Molecular and CellularBiochemistry 103: 15-21, 1991. © 1991Kluwer Academic Publishers. Printedin the Netherlands.

Sex-related differences in the expression of chick enterocyte butyrylcholinesterase during embryonic and post-hatching development Jean-Pierre Sine 1, Raymond Ferrand 2 and Bernard Colas 1 1Laboratoire de Biochimie II, Centre de Recherche de Biologie et Physico-Chimie Cellulaires and 2 Laboratoire de Biologie du D&eloppement, Facult~ des Sciences, 44072 Nantes C~dex 03, France Received30 May 1990; accepted15 October 1990

Key words: cholinesterase, butyrylcholinesterase, mucosal cell, enterocyte, sex-related, chicken intestine

Summary

The butyrylcholinesterase activity of chick enterocytes was studied from day 15 in ovo up to day 90 after hatching. The activities detected in both sexes at the level of jejuno-ileum change in a parallel manner, but the activity is always higher in the female than in the male during embryonic development. After hatching, the differences are less apparent although the study of the enzyme distribution along the intestine showed sex-related variations, mainly at the level of the anterior and middle parts of jejuno-ileum in the young adult. Analysis of butyrylcholinesterase by sucrose gradient centrifugation allowed to identify two globular soluble species (G1 and G4 forms). The G4/(G1 + G4) ratio decreases during the development but this variation in the female does not parallel that observed in the male. Besides, the molecular form distribution along the intestine, studied after hatching, differs according to the sex. Taken together our results lead to hypothesize that the ontogeny and the regulation of the chick enterocyte butyrylcholinesterase depend on hormones. Abbreviations: AChE - Acetylcholinesterase, BuChE - Butyrylcholinesterase

Introduction

Works have shown that the differentiation of the chicken gut, including various enzyme accumulation, depends on hormones [1-4]. The study of the duodenum from hypophysectomized chick embryo, performed by Bellware and Betz [5], led these authors to conclude thatpars distalis hormones probably affect duodenal differentiation. The question arises whether the cholinesterase activity that we have detected in the enterocytes of different vertebrates including mammal and avian species [6-9] is regulated by hormones. Such a process has largely been demonstrated for cholinesterases, essentially in the case of rat liver and serum [10-13]. The cholinesterase activity is modified by castra-

tion [14], adrenalectomy [15] and thyroidectomy [10]. The ontogeny and endocrine regulation of serum cholinesterase appear to be mediated via the hypothalamic-pituitary-gonadal axis [13] and this activity is markedly influenced by androgens and estrogens [16, 17]. As sex-related differences in rat cholinesterase activity levels were reported in the case of serum [11, 12], plasma [18], cervical ganglion [12] and septohippocampal system [19], we investigated whether such differences also exist in the chick intestine. Here we report a comparative study of the butyrylcholinesterase (BuChE) activity and molecular form distribution in the enterocytes of male and female chicks at different embryonic and posthatching stages.

16 Experimental procedures

Animals The eggs, from commercial origin, were incubated at 38 ° C. The experiments were carried out on male and female chick embryos (Gallus gallus, strain JA 657) from day 15 to hatching, and on male and female chickens from hatching to day 90. Rearing was performed in the laboratory poultry-house from hatching till sacrifice.

Chemicals Acetylthiocholine iodide, aprotinin, bacitracin, benzamidine hydrochloride, BW284C51 [1,5,-bis(4 allyl dimethylammonium phenyl) pentan-3-one dibromide], DTNB [5-5' dithio bis-(2-nitrobenzoic acid)], eserine and standard proteins were obtained from Sigma Chemical Co. Triton X-100 was from Aldrich. All other chemicals were of analytical grade.

Removal of the enterocytes The animals were killed by decapitation. The jejuno-ileum was rapidly removed, longitudinally opened and washed with an ice-cooled 10 mM potassium phosphate buffer (pH7.4) containing 150 mM NaC1 and a mixture of protease inhibitors (0.7mM bacitracin, l mM benzamidine and 25 units/ml aprotinin). The mucosal cells were gently scraped off, either using small razor blades under a dissection microscope for the chick embryos and the chickens up to 2 weeks, or using glass slides for older chickens. In the case of embryos and young chickens, the scrapings obtained from 2 or 3 animals were pooled. In addition, for the distribution studies performed after hatching, the whole intestine was excised and the scrapings were performed at the level of the following segments: duodenum, anterior, middle and posterior parts of jejuno-ileum, caeca and rectum. The mucosal cells, corresponding to each segment, were also pooled from 2 or 3 chickens. Homogenization and soluble extract preparations were carried out as described previously [9].

Enzyme assays Cholinesterase activity was measured according to

the colorimetric method of Ellman et al. [20] at 25°C in a 10mM potassium phosphate buffer (pH 7.4) containing 150 mM NaC1. BuChE activity was determined using acetylthiocholine in the presence of 10-5 M BW284C51 (an inhibitor specific for acetylcholinesterase). In all cases, control assays were performed with 10-4 M eserine. The enzymatic unit was defined as the amount of protein which catalyzes the hydrolysis of 1/~mol of substrate/h. Protein concentration was determined according to Lowry et al. [21] using bovine serum albumin as a standard.

Sucrose gradient centrifugation Sedimentation analyses were performed in 2-20% (W/V) linear sucrose gradients prepared with a 10 mM potassium phosphate buffer (pH 7.4) containing 150 mM NaC1. Soluble extracts from mucosal cell homogenates were mixed with sedimentation markers (alkaline phosphatase, 6.1 S; catalase, 11.3S and ~-galactosidase, 16S). The resulting samples were carefully layered onto gradients and centrifuged for 15 h at 119,000 g and 4° C in a Beckman SW 40 Ti rotor. After centrifugation, the gradients were fractionated from the top and fractions of 0.250 ml were collected.

Statistical analysis Results, expressed as mean _ standard deviation, were analyzed by Student's t-test and were considered to be statistically significant when p < 0.05.

Results

Butyrylcholinesterase activity during development In the intestine mucosal cells of male and female chickens, the cholinesterase activity is essentially due to a BuChE (EC 3.1.1.8.) as demonstrated using specific inhibitors [9]. The level of the activity during the development is shown in Fig. 1. As reported previously [22] in the case of the male, during the studied embryonic period, the specific activity peaks at day 19. Then the activity lowers up to day 4 after hatching and again raises, reaching a second maximum at about 2 weeks. In older chicken, the activity slowly decreases, levelling off to the

17

Hatching Z)') 2,5 l -~--I

>

2.0

-r--I

...#

0

1.5

0 LIJ 1,o

JE (.3 D m

o.s

I

-6

I

I

I

-2 0

I

I

I

I

4

8

11,-

//

I

I

I

30

45

90

Dags Fig. 1. Changesin BuChEactivityof male (O--O) and female(O--D) chickenterocytesduringthe developmentfromday 15 in ovo (- 6) up to day90post-hatching.The enterocyteswerescrapedofffromthe wholejejuno-ileum.BuChEactivityis expressedas ~molof substrate hydolyzed/h/mgprotein. The valuesrepresentthe means+ standarddeviationof assaysperformedin duplicatefromthreeto five samples. Femaleactivitydifferssignificantly('Jr) frommale activityat the indicatedstages (p < 0.05). value observed in the adult. In the case of the female, the activity profile parallels that observed in the male. However, an additional peak is observed at day 45. During the embryonic period and around day 45 after hatching the activity is higher in the female than in the male (p < 0.05). By contrast, from hatching up to day 30, the female activity does not significantly differ from that of the male. The same study performed in the intestine mucosal cells of the quail shows that the BuChE activity is also higher in the female than in the male during the studied embryonic period (p < 0.05).

Butyrylcholinesterase activity along the intestine As indicated in 'Experimental procedures', the distribution of BuChE activity along the intestine was only studied from hatching onwards when the scrapings of the different intestinal segments could be performed independently. The highest specific activity was found in the middle part of jejunoileum for both male and female (Fig. 2). At day 4, the sex-related differences remain discrete whatever the segment studied. By contrast, at day 45,

the female exhibits a higher specific activity than that detected in the male, essentially at the level of the anterior and middle parts of jejuno-ileum. No significant difference in activity was observed between sexes in the other segments of the intestine (duodenum, posterior jejuno-ileum, caecum and rectum).

Identification of molecular forms The study of BuChE forms carried out from jejuno-ileum by sucrose gradient centrifugations showed the presence of two globular species corresponding to G1 and G4 forms (Fig. 3) according to the nomenclature proposed by Massouli6 and Bon [23]. Since no shift of sedimentation profiles was observed when the homogenizations and the centrifugations were performed in the presence of 1% Triton X-100, the G1 and G4 globular forms are endogenously soluble. In addition, experiments performed in the presence of 1% Triton X-100 and 1 M NaC1 did not allow to detect any heavy forms corresponding to asymmetric species. In both sexes, the relative amounts of the G1 and

18 0.8 D~0.6 -e--I

>0.4

!

°*--I

-~o.2 0 04.o

I

I

I

I

I

Dag 45

[-~F 3.0 (-~ 2.0 m 1,0 I

Duo

I

I

\RnL

I

Pos/ v J e J u n o - i ]eum Mid

I

Cae

I

Rec

Fig. 2. BuChE activityof male (C)---O) and female (0--0) chick enterocytesalong the intestine at days 4 and 45 after hatching.The studied intestine segments are duodenum (Duo), anterior jejuno-ileum (Ant), middle jejuno-ileum (Mid), posterior jejuno-ileum (Pos), caecum(Cae) and rectum (Rec), BuChE activityis expressedas/xmolof substrate/h/mgprotein. The valuesrepresent the means + standard deviationof assaysperformedin duplicate from four samples. Femaleactivitydifferssignificantly(~') from male activityat the indicated stages (p < 0.05).

G 4 forms change during the chicken development (Fig. 4). The enzymatic activity due to the G4 form is predominant in ovo up to day 19 in the male and during the embryonic period and up to about two weeks after hatching in the female. However, whereas similar levels of G4 activity are present in both sexes at day 17 in ovo (~ 71% for male and 75% for female), this percentage, in the young adult, is much lower in the female than in the male, as shown, for illustration, at day 90 in Fig. 4 ( - 15% and 42% respectively). Distribution of molecular forms A study of the G4 form distribution in the mucosal cells along the intestine was carried out at days 4 and 45 after hatching (Fig. 5). In the posterior jejuno-ileum, the relative amount of the G4 form is identical in both sexes at day 4 (56% of the BuChE total activity) as well as at day 45 (70%). In the other segments, i.e. duodenum, anterior and mid-

die parts of jejuno-ileum, the amount of this molecular species differs according to the sex, it was higher at day 4 and lower at day 45 in the female than in the male.

Discussion

The present results show that, during the development, the BuChE activity found in the chick enterocytes differs according to the sex. This effect is marked during the late embryonic period for which a highest activity was always observed in the female. These sex-related differences seem to disappear in the early post-hatching period. Similar results were obtained in the quail during the embryonic period and after hatching as well. This phenomenom is not modified by the fact that the incubation periods are different in the two species: 21 days for the chick and 16 days for the quail.

19 0.5

hatching

80 70

O.Z 0.3

I

U 5o

0.2

"~ 0 I-

0.1

N ~

4o 3o

,:, iiiiiiil

2o

0 I

I

I

I

I

I

I

b

>"0.3

0

l!i!i i i !ilil --4

> p

--2

O

2

4-

14-

4-5

90

DAYS

0,2

Fig. 4. Changes in the relative amount of the

G 4 form of male (D) and female ([]) chick enterocytes during the development. The amount of each molecular form (G1 and G4) was estimated from sedimentation patterns. The relative amount of the G4 form was calculated as the Gd(G1 + G4) ratio. The values represent the means + standard deviation from three experiments. Differences between male and female are significant(~r) at the indicated stages (p < 0.05).

U 0,1

Ixl I-

U t

~['1 0 . S

I

I

I

I

I

I

I

0.4 0.3 0.2 0.1 0 I

I

I

I

5

10

I

I

I

20

I

30

I

40

FRACTIONS Fig. 3. BuChE forms of male (O--O) and female (@--0) chick enterocytes at day 19 in ovo (A), at days 2 (B) and 45 (C) after

hatching. Centrifugations were performed using soluble extracts obtained from scrapings of the whole jejuno-ileum. Arrows represent the positions of the sedimentation markers. From left to right: alcaline phosphatase (6.1 S), catalase (11.3 S) and ~-galactosidase(16 S).

To our knowledge, no information is available concerning sex-related variations of the enterocyte esterase activities. H o w e v e r , studies p e r f o r m e d on other non-neuronal tissues pointed out that BuChE and/or A C h E exhibit such changes. Plasma BuChE activity of adult female rats was 5.5-fold higher than that of adult male rats [18]. The examination of rat liver [12, 16] and serum [11, 12, 16] showed

that the B u C h E activities were always higher in female than in male. However, it should be noted that sex-related changes in the B u C h E activity are not detected in all tissues. Thus, there is no difference between male and female diaphragm B u C h E in adult rat [12]. Such is also the case in chick and quail intestines during the early post-hatching period. The works about B u C h E characteristics related to sexual differences essentially concern the enzyme activity, the molecular forms being poorly studied. Gel electrophoresis was used to detect the alterations in isozyme patterns of rat serum BuChE. Whereas six bands were identified in the female isozyme pattern, only three bands were obtained in the case of male [11]. Analysis by sucrose gradient centrifugation allowed us to identify two forms, identical for both sexes, in the chick enterocytes. However, the relative amount of these forms, which correspond to soluble globular species (G] and G4), changes during the development. For both sexes, the G4/(G 1 -b G4) ratio decreases during the development period studied. In addition, while this ratio is quite identical at day 17 i n o v o , it obviously differs in the adult, the amount of the G 4

20 80

DAY

70

4

-F

60

T :.:+:.:.:.: z.:.:,:,:+ i !i!i!i~i~i

50 40

¢.:-:.:+: ~ :.:.:.:.:.:. ~ •

::::::::::: !;,?,;?~

I:+:-:.: !:!i!i~i!ii :.: %%. ~,~,° .:.:.:.:..:. :::::::5::::%%* %%'~ :+:+:.:.:

30

iiiiii~iiii:i .:+:+:.:.

i:i:i:J:!:! ~ ?;,,

.:.:.:.:+:.

:.:.:.:.:.: .:.:.:+:, :':':: ~:~..'. ~i! :.:.:,:.:,: ::2:::::: ~**~,*~:

k............., ....... °, ............ ~ ,

20

, •

> 1,1

10