Influence of leptin on in vitro maturation and steroidogenic secretion of cumulus–oocyte complexes through JAK2/STAT3 and MEK 1/2 pathways in the rabbit model

in Reproduction

Extreme body mass indexes may impair reproductive outcome in assisted reproductive technologies. Leptin reflects the amount of body fat and could act as a modulator of oocyte quality through activation of specific transcription factors. The aim of this work was to establish whether: 1) leptin influences meiotic and cytoplasmic oocyte maturation; 2) STAT3 and MAPK mediate the effects of leptin and 3) leptin modulates steroid secretion by cumulus–oocyte complexes (COC) during in vitro maturation (IVM). We confirmed immunolocalisation of leptin receptor in oocytes, cumulus/granulosa cells during the peri-ovulatory period. The confocal study showed that COC supplemented with 1, 10 and 100 ng/ml leptin had a significantly higher metaphase II (MII) percentage than those IVM without leptin (P<0.05) and a similar MII index compared to the group supplemented with 10% FCS. Leptin did not increase the percentage of cytoplasmically matured oocytes in terms of cortical granule migration rate, whereas a significantly higher index was found in the FCS group (P<0.001). Oestradiol concentrations in spent media were higher in the FCS group compared to other treatments (P<0.001). Leptin-stimulated nuclear oocyte maturation was significantly impaired when leptin-induced JAK2/STAT3 and MEK 1/2 activation was suppressed by the inhibitors (P<0.001). Steroid secretion of COC was not affected by leptin activation of JAK2/STAT3 or MEK 1/2 pathways. In conclusion, JAK2/STAT3 and MEK 1/2 pathways mediate the enhancement of nuclear oocyte maturation by leptin; however, neither cytoplasmic oocyte maturation nor steroidogenic response of COC were improved in the present rabbit model.

Abstract

Extreme body mass indexes may impair reproductive outcome in assisted reproductive technologies. Leptin reflects the amount of body fat and could act as a modulator of oocyte quality through activation of specific transcription factors. The aim of this work was to establish whether: 1) leptin influences meiotic and cytoplasmic oocyte maturation; 2) STAT3 and MAPK mediate the effects of leptin and 3) leptin modulates steroid secretion by cumulus–oocyte complexes (COC) during in vitro maturation (IVM). We confirmed immunolocalisation of leptin receptor in oocytes, cumulus/granulosa cells during the peri-ovulatory period. The confocal study showed that COC supplemented with 1, 10 and 100 ng/ml leptin had a significantly higher metaphase II (MII) percentage than those IVM without leptin (P<0.05) and a similar MII index compared to the group supplemented with 10% FCS. Leptin did not increase the percentage of cytoplasmically matured oocytes in terms of cortical granule migration rate, whereas a significantly higher index was found in the FCS group (P<0.001). Oestradiol concentrations in spent media were higher in the FCS group compared to other treatments (P<0.001). Leptin-stimulated nuclear oocyte maturation was significantly impaired when leptin-induced JAK2/STAT3 and MEK 1/2 activation was suppressed by the inhibitors (P<0.001). Steroid secretion of COC was not affected by leptin activation of JAK2/STAT3 or MEK 1/2 pathways. In conclusion, JAK2/STAT3 and MEK 1/2 pathways mediate the enhancement of nuclear oocyte maturation by leptin; however, neither cytoplasmic oocyte maturation nor steroidogenic response of COC were improved in the present rabbit model.

Introduction

Obesity is a rising health problem commonly associated with infertility (Denison 2009) which may impair the outcome of assisted reproductive technologies. Studies performed with infertile women and animals indicate that the optimisation of ovary function is fundamental in the fertility prognosis (Bellver 2006). Different endocrine factors related to the body mass index may affect oocyte quality and therefore reproductive success. Thus, leptin, a 16 kDa peptide hormone encoded by LEP (previously known as the obese gene, OB) and secreted mainly by the adipose tissue, plays an important role in the regulation of food intake, energy expenditure and reproductive success (Keim et al. 1998, Cervero et al. 2006). In fact, a minimum level of leptin is required for maintenance of the reproductive function (Cheung et al. 1997), but high leptin concentrations associated with obesity itself or polycystic ovarian syndrome have been reported to explain partially the negative impact of these pathologies on fertility (Bellver 2006).

Leptin influence on reproduction is mediated by the regulation of the hypothalamus–pituitary axis and the ovarian function through its receptor (Chehab et al. 1996, Brecchia et al. 2006). Earlier studies showed that mice lacking either leptin (OB/OB) or leptin receptors (DB/DB) are both obese and infertile (Zhang et al. 1994, Lee et al. 1996), whereas administration of exogenous leptin has the ability to restore fertility in these animals (Chehab et al. 1996). In the ovary, leptin receptor (LEPR, previously known as OBR) has been detected in granulosa cells of follicles, cumulus cells and oocytes in several species (human: Cioffi et al. 1997; mouse: Antczak & Van Blerkom 1997; mouse: Ryan et al. 2002; pig: Craig et al. 2004; rabbit: Zerani et al. 2004; ewe: Muñoz-Gutiérrez et al. 2005; cattle: Paula-Lopes et al. 2007). The LEPR is a member of the class-I cytokine receptor superfamily with six known isoforms (Tartaglia et al. 1995, Lee et al. 1996). Both long (LEPRb) and short transmembrane isoforms (LEPRa,c,d,f) contain identical extracellular and transmembrane domain, but the cytoplasmic region is different in length (Frühbeck 2006). LEPR usually activates cytoplasmic JAK2 to transmit leptin signals (Frühbeck 2006). LEPRb is highly expressed in the hypothalamus and mediates leptin signalling through activation of both the STAT3 and MAPK (Tartaglia et al. 1995, Lee et al. 1996, Bjørbaek et al. 1997). The short isoforms of the receptor are more widely expressed in peripheral tissues and are capable of signalling through the MAPK pathway (Bjørbaek et al. 1997, Tartaglia 1997). Specifically, MAPK kinases 1–2 (MEK1 and MEK2) are dual-specific protein kinases involved in a MAPK cascade and appear as predominant MAPK in cumulus–oocyte complexes (COC) during in vitro maturation (IVM) in heifers (Van Tol et al. 2008). Although leptin may be integrated in a complex intracellular cross-talk system that regulates specific cell functions (Zhang et al. 1994), there is scarce information about the role of leptin through these pathways in the oocyte of humans and other animal species. In this sense, it is known that human COC are capable to respond to leptin during pre-ovulatory period (Cioffi et al. 1997), and its potential direct effects on oocyte maturation, mainly as a modulator of the oocyte fertility potential, could be through activation of specific transcription factors.

Additionally, interactions between leptin and cumulus cell-enclosed oocytes could modulate steroid production in cumulus cells (Karamouti et al. 2003, Ruiz-Cortés et al. 2003, Swain et al. 2004). It has been reported that follicular cells control oocyte physiology by secreting steroids (Mingoti et al. 1995). Therefore, concentrations of oestradiol (OE2) and progesterone (P4) during the peri-ovulatory period could be implicated in normal meiotic and cytoplasmic oocyte maturation by paracrine or autocrine pathways (Kaji et al. 1987, Younis et al. 1989, Andersen 1993, Tesarik & Mendoza 1997). However, elucidation of leptin's role in such processes has been challenging, with conflicting results reported by several investigators. Understanding these mechanisms is essential because the influence of leptin on oocyte maturation ability has important implications for ovulation induction and could be helpful to find optimal hormone combinations to apply in animal and human infertility therapies.

Rabbits are widely used as an in vitro model in experimental procedures on mammalian oocytes and embryos. As a consequence, taking all these findings together and for the first time in the rabbit oocyte, the objective of this study after the immunolocalisation of the leptin receptor (LEPR) in the ovary throughout the peri-ovulatory period was to investigate: 1) the direct role of different leptin concentrations on meiotic and cytoplasmic in vitro oocyte maturation; 2) the JAK2/STAT3 and MEK 1/2 leptin-dependent intracellular mechanisms by specific pathway inhibition; 3) the steroidogenic response of COC to leptin during the oocyte maturation process.

Results

Immunolocalisation of leptin receptor (LEPR)

Positive immunostaining for LEPR was found in oocytes, cumulus cells and granulosa cells of the follicles at all stages of development in all time points measured during the peri-ovulatory period (Fig. 1). Stroma and corpora lutea exhibited high immunoreactivity for LEPR as well. Theca interna cells showed moderate immunostaining, whereas LEPR was not immunolocalised in theca externa cells. Control tissue samples showed no positive staining.

Figure 1

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Figure 1

Immunolocalisation of leptin receptor (LEPR) in ovarian sections of rabbit does during the peri-ovulatory period. (A) Negative control. (B) Positive staining of LEPR in oocytes and granulosa cells of primordial, primary and secondary follicles. (C) Antral follicle with staining of LEPR in granulosa cells. (D) Immunodetection detail of LEPR in granulosa and theca cells. Theca externa cells show negative immunostaining (o, oocyte; g, granulosa cells; t, theca cells).

Citation: REPRODUCTION 139, 3; 10.1530/REP-09-0309

Effect of leptin on nuclear and cytoplasmic oocyte maturation

As shown in Table 1, nuclear oocyte IVM was significantly improved (P<0.005) when leptin was added to the culture media at all concentrations tested (1, 10, 100 ng/ml) compared with the group without leptin. Groups 1 Lep, 10 Lep and 100 Lep showed similar percentages of metaphase II (MII) compared with the group cultured with FCS. Groups supplemented with 1 and 10 ng/ml leptin showed a lower rate of metaphase I (MI) compared with 0 Lep and FCS groups (P<0.05).

Table 1

Meiotic and cytoplasmic configuration percentages, in terms of cortical granule (CG) migration, observed in rabbit oocytes at 16 h maturation time following the use of different leptin concentrations.

0 ng/ml leptin (0 Lep)1 ng/ml leptin (1 Lep)10 ng/ml leptin (10 Lep)100 ng/ml leptin (100 Lep)10% FCS (FCS)
Nuclear maturation (%)
 Metaphase II54.05a79.35b75.19b66.38b71.62b
 Metaphase I19.8c5.4d7.8d11.2c,d17.6c
 Germinal vesicle17.115.214.025.018.9
 Abnormal chromosome configuration9.0d0.0c3.1c,d7.8d9.5d
Cytoplasmic maturation (%)
 Peripheral distribution of CG7.9c9.3c10.0c2.9c25.7d
 Cortical distribution of CG6.3c,d23.3c8.3c,d20.0c,d2.9d
 Homogeneous distribution of CG82.560.581.777.168.6
 Abnormal distribution of CG3.27.00.00.02.9

Values are mean±s.e.m. Means in rows with different letters differ: a,b(P<0.005) c,d(P<0.05).

Peripheral and cortical distribution of cortical granule (CG) migration (Table 1) was similar for all experimental leptin groups. However, the group supplemented with FCS showed a significantly higher number of cytoplasmically matured oocytes compared to the treatments without serum (P<0.05). No significant differences were found in the percentage of oocytes with homogeneous and abnormal CG distribution between groups.

STAT3 and MAPK leptin-associated signal transduction during IVM

Leptin-stimulated nuclear oocyte maturation in terms of MII rate was significantly impaired (P<0.001) when specific JAK2/STAT3 and MEK 1/2 inhibitors at all concentrations tested were added to the maturation media (Tables 2 and 3 respectively). The percentage of MI and germinal vesicle (GV) was increased in the 10 ng/ml leptin groups supplemented with both the inhibitors. The addition of both inhibitors in the 0 Lep group did not modify MII, MI, GV and abnormal nuclear configuration proportion. The percentage of cytoplasmically matured oocytes was similar in both the leptin IVM media supplemented with JAK2/STAT3 and MEK 1/2 inhibitors (Tables 2 and 3 respectively) and the respective control groups (0 Lep and 10 Lep). The groups supplemented with leptin and 100 μM JAK2/STAT3 or 50 μM MEK 1/2 inhibitor concentrations showed a significant increase in the number of oocytes with abnormal distribution of CG migration (P<0.05).

Table 2

Meiotic and cytoplasmic configuration percentages, in terms of cortical granule (CG) migration, observed in rabbit oocytes matured in vitro with 0 or 10 ng/ml leptin media plus JAK2/STAT3 inhibitors (AG490) at different concentrations.

0 ng/ml leptin (0 Lep)0 ng/ml leptin/10 μM AG498 (0/10)0 ng/ml leptin/100 μM AG490 (0/100)10 ng/ml leptin (10 Lep)10 ng/ml leptin/10 μM AG490 (10/10)10 ng/ml leptin/100 μM AG490 (10/100)
Nuclear maturation (%)
 Metaphase II54.140.045.575.2a38.7b45.9b
 Metaphase I19.824.030.37.8a25.3b17.6a,b
 Germinal vesicle17.124.018.214.0a33.3b27.0b
 Abnormal chromosome configuration9.012.06.13.12.78.1
Cytoplasmic maturation (%)
 Peripheral distribution of CG7.911.817.610.0c,d17.1c2.2d
 Cortical distribution of CG6.323.514.78.317.117.8
 Homogeneous distribution of CG82.564.767.681.7c58.5d64.4c,d
 Abnormal distribution of CG3.20.00.00.0c7.3c,d15.6d

Values are mean±s.e.m. Means in rows with different letters differ: a,b(P<0.005) c,d(P<0.05).

Table 3

Meiotic and cytoplasmic configuration percentages, in terms of cortical granule (CG) migration, observed in rabbit oocytes matured in vitro with 0 or 10 ng/ml leptin media plus MEK 1/2 inhibitors (PD98059) at different concentrations.

0 ng/ml leptin (0 Lep)0 ng/ml leptin/10 μM PD98059 (0/10)0 ng/ml leptin/50 μM PD98059 (0/50)10 ng/ml leptin (10 Lep)10 ng/ml leptin/10 μM PD98059 (10/10)10 ng/ml leptin/50 μM PD98059 (10/50)
Nuclear maturation (%)
 Metaphase II54.151.454.475.2a44.1b49.3b
 Metaphase I19.820.314.77.8a22.6b26.8b
 Germinal vesicle17.123.029.414.0c29.0d22.5c,d
 Abnormal chromosome configuration9.05.41.53.24.31.4
Cytoplasmic maturation (%)
 Peripheral distribution of CG7.97.17.510.010.59.1
 Cortical distribution of CG6.312.52.58.313.211.4
 Homogeneous distribution of CG82.569.687.581.776.368.2
 Abnormal distribution of CG3.210.72.50.0a0.0a11.4b

Values are mean±s.e.m. Means in rows with different letters differ: a,b(P<0.005) c,d(P<0.05).

Steroidogenic response of COC

As shown in Table 4, mean OE2 secretion by COC was significantly higher in the FCS group compared to those oocytes cultured in media supplemented with 0, 1, 10 and 100 ng/ml leptin (P<0.005). No significant differences were found in P4 concentrations at the end of the IVM period between treatments.

Table 4

Steroid production in rabbit cumulus–oocyte complexes (COC) at 16 h maturation time following the use of different leptin concentrations.

0 ng/ml leptin (0 Lep)1 ng/ml leptin (1 Lep)10 ng/ml leptin (10 Lep)100 ng/ml leptin (100 Lep)10% FCS (FCS)
OE2/COC (pg/ml)0.28±0.14a0.22±0.02a0.32±0.14a0.34±0.11a4.13±0.77b
P4/COC (ng/ml)0.03±0.010.02±0.0040.03±0.010.03±0.010.03±0.003

Values are mean±s.e.m. Means in rows with different letters differ: a,b(P<0.005) c,d(P<0.05).

Mean OE2 and P4 secretions by COC were similar when JAK2/STAT3 inhibitors were added to the IVM media compared to the respective control groups. When MEK 1/2 inhibitors were added, a significant increase in the mean OE2 secretion by COC was observed in the 0/10, 0/50 and 10/10 groups as well as similar increases in P4 secretion in 10/10 and 10/50 groups compared to the treatment with 0 and 10 ng/ml leptin (P<0.05; Table 5).

Table 5

Steroid production in rabbit cumulus–oocyte complexes (COC) at 16 h maturation time following the use of different concentrations of JAK2/STAT3 (AG490) and MEK 1/2 (PD98059) leptin-induced pathway inhibitors.

0 ng/ml leptin (0 Lep)0 ng/ml leptin/10 μM AG498 (0/10)0 ng/ml leptin/100 μM AG490 (0/100)10 ng/ml leptin (10 Lep)10 ng/ml leptin/10 μM AG490 (10/10)10 ng/ml leptin/100 μM AG490 (10/100)
OE2/COC (pg/ml)0.18±0.040.23±0.110.10±0.050.39±0.170.44±0.240.14±0.05
P4/COC (ng/ml)0.03±0.010.03±0.000.02±0.000.03±0.000.06±0.030.02±0.00
0 ng/ml leptin (0 Lep)0 ng/ml leptin/10 μM PD98059 (0/10)0 ng/ml leptin/50 μM PD98059 (0/50)10 ng/ml leptin (10 Lep)10 ng/ml leptin/10 μM PD98059 (10/10)10 ng/ml leptin/50 μM PD98059 (10/50)
OE2/COC (pg/ml)0.05±0.01a0.10±0.02b0.11±0.01b0.04±0.01a0.13±0.02b0.07±0.01a
P4/COC (ng/ml)0.02±0.000.03±0.010.05±0.000.01±0.00a0.04±0.00b0.05±0.01b

Values are mean±s.e.m. Means in rows with different letters differ: a,b(P<0.05).

Discussion

The current work shows for the first time the effect of leptin on nuclear and cytoplasmic in vitro oocyte maturation, its underlying intracellular mechanisms and the steroidogenic response using the rabbit oocyte as a model. The results obtained demonstrate that the beneficial influence of leptin on meiotic oocyte maturation is mediated by JAK2/STAT3 and MEK 1/2 pathways.

The presence of leptin and its receptor in the body is essential for reproductive function (Chehab et al. 1996). The universal distribution of leptin receptors between species and their different physiological status are due to the pleiotropic nature of leptin and the highly preserved structure of the LEPR (Tartaglia et al. 1995, Bjørbaek et al. 1997). In the present study, long and short isoforms of the LEPR were immunolocalised in the rabbit oocyte, cumulus and granulosa cells of the ovarian follicles at all stages of development, as well as in theca interna, estroma and corpus luteum cells during the peri-ovulatory period. These findings corroborate those obtained in ovaries of several species (human: Karlsson et al. 1997; rat: Zamorano et al. 1997; mouse: Matsuoka et al. 1999; gilt: Lin et al. 2000, Craig et al. 2004; cattle: Thorn et al. 2007), including pseudo-pregnant rabbits (Zerani et al. 2004), and contribute to the significant body of evidence around the physiological relevance of this hormone, especially in the ovary and during the pre-ovulatory period (Cioffi et al. 1997, Duggal et al. 2002, Archanco et al. 2003).

Oocyte maturation is an essential physiological event for species survival; thus, leptin influence in oocyte quality is of keen clinical interest and potentially important for IVF protocols. In the present work, we have demonstrated that leptin enhances in vitro meiotic oocyte maturation. However, controversial results have been reported in the literature. In some species, leptin seems to promote rearrangement of cytoskeletal elements (Suzuki et al. 2010) that are involved in chromosome segregation and in processes of organelle movement (Sun & Schatten 2006). Other studies have reported that leptin enhances an appropriate spindle assembly during metaphase (Jin et al. 2009) and stimulates meiotic oocyte maturation in mouse (Matsuoka et al. 1999), pig (Craig et al. 2004, Kun et al. 2007, Zhang et al. 2007) and cattle (Paula-Lopes et al. 2007, Van Tol et al. 2008). Nevertheless, Ryan et al. (2002) showed that leptin only induced meiotic resumption when the oocyte was cultured within its normal follicular environment. Other authors have even reported that leptin had no effect on meiotic resumption and development to MII (mouse: Swain et al. 2004; pig: Suzuki et al. 2010).

Regarding cytoplasmic maturation, studies by Runner & Gates (1954) observed that oocytes from OB/OB leptin-deficient mice were able to undergo normal fertilisation and embryo development in vivo, although these animals were obese and showed reproductive failures (Coleman 1978, Zhang et al. 1994). This led to postulate that the sterility defect due to leptin was not in the oocytes (Swain et al. 2004). According to these findings, in the present work, leptin supplementation did not improve oocyte quality in terms of CG migration rate at the concentrations tested compared to the FCS group, which achieved the best results of cytoplasmic maturation, similar to those previously reported in rabbits by our group (Garcia-Garcia et al. 2009). This finding is consistent with that obtained by Paula-Lopes et al. (2007). These authors found a similar proportion of polispermic oocytes when bovine COC were supplemented with leptin or not in IVM media. However, other studies have suggested that leptin only may exert its effect when it is added in vitro at very high concentrations (500–1000 ng/ml), inhibiting microfilament reorganisation in pigs (Suzuki et al. 2010) or improving cytoplasmic maturation in cattle (Van Tol et al. 2008). In turn, these results are not in agreement with the findings obtained by other authors, in which much lower leptin concentrations (1–100 ng/ml) resulted in an increase of developmental competence related to an improvement of oocyte cytoplasmic maturation (bovine: Boelhauve et al. 2005; pig: Craig et al. 2004, Jin et al. 2009). These apparently controversial IVM results reported in the literature are probably dependent on the culture medium used or the species studied. The experiments described in the present work and in Van Tol et al. (2008) were performed in a serum-free medium in absence of any growth factor or hormone other than leptin. However, the other studies usually added gonadotropins, growth factors or follicular fluid to culture media; these additives could have enhanced the sensitivity of COC to leptin or its pathways, showing different responses at lower concentrations. Thus, possible interactions between them and oocyte maturation remain unclear. Further standardised studies are warranted, since leptin may affect oocyte quality, mediating the action of other drugs used in the stimulation protocols of fertility treatments.

The development potential of oocytes is also influenced by the synthesis and release of cumulus- and oocyte-derived factors as steroids (Andersen 1993). Measurements of steroids at the end of the maturation period are commonly used to assess the steroidogenic capacity of COC (Lorenzo et al. 1997, Mingoti et al. 2002, Shirazi & Moalemian 2007). In this sense, the present work shows that leptin, in a serum-free culture medium, did not exert a short-term effect on the final OE2 and P4 concentrations of COC in a dose-dependent manner. Therefore, under our experimental conditions, in rabbit oocytes, leptin stimulation of meiotic maturation is not mediated by an increase of steroid secretion by COC in vitro. Our results agree with previous reports showing that OE2 and P4 do not seem to play a decisive role in oocyte meiotic maturation in vitro (Dode & Graves 2002, Lucidi et al. 2003, Wang et al. 2006). However, contradictory findings exist about stimulatory or inhibitory effects of leptin on steroidogenesis (Zachow & Magoffin 1997, Tsai et al. 2002, Catalano et al. 2003, Ruiz-Cortés et al. 2003, Swain et al. 2004). OE2 secreted by cumulus cells could induce a rapid increase in free intracellular calcium concentration, a mechanism that improves cytoplasmic maturation (bovine: Younis et al. 1989; human: Tesarik & Mendoza 1997). Also, in bovine oocytes, OE2 probably participates in the formation of the polispermic block via CGs (Karlach 1987). This finding is consistent with the increase of the CG migration rate in the FCS group showed in this study, but not in the rest of the leptin groups.

Leptin acts as modulator of oocyte maturation through activation of specific transcription factors. The current work provides the first evidence that leptin uses both JAK2/STAT3 and MEK 1/2 pathways to improve meiotic IVM of rabbit oocytes in agreement with previous results in other species (mouse: Matsuoka et al. 1999; pig: Craig et al. 2004). Matsuoka et al. (1999) first suggested that the function of leptin during nuclear oocyte maturation was due to the activation of the STAT3 signal transduction pathway. In heifers, cumulus cells seem to be more influential in leptin activity than the oocyte itself, mediating the leptin-STAT3 signals to improve oocyte meiotic maturation by the long isoform LEPR (Paula-Lopes et al. 2007). Of the known LEPR isoforms, only the full length one contains the intracellular domains necessary to mediate JAK2/STAT3 and MAPK signal transduction pathways. In contrast with the findings reported by Matsuoka et al. (1999) and Paula-Lopes et al. (2007), other authors showed that leptin did not increase phosphorylated STAT3, but more phosphorylated MEK1 and MEK2 proteins were predominant in COC during IVM (Van Tol et al. 2008). In this sense, Craig et al. (2004) also showed that leptin activates MAPK pathway in the pig oocyte. MAPK enhance maturation promoting factor activity and are involved in post-meiotic resumption events, such as spindle formation, MI to MII transition and MII arrest in several species, including rabbits (Yu et al. 2002), as it was also suggested in the current work.

As we expected, this study also showed the non-suppression of leptin-induced oocyte CG migration by JAK2/STAT3 and MEK 1/2 inhibitors. However, the increase of abnormal distribution when media were supplemented with leptin and the higher JAK2/STAT3 and MEK 1/2 inhibitor concentrations (100 and 50 μM respectively) imply that the oocyte may be a more sensitive cell to higher concentrations of both inhibitors widely used in other cell studies (thymus cells: Mansour et al. 2006; endometrial cancer cells: Sharma et al. 2006; hepatocarcinoma cells: Chen et al. 2007; breast cancer cells: Saxena et al. 2007) or a non-specific effect. In agreement with our cytoplasmic maturation results, it has been reported that although leptin signalling through the STAT3 pathway is beneficial for meiotic maturation (Matsuoka et al. 1999), it does not seem essential for reproduction, at least at the oocyte level (Takeda et al. 1997). Bates et al. (2003) even showed that mice with disrupted LEPRb-STAT3 signalling were obese yet fertile. However, MAPK proteins seem to be necessary for pronuclear formation in parthenogenetically activated oocytes (Posada & Cooper 1992, Liu et al. 1998, Ito et al. 2004) for microfilament formation and thus for organelle distribution and polarity establishment in the oocyte (Verlhac et al. 1993, Sun & Schatten 2006). This discrepancy with our results points to a possible species-specific mode of action of the leptin system on oocyte maturation, or that the MAPK involved in some aspects of cytoplasmic maturation were other than those inhibited in this study.

On the other hand, steroid concentrations were not altered in the 0 or 10 Lep groups with or without inhibitors. This allows us to determine that, in rabbit oocytes, stimulation of meiotic maturation by leptin was not mediated by an increase of steroid secretion induced by activation of JAK2/STAT3 and MEK 1/2 proteins. The higher secretion of steroids found only in some groups treated with leptin and MEK 1/2 inhibitors was unclear. Maybe some compensatory mechanisms could increase steroid secretion when leptin-induced MEK 1/2 pathway was inhibited. However, the reason for this and the potential cross-talk between JAK2/STAT3 and MAPK pathways and steroid secretion in COC units need further research.

In conclusion, the current work provides the first evidence that leptin enhances meiotic oocyte maturation through activation of both JAK2/STAT3 and MEK 1/2 pathways in the rabbit oocyte model. This beneficial effect is not observed on cytoplasmic maturation in terms of CG migration nor it is mediated by an increase of OE2 and P4 secretion by COC. These findings contribute to enhance the knowledge about the relationship between obesity/lean-associated pathologies, leptin and oocyte quality to continue optimising IVF techniques in humans and animals.

Materials and Methods

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Company. Ovaries were obtained from slaughtered adult animals from the abattoir (Jumogar, SL, Tarancón, Spain). Additionally, twelve 18-week-old New Zealand×California white rabbit does (Oryctolagus cuniculus) were used for immunolocalisation of LEPR in different time points during the peri-ovulatory period. Experimental procedures were approved by the Animal Ethics Committee of the Polytechnic University of Madrid (Spain) and were in compliance with the Spanish guidelines for care and use of animals in research (BOE 2005). Animals were housed in individual flat-deck cages under a constant photoperiod of 16 h of light per day, a temperature of 18–22 °C and a relative humidity of 60–75% maintained by a forced ventilation system in the Polytechnic University of Madrid (Spain). Ovulation was induced by i.m. injection of 1 μg buserelin (Suprafact, Hoechst Marion Roussel, SA, Madrid, Spain). Does were killed with 30 mg/kg of i.v. pentobarbital sodium (Dolethal, Vetoquinol, Alcobendas, Spain) and subjected to laparotomy in order to collect their ovaries at different time points during the peri-ovulatory period: 1) before ovulation induction (0 h, n=4); 2) 8 h after ovulation induction (n=4); 3) 16 h after ovulation induction (n=4).

Immunohistochemistry of leptin receptor (LEPR)

After slide deparaffinisation and rehydration in graded alcohol, ovarian sections of each experimental group described above were heat-treated in sodium citrate solution (pH 6) to unmask antigen epitopes. Endogenous peroxidase activity was blocked by incubating the slides with 3% v/v hydrogen peroxide in methanol solution for 30 min. Non-specific binding of immunoglobulins was blocked with normal goat serum (1:10, sc-2043, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS at room temperature (RT) for 30 min. Primary antibody against LEPR (1:10, sc-8391, Santa Cruz Biotechnology) was incubated overnight at room temperature in a humidified chamber. The antibody used in this study specifically recognises long and short forms of human LEPR. In the negative control sections, incubation with primary antibody was replaced by PBS solution. After that, samples were incubated with biotinylated secondary antibody (1:200, biotinylated anti-mouse IgG (H+L) made in goat, Vector Laboratories, Ltd, Peterborough, UK) for 30 min, and subsequently with the avidin–biotin complex (ABC Vector Elite kit, Vector Laboratories, Burlingame, CA, USA) at RT for 30 min. After chromogen incubation (Vector Nova RED substrate Kit for Peroxidase, Vector Laboratories), the sections were counterstained with haematoxylin, photographed and assessed under a light microscope (Olympus BX40, Hamburg, Germany).

Oocyte collection and IVM of COC

Ovaries of slaughtered adult animals were placed in PBS at 37 °C and transported to the laboratory. COC were obtained by aspiration with 2 ml syringe and 25 G needle from ovarian follicles ≥1 mm in size under a stereoscopic microscope. COC with compact cumulus were washed and placed in 500 μl maturation medium in four-well dishes (Nunclon Surface, Nunc, Roskilde, Denmark). They were cultured for 16 h at 38 °C under an atmosphere of 5% CO2 in air with maximum humidity.

Role of leptin on meiotic and cytoplasmic in vitro oocyte maturation

Six replicates were made (n=750 oocytes) to assess the influence of leptin on the oocyte maturation process (Fig. 2A). The maturation medium consisted of tissue culture medium (TCM-199) with 2 mM l-glutamine, 0.1 mg/ml sodium pyruvate, 0.3% w/v BSA supplemented with 0, 1, 10, 100 ng/ml leptin or 10% v/v FCS (0 Lep, 1 Lep, 10 Lep, 100 Lep and FCS groups respectively).

Figure 2

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Figure 2

Experimental design scheme of oocyte in vitro maturation studies. (A) In vitro maturation with different concentrations of leptin (ng/ml). (B) In vitro maturation with 0 and 10 ng/ml leptin plus JAK2/STAT3 (AG490) and MEK 1/2 (PD98059)-pathways inhibitors.

Citation: REPRODUCTION 139, 3; 10.1530/REP-09-0309

Assessment of JAK2/STAT3 and MEK 1/2 leptin-mediated pathways during IVM

In order to assess if leptin acts on oocyte maturation activating STAT3 and MAPK pathways, we have performed a dose–response study in five replicates (n=578 oocytes). For maturation, COC were randomly divided into two groups: control with 0 ng/ml leptin (0 Lep group) and leptin group with 10 ng/ml leptin (10 Lep group). As shown in Fig. 2B, during the whole maturation period, each group (0 Lep or 10 Lep) was further treated with different concentrations of inhibitors for JAK2/STAT3 (Tyrphostin AG490) or MEK 1/2 (PD98059, MEK1 inhibitor, Cell Signalling Technology, Inc., Beverly, MA, USA) pathways. Tyrphostin AG490 is a JAK2 protein tyrosine kinase inhibitor that inhibits leptin action, ranging from 10 to 100 μM in different cell types (Mansour et al. 2006, Sharma et al. 2006, Chen et al. 2007, Saxena et al. 2007). PD98059 [2-(2-amino-3-methoxyphenyl)-4h-1-benzopyran-4-one] has shown to act as a highly selective inhibitor of MEK1 activation and the MAPK cascade (Crews et al. 1992). PD98059 binds to MEK1 and prevents activation by upstream activators such as c-Raf (Rosen et al. 1994). Concentrations for inhibitory activity against MEK1 and MEK2 are 10 and 50 μM respectively.

Study of nuclear and cytoplasmic oocyte maturation by confocal microscopy

After the maturation period, COC were treated for the confocal study. First, cumulus cells were removed in PBS with 2 mM hyaluronidase by gentle pipetting. Next, oocytes were treated with 0.5% w/v pronase in PBS to digest the zonae pellucidae, fixed in PBS containing 4% w/v buffered neutral paraformaldehyde solution (pH 7.2–7.4) and stored in PBS. Oocytes were washed with permeabilisation solution (0.02% v/v Triton X-100 in PBS) and treated for 40 min with blocking solution (7.5% w/v BSA in PBS). They were then incubated for 30 min at room temperature with 100 μg/ml FITC of Lens culinaris for CG staining and for 15 min at 39 °C with 10 μg/ml propidium iodide for nuclear staining. After that, oocytes were mounted between a coverslip and a glass slide supported by columns of paraffin and examined under a confocal laser scanning microscope (Leica, TCS SP5, Wetzlar, Germany). Complete nuclear maturation was measured in terms of MII rate. The rest of nuclear configurations were classified as oocytes that reinitiated the meiosis process (MI index) or not (GV rate). According to a previous work (Arias-Alvarez et al. 2009), CG distribution was classified as follows (Fig. 3): A) peripheral: CG were distributed adjacent to the plasma membrane, since they were cytoplasmically matured; B) cortical: most of the CG were localised in the cortical area of oocytes, thus being considered as partially matured; C) homogeneous: CG were scattered throughout the cytoplasm, since they did not show cytoplasmic maturation; D) non homogeneous or abnormal: anomalous distribution of CG compatible with poor quality or degenerated oocytes.

Figure 3

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Figure 3

Cortical granule distribution in rabbit oocytes after in vitro maturation period under confocal microscopy. (A) Peripheral CG distribution. (B) Cortical distribution. (C) Homogeneous distribution. (D) Abnormal distribution. Oocyte diameter is around 80 μm.

Citation: REPRODUCTION 139, 3; 10.1530/REP-09-0309

Determination of steroids in spent maturation media

Spent maturation media were collected after completion of oocyte maturation period and stored at −32 °C until analysed. Steroid concentrations were measured by ELISA based on the principle of competitive binding using a specific kit (Demeditec Diagnostics GmbH, Kiel, Germany) for OE2 (OE2-sensitive ELISA) and for P4 (P4 ELISA) quantification. Purified anti-OE2 and anti-P4 polyclonal antibodies were used. Intra- and inter-assay coefficients of variation were 5.5 and 6.8% for OE2, and 5.4 and 9.9% for P4, respectively. The assay ranges were 0–200 pg/ml (OE2) and 0–40 ng/ml (P4). Results are expressed as average OE2 (pg/ml) and P4 (ng/ml) concentrations produced by each COC after the IVM period.

Statistical analysis

Data were analysed using the SPSS program for Windows (SPSS 15.0, Inc., Chicago, IL, USA). χ2-test was carried out to compare different categories of nuclear maturation and CG migration rate between experimental groups. Mean OE2 and P4 concentrations in spent media by each COC were analysed with the ANOVA test (post-hoc was Duncan test) for the different IVM groups. All the results are expressed as the mean±s.e.m., and statistical significance was accepted for P<0.05.

Declaration of interest

The authors declare that there is no potential conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by MEC projects AGL08-022283 and UCM-CM research program (920249-2008) and CM/FSE.

Acknowledgements

The authors wish to thank Drs J Contreras and L Revuelta for their support in the experimental part of this work.

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Figures

  • View in gallery

    Immunolocalisation of leptin receptor (LEPR) in ovarian sections of rabbit does during the peri-ovulatory period. (A) Negative control. (B) Positive staining of LEPR in oocytes and granulosa cells of primordial, primary and secondary follicles. (C) Antral follicle with staining of LEPR in granulosa cells. (D) Immunodetection detail of LEPR in granulosa and theca cells. Theca externa cells show negative immunostaining (o, oocyte; g, granulosa cells; t, theca cells).

  • View in gallery

    Experimental design scheme of oocyte in vitro maturation studies. (A) In vitro maturation with different concentrations of leptin (ng/ml). (B) In vitro maturation with 0 and 10 ng/ml leptin plus JAK2/STAT3 (AG490) and MEK 1/2 (PD98059)-pathways inhibitors.

  • View in gallery

    Cortical granule distribution in rabbit oocytes after in vitro maturation period under confocal microscopy. (A) Peripheral CG distribution. (B) Cortical distribution. (C) Homogeneous distribution. (D) Abnormal distribution. Oocyte diameter is around 80 μm.

References

AndersenCY1993Characteristics of human follicular fluid associated with successful conception after in vitro fertilization. Journal of Clinical Endocrinology and Metabolism7712271234.

AntczakMVan BlerkomJ1997Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantaion stage embryo. Molecular Human Reproduction310671086.

ArchancoMMuruzábalFJLopizDGarayoaMGomez-AmbrosiJFrühbeckGBurellMA2003Leptin expression in the rat ovary depends on estrus cycle. Journal of Histochemistry and Cytochemistry5112691277.

Arias-AlvarezMGarcía-GarcíaRMRebollarPGRevueltaLMillánPLorenzoPL2009Influence of endocrine and metabolic status on oocyte quality and follicular characteristics at different postpartum periods in primiparous rabbit does. Theriogenology72612623.

BatesSHStearnsWHDundonTASchubertMTsoAWWangYBanksASLaveryHJHaqAKMaratos-FlierE2003STAT3 signalling is required for leptin regulation of energy balance but not reproduction. Nature421856859.

BellverJ2006Symposium: diet, nutrition and exercise in reproduction. Obesity and assisted reproductive technology outcomes. Reproductive Biomedicine Online12562568.

BjørbaekCUotaniSda SilvaBFlierJS1997Divergent signaling capacities of the long and short isoforms of the leptin receptor. Journal of Biological Chemistry2723268632695.

BoelhauveMSinowatzFWolfEPaula-LopesFF2005Maturation of bovine oocytes in the presence of leptin improves development and reduces apoptosis of in vitro-produced blastocysts. Biology of Reproduction73737744.

Boletin Oficial del Estado (BOE)Real Decreto 1201/2005. Sobre protección de los animales utilizados para experimentación y otros fines científicoscBoletin Oficial del Estado25220053436734391.

BrecchiaGBonannoAGaleatiGFedericiGMaranesiMGobbettiAZeraniMBoitiC2006Hormonal and metabolic adaptation to fasting: effects on the hypothalamic–pituitary–ovarian axis and reproductive performance of rabbit does. Domestic Animal Endocrinology31105122.

CatalanoSMarsicoMGiordanoCMauroLRizzaPPannoMPAndòS2003Leptin enhances, via AP-1, expression of aromatase in the MCF-7 cell line. Journal of Biological Chemistry2782866828676.

CerveroADomínguezFHorcajadasJAQuñoneroAPellicerASimónC2006The role of leptin in reproduction. Current Opinion in Obstetrics and Gynecology18297303.

ChehabFFLimMELuR1996Correction of the sterility defect in homozygous obese female mice by treatment with the humen recombinant leptin. Nature Genetics12318320.

ChenCChangYCLiuCLLiuTPChangKJGuoIC2007Leptin induces proliferation and antiapoptosis in human hepatocarcinoma cell by up-regulating cyclin D1 and down-regulating Bax via a janus kinase 2-linked pathway. Endocrine-Related Cancer14531529.

CheungCCThorntonJEKuijperJLWeigleDSCliftonDKSteinerRA1997Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology138855858.

CioffiJAVan BlerkomJAntczakMShaferAWittmerSSnodgrassHR1997The expression of leptin and its receptors in pre-ovulatory human follicles. Molecular Human Reproduction3467472.

ColemanDL1978Obese and diabetes: two mutant genes causing diabetes–obesity syndromes in mice. Diabetologia14141148.

CraigJZhuHDycePWPetrikJLiJ2004Leptin enhances oocyte nuclear and cytoplasmic maturation via the mitogen-activated protein kinase pathway. Endocrinology14553555363.

CrewsCMAlessandriniAEriksonRL1992The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science258478480.

Denison F 2009 Impact of obesity on maternal reproductive fitness. Proceedings of the Society for Reproduction and Fertility

DodeMANGravesC2002Involvement of steroid hormones on in vitro maturation of pig oocytes. Theriogenology57811821.

DuggalPSWeitsmanSRMagoffinDANormanRJ2002Expression of the long (OB-RB) and short (OB-RA) forms of the leptin receptor throughout the oestrous cycle in the mature rat ovary. Reproduction123899905.

FrühbeckG2006Intracellular signalling pathways activated by leptin. Biochemical Journal393720.

Garcia-GarciaRMArias-AlvarezMRebollarPGRevueltaLLorenzoPL2009Influence of different reproductive rhythms on serum estradiol, testosterone levels, features of follicular population and atresia rate, and oocyte maturation in controlled suckling rabbits. Animal Reproduction Science114423433.

ItoJShimadaMTeradaT2004Mitogen-activated protein kinase kinase inhibitor suppresses cyclin B1 synthesis and reactivation of p34cdc2 kinase, which improves pronuclear formation rate in matured porcine oocytes activated by Ca2+ ionophore. Biology of Reproduction70797804.

JinYXCuiXSHanYJKimNH2009Leptin accelerates pronuclear formation following intracytoplasmic sperm injection of porcine oocytes: possible role for MAP kinase inactivation. Animal Reproduction Science115113148.

KajiEBornslaegerEASchultzRM1987Inhibition of mouse oocyte cyclic AMP phosphodiesterase by steroid hormones: a possible mechanism for steroid hormone inhibition of oocyte maturation. Journal of Experimental Zoology243489493.

KaramoutiMKolliaPKarligiotouEKallitsarisAPrapasNKolliosGSeferiadisKVamvakopoulosNMessinisIE2003Absence of leptin expression and secretion by human luteinized granulosa cells. Journal of Molecular Endocrinology31233239.

KarlachV1987The effect of FSH, LH, oestradiol-17 beta, and progesterone on cytoplasmic maturation of bovine follicular oocytes in vitro. Folia Biologica33258265.

KarlssonCLindellKSvenssonEBerghCLindPBilligHCarlssonLMSCarlssonB1997Expression of functional leptin receptors in the human ovary. Journal of Clinical Endocrinology and Metabolism8241444148.

KeimNLSternJSHavelPJ1998Relation between circulating leptin concentrations and appetite during a prolonged, moderate energy deficit in women. American Journal of Clinical Nutrition68794801.

KunZShaohuaWYufangMYankunLHengxiWXiuzhuSYonghuiZYanLYunpingDLeiZ2007Effects of leptin supplementation in in vitro maturation medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs. Animal Reproduction Science1018596.

LeeGProencaRMontezJMCarrollKMDarvishzadehJGLeeJIFriedmanJM1996Abnormal splicing of the leptin receptor in diabetic mice. Nature379632635.

LinJBarbCRMatteriRLKraelingRRChenXMeinersmannRJRampacekGB2000Long form leptin receptor mRNA expression in the brain, pituitary, and other tissues in the pig. Domestic Animal Endocrinology15361.

LiuLJuJCYangX1998Differential inactivation of maturation-promoting factor and mitogen-activated protein kinase following parthenogenetic activation of bovine oocytes. Biology of Reproduction59537545.

LorenzoPLIlleraJCSilvanGMunroCJIlleraMJIlleraM1997Steroid-level response to insulin-like growth factor-1 in oocytes matured in vitro. Journal of Reproductive Immunology351129.

LucidiPBernabòNTurrianiMBarboniBMattioliM2003Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation. Reproductive Biology and Endocrinology119.

MansourLPereiraFGAraujoEPAmaralMECMorariJFerraroniNRFerreiraDSLorand-MetzeIVellosoL2006Leptin inhibits apoptosis in thymus through a janus kinase-2-independent, insulin receptor substrate-1/phosphatidylinositol-3 kinase-dependent pathway. Endocrinology14754705479.

MatsuokaTTaharaMYokoiTMasumotoNTakedaTYamaguchiMTasakaKKurachiHMurataY1999Tyrosine phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte. Biochemical and Biophysical Research Communications256480484.

MingotiGZGarciaJMRosa-e-SilvaAAM1995The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulose cells. Brazilian Journal of Medical and Biological Research28213217.

MingotiGZGarciaJMRosa-e-SilvaAAM2002Steroidogenesis in cumulus cells of bovine cumulus–oocyte-complexes matured in vitro with BSA and different concentrations of steroids. Animal Reproduction Science69175186.

Muñoz-GutierrezMFindlayPAAdamCLWasGCampbellBKKendallNRKhalidMForsbergMScaramuzziRJ2005The ovarian expresión of mRNAs for aromatase, IGF-I receptor, IGF binding protein-2, -4 and -5, leptin and leptin receptor in cycling ewes after three days of leptin infusion. Reproduction130869881.

Paula-LopesFFBoelhauveMHabermannFASinowatzFWolfE2007Leptin promotes meiotic progression and developmental capacity of bovine oocytes via cumulus cell-independent and -dependent mechanisms. Biology of Reproduction8532541.

PosadaJCooperJA1992Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science255212215.

RosenLBGintyDDWeberMJGreenbergME1994Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras. Neuron1212071221.

Ruiz-CortésZTMartel-KennesYGévryNYDowneyBRPalinMFMurphyBD2003Biphasic effects of leptin in porcine granulosa cells. Biology of Reproduction68789796.

RunnerMNGatesA1954Conception in prepuberal mice following artificially induced ovulation and mating. Nature174222223.

RyanNKWoodhouseCMVan der HoekKHGilchristRBArmstrongDTNormanRJ2002Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation. Biology of Reproduction6615481554.

SaxenaNKVertinoPMAnaniaFASharmaD2007Leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltranferases and mediator complex to cyclin D1 promoter via activation of stat3. Journal of Biological Chemistry2821331613325.

SharmaDSaxenaNKVertinoPMAnaniaFA2006Leptin promotes the proliferative response and invasiveness in human endometrial cancer cells by activating multiple signal-transduction pathways. Endocrine-Related Cancer13629640.

ShiraziAMoalemianZ2007Ovine cumulus cells estradiol-17β production in the presence or absence of oocyte. Animal Reproduction Science101125133.

SunQYSchattenH2006Regulation of dynamic events by microfilaments during oocyte maturation and fertilization. Reproduction131193205.

SuzukiHSasakiYShimizuMMatsuzakiMHashizumeTKuwayamaH2010Ghrelin and leptin did not improve meiotic maturation of porcine oocytes cultured in vitro. Reproduction in Domestic Animals[in press]. DOI: 10.1111/j.1439-0531.2009.01352.x.

SwainJEDunnRLMcConnellDGonzalez-MartinezJSmithGD2004Direct effects of leptin on mouse reproductive function: regulation of follicular, oocyte, and embryo development. Biology of Reproduction7114461452.

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