An antibody for prolonging ovarian function, a culture medium, a method for inhibiting primordial follicle activation, and its application.

Abstract

This invention discloses an antibody for prolonging ovarian function, a culture medium, a method for inhibiting primordial follicle activation, and its application. The antibody is a CD138 antibody. In the technical solution provided by this invention, the CD138 antibody can precisely regulate the transition rhythm of primordial follicles from a quiescent state to a developing state by targeting and inhibiting the primordial follicle activation signaling pathway. This regulatory mechanism can reduce the ineffective activation and loss of follicles in early reproductive years, allowing more primordial follicles to remain dormant, thereby prolonging the physiological lifespan of the follicle pool. This provides biological protection for the even distribution of reproductive cell resources throughout the reproductive cycle in female individuals, effectively avoiding the risk of infertility or premature ovarian failure caused by premature follicle depletion.

Description

Technical Field

[0001] This invention relates to the field of reproductive medicine technology, specifically to an antibody that prolongs ovarian function, a culture medium, a method for inhibiting the activation of primordial follicles, and their applications. Background Technology

[0002] In the female reproductive system, primordial follicles in the ovarian cortex serve as a non-renewable resource of reproductive cells, and their quantity and quality directly determine the duration of fertility. Primordial follicles are like tiny structures embedded in the "framework" of the ovarian cortex, waiting to begin development under appropriate physiological stimulation. However, the total amount of this resource is fixed at birth and is continuously depleted with age or pathological factors. When the primordial follicle pool is excessively depleted, females face serious reproductive health problems such as infertility and premature ovarian failure. Therefore, maintaining the stability of the primordial follicle pool has become a core scientific issue in prolonging ovarian function.

[0003] The size of the primordial follicle pool is determined by the balance of two dynamic processes: maintaining the quiescent state of primordial follicles and regulating the initiation of growth. The mechanism for maintaining the quiescent state ensures that most follicles are in a "dormant" state, avoiding premature activation that could lead to resource depletion; while the initiation of growth is a necessary step for follicle development and maturation. The precise regulation of these two processes is like the meshing of gears in a precision instrument; any imbalance in either will disrupt the timeline of reproductive lifespan.

[0004] The dynamic balance between the quiescent state and the initiation of development of primordial follicles is considered the "golden hub" for regulating ovarian function. The precise maintenance of this balance not only directly determines the size of the primordial follicle pool but also provides core support for expanding the clinical application boundaries of in vitro fertilization technology and ovarian tissue transplantation by optimizing the quality of germ cell reserves. Specifically, when the quiescent-developmental balance of primordial follicles is stably maintained, on the one hand, the overall quantity reserve of the primordial follicle pool can be significantly increased by reducing ineffective activation losses, creating a more ample "reproductive time buffer" for those who wish to delay fertility; on the other hand, the continuous presence of high-quality quiescent follicles can provide better starting material for in vitro culture systems—by simulating the in vivo microenvironment regulation balance mechanism, the success rate of primordial follicles in key stages such as in vitro induction maturation and transplant survival can be significantly improved, thus opening up a new technological path for infertile patients to access a "reproductive bank." This transformation logic from basic mechanisms to application scenarios not only solidifies the theoretical foundation for prolonging ovarian function but also gradually moves the medical concept of "protecting the reproductive future through balanced regulation" towards clinical practice.

[0005] Therefore, understanding how to regulate the dynamic balance between the resting and activated development states of primordial follicles and developing targeted intervention strategies is of great clinical significance for enhancing the application potential of primordial follicles in assisted reproductive technologies. Summary of the Invention

[0006] The main objective of this invention is to propose an antibody, culture medium, method for inhibiting primordial follicle activation, and its application for prolonging ovarian function. The aim is to effectively inhibit the overactivation of primordial follicles by blocking the activity of the PTN-SDC1 pathway, maintain the balance between the resting and developing states of primordial follicles, and reduce the loss of primordial follicles, thus providing technical support for the functional recovery of ovarian transplantation.

[0007] To achieve the above objectives, the present invention proposes an antibody that prolongs ovarian function, wherein the antibody is a CD138 antibody.

[0008] Preferably, the antibody is capable of blocking the activity of the PTN-SDC1 pathway.

[0009] Preferably, the antibody inhibits the overactivation of primordial follicles.

[0010] The present invention also proposes a culture medium containing the antibody as described above, wherein the culture medium comprises the following components: 0.2-0.25 mM pyruvate, 45-55 mg / L streptomycin, 70-80 mg / L penicillin, 0.02-0.04 U / mL FSH (follicle-stimulating hormone), 2-4 mg / mL BSA (bovine serum albumin), and α-MEM culture medium containing 100-130 ng / mL CD138 antibody.

[0011] The present invention also proposes a method for in vitro culturing of ovaries, the method comprising the following steps:

[0012] S1. Add the first culture medium to the cell culture wells;

[0013] S2. Place the activated ovarian tissue block in a Transwell chamber containing a second culture medium;

[0014] S3. Place the Transwell chamber in a cell culture well containing the first culture medium and culture it at 36-38°C in a 4-5% CO2 environment.

[0015] The first culture medium is the culture medium as described above; the second culture medium is α-MEM culture medium.

[0016] This invention also proposes a method for inhibiting the activation of primordial follicles, comprising the following steps:

[0017] Add the CD138 antibody (as described above) at a concentration of 0.004–0.006 mg / mL to the ovarian tissue culture medium and culture the ovarian tissue for 3.5–4.5 h. Then replace the culture medium with ovarian tissue culture medium containing 100–130 ng / mL PTN recombinant protein and 100–130 ng / mL CD138 antibody and continue culturing for 19–21 h.

[0018] The present invention also proposes an application of the antibody as described above in the preparation of a drug for treating premature ovarian failure.

[0019] The present invention also proposes an application of the antibody as described above in the preparation of drugs that delay follicle activation.

[0020] The present invention also proposes an application of the antibody as described above in the preparation of health products that enhance ovarian fertility.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) In the technical solution provided by this invention, the CD138 antibody can precisely regulate the transition rhythm of primordial follicles from a resting state to a developing state by targeting and inhibiting the primordial follicle activation signaling pathway. This regulatory mechanism can reduce the ineffective activation and loss of follicles in the early stages of reproduction, allowing more primordial follicles to remain in a dormant state, thereby prolonging the physiological life cycle of the follicle pool and providing biological protection for the even distribution of reproductive cell resources throughout the reproductive cycle of female individuals, effectively avoiding the risk of infertility or premature ovarian failure caused by premature depletion of follicles.

[0023] (2) The CD138 antibody provided by this invention inhibits the activation of primordial follicles, directly leading to a significant increase in the size of the primordial follicle pool. By maintaining a dynamic balance between resting and developing follicles, this antibody can maintain a higher level of primordial follicles in the ovarian cortex during the natural aging process. This characteristic not only provides a "reproductive time buffer" for healthy women who postpone childbearing age, allowing them to still have a sufficient number of follicles for maturation in their later years; it also has groundbreaking significance for the preservation of fertility in pre-pubertal cancer patients—by using CD138 antibodies to protect the primordial follicle pool before radiotherapy and chemotherapy, the possibility of natural conception or fertility with the help of assisted reproductive technology after adulthood can be greatly increased.

[0024] (3) In the field of in vitro fertilization, the CD138 antibody provided by this invention can maintain the quiescent state of primordial follicles through pretreatment, avoiding spontaneous activation and apoptosis during in vitro culture, thereby improving the efficiency of follicle induction maturation. In the context of ovarian tissue transplantation, CD138 antibody treatment of ovarian tissue before cryopreservation can significantly reduce the activation loss of primordial follicles during cryopreservation-thawing, and improve the functional survival rate of ovarian tissue after transplantation. This technology can help patients who have had their ovaries removed due to disease to rebuild endocrine function and fertility, and even provide cross-species application reference for the preservation of reproductive resources of endangered species. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Figure (A) shows the changes in follicles at various stages of ovarian tissue after freezing and thawing according to the present invention; Figure (B) shows the follicle staining pattern of ovarian tissue after freezing and thawing; Figure (B) shows the statistical chart of the changes in the proportion of follicles in ovarian tissue after freezing and thawing.

[0027] Figure 2 The images provided by this invention show the apoptosis of ovarian tissue after cryopreservation and thawing. Figure (A) is a staining image of the ovarian tissue after cryopreservation and thawing; Figure (B) is a statistical graph of the apoptosis rate of the ovarian tissue after cryopreservation and thawing.

[0028] Figure 3 The electron micrograph of the ovarian tissue after cryopreservation and thawing provided by this invention.

[0029] Figure 4 The figures provided in this invention are follicle proportion diagrams at different time points after mouse ovarian tissue transplantation. Figure (A) shows the overall change in the proportion of follicles at different time points after ovarian tissue transplantation; Figure (BF) shows the change in the proportion of primordial, primary, secondary, antral, and atretic follicles over time after ovarian tissue transplantation. The proportion of follicles at each level after freeze-thaw and without ovarian transplantation serves as the control group for each level of follicle. PriF represents primordial follicles; PF represents primary follicles; SF represents secondary follicles; AntF represents preantral follicles; and AtrF represents corpus luteum.

[0030] Figure 5The present invention provides intercellular communication diagrams of mouse ovarian cells at various stages of tissue transplantation. Figure (A) shows the intercellular communication intensity between different cell subpopulations at different time points (Con, D6, D15) of mouse ovary transplantation, where the circle size, edge width, and edge color represent cell number, communication intensity, and signal source, respectively. Figure (B) is a stacked bar chart of aggregated input and output signal intensities for different cell subpopulations at different time points (Con, D6, D15), with the bar color being the same as the edge color in (A). Figure (C) shows the intercellular communication intensity at different time points (Con, D6, D15). Figure (D15) shows the output signal intensity heatmap of the top 20 signaling pathways for intercellular communication between various cell subpopulations; Figure (DE) shows the input (D) and output (E) signal intensity heatmaps of the top 20 signaling pathways for intercellular communication in mouse stromal cells at different time points (Con, D6, D15). Granulosa, stromal cells; Immune, immune cells; Endothelial, endothelial cells; Epithelial, epithelial cells; Luteal, luteal cells; Smooth Muscle, smooth muscle cells; Theca, membrane cells.

[0031] Figure 6 The following diagram illustrates the effects of the PTN signaling pathway on primordial follicle granulosa cells at different time points provided by this invention. Figure (A) shows the signaling pathway network diagram illustrating the changes in PTN signal intensity in fibroblast-like, proliferative-like, and inflammatory-like stromal cells on primordial follicle granulosa cells at different time points (Con, D6, D15). Figure (B) shows the expression levels of PTN in each stromal cell subset at different time points. Figure (C) shows the expression levels of downstream PTN ligands (Ncl, Sdc1, Sdc3, and Sdc4) in primordial follicle granulosa cells at different time points. Figure (D) shows the UMAP visualization results of Sdc1 in primordial follicle granulosa cells at different time points. Primordial GCs, fibroblast-like SCs, proliferative-like SCs, and inflammatory-like SCs are primordial follicle granulosa cells.

[0032] Figure 7 The images provided by this invention show the activation of primordial follicles in in vitro cultured ovarian tissue after adding different concentrations of PTN protein. Figure (A) is a staining image of primordial follicles in in vitro cultured ovarian tissue after adding different concentrations of PTN protein; Figure (B) is a statistical chart of the activation rate of primordial follicles in in vitro cultured ovarian tissue after adding different concentrations of PTN protein.

[0033] Figure 8This is a diagram showing the activation of primordial follicles in ovarian tissue cultured in vitro after adding PTN protein and SDC1 antibody provided by the present invention. Figure (A) is a staining diagram of primordial follicles in ovarian tissue cultured in vitro after adding PTN protein and SDC1 antibody; Figure (B) is a statistical chart of the activation rate of primordial follicles in ovarian tissue cultured in vitro after adding PTN protein and SDC1 antibody.

[0034] Figure 9 This is a diagram showing the effect of in vivo application of SDC1 antibody on the activation of primordial follicles in transplanted ovaries provided by the present invention. Figure (A) is a staining diagram of the activation of primordial follicles observed at different time points after co-transplanting SDC1 antibody and ovarian tissue into cryopreserved and thawed ovarian tissue; Figure (B) is a statistical chart of the activation rate of primordial follicles at different time points; Figure (C) is a diagram of the follicle count results of ovarian tissue after transplantation.

[0035] The realization, functional characteristics and advantages of the object of the present invention will be further described with reference to the embodiments and the accompanying drawings. Detailed Embodiments

[0036] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on what can be achieved by those of ordinary skill in the art. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0037] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0038] Experimental materials: 8-week-old female C57BL / 6J mice, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. (Experimental Unit License Number: SYXK (E) 2016-0057).

[0039] Experimental reagents: Antibody CD138 is recombinant Anti-Syndecan-1 antibody [EPR6454], ab128936, Abcam (USA)

[0040] Cryosol ①: Leibovitz L-15 medium, 9 V / V % DMSO, 0.8 V / V % HSA, 5 mM N-acetylcysteine; Cryosol ②: Leibovitz L-15 medium, 9 V / V % DMSO, 9 V / V % SSS, 5 mM N-acetylcysteine.

[0041] Resuscitation solution ①: 90V / V% DPBS, 10V / V% HSA, 0.6mol / L sucrose, 5mM N-acetylcysteine; Resuscitation solution ②: 90V / V% DPBS, 10V / V% HSA, 0.4mol / L sucrose, 5mM N-acetylcysteine; Resuscitation solution ③: 90V / V% DPBS, 10V / V% HSA, 0.2mol / L sucrose, 5mM N-acetylcysteine.

[0042] Example 1: Ovarian tissue freezing and thawing

[0043] Eight-week-old female C57BL / 6J mice were used in the experiment. Mice were euthanized by cervical dislocation, and bilateral ovaries were immediately collected. Each ovary was divided into eight portions and processed using a commercial vitrification freezing and thawing kit. Ovarian tissue was placed on copper plates and frozen in a petri dish using the following steps: freezing solution ① for 5 minutes, freezing solution ② for 5 minutes, and freezing solution ③ for 15 minutes. Thawing was performed using the following steps: thawing solution ① for 5 minutes, thawing solution ② for 10 minutes, and thawing solution ③ for 10 minutes. After thawing, follicles were counted to obtain... Figure 1 TUNEL staining was used to detect cell apoptosis. Figure 2 ; and transmission electron microscopy was used to examine the ultrastructure, obtaining Figure 3 And comprehensively evaluate the effects of cryopreservation and resuscitation.

[0044] Figure 1 In the study, PriF represented primordial follicles (Con vs Vitr, 21.4% vs 10.9%), PF represented primary follicles (Con vs Vitr, 21.4% vs 15.1%), SF represented secondary follicles (Con vs Vitr, 28.6% vs 32.7%), AntF represented antral follicles (Con vs Vitr, 17.3% vs 16.8%), and AtrF represented atretic follicles (Con vs Vitr, 11.2% vs 24.4%). Compared to the control group, after ovarian tissue cryopreservation and thawing, the proportions of primordial and primary follicles decreased, while the proportion of atretic follicles increased. This result is consistent with previous literature reviews, indicating that the freeze-thaw process causes some loss of the follicular pool. Follicular damage caused by ovarian tissue cryopreservation and thawing is reasonable and real, and can be used for subsequent experimental observation.

[0045] Primordial Follicle (PriF): Composed of an oocyte and a layer of flattened granulosa cells. (Primordial follicle activation is a continuous process where the growth of granulosa cells influences the growth of the oocyte. It involves changes in a multicellular structure composed of granulosa cells and the oocyte, not a single cell or tissue. We can see that the number and morphology of granulosa cells and oocytes differ among different types of follicles. In this process, the granulosa cells of the primordial follicle change first, followed by the oocyte. Therefore, when analyzing the causes of primordial follicle activation, we focus on the granulosa cells of the primordial follicle. In most experimental descriptions, we observe the proportion of this multicellular structure through ovarian tissue sections to determine whether activation has occurred; simultaneously, FOXO3a staining detects changes in the staining localization of this protein in the oocyte to determine activation.)

[0046] Primary Follicle (PF): The oocyte enlarges, and the surrounding granulosa cells change from a flat shape to a cuboidal shape, forming a layer.

[0047] Secondary Follicle (SF): The oocyte continues to enlarge, and the surrounding granulosa cells increase to multiple layers.

[0048] Antral follicle (AntF): Fluid is secreted between granulosa cells, and the fluid gradually accumulates to form one or more follicular cavities (antral cavities).

[0049] Atretic follicle (AtrF): Granulosa cells die, follicular structure collapses, and oocytes degenerate or disappear.

[0050] Figure 2 The image shows the apoptosis status of ovarian tissues in normal ovarian tissue (Con) and frozen-thawed ovarian tissue (Vitr). FITC (green fluorescent) positive cells are apoptotic cells. The apoptosis rate of the two groups of ovarian tissues was compared: Con vs. Vitr, 1.14% vs. 1.39%, with no difference in apoptosis rate between the two groups.

[0051] Figure 3 Electron microscopy results showed that the follicle structures at all levels of the ovarian tissue were clearly defined after freeze-thaw, and the granulosa cells were arranged in an orderly manner. The yellow arrows in the image indicate the mitochondria of the granulosa cells, which showed a typical double-membrane structure and clearly defined mitochondrial cristae. These results indicate that the freeze-thawed ovarian tissue had normal morphology and good activity, and could be used for subsequent experiments.

[0052] Example 2: Primordial Follicle Activation Experiment

[0053] 1. Preparation of laboratory animals

[0054] 8WC57 mice (Vitalliwa) were used, ensuring the animals were in good health and met the experimental requirements in terms of age and weight. Female mice aged 6-8 weeks and weighing approximately 20-30 grams were typically selected.

[0055] Laboratory animals are housed in a standard animal facility environment, with the temperature maintained at 20-26℃, humidity at 40%-60%, and a 12-hour light / 12-hour dark cycle. They have free access to water and food and are allowed to acclimatize to the environment for 1-2 weeks.

[0056] 2. Organizational processing

[0057] The obtained ovarian tissue blocks were washed in Dulbecco's phosphate-buffered saline containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B to remove blood and other impurities. They were then cut into appropriately sized pieces of 1-2 mm. 3 Organizational block.

[0058] 3. Exposure of the transplant site

[0059] Recipient mice were placed in an anesthesia induction chamber and induced with a mixture of isoflurane and oxygen (isofluorane concentration of 3%-5%). After the mice lost consciousness, they were transferred to the operating table, where isoflurane (concentration of 1.5%-2.5%) was continuously administered through a face mask to maintain anesthesia. The mice were then secured to the operating table, with their limbs restrained with tape or a special fixation device to ensure stability during the surgery.

[0060] Disinfect the abdomen or back of the mouse with povidone-iodine (depending on the transplant site). The disinfection area should be large enough, generally around a radius of about 3-5 cm centered on the surgical incision. Cover the surrounding area with a sterile surgical drape, exposing only the surgical incision site.

[0061] Make a longitudinal incision of about 1-1.5 cm in the flank of the mouse, carefully separating the subcutaneous tissue and muscle layer, avoiding damage to surrounding blood vessels and organs. Gently push the kidney out of the incision, cover it with moist sterile gauze, and keep the kidney moist to avoid dryness and damage. Make a small incision in the renal capsule using micro-forceps and micro-scissors, taking care to avoid damaging the renal parenchyma.

[0062] 4. Ovarian transplantation

[0063] The prepared ovarian tissue block is gently picked up with micro-forceps and slowly placed into the incision under the renal capsule, ensuring that the ovarian tissue block adheres tightly to the renal capsule to facilitate vascular recanalization.

[0064] Carefully suture the renal capsule incision with 7-0 or 8-0 absorbable sutures to fix the primordial follicles under the renal capsule. Ensure the sutures are tight, but not too tight, to avoid compressing tissues and affecting blood circulation.

[0065] 5. Surgical wound suturing and postoperative care

[0066] The kidney was returned to the abdominal cavity, and the muscle layer and subcutaneous tissue were sutured in layers using 5-0 absorbable sutures. Finally, the skin incision was disinfected with povidone-iodine, antibiotic ointment was applied, and the area was bandaged with sterile gauze. Postoperatively, the mouse was placed in a warm, quiet 37°C recovery cage until it recovered. During the first few days postoperatively, the mouse's condition was closely monitored, including its diet, activity, and wound healing. Appropriate postoperative analgesia (e.g., subcutaneous injection of butorphanol at 0.1-0.2 mg / kg) and antibiotics (e.g., subcutaneous injection of cefotaxime sodium at 50 mg / kg) were administered to prevent infection and alleviate pain.

[0067] After the mice have fully recovered their ability to move independently following the surgery, they are transferred to a rearing cage and fed normally until the samples are collected.

[0068] 6. Ovarian follicle count

[0069] Paraffin-embedded ovarian tissue sections were prepared from mice on postoperative days 3 (D3), 6 (D6), 9 (D9), 12 (D12), 15 (D15), and 18 (D18). These sections were stained with hematoxylin and eosin (HE) and eosin (C) for cell counting. Figure 4 Count the stained consecutive sections, taking one section out of every five consecutive sections for counting. The final statistical result is the sum of the counts multiplied by 5.

[0070] Frozen-thawed ovaries were transplanted into the orthotopic ovaries of mice with early-onset ovarian insufficiency (POI), and follicular development was observed at different time points after transplantation. Results showed that the proportion of primordial follicles exhibited a continuous decreasing trend, with the most significant decrease occurring between days 3 and 6. Figure 4 B). The proportion of primary follicles peaks on day 6. Figure 4 C), while secondary follicles ( Figure 4 D) and antral follicles ( Figure 4 E) The proportion decreases significantly at this point. Notably, by day 12, the proportion of primordial follicles continues to decline, the proportion of primary follicles reaches a new small peak, the proportion of secondary follicles decreases again, while the proportion of antral follicles continues to increase and reaches a new peak. From day 15 to day 18, follicular development exhibits a clear regularity and orderliness. This suggests that a large number of primordial follicles are activated on day 6, and the follicles in the transplanted tissue exhibit orderly development on day 15.

[0071] After identifying D6 and D15 as the key time points for primordial follicle activation and orderly follicle development, respectively, we obtained ovarian tissue from frozen thawed tissue before transplantation, on day 6 after transplantation, and on day 15 after transplantation for single-cell transcriptome sequencing to explore the reasons for primordial follicle activation on day 6 after ovarian tissue transplantation.

[0072] 7. An experiment to investigate the causes of primordial follicle activation.

[0073] To investigate the key cell types and molecular pathways regulating primordial follicle activation on day 6 post-implantation, we performed an analysis of intercellular communication in the ovarian cell line. Among all cell types, stromal cells dominated cell signaling output, with their signal transmission exceeding 50% at all time points. Figure 5 A, Figure 5 B); compared to the signals emitted by total ovarian cells ( Figure 5 C), the top TOP5 signaling pathways for both signal transmission and reception in stromal cells are consistent ( Figure 5 D, Figure 5 E) This indicates that stromal cells are key cells regulating ovarian function during ovarian tissue transplantation. Furthermore, it was found that stromal cells emit signals that ( Figure 5 E) A signaling pathway closely related to time changes, the PTN signaling pathway, is observed, showing high expression on day 6 and decreased expression on day 15. This suggests that the PTN signaling pathway may play an important role in the activation of primordial follicles on day 6. Next, we further investigate how PTN signaling regulates granulosa cells of primordial follicles and thus mediates primordial follicle activation.

[0074] 8. PTN signaling regulates the activation of primordial follicles mediated by granulosa cells in primordial follicles.

[0075] Analysis of the PTN signaling pathway network showed that PTN signaling acting on primordial follicle granulosa cells was significantly enhanced on day 6, and then significantly decreased on day 15. Figure 6 A). Meanwhile, the violin plot showed that PTN expression in stromal cells was significantly upregulated on D6 and decreased on D15. Figure 6 B). This indicates that PTN signaling is highly expressed at D6, and its regulation of primordial follicle granulosa cells is also enhanced at D6. Further analysis revealed changes in specific downstream targets after PTN signaling acted on primordial follicle granulosa cells, as shown in the violin diagram. Figure 6 C) and UMAP diagram ( Figure 6D) All showed high expression of downstream SDC1 signaling, indicating that during ovarian tissue transplantation, excessive activation of primordial follicles occurred in the transplanted ovarian tissue on D6, possibly due to PTN signaling regulating SDC1 expression in the granulosa cells of primordial follicles. Next, we will investigate whether adding recombinant PTN protein to cultured ovaries, simulating high PTN signaling in the in vivo transplantation environment, can induce primordial follicle activation in vitro, and whether using an SDC1 antibody can inhibit primordial follicle activation under these conditions.

[0076] Example 3: In vitro culture experiment of ovarian tissue

[0077] (1) Preparation steps of ovarian tissue culture medium: α-MEM (Gibco, USA) culture medium was used as the basic culture medium. The concentrations of other added reagents were 0.23 mM pyruvate (Biofil, China), 50 mg / L streptomycin (Biofil, China), 75 mg / L penicillin (Biofil, China), 0.03 U / mL FSH (Ningbo, China), and 3 mg / mL BSA (Sigma, USA). After preparation, the medium was stored at 4°C and equilibrated to room temperature before use.

[0078] (2) Add 400 μL of ovarian tissue culture medium to each 24-well plate, and then place Millicell inserts (PIHP01250, Merck, USA) into each well to construct a three-dimensional in vitro culture system for ovarian tissue. Place 5-6 small ovaries in each well and add 20 μL of culture medium to the chamber to keep the tissue moist (half of the tissue is immersed in the culture medium and half of the tissue is exposed to the air, which is a semi-dry culture method).

[0079] (3) By adding different concentrations of recombinant PTN protein (0 ng / mL, 50 ng / mL, 125 ng / mL, 250 ng / mL), the optimal conditions for intervention in primordial follicle activation were screened. Ovarian tissue was cultured at 37℃ and 5% CO2. After one day of culture, ovarian tissue was obtained for FOXO3a staining to statistically analyze the primordial follicle activation.

[0080] FOXO3a staining: Prepare paraffin sections of the ovaries before steps (1) to (3) above, and stain them with FOXO3a. The primary antibody used is rabbit anti-FOXO3a polyclonal antibody (FoxO3a(D19A7)Rabbit mAb, 12829, CST, USA, at a concentration of 1:1500). The secondary antibody is biotin-labeled goat anti-rabbit IgG (HRP-labeled goat anti-rabbit, 5220-0336, SeraCare, USA, at a concentration of 1:100).

[0081] The optimal conditions for inducing primordial follicle activation were screened by adding different concentrations of recombinant PTN protein (0 ng / mL, 50 ng / mL, 125 ng / mL, and 250 ng / mL). FOXO3a staining results are shown below. Figure 7 As shown in the figure, red stars indicate unactivated follicles (FOXO3a is located in the cell nucleus), and yellow stars indicate activated follicles (FOXO3a is located ectopically in the cytoplasm). Figure 7 The results showed that, compared with the control group, the activation rate of primordial follicles was significantly increased in the 125 ng / mL PTN recombinant protein group, reaching 65%. Higher concentrations of PTN showed a weakened activation effect on primordial follicles, indicating that the effect of PTN on primordial follicle activation is concentration-dependent and that there exists an optimal concentration. Therefore, we selected 125 ng / mL PTN as the optimal intervention concentration.

[0082] After confirming that 125 ng / mL of recombinant PTN protein had the highest activation effect on primordial follicles, we first cultured ovarian tissue in ovarian tissue culture medium with 0.005 ng / mL of SDC1 antibody (antibody CD138, recombinant Anti-Syndecan-1 antibody [EPR6454], ab128936, Abcam, USA) for 4 hours, then replaced the culture medium with ovarian tissue culture medium containing 125 ng / mL of recombinant PTN protein for another 20 hours. Under these conditions, we compared the activation rates of primordial follicles in ovarian tissue under normal conditions (Con), with those under conditions containing 125 ng / mL of recombinant PTN protein (PTN), and with those under conditions containing both SDC1 antibody and recombinant PTN protein (PTN+Anti-SDC1). Figure 8 In the figure, red stars indicate unactivated follicles (FOXO3a is located in the cell nucleus), and yellow stars indicate activated follicles (FOXO3a is located ectopically in the cytoplasm). The results showed that the activation rate of primordial follicles in a normal ovary was 26.9%, which increased to 64.8% after the addition of recombinant PTN protein. After the addition of SDC1 antibody, the activation of primordial follicles induced by recombinant PTN protein was significantly inhibited, with the activation rate decreasing to 34.8%. This indicates that SDC1 antibody effectively inhibits the over-activation of primordial follicles without inhibiting their activation; the activation rate remains above the normal level of 26.9%.

[0083] Example 4: In vivo study of SDC1 antibody inhibiting primordial follicle activation.

[0084] In this experiment, we co-transplanted SDC1 antibody with ovarian tissue to observe whether the SDC1 antibody could effectively inhibit the massive activation of primordial follicles during transplantation. First, ovarian tissue was obtained and cryopreserved (procedure as before). The thawed ovarian tissue was cultured in ovarian tissue culture medium supplemented with 0.005 mg / mL SDC1 antibody for 4 hours. Next, the cultured ovaries were orthotopically transplanted into mouse ovaries (procedure as before). The activation of primordial follicles at different time points and the follicle count results of the transplanted ovarian tissue were observed to obtain... Figure 9 In the figure, red stars indicate unactivated follicles (FOXO3a is located in the cell nucleus), and yellow stars indicate activated follicles (FOXO3a is located in the cytoplasm).

[0085] FOXO3a staining results showed that on day 6 post-transplantation, compared with the direct transplantation group (primary activation rate 25.4%), the activation rate of primordial follicles in ovarian tissue supplemented with SDC1 antibody was significantly reduced (primary activation rate 13.4%), indicating that SDC1 antibody can effectively inhibit the excessive activation of primordial follicles in transplanted tissue in vivo. However, its activation rate was still higher than that of frozen and thawed mouse ovaries that were not transplanted (Con, primary activation rate 12.57%), indicating that the effect of SDC1 in inhibiting primordial follicle activation is limited. In cases of pathologically high activation of primordial follicles, it can significantly reduce the activation rate, but it cannot completely achieve the normal primordial follicle activation level in transplanted ovarian tissue. This also provides a reference for the safe range of SDC1 antibody regulation of primordial follicle activation. By day 15 post-transplantation, the ovarian tissue entered an orderly development stage, and the activation rate of primordial follicles in the ovarian tissue recovered to near-normal levels. Figure 9 A, Figure 9 B).

[0086] The follicle count results further support this finding. Figure 9 C) At day 6, the proportion of primordial follicles in the SDC1 antibody transplantation group (22.1%) was significantly increased compared to the direct transplantation group (15.7%); at day 15, the proportion of primordial follicles in the SDC1 antibody transplantation group (14.7%) was also increased compared to the direct transplantation group (12.3%). This indicates that SDC1 antibodies can preserve primordial follicles within the ovarian tissue by reducing the activation of primordial follicles in the transplanted ovarian tissue.

[0087] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for in vitro culturing of ovaries, characterized in that, The method includes the following steps: S1. Add the first culture medium to the cell culture wells; S2. Place the activated ovarian tissue block in a Transwell chamber containing a second culture medium; S3. Place the Transwell chamber in a cell culture well containing the first culture medium and culture it at 36–38°C in a 4%–5% CO2 environment. The first culture medium comprises the following components: 0.2–0.25 mM pyruvate, 45–55 mg / L streptomycin, 70–80 mg / L penicillin, 0.02–0.04 U / mL FSH, 2–4 mg / mL BSA, and α-MEM culture medium containing 100–130 ng / mL CD138 antibody; the second culture medium is α-MEM culture medium. The activated ovarian tissue block is an ovarian tissue block activated by PTN recombinant protein, and the CD138 antibody is a recombinant Anti-Syndecan-1 antibody, EPR6454, ab128936, or abcam.

2. A method for inhibiting the activation of primordial follicles, characterized in that, Includes the following steps: Add CD138 antibody at a concentration of 0.004–0.006 mg / mL to ovarian tissue culture medium and culture the ovarian tissue activated by PTN recombinant protein for 3.5–4.5 h. Then replace the culture medium with ovarian tissue culture medium containing 100–130 ng / mL PTN recombinant protein and 100–130 ng / mL CD138 antibody and continue culturing for 19–21 h. The CD138 antibody is a recombinant Anti-Syndecan-1 antibody, EPR6454, ab128936, abcam.

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