The role of IGFs in the regulation of ovarian follicular growth in the brushtail possum (Trichosurus vulpecula)

in Reproduction
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  • 1 Wallaceville Animal Research Centre, AgResearch Ltd, Invermay Agricultural Centre, School of Biological Sciences, Victoria University of Wellington, AgResearch, Upper Hutt 5140, New Zealand

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

Abstract

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

Introduction

The common brushtail possum, a marsupial, has an oestrous cycle of around 26 days with a 16–18-day luteal phase and a 9–11-day follicular phase (Fletcher & Selwood 2000). Extensive follicular growth is observed in both juvenile and adult animals with a single follicle of around 5 mm in diameter ovulating each reproductive cycle in adults during the breeding season (Eckery et al. 2002a, 2002b). In eutherians, ovarian follicular growth is known to be controlled by both extraovarian and intraovarian factors. The pituitary hormones, FSH and LH, are key extraovarian regulators of follicular growth and selection of the ovulatory follicle in monovular eutherian mammals (Webb & Campbell 2007, Mihm & Evans 2008). Downregulation of GNRH with a long acting agonist causes cessation of reproductive cycles in possums suggesting that FSH and/or LH are regulators of follicular development and ovulation in this species (Eymann et al. 2007). However, differences in the expression pattern of LHCGR mRNA during ovarian follicular development indicate that the role of LH in follicular growth differs in marsupials compared with eutherian mammals (Eckery et al. 2002b). Granulosa cells of all healthy antral follicles of the brushtail possum express the LHCGR mRNA, and are responsive to LH. Therefore, LH cannot be playing a central role in selecting and maintaining a single ovulatory follicle in the possum as postulated in eutherian mammals.

Insulin-like growth factor (IGF) is an intraovarian growth factor that is also involved in the regulation of follicular development in eutherian mammals. IGF is known to stimulate granulosa cell proliferation and progesterone production, although these effects can vary across species and stage of follicular development (Mazerbourg et al. 2003, Silva et al. 2009). This effect is driven by binding of either IGF1 or IGF2 to the type 1 IGFR (Mazerbourg et al. 2003, Silva et al. 2009). The regulation of IGF actions is complex, and involves a number of binding proteins (BPs) including IGFBP2, IGFBP4 and IGFBP5 as well as the IGFBP protease pregnancy-associated plasma protein-A (PAPPA), which have been identified as potential key regulators during ovarian follicular development in eutherian mammals (Spicer 2004, Silva et al. 2009). The actions of IGF on regulation of follicular development in marsupials are unknown. Thus, the objectives of these experiments were to characterize the expression pattern of IGF1, IGF2, IGF1R, IGFBP2, IGFBP4, IGFBP5 and PAPPA mRNAs using ovarian sections containing follicles in multiple stages of development, and determine the effects of IGF1 and IGF2 on granulosa cell proliferation and progesterone production in the brushtail possum.

Results

Expression of IGF1, IGF2 and IGF1R mRNA during follicular development

During follicular development, expression of IGF1 mRNA (Fig. 1 and Table 1) was limited to the theca of antral follicles. The majority of atretic antral follicles no longer expressed IGF1 mRNA. Expression was also observed in the area around blood vessels in some animals. In contrast to the relatively sparse expression of IGF1, IGF2 mRNA (Fig. 2 and Table 1) was strongly expressed in granulosa cells of primary, secondary and antral follicles (healthy and atretic). Cells around the blood vessels and some surface epithelial cells also expressed IGF2 mRNA (data not shown). The IGF1R mRNA (Fig. 3 and Table 1) was expressed in follicles of all developmental stages examined. Oocytes of primordial, primary, secondary and antral follicles expressed IGF1R strongly. Secondary and antral (healthy and atretic) follicles also expressed IGF1R mRNA in granulosa, but not theca cells. While the interstitial glands did not express IGF1R mRNA, expression was observed in some stromal cells.

Figure 1
Figure 1

Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from adult brushtail possums following hybridization to IGF1 antisense (A–D) and sense (E and F) RNA. Expression of IGF1 mRNA in the theca (t), but not oocyte (o) or granulosa cells (g) of antral follicles (A and B). Expression of IGF1 mRNA in the cells of the vasculature of the ovary (C and D). Expression was not detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGF1 sense RNA (E and F).

Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0142

Table 1

Expression pattern of mRNAs encoding insulin-like growth factor 1 (IGF1) and IGF2, their receptor (R) (IGF1R) as well as the binding proteins (BPs) IGFBP2, IGFBP4, and IGFBP5 and the protease, pregnancy-associated plasma protein A (PAPPA) during follicular development in the brushtail possum.

Follicle typeIGF1IGF2IGF1RIGFBP2IGFBP4IGFBP5PAPPA
Primordialog
Primarygoggg
Secondarygo, gg, ttg, tg
Antraltgo, gg, tg, ttg
Atretic antralaggg, tg, ttg

–, expression not observed; o, expression observed in oocyte; g, expression observed in granulosa cells; t, expression observed in theca.

Oocytes not observed in atretic follicles.

Figure 2
Figure 2

Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from juvenile and adult brushtail possums following hybridization to IGF2 antisense (A–D) and sense (E and F) RNA. Expression of IGF2 mRNA in granulosa cells (g), but not oocyte (o) or theca (t) of secondary (arrowhead) and antral (arrow) follicles (A–D). Expression was not detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGF2 sense RNA (E and F).

Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0142

Figure 3
Figure 3

Corresponding brightfield (A, C, E and G) and darkfield (B, D, F and H) views of ovaries from juvenile and adult brushtail possums following hybridization to IGF1R antisense (A–F) and sense (G and H) RNA. Oocytes (o) of primordial (#; 2× higher magnification in inset of panel E and F), primary (*), secondary (arrowhead) and antral (arrow) follicles express IGF1R (A–F). Expression of IGF1R mRNA was also observed in granulosa cells (g) but not theca (t) of secondary (arrowhead) and antral (arrow) follicles (A–F). Expression was not detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGF1R sense RNA (G and H).

Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0142

Expression of IGFBP2, IGFBP4, IGFBP5 and PAPPA mRNAs during follicular development

The IGFBP2 mRNA (Fig. 4 and Table 1) was observed in primary, secondary and antral follicles. Both theca and granulosa cells expressed IGFBP2 mRNA but oocytes did not. Atretic antral follicles also expressed IGFBP2 mRNA in both granulosa and theca cells. Interstitial tissue as well as cells of the vasculature expressed IGFBP2 mRNA. IGFPB4 mRNA (Fig. 5 and Table 1) was primarily expressed in the theca of large preantral and antral follicles and in interstitial tissue. Faint hybridization was also observed in the granulosa cells of some antral follicles. The pattern of expression was similar in atretic antral follicles with the expression observed consistently in the theca cells but only in the granulosa cells of some antral follicles. IGFBP5 mRNA (Fig. 6 and Table 1) was observed in granulosa cells of primordial, primary and some secondary follicles. However, granulosa cells of antral follicles no longer expressed IGFBP5 mRNA. Strong expression was also observed in the theca of secondary follicles (when present) and antral follicles (both healthy and atretic). While interstitial glands did not express IGFBP5 mRNA, expression was observed in some stromal cells close to the follicles, as well as blood vessels and some surface epithelial cells. The mRNA encoding PAPPA (Fig. 7 and Table 1) was observed in the granulosa cells of all growing follicles (i.e. starting at the primary stage of development). Atretic antral follicles still expressed PAPPA mRNA, although the signal appeared faint. Neither the interstitial cells nor the surface epithelium expressed PAPPA mRNA.

Figure 4
Figure 4

Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from juvenile and adult brushtail possums following hybridization to IGFBP2 antisense (A–D) and sense (E and F) RNA. Expression of IGFBP2 mRNA in granulosa cells (g) and theca (t), but not oocyte (o) of secondary (arrowhead) and antral (arrow) follicles (A–D). Expression was also detectible in the interstitial tissue (i; C and D). Note the lack of specific signal in an ovary hybridized to the IGFBP2 sense RNA (E and F).

Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0142

Figure 5
Figure 5

Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from juvenile and adult brushtail possums following hybridization to IGFBP4 antisense (A–D) and sense (E and F) RNA. Expression of IGFBP4 was first observed in theca of secondary follicles (A and B; arrowhead). Expression of IGFBP4 mRNA was strongest in the theca (t), with weak signal also seen in the in granulosa cells (g) of some but not all antral follicles (A–D, arrow). Expression was not observed in the oocyte (o) of follicles (A and B). Expression was also detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGFBP4 sense RNA (E and F).

Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0142

Figure 6
Figure 6

Corresponding brightfield (A, C, E and G) and darkfield (B, D, F and H) views of ovaries from juvenile brushtail possums following hybridization to IGFBP5 antisense (A–F) and sense (G and H) RNA. Granulosa cells (g) of primordial (#), primary (*), secondary (arrowhead), but not antral (arrow) follicles express IGFBP5 (A–F). Theca cells also express IGFBP5 mRNA (t; C–F) but interstitial tissue did not (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGFBP5 sense RNA (G and H).

Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0142

Figure 7
Figure 7

Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from juvenile and adult brushtail possums following hybridization to PAPPA antisense (A–D) and sense (E and F) RNA. Expression of PAPPA was not observed in primordial follicles (A and B; #). The granulosa cells, but not theca (t) or oocytes (o), of growing follicles expressed PAPPA mRNA (A–D, arrowhead secondary follicles, arrows antral follicles). Expression was not detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the PAPPA sense RNA (E and F).

Citation: REPRODUCTION 140, 2; 10.1530/REP-10-0142

Effects of IGF1 and IGF2 on granulosa cell function

Treatment of possum granulosa cells with either IGF1 or IGF2 increased cell proliferation. Incorporation of 3H-thymidine was increased (P<0.01) 1.92±0.27- and 1.81±0.33-fold (mean±s.e.m.) above control cells after treatment with IGF1 and IGF2 respectively.

Neither IGF1 (1.26±0.43-fold of controls) nor IGF2 (2.54±1.34-fold of controls; mean±s.e.m.) affected (P>0.05) the concentrations of progesterone in media. The granulosa cells were capable of synthesizing progesterone, although basal concentrations were low, and responding to positive stimuli as treatment with LH increased progesterone concentrations by more than tenfold. Both basal concentrations of progesterone secreted into the media and those following treatment were highly variable between different pools of granulosa cells.

Discussion

Both IGF1 and IGF2 mRNAs, as well as the mRNAs encoding IGF1R and BPs for IGFs, were expressed in ovarian follicles of the brushtail possum. Furthermore, both IGF1 and IGF2 were able to regulate granulosa cell proliferation, causing a near doubling of 3H-thymidine incorporation. Thus, it would appear that IGFs have a local role in regulating follicular development in marsupials as has been observed in eutherian mammals.

There were distinct patterns of expression for IGF1 and IGF2 mRNAs. The potential role that differential expression of IGF1 and IGF2 may play in development of ovarian follicles is unclear. From previous reports, IGF1 and IGF2 seem to have similar effects on granulosa cell function (Silva et al. 2009), and thus, the actions appear to overlap. However, in other tissues, a group of genes regulated specifically by IGF1 or IGF2 as well as those regulated in common by either IGF1 or IGF2 have been identified (Pacher et al. 2007). Thus, IGF1 and IGF2 may have differing roles in regulating ovarian function that have not yet been identified. Given the lack of expression of IGF1 mRNA in growing preantral follicles, IGF2 likely is the predominant IGF regulating growth of the follicle during the early stages of development in the brushtail possum. The pattern of expression of IGFs in preantral follicles appears to differ among species. In cattle, neither IGF1 nor IGF2 mRNA was detected in preantral follicles (Armstrong et al. 2002). In mice, IGF1, but not IGF2, mRNA was expressed in granulosa cells from the primary stage onward (Wandji et al. 1998). In humans, IGF2, but not IGF1, mRNA was observed in oocytes and granulosa cells of preantral follicles (Zhou & Bondy 1993).

Addition of IGFs (IGF1 or IGF2) to cultures of preantral follicles has been shown to affect follicular growth and function. Preantral follicles of multiple species cultured with IGF1 or IGF2 showed increased oocyte and/or follicular diameters when compared with follicles cultured without IGF (Itoh et al. 2002, Mao et al. 2004, Zhou & Zhang 2005, Thomas et al. 2007, Sharma et al. 2009). Addition of IGFs to the media also increased the survival rate of follicles (Zhou & Zhang 2005, Sharma et al. 2009) and increased oestradiol production from preantral follicles (Yuan & Giudice 1999, Demeestere et al. 2004, Thomas et al. 2007). The expression of IGF2 mRNA in preantral follicles as well as expression of mRNA for IGF1R in both granulosa cells and oocytes of preantral follicles in the brushtail possum is consistent with IGF2 being an important regulator of preantral follicular growth in marsupials.

Relatively little is known about the expression of IGFBP mRNA in preantral follicles in eutherian mammals. In mice, cattle, monkeys and humans, IGFBP2 mRNA was expressed in growing preantral follicles (Zhou & Bondy 1993, Wandji et al. 1998, Arraztoa et al. 2002, Thomas et al. 2007), and a similar pattern of expression was observed in the brushtail possum. In addition, expression of IGFBP5 occurred prior to the initiation of follicular growth in the brushtail possum in granulosa cells of primordial follicles as well as growing preantral follicles. This expression pattern is similar to what was observed in mice (Wandji et al. 1998); however, IGFBP5 mRNA was not observed in preantral follicles of monkeys (Arraztoa et al. 2002) or humans (Zhou & Bondy 1993). Somewhat surprisingly, strong expression of PAPPA mRNA was observed in granulosa cells of growing preantral follicles in possums. Studies in rats, cows and pigs have linked the expression of PAPPA to the development of the dominant, ovulatory follicle (Mazerbourg et al. 2001, Hourvitz et al. 2002). In rats and humans, using in situ hybridization, expression was not observed in preantral follicles (Hourvitz et al. 2000, 2002). Collectively, the expression patterns of mRNA encoding IGFs, the IGF1R, IGFBPs as well as an IGFBP protease (PAPPA) are consistent with a complex regulation of the IGF system during preantral follicular growth.

In possums, IGF2 mRNA was strongly expressed in the granulosa of antral follicles, whereas IGF1 mRNA was not expressed in granulosa cells and only weakly expressed in the theca cells. Expression of IGF2 mRNA, with little or no expression of IGF1 mRNA, has been observed in antral follicles of humans (Zhou & Bondy 1993), sheep (Hastie & Haresign 2006, 2008) and cattle (Perks et al. 1995); although in sheep and cattle, expression is limited to the theca cells. In contrast, IGF1, but not IGF2, mRNA was strongly expressed in granulosa cells of rodents (Wandji et al. 1998) and pigs (Liu et al. 2000).

Granulosa cells of antral follicles in the brushtail possum were targets for both IGF1 and IGF2 actions as shown both by the expression of the IGF1R mRNA and stimulation of 3H-thymidine uptake. IGF1R mRNA has commonly been shown to be expressed in the granulosa cells of antral follicles of eutherian mammals (Zhou & Bondy 1993, Perks et al. 1995, Wandji et al. 1998, Liu et al. 2000, Armstrong et al. 2002, Hastie & Haresign 2006). The magnitude of the effects of IGFs on proliferation differs among species and stages of follicular development; however, overall, they seem to promote proliferation and differentiation (Mazerbourg et al. 2003, Silva et al. 2009). In sheep, follicles of a similar developmental stage (based on similar sized follicles and a similar size of follicle at ovulation) IGF1 stimulated proliferation (Monniaux & Pisselet 1992). Similarly, human, cow and pig granulosa cells responded to IGF1/IGF2 with increased cell proliferation (Savion et al. 1981, Baranao & Hammond 1984, Yong et al. 1992).

No effects of IGFs were observed on progesterone secretion in the present study. This is similar to what has been observed in granulosa cells of sheep follicles of similar size (Monniaux & Pisselet 1992). However, granulosa cells from more developed follicles respond to IGF1 with increased progesterone production (Monniaux & Pisselet 1992), and granulosa cells from other species also responded with increased progesterone production (Schams et al. 1988, Yong et al. 1992, Shaw et al. 1993, Armstrong et al. 1996, Spicer & Aad 2007). Thus, IGFs could affect progesterone production in preovulatory follicles in the brushtail possum. However, in the two animals with preovulatory follicles present, these follicles did not express IGFR1 mRNA indicating that preovulatory follicles may lose their ability to respond to IGFs. Moreover, preliminary data obtained with a limited number of animals (n=1–3) support this possibility as the granulosa cells showed no response to IGF1 in thymidine incorporation or progesterone production. IGF1 can also augment the effects of FSH on progesterone production in some species (Adashi et al. 1984, 1989, Baranao & Hammond 1984, Adashi & Resnick 1987). It is important to note, however, that the secretion of progesterone in the cultures in the present study was variable and often at the limit of the detection of the assay. Thus, while factors capable of strongly stimulating progesterone could be identified (i.e. LH), more subtle effects on progesterone secretion may have been below the sensitivity of the assay. Additionally, the interactions between gonadotrophins and IGFs in regulating proliferation and steroidogenesis in marsupials were not examined, and thus, IGFs could synergize with gonadotrophins to stimulate progesterone production as has been observed in other species.

Antral follicles also expressed mRNAs for IGFBPs. Similar to what has been observed in multiple eutherian mammals (Nakatani et al. 1991, Zhou & Bondy 1993, el-Roeiy et al. 1994, Besnard et al. 1996, Liu et al. 2000, Arraztoa et al. 2002, Zhou et al. 2003, Canty et al. 2006, Hastie & Haresign 2006, Llewellyn et al. 2007), theca of antral follicles expressed IGFBPs. However, given the inability to detect IGF1R mRNA in theca cells, the potential role for IGFs as well as autoregulation of IGF action by theca produced IGFBPs is unclear. It is possible that theca produced IGFBPs regulate IGF activity in granulosa cells and/or oocytes. Effects of IGFs on granulosa cells of antral follicles are also likely regulated by locally expressed IGFBPs and the IGFBP protease PAPPA. IGFBP2 mRNA was strongly expressed in granulosa cells of antral follicles. However, expression of IGFBP4 mRNA was weak and inconsistent, and IGFBP5 mRNA was no longer expressed in granulosa cells of antral follicles. In addition, expression of PAPPA mRNA remained strong in the granulosa cells. Thus, expression/actions of the BPs may decrease as the follicle develops in the brushtail possum. This is similar to what has been observed in many eutherian mammals (el-Roeiy et al. 1994, Besnard et al. 1996, Zhou et al. 1996, Wandji et al. 1998, Hourvitz et al. 2000, 2002, Liu et al. 2000, Arraztoa et al. 2002, Canty et al. 2006, Hastie & Haresign 2006, Llewellyn et al. 2007). It is important to note that the follicles examined in the brushtail possum did not include any preovulatory follicles (>3 mm diameter), and that further changes in the expression of IGFBPs as well as PAPPA during the development of the preovulatory follicle are often observed in eutherian mammals (Monget et al. 2002, Spicer 2004, Silva et al. 2009). Whether similar changes in the expression of IGFBPs/PAPPA would also be observed during the development of the preovulatory follicle of the brushtail possum remains to be elucidated.

Atretic antral follicles had similar expression patterns for the IGFBPs, IGF2 and IGF1R as healthy follicles. However, expression of IGF1 in the theca was no longer detectible, and PAPPA expression in granulosa cells did not appear to be as strong as was observed in healthy follicles. Taken together, this is consistent with a decreased bioactivity of IGFs in atretic follicle. In eutherian mammals, decreased bioactivity of IGFs through decreased secretion of ligands, decreased receptor expression and/or increased concentrations of BP activity (either through increased expression of BPs or reduced degradation) has also been implicated in atreasia (Monget et al. 2002, Quirk et al. 2004, Spicer 2004, Hastie & Haresign 2006, Webb & Campbell 2007). Thus, IGFs may play an important role in the regulation of follicular health in marsupials as well as in eutherian mammals.

Expression of IGF1R mRNA in oocytes as well as granulosa cells of preantral and antral follicles indicates that oocytes are also likely targets of IGFs during ovarian follicular development. Expression of IGF1R in oocytes has been observed in other species including cattle (Armstrong et al. 2002), rats (Zhou et al. 1991), humans (Zhou & Bondy 1993) and monkeys (Vendola et al. 1999). Addition of IGF1 or IGF2 to media during in vitro maturation and fertilization increases blastocyst formation indicating that these factors are likely involved in the maturation of the oocyte (Shabankareh & Zandi 2010, Wang et al. 2009).

In conclusion, as has been shown in eutherian mammals, IGFs have the potential to be key local regulators in ovarian follicular development in marsupials. IGF2 may be the dominant IGF regulating follicular development in the brushtail possum and localization of mRNAs encoding IGFBPs and the BP protease, PAPPA in a cell and developmental stage-specific manner indicates that complex regulation of the IGFs may be occurring during follicular development in the brushtail possum as has been observed in eutherian mammals.

Materials and Methods

Collection of tissue samples

Ovaries were collected from juveniles (females that were independent of their mothers but not sexually mature as assessed by their body size and pouch development) and adult females following anaesthesia (Juengel et al. 2002). Animals were sourced from a captive breeding programme or captured from the wild. All experiments performed were with the approval of the Animals Ethics Committee at Wallaceville Animal Research Centre or Invermay Agricultural Centre and in accordance with the 1999 Animal Protection (Codes of Ethical Conduct) Regulations of New Zealand.

Generation of cDNAs of interest

For the generation of the cDNAs of interest, total cellular RNA from ovarian tissue was reverse transcribed using SuperScript preamplification system (Invitrogen NZ Ltd). cDNAs for the genes of interest were obtained using PCR with standard PCR buffer from Qiagen (Biolab Scientific Ltd, Wellington, New Zealand). Hotstart Taq was used for all genes with the following conditions: 1 cycle at 94 °C for 2 min; 40 cycles of denaturing at 94 °C for 20 s, annealing at 58 °C for 15 s, and extension at 72 °C for 50 s; and final extension at 72 °C for 10 min. Comparisons of known sequences for each gene were used to design primers corresponding to conserved areas of the gene. Primers used for genes are shown in Table 2. Confirmation of isolation of the possum homologue of the gene of interest was obtained by sequencing (Waikato DNA Sequencing Facility; The University of Waikato; Hamilton, New Zealand) of the resulting PCR products, which had been ligated into pGEMTeasy vector (Promega; Dade Diagnostics PTY Ltd, Auckland, New Zealand). Sequences (GenBank accession numbers GU250879–GU250885; Table 2) had ≥89% identity with the corresponding region of the gene in other marsupials (Altschul et al. 1990).

Table 2

Primers used for obtaining partial cDNAs for genes of interest and the GenBank accession number of the resulting brushtail possum sequences.

GenesForward primerReverse primerGenBank accession numbers
IGFIAAA AAT CAG CAG TCT TCC AACTTG GGC ATG TCG GTG TGGU250879
IGF2CCT TTG CCT CGT GCT GCGGG ACG GTG ACG CTT GGGU250880
IGFIRGAG TTC AAY TGT CAC CAY GTG GGGG TTR TAC TGC CAG CACGU250881
IGFBP2CTC AAG TCA GGC ATG AAG GAGTA GAA GAG ATG RCA CTC GGGU250882
IGFBP4CCT GGC TGT GGC TGC TGTTG GGG TGG AAG TTG CCGU250883
IGFBP5CCC TGC GAC GAG AAA GCTTC ATC CCR TAC TTG TCC ACGU250884
PAPPACCT TAC AGA GCC TAC TTG GAT GGAT TTG GCG TGA AGG AGT CGU250885

In situ hybridization

In situ hybridization was used to determine the localization of mRNAs in ovarian sections (Tisdall et al. 1999, Juengel et al. 2002). Riboprobes were generated using the Riboprobe Gemini system (Promega). Tissue sections were hybridized overnight at 55 °C with ∼45 000 cpm/μl (48 000 dpm) of 33P-labelled antisense or sense RNA. Tissue sections were viewed and photographed, using both light and dark field illumination, on an Olympus BH-50 microscope comparing hybridization of the antisense and sense riboprobes. As hybridization of the sense RNA over the tissue section was similar or lower in intensity to that observed on the glass of both the sense and antisense hybridized slides, no specific hybridization was considered to have occurred with any of the sense ribroprobes.

Specific hybridization for each gene was then determined for specific stages of follicular development (Juengel et al. 2002). Follicles were considered to be primordial follicles if they contained an oocyte surrounded by a single layer of flattened or mixed flattened and cuboidal granulosa cells. Primary follicles had an oocyte surrounded by 1–<2 layers of cuboidal granulosa cells. Follicles containing at least two layers of granulosa cells without a formed antrum were considered secondary follicles. Follicles containing an antrum were classified as antral. Follicles with several pyknotic nuclei in the granulosa layer were considered to be atretic. As the majority of atretic follicles observed had developed an antrum, only antral atretic follicles were included in the study. These were limited in number, and often did not include the oocyte in the sections examined, and thus, no data for expression of the genes of interest in oocytes of atretic antral follicles are presented. Follicles from a minimum of three juvenile and five adult animals were examined for each gene.

Culture of granulosa cells

Granulosa cells were collected from healthy follicles between 0.5 and 2.5 mm in diameter from adult female possums during the breeding season. Oocytes were removed before cells were cultured in a humidified incubator with 5% CO2 in air at 37 °C. Viability of cells was determined by trypan blue exclusion and averaged 65% at the time of collection. Granulosa cells (quadruplicate wells) were cultured in DMEM:F12 (Invitrogen) supplemented with 15 mM HEPES, 3.15 g/l glucose, 0.1% BSA (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, insulin (10 ng/ml; Sigma), holo-transferrin (5 ng/ml; Invitrogen) and sodium selenite (10 ng/ml; Sigma) containing 0 or 100 ng/ml of IGF1 (Long-R3, Gro-Pep, Adelaide, SA, Australia) or IGF2 (R&D Systems Inc., Minneapolis, MN, USA). Doses of IGF1 and IGF2 were chosen following preliminary experiments testing doses between 0.1 and 100 ng/ml of IGF1 or IGF2 as the dose with maximum effect (data not shown). For determination of proliferation potential, 20 000 cells per well were cultured in a total volume of 125 μl in 96-well microtitre plates. After 18 h of culture, methyl 3H-thymidine (0.4 μCi/well; PerkinElmer, Boston, MA, USA; 20 Ci/ml) was added, and cells were cultured for an additional 6 h. Cells were harvested onto a thin filtermat, and incorporation of 3H-thymidine was determined using a liquid scintillation counter (Wallac Trilux MicroBeta 1450; Biolab). Effects of growth factors on steroid production were determined following culture of 40 000 cells per well in a total volume of 250 μl. A positive control, 1 ng/ml ovine LH (purified in-house), was included in each culture. Every 48 h, 200 μl of medium was removed from each well and replaced with 200 μl of fresh medium containing the respective treatments. Media samples from the last 48 h of treatment were collected on day 6. At the time of collection, media samples were frozen at −20 °C until concentrations of progesterone could be determined by RIA (Asher 1990). The sensitivity of the progesterone assay was 17 pg/ml, and the intra- and inter-assay coefficients of variation were 11.4 and 12.2% respectively. A minimum of three replicate experiments were undertaken for determination of effect of IGF1 or IGF2 on thymidine incorporation and progesterone production. The effects of IGF1 or IGF2 on granulosa cell function were analyzed using a paired t-test comparing untreated control cells to those treated with IGF1 or IGF2.

Declaration of interest

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

Funding

This work was supported by the New Zealand Foundation for Research, Science and Technology (grant number C10X0308).

Acknowledgements

The authors would like to thank Lee-Ann Still and Di Sebelin for the preparation of histological materials and Lloyd Moore for providing purified ovine LH.

References

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    • Search Google Scholar
    • Export Citation
  • Adashi EY, Resnick CE, Svoboda ME & Van Wyk JJ 1984 A novel role for somatomedin-C in the cytodifferentiation of the ovarian granulosa cell. Endocrinology 115 12271229.

    • Search Google Scholar
    • Export Citation
  • Adashi EY, Resnick CE, Hernandez ER, May JV, Purchio AF & Twardzik DR 1989 Ovarian transforming growth factor-β (TGFβ): cellular site(s), and mechanism(s) of action. Molecular and Cellular Endocrinology 61 247256.

    • Search Google Scholar
    • Export Citation
  • Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ 1990 Basic local alignment search tool. Journal of Molecular Biology 215 403410.

  • Armstrong DT, Xia P, de Gannes G, Tekpetey FR & Khamsi F 1996 Differential effects of insulin-like growth factor-I and follicle-stimulating hormone on proliferation and differentiation of bovine cumulus cells and granulosa cells. Biology of Reproduction 54 331338.

    • Search Google Scholar
    • Export Citation
  • Armstrong DG, Baxter G, Hogg CO & Woad KJ 2002 Insulin-like growth factor (IGF) system in the oocyte and somatic cells of bovine preantral follicles. Reproduction 123 789797.

    • Search Google Scholar
    • Export Citation
  • Arraztoa JA, Monget P, Bondy C & Zhou J 2002 Expression patterns of insulin-like growth factor-binding proteins 1, 2, 3, 5, and 6 in the mid-cycle monkey ovary. Journal of Clinical Endocrinology and Metabolism 87 52205228.

    • Search Google Scholar
    • Export Citation
  • Asher GW 1990 Effect of subcutaneous melatonin implants on the seasonal attainment of puberty in female red deer (Cervus elaphus). Animal Reproduction Science 22 145159.

    • Search Google Scholar
    • Export Citation
  • Baranao JL & Hammond JM 1984 Comparative effects of insulin and insulin-like growth factors on DNA synthesis and differentiation of porcine granulosa cells. Biochemical and Biophysical Research Communications 124 484490.

    • Search Google Scholar
    • Export Citation
  • Besnard N, Pisselet C, Monniaux D, Locatelli A, Benne F, Gasser F, Hatey F & Monget P 1996 Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles. Biology of Reproduction 55 13561367.

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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  • View in gallery

    Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from adult brushtail possums following hybridization to IGF1 antisense (A–D) and sense (E and F) RNA. Expression of IGF1 mRNA in the theca (t), but not oocyte (o) or granulosa cells (g) of antral follicles (A and B). Expression of IGF1 mRNA in the cells of the vasculature of the ovary (C and D). Expression was not detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGF1 sense RNA (E and F).

  • View in gallery

    Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from juvenile and adult brushtail possums following hybridization to IGF2 antisense (A–D) and sense (E and F) RNA. Expression of IGF2 mRNA in granulosa cells (g), but not oocyte (o) or theca (t) of secondary (arrowhead) and antral (arrow) follicles (A–D). Expression was not detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGF2 sense RNA (E and F).

  • View in gallery

    Corresponding brightfield (A, C, E and G) and darkfield (B, D, F and H) views of ovaries from juvenile and adult brushtail possums following hybridization to IGF1R antisense (A–F) and sense (G and H) RNA. Oocytes (o) of primordial (#; 2× higher magnification in inset of panel E and F), primary (*), secondary (arrowhead) and antral (arrow) follicles express IGF1R (A–F). Expression of IGF1R mRNA was also observed in granulosa cells (g) but not theca (t) of secondary (arrowhead) and antral (arrow) follicles (A–F). Expression was not detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGF1R sense RNA (G and H).

  • View in gallery

    Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from juvenile and adult brushtail possums following hybridization to IGFBP2 antisense (A–D) and sense (E and F) RNA. Expression of IGFBP2 mRNA in granulosa cells (g) and theca (t), but not oocyte (o) of secondary (arrowhead) and antral (arrow) follicles (A–D). Expression was also detectible in the interstitial tissue (i; C and D). Note the lack of specific signal in an ovary hybridized to the IGFBP2 sense RNA (E and F).

  • View in gallery

    Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from juvenile and adult brushtail possums following hybridization to IGFBP4 antisense (A–D) and sense (E and F) RNA. Expression of IGFBP4 was first observed in theca of secondary follicles (A and B; arrowhead). Expression of IGFBP4 mRNA was strongest in the theca (t), with weak signal also seen in the in granulosa cells (g) of some but not all antral follicles (A–D, arrow). Expression was not observed in the oocyte (o) of follicles (A and B). Expression was also detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGFBP4 sense RNA (E and F).

  • View in gallery

    Corresponding brightfield (A, C, E and G) and darkfield (B, D, F and H) views of ovaries from juvenile brushtail possums following hybridization to IGFBP5 antisense (A–F) and sense (G and H) RNA. Granulosa cells (g) of primordial (#), primary (*), secondary (arrowhead), but not antral (arrow) follicles express IGFBP5 (A–F). Theca cells also express IGFBP5 mRNA (t; C–F) but interstitial tissue did not (i; A and B). Note the lack of specific signal in an ovary hybridized to the IGFBP5 sense RNA (G and H).

  • View in gallery

    Corresponding brightfield (A, C and E) and darkfield (B, D and F) views of ovaries from juvenile and adult brushtail possums following hybridization to PAPPA antisense (A–D) and sense (E and F) RNA. Expression of PAPPA was not observed in primordial follicles (A and B; #). The granulosa cells, but not theca (t) or oocytes (o), of growing follicles expressed PAPPA mRNA (A–D, arrowhead secondary follicles, arrows antral follicles). Expression was not detectible in the interstitial tissue (i; A and B). Note the lack of specific signal in an ovary hybridized to the PAPPA sense RNA (E and F).

  • Adashi EY & Resnick CE 1987 Prolactin as an inhibitor of granulosa cell luteinization: implications for hyperprolactinemia-associated luteal phase dysfunction. Fertility and Sterility 48 131139.

    • Search Google Scholar
    • Export Citation
  • Adashi EY, Resnick CE, Svoboda ME & Van Wyk JJ 1984 A novel role for somatomedin-C in the cytodifferentiation of the ovarian granulosa cell. Endocrinology 115 12271229.

    • Search Google Scholar
    • Export Citation
  • Adashi EY, Resnick CE, Hernandez ER, May JV, Purchio AF & Twardzik DR 1989 Ovarian transforming growth factor-β (TGFβ): cellular site(s), and mechanism(s) of action. Molecular and Cellular Endocrinology 61 247256.

    • Search Google Scholar
    • Export Citation
  • Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ 1990 Basic local alignment search tool. Journal of Molecular Biology 215 403410.

  • Armstrong DT, Xia P, de Gannes G, Tekpetey FR & Khamsi F 1996 Differential effects of insulin-like growth factor-I and follicle-stimulating hormone on proliferation and differentiation of bovine cumulus cells and granulosa cells. Biology of Reproduction 54 331338.

    • Search Google Scholar
    • Export Citation
  • Armstrong DG, Baxter G, Hogg CO & Woad KJ 2002 Insulin-like growth factor (IGF) system in the oocyte and somatic cells of bovine preantral follicles. Reproduction 123 789797.

    • Search Google Scholar
    • Export Citation
  • Arraztoa JA, Monget P, Bondy C & Zhou J 2002 Expression patterns of insulin-like growth factor-binding proteins 1, 2, 3, 5, and 6 in the mid-cycle monkey ovary. Journal of Clinical Endocrinology and Metabolism 87 52205228.

    • Search Google Scholar
    • Export Citation
  • Asher GW 1990 Effect of subcutaneous melatonin implants on the seasonal attainment of puberty in female red deer (Cervus elaphus). Animal Reproduction Science 22 145159.

    • Search Google Scholar
    • Export Citation
  • Baranao JL & Hammond JM 1984 Comparative effects of insulin and insulin-like growth factors on DNA synthesis and differentiation of porcine granulosa cells. Biochemical and Biophysical Research Communications 124 484490.

    • Search Google Scholar
    • Export Citation
  • Besnard N, Pisselet C, Monniaux D, Locatelli A, Benne F, Gasser F, Hatey F & Monget P 1996 Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles. Biology of Reproduction 55 13561367.

    • Search Google Scholar
    • Export Citation
  • Canty MJ, Boland MP, Evans AC & Crowe MA 2006 Alterations in follicular IGFBP mRNA expression and follicular fluid IGFBP concentrations during the first follicle wave in beef heifers. Animal Reproduction Science 93 199217.

    • Search Google Scholar
    • Export Citation
  • Demeestere I, Gervy C, Centner J, Devreker F, Englert Y & Delbaere A 2004 Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice. Biology of Reproduction 70 16641669.

    • Search Google Scholar
    • Export Citation
  • Eckery DC, Juengel JL, Whale LJ, Thomson BP, Lun S & McNatty KP 2002a The corpus luteum and interstitial tissue in a marsupial, the brushtail possum (Trichosurus vulpecula). Molecular and Cellular Endocrinology 191 8187.

    • Search Google Scholar
    • Export Citation
  • Eckery DC, Lun S, Thomson BP, Chie WN, Moore LG & Juengel JL 2002b Ovarian expression of messenger RNA encoding the receptors for luteinizing hormone and follicle-stimulating hormone in a marsupial, the brushtail possum (Trichosurus vulpecula). Biology of Reproduction 66 13101317.

    • Search Google Scholar
    • Export Citation
  • Eymann J, Herbert CA, Thomson BP, Trigg TE, Cooper DW & Eckery DC 2007 Effects of deslorelin implants on reproduction in the common brushtail possum (Trichosurus vulpecula). Reproduction, Fertility, and Development 19 899909.

    • Search Google Scholar
    • Export Citation
  • Fletcher T & Selwood L 2000 Possum reproduction and development. In The Brushtail Possum: Biology, Impact and Management of an Introduced Marsupial, pp 62–81. Ed. TL Montague. Lincoln, New Zealand: Manaaki Whenu Press.

  • Hastie PM & Haresign W 2006 Expression of mRNAs encoding insulin-like growth factor (IGF) ligands, IGF receptors and IGF binding proteins during follicular growth and atresia in the ovine ovary throughout the oestrous cycle. Animal Reproduction Science 92 284299.

    • Search Google Scholar
    • Export Citation
  • Hastie PM & Haresign W 2008 Modulating peripheral gonadotrophin levels affects follicular expression of mRNAs encoding insulin-like growth factors and receptors in sheep. Animal Reproduction Science 109 110123.

    • Search Google Scholar
    • Export Citation
  • Hourvitz A, Widger AE, Filho FL, Chang RJ, Adashi EY & Erickson GF 2000 Pregnancy-associated plasma protein-A gene expression in human ovaries is restricted to healthy follicles and corpora lutea. Journal of Clinical Endocrinology and Metabolism 85 49164920.

    • Search Google Scholar
    • Export Citation
  • Hourvitz A, Kuwahara A, Hennebold JD, Tavares AB, Negishi H, Lee TH, Erickson GF & Adashi EY 2002 The regulated expression of the pregnancy-associated plasma protein-A in the rodent ovary: a proposed role in the development of dominant follicles and of corpora lutea. Endocrinology 143 18331844.

    • Search Google Scholar
    • Export Citation
  • Itoh T, Kacchi M, Abe H, Sendai Y & Hoshi H 2002 Growth, antrum formation, and estradiol production of bovine preantral follicles cultured in a serum-free medium. Biology of Reproduction 67 10991105.

    • Search Google Scholar
    • Export Citation
  • Juengel JL, Whale LJ, Wylde KA, Greenwood P, McNatty KP & Eckery DC 2002 Expression of anti-Mullerian hormone mRNA during gonadal and follicular development in the brushtail possum (Trichosurus vulpecula). Reproduction, Fertility, and Development 14 345353.

    • Search Google Scholar
    • Export Citation
  • Liu J, Koenigsfeld AT, Cantley TC, Boyd CK, Kobayashi Y & Lucy MC 2000 Growth and the initiation of steroidogenesis in porcine follicles are associated with unique patterns of gene expression for individual components of the ovarian insulin-like growth factor system. Biology of Reproduction 63 942952.

    • Search Google Scholar
    • Export Citation
  • Llewellyn S, Fitzpatrick R, Kenny DA, Murphy JJ, Scaramuzzi RJ & Wathes DC 2007 Effect of negative energy balance on the insulin-like growth factor system in pre-recruitment ovarian follicles of post partum dairy cows. Reproduction 133 627639.

    • Search Google Scholar
    • Export Citation
  • Mao J, Smith MF, Rucker EB, Wu GM, McCauley TC, Cantley TC, Prather RS, Didion BA & Day BN 2004 Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro. Journal of Animal Science 82 19671975.

    • Search Google Scholar
    • Export Citation
  • Mazerbourg S, Overgaard MT, Oxvig C, Christiansen M, Conover CA, Laurendeau I, Vidaud M, Tosser-Klopp G, Zapf J & Monget P 2001 Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development. Endocrinology 142 52435253.

    • Search Google Scholar
    • Export Citation
  • Mazerbourg S, Bondy CA, Zhou J & Monget P 2003 The insulin-like growth factor system: a key determinant role in the growth and selection of ovarian follicles? a comparative species study. Reproduction in Domestic Animals 38 247258.

    • Search Google Scholar
    • Export Citation
  • Mihm M & Evans AC Mechanisms for dominant follicle selection in monovulatory species: a comparison of morphological, endocrine and intraovarian events in cows, mares and women Reproduction in Domestic Animals 43 Suppl 2 2008 4856.

    • Search Google Scholar
    • Export Citation
  • Monget P, Fabre S, Mulsant P, Lecerf F, Elsen JM, Mazerbourg S, Pisselet C & Monniaux D 2002 Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals. Domestic Animal Endocrinology 23 139154.

    • Search Google Scholar
    • Export Citation
  • Monniaux D & Pisselet C 1992 Control of proliferation and differentiation of ovine granulosa cells by insulin-like growth factor-I and follicle-stimulating hormone in vitro. Biology of Reproduction 46 109119.

    • Search Google Scholar
    • Export Citation
  • Nakatani A, Shimasaki S, Erickson GF & Ling N 1991 Tissue-specific expression of four insulin-like growth factor-binding proteins (1, 2, 3, and 4) in the rat ovary. Endocrinology 129 15211529.

    • Search Google Scholar
    • Export Citation
  • Pacher M, Seewald MJ, Mikula M, Oehler S, Mogg M, Vinatzer U, Eger A, Schweifer N, Varecka R & Sommergruber W 2007 Impact of constitutive IGF1/IGF2 stimulation on the transcriptional program of human breast cancer cells. Carcinogenesis 28 4959.

    • Search Google Scholar
    • Export Citation
  • Perks CM, Denning-Kendall PA, Gilmour RS & Wathes DC 1995 Localization of messenger ribonucleic acids for insulin-like growth factor I (IGF-I), IGF-II, and the type 1 IGF receptor in the ovine ovary throughout the estrous cycle. Endocrinology 136 52665273.

    • Search Google Scholar
    • Export Citation
  • Quirk SM, Cowan RG, Harman RM, Hu CL & Porter DA Ovarian follicular growth and atresia: the relationship between cell proliferation and survival Journal of Animal Science 82 E Supplement 2004 E40E52.

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    • Export Citation
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