Antibody for prolonging ovarian function, culture solution, method for inhibiting primordial follicle activation and application

Abstract

The invention discloses an antibody for prolonging an ovarian function, a culture solution, a method for inhibiting primordial follicle activation and application. The antibody is a CD138 antibody. In the technical scheme provided by the invention, the CD138 antibody can accurately regulate and control the conversion rhythm of primordial follicles from a resting state to a development state by targeted inhibition of primordial follicle activation signal channels. The regulation and control mechanism can reduce ineffective activation loss of follicles in the early stage of growth, so that more primordial follicles are kept in a dormant state, thereby prolonging the physiological duration of a follicle pool, providing biological guarantee for uniformly distributing germ cell resources in the whole growth cycle of female individuals, and improving the survival rate of the female individuals. And the risk of infertility or premature ovarian failure caused by premature exhaustion of follicles is effectively avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of reproductive medicine, and in particular to an antibody for prolonging ovarian function, a culture medium, a method for inhibiting primordial follicle activation, and applications thereof. Background Art

[0002] In the female reproductive system, the primordial follicles in the ovarian cortex are a non-renewable resource of reproductive cells, and their quantity and quality directly determine the duration of reproductive capacity. Primordial follicles are like tiny structures embedded in the "framework" of the ovarian cortex, waiting to begin development under the stimulation of appropriate physiological signals. 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 over-depleted, females will 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 a balance between two dynamic processes: maintenance of the quiescent state of primordial follicles and regulation of their initiation of growth. The mechanism of maintaining the quiescent state ensures that most follicles remain dormant, preventing premature activation and resource depletion. Meanwhile, initiation of growth is essential for follicle maturation. The precise regulation of these two processes is like the meshing of the gears of a precision instrument; imbalance in either can disrupt the reproductive lifespan.

[0004] The dynamic balance between maintaining the quiescent state of primordial follicles and initiating their development is considered the "golden hub" for regulating ovarian function. Precisely maintaining this balance not only directly determines the size of the primordial follicle pool but also, by optimizing the quality of the germ cell reserve, provides a key support for expanding the clinical application boundaries of in vitro fertilization techniques and ovarian tissue transplantation. Specifically, when the quiescent-developmental balance of primordial follicles is stably maintained, the overall reserve of primordial follicles can be significantly increased by reducing ineffective activation losses, creating a more ample "reproductive time buffer" for those seeking to delay childbearing. Furthermore, the continued presence of high-quality quiescent follicles provides superior starting material for in vitro culture systems. By mimicking the in vivo microenvironmental regulation mechanisms, the success rate of key steps such as in vitro maturation induction and transplant survival can be significantly improved, thereby opening up a new technological path for infertile patients to establish a "fertility bank." This logical transition from fundamental mechanisms to applied scenarios not only strengthens the theoretical foundation for prolonging ovarian function but also allows the medical concept of "protecting the reproductive future through balanced regulation" to gradually move towards clinical practice.

[0005] Therefore, how to regulate the dynamic balance between the resting state and the activated developmental state of primordial follicles and develop targeted intervention strategies are of great clinical significance for enhancing the application potential of primordial follicles in assisted reproductive technology. Summary of the Invention

[0006] The main purpose of the present invention is to propose an antibody, culture medium, method for inhibiting primordial follicle activation and application for prolonging ovarian function, aiming to effectively inhibit the excessive activation of primordial follicles by blocking the activity of the PTN-SDC1 pathway and maintain the balance between the resting and developmental states of primordial follicles, while also reducing the loss of primordial follicles and providing technical support for functional recovery after ovarian transplantation.

[0007] To achieve the above object, the present invention provides an antibody for prolonging 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 overactivation of primordial follicles.

[0010] The present invention also provides 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 provides a method for culturing ovaries in vitro, the method comprising the following steps:

[0012] S1, adding the first culture medium to the cell culture well;

[0013] S2, placing the activated ovarian tissue pieces in a Transwell chamber containing the second culture medium;

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

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

[0016] The present invention also provides a method for inhibiting primordial follicle activation, comprising the following steps:

[0017] The CD138 antibody described above was added to the ovarian tissue culture medium at a concentration of 0.004-0.006 mg / mL, and the ovarian tissue was cultured for 3.5-4.5 hours. The culture medium was then replaced with ovarian tissue culture medium containing 100-130 ng / mL of PTN recombinant protein and 100-130 ng / mL of CD138 antibody and cultured for a further 19-21 hours.

[0018] The present invention also provides an application of the above-mentioned antibody in the preparation of a drug for treating premature ovarian failure.

[0019] The present invention also provides a use of the antibody as described above, which is used in the preparation of a drug for delaying follicle activation.

[0020] The present invention also provides an application of the above-mentioned antibody in the preparation of a health product for improving ovarian fertility.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) In the technical solution provided by the present invention, CD138 antibodies can precisely regulate the rhythm of the transformation of primordial follicles from a resting state to a developing state by targeting and inhibiting the activation signal pathway of primordial follicles. This regulatory mechanism can reduce the ineffective activation loss of follicles in the early reproductive period, allowing more primordial follicles to maintain a dormant state, thereby extending the physiological survival cycle of the follicle pool, providing biological guarantees for the uniform distribution of germ cell resources in female individuals throughout the reproductive cycle, and effectively avoiding the risk of infertility or premature ovarian failure caused by premature follicle exhaustion.

[0023] (2) The inhibitory effect of the CD138 antibody provided by the present invention on the activation of primordial follicles directly leads to a significant increase in the size of the primordial follicle pool. By maintaining a dynamic balance between rest and development, the antibody can keep the number of primordial follicles in the ovarian cortex at a higher level during the natural aging process. This feature not only provides a "reproductive time buffer zone" for healthy women who postpone their childbearing age, so that they still have a sufficient number of follicles for development and maturity at an advanced age; it also has a 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 in adulthood can be greatly increased.

[0024] (3) In the field of in vitro fertilization, the CD138 antibody provided by the present invention can maintain the quiescent state of primordial follicles through pretreatment, avoiding their spontaneous activation and apoptosis during in vitro culture, thereby improving the efficiency of induction of follicle maturation in vitro. In the ovarian tissue transplantation scenario, CD138 antibody treatment of ovarian tissue before freezing can significantly reduce the activation loss of primordial follicles during the freezing-resuscitation process and improve the functional survival rate of ovarian tissue after transplantation. This technology can restore endocrine function and fertility for patients who have had their ovaries removed due to disease, and even provide a cross-species application reference for the preservation of reproductive resources of endangered species. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 Figure 1 is a diagram showing changes in follicles at all levels of ovarian tissue after cryopreservation and thawing. Figure (A) is a stained diagram of ovarian tissue after cryopreservation and thawing; Figure (B) is a statistical diagram showing changes in the proportion of follicles in ovarian tissue after cryopreservation and thawing.

[0027] Figure 2 The present invention provides a graph showing apoptosis of ovarian tissue after cryopreservation. Graph (A) shows a staining graph of ovarian tissue after cryopreservation. Graph (B) shows a statistical graph showing the percentage of apoptosis in ovarian tissue after cryopreservation.

[0028] Figure 3 This is an electron microscope ultrastructure diagram of ovarian tissue after freezing and thawing provided by the present invention.

[0029] Figure 4 Figure 1 is a graph showing the proportion of follicles at different time points after ovarian tissue transplantation in mice provided by the present invention. Figure (A) shows the overall change in the proportion of follicles at different levels 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, where the proportion of follicles at each level in ovaries that were not transplanted after freezing and thawing served as the control group for each level of follicles; PriF, primordial follicle; PF, primary follicle; SF, secondary follicle; AntF, preantral follicle; AtrF, corpus luteum.

[0030] Figure 5The present invention provides a diagram of intercellular communication of mouse ovarian cells at various stages of tissue transplantation. Figure (A) is a diagram of intercellular communication intensity between various cell subpopulations of mouse ovarian transplantation at different time points (Con, D6, D15), where the circle size, edge width, and edge color represent the number of cells, communication intensity, and signal source, respectively. Figure (B) is a stacked bar graph of the aggregated input and output signal intensities of each cell subpopulation at different time points (Con, D6, D15), where the bar color is the same as the edge color in (A). Figure (C) is a stacked bar graph of the aggregated input and output signal intensities of each cell subpopulation at different time points (Con, D6, D15). Figures (D, E) are heat maps of the output signal intensities of the top 20 signal pathways of intercellular communication between cells of each subpopulation; Figures (D, E) are heat maps of the input (D) and output (E) signal intensities of the top 20 signal pathways of intercellular communication of mouse stromal cells at different time points (Con, D6, D15), Granulosa, granulosa cells; Stromal, stromal cells; Immune, immune cells; Endothelial, endothelial cells; Epithelial, epithelial cells; Luteal, luteal cells; Smooth Muscle, smooth muscle cells; Theca, theca cells.

[0031] Figure 6 The present invention provides a diagram of the effect of the PTN signaling pathway on primordial follicle granulosa cells at different time points. Figure (A) is a signal pathway network diagram showing the signal intensity changes of PTN signals in fibrous, proliferative and inflammatory-like stromal cells in primordial follicle granulosa cells at different time points (Con, D6, D15); Figure (B) is a diagram of the expression levels of PTN in each stromal cell subpopulation at different time points; Figure (C) is a diagram of the expression levels of PTN downstream ligands (Ncl, Sdc1, Sdc3 and Sdc4) in primordial follicle granulosa cells at different time points; Figure (D) is a diagram of the UMAP visualization results of Sdc1 in primordial follicle granulosa cells at different time points, Primordial GCs, primordial follicle granulosa cells; Fibroblast-like SCs, fibroblast-like stromal cells; Proliferative-like SCs, proliferative-like stromal cells; Inflammatory-like SCs, inflammatory-like stromal cells.

[0032] Figure 7 The present invention provides a diagram of the activation of primordial follicles of ovarian tissue cultured in vitro after adding different concentrations of PTN protein. Figure (A) is a staining diagram of primordial follicles of ovarian tissue cultured in vitro after adding different concentrations of PTN protein; Figure (B) is a statistical diagram of the activation rate of primordial follicles of ovarian tissue cultured in vitro after adding different concentrations of PTN protein.

[0033] Figure 8The present invention provides a diagram of the activation of primordial follicles in cultured ovarian tissue in vitro after adding PTN protein and SDC1 antibodies. Figure (A) is a staining diagram of primordial follicles in cultured ovarian tissue in vitro after adding PTN protein and SDC1 antibodies; Figure (B) is a statistical diagram of the activation rate of primordial follicles in cultured ovarian tissue in vitro after adding PTN protein and SDC1 antibodies.

[0034] Figure 9 Figure 1 shows the effect of the in vivo application of the SDC1 antibody on the activation of transplanted ovarian primordial follicles provided by the present invention. Figure (A) shows the activation staining of primordial follicles at different time points after adding SDC1 antibody to ovarian tissue after cryopreservation and co-transplantation with the ovarian tissue; Figure (B) shows the statistical graph of primordial follicle activation rates at different time points; and Figure (C) shows the results of ovarian tissue follicle counting after transplantation.

[0035] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0036] In order to make the purpose, 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. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is mutually contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and 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 were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (experimental unit license number: SYXK(E)2016-0057).

[0039] Reagents: Antibody CD138 is a recombinant anti-Syndecan-1 antibody [EPR6454], ab128936, Abcam (USA)

[0040] Freezing solution ①: Leibovitz L-15 medium, 9V / V% DMSO, 0.8V / V% HSA, 5mM N-acetylcysteine; Freezing solution ②: Leibovitz L-15 medium, 9V / V% DMSO, 9V / V% SSS, 5mM 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] The experiment used 8-week-old female C57BL / 6J mice. After the mice were killed by cervical dislocation, the ovaries on both sides were immediately collected. Each ovary was divided into 8 parts and processed using a commercial vitrification and recovery kit. The ovarian tissue was placed on a copper sheet and frozen in a culture dish. The steps were: incubation in freezing solution ① for 5 minutes, incubation in freezing solution ② for 5 minutes, and incubation in freezing solution ③ for 15 minutes. The recovery steps were: incubation in recovery solution ① for 5 minutes, incubation in recovery solution ② for 10 minutes, and incubation in recovery solution ③ for 10 minutes. After the recovery was completed, the follicles were counted and obtained. Figure 1 ; TUNEL staining was used to detect cell apoptosis. Figure 2 ; and transmission electron microscopy to detect the ultrastructure, and obtain Figure 3 , and comprehensively evaluate the effect of freezing and thawing.

[0044] Figure 1 In the study, PriF refers to primordial follicles (Con vs Vitr, 21.4% vs 10.9%), PF refers to primary follicles (Con vs Vitr, 21.4% vs 15.1%), SF refers to secondary follicles (Con vs Vitr, 28.6% vs 32.7%), AntF refers to antral follicles (Con vs Vitr, 17.3% vs 16.8%), and AtrF refers to atretic follicles (Con vs Vitr, 11.2% vs 24.4%). Compared with the control group, the proportion of primordial and primary follicles decreased, while the proportion of atretic follicles increased after cryopreservation of ovarian tissue compared to the control group. This experimental result is consistent with previous literature research, indicating that the freezing and thawing process will partially damage the follicle pool. The follicle damage caused by cryopreservation of ovarian tissue is reasonable and real, and can be used for subsequent experimental observations.

[0045] Primordial follicle (PriF): It is composed of an oocyte and a layer of flat granulosa cells (the activation process of the primordial follicle is a continuous process in which the growth of granulosa cells affects the growth of oocytes. It is a multicellular structure composed of granulosa and oocytes that has changed. It is not a single cell or tissue. We can see that the number and morphology of granulosa cells and oocytes in follicles of different categories are different. In this process, the granulosa cells of the primordial follicles change first, and then the oocytes change. Therefore, when we analyze what causes the activation of primordial follicles, we use the granulosa cells of the primordial follicles as the object of study. In most experimental explanations, we use ovarian tissue sections to observe the proportion of this multicellular structure to determine whether it is activated; at the same time, FOXO3a staining determines activation by the changes in the staining localization of this protein in the oocyte).

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

[0047] Secondary follicle (SF): The oocyte continues to grow and the surrounding granulosa cells increase into multiple layers.

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

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

[0050] Figure 2 The data show the cell apoptosis of normal ovarian tissue (Con) and frozen-thawed ovarian tissue (Vitr). FITC (green fluorescence) positive cells are apoptotic cells. The apoptosis rates of the two groups of ovarian tissues were compared: Con vs Vitr, 1.14% vs 1.39%, and there was no difference in the apoptosis rates between the two groups.

[0051] Figure 3 Electron microscopy results show that the ovarian tissue after freeze-thaw has clear follicle structures at all levels, with orderly arrangement of granulosa cells. The yellow arrows in the image point to granulosa cell mitochondria, which have a typical double membrane structure and clear mitochondrial cristae. These results indicate that the ovarian tissue after freeze-thaw is morphologically normal and viable, suitable for subsequent experiments.

[0052] Example 2 Primordial follicle activation experiment

[0053] 1. Preparation of experimental animals

[0054] 8WC57 mice (Wei Tonglihua) were selected. Ensure that the animals were in good health and that their age and weight met the experimental requirements. Female mice were usually 6-8 weeks old and weighed approximately 20-30 g.

[0055] The experimental animals were housed in a standard animal room environment with a temperature of 20-26°C, a humidity of 40%-60%, a 12-h light / 12-h dark cycle, free access to water and food, and were allowed to acclimate to the environment for 1-2 weeks.

[0056] 2. Tissue processing

[0057] The ovarian tissue blocks obtained above 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. Then, the ovarian tissue blocks were cut into appropriate sizes of 1-2 mm. 3 Organization block.

[0058] 3. Exposure of the transplant site

[0059] The recipient mouse is placed in an anesthesia induction chamber and anesthesia is induced using a mixture of isoflurane and oxygen (isoflurane concentration is 3%-5%). After the mouse loses consciousness, it is transferred to the operating table and anesthesia is maintained by continuous administration of isoflurane (concentration is 1.5%-2.5%) via a mask. The mouse is secured to the operating table, and its limbs are secured with tape or a special fixation device to ensure the animal's stability during the operation.

[0060] Disinfect the mouse's abdomen or back (depending on the transplant site) with iodine tincture. The disinfection area should be large enough, generally centered around the surgical incision with a radius of approximately 3-5 cm. Cover the surrounding area with sterile surgical towels, leaving only the surgical incision site exposed.

[0061] Make a longitudinal incision approximately 1-1.5 cm in the mouse's flank. Carefully separate the subcutaneous tissue and muscle layer, avoiding damage to surrounding blood vessels and internal organs. Gently push the kidney out of the incision and cover it with moistened sterile gauze to keep it moist and prevent desiccation damage. Using microtweezers and microscissors, make a small incision in the renal capsule, taking care to avoid damaging the renal parenchyma.

[0062] 4. Ovarian transplantation

[0063] Gently pick up the prepared ovarian tissue block with microtweezers and slowly place it into the incision under the renal capsule, ensuring that the ovarian tissue block fits tightly against the renal capsule to facilitate revascularization.

[0064] Carefully suture the renal capsule incision with 7-0 or 8-0 absorbable sutures to secure the primordial follicle under the renal capsule. Note that the sutures should be tight, but not too tight to prevent compression of the tissue and blood circulation.

[0065] 5. Surgical wound suture and postoperative care

[0066] Place the kidney back into the abdominal cavity, suture the muscle layer and subcutaneous tissue in layers, and suture with 5-0 absorbable sutures. Finally, disinfect the skin incision with iodine, apply antibiotic ointment, and bandage with sterile gauze. After surgery, place the mouse in a warm, quiet 37°C recovery cage until it wakes up. In the first few days after surgery, closely observe the condition of the mouse, including diet, activity, wound healing, etc. Give appropriate postoperative analgesia (such as subcutaneous injection of butorphanol, dose of 0.1-0.2 mg / kg) and antibiotics (such as ceftriaxone sodium, subcutaneous injection, dose of 50 mg / kg) to prevent infection and relieve pain.

[0067] After the mice fully recovered their independent activities, they were transferred to the breeding cage and continued to be raised normally until the samples were collected.

[0068] 6. Ovarian follicle count

[0069] Paraffin ovarian tissue sections were prepared on the 3rd day (D3), 6th day (D6), 9th day (D9), 12th day (D12), 15th day (D15), and 18th day (D18) after surgery of mice, and HE staining was performed and cell counting was performed to obtain 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 counting results multiplied by 5.

[0070] The frozen-thawed ovaries were transplanted into the ovaries of premature ovarian insufficiency (POI) mice orthotopically, and the development of follicles was observed at different time points after transplantation. The results showed that the proportion of primordial follicles showed a continuous downward trend, with the most obvious decline during D3 to D6 ( Figure 4 B). The proportion of primary follicles reaches its peak on D6 ( Figure 4 C), while the secondary follicles ( Figure 4 D) and antral follicles ( Figure 4 E) ratio decreased significantly at this time point. Notably, by D12, the proportion of primordial follicles continued to decline, the proportion of primary follicles reached a new small peak, the proportion of secondary follicles decreased again, while the proportion of antral follicles continued to increase and reached a new peak. From D15 to D18, follicular development showed a clear regularity and order. This suggests that a large number of primordial follicles were activated on D6, and the follicles in the transplanted tissue showed orderly development on D15.

[0071] After clarifying that D6 and D15 are the key time points for primordial follicle activation and orderly follicle development, we obtained ovarian tissues after freezing and thawing but not transplanted, D6 days after transplantation, and D15 days after transplantation for single-cell transcriptome sequencing to explore the reasons for the activation of primordial follicles on D6 after ovarian tissue transplantation.

[0072] 7. Experiment to explore the causes of primordial follicle activation

[0073] To explore the key cell types and molecular pathways that regulate primordial follicle activation on day 6 after transplantation, we performed ovarian cell-to-cell communication analysis. Among all cell types, stromal cells dominated the cell signaling output, with their signaling volume exceeding 50% at each time point ( Figure 5 A, Figure 5 B); compared with the signaling of total ovarian cells ( Figure 5 C), the top TOP5 signaling pathways for stromal cells to send and receive signals are consistent ( Figure 5 D, Figure 5 E) This suggests that stromal cells are key cells in regulating ovarian function during ovarian tissue transplantation. Figure 5 E) A signaling pathway closely related to temporal changes, the PTN signaling pathway, is highly expressed on Day 6 and decreases on Day 15, suggesting that the PTN signaling pathway may play an important role in the activation of primordial follicles on Day 6. Next, we further explored how PTN signaling regulates granulosa cells in primordial follicles and mediates primordial follicle activation.

[0074] 8. PTN signaling mediates primordial follicle activation by regulating granulosa cells

[0075] The results of the PTN signaling pathway network analysis showed that the PTN signal acting on the granulosa cells of the primordial follicles was significantly enhanced at D6 and decreased significantly at D15 ( Figure 6 A). Meanwhile, the violin plot showed that the expression intensity of PTN in stromal cells was significantly upregulated on D6 and decreased on D15 ( Figure 6 B). This indicates that PTN signaling is highly expressed on D6 and that its regulation on granulosa cells of primordial follicles is also enhanced on D6. We further analyzed the changes in specific downstream targets after PTN signaling acts on granulosa cells of primordial follicles. The violin plot ( Figure 6 C) and UMAP map ( Figure 6D) Both showed high expression of downstream SDC1 signaling, indicating that overactivation of primordial follicles in transplanted ovarian tissue at day 6 during ovarian tissue transplantation is likely due to PTN signaling regulating SDC1 expression in granulosa cells of primordial follicles. Next, we will investigate whether adding recombinant PTN protein to cultured ovaries in vitro, mimicking the in vivo transplantation environment, can activate primordial follicles and whether SDC1 antibodies can inhibit this activation.

[0076] Example 3 Ovarian tissue in vitro culture experiment

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

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

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

[0080] FOXO3a staining: Paraffin sections of ovaries prepared after steps (1) to (3) above were prepared and stained for FOXO3a. The primary antibody used was a rabbit anti-FOXO3a polyclonal antibody (FoxO3a (D19A7) Rabbit mAb, 12829, CST, USA), used at a concentration of 1:1500. The secondary antibody used was a biotinylated goat anti-rabbit IgG (HRP-labeled goat anti-rabbit, 5220-0336, SeraCare, USA), used at a concentration of 1:100.

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

[0082] After confirming that 125ng / mL of PTN recombinant protein had the highest activation effect on primordial follicles, we first used SDC1 antibodies (antibody CD138, recombinant Anti-Syndecan-1 antibody [EPR6454], ab128936, Abcam, USA) added to the ovarian tissue culture medium at a concentration of 0.005ng / mL, cultured the ovarian tissue for 4 hours, and then replaced the culture medium. The ovarian tissue culture medium was replaced with 125ng / mL of PTN recombinant protein and cultured for 20 hours. Under these conditions, the activation rate of primordial follicles of ovarian tissue under normal conditions (Con), under conditions of adding 125ng / mL of PTN recombinant protein (PTN), and under conditions of adding SDC1 antibodies and PTN recombinant protein (PTN+Anti-SDC1) were compared, and the results were obtained. Figure 8 In the figure, red stars indicate inactivated follicles (FOXO3a localized in the nucleus), while yellow stars indicate activated follicles (FOXO3a ectopically located in the cytoplasm). The results showed that the activation rate of primordial follicles in normal ovaries was 26.9%, while the activation rate increased to 64.8% after the addition of recombinant PTN protein. The addition of SDC1 antibodies significantly inhibited the activation of primordial follicles induced by recombinant PTN protein, reducing the activation rate to 34.8%. This percentage indicates that the SDC1 antibody effectively inhibited the overactivation of primordial follicles and had no inhibitory effect on primordial follicle activation, which remained above the normal activation rate of 26.9%.

[0083] Example 4 In vivo experimental study on the inhibition of primordial follicle activation by SDC1 antibodies

[0084] In this experiment, we used SDC1 antibodies to co-transplant with ovarian tissue to observe whether SDC1 antibodies can effectively inhibit the massive activation of primordial follicles during transplantation. First, ovarian tissue was obtained for cryopreservation (the operation steps are the same as before), and the revived ovarian tissue was cultured in ovarian tissue culture medium with 0.005 mg / mL of SDC1 antibodies for 4 hours. Then, the ovaries cultured in vitro were transplanted into mouse ovaries in situ (the operation steps are the same as before), and the activation of primordial follicles at different time points and the ovarian tissue follicle count results after transplantation were observed. Figure 9 In the figure, the red star indicates an unactivated follicle (FOXO3a is localized in the nucleus), and the yellow star indicates an activated follicle (FOXO3a is ectopically located in the cytoplasm).

[0085] The results of FOXO3a staining showed that at D6 after transplantation, the activation rate of primordial follicles in the ovarian tissue with added SDC1 antibodies was significantly decreased (original activation rate 13.4%) compared with the direct transplantation group (original activation rate 25.4%), indicating that SDC1 antibodies can effectively inhibit the excessive activation of primordial follicles in the transplanted tissue in vivo. However, its activation rate is still higher than that of the mouse ovaries that were not transplanted after freezing and thawing (Con, original activation rate 12.57%). This shows that the effect of SDC1 in inhibiting primordial follicle activation is limited. When the primordial follicles are pathologically activated in large quantities, it can significantly reduce the activation rate of primordial follicles, but it fails to completely transplant to the activation level of primordial follicles in normal ovarian tissue. This also provides a reference for the safe range of SDC1 antibodies in regulating primordial follicle activation. By D15 after transplantation, the ovarian tissue entered an orderly development stage, and the activation rate of primordial follicles in the ovarian tissue returned to a level close to normal ( Figure 9 A, Figure 9 B).

[0086] This finding was further supported by follicle count results ( Figure 9 C) At day 6, the proportion of primordial follicles in the SDC1 antibody transplant group (22.1%) was significantly higher than that in the direct transplant group (15.7%). At day 15, the proportion of primordial follicles in the SDC1 antibody transplant group (14.7%) was also higher than that in the direct transplant group (12.3%). This suggests that the SDC1 antibody can preserve primordial follicles in the transplanted ovarian tissue by reducing the activation of primordial follicles.

[0087] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. An antibody for prolonging ovarian function, characterized in that The antibody is CD138 antibody.

2. The antibody according to claim 1, wherein The antibody is capable of blocking the activity of the PTN-SDC1 pathway.

3. The antibody according to claim 1, wherein The antibodies inhibit the over-activation of primordial follicles.

4. A culture solution containing the antibody according to any one of claims 1 to 3, characterized in that: The culture medium comprises the following components: 0.2-0.25 mM pyruvic acid, 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.

5. A method for culturing ovaries in vitro, characterized in that: The method comprises the following steps: S1, adding the first culture medium to the cell culture well; S2, placing the activated ovarian tissue pieces in a Transwell chamber containing the second culture medium; S3. Place the Transwell chamber in the cell culture well containing the first culture medium and culture at 36-38°C in a 4-5% CO2 environment. The first culture medium is the culture medium according to claim 4; the second culture medium is α-MEM culture medium.

6. A method for inhibiting primordial follicle activation, characterized in that: The following steps are involved: The CD138 antibody according to claim 1 was added to the ovarian tissue culture medium at a concentration of 0.004-0.006 mg / mL, and the ovarian tissue was cultured for 3.5-4.5 hours. The culture medium was then replaced with ovarian tissue culture medium containing 100-130 ng / mL PTN recombinant protein and 100-130 ng / mL CD138 antibody, and culture was continued for 19-21 hours.

7. A use of the antibody according to claim 1, characterized in that: It is used in the preparation of drugs for treating premature ovarian failure.

8. A use of the antibody according to claim 1, characterized in that: It is used in the preparation of drugs for delaying follicle activation.

9. A use of the antibody according to claim 1, characterized in that: It is used in the preparation of health products for improving ovarian fertility.

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