Theriogenology 189 (2022) 150e157 Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com Rat age-related benign prostate hyperplasia is concomitant with an increase in the secretion of low ramified a-glycosydic polysaccharides L. Trujillo-Rojas a, b, J.M. Fernandez-Novell c, O. Blanco-Prieto a, d, B. Martí-Garcia e,  T. Rigau a, M.M. Rivera del Alamo a , J.E. Rodríguez-Gil a, * a Dept. Animal Medicine and Surgery, Autonomous University of Barcelona, E-08193, Bellaterra, Cerdanyola del Vall es, Spain b Dept. of Veterinary Medicine, School of Agricultural Sciences, University of Pamplona, Pamplona, Colombia c Dept. Biochemistry and Molecular Biology, University of Barcelona, E-08028, Barcelona, Spain d Dept. Veterinary Medical Sciences, University of Bologne, I-40126, Bologna, Italy e Dept. Animal Safety and Anatomy, Autonomous University of Barcelona, E-08193, Bellaterra, Cerdanyola del Vall es, Spain a r t i c l e i n f o a b s t r a c t Article history: This work analysed the expression of prostate polysaccharides in rats with age-related benign prostatic Received 19 January 2022 hyperplasia (BPH) for a better understanding of the possible relationship between prostate poly- Received in revised form saccharides secretion and BPH onset. For this, prostatic glands from 1 month-old, 3 months-old, 6 9 June 2022 months-old and 12 months-old Sprague-Dawley rats were processed in order to identify their overall Accepted 10 June 2022 polysaccharide content. Additionally, serum testosterone was also determined. One-month old rats Available online 20 June 2022 showed significantly (P < 0.05) lower testosterone levels (0.77 ng/mL±0.12 ng/mL) compared with the other groups, which showed no significant difference among them. PAS staining showed positive Keywords: Prostate hyperplasia polysaccharides markings in both the prostatic lumen and inside of luminal prostatic cells in all groups. Polysaccharide content Semiquantitative analysis of intraluminal PAS showed that one month-old rats had significantly (P < 0.005) lower PAS intensity when compared with all other groups (100.0 ± 0.5, arbitrary units vs. 107.3 ± 0.6, arbitrary units in 3 months-old ones), whereas 12 months-old ones showed significantly (P < 0.005) higher values when compared with all other groups (133.6 ± 3.5, arbitrary units in 12 months-old rats vs. 108.6 ± 1.4, arbitrary units in 6 months-old ones). The PAS þ content practically disappeared when tissues were pre-incubated with either a-amylase or amyloglucosidase, regardless of a previous incubation with proteinase K. Incubation of prostate extracts from 12 months-old rats for 2 h with a-amylase yielded a significantly higher amount of free glucose (1.47 nmol/mg protein±0.23 nmol/ mg protein vs. 0.32 nmol/mg protein±0.01 nmol/mg protein in untreated extracts). Similar results were obtained when extracts were pre-incubated with amyloglucosidase. Contrarily, pre-incubation with N- glycosidase induced a significantly (P < 0.05), much lower increase of free glucose. Pre-treatment with proteinase K did not significantly modify these results, which indicate that BPH is related to an increase in the secretion of low ramified ductal a-glycosydic polysaccharides that were not protected against lysis by any type of protein protective core. These changes seem to not be related with concomitant variations in serum testosterone levels. © 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction that it often develops towards malignant situations such as prostate carcinoma [3]. Thus, a close vigilance of the evolution of BPH is needed Age-related benign prostate hyperplasia (BPH) is a very common to avoid the appearance of derived, malignant processes. condition affecting a very high percentage of men above 50 years old Control of BPH should be based on a good understanding of its and also older male individuals from other species such as rodents pathogenesis. However, currently there are several important as- [1e3]. Although BPH is a benign process, its importance lies in the fact pects regarding BPH pathogenesis that are not well understood, impairing thus an optimal surveillance of the process. Among these, one of the most prominent would be centred in BPH-related * Corresponding author. alterations of prostatic secretions. In this regard, it is well known E-mail address: juanenrique.rodriguez@uab.cat (J.E. Rodríguez-Gil). https://doi.org/10.1016/j.theriogenology.2022.06.011 0093-691X/© 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ). ndez-Novell, O. Blanco-Prieto et al. L. Trujillo-Rojas, J.M. Ferna Theriogenology 189 (2022) 150e157 that one of the important components of prostatic secretions are 20  C. Fixed portions were used for histological analyses. The sec- amyloid bodies (ABs; see Refs. [4,5]). The onset of BPH has been ond portions were immediately frozen in liquid N2 and then they related to a noticeable increase in the ABs content of prostatic se- were stored at 80  C. These portions were used for studies centred cretions [4,5], suggesting thus a relationship between ABs metab- in the analysis of the prostate polysaccharide content. The design of olisms and the onset of BPH. Moreover, the malignant evolution of our experiment did not require any specific code from the Ethical BPH towards carcinoma has also been related to a significant Commission (EC) of the Autonomous University of Barcelona (UAB), change in the composition of ABs. Thus, whereas ABs of both since there was no experimentation procedure linked with the healthy and BPH-affected prostatic glands seem to be mainly work. Despite this, it was specifically approved by the EC of the UAB composed of insoluble amylaceous compounds, those from ma- after checking that it followed all of the Animal Welfare Guidelines lignant carcinoma tissues seem to contain mucin and unidentified approved by the Spanish and Catalan Governments. crystalloids [4e6]. This difference would indicate a significant change in the sugar metabolism linked to the appearance of ma- lignant prostate cells, which would be in accordance with the 2.2. Determination of serum levels of testosterone already described change in metabolism fluxes related to the appearance of malignity in all types of tumours [7]. However, Levels of serum testosterone were determined by means of a despite all of our knowledge, the exact composition of ABs is commercial ELISA kit (Testosterone rat/mouse ELISA kit; ref. currently not well known. Thus, a complete description of the DEV9911; Demeditec Diagnostics GmbH, Kiel, Germany). amylaceous compounds forming BPH-related ABs is lacking, with the exception that they contain glucosamine, galactose and sulphur 2.3. Processing samples for histological procedures [8], which could point to the presence of keratan sulphate [6]. Related to this, it has been also observed that BPH is concomitant As stated, formaldehyde-fixed samples were first thoroughly with an increase in the expression of chondroitin sulphate located washed with PBS and then dehydrated through sequential im- at the basal membrane of epithelial ductal cells and periductal mersion in ethanol dilutions from 70% (v:v) to 100% (v:v), following myocytes [6]. However, it is not known if chondroitin sulphate is a standard practices. Dehydrated samples were then treated with relevant component of BPH-related ABs. xylol for 1 h at 20  C and embedded in paraffin overnight at 58  C. Taking into account the above-described points, the main aim of Paraffined samples were then cooled at 4  C for 1 h until the for- this work was to go in depth in the exact composition of prostate mation of a solid block. These blocks were subsequently sliced by polysaccharide secretions linked with the BPH process. The work means of a microtome (HistoCore Biocat®; Leica Biosystems; was centred on the identification of the overall structure present in Wetzlar, Germany) to obtain slices with a thickness of both 5-mm most prostate polysaccharides through their analysis by both PAS and 7-mm. These slices were then placed onto 25 mm  75 mm staining and selective polysaccharide degradation by using specific xylene-coated slides for their analysis. polysaccharolythic enzymes, namely a-amylase, amyloglucosidase and N-glycosidase, and further testing the possibility of a close structural linking of these polysaccharides with a protein core by 2.4. Histological staining procedures selective digestion with proteinase K. Two separated histological stainings were used in this work. The 2. Material and methods first was a standard haematoxylin-eosin (H-E) staining in order to determine the histological architecture of samples. The second 2.1. Animals staining was carried out through the periodic acid-Schiff (PAS) technique, which analysed the polysaccharide content of the his- The animals used were healthy male Sprague Dawley rats aged 1 tological samples. Both H-E and PAS procedures were carried out month-old (prepuberal, n ¼ 20), 3 months-old (pubertal, n ¼ 20), 6 following previously well-established procedures as shown in months-old (mature, n ¼ 9) and 12 months-old (elder, n ¼ 9). The Refs. [9,10], which were applied to both 5 mm and 7 mm samples. rats were not raised in the university facilities, but they were Focusing on PAS technique, in some cases, slides were previously directly purchased from a commercial supplier (Harlan Interfauna incubated with a proteinase K (Sigma Aldrich; St. Louis, USA) so- rica S.L.; Sant Feliu de Codines, Spain) at the indicated ages. Once Ibe lution in PBS at a final dilution of 30 IU/mL to determine putative at the laboratory, animals were housed in the university facilities in changes in PAS stain related with the presence/absence of proteins. controlled environmental conditions of 12 h/12 h light/darkness Semiquantitative analysis of PAS staining intensity was con- cycle, at 20  C and 40% humidity, fed with a commercial diet and ducted by using the Image J Fiji image processing package [11]. For “ad libitum” access to water. Rats were kept in these conditions for this purpose, images of 9 individuals from each age group were 48 h before euthanasia. Animals were euthanized introducing them analysed. For each animal, 10 circular sections taken at the centre of in a CO2 chamber for 5 min. Following the monitorized death, blood each ductus pertaining to 10 separate intraductal areas were pro- was immediately sampled through intracardiac puncture and ani- cessed to avoid significant bias of the intensity linked to the back- mals were subsequently dissected to perform prostates collection. ground. These circular sections had in all cases a diameter of 5 mm. Regarding blood collection, samples were allowed to clot at 20  C All images were processed with the same setup values of brightness for 20 min and, afterwards, serums were separated by centrifuga- and contrast to avoid artefactual bias of results and mean intensity tion at 1600g at 4  C for 10 min. Subsequently, serums were of the red channel of each section was taken. Afterwards, the mean immediately frozen in liquid N2 and further stored at 80  C until values for the 10 obtained measures for each animal were deter- their analyses were performed. Regarding dissected prostates, only mined. Following this, the obtained mean value from the first an- the ventral prostate was used in the study. For this purpose, ventral imal of the 1 month-old group was transformed to an arbitrary prostates were carefully dissected and then excised into two por- value of 100. Subsequently, all mean values for all other animals, tions. One portion was fixed by immersion in a 4% (v:v) formal- regardless of the age group, were transformed by using the same dehyde solution in phosphate buffered saline (PBS, pH 7.4) at 20  C factor applied to the first individual. Finally, the appropriate sta- by using a volume proportion of 10:1. The minimum time elapsed tistical procedures explained in the appropriate section of this between the start of fixation and the use of samples was 24 h at manuscript were applied to these transformed values. 151 ndez-Novell, O. Blanco-Prieto et al. L. Trujillo-Rojas, J.M. Ferna Theriogenology 189 (2022) 150e157 2.5. Analysis of polysaccharide content of prostatic tissue 2.6. Statistical analysis Only samples from 12 months-old rats were used for this Putative statistical differences among results were carried out analysis, since these animals showed the highest levels of poly- by using a statistical package (SPSS® Ver 25.0 for Windows; IBM saccharides. The analysis was based on the incubation of tissue Corp., Armonk, USA). Analysis of both serum testosterone levels extracts with separated enzymes that had a specific ability to hy- and semiquantitative PAS intensity were performed with the drolyze polysaccharides. The used enzymes were a-amylase, amy- application of the Student-Newman Keuls (SeNeK) test. Other- loglucosidase and N-glycosidase. Incubations were performed wise, data regarding the effects of incubation with glycosidases in either in tissues without any previous treatment or with a first the absence or presence of proteinase K were first tested for normal incubation with proteinase K to determine the possible presence of distribution with the Shapiro Wilks test. In this case, all data protein-trapped polysaccharides that were not hydrolysed in raw showed normal distributions. Afterwards, data was compared by conditions. Procedures were the following: using a two-way analysis of variance (ANOVA), in which the Factor To determine polysaccharide content in raw samples, frozen 1 was the incubation with or without any glycosidase and the samples stored at 80  C were homogenised in an ice-cold 50 mM Factor 2 was the incubation with or without proteinase K. Subse- Tris-HCl homogenization buffer (pH 7.4) containing 1 mM ethylene quently, the ANOVA analysis was followed by a post hoc Sidak test diamino tetraacetic acid (EDTA), 10 mM ethyelenglycol-bis(2- for pair-wise comparisons. aminoether)-N,N,N0 ,N0 tetraacetic acid (EGTA), 25 mM dithyo- treitol (DTT), 1.5% (v:v) Triton X-100®, 1 mM phenylmethyl sulfonil 3. Results fluoride (PMSF), 10 mg/mL leupeptin, 1 mM Na2VO4 and 1 mM benzamidine. Homogenization was performed by using an Ultra- 3.1. Histopathological detection of benign prostatic hyperplasia Turrax® T25 basic device (IKA®-Werke; Staufen, Germany) at 13.500 rpm in ice-cold conditions and with a proportion of 5 mL The histological study of prostate gland in prepubertal rats by buffer:1 mg sample. A 10 mL aliquot of each sample was then taken using the H/E staining procedure showed the presence of a one- in order to evaluate the total protein content of samples through layer ductal tubular epithelium formed by cuboidal cells. This the Bradford technique [12] by using a commercial kit (Protein epithelium was surrounded by mesenchyme which contained Assay Dye Reagent Concentrate; BioRad; London, UK). Afterwards, connective cells and blood vessels (Fig. 1A and B). Furthermore, homogenised samples were centrifuged at 1200g for 10 min at tubuli were filled by an amorphous, acellular, strongly H/E reactive 4  C and resultant supernatants were fractionated in 4 separated content (Fig. 1A and B). This image was only slightly modified in fractions of equal volume. Three portions were added with a 0.07 M pubertal (3 months-old) and mature (6 months-old) rats, although acid acetic-sodium acetate buffer (AA buffer, pH 4.7), whereas the the diameter of tubuli was noticeably greater when compared with remaining fraction was added with PBS. Dilution of all four fractions prepuberal ones (data not shown; Fig. 1C and D). On the other hand, was performed until obtaining a final protein concentration of several 6 months-old animals showed early signs of tubular hy- 2.5 mg/mL. Afterwards, two of the fractions diluted in the AA buffer perplasia. These signs were very focalized and punctual, showing were added with either a-amylase or amyloglucosidase to a final the presence of more than one layer of ductal cells (Fig. 1C,D). Cclear concentration of both enzymes of 0.2 IU/mL, whereas the other signs of hyperplasia were detected in all of the analysed 12 months- fraction diluted in the same buffer was left without any other old rats. In these animals, hyperplasia was detected by the presence addition as a control point. Otherwise, PBS diluted samples were of focal areas of variable size in which ductal epithelium was also added with N-glycosidase until a final activity of 0.2 IU/mL. All composed by more than one layer of cells. Moreover, prostatic samples were incubated for 2 h at 37  C. Then, enzyme reactions mesenchyme of these animals was characterised by a noticeable were stopped by immersion of samples in liquid N2. Samples were increase in the number of blood vessels, especially around and in- subsequently thawed and glucose concentration was determined side the hyperplasia areas (Fig. 1E and F). espectrophotometrically by using a commercial kit (Glucose HK CP ABX, Horiba; Montpellier, France). 3.2. Serum levels of testosterone The determination of polysaccharide content in samples previ- ously treated with proteinase K was performed after homogeni- As shown in Figs. 2 and 1 month-old rats showed the lowest zation of frozen samples stored at 80  C in an ice-cold 50 mM levels of serum testosterone (0.54 ng/mL±0.07 ng/mL; Hepes buffer (pH 5.0) containing 25 mM DTT, 1.5% (v:v) Triton X- mean ± S.E.M.). These levels were significantly (P < 0.05) higher in 100® and 6 mM CaCl2. Homogenization was performed following 3 months-old rats, which showed mean values of 2.71 ng/ the same protocol described above. At this moment, a 10 mL aliquot mL±0.36 ng/mL (mean ± S.E.M.). Finally, serum testosterone levels was taken in order to determine the total protein content by using from both 6 months-old and 12 months-old rats were not signifi- the Bradford technique [12] as described above. Afterwards, pro- cantly different to those obtained in 3 months-old animals (Fig. 2). teinase K (Sigma Aldrich; St. Louis, USA) was added to the homo- genised samples until a final enzyme activity of 30IU/mL. Samples were then incubated for 10 h at 37  C. Proteinase K reaction was 3.3. Analysis of polysaccharide intraductal content by applying the stopped by adding PMSF to samples to a final concentration of PAS staining 5 mM. At this moment samples were centrifuged at 1200g for 10 min at 4  C and resultant supernatants were split in 4 separate The PAS staining showed a positive intraductal content in all the fractions of equal volume. These fractions were diluted and incu- studied rats, from prepubertal animals to 12 months-old ones bated without or with a-amylase, amyloglucosidase or N-glycosi- (FigA. 3). Likewise, epithelial ductal cells also showed a dase following the same protocol described above. Finally, enzyme PAS þ content in all cases (Fig. 3A). This content would be consis- reactions were stopped by immersion of samples in liquid N2. tent with the presence of ABs. Previous incubation of samples with Samples were subsequently thawed and glucose concentration was a-amylase induced a strong decrease and even a complete disap- determined espectrophotometrically by using the Glucose HK CP pearance of the intraductal PAS staining (Fig. 3C). A similar result ABX commercial kit (Horiba; Montpellier, France). was obtained with samples pre-incubated with amyloglucosidase 152 ndez-Novell, O. Blanco-Prieto et al. L. Trujillo-Rojas, J.M. Ferna Theriogenology 189 (2022) 150e157 Fig. 1. Prostatic tissue from 1 month-old, 6 months-old and 12 months-old rats stained with the haematoxylin-eosin technique. Prostatic samples from 1 month-old, 6 months-old and 12 months-old rats were fixed and stained as described in the Material and Methods section. A,B: 1 month old rats. C,D: 6 months-old rats. E,F: 12 months-old rats. A,C,E: images observed at 100X augmentations. B,D,F: images observed at 1000X augmentations. *: intraductal areas. /: interstitial tissue. A: ductal epithelium. Bars in images A, C and E indicate 100 mm length, whereas bars in images B, D and F indicate 20 mm length. Figure shows representative images obtained from twenty 1 month-old rats, nine 6 months-old animals and nine 12 months-old ones. previously to the PAS staining (Fig. 3E). Noticeably, an appreciable months-old one (107.3 ± 0.6, arbitrary units in 3 months-old rats loss of PAS staining was observed when samples were incubated for and 108.6 ± 1.4, arbitrary units in 6 months-old ones; Fig. 4), while 2 h with the dilution medium of both a-amylase and amylogluco- no significant differences between the 3 months-old rats and 6 sidase without enzymes, although this decrease was in all cases months-old ones were detected. Finally, 12 months-old ones much less intense than the observed when enzymes were applied showed significantly (P ˂ 0.005) higher values when compared with (Fig. 3B). The previous incubation with proteinase K before further all other age groups (133.6 ± 3.5, arbitrary units; Fig. 4). incubation with either a-amylase or amyloglucosidase did not modify the above-described results (Fig. 3D and F). 3.4. Free glucose release from prostate gland extracts in the The semiquantitative analysis of the intraductal PAS staining presence of a-amylase, amyloglucosidase or N-glycosidase intensity yielded mean low values in 1 month-old rats (100.0 ± 0.5, arbitrary units; see Fig. 4). These values were significantly (P ˂ 0.05) The incubation of raw prostate gland extracts for 2 h without higher in animals from both the 3 months-old group and the 6 any enzyme yielded a concentration of free glucose of 0.32 nmol/ 153 ndez-Novell, O. Blanco-Prieto et al. L. Trujillo-Rojas, J.M. Ferna Theriogenology 189 (2022) 150e157 increase of testosteronemia linked with the onset of puberty is logical, and, in this way, has been previously reported [13,14]. On the other hand, the lack of great variations in testosteronemia in post-pubertal rats depending on age has been more controversial. Whereas older works found a noticeable drop of testosteronemia in aged rats [15], more recent publications obtained similar results to those shown in this manuscript [16e19]. This discrepancy can be due, on the one hand, to the different methodological techniques applied in each manuscript to detect testosterone and, on the other one, to animal-related differences linked to aspects such as the specific breed and genetic line or even the time of the day in which samples were taken [16e19]. In any case, taking into consideration the utilised technique to determine testosterone and the fact that testes production of testosterone has been described to be main- tained in a stable manner during the entire rat lifetime [20], our results can be considered as entirely logical. In any case, the stable serum testosterone levels suggest that the onset of age-related BPH Fig. 2. Box and whisker plot representation of serum testosterone levels. White bar: 1 is not linked to evident changes in testosteronemia. In this way, the month-old rats. Light grey bar: 3 months-old rats. Medium grey bar: 6 months-old rats. role of testosterone in the induction of age-related rat BPH could be Dark grey bar: 12 months-old rats. Results are obtained from 9 animals in each age more linked to a chronic action of the hormone on prostatic tissue group. Asterisks indicate the mean value for each group, whereas internal lines shown medians. Separate letters indicate significant (P < 0.05) differences when compared by holding testosteronemia high and stable during all the adult life. with the 1 month-old rats group. As stated above, results indicate that the onset of BPH was concomitant with an increase of the polysaccharide prostatic secretion. It is noteworthy that the incubation of prostate gland mg protein ± 0.03 nmol/mg protein (mean ± S.E.M). A similar in- extracts with an enzyme media that did not contain either a- cubation for 2 h with either a-amylase or amyloglucosidase amylase or amyloglucosidase induced by themselves a small, but induced a significant (P < 0.05) increase in free glucose, which significant release of free glucose from tissues. This result would be reached values of 1.47 nmol/mg protein ± 0.03 nmol/mg protein a first indication that the polysaccharide content of the prostate with a-amylase and 1.28 nmol/mg protein ± 0.04 nmol/mg protein gland is mainly composed of substances with high levels of a- with amyloglucosidase (means ± S.E.M.; see Fig. 5). There were no glycosidic bonds. This can be deduced by the fact that a-glycosidic significant differences among values obtained with either a- bonds are highly sensitive to acidic environments [21] and the amylase or amyloglucosidase. On the contrary, the incubation with utilised media had a pH around 4.8. The preponderance of a- N-glycosidase for 2 h induced significantly (P < 0.05) lower glucose glycosidic bonds was further confirmed by the appearance of free levels, with values of 0.41 nmol/mg protein±0.03 nmol/mg protein glucose when prostatic extracts were incubated with either a- (mean ± S.E.M.; see Fig. 5). Free glucose levels of samples previously amylase and amyloglucosidase but not with N-glycosidase. digested with proteinase K without the addition of any other Furthermore, results from both PAS staining and glycosidases enzyme reached values of 0.53 nmol/mg protein±0.03 nmol/mg digestion also suggest that prostatic polysaccharides were mainly protein (mean ± S.E.M.; see Fig. 5). These values were significantly composed of compounds with low ramification levels. The low (P < 0.05) higher than those obtained in raw samples without either ramification was indicated by the fact that the levels of free glucose a-amylase or amyloglucosidase. On the other hand, incubation for obtained after the digestion of prostate gland tissues were not 2 h with either a-amylase or amyloglucosidase of samples previ- significantly different when compared with non-digested ones. In ously digested with proteinase K induced a significant (P < 0.05) this respect, it must be reminded that, whereas both a-amylase and increase in free glucose in both cases, which reached values of amyloglucosidase degrade a-1,4 glycosidic bonds, only amylogly- 1.52 nmol/mg protein±0.03 nmol/mg protein with a-amylase and cosidase is able to hydrolyze a-1,6 ones, which are responsible for 1.56 nmol/mg protein±0.05 nmol/mg protein with amyloglucosi- polysaccharide ramification [22e24]. A similar conclusion has been dase (means ± S.E.M.; see Fig. 5). Again, there were no significant previously suggested [4,25], although in those cases, confirmation differences among values obtained with either a-amylase or amy- of low ramification levels was only suggested indirectly, being our loglucosidase in these conditions, neither when compared with results the first direct indication of this phenomenon. Likewise, the parallel samples incubated without a previous proteinase K diges- lack of an evident increase in the free glucose levels of samples tion (Fig. 5). Finally, the incubation with N-glycosidase for 2 h previously digested with proteinase K, with the exception of con- caused significantly (P < 0.05) lower glucose levels, which reached trol samples, suggests that most polysaccharides present in pros- values of only 0.45 nmol/mg protein±0,07 nmol/mg protein after tatic tissues are not closely linked to a protein structure or nucleus. 2 h of incubation (mean ± S.E.M.; see Fig. 5). This assertion could seem contradictory with the results obtained through the application of PAS techniques in slides previously 4. Discussion incubated with proteinase K. One explanation for this apparent contradiction would be that the polysaccharide content observed in Results obtained in this study strongly suggest that the onset of PAS-stained slides would be held in position with a light protein rat BPH is related to an increase in the synthesis of extracellular, structure that, when digested, throws out the polysaccharide con- ductal a-glycosidic polysaccharides with low ramification. tent from slides after the separate washings included in the PAS Furthermore, these changes seem to not be related with concom- protocol. On the other hand, this light protein structure did not itant variations in serum testosterone levels. In this regard, our impede the lytic action of both a-amylase and amyloglucosidase, results indicate that, although there was a significant increase of maintaining thus similar levels of free glucose in extracts regardless testosteronemia linked to puberty, there were no significant dif- of their pre-incubation with proteinase K or lack of it. Our results, ferences in post-pubertal rats, irrespectively of their age. The however, cannot completely identify what is the main 154 ndez-Novell, O. Blanco-Prieto et al. L. Trujillo-Rojas, J.M. Ferna Theriogenology 189 (2022) 150e157 Fig. 3. Prostatic tissue from 3 months-old rats stained with the PAS technique. Prostatic samples were fixed and stained as described in the Material and Methods section. A: samples stained without incubations. B: samples incubated for 2 h with the dilution medium for both a-amylase and amyloglucosidase. C,D: samples incubated for 2 h with a- amylase. E,F: samples incubated for 2 h with amyloglucosidase. D,F: samples previously incubated with proteinase K for 8 h. All images are observed at 400X augmentations. Bars in images indicate 20 mm length. Selected images show representative results obtained in 3 months-old rats. These results are representative not only for the 3 months-old group, but for all of the other age-classified ones. Rats analysed were twenty in both 1 month-old group and 3 months-old one and nine in 6 months-old group and 12 months-old one. polysaccharide content of prostatic tissue. Despite this, several 5. Conclusions conclusions can be reached. First, glycogen can be discarded as the main compound since it is highly ramified, containing a great Our results indicate that age-related benign prostate hyperpla- number of a-1,6 glycosidic bonds [26]. In this way, results are sia (BPH) in rats is concomitant with an increase in the accumu- pointing to an amylaceous polysaccharide since these compounds lation of low-ramified polysaccharide content with predominance are characterised by low ramification levels as well as by very low of a-glycosidic bonds inside of prostate ducts. This polysaccharide or totally absent presence of N-glycosidic bonds [27]. Likewise, this content was not linked with any protein structure that protects it hypothesis is concomitant with results obtained by other authors against lithic enzymes. In summary, results suggest that the onset [28,29]. In this way, amylaceous compounds, mostly with a free- of rat BHP would be concomitant with local alterations of the protein content, would be the most probable candidates to inte- overall polysaccharide metabolism leading to an imbalance in both grate the main polysaccharide component of prostatic tissue of old, polysaccharide prostate secretion and cell glycogen metabolism BPH-affected rats. modulation. 155 ndez-Novell, O. Blanco-Prieto et al. L. Trujillo-Rojas, J.M. Ferna Theriogenology 189 (2022) 150e157 and obtaining funding. M.M. R A: Writing e original draft, Formal analysis, contributed to the analysis and interpretation of data as well as in the final writing of the manuscript. J.E.R-G: performed the experimental design, interpretation of data and Writing e original draft, final writing of manuscript as well as contributed to obtain funding. Acknowledgments We would like to thank Dr. Manel Bosch (Advanced Optical Microscopy Facilities, University of Barcelona) for his invaluable advice and collaboration to obtain microscope images of both Haematoxylin-Eosin-stained samples and PAS ones. No disclosure was applicable on this work. Likewise, there is no conflict of interest related with this manuscript. Furthermore, no specific Funding was afforded for this work. Fig. 4. Box and whisker plot representation of semiquantitative PAS intensity of prostate ductal content in rats of separate ages. The semiquantitative analysis of PAS References staining intensity was conducted as explained in the Material and Methods section. White bar: 1 month-old rats. Light grey bar: 3 months-old rats. Medium grey bar: 6 [1] Golomb E, Rosenzweig N, Eilam R, Abramovici A. Spontaneous hyperplasia of months-old rats. Dark grey bar: 12 months-old rats. Results are obtained from 9 ani- the ventral lobe of the prostate in aging genetically hypertensive rats. J Androl 2000;21:58e64. https://doi.org/10.1002/j.1939-4640.2000.tb03276.x. mals in each age group. Asterisks indicate the mean value for each group, whereas [2] Mahapokai W, Van Sluijs F, Schalken J. Model for studying benign prostatic internal lines shown medians. Separate letters indicate significant (P < 0.05) differ- hyperplasia. Prostate Cancer P D 2000;3:28e33. https://doi.org/10.1038/ ences among groups. sj.pcan.4500391. [3] Lepor H. Pathophysiology, epidemiology and natural history of benign pros- tatic hyperplasia. Rev Urol 2004;6:S3e10. [4] Cohen RJ, McNeal JE, Redmond SL, Meehan K, Thomas R, Wilce M, et al. Luminal contents of benign and malignant prostatic glands: correspondence to altered secretory mechanisms. Hum Pathol 2000;31:94e100. https:// doi.org/10.1016/s0046-8177(00)80204-x. [5] Sfanos KS, Wilson BA, De Marzo AM, Issacs WB. Accute inflammatory proteins constitute the organic matrix of proststic corpora amylacea and calculi in men with pre state cancer. Proc Natl Acad Sci USA 2009;106:3443e8. https:// doi.org/10.1073/pnas.0810473106. [6] Cardoso L, Falcao P, Sampaio F. Increased and localized accumulation of chondroitin sulfate proteoglycans in the hyperplastic human prostate. BJU Int 2004;93:532e8. https://doi.org/10.1111/j.1464-410x.2003.04688.x. [7] Günther UL, Chong MG, Volpari T, Koczula KM, Atkins K, Bunce CM, et al. Metabolic fluxes in cancer metabolism. In: Mazurek S, Shoshan M, editors. Tumor cell metabolism. Vienna: Springer; 2015. p. 5784e92. https://doi.org/ 10.1007/978-3-7091-1824-5_14. [8] Ginja M, Pires MJ, Gonzalo-Orden JM, Seixas F, Correia-Cardoso M, Ferreira R, Fardilha M, Oliveira PA, Faustino Rocha AI. Anatomy and imaging of rat prostate: practical monitoring in experimental cancer-induced protocols. Di- agnostics 2019;9:68. https://doi.org/10.3390/diagnostics9030068. [9] Pearse GT. Carbohydrates and mucosubstances. In: Pearse AGE, editor. His- tochemistry, theoretical and applied, vol. 2. Edinburgh: Churchill Livingston; 1985. p. 675e785. [10] Feldman AT, Wolfe D. Tissue processing and hematoxylin eosin staining. Meth Mol Biol 2014;1180:844e53. https://doi.org/10.1007/978-1-4939-1050-2_3. Fig. 5. Box and whisker plot representation of free glucose levels of prostatic extracts [11] Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B. Fiji: an open-source platform for incubated with a-amylase, amyloglucosidase or N-glycosidase without or with a pre- biological-image analysis. Nat Methods 2012;9:676e82. https://doi.org/ vious proteinase K treatment. Processing of samples has been described in the Mate- 10.1038/nmeth.2019. 2019. rials and Methods . Samples were incubated for 2 h without any enzyme (Control [12] Bradford MM. A rapid and sensitive method for the quantitation of microgram group, white bars) or with a-amylase (light grey bars), amyloglucosidase (dark grey quantities of protein utilizing the principle of protein-dye binding. Anal Bio- bars) or N-glycosidase (black bars). These incubations were performed in untreated chem 1976;72:248e54. https://doi.org/10.1006/abio.1976.9999. tissues extracts (Without proteinase K group) or after a previous incubation for 1 h [13] Moger W. Serum 5a-androstane-3a, 17/3 diol, androsterone and testosterone with proteinase K (With proteinase K group). Results are obtained from 7 samples in concentrations in the male rat. Influence of age and gonadotrophin stimula- each group. Asterisks indicate the mean value for each group, whereas internal lines tion. Endocrinology 1977;100:1027e32. https://doi.org/10.1210/endo-100-4- shown medians. Separate letters indicate significant (P < 0.05) differences when 1027. compared with the Control group within either the “Without proteinase K00 or the [14] Gray GD, Smith ER, Dorsa DM, Davidson JM. Sexual behavior and testosterone “With proteinase K00 ones. Separate numbers indicate significant (P < 0.05) differences in middle-aged male rats. Endocrinology 1981;109:1597e604. https:// when compared parallel groups (Control, a-amylase, amyloglucosidase and N-glyco- doi.org/10.1210/endo-109-5-1597. sidase) between the “Without proteinase K00 and the “With proteinase K00 ones. [15] Ghanadian R, Lewis JG, Chisholm GD. Serum testosterone and dihy- drotestosterone changes with age in rat. Steroids 1975;25:753e62. https:// doi.org/10.1038/bjc.1979.122. [16] Grünewald DA, Naai MA, Marck BT, Matsumoto AM. Age-related decrease in Author contribution hypothalamic gonadotropin-release hormone (GnRH) gene expression, but not pituitary responsiveness to GnRH, in the male Brown Norway rat. J Androl L T-R: Formal analysis, performed all experimental analyses, O 2000;21:72e84. https://doi.org/10.1002/j.1939-4640.2000.tb03278.x. [17] Wu D, Gore AC. Sexual experience changes sex hormones buy not hypotha- B-P: Formal analysis, performed all experimental analyses and B lamic steroid hormone receptor expression in young and middle-aged male M-G: Formal analysis, performed all experimental analyses. JM F- rats. Horm Behav 2009;56:299e308. https://doi.org/10.1016/ N: Formal analysis, contributed with his advice for the correct j.yhbeh.2009.06.007. [18] Wu D, Gore AC. Changes in androgen receptor, estrogen receptor alpha, and performance of experimental analyses as well as in obtaining sexual behavior with aging and testosterone in male rats. Horm Behav funding. T R: Formal analysis, contributed to the analysis of data 2010;58:306e16. https://doi.org/10.1016/j.yhbeh.2010.03.001. 156 ndez-Novell, O. Blanco-Prieto et al. L. Trujillo-Rojas, J.M. Ferna Theriogenology 189 (2022) 150e157 [19] Green MR, Zeidan M, Hodges TE, McCormick CM. Age-dependent regulation [25] Martikainen P, Malmi R, Suominen J. Distribution of glycoconjugates in by androgens of gene expression in the anterior hypothalamus and stress- normal rat ventral prostate and their use as markers of androgen-controlled induced release of adrenal hormones in adolescent and adult male rats. secretory function in culture. Prostate 1986;8:37e49. https://doi.org/ J Neuroendocrinol 2019;31:e12714. https://doi.org/10.1111/jne.12714. 10.1002/pros.2990080106. [20] Frankel AI, Mock EJ. Time course of hormonal response to sexual behavior in [26] Manners DJ. Recent developments in our understanding of glycogen structure. aging male rats. Exp Gerontol 1981;16:363e9. https://doi.org/10.1016/0531- Carbohyd Polym 1991;16:37e82. https://doi.org/10.1016/0144-8617(91) 5565(81)90057-7. 90071-J. [21] Okuyama M, Saburi W, Mori H, kimura A. a-glucosidases and a-1,4-glucan [27] Selvakumar P, Ashakumary L, Helen A, Pandey A. Purification and character- lyases: structures, functions and physiological actions. Cell Mol Sci 2016;73: ization of glucoamylase produced by Aspergillus niger in solid state fermen- 2727e51. https://doi.org/10.1007/s00018-016-2247-5. tation. Lett Appl Microbiol 1996;23:403e6. https://doi.org/10.1111/j.1472- [22] Preece I, Shadaksharaswamy M. Reducing-group production from starch by 765x.1996.tb01346.x. the action of a- and b-amylases of barley malt. Activity of a- and b-amylases. [28] Munkley J. Glycosylation is a global target for androgen control in prostate Biochem J 1949;44:270e4. https://doi.org/10.1042/bj0440270. cancer cells. Endocr Relat Cancer 2017;24:R49e64. https://doi.org/10.1530/ [23] Colonna WJ, Magee P. Glycogenolytic enzymes in sporulating yeast. J Bacteriol ERC-16-0569. 1978;134:844e53. https://doi.org/10.1128/jb.134.3.844-853.1978. [29] Ojalill M, Rappu P, Siljam€ € m P, Heino J. The composition aki E, Taimen P, Bostro [24] Krisman CR, Tolmasky DS, Raffo S. Branching enzyme assay: selective quan- of prostate core matrisome in vivo and in vitro unveiled by mass spectro- titation of the a-1,6-linked glucosyl residues involved in the branching point. metric analysis. Prostate 2018;78:583e94. https://doi.org/10.1002/ Anal Biochem 1985;147:491e6. https://doi.org/10.1016/0003-2697(85) pros.23503. 90303-3. 157