Metabolic marker for regulating and controlling Hu sheep follicular granular cell proliferation and steroid hormone secretion and application

Abstract

The invention relates to a metabolic marker for regulating follicular granule cell proliferation and steroid hormone secretion and application thereof, and belongs to the technical field of biology. According to the regulation and control method of the granulosa cells and the product for regulating and controlling the granulosa cells, the content of NAD and ATP in the granulosa cells can be effectively increased, proliferation and apoptosis of the granulosa cells can be regulated and controlled, and secretion of reproductive hormone related to dominant follicle development can be regulated and controlled. Meanwhile, based on the regulation effect of granular cells on follicle and ovary development, the granular cells can be further applied to regulation of follicle and ovary development, or can be used for preparing products for regulating dominant follicle and ovary development, so that the regulation effect on dominant follicle and / or ovary in vivo or in vitro is realized; the method and product guarantee is provided for research on development and physiological and biochemical functions of granular cells, follicles and ovary.

Description

Technical Field

[0001] The invention relates to a metabolic marker for regulating the proliferation of Hu sheep follicular granulosa cells and the secretion of steroid hormones and an application thereof, belonging to the field of biotechnology. Background Art

[0002] Fertility is the main economic trait of sheep. With the rapid development of the scale and barn feeding of the mutton sheep industry, higher requirements are placed on the reproductive performance of ewes. Therefore, selecting excellent breeds and improving the reproductive capacity of mutton sheep are crucial to improving the economic benefits of large-scale barn feeding of sheep. Hu sheep, known as the national treasure, originated in the Taihu Lake Basin. It is famous for "year-round estrus, multiple births, tolerance to roughage, and strong adaptability". It has been widely introduced to all parts of the country. It is not only the most important maternal breed for domestic sheep hybrid breeding and mutton sheep production, but also an ideal object for studying multiple birth performance and regulatory mechanisms. The reproductive performance of animals is closely related to the development of follicles, especially the number of dominant follicles. After recruitment and selection during the follicular period, generally only one dominant follicle can reach full maturity and discharge eggs. The development of the dominant follicle is accompanied by the continuous increase of follicular fluid and the continued expansion of the follicular cavity. The oocyte and some surrounding follicular cells are squeezed to one side of the follicle and protrude into the follicular cavity, called the cumulus, and the cumulus granulosa cells proliferate rapidly. The remaining follicular cells that constitute the egg wall are densely arranged in several layers, called the granulosa layer, which contains a large number of follicular wall granulosa cells. Follicular fluid granulosa cells secrete growth factors and hormones by directly contacting the follicular wall granulosa cells. Estrogen and other endocrine regulatory substances produced by granulosa cells trigger oocyte processes such as meiosis, steroidogenesis, follicular development, cumulus expansion, and luteinization, ultimately leading to mature ovulation. Therefore, the proliferation, apoptosis, and differentiation of granulosa cells become the key to follicular development. The number of ovulations plays a decisive role in the number of lambs born. The proliferation of granulosa cells determines the growth and development of oocytes, which in turn affects the ovulation of oocytes and the number of lambs born. Granulosa cell apoptosis eliminates redundant or damaged cells, promotes the selection of dominant follicles, and regulates the ovulation process. In the selection of dominant follicles and the pre-ovulatory maturation stage, the apoptosis rate of granulosa cells is high, so it is of great scientific significance to explore the molecular mechanism of regulating apoptosis of follicular fluid granulosa cells. Lipopolysaccharide (LPS) is a common bacterial exotoxin that can induce inflammatory and immune responses. Studies have shown that LPS can promote apoptosis of ovarian granulosa cells, trigger inflammatory responses in cells, and lead to loss of granulosa cell function. Maintaining the survival of ovarian granulosa cells is essential for follicular development. Follicular fluid is rich in steroid hormones, proteins, metabolites and antioxidants, which have physiological significance in terms of the nutritional source of oocytes and granulosa cells. Metabolites in follicular fluid can also transmit signals to granulosa cells as signal molecules. Some studies have identified key biomarkers affecting human infertility through follicular fluid metabolomics. Whether there are molecular substances in the dominant follicular fluid that regulate reproductive activity is still unknown. This study screened out the differential metabolite nicotinamide in the dominant follicular fluid of high- and low-breeding Hu sheep through transcriptome and metabolome, and further analyzed its regulatory mechanism by culturing follicular fluid granulosa cells in vitro. Nicotinamide is an important compound in the body, as a precursor of β-nicotinamide mononucleotide (NMN).NMN is converted to NAD+ in the body and then to nicotinamide, which highlights the importance of nicotinamide in maintaining cellular health and supporting various physiological processes. There is increasing evidence that NAD is a mediator of antiviral and anti-inflammatory mechanisms. NAD+ plays an important role as a coenzyme and substrate of SIRT and PARP1 in the body's energy metabolism, immunity and oxidative stress. The level of NAD+ in the body decreases with age, which is closely related to reduced mitochondrial energy production, oxidative stress, DNA damage and inflammatory response. NMN is a biologically active nucleotide that can be synthesized endogenously. Generally, NMN exists naturally in two irregular forms, α and β, and the β isomer is the active form of NMN. As a precursor of NAD, NMN is a key intermediate in the biosynthesis of NAD. It has been reported that exogenous NMN supplementation can reduce oxidative stress and promote vascular endothelial formation by participating in mitochondrial regeneration, anti-inflammatory and anti-apoptotic pathways to restore NAD levels. Although multiple pathways affected by NMN supplementation have been demonstrated, such as SIRT1 / PGC-1α, Wnt / β-catenin, and mTOR, the underlying mechanisms are still not fully understood. Summary of the invention

[0003] The purpose of the present invention is to address the defects of the prior art and propose a metabolic marker and application for regulating the proliferation of granulosa cells and the secretion of steroid hormones, thereby providing a method and product guarantee for the study of the development and physiological and biochemical functions of granulosa cells, follicles and ovaries.

[0004] The first purpose of the present invention is to provide a metabolite β-nicotinamide mononucleotide that is different in the dominant follicular fluid of high- and low-breed Hu sheep screened by combining phenotype and genotype, and is closely related to the function of granulosa cells.

[0005] The second purpose of the present invention is to provide an application of NMN in the preparation of a product for regulating granulosa cells, so that the prepared product for regulating granulosa cells has at least one of the functions of regulating granulosa cell apoptosis, hormone secretion and gene expression.

[0006] The third object of the present invention is to provide a product for regulating granulosa cells.

[0007] The fourth object of the present invention is to provide a method for regulating granulosa cells.

[0008] The fourth object of the present invention is to provide the application of the above-mentioned product for regulating granulosa cells or the above-mentioned method for regulating granulosa cells.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions: According to one aspect of the present invention, the present invention provides the use of the metabolite NMN in the preparation of a product for regulating granulosa cells; the regulating granulosa cells includes: regulating the energy metabolism level in granulosa cells, regulating cell proliferation and apoptosis, regulating the expression of granulosa cell genes, and regulating at least one of the secretion of hormones by granulosa cells.

[0010] The regulating energy metabolism level in granulosa cells includes at least one of NAD and ATP levels; the regulating expression of granulosa cell genes includes regulating at least one of PCNA gene, BAX gene, and BCL2 gene; the hormone includes at least one of E2 and P4. According to another aspect of the present invention, the present invention also provides a product for regulating granulosa cells, the product includes a substance for increasing the NAD content. According to another aspect of the present invention, the present invention also provides a method for regulating granulosa cells, the method for regulating includes up-regulating or down-regulating energy metabolism in granulosa cells including NAD and ATP levels. According to another aspect of the present invention, the present invention also provides at least one application of the product for regulating granulosa cells or the method for regulating granulosa cells in regulating the development of dominant follicles, regulating ovarian development, preparing a product for regulating the development of dominant follicles, and preparing a product for regulating ovarian development.

[0011] Compared with the prior art, the present invention has the following beneficial effects: Based on the regulatory effect of NMN on granular cells discovered in the experiments of the present invention, the present invention provides the use of NMN in the preparation of products for regulating granular cells, products for regulating granular cells, methods for regulating granular cells and their applications.

[0012] The present invention has found through experiments that NMN supplementation can significantly increase the levels of NAD and ATP in granulosa cells; NMN can improve LPS-induced apoptosis of granulosa cells and promote the proliferation of granulosa cells; NMN is also related to the expression of PCNA gene, BAX gene, and BCL2 gene in granulosa cells, and NMN can promote the expression of PCNA gene, BAX gene, and BCL2 gene. The present invention has also found through experiments that NMN can regulate the secretion of hormones by granulosa cells, wherein the hormones mainly include estradiol (E2) and progesterone (P4), and NMN can promote the secretion of E2 and P4.

[0013] Based on the regulatory effect of the above-mentioned NMN on granulosa cells discovered by the present invention, the method for regulating granulosa cells and the product for regulating granulosa cells provided by the present invention can effectively increase the NAD and ATP content in granulosa cells, regulate the proliferation and apoptosis of granulosa cells, and regulate the secretion of reproductive hormones related to the development of dominant follicles. At the same time, based on the regulatory effect of granulosa cells on the development of follicles and ovaries, the above-mentioned method for regulating granulosa cells and the product for regulating granulosa cells can be further applied to regulating the development of follicles and ovaries, or used to prepare products for regulating the development of dominant follicles and ovaries, to achieve in vivo or in vitro regulatory effects on dominant follicles and / or ovaries, and provide methods and product guarantees for the study of the development and physiological and biochemical functions of granulosa cells, follicles and ovaries.

[0014] The method for regulating granulosa cells provided by the present invention, the products for regulating granulosa cells and their applications can deepen the understanding of the mechanism of oocyte maturation in the late stage of follicular development, further explore the factors related to follicular development, improve the signal pathways related to follicular development, reveal the mechanism of follicular development, and optimize the in vitro culture system of oocytes. This is of great significance for further developing superovulation of mammals, breeding of multiple births, improving abnormal follicular development and treating infertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the difference in the metabolome of the dominant follicular fluid between high- and low-breed Hu sheep in Example 1 of the present invention.

[0016] Figure 2 This is the effect of different concentrations of LPS and NMN on the viability of granulosa cells in Example 2 of the present invention.

[0017] Figure 3 This is the effect of NMN on the NAD content in LPS-induced granulosa cells in Example 2 of the present invention.

[0018] Figure 4 This is the effect of NMN on ATP levels in LPS-induced granulosa cells in Example 2 of the present invention.

[0019] Figure 5 This is the effect of NMN on LPS-induced granulosa cell proliferation in Example 3 of the present invention.

[0020] Figure 6 This is the effect of NMN on LPS-induced granulosa cell gene expression in Example 3 of the present invention.

[0021] Figure 7 This is the effect of NMN on LPS-induced granulosa cell apoptosis in Example 3 of the present invention.

[0022] Figure 8 This is the effect of NMN on LPS-induced E2 secretion in granulosa cells in Example 4 of the present invention. DETAILED DESCRIPTION

[0023] The following examples provide the use of NMN in the preparation of products for regulating granulosa cells, wherein regulating granulosa cells includes regulating the content of ATP and NAD in granulosa cells, affecting proliferation and apoptosis, regulating the expression of granulosa cell genes, and regulating at least one of the secretion of hormones by granulosa cells. The regulation of the expression of granulosa cell genes includes regulating at least one of the PCNA gene, the BAX gene, and the BCL2 gene; the hormone includes at least one of E2 and P4.

[0024] Mutations or inactivation of FecB, GDF9, and BMP15 genes affect livestock reproductive traits. Follicular fluid granulosa cells are sensitive to increased ovulation numbers, which indirectly affects litter size and regulates animal reproductive activity. Oxidative stress-induced granulosa cell death is a common cause of follicular atresia, but key metabolic molecules need to be further elucidated. Therefore, this study used high- and low-breeding Hu sheep as the research objects, detected the differences in metabolites in the ovarian follicular fluid of high- and low-breeding Hu sheep through metabolomics, and combined with follicular fluid granulosa cell microtranscriptomics to further explore the mechanism of action of the key metabolite nicotinamide (β-Nicotinamide mononucleotide, NMN), providing a theoretical basis for the study of sheep's multiple birth traits.

[0025] On the basis of the existing litter size phenotype, blood samples of Hu sheep with records of the first and second litters were collected, DNA was extracted and then amplified by PCR, RFLP and SSCP were used for genotyping, and high- and low-breed Hu sheep with consistent genotypes and phenotypes were selected as research objects. After synchronous estrus treatment, they were slaughtered in the first estrus period, and follicular fluid was collected for metabolomics detection, and granulosa cells were collected for micro-transcriptome detection. Ovaries of sheep in the slaughterhouse were used to collect follicular fluid granulosa cells. After cell identification, an LPS-induced injury model was established in vitro. NMN was added to this model, and cell viability was detected by CCK-8, changes in cell apoptosis were analyzed by flow cytometry, and the secretion of steroid hormones was detected by ELISA. Kits were used to detect intracellular ATP levels and NAD content. At the same time, qPCR and Western blot were used to detect the expression of genes and proteins related to proliferation, apoptosis and hormone secretion.

[0026] The key metabolite NMN is differentially expressed in the follicular fluid of high- and low-breeding Hu sheep, and presents a similar or opposite expression pattern to the differential genes in the granulosa cells of the follicular fluid. In vitro cell validation experiments found that NMN increased the content of NAD+ and ATP in granulosa cells, inhibited LPS-induced granulosa cell apoptosis, increased the expression of the anti-apoptotic gene BCL2 and the proliferation marker gene PCNA, and inhibited the abundance of the pro-apoptotic gene BAX mRNA transcript. Exposure of LPS-stimulated granulosa cells to NMN improved the reduced P4 level. In summary, NMN inhibits LPS-induced apoptosis of granulosa cells, restores mitochondrial function, and restores cell function to normal. The above studies help to understand the potential molecular mechanisms of follicular development in the reproductive process and lay the foundation for the mechanism of folliculogenesis. The present invention studies the application of NMN through the following examples. The sheep ovarian follicular fluid granulosa cells used in the following examples were purchased from a slaughterhouse and obtained by centrifugation of healthy ovarian follicular fluid, which will not be repeated.

[0027] Example 1 This example is an analysis of the metabolomics characteristics of the dominant follicular fluid of high- and low-breed Hu sheep 200 3-4 year old, uniform body shape and similar reproductive physiological state multiparous empty-breasted Hu sheep with lambing records were selected as research subjects. The multiple lambing rate was 300%, while the single lambing rate was 100%. Then the blood of the test sheep was collected and the whole genome DNA of the blood was extracted. The DNA bands were clear, single and without dragging phenomenon, indicating that the integrity of the blood genome DNA of the test sheep was good. PCR amplification was performed on the genomes of different sheep for the major genes of multiple births BMPR-IB, GDF9 and BMP15 in Hu sheep. The reaction system was 20 uL, including Mix 10 uL, upstream and downstream primers 0.8 uL, ddH2O 7.4 uL, DNA 1 uL, and the PCR reaction cycle was set as 94 ℃ 3 min, 94 ℃ 30 s, 60 ℃ 30 s, 72 ℃ 1 min, 30 cycles, 72 ℃ 5 min, 4℃ ∞; the size of the obtained product was consistent with the size of the target fragment, and the bands were clear and free of mixed bands, which could be used for sequencing reactions. PCR-SSCP technology was used to detect that the AG mutation occurred at 152bp in the promoter region of GDF9 in the sheep population, and the first exon of the BMP15 gene (AF236078) had wild-type AA and mutant BB. The FecB mutation of the BMPR-1B gene was detected in the sheep population by PCR-SSCP and PCR-RFLP techniques, and there were two genotypes: wild homozygous (AA) and heterozygous mutant (AG). After matching the phenotypes and genotypes of Hu sheep one by one, the high-breeding and low-breeding groups of Hu sheep with different genotypes were determined: high-breeding group Hu sheep (FecBBB, GDF9GG and BMP15AA) and low-breeding group Hu sheep (FecBB+, GDF9AT and BMP15BB), with 4 sheep in each group. The genotypes were identified by PCR-SSCP and PCR-RFLP methods. For PCR-SSCP typing, the upstream primer sequence of FecB is shown in SEQ ID No.1, the downstream primer sequence is shown in SEQ ID No.2, and the product sequence is shown in SEQ ID No.3, SEQ ID No.4. The upstream primer sequence of BMP15 is shown in SEQ ID No.5, the downstream primer sequence is shown in SEQ ID No.6, and the product sequence is shown in SEQ ID No.7, SEQ ID No.8. The upstream primer sequence of GDF9 is shown in SEQ ID No.9, the downstream primer sequence is shown in SEQ ID No.10, and the product sequence is shown in SEQ ID No.11-SEQ ID No.16.

[0028] After the sheep in the experimental group were slaughtered, their ovaries were first collected, the dominant follicles (3-5 mm) were peeled off, and the follicular fluid was collected for metabolome sequencing. Figure 1LP_B_FF and LP_B_FF are the follicular fluid samples of low-breeding and high-breeding Hu sheep, respectively. Figure 1 This is a heat map of the relative abundance of differential metabolites in the metabolome of the dominant follicular fluid of high- and low-breed Hu sheep. It was found that there were significant metabolic differences in the dominant follicular fluid of high- and low-breed Hu sheep, especially in metabolites related to energy metabolism, which may affect the energy balance in the follicles, thereby affecting the development quality of the follicles and the maturation of the oocytes. This discovery provides a new strategy for screening potential biomarkers for predicting or improving the reproductive performance of sheep, or for sheep breeding.

[0029] Example 2 This example shows the effect of NMN on the activity and energy metabolism of Hu sheep granulosa cells induced by LPS Establishment of LPS-induced Hu sheep ovarian granulosa cell model: Use CCK8 method to detect the effects of different LPS addition concentrations and different time points (0, 12, 24 h) on the viability of ovarian granulosa cells, and explore the most suitable concentration of LPS added to granulosa cells; Exploration of the optimal concentration of MN in granulosa cells: Purchase commercial NMN reagent (MCE), first design different concentration gradients according to its instructions and existing references, and after treating ovarian granulosa cells, use CCK-8 method to detect its effect on cell viability, and screen the most suitable concentration of NMN added to ovarian granulosa cells. Figure 2 The effects of different concentrations of LPS on the viability of granulosa cells. Figure 2 In A, HP is the dominant follicular fluid sample of high-breeding Hu sheep, and LP is the dominant follicular fluid sample of low-breeding Hu sheep. ** indicates significant difference (P<0.01). Figure 2 B and 2C show the effects of different addition amounts of NMN and LPS on granulosa cell viability in Hu sheep granulosa cells, respectively, indicating that exogenous addition of NMN and LPS affects cell viability, demonstrating effectiveness.

[0030] Based on the above experiment, appropriate concentrations of LPS and NMN were added to granulosa cells, the cells were collected after 48 h, and commercial kits were used to detect changes in ATP and NAD levels in granulosa cells. Figure 3 and Figure 4 The results showed that the ATP and NAD levels in the LPS-treated group were significantly lower than those in the control group, while NMN treatment rescued the low energy level caused by LPS, indicating that it affected the energy metabolism in granulosa cells.

[0031] Example 3 This example shows the effect of NMN on LPS-induced proliferation and apoptosis of Hu sheep granulosa cells and related genes RNA samples were collected 24 h after the cells were treated, extracted using RTIZOL reagent, and reverse transcribed and fluorescent quantitative PCR was performed using a commercially available kit to detect the expression changes of genes related to proliferation and apoptosis, such as PCNA, BAX, and BCL2, in granulosa cells. Figure 5 As shown. The upstream primer sequence of the internal reference gene ACTB is shown in SEQ ID No.17, the downstream primer sequence is shown in SEQ ID No.18, and the product sequence is shown in SEQ ID No.25. The upstream primer sequence of PCNA is shown in SEQ ID No.19, the downstream primer sequence is shown in SEQ ID No.20, and the product sequence is shown in SEQ ID No.26. The upstream primer sequence of BAX is shown in SEQ ID No.21, the downstream primer sequence is shown in SEQ ID No.22, and the product sequence is shown in SEQ ID No.27. The upstream primer sequence of BCL2 is shown in SEQ ID No.23, the downstream primer sequence is shown in SEQ ID No.24, and the product sequence is shown in SEQ ID No.28.

[0032] Cell treatment method: The separated and frozen Hu sheep granulosa cells were revived to a 6-well plate. When the cell confluence reached 60-70%, the dissolved β-nicotinamide mononucleotide was diluted to 100 uM with complete culture medium, and the lipopolysaccharide LPS was diluted to 80 ug / mL. Then, according to the existing literature, the cells were pretreated with LPS for 3 h, and then β-nicotinamide mononucleotide was added. RNA was collected after 24 h of treatment, and protein was collected after 48 h. The proliferation and apoptosis of sheep granulosa cells treated with NMN and LPS were detected by EDU kit and flow cytometry apoptosis detection. The results are as follows: Figure 6 and Figure 7 As shown, CON is the control group, NMN is the group with appropriate NMN concentration, LPS is the group with appropriate LPS concentration, and LPS+NMN group is the group with LPS and NMN co-treatment. The addition of NMN can rescue the granulosa cell death caused by LPS to a certain extent, indicating that it affects the apoptosis level in granulosa cells and has a promoting effect on the function of granulosa cells.

[0033] Example 4 Effect of NMN on LPS-induced P4 secretion in Hu sheep granulosa cells The cell supernatant was obtained 48 hours after cell treatment, and the secretion level of P4 in the cell supernatant of different groups was detected using the sheep P4 ELISA kit. Cell treatment method: The separated and frozen Huyang granulosa cells were revived to a 6-well plate. When the cell confluence reached 60-70%, the dissolved β-nicotinamide mononucleotide was diluted to 100uM with complete culture medium, and the lipopolysaccharide LPS was diluted to 80 ug / mL. Then, according to the existing literature, the cells were pretreated with LPS for 3 hours, and then β-nicotinamide mononucleotide was added. After 48 hours of treatment, the cell supernatant was collected. Figure 8 This is the effect of NMN on LPS-induced granulosa cell P4 secretion in Example 4 of the present invention. Progesterone, as a key type of steroid hormone, is a key factor in the function of granulosa cells during follicular development. This study found that NMN can control the secretion of steroid hormones in the granulosa of Hu sheep follicles, which may affect follicular development and is a metabolic marker.

[0034] In addition to the above implementations, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A metabolic marker for regulating follicular granulosa cell proliferation and steroid hormone secretion, characterized in that: The metabolic marker is a metabolite β-nicotinamide mononucleotide that is different in the dominant follicular fluid of high- and low-breed Hu sheep screened by combining phenotype and genotype.

2. The use of the metabolic marker for regulating follicular granulosa cell proliferation and steroid hormone secretion according to claim 1, comprising preparing β-nicotinamide mononucleotide for use in regulating Hu sheep granulosa cell products.

3. The use of the metabolic marker for regulating follicular granulosa cell proliferation and steroid hormone secretion according to claim 2, characterized in that: Regulating Hu sheep granulosa cells includes regulating at least one of the following: regulating the energy metabolism level in granulosa cells, regulating cell proliferation and apoptosis, regulating the expression of granulosa cell genes, and regulating the secretion of hormones by granulosa cells.

4. The use of the metabolic marker for regulating follicular granulosa cell proliferation and steroid hormone secretion according to claim 3, characterized in that: The energy metabolism level in the regulated granulosa cells includes at least one of NAD and ATP levels.

5. The use of the metabolic marker for regulating follicular granulosa cell proliferation and steroid hormone secretion according to claim 3, characterized in that: The regulating the expression of granulosa cell genes includes regulating at least one of the PCNA gene, the BAX gene, and the BCL2 gene.

6. The use of the metabolic marker for regulating follicular granulosa cell proliferation and steroid hormone secretion according to claim 3, characterized in that: The hormone includes at least one of estradiol E2 and progesterone P4.

7. The use of the metabolic marker for regulating follicular granulosa cell proliferation and steroid hormone secretion according to claim 2, characterized in that: The product is used to increase the content of nicotinamide adenine dinucleotide.

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