The role of chitosan oligosaccharides in regulating the function of ovarian reproductive stem cells and protecting against premature ovarian failure

In recent years, the discovery of ovarian germ stem cells (OGSCs) has provided a new research direction for the treatment of ovarian failure. The ovarian microenvironment affects the proliferation and differentiation of OGSCs, and immune cells and related cytokines are important components of the microenvironment. However, whether improving the ovarian microenvironment can regulate the proliferation of OGSCs and remodel ovarian function has not been reported. In this study, we linked chitosan oligosaccharide (COS) with fluorescein isothiocyanate (FITC) to select the best route of administration. COS was given to mice through the best route of administration, and the changes in ovarian and immune function were observed using assays of organ index, follicular growth, serum estrogen (E 2 ) and anti-Mullerian hormone (AMH) levels, and the expression of IL-2 and TNF-α in the ovaries. COS significantly increased the weight of the ovary and immune organs, reduced the rate of follicular atresia, increased the levels of E 2 and AMH hormones, and increased the protein expression of IL-2 and TNF-α in the ovary. Then, COS and OGSCs were cocultured to observe the entry of COS into OGSCs and to measure the survival rate of OGSCs. With increasing time, COS gradually entered the cell, and the cytokines IL-2 and TNF-α significantly promoted OGSCs promotion. In conclusion, COS significantly improved the ovarian and immune function of mice with pathological ovarian aging, and improved the survival rate of OGSCs, which provided a preliminary blueprint for further exploring the mechanism of COS in anti ovarian aging.


Introduction
Ovarian dysfunction includes a "natural" decline in ovarian function caused by age factors and pathological ovarian function decline caused by pathogenic factors such as radiotherapy, chemotherapy and surgery. Menopause is regarded as a landmark event. Its essence is ovarian follicle depletion, and the resulting low estrogen levels in the ovary induce the occurrence and development of many other diseases [1]. Women under 40 years of age, who have hypomenorrhea or amenorrhea for at least 4 months, and whose follicle stimulating hormone (FSH) level are greater than 25U/L, are defined as having premature ovarian insufficiency [2,3].The incidence rate of POI increases yearly. Because of the unclear pathogenesis, estrogen and progestogen replacement therapy is currently used to improve other diseases caused by low estrogen levels, however, adverse reactions are more frequent, and the effects are not good.
In 2004, Johnson [4] and others found that the atresia rate of mouse follicles was significantly higher than the decrease of non-atretic follicles in follicle count. Using double-immunofluorescence staining, it was found that ovoid cells in the epithelial layer of the ovary simultaneously expressed the germ cell specific marker MVH and the proliferation cell marker BrdU. This discovery invalidated the "fixation theory of primordial follicle pool" and suggested for the first time the hypothesis that reproductive stem cells also exist in the ovary. Niikura [5] and others observed germ cells expressing Stra8 (stimulated by retinoic acid gene 8) in the ovarian surface epithelium, indicating that there may be germ cells with meiotic function in the ovary.
Subsequently, Wu et al. [6] successfully isolated OGSCs from the ovaries of suckling mice and adult female mice for the first time, transfected them with GFP virus in an appropriate environment in vitro, and then transplanted them into the ovaries of model sterile mice. New oocytes were produced and normal GFP-positive offspring were produced after mating with male mice. At present, it is generally believed that OGSCs come from the ovarian cortex, which express stem cells and have the ability to develop into oocytes [7][8][9]. Some scholars have proposed that the loss of OGSCs in vivo and abnormal function may be related to ovarian aging [10][11][12][13]. It is worth noting that the proliferation and differentiation of stem cells cannot be separated from the surrounding microenvironment, and immune system related cells are an important part of the microenvironment of the OGSCs nest [14]. When OGSC nests are destroyed, ovarian dysfunction occurs [15,16]. Some studies have shown that POI may occur in neonatal mice with thymectomy [17]. In short, once the mechanism of OGSCs has been thoroughly studied, there will be an unlimited source of eggs for the clinical treatment of infertility patients, and the problem of infertility will be solved.
COS is the only basic amino oligosaccharide in natural sugar. It is derived mainly from small molecular oligosaccharides with amino groups that are degraded after deacetylation of chitosan from crustaceans such as shrimp and crab. COS is formed by polymerization of 2 -10 identical or different monosaccharides by glycosidic bonds, and its content in nature is second only to cellulose [18][19][20]. It has been confirmed that COS administration by intraperitoneal injection and intragastric administration significantly improved the phagocytic function of mouse peritoneal macrophages, increased the index of immune organs such as thymus and spleen, stimulated lymphocytes to secrete IL-2 cytokines, and enhanced the activity of NK cells [21,22]. COS stimulated the secretion of other cytokines by activating macrophages, to then produce a cascade reaction [23]. Several studies showed that it improves the body's immunity and play the role of antioxidant, and is also recognized as an immune enhancer [24]. By observing the effect of COS on the reproductive capacity of male mice, some scholars found that COS significantly improved the reproductive capacity of male mice, improved the antioxidant capacity of testes, and promoted sperm formation [25]. However, it remains unclear whether the immunopotentiation of chitooligosaccharides can improve the ovarian microenvironment, promoting the proliferation of OGSCs and reshaping ovarian function.
In this study, we used a chemical modification method to connect fluorescein isothiocyanate (FITC) and COS to form stable FITC-COS labeled products, which were analyzed using high performance liquid chromatography [26,27]. Then FITC-COS was given to mice in various ways, and the best way to reach the ovary was screened using a spectrophotometer [28][29][30]. We also constructed a mouse model of pathological ovarian decline, and then we administered COS to mice to evaluate the changes in ovarian function and immune function in mice, and to further study whether COS can directly regulate the immune factors IL-2 and TNF -α to promote the proliferation of OGSCs, to gain a new understanding of ovarian dysfunction with limited therapeutic effects.

Animals and Treatment
In this experiment, clean-grade Kunming female mice aged 4 weeks, weighing 16 -25g, and female Kunming suckling mice aged 3 -5 days were selected and purchased from the Medical Animal Center of Nanchang University. Animal certificate number: SYXK (Gan) 2010-0002. Mice aged 4 weeks were randomly divided into the oral gavage group, intraperitoneal injection group and tail vein injection group. According to the sampling time, each group was divided into eight time periods: 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. Eight mice were randomly assigned to each time period, and were fasted for 12 h before the experiment. Then, FITC-COS solution was administered to mice by different administration methods (10 mg/mL, 0.5 mL). When the mice were sacrificed at the corresponding times, the ovaries were immediately removed and washed with normal saline; the surface was dried with filter paper, weighed, and made into tissue homogenates according to the corresponding proportion. The supernatants were centrifuged at 6000 rpm for 10 min to measure the absorbance. In addition, Kunming female mice were randomly divided into the blank control group, saline group, and chitooligosaccharide treatment group (COS group) after modeling. A model of infertility was established by intraperitoneal injection of cyclophosphamide (120 mg/kg)/busulfan (30 mg/kg) according to the references. The control group and saline group were given the same amount of saline continuously, and the COS treatment group was given 300 mg/kg.d COS continuously by the best administration method.
After 21 days, the ratio of the bilateral ovaries, thymus and spleen was calculated.
Under sterile conditions, suckling mice aged 3 -5 days were sacrificed, ovaries were extracted, ovarian appendages were carefully removed, and intact ovaries were washed in D-Hanks solution for later use. The temperature of the rat house was controlled at 22±2 ℃, the relative humidity was maintained at approximately 50%, ensuring 12 h light and 12 h darkness, and the mice had free access to food and drinking water. All experiments were carried out according to the guidelines of the Institutional Animal Ethics Committee (IAEC) of Nanchang University (Nanchang, P. R. China).

OGSC primary isolation and culture
Ten to sixteen ovaries of 3-to 5-day-old suckling mice were selected and cleaned with D-HANKS solution, and 5 ml collagenase was prepared. The intact ovarian tissue was removed by forceps and placed into collagenase. Under the condition of avoiding light, the ovaries were bathed at 37 ℃ for 13 min at 2000 rpm. The solution was centrifuged for 10 min and remove the supernatant, and 5 ml pancreatin was aspirated with a pipette gun and added into a centrifuge tube, which was fully shaken and kept in a constant temperature water bath at 37 ℃ for 3 min. In addition, DMEM cell culture medium containing 10% fetal bovine serum was prepared to stop trypsin in subsequent experiments at 2000 rpm, centrifuged for 10 min and supernatants were removed. Then 1 ml of OGSC culture medium was added, and the cells were blown and mixed well. After standing for 3 seconds, when the ovary was about to precipitate to the bottom of the centrifuge tube, the cell suspension was added to the 48-well cell culture plate, and the remaining ovarian tissue was inhaled into another well. After adding the culture medium again, the cell plate was put into a 37 ℃ 5% CO 2 incubator for further culture.

FITC standard curve
A certain amount of FITC powder was accurately weighed, and dissolved in anhydrous methanol, and 1 mg/ml FITC methanol solution was prepared, which was shaken and mixed evenly. Then 0.1 mol/l acetic acid was prepared to gradually dilute FITC methanol solution into six series of standard FITC solutions with concentrations of 0.001 mg/ml, 0.004 mg/ml, 0.006 mg/ml, 0.008 mg/ml, 0.01 mg/ml and 0.016 mg/ml, and then mixed well and kept away from light. The fluorescence intensity a was determined at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The absorbance value was used as the ordinate of the standard curve and the FITC concentration (mg/ml) was used as the abscissa.

Labeling of chitooligosaccharides
Fifty milliliters of FITC-anhydrous methanol solution (1 mg/mL) and 20 mL of COS aqueous solution (15%, m/V) were configured, and the pH value of the COS aqueous solution was adjusted to 9. This solution was thoroughly mixed and magnet-stirred 3 -24 h at room temperature in the dark (to ensure that the mass ratio of COS to FITC was controlled at 60: 1). Then, the solution was poured into dialysis bags and dialyzed with ddH 2 O for 5 days until the solution outside the dialysis bag was found to be nonfluorescent. Then, the dialyzed solution was washed with excess ethanol three times (5 -6 times the volume of anhydrous ethanol was slowly poured in). When no fluorescence absorption was detected in the supernatant, the liquid was freeze-dried in vacuum to obtain the labeled product FITC-COS, which was stored away from light. FITC-COS powder was prepared into a 1 mg/ml FITC-COS methanol solution, and then diluted to various multiples using 0.1 mol/L acetic acid solution, and the absorbance of the solution was determined. The absorbance value was substituted into the FITC-COS standard curve to calculate the content of FITC in the FITC-COS sample using the formula: Labeling rate(%)=FITC(mg)/FITC-COS (mg) ×100%, the labeling rate of the sample was calculated.

Precision, accuracy and stability of FITC-COS
FITC-COS, was freeze-dried, and dissolved in 0.1 mol/L acetic acid solution and prepared into an initial solution with a concentration of 1 mg/mL. Then, 0.1 mol/L acetic acid was further diluted to standard solutions of different concentrations.
Normal mouse ovarian tissue (10 mg) and PBS 600 μL were added to the standard solution, and the tissue homogenate was fully ground and centrifuged at 6500 rpm for 10 min. The supernatants were taken to determine the fluorescence intensity A value, and the standard liquid concentration was taken as the X-axis and absorbance A as the Y-axis. The linear regression equation was calculated and the curve was drawn. The absorbance of FITC-COS solution (25 μg/mL) was measured immediately and after one week of storage (in the dark at 4 ℃), the light absorption values of the two were compared. An ovarian tissue homogenate solution containing FITC-COS (25 μg/mL) was also prepared. The absorbance was measured immediately and at room temperature for 4 h, and the absorbance was compared. FITC-COS tissue samples were prepared with low (0.78 μg/mL), medium (12.5 μg/mL) and high (50 μg/mL) concentrations. In the same operation as above, the absorbance A value was determined, and then the concentration of FITC-COS in each sample solution was obtained by plugging it into the associated standard curve, and the accuracy rate (%) was divided by the added concentration. Five samples were measured for each concentration, and the experiment was repeated three times to calculate the intrabatch precision and interbatch precision.

Detection of serum estrogen and anti-Mullerian hormone
The estrous cycle of mice was observed by vaginal smear. After anesthesia, blood was taken from the eyeball and left for 30 min. After centrifugation at 3500 rpm, the supernatant was taken and the hormone indexes were measured by ELISA kit.

H (hematoxylin) and E (eosin) staining
The ovaries were soaked overnight in 4% paraformaldehyde fixative solution, paraffin-embedded, sliced and stained. The morphological changes of ovaries were observed under 200X magnification.

Reverse Transcription-PCR
According to the manufacturer's instructions, total RNA was extracted from OGSCs by the Trizol method(Invitrogen,Germany), and the concentration and purity of total RNA were detected by a nanometer photometer(IMPLEN, Germany). The measured optical density was 1.8<A260/A280< 2.2.RNA expression was detected by 2% agarose gel electrophoresis(Bio-Rad，USA). Table 1 contains the list of primers used in this study.

Cell proliferation assay
The cell suspension was evenly tiled onto 96-well cell plates (100 µL /well) and cultured in an incubator at 37 ℃ and 5 % CO 2 for 24-72 h to make the cells adherent to the wall. Then, 100 µL of drugs of different concentrations (0 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL and 20 ng/mL) were added to each well cell, and each group was reseeded for 72 h. Before adding CCK-8, we replaced fresh culture medium to remove the influence of the drug, added 10 µL of CCK-8 solution (Transgen BioTECH, China) to each well, and incubated in the incubator in the dark for 1 h. The absorbance OD value was measured at a wavelength of 450 nm.

Laser confocal observation of the distribution
FITC-COS was dissolved in ultrapure water and filtered using a 0.2 μm filter membrane. Chitosan oligosaccharide (50 μg/mL) and ovarian germline stem cells were cultured in a CO 2 incubator for 10 min, 30 min, 1 h and 2 h. The culture dish was removed from the CO 2 incubator, washed twice with PBS, and new OGSC culture medium was added, which was stored away from light. The images were observed and collected under a laser confocal microscope as soon as possible.
2.14 Statistical analysis The experimental results were statistically analyzed using Graph Pad Prism 7.0.
Statistical differences between sets of data were detected using one-way ANOVA.
All data were expressed as the mean ± standard deviation of at least three independent experiments. P< 0.05 was regarded as statistically significant, and P< 0.01 was considered extremely statistically significant.

Preparation and chromatographic analysis of FITC-chitosan oligosaccharides
We plotted the standard curve of FITC solution. According to the experimental results, the standard curve of FITC solution had a good linear relationship, in the range of 0.001-0.016 mg/ml, and the R value was 0.9985. According to the curve, the labeling rate of FITC-COS measured was 3.5% (Fig.1A). The FITC-COS standard curve of the ovarian tissue measured had a good linear relationship in the range of 0.78-50 μg/mL, and the R value was 0.9991 (Fig.1B). According to the HPLC chromatogram results, the prepared chitosan oligosaccharides and FITC were combined after a sufficient reaction, the retention time was 15.753 min (Fig.1D), and the retention time of FITC was 17.685 min (Fig.1C). Figure 1D has no peak in the period of 17.685 min, indicating that the fitC-COS preparation did not have free luciferin, which can exclude the interference effect of free FITC luciferin in subsequent experiments.

Precision, accuracy and stability of FITC-COS in ovaries
The intrabatch precision, interbatch precision and accuracy of the ovarian tissue homogenates containing FITC-COS are shown in Table 2. Single factor analysis of variance (ANOVA) was used to calculate intrabatch precision and interbatch precision.
Among them, the coefficient of intrabatch variation was 1.90% -4.05%, and the coefficient of interbatch variation was 2.23% -5.33%. Both values were less than 10%. The accuracy of FITC-COS with high, medium and low concentrations in the ovarian samples was measured as 99.53% -104.42%, and the results were all within the range of 80% -120%. Therefore, this experiment conforms to the pharmacokinetic study of drug distribution detection in vivo. The stability of the FITC standard solution and the ovarian sample solution containing FITC-COS after being placed at room temperature for a period of time is shown in Table 3. The results showed that the fluorescence values of the newly configured solutions were changed, but the RSD was less than 10%.

The distribution of chitooligosaccharides in the ovary
Mice in each group were sacrificed at each time period, and the ovarian tissue was removed to measure the drug concentration. The measured results are shown in Figure 2. The retention time of intraperitoneal injection was the longest in the ovaries, and fluorescence was detected from 0.5 to 12 h ( Fig.2A). The administration of chitosan oligosaccharide through tail vein injection takes the fastest time to reach the ovaries; however, the retention time of the ovaries is not as long as that of intraperitoneal injection, and the administration method was more difficult (Fig.2C).
Only a small portion of the chitosan oligosaccharides reached the ovaries by gavage administration and the retention time was relatively short (Fig.2B). In summary, intraperitoneal injection was the best method for the delivery of chitooligosaccharides to the ovaries. To further verify the distribution of chitosan oligosaccharides in ovaries after intraperitoneal administration, the fluorescence labeled chitosan oligosaccharides were distributed in the ovarian cortex through tissue embedding and paraffin sections, while in the kidney group, chitooligosaccharides were distributed in the parenchyma, and no fluorescence was observed in the unlabeled chitooligosaccharide control group (Fig.2D).

Protective effects of chitooligosaccharides on reproductive and immune function
Organ indexes of ovary, thymus and spleen of mice in the cyclophosphamide/Busulfex model group were significantly lower than those in the control group, while the COS treatment group was able to improve the decreased levels of the ovary, thymus and spleen indexes in mice compared with the CY/BUS group ( Fig. 3A-C). The results showed that COS increased the weight of ovaries and immune organs in the cyclophosphamide/Busulfex model mice. In the CY/BUS model group, the ovarian structure of mice was completely destroyed, and a large number of follicles were atretic, while in the COS treatment group, chitooligosaccharides significantly repaired the damaged ovarian structure, increased the number of primordial and primary follicles, and reduced the number of atresia follicles (Fig.3D). The levels of AMH and E 2 in the peripheral blood of mice in the CY/BUS group were significantly lower than those of the control group, while the levels of AMH and E 2 in the COS group were significantly higher (Fig. 3E, F)  approximately 10 -20 μm in diameter. The number of freshly isolated stem cells was small; however, but after 24 h of self-proliferation and division, the number of cells increased continuously and finally showed a beadlike shape, which was similar to the growth pattern of spermatogenic stem cells (Fig.4A,B). Alkaline phosphatase (ALP) test results showed that OGSCs were weakly positive (Fig.4C). mRNA of ovarian germ stem cells and the whole ovary after 7 days was extracted to detect the expression of related markers by RT-PCR, and the results showed that OGSCs expressed MVH (germ cell specific marker), fragilis (germ cell specific marker), DAZL (germ cell specific marker), OCT4 (stem cell specific marker) and Stella (markers of pluripotent cells and germ cells) (Fig.4D). MVH, OCT4, and EdU (cell proliferation marker) were used for simultaneous fluorescence double-labeling. The results showed that OGSCs were all expressed, confirming that OGSCs were proliferative ovarian reproductive stem cells, rather than formations of multiple cells adhering to one another (Fig. 4E,F).

The distribution of chitooligosaccharides in cells at different times
FITC-COS (50 ug/mL) and OGSCs were cultured in a CO 2 incubator at 37 ℃ for 10 min, 30 min, 60 min and 120 min, respectively, and the fluorescence distribution in the cells changed with increasing time. When cultured for 10 min, a small amount of fluorescence was distributed in the cell membrane; when cultured for 30 min, a small amount of fluorescence granules could be found in the cell; when cultured for 60 min, green fluorescence could be seen in almost all cells, and fluorescence aggregation could be clearly seen in the cell at 120 min (Fig. 5).

Effects of cytokines IL-2 and TNF-α on OGSC proliferation
Cytokines IL-2 and TNF-α at various concentrations (1 ng/ml, 5 ng/ml, 10 ng/ml, and 20 ng/ml) were cultured with OGSCs for 72 h (combined with previous laboratory results), and the proliferative activity of each group was measured using CCK-8. Compared with the control group,the IL-2 group (20 ng/ml) and TNF-α group (10 ng/ml and 20 ng/ml) had a significant effect on OGSC proliferation (Fig. 6A,   B).The results indicated that the cytokines IL-2 and TNF-α significantly promoted OGSC proliferation.

Discussion
Poor To analyze the differences in tissue distribution of COS through different administration routes to the ovary in vivo, FITC and COS were chemically linked in this experiment, FITC-COS with a labeling rate of 3.5% was prepared, and the prepared samples were confirmed to have no free FITC fluorescein by HPLC. The study shows that the labeling rate of approximately 4% has no effect on the structure and properties of COS, which can better reflect the distribution of substances in the body [28]. In this experiment, fluorescently labeled FITC-COS was administered to mice through the abdominal cavity, gavage and tail vein. Ovarian tissues were taken at specified times to make tissue homogenates, and absorbance was measured and aggregates. Because COS is water-soluble, consideration may be to enter the cell by facilitated diffusion or by swallowing.However, the sublocalization of COS in cells to which organelles, through which carrier proteins into cells, and the specific mechanism of action need further study.
Immune-related cytokines are important components in the microenvironment of OGSC nests, which participate in and affect the asymmetric division of OGSCs, promote the formation of dominant follicles, and thus maintain the normal physiological function of ovaries [17,50]. Previous laboratory studies have found that using macrophages as trophoblasts, COS can significantly promote the proliferation of OGSCs and inhibit their senescence. The supernatants of cells were assayed, and the results showed that the secretion of the immune cytokines IL-2 and TNF-α increased. TNF-α is produced by activated monocytes/macrophages, that promote cell proliferation and differentiation and also synergize with epi-epidermal growth factor and insulin to promote the expression of the EGF receptor and produce growth factor-like effects on some tumor cells [53,54]. Studies have shown that [55] there is a biphasic activity of TNF-α in the follicles before ovarian ovulation, which not only inhibits the production of prostaglandin F2a (PGF2a), but also stimulates the temporary elevation of PGF2a before ovulation to promote the occurrence of ovulation. IL-2 is an interleukin produced by T cells or T cell lines. It stimulates T cells to secrete B cell proliferation and differentiation factors, promoting thymocytes to enter S phase after activation by antigen stimulation, and then maintaining cell proliferation.Some studies have shown that [56] IL-2 and IL-18 combined stimulation can effectively promote NK cell proliferation, IL-18 induces NK cells to enhance IL-2R, especially CD25, to make cells respond to IL-2, activating STAT3 and STAT5, increasing cycliln B1 expression and thus promoting cell proliferation. Therefore, it is speculated that COS promotes the proliferation of OGSCs by increasing the secretion of TNF-α and IL-2.
To further explore whether the proliferation of OGSCs is regulated by immune cytokines, CCK-8 experiments were designed. The results of CCK-8 assays showed that TNF-α and IL-2 at various concentrations (1 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml) interfered with the cells, and the results showed that both cytokines promoted the proliferation of OGSCs. These results are consistent with those of previous laboratory studies. Combined with the preliminary laboratory results, it is suggested that COS may have a certain proliferative effect on OGSCs; however, but it mainly relies on promoting the secretion of immune cytokines to indirectly promote the proliferation of OGSCs.
In summary, through the study of this subject, COS was successfully combined with FITC using a chemical method and free fluorescein was removed. The optimal administration mode of COS to the ovary was screened using spectrophotometry and HPLC technology. The remodeling function of COS in pathological ovaries was confirmed by HE, serum sex hormone level, organ index and other methods. In addition, the observation of COS entry into OGSCs using laser confocal microscopy provides a preliminary basis for future studies on the sublocalization of COS within cells. Further studies in cell experiments confirmed that COS indirectly promoted the proliferation of OGSCs by regulating the secretion of the immune factors IL-2 and TNF-α, thereby improving ovarian function. This will provide a new idea and method for further study of specific immune regulation mechanisms, sublocalization of COS combined with OGSCs and its mechanism of action, and whether COS can maintain local concentration stability and increase through targeted nanotechnology, laying a theoretical and experimental foundation.

5.Conclusion
In conclusion, this study enriched the potential mechanism of delaying ovarian function decline. We found that COS can promote the proliferation of OGSC and reshape the ovarian function by improving the ovarian microenvironment and stimulating the secretion of immune related factors. In the future, we will construct the ovarian targeted COS nano delivery system, and observe the effect of targeted nano COS on the proliferation and differentiation of OGSC and the recovery of damaged ovaries through in vitro organ and cell level. The molecular mechanism of COS delaying ovarian function decline is still unclear, which needs further basic research to prove.