- Email: [email protected]
Secretion of IGF-1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone
Animal Reproduction Science 58 Ž2000. 261–272 www.elsevier.comrlocateranireprosci
Secretion of IGF-1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone M. Khalid a,) , W. Haresign b, M.R. Luck c a
c
Department of Farm Animal and Equine Medicine, The Royal Veterinary College, UniÕersity of London, Hawskshead Lane, North Hymms, Harfield, Hertfordshire AL97TA, UK b Animal Sciences Group, Welsh Institute of Rural Studies, UniÕersity of Wales Aberystwyth, Llanbadarn Campus, Aberystwyth SY23 3AL, UK DiÕision of Animal Physiology, School of Biological Sciences, UniÕersity of Nottingham, Sutton Bonington Campus, Loughborough L.C. LE12 5RD, UK Received 13 January 1999; received in revised form 9 August 1999; accepted 11 October 1999
Abstract Insulin-like growth factor-1 ŽIGF-1. is implicated in follicle development and is considered to mediate the actions of growth hormone ŽGH. and gonadotrophins at the ovarian level. However, the expression and secretion of IGF-1 by the ovary are controversial, partly because of species and cell-type specificity. The present study investigated whether IGF-1 is produced by ovine granulosa cells and whether its production is regulated by GH and follicle stimulating hormone ŽFSH.. Follicles ŽG 4.0 mm. were obtained from ewes during seasonal anoestrus. Granulosa cells were cultured for a total period of 96 h in Dulbecco’s modified Eagle’s medium ŽDMEM.rHam’s F-12 medium supplemented with BSA Ž0.1%, w:v., transferrin Ž0.5 mgrml. and testosterone Ž100 ngrml.. In the first set of experiments, cells were incubated in the presence of bovine calf serum ŽBCS. Ž2.5%. for the initial 48 h of culture. The cells were then cultured for the next 48 h in medium without BCS, but containing either GH Ž0, 2, 20, and 200 ngrml. or FSH Ž0, 20, 200, and 2000 ngrml.. The medium was assayed for oestradiol ŽE., progesterone ŽP. and IGF-1. There were six wells per treatment and the experiment was carried out four times. Control granulosa cells maintained both IGF-1 and E secretion, with only low levels of progesterone output. In all experiments, both GH and FSH produced significant Ž P - 0.001. dose-related increases in E, IGF-1 and P secretion into the medium. The maximum responses to GH Ž20 or 200 ngrml. were 402% for E and 528% for IGF-1 compared with controls. The maximum responses to FSH Ž200 or
)
Corresponding author. Tel.: q44-1707-666-333, ext. 2485; fax: q44-1707-660-671. E-mail address: [email protected] ŽM. Khalid..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 7 5 - 5
262
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
2000 ngrml. were 460% for E and 514% for IGF-1. The objective of the second set of experiments was to determine the effect of the progestogenic status of cells on IGF-1 production. Granulosa cells were cultured both in the presence and absence of BCS Ž2.5% in the medium. during the initial 48 h of culture. For the next 48 h, cells were cultured in serum-free medium. Addition of BCS to the medium during the initial 48 h of culture stimulated progesterone production. However, it did not affect either IGF-1 or oestradiol secretion between 49 and 96 h of culture, or the cell numbers at the end of culture. In conclusion, Ž1. IGF-1 is secreted by granulosa cells irrespective of their progestogenic status and Ž2. concomitant increases in E and IGF-1 production by granulosa cells as a result of GH andror FSH treatment suggest a role for GH and FSH in the regulation of ovarian function. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Sheep; Growth hormone; FSH; Granulosa cells; IGF-1 synthesis
1. Introduction Evidence from in vivo and in vitro studies implicates insulin-like growth factor-1 ŽIGF-1. in the control of follicle growth and development. Follicular development is arrested in IGF-1 knockout mice, and such animals do not ovulate even after administration of gonadotrophins ŽBaker et al., 1996.. Moreover, IGF-1 enhances follicle stimulating hormone ŽFSH.-stimulated oestrogen andror progesterone production in murine ŽAdashi et al., 1985; Davoren et al., 1985., bovine ŽSchams, 1987., porcine ŽVeldhuis et al., 1987; Maruo et al., 1988. and ovine ŽMonniaux and Pisselet, 1992; Campbell et al., 1996. granulosa cells. Relationships between follicular fluid IGF-1 concentrations and stage of follicle development have also been documented for a number of species, including cattle ŽSpicer and Enright, 1991; Spicer et al., 1988; Echternkamp et al., 1990., pigs ŽMeurer et al., 1991. and sheep ŽMonget et al., 1993; Khalid and Haresign, 1996.. IGF-1 plays an obligatory role in amplifying the effect of FSH andror growth hormone ŽGH. in rat ŽAdashi et al., 1985, 1988, 1991., pig ŽHammond et al., 1991. and sheep ŽMonniaux and Pisselet, 1992. granulosa cells. Increased levels of IGF-1 Žboth plasma and intrafollicular. as a result of GH treatment are associated with an increased number of small follicles in both cattle ŽGong et al., 1991. and sheep ŽGong et al., 1996a., as well as the number of oestrogenic follicles in FSH-treated ewes ŽJoyce et al., 1998.. In addition, a significant increase in the superovulatory response to FSH has been reported as a result of GH treatment in cattle ŽGong et al., 1996b.. On the other hand, decreased serum andror intra-follicular IGF-1 concentrations as a result of immunization against GH releasing factor or chronic feed restriction impairs the ability of the ovary to synthesize preovulatory concentrations of oestradiol, and delays the maturation of preovulatory follicles and the onset of puberty in heifers ŽSchoppee et al., 1996.. Immunoreactive IGF-1 is secreted by porcine ŽHammond et al., 1985., rat ŽAdashi et al., 1991. and human ŽGiudice, 1992. granulosa cells. However, there are conflicting reports regarding the secretion of IGF-1 by ovine granulosa cells. Incubating ovine granulosa cells with tritiated amino acids, a de novo synthesis has been shown by Leeuwenberg et al. Ž1996., whereas the data of Wathes et al. Ž1995. suggest that ovine granulosa cells do not secrete IGF-1, although there was a low level of production after they became luteinised.
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
263
While there is a clear expression of IGF-1 mRNA in rat ŽOliver et al., 1989. and pig ŽSamaras et al., 1993. granulosa cells, expression of IGF-1 mRNA in the ovine ovary is controversial. Using ovine and human riboprobes, Leeuwenberg et al. Ž1995. reported IGF-1 mRNA in both granulosa and theca cells, whereas Perks et al. Ž1995., using an oligo-probe, were unable to detect any IGF-1 mRNA in ovine follicles. Using RTPCR, Spicer et al. Ž1995. reported the presence of IGF-1 mRNA in ovine ovarian follicle walls, although to a lower extent than IGF-2 mRNA. The situation with regard to the ability of the sheep ovary to produce IGF-1 is therefore confusing and requires clarification. One way in which this might be achieved is to use cell culture techniques. The present study was therefore performed to investigate whether IGF-1 is produced by ovine granulosa cells and if so, whether its production is regulated by GH andror FSH ŽExperiment 1.. Another objective of this study was to investigate whether IGF-1 production is related to the progestogenic status of cultured granulosa cells ŽExperiment 2..
2. Materials and methods 2.1. Culture of oÕine granulosa cells All the reagents were obtained from Sigma ŽPoole, Dorset, UK., unless otherwise stated. Ovaries were obtained from seasonally anoestrous ewes of mixed breeds, slaughtered at a commercial slaughterhouse. They were collected into culture medium Žbicarbonate-buffered Dulbecco’s modified Eagle’s medium ŽDMEM.rHam’s F-12 Ž1:1 v:v. supplemented with BSA Ž0.15%, wrv., transferrin Ž0.5 mgrml., testosterone Ž30 mM. and antibiotics Žpenicillin and streptomycin; Gibco Life Technologies, Paisley, Strathclyde, UK. at 378C and transported to the laboratory within 1 h after slaughter. Visually normal healthy follicles Žwith good vascular supply and clearrtransparent follicular fluid. of G 4.0 mm in diameter were dissected out and granulosa cells isolated as follows. After aspiration of follicular fluid, each follicle was sliced open in culture medium and granulosa cells were removed by gently scrapping the interior surface of the follicle wall with an inoculation loop. Granulosa cell suspensions were filtered through nylon hose mesh and centrifuged at 300 = g for 10 min. The supernatant was discarded and the resulting pellet was resuspended in the medium. The number of cells was estimated by measuring the amount of DNA in a sonicated aliquot of the granulosa cell suspension. Granulosa cells were plated at a density of 1.4 = 10 6 cellsrwell on collagen type 1 Ž1%.-pre-treated 48-well plates ŽFalcon; Becton Dickinson UK. as described by Luck Ž1989.. The cells were then cultured in 0.5 ml of medium in a humidified incubator at 378C in atmospheric air with 5% CO 2 . 2.1.1. Experiment 1 The objective of this experiment was to investigate whether the ovine granulosa cells produce IGF-1 and if so, whether this is regulated by GH andror FSH. To obtain optimal attachment, cells were maintained in the presence of 2.5% bovine calf serum
264
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
ŽBCS; Life Technologies. in the culture medium for the first 48 h. After this time, granulosa cells were washed with 0.5 ml serum-free medium and incubations were then continued for the next 48 h in serum-free medium containing ovine GH Ž0, 2, 20, and 200 ngrml. or ovine FSH Ž0, 20, 200, and 200 ngrml.. Both ovine GH ŽoGH-15. and ovine FSH ŽoFSH-19-SIAFP. were obtained from NIDDK, National Hormone and Pituitary Programme, Baltimore, MA, USA. At the end of the 96-h incubation period, medium was collected and stored at y208C until assayed for oestradiol, progesterone and IGF-1. After removal of the medium, cells in each well were gently washed and stored in 500 ml of DNA assay buffer Ž0.05 M phosphate, 0.9% wrv NaCl, 2 mM EDTA, pH 7.4. at y208C until the measurement of their DNA content. 2.1.2. Experiment 2 The objective of this experiment was to investigate whether there was any relationship between progestogenic status of cultured granulosa cells and IGF-1 production. Granulosa cells were cultured either with or without 2.5% BCS for the first 48 h in order to alter their basal level of progesterone secretion. The aim of culturing granulosa cells without BCS was to reducerblock the process of spontaneous luteinisation. The cells were then cultured for the next 48 h in serum-free medium. At the end of the second incubation period, the medium was collected and analysed, and cells counted as described for Experiment 1. 2.2. DNA assay The DNA content of granulosa cell suspensions was measured by the rapid fluorescence technique of Labarca and Paigen Ž1980. using a fluorimeter ŽJENWAY 6200.. Briefly, cell suspensions were sonicated and 250-ml aliquots of sonicated cells were mixed with fluorescence reagent Žbisbenzimide, B2883, Sigma Chemical Company, Poole, Dorset, UK. and analyzed spectrofluorometrically Žexcitation: 356 nm; emission: 458 nm.. A standard curve was made using calf thymus DNA ŽSigma.. 2.3. Oestradiol assay Measurements of oestradiol concentrations were carried out in unextracted samples of culture medium using the method validated by Foxcroft et al. Ž1987.. The limit of sensitivity of the assay was 16 pgrml. The inter- and intra-assay coefficients of variation were 12.1% and 8.9%, respectively. 2.4. Progesterone assay The concentrations of progesterone in culture medium samples were determined in a direct assay described by Hunter et al. Ž1988.. Culture medium blanks included in the assay showed no cross-reactivity and had progesterone values below the limit of sensitivity of the assay Ž0.12 ngrml within the present study.. The inter- and intra-assay coefficients of variation were 7.6% and 8.1%, respectively.
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
265
2.5. IGF-1 assay Samples of the culture medium Ž500 ml. were freeze-dried and then re-constituted in 50-ml distilled water. IGF-1 concentrations were measured using the radioimmunoassay technique described previously ŽKhalid and Haresign, 1996. and modified recently in our laboratory ŽJoyce et al., 1998.. The antiserum for IGF-1 used in this study had essentially no cross-reactivity with IGF-2 Ž0.68%.. Briefly, acid–ethanol mixture was added to the samples in a 4:1 ratio, incubated for 30 min and centrifuged at 2100 = g and 48C. The supernatant was then decanted and mixed with 30 ng of IGF-2 ŽPeninsula Europe Laboratories, St. Helens, Merseyside, UK., neutralised with 0.855 M Tris buffer, and incubated for 30 min prior to assay. Using this approach, serial dilutions of extracted plasma, follicular fluid and culture medium samples showed parallelism with the IGF-1 standard curve. This method gave a recovery of cold IGF-1 in medium of 85–100%. For every 0.5-, 1.5-, 5.0- and 15.0-ng IGF-1 added in the extracted medium, 0.45-, 1.43-, 5.0- and 14.85-ng IGF-1 were recovered, respectively. The inter- and intra-assay coefficients of variation were 9.8% and 11.4%, respectively, while the limit of sensitivity of the assay was 0.20 ngrtube. 2.6. Statistical analysis There were six replicate wells per treatment, and Experiments 1 and 2 were carried out three and four times, respectively. Similar results were obtained within different replicates of a particular experiment, despite variations in the absolute levels of hormones secreted by different batches of cells. Differences in hormone concentrations between different treatment groups were assessed by analysis of variance ŽExperiment 1. or Student’s t-test ŽExperiment 2.. All hormone concentrations are normalised for cell number, based on the DNA remaining in the well at the end of the experiment.
3. Results 3.1. Experiment 1 In all the three cultures, similar results were obtained for all parameters studied; a representative set of data is shown in Fig. 1. Both ovine GH and FSH produced significant Ž P - 0.001. dose-dependent increases in oestradiol production by cultured granulosa cells. The maximum responses were observed at the highest doses used, and were 402% ŽGH. and 460% ŽFSH. of control values. However, differences in oestradiol secretion between the 20 and 200 ngrml GH treatments and between the 200 and 2000 ngrml of FSH treatments were not significant. GH was a weak stimulator but significantly Ž P - 0.05. increased progesterone production only at the highest dose level used, whereas FSH stimulated progesterone production in a dose-dependent manner up to 200 and 2000 ngrml. Both ovine GH and FSH significantly Ž P - 0.001. increased IGF-1 production at all dose levels used. The maximum responses to GH and FSH were 514% and 528%,
266
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
Fig. 1. IGF-1, oestradiol and progesterone production by cultured ovine granulosa cells expressed as a function of the DNA remaining Žattached cells. at the end of the culture. Granulosa cells were incubated in medium supplemented with BCS Ž2.5%. during the initial 48 h of culture. They were then cultured for the next 48 h under serum-free conditions but in medium containing different doses of GH and FSH. The data are expressed as means"SEM of different hormones secreted during 49–96 h of culture. DNA content of cells was measured at the end of the second period of culture. There were six replicate wells per treatment and the experiment was carried out three times. Significant differences from control cultures are shown: A s P - 0.05, Bs P - 0.01 and C s P - 0.001; NSs non-significant.
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
267
respectively, compared with controls. However, in contrast to oestradiol production, the magnitude of the stimulation of IGF-1 production did not appear to be dose-dependent. The number of cells was significantly Ž P - 0.001. reduced as a result of both GH and FSH treatments in a dose-dependent manner. With both hormonal treatments, no difference was observed in cell numbers at the end of the culture between the two highest doses used.
Fig. 2. Effects of 2.5% BCS on oestradiol, progesterone and IGF-1 production by cultured ovine granulosa cells expressed as a function of the DNA remaining Žattached. at the end of the culture. Granulosa cells were cultured either in the presence Žq. or absence Žy. of BCS. Medium was supplemented with BCS only during the initial 48 h of incubation. Cells were cultured for the next 48 h under serum-free conditions. Data are expressed as means"SEM for each hormone secreted during 49–96 h of culture. DNA content of cells was measured at the end of the second period of culture. There were six replicate wells per treatment and the experiment was carried out four times.
268
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
3.2. Experiment 2 Granulosa cells secreted measurable quantities of all of the hormones Žoestradiol, progesterone and IGF-1. studied, both in the presence and absence of BCS treatment. While there was no significant difference in the quantities of oestradiol and IGF-1 produced, supplementation of the medium with BCS during the first 48 h of culture significantly Ž P - 0.01. increased progesterone output by the cells. However, the cell numbers at the end of culture were essentially the same in both treatment groups ŽFig. 2..
4. Discussion The present study suggests that ovine granulosa cells from seasonally anoestrous ewes can secrete IGF-1, and that both GH and FSH stimulate IGF-1 production in a dose-dependent manner. Moreover, the lack of difference between treatment groups in Experiment 2 Žwith and without BCS supplementation for the initial 48 h of incubation. indicates that BCS supplementation of the medium is not necessary for IGF-1 and oestradiol secretion by ovine granulosa cells, and that IGF-1 production is not related to the progestogenic status of cells. It is not clear whether GH andror FSH increased oestradiol concentrations by granulosa cells directly, or if their effects were mediated through increased IGF-1 production. However, considering that decreased serum andror intra-follicular IGF-1 concentrations impair the ability of the ovary to synthesize oestradiol ŽSchoppee et al., 1996., and that IGF-1 can stimulate oestradiol production by ovine granulosa cells ŽCampbell et al., 1996., it seems likely that the increases in IGF-1 concentrations contribute to the stimulatory effects of both GH and FSH on oestradiol production. However, direct effects of these two hormones cannot be ruled out because granulosa cells are known to have receptors for both GH and FSH ŽEckery et al., 1997.. IGF-1 was only measured after separation from its binding proteins; IGFBPs are known to be also produced by ovine granulosa cells ŽArmstrong et al., 1996., and is not clear what proportion of the secreted IGF-1 was biologically available to carry out its actions. The results of experiment show that granulosa cells produce IGF-1. An important question that arises from these results concerns the status of the cells which were capable of secreting IGF-1 when cultured in vitro: were they predominantly oestrogenic Žfollicular in phenotype. or predominantly progestogenic Žluteal in phenotype.? Previous studies in the ewe have shown that IGF-1 is produced by cultured granulosa cells, but that this occurs only after luteinisation. The increase in IGF-1 concentrations in the medium was linked to that of progesterone so far as IGF-1 concentrations started to rise only after progesterone levels in the medium had begun to decline ŽWathes et al., 1995.. To clarify whether IGF-1 production by cultured granulosa cells is a function only of luteinised cells and how the progestogenic status of the cells affects IGF-1 production, cells were cultured both in the presence and absence of BCS for the first 48 h of culture ŽExperiment 2.. Evidence from bovine studies suggests that addition of serum to granulosa cultures either initiates terminal differentiation towards luteinisation ŽGutier-
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
269
rez et al., 1995. or reduces the expression of the luteal phenotype ŽLuck, 1989; Luck et al., 1990. depending on the exact nature of the culture conditions. There is no equivalent information on the response of cultured anoestrous ovine cells to serum. In the present study, pre-treatment with serum for the first 48 h of culture resulted in an elevated progesterone secretion but allowed the cells to retain their oestrogenic response to FSH. Since luteinised ovine granulosa cells are known to produce little oestradiol ŽNiswender et al., 1994., this indicates that increased progesterone did not represent significant terminal differentiation towards luteinisation. The results clearly demonstrate that IGF-1 production was not at all affected by the degree of luteinisation. While such an observation of IGF-1 production by cultured granulosa cells accords with reports of IGF-1 mRNA expression by ovine granulosa cells ŽLeeuwenberg et al., 1995. and follicle walls ŽSpicer et al., 1995., it contrasts the conclusions of Perks et al. Ž1995. and Wathes et al. Ž1995.. Differences in results between the present study and that of Wathes et al. Ž1995. are difficult to explain directly on the basis of the animal model Žcyclic versus seasonally anoestrous ewes. because neither the number of preovulatory follicles nor their ability to synthesize oestradiol differ between the anoestrous and breeding season ŽMcNatty et al., 1984.. However, other differences between the studies may relate to the type of follicle from which the granulosa cells were harvested. Being from the anoestrous season, the follicles in the present study had never been exposed to high levels of progesterone, whereas those used by Wathes et al. Ž1995. were collected during the preovulatory phase of the oestrous cycle and would have been exposed to luteal phase concentrations of progesterone. The significance of such differences remains unclear at present. Whatever the physiological basis of such differences, the results of the present experiments clearly demonstrate local production of IGF-1 by granulosa cells harvested from follicles with the potential to mature successfully under appropriate gonadotrophic stimulation, and that this IGF-1 production can be increased by both FSH and GH. Involvement of GH in ovine follicle growth and development is documented ŽGong et al., 1996a; Joyce et al., 1998.. Moreover, it has also been shown that rGH treatment of ewes in vivo significantly increases the secretion of IGF-1 by ovarian follicles in vitro ŽGong et al., 1996a.. However, relatively few studies have shown direct effects of GH on cultured ovine ovarian cells. The present study demonstrates that ovine granulosa cells produce IGF-1 when cultured in vitro and suggests that local production of IGF-1 may contribute to an autocrine andror paracrine system, which enhances follicular growth and gonadotrophin or GH-induced differentiation. Treatments with GH and FSH reduced the number of granulosa cells at the end of incubations. It is not clear whether this was due to loss through apoptosis, necrosis or just reduced attachment in culture. Further experiments would be needed to examine these possibilities over the course of culture using a different experimental design. However, the possibility that the cell density used in these cultures might have been the reason for this, by rendering the medium acidic, can be ruled out as the number of cells plated was well within the range of the buffering capacity of the medium, and no change in the colour of the medium whatsoever was noticed at the end of incubations. Notwithstanding these cell losses, expressing hormone secretion on a per cell basis Žcells remaining at the end of culture. gives the best indication of changes in output per cell at
270
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
any time in the culture. The hormone assays were carried out on samples from the last 48 h of culture, following the last medium change, and over the period when GH and FSH treatments were applied; the data generated therefore represent the amount of the hormone produced by the cells in the dish over that time, irrespective of their condition or state of attachment. The contribution of unattached or loosely attached cells to the secretion is not known. Nevertheless, reduction in cell number and an apparent significant increase in the oestradiol output per cell suggests the presence of an inverse relationship between granulosa cell proliferation and steroidogenesis, which has already been documented for cultured ovine granulosa cells ŽMonniaux et al., 1994.. Similar effects of GH and FSH on proliferation ŽGong et al., 1993. and differentiation ŽGong et al., 1994. have also been reported for bovine granulosa cells. Other studies have also indicated that the incidence of mitosis Žcell proliferation. is inversely related to the stage of differentiation and hence steroidogenic capacity of sheep granulosa cells ŽCarson et al., 1989.. In summary, the results of the present study demonstrate that ovine granulosa cells can produce IGF-1 irrespective of their progestogenic status. Moreover, IGF-1 secretion by granulosa cells can be regulated by both GH and FSH in the ewe.
Acknowledgements The authors wish to thank the Ministry of Agriculture, Fisheries and Food for financial support, Dr. S. Spencer ŽMAF Tech, New Zealand. for the supplies of IGF-1 antiserum and NIDDK, National Hormone and Pituitary Programme, Baltimore, MA, USA for ovine GH and FSH.
References Adashi, E.Y., Resnick, C.E., D’Ercole, A.J., Svoboda, M.E., van Wyk, J.J., 1985. Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr. Rev. 6, 400–420. Adashi, E.Y., Resnick, C.E., Hernandez, E.R., May, J.V., Knecht, M., Svoboda, M.E., van Wyk, J.J., 1988. Insulin-like growth factor-I as an amplifier of follicle-stimulating hormone action: studies on mechanismŽs. and siteŽs. of action in cultured rat granulosa cells. Endocrinology 122, 1583–1591. Adashi, E.Y., Resnick, C.E., Hurwitz, A., Ricciarelli, E., Hernandez, E.R., Roberts, C.T., Leroith, D., Rosenfeld, R., 1991. Insulin-like growth factors: the ovarian connection. Hum. Reprod. 6, 1213–1219. Armstrong, D.G., Hogg, C.O., Campbell, B.K., Webb, R., 1996. Insulin-like growth factor ŽIGF.-binding protein production by primary cultures of ovine granulosa and theca cells. The effects of IGF-I, gonadotropin, and follicle size. Biol. Reprod. 55, 1163–1171. Baker, J., Hardy, M.P., Zhou, J., Bondy, C., Lupu, F., Bellve, A.R., Efstratiadis, A., 1996. Effects of an IGF-1 gene null mutation on mouse reproduction. Mol. Endocrinol. 10, 903–918. Campbell, B.K., Scaramuzzi, R.J., Webb, R., 1996. Induction and maintenance of oestradiol and immunoreactive inhibin production with FSH by ovine granulosa cells cultured in serum-free media. J. Reprod. Fertil. 106, 7–16. Carson, R.S., Zhang, Z., Hutchinson, L.A., Herington, A.C., Findlay, J.K., 1989. Growth factors in ovarian function. J. Reprod. Fertil. 85, 735–746. Davoren, J.B., Hsueh, A.J.W., Li, C.H., 1985. Somatomedin C augments FSH-induced differentiation of cultured rat granulosa cells. Am. J. Phys. 249, E26–E33.
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
271
Echternkamp, S.E., Spicer, L.J., Gregory, K.E., Canning, S.F., Hammond, J.M., 1990. Concentrations of insulin-like growth factor-1 in blood and ovarian follicular fluid of cattle selected for twins. Biol. Reprod. 43, 8–14. Eckery, D.C., Moeller, C.L., Nett, T.M., Sawyer, H.R., 1997. Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, and insulin-like growth factor-1 in sheep ovarian follicles. Biol. Reprod. 57, 507–513. Foxcroft, G.R., Shaw, H.J., Hunter, M.G., Booth, P.J., Lancaster, R.T., 1987. Relationships between luteinizing hormone, follicle stimulating hormone and prolactin secretion and ovarian follicular development in the weaned sow. Biol. Reprod. 36, 175–191. Giudice, L.C., 1992. Insulin-like growth factors and ovarian follicular development. Endocr. Rev. 13, 641–669. Gong, J.G., Bramley, T.A., Webb, R., 1991. The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones. Biol. Reprod. 45, 941–949. Gong, J.G., Campbell, B.K., Bramley, T.A., Webb, R., 1996a. Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes. Anim. Reprod. Sci. 41, 13–26. Gong, J.G., McBride, D., Bramley, T.A., Webb, R., 1993. Effects of recombinant bovine somatotrophin, insulin-like growth factor-1 and insulin on the proliferation of bovine granulosa cell steroidogenesis. J. Endocrinol. 139, 67–75. Gong, J.G., McBride, D., Bramley, T.A., Webb, R., 1994. Effects of recombinant bovine somatotrophin, insulin-like growth factor-1 and insulin on bovine granulosa cell steroidogenesis in vitro. J. Endocrinol. 143, 157–164. Gong, J.G., Wilmut, I., Bramley, T.A., Webb, R., 1996b. Pretreatment with recombinant bovine somatotropin enhances the superovulatory response to FSH in heifers. Theriogenology 45, 611–622. Gutierrez, C.G., Campbell, B.K., Bramley, T.A., Webb, R., 1995. Comparison of ultrastructural characteristics of bovine granulosa cells cultured in the presence or absence of serum. J. Reprod. Fertil. Abstr. Ser. 16, abstract number 20,10. Hammond, J.M., Lino, J., Baranao, S., Skaleris, D., Knight, A.B., Romanus, J.A., Rechler, M.M., 1985. Production of insulin-like growth factors by ovarian granulosa cells. Endocrinology. 117, 2553–2555. Hammond, J.M., Mondschien, J.S., Samaras, S.E., Canning, S.F., 1991. The ovarian insulin-like growth factors, a local amplification mechanism for steroidogenesis and hormone action. J. Steroid Biochem. Mol. Biol. 40, 411–416. Hunter, M.G., Southee, J.A., Lamming, G.E., 1988. Function of abnormal corpora lutea in vitro after GnRH-induced ovulation in the anoestrous ewe. J. Reprod. Fertil. 84, 139–148. Joyce, I.M., Khalid, M., Haresign, W., 1998. Growth hormone priming as an adjunct treatment in superovulatory protocols in the ewe alters follicle development but has no effect on ovulation rate. Theriogenology 50, 873–884. Khalid, M., Haresign, W., 1996. Relationships between concentrations of ovarian steroids, insulin-like growth factor-1 and IGF-binding proteins during follicular development in the ewe. Anim. Reprod. Sci. 41, 119–129. Labarca, C., Paigen, K., 1980. A simple, rapid and sensitive DNA procedure. Anal. Biochem. 102, 344–352. Leeuwenberg, B.R., Hudson, N.L., Moore, L.G., Hurst, P.R., McNatty, K.P., 1996. Peripheral and ovarian IGF-I concentrations during the ovine oestrous cycle. J. Endocrinol. 148, 281–289. Leeuwenberg, B.R., Hurst, P.R., McNatty, K.P., 1995. Expression of IGF-I mRNA in the ovine ovary. J. Mol. Endocrinol. 15, 251–258. Luck, M.R., 1989. Greatly elevated and sustained secretion of oxytocin by bovine granulosa cells in serum-free culture. J. Exp. Zool. 251, 361–366. Luck, M.R., Rodgers, R.J., Findlay, J.K., 1990. Secretion and gene expressions of inhibin, oxytocin and steroid hormones during the in vitro differentiation of bovine granulosa cells. Reprod. Fertil. Dev. 2, 11–25. Maruo, T., Hayashi, M., Matsuo, H., Ueda, Y., Morikawa, H., Mochizuki, M., 1988. Comparison of the facilitative roles of insulin and insulin-like growth factor-1 in the functional differentiation of granulosa cells, in vitro porcine model. Acta Endocrinol. ŽCopenhagen. 117, 230–240. McNatty, K.P., Hudson, N.L., Henderson, K.M., Lun, S., Heath, D.A., Gibb, M., Ball, K., McDiarmid, J.M.,
272
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
Thurley, D.C., 1984. Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep through the year. J. Reprod. Fertil. 70, 309–321. Meurer, K.A., Cox, N.M., Matamoros, I.A., Tubbs, R.C., 1991. Decreased follicular steroids and insulin-like growth factor-1 and increased atresia in diabetic gilts during follicular growth stimulated with PMSG. J. Reprod. Fertil. 91, 187–196. Monget, P., Monniaux, D., Pisselet, C., Durand, P., 1993. Changes in insulin-like growth factor-1 ŽIGF-I., IGF-II, and their binding proteins during growth and atresia of ovine ovarian follicles. Endocrinology 132, 1438–1446. Monniaux, D., Pisselet, C., 1992. Control of proliferation and differentiation of ovine granulosa cells by insulin-like growth factor-1 and follicle-stimulating hormone in vitro. Biol. Reprod. 46, 109–119. Monniaux, d., Pisselet, C., Fontaine, J., 1994. Uncoupling between proliferation and differentiation of ovine granulosa cells in vitro. J. Endocrinol. 142, 497–510. Niswender, G.D., Juengel, J.L., McGuire, W.J., Belfiore, C.J., Wiltbank, M.C., 1994. Luteal function: the estrous cycle and early pregnancy. Biol. Reprod. 50, 239–247. Oliver, J.E., Aitman, T.J., Powell, J.F., Wilson, C.A., Clayton, R.N., 1989. Insulin-like growth factor I gene expression in the rat ovary is confined to the granulosa cells of developing follicles. Endocrinology 124, 2671–2679. Perks, C.M., Denning-Kendall, P.A., Gilmour, R.S., Wathes, C.D., 1995. Localization of messenger ribonucleic acids for insulin-like growth factor-I ŽIGF-I., IGF-II and the type 1 IGF receptor in the ovine ovary throughout the estrous cycle. Endocrinology 136, 5266–5273. Samaras, S.E., Guthrie, H.D., Barber, J.A., Hammond, J.M., 1993. Expression of mRNAs for the insulin-like growth factors and their binding proteins during development of porcine ovarian follicles. Endocrinology 133, 2395–2398. Schams, D., 1987. Luteal peptides and intracellular communication. J. Reprod. Fertil., Suppl. 34, 87–99. Schoppee, P.D., Armstrong, J.D., Harvey, R.W., Whitacre, M.D., Felix, A., Campbell, R.M., 1996. Immunization against growth hormone releasing factor or chronic feed restriction initiated at 3.5 months of age reduces ovarian response to pulsatile administration of gonadotropin-releasing hormone at 6 months of age and delays onset of puberty in heifers. Biol. Reprod. 55, 87–98. Spicer, L.J., Echternkamp, S.E., Canning, S.F., Hammond, J.M., 1988. Relationships between concentrations of immunoreactive insulin-like growth factor-1 in follicular fluid and various biochemical markers of differentiation in bovine antral follicles. Biol. Reprod. 39, 573–580. Spicer, L.J., Echternkamp, S.E., Wong, E.A., Hamilton, D.T., Vernon, R.K., 1995. Serum hormones, follicular fluid steroids, insulin-like growth factors and their binding proteins, and ovarian IGF mRNA in sheep with different ovulation rates. J. Anim. Sci. 73, 1152–1163. Spicer, L.J., Enright, W.J., 1991. Concentrations of insulin-like growth factor-1 in follicular fluid of bovine ovarian follicles: effect of daily injections of a growth hormone-releasing factor analog andŽor. thyrotropin-releasing hormone. J. Anim. Sci. 69, 1133–1139. Veldhuis, J.D., Rodgers, R.J., Azimi, P., Garmey, J., Juchter, D., May, W., 1987. Mechanisms subserving the steroidogenic synergism between follicle-stimulating hormone and insulin-like growth factor-1 Žsomatomedin C.: alterations in cellular sterol metabolism in swine granulosa cells. J. Biol. Chem. 262, 7658–7664. Wathes, D.C., Perks, C.M., Davis, A.J., Denning-Kendall, P.A., 1995. Regulation of insulin-like growth factor-I and progesterone synthesis by insulin and growth hormone in the ovine ovary. Biol. Reprod. 53, 882–889.
Secretion of IGF-1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone M. Khalid a,) , W. Haresign b, M.R. Luck c a
c
Department of Farm Animal and Equine Medicine, The Royal Veterinary College, UniÕersity of London, Hawskshead Lane, North Hymms, Harfield, Hertfordshire AL97TA, UK b Animal Sciences Group, Welsh Institute of Rural Studies, UniÕersity of Wales Aberystwyth, Llanbadarn Campus, Aberystwyth SY23 3AL, UK DiÕision of Animal Physiology, School of Biological Sciences, UniÕersity of Nottingham, Sutton Bonington Campus, Loughborough L.C. LE12 5RD, UK Received 13 January 1999; received in revised form 9 August 1999; accepted 11 October 1999
Abstract Insulin-like growth factor-1 ŽIGF-1. is implicated in follicle development and is considered to mediate the actions of growth hormone ŽGH. and gonadotrophins at the ovarian level. However, the expression and secretion of IGF-1 by the ovary are controversial, partly because of species and cell-type specificity. The present study investigated whether IGF-1 is produced by ovine granulosa cells and whether its production is regulated by GH and follicle stimulating hormone ŽFSH.. Follicles ŽG 4.0 mm. were obtained from ewes during seasonal anoestrus. Granulosa cells were cultured for a total period of 96 h in Dulbecco’s modified Eagle’s medium ŽDMEM.rHam’s F-12 medium supplemented with BSA Ž0.1%, w:v., transferrin Ž0.5 mgrml. and testosterone Ž100 ngrml.. In the first set of experiments, cells were incubated in the presence of bovine calf serum ŽBCS. Ž2.5%. for the initial 48 h of culture. The cells were then cultured for the next 48 h in medium without BCS, but containing either GH Ž0, 2, 20, and 200 ngrml. or FSH Ž0, 20, 200, and 2000 ngrml.. The medium was assayed for oestradiol ŽE., progesterone ŽP. and IGF-1. There were six wells per treatment and the experiment was carried out four times. Control granulosa cells maintained both IGF-1 and E secretion, with only low levels of progesterone output. In all experiments, both GH and FSH produced significant Ž P - 0.001. dose-related increases in E, IGF-1 and P secretion into the medium. The maximum responses to GH Ž20 or 200 ngrml. were 402% for E and 528% for IGF-1 compared with controls. The maximum responses to FSH Ž200 or
)
Corresponding author. Tel.: q44-1707-666-333, ext. 2485; fax: q44-1707-660-671. E-mail address: [email protected] ŽM. Khalid..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 7 5 - 5
262
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
2000 ngrml. were 460% for E and 514% for IGF-1. The objective of the second set of experiments was to determine the effect of the progestogenic status of cells on IGF-1 production. Granulosa cells were cultured both in the presence and absence of BCS Ž2.5% in the medium. during the initial 48 h of culture. For the next 48 h, cells were cultured in serum-free medium. Addition of BCS to the medium during the initial 48 h of culture stimulated progesterone production. However, it did not affect either IGF-1 or oestradiol secretion between 49 and 96 h of culture, or the cell numbers at the end of culture. In conclusion, Ž1. IGF-1 is secreted by granulosa cells irrespective of their progestogenic status and Ž2. concomitant increases in E and IGF-1 production by granulosa cells as a result of GH andror FSH treatment suggest a role for GH and FSH in the regulation of ovarian function. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Sheep; Growth hormone; FSH; Granulosa cells; IGF-1 synthesis
1. Introduction Evidence from in vivo and in vitro studies implicates insulin-like growth factor-1 ŽIGF-1. in the control of follicle growth and development. Follicular development is arrested in IGF-1 knockout mice, and such animals do not ovulate even after administration of gonadotrophins ŽBaker et al., 1996.. Moreover, IGF-1 enhances follicle stimulating hormone ŽFSH.-stimulated oestrogen andror progesterone production in murine ŽAdashi et al., 1985; Davoren et al., 1985., bovine ŽSchams, 1987., porcine ŽVeldhuis et al., 1987; Maruo et al., 1988. and ovine ŽMonniaux and Pisselet, 1992; Campbell et al., 1996. granulosa cells. Relationships between follicular fluid IGF-1 concentrations and stage of follicle development have also been documented for a number of species, including cattle ŽSpicer and Enright, 1991; Spicer et al., 1988; Echternkamp et al., 1990., pigs ŽMeurer et al., 1991. and sheep ŽMonget et al., 1993; Khalid and Haresign, 1996.. IGF-1 plays an obligatory role in amplifying the effect of FSH andror growth hormone ŽGH. in rat ŽAdashi et al., 1985, 1988, 1991., pig ŽHammond et al., 1991. and sheep ŽMonniaux and Pisselet, 1992. granulosa cells. Increased levels of IGF-1 Žboth plasma and intrafollicular. as a result of GH treatment are associated with an increased number of small follicles in both cattle ŽGong et al., 1991. and sheep ŽGong et al., 1996a., as well as the number of oestrogenic follicles in FSH-treated ewes ŽJoyce et al., 1998.. In addition, a significant increase in the superovulatory response to FSH has been reported as a result of GH treatment in cattle ŽGong et al., 1996b.. On the other hand, decreased serum andror intra-follicular IGF-1 concentrations as a result of immunization against GH releasing factor or chronic feed restriction impairs the ability of the ovary to synthesize preovulatory concentrations of oestradiol, and delays the maturation of preovulatory follicles and the onset of puberty in heifers ŽSchoppee et al., 1996.. Immunoreactive IGF-1 is secreted by porcine ŽHammond et al., 1985., rat ŽAdashi et al., 1991. and human ŽGiudice, 1992. granulosa cells. However, there are conflicting reports regarding the secretion of IGF-1 by ovine granulosa cells. Incubating ovine granulosa cells with tritiated amino acids, a de novo synthesis has been shown by Leeuwenberg et al. Ž1996., whereas the data of Wathes et al. Ž1995. suggest that ovine granulosa cells do not secrete IGF-1, although there was a low level of production after they became luteinised.
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
263
While there is a clear expression of IGF-1 mRNA in rat ŽOliver et al., 1989. and pig ŽSamaras et al., 1993. granulosa cells, expression of IGF-1 mRNA in the ovine ovary is controversial. Using ovine and human riboprobes, Leeuwenberg et al. Ž1995. reported IGF-1 mRNA in both granulosa and theca cells, whereas Perks et al. Ž1995., using an oligo-probe, were unable to detect any IGF-1 mRNA in ovine follicles. Using RTPCR, Spicer et al. Ž1995. reported the presence of IGF-1 mRNA in ovine ovarian follicle walls, although to a lower extent than IGF-2 mRNA. The situation with regard to the ability of the sheep ovary to produce IGF-1 is therefore confusing and requires clarification. One way in which this might be achieved is to use cell culture techniques. The present study was therefore performed to investigate whether IGF-1 is produced by ovine granulosa cells and if so, whether its production is regulated by GH andror FSH ŽExperiment 1.. Another objective of this study was to investigate whether IGF-1 production is related to the progestogenic status of cultured granulosa cells ŽExperiment 2..
2. Materials and methods 2.1. Culture of oÕine granulosa cells All the reagents were obtained from Sigma ŽPoole, Dorset, UK., unless otherwise stated. Ovaries were obtained from seasonally anoestrous ewes of mixed breeds, slaughtered at a commercial slaughterhouse. They were collected into culture medium Žbicarbonate-buffered Dulbecco’s modified Eagle’s medium ŽDMEM.rHam’s F-12 Ž1:1 v:v. supplemented with BSA Ž0.15%, wrv., transferrin Ž0.5 mgrml., testosterone Ž30 mM. and antibiotics Žpenicillin and streptomycin; Gibco Life Technologies, Paisley, Strathclyde, UK. at 378C and transported to the laboratory within 1 h after slaughter. Visually normal healthy follicles Žwith good vascular supply and clearrtransparent follicular fluid. of G 4.0 mm in diameter were dissected out and granulosa cells isolated as follows. After aspiration of follicular fluid, each follicle was sliced open in culture medium and granulosa cells were removed by gently scrapping the interior surface of the follicle wall with an inoculation loop. Granulosa cell suspensions were filtered through nylon hose mesh and centrifuged at 300 = g for 10 min. The supernatant was discarded and the resulting pellet was resuspended in the medium. The number of cells was estimated by measuring the amount of DNA in a sonicated aliquot of the granulosa cell suspension. Granulosa cells were plated at a density of 1.4 = 10 6 cellsrwell on collagen type 1 Ž1%.-pre-treated 48-well plates ŽFalcon; Becton Dickinson UK. as described by Luck Ž1989.. The cells were then cultured in 0.5 ml of medium in a humidified incubator at 378C in atmospheric air with 5% CO 2 . 2.1.1. Experiment 1 The objective of this experiment was to investigate whether the ovine granulosa cells produce IGF-1 and if so, whether this is regulated by GH andror FSH. To obtain optimal attachment, cells were maintained in the presence of 2.5% bovine calf serum
264
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
ŽBCS; Life Technologies. in the culture medium for the first 48 h. After this time, granulosa cells were washed with 0.5 ml serum-free medium and incubations were then continued for the next 48 h in serum-free medium containing ovine GH Ž0, 2, 20, and 200 ngrml. or ovine FSH Ž0, 20, 200, and 200 ngrml.. Both ovine GH ŽoGH-15. and ovine FSH ŽoFSH-19-SIAFP. were obtained from NIDDK, National Hormone and Pituitary Programme, Baltimore, MA, USA. At the end of the 96-h incubation period, medium was collected and stored at y208C until assayed for oestradiol, progesterone and IGF-1. After removal of the medium, cells in each well were gently washed and stored in 500 ml of DNA assay buffer Ž0.05 M phosphate, 0.9% wrv NaCl, 2 mM EDTA, pH 7.4. at y208C until the measurement of their DNA content. 2.1.2. Experiment 2 The objective of this experiment was to investigate whether there was any relationship between progestogenic status of cultured granulosa cells and IGF-1 production. Granulosa cells were cultured either with or without 2.5% BCS for the first 48 h in order to alter their basal level of progesterone secretion. The aim of culturing granulosa cells without BCS was to reducerblock the process of spontaneous luteinisation. The cells were then cultured for the next 48 h in serum-free medium. At the end of the second incubation period, the medium was collected and analysed, and cells counted as described for Experiment 1. 2.2. DNA assay The DNA content of granulosa cell suspensions was measured by the rapid fluorescence technique of Labarca and Paigen Ž1980. using a fluorimeter ŽJENWAY 6200.. Briefly, cell suspensions were sonicated and 250-ml aliquots of sonicated cells were mixed with fluorescence reagent Žbisbenzimide, B2883, Sigma Chemical Company, Poole, Dorset, UK. and analyzed spectrofluorometrically Žexcitation: 356 nm; emission: 458 nm.. A standard curve was made using calf thymus DNA ŽSigma.. 2.3. Oestradiol assay Measurements of oestradiol concentrations were carried out in unextracted samples of culture medium using the method validated by Foxcroft et al. Ž1987.. The limit of sensitivity of the assay was 16 pgrml. The inter- and intra-assay coefficients of variation were 12.1% and 8.9%, respectively. 2.4. Progesterone assay The concentrations of progesterone in culture medium samples were determined in a direct assay described by Hunter et al. Ž1988.. Culture medium blanks included in the assay showed no cross-reactivity and had progesterone values below the limit of sensitivity of the assay Ž0.12 ngrml within the present study.. The inter- and intra-assay coefficients of variation were 7.6% and 8.1%, respectively.
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
265
2.5. IGF-1 assay Samples of the culture medium Ž500 ml. were freeze-dried and then re-constituted in 50-ml distilled water. IGF-1 concentrations were measured using the radioimmunoassay technique described previously ŽKhalid and Haresign, 1996. and modified recently in our laboratory ŽJoyce et al., 1998.. The antiserum for IGF-1 used in this study had essentially no cross-reactivity with IGF-2 Ž0.68%.. Briefly, acid–ethanol mixture was added to the samples in a 4:1 ratio, incubated for 30 min and centrifuged at 2100 = g and 48C. The supernatant was then decanted and mixed with 30 ng of IGF-2 ŽPeninsula Europe Laboratories, St. Helens, Merseyside, UK., neutralised with 0.855 M Tris buffer, and incubated for 30 min prior to assay. Using this approach, serial dilutions of extracted plasma, follicular fluid and culture medium samples showed parallelism with the IGF-1 standard curve. This method gave a recovery of cold IGF-1 in medium of 85–100%. For every 0.5-, 1.5-, 5.0- and 15.0-ng IGF-1 added in the extracted medium, 0.45-, 1.43-, 5.0- and 14.85-ng IGF-1 were recovered, respectively. The inter- and intra-assay coefficients of variation were 9.8% and 11.4%, respectively, while the limit of sensitivity of the assay was 0.20 ngrtube. 2.6. Statistical analysis There were six replicate wells per treatment, and Experiments 1 and 2 were carried out three and four times, respectively. Similar results were obtained within different replicates of a particular experiment, despite variations in the absolute levels of hormones secreted by different batches of cells. Differences in hormone concentrations between different treatment groups were assessed by analysis of variance ŽExperiment 1. or Student’s t-test ŽExperiment 2.. All hormone concentrations are normalised for cell number, based on the DNA remaining in the well at the end of the experiment.
3. Results 3.1. Experiment 1 In all the three cultures, similar results were obtained for all parameters studied; a representative set of data is shown in Fig. 1. Both ovine GH and FSH produced significant Ž P - 0.001. dose-dependent increases in oestradiol production by cultured granulosa cells. The maximum responses were observed at the highest doses used, and were 402% ŽGH. and 460% ŽFSH. of control values. However, differences in oestradiol secretion between the 20 and 200 ngrml GH treatments and between the 200 and 2000 ngrml of FSH treatments were not significant. GH was a weak stimulator but significantly Ž P - 0.05. increased progesterone production only at the highest dose level used, whereas FSH stimulated progesterone production in a dose-dependent manner up to 200 and 2000 ngrml. Both ovine GH and FSH significantly Ž P - 0.001. increased IGF-1 production at all dose levels used. The maximum responses to GH and FSH were 514% and 528%,
266
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
Fig. 1. IGF-1, oestradiol and progesterone production by cultured ovine granulosa cells expressed as a function of the DNA remaining Žattached cells. at the end of the culture. Granulosa cells were incubated in medium supplemented with BCS Ž2.5%. during the initial 48 h of culture. They were then cultured for the next 48 h under serum-free conditions but in medium containing different doses of GH and FSH. The data are expressed as means"SEM of different hormones secreted during 49–96 h of culture. DNA content of cells was measured at the end of the second period of culture. There were six replicate wells per treatment and the experiment was carried out three times. Significant differences from control cultures are shown: A s P - 0.05, Bs P - 0.01 and C s P - 0.001; NSs non-significant.
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
267
respectively, compared with controls. However, in contrast to oestradiol production, the magnitude of the stimulation of IGF-1 production did not appear to be dose-dependent. The number of cells was significantly Ž P - 0.001. reduced as a result of both GH and FSH treatments in a dose-dependent manner. With both hormonal treatments, no difference was observed in cell numbers at the end of the culture between the two highest doses used.
Fig. 2. Effects of 2.5% BCS on oestradiol, progesterone and IGF-1 production by cultured ovine granulosa cells expressed as a function of the DNA remaining Žattached. at the end of the culture. Granulosa cells were cultured either in the presence Žq. or absence Žy. of BCS. Medium was supplemented with BCS only during the initial 48 h of incubation. Cells were cultured for the next 48 h under serum-free conditions. Data are expressed as means"SEM for each hormone secreted during 49–96 h of culture. DNA content of cells was measured at the end of the second period of culture. There were six replicate wells per treatment and the experiment was carried out four times.
268
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
3.2. Experiment 2 Granulosa cells secreted measurable quantities of all of the hormones Žoestradiol, progesterone and IGF-1. studied, both in the presence and absence of BCS treatment. While there was no significant difference in the quantities of oestradiol and IGF-1 produced, supplementation of the medium with BCS during the first 48 h of culture significantly Ž P - 0.01. increased progesterone output by the cells. However, the cell numbers at the end of culture were essentially the same in both treatment groups ŽFig. 2..
4. Discussion The present study suggests that ovine granulosa cells from seasonally anoestrous ewes can secrete IGF-1, and that both GH and FSH stimulate IGF-1 production in a dose-dependent manner. Moreover, the lack of difference between treatment groups in Experiment 2 Žwith and without BCS supplementation for the initial 48 h of incubation. indicates that BCS supplementation of the medium is not necessary for IGF-1 and oestradiol secretion by ovine granulosa cells, and that IGF-1 production is not related to the progestogenic status of cells. It is not clear whether GH andror FSH increased oestradiol concentrations by granulosa cells directly, or if their effects were mediated through increased IGF-1 production. However, considering that decreased serum andror intra-follicular IGF-1 concentrations impair the ability of the ovary to synthesize oestradiol ŽSchoppee et al., 1996., and that IGF-1 can stimulate oestradiol production by ovine granulosa cells ŽCampbell et al., 1996., it seems likely that the increases in IGF-1 concentrations contribute to the stimulatory effects of both GH and FSH on oestradiol production. However, direct effects of these two hormones cannot be ruled out because granulosa cells are known to have receptors for both GH and FSH ŽEckery et al., 1997.. IGF-1 was only measured after separation from its binding proteins; IGFBPs are known to be also produced by ovine granulosa cells ŽArmstrong et al., 1996., and is not clear what proportion of the secreted IGF-1 was biologically available to carry out its actions. The results of experiment show that granulosa cells produce IGF-1. An important question that arises from these results concerns the status of the cells which were capable of secreting IGF-1 when cultured in vitro: were they predominantly oestrogenic Žfollicular in phenotype. or predominantly progestogenic Žluteal in phenotype.? Previous studies in the ewe have shown that IGF-1 is produced by cultured granulosa cells, but that this occurs only after luteinisation. The increase in IGF-1 concentrations in the medium was linked to that of progesterone so far as IGF-1 concentrations started to rise only after progesterone levels in the medium had begun to decline ŽWathes et al., 1995.. To clarify whether IGF-1 production by cultured granulosa cells is a function only of luteinised cells and how the progestogenic status of the cells affects IGF-1 production, cells were cultured both in the presence and absence of BCS for the first 48 h of culture ŽExperiment 2.. Evidence from bovine studies suggests that addition of serum to granulosa cultures either initiates terminal differentiation towards luteinisation ŽGutier-
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
269
rez et al., 1995. or reduces the expression of the luteal phenotype ŽLuck, 1989; Luck et al., 1990. depending on the exact nature of the culture conditions. There is no equivalent information on the response of cultured anoestrous ovine cells to serum. In the present study, pre-treatment with serum for the first 48 h of culture resulted in an elevated progesterone secretion but allowed the cells to retain their oestrogenic response to FSH. Since luteinised ovine granulosa cells are known to produce little oestradiol ŽNiswender et al., 1994., this indicates that increased progesterone did not represent significant terminal differentiation towards luteinisation. The results clearly demonstrate that IGF-1 production was not at all affected by the degree of luteinisation. While such an observation of IGF-1 production by cultured granulosa cells accords with reports of IGF-1 mRNA expression by ovine granulosa cells ŽLeeuwenberg et al., 1995. and follicle walls ŽSpicer et al., 1995., it contrasts the conclusions of Perks et al. Ž1995. and Wathes et al. Ž1995.. Differences in results between the present study and that of Wathes et al. Ž1995. are difficult to explain directly on the basis of the animal model Žcyclic versus seasonally anoestrous ewes. because neither the number of preovulatory follicles nor their ability to synthesize oestradiol differ between the anoestrous and breeding season ŽMcNatty et al., 1984.. However, other differences between the studies may relate to the type of follicle from which the granulosa cells were harvested. Being from the anoestrous season, the follicles in the present study had never been exposed to high levels of progesterone, whereas those used by Wathes et al. Ž1995. were collected during the preovulatory phase of the oestrous cycle and would have been exposed to luteal phase concentrations of progesterone. The significance of such differences remains unclear at present. Whatever the physiological basis of such differences, the results of the present experiments clearly demonstrate local production of IGF-1 by granulosa cells harvested from follicles with the potential to mature successfully under appropriate gonadotrophic stimulation, and that this IGF-1 production can be increased by both FSH and GH. Involvement of GH in ovine follicle growth and development is documented ŽGong et al., 1996a; Joyce et al., 1998.. Moreover, it has also been shown that rGH treatment of ewes in vivo significantly increases the secretion of IGF-1 by ovarian follicles in vitro ŽGong et al., 1996a.. However, relatively few studies have shown direct effects of GH on cultured ovine ovarian cells. The present study demonstrates that ovine granulosa cells produce IGF-1 when cultured in vitro and suggests that local production of IGF-1 may contribute to an autocrine andror paracrine system, which enhances follicular growth and gonadotrophin or GH-induced differentiation. Treatments with GH and FSH reduced the number of granulosa cells at the end of incubations. It is not clear whether this was due to loss through apoptosis, necrosis or just reduced attachment in culture. Further experiments would be needed to examine these possibilities over the course of culture using a different experimental design. However, the possibility that the cell density used in these cultures might have been the reason for this, by rendering the medium acidic, can be ruled out as the number of cells plated was well within the range of the buffering capacity of the medium, and no change in the colour of the medium whatsoever was noticed at the end of incubations. Notwithstanding these cell losses, expressing hormone secretion on a per cell basis Žcells remaining at the end of culture. gives the best indication of changes in output per cell at
270
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
any time in the culture. The hormone assays were carried out on samples from the last 48 h of culture, following the last medium change, and over the period when GH and FSH treatments were applied; the data generated therefore represent the amount of the hormone produced by the cells in the dish over that time, irrespective of their condition or state of attachment. The contribution of unattached or loosely attached cells to the secretion is not known. Nevertheless, reduction in cell number and an apparent significant increase in the oestradiol output per cell suggests the presence of an inverse relationship between granulosa cell proliferation and steroidogenesis, which has already been documented for cultured ovine granulosa cells ŽMonniaux et al., 1994.. Similar effects of GH and FSH on proliferation ŽGong et al., 1993. and differentiation ŽGong et al., 1994. have also been reported for bovine granulosa cells. Other studies have also indicated that the incidence of mitosis Žcell proliferation. is inversely related to the stage of differentiation and hence steroidogenic capacity of sheep granulosa cells ŽCarson et al., 1989.. In summary, the results of the present study demonstrate that ovine granulosa cells can produce IGF-1 irrespective of their progestogenic status. Moreover, IGF-1 secretion by granulosa cells can be regulated by both GH and FSH in the ewe.
Acknowledgements The authors wish to thank the Ministry of Agriculture, Fisheries and Food for financial support, Dr. S. Spencer ŽMAF Tech, New Zealand. for the supplies of IGF-1 antiserum and NIDDK, National Hormone and Pituitary Programme, Baltimore, MA, USA for ovine GH and FSH.
References Adashi, E.Y., Resnick, C.E., D’Ercole, A.J., Svoboda, M.E., van Wyk, J.J., 1985. Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr. Rev. 6, 400–420. Adashi, E.Y., Resnick, C.E., Hernandez, E.R., May, J.V., Knecht, M., Svoboda, M.E., van Wyk, J.J., 1988. Insulin-like growth factor-I as an amplifier of follicle-stimulating hormone action: studies on mechanismŽs. and siteŽs. of action in cultured rat granulosa cells. Endocrinology 122, 1583–1591. Adashi, E.Y., Resnick, C.E., Hurwitz, A., Ricciarelli, E., Hernandez, E.R., Roberts, C.T., Leroith, D., Rosenfeld, R., 1991. Insulin-like growth factors: the ovarian connection. Hum. Reprod. 6, 1213–1219. Armstrong, D.G., Hogg, C.O., Campbell, B.K., Webb, R., 1996. Insulin-like growth factor ŽIGF.-binding protein production by primary cultures of ovine granulosa and theca cells. The effects of IGF-I, gonadotropin, and follicle size. Biol. Reprod. 55, 1163–1171. Baker, J., Hardy, M.P., Zhou, J., Bondy, C., Lupu, F., Bellve, A.R., Efstratiadis, A., 1996. Effects of an IGF-1 gene null mutation on mouse reproduction. Mol. Endocrinol. 10, 903–918. Campbell, B.K., Scaramuzzi, R.J., Webb, R., 1996. Induction and maintenance of oestradiol and immunoreactive inhibin production with FSH by ovine granulosa cells cultured in serum-free media. J. Reprod. Fertil. 106, 7–16. Carson, R.S., Zhang, Z., Hutchinson, L.A., Herington, A.C., Findlay, J.K., 1989. Growth factors in ovarian function. J. Reprod. Fertil. 85, 735–746. Davoren, J.B., Hsueh, A.J.W., Li, C.H., 1985. Somatomedin C augments FSH-induced differentiation of cultured rat granulosa cells. Am. J. Phys. 249, E26–E33.
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
271
Echternkamp, S.E., Spicer, L.J., Gregory, K.E., Canning, S.F., Hammond, J.M., 1990. Concentrations of insulin-like growth factor-1 in blood and ovarian follicular fluid of cattle selected for twins. Biol. Reprod. 43, 8–14. Eckery, D.C., Moeller, C.L., Nett, T.M., Sawyer, H.R., 1997. Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, and insulin-like growth factor-1 in sheep ovarian follicles. Biol. Reprod. 57, 507–513. Foxcroft, G.R., Shaw, H.J., Hunter, M.G., Booth, P.J., Lancaster, R.T., 1987. Relationships between luteinizing hormone, follicle stimulating hormone and prolactin secretion and ovarian follicular development in the weaned sow. Biol. Reprod. 36, 175–191. Giudice, L.C., 1992. Insulin-like growth factors and ovarian follicular development. Endocr. Rev. 13, 641–669. Gong, J.G., Bramley, T.A., Webb, R., 1991. The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones. Biol. Reprod. 45, 941–949. Gong, J.G., Campbell, B.K., Bramley, T.A., Webb, R., 1996a. Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes. Anim. Reprod. Sci. 41, 13–26. Gong, J.G., McBride, D., Bramley, T.A., Webb, R., 1993. Effects of recombinant bovine somatotrophin, insulin-like growth factor-1 and insulin on the proliferation of bovine granulosa cell steroidogenesis. J. Endocrinol. 139, 67–75. Gong, J.G., McBride, D., Bramley, T.A., Webb, R., 1994. Effects of recombinant bovine somatotrophin, insulin-like growth factor-1 and insulin on bovine granulosa cell steroidogenesis in vitro. J. Endocrinol. 143, 157–164. Gong, J.G., Wilmut, I., Bramley, T.A., Webb, R., 1996b. Pretreatment with recombinant bovine somatotropin enhances the superovulatory response to FSH in heifers. Theriogenology 45, 611–622. Gutierrez, C.G., Campbell, B.K., Bramley, T.A., Webb, R., 1995. Comparison of ultrastructural characteristics of bovine granulosa cells cultured in the presence or absence of serum. J. Reprod. Fertil. Abstr. Ser. 16, abstract number 20,10. Hammond, J.M., Lino, J., Baranao, S., Skaleris, D., Knight, A.B., Romanus, J.A., Rechler, M.M., 1985. Production of insulin-like growth factors by ovarian granulosa cells. Endocrinology. 117, 2553–2555. Hammond, J.M., Mondschien, J.S., Samaras, S.E., Canning, S.F., 1991. The ovarian insulin-like growth factors, a local amplification mechanism for steroidogenesis and hormone action. J. Steroid Biochem. Mol. Biol. 40, 411–416. Hunter, M.G., Southee, J.A., Lamming, G.E., 1988. Function of abnormal corpora lutea in vitro after GnRH-induced ovulation in the anoestrous ewe. J. Reprod. Fertil. 84, 139–148. Joyce, I.M., Khalid, M., Haresign, W., 1998. Growth hormone priming as an adjunct treatment in superovulatory protocols in the ewe alters follicle development but has no effect on ovulation rate. Theriogenology 50, 873–884. Khalid, M., Haresign, W., 1996. Relationships between concentrations of ovarian steroids, insulin-like growth factor-1 and IGF-binding proteins during follicular development in the ewe. Anim. Reprod. Sci. 41, 119–129. Labarca, C., Paigen, K., 1980. A simple, rapid and sensitive DNA procedure. Anal. Biochem. 102, 344–352. Leeuwenberg, B.R., Hudson, N.L., Moore, L.G., Hurst, P.R., McNatty, K.P., 1996. Peripheral and ovarian IGF-I concentrations during the ovine oestrous cycle. J. Endocrinol. 148, 281–289. Leeuwenberg, B.R., Hurst, P.R., McNatty, K.P., 1995. Expression of IGF-I mRNA in the ovine ovary. J. Mol. Endocrinol. 15, 251–258. Luck, M.R., 1989. Greatly elevated and sustained secretion of oxytocin by bovine granulosa cells in serum-free culture. J. Exp. Zool. 251, 361–366. Luck, M.R., Rodgers, R.J., Findlay, J.K., 1990. Secretion and gene expressions of inhibin, oxytocin and steroid hormones during the in vitro differentiation of bovine granulosa cells. Reprod. Fertil. Dev. 2, 11–25. Maruo, T., Hayashi, M., Matsuo, H., Ueda, Y., Morikawa, H., Mochizuki, M., 1988. Comparison of the facilitative roles of insulin and insulin-like growth factor-1 in the functional differentiation of granulosa cells, in vitro porcine model. Acta Endocrinol. ŽCopenhagen. 117, 230–240. McNatty, K.P., Hudson, N.L., Henderson, K.M., Lun, S., Heath, D.A., Gibb, M., Ball, K., McDiarmid, J.M.,
272
M. Khalid et al.r Animal Reproduction Science 58 (2000) 261–272
Thurley, D.C., 1984. Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep through the year. J. Reprod. Fertil. 70, 309–321. Meurer, K.A., Cox, N.M., Matamoros, I.A., Tubbs, R.C., 1991. Decreased follicular steroids and insulin-like growth factor-1 and increased atresia in diabetic gilts during follicular growth stimulated with PMSG. J. Reprod. Fertil. 91, 187–196. Monget, P., Monniaux, D., Pisselet, C., Durand, P., 1993. Changes in insulin-like growth factor-1 ŽIGF-I., IGF-II, and their binding proteins during growth and atresia of ovine ovarian follicles. Endocrinology 132, 1438–1446. Monniaux, D., Pisselet, C., 1992. Control of proliferation and differentiation of ovine granulosa cells by insulin-like growth factor-1 and follicle-stimulating hormone in vitro. Biol. Reprod. 46, 109–119. Monniaux, d., Pisselet, C., Fontaine, J., 1994. Uncoupling between proliferation and differentiation of ovine granulosa cells in vitro. J. Endocrinol. 142, 497–510. Niswender, G.D., Juengel, J.L., McGuire, W.J., Belfiore, C.J., Wiltbank, M.C., 1994. Luteal function: the estrous cycle and early pregnancy. Biol. Reprod. 50, 239–247. Oliver, J.E., Aitman, T.J., Powell, J.F., Wilson, C.A., Clayton, R.N., 1989. Insulin-like growth factor I gene expression in the rat ovary is confined to the granulosa cells of developing follicles. Endocrinology 124, 2671–2679. Perks, C.M., Denning-Kendall, P.A., Gilmour, R.S., Wathes, C.D., 1995. Localization of messenger ribonucleic acids for insulin-like growth factor-I ŽIGF-I., IGF-II and the type 1 IGF receptor in the ovine ovary throughout the estrous cycle. Endocrinology 136, 5266–5273. Samaras, S.E., Guthrie, H.D., Barber, J.A., Hammond, J.M., 1993. Expression of mRNAs for the insulin-like growth factors and their binding proteins during development of porcine ovarian follicles. Endocrinology 133, 2395–2398. Schams, D., 1987. Luteal peptides and intracellular communication. J. Reprod. Fertil., Suppl. 34, 87–99. Schoppee, P.D., Armstrong, J.D., Harvey, R.W., Whitacre, M.D., Felix, A., Campbell, R.M., 1996. Immunization against growth hormone releasing factor or chronic feed restriction initiated at 3.5 months of age reduces ovarian response to pulsatile administration of gonadotropin-releasing hormone at 6 months of age and delays onset of puberty in heifers. Biol. Reprod. 55, 87–98. Spicer, L.J., Echternkamp, S.E., Canning, S.F., Hammond, J.M., 1988. Relationships between concentrations of immunoreactive insulin-like growth factor-1 in follicular fluid and various biochemical markers of differentiation in bovine antral follicles. Biol. Reprod. 39, 573–580. Spicer, L.J., Echternkamp, S.E., Wong, E.A., Hamilton, D.T., Vernon, R.K., 1995. Serum hormones, follicular fluid steroids, insulin-like growth factors and their binding proteins, and ovarian IGF mRNA in sheep with different ovulation rates. J. Anim. Sci. 73, 1152–1163. Spicer, L.J., Enright, W.J., 1991. Concentrations of insulin-like growth factor-1 in follicular fluid of bovine ovarian follicles: effect of daily injections of a growth hormone-releasing factor analog andŽor. thyrotropin-releasing hormone. J. Anim. Sci. 69, 1133–1139. Veldhuis, J.D., Rodgers, R.J., Azimi, P., Garmey, J., Juchter, D., May, W., 1987. Mechanisms subserving the steroidogenic synergism between follicle-stimulating hormone and insulin-like growth factor-1 Žsomatomedin C.: alterations in cellular sterol metabolism in swine granulosa cells. J. Biol. Chem. 262, 7658–7664. Wathes, D.C., Perks, C.M., Davis, A.J., Denning-Kendall, P.A., 1995. Regulation of insulin-like growth factor-I and progesterone synthesis by insulin and growth hormone in the ovine ovary. Biol. Reprod. 53, 882–889.