Differential expression of GDF-9 and BMP- 15 during follicular development in canine ovaries evaluated by flow cytometry
Introduction
Oocyte development and follicle growth are closely regulated by an ordered and complex series of signaling events throughout folliculogenesis. The interplay between oocytes and the somatic cells of ovarian follicles determines the developmental ability of these cells. During this process, meiotic competence of the oocyte is gained gradually (Eppig, 2001, Fair, 2003, Hussein et al., 2006) and gap junctional communication within the developing follicle, both between oocytes and granulosa cells and among the follicular cells themselves, maintains the follicle in a functionally integrated state (Kidder and Vanderhyden, 2010, Huiyu et al., 2013). However, little is known about the physiological control of follicular growth in canines, despite being of pivotal importance in oocyte development in vivo and in vitro. The subsequent in vitro maturation (IVM) of oocytes, that remains unsuccessful in this species (De los Reyes et al., 2005, De los Reyes et al., 2011, Chastant-Maillard et al., 2011), depends strongly on its prior period inside the follicles. In fact, the compromised developmental competence of IVM in canines can be attributed to the lack of knowledge of the ability of the oocyte’s intrinsic requirements during follicular maturation.
The communication of oocyte and follicular cells is characterized by the secretion of several growth factors such as those members of transforming growth factor beta superfamily (TGF-β), which plays an important function in proliferation and differentiation of a variety of cell types. Many studies have shown that members of the TGF-β superfamily can regulate granulosa cell proliferation and differentiation (Sudiman et al., 2014, Cheng et al., 2015). Growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 are members of the TGF-β superfamily of proteins, and thus are similar at a structural level (Knight and Glister, 2006, Otsuka et al., 2011). Studies indicate that GDF-9 and BMP-15 are both present in follicles throughout different stages of follicular development and in many species these factors influence ovarian follicular growth. Findings in sheep, humans and rodents show that BMP-15 and GDF-9 can be considered to be new targets for fertility regulation in mammals (Gilchrist et al., 2006, Persani et al., 2014), as they participate in signaling pathways that control the development of ovarian follicles. These proteins are thought to affect granulosa cell proliferation independently or synergistically from small follicles (Fenwick et al., 2013) and the growth-promoting actions of oocytes are mediated, at least in part, by these factors (Su et al., 2004, Hussein et al., 2006), although the signals exchanged between the oocyte and the surrounding cells are far from being fully understood. Studies in mice and sheep, as well as evidences from in vitro studies in other species, have demonstrated that cooperative interactions between GDF-9 and BMP-15 occur in many functions (McNatty et al., 2005, Mottershead et al., 2012). GDF9 promotes the expansion of cumulus cells by induction of expression of Has2, Tnfaip6, Ptx3, and Ptgs2 (Varani et al., 2002); BMP-15 also is involved in this process (Gueripel et al., 2006), and both factors promote proliferation of granulosa cells (Kidder and Vanderhyden, 2010).
It has been reported that BMP-15 and GDF-9 are expressed only in oocytes in rodents (Gilchrist et al., 2008), however, these factors are expressed also in cumulus and mural granulosa cells in many other mammals (Hosoe et al., 2011, Lim et al., 2014), including canine (De los Reyes et al., 2013, Maupeu et al., 2015), demonstrating that the expression patterns and biological functions may differ among species. Therefore, many species differences have already emerged (Galloway et al., 2000, Hussein et al., 2006, Sun et al., 2008). In previous studies we suggested that oocyte-produced GDF-9 in vitro may be insufficient for promoting cumulus cell expansion in canine (De los Reyes et al., 2013), which could be related to delay for resumption of meiosis. However, the levels of GDF-9 and BMP-15 expression in canine granulosa cells during follicular development are largely unknown. As GDF-9 and BMP-15 are both possibly present in follicles throughout most stages of follicular growth, it is important to evaluate whether these paracrine factors are differentially expressed in these follicles. The knowledge of the physiology of GDF-9 and BMP-15 factors in canine could contribute to understanding the special reproductive characteristics of this species and also potentially helps to improve protocols that may to overcome the low success of IVM in canine oocytes.
Flow cytometry is a powerful analytical instrument for rapid evaluation of high numbers of cells; it detects labeling by multiple fluorochromes associated with individual cells flowing in a flowing stream (Hirshfield et al., 1988). This technique has been successfully used for granulosa cell analysis (Rao et al., 1991, De Neubourg et al., 1996, Douville and Sirard, 2014, Regan et al., 2015), providing an alternative method for assessing protein expression in follicular cells. Therefore, the experiments reported in the present study were undertaken to evaluate by flow cytometric methods the expression of GDF-9 and BMP-15 in canine follicular cells during follicular development.
Section snippets
Material and methods
This study was approved by the Bioethics Committee, Faculty of Veterinary Sciences, University of Chile and the Research Ethics Committee of the Chilean National Commission for Scientific and Technological Research (FONDECYT).
Results
The ELISA test corroborated the physiological changes in progesterone concentration according the different phases of estrous cycle in accordance with the predominant ovarian structures. Individual progesterone values of bitches submitted to ovariohysterectomy were 0.01–0.1 ng/mL in anestrus phase; 0.33–14.83 ng/mL in proestrous/estrus and 17.91–31.07 ng/mL in diestrus.
All antral follicles were classified according to size and reproductive phase of the donor (Table 1).
Flow cytometry experiments
Discussion
Flow cytometry gave a fast and accurate approach to the investigation of follicular cells in the canine ovarian follicles. Antral follicles yield enough follicular cells to perform a good flow cytometry analysis, providing a visual representation of the number of cells as a function of GDF-9 and BMP-15 content and, thus, the proportion of these proteins in the follicle cells in each phase of the reproductive cycle and development. Therefore, it was possible to identify the presence of these two
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgments
This research was supported by Grant 1140658 (FONDECYT) and by Grant EQM120156 (FONDEQUIP), from The National Commission for Scientific and Technological Research, Chile (CONICYT).
We thank to veterinarians from El Roble Veterinary Hospital for procuring bitch ovaries for this study and also we are grateful to Prof. Donald L Palmquist for his helpful suggestions and comments on this manuscript.
References (60)
Reproductive cycles of the domestic bitch
Anim. Reprod. Sci.
(2011)- et al.
Temporal relations between plasma concentrations of luteinizing hormone, follicle-stimulating hormone, estradiol-17β, progesterone, prolactin, and a-melanocyte- stimulating hormone during the follicular, ovulatory, and early luteal phase in the bitch
Theriogenology
(2006) - et al.
Effect of human chorionic gonadotrophin supplementation during different culture periods on in vitro maturation of canine oocytes
Theriogenology
(2005) - et al.
Expression of growth differentiation factor 9 (GDF-9) during in vitro maturation in canine oocytes
Theriogenology
(2013) - et al.
Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene
Am. J. Hum. Genet.
(2004) Follicular oocyte growth and acquisition of developmental competence Anim
Reprod. Sci.
(2003)- et al.
Oocytes secreted factors enhance oocyte developmental competence
Development
(2006) - et al.
Bone morphogenetic protein-15. Identification of target cells and biological functions
J. Biol. Chem.
(2000) - et al.
Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression
J. Biol. Chem.
(2001) - et al.
Synergistic roles of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte–granulosa cell regulatory loop
Dev. Biol.
(2004)
Use of enzyme-immunoassay for oestradiol-17β and progesterone quantification in canine serum
Anim. Reprod. Sci.
Cloning of GJA1 (connexin43) and its expression in canine ovarian follicles throughout the estrous cycle
Gene Expr. Patterns
BMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells
Mol. Endocrinol.
Oocyte-derived BMP15 but not GDF9 down-regulates connexin43 expression and decreases gap junction intercellular communication activity in immortalized human granulosa cells
Mol. Hum. Reprod.
The canine oocyte: uncommon features of in vivo and in vitro maturation
Reprod. Fertil. Dev.
TGF-β1 Up-regulates connective tissue growth factor expression in human granulosa cells through smad and ERK1/2 signaling pathways
PLoS One
Endocrinologic control of normal canine ovarian function
Reprod. Domest. Anim.
Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation
Theriogenology
Dynamic expression of bone morphogenetic protein 15 (BMP-15) during in vitro maturation of canine oocytes
Flow cytometric analysis of granulosa cells from follicular fluid after follicular stimulation
Hum. Reprod.
Changes in granulosa cells gene expression associated with growth, plateau and atretic phases in medium bovine follicles
J. Ova Res.
Oocyte control of ovarian follicular development and function in mammals
Reproduction
Investigations of TGF-beta signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15
Endocrinology
Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner
Nat. Genet.
Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality
Hum. Reprod. Update
Molecular basis of oocyte–paracrine signaling that promotes granulosa cell proliferation
J. Cell Sci.
Interplay between paracrine signaling and gap junctional communication in ovarian follicles
J. Cell Sci.
Oocyte bone morphogenetic protein 15 but not growth differentiation factor 9, is increased during gonadotropin-induced follicular development in the immature mouse and is associated with cumulus oophorus expansion
Biol. Reprod.
Flow cytofluorometric analysis of granulosa cell proliferation in rats
J. Reprod. Fertil.
Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries
Reprod. Biol. Endocrinol.
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