist (ICI 182780), cauda epididymis sperm number, sperm motility and sperm fertilizing ability were reduced compared to intact/control animals [9]. Boars treated weekly with letrozole, an aromatase inhibitor, showed a reduced cauda sperm fertilizing ability and sperm number [10]. Morphological development of the caput and corpus epididymis was delayed in letrozole-treated boars [11].
* Corresponding author. E-mail address: [email protected] (S. Zdun´czyk). 1642-431X/$ – see front matter # 2012 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. http://dx.doi.org/10.1016/j.repbio.2012.10.012
reproductive biology 12 (2012) 374–378
In the male reproductive tract, the main source of estrogens is testes. Estrogens are delivered from the testis to the epididymis through the tubules and via the blood. Epididymal cells [12–14] and spermatozoa [15,16] can also be a potential source of estrogens. Estrogens affect cells by binding to their specific receptors in target tissues. Both ERa and ERb were found to be expressed in all three epididymal regions (caput, corpus and cauda) in boars from 2 to 8 months of age. However, in the older boars (1.5–2.5 years) ERb was not demonstrated in the cauda [17]. Since only free estrogens are biologically active, whilst conjugated estrogens do not interact with the receptor [18], it was hypothesized that the availability of biologically active estrogens depends partially on locally expressed steroid sulphatase (StS) and estrogen sulphotransferase (EST [3,19]). StS catalyzes desulphation (i.e. activation of conjugated estrogens) and EST catalyzes sulphoconjugation (i.e. inactivation of free estrogens). The concentration of free estrogens in the epididymal fluid decreased from caput to cauda epididymis of boars [20]. The tissue concentration of free estradiol did not significantly differ between the epididymal regions, however the regional differences in the concentration of estrogen conjugates in epididymal tissue were observed [17]. Studies on the activity of StS and EST in the boar epididymis are limited. High conversion level of free estrogen to estrogen conjugates was demonstrated for the epididymis of two 2-year-old boars [21]. Activities of StS and EST were detected in the epididymal tissue (caput and corpus together) of three 200-day-old boars [3]. Boars become sexually mature between 5 and 9 months of age [22]. After reaching sexual maturity, the reproductive system undergoes so called ‘‘biochemical maturation’’ lasting to the age of 18 months. During this period the increase of protein content and the activity of the antioxidant system were observed in seminal plasma [23,24]. Epididymal protein secretion increased from 1-month-old to more than 8-month-old boars [25]. The biochemical changes seem to be essential for the production of semen with high biological properties. There is no data concerning desulphation and sulphoconjugation of estrogens in different epididymal regions of the boar during the postpubertal period. Therefore, the aim of this study was to determine the activity of StS and EST in all three epididymal regions of 8-, 12- and 16-month-old boars.
2.
Materials and methods
The investigation was carried out on 18 Polish Landrace boars in a pig insemination station in the north-east of Poland. The animals were kept in individual pens and fed on standard diet. The animals were divided into three groups (n = 6) according to age (8, 12 and 16 months). The boars were anesthetized with azaperone (20 mg/kg i.m.) and ketamine (20 mg/kg i.v.) and castrated. The protocol was approved by the Local Ethics Commission for Animal Experiments. After castration, the epididymides were separated from the testes, placed in icecold phosphate buffer (PBS) and transported to the laboratory. The epididymides were divided into three regions (caput, corpus and cauda [25]). The tissue samples of each epididymal region were collected and washed with saline to remove
375
spermatozoa. The samples were cut into small pieces and washed with Ringer solution (Polfa, Warsaw, Poland) until a clear supernatant was obtained following centrifugation. After the final wash, 5 g of the tissue pulp was homogenized in 15 ml Ringer–Hepes buffer, consisting with Ringer solution and Hepes buffer (39:1; Sigma–Aldrich Co., St. Louis, MO, USA). The homogenate was filtered to remove connective tissue. All steps were performed on ice. Activities of steroid sulphatase (StS) and estrogen sulphotransferase (EST) were assessed as was described for bovine placenta [19]. For determination of the StS activity, 1 ml of incubation medium (Ringer–Hepes buffer with 0.1% BSA; Sigma–Aldrich Co., St. Louis, MO, USA), the substrate (0.6 pmol 3 H-estrone sulphate [3H-E1S; NEN Science Products Inc., Brussels, Belgium] dissolved in 0.1 ml Ringer solution with 0.1% BSA) and 0.2 ml homogenate was added to 15 ml glass tubes (Wheaton Science Products, Millville, NJ, USA). The samples were incubated at 37 8C in a water bath with shaking for 1, 5, 15, 30 and 60 min. Control samples contained 1 ml incubation medium, the substrate and 0.2 ml incubation buffer instead of homogenate (medium blank) or heat inactivated homogenate (tissue blank). The controls were incubated for 60 min and the incubates were stored at 20 8C until assay. Each reaction was run in duplicates and the experiments were repeated four times. The formed free 3H-E1 was extracted from the whole samples (incubates) with 2 ml 3 ml toluene (Sigma–Aldrich Co., St. Louis, MO, USA). The samples were mixed on a overhead shaker for 20 min. The aqueous phase was frozen and the toluene phase was decanted. The pooled extracts containing 3H-E1 were dried and dissolved in scintillator (Perkin Elmer Inc., Waltham, MA, USA). The 3H-radioactivity was measured with a liquid scintillation counter (LSA Tri-Carb 2800TR, Perkin Elmer Inc., Waltham, MA, USA). The 3 H-radioactivity in the remaining aqueous phase (3H-E1S) was also counted. The extraction efficiency was 87.9%. The activity of StS was expressed as the percentage of substrate conversion. The total 3H-radioactivity in toluene extracts and aqueous phases was set as 100% value for calculation of conversion rate. For determination of EST activity, 3H-E1 (0.6 pmol; NEN Science Products Inc., Brussels, Belgium) was used as substrate. Additionally, 0.5 mmol of 4-nitrophenylsulphatepotassium salt (Sigma–Aldrich Co., St. Louis, MO, USA) for inhibition of StS and 50 mmol of 30 -phosphoadenosine-50 phosphosulphate (Sigma–Aldrich Co., St. Louis, MO, USA) as a co-factor were used for the reaction. The formed 3H-E1S was measured after removal of free 3H-E1 by extraction with toluene (Sigma–Aldrich Co., St. Louis, MO, USA). The samples were then hydrolyzed with 0.3 standard units of arylsulphatase (Merck KGaA, Darmstadt, Germany) and again extracted as described above. Statistical analysis was performed on raw data using GraphPad PRISM program (GraphPad Software Inc., San Diego, CA, USA). The data were shown as a percentage of added radioactivity (mean SEM). The statistical significance of differences among various incubation times, epididymal regions or age groups was assessed using one-way ANOVA followed by Bonferroni’s multiple comparison test. The level of significance was set at p < 0.05 for all analyses.
376
3.
reproductive biology 12 (2012) 374–378
[(Fig._1)TD$IG] Results
The activities of StS and EST in epididymal homogenates increased gradually along with the incubation time. The desulphation rate of 3H-E1S into 3H-E1 was significantly ( p < 0.05) higher compared to tissue blank after 15 min of incubation, whereas the sulphoconjugation rate of 3H-E1 into 3 H-E1S was higher ( p < 0.05) after 5 min of incubation. The substrate conversion rates after 60 min of incubation were 51.25% for 3H-E1S and 45.65% for 3H-E1. The activity of StS was significantly ( p < 0.05) higher in the caput epididymis compared to the cauda epididymis after 30 and 60 min of incubation. The activity of EST was also higher in the caput epididymis than in cauda epididymis, but it was dependent on age and time of incubation. The difference between the epididymal regions was statistically significant ( p < 0.05) for boars aged 8 months after 5, 15, 30 and 60 min of incubation, for boars aged 12 months after 5 and 15 min of incubation, for boars aged 16 months after 5 and 60 min of incubation (Figs. 1 and 2). A significant age-dependent increase of StS and EST activities was observed. The activity of StS was significantly ( p < 0.05) higher in boars aged 16 months compared to 8-month-old animals after 15 and 30 min of incubation (caput and corpus) and after 30 and 60 min (cauda). The activity of EST was significantly ( p < 0.05) higher in boars aged 16 months compared to 8-month-old animals after 5, 15, 30 and 60 min of incubation, except for corpus after 15 min of incubation (Figs. 1 and 2).
4.
Discussion
The results of our study showed the high epididymal activities of StS and EST. Data on the StS and EST activities in the reproductive tract of boar are scarce. Activity of StS was examined in the boar testis [3,26,27] and epididymis (head together with corpus [3]). Low activity of EST in the boar testis and high in the epididymis was reported [3]. Our data confirmed the observations of Hoffmann et al. [3], which were, however, limited to the head/corpus epididymis. The activities of StS and EST in our study were significantly higher ( p < 0.05) in the caput than in the cauda epididymis. This is consistent with the results of Claus et al. [20] who found decreased concentrations of estrogens in the epididymal fluid from caput to cauda epididymis of the boar. StS and EST catalyze opposite reactions, leading to the activation of sulphoconjugated estrogens and to the inactivation of free estrogens, respectively. StS increases concentrations of free estrogens, while EST protects the epithelium from excess of estrogens. EST knockout mice showed reduced epididymis weight and reduced motility of sperm isolated from caudal epididymis [28]. High StS and EST activities in the epididymis suggest that the local availability of biologically active estrogens in the epididymis may result also from an interplay between estrogen supply and their metabolism. Pearl et al. [17] found similar tissue concentration of estradiol throughout the epididymis and suggested that the regional differences in estrogen action are likely due to regional differences in their receptor expression.
Fig. 1 – Desulphation (%) of 3H-E1S into 3H-E1 by epididymal homogenates of boars (n = 6); MB: medium blank, TB: tissue blank; a,bdifferent letters depict significant differences ( p < 0.05) between the particular incubation time and TB (within one epididymal region and one age group); c,ddifferent letters depict significant differences ( p < 0.05) among age groups (within one incubation time and one epididymal region); *significant differences ( p < 0.05) between cauda and caput epididymis (within one incubation time and one age group).
We found significant increases in StS and EST activities from eight to 16 months of age. There are no studies on the effect of age on activities of StS and EST in the boar epididymis. The increase in activities of both enzymes corresponded with the increase of estrogen concentration in blood plasma and seminal plasma of boars during this period [4]. ‘‘Biochemical maturation’’ of the reproductive system occurs in boars after
reproductive biology 12 (2012) 374–378
[(Fig._2)TD$IG]
377
In conclusion, high activities of StS and EST in epididymal homogenates suggest that the availability of estrogens in the boar epididymis may be locally controlled also by StS and EST. The increase in StS and EST activities from 8 to 16 months of age may be related to the process of ‘‘biochemical maturation’’ of reproductive system during the postpubertal period.
Acknowledgement This research was supported by a Grant No. N308242935 of the Polish Ministry of Sciences and Higher Education.
references
Fig. 2 – Sulphoconjugation (%) of 3H-E1 into 3H-E1S by epididymal homogenates of boars (n = 6); MB: medium blank, TB: tissue blank; a,bdifferent letters depict significant differences ( p < 0.05) between the particular incubation time and TB (within one epididymal region and one age group); c,ddifferent letters depict significant differences ( p < 0.05) among age groups (within one incubation time and one epididymal region); *significant differences ( p < 0.05) between cauda and caput epididymis (within one incubation time and one age group).
reaching sexual maturity at the age of 18 months. The development of the boar reproductive tract is accompanied by an increased secretory capacity of the accessory sex glands [23,24] and epididymis [25]. The biochemical components of the seminal plasma play an important role in sperm physiological function [24]. Our study demonstrated significant increase in estrogen metabolism in the boar epididymis during the postpubertal period. Estrogens are essential for the function of epididymis and production of semen with high biological properties [7–11].
[1] Claus R, Hoffmann B. Oestrogens, compared to other steroids of testicular origin in blood plasma of boars. Acta Endocrinologica 1980;94(3):404–11. [2] Claus R, Schopper D, Wagner HG. Seasonal effect on steroids in blood plasma and seminal plasma of boars. Journal of Steroid Biochemistry 1983;19(2):725–9. [3] Hoffmann B, Rostalski A, Mutembei M, Goericke-Pesch S. Testicular steroid hormone secretion in the boar and expression of testicular and epididymal steroid sulphatase and estrogen sulphotransferase activity. Experimental and Clinical Endocrinology & Diabetes 2010;118(4):274–80. [4] Zdun´czyk S, Janowski T, Ras´ A, Baran´ski W. Concentrations of oestrogens in blood plasma and seminal plasma of boars during the postpubertal period. Polish Journal of Veterinary Sciences 2011;14(4):539–44. [5] Hess RA, Carnes K. The role of oestrogen in testis and male reproductive tract – a review. Animal Reproduction 2004;1(1):5–30. [6] Zdun´czyk S, Janowski T. Role of oestrogens in the regulation of reproductive tract functions in the boar. Medycyna Weterynaryjna 2009;65(8):521–5 [in Polish]. [7] Hess RA, Bunick D, Lubahn BD, Zhou Q, Bouma J. Morphological changes in the efferent ductules and epididymis in oestrogen knock out mice. Journal of Andrology 2000;21(1):107–21. [8] Zhou LJ, Clarke L, Nie R, Carnes K, Lai LW, Lien YH, et al. Estrogen action and male fertility: roles of sodium/ hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proceedings of the National Academy of Sciences of the United States of America 2001;98(21):14132–7. [9] Cho HW, Nie R, Carnes K, Zhou Q, Sharief NA, Hess RA. The antiestrogen ICI 182, 780 induces early effects on the adult male mouse reproductive tract and long-term decreased fertility without testicular atrophy. Reproductive Biology and Endocrinology 2003;1:57. [10] McCarthy MJ, At-Taras EE, Pearl CA, Nitta-Oda BS, Roser JF, Conley AK, et al. Suppression of endogenous estrogen during development effects porcine epididymal sperm maturation. Molecular Reproduction & Development 2006;73(9):1122–8. [11] Pearl CA, At-Taras E, Berger T, Roser JF. Reduced endogenous estrogen delays epididymal development but has no effect on efferent duct morphology in boars. Reproduction 2007;134(4):593–604. [12] Pereyra-Martinez AC, Roselli CE, Stadelman HL, Resko JA. Cytochrome P450 aromatase in testis and epididymis of male rhesus monkeys. Endocrine 2001;16(1):15–9.
378
reproductive biology 12 (2012) 374–378
[13] Shayu D, Rao AJ. Expression of functional aromatase in the epididymis: role of androgen and LH in modulation of expression and activity. Molecular and Cellular Endocrinology 2006;249(1–2):40–50. [14] Wiszniewska B. Primary culture of rat epididymal epithelial cells as a source of estrogen. Andrologia 2002;34(3):180–7. [15] Rago V, Bilin´ska B, Palma A, Ando S, Carpino A. Evidence of aromatase localization in cytoplasmic droplet of human immature ejaculated spermatozoa. Folia Histochemica et Cytobiologica 2003;41(1):23–8. [16] Rago VF, Aquilla S, Panza R, Carpino A. Cytochrome P450arom, androgen and estrogen receptors in pig sperm. Reproductive Biology and Endocrinology 2007;5:23. [17] Pearl CA, Berger T, Roser JF. Estrogen and androgen receptor expression in relation to steroid concentrations in the adult boars epididymis. Domestic Animal Endocrinology 2007;33(4):451–9. [18] Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nolsson J, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 1997;138(3):863–70. [19] Hoffmann B, Falter K, Vielemeier A, Failing K, Schuler G. Investigations on the activity of bovine placental oestrogen sulfotransferse and -sulfatase from midgestation to parturition. Experimental and Clinical Endocrinology & Diabetes 2001;109(5):284–301. [20] Claus R, Schopper D, Hoang-Vu C. Contribution of individual compartments of the genital tract to oestrogen and testosterone concentrations in ejaculates of the boar. Acta Endocrinologica 1985;109(2):281–8.
[21] Raeside JI, Christie HL, Renaud RL. Androgen and oestrogen metabolism in the reproductive tract and accessory sex glands of the domestic boar (Sus scrofa). Biology of Reproduction 1999;61(5):1242–8. [22] Cameron RDA. Sexual development and semen production in boars. Pig News and Information 1987;8(4):389–92. [23] Kowalowka M, Wysocki P, Fraser L, Strzezek J. Extracellular superoxide dismutase of boar seminal plasma. Reproduction in Domestic Animals 2008;43(4): 490–6. [24] Strzezek J, Saiz-Cidoncha F, Wysocki P, Tyszkiewicz A, Jastrze˛bski M. Seminal plasma proteins as markers of biological value of boar semen. Animal Science Papers and Reports 2002;20(4):255–66. [25] Syntin P, Dacheux JL, Dacheux F. Postnatal development and regulation of proteins secreted in the boar epididymis. Biology of Reproduction 1999;61(6):1622–35. [26] Mutembei H, Kowalewski M, Ugele B, Schuler G, Hofmann B. Expression and activity of steroid sulphatase in the boar testis. Reproduction in Domestic Animals 2009;44(1):17–23. [27] Yamamoto K, Handa S, Yamakawa T. Purification of arylsulfatase A from boar testis and its activities toward seminolipid and sulfatide. Journal of Biochemistry 1997;75:1241–7. [28] Qian YM, Sun XJ, Tong MH, Li XP, Richa J, Song W-C. Targeted disruption of the mouse estrogen sulfotransferase gene reveals a role of estrogen metabolism in intracrine and paracrine estrogen regulation. Endocrinology 2001;142(12):5342–50.
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/repbio
Original research article
Activity of steroid sulphatase and estrogen sulphotransferase in the boar epididymis during the postpubertal period Sławomir Zdun´czyk *, Tomasz Janowski, Andrzej Ras´, Wojciech Baran´ski Department of Obstetrics and Pathology of Reproduction, Faculty of Veterinary Medicine, University of Warmia and Mazury, Olsztyn, Poland
article info
abstract
Article history:
The activities of steroid sulphatase (StS) and estrogen sulphotransferase (EST) were deter-
Received 26 July 2011
mined in the epididymis of 18 boars. The animals were divided into three groups (n = 6)
Accepted 30 March 2012
according to age (8, 12 and 16 months). The boars were anesthetized and castrated. The tissue samples of different epididymal parts (caput, corpus and cauda) were taken and
Keywords:
homogenized. Activities of StS and EST were assessed using 3H-estrone sulphate (3H-E1S)
Boar
and free 3H-oestrone (3H-E1) as substrates, respectively. The substrate conversion rates after
Epididymis
60 min of incubation were 51.25% for 3H-E1S and 45.65% for 3H-E1. The activities of both
Steroid sulphatase
enzymes were significantly higher in the caput epididymis compared to the cauda epididy-
Estrogen sulphotransferase
mis ( p < 0.05). A significant age-dependent increase of StS and EST activities ( p < 0.05) was observed. These results suggest that the availability of estrogens in the boar epididymis may be locally controlled also by StS and EST. The age-dependent increase of StS and EST activities may be related to the process of ‘‘biochemical maturation’’ of the reproductive system during the postpubertal period. # 2012 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
1.
Introduction
The spermatozoa become motile and gain the fertilizing ability during epididymal storage and transit. The boar testes produce large amounts of estrogens, which also may be found in semen. The main estrogen secreted by testis is estrone, predominantly in its sulphoconjugated form [1–4]. Estrogens play an important role in the regulation of development and function of the reproductive tract in the boar, for review see [5,6]. Estrogens in mice are involved in
reabsorption of epididymal fluid, a process essential for the maintenance of normal male fertility [7,8]. In estrogen receptor alpha knockout (aERKO) mice and mice treated with estrogen receptor (ER) antagonist (ICI 182780), cauda epididymis sperm number, sperm motility and sperm fertilizing ability were reduced compared to intact/control animals [9]. Boars treated weekly with letrozole, an aromatase inhibitor, showed a reduced cauda sperm fertilizing ability and sperm number [10]. Morphological development of the caput and corpus epididymis was delayed in letrozole-treated boars [11].
* Corresponding author. E-mail address: [email protected] (S. Zdun´czyk). 1642-431X/$ – see front matter # 2012 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. http://dx.doi.org/10.1016/j.repbio.2012.10.012
reproductive biology 12 (2012) 374–378
In the male reproductive tract, the main source of estrogens is testes. Estrogens are delivered from the testis to the epididymis through the tubules and via the blood. Epididymal cells [12–14] and spermatozoa [15,16] can also be a potential source of estrogens. Estrogens affect cells by binding to their specific receptors in target tissues. Both ERa and ERb were found to be expressed in all three epididymal regions (caput, corpus and cauda) in boars from 2 to 8 months of age. However, in the older boars (1.5–2.5 years) ERb was not demonstrated in the cauda [17]. Since only free estrogens are biologically active, whilst conjugated estrogens do not interact with the receptor [18], it was hypothesized that the availability of biologically active estrogens depends partially on locally expressed steroid sulphatase (StS) and estrogen sulphotransferase (EST [3,19]). StS catalyzes desulphation (i.e. activation of conjugated estrogens) and EST catalyzes sulphoconjugation (i.e. inactivation of free estrogens). The concentration of free estrogens in the epididymal fluid decreased from caput to cauda epididymis of boars [20]. The tissue concentration of free estradiol did not significantly differ between the epididymal regions, however the regional differences in the concentration of estrogen conjugates in epididymal tissue were observed [17]. Studies on the activity of StS and EST in the boar epididymis are limited. High conversion level of free estrogen to estrogen conjugates was demonstrated for the epididymis of two 2-year-old boars [21]. Activities of StS and EST were detected in the epididymal tissue (caput and corpus together) of three 200-day-old boars [3]. Boars become sexually mature between 5 and 9 months of age [22]. After reaching sexual maturity, the reproductive system undergoes so called ‘‘biochemical maturation’’ lasting to the age of 18 months. During this period the increase of protein content and the activity of the antioxidant system were observed in seminal plasma [23,24]. Epididymal protein secretion increased from 1-month-old to more than 8-month-old boars [25]. The biochemical changes seem to be essential for the production of semen with high biological properties. There is no data concerning desulphation and sulphoconjugation of estrogens in different epididymal regions of the boar during the postpubertal period. Therefore, the aim of this study was to determine the activity of StS and EST in all three epididymal regions of 8-, 12- and 16-month-old boars.
2.
Materials and methods
The investigation was carried out on 18 Polish Landrace boars in a pig insemination station in the north-east of Poland. The animals were kept in individual pens and fed on standard diet. The animals were divided into three groups (n = 6) according to age (8, 12 and 16 months). The boars were anesthetized with azaperone (20 mg/kg i.m.) and ketamine (20 mg/kg i.v.) and castrated. The protocol was approved by the Local Ethics Commission for Animal Experiments. After castration, the epididymides were separated from the testes, placed in icecold phosphate buffer (PBS) and transported to the laboratory. The epididymides were divided into three regions (caput, corpus and cauda [25]). The tissue samples of each epididymal region were collected and washed with saline to remove
375
spermatozoa. The samples were cut into small pieces and washed with Ringer solution (Polfa, Warsaw, Poland) until a clear supernatant was obtained following centrifugation. After the final wash, 5 g of the tissue pulp was homogenized in 15 ml Ringer–Hepes buffer, consisting with Ringer solution and Hepes buffer (39:1; Sigma–Aldrich Co., St. Louis, MO, USA). The homogenate was filtered to remove connective tissue. All steps were performed on ice. Activities of steroid sulphatase (StS) and estrogen sulphotransferase (EST) were assessed as was described for bovine placenta [19]. For determination of the StS activity, 1 ml of incubation medium (Ringer–Hepes buffer with 0.1% BSA; Sigma–Aldrich Co., St. Louis, MO, USA), the substrate (0.6 pmol 3 H-estrone sulphate [3H-E1S; NEN Science Products Inc., Brussels, Belgium] dissolved in 0.1 ml Ringer solution with 0.1% BSA) and 0.2 ml homogenate was added to 15 ml glass tubes (Wheaton Science Products, Millville, NJ, USA). The samples were incubated at 37 8C in a water bath with shaking for 1, 5, 15, 30 and 60 min. Control samples contained 1 ml incubation medium, the substrate and 0.2 ml incubation buffer instead of homogenate (medium blank) or heat inactivated homogenate (tissue blank). The controls were incubated for 60 min and the incubates were stored at 20 8C until assay. Each reaction was run in duplicates and the experiments were repeated four times. The formed free 3H-E1 was extracted from the whole samples (incubates) with 2 ml 3 ml toluene (Sigma–Aldrich Co., St. Louis, MO, USA). The samples were mixed on a overhead shaker for 20 min. The aqueous phase was frozen and the toluene phase was decanted. The pooled extracts containing 3H-E1 were dried and dissolved in scintillator (Perkin Elmer Inc., Waltham, MA, USA). The 3H-radioactivity was measured with a liquid scintillation counter (LSA Tri-Carb 2800TR, Perkin Elmer Inc., Waltham, MA, USA). The 3 H-radioactivity in the remaining aqueous phase (3H-E1S) was also counted. The extraction efficiency was 87.9%. The activity of StS was expressed as the percentage of substrate conversion. The total 3H-radioactivity in toluene extracts and aqueous phases was set as 100% value for calculation of conversion rate. For determination of EST activity, 3H-E1 (0.6 pmol; NEN Science Products Inc., Brussels, Belgium) was used as substrate. Additionally, 0.5 mmol of 4-nitrophenylsulphatepotassium salt (Sigma–Aldrich Co., St. Louis, MO, USA) for inhibition of StS and 50 mmol of 30 -phosphoadenosine-50 phosphosulphate (Sigma–Aldrich Co., St. Louis, MO, USA) as a co-factor were used for the reaction. The formed 3H-E1S was measured after removal of free 3H-E1 by extraction with toluene (Sigma–Aldrich Co., St. Louis, MO, USA). The samples were then hydrolyzed with 0.3 standard units of arylsulphatase (Merck KGaA, Darmstadt, Germany) and again extracted as described above. Statistical analysis was performed on raw data using GraphPad PRISM program (GraphPad Software Inc., San Diego, CA, USA). The data were shown as a percentage of added radioactivity (mean SEM). The statistical significance of differences among various incubation times, epididymal regions or age groups was assessed using one-way ANOVA followed by Bonferroni’s multiple comparison test. The level of significance was set at p < 0.05 for all analyses.
376
3.
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[(Fig._1)TD$IG] Results
The activities of StS and EST in epididymal homogenates increased gradually along with the incubation time. The desulphation rate of 3H-E1S into 3H-E1 was significantly ( p < 0.05) higher compared to tissue blank after 15 min of incubation, whereas the sulphoconjugation rate of 3H-E1 into 3 H-E1S was higher ( p < 0.05) after 5 min of incubation. The substrate conversion rates after 60 min of incubation were 51.25% for 3H-E1S and 45.65% for 3H-E1. The activity of StS was significantly ( p < 0.05) higher in the caput epididymis compared to the cauda epididymis after 30 and 60 min of incubation. The activity of EST was also higher in the caput epididymis than in cauda epididymis, but it was dependent on age and time of incubation. The difference between the epididymal regions was statistically significant ( p < 0.05) for boars aged 8 months after 5, 15, 30 and 60 min of incubation, for boars aged 12 months after 5 and 15 min of incubation, for boars aged 16 months after 5 and 60 min of incubation (Figs. 1 and 2). A significant age-dependent increase of StS and EST activities was observed. The activity of StS was significantly ( p < 0.05) higher in boars aged 16 months compared to 8-month-old animals after 15 and 30 min of incubation (caput and corpus) and after 30 and 60 min (cauda). The activity of EST was significantly ( p < 0.05) higher in boars aged 16 months compared to 8-month-old animals after 5, 15, 30 and 60 min of incubation, except for corpus after 15 min of incubation (Figs. 1 and 2).
4.
Discussion
The results of our study showed the high epididymal activities of StS and EST. Data on the StS and EST activities in the reproductive tract of boar are scarce. Activity of StS was examined in the boar testis [3,26,27] and epididymis (head together with corpus [3]). Low activity of EST in the boar testis and high in the epididymis was reported [3]. Our data confirmed the observations of Hoffmann et al. [3], which were, however, limited to the head/corpus epididymis. The activities of StS and EST in our study were significantly higher ( p < 0.05) in the caput than in the cauda epididymis. This is consistent with the results of Claus et al. [20] who found decreased concentrations of estrogens in the epididymal fluid from caput to cauda epididymis of the boar. StS and EST catalyze opposite reactions, leading to the activation of sulphoconjugated estrogens and to the inactivation of free estrogens, respectively. StS increases concentrations of free estrogens, while EST protects the epithelium from excess of estrogens. EST knockout mice showed reduced epididymis weight and reduced motility of sperm isolated from caudal epididymis [28]. High StS and EST activities in the epididymis suggest that the local availability of biologically active estrogens in the epididymis may result also from an interplay between estrogen supply and their metabolism. Pearl et al. [17] found similar tissue concentration of estradiol throughout the epididymis and suggested that the regional differences in estrogen action are likely due to regional differences in their receptor expression.
Fig. 1 – Desulphation (%) of 3H-E1S into 3H-E1 by epididymal homogenates of boars (n = 6); MB: medium blank, TB: tissue blank; a,bdifferent letters depict significant differences ( p < 0.05) between the particular incubation time and TB (within one epididymal region and one age group); c,ddifferent letters depict significant differences ( p < 0.05) among age groups (within one incubation time and one epididymal region); *significant differences ( p < 0.05) between cauda and caput epididymis (within one incubation time and one age group).
We found significant increases in StS and EST activities from eight to 16 months of age. There are no studies on the effect of age on activities of StS and EST in the boar epididymis. The increase in activities of both enzymes corresponded with the increase of estrogen concentration in blood plasma and seminal plasma of boars during this period [4]. ‘‘Biochemical maturation’’ of the reproductive system occurs in boars after
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[(Fig._2)TD$IG]
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In conclusion, high activities of StS and EST in epididymal homogenates suggest that the availability of estrogens in the boar epididymis may be locally controlled also by StS and EST. The increase in StS and EST activities from 8 to 16 months of age may be related to the process of ‘‘biochemical maturation’’ of reproductive system during the postpubertal period.
Acknowledgement This research was supported by a Grant No. N308242935 of the Polish Ministry of Sciences and Higher Education.
references
Fig. 2 – Sulphoconjugation (%) of 3H-E1 into 3H-E1S by epididymal homogenates of boars (n = 6); MB: medium blank, TB: tissue blank; a,bdifferent letters depict significant differences ( p < 0.05) between the particular incubation time and TB (within one epididymal region and one age group); c,ddifferent letters depict significant differences ( p < 0.05) among age groups (within one incubation time and one epididymal region); *significant differences ( p < 0.05) between cauda and caput epididymis (within one incubation time and one age group).
reaching sexual maturity at the age of 18 months. The development of the boar reproductive tract is accompanied by an increased secretory capacity of the accessory sex glands [23,24] and epididymis [25]. The biochemical components of the seminal plasma play an important role in sperm physiological function [24]. Our study demonstrated significant increase in estrogen metabolism in the boar epididymis during the postpubertal period. Estrogens are essential for the function of epididymis and production of semen with high biological properties [7–11].
[1] Claus R, Hoffmann B. Oestrogens, compared to other steroids of testicular origin in blood plasma of boars. Acta Endocrinologica 1980;94(3):404–11. [2] Claus R, Schopper D, Wagner HG. Seasonal effect on steroids in blood plasma and seminal plasma of boars. Journal of Steroid Biochemistry 1983;19(2):725–9. [3] Hoffmann B, Rostalski A, Mutembei M, Goericke-Pesch S. Testicular steroid hormone secretion in the boar and expression of testicular and epididymal steroid sulphatase and estrogen sulphotransferase activity. Experimental and Clinical Endocrinology & Diabetes 2010;118(4):274–80. [4] Zdun´czyk S, Janowski T, Ras´ A, Baran´ski W. Concentrations of oestrogens in blood plasma and seminal plasma of boars during the postpubertal period. Polish Journal of Veterinary Sciences 2011;14(4):539–44. [5] Hess RA, Carnes K. The role of oestrogen in testis and male reproductive tract – a review. Animal Reproduction 2004;1(1):5–30. [6] Zdun´czyk S, Janowski T. Role of oestrogens in the regulation of reproductive tract functions in the boar. Medycyna Weterynaryjna 2009;65(8):521–5 [in Polish]. [7] Hess RA, Bunick D, Lubahn BD, Zhou Q, Bouma J. Morphological changes in the efferent ductules and epididymis in oestrogen knock out mice. Journal of Andrology 2000;21(1):107–21. [8] Zhou LJ, Clarke L, Nie R, Carnes K, Lai LW, Lien YH, et al. Estrogen action and male fertility: roles of sodium/ hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proceedings of the National Academy of Sciences of the United States of America 2001;98(21):14132–7. [9] Cho HW, Nie R, Carnes K, Zhou Q, Sharief NA, Hess RA. The antiestrogen ICI 182, 780 induces early effects on the adult male mouse reproductive tract and long-term decreased fertility without testicular atrophy. Reproductive Biology and Endocrinology 2003;1:57. [10] McCarthy MJ, At-Taras EE, Pearl CA, Nitta-Oda BS, Roser JF, Conley AK, et al. Suppression of endogenous estrogen during development effects porcine epididymal sperm maturation. Molecular Reproduction & Development 2006;73(9):1122–8. [11] Pearl CA, At-Taras E, Berger T, Roser JF. Reduced endogenous estrogen delays epididymal development but has no effect on efferent duct morphology in boars. Reproduction 2007;134(4):593–604. [12] Pereyra-Martinez AC, Roselli CE, Stadelman HL, Resko JA. Cytochrome P450 aromatase in testis and epididymis of male rhesus monkeys. Endocrine 2001;16(1):15–9.
378
reproductive biology 12 (2012) 374–378
[13] Shayu D, Rao AJ. Expression of functional aromatase in the epididymis: role of androgen and LH in modulation of expression and activity. Molecular and Cellular Endocrinology 2006;249(1–2):40–50. [14] Wiszniewska B. Primary culture of rat epididymal epithelial cells as a source of estrogen. Andrologia 2002;34(3):180–7. [15] Rago V, Bilin´ska B, Palma A, Ando S, Carpino A. Evidence of aromatase localization in cytoplasmic droplet of human immature ejaculated spermatozoa. Folia Histochemica et Cytobiologica 2003;41(1):23–8. [16] Rago VF, Aquilla S, Panza R, Carpino A. Cytochrome P450arom, androgen and estrogen receptors in pig sperm. Reproductive Biology and Endocrinology 2007;5:23. [17] Pearl CA, Berger T, Roser JF. Estrogen and androgen receptor expression in relation to steroid concentrations in the adult boars epididymis. Domestic Animal Endocrinology 2007;33(4):451–9. [18] Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nolsson J, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 1997;138(3):863–70. [19] Hoffmann B, Falter K, Vielemeier A, Failing K, Schuler G. Investigations on the activity of bovine placental oestrogen sulfotransferse and -sulfatase from midgestation to parturition. Experimental and Clinical Endocrinology & Diabetes 2001;109(5):284–301. [20] Claus R, Schopper D, Hoang-Vu C. Contribution of individual compartments of the genital tract to oestrogen and testosterone concentrations in ejaculates of the boar. Acta Endocrinologica 1985;109(2):281–8.
[21] Raeside JI, Christie HL, Renaud RL. Androgen and oestrogen metabolism in the reproductive tract and accessory sex glands of the domestic boar (Sus scrofa). Biology of Reproduction 1999;61(5):1242–8. [22] Cameron RDA. Sexual development and semen production in boars. Pig News and Information 1987;8(4):389–92. [23] Kowalowka M, Wysocki P, Fraser L, Strzezek J. Extracellular superoxide dismutase of boar seminal plasma. Reproduction in Domestic Animals 2008;43(4): 490–6. [24] Strzezek J, Saiz-Cidoncha F, Wysocki P, Tyszkiewicz A, Jastrze˛bski M. Seminal plasma proteins as markers of biological value of boar semen. Animal Science Papers and Reports 2002;20(4):255–66. [25] Syntin P, Dacheux JL, Dacheux F. Postnatal development and regulation of proteins secreted in the boar epididymis. Biology of Reproduction 1999;61(6):1622–35. [26] Mutembei H, Kowalewski M, Ugele B, Schuler G, Hofmann B. Expression and activity of steroid sulphatase in the boar testis. Reproduction in Domestic Animals 2009;44(1):17–23. [27] Yamamoto K, Handa S, Yamakawa T. Purification of arylsulfatase A from boar testis and its activities toward seminolipid and sulfatide. Journal of Biochemistry 1997;75:1241–7. [28] Qian YM, Sun XJ, Tong MH, Li XP, Richa J, Song W-C. Targeted disruption of the mouse estrogen sulfotransferase gene reveals a role of estrogen metabolism in intracrine and paracrine estrogen regulation. Endocrinology 2001;142(12):5342–50.