Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat
0
Citation
189
Reference
10
Related Paper
Abstract:
<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>Keywords:
Antral follicle
Table S1. Total identified proteins in Our study. Table S2. Identified proteins in Pool. Table S3. Identified proteins in Individual samples Table S4. List of proteins identified in human ovarian follicular fluid (until 2018 and including our study). Table S5. High abundant proteome of FF from hSAF compared with proteins identified by Pouslen L. et al.,2019 in big follicles. Non-depleted samples. Table S6. Proteins potentially more concentrated or accessible by MS in FF from hSAF Table S7. Functional annotation clustering carried out with 226 proteins
Antral follicle
Proteome
Cite
Citations (0)
Is it possible to identify by mass spectrometry a wider range of proteins and key proteins involved in folliculogenesis and oocyte growth and development by studying follicular fluid (FF) from human small antral follicles (hSAF)?The largest number of proteins currently reported in human FF was identified in this study analysing hSAF where several proteins showed a strong relationship with follicular developmental processes.Protein composition of human ovarian FF constitutes the microenvironment for oocyte development. Previous proteomics studies have analysed fluids from pre-ovulatory follicles, where large numbers of plasma constituents are transferred through the follicular basal membrane. This attenuates the detection of low abundant proteins, however, the basal membrane of small antral follicles is less permeable, making it possible to detect a large number of proteins, and thereby offering further insights in folliculogenesis.Proteins in FF from unstimulated hSAF (size 6.1 ± 0.4 mm) were characterised by mass spectrometry, supported by high-throughput and targeted proteomics and bioinformatics. The FF protein profiles from hSAF containing oocytes, capable or not of maturing to metaphase II of the second meiotic division during an IVM (n = 13, from 6 women), were also analysed.We collected FF from hSAF of ovaries that had been surgically removed from 31 women (∼28.5 years old) undergoing unilateral ovariectomy for fertility preservation.In total, 2461 proteins were identified, of which 1108 identified for the first time in FF. Of the identified proteins, 24 were related to follicular regulatory processes. A total of 35 and 65 proteins were down- and up-regulated, respectively, in fluid from hSAF surrounding oocytes capable of maturing (to MII). We found that changes at the protein level occur already in FF from small antral follicles related to subsequent oocyte maturation.A possible limitation of our study is the uncertainty of the proportion of the sampled follicles that are undergoing atresia. Although the FF samples were carefully aspirated and processed to remove possible contaminants, we cannot ensure the absence of some proteins derived from cellular lysis provoked by technical reasons.This study is, to our knowledge, the first proteomics characterisation of FF from hSAF obtained from women in their natural menstrual cycle. We demonstrated that the analysis by mass spectrometry of FF from hSAF allows the identification of a greater number of proteins compared to the results obtained from previous analyses of larger follicles. Significant differences found at the protein level in hSAF fluid could predict the ability of the enclosed oocyte to sustain meiotic resumption. If this can be confirmed in further studies, it demonstrates that the viability of the oocyte is determined early on in follicular development and this may open up new pathways for augmenting or attenuating subsequent oocyte viability in the pre-ovulatory follicle ready to undergo ovulation.The authors thank the financial support from ReproUnion, which is funded by the Interreg V EU programme. No conflict of interest was reported by the authors.N/A.
Antral follicle
Cite
Citations (24)
Corona radiata (embryology)
Cite
Citations (33)
The ontogeny of glycodelin in human ovarian follicles during folliculogenesis was studied. Glycodelin immunoreactivity began to be detected in the granulosa cells and thecal cells of late secondary follicles. Immunoreactivity was also found in both the luteinized granulosa cells and cumulus cells obtained from women undergoing the assisted reproduction treatment. However, only the luteinized granulosa cells, and not the cumulus cells, expressed glycodelin mRNA. Results also showed that the cumulus cells took up radiolabelled glycodelin and partially deglycosylated some of it. Glycodelin (and a partially deglycolsylated form of glycoldelin) appeared to complex with two cytoplasmic or membrane components of the cumulus cells. The data also demonstrated that ZIF-1, a glycoprotein isolated from human follicular fluid, was immunologically similar to glycodelin. In conclusion, we suggest that glycodelin is synthesized in the granulosa cells of ovarian follicles at late secondary follicle stage. It then may be released into the follicular fluid from where it is taken up and partially modified by the cumulus cells.
Follicular fluid
Granulosa cell
Cite
Citations (50)
Table S1. Total identified proteins in Our study. Table S2. Identified proteins in Pool. Table S3. Identified proteins in Individual samples Table S4. List of proteins identified in human ovarian follicular fluid (until 2018 and including our study). Table S5. High abundant proteome of FF from hSAF compared with proteins identified by Pouslen L. et al.,2019 in big follicles. Non-depleted samples. Table S6. Proteins potentially more concentrated or accessible by MS in FF from hSAF Table S7. Functional annotation clustering carried out with 226 proteinsTable S8. Proteins that correlated significatly with MDK or VIM.
Table S9. MRM results
Table S9. MRM results
Antral follicle
Proteome
Cite
Citations (0)
Antral follicle
Cite
Citations (30)
The mammalian cumulus-oocyte complex (COCs) promotes oocyte growth and development during long stages of folliculogenesis and oogenesis. Before ovulation, the follicle is formed by a variety of fully differentiated cell populations; cumulus cells (CCs) that tightly surround the female gamete, granulosa cells (GCs) and theca cells (TCs) which build the internal and external mass of the follicular wall. It is well documented that CCs surrounding the oocyte are necessary for resumption of meiosis and full maturation of the gamete. However, the role of the granulosa cells in acquisition of MII stage and/or full fertilization ability is not yet entirely known. In this article, we present an overview of mammalian oocytes and their relationship to the surrounding cumulus and granulosa cells. We also describe the processes of GCs differentiation and developmental capacity. Finally, we describe several markers of mammalian GCs, which could be used for positive identification of isolated cells. The developmental capacity of oocytes and surrounding somatic cells a fingerprint of folliculogenesis and oogenesis.
Granulosa cell
Gamete
Growth differentiation factor-9
Cite
Citations (7)
Table S1. Total identified proteins in Our study. Table S2. Identified proteins in Pool. Table S3. Identified proteins in Individual samples. Table S4. Proteins identified in hSAF fluid that contain oocyte capable or not to reach metaphase II. Table S5. List of proteins identified in proteomics studies performed in human ovarian follicular fluid (up to 2020 and including our study). Table S6. High abundant proteome of FF from hSAF compared with proteins identified by Pouslen L. et al.,2019 in big follicles. Non-depleted samples. Table S7. Proteins potentially more concentrated or accessible by MS in FF from hSAF. Table S8. Functional annotation clustering carried out with 226 proteinsTable S9. Proteins that correlated significatly with MDK or VIM.
Table S10. Functional annotation clustering carried out with top 100 dysregulated proteins in FF that contained oocyte capable to reach MII.
Table S11. List of proteins that correlated significatly with secreted proteins dysregulated in FF that contained oocyte capable to reach MII.
Table S10. Functional annotation clustering carried out with top 100 dysregulated proteins in FF that contained oocyte capable to reach MII.
Table S11. List of proteins that correlated significatly with secreted proteins dysregulated in FF that contained oocyte capable to reach MII.
Antral follicle
Cite
Citations (0)
The main biological role of the anti-Mullerian hormone (AMH) is to induce the involution of the Muller ducts in embryos during differentiation of masculine gender. In case of women, AMH is produced in granular cells of primary, preantral and antral follicles. The expression of AMH initiates at the moment of the follicle recruitment and it lasts until the stage of an antral follicle. The level of this hormone decreases with age and in postmenopausal period is undetectable in blood. Therefore, AMH could be a useful marker of ovarian reserve. Multiple investigations have revealed higher AMH levels in the blood of PCOS patients. It is believed to be the consequence of the increased amount of small antral follicles. AMH is considered to have an essential role in folliculogenesis. It inhibits the process of recruitment of primordial follicles and modifies the growth of preantral and antral follicles by diminishing the sensitivity of follicles for FSH stimulation. The paper is a review of the present knowledge of the structure and activity of AMH. AR gene and protein. Participation of AMH in folliculogenesis and changes of AMH levels depending on structure and age of the ovary have also been discussed. Recent findings concerning the possibility of using AMH to assess ovarian reserve and efficiency of the stimulation of ovulation in infertile women have been presented. It is believed that increased knowledge concerning AMH might improve the diagnosis and treatment of infertility caused by lack of ovulation.
Antral follicle
Anti-Müllerian hormone
Ovarian Reserve
Cite
Citations (9)
The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage.Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined.All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis.ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.
Zona pellucida glycoprotein
Cite
Citations (71)