The action of hormones plays an essential role in the formation and reconstruction of reproductive organs, which is known to determine the reproductive cycle through the smooth action of the feedback loop. In particular, progesterone (P4) and estradiol (E2), which are essential steroid hormones for the typical estrus system, affect the morphological and physiological changes of the uterus and influence the maintenance of estrus and the return of the uterus (Kumar et al., 2018). In the case of P4, it reproduces by receptors containing progesterone receptor (PGR) A and PGRB. It affects endometrial cells to maximize uterine proliferation and blood vessel formation, thereby composing a state where implantation and pregnancy are possible (Rekawiecki et al., 2016). In addition, in the case of E2, it functions by binding to a receptor composed of two subtypes of estrogen receptor (ER) α and ERβ, thereby reconstructing the uterus to its initial estrus state (Kumar et al., 2018). In general, hormone signaling for the morphological determination of the reproductive system appears to play a significant role in the action of P4 and E2. It has been considered that the influence of other hormones is not significantly involved.

A recent study by Kim et al. (2020), suggested that the hormonal hypersecretory action of PMSG could affect endometrial cell remodeling and angiogenesis, which could induce functional and morphological changes due to abnormal endometrial changes. In addition, it is suggested that the action of LH/hCG receptors in endometrial cells in the early estrus period can be activated to form a feedback loop that can affect endometrial cell growth (Reshef et al., 1990). A common disease formed from hormonal imbalance is the induction of polycystic ovary syndrome (PCOS), which aggravates ovarian dysfunction, pregnancy complications, endometritis and venous thrombosis in the uterus (Mahmood and Templeton 1991), and abnormal expression of the hormone due to the abnormal action of PGR (Margarit et al., 2010; Hu et al., 2018).

In addition, some studies suggest that high concentrations of androgens (or testosterone) may increase the onset of PCOS (Kumar et al., 2005; Azziz et al., 2009). In other words, it can be said that the critical action on the morphological change of the uterus depends on the secretion of hormones. In particular, the action of testosterone, a high androgen formed from the adrenal gland, in the case of a decrease in female fertility and the problem of hormonal feedback mechanisms (Kumar et al., 2005), acts directly on the endometrium of women and affects the proliferation, differentiation, and tissue reorganization of endometrial cells (Milne et al., 2005).

However, in many studies, the morphological changes of the reproductive organs due to the hypersecretion of each hormone have been limited to the ovaries and the uterus, and there are very few studies on the morphological changes of the vagina plays an essential role in the external genitalia. Therefore, this study aimed to analyze the effects of hormone hypersecretion on the morphological changes of the vaginal tissue and to analyze abnormal growth in the reproductive organs caused by hormone hypersecretion.

Preparation and certification of animals

All experiments applied the experimental method of Kim et al. (2020). All animal procedures followed the protocol approved by the Animal Experimental Ethics Committee of Hankyong National University (Permission No.: 2019-1). All surgeries were performed under pentobarbital sodium anesthesia and every effort was made to minimize pain. All ovaries were treated to stimulate ovulation and then used in the experiment (Kim et al., 2020). The experimental group was injected with 5 IU of the hormone every 2 days for 14 days. Six female mice were randomly divided into three groups, including: the normal control group (NC), hCG injected group (hCG group), progesterone group (P4 group), and testosterone group (Te group). The mice of each group collected the vagina tissue according to the dissection method (Kim et al., 2020).

Histological of the vagina tissue

Each group vagina tissue was collected and fixed in 70% Diethyl pyrocarbonate (DEPC)-ethanol, dehydrated, paraffin-embedded, and sectioned at 5 um thickness. After representative sections from each uterus paraffin-block in the treatment group was randomly selected, and routine hematoxylin and eosin (H&E) staining and DAPI fluorescence (V11324, Thermo Fisher Scientific Solutions, Massachusetts, USA) staining was performed for histological inspection with optical micro-scope (×200, ×400) (Kim et al., 2018).

In-situ Zymography

In order to perform an in-situ Zymography experiment, Deparaffinize / hydrate was firstly repeated twice in Xylene, 100% Ethanol, 95% Ethanol for 10 minutes, washing in ddW for 5 minutes, and boiling in 10 mM sodium citrate for 10 minutes. After that, emulsion (ddW, 10% SDS, 2% Glycerol) and zymography reaction buffer were mixed at a 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 was completed, the slide was dried at 37℃, stained with Hematoxylin and Eosin (Kim et al., 2018). And then overwritten with cover glass and analyzed with an optical microscope (×200, ×400).

Immuno-detected of vagina tissue protein

Casp-3 (Abcam, Cambridge, UK) primary antibodies and proteins were applied to 96-well ELISA plates and activated at 4℃ for 1 day to evaluate specific proteins in each cell. In the next step, after immune reactions were made using secondary antibodies (rab-bit IgG-HRP antibody, Santa cruz biotechnology) for 2 h at room temperature, a substrate solution (R&D Systems, MN, USA) was added. After that, absorbance was measured at 450 nm.

Statistical analysis

ELISA data were tested for significance (Duncan and General Linear Model) using Statistical Analysis System software (SAS Institute, version 9.4, NC, USA). Statistical significance was established at < 0.05.

Morphological changes in vaginal tissue due to hormonal hypersecretion

Fig. 1 shows the analysis results of changes in vaginal lining cells and secretory tissue in each hormone hypersecretion state. Changes in the mucosa zone and the formation of CT papillae, which are essential sections for confirming the morphological changes of vaginal tissue, can be considered to affect the secretion and growth of vaginal mucus. This result shows that the basal membrane is affected at the time of hormone hypersecretion, compared to the CT papillae with a sharp curve seen in the vaginal lining during normal estrus. In the case of the hCG group, the mucosal layer of the mucosa zone of the vaginal lining was not evenly distributed compared to the normal group, and the formation of CT papillae was not high. However, it was confirmed that the growth of the Mucularis and the Adventitia zone increased. In the case of the progesterone (P4) group, the formation of the mucosa layer in the mucosa zone was evenly distributed, and it was confirmed that the mucosa zone section was maintained typically. However, in the case of the testosterone (Te) group, it was confirmed that the Adventitia and Mucularis sections were expanded, the cell invasion density was low, and the mucosa zone was regressed. In addition, the formation of CT papillae was very low, and abnormal cell deposition was confirmed in the basal membrane.

Figure 1.Histological characterization of the vagina in the mouse. (A) Normal group, (B) hcg group, (C) P4 group, (D) Te group. 1: Squamous epithelium cell zone, 2: Lamina prepria (bar = 100 μm; magnification: Large picture ×200, small picture ×400).

Analysis of cell distribution in vaginal lining tissue

Fig. 2 shows the results of the analysis of cell sedimentation density in the vaginal mucosa zone, Mucularis zone, and Adventitia zone. In the case of the normal and hCG groups, cell growth and cell growth in the mucosa zone were increased, whereas in the P4 group, the cell development rate in the Mucularis and Adventitia zones was increased. In the case of the mucosa zone, it was confirmed that the cell invasion density was low, but the cytoplasmic distribution was increased. However, in the case of the Te group, overall cell invasion density and cell number were significantly lower than those of the other groups, and abnormal cell generation in the basal membrane zone could be confirmed.

Figure 2.Cellular distribution analysis of mouse vagina tissue. (A) Normal group, (B) hcg group, (C) P4 group, (D) Te group (bar = 100 μm; magnification: ×400).

Analysis of MMP activity and Casp-3 expression pattern in the vaginal tissue

Fig. 3 shows the results of analyzing the activity of MMPs for each section of the vaginal tissue. In the case of the normal group, MMPs activity in Mucularis and Adventitia zone, which are internal tissue systems, was generally low. In contrast, MMPs activity in mucosa zone was very low. The hCG group did not differ much from the normal group, but some MMPS activity was observed in the mucosa zone. In the case of group P4, the activity of MMPs was increased in the Mucularis and Adventitia zone, which are the internal tissues of the uterus, and it was further increased in the basal membrane. In the case of the Te group, it was confirmed that the activity of MMPs in the mucosa zone was very high compared to the other groups, the activity in the basal membrane was very low, and the activity of MMPs in the Mucularis and the Adventitia zone was low. As a result of analyzing the expression pattern of Casp-3, it was significantly increased in the Te group and very low in the P4 group.

Figure 3.MMPs activation analysis of mouse vagina tissue. (A, B) In-situ zymography. (A) hcg group, (B) P4 group, (C) Te group. Black arrow is MMPs activation zone. (D) ELISA analysis (bar = 100 μm; magnification: Large picture ×400, The small picture is a 10× magnification of the large picture). *Different letters within the same column represent a significant difference (p < 0.05).

Smooth hormonal action changes the uterus on time, leading to successful implantation and return of estrus (Kumer et al., 2018). In addition, the occurrence of PCOS due to hormone hypersecretion can lead to widespread diseases of the reproductive organs and disrupt the normal estrus cycle by inhibiting the action of the feedback loop (Hu et al., 2018). Furthermore, recent studies have shown that the effects of PMSG, hCG, progesterone, and testosterone, which regulate the overall period of estrus, affect reproductive organs and the period of estrus (Cousins et al., 2014; Rekawiecki et al., 2016; Kim et al., 2020), the action of each hormone receptor can be seen to affect throughout the reproductive organs. However, little is known yet about whether hormone hypersecretion affects women’s vaginal morphological changes and cells, and studies on physiological changes are insufficient.

This study measured the change in the vagina after maintaining each hormone in an excess state for a certain period in a mouse with estrus synchronized (synchronization) and analyzed the activity of MMPs to determine the final tissue change. The normal vaginal formation is in the growth of the Mucosa zone, and it was confirmed that sufficient maturation was achieved during estrus. However, as the hypersecretion of hormones was maintained, there was no significant difference in hCG, and changes in the mucosa and mumularis zones were also not significantly different, indicating that although it was different from the results of Papanikolaou et al. (2013), it affected the formation of mucosa and basal membrane and CT papillae sections.

In the case of P4, it appears to grow in a form similar to that of uterine changes, as in the results of Kim et al. (2020), so it is thought to affect the squamous epithelium cell zone of the vagina, and it is also thought to affect the change in the cytoplasm (Cousins et al., 2014; Rekawiecki et al., 2016). However, unlike the study results of Kim et al. (2014), that progesterone affects the rapid growth of cells throughout the uterine tissue, no distinct growth action was observed in the vagina. Testosterone, which is excessively secreted by abnormal hormonal action in women, is known to cause atrophy and morphological changes in the uterus (Kumar et al., 2005). Specifically, testosterone allows direct action in women’s endometrium and can affect endometrial cell proliferation, differentiation during pregnancy establishment, and tissue reconstruction during menstruation (Milne et al., 2005; Zang et al., 2008).

However, the results of our study are thought to increase the expression of MMPs in the vagina as it affects the morphological change of the uterus, as in the results of Kim et al. (2018), and to increase the expression of Casp-3, an apoptotic factor, and give abnormal signals to the functional physiological action of the uterus. Therefore, the results of this study believe that hormone hypersecretion affects the reproductive organs as a whole and that the action of progesterone and testosterone can affect other organs besides the uterus and ovaries.

As a result of analyzing changes in mouse vagina according to hormone hypersecretion, hCG showed a similar pattern to normal in hypersecretion action, and progesterone showed the same effect as endometrial growth. However, in the case of testosterone, it was confirmed that the regression (degradation) of vaginal squamous epithelium cells and the overall abnormal development of the Adventitia zone, including mucularis zone, were formed. Therefore, it is considered that abnormal signaling systems of hormone receptors can be established in the vagina, unlike studies that have focused on diseases of the uterus and ovaries by affecting feedback loop action.

None.

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

None.

All animal procedures followed the protocol approved by the Animal Experimental Ethics Committee of Hankyong National University (Permission No.: 2019-1).

Not applicable.

Yes.

Not applicable.

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