CN111690726A - Method for activating primordial follicles by BTC growth factor - Google Patents
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- CN111690726A CN111690726A CN202010628723.1A CN202010628723A CN111690726A CN 111690726 A CN111690726 A CN 111690726A CN 202010628723 A CN202010628723 A CN 202010628723A CN 111690726 A CN111690726 A CN 111690726A
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Abstract
The invention discloses a method for activating primordial follicles by BTC growth factors, which comprises the following steps: step one, finding out target research factors Btc by applying an injury model and an ovulation induction model; step two, Btc expression and location of growth factor and its receptor in ovary; step three, exploring Btc the activation effect of the growth factor on primordial follicles; step four, verifying Btc whether the factor regulates the primordial follicle activation through a PI3K and/or mTOR signaling pathway; and step five, analyzing Btc the change of the transcriptome level induced by the growth factor treatment, and finding out which key molecules activate primordial follicles. The invention relates to the technical field of follicle activation. According to the method for activating the primordial follicle by the BTC growth factor, the initiator and key molecules of primordial follicle initiation recruitment under physiological conditions are searched by deep mining of RNA-seq data of an ovarian injury animal model, so that a foundation is laid for the subsequent development of related researches, and a new thought is provided for further improving the primordial follicle activation efficiency.
Description
Technical Field
The invention relates to the technical field of follicle activation, in particular to a method for activating primordial follicles by BTC growth factors.
Background
Improving reproductive health level is one of the core contents of the population strategy in China. In recent years, the incidence of infertility has increased year by year, reaching 10 to 15% among couples of childbearing age. With environmental pollution and increased living pressure, premature ovarian failure is aged, the incidence rate of premature ovarian failure accounts for 1 percent of women in childbearing age, and the physical and mental health of women in childbearing age is seriously influenced. Premature ovarian failure is mainly caused by premature exhaustion of primordial follicle banks due to various reasons, so that the exploration of a molecular regulation mechanism of primordial follicle formation and development has important significance for researching the pathogenesis of premature ovarian failure and developing in-vitro ovarian activation (IVA).
Primordial follicles, which serve as the source of the growing follicles at all levels in the ovary, are formed and in constant numbers before and after birth in mammals. Primordial follicles undergo transformation into primary follicles, a process called initial recruitment. The follicle then develops into a secondary follicular stage, which begins to receive hormonal regulation. After the onset of recruitment, the number of primordial follicles is reduced until depletion. The number and rate of depletion of the primordial follicle pool directly determine the fertility and length of reproductive life of the female. Premature ovarian failure clinically refers to the serious shortage of primordial follicle reserve in female ovaries, ovarian function decline before the age of 40, and the clinical manifestations are primary or secondary amenorrhea, decline of serum estrogen level, rise of gonadotropin level, thereby causing the problems of ovulation failure, infertility and the like. However, the mechanism of follicle recruitment is not clear to date.
The initial recruitment of primordial follicles is a finely regulated process by various factors from the peripheral environment, the in vivo environment, the local internal environment of the ovary, which interact to regulate the homeostasis of resting and activation of the primordial follicle pool. In addition to the regulatory factors within the ovary, the age and estrus cycle of the animal also determine to some extent the rate of initial recruitment, and studies have shown that primordial follicles in the prophase of estrus are more active than those in the interestrus phase; the activation speed of the human primordial follicles is inversely proportional to the size of the primordial follicle bank, and the consumption speed of the primordial follicles before and after menopause is obviously accelerated. In addition, external environmental factors can also disrupt the homeostasis of the primordial follicle library. For example, environmental endocrine disruptors DEHP and bisphenol A are the main components of plastic products in our daily life, and research shows that the compounds can promote the consumption of original follicles in animal models. It follows that, however, under physiological conditions, the process of initial recruitment of follicles in the ovary is not readily observable, and establishing a model that can activate primordial follicles in vivo for a short period of time and in large quantities would be advantageous for studying the above-mentioned problem.
In past studies, scientists have revealed an important role for PI3K and mTOR signaling pathways in oocytes and mTOR signaling pathways in granulosa cells in regulating primordial follicle activation by constructing systemic knockout or conditional knockout mouse models. Further studies have shown that these two signal pathways in oocytes are not simply up-and-down regulated but rather have some synergy. There is a very complex mechanism for regulating the PI3K signaling pathway and the mTOR signaling pathway in oocytes, which is involved in regulating the homeostasis of primordial follicles in the primordial follicle library with activation. Recent studies have shown that mTORC1 signaling pathway in granulosa cell precursors is also involved in regulating primordial follicle activation. When the inhibitory molecule Tsc1 of mTOR in the precursor granulosa cells is specifically knocked out, the mTOR signaling pathway in the precursor granulosa cells is activated, thereby promoting Kit ligand expression in the granulosa cells. The Kit ligand binds to the cKit receptor on the oocyte membrane, transmits a signal to the oocyte, subsequently activates the PI3K signaling pathway in the oocyte and finally activates the primordial follicle. When the Rictor gene, an important component of mTORC1, in the precursor granulosa cell is conditionally knocked out, the precursor granulosa cell in the primordial follicle can not differentiate into granulosa cells, so that the primordial follicle can keep a resting state for a short time, but the resting state can not be maintained for a long time, and the oocyte can die after a while. It follows that mTOR signaling activity in both oocytes and granulosa cells is critical in regulating primordial follicle development. In addition, due to the lack of gonadotropin receptor expression, primordial follicle activation is not regulated by the hypothalamic-pituitary-gonadal axis, but by cytokines and growth factors produced locally in the ovary. Many cytokines and growth factors such as neurotropins, kit ligand, LIF, KGF, bFGF and BMP family factors have been shown to promote initial recruitment of primordial follicles by autocrine or paracrine means. While other factors such as AMH (Anti-Mullerian hormone), somatotatin, CXCL12, etc. inhibit primordial follicle activation. Although the above studies reveal a significant role for extracellular factors and intracellular signaling pathways in regulating primordial follicle activation. The mechanism of how primordial follicles are selectively activated under physiological conditions is not clear.
In early studies, it was thought that the ovulation process caused an acute inflammatory response in the ovaries, and thus, the ovulation process was also considered to be a periodic inflammatory response process in the ovarian tissue. In previous studies, we constructed a mouse model of ovarian injury and found that the injury induces the expression of Nerve Growth Factor (NGF) in local stromal cells, which ultimately activates primordial follicles near the incision by upregulation of a range of mTOR signaling pathways from stromal cells to granulocytic precursors to oocytes. Following ovarian injury, both transcriptome and translatome sequencing results showed significant changes in gene expression. In addition to the drastic changes of the functional genes related to the nerve active receptor-ligand (such as NGF), we also found other growth factors which are not reported in the previous researches, and the enrichment of the growth factors provides new clues and bases for the research of the regulation mechanism of primordial follicle activation.
During normal ovulation, LH hormone stimulation promotes the expression of Areg (ampheregulin, amphiregulin), Ereg (epiregulin), Btc (beta-cellulin) of the EGF growth factor family in granulosa cells, and these factors are involved in inducing cumulus cells to express HAS2, Tnfaip6, Ptgs2, thereby promoting the expansion of cumulus cells and the maturation of oocytes. In a synchronous activation effect model of a large number of primordial follicles caused by ovarian damage, the gene is obviously up-regulated after the ovarian damage. Since granulosa cell expansion of the follicles and oocyte maturation are not involved in ovarian damage, the specific roles played by these molecules in these processes and whether they would be involved in regulating primordial follicle activation remain to be investigated. Of the three EGF family members up-regulated following ovarian injury, Ereg systemic knockout mice resulted in down-regulation of Areg expression, but normal follicular development and ovulation numbers. Indicating that Ereg deletion and Areg down-regulation did not have a major effect on the development of the primordial follicle to the preovulatory follicle. Btc has not been reported to play a role in regulating follicular development.
Therefore, the present invention focuses on exploring the role of EGF family member Btc in primordial follicle activation and delves into Btc a possible mechanism of action in primordial follicle activation. Whether growth factors generated in the normal periodic ovulation process participate in the regulation and control on the initial recruitment of peripheral primordial follicles is searched, so that the relation between the ovulation process and primordial follicle activation is established, the influence of the epidermal growth factor Btc on the primordial follicle activation is discussed, the regulation and control mechanism is researched, and a new mechanism for selectively activating the primordial follicles is provided.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for activating primordial follicles by a BTC growth factor, which searches for a starting factor and a key molecule of primordial follicle initiation recruitment under physiological conditions by deep mining of RNA-seq data of an ovary injury animal model, lays a foundation for subsequent research on relevant aspects, and provides a new idea for further improving primordial follicle activation efficiency.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a method of activating primordial follicles by BTC growth factor, comprising the steps of:
step one, finding out target research factors Btc by applying an injury model and an ovulation induction model;
step two, Btc expression and location of growth factor and its receptor in ovary;
step three, Btc activation of the growth factor on primordial follicles, after Btc is applied to short-term culture of the ovary of a newborn mouse in vitro, the influence of Btc on primordial follicle activation is evaluated through the ovarian development condition;
step four, verifying Btc whether the factor regulates the primordial follicle activation through a PI3K and/or mTOR signaling pathway;
step five, analyzing Btc the change of the transcriptome level induced by the growth factor treatment, and finding out Btc which key molecules activate primordial follicles through regulation.
Preferably, in the first step, the differentially expressed genes in the Ovarian surgery model and the dynamic expression pattern in the ovulation process in the Ovarian Kaleidoscope Database are screened by RNA-seq.
Preferably, the expression and localization of Btc in the ovary of the newborn mouse are detected by RT-qPCR, Western blot and immunohistochemistry experiments from the mRNA and protein levels respectively in the second step.
Preferably, the indexes tested for evaluating ovarian development condition in the third step comprise: follicular count, apoptosis and proliferation, changes in follicular activation and expression of developmental marker molecules, and the like.
Preferably, on the basis of the third step, an aging mouse model is selected to simulate the situation of ovarian failure, and after Btc is injected into the ovarian cyst, the growth and development conditions of all levels of follicles of the aging mouse are observed and the fertility is identified.
Preferably, in step four, the ovaries of newborn mice are treated with PI3K and/or the mTOR signaling pathway specific blockers Wortmannin and Rapamycin and Btc receptor EGFR antagonists in combination with Btc.
Preferably, in the fourth step, the Western-blot method is used for verifying the specificity of the signal pathway inhibitor, and the immunohistochemistry method is used for detecting whether the expression of the primary follicle activation marker molecule in the ovary is changed after the inhibitor is added.
Preferably, in step four, serial section and follicle count are performed by collecting ovarian tissue, the effect of the inhibitor is further determined by follicular development, and Btc is found to be dependent on which signaling pathway regulates primordial follicle activation.
Advantageous effects
The invention provides a method for activating primordial follicles by BTC growth factors. The method has the following beneficial effects:
the method of this BTC growth factor for primordial follicle activation, through the above preliminary studies, using ovarian injury models and normal ovulation models, we found that EGF family changes during ovulation and injury are highly consistent, and these factors might explain how normal ovulation mediates initial recruitment of primordial follicles. Based on the established primordial follicle activation platform, we found Btc was able to activate primordial follicles and promote follicle development through mTORC1 signaling pathway. Future studies will focus on how to maintain the development of Btc-activated secondary follicles and whether, in combination with existing activators, a better activation is obtained than when they are used alone, enabling maximal activation of primordial follicles and preliminary exploration of their feasibility as a clinical application protocol.
Drawings
FIG. 1 is a schematic diagram of the steps of the method of the present invention;
FIG. 2 is a graph showing EGF family molecular changes during ovarian surgery and ovulation according to the present invention:
FIG. 2A shows the differential gene distribution of transcriptome and translatome sequencing screens after ovarian surgery, with consistent red, inconsistent green and no blue changes;
FIG. 2B shows the gene expression (FPKM) of EGF family Btc, Areg and Ereg and their downstream HAS2, Tnfaip6, Ptgs2 in transcriptome and translatome, and the results of qPCR validation after injury;
fig. 2C shows the location of Foxo3a in the operated ovary. Upper panel, near the surgical side ovarian incision, arrow, expression of Foxo3a transferred from the nucleus to the cytoplasm; lower panel, distal to the surgical side ovarian incision, arrows, with expression of Foxo3a distributed in the nucleus. Scale bar 100 μm;
FIG. 2D shows the dynamic expression changes of EGF family Btc, Areg and Ereg and their downstream HAS2, Tnfaip6, Ptgs2 during superovulation, at 0h, P.I. PMSG was injected; at 48h, hCG was injected intraperitoneally.
Data comes to an OKDB database;
FIG. 3 is a graph of Btc expression of its receptor in ovaries at various time points;
figure 3A is Btc and the mRNA dynamics of the receptors Egfr, Erbb2, Erbb3, Erbb4 in E17.5, D0.5, D2.5, D5.5 ovaries, ×, p < 0.01;
fig. 3B is Btc and the dynamic changes in the receptor Egfr, Erbb2 protein levels in the E17.5, D0.5, D2.5, D5.5 ovary, as well as changes in the activity of the mTORC1 signaling pathway;
FIG. 3C is the expression localization of Btc in D0.5 and D5.5 mouse ovaries. Green, Btc; blue, DAPI; scale bar 100 μm.
FIG. 4 shows Btc follicle development promotion by in vitro culture, (n >5), follicle count statistics after serial sectioning and H & E staining;
FIG. 4A shows that after Btc in vitro D2.5 mice ovaries treated for 96h, Btc treated groups had more secondary follicles. Scale bar 100 μm;
fig. 4B is a graph of statistical analysis of follicle counts. primordial, primordial follicles; primary, primary follicle; secondary, secondary follicle;
FIG. 4C is a graph showing analysis of egg cell diameter in ovaries. P < 0.01; D) btc transplantation of 24h newborn mouse ovaries under the kidney capsule can promote the development of ovaries.
Fig. 4E is H & E staining of ovarian sections. Red arrow, luminal follicle from group Btc;
FIG. 4F shows the statistical results of the serial section of ovaries, the count of follicles, the percentage of follicles in each developmental stage to the total number of follicles, primordial follicles; primary, primary follicle; secondary, secondary follicle; antral, antral follicles; p < 0.01;
FIG. 5 is a graph of the effect of short-term Btc treatment on the PI3K/mTORC1 signal pathway;
FIG. 5A shows the result of Western blot analysis, wherein Btc shows that the phosphorylation levels of marker molecules of PI3K/mTORC1 signal channel in ovary are changed at 1h, 3h and 6h when mouse ovary is treated;
fig. 5B is qPCR analysis, Btc treatment induced up-regulation of mRNA of Kitl,. P < 0.01. C) Localization of the primordial follicle activation marker molecule Foxo3a in the ovary, scale 100 μm;
fig. 5D shows the coring ratio of Foxo3a, P < 0.01.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-5, the present invention provides a technical solution: a method of activating primordial follicles by BTC growth factor, comprising the steps of:
step one, finding out target research factors Btc by applying an injury model and an ovulation induction model; in the first step, differential expression genes in an Ovarian surgery model and a dynamic expression map in an ovulation process in an OKDB (Ovarian Kaleidoscope Database, http:// OKDB. appliedbioinfo. net /) Database are screened out through RNA-seq. According to published articles, differential expression genes after ovarian injury are screened out. The gene which is severely changed in the ovulation process and the Ovarian injury simultaneously is screened by mainly selecting growth factor related molecules from the gene and combining an OKDB (Ovarian Kaleidosipe Database, http:// OKDB. applidobio-info. net /) Database, and the growth factors and the cytokines are focused. We found that expression of 3 molecules Areg (ampheregulin, amphiregulin), Ereg (epiregulin), Btc (beta-cellulin) of the EGF family was significantly altered in the ovarian injury model. In combination with unpublished MicroArray results, we verified the high similarity of expression patterns of 3 genes Areg, Ereg, Btc during ovarian injury and ovulation by qPCR. Further according to the existing literature report, the research range is narrowed, and finally Btc is selected for subsequent research.
Step two, Btc expression and location of growth factor and its receptor in ovary; btc expression and localization in the newborn mouse ovary were detected from mRNA and protein levels by RT-qPCR, Westernblot and immunohistochemistry experiments, respectively. Btc are members of the EGF family, highly expressed in the pancreas, and their receptors include EGFR, Erbb2, Erbb3, Erbb 4. Btc and receptors, regulate the vital activities of cells. In ovarian ovulation, the secretion of Btc factor by granulosa cells is induced under the stimulation of LH hormone. The factor Btc acts on the cumulus cells to promote the expression of proteins such as Tnfaip6, HAS2 and Ptgs2 by the cumulus cells, thereby regulating the expansion of the cumulus and the maturation of the oocyte. In previous studies, no function of Btc in early follicles was reported. Here we first performed qPCR detection of mRNA and detection of protein for the Btc and Btc receptors. It was determined that it was also expressed in early ovaries and that important regulatory functions may also be present.
Step three, Btc activation of the growth factor on primordial follicles, after Btc is applied to short-term culture of the ovary of a newborn mouse in vitro, the influence of Btc on primordial follicle activation is evaluated through the ovarian development condition; the indexes for evaluating and detecting the ovarian development condition comprise: follicular count, apoptosis and proliferation, changes in follicular activation and expression of developmental marker molecules, and the like. On the basis, an aging mouse model is selected to simulate the situation of ovarian failure, Btc is injected into the ovarian cyst, the growth and development conditions of all levels of follicles of the aged mouse are observed, and the fertility is identified. To verify the paracrine regulatory effect of factor Btc on primordial follicle activation, neonatal mouse (D2.5) ovaries were selected for in vitro culture experiments. Since the ovary of the mouse at this stage mainly contains primordial follicles, the influence on the activation and development of primordial follicles can be evaluated by adding key factors to the culture system. Collecting ovaries 24h after the key factors are added, and detecting the expression of a follicle development important factor Kitl through RT-qPCR; detecting the phosphorylation level of a related signal pathway (PI3K or mTOR signal pathway) by Western-blot; immunohistochemistry was performed to detect changes in expression of primordial follicle activation marker molecules, Foxo3a and p-rpS6, to determine primordial follicle activation. On the other hand, functional blocking antibodies or inhibitors of key factors are added into the culture system, and after long-term in vitro culture (5 days), the influence of endogenous key factors on primordial follicle activation and development is determined by evaluating the development of follicles. Also, we treated with Btc briefly and transplanted it under the kidney tunica mucosa of adult recipient female mice for 2 weeks. In order to remove the influence of hormone secretion of the own ovary of the recipient mouse, the bilateral ovary of the recipient mouse is removed. The recipient mice need to be injected with FSH at all times to maintain the development of the transplanted ovaries. The duration of ovarian transplantation is determined by the purpose of the test: ovaries around two weeks were transplanted for evaluation of follicular development to determine the optimal in vitro activation protocol on the one hand and to verify the specificity of the signalling pathway activator for primordial follicular activation by blocking the addition of inhibitors of this signalling pathway upon transient treatment in vitro at Btc on the other hand. On the basis, an aging mouse model is selected to simulate the situation of ovarian failure, Btc is injected into the ovarian cyst, the growth and development conditions of all levels of follicles of the aged mouse are observed, and the fertility is identified. It is studied whether Btc can activate primordial follicle in ovary of old mouse after administration, improve fertility and prolong reproductive life.
And fourthly, verifying Btc whether factor regulates primordial follicle activation through a PI3K and/or mTOR signaling pathway, treating the ovary of a newborn mouse with PI3K and/or mTOR signaling pathway specific blockers Wortmannin and Rapamycin and Btc receptor EGFR antagonist and Btc, verifying the specificity of a signaling pathway inhibitor by using a Western-blot method, detecting whether the expression of primordial follicle activation marker molecules in the ovary is changed by using an immunohistochemical method after the inhibitor is added, performing continuous section and follicle counting by collecting ovarian tissues, further determining the action of the inhibitor through the development condition of follicles, and finding a Btc growth factor depending on which signaling pathway regulates primordial follicle activation. Activation of the PTEN-PI3K-Akt-Foxo3a signaling pathway in oocytes during primordial follicle activation phosphorylates the transcription factor Foxo3a and results in intracellular translocation of Foxo3a from the nucleus to the cytoplasm (Li J et al, 2010). In addition, the use of an mTOR signaling pathway activator in our previous studies also promoted the expression of the downstream molecule p-rpS6 of this pathway in activated oocytes (Sun X et al, 2015). Therefore, the primordial follicle activation marker molecules Foxo3a and p-rpS6 were selected in this study and examined for their expression changes in primordial follicles shortly after ovarian damage by immunohistochemical methods for determining primordial follicle activation.
In addition, since primordial follicles are activated and then enter a growth state, the ratio of follicles at each stage is also a good index for evaluating whether primordial follicles are activated by collecting ovaries 2 weeks after kidney capsule transplantation and comparing the number of follicles at each stage. The follicle at each stage is divided into primordial follicle, primary follicle, secondary follicle and antral follicle, the ratio of the number of follicles at each stage to the total number of follicles is counted by continuous ovarian section, and then the number is compared with the number of follicles at each stage to determine whether the primordial follicle is activated.
Step five, analyzing Btc the change of the transcriptome level induced by the growth factor treatment, and finding out Btc which key molecules activate primordial follicles through regulation. Primordial follicle activation is accompanied by changes in gene transcription levels. We investigated Btc the alteration of the transcriptome of the ova in primordial follicles by a technique using single-cell RNA-seq. Comparing and screening differential gene data obtained by RNA-seq, firstly verifying the consistency of the differential gene data with the RNA-seq data by methods such as real time-qPCR, western-blot, immunohistochemistry and the like; and carrying out preliminary bioinformatics analysis on the screened differential genes to establish a gene regulation and control interaction network. And 3-4 key molecules for Btc regulation were screened. The method can better screen key molecules for primordial follicle activation.
The above technical solution is further explained below by combining the existing data with experimental data:
1) in a published article of ovarian injury, we constructed a sequencing method for the translational set-polyribosome-bound RNA-Seq (He Y et al, 2017). In eukaryotic cells, due to the existence of numerous post-transcriptional regulatory mechanisms, the intracellular transcriptome and mRNA into the translational set are not completely consistent, and the level of the transcriptome mRNA does not completely represent the change of protein expression in the cell at a certain time. Although proteomics provides the most direct assessment of gene expression, there are sensitivity limitations. Thus, the transcriptome acts as an intermediate bridge between transcriptomes and proteomes, and it is more accurate than transcriptomes in assessing gene expression, while having higher sensitivity than proteomics (Ingolia et al, 2009). We mined transcriptome and translational set sequencing results deeply. Screening for highly consistent transcriptome and translational sets of data. We mainly aimed at the analysis of factors related to growth regulation. We note that all 3 factors of the epidermal growth factor family (EGF family), Btc, Areg and Ereg, are clearly up-regulated after injury (as shown in figure 2A). EGF is a multifunctional growth factor that, by binding to the receptor EGFR, induces cell growth, migration, and promotes expression of differentiation genes. In the process of ovarian ovulation of the mouse, stimulated by LH hormone, the ovarian cyst is induced to secrete Btc, Areg and Ereg factors. And then acting on the cumulus cells to promote the expression of proteins such as Tnfaip6, HAS2 and Ptgs2 of the cumulus cells, thereby regulating the expansion of the cumulus cells and the maturation of the oocytes. Interestingly, when the ovary was damaged, the expression of these 3 genes increased rapidly, and the polysome-related RNA-seq also increased significantly, indicating that there was also a significant increase in these 3 factors at the protein level. Even more surprisingly, significant expression increases were observed in the downstream regulated genes Tnfaip6, HAS2 and Ptgs2 (fig. 2B). We verified the expression changes corresponding to 6 genes by qPCR (shown in figure 2B).
Our past studies have found that ovarian surgery can activate primordial follicles near the incision. We verified by the marker molecule Foxo3a for primordial follicle activation. After 6h of ovarian injury, we found that Foxo3a from primordial follicles near the incision was transported from the nucleus to the cytoplasm, while Foxo3a from primordial follicles far from the incision was still localized in the nucleus (as shown in FIG. 2C). We also found a similar situation in staining of ovulated Foxo3a in normal mice, i.e. more of the primordial follicle Foxo3a surrounding the ovulatory point was localized in the cytoplasm (data not shown). We speculate that there is a direct link between ovulation and ovarian damage.
The OKDB database has an unpublished set of microarray data. One set of samples was collected every 2 hours after PMSG injection and every 1 hour after hCG injection, and dynamic expression of genes was observed throughout the superovulation process. We analyzed 3 molecules of the EGF family Btc, Areg and Ereg as well as downstream regulated HAS2, Tnfaip6, Ptgs 2. Btc, Areg and Ereg were slightly upregulated after PMSG injection, Btc, Areg returned to baseline 8h after PMSG injection, Ereg was returned to baseline 16h, and downstream thereof, regulated HAS2, Tnfaip6, Ptgs2 had similar expression changes. Btc, Areg and Ereg were rapidly upregulated more than 1000-fold following hCG injection, with downstream rapid upregulation of the regulated HAS2, Tnfaip6, Ptgs2 (as shown in FIG. 2D).
Btc, Areg and Ereg rise rapidly during ovulation, and regulated HAS2, Tnfaip6 and Ptgs2 are also up-regulated rapidly, and the expression pattern is extremely similar to that in an ovarian injury model. This may mean that the mechanisms found for ovarian damage are also present during normal ovulation. We performed a rigorous screen for Btc, Areg and Ereg, in which Ereg systemic knockout mice, Areg, were also severely down-regulated, with some effect on fertility, but in which the egg cells developed normally (Kim, k., et., 2011). We finally selected Btc as the target for downstream studies.
2) Previous studies of Btc in the ovary have focused primarily on the process of ovulation, where cumulus cells secrete Btc. Here we sought to study Btc the activation effect on primordial follicles. We first needed to detect whether Btc molecule is expressed in ovaries when there are no cumulus cells in the early stages of ovaries. We selected the embryo E17.5, postnatal D0.5, D2.5, D6.5 mouse ovary as the subject. Our qPCR detected the expression of Btc, as well as Btc receptors in early ovaries. Subsequently, we also examined the expression of protein levels. Fortunately, at the mRNA level, we found Btc, and its receptor, to be expressed in early ovaries, as shown in figure 3A. Wherein Btc, EGFR and Erbb2 are expressed in ovary at D5.5 days obviously higher than E17.5, D0.5 and D2.5 times. The main difference from day D2.5 to day D5.5 is that there is a large number of primordial follicles developing into primary follicles. We speculate that Btc and its receptors EGFR and Erbb2 may be involved in regulating primordial follicle activation. Subsequently, the Westernbolt experiment also confirmed this result as shown in figure 3A. Btc protein was significantly higher than the E17.5, D0.5, D2.5 period at D5.5 days, whereas EGFR and Erbb2 were higher than E17.5 and D5.5 than D2.5 at D0.5 days, this trend is consistent with the activity of mTORC1, and we found that the phosphorylation levels of proteins P70S6k and Rps6 downstream of mTORC1 were also increased at the corresponding periods. The period of onset of the formation of large primordial follicles at D0.5 day, and the period of conversion of large primordial follicles to primary follicles at D5.5 day. The involvement of mTORC1 signaling in regulating primordial follicle formation and activation has been recognized (Sun X et al, 2015; adikari, D et al, 2010; adikari, D et al, 2009; Zhang, J et al; 2017.). At the same time we also tested Btc for localization in the ovary, as shown in FIG. 3C. At D0.5, it is localized mainly in epidermal cells surrounding the ovary, and in the oocyte cytoplasm in cytt. There was a clear positional change during development, and at D5.5 we found Btc to be predominantly localized in both the egg cytoplasm and nucleus. There is also a clear localization in granulosa cells that develop into secondary follicles. However, the current Btc antibody is less available for immunohistochemistry, and the results need to be tested again to determine the specificity of the antibody, what is the effect of Btc in the nucleus, as we found? Therefore, we can speculate that Btc and its receptor may have multiple functions on ovarian development, and may be involved in primordial follicle formation and primordial follicle activation in addition to the regulation of cumulus cell expansion and oocyte maturation in previous studies.
3) We found Btc and its receptor to be expressed dynamically in early ovaries, so in further experiments we chose to add Btc in vitro to observe its effect on primordial follicle development. We took the D2.5 mouse ovary, and the largest number of primordial follicles were present at this time point. After 5 days of continuous culture in vitro, the ovary volume was increased, and then the follicle development of the Btc-treated group was better than that of the control group by observing the HE staining of the continuous section, as shown in FIGS. 4A and 4B. The control group had a primordial follicle percentage of 75.21% and a primary follicle percentage of 20.13%, whereas the Btc-treated group had only 64.32% and the primary follicle percentage of 25.25%. Most notably, we found that there were more secondary follicles of larger diameter in the treatment group. Also by measuring the egg cell diameters, it was found that the two groups of egg cell diameters were mainly concentrated between 20-25 μm, as shown in fig. 4A, 4C, which is mainly primordial and primary follicles, while in the Btc treated ovary, more secondary follicles were present. Here we used the established platform for primordial follicle activation in vitro and demonstrated Btc activation of primordial follicles as well as promotion of transformation to secondary follicles. After the Btc-treated ovaries were transplanted under the renal tunica mucosa of recipient mice, the development degree of ovaries was improved significantly, as shown in fig. 4D and 4E, but in the result of follicle counting, as shown in fig. 4F, we did not find a large increase in primary follicles and secondary follicles, and only the proportion of antral follicles was higher than that of the control group, and was statistically significant. This is not consistent with our results from 5 days of in vitro culture. Returning again to the experiments in vitro culture, we found that although a large number of primordial follicles were activated and developed into primary and secondary follicles, granulosa cells from secondary follicles did not proliferate in large amounts, mostly remaining encapsulated in 2-3 layers of granulosa cells. Therefore, we speculate that primordial follicles are activated into the growth phase, but the subsequent development of secondary follicles, especially granulosa cell proliferation, is not well supported, leading to certain problems in the development of the final secondary follicles. In response to this problem we will subsequently further investigate Btc the effect on granulosa cells in the follicles as well as on oocytes.
4) We added Btc in vitro and selected Btc transiently treated ovaries for study of 2 signaling pathways associated with primordial follicle activation, the PI3K-Akt signaling pathway, and the mTORC1-P70S6k signaling pathway. We collected ovarian tissues 1h, 3h, 6h after Btc addition for Western-blot analysis. As shown in FIG. 5A, activation of both the PI3K-Akt signal path and the mTORC1-P70S6k signal path occurs. The phosphorylation of Akt reached a maximum at 1h and returned to baseline after 3h, whereas p70S6K, rpS6 downstream of mTORC1 appeared to rise at 1h and was at a higher phosphorylation level after 6 h. Therefore, we speculate that Btc is a short-term effect on the activation of PI3K, while activation of mTORC1 may be a persistent effect. qPCR results found that Kitl mRNA was upregulated 24h after Btc treatment, as shown in FIG. 5B, whereas recent studies found that Kitl upregulation in granulosa cells could interact with the oocyte c-kit to activate the oocyte PI3K/mTORC1 signaling pathway and thus primordial follicles. Foxo3a is phosphorylated during primordial follicle activation and is transferred from the nucleus to the cytoplasm. We found through immunohistochemistry that Foxo3a showed a significant increase in the nucleus-forming ratio in Btc-treated group, only 23% in control group, and 42% in Btc-treated group, indicating that Btc could promote nucleus-forming of Foxo3 a. The above results indicate that Btc treatment activates primordial follicles.
In summary, from the preliminary studies described above, using an ovarian injury model and a normal ovulation model, we have found that the EGF family varies highly uniformly during ovulation and injury, factors that may explain how the normal ovulation process mediates initial recruitment of primordial follicles. Based on the established primordial follicle activation platform, we found Btc was able to activate primordial follicles and promote follicle development through mTORC1 signaling pathway. Future studies will focus on how to maintain the development of Btc-activated secondary follicles and whether, in combination with existing activators, a better activation is obtained than when they are used alone, enabling maximal activation of primordial follicles and preliminary exploration of their feasibility as a clinical application protocol.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation. The use of the phrase "comprising one of the elements does not exclude the presence of other like elements in the process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A method for activating primordial follicles by BTC growth factor, comprising the steps of:
step one, finding out target research factors Btc by applying an injury model and an ovulation induction model;
step two, Btc expression and location of growth factor and its receptor in ovary;
researching the activation effect of Btc growth factors on primordial follicles, and evaluating the influence of Btc on primordial follicle activation through the ovarian development condition after short-term culture is carried out on the ovary of a newborn mouse in vitro by Btc;
step four, verifying Btc whether the factor regulates the primordial follicle activation through a PI3K and/or mTOR signaling pathway;
step five, analyzing Btc the change of the transcriptome level induced by the growth factor treatment, and finding out Btc which key molecules activate primordial follicles through regulation.
2. The method of claim 1, wherein the activation of primordial follicles by a BTC growth factor is performed by: in the first step, differential expression genes in an ovarian surgery model and a dynamic expression map in an ovulation process in an ovarian Kaleidoscope Database are screened out through RNA-seq.
3. The method of claim 1, wherein the activation of primordial follicles by a BTC growth factor is performed by: in the second step, the expression and location of Btc in the ovary of the newborn mouse are detected from the mRNA and protein level respectively by RT-qPCR, Westernblot and immunohistochemistry experiments.
4. The method of claim 1, wherein the activation of primordial follicles by a BTC growth factor is performed by: the indexes for evaluating and detecting the ovarian development condition in the step three comprise: follicular count, apoptosis and proliferation, changes in follicular activation and expression of developmental marker molecules, and the like.
5. The method of claim 1, wherein the activation of primordial follicles by a BTC growth factor is performed by: and on the basis of the third step, selecting an aging mouse model to simulate the ovarian failure condition, injecting Btc into the ovarian cyst, observing the growth and development conditions of all levels of follicles of the old mouse, and identifying the fertility.
6. The method of claim 1, wherein the activation of primordial follicles by a BTC growth factor is performed by: in step four, the ovaries of newborn mice were treated with PI3K and/or the specific blockers of the mTOR signaling pathway Wortmannin and Rapamycin, as well as Btc receptor EGFR antagonists, in combination with Btc.
7. A method of activating primordial follicles as claimed in claim 6, wherein: in the fourth step, a Western-blot method is used for verifying the specificity of the signal channel inhibitor, and an immunohistochemical method is used for detecting whether the expression of the primary follicle activation marker molecules in the ovary is changed after the inhibitor is added.
8. A method of activating primordial follicles as claimed in claim 7, wherein: in step four, serial section and follicle count were performed by collecting ovarian tissue, the effect of the inhibitor was further determined by follicular development, and Btc was found to be dependent on which signaling pathway regulates primordial follicle activation.
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| CN103387960A (en) * | 2013-07-22 | 2013-11-13 | 南京医科大学 | mTOR signaling pathway activator and applications thereof in in-vitro primordial follicle activation |
| CN108795849A (en) * | 2018-07-05 | 2018-11-13 | 中国农业大学 | Application of the CDC42 signal paths in activating primordial follicle growth and development |
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| CN103387960A (en) * | 2013-07-22 | 2013-11-13 | 南京医科大学 | mTOR signaling pathway activator and applications thereof in in-vitro primordial follicle activation |
| CN108795849A (en) * | 2018-07-05 | 2018-11-13 | 中国农业大学 | Application of the CDC42 signal paths in activating primordial follicle growth and development |
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