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Journal of Animal Reproduction and Biotechnology 2023; 38(4): 254-262

Published online December 31, 2023

https://doi.org/10.12750/JARB.38.4.254

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

Morphological differences according to uterine tissue remodeling during estrus between sika deer and water deer

Min-Gee Oh1,4,# , Yong-Su Park2,# and Sang-Hwan Kim1,3,4,*

1General Graduate School of Animal life Convergence Science, Hankyong National University, Ansung 17579, Korea
2Research Center for Endangered Species, National Institute of Ecology, Seocheon 33657, Korea
3School of Animal Life Convergence Science, Hankyong National University, Ansung 17579, Korea
4Institute of Applied Humanimal Science, Hankyong National University, Ansung 17579, Korea

Correspondence to: Sang-Hwan Kim
E-mail: immunoking@hknu.ac.kr

#These authors contributed equally to this work.

Received: December 4, 2023; Revised: December 8, 2023; Accepted: December 9, 2023

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Water deer and sika deer, which breed in the wild environment, are known to have similar reproductive physiology mechanisms. Therefore, this study aimed to analyze the differences in uterine development between water deer and sika deer during estrus.
Methods: MMPs and uterine development-related factors were analyzed and morphological differences were compared in the uterus of sika deer captured near Russia near Korea and water deer captured in the wild in Korea.
Results: In terms of morphological differences in the uterus, the glands that form villus within the endometrium of the water deer were newly developed, and the formation of small glands was high, but the villus and glands of the sika deer were expanded, and the stroma zone in the myometrium was higher than that of the water deer. Development has increased. Additionally, the expression of PAPP-A and VEGF factors was increased in the endometrium of water deer than in sika deer, but the actions of MMPs were increased in sika deer.
Conclusions: As a result of this study, there is a significant difference in the development of glands in the endometrium of water deer and sika deer during estrus, and it is believed that there is a significant difference in the development of the uterus due to the physiological effects of estrus between water deer and sika deer. Additionally, it is believed that there will be differences in the timing at which pregnancy can be decided.

Keywords: estrus cycle, sika deer, uterus, VEGF, water deer

The reproductive physiology of deer and elk is characterized by ‘silent ovulations’ that occur for about 8 to 10 days when they exist in a herd during the breeding season. This phenomenon differs from Artiodactyla animals, which tend to ovulate without showing clear signs of estrus, such as monovulatory species, and have a general reorganization of the uterus or corpus luteum (Asher et al., 1999a; Asher et al., 1999b; McCorkell et al., 2006). In addition, the uteri of elk and deer undergo rapid changes, and the branching state of fetal villi is known to vary among Cervidae families depending on the environment (Hamasaki et al., 2001; Chen et al., 2015; Kotlarczyk et al., 2021).

During estrus in common artiodactyls, the uterus undergoes morphological changes depending on the specific period under the influence of estrogen and progesterone, and the tissue is reorganized to a state capable of pregnancy. In particular, during the estrous cycle, the uterus undergoes morphological and physiological changes, such as the development and secretion of uterine glands and changes in the thickness of the uterine wall, and is affected by various factors such as sex hormones such as estrogen and progesterone (P4), prostaglandin (PG), and interleukin (IL). It is known to be controlled (Chen et al., 2015; Kumar et al., 2018; Kotlarczyk et al., 2021).

What influences uterine changes during this period is Matrix metalloproteinase (MMPs) secreted in the endometrium, which play an important role in reorganizing various physiological tissues, such as ovarian and uterine functions, according to hormonal signals and biological changes. However, an abnormal hormonal feedback loop leads to uterine degeneration and uterine inflammation, resulting in dramatic tissue remodeling and abnormal angiogenesis. To solve this problem, the uterus appears to control abnormally active tissues by increasing the expression of MMPs at appropriate times, destroying the basement membrane of abnormal cells, and maintaining uterine tissue (Tsafriri, 1995; Kim et al., 2014a).

In particular, as is known so far, water deer have similar habits to sika deer, and their estrous and gestation periods are very similar (Kim et al., 2014a; Kotlarczyk et al., 2021).

However, it can be confirmed that there are differences in the morphological findings of water deer and sika deer in the uterus and placenta and in the shape of the uteroplacental junction that spreads from the uterus to the placenta (Kotlarczyk et al., 2021).

In other words, the results show that the reorganization and function of the uterus during the estrus phase of water deer and sika deer may be different, and there is an opinion that there may be differences in the endometrial reorganization, placenta formation, and uterine shape in non-invasive placentation (Sohn and Kimura, 2012).

Therefore, this study analyzed the morphological and physiological differences between the uteri of water deer and sika deer in estrus and controlled tissue reorganization by decomposing collagen or gelatin, the main components of the extracellular matrix (Oh and Kim, 2022). This study was conducted to provide basic data for research on reproductive physiology in wild animals by analyzing the effects of MMPs and apoptosis on uterine reorganization during estrus.

Animals

The estrus phase uterus of water deer was collected in Ansan-si, Gyeonggi-do, Korea, in accordance with Article 19, paragraph 1 of the Wild Animal Protection and Management Act and under the guidance of the Institutional Animal Care and Use Committee of the Wildlife Conservation and Research Center (Permittion No. 2013-1).

In the case of sika deer, the estrus uterus was collected from an individual presumed to be a sika deer living in adjacent areas of Russia and the Korean Peninsula (in prior research, an MOU signed with the Primorskaya State Agricultural Academy (date: 2014.11.25). All experiments followed the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For each uterus, the fetal villi of the caruncle and the trophoblast cells/glandular epithelial cells of the endometrium were excised and used (Lee et al., 2023) (Fig. 1). The samples used in the experiment were three water deer and three sika deer.

Figure 1. Different parts of genitalia for biometric recording of water deer and sika deer uterus. (A) Sika deer. (B) Water deer. (a) Uterus parts of sika deer. (b) Uterus parts of water deer.

Sampling of uterus tissue

Uterus tissue was fixed in 70% DEPC EtOH for 24 hrs, sequentially dehydrated and transparent in 95% EtOH- 100% EtOH-xylene solution (Sigma Aldrich, St. Louis, MO, USA), and then embedded in paraffin for storage. Before the experiment, slides were produced by cutting them into thicknesses of 5 μm and 10 μm and used in the experiment after deparaffinization and hydration.

H&E staining

Hematoxylin and eosin were stained on slides of each uterus, and the slides were analyzed by an optical microscope (AX70, Olympus, Tokyo, Japan) (Kim et al., 2014b).

Immunohistochemistry

Antigen retrieval was performed by heating 10 mM sodium citrate at 95℃ for 5 min. Antigen unmasking was reacted with 3% hydrogen peroxide for 5 min at room temperature, and blocking was incubated in blocking buffer (3% Bovine Albumin Serum in 1XPBS) for 1 hr. Primary antibodies insulin-like growth factor-receptor (IGF1-r; ab263903; Abcam, Cambridge, UK), Pregnancy associated plasma protein A (PAPP-A; ab249813; Abcam) and Tissue inhibitor of metalloproteinases 2 (TIMP-2; ab230511; Abcam) were each diluted in blocking buffer and incubated at room temperature for 2 hrs. Washed in 1XPBS and incubated for 1 hr with Secondary HRP Anti-Rabbit IgG secondary antibody (ab288151; Abcam) or HRP Anti-Mouse IgG secondary antibody (ab47827; Abcam). Washed and reacted for 10 min using ABC detection kit (Vector, CA, USA) and confirmed the reaction using Dia-minbenzidine (Vector, CA, USA) after staining with a hematoxylin solution containing 4% acetic acid and PAS. The samples were analyzed under an optical microscope.

In-situ zymography

Boiling in 10 mM sodium citrate for 10 min after deparaffinize and hydrate, then emulsion (ddW, 10% SDS, 2% Glycerol) and zymography reaction buffer were mixed at a ratio of 1: 2 and raised to slide, followed by enzymatic reaction at 37℃ for 48 h in a slide box filled with 1 M Tris. After the reaction, routine hematoxylin and eosin (H&E) staining was performed for histological inspection with optical microscope (Oh and Kim, 2022).

Immunofluorescence

Paraffin blocks of uterine and caruncle tissues in each group were sectioned to a thickness of 5 μm. Tissue slides were prepared following deparaffinization, hydration using xylene and ethanol, and permeabilization at -20℃ with 0.1% Triton X-100 in 1 × PBS (PBS-T). The samples were blocked at room temperature (RT) for 30 min in TPBS (1 × PBS with 0.01% Tween-20) containing 5% normal horse serum (NHS) and 1% normal goat serum (NGS). All tissue slides were incubated in a dark room at RT for 1 hr, with primary antibody vascular endothelial growth factor (VEGF; 2893S; Cell signaling, United States, USA) antibody diluted 1:200 in blocking solution. After that, to induce secondary antibody conjugation, incubated in the dark with a blocking solution for 30 min. After that, all slides were incubated with Alexa 594-conjugated anti-rabbit secondary antibody (ab150080; Abcam) diluted 1:300 in blocking solution in the darkroom at RT for 1 hr. Decant the second secondary antibody solution and wash it three times with PBS for 5 min each in the dark.

In situ apoptosis detection

Annexin V staining was performed using PE Annexin V Apoptosis Detection Kit Ⅰ (BD Biosciences, San Jose, CA). After washing three times with 1XPBS, 5 uL each of Annexin V and 7-AAD into 100 uL of 1X binding buffer, staining was performed for 1 hr and 30 min at room temperature for analysis.

Morphology and Ca2+ analysis of water deer and sika deer uterus on the estrus

The results of confirming the morphological differences between the endometrium and myometrium of water deer and sika deer during estrus are shown in Fig. 2. It was confirmed that the size of the fetus villus cytotrophoblast zone in the endometrium of the water deer is smaller and more concentrated than that of the sika deer and that the cells of the myometrium have a lower cell density than those of the sika deer. In the case of sika deer, the size of the fetus villus cytotrophoblast zone in the endometrium is larger, and the density is lower than that of water deer, while the cell density appears to be high in the myometrium. Alizarin Red Staining was performed to check cell activity, and overall, water deer showed higher activity than sika deer. Endometrium showed high activity in the fetus villus of water deer, and myometrium was similarly highly active overall in the cells of water deer.

Figure 2. Morphological and physiological analysis of the estrous uterus of water deer and sika deer. Activation of Ca2+ was concentrated in the uterus zones and activated in the glands in both water deer and sika deer.

Analysis of protein expression pattern and MMPs activation in water deer and sika deer uterus on the estrus

The results of comparing the expression patterns of growth-related genes in the ovarian uterus of water deer and sika deer are shown in Fig. 3. The expression of PAPP-A was highly expressed in the fetus villus zone of water deer, and in the case of myometrium, it was confirmed that it was expressed relatively higher in sika deer than in water deer. On the other hand, the expression of IGF-r is generally higher in sika deer than in water deer, and it can be confirmed that it is expressed higher in the fetus villi than in the stroma cell zone. Comparing the activity of MMPs that promote changes in the Extracellular Matrix (ECM) to measure uterine remodeling revealed that the overall activity was much higher in sika deer than in water deer. Additionally, the expression of TIMP-2 showed no difference in both stags and sika deer (Fig. 4).

Figure 3. Tissue immunohistochemical analysis of BCL-2, E2-r, PAPP-A, and IGF1-r protein expression in the uterus. Black arrows indicate the expression location of the protein. The endometrium is expressed in the glands, and ectopy is expressed in the stroma and basal layer.
Figure 4. Analysis of MMP activity and TIMP-2 expression in uterine tissues. In the in-situ zymography, water deer generally expressed MMPs in the stromal zone, whereas sika deer expressed MMPs in the glands zone. Magnification = 200×.

Detection of VEGF protein on estrus uterus

As a result of analyzing the expression pattern of VEGF protein, which affects angiogenesis (Fig. 5), the expression of VEGF was high in the villus zone of the endometrium zone. In the endometrium, sika deer had overall increased VEGF expression than water deer. However, in the myometrium zone, VEGF expression appears more widespread in water deer than in sika deer, but the expression level was low. Regarding the overall expression pattern, higher expression of VEGF was detected in the endometrium of sika deer compared to water deer.

Figure 5. Detection of VEGF protein expression patterns in the uterus tissues. Green fluorescence indicates gene expression. It is expressed mainly at very high levels in the uterus of water deer and is relatively low in sika deer. White arrows indicate the expression location of the protein. Magnification = 200×.

Apoptosis activation of the uterus on the estrus

Fig. 6 shows the results of apoptosis detection analysis to confirm apoptosis, which acts on cell reorganization and uterine maintenance. As a result of confirming apoptosis throughout the uterus, apoptosis was relatively higher in the specific villus cytotrophoblast zone of sika deer than in water deer. In the case of water deer, there was no overall expression of apoptosis, but it was confirmed that it was expressed in blood vessels.

Figure 6. Detection of DNA fragments in water deer and sika deer uterus tissue. The apoptosis detection assay used terminal deoxynucleotidyl transferase to label the 3-OH termini of DNA fragments generated during apoptosis. White arrows indicate areas with distinct expressions. Magnification = 200×.

In the endometrial follicular phase, estrogen induces proliferation of the endometrial epithelium. In particular, the endometrium, which undergoes biochemical and morphological changes, is a target tissue for progesterone and estrogen. During the estrous cycle, the bovine uterus undergoes morphological and physiological changes (Chen et al., 2015), such as the development and secretion of uterine glands and changes in the thickness of the uterine wall. In our study, the development of villus cytotrophoblast of endometrium in sika deer was lower, and the size was lower than in water deer. Although it is relatively large, the deposition rate of Ca2+ is generally low, and in the myometrium, it can be seen that the overall cell density of sika deer is high. However, as in the endometrium, the deposition rate of Ca2+ is high in water deer, so the metabolic maturation of villus acts relatively differently, and it is predicted that metabolic activity and cellular changes will be higher in sika deer than in water deer (Spencer et al., 2005; Kotlarczyk et al., 2021).

In addition, the activation and morphological changes of the uterus at the same time show a big difference in sika deer and water deer, which is thought to be able to rapidly change the composition of the uterus for pregnancy as the activity of PAPP-A and estradiol receptor (E2-r) increases high in the villus cytotrophoblast of water deer (Yanagawa et al., 2008; Lee et al., 2023). However, in the analysis of histological changes, the development of villus cytotrophoblast in the uterus of sika deer seemed to be higher than that of water deer, which is expected to have acted differently depending on the influence of progesterone, which accumulates in high concentrations during the period of change in the uterus of sika deer. In general, the target tissues of P4 and E2 are activated by estrogen receptors for biochemical and morphological changes and activate histological changes by inducing the action of MMPs released from uterine cells (Kotlarczyk et al., 2021). In particular, the activity of MMP-2, which dissolves collagen and elastic fibers in the extracellular matrix and is involved in angiogenesis, tissue reorganization, and activation of growth factors, can be seen to be high in sika deer.

In addition, VEGF activity, which plays an essential role in the tissue remodeling process, increases in the villus cytotrophoblast zone. The extravascular zone of the myometrium of water deer and sika deer is most highly active in the endometrium of sika deer, thereby increasing the activity of vascular epithelial cells surrounding tissues in the uterus (Kim et al., 2014a, 2014b, 2018). Promotes the proliferation of in other words, it is thought that changes in the uterus of the sika deer stabilize the villus earlier than changes in the uterine tissue of the water deer. According to the research results of Sohn et al., 2021, in the case of water deer, the placenta is classified as oligocotyledon, similar to other plumids with 6-8 placentas, and the villus is well developed, so a distinct glands zone is visible in each villus (Yanagawa et al., 2008).

In the case of the sika deer, the overall shape is similar to that of the water deer, but the branching state of fetal villi in the convex-shaped placenta is known to be different for each Cervidae family (Hamasaki et al., 2001; Sohn and Kimura, 2012). In other words, the morphological findings of water deer and sika deer are similar regarding the shape of the uterus and placenta. However, it can be confirmed that there is a difference in the shape of the uteroplacental junction that spreads from the uterus to the placenta. These morphological differences in the uterus and the overall tissue reorganization formed during the estrus period of the sika deer can be seen as a unique shape unique to the sika deer, and may differ depending on the Cervidae family (Wooding et al., 2018; Sohn et al., 2021). However, most sika deers’ wombs rapidly remodel uterine tissues compared to water deers. Through the expression of high concentrations of progesterone and the cooperation of MMPs formed in tissues, they quickly restore the condition of the uterus when pregnancy fails.

Therefore, through this study, it is believed that the action of hormones related to the estrus period of sika deer and the activity of genes related to uterine remodeling act differently depending on the presence or absence of pregnancy, which will play a role in determining the timing of pregnancy in sika deer in the future. In addition, since samples of the sika deer, which has become extinct in Korea, can no longer be collected due to international issues, our research can be valuable data for future species restoration.

This study analyzed changes in the uterus compared to the physiological state of water deer during estrus to confirm the physiological state of smooth pregnancy in sika deer as a domestic animal. As a result of the study, we confirmed that the state of the uterus during estrus shows a significant difference from that of water deer, which has similar reproductive physiology, and that there is a significant difference in the action of MMPs to reorganize tissues and the resulting morphological changes. In particular, the sika deer uterus has a similar shape to the pregnant uterus during estrus. Therefore, functional development associated with the conception is quite different from that of the water deer, and that there will be a unique reproductive action of the sika deer of the Cervidae family.

The authors sincerely thank the Research Center for Endangered Species and the National Institute of Ecology for their support in conducting this study.

Conceptualization, S-H.K.; data curation, S-H.K.; formal analysis, Y-S.P., M-G.O.; funding acquisition, Y-S.P., S-H.K.; investigation, S-H.K., M-G.O.; methodology, M-G.O.; project administration, S-H.K.; resources, S-H.K.; supervision, S-H.K.; roles/writing - original draft, Y-S.P., M-G.O.; writing - review & editing, Y-S.P., S-H.K.

All experiment were conduced in accordance with the guidelines established by the Institutional Animal Care and Use Committee of Sangji University approved protocol (#2021-23).

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Article

Original Article

Journal of Animal Reproduction and Biotechnology 2023; 38(4): 254-262

Published online December 31, 2023 https://doi.org/10.12750/JARB.38.4.254

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

Morphological differences according to uterine tissue remodeling during estrus between sika deer and water deer

Min-Gee Oh1,4,# , Yong-Su Park2,# and Sang-Hwan Kim1,3,4,*

1General Graduate School of Animal life Convergence Science, Hankyong National University, Ansung 17579, Korea
2Research Center for Endangered Species, National Institute of Ecology, Seocheon 33657, Korea
3School of Animal Life Convergence Science, Hankyong National University, Ansung 17579, Korea
4Institute of Applied Humanimal Science, Hankyong National University, Ansung 17579, Korea

Correspondence to:Sang-Hwan Kim
E-mail: immunoking@hknu.ac.kr

#These authors contributed equally to this work.

Received: December 4, 2023; Revised: December 8, 2023; Accepted: December 9, 2023

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: Water deer and sika deer, which breed in the wild environment, are known to have similar reproductive physiology mechanisms. Therefore, this study aimed to analyze the differences in uterine development between water deer and sika deer during estrus.
Methods: MMPs and uterine development-related factors were analyzed and morphological differences were compared in the uterus of sika deer captured near Russia near Korea and water deer captured in the wild in Korea.
Results: In terms of morphological differences in the uterus, the glands that form villus within the endometrium of the water deer were newly developed, and the formation of small glands was high, but the villus and glands of the sika deer were expanded, and the stroma zone in the myometrium was higher than that of the water deer. Development has increased. Additionally, the expression of PAPP-A and VEGF factors was increased in the endometrium of water deer than in sika deer, but the actions of MMPs were increased in sika deer.
Conclusions: As a result of this study, there is a significant difference in the development of glands in the endometrium of water deer and sika deer during estrus, and it is believed that there is a significant difference in the development of the uterus due to the physiological effects of estrus between water deer and sika deer. Additionally, it is believed that there will be differences in the timing at which pregnancy can be decided.

Keywords: estrus cycle, sika deer, uterus, VEGF, water deer

INTRODUCTION

The reproductive physiology of deer and elk is characterized by ‘silent ovulations’ that occur for about 8 to 10 days when they exist in a herd during the breeding season. This phenomenon differs from Artiodactyla animals, which tend to ovulate without showing clear signs of estrus, such as monovulatory species, and have a general reorganization of the uterus or corpus luteum (Asher et al., 1999a; Asher et al., 1999b; McCorkell et al., 2006). In addition, the uteri of elk and deer undergo rapid changes, and the branching state of fetal villi is known to vary among Cervidae families depending on the environment (Hamasaki et al., 2001; Chen et al., 2015; Kotlarczyk et al., 2021).

During estrus in common artiodactyls, the uterus undergoes morphological changes depending on the specific period under the influence of estrogen and progesterone, and the tissue is reorganized to a state capable of pregnancy. In particular, during the estrous cycle, the uterus undergoes morphological and physiological changes, such as the development and secretion of uterine glands and changes in the thickness of the uterine wall, and is affected by various factors such as sex hormones such as estrogen and progesterone (P4), prostaglandin (PG), and interleukin (IL). It is known to be controlled (Chen et al., 2015; Kumar et al., 2018; Kotlarczyk et al., 2021).

What influences uterine changes during this period is Matrix metalloproteinase (MMPs) secreted in the endometrium, which play an important role in reorganizing various physiological tissues, such as ovarian and uterine functions, according to hormonal signals and biological changes. However, an abnormal hormonal feedback loop leads to uterine degeneration and uterine inflammation, resulting in dramatic tissue remodeling and abnormal angiogenesis. To solve this problem, the uterus appears to control abnormally active tissues by increasing the expression of MMPs at appropriate times, destroying the basement membrane of abnormal cells, and maintaining uterine tissue (Tsafriri, 1995; Kim et al., 2014a).

In particular, as is known so far, water deer have similar habits to sika deer, and their estrous and gestation periods are very similar (Kim et al., 2014a; Kotlarczyk et al., 2021).

However, it can be confirmed that there are differences in the morphological findings of water deer and sika deer in the uterus and placenta and in the shape of the uteroplacental junction that spreads from the uterus to the placenta (Kotlarczyk et al., 2021).

In other words, the results show that the reorganization and function of the uterus during the estrus phase of water deer and sika deer may be different, and there is an opinion that there may be differences in the endometrial reorganization, placenta formation, and uterine shape in non-invasive placentation (Sohn and Kimura, 2012).

Therefore, this study analyzed the morphological and physiological differences between the uteri of water deer and sika deer in estrus and controlled tissue reorganization by decomposing collagen or gelatin, the main components of the extracellular matrix (Oh and Kim, 2022). This study was conducted to provide basic data for research on reproductive physiology in wild animals by analyzing the effects of MMPs and apoptosis on uterine reorganization during estrus.

MATERIALS AND METHODS

Animals

The estrus phase uterus of water deer was collected in Ansan-si, Gyeonggi-do, Korea, in accordance with Article 19, paragraph 1 of the Wild Animal Protection and Management Act and under the guidance of the Institutional Animal Care and Use Committee of the Wildlife Conservation and Research Center (Permittion No. 2013-1).

In the case of sika deer, the estrus uterus was collected from an individual presumed to be a sika deer living in adjacent areas of Russia and the Korean Peninsula (in prior research, an MOU signed with the Primorskaya State Agricultural Academy (date: 2014.11.25). All experiments followed the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For each uterus, the fetal villi of the caruncle and the trophoblast cells/glandular epithelial cells of the endometrium were excised and used (Lee et al., 2023) (Fig. 1). The samples used in the experiment were three water deer and three sika deer.

Figure 1.Different parts of genitalia for biometric recording of water deer and sika deer uterus. (A) Sika deer. (B) Water deer. (a) Uterus parts of sika deer. (b) Uterus parts of water deer.

Sampling of uterus tissue

Uterus tissue was fixed in 70% DEPC EtOH for 24 hrs, sequentially dehydrated and transparent in 95% EtOH- 100% EtOH-xylene solution (Sigma Aldrich, St. Louis, MO, USA), and then embedded in paraffin for storage. Before the experiment, slides were produced by cutting them into thicknesses of 5 μm and 10 μm and used in the experiment after deparaffinization and hydration.

H&E staining

Hematoxylin and eosin were stained on slides of each uterus, and the slides were analyzed by an optical microscope (AX70, Olympus, Tokyo, Japan) (Kim et al., 2014b).

Immunohistochemistry

Antigen retrieval was performed by heating 10 mM sodium citrate at 95℃ for 5 min. Antigen unmasking was reacted with 3% hydrogen peroxide for 5 min at room temperature, and blocking was incubated in blocking buffer (3% Bovine Albumin Serum in 1XPBS) for 1 hr. Primary antibodies insulin-like growth factor-receptor (IGF1-r; ab263903; Abcam, Cambridge, UK), Pregnancy associated plasma protein A (PAPP-A; ab249813; Abcam) and Tissue inhibitor of metalloproteinases 2 (TIMP-2; ab230511; Abcam) were each diluted in blocking buffer and incubated at room temperature for 2 hrs. Washed in 1XPBS and incubated for 1 hr with Secondary HRP Anti-Rabbit IgG secondary antibody (ab288151; Abcam) or HRP Anti-Mouse IgG secondary antibody (ab47827; Abcam). Washed and reacted for 10 min using ABC detection kit (Vector, CA, USA) and confirmed the reaction using Dia-minbenzidine (Vector, CA, USA) after staining with a hematoxylin solution containing 4% acetic acid and PAS. The samples were analyzed under an optical microscope.

In-situ zymography

Boiling in 10 mM sodium citrate for 10 min after deparaffinize and hydrate, then emulsion (ddW, 10% SDS, 2% Glycerol) and zymography reaction buffer were mixed at a ratio of 1: 2 and raised to slide, followed by enzymatic reaction at 37℃ for 48 h in a slide box filled with 1 M Tris. After the reaction, routine hematoxylin and eosin (H&E) staining was performed for histological inspection with optical microscope (Oh and Kim, 2022).

Immunofluorescence

Paraffin blocks of uterine and caruncle tissues in each group were sectioned to a thickness of 5 μm. Tissue slides were prepared following deparaffinization, hydration using xylene and ethanol, and permeabilization at -20℃ with 0.1% Triton X-100 in 1 × PBS (PBS-T). The samples were blocked at room temperature (RT) for 30 min in TPBS (1 × PBS with 0.01% Tween-20) containing 5% normal horse serum (NHS) and 1% normal goat serum (NGS). All tissue slides were incubated in a dark room at RT for 1 hr, with primary antibody vascular endothelial growth factor (VEGF; 2893S; Cell signaling, United States, USA) antibody diluted 1:200 in blocking solution. After that, to induce secondary antibody conjugation, incubated in the dark with a blocking solution for 30 min. After that, all slides were incubated with Alexa 594-conjugated anti-rabbit secondary antibody (ab150080; Abcam) diluted 1:300 in blocking solution in the darkroom at RT for 1 hr. Decant the second secondary antibody solution and wash it three times with PBS for 5 min each in the dark.

In situ apoptosis detection

Annexin V staining was performed using PE Annexin V Apoptosis Detection Kit Ⅰ (BD Biosciences, San Jose, CA). After washing three times with 1XPBS, 5 uL each of Annexin V and 7-AAD into 100 uL of 1X binding buffer, staining was performed for 1 hr and 30 min at room temperature for analysis.

RESULTS

Morphology and Ca2+ analysis of water deer and sika deer uterus on the estrus

The results of confirming the morphological differences between the endometrium and myometrium of water deer and sika deer during estrus are shown in Fig. 2. It was confirmed that the size of the fetus villus cytotrophoblast zone in the endometrium of the water deer is smaller and more concentrated than that of the sika deer and that the cells of the myometrium have a lower cell density than those of the sika deer. In the case of sika deer, the size of the fetus villus cytotrophoblast zone in the endometrium is larger, and the density is lower than that of water deer, while the cell density appears to be high in the myometrium. Alizarin Red Staining was performed to check cell activity, and overall, water deer showed higher activity than sika deer. Endometrium showed high activity in the fetus villus of water deer, and myometrium was similarly highly active overall in the cells of water deer.

Figure 2.Morphological and physiological analysis of the estrous uterus of water deer and sika deer. Activation of Ca2+ was concentrated in the uterus zones and activated in the glands in both water deer and sika deer.

Analysis of protein expression pattern and MMPs activation in water deer and sika deer uterus on the estrus

The results of comparing the expression patterns of growth-related genes in the ovarian uterus of water deer and sika deer are shown in Fig. 3. The expression of PAPP-A was highly expressed in the fetus villus zone of water deer, and in the case of myometrium, it was confirmed that it was expressed relatively higher in sika deer than in water deer. On the other hand, the expression of IGF-r is generally higher in sika deer than in water deer, and it can be confirmed that it is expressed higher in the fetus villi than in the stroma cell zone. Comparing the activity of MMPs that promote changes in the Extracellular Matrix (ECM) to measure uterine remodeling revealed that the overall activity was much higher in sika deer than in water deer. Additionally, the expression of TIMP-2 showed no difference in both stags and sika deer (Fig. 4).

Figure 3.Tissue immunohistochemical analysis of BCL-2, E2-r, PAPP-A, and IGF1-r protein expression in the uterus. Black arrows indicate the expression location of the protein. The endometrium is expressed in the glands, and ectopy is expressed in the stroma and basal layer.
Figure 4.Analysis of MMP activity and TIMP-2 expression in uterine tissues. In the in-situ zymography, water deer generally expressed MMPs in the stromal zone, whereas sika deer expressed MMPs in the glands zone. Magnification = 200×.

Detection of VEGF protein on estrus uterus

As a result of analyzing the expression pattern of VEGF protein, which affects angiogenesis (Fig. 5), the expression of VEGF was high in the villus zone of the endometrium zone. In the endometrium, sika deer had overall increased VEGF expression than water deer. However, in the myometrium zone, VEGF expression appears more widespread in water deer than in sika deer, but the expression level was low. Regarding the overall expression pattern, higher expression of VEGF was detected in the endometrium of sika deer compared to water deer.

Figure 5.Detection of VEGF protein expression patterns in the uterus tissues. Green fluorescence indicates gene expression. It is expressed mainly at very high levels in the uterus of water deer and is relatively low in sika deer. White arrows indicate the expression location of the protein. Magnification = 200×.

Apoptosis activation of the uterus on the estrus

Fig. 6 shows the results of apoptosis detection analysis to confirm apoptosis, which acts on cell reorganization and uterine maintenance. As a result of confirming apoptosis throughout the uterus, apoptosis was relatively higher in the specific villus cytotrophoblast zone of sika deer than in water deer. In the case of water deer, there was no overall expression of apoptosis, but it was confirmed that it was expressed in blood vessels.

Figure 6.Detection of DNA fragments in water deer and sika deer uterus tissue. The apoptosis detection assay used terminal deoxynucleotidyl transferase to label the 3-OH termini of DNA fragments generated during apoptosis. White arrows indicate areas with distinct expressions. Magnification = 200×.

DISCUSSION

In the endometrial follicular phase, estrogen induces proliferation of the endometrial epithelium. In particular, the endometrium, which undergoes biochemical and morphological changes, is a target tissue for progesterone and estrogen. During the estrous cycle, the bovine uterus undergoes morphological and physiological changes (Chen et al., 2015), such as the development and secretion of uterine glands and changes in the thickness of the uterine wall. In our study, the development of villus cytotrophoblast of endometrium in sika deer was lower, and the size was lower than in water deer. Although it is relatively large, the deposition rate of Ca2+ is generally low, and in the myometrium, it can be seen that the overall cell density of sika deer is high. However, as in the endometrium, the deposition rate of Ca2+ is high in water deer, so the metabolic maturation of villus acts relatively differently, and it is predicted that metabolic activity and cellular changes will be higher in sika deer than in water deer (Spencer et al., 2005; Kotlarczyk et al., 2021).

In addition, the activation and morphological changes of the uterus at the same time show a big difference in sika deer and water deer, which is thought to be able to rapidly change the composition of the uterus for pregnancy as the activity of PAPP-A and estradiol receptor (E2-r) increases high in the villus cytotrophoblast of water deer (Yanagawa et al., 2008; Lee et al., 2023). However, in the analysis of histological changes, the development of villus cytotrophoblast in the uterus of sika deer seemed to be higher than that of water deer, which is expected to have acted differently depending on the influence of progesterone, which accumulates in high concentrations during the period of change in the uterus of sika deer. In general, the target tissues of P4 and E2 are activated by estrogen receptors for biochemical and morphological changes and activate histological changes by inducing the action of MMPs released from uterine cells (Kotlarczyk et al., 2021). In particular, the activity of MMP-2, which dissolves collagen and elastic fibers in the extracellular matrix and is involved in angiogenesis, tissue reorganization, and activation of growth factors, can be seen to be high in sika deer.

In addition, VEGF activity, which plays an essential role in the tissue remodeling process, increases in the villus cytotrophoblast zone. The extravascular zone of the myometrium of water deer and sika deer is most highly active in the endometrium of sika deer, thereby increasing the activity of vascular epithelial cells surrounding tissues in the uterus (Kim et al., 2014a, 2014b, 2018). Promotes the proliferation of in other words, it is thought that changes in the uterus of the sika deer stabilize the villus earlier than changes in the uterine tissue of the water deer. According to the research results of Sohn et al., 2021, in the case of water deer, the placenta is classified as oligocotyledon, similar to other plumids with 6-8 placentas, and the villus is well developed, so a distinct glands zone is visible in each villus (Yanagawa et al., 2008).

In the case of the sika deer, the overall shape is similar to that of the water deer, but the branching state of fetal villi in the convex-shaped placenta is known to be different for each Cervidae family (Hamasaki et al., 2001; Sohn and Kimura, 2012). In other words, the morphological findings of water deer and sika deer are similar regarding the shape of the uterus and placenta. However, it can be confirmed that there is a difference in the shape of the uteroplacental junction that spreads from the uterus to the placenta. These morphological differences in the uterus and the overall tissue reorganization formed during the estrus period of the sika deer can be seen as a unique shape unique to the sika deer, and may differ depending on the Cervidae family (Wooding et al., 2018; Sohn et al., 2021). However, most sika deers’ wombs rapidly remodel uterine tissues compared to water deers. Through the expression of high concentrations of progesterone and the cooperation of MMPs formed in tissues, they quickly restore the condition of the uterus when pregnancy fails.

Therefore, through this study, it is believed that the action of hormones related to the estrus period of sika deer and the activity of genes related to uterine remodeling act differently depending on the presence or absence of pregnancy, which will play a role in determining the timing of pregnancy in sika deer in the future. In addition, since samples of the sika deer, which has become extinct in Korea, can no longer be collected due to international issues, our research can be valuable data for future species restoration.

CONCLUSION

This study analyzed changes in the uterus compared to the physiological state of water deer during estrus to confirm the physiological state of smooth pregnancy in sika deer as a domestic animal. As a result of the study, we confirmed that the state of the uterus during estrus shows a significant difference from that of water deer, which has similar reproductive physiology, and that there is a significant difference in the action of MMPs to reorganize tissues and the resulting morphological changes. In particular, the sika deer uterus has a similar shape to the pregnant uterus during estrus. Therefore, functional development associated with the conception is quite different from that of the water deer, and that there will be a unique reproductive action of the sika deer of the Cervidae family.

Acknowledgements

The authors sincerely thank the Research Center for Endangered Species and the National Institute of Ecology for their support in conducting this study.

Author Contributions

Conceptualization, S-H.K.; data curation, S-H.K.; formal analysis, Y-S.P., M-G.O.; funding acquisition, Y-S.P., S-H.K.; investigation, S-H.K., M-G.O.; methodology, M-G.O.; project administration, S-H.K.; resources, S-H.K.; supervision, S-H.K.; roles/writing - original draft, Y-S.P., M-G.O.; writing - review & editing, Y-S.P., S-H.K.

Funding

None.

Ethical Approval

All experiment were conduced in accordance with the guidelines established by the Institutional Animal Care and Use Committee of Sangji University approved protocol (#2021-23).

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.

Availability of Data and Materials

Not applicable.

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Fig 1.

Figure 1.Different parts of genitalia for biometric recording of water deer and sika deer uterus. (A) Sika deer. (B) Water deer. (a) Uterus parts of sika deer. (b) Uterus parts of water deer.
Journal of Animal Reproduction and Biotechnology 2023; 38: 254-262https://doi.org/10.12750/JARB.38.4.254

Fig 2.

Figure 2.Morphological and physiological analysis of the estrous uterus of water deer and sika deer. Activation of Ca2+ was concentrated in the uterus zones and activated in the glands in both water deer and sika deer.
Journal of Animal Reproduction and Biotechnology 2023; 38: 254-262https://doi.org/10.12750/JARB.38.4.254

Fig 3.

Figure 3.Tissue immunohistochemical analysis of BCL-2, E2-r, PAPP-A, and IGF1-r protein expression in the uterus. Black arrows indicate the expression location of the protein. The endometrium is expressed in the glands, and ectopy is expressed in the stroma and basal layer.
Journal of Animal Reproduction and Biotechnology 2023; 38: 254-262https://doi.org/10.12750/JARB.38.4.254

Fig 4.

Figure 4.Analysis of MMP activity and TIMP-2 expression in uterine tissues. In the in-situ zymography, water deer generally expressed MMPs in the stromal zone, whereas sika deer expressed MMPs in the glands zone. Magnification = 200×.
Journal of Animal Reproduction and Biotechnology 2023; 38: 254-262https://doi.org/10.12750/JARB.38.4.254

Fig 5.

Figure 5.Detection of VEGF protein expression patterns in the uterus tissues. Green fluorescence indicates gene expression. It is expressed mainly at very high levels in the uterus of water deer and is relatively low in sika deer. White arrows indicate the expression location of the protein. Magnification = 200×.
Journal of Animal Reproduction and Biotechnology 2023; 38: 254-262https://doi.org/10.12750/JARB.38.4.254

Fig 6.

Figure 6.Detection of DNA fragments in water deer and sika deer uterus tissue. The apoptosis detection assay used terminal deoxynucleotidyl transferase to label the 3-OH termini of DNA fragments generated during apoptosis. White arrows indicate areas with distinct expressions. Magnification = 200×.
Journal of Animal Reproduction and Biotechnology 2023; 38: 254-262https://doi.org/10.12750/JARB.38.4.254

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