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Proliferation patterns in the canine endometrium during the estrous cycle
Theriogenology 62 (2004) 631–641
Proliferation patterns in the canine endometrium during the estrous cycle S. Van Cruchten*, W. Van den Broeck, M. D’haeseleer, P. Simoens Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 17 July 2003; received in revised form 10 October 2003; accepted 14 November 2003
Abstract The proliferative activity in the endometrium of 58 bitches in different stages of the estrous cycle was assessed by immunohistochemical detection of the Ki-67 proliferation associated nuclear antigen and by counting mitotic figures. The Ki-67 labelling index and the mitotic index were determined in the surface epithelium, the stroma, the crypts and the basal glands by calculating the percentage of Ki-67 positive cells and mitotic figures, respectively, on a total of 500 cells of each category. Endometrial vascular proliferation was also verified by counting the number of Ki-67 positive cells on a total of 100 endothelial cells. The present study showed two proliferation peaks involving different cell groups. In the surface epithelium, the stroma, the blood vessels and the crypts, the highest labelling and mitotic indexes were noticed during proestrus, whereas for the basal glands these indexes significantly increased (P < 0:05) during estrus compared to late metestrus and anestrus. Furthermore, a slightly positive correlation (P < 0:05) was found between the labelling index in the basal glands and the serum progesterone levels, whereas the labelling indexes in the other cell groups were positively correlated with the estradiol-17b levels, although not always significantly. These findings suggest that regulation of the proliferation in the surface epithelium, the stroma, the blood vessels and the crypts is different from the proliferation in the basal glands. # 2004 Elsevier Inc. All rights reserved. Keywords: Ki-67; DAPI; Endometrium; Canine; Estrous cycle
1. Introduction During the estrous cycle, the canine endometrium undergoes manifest morphological and biochemical changes [1–4]. Recent studies have demonstrated the role of steroid hormone receptors [5,6], matrix metalloproteinases [7] and apoptosis [8,9] in the regulation * Corresponding author. Tel.: þ32-9-2647714; fax: þ32-9-2647790. E-mail address: [email protected] (S. Van Cruchten).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.11.015
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of these cyclic endometrial changes. However, in contrast to other species such as man [10], the horse [11], the rabbit [12], the rat [13,14] and the mouse [15,16], proliferation patterns in the cyclic canine endometrium and their relation to the steroid hormone levels remain enigmatic. Barrau et al. investigated the proliferation of the canine endometrial glands during the estrous cycle [4]. In this study two phases of glandular growth were detected, viz. the first at the end of anestrus until the end of proestrus in the crypts and the second during estrus in the basal glands [4]. In contrast, Spanel-Borowski et al., who verified proliferation in different cell groups of the canine uterine body, observed only one peak of proliferation in all cell groups, viz. during early proestrus [17]. However, in the latter study only one sample per cycle stage was investigated and no data of dogs in anestrus were included. Therefore, the aim of the present study was to verify endometrial proliferation in several dogs during the different cycle stages by using immunohistochemical detection of Ki-67 expression, which is a non-histone protein exclusively expressed in the nuclei of cycling cells [11,18–20], and by counting mitotic figures. Furthermore, the role of the serum estradiol-17b and progesterone levels in this process has also been assessed. Knowledge of the proliferation patterns in the normal cyclic endometrium of the dog might lead to a better understanding of tissue changes during pregnancy and in pathological conditions such as the cystic endometrial hyperplasia–pyometra complex [21].
2. Materials and methods 2.1. Animals Samples of both ovaries and the left uterine horn were obtained from 58 healthy adult dogs presented for ovariohysterectomy or euthanasia at the Faculty of Veterinary Medicine, Ghent University, and at four veterinary clinics. All dogs had been used in our previous study on apoptosis in the cyclic canine endometrium [9] and from seven of these animals also a sample of the uterine body was taken to verify possible differences in proliferation activity between this part of the uterus and the uterine horns. For each dog data concerning the anamnesis, age, breed, litters and last proestrous bleeding were recorded. The dogs varied in age from 1 to 8.5 years and included different breeds. 2.2. Tissue processing Immediately after excision samples were fixed in a phosphate buffered 3.5% (w/v) paraformaldehyde solution (pH 6.7) for 24–48 h. All tissue samples were embedded in paraffin and 5 mm thin sections were cut, mounted on 3-aminopropyl-triethoxysilanecoated slides (APES, Sigma, St. Louis, MO, USA), dried for 1 h at 60 8C on a hot plate and further dried overnight at 37 8C. 2.3. Serology Blood samples were taken immediately before surgery or euthanasia for determination of the serum levels of the sex steroids using a radio-immunoassay technique. After clotting
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and centrifugation at 1500 g, serum was separated and stored at 20 8C until assayed. The concentration of estradiol-17b was measured after extraction with diethylether without further purification using an antiserum against 17b-estradiol-3-hemisuccinate-BSA raised in sheep [22]. The concentration of progesterone was measured after extraction with petroleumether without further purification using an antiserum against progesterone-11hemisuccinate-BSA raised in sheep [22]. The detection limit for estradiol-17b was 5 pg per tube and for progesterone 0.005 ng per tube. The inter- and intra-assay variations for estradiol-17b were 5.75 and 8.30%, whereas for progesterone these variations were 7.05 and 8.75%. 2.4. Cycle stage determination The stage of the estrous cycle was determined like in our previous studies using histologic and serological parameters [23,24]. The animals were first sorted by macroscopic evaluation of the genital tract combined with the histologic examination of the ovaries and the uterus. After this preliminary morphological classification the animals were further classified according to their serum progesterone levels. Animals with uterine tissue at a proliferative stage and a progesterone level lower than 1 ng/ml were classified as being in proestrus. Animals with proliferative uterine tissue, developing corpora lutea and progesterone levels between 1 and 15 ng/ml were classified as being in estrus. Dogs were classified in early metestrus when uterine tissue was proliferative and progesterone levels were above 15 ng/ml in the presence of growing or fully developed corpora lutea, or above 10 ng/ml when regressing corpora lutea were present. Animals with uterine tissue at a secretory stage and progesterone levels lower than 10 ng/ml and higher than 0.5 ng/ml were classified as being in late metestrus. When uterine tissue was at rest and progesterone levels were basal (0.5 ng/ml) the dogs were in anestrus. Eight dogs were in proestrus, 10 in estrus, 9 in early metestrus, 15 in late metestrus and 16 in anestrus. 2.5. Immunohistochemical detection of Ki-67 After rehydration, the uterine sections were pretreated in an Antigen Retrieval Citra Solution (BioGenex, San Ramon, USA). This pretreatment consisted of microwaving the slides for 2 min at 700 W and then again for 3, 5 and 5 min at 200 W with 5 min rest in between. After cooling for 30 min at 4 8C and rinsing in distilled water, the slides were incubated for 5 min with 50 ml of a 3% (v/v) hydrogen peroxide–methanol solution to quench endogenous peroxidase activity. All incubations were carried out in a humidified environment. Then the slides were rinsed in TBS and incubated consecutively with normal rabbit serum (1:3) for 30 min at 37 8C to reduce nonspecific staining. The actual immunohistochemical detection of Ki-67 was performed using a monoclonal mouse anti-human antibody, which cross-reacts with the canine Ki-67. All sections were incubated with 50 ml of a 1:10 concentrated monoclonal mouse anti-human NCL-Ki67-MM1 antibody (Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) in TBS. After rinsing in TBS the sections were incubated for 30 min at room temperature with 50 ml of a secondary biotinylated rabbit anti-mouse antibody (1:250) (DAKO A/S,
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Table 1 Ki-67 labelling indexes (LI) and mitotic indexes (MI) in the superficial, middle and deep parts of the stroma and in blood vessels of the canine endometrium during proestrus, estrus, early metestrus, late metestrus and anestrus Cycle stage
Superficial stroma LI
Proestrus Estrus Early metestrus Late metestrus Anestrus
2.00 1.15 0.33 0.20 0.50
Middle stroma
MI
a
0.93 0.43a,b 0.19a,b 0.65b 0.3a,b
LI a
0.31 0.19 0.10 0.10a,b 0.06 0.06a,b 0b 0b
Deep stroma MI
a
1.50 0.71 0.20 0.13b 0b 0b 0.19 0.14b
a
0 0a 0a 0a 0a
LI 2.81 0.95 0.39 0.10 0.19
Blood vessels MI
a
0.99 0.43b 0.28b 0.05b 0.06b
LI a
0.12 0.08 0b 0b 0b 0b
5.00 1.40 0.33 0.13 0.44
MI
a
1.27 0.40b 0.17b 0.13b 0.20b
– – – – –
Values are means S:E:M. Values within columns with different superscripts (a and b) are significantly different (P < 0:05). (–): not analysed.
Glostrup, Denmark). The specimens were rinsed in TBS and incubated for 30 min at room temperature with streptavidine–HRP (1:300) (DAKO A/S, Glostrup, Denmark). Finally, after rinsing in TBS, 50 ml of DAB chromogen substrate (DAKO, Carpinteria, USA) was administered for 5 min. Mayer’s haematoxylin was applied during 30 s as a nuclear counter-stain. In every staining procedure positive and negative controls were included. The positive control was a canine duodenum section with many proliferating crypt cells. The negative controls were a uterine tissue section incubated with monoclonal mouse antibodies directed against Helicobacter pylori (Abcam Ltd., Cambridge, UK) instead of the primary antibody at an equivalent IgG1 concentration, viz. 5.6 mg/ml, and a uterine tissue section incubated without the primary and secondary antibodies. Ki-67 expression was evaluated in the surface epithelium, the stroma, the blood vessels, the crypts and the basal glands. The mean percentage of Ki-67 positive cells, represented as the labelling index (LI) in Fig. 2 and Table 1, was estimated by counting 100 cells of each category in five random areas of each uterine section. However, for the blood vessels only 100 endothelial cells were counted, and for the stroma a further classification into a superficial part surrounding the crypts, a middle part surrounding the glandular ducts and a deep part surrounding the basal glands has been used to verify possible regional differences in proliferation. In these latter subgroups 200, 100 and 200 cells were counted, respectively. 2.6. DAPI staining Mitotic figures were identified by means of 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich Inc., St. Louis, USA), a cell permeable fluorescent DNA binding probe. After rehydration and rinsing in PBS, the uterine sections were incubated with DAPI (5 mg/ml PBS) for 12 min at room temperature in a humidified environment. After incubation, sections were rinsed in PBS and mounted with a mounting solution for fluorescent microscopy supplied by Sigma-Aldrich Inc. Finally, stained sections were observed with an Olympus BX61 fluorescence microscope using the UV filter cube U-MNU2 (Olympus Optical Co. Ltd., Tokyo, Japan).
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The mean percentage of mitotic figures was estimated in all endometrial cell types as described for Ki-67 expression and is schematically represented in Fig. 2 and Table 1 as the mitotic index (MI). However, for the blood vessels no mitotic figures were counted as DAPI stained nuclei of endothelial cells were difficult to differentiate from stromal cell nuclei. 2.7. Statistical analysis In all cell types, overall differences between the cycle stages were assessed by using the non-parametric Kruskall–Wallis test, whereas the Tukey’s test was used to determine between which particular cycle stages significant differences were present (P < 0:05). Significant differences in the various parts of the stroma and in the blood vessels were listed in Table 1, whereas the significant differences in the other cell groups and in the stroma in general are shown in Fig. 2. The Spearman rank correlation test was used for determination of correlations between the LI and the serum estradiol-17b and progesterone levels. This test was also used to verify a possible correlation between the LI of the left uterine horn and that of the uterine body.
3. Results Cycle-dependent differences of both Ki-67 expression and mitotic figures were present in all endometrial cell groups, except in the middle part of the stroma in which no mitotic figures were found (Table 1, Figs. 1 and 2). During proestrus, both the LI and MI increased in the surface epithelial cells compared to early and late metestrus, respectively (Figs. 1a, b and 2). This increase was also present in the stroma in which both indexes were significantly higher than during any other cycle stage (Table 1, Figs. 1a, b and 2) and was even more prominent in the crypts (Figs. 1a, b and 2). An increase of the LI was also observed in the blood vessels (Table 1), in contrast to the low LI and MI in the basal glands (Figs. 1c, d and 2). During estrus, the LI decreased in the crypts and in the blood vessels (Table 1, Figs. 1e, f and 2) compared to proestrus, whereas both indexes also decreased in the middle and deep parts of the stroma, but not yet in the superficial part (Table 1). In contrast, the LI and MI peaked to their highest level in the basal glands (Figs. 1g, h and 2). In early metestrus, the LI and MI had further decreased in the surface epithelium and the crypts, respectively, whereas the LI and MI in the basal glands remained relatively high during that period (Table 1, Fig. 2). During late metestrus and anestrus the LI and MI remained rather low in the surface epithelium, stroma and crypts, and a clear decrease was also noticed in the basal glands (Table 1, Fig. 2). A positive correlation was observed between the serum estrogen levels and the LI in the stroma (r ¼ 0:46; P < 0:05) and the blood vessels (r ¼ 0:82; P < 0:01), whereas the serum progesterone levels were negatively correlated to the LI in the surface epithelium (r ¼ 0:31; P < 0:05) and the crypts (r ¼ 0:43; P < 0:01). In contrast, progesterone
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Fig. 1. Ki-67 labelling (a, c, e, g) and DAPI staining (b, d, f, h) during proestrus (a–d) and estrus (e–h) in the surface epithelium (1), stroma (2), blood vessels (3), crypts (4) and basal glands (5) of the canine endometrium. (a) Ki-67 positive cells and (b) mitotic figures were prominently present in the surface epithelium (small arrows),
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Fig. 2. (a) Ki-67 labelling indexes (LI) and (b) mitotic indexes (MI) in the surface epithelium, stroma, crypts and basal glands of canine uterine specimens during proestrus (n ¼ 8), estrus (n ¼ 10), early metestrus (n ¼ 9), late metestrus (n ¼ 15) and anestrus (n ¼ 16). Within each cell group, index values are significantly different (P < 0:05) when indicated by different letters (a and b). (8): mean value for all segments of the stroma; (): zero value.
levels were positively correlated to the LI in the basal glands (r ¼ 0:27; P < 0:05), but all coefficients were very low. For each of the seven dogs investigated, the LI in the uterine horns was strongly correlated with that of the corresponding uterine body (r ¼ 0:98; P < 0:01). the stroma (arrowheads) and the crypts (large arrows) in proestrus. (c) Ki-67 positivity and (d) mitotic figures were rare in the basal glands during proestrus and only some periglandular stromal cells (black arrowheads) and endothelial cells (black arrow) showed positivity. (e) Practically no Ki-67 positive cells nor (f) mitotic figures were noticed in the surface epithelium, the stroma, the blood vessels and the crypts in estrus. (g) Ki-67 positive cells and (h) mitotic cells (arrows) were prominently present in the basal glands during estrus. Scale bars represent 50 mm.
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4. Discussion In the present study, proliferation in the canine endometrium was assessed by using two techniques, viz. immunohistochemical detection of Ki-67 expression and counting of mitotic figures. Both techniques showed two peaks of proliferation, i.e. one during proestrus in the surface epithelium, the stroma, the blood vessels and the crypts and the other during estrus in the basal glands. These findings correspond with earlier data on canine endometrial proliferation by Barrau et al. [4] and with observations in other species such as the horse [11], the rat [25] and the mouse [16], although the regional differences in stromal proliferation described in these species were not observed in the dog. In contrast, Spanel-Borowski et al. found only one proliferation peak in the uterine body of the dog, i.e. during early proestrus [17]. This discrepancy can hardly be explained by regional differences as proliferation in the uterine horn and in the uterine body were strongly correlated in the present study. Possibly, the second proliferation peak has been missed by Spanel-Borowski et al. who only examined one sample per cycle stage [17]. In humans [25], rats [26–29] and mice [15,30–33] endometrial proliferation has been clearly related to the serum steroid hormone levels, but species differences are present. Progesterone caused a prominent stop of endometrial proliferation in the rat [26] and the mouse [30], whereas high proliferation rates were noticed in rabbits [12]. In the present study on the dog, proliferation in the surface epithelium, the stroma, the blood vessels and the crypts was slightly positively correlated with the estradiol-17b levels, whereas proliferation in the basal glands tended to peak when progesterone levels were increasing. Therefore, it is tempting to speculate that proliferation in the former and latter cell groups is mediated by estradiol-17b and progesterone, respectively. However, Gerstenberg et al. who found similar results in the horse suggested that proliferation of the basal glands is rather due to a delayed effect of estrogens [11], as has been shown in rodents [26,30], than being caused by progesterone. This hypothesis was further strengthened by findings of Allen et al. who observed a lack of increase in the mitotic rate of any endometrial cell type in mares that were under long and high progesterone influence due to a failure of corpus luteum regression [34]. In mice and rats, the delayed effect of estrogens on proliferation of the endometrial glands is reflected by differences in expression of estrogen receptors (ER) in this cell group compared to the other endometrial cell groups. In rodents, ER are highly concentrated in the surface epithelium and stroma during proestrus, whereas ER reach their maximum levels in the basal glands during early diestrus [35,36]. However, no such differences in distribution of ER between the basal glands and the other cell groups have been described for the dog [5] or the horse [37]. In contrast, progesterone receptor (PR) levels in these species remain high in the basal glands during estrus [6] and in early diestrus [37] when progesterone levels are increasing and high proliferation rates are noticed. The lack of increase in endometrial gland proliferation in mares with impaired regression of the corpus luteum [34] is probably due to a negative feedback mechanism in which the PR expression is downregulated by high progesterone levels [25]. These findings favour our hypothesis that proliferation in the basal glands is mediated by progestins rather than by a delayed effect of estrogens, but this item should be further investigated by experimental research. The present study indicates that these proliferation patterns play an important role in endometrial remodelling in the dog as they do in other species [10,11,14]. As homeostasis
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in reproductive tissues is based on a delicate balance between cellular proliferation and cell death, we compared the present findings with a previous study on apoptosis in the cyclic canine endometrium [9]. As expected, proliferation present during the first cycle stages was counteracted by apoptosis in later cycle stages, but throughout the estrous cycle the LI was two to three times higher than the AI for all cell groups, whereas the MI was about a 10-fold lower than the AI. Although various factors might be responsible for this discrepancy, differences in the kinetics of both phenomena seem most likely. Ki-67 is expressed in the nucleus during approximately 24 h as it is present during the entire active cell cycle that consists of the G1-, S-, G2- and M-phase of which the latter comprises less than 1 h [18,38]. For apoptosis, data on kinetics are sparse but the apoptotic process in vivo is generally considered to last only some hours as apoptotic cells are quickly recognised and engulfed by macrophages [39]. When no macrophages are present, apoptotic characteristics such as DNA fragmentation and active caspase-3 may be observed for a longer time. This latter fact might also explain why the discrepancy between the LI and the previously studied AI [9] was stronger in the stroma than in the crypts and the basal glands. Apoptotic cells of the stroma are immediately recognised and engulfed by macrophages, whereas elimination of apoptotic cells in the crypts and the basal glands seemed to be mainly directed to the glandular lumen not involving macrophages [9], resulting in a prolonged detection of caspase-3 and consequently in a higher AI. Luminal elimination is also the most likely way of apoptotic cell removal in the surface epithelium. Still, the discrepancy between the LI and the previously studied AI [9] was higher in this cell group than in the crypts and in the basal glands, which might have been caused by flushing of the uterine lumen with PBS before fixation. Detailed knowledge of both proliferation and regression patterns in the canine endometrium is important as a basis for understanding mechanisms involving pregnancy and pathological conditions such as the pyometra-cystic endometrial hyperplasia complex (CEH), a common disease in older nulliparous dogs [21]. During early pregnancy, Barrau et al. [40] already described high proliferative activity in the canine endometrial crypts and low proliferative activity in the basal glands, but no data are available on proliferation in the surface epithelium, the stroma and the blood vessels nor on endometrial apoptosis at the beginning of gestation. As remarkable similarities between pregnancy and the pyometra– CEH complex have recently been found in the dog [41,42], it would be interesting to complete our data on Ki-67 expression and apoptosis in the normal canine endometrium with the corresponding data in these two conditions. We can conclude that the present study showed two peaks of proliferation in the canine endometrium, i.e. one during proestrus in the surface epithelium, stroma, blood vessels and crypts, and the other during estrus in the basal glands. Furthermore, proliferation in the former cell groups seems to be under estrogenic influence, whereas proliferation in the latter cell group is likely to be regulated by progestins.
Acknowledgements The authors thank Dr. A. Boone, Dr. P. Herbots and Dr. I. Christiaens for assistance with sample collections, L. De Bels and L. Standaert for excellent technical assistance and Ghent University for financial support (BOF #011B4101).
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Proliferation patterns in the canine endometrium during the estrous cycle S. Van Cruchten*, W. Van den Broeck, M. D’haeseleer, P. Simoens Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 17 July 2003; received in revised form 10 October 2003; accepted 14 November 2003
Abstract The proliferative activity in the endometrium of 58 bitches in different stages of the estrous cycle was assessed by immunohistochemical detection of the Ki-67 proliferation associated nuclear antigen and by counting mitotic figures. The Ki-67 labelling index and the mitotic index were determined in the surface epithelium, the stroma, the crypts and the basal glands by calculating the percentage of Ki-67 positive cells and mitotic figures, respectively, on a total of 500 cells of each category. Endometrial vascular proliferation was also verified by counting the number of Ki-67 positive cells on a total of 100 endothelial cells. The present study showed two proliferation peaks involving different cell groups. In the surface epithelium, the stroma, the blood vessels and the crypts, the highest labelling and mitotic indexes were noticed during proestrus, whereas for the basal glands these indexes significantly increased (P < 0:05) during estrus compared to late metestrus and anestrus. Furthermore, a slightly positive correlation (P < 0:05) was found between the labelling index in the basal glands and the serum progesterone levels, whereas the labelling indexes in the other cell groups were positively correlated with the estradiol-17b levels, although not always significantly. These findings suggest that regulation of the proliferation in the surface epithelium, the stroma, the blood vessels and the crypts is different from the proliferation in the basal glands. # 2004 Elsevier Inc. All rights reserved. Keywords: Ki-67; DAPI; Endometrium; Canine; Estrous cycle
1. Introduction During the estrous cycle, the canine endometrium undergoes manifest morphological and biochemical changes [1–4]. Recent studies have demonstrated the role of steroid hormone receptors [5,6], matrix metalloproteinases [7] and apoptosis [8,9] in the regulation * Corresponding author. Tel.: þ32-9-2647714; fax: þ32-9-2647790. E-mail address: [email protected] (S. Van Cruchten).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.11.015
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of these cyclic endometrial changes. However, in contrast to other species such as man [10], the horse [11], the rabbit [12], the rat [13,14] and the mouse [15,16], proliferation patterns in the cyclic canine endometrium and their relation to the steroid hormone levels remain enigmatic. Barrau et al. investigated the proliferation of the canine endometrial glands during the estrous cycle [4]. In this study two phases of glandular growth were detected, viz. the first at the end of anestrus until the end of proestrus in the crypts and the second during estrus in the basal glands [4]. In contrast, Spanel-Borowski et al., who verified proliferation in different cell groups of the canine uterine body, observed only one peak of proliferation in all cell groups, viz. during early proestrus [17]. However, in the latter study only one sample per cycle stage was investigated and no data of dogs in anestrus were included. Therefore, the aim of the present study was to verify endometrial proliferation in several dogs during the different cycle stages by using immunohistochemical detection of Ki-67 expression, which is a non-histone protein exclusively expressed in the nuclei of cycling cells [11,18–20], and by counting mitotic figures. Furthermore, the role of the serum estradiol-17b and progesterone levels in this process has also been assessed. Knowledge of the proliferation patterns in the normal cyclic endometrium of the dog might lead to a better understanding of tissue changes during pregnancy and in pathological conditions such as the cystic endometrial hyperplasia–pyometra complex [21].
2. Materials and methods 2.1. Animals Samples of both ovaries and the left uterine horn were obtained from 58 healthy adult dogs presented for ovariohysterectomy or euthanasia at the Faculty of Veterinary Medicine, Ghent University, and at four veterinary clinics. All dogs had been used in our previous study on apoptosis in the cyclic canine endometrium [9] and from seven of these animals also a sample of the uterine body was taken to verify possible differences in proliferation activity between this part of the uterus and the uterine horns. For each dog data concerning the anamnesis, age, breed, litters and last proestrous bleeding were recorded. The dogs varied in age from 1 to 8.5 years and included different breeds. 2.2. Tissue processing Immediately after excision samples were fixed in a phosphate buffered 3.5% (w/v) paraformaldehyde solution (pH 6.7) for 24–48 h. All tissue samples were embedded in paraffin and 5 mm thin sections were cut, mounted on 3-aminopropyl-triethoxysilanecoated slides (APES, Sigma, St. Louis, MO, USA), dried for 1 h at 60 8C on a hot plate and further dried overnight at 37 8C. 2.3. Serology Blood samples were taken immediately before surgery or euthanasia for determination of the serum levels of the sex steroids using a radio-immunoassay technique. After clotting
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and centrifugation at 1500 g, serum was separated and stored at 20 8C until assayed. The concentration of estradiol-17b was measured after extraction with diethylether without further purification using an antiserum against 17b-estradiol-3-hemisuccinate-BSA raised in sheep [22]. The concentration of progesterone was measured after extraction with petroleumether without further purification using an antiserum against progesterone-11hemisuccinate-BSA raised in sheep [22]. The detection limit for estradiol-17b was 5 pg per tube and for progesterone 0.005 ng per tube. The inter- and intra-assay variations for estradiol-17b were 5.75 and 8.30%, whereas for progesterone these variations were 7.05 and 8.75%. 2.4. Cycle stage determination The stage of the estrous cycle was determined like in our previous studies using histologic and serological parameters [23,24]. The animals were first sorted by macroscopic evaluation of the genital tract combined with the histologic examination of the ovaries and the uterus. After this preliminary morphological classification the animals were further classified according to their serum progesterone levels. Animals with uterine tissue at a proliferative stage and a progesterone level lower than 1 ng/ml were classified as being in proestrus. Animals with proliferative uterine tissue, developing corpora lutea and progesterone levels between 1 and 15 ng/ml were classified as being in estrus. Dogs were classified in early metestrus when uterine tissue was proliferative and progesterone levels were above 15 ng/ml in the presence of growing or fully developed corpora lutea, or above 10 ng/ml when regressing corpora lutea were present. Animals with uterine tissue at a secretory stage and progesterone levels lower than 10 ng/ml and higher than 0.5 ng/ml were classified as being in late metestrus. When uterine tissue was at rest and progesterone levels were basal (0.5 ng/ml) the dogs were in anestrus. Eight dogs were in proestrus, 10 in estrus, 9 in early metestrus, 15 in late metestrus and 16 in anestrus. 2.5. Immunohistochemical detection of Ki-67 After rehydration, the uterine sections were pretreated in an Antigen Retrieval Citra Solution (BioGenex, San Ramon, USA). This pretreatment consisted of microwaving the slides for 2 min at 700 W and then again for 3, 5 and 5 min at 200 W with 5 min rest in between. After cooling for 30 min at 4 8C and rinsing in distilled water, the slides were incubated for 5 min with 50 ml of a 3% (v/v) hydrogen peroxide–methanol solution to quench endogenous peroxidase activity. All incubations were carried out in a humidified environment. Then the slides were rinsed in TBS and incubated consecutively with normal rabbit serum (1:3) for 30 min at 37 8C to reduce nonspecific staining. The actual immunohistochemical detection of Ki-67 was performed using a monoclonal mouse anti-human antibody, which cross-reacts with the canine Ki-67. All sections were incubated with 50 ml of a 1:10 concentrated monoclonal mouse anti-human NCL-Ki67-MM1 antibody (Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) in TBS. After rinsing in TBS the sections were incubated for 30 min at room temperature with 50 ml of a secondary biotinylated rabbit anti-mouse antibody (1:250) (DAKO A/S,
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Table 1 Ki-67 labelling indexes (LI) and mitotic indexes (MI) in the superficial, middle and deep parts of the stroma and in blood vessels of the canine endometrium during proestrus, estrus, early metestrus, late metestrus and anestrus Cycle stage
Superficial stroma LI
Proestrus Estrus Early metestrus Late metestrus Anestrus
2.00 1.15 0.33 0.20 0.50
Middle stroma
MI
a
0.93 0.43a,b 0.19a,b 0.65b 0.3a,b
LI a
0.31 0.19 0.10 0.10a,b 0.06 0.06a,b 0b 0b
Deep stroma MI
a
1.50 0.71 0.20 0.13b 0b 0b 0.19 0.14b
a
0 0a 0a 0a 0a
LI 2.81 0.95 0.39 0.10 0.19
Blood vessels MI
a
0.99 0.43b 0.28b 0.05b 0.06b
LI a
0.12 0.08 0b 0b 0b 0b
5.00 1.40 0.33 0.13 0.44
MI
a
1.27 0.40b 0.17b 0.13b 0.20b
– – – – –
Values are means S:E:M. Values within columns with different superscripts (a and b) are significantly different (P < 0:05). (–): not analysed.
Glostrup, Denmark). The specimens were rinsed in TBS and incubated for 30 min at room temperature with streptavidine–HRP (1:300) (DAKO A/S, Glostrup, Denmark). Finally, after rinsing in TBS, 50 ml of DAB chromogen substrate (DAKO, Carpinteria, USA) was administered for 5 min. Mayer’s haematoxylin was applied during 30 s as a nuclear counter-stain. In every staining procedure positive and negative controls were included. The positive control was a canine duodenum section with many proliferating crypt cells. The negative controls were a uterine tissue section incubated with monoclonal mouse antibodies directed against Helicobacter pylori (Abcam Ltd., Cambridge, UK) instead of the primary antibody at an equivalent IgG1 concentration, viz. 5.6 mg/ml, and a uterine tissue section incubated without the primary and secondary antibodies. Ki-67 expression was evaluated in the surface epithelium, the stroma, the blood vessels, the crypts and the basal glands. The mean percentage of Ki-67 positive cells, represented as the labelling index (LI) in Fig. 2 and Table 1, was estimated by counting 100 cells of each category in five random areas of each uterine section. However, for the blood vessels only 100 endothelial cells were counted, and for the stroma a further classification into a superficial part surrounding the crypts, a middle part surrounding the glandular ducts and a deep part surrounding the basal glands has been used to verify possible regional differences in proliferation. In these latter subgroups 200, 100 and 200 cells were counted, respectively. 2.6. DAPI staining Mitotic figures were identified by means of 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich Inc., St. Louis, USA), a cell permeable fluorescent DNA binding probe. After rehydration and rinsing in PBS, the uterine sections were incubated with DAPI (5 mg/ml PBS) for 12 min at room temperature in a humidified environment. After incubation, sections were rinsed in PBS and mounted with a mounting solution for fluorescent microscopy supplied by Sigma-Aldrich Inc. Finally, stained sections were observed with an Olympus BX61 fluorescence microscope using the UV filter cube U-MNU2 (Olympus Optical Co. Ltd., Tokyo, Japan).
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The mean percentage of mitotic figures was estimated in all endometrial cell types as described for Ki-67 expression and is schematically represented in Fig. 2 and Table 1 as the mitotic index (MI). However, for the blood vessels no mitotic figures were counted as DAPI stained nuclei of endothelial cells were difficult to differentiate from stromal cell nuclei. 2.7. Statistical analysis In all cell types, overall differences between the cycle stages were assessed by using the non-parametric Kruskall–Wallis test, whereas the Tukey’s test was used to determine between which particular cycle stages significant differences were present (P < 0:05). Significant differences in the various parts of the stroma and in the blood vessels were listed in Table 1, whereas the significant differences in the other cell groups and in the stroma in general are shown in Fig. 2. The Spearman rank correlation test was used for determination of correlations between the LI and the serum estradiol-17b and progesterone levels. This test was also used to verify a possible correlation between the LI of the left uterine horn and that of the uterine body.
3. Results Cycle-dependent differences of both Ki-67 expression and mitotic figures were present in all endometrial cell groups, except in the middle part of the stroma in which no mitotic figures were found (Table 1, Figs. 1 and 2). During proestrus, both the LI and MI increased in the surface epithelial cells compared to early and late metestrus, respectively (Figs. 1a, b and 2). This increase was also present in the stroma in which both indexes were significantly higher than during any other cycle stage (Table 1, Figs. 1a, b and 2) and was even more prominent in the crypts (Figs. 1a, b and 2). An increase of the LI was also observed in the blood vessels (Table 1), in contrast to the low LI and MI in the basal glands (Figs. 1c, d and 2). During estrus, the LI decreased in the crypts and in the blood vessels (Table 1, Figs. 1e, f and 2) compared to proestrus, whereas both indexes also decreased in the middle and deep parts of the stroma, but not yet in the superficial part (Table 1). In contrast, the LI and MI peaked to their highest level in the basal glands (Figs. 1g, h and 2). In early metestrus, the LI and MI had further decreased in the surface epithelium and the crypts, respectively, whereas the LI and MI in the basal glands remained relatively high during that period (Table 1, Fig. 2). During late metestrus and anestrus the LI and MI remained rather low in the surface epithelium, stroma and crypts, and a clear decrease was also noticed in the basal glands (Table 1, Fig. 2). A positive correlation was observed between the serum estrogen levels and the LI in the stroma (r ¼ 0:46; P < 0:05) and the blood vessels (r ¼ 0:82; P < 0:01), whereas the serum progesterone levels were negatively correlated to the LI in the surface epithelium (r ¼ 0:31; P < 0:05) and the crypts (r ¼ 0:43; P < 0:01). In contrast, progesterone
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Fig. 1. Ki-67 labelling (a, c, e, g) and DAPI staining (b, d, f, h) during proestrus (a–d) and estrus (e–h) in the surface epithelium (1), stroma (2), blood vessels (3), crypts (4) and basal glands (5) of the canine endometrium. (a) Ki-67 positive cells and (b) mitotic figures were prominently present in the surface epithelium (small arrows),
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Fig. 2. (a) Ki-67 labelling indexes (LI) and (b) mitotic indexes (MI) in the surface epithelium, stroma, crypts and basal glands of canine uterine specimens during proestrus (n ¼ 8), estrus (n ¼ 10), early metestrus (n ¼ 9), late metestrus (n ¼ 15) and anestrus (n ¼ 16). Within each cell group, index values are significantly different (P < 0:05) when indicated by different letters (a and b). (8): mean value for all segments of the stroma; (): zero value.
levels were positively correlated to the LI in the basal glands (r ¼ 0:27; P < 0:05), but all coefficients were very low. For each of the seven dogs investigated, the LI in the uterine horns was strongly correlated with that of the corresponding uterine body (r ¼ 0:98; P < 0:01). the stroma (arrowheads) and the crypts (large arrows) in proestrus. (c) Ki-67 positivity and (d) mitotic figures were rare in the basal glands during proestrus and only some periglandular stromal cells (black arrowheads) and endothelial cells (black arrow) showed positivity. (e) Practically no Ki-67 positive cells nor (f) mitotic figures were noticed in the surface epithelium, the stroma, the blood vessels and the crypts in estrus. (g) Ki-67 positive cells and (h) mitotic cells (arrows) were prominently present in the basal glands during estrus. Scale bars represent 50 mm.
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4. Discussion In the present study, proliferation in the canine endometrium was assessed by using two techniques, viz. immunohistochemical detection of Ki-67 expression and counting of mitotic figures. Both techniques showed two peaks of proliferation, i.e. one during proestrus in the surface epithelium, the stroma, the blood vessels and the crypts and the other during estrus in the basal glands. These findings correspond with earlier data on canine endometrial proliferation by Barrau et al. [4] and with observations in other species such as the horse [11], the rat [25] and the mouse [16], although the regional differences in stromal proliferation described in these species were not observed in the dog. In contrast, Spanel-Borowski et al. found only one proliferation peak in the uterine body of the dog, i.e. during early proestrus [17]. This discrepancy can hardly be explained by regional differences as proliferation in the uterine horn and in the uterine body were strongly correlated in the present study. Possibly, the second proliferation peak has been missed by Spanel-Borowski et al. who only examined one sample per cycle stage [17]. In humans [25], rats [26–29] and mice [15,30–33] endometrial proliferation has been clearly related to the serum steroid hormone levels, but species differences are present. Progesterone caused a prominent stop of endometrial proliferation in the rat [26] and the mouse [30], whereas high proliferation rates were noticed in rabbits [12]. In the present study on the dog, proliferation in the surface epithelium, the stroma, the blood vessels and the crypts was slightly positively correlated with the estradiol-17b levels, whereas proliferation in the basal glands tended to peak when progesterone levels were increasing. Therefore, it is tempting to speculate that proliferation in the former and latter cell groups is mediated by estradiol-17b and progesterone, respectively. However, Gerstenberg et al. who found similar results in the horse suggested that proliferation of the basal glands is rather due to a delayed effect of estrogens [11], as has been shown in rodents [26,30], than being caused by progesterone. This hypothesis was further strengthened by findings of Allen et al. who observed a lack of increase in the mitotic rate of any endometrial cell type in mares that were under long and high progesterone influence due to a failure of corpus luteum regression [34]. In mice and rats, the delayed effect of estrogens on proliferation of the endometrial glands is reflected by differences in expression of estrogen receptors (ER) in this cell group compared to the other endometrial cell groups. In rodents, ER are highly concentrated in the surface epithelium and stroma during proestrus, whereas ER reach their maximum levels in the basal glands during early diestrus [35,36]. However, no such differences in distribution of ER between the basal glands and the other cell groups have been described for the dog [5] or the horse [37]. In contrast, progesterone receptor (PR) levels in these species remain high in the basal glands during estrus [6] and in early diestrus [37] when progesterone levels are increasing and high proliferation rates are noticed. The lack of increase in endometrial gland proliferation in mares with impaired regression of the corpus luteum [34] is probably due to a negative feedback mechanism in which the PR expression is downregulated by high progesterone levels [25]. These findings favour our hypothesis that proliferation in the basal glands is mediated by progestins rather than by a delayed effect of estrogens, but this item should be further investigated by experimental research. The present study indicates that these proliferation patterns play an important role in endometrial remodelling in the dog as they do in other species [10,11,14]. As homeostasis
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in reproductive tissues is based on a delicate balance between cellular proliferation and cell death, we compared the present findings with a previous study on apoptosis in the cyclic canine endometrium [9]. As expected, proliferation present during the first cycle stages was counteracted by apoptosis in later cycle stages, but throughout the estrous cycle the LI was two to three times higher than the AI for all cell groups, whereas the MI was about a 10-fold lower than the AI. Although various factors might be responsible for this discrepancy, differences in the kinetics of both phenomena seem most likely. Ki-67 is expressed in the nucleus during approximately 24 h as it is present during the entire active cell cycle that consists of the G1-, S-, G2- and M-phase of which the latter comprises less than 1 h [18,38]. For apoptosis, data on kinetics are sparse but the apoptotic process in vivo is generally considered to last only some hours as apoptotic cells are quickly recognised and engulfed by macrophages [39]. When no macrophages are present, apoptotic characteristics such as DNA fragmentation and active caspase-3 may be observed for a longer time. This latter fact might also explain why the discrepancy between the LI and the previously studied AI [9] was stronger in the stroma than in the crypts and the basal glands. Apoptotic cells of the stroma are immediately recognised and engulfed by macrophages, whereas elimination of apoptotic cells in the crypts and the basal glands seemed to be mainly directed to the glandular lumen not involving macrophages [9], resulting in a prolonged detection of caspase-3 and consequently in a higher AI. Luminal elimination is also the most likely way of apoptotic cell removal in the surface epithelium. Still, the discrepancy between the LI and the previously studied AI [9] was higher in this cell group than in the crypts and in the basal glands, which might have been caused by flushing of the uterine lumen with PBS before fixation. Detailed knowledge of both proliferation and regression patterns in the canine endometrium is important as a basis for understanding mechanisms involving pregnancy and pathological conditions such as the pyometra-cystic endometrial hyperplasia complex (CEH), a common disease in older nulliparous dogs [21]. During early pregnancy, Barrau et al. [40] already described high proliferative activity in the canine endometrial crypts and low proliferative activity in the basal glands, but no data are available on proliferation in the surface epithelium, the stroma and the blood vessels nor on endometrial apoptosis at the beginning of gestation. As remarkable similarities between pregnancy and the pyometra– CEH complex have recently been found in the dog [41,42], it would be interesting to complete our data on Ki-67 expression and apoptosis in the normal canine endometrium with the corresponding data in these two conditions. We can conclude that the present study showed two peaks of proliferation in the canine endometrium, i.e. one during proestrus in the surface epithelium, stroma, blood vessels and crypts, and the other during estrus in the basal glands. Furthermore, proliferation in the former cell groups seems to be under estrogenic influence, whereas proliferation in the latter cell group is likely to be regulated by progestins.
Acknowledgements The authors thank Dr. A. Boone, Dr. P. Herbots and Dr. I. Christiaens for assistance with sample collections, L. De Bels and L. Standaert for excellent technical assistance and Ghent University for financial support (BOF #011B4101).
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