Prolactin acts as a potent survival factor against C2-ceramide-induced apoptosis in human granulosa cells

C.M. Perks1,4, P.V. Newcomb1, M. Grohmann1, R.J. Wright2, H.D. Mason3 and J.M.P. Holly1

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 George’s Hospital Medical School, London SW17 0RE, UK

4 To whom correspondence should be addressed. e-mail: Claire.M.Perks{at}bristol.ac.uk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The role of prolactin in the regulation of ovarian folliculogenesis and corpus luteal function and in particular its relationship to atresia in these structures is as yet unclear. We established a model of apoptosis in which to examine the actions of prolactin. METHOD: Granulosa cells collected from IVF-flush were cultured at 0.1–0.3x106 cells/well in growth media for 48 h, placed into serum-free media for 24 h prior to dosing for 24 h. Dose responses to C2-ceramide and prolactin were performed. Cells were then treated with an apoptotic dose of C2-ceramide alone, prolactin (100 ng/ml) alone or a combination of the two. Cell death was assessed by Trypan Blue cell counting and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl Blue] assay and apoptosis confirmed by morphological assessment and flow cytometry. RESULTS: C2-ceramide (0–40 µmol/l) induced a dose-dependent increase in cell death (63.8% increase at 40 µmol/l) and, morphologically, cells exhibited classical features of apoptosis. Prolactin alone had no effect on metabolic activity or total cell number. On co- incubation, prolactin alone had no effect on cell death, whereas C2-ceramide induced an ~62.6% increase in apoptosis, which was inhibited in the presence of prolactin. CONCLUSIONS: Prolactin may contribute significantly to early corpus luteum formation and survival by acting as a potent antiapoptotic factor for human granulosa cells.

Key words: apoptosis/C2-ceramide/granulosa cells/prolactin


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Follicular selection in monovular species is a complex process, the mechanism of which is still not completely understood. The process involves the development of one follicle, which possesses the ability to ovulate from a cohort of growing follicles. This follicle then undergoes transformation into a corpus luteum, which in the absence of pregnancy later also undergoes atresia. More than 99% of ovarian follicles are lost by a degenerative process known as atresia, a phenomenon characterized by apoptosis of granulosa cells (Ginther et al., 2001Go). Apoptosis is a highly regulated mode of cell death characterized by a number of morphological and biochemical features including blebbing of the plasma membrane, cell shrinkage, chromatin condensation and DNA fragmentation (Wyllie et al., 1980Go).

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., 1993Go). Ceramide has been used previously to induce apoptosis in granulosa cells from a number of different species including rat (Kim, J.M. et al., 1999Go), mice (Kim, J.H. et al., 1999Go) and hen (Witty et al., 1996Go).

Prolactin is synthesized in the anterior pituitary but other prolactin sources have now been identified including the endometrium (Prigent-Tessier et al., 2001Go) and prostate (Nevalainen et al., 1997Go). 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., 2001Go). 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, 1995Go), rat mammary (Travers et al., 1996Go) and rat dorsal and lateral prostate epithelial cells (Ahonen et al., 1999Go).

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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
The ceramide analogue, C2-ceramide, and prolactin, purified from human pituitary glands, were obtained from Sigma (UK). Tissue culture plastics were obtained from Nunc, Gibco Life Technologies (UK).

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 Dulbecco’s modified Eagle’s 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 Ham’s 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 ~30–40% 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., 1996Go).

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 (0.1–0.2x106) 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-V–FITC staining
Annexin staining of C2-treated and untreated cells was performed using an annexin-V–fluorescein isothiocyanate (FITC) kit according to the manufacturer’s instructions (Biowhittaker UK Ltd–Boehringer 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 Wright’s stain in an automated stainer (Lillie et al., 1977Go). 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 sulphate–polyacrylamide 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 manufacturer’s 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 Student’s t-test. P < 0.05 was considered to be statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of C2-ceramide on apoptosis
Figure 1A demonstrates that C2-ceramide induced a dose-dependent increase in the level of dead cells with a 63.8% increase at 40 µmol/l C2. Flow cytometric analysis confirmed that this induction of cell death was apoptotic with a dose-dependent increase in the number of cells undergoing programmed cell death (Figure 1B) (29.2% increase at 40 µmol/l relative to controls). Granulosa cells stained with annexin-V are shown in Figure 2 and the upper panel represents untreated granulosa cells (control), where a small proportion of cells have stained positive (white staining) for annexin-V. This degree of staining corresponds to the basal levels of cell death that were detected when Trypan Blue cell counting was performed. Following C2-ceramide treatment, there is a marked increase in the number of apoptotic cells illustrated by an increase in positive staining for annexin-V (lower panel).



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Figure 1. The effects of treatment with C2-ceramide (0–40 µmol/l) for 24 h on cell death and apoptosis of granulosa cells. Graphs represent (A) percentage dead cells and (B) percentage cells in pre-G1 of the cell cycle as measured by Trypan Blue cell counting and flow cytometry respectively. Data represent the mean of three experiments ± SEM.

 


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Figure 2. Cytochemical and morphological assessment of C2-ceramide-induced apoptosis. Cells dosed ± an apoptotic dose of C2-ceramide were treated with an apoptotic marker, annexin-V–FITC (as per manufacturer’s specifications) followed by propidium iodide (1 µg/ml), centrifuged and photomicrographs were taken using an Olympus BX40 fluorescent microscope under oil immersion at a magnification of x40. (A) Upper panel represents untreated granulosa cells (control), where a small proportion of cells have stained positive (white staining) for annexin-V. Following C2-ceramide treatment there is a marked increase in the number of apoptotic cells illustrated by an increase in positive staining for annexin-V (lower panel). (B) Photomicrographs of centrifuged aliquots of cells dosed ± an apoptotic dose of C2-ceramide and stained with Wright’s stain illustrating some of the classical morphological features of apoptosis including the formation of apoptotic bodies.

 
Photomicrographs of C2-treated and control granulosa cells stained with Wright’s stain are shown in Figure 2B, which illustrates some of the classical morphological features of apoptosis including the formation of apoptotic bodies and membrane blebbing.

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 (0–200 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).



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Figure 3. Represents the effects of prolactin (0–200 ng/ml) treatment for 24 h on (A) the metabolic activity and (B) cell growth of granulosa cells as measured by MTT assay and cell counting respectively. Graphs represent the mean of three experiments ± SEM. (C) Western immunoblot of prolactin peptide detected in supernatants and cell lysates of untreated granulosa cells.

 
Effects of prolactin on progesterone production
Progesterone levels ranged from 50 to 160 nmol/1000 cells/h in the culture. There was no consistent effect of prolactin on progesterone production in our experiments.

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., 2001Go; Gobello et al., 2001Go). It is important to note that such preparations of granulosa cells are known to contain a proportion of white blood cells (Best et al., 1994Go), which are known to produce prolactin (Buckley, 2001Go). 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).




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Figure 4. Effects of prolactin on C2-ceramide-induced apoptosis. (A) The percentage of dead cells following treatment for 24 h with an apoptotic dose of C2-ceramide, or prolactin (100 ng/ml), or the combination of the two. Experiments represent the mean of three experiments ± SEM, where C2 > control, and C2 & prolactin < C2 (***P < 0.001). (B) Photomicrographs of granulosa cells with the same treatments as above to assess levels of cell death.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have established a model of inducible apoptosis in human granulosa cells, using an analogue of ceramide, C2-ceramide. Ceramide is the product of sphingomyelin hydrolysis, which is recognized as an important pathway of signal transduction. Ceramide can act as a second messenger for the induction of apoptosis (Obeid et al., 1993Go) and acts as such for a number of different extracellular agents including cytokines, chemotherapy and radiotherapy (Obeid and Hannun, 1995Go; Witty et al., 1996Go). Using this model of programmed cell death, we demonstrated that whilst prolactin alone had no effect on metabolic activity or total cell number of human granulosa cells, it did act as a potent survival factor against C2-ceramide-induced apoptosis.

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., 2001Go). 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., 1992Go) but is in agreement with more recent data, which demonstrated that prolactin gene expression was evident in homogenized whole ovarian samples (Schwarzler et al., 1997Go) and in human luteinized granulosa cells (Phelps et al., 2003Go). 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., 1975Go). 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., 1992Go). In an earlier study (McNatty et al., 1974Go), 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, 1992Go). 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., 2000Go) and to increase the production of insulin-like growth factor-II in human granulosa cells (Ramasharma and Li, 1987Go). 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., 1974Go). 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 McNatty’s 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., 1991Go; Wise et al., 1994Go). 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., 1998Go) 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., 1991Go; Mendes et al., 2001Go). However, other studies found no differences in follicluar fluid prolactin concentrations between patients with high and low fertilization rates (Bohnet and Baukloh, 1985Go; Lee et al., 1987Go).

It is clear that a number of factors, such as estradiol, regulate prolactin synthesis (Kicovic et al., 1981Go) 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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 30, 2003; accepted on September 2, 2003.