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Division of Development and Reproduction (D.G.A., G.B., C.G.G., C.O.H., A.L.G.), Roslin Institute, (Edinburgh), Roslin, Midlothian, EH25 9PS, Scotland, United Kingdom; University of Edinburgh (B.K.C., T.A.B.), Department of Obstetrics and Gynaecology, Centre of Reproductive Biology, Edinburgh, EH3 9EW, Scotland, United Kingdom; and University of Nottingham (R.W.), Division of Agriculture and Horticulture, School of Biological Sciences, Sutton-Bonington Campus, Loughborough, Leicstershire, LE12 5RD, United Kingdom
Address all correspondence and requests for reprints to: D. G. Armstrong, Division of Development and Reproduction, Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, Scotland, United Kingdom. E-mail: david.armstrong{at}bbsrc.ac.uk
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most of the early concepts on the intraovarian control of ovarian function (particularly those related to the IGF system) were developed using in vitro rodent models (4). However, there is considerable species variation in both the temporal and spatial patterns of growth factor expression during folliculogenesis, and it would be wrong to extrapolate observations made in rats and mice to other species, many of which show considerable differences in their patterns of follicle growth. For example, in rodents (5) and pigs (6), the expression of messenger RNA (mRNA) encoding IGF-I is confined to granulosa tissue, whereas in humans, mRNA encoding IGF-II, but not IGF-I, is localized to granulosa tissue (7). We have detected the expression of mRNA encoding IGF-II in thecal tissue of bovine ovarian follicles (2), and a similar spatial distribution has been described in sheep (8). The expression of mRNA encoding IGF-I in ruminants remains controversial. Leeuwenberg et al. (9) detected IGF-I mRNA in ovine granulosa and theca tissue, whereas Perks et al., (8) failed to detect the expression of mRNA encoding IGF-I in ovarian follicles from the ewe. We have recently demonstrated that nonluteinized bovine granulosa cells do not produce IGF-I in serum-free cultures (10).
The bioactivity of IGFs is controlled by their association with a family of, at least six, specific IGF binding proteins (IGFBPs), which have been characterized, after purification and/or complementary DNA (cDNA) isolation and sequencing (11, 12). As with IGFs, the spatial expression of these binding proteins within ovarian follicles is species-specific. In the sheep, for example, IGFBP-4 is produced by theca cells (13, 14), whereas in the pig, this binding protein is expressed in granulosa cells (15). In contrast, IGFBP-4 is expressed in both granulosa and thecal tissue in women (16).
In all ovarian cell culture systems examined, so far, IGFBPs attenuate the actions of IGFs (17, 18, 19). A decrease in follicular IGFBP production, therefore, would be expected to result in increased biological activity of locally produced IGF and thus increase the response of the follicle to gonadotropins. Although the observed changes in the concentration of IGFBPs in follicular fluid during folliculogenesis agree with this hypothesis (14, 17), the precise role of individual binding proteins within the developing follicle remains obscure.
We are currently analyzing the IGF-system in the bovine ovary, and we are particularly interested in how IGFBPs regulate the development and maintenance of follicular dominance. The cow is a monoovulatory species and provides a particularly useful model to study dominance (20, 21, 22). Greater than 96% of cows produce a single ovulation per estrous cycle, and mechanisms regulating dominance in this species, therefore, are presumed to be more prominent than in polyovulating species. Follicle growth in cattle occurs in a wave-like pattern, with two or three waves per estrous cycle. Each wave is associated with the selection of a large ovulatory, gonadotropin-dependent follicle from a cohort of growing, gonadotropin-sensitive follicles. This so-called dominant follicle suppresses the growth of the remaining follicles within the cohort (subordinate follicles) and will eventually ovulate if its selection coincides with the luteolytic phase of the cycle.
We have developed a serum-free primary cell culture system for ruminant granulosa and theca cells in which the follicular phenotype is maintained for up to 6 days in culture (23, 24). Our previous work, using ovine cultures, indicated that IGFBP-2 and -4 are the major binding proteins produced by ruminant granulosa and thecal cells (14). In an attempt to define the mechanisms by which IGFBPs modulate the bioactivity of IGFs in the intact bovine ovarian follicle more precisely, we have examined the effects of gonadotropin on IGFBP-2 and -4 mRNA expression in vitro and have compared this with the spatial/temporal changes in IGFBP-2 and -4 mRNA expression during the development of small (1–4 mm) gonadotropin-sensitive follicles into medium-sized (4–8 mm) and large (>8 mm) gonadotropin-dependent follicles, using in situ hybridization. From the results of this investigation, we have developed a hypothesis for the role of IGFBP-2 and-4 in the development of follicular dominance in the cow.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Collection of tissue and characterization of follicles
Ovaries were obtained from the local abattoir. They were either
processed directly for cell cultures or were divided into blocks, had
follicle diameters measured, were frozen in liquid nitrogen, and stored
at -70 C until required for subsequent in situ
hybridization or immunohistochemistry. Follicles were classified
morphologically as healthy, atretic, or grossly atretic. Healthy
follicles had an intact basement membrane and a healthy granulosa cell
layer. Atretic follicles had fewer granulosa cells with local
disruption in the basement membrane. Cells with pycnotic nuclei were
identified within the granulosa layer. Follicles with a more
extensively disrupted basement membrane, with a significant reduction
in the number of granulosa cells and an increase in the number of
pycnotic nuclei, were classified as grossly atretic (25).
Granulosa and theca primary cell cultures
Granulosa and theca cells from medium-sized (4–8 mm)
gonadotropin-dependent follicles were isolated and pooled. Three
million cells were cultured in duplicate in 10 ml of the appropriate
culture medium for up to 96 h, as described previously (24).
Granulosa cell cultures were supplemented with 10 ng/ml of insulin and
with 0, 1, or 50 ng/ml FSH or 0 or 100 ng/ml LH. Under those conditions
FSH, at a dose of 1 ng/ml, maintained estradiol production for up to 5
days in culture. The 50 ng/ml dose of FSH inhibited estradiol
production and increased progesterone production. LH had no effect on
the production of either estradiol or progesterone by granulosa cells
under the conditions described here.
After isolation of the granulosa cells, the remainder of the follicle wall was dispersed by enzymatic digestion to isolate the theca cells (26). Briefly, theca shells were digested at 37 C in an enzymatic mixture (Sigma Chemical Co. Ltd., Poole, UK) containing collagenase (type 1A) (5 mg/ml), pronase E (1 mg/ml), and hyaluronidase (1 mg/ml) in 20 ml of Dulbecco’s PBS without calcium chloride and magnesium chloride. After 20 min, 200 µl DNA (2 mg/ml) was added, and the incubation continued. The reaction was stopped by addition of fetal donor serum (1 ml) and the solution containing the cells was transferred to centrifuge tubes, where it was diluted with an equal volume of theca cell culture medium consisting of DMEM:Hams F12 (1:1) media supplemented with bicarbonate, 15 mM HEPES, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 3 mM L-glutamine, 0.1% BSA, 20 ng/ml estradiol, 2.5 µg/ml transferrin, and 4 ng/ml selenium; and the solution was centrifuged. After a second wash, the cells were resuspended in 15 ml of theca culture medium, and the cell number and viability were estimated in a hemocytometer using Trypan blue exclusion. Aliquots containing 3 x 106 viable theca cells were then cultured in the theca culture medium, supplemented with insulin (100 ng ml) and 0 or 100 ng/ml LH, in a humidified atmosphere with 3.8% CO2 at 37 C replacing media every 48 h. Under those conditions, LH significantly increased the production of both androstenedione and progesterone.
RT-PCR
RNA was isolated from granulosa and theca cells after 96 h
of culture using RNeasy spin columns (Qiagen Ltd., Crawley, UK),
according to the manufacturer’s instructions. Concentrations were
estimated by absorbance at 260 nm, and the samples were stored in
RNase-free 10 mM Tris buffer, pH 74, containing 1
mM EDTA at -80 C until required. The
A260/A280 ratio for all RNA samples was more
than 1.8. First-strand cDNA synthesis was carried out using SUPERSCRIPT
II RT (Life Technologies), using a modification of the method described
previously (27). Briefly, total RNA (1 µg) was reverse transcribed in
20 µl Tris buffer (50 mmol/liter; pH 8.3) supplemented with KCl (75
mmol/liter), MgCl2 (3 mmol/liter), dithiothreitol (10
mmol/liter), deoxynucleotide triphosphates (0.5 µmol/liter),
acetylated BSA (0.1 mg/ml), random hexamers (50 µmol/liter),
recombinant RNasin ribonuclease inhibitor (Promega, Southampton, UK) (4
U), and SUPERSCRIPT II RT (13.5U) at 37 C for 60 min. The PCR reaction
was carried out using 2 µl of the RT reaction (equivalent to 0.1 µg
of the original total RNA) in 8 µl PCR buffer. The primers used for
the PCRs are shown in Table 1
. Samples
were heated to 94 C for 3 min and amplified for 25 cycles (94 C for 1
min; 55 C for 1 min, and 72 C for 1 min). The final 72 C incubation was
continued for a further 4 min. RT blanks, RNA blanks, and PCR blanks
(no cDNA products) and positive controls were included in each
analysis. Products were visualized by electrophoresis on 4% agarose
gels (NuSieve GTG Agarose; FMC Bioproducts, Rockland, ME). In all
experiments, RT blanks, RNA blanks, and PCR blanks were negative.
Identities of the PCR products were confirmed by restriction
endonuclease digestion and by DNA sequencing of representative
samples.
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IGFBP-2 and -4 RNA probes
A plasmid containing the bovine IGFBP-2 probe was a gift from
Dr. M. Lucy (University of Missouri). The probe corresponded to
positions 1084–1192 of a bovine IGFBP-2 cDNA (28). The IGFBP-4 probe
was prepared by RT-PCR (see previous section) using the oligos
described in Table 1
. The 227-bp amplified fragment was cloned into
pGEM-T (Promega Ltd., Southampton, UK) using standard procedures, and
it was sequenced to confirm its identity and orientation within the
plasmid. The construct was linearized with NcoI and was used
as a template to generate 35S-labeled antisense RNA, using
SP6 DNA-dependent RNA polymerase. A sense RNA probe was prepared, using
T7 DNA-dependent RNA polymerase, after linearization with
SalI.
In situ hybridization
Frozen sections (14 µm) were dehydrated, fixed, and probed
with 35S-labeled IGFBP-2 and -4 riboprobes, according to
the method described by Xu et al. (29). After the final high
stringency wash, the sections were dipped in autoradiographic K2
photographic emmulsion (Ilford Limited, Mobberley, UK) and exposed for
3 weeks at 4 C. Sections were then developed (Kodak D-19) (IBI Limited,
Cambridge, UK) and fixed (Ilford Hypam fixer) before staining in
hematoxylin and eosin. The sections were finally mounted in DPX
mountant, before microscopic examination, using both light- and
dark-field illumination. Each follicle was examined in six serial
sections. The antisense RNA probes for IGFBP-2 and -4 were each
hybridized to two of the sections, and the remaining two slides were
hybridized with the sense RNA probe for each binding protein.
Image analysis
The intensity of the in situ hybridization signal was
analyzed using an NIH-Image analysis system (NIH, Bethesda, MD). The
number of graphic pixels occupied by silver grains (identified by a set
gray threshold) within a defined area of the tissue section was counted
and presented as a percentage of the total pixel number within the
defined area. The hybridization intensity, therefore, is presented as
the percentage of occupied pixels to total pixels within a defined area
of the tissue. Background hybridization intensity, measured with the
sense RNA probes, was subtracted from the measurements obtained with
the antisense probes to give the final hybridization signal. Within
each follicle, three separate fields were analyzed for each probe.
There was no significant difference (P > 0.05) in
hybridization intensity obtained with antisense and sense RNA probes
within a nonexpressing region of a tissue section. Under the conditions
described here, the hybridization signal was proportional to the length
of time the slides were exposed to photographic emmulsion (for up to 3
weeks).
Immunohistochemistry
Frozen sections (10 µm) were immersed in blocking buffer (50
mmol/liter Tris buffer, pH 7.2, supplemented with 5% BSA (RIA grade)
and 1% Tween 20) for 2 h at room temperature. The solution was
removed, and the sections were immersed in blocking buffer containing
the primary antibody (rabbit antibovine IGFBP-2 or rabbit antihuman
IGFBP-4; Upstate Biotechnology Incorporated, Lake Placid, NY) diluted
1:1000 and 1:500 in blocking buffer for IGFBP-2 and -4 antibodies,
respectively, overnight at 4 C. The sections were washed and stained
using an antirabbit IgG biotin-avidin, peroxidase kit (Sigma),
according to the manufacturer’s instructions.
The IGFBP-2 antisera cross-reacted with a single protein band (Mr = 34 x 103) in immunoblots of bovine follicular fluid obtained from dominant follicles. In contrast, the IGFBP-4 antiserum was less specific, and it recognized three proteins, two of which (Mr = 25 and 29 x 103) were assumed to be the nonglycosylated and glycosylated forms of IGFBP-4. The third protein (Mr = 34 x 103) was identical in size to that detected by the IGFBP-2 antibody (results not shown).
Steroid measurement
Androstenedione and progesterone were measured by RIA (30).
Estradiol-17ß was also measured by RIA, according to the method
described by Webb et al (31).
Statistical analysis
Steroid production by cell cultures and band intensities of PCR
products are presented as mean ± SEM (three cultures
with two replicates). Comparison between treatments was assessed by
repeated-sample ANOVA. The effects of FSH or LH (fixed effects) were
analyzed using the mixed models, allowing for variation caused by
culture and different pools of cells (random effects).
The effect of follicle size and health status on the intensity of the
in situ hybridization signal was determined by ANOVA and the
2 test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, were identical to the expected bovine IGFBP-2 and
-4 sequences (results not shown). Expression was measured in cells
cultured for 4 days in the presence and absence of either FSH
(granulosa) or LH (theca). Typical agarose gels are shown in the
insets to Fig. 1. The results
from three separate experiments, obtained after image analysis of the
PCR gels, are presented. Granulosa IGFBP-2 mRNA expression (Fig. 1a)
was significantly reduced (P < 0.001) after treatment
with FSH (50 ng/ml) for 4 days. IGFBP-4 mRNA expression (Fig. 1b) was
increased significantly (P < 0.05) in thecal cells
cultured in the presence of LH (100 ng/ml). There was no change in the
amount of the amplified cDNA of the bands obtained with primers for
ATPase between all the incubations.
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IGFBP-2 mRNA was located in granulosa cells from small and medium-sized
healthy antral follicles (1–8 mm in diameter) (Fig. 3, a and b). The granulosa cells closest
to the basement membrane showed the greatest mRNA expression. In
contrast, both the cumulus and the outermost layer of the mural
granulosa cells did not contain IGFBP-2 mRNA. The proportion of large
healthy (>8 mm) follicles expressing IGFBP-2 mRNA in granulosa tissue
was significantly less (2 = 17.5; P <
0.001), compared with small (1–4 mm) and medium-sized (4–8 mm)
follicles (Table 2). In contrast to
healthy follicles, IGFBP-2 mRNA expression was detected in theca cells
from atretic follicles (Fig. 2).
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, d and e). In this case, there was no
difference in expression between small, medium, and large follicles
(P > 0.05). In atretic follicles of all sizes, low
(but detectable) amounts of IGFBP-4 mRNA were detected in granulosa
cells. Grossly atretic follicles did not express significant levels of
mRNA encoding IGFBP-2 or -4 (Fig. 2).
Immunohistochemical localization of IGFBP-2 and -4
The spatial distribution IGFBP-2 and -4 immunoactivity was
examined in six ovaries, three of which contained a healthy follicle
greater than 8 mm in diameter. A representative sample of the spatial
distributions observed within healthy follicles is shown in Fig. 4. IGFBP-2 immunoactivity was detected in
granulosa tissue and the basement membrane of healthy antral follicles
ranging in size from 1–8 mm in diameter (Fig. 4, a and c). It was not
detected in large (>8 mm), healthy follicles (Fig. 4c).
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). Because of the
cross-reactivity of the IGFBP-4 antisera with IGFBP-2, a proportion of
the signal observed in granulosa tissue using this antiserum would be
expected to be caused by the presence of IGFBP-2. Nevertheless, the
presence of IGFBP-4 immunoreactivity in granulosa tissue of all
follicles examined, including large (>8 mm in diameter), healthy
follicles, which did not cross-react with IGFBP-2 antiserum, provides
evidence supporting the presence of IGFBP-4 immunoreactivity in
granulosa tissue. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By combining the results of our in vivo and in vitro studies, we are now able to shed light on three basic questions concerning the role of IGFBP-2 and -4 in regulating follicle development in the cow: 1) how the patterns of IGFBP mRNA expression in granulosa and theca tissue relate to the changes in the concentration of IGFBPs in bovine follicular fluid during folliculogenesis; 2) what mechanisms are involved in the regulation of the expression of IGFBP-2 and -4 in granulosa and theca tissue; and 3) what the roles of IGFBP-2 and -4 are in the regulation of follicle growth and the development of dominance.
The changes in IGFBP concentration in bovine follicular fluid during follicle growth and the development of dominance have been well documented (33, 34, 35) and are similar to those occurring in sheep (14, 17) and pigs (36, 37). Dominance was associated with a decrease in the concentrations of IGFBP-2 and -4 in follicular fluid. The changes in the IGFBP content of follicular fluid could arise through the regulation of IGFBP production, by changes in the activity of specific IGFBP proteases, or by changes in the selective uptake of IGFBPs from the circulation during follicle growth. In the case of IGFBP-2 mRNA expression in bovine follicles, there was no difference in expression between small (1–4 mm) and medium-sized (4–8 mm) healthy follicles. However, 60% of the large (>8 mm) healthy follicles examined did not express IGFBP-2 mRNA, and the remaining 40% expressed low levels of IGFBP-2 mRNA. This observation was supported by the absence of IGFBP-2 immunoactivity in the large (>8 mm) healthy follicles examined in this study. We conclude that the reduction in IGFBP-2 concentration in follicular fluid during the final stages of follicular growth is caused by a decrease in IGFBP-2 mRNA expression. In contrast to IGFBP-2, IGFBP-4 mRNA expression did not change during follicle growth, and the decrease in IGFBP-4 concentration in follicular fluid, in this case, is most likely to be caused by the action of specific proteases, as suggested previously by Besnard et al. (38). This group described an IGFBP-4 protease in follicular fluid of the ewe, the levels and/or activity of which increase during follicle growth.
The spatial distribution of IGFBP-2 and -4 mRNA expression between granulosa and theca tissue, described here, was similar to that described in the ewe (13, 14), but differed from that in pigs (39), rodents (40), and humans (7). Our results also highlight the variation between different subtypes of granulosa cells in relation to IGFBP-2 mRNA expression. The inner layers of the mural granulosa cells contained significantly higher amounts of IGFBP-2 mRNA, relative to the outer layer of the mural and cumulus cells. The significance of this observation, however, remains obscure.
The regulation of IGFBP-2 and -4 mRNA expression in granulosa and theca cells, respectively, was analyzed using a serum-free cell culture system. Steroid production by the primary cultures of granulosa cells was similar to that described recently (24) and indicated that the follicular phenotype was maintained for up to 4 days without significant luteinization. FSH (1 ng/ml) stimulated granulosa cell differentiation, as judged by the increase in estradiol production. However, when the cells were exposed to high (50 ng/ml) doses of FSH, estradiol production was inhibited, whereas progesterone production increased, suggesting that under those conditions, the cells were luteinized. The absence of an effect of LH on steroid production by granulosa cells isolated from medium-sized follicles suggested that these cells had yet to develop a functional LH receptor. Using this cell culture system, FSH concentrations of 50 ng/ml were shown to switch off the expression of mRNA encoding IGFBP-2 in granulosa cells. This result supports previous observations, using ovine granulosa cells, that showed decreased IGFBP-2 production in the presence of FSH (14).
The steroid profiles of thecal cell cultures used in this study indicate that the follicular phenotype of these cells can be maintained also under the culture conditions described here. Doses of LH that luteinize the cells stimulated the expression of mRNA encoding IGFBP-4. Similarly, using ovine theca cells, we have shown that LH stimulates the production of IGFBP-4 in cell-conditioned medium (14).
IGFBPs control IGF bioactivity by regulating its availability to IGF
receptors (41). Immunohistochemical localization of IGFs has shown that
they codistribute with the IGFBPs (42), which are often found in
association with the extracellular matrix (ECM), providing an
extracellular store of IGFs that can be accessed by the action of IGFBP
proteases (43). Our immunohistochemical observations demonstrated the
presence of both IGFBP-2 and -4 in granulosa tissue (Fig. 4).
Immunocytochemistry revealed the presence of both these binding
proteins on the plasma membranes of granulosa cells and within the ECM
surrounding these cells (44). The presence of IGFBP-2 and -4 in the ECM
surrounding granulosa tissue provides evidence for the existence of
biologically-distinct pools of IGF, which can be differentially
regulated by the action of specific proteases. In this respect, IGFBP-2
and -4 proteases with molecular weights of 36,000 and 48,000,
respectively, have been shown to be produced by porcine aortic
smooth-muscle cells in culture (45). Specific IGBP-2 and -4 proteases
also have been demonstrated in ovine follicular fluid (38). Thus,
changes in the expression of mRNA for IGFBP-2 and -4 during
folliculogenesis, coupled with changes in the activity of
IGFBP-specific proteases, provide a mechanism to regulate the
bioavailability of IGF during follicle growth.
A major mechanistic problem in the development of dominance is how the
selected follicle increases its capacity to produce estrogen during a
period when circulating FSH concentrations decrease. Granulosa
aromatase activity is regulated by the synergistic actions of IGF-I and
FSH (1, 3, 4, 46), and hence, regulation of the intrafollicular
bioactivity of IGFs may be a key mechanism in the development of
dominance. Although IGFBPs inhibit IGF action on granulosa cells
in vitro, our results suggest the existence of different
roles for IGFBP-2 and -4 in the developing follicle in vivo.
We hypothesize that IGFBP-4, produced by thecal cells, under the
control of LH, is transported in association with IGFs to the ECM
surrounding granulosa cells, where it acts as an extracellular storage
site for IGF. Supporting this hypothesis was the observation that
although mRNA encoding IGFBP-4 was confined to theca, immunoreactive
protein was detected in both theca and granulosa tissue. We suggest
that the IGFs associated with IGFBP-4 can then be accessed by granulosa
cells through the activation/production of specific IGFBP-4 proteases
(38). In contrast to IGFBP-4, IGFBP-2 produced by granulosa tissue may
play an inhibitory role. After binding IGF, the affinity of IGFBP-2 for
heparin-like molecules in the ECM surrounding granulosa cells increases
(47). Hence, IGFBP-2 could sequester free IGF from the immediate
vicinity of granulosa cells. Granulosa IGFBP-2 mRNA expression is
inhibited by FSH (Fig. 2a), resulting in the absence of IGFBP-2 mRNA
and immunoreactive IGFBP-2 in dominant follicles (Figs. 3 and 4,
respectively). This, combined with a corresponding increase in IGFBP-4
protease activity in large follicles (36), may increase the biological
activity of intrafollicular IGFs and, in turn, increase the sensitivity
of the granulosa cells to FSH, allowing the follicle to maintain
responsiveness to FSH during the period when systemic FSH
concentrations decrease (establishing this follicle as the dominant
follicle).
The present study has described temporal and spatial changes in IGFBP-2 and -4 mRNA expression during follicular growth. The effects of LH on thecal IGFBP-4 production, and FSH on granulosa IGFBP-2 production, are described; and the importance of decreased IGFBP-2 mRNA expression in large healthy follicles, as a mechanism for the maintenance of follicle growth, is highlighted. These results will contribute to the development of hypotheses relating to the specific roles of IGFBPs in the bovine ovarian follicle.
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Footnotes |
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2 Present address: Institute of Pediatrics, East Siberian Branch of
the Russian Academy of Medical Science, Timirjazeva 16, 664003,
Irkutsk, Russia.
Received September 3, 1997.
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References |
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-hydroxylase, and cytochrome P450 aromatase in bovine follicles
during the first follicular wave. Endocrinology 136:981–989[Abstract]
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M.A. Beg, D.R. Bergfelt, K. Kot, and O.J. Ginther Follicle Selection in Cattle: Dynamics of Follicular Fluid Factors During Development of Follicle Dominance Biol Reprod, January 1, 2002; 66(1): 120 - 126. [Abstract] [Full Text]
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 A. Hoeflich, R. Reisinger, H. Lahm, W. Kiess, W. F. Blum, H. J. Kolb, M. M. Weber, and E. Wolf Insulin-like Growth Factor-binding Protein 2 in Tumorigenesis: Protector or Promoter? Cancer Res., December 1, 2001; 61(24): 8601 - 8610. [Full Text] [PDF]
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O.J. Ginther, M.A. Beg, D.R. Bergfelt, F.X. Donadeu, and K. Kot Follicle Selection in Monovular Species Biol Reprod, September 1, 2001; 65(3): 638 - 647. [Abstract] [Full Text] [PDF]
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J.E. Fortune, G.M. Rivera, A.C.O. Evans, and A.M. Turzillo Differentiation of Dominant Versus Subordinate Follicles in Cattle Biol Reprod, September 1, 2001; 65(3): 648 - 654. [Abstract] [Full Text] [PDF]
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G.M. Rivera, Y.A. Chandrasekher, A.C.O. Evans, L.C. Giudice, and J.E. Fortune A Potential Role for Insulin-Like Growth Factor Binding Protein-4 Proteolysis in the Establishment of Ovarian Follicular Dominance in Cattle Biol Reprod, July 1, 2001; 65(1): 102 - 111. |
