Effect of samul-tang on female fertility via RAS signaling pathway in ovaries of aged mice

SM improved age-related decline of serum AMH and FSH level

Body weight changes in mice of each group were measured for four weeks since SM administration (Figure 1A). SM had no effect on the body weight (Figure 1B). However, decreased ovary weight associated with aging was regained by SM administration for four weeks (Figure 1C).

Figure 1. Body weight changes and serum hormone levels in mice after Samul-tang (SM) administration. (A) Eight-week-old mice were orally administered distilled water (n=6, YC group). Forty-week-old mice were orally administered distilled water (n=6, OC group) or 2.5 g/kg of SM (n= 7, OC+SM group) five times a week for four weeks. Post SM administration, the mice were weighed and hormonal assessment was performed. (B) Body weight changes. (C) Ovary weight. (D) Serum levels of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), and luteinizing hormone(LH). Data are presented as mean ± standard error of the mean. Statistical analysis was performed using the Student’s t-test.

Next, we measured the serum levels of anti-Müllerian hormone (AMH), FSH, and luteinizing hormone (LH) in each mouse group. OC mice (40-week-old mice orally administered distilled water) exhibited low AMH levels resulting in a decreased ovarian reserve (Figure 1D). SM administration in OC+SM mice significantly enhanced the serum AMH levels. Although serum LH levels did not change, FSH levels increased significantly in OC+SM mice compared to those in OC mice (Figure 1D). It is likely that the production of more FSH directly stimulates the granulosa cells for follicular growth and development [20].

SM prevented aged-related ovarian follicle loss

To investigate the protective effects of SM on ovarian follicle growth under aging conditions, a histological analysis of the ovaries excised from the mice after SM administration was performed (Figure 2A). Histological changes in YC (8-week-old mice orally administered distilled water), OC, and OC+SM mice were observed in ovarian tissues (Figure 2B). Compared to the YC mice, aged mice showed an overall reduction in ovarian follicles. OC+SM mice showed higher number of follicles at all stages than the OC mice (Figure 2C). These results suggest that SM administration prevents depletion of the primordial follicle pool.

Figure 2. Histological analysis of ovarian follicles in mice after Samul-tang (SM) administration. (A) Eight-week-old mice were orally administered distilled water (n = 6, YC group). Forty-week-old mice were orally administered distilled water (n = 6, OC group) or 2.5 g/kg of SM (n = 7, OC+SM group) five times a week for four weeks. (B) Post SM administration, both mouse ovaries were assessed histologically. (C) Number of ovarian follicles in different stages and the total number of ovarian follicles. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using the Student’s t-test.

SM ameliorated aged-related impairment of oocyte quality

To investigate the protective effects of SM on oogenesis under aging conditions, mice were hormonally superovulated to collect the oocytes (Figure 3A). Thereafter, the quantity and quality of oocytes were assessed (Figure 3B). Number of total oocytes retrieved from the aged mice was significantly lower than that retrieved from the YC mice (P< 0.05; Figure 3C). However, the number of mature metaphase II (MII) oocytes, with normal chromosomes and well-organized spindle alignments, was significantly higher in OC+SM mice than in OC mice (Figure 3D, 3E). These results indicate that SM administration could improve the quality of oocytes in aged mice.

Figure 3. Quality and quantity of mouse oocytes retrieved after Samul-tang (SM) administration. (A) Eight-week-old mice were orally administered distilled water (n = 6, YC group). Forty-week-old mice were orally administered distilled water (n = 10, OC group) or 2.5 g/kg of SM (n = 10, OC+SM group) five times a week for four weeks. Post SM administration, the mice were superovulated via hormonal stimulation. (B) Oocytes retrieved from the YC, OC, and OC+SM mice at 18 h after hCG injection. Number of retrieved oocytes (C), mature metaphase II (MII) oocytes (D), and MII oocytes with normal chromosomal and spindle alignment (E), retrieved from the three different groups of mice. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using the Student’s t-test.

SM reversed aged-induced changes in mRNA expression in mouse ovaries

QuantSeq 3’ messenger RNA (mRNA) sequencing was performed to compare the mRNA expression patterns in ovulated ovaries obtained from YC, OC, and OC+SM mice (Figure 4A). Hierarchical clustering analysis revealed 2,389 marked differentially expressed genes (DEGs) in the three mice groups, with fold changes >1.5 (P< 0.05; Figure 4B). We specifically focused on 393 DEGs in the OC+SM vs. OC group (Figure 4C; blue circle). Bioinformatic analysis was performed for 122 upregulated genes (31.0%) and 271 downregulated genes (69.0%) (Supplementary Tables 1, 2). Kyoto Encyclopedia of Genes and Genomes pathway analysis of OC and OC+SM mice datasets revealed that the DEGs were involved in the RAS signaling pathway (Table 1). Expression of nine genes associated with the RAS signaling pathway was analyzed. The genes include fibroblast growth factor receptor 2 (Fgfr2), guanine nucleotide binding protein gamma 8 (Gng8), RAS protein activator like 1 (Rasal1), RAS protein-specific guanine nucleotide-releasing factor 1 and 2 (Rasgrf1 and Rasgrf2), neurofibromatosis 1 (Nf1), fibroblast growth factor 13 (Fgf13), Src homology 2 domain-containing transforming protein C3 (Shc3), and epidermal growth factor (Egf). Fgfr2, Rasgrf2, and Egf were downregulated in OC mice; however, their expression levels were restored with SM administration (Figure 5A). Likewise, SM administration also restored the expression levels of Shc3 and Rasal1, which were initially upregulated in OC mice (Figure 5B). Expression of Nf1 and Fgf13 in the ovaries of OC+SM mice decreased significantly compared to that in the ovaries of YC and OC mice (Figure 5C). Gng8 and Rasgrf1 expressions increased significantly in OC+SM mice than in the ovaries of OC mice (Figure 5C). These genes were validated by quantitative polymerase chain reaction (qPCR) using the fluorescent probe-based TaqMan assay (Figure 6). Different expression patterns of Gng8 and Rasgrf1 were observed in sequencing data. Given the filtering of transcriptomics technologies for quality measures, including fold-change >1.5 and P < 0.05, this study obtained varying results from sequencing and qPCR analysis [21]. These findings suggest that SM administration ameliorates the age-related deterioration of ovarian function via genetic regulation.

Figure 4. Hierarchical clustering and analysis of differentially expressed messenger RNAs (mRNAs). (A) QuantSeq 3’ mRNA analysis was performed to compare the gene expression in ovulated ovaries of YC (n = 6), OC (n = 6), and OC+SM (n = 6) mice. (B) Hierarchical clustering among the mRNA expression profiles showing 2,389 differentially expressed mRNAs in the three groups, with a fold-change >1.5 and P< 0.05. (C) Venn diagram presenting the numbers of differentially expressed mRNAs between OC vs. YC and OC+SM vs. OC pairs. YC: 8-week-old control mice; OC: 40-week-old mice; OC+SM: 40-week-old mice orally administered Samul-tang. Up, upregulated genes between compared sets; Contra, contraregulated genes between compared sets; Down, downregulated genes between compared sets.

Figure 5. Differentially expressed mRNAs involved in RAS signaling pathway. Fluorescence intensities indicating the expression levels of Fgfr2, Rasgrf1, Egf, Rasal1, Sch3, Nf1, Fgf13, Gng8, and Rasgrf2 in the ovaries of YC, OC, and OC+SM mice. (A) Aging-induced downregulated expression of Fgfr2, Rasgrf2 and Egf were restored with SM administration. (B) Aging-induced upregulated expression of Rasal1 and Sch3 were restored with SM administration. (C) Expression of Nf1, Fgf13, Gng8, and Rasgrf1 were changed by SM administration in OC mice. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using the Student’s t-test. YC: 8-week-old control mice; OC: 40-week-old mice; OC+SM: 40-week-old mice orally administered Samul-tang.

Figure 6. Validation of expression of differentially expressed mRNAs involved in ovarian function. Quantitative polymerase chain reaction was performed to validate the expression of Fgfr2, Rasgrf1, Egf, Rasal1, Sch3, Nf1, Fgf13, Gng8, and Rasgrf2 in the ovaries of YC, OC and OC+SM mice. (A) Aging-induced downregulated expression of Fgfr2, Rasgrf2 and Egf were restored with SM administration. (B) Aging-induced upregulated expression of Rasal1 and Sch3 were restored with SM administration. (C) Expression of Nf1, Fgf13, Gng8, and Rasgrf1 were changed by SM administration in OC mice. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using the Student’s t-test. YC: 8-week-old control mice; OC: 40-week-old mice; OC+SM: 40-week-old mice orally administered Samul-tang.

SM improved aged-induced decline in blastocyst developmental competence

To investigate whether SM enhances the developmental competence under aging conditions, superovulated mice were mated with fertile males. At 1.5 days post coitum (dpc), 2-cell embryos were flushed from the oviduct and cultured to obtain blastocysts for three days. At 4.5 dpc, blastomeres and DNA fragmentation in blastocysts were stained to assess developmental competence (Figure 7A).

Figure 7. Quality and quantity of mouse blastocysts retrieved after Samul-tang (SM) administration. Forty-week-old mice were orally administered distilled water (n = 13, OC group) or 2.5 g/kg of SM (n =13, OC+SM group) five times a week for four weeks. Post SM administration, the mice were superovulated via hormonal stimulation and mated with fertile males. At 1.5 days post coitum (dpc), 2-cell embryos were collected and cultured up to blastocyst stage for three days. (A) Blastocyst quality was evaluated by assessing the stained nuclei and DNA fragmentation in blastocysts from each mice group. Number of collected 2-cell embryos at 1.5 dpc (B) and cultured blastocysts at 4.5 dpc (C). (D–F) Blastocyst developmental competence was assessed from the number of blastomeres and apoptotic nuclei, and apoptotic index. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using the Student’s t-test.

No significant difference in the number of 2-cell embryos and developed blastocysts was observed between 1.5 to 4.5 dpc (Figure 7B, 7C). However, the number of blastomeres per blastocyst increased significantly. Number of apoptotic cells was lower in OC+SM mice than in OC mice (Figure 7D, 7E). Apoptotic index decreased in blastocysts obtained from OC+SM mice compared to OC mice (Figure 7F). These results indicate that blastocysts with elevated total cell number and decreased apoptotic index could affect the implantation potential in vivo and pregnancy outcomes.

SM improved implantation potential in aged mice

To investigate whether SM enhances the implantation potential in vivo under aging conditions, superovulated mice were mated with fertile males. Pregnancy rate, number and weight of litter were assessed at 9.5 dpc (Figure 8A).

Figure 8. In vivo implantation potential after Samul-tang (SM) administration. Forty-week-old mice were orally administered distilled water (n = 20, OC group) or 2.5 g/kg of SM (n = 10, OC+SM group) five times a week for four weeks. Post SM administration, the mice were superovulated via hormonal stimulation and mated with fertile males. (A) At 9.5 days post coitum (dpc), the uterus from mice of both the groups was collected to assess the implantation potential. (B) Pregnancy rates. (C) Number of litters. (D) Weight of litters. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using the Student’s t-test. The significance of difference in pregnancy rates was determined using Fisher’s exact test.

The pregnancy rate was significantly higher in OC+SM mice than in OC mice (Figure 8B). Furthermore, SM administration enhanced the number of litters having weight similar to OC mice weight (Figure 8C, 8D).

Mice

All experiments and analyses were conducted in accordance with the relevant guidelines and regulations. Experimental protocols concerning animals were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine, Daejeon, Korea (approval number 19-061). Female BALB/c mice aged 8 and 40 weeks (Central Lab Animal Inc., Seoul, Korea) were housed under specific pathogen-free conditions. To investigate the effect of SM (Hanpoong, Iksan, Korea), the old mice were orally administered either distilled water (OC group) or 2.5 g/kg of SM (OC+SM group) five times a week for four weeks.

Mice from the OC and OC+SM groups were separated within the same cage to synchronize their hormonal cycles [48]. All mice were sacrificed after completion of SM administration. Ovaries were removed, weighed, and immediately placed in 4% paraformaldehyde (Biosesang, Seongnam, Korea) or liquid nitrogen for histological or RNA sequencing, respectively.

Enzyme-linked immunosorbent assay (ELISA) for hormonal assessment

Post SM administration, blood samples were collected from the mice and sera were separated and stored at -80 ° C until analysis. The serum concentrations of AMH, FSH, and LH were measured using hormone-specific ELISA kits from Ansh Lab (Webster, TX, USA), Cusabio Biotech Co. (Wuhan, China), and Endocrine technologies (Newark, CA, USA), respectively, according to the standard protocols and manufacturers’ instructions. For AMH, the inter-assay coefficients of variation (CV) was <10%, with a sensitivity of 0.06 ng/mL. For FSH, both intra- and inter-assay CVs were <15% with a sensitivity of 2.5 mIU/mL. For LH, the intra- and inter-assay CVs were 7% and 15%, respectively, and the functional sensitivity was 5.2 mIU/mL.

Histological assessment of ovarian follicles

Post SM administration, ovaries from YC, OC and OC+SM groups were serially sectioned to obtain 5-μm-thick tissue sections. These sections were then subjected to hematoxylin and eosin staining. Primordial, primary, secondary, and preovulatory follicles, with visible oocytes, were counted in every tenth stained section to avoid repeated counting of the same follicle. The follicular stages were classified as previously described [49]: primordial follicles, with a single flat layer of granulosa cells surrounding the oocyte; primary follicles, with a single cuboidal granulosa cell layer; secondary follicles, with at least two granulosa cell layers and a theca layer; and preovulatory follicles, with a complete antrum and theca layer.

Assessment of oocyte quantity and quality

Post SM administration, the mice were superovulated with an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (PMSG; Prospec, Rehovot, Israel). Another injection of 5 IU of human chorionic gonadotropin (hCG; Prospec) was administered 48 h later. Oocytes were collected 18 h post-hCG injection in preincubated human tubal fluid medium (Irvine Scientific, CA, USA). Oocytes were fixed with 4% paraformaldehyde (Biosesang), permeabilized with 0.5% Triton X-100 (Sigma–Aldrich, St. Louis, MO, USA) for 10 min, and blocked with phosphate-buffered saline containing 3% bovine serum albumin (GenDEPOT, TX, USA). Thereafter, the oocytes were incubated with a rabbit anti-α-tubulin antibody (1:200; Cell Signaling Technology, MA, USA) and subsequently mounted on slides using VECTASHIELD antifade mounting medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Peterborough, UK) to visualize the chromosomes using a fluorescence microscope (BX51; Olympus, Tokyo, Japan). Oocytes with well-organized bipolar spindles and tightly aligned chromosomes at metaphase were scored as normal.

RNA sequencing for mRNA expression

Post SM administration, ovaries were collected from the mice post-ovulation, and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturers’ protocol. The purity and integrity of the extracted RNA were evaluated using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands). All samples showed high purity (optical density (OD)₂₆₀/OD₂₈₀> 1.80) and integrity (RNA integrity number >7.0). Sequencing was performed using the Illumina NextSeq 500 platform. A fold-change value >1.5 and a P-value <0.05 were considered thresholds to identify DEGs.

Validation of selected DEGs in the ovaries

To confirm the mRNA microarray results, validation was performed on significant genes of interest (Fgfr2, Gng8, Rasal1, Rasgrf1, Rasgrf2, NF1, Fgf13, Shc3, and Egf) using real-time qPCR. Complementary DNA (cDNA) was synthesized from the extracted total RNA using iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturers’ protocol. qPCR was performed in a final reaction volume of 20 μL using a QuantStudio 6 Flex Real-time PCR system with fluorescent probe-based TaqMan assays, according to the manufacturers’ protocol (Thermo Fisher Scientific). The cycle threshold was normalized and compared using glyceraldehyde 3-phosphate dehydrogenase as the internal standard.

Assessment of in vitro developmental competence

Post SM administration, the mice were superovulated with an intraperitoneal injection of 5 IU of PMSG, followed by 5 IU of hCG 48 h later. Superovulated mice were mated with fertile male mice, and the day of vaginal plugging was designated as 0.5 dpc. At 1.5 dpc, 2-cell embryos were collected and cultured up to the blastocyst stage for three days. DNA fragmentation was determined by transferase dUTP nick end labeling (Promega, WI, USA). Blastocyst quality was assessed using the following three parameters: number of blastomeres, DNA fragmentation, and apoptotic index per blastocyst. The apoptotic index was calculated for each blastocyst as follows: apoptotic index= (number of TUNEL-positive nuclei/total number of nuclei) × 100.

Assessment of in vivo implantation potential

Post SM administration, superovulation and mating were achieved as described above. The day of vaginal plugging was designated as 0.5 dpc. At 9.5 dpc, pregnant mice were sacrificed to assess the implantation potential following three parameters: pregnancy rate, total number of litter, and litter weight.

Statistical analysis

Data are presented as mean ± standard error of the mean. The significance of difference between two groups was determined by a Student’s t-test using GraphPad Prism version 8.4.0 (GraphPad Software, La Jolla, CA, USA). The significance of difference between the expected frequencies was determined using Fisher’s exact test. A P-value < 0.05 was considered statistically significant.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.