Analysis of glycoconjugates and

morphological characterization of the

descending colon and rectum of the
plains viscacha, Lagostomus maximus
Mara Florencia Tano De La Hoz & Mirta
Alicia Flamini & Enrique Leo Portiansky
& Alcira Ofelia Daz
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Accepted Manuscript

Title: Analysis of glycoconjugates and morphological

characterization of the descending colon and rectum of the
plains viscacha, Lagostomus maximus

Authors: Marı́a Florencia Tano de la Hoz, Mirta Alicia

Flamini, Enrique Leo Portiansky, Alcira Ofelia Dı́az

PII: S0944-2006(18)30240-X
DOI: https://doi.org/10.1016/j.zool.2019.06.001
Reference: ZOOL 25691

To appear in:

Received date: 21 December 2018

Revised date: 29 May 2019
Accepted date: 3 June 2019

Please cite this article as: de la Hoz MFT, Flamini MA, Portiansky EL, Dı́az AO,
Analysis of glycoconjugates and morphological characterization of the descending
colon and rectum of the plains viscacha, Lagostomus maximus, Zoology (2019),
https://doi.org/10.1016/j.zool.2019.06.001

This is a PDF file of an unedited manuscript that has been accepted for publication.
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apply to the journal pertain.
Analysis of glycoconjugates and morphological characterization of the descending

colon and rectum of the plains viscacha, Lagostomus maximus

Short title: Descending colon and rectum of L. maximus.

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María Florencia Tano de la Hoz a,d,*, Mirta Alicia Flamini b, Enrique Leo Portiansky c,d, and

Alcira Ofelia Díaz a

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a N
Instituto de Investigaciones Marinas y Costeras (IIMyC), Departamento de Biología,
A
FCEyN, CONICET-Universidad Nacional de Mar del Plata, Funes 3250 (7600), Mar del
M

Plata, Argentina.
b
Laboratorio de Histología y Embriología Descriptiva, Experimental y Comparada,
D

Departamento de Ciencias Básicas, Facultad de Ciencias Veterinarias, 60 y 118 (1900),

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Universidad Nacional de La Plata, La Plata, Argentina.

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c
Laboratorio de Análisis de Imágenes, Facultad de Ciencias Veterinarias, Universidad

Nacional de La Plata (LAI, FCV-UNLP), 60 y 118 (1900), La Plata, Argentina.

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d
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
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*
Corresponding author: M.F. Tano de la Hoz, IIMyC, FCEyN, CONICET-Universidad

Nacional de Mar del Plata. Funes 3250 3° piso, 7600 Mar del Plata, Buenos Aires,

Argentina. E-mail: mftano@mdp.edu.ar

1
Graphical abstract

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Comparative analysis of the morphology, ultrastructure and glycosylation pattern of the

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descending colon and rectum of Lagostomus maximus. Histochemical results (HQ) revealed

that in both sectors of the large intestine, there are goblet cells (GC) with different

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glycosylation pattern (GCa, GCb and GCc) within a morphologically homogeneous cell
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population. Specific differences between both intestinal segments were revealed by lectin
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histochemistry (LHQ). Arrow, glycocalyx; *, lumen. Scale bar: 300 µm (B); 20 µm (A, C).
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D
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Highlights

 Goblet cells form a homogeneous cell population at ultrastructural level.

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 The glycosylation pattern of goblet cells depends on the intestinal region studied.


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Sulphates are the main group responsible in determining the acid gradient of mucus.

 The glycocalyx of both anatomical regions showed carboxylated glycoconjugates.

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2
Abstract

Herbivores exhibit specializations at the intestinal level that facilitate the bacterial

fermentation. The available information on the digestive physiology of Lagostomus

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maximus makes this rodent an interesting model to evaluate morpho-functional adaptations

to herbivory. The general objective of this work was centered on the study of the

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morphology and histochemistry of the descending colon and rectum of L. maximus. To do

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so, a comparative analysis of the morphology, ultrastructure and glycosylation pattern of

both anatomical regions was carried out. Histochemical results revealed that in both sectors

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of the large intestine, there are goblet cells with different glycosylation pattern within a

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morphologically homogeneous cell population. The main difference between both intestinal
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segments lay in the fact that the most distal region of the large intestine showed a greater
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proportion of sialomucins, characterized by being slightly O-acetylated. Further specific

differences were revealed by lectin histochemistry. These data allowed to perform a

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functional interpretation of the cell types and secreted substances, thus contributing to a
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better understanding of the role of mucins in the intestinal tract functioning.

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Key words: Hystricognathi, large intestine, mucins, histochemistry, morphology.

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3
1. Introduction

Herbivores depend on microbial fermentation to use the cellulose as their main source of

energy. Many herbivorous vertebrate species possess specialized regions in the digestive

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tract, such as fermenting chambers, where symbiotic microorganisms degrade cellulose

(Kardong, 2015). Whenever digestion of homopolysaccharides is carried out in a

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specialized stomach, it is said to be a gastric fermentation, while the microbial digestion

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centered in the intestine is called intestinal fermentation (Sakaguchi, 2003; Kardong, 2015).

A low bacterial density is present in the intestinal fermenters of the small intestine in

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comparison to the large intestine due to the efficient trapping of bacteria by mucus and its

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rapid transport to the distal portion of the intestinal tract (Hansson, 2012).
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The mucus forms a highly hydrated gel over the intestinal mucosa. It is mainly
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composed by mucins, and other minor components such as salts, immunoglobulins and

growth factors. Mucins are high molecular weight glycoproteins with a high content of O-
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linked oligosaccharides and a smaller proportion of N-linked oligosaccharides. Their main

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functions in the large intestine are to limit the contact of bacteria with the epithelium and to
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facilitate their transport toward the distal region of the intestinal tract (Robbe et al., 2004;

Kim and Ho, 2010).

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Comparative studies in diverse vertebrate species have demonstrated that the

morphology of the digestive tract varies significantly, even among related groups, by
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exhibiting structural modifications adapted to specialized diets (Kardong, 2015). In

particular, rodents present noticeable differences in their intestinal anatomy, mainly in the

caecum and colon (Kotzé et al., 2010).

4
 Plains viscachas, Lagostomus maximus, are Hystricognathi (Rodentia, Caviomorpha) in

the Chinchillidae family, that inhabit Argentina, Bolivia and Paraguay. They are

exclusively herbivore rodents that in their natural environment feed on a great variety of

plant species, showing preference for grasses and dicotyledons (Puig et al., 1998). The

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structural organization of the intestinal tract of L. maximus is similar to that described in

other hystricognath rodents, by presenting a voluminous caecum and a particularly long

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colon (Clauss et al., 2007; Hagen et al., 2015). Moreover, using markers for digestibility

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essays it was shown that L. maximus practices caecotrophy and presents a colonic groove

along the mesenteric side of the ascending colon (Clauss et al., 2007; Hagen et al., 2015).

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In this way, this hystricomorph rodent exhibits, as other herbivores, specializations at the

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intestinal level that facilitate the bacterial fermentation. The available information on the
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digestive physiology of L. maximus makes this rodent an interesting model to evaluate
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morpho-functional adaptations to herbivory.

In the last years, our research group has studied the histochemical profile of the goblet
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cells along the small intestine of L. maximus (Tano de la Hoz et al., 2014; 2016). Also, we
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have recently demonstrated that the glycosylation pattern of mucus has a key role in the
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functioning of the colonic groove of L. maximus (Tano de la Hoz et al., 2017). To further

studying the large intestine, the main goal of this work was centered on the morphology and
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histochemistry of the descending colon and rectum of L. maximus. Thus, a comparative

analysis of the morphology, ultrastructure and glycosylation pattern of both anatomical

A

regions was carried out.

2. Materials and methods

2.1 Animals

5
 Wild adult plains viscachas, Lagostomus maximus, Desmarest, 1817 of both sexes (n=

14; 8 females and 6 males) from the Estación de Cría de Animales Silvestres (ECAS),

Ministry of Agroindustry of the Province of Buenos Aires (Argentina) were used.

Captures were made during the periods of March-April, July-August and December-

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January, years 2009-2014. Captured animals were anesthetized with xylazine (8 mg/kg

body weight), followed by ketamine (50 mg/kg body weight) by intramuscular via

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(Ketanest, Laboratorio Scott Cassara). Each anesthetized animal was weighed and sexed by

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the traditional anus-genital distance method. Average body weights of females were 4-5.5

kg, while males weighed between 7-8.5 kg. Sanitary status of animals was evaluated,

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especially observing the hairy cover condition and the presence of ectoparasites and

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tegumentary injuries. Only healthy animals were used for this study. Once they reached the
A
deep plane of anesthesia, intracardiac perfusion was conducted using physiological saline
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solution, followed by paraformaldehyde 4% in phosphate buffer 0.1M. The method used

was approved by the Institutional Committee for the Caring and Use of Laboratory Animals
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(CICUAL) of the School of Veterinary Sciences, National University of La Plata (52-4-

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15T) and is in accordance with the international recommendations for the use of
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experimental animals (National Research Council, 2011).

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2.2 Sampling and histological processing

Necropsies were carried out immediately after euthanasia. For histological and
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histochemical analyses, three samples from the cranial portion of the descending colon and

rectum were taken per animal. Samples were processed for inclusion in paraffin and 4µm

thick sections were stained with hematoxylin-eosin (H-E) and Masson’s trichrome. From

each sample, at least forty-two serial cross sections were obtained. Each technique was
6
repeated in duplicates for each sample. Images were taken using a trinocular microscope

(CH30, Olympus, Japan).

2.3. Ultrastructural study

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To perform an ultrastructural study of the intestinal epithelium, 0.5-1 mm3 samples from

the descending colon and rectum of L. maximus were taken. Sections were fixed in cold

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(4°C) 2% glutaraldehyde in 0.1 M phosphate buffer solution, pH 7.3, at 4°C during 2 h.

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Afterwards, the samples were exposed to three folds washings with 0.1 M phosphate-

buffered saline (PBS), pH 7.2, during 20 min each. Samples were then post fixed in 1%

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osmium tetroxide at 4°C during 1 h, dehydrated in ethanol solution at increasing
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concentrations and included in Epon 812. To select the area of study semi thin cuts were
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stained with toluidine blue for optical microscope observation. Then, ultra-fine cuts were
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contrasted with uranyl acetate and 1% plumb citrate. Samples were examined using a JEOL
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JEM 1200EX II electronic transmission microscope (TEM) and photographed with a digital
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camera (ES 500W Erlangshen Gatan CCD).

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2.4 Histochemical study for the determination of glycoconjugates

The histological sections were also exposed to the following histochemical techniques to
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characterize glycoconjugates (GCs) and to evaluate their distribution and possible

A

physiological roles:

2.4.1 Periodic acid-Schiff’s reagent (PAS). It is used to evidence GCs with oxidizable

vicinal diols and/or glycogen. For this purpose, sections were treated with 1% periodic acid

7
during for 15min. Then, they were washed with running water and dyed during 2 min with

the Schiff reactive (Mc Manus, 1948).

2.4.2 α-amylase-PAS. Sections were subjected to an enzymatic digestion with α-amylase

in a 36º moist chamber during 45 min to identify glycogen. Then, the PAS technique was

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applied (Pearse, 1985).

2.4.3 KOH/PA*S (saponification-selective periodic acid-Schiff’s reagent). This

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technique characterizes GCs with sialic acid residues. The saponification reaction (KOH)

SC
was performed with 0.5% potassium hydroxide in 70% ethanol for 30 min at room

temperature. Before staining with the Schiff’s reagent, sections were subjected to a

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selective oxidation with 0.4 mM periodic acid in 1 M hydrochloric acid at 4ºC (Culling et

al., 1976). N
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2.4.4 PA/Bh/KOH/PAS (periodic acid-reduction with borohydride- saponification-
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periodic acid-Schiff’s reagent). This technique demonstrates the presence of CGs with

sialic acid residues with O-acyl substitutions in 7C, 8C o 9C and O-acyl sugars. Sections
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were subjected to a room temperature oxidation with 1% periodic acid for 2 h. The
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aldehydes generated by the initial oxidation were reduced to primary alcohols with sodium
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borohydride. After saponification (KOH) the PAS technique was applied (Reid et al.,

1973).
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2.4.5 KOH/PA*/Bh/PAS (saponification-selective periodic acid-reduction with

borohydride-periodic acid-Schiff’s reagent). This technique allows the identification of

A

neutral sugars. Sections were treated with 0.5 % potassium hydroxide in 70 % ethanol for

15 min at ambient temperature. Before issuing the PAS technique a selective periodic

oxidation at 4ºC during 1 h followed by a reduction with sodium borohydride was

performed (Volz et al., 1987).

8
 2.4.6 Alcian Blue (AB). AB solutions at different pHs were used to selectively

differentiate color subgroups of acid mucins. Glycoconjugates with carboxylic groups and

O-sulphated esters became evident with a pH 2.8 solution, while sulpho-mucins and highly

sulphated GCs were identified with pH 1.0 and 0.5 solutions, respectively (Lev and Spicer,

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1964).

2.4.7 AB/ PAS (Alcian Blue/ Periodic Acid-Schiff’s Reagent). This combined technique

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allows the identification of acid (AB positive), neutral (PAS-positive) and mixed (AB/PAS

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positive) GCs in the same cut. Sections were exposed to the AB technique followed by the

PAS technique. The AB solution was used at pHs 2.8 and 1.0 to identify carboxylated and

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sulphated GCs, and GCs with O-sulphated esters, respectively (Mowry, 1963).

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2.4.8 Toluidine Blue (TB). The TB solution was used at pHs 5.6 and 4.2 to identify both
A
carboxylated and sulphated GCs, and O-sulphated esters CGs, respectively. In both cases,
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the CGs showing high concentrations of anionic groups stain red-purple (metachromatia);

in the absence of polyanions they stain blue (orthochromasia) (Lison, 1953).

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Results were evaluated by independent observers at a semi-quantitative scale to

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determine the intensity of the reactions (0, negative; 1, light; 2, moderate; 3, strong). This
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classification was established according to previous histochemical studies (Tano de la Hoz

et al., 2014; 2017).

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2.5 Lectin histochemical Study

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A battery of seven biotinylated lectins (Vector Laboratories, Inc. Burlingame, CA, USA)

was used to identify specific sugar residues (Table 1). Paraffin sections were mounted on

slides treated with poly-L-lysine (Sigma Diagnostics, St Louis, MO, USA), deparaffinized

with xylol and incubated in a 0.3% H2O2 solution in methanol for 30 min at room
9
temperature. Then, sections were hydrated, washed with 0.01 M PBS, pH 7.6 and incubated

with a bovine serum albumin in PBS for 20 min. Next, they were independently incubated

with each of the biotinylated lectins for 30 min at room temperature and treated with the

avidine-biotine-peroxidase complex (ABC) during 45 min (Vector Laboratories, Inc). The

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peroxidase complex was activated after 4-10 min incubation with a Tris-HCl 0.05 M, pH

7.6 buffered solution containing 0.02% diaminobenzidine (DAB) (Dako, Carpinteria, CA,

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EE.UU.) and 0.05% H2O2. All lectins were used at a concentration of 30 mg ml-1 in PBS

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dilution, except for PNA that was prepared as 10 mg ml-1.

A semi-quantitative evaluation of the results using the same scale described at 2.4 was

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performed. Two types of controls were used: (1) the lectin solution replaced by PBS and (2)

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lectins pre-incubated for 1 h at room temperature in the presence of the appropriated
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blocking sugars.
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3. Results
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3.1 Morphological and ultrastructural characteristics

The histological structure of the descending colon and rectum of L. maximus showed the
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four typical tunica of the digestive tube. From the luminal surface to the exterior, mucosa,
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submucosa, muscular and serosa tunica were identified. The most conspicuous

characteristic of the descending colon and rectum was the large quantity of folds in the
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wall, formed by a submucosa center, which gives an irregular aspect to the intestinal lumen

of both organs (Fig. 1A, B).

The ultrastructural study allowed the characterization of absorptive and goblet cells from

the intestinal epithelium (Fig. 2). The absorptive cells of both anatomical regions presented

10
a well-defined morphological polarity through the display of microvilli in their apical

surface and junctional complexes in the lateral region of the membrane (Fig. 2A, C). All the

enterocytes presented an electron-lucid cytoplasm and displayed an euchromatic nucleus

and cisterns from the rough endoplasmic reticulum in the basal third. Mitochondria were

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located in the apical cytoplasm between the terminal veil and the nucleus (Fig. 2A-C).

Goblet cells were distributed all along the crypt axis, although they predominated in the

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deep portion of the intestinal gland (Fig. 2D, E). These unicellular glands showed

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microvilli and abundant electron-lucid mucinogen granules concentrated in the apical

cytoplasm.

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3.3 Histochemical study N
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All goblet cells from the descending colon showed carboxylated and sulphated GCs
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(Figs. 3A, B). Even though, neutral secretion cells were identified through the AB pH

2.8/PAS method, mucins of most goblet cells exhibited an acid component (AB positive)
D

(Fig. 3C). Cells of acid and mixed secretions were identified in the middle and inferior
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region of the Lieberkühn crypts, while positive PAS cells were found only in the upper
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third of the intestinal glands (Fig. 3C). The KOH/PA*/Bh/PAS technique also allowed to

corroborate that the pattern of neutral mucins varies along the intestinal crypt axis, being
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the cells of the upper region of the glands those which presented a more intense reaction

(Fig. 3D). The AT method at both pH values revealed the presence of metachromatic goblet
A

cells all along the intestinal gland axis (Fig. 3E, F). Also, scarce sialic acid residues slightly

O-acetylated were identified in the goblet cells of this anatomical region (Fig. 3G, H).

The glycosylation pattern of the rectum was similar to that of the descending colon (Fig.

4A-E); the main difference lay in the fact that the most distal region of the large intestine
11
showed a greater proportion of sialomucins, characterized by being slightly O-acetylated

(Fig. 4F). The glycocalyx of both anatomical regions showed a moderate reaction with the

AB pHs 2.8 and 0.5 techniques. Therefore, it presents carboxylated GCs and sulphomucins

(Figs. 3A-C and 4B-D).

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The histochemical profile of the descending colon and rectum are shown in Tables 2 and

3, respectively. Differences between sexes and capture time were not observed.

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3.4 Lectin histochemical study

The lectin histochemical method evidenced different sugar residues present in the

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glycocalyx, enterocytes and goblet cells from the descending colon and rectum of L.

maximus (Table 4). N

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In both anatomical regions, the glycocalyx presented an intense positive reaction with
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lectins WGA, RCA-I and PNA, while it exhibited no positive staining with UEA-I (Fig.5

A-H). On the contrary, the affinity of Con-A, DBA and SBA varied according to the
D

studied intestinal region. Lectins DBA and SBA showed affinity only for the descending
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colon glycocalyx (Fig. 6A-D), whereas Con-A marked the rectum glycocalyx more
EP

intensely (Fig. 6E, F).

The enterocytes showed a similar glycosylation pattern in both anatomical sectors. The
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most intense marking was detected with lectins WGA and SBA in the supranuclear region

of the cytoplasm (Figs. 5b and 6a).

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Goblet cells of both intestinal regions did not stain with lectins Con-A, DBA and UEA-I.

Therefore, their secreting mucins lack the terminal α-mannose, α-glucose, N-acetyl

galactosamine and L-fucose residues (Figs. 5G, H and 6C-F). Except for WGA, the

remaining lectins demonstrated that the lectin histochemical profile of goblet cells varies
12
according to the intestinal region under study. The main differences encountered between

both anatomical regions were detected in the β-D-N acetylgalactosamine and β-galactose

residues (Figs. 5C, D and 6A, B).

Lectin labelling was completely inhibited when the lectins were omitted from the

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incubation medium or when they were pre-incubated with the appropriate hapten sugar

(Figs. 6G, H). Differences between sexes and capture time were not observed.

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4. Discussion

4.1 Glycosylation pattern of intestinal mucins

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The morphology and ultrastructure of the small and large intestines have been studied in

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several mammalian species (Hosoyamada and Sakai, 2007, Zanuzzi et al., 2008, Hansen et
A
al., 2009, Mantani et al., 2014, Vásquez Cachay et al., 2014). In these studies, it was
M

demonstrated that the epithelium has highly specialized cells, a fact that is related to the

multiple digestive, absorptive, endocrine and immunological functions of the intestinal

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mucosa.
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In L. maximus, as in other vertebrates, the absorptive cells or enterocytes exhibited a

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marked cellular polarity, since they presented uniform microvilli in the apical region and

junctional complexes and interdigitated cytoplasmic processes on their lateral surfaces.

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This shows that, as suggested by Takashima et al., (2013), the membrane specializations

present in this cell type are highly conserved and are associated with absorption and
A

transport processes.

Comparing the ultrastructural characteristics of the epithelial cells of the small and large

intestines of L. maximus, the main differences were observed in the enterocytes. In some

sectors of the small intestine two morphological types of enterocytes were described that

13
differed mainly in the electron-density of their cytoplasm (Tano de la Hoz et al., 2016). In

contrast, in the descending and rectum colon only absorptive cells with electron-lucid

cytoplasm were identified. Although in mammals different types of absorptive cells have

not been identified at the ultrastructural level, in humans, molecular studies have described

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two types of enterocytes in the small intestine that were classified as non-absorptive and

absorptive enterocytes (Gassler et al., 2006). Although future studies are required, the

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results described at the small intestine of L. maximus could offer ultrastructural support to

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the molecular characterization carried out by Gassler et al. (2006). Therefore,

morphological differences found between the enterocytes in the present study could be

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indicative of the existence of cells with different physiological states in the small intestine,

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while in the large intestine of L. maximus this cell type would only fulfill absorptive
A
functions.
M

The intestinal mucins are synthesized and secreted mainly by unicellular epithelial

glands called goblet cells. For this reason, studies carried out in recent years have focused
D

on the knowledge of their morphology, function, distribution and glycosylation patterns

TE

(McDole et al., 2012). As in all mammals, the goblet cells of L. maximus were distributed
EP

throughout the epithelium of the intestinal tract, their number being greater in the large

intestine (Boonzaier et al., 2013; Tano de la Hoz et al., 2014; 2016). This observation using
CC

TEM confirmed the presence of a large accumulation of mucus granules in the apical

cytoplasm, which distends this region of the cell. The presence of abundant mucus granules
A

is an ultrastructural characteristic that is currently used as a criterion to identify cells in a

mature secretory stage (Birchenough et al., 2015). In the large intestine of L. maximus,

mature goblet cells in the lower third of Lieberkühn's glands were characterized by

histochemical and TEM techniques. These observations corroborate that goblet cells of this
14
region of the intestine of L. maximus begin to mature in the area of cellular replication, as is

observed in other vertebrates.

The glycoconjugate analysis demonstrated that the histochemical profile of goblet cells

varies between the descending colon and the rectum of L. maximus, and that it remains

PT
constant in all the studied individuals. These results agree with previous studies describing

gradual variations along the small intestine of L. maximus, and even abrupt changes at the

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level of the colonic groove of the ascending colon were identified (Tano de la Hoz et al.,

SC
2014; 2016; 2017). Altogether, these results demonstrate that each anatomical region of the

intestinal tract of L. maximus shows a particular glycosylation pattern that in turn stays

U
highly preserved between members of the same species. Histochemical and proteomic

N
studies have described similar results for diverse mammal species (Robbe et al., 2004;
A
Boonzaier et al., 2013). Since the histochemical profile of intestinal mucins does not vary
M

among individuals of the same species, some authors have recently proposed that such a

pattern can act as a selection mechanism of the intestinal microbiota (Holmén Larsson et
D

al., 2009; Hansson, 2012). In this way, the glycosylation pattern of the intestinal mucins
TE

would probably be the result of a co-evolutive process between the bacterial flora and the
EP

host in order to maintain an optimal symbiotic relation (Johansson et al., 2011a; 2011b),

being, in turn, a system susceptible to alterations according to age, diet and certain
CC

physiological and pathological states (Beyaz and Liman, 2009; Liquori et al., 2012;

Mastrodonato et al., 2014).

A

As in previous histochemical studies, the AB pH 2.8/PAS technique allowed to evaluate

the existence of goblet cells with different glycosylation patterns (Tano de la Hoz et al.,

2014; 2016; 2017). Thus, in both sectors of the large intestine of L. maximus, cells with

three different histochemical profiles – neutral, acid and mixed– were identified, those with
15
acid secretions being in the largest proportion. These results, together with previous studies,

demonstrate that along the intestinal tract of L. maximus an increasing gradient of

sulphomucins exists, associated to a decreasing gradient of mucins with oxidizable vicinal

diols (Tano de la Hoz et al., 2014; 2016; 2017). Studies on different mammal species have

PT
described a glycosylation pattern similar to that of L. maximus. These researches agree that

there is a predominance of acid mucins, sulphated as well as carboxylated, in the different

RI
large intestine segments (Robbe et al., 2004; Boonzaier et al., 2013). On the other hand, the

SC
existence of goblet cells with different glycosylation patterns demonstrates that there are

functionally different subclasses of goblet cells within a morphologically homogeneous cell

U
population, both at histological and ultrastructural levels. These subpopulations of goblet

N
cells have also been described in the human and rat colonic epithelium by using antibodies
A
against diverse types of intestinal mucins (Podolsky et al., 1986; Gouyer et al., 2011).
M

Since the glycosylation pattern of the intestinal tract acts as a dynamic system able to

adapt to local physiological requirements, including changes in the intestinal microflora

D

(Moran et al., 2011; Pelaseyed et al., 2014), the sulphomucin gradient found along the
TE

intestinal tract of L. maximus would probably be linked to differences in the microbial

EP

charge between the small and large intestine. In this way, these results would also agree

with other studies on rodents, where it was demonstrated that the microflora modulates
CC

specifically the intestinal glycosylation pattern, both at the cellular and subcellular levels

(Freitas et al., 2002; Moran et al., 2011).

A

In mammals, the negative charge of mucins is chiefly determined by sulphate groups

and terminal sialic acid residues (Liquori et al., 2012; Mastrodonato et al., 2014). The high

density of negative charges in intestinal mucins attracts water, thus forming a highly

hydrated gel with viscoelastic properties (Accili et al., 2008). Sialomucins have been
16
identified in the descending colon as well as in the rectum of L. maximus, although in

smaller proportion than sulphomucins. Similar results were described for the mice intestinal

tract by Mastrodonato et al. (2013). Studies on humans instead have reported intestinal

mucins highly sialylated, with little sulphated residues (Robbe et al., 2004). Unlike the

PT
pattern described in humans, our results indicate that the main groups responsible of

determining the acid gradient of mucus along the intestinal tract are those of sulphates.

RI
The lectin histochemical technique has been applied to in situ locate and identify

SC
different terminal and subterminal residues of monosaccharide sugars present in the GCs of

diverse species (Scillitani et al., 2007; Fayed et al., 2010; Mastrodonato et al., 2014). This

U
technique allowed us to characterize the lectin histochemical pattern of the glycocalyx, the

enterocytes, goblet cells and ICC of L. maximus. N

A
In the present study, the goblet cells showed a negative reaction with lectins DBA and
M

UEA-I, regardless of the anatomical region taken into account, which indicates that both

regions lack α-N-acetylgalactosamine and L-fucose terminal residues. Instead, the affinity
D

of the rest of the lectins employed by these cells varied considerably according to the
TE

intestinal segment. In agreement with our results, Galotta et al. (2009) demonstrated that
EP

the lectinhistochemical pattern of goblet cells showed a large variation depending on the

intestinal region studied.

CC

In mammals, the presence of N-acetylglucosamine (GlcNAc) is particularly associated

with the acidification degree of mucins, for the sulphate groups are generally linked to this
A

monosaccharide (Liquori et al., 2012). Lectin WGA revealed terminal residues in goblet

cells in the entire intestinal tract of L. maximus, having the ascending colon and the rectum

the greatest marking intensity (Tano de la Hoz et al., 2014; 1016; 2017). In this manner, in

17
the present study a correspondence between the acid gradient of secreting mucins and the

distribution of N-acetylglucosamine terminal residues was found.

The secreting function of the enterocytes consists of the synthesis of glycoprotein

enzymes that will be inserted in the apical cell membrane. Biosynthesis of glycan chains

PT
mainly occur in compartments of the rough endoplasmic reticulum - Golgi in reactions

implying the sequential activity of glycosidases and glycosyltransferases (Moran et al.,

RI
2011). In the present study, the enterocytes of most of the intestinal sectors evidenced a

SC
positive supranuclear marking with some of the used lectins. As demonstrated in other

lectin histochemical studies, it is probable that this marking pattern be indicative of the

U
glycosylation process undergone by CGs within the endomembrane system (Gabrielli and

N
Tomassoni, 2018). As a morphological correlate, in these absorptive cells, Golgi cisternae
A
in the supranuclear region and a well-developed rough endoplasmic reticulum were
M

observed.
D

Declarations of interest
TE

None.
EP

Acknowledgments
CC

This research was supported by a Grant from Universidad Nacional de Mar del Plata

(EXA 765/16), Buenos Aires, Argentina. We would like to thank to the staff of Estación de
A

Cría de Animales Silvestres (ECAS), Ministerio de Agroindustria de la Provincia de

Buenos Aires.

References
18
Accili, D., Menghi, G., Gabrielli, M.G., 2008. Lectin histochemistry for in situ profiling of

rat colon sialoglycoconjugates. Histol. Histopathol. 23, 863–875.

Beyaz, F., Liman, N., 2009. The Pprenatal Ddevelopment and Hhistochemistry of the Iileal

Mmucins in the Bbovine Ffetuses. Anat. Histol. Embryol. 38, 436–442.

PT
Birchenough, G.M.H., Johansson, M.E.V., Gustafsson, J.K., Bergström, J.H., Hansson,

G.C., 2015. New developments in goblet cell mucus secretion and function. Mucosal

RI
Immunol. 8, 712–719.

SC
Boonzaier, J., Van der Merwe, E.L., Bennett, N.C., Kotzé, S.H., 2013. A comparative

histochemical study of the distribution of mucins in the gastrointestinal tracts of three

U
insectivorous mammals. Acta Histochem. 115, 549–556.

N
Clauss, M., Besselmann, D., Schwarm, A., Ortmann, S., Hatt, J.M., 2007. Demonstrating
A
coprophagy with passage markers? The example of the plains viscacha (Lagostomus
M

maximus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 147(2), 453–459.

Culling, C.F.A., Reid, P.E., Dunn, W.L., 1976. A new histochemical method for the
D

identification and visualization of both side-chain acylated and non-acylated sialic acids.
TE

J Histochem Cytochem. 24, 1225–1230.

EP

Fayed, M.H., Elnasharty, M., Shoaib, M., 2010. Localization of sugar residues in the

stomach of three species of monkeys (Tupaiidae glis, Nycticebus cocang and Callithrix
CC

jacchus) by lectin histochemistry. Int. J Morphol. 28, 111–120.

Freitas, M., Axelsson, L.G., Cayuela, C., Midtvedt, T., Trugnan, G., 2002. Microbial–host
A

interactions specifically control the glycosylation pattern in intestinal mouse mucosa.

Histochem. Cell Biol. 118, 149–161.

Gabrielli, M.G., Tomassoni, D., 2018. Starch-enriched diet modulates the glucidic profile

in the rat colonic mucosa. Eur Jour Nutr, 57(3), 1109–1121.

19
Another random document with
no related content on Scribd:
 Mutta niinkauvan, kun hän jaksoi istua, sai rohdin kuontalokin
kulkea hänen koukistuneitten sormiensa läpi rullalle ja sitä tietä ne
menivät pappilan ruustinnan karvamattolangatkin. —

Pahemmin ei voinut kukaan loukata Miinaa, kun puhumalla

hänelle vaivaishoidon avusta. —

"Häpeäisit!" sanoi hän vihaisesti.

"Ota itse, jos haluat, — mie ennen vaikka kuolen…"

Ja niin kai hän olisi tehnytkin, säästöjensä loputtua, jos ei

tyttärensä olisi saanut häntä houkutelluksi luokseen, nähdessään
että vanhuksen oli mahdoton tulla toimeen yksinäisessä
tupasessaan.

Mutta ei hän sieltä sittenkään aivan mielisuosiolla lähtenyt. —

Pettymyksiä.

Kaarlo heitti pallon huimaavan korkealle ilmaan.

Suuren lapsijoukon käsivarret kohosivat kuin jännitetyt jouset, ne

tempoilivat ja värähtelivät malttamattomuudesta sillä itsekukin oli
mielestään juuri se, jonka käsiin pallon oli määrä pudota… Ja
huudot: minä, minä, minä, raikuivat kilvan puutarhan nurmikolla!

Pojat, jotka luonto on varustanut vahvemmilla käsivarsilla,

rynnistivät etualalle, työntäen kilpailussa heikommat tytöt syrjään.
Mutta yksi niistä — pienin ehkä — vilkas ja vikkelä kuin orava,
pujottausi vikkelästi joukon läpi, ennätti edelle ja ojensi itsepäisesti
pienet, valkoisen pitsin verhoamat käsivartensa ylös, kohti putoavaa
palloa, joka kiisi kuin nuoli suoraan ja empimättä pieniin, pehmoisiin
tytön käsiin.

"Minä!" kajahti riemuhuuto lapsen suusta ja ylpeänä voitostaan

keikautti hän valkohiuksista päätänsä.

Pojat katselivat vähän nyrpeissään sivu suun mennyttä voittoa ja

sieltä täältä kuului ääniä, jotka sanoivat:

"Eihän se Elli olisi voittanut, jos —"

"Eikähän se Elli oikeastaan olisi saanut —" mutta lauseitten

loppuosat haipuivat jostakin syystä niin hiljaisiksi että ainoastaan
vierustoveri saattoi kuulla ne.

Vaan kun Ellin suurien silmien voitonriemu kävi aivan

sietämättömäksi, sanoi suurin pojista kuuluvammalla äänellä: "Eikä
tämä peli oikeastaan tytöille ollutkaan!"

Kun pallopeli alkoi ikävystyttää, heittäytyivät tytöt piiritanssiin

pihamaalle ja pojat lähtivät kiipeilemään leveitä tikapuita ylös
rakennuksen katolle.

"Hei vaan, kuinka kauvas täällä näkee!" huusi kehoittavasti se,

joka ensinnä perille ehti ja sen kuultuaan nekin jotka vielä tikapuitten
juurella seisoivat kapusivat kilvassa ylös. "Ai ihmettä!" kuului
reippaasti ylhäältä.

"Kuinka kaunista!" — "Kuinka hauskaa!" — "Melkein päätä

pyörryttää!" Pääskysparven lailla viserteli poikalauma räystäällä ja
uteliaina, ihaillen korottivat tytöt kaulojansa, silmääräpäyttämättä
seuraten toverien liikkeitä.

"Kiivetkäämme mekin sinne! Yks', kaks', kolme!" huudahti Elli ja

silmänräpäyksessä oli hän jo puolitiessä portailla.

"Ei, ei Elli!" huusivat silloin isä, äiti ja kaikki muut aikaihmiset

verannalta, kauhistunein liikkein vaatien yltiöpäätä heti
laskeutumaan alas. Mutta yritteliä vitkasteli… Siitäkin — puolitiestä
— näki hän jo laajenneen, loistavan maiseman, joka tältä
korkeammalta asteelta katsottuna näytti tuhat kertaa
ihmeellisemmältä, viehättävämmältä, kun esimerkiksi verannan
kaidepuun takaa, taikkapa kellarin katoltakin, — mihinkä joskus
luvallakin oli saanut kiivetä.

"Tule alas! Kuuletko, hetipaikalla alas, sinä pahankurinen lapsi!" Ja

pahankurinen lapsi totteli, tullen alas vaikka verkkaan ja nyrpeän
näköisenä.

"Miks'en minä saa mennä sinne, yhtähyvin kun pojatkin?" nurkui

hän.

"Siksi että se ei sovi tytöille — muista se!" —

Elli painui verannan alimmalle rappuselle istumaan. Äsken niin

iloinen, toiminnanhaluinen lapsensielu oli sumennettu ja
sulkeutuneena mietti se arvoitusta: miksi en minä yhtähyvin kun
pojatkin…

*****

"Se on siis päätetty", sanoi isä tyytyväisesti. "Ja minä toivon

kaikesta sydämestäni, että menestyt lääkäriurallasi Kaarloseni! —
Mutta entäs Esko? On jo aika sinunkin vakiintua siitä, miksi aijot
maailmassa."

"Siitä olen jo kauvan ollut selvillä, isä. Kun poikasena kerran tein
huvihuoneen piirustukset, sanoit sinä että näyttää siltä kun Eskolla
olisi tulevaisuutta tällä alalla ja sitä minulla varmasti onkin! Saat
uskoa, isä, että minä kerran vien arkkitehtuuriamme loistavan
askeleen eteenpäin."

Isä hymyili. "Hyvä, olen siis selvillä ja tyytyväinen lasteni

tulevaisuudesta."

"Mutta minuthan sinä aivan syrjäytät, isä", muistutti Elli "vai eikö
sinua ollenkaan huvita tietää miksi minä aijon?

"Ah sinä! Noo — sinusta kaiketi tulee kunnianarvoisa pikku rouva,

kunhan ennättää — ellet juuri vanhaksi piiaksi aijo", nauroi isä.

Ellin raikkaat kasvot peittyivät synkkiin pilviin ja silmissään loukattu

ihmisarvo salamoi…

"Niin, tuomari Arvolahan on sinuun pahoin pikeytynyt. Ja,

luullakseni, on hänen kainalossaan tulevaisuutesi turvattu", täydensi
Kaarlo.

"Oi, kuinka te olette ilkeitä!" huudahti Elli. "Sekö se siis on tyttöjen

päämäärä ja siihenkö heillä vaan on lupa pyrkiä? 'Tulevaisuus
turvattu'… on se sekin yks' rääsyinen sana! Ikäänkuin meillä ei olisi
oikeutta, niinhyvin kuin velvollisuuttakin, itse, omintakeisesti turvata
tulevaisuuttamme!"

"Sillä tavallahan se asia kuitenkin tavallisesti tässä Jumalan

luomassa maailmassa menee —"
 "Tässä miesten muokkaamassa maailmassa — niin olemme
epäilemättä lähempänä totuutta. Vai luuletteko, uskallatteko luulla
Jumalasta jotain niin alhaista, äärimmäisen ilkeämielistä, että hän
olisi sysännyt naisen siihen alennustilaan, missä tämä vuosituhansia
on saanut värjötellä?"

"Elli, Elli — elä pidä noin suurta suuta. Ehkäpä ne vuosituhannet

juuri kumoavat väitteesi —"

"Mahdollisesti, jos naisasia olisi vieläkin siinä pisteessä, missä se

Aabrahamin aikoina oli — mutta Jumalan kiitos, ne samaiset
vuostuhannet ovat kuitenkin olleet kehityksen, vapautuksen aikoja ja
kieltämättä me olemme matkalla oikeutta, täydellistä tasa-arvoa
kohden, olkoonpa tämä matkamme viimeinen virstapylväs vielä
kuinka kaukana tahansa."

"Sinä olet oikea naisasian agitaattori, Elli!" pisti Kaarlo väliin,

hymyillen ainaista ylimielistä hymyänsä.

"Minulla on kunnia olla! Mutta elä sinä yhtään tuolla tavalla

hymyile… Kuuleppas, se aika on onneksi mennyt, jolloin naisasialle
voitiin vaan hymyillä ja etäällä, etäällä — vaikka tulevassa — on
sekin, jolloin sille enään vaan tarvitaan hymyillä. Nyt on toiminnan,
työn ja taistelun aika!"

"Kiitä Luojaa, lapseni, että elät ajassa, joka sallii sinun nauttia työsi
hedelmiä", lausui äiti. "Minun nuoruudessani saimme tyytyä — ah
emmehän kaikkea uskaltaneet uneksiakaan — mitä te nyt saatte
todellisuudessa omistaa ja nauttia…"

"Ja kuitenkaan, äiti, ei vielä meilläkään ole läheskään kaikkea,

mitä pitäisi olla! — No niin, isä, minä olen päättänyt lukea lääkäriksi
kuten Kaarlokin." —

"Vai päättänyt? Sehän kuuluu erittäin juhlalliselta… Mutta minulta

ei totisesti riitä rahoja siihen, — saan luvan ilmoittaa."

"Vai niin, isä rukka! Olet siis ikuisessa kiitollisuuden velassa

kohtalolle, joka salli kolmannen lapsesi syntyä tytöksi, säästääkseen
kukkaroasi ja estääkseen sen julmuuden tapahtumasta, mikä olisi
ollut välttämätön, jos se kolmaskin olisi ollut poika!"

Isä näytti harmistuneelta, eikä löytänyt pitkään aikaan sanoja

vastineeksi.

"Et sinä toki henno särkeä Ellin tulevaisuutta", sanoi viimein äiti
pyytävän hellästi niinkuin ainakin.

"Te naiset olette niin hiton itsepäisiä toisinaan…"

"Ei isä, kyllä sinulta pitää riittää rahoja Ellillekin, Kaarlon ja minun
on elettävä sitä myöten. Muuten minäkin olen aivan onneton",
lämpeni Esko. "Sehän olisi muuten samaa, kuin jos riistettäisiin Elliltä
vaatteet meille pojille — ja hän työnnettäisiin alastomana pakkaseen!
Se ei saa tapahtua, isä!" —

"Voi tuota Eskoa, sehän on itse olennoitu oikeus ja hyvyys!"

riemuitsi Elli. — "Enkös olekin aina sanonut, että Esko se on, joka
meidän kotimme piirissä edustaa tulevaisuuden herttaista, ylevää,
täydellistynyttä miestä. Tuollaisesta veljestä kannattaa ylpeilläkin!"

Mutta isä vääntelihe nyrpeänä tuolissaan. Hän muistutti suurta

mehiläistä, joka on lentänyt kärpäspaperille ja koettaa turhaan
irroittautua siitä…
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