254 E.M. Shores, M.G. Hunter Animal Reproduction Science 64 2000 247–258
Table 1 Hormone production and viable cell number of porcine theca cells following the removal of red blood cells
a,b
a Hormone production at 144 h
No IGF-1 100 ngml IGF-1
pg10
3
viable cells48 h Whole
− rbc
S.E.D. Whole
− rbc
S.E.D. Log oestradiol
0.43
c
0.31
d
0.06 1.35
1.45 0.05
Log androstenedione 1.64
1.50 0.39
2.06 2.56
0.41 b Viable cell number at 144 h
× 10
3
cells Whole
− rbc
S.E.D. No IGF-1
8.9
c
11.3
d
0.9 With IGF-1
7.9 7.6
0.6
a
Porcine theca cells were seeded at 50 × 10
3
viable cells per well in DMEMHam’s F12 with 0.01 ngml LH and 10 ngml insulin. Cells were cultured with or without 100 ngml IGF-1. Hormone production was expressed
per 1000 viable cells and log transformed prior to ANOVA.
b
Values represent the mean of three separate cultures each containing four wells per treatment. Whole: all cell types present; −rbc: red blood cell removed with lysing buffer prior to culture.
c,d
Values with the same superscript are not significantly different P0.05.
PDGF dose increased the number of viable cells at 144 h. However, the value was not significantly more than that obtained with 1 ngml PDGF Fig. 3b. An interaction between
PDGF dose and the cell types present was observed in the absence of IGF-1 P = 0.02. Macrophage-free cultures treated with 10 ngml PDGF contained almost twice as many
viable cells after 144 h as whole cell preparations with no PDGF.
3.6. Removal of red blood cells Red blood cells were completely removed using the lysing buffer. The removal of red
blood cells with lysing buffer decreased the theca cell number by approximately 40 P = 0.003, but did not affect cell viability at the start of culture P = 0.19 paired t-
test. The removal of red blood cells reduced oestradiol output by 35 at 144 h in the absence
of IGF-1 P = 0.04, Table 1a but had no significant effect on oestradiol production in the presence of IGF-1 P 0.05, Table 1a. Red blood cell removal did not influence
androstenedione production either with or without IGF-1 P 0.05 at all points, Table 1a. Red blood cell removal increased viable cell number by 25 at 144 h but only in the absence
of IGF-1 P = 0.02, Table 1b. In the presence of IGF-1 viable cell number was similar in the presence or absence of the red blood cells P = 0.5, Table 1b.
4. Discussion
The results of this study clearly show that theca cells and macrophages interact to influ- ence thecal steroidogenesis and viable cell number during culture. Removal of macrophages
decreased oestradiol output, but enhanced androstenedione output. This raises the pos-
E.M. Shores, M.G. Hunter Animal Reproduction Science 64 2000 247–258 255
sibility that macrophage removal decreased P450 aromatase enzyme activity which led to accumulation of androgen substrate. However, this cannot be the only explanation
as oestradiol levels declined by only 21–39 compared with androstenedione level in- creases of 214–238. The absolute amounts of androstenedione and oestradiol produced
vary considerably androstenedione up to 10-fold higher than oestradiol. Therefore, even similar percentage changes in the two steroids would reflect a much larger change in
the absolute amount of androstenedione synthesised than oestradiol. This provides fur- ther strong support for a theca cellmacrophage interaction. Since only theca cells pro-
duce androgen macrophage removal must directly affect theca cell function. The action of the macrophage products is 2-fold. Firstly, it inhibits androstenedione production
possibly by decreasing P450
17
a
activity, which could be tested by measuring proges- terone output. Accumulation of progesterone would suggest that macrophage removal
reduced P450
17
a
activity. Secondly, it enhances oestradiol synthesis suggesting a direct action on P450 aromatase. These data may reflect the influence of a single factor act-
ing at two points in the steroidogenic pathway or two distinct factors each acting at one point.
The altered steroidogenesis observed here cannot be accounted for by the removal of other contaminating cell types such as granulosa cells or stromal cells. The current theca
cell preparation method results in negligible granulosa cell contamination Shores et al., 2000. Histochemical staining for 3b-HSD data not shown confirmed that contamination
by non-steroidogenic cells, such as stromal cells was also minimal.
Viable cell number after 144 h was increased by 15–20 in macrophage deficient cultures compared to whole preparations. It is possible that some cells counted as theca cells in whole
preparations are in fact leukocytes. If this is the case macrophage removal would increase the proportion of true theca cells seeded at the start of culture but by less than 1 based
on the proportion of macrophages one macrophage to 300 theca cells observed here. This cannot account for the increase in viable cell number and therefore, implicates a macrophage
product in reducing the number of viable theca cells present at the end of culture.
Two reports have examined the number of macrophages per follicle and suggested values of 30 macrophages per 9 mm
2
pig Standaert et al., 1991 and 5 per 10 mm
2
chicken Barua et al., 1998. The surface area of the theca layer of a 7–8 mm diameter follicle is
approximately 500 mm
2
giving 250–1700 macrophages per follicle compared to 1000 per follicle estimated in the current study.
PDGF was hypothesised to mediate macrophagetheca cell interactions. Not only is PDGF secreted by macrophages but is also a key mitogen for mesenchymal cells Ross et al.,
1986 and theca cells possess PDGF receptors Duleba et al., 1999. It has not been possible to find any reports of precisely how much PDGF macrophages secrete. PDGF has been
detected in human follicular fluid at an average level of 0.37 ngml McWilliam et al., 1995. Westermark et al. 1983 reported 30–40 ngml PDGF in platelet-poor plasma. If all this
PDGF were due to macrophage production it would equate to 3 × 10
− 4
ngmacrophage. In the current study there were an estimated 166 macrophages per well. They would therefore,
produce 0.05 ng of PDGF. This is similar to the lowest dose of PDGF used in the study, i.e. 0.1 ngml which is equivalent to 0.025 ngwell.
The current experiments were carried out in the presence and absence of an optimal dose of IGF-1 to remove the possibility of the optimum culture system Shores et al., 2000
256 E.M. Shores, M.G. Hunter Animal Reproduction Science 64 2000 247–258
masking any effect of PDGF. Differences in the response of the cells to PDGF were ob- served with and without IGF-1. After 144 h, oestradiol output was increased by 0.1 ngml
PDGF but only in the presence of IGF-1. This is consistent with reports by May et al. 1992 who showed synergism between IGF and PDGF using porcine theca cells. PDGF
did not affect androstenedione synthesis in the presence of IGF-1. In the absence of IGF-1, PDGF generally stimulated androstenedione production. Since macrophage removal en-
hanced androstenedione production, a macrophage product must decrease androstenedione output. Therefore, PDGF cannot mediate the theca cellmacrophage interaction in terms of
androstenedione production.
PDGF increased DNA synthesis by porcine granulosa cells Hammond and English, 1987 and was mitogenic to porcine theca cells collected from small 1–4 mm follicles
May et al., 1992. PDGF also stimulated porcine theca cell proliferation and activated kinase signalling pathways in a culture system containing serum Taylor, 2000. Recent
work using rat theca-interstitial cells showed 30–136 increases in DNA synthesis with PDGF doses of 3–30 ngml Duleba et al., 1999. Lack of IGF-1 was previously found to be
detrimental to the number of viable theca cells Shores et al., 2000. PDGF was therefore, able to compensate to some extent for the lack of IGF-1 by increasing viable cell number
when a dose of 10 ngml was used. In the presence of IGF-1, PDGF treatment increased viable cell number but doses of 1 and 10 ngml were not different. This is in agreement with
Duleba et al. 1999 who observed additive effects of PDGF and IGF-1 on rat theca cell DNA synthesis. Macrophage removal increased viable cell number at 144 h and therefore,
a macrophage product suppressed viable cell number. PDGF cannot mediate this theca cellmacrophage interaction since PDGF increased viable cell number.
In the current study, macrophage products increased oestradiol production, decreased androstenedione output and reduced viable theca cell number. These interactions could not
be mimicked by PDGF alone. Other potential candidates include TNFa, which acts di- rectly on P450
17
a
to decrease androstenedione production Zachow and Terranova, 1994. TGFb inhibits ovarian cell growth in several species pig May et al., 1994; Gangrade
and May, 1990; cow Roberts and Skinner, 1991; rat Skinner et al., 1987 and stimu- lates oestradiol synthesis and inhibits androgen production by porcine theca cells Caubo
et al., 1989. Macrophage produced TGFb Adashi, 1990, in addition to that produced by porcine granulosa and theca cells May et al., 1994, could be an important intraovarian
factor and further work using the current physiologically relevant culture system would be valuable.
The presence of red blood cells in theca cell cultures is an inevitable consequence of the blood supply to the theca layer. Viable theca cell number and steroidogenesis were
previously shown to be lower at high initial cell density Shores et al., 2000. Red blood cell removal did not effect thecal steroidogenesis or viable cell number when the cells were
cultured under optimal conditions, i.e. in the presence of IGF-1. Oestradiol production was reduced at 144 h and viable cell number increased by red blood cell removal but only in the
absence of IGF-1. Androstenedione production was unaffected by red blood cell removal at all time points, either with or without IGF-1. These data suggest that red blood cells do not
contribute to the overall cell density effect and their presence does not need to be considered in future theca cell culture experiments, particularly when cells are cultured in the presence
of IGF-1.
E.M. Shores, M.G. Hunter Animal Reproduction Science 64 2000 247–258 257
5. Conclusion
