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1
Hussein, Tamer S., Jeremy G. Thompson, and Robert B. Gilchrist. "Oocyte-secreted factors enhance oocyte developmental competence." Developmental Biology 296, no. 2 (August 2006): 514–21. http://dx.doi.org/10.1016/j.ydbio.2006.06.026.
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Gilchrist, Robert B., Michelle Lane, and Jeremy G. Thompson. "Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality." Human Reproduction Update 14, no. 2 (January 5, 2008): 159–77. http://dx.doi.org/10.1093/humupd/dmm040.
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Vanderhyden, Barbara C. "Oocyte-secreted factros regulate granulosa cell steroidogenesis." Zygote 4, no. 04 (November 1996): 317–21. http://dx.doi.org/10.1017/s0967199400003324.
Повний текст джерелаАнотація:
Investigations of strains of mice defective in germ cell development have revealed the importance of oocytes for the initial stages of folliculogenesis (Pellaset al., 1991; Huanget al., 1993). Various aspects of follicular development are dependent upon and/or influenced by the presence of oocytes, including granulosa cell proliferation (Vanderhydenet al., 1990, 1992) and cumulus expansion (Buccioneet al., 1990; Salustriet al., 1990; Vanderhydenet al., 1990; Vanderhyden, 1993). We are investigating the possibility that oocytes influence one of the primary functions of granulosa cells: steroidogenesis. In many species, granulosa cells removed from preovulatory follicles luteinisein vitro(Channinget al., 1982), presumably due to loss of contact with follicular luteinisation inhibitory factor(s). Indeed, follicular fluid can prevent granulosa cell luteinisationin vitro(Ledwitz-Rigbyet al., 1977). Follicular fluid, however, may simply be the medium for transport of factors secreted by oocytes to regulate granulosa cell activities.
4
Gilchrist, Robert B. "Actions of Oocyte-Secreted TGFbeta Superfamily Ligands." Biology of Reproduction 78, Suppl_1 (May 1, 2008): 279–80. http://dx.doi.org/10.1093/biolreprod/78.s1.279b.
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da Silveira, Juliano C., Ana Clara F. C. M. de Ávila, Hannah L. Garrett, Jason E. Bruemmer, Quinton A. Winger, and Gerrit J. Bouma. "Cell-secreted vesicles containing microRNAs as regulators of gamete maturation." Journal of Endocrinology 236, no. 1 (January 2018): R15—R27. http://dx.doi.org/10.1530/joe-17-0200.
Повний текст джерелаАнотація:
Mammalian gamete maturation requires extensive signaling between germ cells and their surrounding somatic cells. In the ovary, theca cells, mural granulosa cells, cumulus cells and the oocyte all secrete factors throughout follicle growth and maturation that are critical for ovulation of a high-quality oocyte with the competence to develop into an embryo. Similarly, maturation of sperm occurs as it transits the epididymis during which epididymal epithelium and sperm exchange secretory factors that are required for sperm to gain motility and fertility. Recent studies in a variety of species have uncovered the presence of cell-secreted vesicles in follicular fluid (microvesicles and exosomes) and epididymal fluid (epididymosomes). Moreover, these cell-secreted vesicles contain small non-coding regulatory RNAs called microRNAs, which can be shuttled between maturing gametes and surrounding somatic cells. Although little is known about the exact mechanism of how microRNAs are loaded into these cell-secreted vesicles or are transferred and modulate gene expression and function in gametes, recent studies clearly suggest that cell-secreted vesicle microRNAs play a role in oocyte and sperm maturation. Moreover, a role for cell-secreted vesicular microRNAs in gamete maturation provides for novel opportunities to modulate and discover new diagnostic markers associated with male or female fertility. This manuscript provides an overview of cell-secreted vesicles in ovarian follicular fluid and epididymal fluid and microRNAs and discusses recent discoveries on the potential function of cell-secreted vesicles as carriers of microRNAs in oocyte and sperm maturation.
6
Cakmak, Hakan, Federica Franciosi, A. Musa Zamah, Marcelle I. Cedars, and Marco Conti. "Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells." Proceedings of the National Academy of Sciences 113, no. 9 (February 10, 2016): 2424–29. http://dx.doi.org/10.1073/pnas.1519990113.
Повний текст джерелаАнотація:
The differentiation of the female gamete into a developmentally competent oocyte relies on the protected environment of the ovarian follicle. The oocyte plays a key role in establishing this microenvironment by releasing paracrine factors that control the functions of surrounding somatic cells. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are secreted during follicle growth and play pivotal roles in this local regulation. The current view is that the function of these secreted factors declines in the periovulatory period when the oocyte reenters the meiotic cell cycle. Here, we provide evidence that oocyte reentry into meiosis is instead associated with a shift in the pattern of secretion with a new set of bioactive molecules synthesized before ovulation. Using interleukin 7 (IL7) as a prototypic secreted factor, we show that its secretion is dependent on activation of mRNA translation in synchrony with the cell cycle and that its translation is under the control of somatic cells. IL7 is part of a local feedback loop with the soma because it regulates cumulus cell replication. Similar conclusions are reached when IL7 secretion is measured in human follicular fluid during in vitro fertilization cycles. IL7 concentration in the follicular fluid correlates with the oocyte ability to reach the MII stage of maturation. These findings are consistent with the hypothesis that a new set of local factors is secreted by the oocyte during ovulation. These dynamic secretions are likely critical for promoting the final stages of maturation and oocyte developmental competence.
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Yan, Changning, Frank L. Pendola, Renu Jacob, Anthony L. Lau, John J. Eppig, and Martin M. Matzuk. "Oosp1 encodes a novel mouse oocyte-secreted protein." genesis 31, no. 3 (2001): 105–10. http://dx.doi.org/10.1002/gene.10010.
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RICHANI, Dulama, Yiqing ZHAO, Xiuhua LIAO, Jared M. CAMPBELL, Abbas HABIBALAHI, William A. STOCKER, Ewa M. GOLDYS, Craig A. HARRISON, and Robert B. GILCHRIST. "Novel Oocyte-Secreted Factors Improve Mouse IVM Outcomes." Fertility & Reproduction 04, no. 03n04 (September 2022): 132. http://dx.doi.org/10.1142/s2661318222740449.
Повний текст джерелаАнотація:
Background: In vitro maturation (IVM) is a technology designed to obtain mature oocytes following culture of immature cumulus–oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for patients with excessive AFC or those requiring immediate fertility preservation. However, the clinical uptake of IVM has been slow, primarily due to lower embryo yield and live birth rate relative to IVF, therefore improving IVM culture is required. Aim: To assess whether supplementation of IVM culture medium with the novel in-house engineered TGFβ proteins cumulin and super-GDF9 improves subsequent embryo development. Method: Immature mouse COCs were cultured by standard IVM or bi-phasic IVM ± cumulin or super-GDF9. Following IVM, cumulus expansion was scored and COCs were fertilized, and cultured to assess embryo development. Differential staining was performed on day 6 blastocysts following bi-phasic IVM to assess cell allocation. In a separate experiment, hyperspectral imaging of autofluorescence was carried out on oocytes and cumulus cells following standard IVM ± cumulin to assess the molecular composition of these cells. Results: Both cumulin and super-GDF9 in standard IVM significantly increased cumulus expansion (P<0001; n=104-115 COCs) and blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; P=0.006; n=382-406 oocytes). Hyperspectral imaging showed that oocytes (n=115-158) and cumulus cells (n=532-600) exposed to cumulin during IVM had a distinct spectral profile that varied dramatically (P<0.005) from untreated cells, demonstrating that cumulin has a major impact on the molecular composition of these cells, likely contributing to the improved oocyte quality. In bi-phasic IVM, cumulin did not significantly alter embryo yield (n=387-424 oocytes) or blastocyst cell number or allocation (n=84-112 blastocysts). Conclusion: Cumulin did not provide an additional beneficial effect in bi-phasic IVM, however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence suggesting that their effects in human IVM should be investigated.
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Dragovic, Rebecca A., Lesley J. Ritter, Samantha J. Schulz, Fred Amato, David T. Armstrong, and Robert B. Gilchrist. "Role of Oocyte-Secreted Growth Differentiation Factor 9 in the Regulation of Mouse Cumulus Expansion." Endocrinology 146, no. 6 (June 1, 2005): 2798–806. http://dx.doi.org/10.1210/en.2005-0098.
Повний текст джерелаАнотація:
Abstract Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex, which is necessary for ovulation. Oocyte-secreted growth differentiation factor 9 (GDF9) signals through the bone morphogenetic protein receptor II and is currently the primary candidate molecule for the cumulus-expansion enabling factor. This study was conducted to determine whether GDF9 is the mouse cumulus-expansion enabling factor. Cumulus-oocyte complexes were collected from mice, and the oocyte was microsurgically removed to generate an oocytectomized (OOX) complex. OOX complexes treated with FSH alone or recombinant mouse GDF9 alone failed to expand, whereas expansion was induced in the presence of FSH by GDF9, TGFβ1, or coculture with oocytes. A specific GDF9-neutralizing antibody, mAb-GDF9–53, neutralized the expansion of OOX complexes in response to GDF9 but not the expansion of OOX complexes cocultured with oocytes. Using real-time RT-PCR, hyaluronan synthase 2 (HAS2) mRNA expression by OOXs was up-regulated 4- to 6-fold by oocytes and GDF9. Monoclonal neutralizing antibody-GDF9–53 attenuated GDF9-induced OOX HAS2 expression but not oocyte-induced HAS2 expression. A TGFβ antagonist neutralized TGFβ-induced, but not oocyte-induced, expansion of OOX complexes, and when combined with monoclonal neutralizing antibody-GDF9–53 also failed to neutralize oocyte-induced expansion. Furthermore, a soluble portion of the bone morphogenetic protein receptor II extracellular domain, which is a known GDF9 antagonist, completely antagonized GDF9-induced expansion but only partially neutralized oocyte-induced expansion. This study provides further evidence that like TGFβ, GDF9 can enable FSH-induced cumulus expansion, but more importantly, demonstrates that neither GDF9 nor TGFβ alone, nor the two in unison, account for the critical oocyte-secreted factors regulating mouse cumulus expansion.
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Hussein, Tamer S., Melanie L. Sutton-McDowall, Robert B. Gilchrist, and Jeremy G. Thompson. "Temporal effects of exogenous oocyte-secreted factors on bovine oocyte developmental competence during IVM." Reproduction, Fertility and Development 23, no. 4 (2011): 576. http://dx.doi.org/10.1071/rd10323.
Повний текст джерелаАнотація:
We investigated whether paracrine signalling between the bovine oocyte and cumulus cells is altered during the course of in vitro maturation (IVM). Bovine COCs were cocultured with denuded oocytes or treated with specific oocyte-secreted factors, namely recombinant bone morphogenetic protein (BMP)-15 or growth differentiation factor (GDF)-9, beginning from 0 or 9 h IVM. To generate a 9-h denuded oocyte (DO) group, COCs were cultured intact for the first 9 h of IVM and then denuded. Coculturing intact COCs with DOs denuded immediately after collection or following 9 h of maturation did not affect cleavage rate, but improved blastocyst yield (P < 0.05) on Day 8 (51 and 61%, respectively; P < 0.05) and cell number compared with COCs cultured alone (41%). Significantly, we observed higher levels of endogenous GDF-9 and BMP-15 protein in oocytes of COCs matured for 9 h compared with no incubation. The addition of 175 ng mL–1 GDF-9 or 10% v/v BMP-15 from partially purified transfected 293H cell supernatant for 24 h IVM significantly enhanced development to the blastocyst stage from 40% (control) to 51 and 47%, respectively (P < 0.05). However, treatment of COCs with GDF-9 or BMP-15 between 9 and 24 h of IVM did not increase blastocyst yield. These results provide evidence of quantitative and possibly qualitative temporal changes in oocyte paracrine factor production during IVM.
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Su, Jianmin, Yongsheng Wang, Lei Zhang, Bo Wang, Jun Liu, Yan Luo, Zekun Guo, Fusheng Quan, and Yong Zhang. "Oocyte-secreted factors in oocyte maturation media enhance subsequent development of bovine cloned embryos." Molecular Reproduction and Development 81, no. 4 (February 18, 2014): 341–49. http://dx.doi.org/10.1002/mrd.22302.
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12
Hussein, T. S., R. B. Gilchrist, and J. G. Thompson. "327 OOCYTE-SECRETED FACTORS DIRECTLY AFFECT OOCYTE DEVELOPMENTAL COMPETENCE DURING IN VITRO MATURATION OF THE BOVINE CUMULUS - OOCYTE COMPLEX." Reproduction, Fertility and Development 18, no. 2 (2006): 271. http://dx.doi.org/10.1071/rdv18n2ab327.
Повний текст джерелаАнотація:
Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.
13
Hussein, T. S., D. A. Froliand, J. G. Thompson, and R. B. Gilchrist. "252.Oocyte-secreted factor(s) regulate apoptosis of bovine cumulus cells." Reproduction, Fertility and Development 16, no. 9 (2004): 252. http://dx.doi.org/10.1071/srb04abs252.
Повний текст джерелаАнотація:
Paracrine factors secreted by the oocyte affect cumulus cell proliferation and differentiation. These factors may also act in an anti-apoptotic manner, maintaining the low incidence of cellular apoptosis within cumulus cells. The purpose of this study was to determine whether the incidence of apoptosis within cumulus cells is regulated by oocyte-secreted factors (OSF). Bovine cumulus-oocyte complexes (COC) aspirated from abattoir-derived ovaries were randomly allocated to 3 treatments: (1) groups of 5 intact COC; (2) groups of 5 oocytectomised complexes (OOX), where oocytes were removed microsurgically; and (3) groups of 5 OOX co-cultured with 25 denuded oocytes (DO). To examine a dose effect of OSF, OOX were also cultured with increasing numbers of DO (OOX+5DO, OOX+25DO, OOX+50DO). In both experiments, complexes were cultured in their respective treatments for 24 h in 50 �l of oocyte maturation medium (+/– rFSH; 0.1 IU/mL). Apoptosis was assessed using TUNEL, with all nuclei counterstained with propidum iodide (PI). Nikon TE2000 laser confocal scanning microscopy was used to visualise and quantify the incidence of apoptosis (TUNEL/PI). The proportion of apoptotic cells was determined by image analysis. Oocyte removal lead to a significant increase in cumulus cell apoptosis (OOX, 67% apoptotic; COC, 15%; P�<�0.001). However, the incidence of apoptosis in OOX was restored to COC levels when co-cultured with DO (18%; P�>�0.05, COC v. OOX+DO). FSH prevented apoptosis in all treatment groups (P�<�0.05) decreasing the incidence by 27% in OOX and by 12% in COC. Cumulus cell apoptosis within OOX (+/– FSH) was reduced in a dose dependent manner by treating with increasing numbers of DO. These results indicate for the first time that oocyte-secreted factor(s) regulate the incidence of apoptosis within cumulus cells.
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Thompson, Jeremy, Tamer Hussein, and Robert Gilchrist. "DIFFERENTIAL AND TEMPORAL EFFECTS OF NATIVE AND RECOMBINANT OOCYTE-SECRETED FACTORS ON OOCYTE DEVELOPMENTAL CAPACITY." Biology of Reproduction 77, Suppl_1 (July 1, 2007): 222. http://dx.doi.org/10.1093/biolreprod/77.s1.222a.
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Buratini, J., and C. A. Price. "Follicular somatic cell factors and follicle development." Reproduction, Fertility and Development 23, no. 1 (2011): 32. http://dx.doi.org/10.1071/rd10224.
Повний текст джерелаАнотація:
Considerable attention is currently paid to oocyte-derived secreted factors that act upon cumulus and granulosa cells. Also important for follicle development are somatic cell-derived secreted factors. This is illustrated by the ability of granulosa cell-derived Kit ligand (KITL) to promote primordial follicle activation, and the loss of follicle development that accompanies KITL gene disruption. This review summarises our current understanding of somatic cell factors during both preantral and antral follicle growth, involving not only signalling from granulosa cells to the oocyte, but also signalling between granulosa and theca cells. Principal granulosa cell-derived factors include activin, anti-Müllerian hormone (AMH), bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Theca cells also secrete BMPs and FGFs. The interplay between these factors is equally important for follicle growth as the activity of oocyte-derived factors.
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Buratini, Jose, Thaisy Tino Dellaqua, Mariabeatrice Dal Canto, and Mario Mignini Renzini. "OOCYTE SECRETED FACTORS REGULATE AND mRNA LEVELS IN CUMULUS CELLS." Fertility and Sterility 114, no. 3 (September 2020): e440. http://dx.doi.org/10.1016/j.fertnstert.2020.08.1277.
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Dalbies-Tran, Rozenn, Véronique Cadoret, Alice Desmarchais, Sébastien Elis, Virginie Maillard, Philippe Monget, Danielle Monniaux, Karine Reynaud, Marie Saint-Dizier, and Svetlana Uzbekova. "A Comparative Analysis of Oocyte Development in Mammals." Cells 9, no. 4 (April 17, 2020): 1002. http://dx.doi.org/10.3390/cells9041002.
Повний текст джерелаАнотація:
Sexual reproduction requires the fertilization of a female gamete after it has undergone optimal development. Various aspects of oocyte development and many molecular actors in this process are shared among mammals, but phylogeny and experimental data reveal species specificities. In this chapter, we will present these common and distinctive features with a focus on three points: the shaping of the oocyte transcriptome from evolutionarily conserved and rapidly evolving genes, the control of folliculogenesis and ovulation rate by oocyte-secreted Growth and Differentiation Factor 9 and Bone Morphogenetic Protein 15, and the importance of lipid metabolism.
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Dragovic, R. A., L. J. Ritter, F. Amato, S. J. Scott, M. Cranfield, N. P. Groome, D. T. Armstrong, and R. B. Gilchrist. "251.Regulation of mouse cumulus expansion by oocyte-secreted growth differentiation factor-9 (GDF-9)." Reproduction, Fertility and Development 16, no. 9 (2004): 251. http://dx.doi.org/10.1071/srb04abs251.
Повний текст джерелаАнотація:
Oocyte paracrine signalling is necessary for mouse cumulus cell expansion, an important preovulatory process. The oocyte-secreted factor growth differentiation factor-9 (GDF-9) signals through the bone morphogenetic protein receptor-II (BMPR-II) and is currently the primary candidate molecule for the cumulus expansion enabling factor (CEEF). The present study was conducted to determine whether in the mouse GDF-9 is the CEEF. Cumulus oocyte complexes (COC) were collected from eCG-primed mice and the oocyte was microsurgically removed to generate an oocytectomised complex (OOX). An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P�<�0.05) induced by either recombinant mouse GDF-9 (score; mean +/– SEM: 2.7 +/– 0.1), recombinant TGF-μ1 (score: 2.6 +/– 0.2) or co-culture with oocytes (score: 2.3 +/– 0.2). A GDF-9 neutralising antibody mAb-53, raised against hGDF-9, was effective in neutralising the response of OOX complexes to GDF-9 (score: 0.1 +/– 0.1), but had no significant effect on the expansion of OOX complexes co-cultured with oocytes (score: 2.3 +/– 0.2). Likewise, a TGF-μ antagonist neutralised (P�<�0.05) TGF-μ-induced, but not oocyte-induced, expansion of OOX complexes. A soluble portion of the BMPR-II ectodomain, a known GDF-9 antagonist, failed to neutralise oocyte-induced cumulus expansion (P�>�0.05) at the highest dose implying that BMPR-II is not a critical receptor involved in regulating cumulus expansion. Using real-time RT-PCR, hyaluronan synthase-2 (HAS2) mRNA expression by OOXs was upregulated 6- to 7-fold by oocytes and GDF-9. The GDF-9 neutralising antibody mAb-53, partially neutralised GDF-9-induced OOX HAS2 expression, but not oocyte-induced HAS2 expression. This study provides evidence that like TGF-μ1, GDF-9 can enable FSH-induced cumulus expansion, however more importantly demonstrates that neither GDF-9 nor TGF-μ1 alone account for the crucial oocyte-secreted factor regulating cumulus expansion in the mouse.
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Gupta, Swati, Sriti Pandey, Mehtab S. Parmar, Anjali Somal, Avishek Paul, Bibhudatta S. K. Panda, Irfan A. Bhat, et al. "Impact of oocyte-secreted factors on its developmental competence in buffalo." Zygote 25, no. 3 (June 2017): 313–20. http://dx.doi.org/10.1017/s0967199417000156.
Повний текст джерелаАнотація:
SummaryOocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus–oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, <6 mm) were collected and matured in vitro either in the presence of GDF9 or BMP15, or both, or with the denuded oocytes (DOs) as a source of native OSFs. Cleavage and blastocyst rates were significantly (P < 0.05) higher in LF-derived than SF-derived oocytes. Cleavage and blastocyst rates were significantly higher (P < 0.05) in the DOs and the combination groups compared with the control, GDF9 alone and BMP15 alone groups, both in LF-derived and SF-derived oocytes, although the cleavage and blastocyst rates did not differ significantly (P > 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.
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Dunning, K. R., L. N. Watson, J. G. Thompson, R. L. Robker, and D. L. Russell. "146. MOLECULAR FILTRATION PROPERTIES OF THE EXPANDED CUMULUS MATRIX: CONTROLLED SUPPLY OF METABOLITES AND EXTRACELLULAR SIGNALS TO CUMULUS CELLS AND THE OOCYTE." Reproduction, Fertility and Development 22, no. 9 (2010): 64. http://dx.doi.org/10.1071/srb10abs146.
Повний текст джерелаАнотація:
Cumulus matrix genes are positively correlated with oocyte competence [1]. Formation of the expanded cumulus matrix during oocyte maturation is well described; however its function remains elusive. We investigated whether cumulus matrix acts as a molecular filter, based on recognised filtration properties of analogous matrices. We found that cumulus matrix controls metabolite supply to the oocyte and retains prostaglandin E2 (PGE2), which is critical in oocyte maturation. The uptake of fluorescently labelled hydrophilic and hydrophobic metabolites showed that cumulus matrix formation significantly impeded diffusion to the oocyte. Expanded in vivo matured cumulus oocyte complexes (COCs, eCG+hCG16h) resisted uptake of glucose and cholesterol compared to unexpanded (eCG44h, P < 0.05), as assessed by confocal microscopy and spatial quantitation of fluorescence (P < 0.05). In vitro maturation (IVM) results in pronounced compositional deficiency of cumulus matrix proteins [2] and poor oocyte quality. Glucose and cholesterol were transported more readily into cumulus cells and the oocyte of IVM COCs (matured in αMEM/5% FCS/50 mIU/mL FSH, 16 h) compared to in vivo matured COCs (P < 0.05 and P = 0.08, respectively). Taking the inverse approach we found that PGE2 synthesised by cumulus cells is retained within the matrix compartment of in vivo matured COCs but IVM COCs did not retain PGE2 and secreted 4.3-fold more into the media. The relationship of retained to secreted PGE2 was significantly higher after in vivo maturation vs IVM COCs (P < 0.0001). This property of the COC matrix reveals a potential mechanism whereby the prostaglandin signal intensifies through a physicochemical mechanism rather than gene regulation. This is the first demonstration that cumulus matrix regulates diffusion toward and secretion from the COC, thus excluding glucose, known to negatively affect oocyte quality, and trapping factors, including PGE2, with critical roles in oocyte maturation and fertilisation. Thus, IVM may reduce oocyte quality due to poor trafficking of metabolites and signalling molecules. (1) McKenzie LJ, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod 2004; 19: 2869–2874.(2) Dunning KR, et al. Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Hum Reprod 2007; 22: 2842–2850.
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Roth, Siegfried. "The origin of dorsoventral polarity in Drosophila." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 358, no. 1436 (August 29, 2003): 1317–29. http://dx.doi.org/10.1098/rstb.2003.1325.
Повний текст джерелаАнотація:
In Drosophila dorsoventral (DV) polarity arises during oogenesis when the oocyte nucleus moves from a central posterior to an asymmetrical anterior position. Nuclear movement is a symmetry–breaking step and establishes orthogonality between the anteroposterior and the DV axes. The asymmetrically anchored nucleus defines a cortical region within the oocyte which accumulates high levels of gurken messenger RNA (mRNA) and protein. Gurken is an ovarian–specific member of the transforming growth factor–α (TGF–α) family of secreted ligands. Secreted Gurken forms a concentration gradient that results in a dorsal–to–ventral gradient of EGF receptor activation in the follicle cells surrounding the oocyte. This leads to concentration–dependent activation or repression of target genes of the EGF pathway in the follicular epithelium. One outcome of this process is the restriction of pipe expression to a ventral domain that comprises 40% of the egg circumference. Pipe presumably modifies extracellular matrix components that are secreted by the follicle cells and are present at the ventral side of embryo after egg deposition. Here, they activate a proteolytic cascade that generates a gradient of the diffusible ligand, Spätzle. Spätzle activates the Toll receptor at the surface of the embryo that stimulates the nuclear uptake of the transcription factor Dorsal. This leads to a nuclear concentration gradient of Dorsal that specifies the cell types along the DV axis of the embryo.
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APPELTANT, Ruth, Tamás SOMFAI, Dominiek MAES, Ann VAN SOOM, and Kazuhiro KIKUCHI. "Porcine oocyte maturation in vitro: role of cAMP and oocyte-secreted factors – A practical approach." Journal of Reproduction and Development 62, no. 5 (2016): 439–49. http://dx.doi.org/10.1262/jrd.2016-016.
Повний текст джерела
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Hussein, T. S., A. N. Filby, R. B. Gilchrist, and M. Lane. "114. HUMAN CUMULUS - OOCYTE COMPLEXES SECRETE CUMULUS EXPANSION ENABLING FACTOR(S)." Reproduction, Fertility and Development 21, no. 9 (2009): 33. http://dx.doi.org/10.1071/srb09abs114.
Повний текст джерелаАнотація:
Interactions between the oocyte and its companion somatic cells are crucial to establish and maintain a highly specialized microenvironment required for oocyte viability. Specifically, cumulus cell expansion in the mouse is reliant on oocyte-secreted factors (OSF). Little is know about factors secreted by the human oocyte and how they may interact with cumulus cells. Therefore, the aim of this study was to establish whether human cumulus oocyte complexes (COC) produce OSF that induces cumulus expansion. COC of patients undergoing routine clinical IVF were cultured individually for 6h following oocyte retrieval. The human oocyte conditioned medium (HOCM) was collected. The bioactivity of OSF in the HOCM was assessed using an established assay of cumulus expansion of mouse oocytectomized complexes (OOX). Cumulus expansion was assessed blinded using the scoring system; 1 (no expansion) to 4 (maximally expanded) and gene expression was assessed by real time RT-PCR. Culture of OOX in control media with or without FSH did not induce expansion. Similarly, OOX cultured in HOCM without FSH did not expand. However, culture of OOX in HOCM with FSH significantly induced expansion (2.4±0.1 compared with control 1.1±0.04, P<0.05). Furthermore, this expansion was not different to OOX co-cultured with human (2.9±0.1) or mouse (2.6±0.1) denuded oocytes. Cumulus/OOX gene expression of hyaluronan synthase-2 and cyclooxygenase-2 was significantly up-regulated 4-5 fold when OOX were cultured in HOCM compared to control (P<0.05). Interestingly, different patients produced HOCM which resulted in different levels of expansion (range from 1.5-3.7). This study has established that human COC secrete paracrine factor(s) that enable cumulus expansion. This expansion was dependent on the presence of FSH. The identity of these factor(s) are currently unknown however it appears that COC from different patients produce differing levels of these cumulus expansion enabling factor(s).
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Fujihara, Yoshitaka, Yonggang Lu, Taichi Noda, Asami Oji, Tamara Larasati, Kanako Kojima-Kita, Zhifeng Yu, Ryan M. Matzuk, Martin M. Matzuk, and Masahito Ikawa. "Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice." Proceedings of the National Academy of Sciences 117, no. 17 (April 15, 2020): 9393–400. http://dx.doi.org/10.1073/pnas.1917060117.
Повний текст джерелаАнотація:
Sperm–oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm–oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm–oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm–oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm–oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm–oocyte fusion process.
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Lo, Belinda K. M., Agnes Archibong-Omon, Panayiota Ploutarchou, Anthony J. Day, Caroline M. Milner, and Suzannah A. Williams. "Oocyte-specific ablation of N- and O-glycans alters cumulus cell signalling and extracellular matrix composition." Reproduction, Fertility and Development 31, no. 3 (2019): 529. http://dx.doi.org/10.1071/rd18209.
Повний текст джерелаАнотація:
Cumulus–oocyte complex (COC) expansion is essential for ovulation and fertilisation and is linked to oocyte quality. Hyaluronan (HA), the major matrix constituent, is cross-linked via inter-α-inhibitor heavy chains (HCs), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene 6 (TSG-6). All except HCs are secreted by cumulus cells in response to oocyte-secreted factors, which signal via SMAD pathways. The double mutant (DM) mouse generates oocytes lacking complex N- and O-glycans due to oocyte-specific deletion of core 1 β1,3-galactosyltransferase (C1galt1) and N-acetylglucosaminyltransferase I (Mgat1) and has modified cumulus expansion. We compared COCs before expansion (48 h-post-pregnant mare serum gonadotrophin (PMSG)) and at late-stage expansion (9 h-post-human chorionic gonadotrophin (hCG); control n=3 mice, DM n=3 per group). Using histochemistry the levels of HA, HCs, PTX3, TSG-6 and phosphorylated-SMAD1/5/8 and -SMAD2 (12–25 COCs per group) were assessed. DM COCs did not differ from Controls in cumulus size or cell density at 9 h-post-hCG; however, HA and HC levels and phosphorylated-SMAD1/5/8 were reduced. Furthermore, no correlations were found between the levels of matrix molecules and cumulus area in DM or Control samples. These data suggest that HA and HCs can support cumulus expansion provided that they are present above minimum threshold levels. We propose that oocyte-specific ablation of C1galt1 and Mgat1 may affect bone morphogenetic protein 15 synthesis or bioactivity, thereby reducing SMAD1/5/8 phosphorylation and HA production.
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Watson, Laura N., David G. Mottershead, Kylie R. Dunning, Rebecca L. Robker, Robert B. Gilchrist, and Darryl L. Russell. "Heparan Sulfate Proteoglycans Regulate Responses to Oocyte Paracrine Signals in Ovarian Follicle Morphogenesis." Endocrinology 153, no. 9 (September 1, 2012): 4544–55. http://dx.doi.org/10.1210/en.2012-1181.
Повний текст джерелаАнотація:
In the ovarian follicle, oocyte-secreted factors induce cumulus-specific genes and repress mural granulosa cell specific genes to establish these functionally distinct cell lineages. The mechanism establishing this precise morphogenic pattern of oocyte signaling within the follicle is unknown. The present study investigated a role for heparan sulphate proteoglycans (HSPG) as coreceptors mediating oocyte secreted factor signaling. In vitro maturation of cumulus oocyte complexes in the presence of exogenous heparin, which antagonizes HSPG signaling, prevented cumulus expansion and blocked the induction of cumulus-specific matrix genes, Has2 and Tnfaip6, whereas conversely, the mural granulosa-specific genes, Lhcgr and Cyp11a1, were strongly up-regulated. Heparin also blocked phosphorylation of SMAD2. Exogenous growth differentiation factor (GDF)-9 reversed these heparin effects; furthermore, GDF9 strongly bound to heparin sepharose. These observations indicate that heparin binds endogenous GDF9 and disrupts interaction with heparan sulphate proteoglycan coreceptor(s), important for GDF9 signaling. The expression of candidate HSPG coreceptors, Syndecan 1–4, Glypican 1–6, and Betaglycan, was examined. An ovulatory dose of human chorionic gonadotropin down-regulated Betaglycan in cumulus cells, and this regulation required GDF9 activity; conversely, Betaglycan was significantly increased in luteinizing mural granulosa cells. Human chorionic gonadotropin caused very strong induction of Syndecan 1 and Syndecan 4 in mural granulosa as well as cumulus cells. Glypican 1 was selectively induced in cumulus cells, and this expression appeared dependent on GDF9 action. These data suggest that HSPG play an essential role in GDF9 signaling and are involved in the patterning of oocyte signaling and cumulus cell function in the periovulatory follicle.
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Soares, Ana Caroline S., Valentina Lodde, Rodrigo G. Barros, Christopher A. Price, Alberto M. Luciano, and José Buratini. "Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte–cumulus communication and to improve the quality of in vitro-produced embryos in cattle." Reproduction, Fertility and Development 29, no. 11 (2017): 2217. http://dx.doi.org/10.1071/rd16320.
Повний текст джерелаАнотація:
In vivo, oocyte maturation is triggered by the ovulatory LH surge, whereas in vitro it is precociously induced when the cumulus–oocyte complex is removed from the follicle. Natriuretic peptide C (NPPC) delays germinal vesicle breakdown (GVBD) while increasing oocyte–cumulus communication during in vitro maturation (IVM) in cattle. In the present study we first tested the hypothesis that steroids secreted by the follicle (17β-oestradiol, progesterone and androstenedione) interact with NPPC to delay GVBD and to maintain oocyte–cumulus communication as assessed by transfer of a dye (Lucifer Yellow) from the oocyte to cumulus cells. Then, we assessed the effects of steroid hormones and NPPC, alone and in combination in a pre-IVM culture, on embryo production. The combination of NPPC with steroids delayed GVDB, increased natriuretic peptide receptor 2 (NPR2) mRNA abundance in cumulus cells during culture, and maintained oocyte–cumulus communication at levels not different from non-cultured controls. The addition of steroids and/or NPPC to a pre-IVM culture did not alter blastocyst rates after IVF, but supplementation with steroids increased blastocyst total cell number. The present study provides evidence, for the first time in cattle, that steroids interact with NPPC to regulate oocyte nuclear maturation and oocyte–cumulus communication, and improve oocyte developmental competence.
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Varani, Simona, Julia A. Elvin, Changning Yan, Janet DeMayo, Francesco J. DeMayo, Heidi F. Horton, Michael C. Byrne, and Martin M. Matzuk. "Knockout of Pentraxin 3, a Downstream Target of Growth Differentiation Factor-9, Causes Female Subfertility." Molecular Endocrinology 16, no. 6 (June 1, 2002): 1154–67. http://dx.doi.org/10.1210/mend.16.6.0859.
Повний текст джерелаАнотація:
Abstract The ovulatory process is tightly regulated by endocrine as well as paracrine factors. In the periovulatory period, extensive remodeling of the follicle wall occurs to allow the extrusion of the oocyte and accompanying cumulus granulosa cells. Growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) are secreted members of the TGFβ superfamily that are expressed beginning in the oocyte of small primary follicles and through ovulation. Besides its critical role as a growth and differentiation factor during early folliculogenesis, GDF-9 also acts as a paracrine factor to regulate several key events in preovulatory follicles. By analyzing GDF-9-regulated expression profiles using gene chip technology, we identified TNF-induced protein 6 (Tnfip6) and pentraxin 3 (Ptx3 or PTX3) as novel factors induced by GDF-9 in granulosa cells of preovulatory follicles. Whereas Tnfip6 is induced in all granulosa cells by the LH surge, Ptx3 expression in the ovary is specifically observed after the LH surge in the cumulus granulosa cells adjacent to the oocyte. PTX3 is a member of the pentraxin family of secreted proteins, induced in several tissues by inflammatory signals. To define PTX3 function during ovulation, we generated knockout mice lacking the Ptx3 gene. Homozygous null (Ptx3−/−) mice develop normally and do not show any gross abnormalities. Whereas Ptx3−/− males are fertile, Ptx3−/− females are subfertile due to defects in the integrity of the cumulus cell-oocyte complex that are reminiscent of Bmp15−/−Gdf9+/− double mutant and BMP type IB receptor mutant mice. These studies demonstrate that PTX3 plays important roles in cumulus cell-oocyte interaction in the periovulatory period as a downstream protein in the GDF-9 signal transduction cascade.
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Ebeling, S., D. Töpfer, J. M. Weitzel, and B. Meinecke. "Bone morphogenetic protein-6 (BMP-6): mRNA expression and effect on steroidogenesis during in vitro maturation of porcine cumulus oocyte complexes." Reproduction, Fertility and Development 23, no. 8 (2011): 1034. http://dx.doi.org/10.1071/rd11027.
Повний текст джерелаАнотація:
Oocyte secreted factors (OSFs) have emerged as important factors for follicular development. The present study investigated the effect of the potential OSF bone morphogenic protein (BMP)-6 on steroidogenesis in porcine cumulus oocyte complexes during in vitro maturation. Cumulus oocyte complexes (COCs), cumulus complexes (CCs) without oocytes and CCs with supplemented BMP-6 were cultured for 0, 5, 26 or 46 h. BMP-6 transcripts were detected in oocytes and cumulus cells at all time points. In both cell types the mRNA expression was most intense after 5 h, and decreased during further maturation. After 26 and 46 h of culture, CCs secreted significantly less 17β-estradiol than COCs. This effect was reversed by adding BMP-6 to CCs cultures. In addition, a down-regulation of Cyp19A1, the rate-limiting enzyme of 17β-estradiol synthesis, was detected in CC cultures after 5 h. As seen for 17β-estradiol secretion, the addition of BMP-6 caused a significant increase in Cyp19A1 mRNA levels after 5, 26 and 46 h of culture. Progesterone secretion and transcripts of steroidogenic marker proteins StAR and 3β-HSD were not affected considerably by oocyte removal or addition of BMP-6. Furthermore, BMP-6 did not affect the activity of the mitogen-activated protein kinase. The results indicated that BMP-6 is a potential OSF and is involved in the prevention of premature luteinisation in cumulus cells via enhancing 17β-estradiol synthesis.
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Roberts, S. J., D. S. Leaf, H. P. Moore, and J. C. Gerhart. "The establishment of polarized membrane traffic in Xenopus laevis embryos." Journal of Cell Biology 118, no. 6 (September 15, 1992): 1359–69. http://dx.doi.org/10.1083/jcb.118.6.1359.
Повний текст джерелаАнотація:
Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin was polarized. Oogenic prolactin was secreted only into the blastocoel (from the cleavage membrane), none could be detected in the external medium (from the original oocyte membrane). These results provide the first direct evidence that the oocyte synthesizes a cache of vesicles for specific recruitment to the embryonic cleavage membranes which are polarized beginning with the first cleavage division.
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Ghiglione, Christian, Erika A. Bach, Yolande Paraiso, Kermit L. Carraway, Stéphane Noselli та Norbert Perrimon. "Mechanism of activation of theDrosophilaEGF Receptor by the TGFα ligand Gurken during oogenesis". Development 129, № 1 (1 січня 2002): 175–86. http://dx.doi.org/10.1242/dev.129.1.175.
Повний текст джерелаАнотація:
We have analyzed the mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor (TGF) α-like molecule, Gurken (Grk). Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. We show that expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. We find that the two transmembrane proteins Star and Brho potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium.
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Tremblay, Karine, Christian Vigneault, Serge McGraw, Guillaume Morin, and Marc-André Sirard. "Identification and characterization of a novel bovine oocyte-specific secreted protein gene." Gene 375 (June 2006): 44–53. http://dx.doi.org/10.1016/j.gene.2006.02.006.
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Hobeika, E., M. Armouti, M. Fierro, N. Winston, H. Scoccia, A. M. Zamah, and C. Stocco. "Oocyte secreted factors regulate aromatase expression in human primary cumulus granulosa cells." Fertility and Sterility 110, no. 4 (September 2018): e99. http://dx.doi.org/10.1016/j.fertnstert.2018.07.299.
Повний текст джерела
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Romaguera, R., A. R. Jiménez-Macedo, D. Izquierdo, R. Morató, M. Català, M. Roure, M. J. Palomo, and M. T. Paramio. "354 EFFECT OF OOCYTE-SECRETED FACTORS (OSF) ON EMBRYO DEVELOPMENT OF OOCYTES FROM SMALL FOLLICLES OF PREPUBERTAL GOATS." Reproduction, Fertility and Development 22, no. 1 (2010): 333. http://dx.doi.org/10.1071/rdv22n1ab354.
Повний текст джерелаАнотація:
Prepubertal goat ovaries contain a great number of small follicles, between 2.5 and 3 mm in diameter (Martino A et al. 1994 Theriogenology 41, 969-980). Oocyte-secreted factors (OSFs) act on granulosa cells to perform multiple functions required for an appropriate development of the oocyte. In bovines, co-culture of COCs with denuded oocytes (DOs) during IVM improved COCs embryo development dueto OSFs secreted byDOs (Hussein T et al. 2006 Dev Biol. 296, 514-521). In each step of follicle growth, the OSFs secreted are different (Eppig JJ et al. 2001 Reproduction. 122, 829-838). Our hypothesis is that oocytes from small follicles could improve their embryo development by co-culturing with DOs which secrete additional OSFs. Oocytes from prepubertal goats were classified according to their follicular origin: large follicles (>3 mm), small follicles (<3 mm), and control (oocytes recovered by slicing technique and selected by their morphology). The COCs from small follicles were divided in 3 groups and each one transferred into 150-μL droplets of IVM (TCM-199 with serum, hormones and cysteamine): 1) 30 COCs alone (small group); 2) 30 COCs co-cultured with 75 denuded oocytes from small follicles (SDO group); 3) 30 COCs co-cultured with 75 denuded oocytes from large follicles (LDO group). After 27 h of IVM, COCs were fertilized in vitro and the presumptive zygotes were cultured for 8 days in SOF with 10% FCS. Two samples of oocytes were used as control groups: oocytes fertilized (IVF group) and oocytes activated (activated group).At 48 h and 8 days post-insemination (pi), respectively, cleavage and blastocyst rates were recorded. Results are shown in Table 1. Differences between treatment groups were assessed using Fisher’s exact test (Graph-Pad software, San Diego, CA, USA). Values with P < 0.05 were considered statistically significant. Oocytes from small follicle had a significantly lower cleavage and blastocyst rates than IVF and activation oocyte groups. The co-cultured of COCs with SDOs and LDOs increased significantly the blastocyst rate compared to COCs from small follicles. In conclusion, additional OSFs secreted by DOs, improved blastocyst yield of oocytes coming from small follicles. However, not differences were found between OSFs secreted by small or large follicles of prepubertal goat oocytes. Table 1.
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Lopes, EF, LS Marques, RG Duranti, ATD de Oliveira, RFF Lopes, and JL Rodrigues. "Gene Expression of Monocarboxylate Transporters and Oocyte-secreted Factors in Bovine Cumulus-oocyte Complexes Selected by Brilliant Cresyl Blue." Reproduction in Domestic Animals 50, no. 5 (August 17, 2015): 763–70. http://dx.doi.org/10.1111/rda.12585.
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Lange-Consiglio, Anna, Claudia Perrini, Giulia Albini, Silvia Modina, Valentina Lodde, Eleonora Orsini, Paola Esposti, and Fausto Cremonesi. "Oviductal microvesicles and their effect on in vitro maturation of canine oocytes." Reproduction 154, no. 2 (August 2017): 167–80. http://dx.doi.org/10.1530/rep-17-0117.
Повний текст джерелаАнотація:
The effect of conditioned medium (CM) or microvesicles (MVs), secreted by multicellular spheroids of oviductal cells, and the involvement of some microRNAs (miRNAs) were investigated in canine oocyte maturation. To generate CM, spheroids were cultured for 3 days. MVs were obtained by ultracentrifugation of CM at 100,000 gand measured for size and concentration by NanoSight instrument. Cumulus-oocyte complexes (COCs) were matured at 38.5°C with 5% CO2and 5% of O2in synthetic oviductal fluid (SOF) in biphasic systems: for 24 h, with 5.0 μg/mL of LH and for other 48 h with 10% oestrous bitch serum. SOF was used as control (CTR) or supplemented with 10% CM or 25–50–75–100–150 × 106 MVs/mL labeled with PKH-26. Results show that multicellular aggregates secreted shedding vesicles. By fluorescence microscopy, the incorporation of labeled MVs was visible only at 72 h in oocyte cytoplasm. These MVs had a positive effect (P < 0.05) on maturation rate (MII) at the concentration of 75 and 100 × 106 MVs/mL compared to CM and CTR (20.34% and 21.82% vs 9.09% and 8.66% respectively). The concentration of 150 × 106 MVs/mL provided only 9.26% of MII. The expression of three specific miRNAs (miR-30b, miR-375 and miR-503) was studied. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375. In conclusion, the oviductal MVs could be involved in cellular trafficking during oocyte maturation and their possible usein vitrocould facilitate the exploitment of canine reproductive biotechnologies.
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Gilchrist, R. B., L. J. Ritter, S. Myllymaa, N. Kaivo-Oja, F. Amato, O. Ritvos, and D. G. Mottershead. "247.Molecular basis of oocyte - paracrine signalling that promotes mouse granulosa cell proliferation." Reproduction, Fertility and Development 16, no. 9 (2004): 247. http://dx.doi.org/10.1071/srb04abs247.
Повний текст джерелаАнотація:
Oocytes regulate follicle growth and development by secreting paracrine growth factors that act on granulosa cells (GC). We have recently determined that growth differentiation factor-9 (GDF-9) accounts for ~50% of the total mitogenic activity of oocytes, the remaining portion is as yet uncharacterised. This study was conducted to identify the receptor/signalling system utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded oocytes are co-cultured with primed-mouse mural GC. In this system, oocytes, GDF-9, TGF-b1 and activin-A all promoted GC DNA synthesis in a dose-dependent manner, but bone-morphogenetic protein-6 (BMP-6) and BMP-7 did not. The type-II receptor for GDF-9 is BMPRII and using real-time RT-PCR, cumulus cells (CC) and mural GC were found to express equivalent levels of BMPRII mRNA. We tested the capacity of the receptor ectodomain (ECD) to neutralise oocyte-stimulated mural GC proliferation. The BMPRII ECD antagonised both oocyte and GDF-9 bioactivity in a dose-dependent manner, completely abolishing activity of both mitogens at 1 ug/mL. The BMPRII ECD did not antagonise TGF-β and partially antagonised activin-A bioactivity, demonstrating its specificity. The TGFβR-II ECD, activin R-II ECD and activin R-IIB ECD all failed to neutralise oocyte- or GDF-9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective ligands. The BMPRII ECD also completely antagonised oocyte-stimulated CC DNA synthesis. Using this oocyte-factor bioassay with mural GC transfected with Smad luciferase reporter constructs, we found that oocytes, GDF-9 and TGF-β (but not BMP-6) activated the Smad2/3 pathway. Consistent with this, oocytes and GDF-9 led to phosphorylation of GC Smad2 molecules as detected by Western blot. Conversely the Smad1/5/8 pathway was activated by BMP-6, but not by GDF-9, TGF-β nor surprisingly by oocytes. This study provides evidence that BMPRII is a key receptor for transmitting the paracrine actions of oocytes in GC. However, oocyte-secreted factors do not activate the BMP intracellular signalling pathway but rather the TGF-β/activin intracellular pathway.
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de Ávila, A. C. F. C. M., and J. C. da Silveira. "Role of extracellular vesicles during oocyte maturation and early embryo development." Reproduction, Fertility and Development 32, no. 2 (2020): 56. http://dx.doi.org/10.1071/rd19389.
Повний текст джерелаАнотація:
The follicle is a dynamic microenvironment in the ovary where the oocyte develops. Intercellular communication between somatic cells and the oocyte inside the follicle is essential to generate a competent gamete. Extracellular vesicles are nanoparticles secreted by cells that mediate cell-to-cell communication in the follicle microenvironment and can be obtained from the follicular fluid. These extracellular vesicles have been studied as biomarkers and supplementation tools to mimic physiological conditions during assisted reproductive techniques because they are vehicles of bioactive molecules. Therefore, this paper reviews the importance of changes in the ovarian follicle and the effects of extracellular vesicles from follicular fluid during oocyte maturation and early embryo development. Finally, we propose that is important to consider the source of the extracellular vesicles to improve diagnostic methods and to increase invitro embryo production.
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McNatty, K. P., L. G. Moore, N. L. Hudson, L. D. Quirke, S. B. Lawrence, K. Reader, J. P. Hanrahan, et al. "The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology." Reproduction 128, no. 4 (October 2004): 379–86. http://dx.doi.org/10.1530/rep.1.00280.
Повний текст джерелаАнотація:
Ovulation rate in mammals is determined by a complex exchange of hormonal signals between the pituitary gland and the ovary and by a localised exchange of hormones within ovarian follicles between the oocyte and its adjacent somatic cells. From examination of inherited patterns of ovulation rate in sheep, point mutations have been identified in two oocyte-expressed genes, BMP15 (GDF9B) and GDF9. Animals heterozygous for any of these mutations have higher ovulation rates (that is, + 0.8–3) than wild-type contemporaries, whereas those homozygous for each of these mutations are sterile with ovarian follicular development disrupted during the preantral growth stages. Both GDF9 and BMP15 proteins are present in follicular fluid, indicating that they are secreted products. In vitro studies show that granulosa and/or cumulus cells are an important target for both growth factors. Multiple immunisations of sheep with BMP15 or GDF9 peptide protein conjugates show that both growth factors are essential for normal follicular growth and the maturation of preovulatory follicles. Short-term (that is, primary and booster) immunisation with a GDF9 or BMP15 peptide-protein conjugate has been shown to enhance ovulation rate and lamb production. In summary, recent studies of genetic mutations in sheep highlight the importance of oocyte-secreted factors in regulating ovulation rate, and these discoveries may help to explain why some mammals have a predisposition to produce two or more offspring rather than one.
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Gomez, Ma Ninia L., Jung Taek Kang, Ok Jae Koo, Su Jin Kim, Dae Kee Kwon, Sol Ji Park, Mohammad Atikuzzaman, So Gun Hong, Goo Jang, and Byeong Chun Lee. "Effect of oocyte-secreted factors on porcine in vitro maturation, cumulus expansion and developmental competence of parthenotes." Zygote 20, no. 2 (July 27, 2011): 135–45. http://dx.doi.org/10.1017/s0967199411000256.
Повний текст джерелаАнотація:
SummaryThe oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus–oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up-regulation.
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Riepsamen, Angelique H., Karen Chan, Shelly Lien, Prudence Sweeten, Mark W. Donoghoe, Glenda Walker, Eloïse H. J. Fraison, et al. "Serum Concentrations of Oocyte-Secreted Factors BMP15 and GDF9 During IVF and in Women With Reproductive Pathologies." Endocrinology 160, no. 10 (June 18, 2019): 2298–313. http://dx.doi.org/10.1210/en.2019-00264.
Повний текст джерелаАнотація:
Abstract Oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are critical for folliculogenesis and fertility. This study developed ELISAs for the measurement of BMP15 and GDF9 in serum and investigated their usefulness as biomarkers of female reproductive function. Serum samples were obtained from women undergoing infertility treatments (n = 154) and from perimenopausal and postmenopausal women (n = 28). Serum concentrations of BMP15 and GDF9 were analyzed in women relative to age, anti-Müllerian hormone, number of oocytes retrieved, and polycystic ovary syndrome (PCOS) after superovulation for in vitro fertilization. BMP15 and GDF9 immunoassays were validated for specificity, sensitivity (24 and 26 pg/mL, respectively), and reproducibility. BMP15 and GDF9 were detectable in 61% and 29% of women, respectively. BMP15 and GDF9 varied 64-fold and 15-fold, respectively, between women, but they did not change within subjects following ovarian stimulation with gonadotropins. Serum GDF9 concentration, but not BMP15 concentration, was associated with oocyte number retrieved in patients without PCOS (P = 0.018). GDF9 and BMP15 associations with oocyte number differed significantly (P < 0.05) with PCOS status. GDF9 concentrations were lower in poor responders (women with fewer than four oocytes retrieved or with cancelled cycles; P = 0.020). Serum BMP15, but not GDF9, was lower in women >55 years of age, compared with women of reproductive age (P < 0.01). This study develops and validates immunoassays to quantitate BMP15 and GDF9 in human serum and to correlate concentrations with female reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine.
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Ritter, Lesley J., Satoshi Sugimura, and Robert B. Gilchrist. "Oocyte Induction of EGF Responsiveness in Somatic Cells Is Associated With the Acquisition of Porcine Oocyte Developmental Competence." Endocrinology 156, no. 6 (June 1, 2015): 2299–312. http://dx.doi.org/10.1210/en.2014-1884.
Повний текст джерелаАнотація:
Abstract Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (<4 mm) vs medium sized (>4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.
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Hussein, T. S., D. A. Froiland, J. G. Thompson, and R. B. Gilchrist. "232. Oocytes prevent bovine cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins." Reproduction, Fertility and Development 17, no. 9 (2005): 91. http://dx.doi.org/10.1071/srb05abs232.
Повний текст джерелаАнотація:
Paracrine factors secreted by the oocyte regulate a broad range of cumulus cell (CC) functions. Previously we have shown that the low incidence of apoptosis in CCs is due to unidentified oocyte-secreted factors (OSF) acting in an anti-apoptotic manner. Here we examine the nature of the paracrine network of oocyte BMP growth factors and their binding proteins regulating CC apoptosis. Bovine cumulus–oocyte complexes (COC) were aspirated from abattoir-derived ovaries and oocytes microsurgically removed to create oocytectomized (OOX) complexes. OOX were treated with denuded oocytes (DO) or various growth factors for 24 h, then CC apoptosis was assessed using TUNEL together with confocal microscopy plus image analysis and by Western blotting for Bcl-2 and Bax. CC apoptosis was significantly (P < 0.001) reduced by DO, bone morphogenetic protein 15 (BMP15), BMP6 or BMP7 as assessed by TUNEL. Accordingly, expression of anti-apoptotic Bcl-2 was high in OOX+DO and OOX+BMP15, and low with OOX+GDF9 and OOX alone, whereas the reverse was observed for pro-apoptotic Bax. Combined treatment of OOXs with BMP6 and BMP15 did not further decrease apoptosis levels beyond that of BMP15 alone (P > 0.05), suggesting no additive effect of these two BMPs. Follistatin (FS) effectively antagonized BMP15 anti-apoptotic effects, and likewise, a BMP6 neutralizing antibody (NAb) antagonized the inhibitory effect of BMP6. Gremlin blocked BMP7 anti-apoptotic effects on CCs, but had no significant effect on BMP15. FS or BMP6. NAb antagonized ~50% of the anti-apoptotic activity of oocytes; however, these effects were not additive suggesting the additional involvement of other OSF. These results indicate for the first time that OSF (BMP15 and BMP6 in particular) maintains the low incidence of CC apoptosis by establishing a localized morphogenic gradient of bone morphogenetic proteins.
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Schuetz, Allen W. "Extrafollicular mediation of oocyte maturation by radial nerve factor in starfish Pisaster ochraceus." Zygote 8, no. 4 (November 2000): 359–68. http://dx.doi.org/10.1017/s0967199400001155.
Повний текст джерелаАнотація:
In starfish ovaries follicle cells that envelop each oocyte are thought to mediate the production of a maturation inducing substance (MIS), identified as 1-methyladenine, that induces maturation and spawning of oocytes after exposure to a gonadotropic substance secreted by the radial nerve (RNF). Studies were carried out to assess the possible role of extrafollicular cells within the ovarian wall in mediating this signal transduction process in the ovary of Pisaster ochraceus. Oocyte maturation and spawning occurred following the addition of RNF to intact ovarian tissue in vitro whereas no maturation occurred following the addition of RNF to germinal vesicle (GV) oocytes or GV oocytes surrounded by follicle cells. In contrast, oocyte maturation occurred when small ovarian wall fragments, lacking mature follicles, were incubated with GV oocytes and RNF. Neither actinomycin D nor cycloheximide altered RNF induction of oocyte maturation in the presence of the ovarian wall tissue whereas preheating (boiling water for 5 min) the tissue obliterated its response to RNF. Non-ovarian tissues failed to produce MIS in response to RNF. Results suggest that ovarian components other than the follicle cells that envelop fully grown immature oocyte are responsive to RNF and represent a significant and previously unrecognised intra-ovarian source of MIS.
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Mester, Brigitta, Lesley J. Ritter, Janet L. Pitman, Adrian H. Bibby, Robert B. Gilchrist, Kenneth P. McNatty, Jennifer L. Juengel, and C. Joy McIntosh. "Oocyte expression, secretion and somatic cell interaction of mouse bone morphogenetic protein 15 during the peri-ovulatory period." Reproduction, Fertility and Development 27, no. 5 (2015): 801. http://dx.doi.org/10.1071/rd13336.
Повний текст джерелаАнотація:
Bone morphogenetic protein 15 (BMP15) is a key intraovarian growth factor regulating mammalian fertility, yet expression and localisation of different BMP15 protein forms within ovarian follicles around the time of the preovulatory LH surge remains unclear. Using immunoblotting and immunocytochemistry, the present study identified that post-translationally processed BMP15 proregion and mature proteins are increasingly expressed and localised with cumulus and granulosa cells from mice treated with pregnant mare’s serum gonadotropin (PMSG) + human chorionic gonadotrophin (hCG). However, this increased expression was absent in cumulus–oocyte complexes matured in vitro. Pull-down assays further revealed that the recombinant BMP15 proregion is capable of specific interaction with isolated granulosa cells. To verify an oocyte, and not somatic cell, origin of Bmp15 mRNA and coregulated growth differentiation factor 9 (Gdf9), in situ hybridisation and quantitative polymerase chain reaction results confirmed the exclusive oocyte localisation of Bmp15 and Gdf9, regardless of treatment or assay method. Relative oocyte expression levels of Bmp15 and Gdf9 decreased significantly after PMSG + hCG treatment; nevertheless, throughout all treatments, the Bmp15 : Gdf9 mRNA expression ratio remained unchanged. Together, these data provide evidence that the preovulatory LH surge leads to upregulation of several forms of BMP15 protein secreted by the oocyte for putative sequestration and/or interaction with ovarian follicular somatic cells.
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Fatehi, A. Nader, Bernard A. J. Roelen, Ben Colenbrander, Eric J. Schoevers, Bart M. Gadella, Mart M. Bevers, and Rob van den Hurk. "Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development." Zygote 13, no. 2 (May 2005): 177–85. http://dx.doi.org/10.1017/s0967199405003126.
Повний текст джерелаАнотація:
The present study was conducted to evaluate the function of cumulus cells during bovine IVF. Oocytes within cumulus–oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.
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Hornick, J. E., F. E. Duncan, L. D. Shea, and T. K. Woodruff. "Multiple follicle culture supports primary follicle growth through paracrine-acting signals." REPRODUCTION 145, no. 1 (January 2013): 19–32. http://dx.doi.org/10.1530/rep-12-0233.
Повний текст джерелаАнотація:
In vitro follicle growth in alginate hydrogels is a unique and versatile method for studying ovarian and follicle biology that may also have implications for fertility preservation. Current culture systems support the development of isolated mouse follicles from the secondary stage onward. However, it has been a challenge to grow smaller follicles in vitro due to the dissociation of the oocyte from companion somatic cells. Recent work has demonstrated that coculturing primary follicles with mouse embryonic fibroblasts or ovarian stromal cells supports follicle survival and growth. In this study, we demonstrate that follicles themselves can exert a beneficial coculture effect. When primary follicles were cultured in groups of five or ten (multiple follicle culture), there was increased growth and survival. The multiple follicle culture approach maintained follicle integrity and resulted in the formation of antral stage follicles containing meiotically competent gametes. The growth and survival of primary follicles were highly number dependent, with the most significant enhancement observed when the largest number of follicles was grown together. Our data suggest that the follicle unit is necessary to produce the secreted factors responsible for the supportive effects of multiple follicle culture, as neither denuded oocytes, oocyte-secreted factors, nor granulosa cells alone were sufficient to support early follicle growth in vitro. Therefore, there may be signaling from both the oocyte and the follicle that enhances growth but requires both components in a feedback mechanism. This work is consistent with current in vivo models for follicle growth and thus advances the movement to recapitulate the ovarian environment in vitro.
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Burns, G., P. F. Suchodolski, A. J. Pearks Wilkerson, and C. Long. "248 PRODUCTION OF FLAGGED RECOMBINANT BOVINE BMP15 TO IMPROVE BOVINE IN VITRO EMBRYO PRODUCTION SYSTEMS." Reproduction, Fertility and Development 23, no. 1 (2011): 222. http://dx.doi.org/10.1071/rdv23n1ab248.
Повний текст джерелаАнотація:
Current in vitro systems for bovine embryo production are inefficient and produce embryos with lower viability than their in vivo-derived counterparts. Recent reports demonstrate that in vitro bovine oocyte maturation systems could benefit from the addition of oocyte-secreted factors, specifically GDF9 and BMP15 (Gilchrist et al. 2007 Theriogenology 67, 6–15). The long-term goal of this work is to produce species-specific recombinant oocyte-secreted factors capable of improving bovine embryo production in vitro. In the current project, the objective was to produce a cell line that expresses recombinant bovine BMP15. This protein is first translated as a large precursor peptide consisting of propeptide and mature regions, which are enzymatically cleaved to form the active mature protein. The wild-type BMP15 gene was cloned using reverse transcriptase PCR with RNA obtained from bovine ovarian tissue. For improved detection and purification of the active form of the recombinant protein, a detectable FLAG tag sequence (DYKDDDDK) was incorporated into the wild-type BMP15 gene by PCR and cloned into pCDNA expression vector. The FLAG tag was introduced immediately 3′ of the cleavage site at the N-terminal portion of the mature protein to produce recombinant FLAG-tagged BMP15 (rbFL-BMP15). To ensure efficient production of the mature protein, a Kozak sequence was inserted 5′ of the start ATG and the cleavage site altered to be recognised by PACE/furin enzymes, which are endogenously expressed in most mammalian cells including HEK-293 cells (Li et al. 2009 Mol. Hum. Reprod. 15, 779–788). Following sequencing to verify transcript fidelity, pCDNA-rbFL-BMP15 was transfected into HEK-293 cells, and mature protein production was detected by Western blot analysis. Cells plated at 85% confluency were transfected with Lipofectamine 2000, and lysates were harvested 48 h post-transfection. The presence of bovine rbFL-BMP-15 in cell lysates was confirmed by Western blot using the anti-FLAG antibody. Ongoing experiments will test the bioactivity of the purified rbFL-BMP15 by evaluating activation of the SMAD 1/5 pathway via Western blot for phosphorylated SMAD 1/5. After a biologically active protein is confirmed, purified protein will be collected for testing during in vitro maturation of bovine oocytes. We anticipate the species-specific form of oocyte-secreted factors will further enhance in vitro embryo production systems beyond that reported using heterologous factors.
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Heath, Derek A., Janet L. Pitman, and Kenneth P. McNatty. "Molecular forms of ruminant BMP15 and GDF9 and putative interactions with receptors." Reproduction 154, no. 4 (October 2017): 521–34. http://dx.doi.org/10.1530/rep-17-0188.
Повний текст джерелаАнотація:
Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted factors with demonstrable effects on ovarian follicular development and ovulation rate. However, the molecular forms of BMP15 and GDF9 produced by oocytes remain unclear. The aims herein, using Western blotting (WB) procedures with specific monoclonal antibodies (mabs), were to identify the molecular forms of BMP15 and GDF9 synthesised and secreted by isolated ovine (o) and bovine (b) oocytes in vitro. The mabs were known to recognise the biological forms of BMP15 or GDF9 since they had previously been shown to inhibit their bioactivities in vitro and in vivo. Using recombinant variants of oBMP15 and oGDF9, including a cysteine mutant form of oBMP15 (S356C) and a human (h) BMP15:GDF9 heterodimer (cumulin), it was established that the mabs were able to identify monomeric, dimeric, promature and higher-molecular-weight forms of BMP15 and GDF9 and cumulin (GDF9 mab only). After using non-reducing, reducing and reducing + cross-linking conditions, the major oocyte-secreted forms of o and b BMP15 and GDF9 were the cleaved and uncleaved monomeric forms of the promature proteins. There was no evidence for dimeric or heterodimeric forms of either mature BMP15 or GDF9. From in silico modelling studies using transforming growth factor beta (TGFB), activin or BMP crystal templates, and both present and previously published data, a model is proposed to illustrate how the monomeric forms of BMP15 and GDF9 may interact with their type II and type I cell-surface receptors to initiate the synergistic actions of these growth factors.
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Vanderhyden, B. C. "Species differences in the regulation of cumulus expansion by an oocyte-secreted factor(s)." Reproduction 98, no. 1 (May 1, 1993): 219–27. http://dx.doi.org/10.1530/jrf.0.0980219.
Повний текст джерела