Background
Fully grown oocytes in antral follicles (more than 2 mm in diameter) are an important source of in vitro embryo production in cattle [
1,
2]. However, the majority of oocytes in an ovary are small oocytes that are either dormant or at various growing stages. It is, therefore, necessary to utilize these small oocytes to make better use of ovaries, especially for species for which ovary samples are extremely rare. Although complete in vitro development of oocytes from primordial follicles has been demonstrated in mice [
3,
4], it has not been achieved in other mammals. Among follicles in developmental stages, early antral follicles show the potential to be a supplemental source of oocytes because they are most similar in size to late antral follicles. Several research groups have successfully produced live calves from oocytes released from early antral follicles (less than 1 mm in diameter) after in vitro growth (IVG), in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) [
5‐
7]. Compared to oocytes grown in vivo, however, the meiotic and developmental competence of IVG oocytes are generally lower [
5,
6,
8]. Therefore, it is necessary to improve the IVG culture system.
Theca cells are an essential component of growing follicles, supporting follicle growth and development not only by delivering nutrients and providing the androgens required for conversion into estrogens by granulosa cells (GCs) but also by producing growth factors that can promote follicular development [
9]. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily of extracellular signaling molecules, which play multiple roles in the regulation of the growth, differentiation and apoptosis of numerous cell types. Theca-derived BMP-4 has been shown to be capable of regulating the growth and function of GCs by suppressing apoptosis and enhancing the secretion of estradiol, while reducing progesterone (P
4) secretion in vitro, an action consistent with a delay of luteinization and/or atresia [
10,
11]. Furthermore, previous in vivo studies have shown that BMP-4 mRNA was expressed at high levels in the theca of developing dominant follicles, while it was very low or undetectable in atretic follicles [
12]. This evidence suggest the possibility that theca-derived BMP-4 is related to the emergence and/or maintenance of dominant follicles; thus, it may contribute to IVG, which is aimed at producing healthy oocytes similar to those grown in the dominant follicles. However, the effects of BMP-4 on oocyte growth and subsequent developmental competence are unknown because GCs alone were studies in previous studies [
10,
13,
14].
The current IVG protocol for bovine oocytes only uses theca-free oocyte-granulosa cell complexes (OGCs). As a result, the products from theca cells, especially growth factors, such as BMP-4, are absent. Furthermore, a previous study showed P
4 concentration (68 ng/ml) in the follicular fluid of the growing antral follicles with a low antral follicle count (AFC) were double that in follicles with a high AFC (32 ng/ml) [
15]. It was also reported that oocytes derived from follicles with a low AFC had a much lower blastocyst formation rate [
15]. A low AFC is associated with diminished ovarian function and fertility in both human and cow [
16,
17]. Our preliminary study found P
4 concentration in the IVG culture medium soared above 60 ng/ml, a high level comparable to that in follicles with a low AFC. Therefore, in an attempt to compensate for the missing roles of theca cells and reduce P
4 production during IVG culture, the aim of our study was clarifying the functions of BMP-4 produced by theca cells. We asked whether BMP-4 adding in the IVG culture medium affects OGC growth, P
4 production and subsequent developmental competence acquisition in the present study.
Discussion
In the current study, BMP-4 suppressed P
4 production from GCs, in agreement with the results of other in vitro studies [
13,
14]. It has been reported that the inhibition of the acetylation of histone H3 associated with the steroidogenic acute regulatory protein promoter region by BMP-4 may be one of the underlying molecular mechanisms of the inhibition of P
4 synthesis in GCs [
13]. Because increased P
4 synthesis is a characteristic of the GC luteinization process [
24], BMP-4 showed an effect of anti-luteinization. This effect was further verified by the significantly smaller diameter (~11 μm) of GCs in the BMP-4 treated groups after IVG culture compared to non-BMP-4 treated control (12.0 μm). It has been reported that both in vivo-grown large luteal cells originated from GCs and in vitro-luteinized GCs have similar sizes, which are larger than that of GCs in pre-ovulatory follicles (mean diameter, 38.4 vs.10.6 μm, respectively) [
25,
26]. Therefore, the delay in GC enlargement observed in the present study may be attributed to delayed luteinization, which is associated with reduced P
4 synthesis in GCs treated with BMP-4. It is known that GCs become luteinized and P
4 secretion from GCs increases in atretic follicles [
27,
28]. In the present study, however, BMP-4 treatment reduced P
4 to a level (~33 ng/ml) close to that of follicular fluid (~32 ng/ml) in growing antral follicles with high AFC [
17]. These data indicate the possibility for developing an in vitro model mimicking the growth of bovine oocytes in healthy follicles.
Despite the similarity in the ability to delay luteinization, the 50 ng/mL BMP-4 treatment caused a decrease in the GC number relative to the controls, while 10 ng/mL BMP-4 did not. It appears that BMP-4 impaired proliferation, but did not induce apoptosis of GCs, as the viability of GCs was high (more than 90 %) in all of the experimental groups. This finding is inconsistent with previous studies in which 50 or 100 ng/mL BMP-4 did not affect GC proliferation over 6 or 4 days of GC culture, respectively [
13,
14]. We didn’t expected the GC number to decrease with BMP-4 treatment because it has been demonstrated that BMP-4 could reduce apoptosis of GCs by suppressing the action of caspase-activated DNase induced by Survivin, a member of the inhibitor of apoptosis family [
10]. One major difference between the present and the previous studies is that we cultured OGCs, rather than GCs alone. The involvement of oocytes complicates the culture system and may be responsible for the discrepancies. It is now widely recognized that oocyte secreted factors (OSFs), such as growth differentiation factor-9 (GDF-9) and BMP-15, direct the functions of their surrounding GCs, including the promotion of cell growth and prevention of cell death and luteinization [
29‐
32]. Because the types of receptors for the transforming growth factor-β superfamily are limited, ligands of the BMP/GDF subfamily bind to receptors in a shared manner [
33]. Among the limited receptors, BMP type-II receptor is the sole type-II receptor presenting in GCs for GDF-9 and BMP-15 and is also an important receptor for BMP-4 [
34]. In follicles, GDF-9 and BMP-4 could induce GCs to produce Gremlin, which is known to be effective at antagonizing BMP-4 actions without affecting oocyte-derived GDF-9 and BMP-15 [
31,
35,
36]. As a result, it is proposed that oocyte could maintain its surrounding microenvironment, which is important for oocyte development, from the actions of theca-derived BMPs [
31]. Take into consideration of the fact that a 20-fold excess of gremlin to BMP-4 was needed to completely block BMP-4 action [
35], exogenous BMP-4 at the doses studied (10 and 50 ng/ml) may be too high and probably impaired the function of OSFs by competitively binding receptors against OSFs, resulting in decreased GC proliferation in a dose-dependent manner. A smaller number of GCs in the 50 ng/mL BMP-4 treated group, in turn, may lead to the decreased OGC viability when consider the fact that oocytes also relay on the support of GCs for long-term growth.
In the present study, BMP-4 treatment did not promote oocyte growth. However, BMP-7, another theca-derived growth factor, increased the oocyte diameter and volume during IVG, in which OGCs were cultured in groups on membrane inserts [
37]. The culture system may partially cause the different results, but unknown differences in the biological function between BMP-4 and–7 may also be responsible [
34]. Our study showed that BMP-4 impaired embryo development of IVG oocytes without affecting oocyte nuclear maturation and fertilization, indicating that BMP-4 may inhibit the cytoplasmic maturation of oocytes. Another study reported that BMP-4 addition during IVM of COC had no effect on bovine oocyte nuclear maturation and subsequent embryo development [
38]. The differences in the growth stage of oocytes and exposure time to BMP-4 may have led to the discrepancy. Through OSFs, oocytes appear to control their neighboring somatic cells, directing them to perform functions required for the appropriate development of oocytes. The presence of this regulatory loop was demonstrated by the fact that the neutralization of OSFs by antagonists of BMP-15 and GDF-9 during IVM impaired the developmental competence of COCs [
39]. Although there was no BMP-4 addition during pre-IVM and IVM in the present study, the presumed interruption of the regulatory loop by BMP-4 during IVG appeared to cause a lasting adverse effect, as shown in the subsequent developmental competence of oocytes. Further investigations are necessary to verify this type of receptor competition between BMP-4 and OSFs and its consequential effects.
Compared to in vivo-grown oocytes, IVG oocytes showed inferior competences for fertilization and development to blastocyst, although their nuclear maturation was similar to in vivo-grown ones, indicating that the cytoplasmic maturation of IVG oocytes might be inadequate.
To our knowledge, this is the first study utilizing OGCs model to investigate the BMPs on growth, steroidogenesis and subsequent developmental competence of OGCs derived from bovine early antral follicles. This model will be helpful for studying the function of BMPs or other growth factors in growing antral follicles in a more comprehensive way.
Competing interests
The authors have no competing interest in publishing findings of this research.
Authors’ contributions
YY carried out the most studies, analyzed data and wrote the manuscript. CK helped the oocyte collection. WH and SK performed IVM and IVF studies of in vivo-grown oocyte. YY and MN guided the interpretation of data and revised the manuscript. All authors read and approved the final manuscript.