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Departments of Biochemistry (R.V., G.E.D.), Oncology (G.E.D.), Physiology (H.D., G.W.) and Obstetrics/Gynecology (G.E.D.) in the Faculty of Medicine and Dentistry, The University of Western Ontario, and the London Regional Cancer Center (G.E.D.), London, Ontario, Canada
Address all correspondence and requests for reprints to: Dr. Gabriel DiMattia, London Regional Cancer Centre, 790 Commissioners Road, London, Ontario N6A 4L6. E-mail: dimattia{at}julian.uwo.ca
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the mouse, we have shown that low levels of STC mRNA are found in a number of different tissues, whereas the highest levels occur in ovary, specifically in the theca-interstitial compartment (11). Interestingly, STC protein appears to be sequestered in luteal cells. This suggests that theca-derived STC is acting in a paracrine manner to regulate corpora lutea function (11). Others have shown that STC gene expression in humans is also high in ovary (1). These intriguing observations imply that STC has an important role in mammalian ovarian physiology and reproduction. As a result we have chosen to focus much of our effort on understanding the regulation and function of STC in mammalian ovary using mouse as a model. Therefore, in this report we have monitored the patterns of STC gene expression in the mouse ovary over a number of different experimental paradigms; postnatal development, estrous, superovulation, gestation, and lactation. The results of these studies serve to further strengthen the notion that STC may play a role in ovarian physiology during pregnancy and lactation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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STC gene expression during gestation
To examine STC gene expression in the maternal ovary during
gestation and lactation, C57BL/6J x CBA mice at 6, 8, 10, 12, 14,
16, and 18 dpc and at 2, 10, and 21 days postpartum were
killed by CO2 asphyxiation. Blood samples were
collected by cardiac puncture for measurement of serum STC by RIA on 50
µl aliquots of serum in triplicate as described previously (13). The
coefficient of variation for intraassay variability was 8.4% (n =
36), whereas interassay variability was 13.2% (n = 11). Ovaries
and kidneys were dissected, frozen on dry ice, and stored at -80 C.
RNA and protein were then isolated from ovaries using Trizol
(Life Technologies, Inc., Grand Island, NY) according to
the manufacturer’s instructions. Five micrograms of total RNA from
each pairs of ovaries were pooled from each experimental set of five
mice and size fractionated on a 1% agarose, 3% formaldehyde gel.
Northern blots were then prepared and hybridized as described
previously (11) with [
-32P]deoxy-CTP (3000
Ci/mmol) labeled mouse STC complementary DNA (cDNA) probe.
Autoradiography was performed using Kodak BIOMAX MS film
with a transcreen (Eastman Kodak Co., Rochester, NY) at
-80 C for 24–96 h. For quantitation of the STC mRNA signal, Northern
blots were also exposed to phosphor screens and densitometrically
analyzed using a PhosphorImager SI (Molecular Dynamics, Inc., Sunnyvale, CA). Northern blots were then hybridized to a
rat cyclophilin cDNA probe (obtained from Dr. E. Turner, Graduate
Program in Neurosciences, UCSD) and the resultant signal quantitated as
above to standardize the STC mRNA signal as done previously (11). The
results were expressed as STC/cyclophilin mRNA ratios.
STC gene expression profiles during superovulation of immature
mice
Two-week-old immature mice were treated with 5 IU of pregnant
mare serum gonadotropin (PMSG) and then 8 mice were killed at 12, 24,
and 48 h after PMSG treatment alone. At 48 h the remaining
mice were treated with 5 IU of human CG (hCG) and then killed at 60,
72, and 96 h after the initial PMSG injection. The ovaries were
collected as outlined above and subjected to Northern analysis.
STC gene expression profiles during lactation
Two experimental groups were established for this study. One
group of mothers were allowed to nurse their pups for 21 days while the
second group had the pups removed immediately after birth. In both
cases the mothers were then killed at 2, 10, and 21 days postpartum and
ovary RNA was subjected to Northern analysis as outlined above.
In situ hybridization (ISH) and immunocytochemistry (ICC)
STC mRNA was localized by in situ hybridization using
35S-UTP (Amersham Pharmacia Biotech
Inc., Oakville, Ontario, Canada) labeled sense and antisense riboprobes
prepared by in vitro transcription of mouse STC cDNA as
described previously (11). Five micron paraffin sections were mounted
on Superfrost slides (Fisher Scientific). A
polyclonal antiserum against human STC (1:1000 dilution) was used for
immunocytochemistry in conjunction with the Avidin-Biotin peroxidase
method (Vectastain, Vector Laboratories, Inc., Burlingame, CA) as previously described (11) on five
micron serial sections mounted on superfrost slides (Fisher Scientific). The sites of antigen-antibody binding were
visualized with 3,3' diaminobenzidine tetrachloride
(Sigma, St Louis, MO) as the chromagen. Control procedures
included the application of preimmune rabbit serum or antiserum
preabsorbed with hSTC (1 µg of hSTC/ml of 1:1000 diluted hSTC
antiserum) in lieu of antiserum alone. Slides were counterstained with
Carazzi’s hematoxylin and mounted with Micromount mounting media
(Surgipath Canada, Winnipeg, Manitoba). The results of ISH and ICC
staining were photographed with a SPOT2 digital camera.
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Results |
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. In
1-day-old mice, there was clear-cut staining for STC protein in the
primordial follicle cells and the oocytes within. STC immunoreactivity
(STCir) was also present at very low levels in the undifferentiated
stroma between the nests of oocytes and in the stroma invading the
center of the ovary (Fig. 1A). A higher magnification inset is provided
to emphasize the differential STCir that is present between
morphologically different cells (Fig. 1A, inset). In
contrast to the distinct protein staining, the pattern of STC gene
expression was diffuse throughout the ovary such that it appeared to be
present in both the stroma and primordial oocytes (Fig. 1B).
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). Ovarian stromal
cells that eventually give rise to the theca and interstitial cells
contained moderate levels of STC protein. There was also an intriguing
pattern of staining in granulosa cells of primary follicles. In some
follicles, the granulosa cells were completely devoid of STCir, whereas
in others they were distinctly stained (compare lower left
and upper right in Fig. 1D). The higher magnification image
of maturing follicles in the day 5 ovary shows this heterogeneity in
STCir even within a single follicle (Fig. 1D). The pattern of STC gene
expression was markedly different than the protein and beginning to
resemble the pattern seen in adult ovary. STC gene activity was now
uniquely restricted to stromal cells surrounding uni and multilaminar
primary follicles (Fig. 1E), whereas the stroma surrounding nests of
primordial follicles showed little evidence of STC mRNA. Interestingly,
the oocytes that stained most intensely for STC protein (Fig. 1C) were
devoid of STC mRNA, as were the granulosa cells that contained STCir.
Control immunocytochemical sections were carried out using normal
rabbit serum or antiserum preabsorbed with STC in place of STC
antiserum (Fig. 1F). Similar immunostaining controls were carried out
for each time-point and no consistent background staining was observed.
When sense probes were used in lieu of antisense probes the level of
ISH staining proved to be negligible (Fig. 1G).
By 14 days of age, primary oocytes of uni- and multilaminar follicles
continued to have the highest levels of STCir protein, whereas theca
and interstitial cells were only moderately stained (Fig. 1H).
Granulosa cells were essentially devoid of STCir (Fig. J). STC gene
expression continued on its adult course and was now highest in the
theca and interstitial cells, both of which were now fully
differentiated from the ovarian stroma (Fig. 1I). There continued to be
no evidence of STC mRNA in the oocytes but a low level of expression
was apparent in granulosa cells. Northern blot analysis of ovary RNA
from 5, 14, 21, and 28 day mice confirmed the presence of the
approximately 4 kb STC mRNA at each day of prepubertal development
(data not shown).
Ovarian STC expression during the estrous cycle and
superovulation
When steady-state levels of STC mRNA were examined at different
stages of the estrous cycle no changes were observed (Fig. 2). Nonetheless, the spatial pattern of
expression over the estrous cycle was also examined to determine
whether there were changes in the types of cells that produced or
contained STC, and if the level of expression was altered in specific
cellular compartment. Our results, which are shown in Fig. 3, revealed no changes in the pattern of
STC gene expression, but distinct changes in the pattern of STCir was
observed in corpora lutea.
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). Luteal cell staining varied greatly
between CLs, suggesting there might be an inherent difference amongst
them. Granulosa cells did not exhibit STCir. STC protein staining was
most intense during metestrus I particularly in the CLs (Fig. 3B). The
oocytes of preantral follicles and interstitial cells were moderately
stained while granulosa cells continued to be devoid of STCir. During
metestrus II and diestrous the level of STCir declined remarkably but
remained most apparent in the CLs as well as thecal and interstitial
cells surrounding peripheral follicles (Fig. 3C). The homogeneity
within a single CL and the range of difference between CLs STCir
staining is shown in Fig. 3F. At higher magnification, it is evident
that STC protein is localized to the cytoplasm of cells that constitute
the CL without distinction between large and small luteal cells. A
representative ISH from metestrus II shows that STC gene expression was
high in the theca surrounding peripheral follicles with lower levels of
expression in interstitial cells (Fig. 3E). This pattern of gene
expression was recapitulated at each stage of the estrous cycle. A low
level of gene expression was also detected over the CLs throughout
estrous, whereas oocytes and granulosa cells did not appear to express
the gene.
As one approach to assessing potential changes in STC expression during
the ovulatory process, we chose superovulation of immature mice using
PMSG and hCG treatments. This system provides a relatively simple model
with which to examine changes in gene expression associated with
ovulation in the absence of corpora lutea from previous cycles. The
results of this study are shown in (Fig. 4). We found that STC levels did not
increase in response to PMSG alone (Fig. 4A). Subsequent treatment with
hCG, 48 h following PMSG priming, resulted in a small and
reproducible increase (2.5-fold) in STC mRNA levels that returned to
normal within 24 h (Fig. 4B). The expression of the P450
17-hydroxylase/C17–20 lyase gene (CYP17), which is known to be
responsive to PMSG (14), was also examined to ensure that the mice
responded to the superovulation regime as expected. The level of CYP17
mRNA increased steadily following PMSG injection and reached a maximum
just before hCG treatment. Following hCG treatment the level of CYP17
mRNA decreased rapidly in accordance with previous studies
demonstrating that high levels of LH (hCG) inhibit
transcription of the CYP17 gene (15, 16, 17). Hence, CYP17 and STC
were differentially regulated in the mouse model of superovulation.
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). STC
mRNA levels continued to increase until day 10 of pregnancy where they
peaked at 15 times the levels seen in normal cycling mice, and remained
elevated between 10–14 days, then dropped sharply at parturition (Fig. 5B). Following parturition the level of STC expression increased again
and remained elevated, at least by a factor of 6, over the next 10
days. Hence, the levels of gene expression peaked and fell over
gestation but rose again postpartum. Serum STC levels, which were also
assessed in the maternal circulation, rose and fell in concert with
gene expression. Although normally undetectable in the nonpregnant
mouse, serum STC rose to a maximum of 1.6 ng/ml on day 14 of gestation
(hormone levels essentially reached a plateau between days 10 and 16).
The levels then decreased to 1 ng/ml before parturition but remained
detectable throughout the entire postpartum period. To the best of our
knowledge, this is the first indication of STC in the circulation of
any mammal. Northern analysis of kidney STC gene expression during
gestation revealed no changes in the level of mRNA (data not shown)
indicating that changes seen in the ovary were organ specific.
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. STC mRNA was readily detectable in the
interstitium and theca compartment of the ovary at day 5.5 of gestation
(Fig. 6A). A noticeable increase in gene expression was observed at day
8.5 of gestation particularly in the interstitium (Fig. 6C). This trend
continued to day 14.5 of pregnancy where an extremely strong
hybridization signal was seen in both interstitial and theca cells
(Fig. 6E). By day 18.5 of gestation, however, there was a marked
reduction in signal in both of these cellular compartments (data not
shown). In lactating mothers, moderate levels of STC mRNA were found in
interstitial cells whereas the level of expression in theca cells was
now low (Fig. 6G). The CL also exhibited a weak signal at each
gestational stage examined.
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). The pattern of staining was
unchanged on day 8.5 (Fig. 6D). By day 14.5 of gestation, coincident
with the peak of gene expression, the CLs were more strongly stained
than at any other stage in gestation. The theca and interstitial
compartments were also most highly immunoreactive at this gestational
stage (Fig. 6F). By comparison, the ovaries of postpartum lactating
mice showed distinctly less STC staining in the interstitial and theca
compartments although luteal cells and oocytes of primary, secondary,
and graafian follicles continued to show moderate staining (Fig. 6H).
Collectively, the ISH and ICC results concurred with the northern
analysis indicating that STC production gradually rose and peaked at
mid-pregnancy.
STC gene expression in the ovary appears to be regulated during
lactation
Because the levels of ovarian STC mRNA remained elevated during
lactation, we explored the possibility that the presence of a nursing
litter might be an important factor in regulating STC mRNA levels
postpartum. Northern analysis revealed that the levels of STC mRNA were
indeed elevated in nursing mothers, peaking day 10 postpartum, and then
decreased thereafter to nonpregnant levels by day 21 when the pups were
weaned (Fig. 7A). In contrast, the level
of expression was unchanged in nonnursing mothers (Fig. 7B).
Consequently, the presence of a nursing litter had a stimulatory effect
on the production of STC in the maternal ovary, implying that STC has a
role in the physiology of lactation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With regard to postnatal development, the mouse ovary displays a degree of regionalization with respect to the localization of primordial follicles and stroma. Those oocytes destined to become the first primordial follicles are completely surrounded by a small layer of presumptive granulosa cells, ringed by basement membrane, and are located in the outer cortical layer of the newborn ovary, while the medulla or central portion of the ovary is primarily composed of stromal tissue (18, 19, 20, 21, 22). Our ISH analysis of newborn ovary consistently showed a diffuse signal over the entire organ. Because the ovary does not develop a significant complement of primordial follicles until postnatal day 3, it is not surprising that the hybridization signal was not regionalized in newborns. Precisely which cells are responsible for STC gene expression at this stage cannot be resolved and could include oocytes, follicle cells and stroma destined to be theca. The newborn ovary contains abundant stromal strands that emanate from the cortical region and reach the central part of the ovary; therefore, it is possible that the stroma or presumptive theca compartment is the primary source of STC at this time as it is in adults (11). ICC showed distinct staining in cells throughout the ovary, with patches of stronger staining in the cortical regions. These stronger stained cells appear to be oocytes and scattered follicle cells. Less intense staining was present in the strands of undifferentiated stroma in the cortical region of the ovary. Hence, it is clear at this stage of ovarian development that STC expression does not exhibit the cell-specificity of later stages. However, within the next few days primordial follicles form, consisting of one or more layers of granulosa cells and primary theca begins to develop around secondary follicles by day 5 (20). At this stage, the adult pattern of STC gene expression becomes recognizable as distinctly compartmentalized to the stroma surrounding the growing follicles. At the protein level, STC staining was most intense in the oocyte cytoplasm but was also present in some granulosa cells, both of which did not appear to express the STC gene. The primary theca surrounding each follicle stained for STC consistent with production by these cells. Therefore, it is clear that the adult pattern of STC gene expression and protein sequestration is established as early as 5 days after birth, coinciding with the biochemical markers of steroidogenesis such as LH receptors, CYP17, and P450 side-chain cleavage enzyme activity (23). Thus, it can be concluded that STC gene expression precedes thecal differentiation because it is expressed in the interfollicular stroma predestined to form the theca-interstitial cell (TIC) compartment of the ovary (24, 25).
At 2 weeks, STC gene expression remained highest in the TIC
compartment, although a low level of expression was also apparent in
granulosa cells. However, ICC on the same ovaries clearly showed the
strongest STCir staining in oocytes. This was reminiscent of the
pattern seen for GDF-9 except that oocytes also produce GDF-9, which
does not appear to be the case for STC (26, 27). We also observed lower
levels of STCir staining in theca cells and a complete lack of staining
in surrounding granulosa cells. An interpretation of these results
could be that STC is sequestered by oocytes, and this is mediated by a
specific receptor not present on granulosa cells as suggested by
the absence of STCir staining. Because ISH indicated that TICs are the
major source of ovarian STC, it is possible that the oocyte-sequestered
STC comes from that secreted by TICs. There are similar examples of
thecal-paracrine action on the follicle including BMP-4 and -7, which
are produced in theca, whereas their receptors are found on oocytes and
granulosa cells (28). TGF and EGF are also produced almost
exclusively in the TIC compartment, yet the cognate receptor is found
on granulosa cells and TGF accumulates in follicular fluid (29, 30).
Alternatively, the intense immunostaining for STC in the oocyte could
be due to a low level of gene expression coupled with low protein
turnover.
In an effort to determine if STC production was significantly modulated with a specific stage of the estrous cycle and therefore point to its potential involvement as a regulatory factor, we examined its expression in staged mouse ovaries. Our analysis of ovary STC mRNA levels by Northern blot and ISH indicated that STC gene expression was primarily found in the TIC compartment of follicles and did not vary over the estrous cycle. At the protein level, the most obvious finding was that STC was highly concentrated in CLs and oocytes. The most intense level of immunostaining occurred in the CLs at metestrus I; however, it was also clear that some CLs remained unstained. In STCir CLs the protein was concentrated in the cytoplasm of both small or large luteal cells. Variability in CL staining may correlate with CL age because the ovary of a normal cycling mouse contains CL from as many as 4 cycles (31). Variability in follistatin immunoreactivity has been correlated with CL maturation during estrous (32). Variability in CL staining has also been reported for relaxin and the relaxin-like factor (33, 34).
To examine STC gene expression in a more controlled manner during the maturation of follicles, we employed the superovulation model of immature mice. Northern blot analysis revealed a small but specific transient increase in steady-state STC mRNA levels 12 h after administration of hCG in PMSG primed mice. It is well established that the hCG treatment mimics the LH surge and that ovulation is initiated approximately 12 h after its administration (35, 36). Hence, the induction of STC expression coincident with the ovulatory period implies that STC is regulated by LH. That these same changes were not observed over the estrous cycle could be due to the fact that hCG treatment induces massive hypertrophy and differentiation of the thecal-interstitial compartment not normally seen in a normal cycling mouse (35). Therefore, the rise in STC expression during superovulation may simply have reflected an enhancement of thecal cell activity upon ovulation of an abnormally large number of follicles
The most dramatic changes in gene expression were revealed during gestation where steady-state level of STC mRNA increased 15-fold and was accompanied by the appearance of STC in the circulation. Normally, immunoreactive STC is not detectable in the serum of humans or rodents, which appears to be a result of its extremely short half-life and/or rapid degradation (13); therefore, pregnancy is unique in this regard. One could easily postulate roles for ovary-derived STC at sites such as the uterus, mammary gland, placenta, pituitary, and the embryo proper. Equally intriguing is how the clearance/degradation of STC could be altered during pregnancy to allow for its accumulation in the serum. Analogous to the increase in both GH and leptin during pregnancy, the rise in serum STC levels may be associated with soluble binding protein (37, 38). Interaction with soluble binding proteins has been proposed as a mechanism to inactivate high circulating levels of GH and leptin and regulate availability to target tissues during pregnancy.
Enhanced production of STC during gestation does not appear to due to an increase in TICs or their activity or production by other structures of the ovary. According to Pedersen and Peters (39), ovarian function during pregnancy is not significantly altered with regard to follicle maturation because it occurs continuously throughout gestation but with significantly fewer follicles than normal. Moreover, Choudary and Greenwald (40) describe a sharp increase in follicle atresia between days 10–14 of gestation, which coincides with the rise in ovarian STC production. Hypertrophy or proliferation of the maternal ovarian stroma is not a hallmark of gestation (39, 40) and cannot completely account for the massive induction of STC production during this time. The maternal ovarian weight increases during the second half of pregnancy due to continued growth of the CLs and renewed growth of antral follicles (39, 40, 41) but our studies indicate that the CLs do not produce significant amounts of STC. In fact, before parturition, the development of antral follicles and the associated increase in TICs correlated with a drop in ovarian STC production and regression of the CL of pregnancy (41). Consequently, it appears that induction of ovarian STC synthesis cannot simply be accounted for by significant changes in the population of TICs that produce the hormone.
It is more likely that a regulatory event mediated by a local ovarian factor may be responsible for strong induction of STC gene expression during pregnancy. Rescue of the CL of estrous to the functional CL of pregnancy is accompanied by a decrease in circulating LH and an increase in progesterone (P4), ether of which could be a trigger for STC induction (44, 45). Enhanced STC synthesis during pregnancy also correlates with the production of luteotropic PRL-related molecules such as PL-I by the fetal placenta (42, 43); however, direct effects of PL-I on the ovarian stroma have not been documented. Nevertheless, the peak in ovarian STC synthesis during gestation does correlate with the switch from pituitary to placenta in terms of production of luteotropic factors and this process (42, 43) may regulate STC production.
Interestingly, normal postpartum expression of ovarian STC appears to be coupled to the presence of a nursing litter or perhaps the suckling stimulus. Removal of a nursing litter correlated with a significant decrease in the steady-state level of maternal ovarian STC mRNA. Ovarian progesterone and pituitary PRL production are directly related to the number of suckling pups in the rat and they function to suppress follicular maturation during the lactation period (44, 45). During the first half of lactation, suckling induces circulating PRL that is luteotropic, thereby, maintaining high circulating progesterone. It is possible that PRL regulates ovarian STC production during lactation either directly or indirectly through induction of high progesterone levels. The fact that STC production is significantly up-regulated during lactation is suggestive of an affect on milk production and/or mammary gland morphogenesis as has been shown for PTHrP (46, 47). In any case, this is the first demonstration of an ovarian thecal cell product whose production is regulated by the presence of a litter perhaps through endocrine effects or exteroceptive stimuli.
Stanniocalcin, to our knowledge, is unique because no other secreted proteins produced by the ovarian thecal-interstitial compartment are significantly stimulated during mouse pregnancy. Only relaxin, produced by the corpora lutea, is significantly up-regulated in the pregnant mouse ovary (33). Therefore, this is the first demonstration of a theca-interstitial product that is regulated by the pregnant state. In summary, our data provide evidence for the active regulation of STC expression in the ovary during gestation and lactation. It is therefore reasonable to postulate that STC could be involved in gestation and lactation. Future experiments will focus on determining whether STC is a new regulator of the gestational and nursing state.
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Footnotes |
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2 These authors should be considered co-first authors.
Received February 29, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, cytochrome P450scc, and
3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian
follicles and corpora lutea: correlation with theca cell
steroidogenesis. Biol Reprod 37:211–223[Abstract]
-hydroxylase and C-17-C-20 lyase is due to decrease of microsomal
cytochrome P-450 through de novo synthesis of ribonucleic acid and
protein. Endocrinology 113:1985–1991[Abstract]
-hydroxylase and P450
aromatase in preovulatory bovine follicles decrease after the
luteinizing hormone surge. Endocrinology 132:2239–2245[Abstract]
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C. R. McCudde |
.
Inset, A higher magnification of a section of panel A. B,
ISH darkfield micrograph of an adjacent section to panel A, shows a
diffuse pattern of STC gene expression over the entire ovary. C, ICC
staining of a day 5 ovary shows high levels of STCir in the oocytes of
primordial, unilaminar, and multilaminar follicles and lower levels in
the stroma. D, Higher magnification of day 5 ICC staining demonstrating
heterogeneity in granulosa cell STCir staining within follicles. E, ISH
darkfield micrograph of an adjacent section to panel C, shows the
transition from an indistinct pattern of STC expression in a newborn ovary to one of clear,
strong circular patterns of STC expression in stromal cells 
. D,
During diestrus the pattern of STCir staining was similar to that seen
at other stages with the CL being the most prominent structure stained.
E, Correlative ISH darkfield micrograph of metestrus II ovary shows
high levels of STC mRNA in the theca surrounding each peripheral
follicle and moderate levels in the interstitium. This pattern of
expression was typical for all stages of the estrous cycle examined. F,
A higher magnification of 2 CLs from panel B (box) highlighting the
variation in staining pattern that was often observed. cl, Corpus
luteum; af, antral follicle; i, interstitium; pa, preantral follicle;
t, theca.
