Successful development of follicles is critical to the production of offspring and the maintenance of pregnancy. In particular, it also reconstructs the body through the production of estrogen and progesterone and the feedback action that can control them (Selam and Arici, 2000; Bulun et al., 2002). The primary cells organized in the ovaries are Granulose cells, theca cells, and corpus luteum cells, especially corpus luteum cells, which form luteum cells after ovulation and are remodeled from theca cells and granulosa cells to the function of corpus luteum (Kim et al., 2014a; 2019). The functional normality of the ovary allows normal physiological functions such as inducing estrus and maintaining pregnancy at the correct time. It is known that most mammals produce offspring with this phenomenon. The Water deer known so far have similar habits to Sika deer, and their estrus and pregnancy periods are also very similar. The development of follicles in Sika deer on the estrus period is known to go through at least 5 to 8 cycles, and ‘silent ovulations’ are sometimes formed like monovulatory species (Asher et al., 1997; McCorkell et al., 2006; Asher, 2011).

In visual difference of ovaries, the ovaries of Water deer and Sika deer have very flat surfaces, and it is difficult to observe protruding ovaries compared to the ovaries of sheep (Hamasaki et al., 2001; Menchaca and Rubianes, 2004) or goats (Menchaca et al., 2007), which have irregular the surface due to severe protruding follicles and corpus luteum (Casida et al., 1966; Pérez et al., 2013). In addition, the estrus cycle patterns of Water deer and Sika deer are very similar, and it is known that ovulation occurs about 5 to 8 times on the estrus, which can be seen as having rapid changes in the state of the ovary (Monfort et al., 1990). ‘Silent ovulations’ occurs for about 8 to 10 days in groups of Water deer and Sika deer on breeding season, which is similar to monovulatory species that ovulate without apparent signs of estrus, and it is that corpus luteum remodeling is different from Artiodactyla animals, and growth and regression of discontinuous follicle on the estrus forms 2 to 4 dominant follicles (Asher et al., 1997; 1999; McCorkell et al., 2006).

The cortex of the ovary contains numerous primordial follicles from birth, and the appropriate number of primordial follicles in each sexual cycle become mature follicles through primary follicles, secondary follicles, tertiary follicles, and ovulate (Day, 1991; Braw-Tal and Yossefi, 1997). Most of the follicles that grow in every sexual cycle are denatured in the middle, which becomes fibroblasts, and only a few follicles grow and mature and ovulate, and in that area, change corpus rebrum, corpus luteum, corpus albicans, and disappear (Day, 1991). In general, abnormal reproductive physiology occurs when the enlarged follicle or corpus luteum does not disappear or is delayed (Rayos et al., 1986; Farin et al., 1992). Although there are no accurate reports that in the coexistence of large follicles and large corpus luteum on the individual, follicles grow into large follicles in the sexual cycle, the follicles grow the maximum size, ovulate in 19-21 days of the sexual cycle, and corpus luteum maintains maximum size until 17 days and degenerates after 18 days, which completely degenerates on 1-2 days of estrus.

Species belonging to the Sika deer family have a similar pattern to the estrus cycle of the Artiodactyla, and the development of follicles and formation of corpus luteum is expected to be similar. It is known that Water deer have a breeding season from November to January, 210 days of pregnancy period (Brown, 1991), Sika deer and red deer have a breeding season from September to December, 234 days of pregnancy period (Spector, 1956). Most of the research results on deer species are reported on ecological studies, and physiological and morphological studies on species reproduction are insufficient, especially reproductive physiological research for restoration of Water deer and Sika deer, which are designated as endangered and extinct animals, is insufficient. Therefore, this study aims to histologically investigate the difference in development between follicles and corpus luteum and hormone receptors in the sexual cycle.

Animals

In order to collect the ovaries of Water deer, which are endangered wild animals, and Sika deer, which is known extinct animals in Korean peninsula, Water deer were captured when it was possible to catch them in Korea peninsula. In accordance with Article 19 paragraph 1 of the wildlife protection and Management Act, the Water deer was captured in Ansan, Gyeonggi-do, Korea by the guidance of the Institutional Animal Management and Use Committee of the Wildlife Conservation and Research Center (Capture Permit No. 2013-1) from November to January in estrus, in case of pregnancy, Water deer which completed placenta formation captured from March to May. Sika deer captured individuals which presumed to be Korean Peninsula deer living in Primorskaya State Agricultural Academy and the border of Primorskaya and North Korea, and used ovaries obtained in similar time to Water deer. And divided estrus and pregnancy (in the previous study, Primorskaya State Agricultural Academy signed an MOU [date : 11.25.2014]).

Paraffin block and H&E staining

Each group’s ovaries were collected and fixed in 70% ethanol, dehydrated, paraffin embedded, and sectioned at 10 mm thickness. After representative sections from each ovary paraffin-block were randomly selected, hematoxylin and eosin (H&E) staining was performed to facilitate histological examination using an optical microscope (AX70, Olympus, JAP).

Immunohistochemistry

These samples were then sequentially treated with xylene, 100% ethanol, 95% ethanol, and ddW and then boiled in 10 mM sodium citrate for 5 minutes. Antigen retrieval was accomplished by heating each sample in 10 mM sodium citrate buffer (pH 6.0). After rapid cooling, endogenous peroxidase activity was validated using a 0.3% hydrogenperoxide solution. After three washes in 1×phosphate buffered saline (PBS), each slide was blocked using 1% goat serum and 5% horse serum. Primary antibodies against LH-r (ab125214; Abcam, Cambridge, UK) and Estrogen-r (ab66102; Abcam) and VEGF-r1 (ab32152; Abcam) were then applied and left to incubate overnight at 4℃. Following a 5-minute wash using 1×PBS, HRP Anti-Rabbit IgG secondary antibody (ab288151; Abcam) was added, and the slides were incubated for 2 hours at room temperature. Each sample was washed three times for 5 minutes each with 1×PBS, and then 200 μL of reaction substrate (3,3’diaminobenzidine, DAB) was added, and the reaction was allowed to proceed for up to 10 minutes before washing with ddW. Counter staining with a hematoxylin solution completed the processing, and each sample was covered with a dehydration, clarification, and permount solution (Thermo Fisher Scientific, Waltham, MA, USA) and observed using an optical microscope (Nikon, Tokyo, Japan).

Alizarin red staining for Ca²⁺ analysis

In order to perform the Alizarin staining, sections were deparaffinized and dehydrated using xylene, 100% ethanol and 95% ethanol, washed in ddW for 5 minutes, and then stained in Alizarin Red (A5533, Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes. After washing, dehydration permounting was performed.

Morphological and Ca²⁺ analysis of ovary in Water deer and Sika deer

The results of analyzing the ovary morphology and Ca²⁺ reaction of Water deer and Sika deer on the estrus and pregnancy are shown in Fig. 1 and 2. It was difficult to observe clear morphological changes in Water deer and Sika deer on the estrus and pregnancy. However, morphological differences in many areas were observed in Water deer and Sika deer. Water deer have large follicular and corpus luteum except for development follicular (Fig. 1). In particular, the development of theca cells around the follicle was high, and it was confirmed that the reaction of Ca²⁺ in Water deer ovary was expressed from extra to intra section of the theca cells. The corpus luteum of Water deer was observed large or small lutein cells, and the reaction of Ca²⁺ was very lowly in the large lutein cell zone on pregnancy. In the corpus luteum on the estrus, the reaction of Ca²⁺ was confirmed to affect corpus luteum remodeling as it was expressed from connecting tissue to the extra zone. Unlike the protruding corpus luteum of Water deer, the corpus luteum of Sika deer was not observed to be slightly modified resistant to Water deer. Also, early development follicular was mainly observed rather than large follicular contrary to Water deer. The distribution of stroma cells composed of the entire ovary was extensive, indicating the high development of granulosa cells and theca cells in follicles. The reaction of Ca²⁺ in the Sika deer ovary on pregnancy showed a similar pattern to Water deer at the follicles (Fig. 2). However, the reaction in follicles on the estrus was expressed contrary to Water deer. The corpus luteum of Sika deer showed many differences in morphological changes and cell shape compared to that of Water deer. It was confirmed that the reaction of Ca²⁺ was very low on the estrus especially.

Figure 1.Histological characterization analysis of follicular and corpus luteum during estrus and pregnancy in Water and Sika deers. This figure shows the histological differences between Water deer and Sika deer corpus luteum by H&E analysis. eDFC, ealy Deveropmental Follicular cells; LFC, Large Follicular cells; GC, Glanulosa cells; CL, Corpus luteum. (bar = 100 μm; The microscope magnification is ×100; Magnification of corpus luteum cells is ⅹ400).

Figure 2.Alizarin Red staining. Overall, calcium concentrations were higher in non-pregnant corpus luteum cells of Water deer and higher in pregnant corpus luteum of Sika deer. FC, Follicular cells; GC, Glanulosa cells; CL, Corpus luteum. (bar = 100 μm; The microscope magnification is ×100).

Expression patterns of hormone receptors and VEGF in ovaries on estrus and pregnancy

The results of analyzing hormone receptors and vascular endothelial growth factor (VEGF) in Water deer and Sika deer on the estrus and pregnancy are shown in Fig. 3 and 4. As a result of analyzing the estradiol and luteinizing hormone (LH) receptors of the ovary in Water deer and Sika deer, the ovary in the estrus was higher than that in the pregnancy. Estradiol receptor was expressed in theca and granulosa cell layers of follicles in Water deer and Sika deer on the estrus. Low expression was shown in the Sika deer corpus luteum on the estrus, and the corpus luteum of Water deer expressed lutein cells and theca cells, especially high expression in connecting tissue. In addition, the expression of atretic follicles, theca cells, and some sections of corpus luteum was very high in Sika deer on pregnancy (Fig. 3). The expression patterns of the luteinizing hormone receptor in Water deer and Sika deer on the estrus were higher than that on the pregnancy. The expression of some corpus lutein cell sections in Water deer on pregnancy was high. The expression in Water deer was higher than in Sika deer on estrus corpus luteum, and lutein cell and connect tissue sections were generally highly expressed in Water deer, and connect tissue and theca cell sections were mainly expressed in Sika deer. Also, LH receptor expression was rarely observed in Sika deer corpus luteum tissue (Fig. 3). The expression of VEGF was higher in the corpus luteum on the estrus and pregnancy. Water deer tissue was higher expression patterns than Sika deer, especially the corpus luteum of Water deer. In the case of Sika deer tissue, connecting tissue and vesicular vessels were more highly expressed than Sika deer ovary (Fig. 4).

Figure 3.Immunohistochemistry of Estrogen-receptor and Luteinizing Hormone-receptor protein detected in the follicular cell and corpus luteum during estrus and pregnancy. The difference in the expression of E2, and LH receptor, was confirmed in Water and Sika deer’s. The localization image detected in brown the target protein’s degree of detection. Black arrows indicate gene expression (bar = 100 μm; The microscope magnification is ×100).

Figure 4.VEGF-r1 protein detection in ovarian tissues. VEGF-r1 protein expression was highest in the corpus luteum tissue of Water deer, and very lowly in other regions (bar = 100 μm; The microscope magnification is ×100).

The cortex of the ovary have numerous primordial follicles from birth, and appropriate number of primordial follicles in each sexual cycle become mature follicles through primary follicles, secondary follicles, and tertiary follicles, and ovulate (Day, 1991; Braw-Tal and Yossefi, 1997). Change of the sexual cycle are influenced by hormones and morphological changes on development of follicles in ovary is seen. Morphological changes in the ovary affect reproductive physiological functions and are very important in determining the normal estrus cycle through endocrine activity (Adriaens et al., 2004; Kim et al., 2014a). In particular, the formation of the corpus luteum after ovulation is an important organ that produces progesterone for pregnancy maintenance and tissue remodeling for estrus relapse. It also affects the physiology of individual reproduction and performs a successful hormonal feedback (Kawate et al., 2000; Kim et al., 2021). However, in some seasonal animals, the development and regression of follicles occurs rapidly during estrus and pregnancy, and silent ovulation are sometimes formed, which leads to unstable reproductive physiology (Asher, 2011). That is, the morphological changes of the ovaries on estrus and pregnancy showed a different pattern from that of domesticated cattle, especially, it is thought to show differences from function of cattle corpus luteum on estrus and pregnancy (Pérez et al., 2013; Kim et al., 2014b; 2019). This study aims to analyze the difference in morphological changes and sexual hormones in ovaries of endangered or huntable in the Korean Peninsula type Sika deer near Russia and Water deer, and in the future, it is intended to be used as a basic study to judge changes on developmental field in restoration of Sika deer, endangered species. According to the research of Brown (1991), there was no significant difference of breeding season between Water deer and Sika deer, and it was thought that ecological action of Sika deer would be similar to that of Water deer. The Water deer has a breeding season from November to January, and 210 days of pregnancy period (Brown, 1991), Sika deer and red deer have a breeding season from September to December and give birth from April to July after 234 days of pregnancy (Spector, 1956). These two species have similar reproductive cycles, and are known to have simultaneous estrus in the groups, and to have sexual cycle from 5 to 8 times on the breeding season according to follicle development (Menchaca and Rubianes, 2004; Menchaca et al., 2007). However, in this study, a comparative analysis of the ovaries of these two species confirmed that were many difference. In this study, as a result of the study on the functional mechanism of the corpus luteum of Water deer and Sika deer, in morphological analysis, Water deer was observed formation of at least one graffian follicles and development of developmental follicles after the corpus luteum was formed (Kim et al., 2014b). On the other hand, in Sika deer, the development of developmental follicles was represented, graffian follicles was not represented, and the shape of corpus luteum tissue was less than Water deer. The estrus ovaries formed a smaller follicles and a corpus luteum than observed in other small ruminants as shown in study of Pérez et al., 2013, and it was seem to having different morphological change in Water deer, compared in our study. Calcium is stored or released into the cytoplasm through mitochondria and endoplasmic reticulum, cell organelles, and is known as a secondary messenger that causes various cellular reactions (Malcuit et al., 2006). In addition, it is known that intracellular calcium ions can affect the activity of mitochondrial enzymes related to metabolism located on the outer surface of the inner membrane of mitochondria (Contreras et al., 2010). In other words, calcium, which affects ovarian development and metabolic activity in the corpus luteum, seems to act differently in sika deer and water deer. In particular, calcium reaction in Sika deer was low in both pregnancy and estrus, the reaction in Water deer was very low in large lutein cell zone on pregnancy, and was high expression from connect tissue to extra zone in corpus luteum on the estrus, and it is seen that calcium activity affected the corpus luteum remodeling (Malcuit et al., 2006; Contreras et al., 2010). The higher expression of estradiol receptors in Water deer and Sika deer on the estrus than on pregnancy was appeared according to development of follicles. The expression of the luteinizing hormone receptor was higher on the estrus of the Water deer, especially expression of VEGF receptor was high in corpus luteum tissue in Water deer, and it is considered to maintenance and formation of corpus luteum according to ovulation. Really, it is considered that the formation and regression of follicles depend on the presence of corpus luteum, which is that secretion of progesterone causes the functional degradation of FSH and high LH maintains continuously corpus luteum, so development of follicles is inhibited by insufficient FSH (Stocco et al., 2017). Hormonal changes according to the estrus cycle affected formation and maintenance of corpus luteum and follicle development, as results of studies by Lee et al. (2023) and others, the results of analyzing the ovarian cycle change according to season of horse, which seasonal breeding such as deer families, are ovarian cycle delay type I, which corpus luteum maintains, ovarian cycle delay type II, which ovulation delay, and ovarian cycle delay type III, which ovarian cycle is prolonged. The function of the ovary is evaluated by the structure in the ovary. Therefore, the results of study were shown that the composition of the corpus luteum of Sika deer is not clear compared to Water deer, and there are many differences in the functional part and morphological formation of the corpus luteum, and suggested that the function of the ovary involved in the pregnancy and maintenance of Sika deer is also affected.

None.

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

None.

Not applicable.

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