Expression of growth differentiation factor 9 (GDF-9) during in vitro maturation in canine oocytes

Abstract

The aim of this study was to characterize in canine oocytes and cumulus cells the dynamic expression of growth differentiation factor 9 (GDF-9) in relation to meiotic development and cumulus expansion throughout in vitro maturation (IVM). Cumulus oocytes complexes (COCs) from ovaries of adult bitches were cultured intact for IVM during 0, 48, 72, and 96 hours. At 0 hours or after IVM, COCs were divided into two groups: one group remained with their cumulus cells and in the other group the cumulus cells were extracted. The expression levels of GDF-9 were determined in both groups using indirect immunofluorescence and Western blot analysis. For immunofluorescence assay, in vivo-matured oocytes collected from oviducts were also used as a positive control. The nuclear stage was analyzed in parallel with 4′-6-diamidino-2-phenylindole staining in denuded oocytes from all maturing groups. The intensity of fluorescence, indicative of GDF-9 expression level, decreased with time (P < 0.05). High expression was observed only in germinal vesicle nonmature oocytes; in contrast, second metaphase oocytes showed only low expression. Western blot analysis showed bands of approximately 56 kd and a split band of approximately 20 kd representing the proprotein and possibly two mature protein forms of GDF-9, respectively. The proprotein was detected in all samples, and it was highly expressed before IVM and in a lesser degree, during the first 48 hours, declining thereafter in coincidence with the expansion of the cumulus cell (P < 0.05). There was a negative correlation (r = −0.97; P < 0.05) between the expression level of GDF-9 and mucification. Mature forms were evident only in COCs, before culture and up to 48 hours of IVM. It was concluded that GDF-9 is expressed in canine oocytes and cumulus cells, mainly in the early developmental states, with low levels in mature oocytes in vitro and in vivo, representing the first approach of GDF-9 dynamic in dog oocyte maturation.

Introduction

Impaired oocyte meiotic maturation in dog has been the major obstacle to fertilization and embryo development in culture. Different in vitro maturation (IVM) protocols in canine have been established to study oocyte development; although significant progress has been achieved in recent years, the current IVM systems are far from optimal in terms of the percentage of mature oocytes obtained, and fertilization and in vitro embryo development.

A well-coordinated maturation between cytoplasm and nucleus depends on a complex interplay of endocrine and paracrine events, and direct communication between oocytes and neighboring somatic cells [1], [2]. In our recent studies, we have long observed that during culture some parameters of cytoplasmic maturation in canine oocytes, including cortical granules migration [3], mitochondria distribution [4], and the ability of the ovum cytoplasm to induce sperm nuclear decondensation [5], can be completed in a manner similar to that in vivo; however, nuclear maturation is not assured at high values and is not coordinated with cytoplasmic changes. Many IVM dog oocytes that can support cytoplasmic changes are only partially competent to undergo nuclear maturation [3], [4], [5].

Meiotic competence is gained gradually during folliculogenesis and oocyte growth [1], [6]. Several studies have demonstrated the importance of paracrine signals from oocytes via receptors located in the gap junction of neighboring granulosa cells. This cooperativity between these cells has been described to be under the control of the oocytes and it is mediated by factors, such as growth differentiation factor 9 (GDF-9), a member of the transforming growth factor beta superfamily [7], [8], [9]. This factor can activate the signaling pathways in cumulus cells to mediate the development of their neighboring oocytes through gap junctions channels [10], [11] or connexins (CXs) [12], with CX37 and CX43 being important in this regard [13]. In fact, granulosa cells must be coupled via the CX43 gap junction in order to respond optimally to GDF-9 [14].

In rodents, GDF-9 is involved in proliferation of granulosa cells from small follicles [15], and the growth-promoting actions of oocytes are mediated, at least in part, by GDF-9 [2], [16]. This protein also regulates cumulus expansion [17], [18] through induction of expression of Has2, Tnfaip6, Ptx3, and Ptgs2 [19], [20]. Hence, it is able to regulate oocyte meiotic resumption in preovulatory oocytes, activating the mitogen-activated protein kinase pathway by modification of cumulus cell function after the preovulatory LH surge [14]. In addition, the concentration of GDF-9 in follicular fluid has been significantly correlated with the nuclear maturation of human oocytes [21].

Cumulus cells control mitogen-activated protein kinase activation and phosphorylation in blue fox oocytes [22], confirming the influence of cumulus cells and oocyte interaction in regulating meiotic development in canine. In fact, the loss of cumulus gap junctions and thus, cumulus expansion has been associated with resumption of meiosis (germinal vesicle breakdown stage) in dog [23]. In the bitch, the mechanisms responsible for the delay of meiotic resumption after ovulation are not well known, but it has been suggested that the persistence of close relations between the oocyte and the cumulus cells might contribute to this delay [24].

It has been demonstrated that GDF-9 also inhibits granulosa cell LH receptor expression [1] and thus regulates progesterone production in murine [25], porcine [26], and bovine cumulus cells [27], [28], both indicative of a role in suppression of luteinization. In the bitch, follicular luteinization before ovulation is a typical feature [29], [30] and in contrast to other mammalian species, with increasing concentration of progesterone at the moment of ovulation [31]. Therefore, these progesterone-inhibiting actions of GDF-9 might be insufficient in dogs to prevent lutenization.

The relationship between cumulus cells, GDF-9, and other growing factors might have different functions depending on the species. The different phenotypes exhibited by mice and sheep with inactivating mutations in GDF-9 [32] are evidence of species differences in the actions of this factor, especially considering the peculiar aspects of dog reproductive physiology. To our knowledge, the presence of GDF-9 in canine oocytes or cumulus oocytes complexes (COCs) has not been described, and its influence on the canine oocyte and the surrounding cumulus cells could be relevant throughout maturation, because the oocyte-cumulus communication in the bitch is longer than in other animals because the cumulus cells remain strongly attached to the oocyte for several days after the LH peak and ovulation [33]. Therefore, this uncommon pattern of cumulus expansion in canine oocytes might be related to a different expression pattern of GDF-9, which would modulate among other proteins, granulosa cell functions involved in oocyte developmental ability. Therefore, in the present study we evaluated the dynamic expression of GDF-9 in relation to nuclear maturation and cumulus expansion throughout culture time in canine oocytes using indirect immunofluorescence and Western blot analysis.