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.
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 type | IGF1 | IGF2 | IGF1R | IGFBP2 | IGFBP4 | IGFBP5 | PAPPA |
---|---|---|---|---|---|---|---|
Primordial | – | – | o | – | – | g | – |
Primary | – | g | o | g | – | g | g |
Secondary | – | g | o, g | g, t | t | g, t | g |
Antral | t | g | o, g | g, t | g, t | t | g |
Atretic antrala | – | g | g | g, t | g, t | t | g |
–, 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.
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.
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).
Primers used for obtaining partial cDNAs for genes of interest and the GenBank accession number of the resulting brushtail possum sequences.
Genes | Forward primer | Reverse primer | GenBank accession numbers |
---|---|---|---|
IGFI | AAA AAT CAG CAG TCT TCC AAC | TTG GGC ATG TCG GTG TG | GU250879 |
IGF2 | CCT TTG CCT CGT GCT GC | GGG ACG GTG ACG CTT GG | GU250880 |
IGFIR | GAG TTC AAY TGT CAC CAY GTG G | GGG TTR TAC TGC CAG CAC | GU250881 |
IGFBP2 | CTC AAG TCA GGC ATG AAG GA | GTA GAA GAG ATG RCA CTC GG | GU250882 |
IGFBP4 | CCT GGC TGT GGC TGC TG | TTG GGG TGG AAG TTG CC | GU250883 |
IGFBP5 | CCC TGC GAC GAG AAA GC | TTC ATC CCR TAC TTG TCC AC | GU250884 |
PAPPA | CCT TAC AGA GCC TAC TTG GAT G | GAT TTG GCG TGA AGG AGT C | GU250885 |
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.
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