1 Division of Surgery, Department of Hospital Medicine, Bristol Royal Infirmary, Bristol BS2 8HW, 2 Centre for Cardiovascular Genetics, BHF Laboratories, Department of Medicine, The Rayne Building, 5 University Street, London WC1E 6JF and 3 Departments of Basic Medical Sciences and Clinical Developmental Sciences, St Georges Hospital Medical School, London SW17 0RE, UK
4 To whom correspondence should be addressed. e-mail: Claire.M.Perks{at}bristol.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: apoptosis/C2-ceramide/granulosa cells/prolactin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The plasma membrane is the site of sphingomyelin hydrolysis, which is now recognized as an important pathway of signal transduction. Ceramide is one product of sphingomyelin hydrolysis and has been implicated as an important mediator of cell death (Obeid et al., 1993). Ceramide has been used previously to induce apoptosis in granulosa cells from a number of different species including rat (Kim, J.M. et al., 1999
), mice (Kim, J.H. et al., 1999
) and hen (Witty et al., 1996
).
Prolactin is synthesized in the anterior pituitary but other prolactin sources have now been identified including the endometrium (Prigent-Tessier et al., 2001) and prostate (Nevalainen et al., 1997
). It is not yet conclusively determined if prolactin is synthesized locally within the ovary; however, prolactin receptors have been immunolocalized to the cell membrane of luteinized granulosa cells and in paraffin-embedded preparations of isolated human granulosa cells (Vlahos et al., 2001
). The absence of prolactin receptors in secondary and early antral follicles in these studies perhaps suggests a role for prolactin within the ovary at the time of ovulation and beyond. Prolactin has established roles in both cellular proliferation and differentiation and has also been identified as an anti-apoptotic agent for a number of different cell types including ratNb2 lymphoma cells (LaVoie and Witorsch, 1995
), rat mammary (Travers et al., 1996
) and rat dorsal and lateral prostate epithelial cells (Ahonen et al., 1999
).
Despite the regulated expression of prolactin receptors on human granulosa cells, the potential role for prolactin in modulating human follicular atresia and its role in corpus luteum function, whether its source is local or from the circulation, is as yet unknown. The aim of this study was to establish a model of ceramide-induced apoptosis in human ovarian granulosa cells in which to examine the role of prolactin.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and culture of granulosa cells
Cells were collected at the time of aspiration of follicles from women undergoing IVF. The cells were recovered by centrifugation at 444 g for 20 min and granulosa cells were then purified on a Percoll gradient by centrifugation at 367 g for 25 min. Granulosa cells were grown in a humidified 5% CO2 atmosphere at 37°C. Cells were maintained in Dulbeccos modified Eagles medium (DMEM) with glutamax-1 supplemented with 10% fetal calf serum, penicillin (50 IU/ml) and streptomycin (5 mg/ml) growth media (GM). Experiments were performed on cells in Phenol Red-free, serum-free DMEM and Hams nutrient mix F-12 (SFM) with sodium bicarbonate (0.12%), bovine serum albumin (0.2 mg/ml), transferrin (0.01 mg/ml) and supplemented as before.
Dosing protocol
Granulosa cells were grown in GM for 48 h before switching to SFM for a further 24 h prior to dosing. Cells were either (i) incubated with increasing doses of C2-ceramide or prolactin for 24 h or (ii) co-incubated with an apoptotic dose of C2-ceramide with or without prolactin for 24 h. An apoptotic dose of C2-ceramide was chosen to give 3040% cell death to allow any modulation by prolactin to be evident.
Measurement of progesterone
Progesterone was measured in the overlying medium by radioimmunoassay as described previously (Willis et al., 1996).
MTT assay
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl Blue] reagent is converted into a coloured, water-insoluble, formazan salt by the metabolic activity of viable cells and can be used as a crude measure of cell viability. Cells were seeded at 2.5x104/ml (150 µl growth medium) in 96-well plates and were allowed to grow as outlined above. Growth medium was replaced with SFM (100 µl) 24 h prior to dosing. MTT reagent (7.5 mg/ml) in phosphate-buffered saline (PBS) was added to the cells (10 µl/well) and the cultures were incubated for 30 min at 37°C. The reaction was stopped by the addition of acidified Triton buffer [0.1 mol/l HCl, 10% (v/v) Triton X-100; 50 µl/well] and the tetrazolium crystals were dissolved by mixing on a Titertek plate shaker for 20 min at room temperature. The samples were measured on a Biorad 450 plate reader at a test wavelength of 595 nm and a reference wavelength of 650 nm.
Trypan Blue dye exclusion
The percentage of dead cells was calculated by Trypan Blue dye exclusion.
Flow cytometry
This technique was used to confirm and measure the presence and quantity of apoptosis in a given sample of cells. In apoptotic cells, fragmented DNA is washed out of fixed cells resulting in lower DNA staining of the cells which appear as a pre-G1 peak following cell cycle analysis. Cells (0.10.2x106) were washed in PBS and fixed for 30 min by the addition of 70% ethanol (1 ml). Cells were pelleted (720 g; 5 min) and washed with PBS. The supernatant was removed and the cells were resuspended in reaction buffer (propidium iodide, 0.05 mg/ml; sodium citrate, 0.1%; RNAse A, 0.02 mg/ml; Nonidet P-40, 0.3%; pH 8.3) and incubated at 4°C for 30 min. All cells were then measured on a FACS Calibur flow cytometer (Becton Dickinson, UK) with an argon laser at 488 nm for excitation and analysed using Cell Quest (Becton Dickinson).
Annexin-VFITC staining
Annexin staining of C2-treated and untreated cells was performed using an annexin-Vfluorescein isothiocyanate (FITC) kit according to the manufacturers instructions (Biowhittaker UK LtdBoehringer Ingelheim Bioproducts Partnership, Germany) to confirm that C2-ceramide-induced apoptotic cell death. When cells undergo apoptosis, a phosphatidylserine residue normally on the inside of the plasma membrane flips to the outside and is specifically recognized by annexin-V. Briefly following treatment, the supernatant was removed and any cells were pelleted and then resuspended in annexin-V-conjugated with FITC (1:40 dilution in binding buffer as supplied in kit), which was then added back to the attached cells for 15 min at room temperature. The cells were trypsinized and all cells were resuspended in propidium iodide (final concentration of 1 µg/ml). Cells were then centrifuged (720 g) and photomicrographs were taken using an Olympus BX40 fluorescent microscope under oil immersion at a magnification of x40.
Morphological assessment
To establish that C2-induced cell death gave rise to the classical morphological features associated with apoptosis, aliquots of treated and untreated cells were cytospun and stained with Wrights stain in an automated stainer (Lillie et al., 1977). Photomicrographs of cells were taken under oil immersion at a magnification of x100.
To assess changes in the levels of apoptosis, cells were viewed under phase contrast with x20 objective optics and photomicrographs were captured using JVC TK 1281 colour video camera coupled to a time lapse video recorder using Adobe Premiere 4.1.
Western immunoblot analysis
Proteins from granulosa cell lysates and supernatants were separated by 12% sodium dodecyl sulphatepolyacrylamide gel electrophoresis and then transferred onto a nylon membrane. Non-specific binding sites were blocked (5% milk in TBST) and the membrane was then probed with anti-prolactin (1 µg/ml; Upstate Biotechnology, USA) overnight. Following the removal of excess unbound antibody, an anti-mouse antibody conjugated to peroxidase (1:2000) was added for 1 h. Binding of the peroxidase was visualized by enhanced chemiluminescence according to the manufacturers instructions (Amersham International, UK).
Statistical analysis
The data were analysed using the Microsoft Excel 5 software package. Significance was determined using analysis of variance and Students t-test. P < 0.05 was considered to be statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Effects of prolactin on the metabolic activity or total cell number of granulosa cells
Having terminally differentiated following exposure to hCG, we confirmed as anticipated that prolactin (0200 ng/ml) had no effect on the metabolic activity of the cells relative to controls and at 100 ng/ml also had no effect on overall cell number (Figure 3A and B).
|
Detection of prolactin in both granulosa cell supernatants and lysates
We detected prolactin peptide in both the supernatants and lysates of untreated granulosa cells. Endogenously detected prolactin appeared as primarily a non-glycosylated form but a glycosylated form was also evident (Figure 3C). This is consistent with the literature in which prolactin can be differentially glycosylated (Bollengier et al., 2001; Gobello et al., 2001
). It is important to note that such preparations of granulosa cells are known to contain a proportion of white blood cells (Best et al., 1994
), which are known to produce prolactin (Buckley, 2001
). However, their exact numbers and the length of time they remain in the cultures is not known. Regardless of how much they contribute to the levels of prolactin that we have observed in granulosa cell supernatants and lysates, they still represent a local source of prolactin within the ovary.
Effects of prolactin on C2-ceramide-induced apoptosis
We next investigated if prolactin acted as a survival factor against C2-ceramide-induced apoptosis. Prolactin (100 ng/ml) had no effect on cell death alone (Figure 4A). Treatment with C2-ceramide induced a significant (P < 0.001) increase in the level of dead cells. This induction of cell death by C2-ceramide was significantly (P < 0.001) inhibited back to control levels in the presence of prolactin (Figure 4A). Photomicrographs of the cells during culture in each condition revealed distinct rounding of the cells and a reduction in overall number due to cell detachment in the C2-treated cultures. On co-incubation with prolactin, levels of cell death were comparable to those seen in the control wells and in those treated with prolactin alone (Figure 4B).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Granulosa cells collected at the time of oocyte aspiration for IVF have been exposed to high levels of hCG and are therefore luteinized. In this state, they are undergoing the granulosa to granulosa luteal transition. These cells would not therefore be expected to be undergoing high levels of apoptosis, as this occurs more frequently in smaller non-selected follicles undergoing atresia or in late luteal cells. It is interesting therefore that it is precisely at this time that the prolactin receptors first appear on these cells (Vlahos et al., 2001). This indicates a physiological role for prolactin at this time and our data suggest that this may be to protect the cells from apoptosis during the early part of the luteal phase or during the luteinization.
A pituitary, endocrine source of prolactin was traditionally thought to be responsible for the observed effects of this peptide on reproductive processes. However, our data demonstrating the presence of prolactin peptide in lysates and supernatants of granulosa cells suggest that the ovary could be an additional extrapituitary source of prolactin. This finding is in contrast to an earlier study (Ohwaki et al., 1992) but is in agreement with more recent data, which demonstrated that prolactin gene expression was evident in homogenized whole ovarian samples (Schwarzler et al., 1997
) and in human luteinized granulosa cells (Phelps et al., 2003
). If prolactin is a local product, then its levels might be expected to exceed those in serum, and indeed the levels were found to range from 9 to 180% of serum levels in a range of matched follicles (McNatty et al., 1975
). However, reports comparing the concentration of prolactin in follicular fluid to that in serum are contradictory. In unstimulated ovaries, the concentration of prolactin in follicular fluid correlated positively with that in serum (Ohwaki et al., 1992
). In an earlier study (McNatty et al., 1974
), in follicular fluid from follicles collected across the luteal phase, prolactin levels rose to a peak in the mid-luteal phase and then fell. It is unclear whether this represents local production, as across the menstrual cycle, circulating levels of prolactin are generally higher in the luteal than follicular phase (Brumstead and Riddick, 1992
). Exposure of the corpus luteum to these higher mid-luteal levels would, however, be consistent with a physiological role for prolactin in protecting the corpus luteum from degeneration until the end of the luteal phase.
The role of prolactin in human ovarian physiology is unclear, although it does now seem likely to be an ovarian product. In terms of its endocrinogical role in the ovary, prolactin has been shown to amplify the stimulatory effects of FSH on the acquistion of the FSH receptor and progesterone production in porcine granulosa cells (Porter et al., 2000) and to increase the production of insulin-like growth factor-II in human granulosa cells (Ramasharma and Li, 1987
). Interestingly, in the human it was found that prolactin had a dose-dependent effect on steroidogenesis with doses <100 ng/ml stimulating, and higher doses inhibiting, progesterone production (McNatty et al., 1974
). Therefore, in addition to its effects on apoptosis the prolactin present in the follicular fluid might also be expected to stimulate progesterone and assist in the maintainance of production of this major product of the corpus luteum.
We observed no consistent effect of prolactin on progesterone production in our cultures, but this may be due to the fact that our cells were luteinized, whereas those used in McNattys study were from small to medium-sized antral follicles collected from normally cycling women.
It has been demonstrated in bovine and human follicles that follicular fluid prolactin levels increase with oocyte maturation, and this was particularly evident in medium and large follicles (Subramanian et al., 1991; Wise et al., 1994
). In support of our hypothesis, a decline in follicular fluid prolactin concentrations in bovine ovarian follicles was associated with an increase in apoptosis (Lebedeva et al., 1998
) and there were lower levels of apoptosis in the granulosa cells of patients undergoing IVF who conceived. It is not clear if these results are due to changes in prolactin levels, as a number of IVF studies have been conducted to determine if success rates are linked with prolactin modulations, but the results are inconclusive. Patients with high follicular fluid concentrations of prolactin presented with a greater number of follicles and mature oocytes and had a better better IVF success rate than patients with lower levels in two studies (Irahara et al., 1991
; Mendes et al., 2001
). However, other studies found no differences in follicluar fluid prolactin concentrations between patients with high and low fertilization rates (Bohnet and Baukloh, 1985
; Lee et al., 1987
).
It is clear that a number of factors, such as estradiol, regulate prolactin synthesis (Kicovic et al., 1981) and so the ultimate action of prolactin within the ovary will depend upon precise levels and combinations of such factors. Any slight alteration in any one component could have profound influences by critically modulating the amount of prolactin made available to its receptor. This may explain contrasting observations described in the literature.
In summary, we have shown that prolactin, which appears to be produced locally within the ovary, acts as a potent survival factor for human granulosa cells. These data add to the growing literature suggesting that prolactin plays a more significant role in folliculogenesis than has previously been reported.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Best, C.L., Pudney, J., Anderson, D.J. and Hill, J.A. (1994) Modulation of human granulosa cell steroid production in vitro by tumor necrosis factor alpha: implications of white blood cells in culture. Obstet. Gynecol., 84, 121127.[Abstract]
Bohnet, H.G. and Baukloh, V. (1985) Prolactin concentrations in follicular fluid following ovarian hyperstimulation for in vitro fertilization. Horm. Res., 22, 189195.[ISI][Medline]
Bollengier, F., Mahler, A., Braet, C., Claeyssens, M. and Vanhaelst, L. (2001) Glycosylated rat prolactin: isolation and structural characterization. Arch. Physiol. Biochem., 109, 180190.[CrossRef][Medline]
Brumstead, J.R. and Riddick, D.H. (1992) Prolactin and the human menstrual cycle. Semin. Reprod. Endocrinol., 10, 220227.[ISI]
Buckley, A.R. (2001) Prolactin, a lymphocyte growth and survival factor. Lupus, 10, 684690.[ISI][Medline]
Ginther, O.J., Beg, M.A., Bergfelt, D.R., Donadeu, F.X. and Kot, K. (2001) Follicle selection in monovular species. Biol. Reprod., 65, 638647.
Gobello, C., Colombani, M., Scaglia, H., De La Sota, R.L. and Goya, R.G. (2001) Heterogeneity of circulating prolactin in the bitch. Reprod. Nutr. Dev., 41, 505511.[CrossRef][ISI][Medline]
Irahara, M., Azuma, K., Yamano, S. and Aono, T. (1991) Effect of prolactin in serum and follicular fluid on fertilization and cleavage of human oocyte. Horm. Res., 35 (Suppl. 1), 4546.[ISI]
Kicovic, P.M., Franchi, F. and Lusi, M. (1981) Correlation between incremental changes of oestradiol-17 beta and prolactin during the menstrual cycle by means of multiple plasma sampling. Reproduccion, 5, 4348.[Medline]
Kim, J.H., Han, J.S. and Yoon, Y.D. (1999) Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells. Tissue Cell, 31, 531539.[CrossRef][ISI][Medline]
Kim, J.M., Yoon, Y.D. and Tsang, B.K. (1999) Involvement of the Fas/Fas ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia. Endocrinology, 140, 23072317.
LaVoie, H.A. and Witorsch, R.J. (1995) Investigation of intracellular signals mediating the anti-apoptotic action of prolactin in Nb2 lymphoma cells. Proc. Soc. Exp. Biol. Med., 209, 257269.[Abstract]
Lebedeva, I., Denisenko, Y., Lebedev, V.A. and Kuzmina, T.I. (1998) Prolactin in follicular fluid and intracellular store calcium in follicular cells are related to morphological signs of ovarian follicle atresia in cows: work in progress. Theriogenology, 49, 509519.[CrossRef][ISI][Medline]
Lee, M.S., Ben-Rafael, Z., Meloni, F., Mastroianni, L. Jr and Flickinger, G.L. (1987) Relationship of human oocyte maturity, fertilization and cleavage to follicular fluid prolactin and steroids. J. In Vitro Fertil. Embryo Transfer, 4, 168172.[ISI][Medline]
Lillie, R.D., Donaldson, P.T., Vacca, L.L., Pizzolato, P.P. and Jirge, S.K. (1977) Reduction and azo coupling of quinones. A histochemical study of human cutaneous melanin and adrenochrome. Histochemistry, 51, 141152.[ISI][Medline]
McNatty, K.P. and Sawers, R.S. (1975) Relationship between the endocrine environment within the Graafian follicle and the subsequent rate of progesterone secretion by human granulosa cells in vitro. J. Endocrinol., 66, 391400.[Abstract]
McNatty, K.P., Sawers, R.S. and McNeilly, A.S. (1974) A possible role for prolactin in control of steroid secretion by the human Graafian follicle. Nature, 250, 653655[ISI][Medline]
Mendes, M.C., Ferriani, R.A., Sala, M.M., Moura, M.D., Carrara, H.H. and de Sa, M.F. (2001) Effect of transitory hyperprolactinemia on in vitro fertilization of human oocytes. J. Reprod. Med., 46, 444450.[ISI][Medline]
Nevalainen, M.T., Valve, E.M., Ahonen, T., Yagi, A., Paranko, J. and Harkonen, P.L. (1997) Androgen-dependent expression of prolactin in rat prostate epithelium in vivo and in organ culture. FASEB J., 11, 12971307.
Obeid, L.M. and Hannun, Y.A. (1995) Ceramide: a stress signal and mediator of growth suppression and apoptosis. J. Cell. Biochem., 58, 191198.[ISI][Medline]
Obeid, L.M., Linardic, C.M., Karolak, L.A. and Hannun, Y.A. (1993) Programmed cell death induced by ceramide. Science, 259, 17691771.[ISI][Medline]
Ohwaki, M., Suganuma, N., Seo, H., Nawa, A., Kikkawa, F., Narita, O., Matsui, N. and Tomoda, Y. (1992) Source of prolactin in human follicular fluid. Endocrinol. Jpn, 39, 601607.[Medline]
Phelps, J.Y., Bugg, E.M., Shamblott, M.J., Vlahos, N.P., Whelan, J. and Zacur, H.A. (2003) Prolactin gene expression in human ovarian follicular cells. Fertil. Steril., 79, 182185.[CrossRef][ISI][Medline]
Porter, M.B., Brumsted, J.R. and Sites, C.K. (2000) Effect of prolactin on follicle-stimulating hormone receptor binding and progesterone production in cultured porcine granulosa cells. Fertil. Steril., 73, 99105.[CrossRef][ISI][Medline]
Prigent-Tessier, A., Barkai, U., Tessier, C., Cohen, H. and Gibori, G. (2001) Characterization of a rat uterine cell line, U(III) cells: prolactin (PRL) expression and endogenous regulation of PRL-dependent genes; estrogen receptor beta, alpha(2)-macroglobulin and decidual PRL involving the Jak2 and Stat5 pathway. Endocrinology, 142, 12421250.
Ramasharma, K. and Li, C.H. (1987) Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells. Proc. Natl Acad. Sci. USA, 84, 26432647.[Abstract]
Schwarzler, P., Untergasser, G., Hermann, M., Dirnhofe, S., Abendstein, B. and Berger, P. (1997) Prolactin gene expression and prolactin protein in premenopausal and postmenopausal human ovaries. Fertil. Steril., 68, 696701.[CrossRef][ISI][Medline]
Subramanian, M.G., Sacco, A.G., Moghissi, K.S., Lawson, D.M. and Gala, R.R. (1991) Prolactin size heterogeneity in human follicular fluid: a preliminary study. Int. J. Fertil., 36, 367371.[ISI][Medline]
Travers, M.T., Barber, M.C., Tonner, E., Quarrie, L., Wilde, C.J. and Flint, D.J. (1996) The role of prolactin and growth hormone in the regulation of casein gene expression and mammary cell survival: relationships to milk synthesis and secretion. Endocrinology, 137, 15301539.[Abstract]
Vlahos, N.P., Bugg, E.M., Shamblott, M.J., Phelps, J.Y., Gearhart, J.D. and Zacur, H.A. (2001) Prolactin receptor gene expression and immunolocalization of the prolactin receptor in human luteinized granulosa cells. Mol. Hum. Reprod., 7, 10331038.
Willis, D.S., Mason, H.D., Gilling-Smith C. and Franks, S. (1996) Modulation by insulin of follicle stimulating hormone and luteinising hormone actions in human granulosa cells of normal and polycystic ovaries. J. Clin. Endocrinol. Metab., 81, 302309.[Abstract]
Wise, T., Suss, U., Stranzinger, G., Wuthrich, K. and Maurer, R.R. (1994) Cumulus and oocyte maturation and in vitro and in vivo fertilization of oocytes in relation to follicular steroids, prolactin and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge, no detectable LH surge and progestin inhibition of LH surge. Domest. Anim. Endocrinol., 11, 5986.[CrossRef][ISI][Medline]
Witty, J.P., Bridgham, J.T. and Johnson, A.L. (1996) Induction of apoptotic cell death in hen granulosa cells by ceramide. Endocrinology, 137, 52695277.[Abstract]
Wyllie, A.H., Kerr, J.F. and Currie, A.R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol., 68, 251306.[Medline]
Submitted on May 30, 2003; accepted on September 2, 2003.