1 Huntington Reproductive Center, 1220 La Venta Drive, Westlake Village, CA 91361 and 2 Department of OB/GYN, Stanford University, Stanford, CA 94305, USA
3 To whom correspondence should be addressed. E-mail: whbdf{at}yahoo.com
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Abstract |
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Key words: CD45/CYP17/follicular development/GM-CSF/in vivo
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Introduction |
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Expression and selective cellular localization of GM-CSF and its receptor in ovarian tissue imply an autocrine/paracrine role in ovarian function. Moreover, evidence indicating a functional role for GM-CSF in ovarian follicular cells has been provided by studies with GM-CSF knockout (GM/) mice (Gilchrist et al., 2000), which suggest GM-CSF influences events associated with murine follicular maturation. Cumulusoocyte complexes recovered from GM/ mice had approximately twice the number of cumulus cells per cumulusoocyte complex than did those of GM+/+ mice. GM-CSF deficiency was associated with significantly lower progesterone production by mural granulosa cells recovered from GM/ compared with those recovered from wild-type mice. In the present study, we examine the effect of GM-CSF on follicular development in vivo by administrating exogenous GM-CSF to immature rats. In addition, we performed immunostaining of CD45, a common leukocyte antigen (Jasper et al., 2000
), to evaluate the alteration of distribution of leukocytes in ovaries after GM-CSF treatment in vivo. Moreover, we examined the expression of ovarian CYP17, a marker for the theca cells (Gelety and Magoffin, 1997
), in theca-interstitial (T-I) cells in vivo and in in-vitro cell culture after exogenous GM-CSF treatment.
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Materials and methods |
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To assess the role of GM-CSF on follicular development, female rats at 5 days of age, when their ovaries contain mainly primary and primordial follicles (Vitt et al., 2000), were injected intraperitoneally daily with 0.1 µg rat GM-CSF in 100 µl saline or with FSH (10 IU). Both the GM-CSF and the FSH dose corresponded to ~100 times the concentration needed for optimal in-vitro bioactivity (McGee et al., 1997
; Tamura and Kogo, 1999
; Vitt et al., 2000
). Control animals were injected with saline (100 µl). Rats (each group has five to seven rats) were injected daily for either 5 or 10 days. The ovaries were weighed individually using a scale sensitive for µg ranges (Mettler balance; Mettler Instrument Corp., Hightstown, NJ, USA). At the end of the experiments, ovaries of each animal were fixed in 4% paraformaldehyde and sectioned for histological examination.
In addition, to evaluate the effects of GM-CSF on T-I cells in vitro, same strain rats at 25 days were sacrificed to obtain the T-I cells for culture.
Histological evaluation
Ovaries (five to seven ovaries per group) were fixed in 4% paraformaldehyde and then embedded in paraffin, and serially sectioned at 6-µm intervals. The sections were mounted on glass slides and the even number slides were stained with haematoxylin and eosin. Follicles were counted using the dissector and fractionator principles (Vitt et al., 2000). Every sixth section of each ovary was chosen for analysis. Follicle stages were determined in a manner similar to the classification used by Flaws et al. (1997)
. Only follicles with a visible nucleus in the oocyte were considered, thus avoiding the counting of atretic follicles. Follicles were counted at a magnification of 200x.
Immunofluorescent staining for CYP17 and CD45
Immunofluorescent staining was performed to localize CYP17, a marker of thecal cells, and CD45, a marker for leukocytes, in ovaries in the three groups 5 days after different treatment. After the ovarian sections were deparaffinized, normal goat serum was applied for 30 min at room temperature to minimize non-specific binding. Rabbit polyclonal anti-rat CYP17 (kindly provided by Dr Anita Payne, Stanford University School of Medicine) and Rabbit polyclonal anti-rat CD45 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied as the primary antibody. Sections were incubated with the CYP17 and CD45 antibody in a sealed, humidified chamber for 1 h at 37°C and then washed with phosphate-buffered saline (PBS) to remove excess unbound antibody. Specimens were incubated with FITC-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology) at a dilution of 1:200 for 40 min at room temperature. After thorough rinsing, sections were mounted with Fluoromount (Vector Laboratories, Inc., Burlingame, CA, USA) and evaluated under Zeiss-Axioskop 40 microscope equipped with epifluorescent illumination. These images were stored in the computer. Raw images were analysed using NIH Image Software (ImageJ, 1.28u) by quantifying the average pixel intensities in the ovarian images (Seino et al., 2002). We randomly selected four follicles in each quadrant of every section to get the average intensity (density/follicle). The average intensity represents the intensity of CYP17 and CD45 staining. Negative controls were performed simultaneously by replacing the primary antibodies with PBS.
Culture of T-I cells
T-I cells were isolated from 25-day-old rats using the method of Foghi et al. (1998) with modifications. Twenty-five-day-old female SpragueDawley rats (n = 5) were killed by CO2 asphyxiation, and the ovaries were collected in McCoys 5A medium (Gibco-BRL) containing 25 mmol/l HEPES (pH 7.4), 2 mmol/l l-glutamine and 1 mg/ml bovine serum albumin (BSA). The ovaries were punctured with a needle to remove follicular fluid and granulosa cells, cut into small pieces, washed and incubated for 90 min at 37°C in medium containing collagenase (2.5 mg/ml) and DNase (10 µg/ml). Digested pieces of ovarian tissue were further dispersed by 20 passages through a Pasteur pipette and centrifuged at 90 g for 5 min. The pellet of this centrifugation step was resuspended, and large cell aggregates and vascular elements were removed by sedimentation under unit gravity for 5 min. The supernatant containing small cell aggregates and single cells was centrifuged at 90 g for 5 min, resuspended and plated in culture dishes in McCoys 5A medium containing 2 mmol/l l-glutamine, 1 mg/ml BSA, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco-BRL) at a density that generated subconfluent cultures. T-I cells, prepared as described, have been characterized morphologically and biochemically under a variety of culture conditions, which indicated that thecal/interstitial cell cultures with granulosa cells was <5% (Foghi et al., 1998
).
Extraction of total RNA and protein from T-I cells
After 4 days of culture, T-I cells were treated with different concentrations of GM-CSF (0, 1, 3, 10 and 30 ng/ml). Total RNA was isolated from the T-I cell cultures using TRIzol reagent (Life Technologies, Inc.) 8 h after GM-CSF treatment. For protein extraction, cells were harvested in ice-cold lysis buffer [PBS, Nonidet P-40 (1%; v/v), sodium deoxycholate (0.05%; w/v), SDS (0.1%; w/v)] containing kinase inhibitors [phenylmethylsulfonyl fluoride (10 µmol/l), aprotinin (50 µg/ml), leupeptin (5 µg/ml)]. The supernatant was collected after the cells were sonicated and centrifuged. Protein content of the extracts was determined with the DC protein assay reagent (Bio-Rad Laboratories, Inc.).
Analysis CYP17 mRNA by semi-quantitative reverse transcriptasePCR
The analysis of CYP mRNA by semi-quantitative reverse transcriptase (RT)PCR has been reported in previous studies (Dalla Valle et al., 1995). Briefly, 1 µg of sample was reverse transcribed in 20 µl of reaction mixture containing 20 pmol oligo (dT); 40 U/µl ribonuclease inhibitor, 10 U/µl avian myeloblastosis virusRT and buffer. The RT reaction was followed by a PCR conducted in a total volume of 100 µl containing 10x PCR buffer, 10 µmol/l each of 5' and 3' primers (30 cycles at 95°C for 1 min, 60°C for 1 min and 72°C for 1.5 min). The primers used for amplification of CYP17 and
-actin have been described previously (Dalla Valle et al., 1995
). The PCR products generated for CYP17 and
-actin were 449 and 421 bp, respectively. PCR products and molecular weight markers were separated on agarose gels containing ethidium bromide (10 mg/ml) and visualized by UV light. The intensity of each band was normalized to its corresponding
-actin band to semi-quantitatively compare values between samples.
Western blotting for CYP17
Fourty micrograms of extract protein were resolved over a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. The blot was preincubated in blocking buffer (5% non-fat dry milk, 1% Tween 20, in 20 mmol/l Tris-buffered saline, pH 8.0) overnight at 4°C, then incubated with rabbit anti-rat CYP17 primary antibody in blocking buffer for 1 h at room temperature, followed by incubation with anti-rabbit secondary antibody conjugated with horseradish peroxidase and detected by chemiluminescence and autoradiography using X-ray film. The intensity of protein of interest was densitometrically determined using Molecular Analyst software version 1.4 (Bio-Rad Laboratories, Inc).
Statistical analysis
Differences in ovarian weight, the number of follicles, and the relative expression of CYP17 mRNA and protein between treatment groups were evaluated by ANOVA and followed by Tukey HSD test. Significant differences were assigned at P < 0.05.
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Results |
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In-vivo treatment with GM-CSF increases the number of large preantral follicles
Follicles were counted in ovarian sections stained with haematoxylin and eosin. There was no difference in follicle morphology, as judged by criteria published by Pedersen and Peters (1968). In the present study, we counted only the growing follicles that included primary, small preantral and large preantral stages according to the criteria described previously (Vitt et al., 2000
). Briefly, primary follicles are identified as a single layer of cubiodal granulosa cells surrounding the oocyte. Follicles with a single granulosa layer that consisted of both flattened and cuboidal cells were considered to be in transition from primordial to primary follicles and also scored as primary. Small preantral follicles have more than one but less than four layers of granulosa cells and large preantral follicles have more than four layers of granulosa cells (Figure 1).
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After 5 days of treatment with GM-CSF, the number of large preantral follicles was significantly increased compared with the control group (P < 0.05). However, no effect of GM-CSF on primary follicles and small preantral follicles was observed (P > 0.05). Similarly, treatment with FSH had no effect on the number of primary follicles but increased the number of small preantral and large preantral follicles by 1.2-fold and 6.7-fold, respectively (Figure 2A).
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After 10 days of treatment with GM-CSF, the number of small and large preantral follicles was significantly increased compared with the control group (P < 0.05). Similarly, treatment with FSH increased the number of small preantral and large preantral follicles (P < 0.05). After treatment with either GM-CSF or FSH, the number of primary follicles was lower than in the control group, but did not achieve significance (P > 0.05) (Figure 2B).
The different effects between GM-CSF and FSH treatments on follicle progression can also be seen in representative ovarian sections of the three groups analysed (Figure 3). The ovaries of the control group contained multiple primordial and primary follicles in the ovarian lobe region, while the GM-CSF-treated group showed many large preantral follicles. Similarly, ovaries from the FSH-treated group contained mainly small preantral and large preantral follicles. These morphological observations may indicate that GM-CSF has an effect similar to FSH enhancing preantral follicle progression.
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In-vivo treatment with GM-CSF increases ovarian CYP17 expression
CYP17 staining was found distributed in the thecal cells but not stromal and granulosa cells. Some of the preantral follicles were also found staining for CYP17. Using the NIH image software ImageJ, analysis of the intensity of CYP17 staining in ovaries was performed. The average intensity of CYP17 staining in ovaries from GM-CSF treatment group was 126.86 ± 25.55, which was significantly different compared with the FSH group and the control group (70.43 ± 14.78 and 67 ± 12.40, respectively) (P < 0.05). Immunofluorescent staining showed that CYP17 expression in ovarian T-I cells was significantly increased 5 days after GM-CSF treatment compared with the control group. In contrast, CYP17 staining in rats of the FSH treatment group was very weak, appearing no different from the control group (Figure 4).
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In-vivo treatment with GM-CSF did not alter the CD45 distribution in ovaries
CD45, leukocyte common antigen-positive cells were localized predominantly within stromal tissue and surrounding blood vessels but were also found within the theca cell layer of follicles, adjacent to the basement membrane. Seldom were leukocytes found within the granulosa cell layer of follicles, and in such cases these follicles exhibited atretic characteristics (Figure 5). Using the NIH image software ImageJ, analysis of the intensity of CD45 staining in ovaries was performed. The average intensity of CD45 staining in ovaries from the GM-CSF treatment group was 123.98 ± 23.15, which was noi different compared with the FSH group and the control group (117.36 ± 27.83 and 133 ± 37.56, respectively) (P > 0.05). It was demonstrated that the distribution of leukocytes in rat ovaries was not affected by the GM-CSF and FSH treatment.
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In-vitro treatment of GM-CSF increased CYP17 mRNA and protein expression in T-I cells
Total RNA was extracted from T-I cells for semi-quantitative RTPCR analysis of CYP17. The results showed that CYP17 mRNA was increased by 4.8-fold in the presence of 1 ng/ml GM-CSF in the culture medium compared with the group without GM-CSF (P < 0.05) (Figure 6). Similarly, increased CYP17 protein (by 6.8-fold) was found in the presence of 1 ng/ml GM-CSF compared with the group without GM-CSF treatment (Figure 7).
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Discussion |
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GM-CSF is synthesized within the ovary. The rate of synthesis displays temporal fluctuations across the oestrous cycle, with peak values reached near the time of ovulation (Brannstrom et al., 1994). Gonadotrophins and ovarian steroid hormones may regulate GM-CSF production (Gilchrist et al., 2000
). GM-CSF mediates its biological activity through two interacting receptor subunits, the
and
. The GM-CSF receptor
is located in cells throughout the ovary and the GM-CSF receptor
is prominently expressed in the corpus luteum and thecal layer of the follicle, the expression both increasing in theca and granulosa cells with increasing follicle size (Jasper et al., 1996
; Tamura et al., 1998
).
After 5 days of treatment with GM-CSF, the number of large preantral follicles was significantly increased compared with the control group (P < 0.05). However, no effect of GM-CSF on primary follicles and small preantral follicles was observed (P > 0.05). After 10 days of treatment with GM-CSF, the number of small and large preantral follicles was significantly increased compared with the control group (P < 0.05). The observed stimulatory effect of GM-CSF on the increase in number of small and large preantral follicles but not the number of primary follicles is consistant with the increased GM-CSF receptors in preantral follicles with increasing size. On the other hand, it seems unlikely that the primordial to primary transition is altered under the stimulation of GM-CSF. Similarly, treatment with FSH increased the number of small preantral and large preantral follicles (P < 0.05) in both 5- and 10-day treatment groups. However, after treatment with either GM-CSF or FSH, the number of primary follicles was lower than in a previous report that treatment with FSH had no effect on the number of primordial and primary follicles in immature rats (Vitt et al., 2000).
Of interest, GM-CSF knockout mice exhibit impaired reproductive capacity, having 25% smaller litters at weaning than wild-type mice owing to fetal death late in gestation and early in postnatal life. It is possible that deficiencies in folliculogenesis leading to oocyte defects may contribute to diminished developmental competence, since there were clear differences in the number of cumulus cells per cumulusoocyte complex between GM/ and GM+/+ mice (Robertson et al., 1999; Gilchrist et al., 2000; Jasper et al., 2000
). Moreover, Jasper et al. (2000)
found that no mitogenic effect on cumulus or granulosa cells was observed when GM-CSF was added in vitro to ether cumulus cells or cumulusoocyte complexes of either genotype, despite the presence of GM-CSF receptors. Interestingly, the granulosa cells of GM/ mice showed lower progesterone secretion than the granulosa cells of GM+/+ mice. Ovaries of GM/ mice perfused in vitro also produced less progesterone per oocyte ovulated compared with GM+/+ mice (Jasper et al., 2000
). Progesterone secretion by granulosa cells is an indicator of their differentiation status, with higher values observed from terminally differentiated granulosa cells or luteal cells. Taken together, its role in the differentiation and maturation of follicular cells in vivo may be mediated indirectly, perhaps through the agency of an intermediate cell (T-I) population (Gilchrist et al., 2000
).
T-I cells control follicle growth and atresia, regulate ovarian steroidogenesis and may provide mechanical support for ovarian follicles (Parrott and Skinner, 1998). Normal follicular development requires accurate regulation of T-I function through extra- and intraovarian mechanisms. Growth, differentiation and function of T-I cells are modulated by a network of intraovarian and circulating growth factors and cytokines. Duleba et al. (1999)
found that platelet-derived growth factor and insulin-like growth factor-1 induced selective proliferation of specialized subpopulations of T-I cells, which may lead to selective development of ovarian stromal, theca external and the internal. CYP17 is present in steroidogenic theca internal cells and is found in immature rat ovaries (Doody et al., 1991
). In the present study, we also observed increased CYP17 expression in ovaries of rats treated with GM-CSF, which may demonstrate enhancement of the ovarian CYP17 content in theca cells by GM-CSF in vivo. Few theca cells are present during the initiation of growth of primordial follicles and primary follicles contain only one layer of theca cells. These cells proliferate and the number of theca cell layers increases with follicular progression, likely in response to intraovarian factors (Duleba et al., 1999
). The increase in ovary CYP17 content could be due to either a higher abundance of preantral follicles and therefore more follicles with more layers of theca cells, and/or to a specific increase in CYP17 levels per cell. However, there was no significant increase of CYP17 expression in rats of FSH treatment, even though there were more preantral follicles compared with the control. It may suggest that GM-CSF promotes follicle development in vivo through the increased steroidogenic activity of T-I cells by up-regulating CYP17 expression.
It is not known whether CYP17 expression in T-I cells is regulated by GM-CSF directly or indirectly. Various cytokines produced mainly by resident and infiltrating leukocytes in the ovary may be involved in the regulation of ovarian functions. In uterine endometrium, GM-CSF has been observed mediating the recruitment and potentially modifying the sbehaviour of uterine leukocytes during the post-mating inflammatory response in mice (Robertson et al., 2000). Also, alteration of distribution of leukocytes in mouse ovaries in GM-CSF knockout mouse was reported by Jasper et al. (2000)
. To evaluate the effects of GM-CSF on the distribution of leukocytes in the ovary after GM-CSF treatment in vivo, immunofluorescence of CD45, a leukocyte common antigen, was performed. The results showed that there was no significant difference of CD45 staining intensity in the GM-CSF treatment group compared with the FSH and the control groups.
To demonstrate the action of GM-CSF directly on T-I cells, T-I cells were collected and cultured with or without GM-CSF. Interestingly, CYP17 mRNA and protein expression was significantly increased in the presence of 1 ng/ml GM-CSF in the cultured T-I cells. In addition, the expression of CYP17 mRNA was inhibited with increasing concentration of GM-CSF. A previous study showed that GM-CSF down-regulates its receptor in cultured human mast cells (Welker et al., 2001). The negative regulation of GM-CSF versus GM-CSF receptor may be the explanation for why increased concentrations of GM-CSF decrease the expression of CYP17. Similarly, Tamura et al. (1998) found that GM-CSF may be involved in the direct regulation of ovarian histamine secretion in mast cells, partially by enhancing interleukin-1
induced histamine release in the process of ovulation. Taken together, GM-CSF may not act on follicular somatic cells to influence their function, but instead may act directly via T-I cells in vivo. A striking feature of the ovaries as endocrine targets is the shifting pattern of gonadotropin sensitivity displayed by individual ovarian cell types. Much of this can be explained by the local production of growth factors such as GM-CSF, steroids and other extracellular substances that mediate and modulate gonadotropin action (Erickson and Shimasaki, 2001
).
Thus, GM-CSF may play a role in follicular development complementary to that of FSH. Our data provides additional information about the control of folliculogenesis in the rat and could provide insight into an alternative approach to follicular stimulation.
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Submitted on March 4, 2005; resubmitted on April 25, 2005; accepted on May 9, 2005.
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