Powdered UKT extracts (lot nos. 362187700 and 382037400) were manufactured and supplied by Tsumura & Co. (Tokyo, Japan). UKT comprises 12 crude drugs in the following composition: four parts ophiopogon root (root of Ophiopogon japonicus Ker-Gawler, Liliaceae), four parts pinellia tuber (tuber of Pinellia ternata Breitenbach, Araceae), three parts Japanese angelica root (root of Angelica acutiloba Kitagawa, Umbelliferae), two parts glycyrrhiza (root of Glycyrrhiza uralensis Fischer, Leguminosae), two parts cinnamon bark (bark of Cinnamomum cassia J. Presl, Lauraceae), two parts peony root (root of Paeonia lactiflora Pallas, Paeoniaceae), two parts cnidium rhizome (rhizome of Cnidium officinale Makino, Umbelliferae), two parts ginseng (root of Panax ginseng C. A. Meyer, Araliaceae), two parts moutan bark (root bark of Paeonia suffruticosa Andrews, Paeoniaceae), one part euodia fruit (fruit of Euodia ruticarpa Hooker filius et Thomson, Rutaceae), one part ginger (rhizoma of Zingiber officinale Roscoe, Zingiberaceae), and two parts gelatin (i.e., alternative to Asini Corii Collas). A mixture of the constituent crude drugs was extracted using purified hot water for 1 h and the dried extract powder was obtained by spray drying. DHT was purchased from Tokyo Chemical Industry (Tokyo, Japan).

Female Wistar rats (pregnant [15 rats] or 3-week-old [35 rats]) were purchased from Charles River Laboratories (Yokohama, Japan). All animals were housed in cages and lighting (12-h light/dark cycle) and temperature (20–25°C) were controlled. They were provided ad libitum access to water and an animal diet (CE-2; CLEA Japan, Tokyo, Japan, or MF; Oriental Yeast, Tokyo, Japan) during acclimatization.

Pregnant rats (gestational day 16) were subcutaneously administered 3 mg DHT for 4 days. Their female offspring were used as a prenatally DHT-induced rat model of PCOS, as reported previously (12, 13).

CE-2 diet (normal diet) and CE-2 diet containing 3% UKT (UKT diet) were prepared by CLEA. After weaning on postnatal day (PND) 21, female offspring were randomly divided into two groups of DHT-induced PCOS rats fed CE-2 diet (DHT group) and DHT-induced PCOS rats fed UKT diet (DHT+UKT group).

All protocols were approved by the Animal Experiment Committee of Nagoya University Graduate School of Medicine (approval no. M210146) and Tsumura & Co. (approval nos. 19-052, 19-059, and 20-035). The care and use of the rats followed the guidelines of the Animal Experiment Committee of the Nagoya University Graduate School of Medicine and Tsumura & Co., as well as other relevant guidelines and regulations.

Vaginal smears of all rats were examined daily from PND 56 to PND 67 to assess the estrous cycle. Vaginal smear cells from each animal were viewed and classified as diestrus (D), proestrus (P), estrus (E), or metestrus (M) based on their shape and density, as previously described (14). The length of each estrous cycle was determined as the number of consecutive days from the one-cornified cell phase to the day before the next cornified cell phase. According to the classification detailed by Ishii et al. (15), we classified the estrous cycles as follows: normal estrous cycle (4-day or 5-day cycle, i.e., PEXX, PPXX, EEXX, or PEEXX); irregular estrous cycles, including shortened estrous cycle (3-day cycle, i.e., PXX or EXX); persistent estrous cycle (at least 4 days of sequential P or E), and persistent diestrus (at least 3 days of sequential M or D). XX indicates DD, DM, MD, or MM. Sequential vaginal smear scores were defined as a combination of normal 4- and 5-day cycles, shortened 3-day cycles, persistent estrus, or persistent diestrus (0, at least two normal cycles; 1, at least one normal cycle and at least one shortened cycle; 2, at least two shortened cycles; 3, at least one normal or shortened cycle and persistent diestrus; 4, no cycles with at least one persistent estrus, and 5, no cycles with at least one persistent diestrus).

The rats were euthanized under isoflurane anesthesia during diestrus or metestrus on PND 70–90. Blood samples were collected immediately from the heart by needle aspiration. The serum was isolated by centrifugation at 3000 × g for 10 min and stored at -80°C until measurement. One ovary was removed from each animal, fixed in 10% formalin, embedded in paraffin, and stored at -80°C.

LH and FSH concentrations were determined using rat LH and FSH enzyme-linked immunosorbent assay (ELISA) kits (FUJIFILM Wako Shibayagi Corporation, Gunma, Japan), respectively. Serum concentrations of estradiol (E2), progesterone (P4), and testosterone (T) were measured using an electrochemiluminescence immunoassay (SRL, Tokyo, Japan).

Ovarian tissues were fixed in 10% neutral-buffered formalin and the paraffin-embedded tissue sections (6-µm) were stained with hematoxylin and eosin. Ovarian follicles were counted in the DHT (n = 6) and DHT+UKT (n = 5) groups. Follicles were classified as follows: preantral follicles (oocyte with two to five layers of cuboidal GCs), small antral follicles (oocyte surrounded by more than five layers of GCs, one or two small areas of follicular fluid, or both), large antral follicles (containing a single large antral cavity), preovulatory follicles (possessing a single large antrum and an oocyte surrounded by cumulus cells at the end of a stalk of mural GCs), and atretic cyst-like follicles (large fluid-filled cyst with an attenuated GC layer and dispersed theca cell layer) (16, 17). Follicle distribution was recorded as the follicle type for each ovary. Images of the entire ovary were examined side-by-side to prevent over-counting of corpora lutea and large antral, preovulatory, and atretic cyst-like follicles across sections. Seven representative sections per ovary, at least 300 µm apart, were analyzed to count the total number of preantral and small antral follicles using a light microscope (Axio Imager A1; Carl Zeiss, Oberkochen, Germany).

Immunohistochemical staining of ovarian tissue sample sections was performed using the avidin-biotin immunoperoxidase method and a Histofine SAB-PO kit (Nichirei Biosciences, Tokyo, Japan). Endogenous peroxidase activity and nonspecific IgG binding were blocked by room temperature incubation in 0.3% hydrogen peroxide in methanol for 20 min at 20–25°C and 10% normal goat serum in phosphate-buffered saline for 10 min, respectively. Tissue sections were incubated for 16 h at 4°C with polyclonal rabbit anti-FSHR antibody (bs-0895R; 1:100 dilution; Bioss Antibodies, Woburn, Massachusetts, USA), followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 10 min at 20–25°C. All slides were incubated with 3,3′-diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, dehydrated, and mounted. For semi-quantification, FSHR-positive GCs on each slide were counted using the ImageJ software (https://imagej.nih.gov/ij/) and a modified H-score (18). The score for the FSHR-positive area was calculated by classifying the staining intensity in the GC layer fields on a four-point scale (0 = negative; 1, weak; 2, moderate, and 3 = strong). Representative images of the FSHR-positive area (score: 1+ [weak] and 3+ [strong]) are shown in Supplementary Figure 1 . The percentage of GCs at each intensity level was calculated in a possible range of 0–300, using the following formula: [1 × (% cells with intensity 1+) + 2 × (% cells with intensity 2+) + 3 × (% cells with intensity 3+)]. Slides were imaged using a BZ-9000 microscope (Keyence Corporation, Osaka, Japan). The results of staining without primary antibody as negative control are shown in Supplementary Figure 2 .

GCs from rat ovaries were prepared as previously described with slight modifications (19). Briefly, female Wistar rats (3-week-old) and female offspring rats (PND 21) treated prenatally with vehicle or DHT were injected with 10 IU pregnant mare serum gonadotropin (Aska Animal Health, Tokyo, Japan). After 48 h, the ovaries were obtained from the rats after euthanasia by exsanguination under deep isoflurane anesthesia. The adipose tissue and capsule surrounding the ovaries were removed. Primary GCs were isolated using a 27G needle puncture and visualized with a stereomicroscope (Nikon, Tokyo, Japan). After filtration through 100-μm and 40-μm cell strainers (Corning, NY, USA), GCs were seeded on 96-well plates, 6-well plates, or 60-mm dishes (Corning Primaria, Corning) at a density of 4×10⁴ cells/cm². The plates were incubated overnight at 37°C with 5% carbon dioxide in phenol red-free medium (i.e., a mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium [DMEM/F12; Thermo Fisher Scientific, Waltham, MA, USA]) supplemented with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS, USA) and antibiotic-antimycotic (100 μg/mL streptomycin and 100 IU/mL penicillin; Thermo Fisher Scientific). After incubation, the medium was replaced with a serum-free medium (i.e., DMEM/F12 supplemented with 0.1% bovine serum albumin [Sigma-Aldrich, Saint Louis, MO, USA] containing 1% non-essential amino acid solution [MP Biomedicals, Irvine, CA, USA], 2.5 μg/mL transferrin [Nacalai Tesque, Kyoto, Japan], 4 ng/mL sodium selenite [Wako, Osaka, Japan], and 10 ng/mL insulin [Sigma-Aldrich]) to prevent luteinization. GCs were cultured for 24 h before experiments. The medium was replaced with serum-free medium (supplemented with 100 nM androstenedione [i.e., a synthetic source of E2] [Tokyo Chemical Industry], in addition to the aforementioned composition). Cultured GCs were treated with UKT (63–500 μg/mL) and FSH (100 mIU/mL; Merck Serono, Genève, Switzerland), alone or in combination, for 48 h.

Total RNA was extracted from GCs or rat ovarian tissues using QIAzol (Qiagen, Venlo, Netherlands), the RNeasy Universal Tissue Kit (Qiagen), or the AllPrep DNA/RNA/Protein Mini Kit (Qiagen), and reverse-transcribed to cDNA using the TaqMan reverse transcription reagent (Applied Biosystems, Waltham, MA, USA) or ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan).

mRNA levels were analyzed using real-time PCR with the TaqMan Fast Advanced Master Mix (Applied Biosystems) and the Prism 7900HT Sequence Detection System (Applied Biosystems) and normalized to that of the endogenous control Gapdh. All oligonucleotide primers and fluorogenic probe sets were manufactured by Applied Biosystems (Fshr, Rn01648507_m1; Bmp2, Rn00567818_m1; Bmp6, Rn00432095_m1; Cyp19a1, Rn00567222_m1; Hsd17b, Rn00563388_m1; Star, Rn00580695_m1; Cyp11a1, Rn00568733_m1; Hsd3b, Rn01789220_m1, and Gapdh, Rn01775763_g1).

The concentration of E2, bone morphogenetic protein (BMP)-2, or BMP-6 in the medium was determined using a 17β-Estradiol high-sensitivity ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA), Quantikine ELISA BMP-2 Immunoassay (R&D systems, Minneapolis, MN, USA) or Rat BMP-6 ELISA Kit (Novus Biologicals, Centennial, CO, USA). The concentration of cyclic adenosine monophosphate (cAMP) or total protein in the cell lysate was determined using a cAMP complete ELISA kit (Enzo Life Sciences) or a DC Protein Assay kit (Bio-Rad, Hercules CA, USA).

Statistically significant differences between the two groups were determined using the Student’s t-test or Mann–Whitney U test; the latter was used when the variables were not normally distributed. One- or two-way analysis of variance with Dunnett’s multiple comparison test or Holm–Sidak’s multiple comparison tests was used to determine the statistical differences between multiple groups. Values were expressed as dot plots. Means or medians were expressed as horizontal bars or bar graphs. A P-value less than 0.05 was considered statistically significant. All analyses were performed using the GraphPad Prism software, version 8.1.2 (GraphPad Software, San Diego, CA, USA).

The time schedule of the experimental design for drug administration and phenotype evaluation of the rat models is illustrated in Figure 1 . Although none of the rats in the DHT group had a regular cycle, some rats in the DHT+UKT group showed a better estrous cycle ( Figure 2A ). The smear scores of the DHT+UKT group significantly improved compared to those of the DHT group ( Figure 2B ).

We assessed the follicle count, as described previously (12), and found that the number of small antral and preovulatory follicles in the DHT+UKT group were significantly increased compared to those in the DHT group ( Figures 2C, D ). Although the number of large antral follicles and corpora lutea also tended to increase in the DHT+UKT group, this difference was not statistically significant.

Serum sex steroid hormone and gonadotropin levels were measured during diestrus and metoestrus. Some samples were below the limit of quantitation for LH and FSH. Serum P4 levels were higher in the DHT+UKT group than in the DHT group, although the difference was not statistically significant. Serum E2, T, LH, FSH, and LH/FSH ratios were similar between the two groups ( Figure 3 ).

We determined the mRNA expression levels of Fshr, Bmp2, and Bmp6 in the ovaries, which were significantly higher in the DHT+UKT group than in the DHT group ( Figure 4A ). Among the expressions of the other genes involved in follicle development and steroid synthesis, growth and differentiation factor (Gdf9), inhibin subtypes (Inha, Inhba, and Inhbb), Star, Cyp11a1, Cyp19a1, and Hsd3b, showed no significant difference between DHT and DHT+UKT groups ( Supplementary Figure 3 ).

Next, we assessed the FSHR protein expression levels in the ovaries using western blot analysis and the GCs of each follicle type by IHC. The DHT+UKT group showed a slight increase in ovarian FSHR expression ( Supplementary Figure 4 ). Representative images and FSHR-positive area ratio scores (P-scores) are shown in Figure 4B . In the small antral follicles, the P-scores of the DHT+UKT group were higher than those of the DHT group. By contrast, the P-scores for the other follicle types did not differ between the two groups.

We examined whether UKT affected the expressions of Fshr, Bmp2, and Bmp6 in primary cultured GCs derived from normal rats. UKT treatment for 48 h increased expressions of Fshr, Bmp2, and Bmp6 in a dose-dependent manner ( Figure 5A ). The levels of BMP-2 and BMP-6 in the culture medium were increased following UKT treatment, indicating stimulation of BMP-2 and BMP-6 secretion ( Figure 5B ).

We also assessed the effect of FSH responsiveness, as evidenced by FSH-induced E2 production and related gene expression. FSH treatment for 48 h significantly increased E2 production compared to that in the control, whereas UKT enhanced FSH-induced E2 production in a dose-dependent manner ( Figure 6A ). By contrast, UKT treatment alone did not alter E2 production. Similar results were observed for the expression of Cyp19a1 and Hsd17b mRNA ( Figure 6B ). Concerning the genes involved in P4 production, UKT enhanced the levels of Star and Cyp11a1 mRNA but not Hsd3b in FSH-treated GCs ( Figure 6C ).

We examined the mRNA expressions of Fshr, Bmp2, and Bmp6 in the GCs of control and DHT rats and found them to be lower in the GCs of DHT rats than in those of the control rats. However, UKT upregulated the mRNA expression levels in the GCs of DHT rats ( Figure 7 ). UKT also increased the FSHR protein expression in GCs ( Supplementary Figure 5 ). FSH-induced cAMP production, an index of FSH responsiveness, was lower in the GCs of DHT rats than in control rats and was restored following UKT treatment in the GCs of DHT rats ( Figure 8 ).

Next, we examined FSH-induced expression of genes involved in sex steroid hormone synthesis ( Figure 9 ). No significant differences were observed for Cyp19a1 and Hsd17b in the GCs of DHT and control rats, whereas the expressions of Star, Cyp11a1, and Hsd3b were downregulated in the GCs of DHT rats compared with those of control rats. An enhanced effect of UKT on FSH-induced Cyp19a1 and Star mRNA expression was observed in the GCs of DHT rats.