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Experimental Studies |
Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Dr. R. L. Stouffer, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascular endothelial growth factor (VEGF), an endothelial-specific mitogenic, chemotactic, and permeability agent, is a key agent associated with rapid vascular growth in a variety of physiological and pathological conditions (11, 12). In situ hybridization and Northern analyses detected VEGF messenger ribonucleic acid (mRNA) expression within the dominant follicle and developing corpus luteum of both nonprimate (13) and primate (14, 15) species. Increased VEGF mRNA expression in the rat ovary after a bolus of hCG provides indirect evidence that VEGF production and secretion by periovulatory follicles is stimulated by the midcycle gonadotropin surge (16). Additionally, decreased VEGF mRNA expression in the monkey corpus luteum (15) after the administration of a GnRH antagonist suggests that VEGF production is also dependent on tonic gonadotropin secretion during the ovarian cycle. Recently, VEGF was localized by immunocytochemistry to human granulosa cells in the preovulatory follicle and to luteal cells within the corpus luteum (17, 18). However, studies directly examining VEGF production and its regulation by gonadotropins in granulosa/luteal cells of primate species have not been reported. The objectives of this study were 1) to determine whether macaque granulosa cells produce VEGF in response to an in vivo ovulatory surge of FSH- or LH/CG-like gonadotropins, and 2) to examine the direct effects of pure human (h) gonadotropins [i.e. recombinant hFSH (r-hFSH) and r-hLH] on VEGF production by preovulatory (nonluteinized) and periovulatory (luteinized) granulosa cells in culture. In addition, VEGF production was correlated with progesterone synthesis by granulosa cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rhesus monkeys (Macaca mulatta) were maintained at
the Oregon Regional Primate Research Center as previously described
(19). Animal protocols were approved by the Oregon Regional Primate
Research Center Animal Care and Use Committee, and studies were
conducted in accordance with the NIH Guide for the Care and Use of
Laboratory Animals. Adult female monkeys exhibiting regular menstrual
cycles (
28 days) were checked daily for menses. Beginning at menses,
monkeys received human gonadotropins to promote the development of
multiple preovulatory follicles (20, 21). In study 1, animals either
received twice daily injections of urinary gonadotropins \[30 IU hFSH,
twice daily (Metrodin, Serono Laboratories, Norwell, MA) for 6 days
followed by 30 IU hFSH and 30 IU hLH twice daily (Pergonal, Serono
Laboratories) for 2–3 days\] followed by no ovulatory stimulus or the
standard ovulatory stimulus (1000 IU hCG; Profasi, Serono Laboratories;
n = 6/group) (20), or monkeys received r-hFSH (30 IU, twice daily;
Laboratoires Serono, Aubonne, Switzerland) for 12 days followed by a
bolus of 100 or 1000 IU r-hCG (Laboratoires Serono) or 2500 IU r-hFSH
(n = 5/group) (21). In study 2, monkeys received r-hFSH (30 IU,
twice daily) for 6 days and r-hFSH (30 IU) plus r-hLH (30 IU) twice
daily for 3 days followed by no ovulatory stimulus or 1000 IU of hCG
(n = 6/group). Monkeys in both studies received a GnRH antagonist,
antide (1 mg/kg BW·day; Laboratoires Serono), throughout the
follicular stimulation protocol to block endogenous secretion of
pituitary gonadotropins (22). Daily blood samples were obtained from
unanesthetized animals by saphenous venipuncture from the beginning of
gonadotropin treatment until 4 days after follicular aspiration. Serum
estradiol or progesterone concentrations were determined by RIA (23, 24). Follicular growth/maturation was monitored by serum estrogen
levels and ultrasonography (22). Baseline levels (
0.1 ng/mL) of serum
progesterone in samples collected after follicle aspiration confirmed
that animals receiving no ovulatory stimulus lacked an endogenous surge
of pituitary gonadotropins.
Cell preparation and culture
Granulosa cells were obtained by follicle aspiration during laparoscopy of anesthetized animals (25) either the morning after the last FSH plus LH treatment (nonluteinized granulosa cells; NLGC) or 27 h after administration of the bolus of hCG and/or FSH (luteinized granulosa cells; LGC). After oocyte removal, enriched preparations of granulosa cells from individual monkeys were obtained as previously described (26). Pooled follicular aspirates were centrifuged at 290 x g for 10 min to obtain a cell pellet that was resuspended in Ham’s F-10 medium (Life Technologies, Grand Island, NY) supplemented with penicillin G (20 mg/L), streptomycin sulfate (100 mg/L), sodium bicarbonate (1.2 g/L), HEPES buffer (4.7 g/L), and BSA (1 g/L; Sigma Chemical Co., St. Louis, MO), pH 7.4. The cell suspension was layered on a 40% Percoll (Sigma Chemical Co.) and 60% Hanks’ Balanced Salt Solution containing 0.1% BSA and centrifuged at 470 x g for 30 min (26). The resulting layer of granulosa cells was resuspended in culture medium, cell numbers were determined using a hemocytometer, and cell viability was assessed by trypan blue exclusion.
In study 1, granulosa cells collected after the urinary hFSH plus hLH stimulation protocol were plated on fibronectin (Sigma)-coated eight-well glass chamber slides (Nunc, Naperville, IL) at 8 x 104 cells/well and cultured in DMEM-Ham’s F-12 plus ITSA [insulin (2 mg/L), transferrin (5 mg/L), H2SeO3 (0.25 nmol), and aprotinin (25 g/L); Sigma] medium containing 25 µg/mL human low density lipoprotein (Sigma) (26). Granulosa cells collected after the r-hFSH stimulation protocol were plated at 5 x 104 cells/well in 48-well plastic plates and cultured in Ham’s F-10 medium and 10% amenorrheic monkey serum (AMS) (21). In both cases, granulosa cells were incubated at 37 C in a 5% CO2-95% air environment for 24 h in the presence and absence of 100 ng/mL hCG (CR123-NIH) (26). Media were collected daily and frozen at -20 C until assayed for VEGF and progesterone. Cells were cultured in triplicate for each treatment group, and each experiment used a cell preparation from one monkey.
In study 2, granulosa cells were plated at 8 x 104 cells/well on fibronectin-coated eight-well chamber slides and cultured in DMEM-Ham’s F-12, ITSA, and 25 µg/mL hLDL (i.e. control medium) for 6 days, with medium changed daily and frozen until assayed for VEGF and progesterone. Preparations of NLGC (n = 3) and LGC (n = 3) were cultured in control medium alone or in medium containing 0.1, 1, 10, and 100 ng/mL r-hFSH and r-hLH or 100 ng/mL hCG (CR-123). Again, in vitro treatments were tested in triplicate for each cell preparation (animal). To examine plating efficiency, cell proliferation, and the effects of combined LH and FSH treatment, additional groups of NLGC (n = 3) and LGC (n = 3) were cultured in control medium or in the presence of 100 ng/mL r-hFSH, r-hLH, hCG, and the combination of r-hFSH and r-hLH (100 ng/mL of each) for 4 days. Cells on slides 1 and 2 were methanol fixed on days 1 and 4 in preparation for determination of cell number via the crystal violet assay (26).
Assays
VEGF concentrations in culture medium were quantitated by enzyme-linked immunosorbent assay (ELISA; Quantikine-human VEGF Immunoassay, R&D Systems, Minneapolis, MN). This highly specific sandwich assay recognizes human VEGF and exhibits negligible cross-reactivity with more than 90 other cytokines/growth factors (R&D Systems). Our laboratory (27) and that of Shima et al. (28) demonstrated that rhesus and cynomolgus macaque VEGF mRNA encode for a protein that is 100% homologous to human VEGF (27). A 96-well plate reader (Molecular Devices, Menlo Park, CA) set to read 450 nm emission was used to quantitate assay results. r-hVEGF (50 and 250 pg) was added to three different samples of spent culture medium, and recovery was 102%. To examine assay linearity, serial dilutions of samples were tested; correction for dilution of the culture medium indicated that the sample concentration had a coefficient of variation (CV) of less than 4%. We also compared Ham’s F-10 and 10% AMS medium alone and with added VEGF. Ham’s F-10 and 10% AMS contained no detectable VEGF, and the recovery averaged 95%. Standards included in each of the nine assays indicated that intra- and interassay CVs were 4.6% and 7.2%, respectively.
Basic fibroblast growth factor (bFGF) concentrations in culture medium were determined by ELISA (R&D Systems) for the control and 100 ng/mL r-hLH-treated cell cultures in study 2. The addition of bFGF to medium samples indicated that recovery was 97%, and serial dilution of three medium samples indicated that the assay was linear; the calculated CV for the dilutions was 8.9%. Medium progesterone concentrations were determined by RIA as previously validated in our laboratory (23). The intra- and interassay CVs for the progesterone assay were 6.6% and 12.2%, respectively.
Statistical analyses
Statistical tests were performed using the Statpak (Northwest Analytical, Portland, OR) computer program. To correct for heterogeneity of variance as determined by Bartlett’s test, log transformation of the VEGF (logX), bFGF (logX), and progesterone (1/logX 4) data was performed before analysis. In study 1, differences in medium VEGF and progesterone content were determined by two-way ANOVA, using a completely randomized split-plot design with animals assigned to in vivo treatments (main plots) and cells assigned to in vitro treatments (split plots). Unpaired t tests were used to detect differences between basal and hCG treatments within an in vivo treatment group (i.e. NLGC vs. LGC). In study 2, VEGF, bFGF, and progesterone data were analyzed using a completely randomized split-split plot design, with animals assigned to the in vivo treatments (main plots) and the cells assigned to in vitro treatments (split plots) examined over days (split-split plots). r-hFSH and r-hLH dose responses were analyzed separately on days 1 and 4, using a completely randomized design with cells assigned to five different doses. After a significant (P < 0.05) F test, Duncan’s multiple range tests were used to determine differences between means. All data are expressed as nontransformed values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF production by nonluteinized and luteinized granulosa cells
(i.e. NLGC and LGC) during short term incubations (24 h) in
the presence and absence of hCG are depicted in Fig. 1
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Cells obtained from monkeys that did not receive an ovulatory stimulus
(i.e. NLGC) produced approximately 8-fold lower
(P < 0.05) levels of VEGF than cells from animals that
received a standard gonadotropin bolus (1000 IU hCG; i.e.
LGC) 27 h earlier to initiate periovulatory events. In
vitro hCG treatment of NLGC increased (P < 0.05)
VEGF 4-fold over control levels, to concentrations that were not
different from LGC controls. In contrast, in vitro hCG
treatment of LGC only modestly increased (P < 0.07)
VEGF production compared to its control values. Progesterone levels
produced by NLGC (55 \ 13 ng/mL) also were much lower
(P < 0.05) than those produced by LGC (519 \ 177
ng/mL) under control conditions. In vitro hCG treatment
markedly increased (P < 0.05) progesterone secretion
by NLGC (16-fold), but only modestly increased that by LGC (1.5-fold;
P < 0.05). As noted for VEGF levels, progesterone
concentrations were not different between NLGC treated with hCG
in vitro and LGC exposed to hCG in vivo and
incubated under control conditions.
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depicts VEGF production by incubated granulosa
cells obtained after different in vivo ovulatory stimuli
(i.e. 100 vs. 1000 IU r-hCG and 2500 IU r-hFSH).
VEGF synthesis by cells from monkeys that received a 10-fold lower dose
(100 IU) of hCG was similar to that by cells from animals receiving the
standard ovulatory bolus (1000 IU hCG). Furthermore, comparison of VEGF
levels produced by LGC after in vivo exposure to 1000 IU
r-hCG (Fig. 2) or urinary hCG (Fig. 1
) indicated no differences. Cells
obtained from monkeys that received a bolus of r-hFSH (2500 IU) also
produced similar VEGF levels under control conditions as LGC obtained
after a hCG bolus. In vitro hCG treatment increased
(P < 0.05) VEGF production by cells collected after
all three in vivo ovulatory stimuli compared to their
respective control values. Progesterone production after all three
ovulatory stimuli was not different and averaged 801 \ 166 ng/mL
when incubated in control medium. In vitro hCG treatment
increased (P < 0.05) progesterone production for all
groups and averaged 1726 \ 443 ng/mL.
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Figure 3 depicts VEGF (A) and progesterone (B)
levels produced by NLGC cultured for 6 days with or without (control)
100 ng/mL of various gonadotropins. Cells cultured in control medium
had low levels of VEGF (140 \ 34 pg/mL) on day 1, which declined
(P < 0.05) progressively to nondetectable levels (<15
pg/mL) by day 5 of culture. r-hFSH, r-hLH, and hCG markedly increased
(P < 0.05) VEGF production above that observed in
control medium alone throughout the culture interval. No significant
differences were observed among the three gonadotropin treatments.
Gonadotropin exposure progressively increased VEGF concentrations from
day 1 through days 4–5 of culture; levels remained at this level
through day 6. Progesterone production by NLGC in the presence and
absence of gonadotropins exhibited a pattern similar to that observed
for VEGF. NLGC treated with a combination of 100 ng/mL FSH and LH
produced levels of VEGF and progesterone not different from those
elicited by either gonadotropin alone (data not shown).
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depicts VEGF and progesterone production by LGC
during 6 days of culture in the presence or absence of 100 ng/mL of
various gonadotropins. VEGF levels (Fig. 4A) produced under control
conditions were high and did not change throughout culture. Treatment
with r-hFSH or r-hLH, but not hCG, increased (P <
0.05) VEGF concentrations on day 1 of culture. Thereafter, VEGF levels
produced by r-hFSH-, r-hLH-, and hCG-treated LGC were not different
from each other and were 3- to 4-fold greater than (P
< 0.05) control levels. Progesterone concentrations (Fig. 4B) in
control cultures averaged 473 ng/mL over the first 4 days of culture
before declining (P < 0.05) to 63 ng/mL on days 5–6.
On day 1, r-hLH and r-hFSH, but not hCG, treatment increased
(P < 0.05) progesterone levels compared to those in
control medium. On days 2–6, all three gonadotropin treatments
increased (P < 0.05) progesterone concentrations to a
similar extent compared to control values. LGC treated with both 100
ng/mL FSH and LH produced levels of VEGF and progesterone not different
from those elicited by either gonadotropin alone (data not shown).
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. r-hLH caused a dose-dependent increase
(P < 0.05) in VEGF and progesterone production by
NLGC, with the lowest dose (0.1 ng/mL) eliciting no effect and the
remaining doses producing elevated levels compared to control values.
On day 1 of culture, similar dose-response curves for VEGF and
progesterone production by NLGC were observed, except the relative
stimulation by r-hLH was lower than that on day 4 (data not shown). In
contrast, when LGC were cultured with various doses of r-hLH, only the
highest concentrations increased VEGF (100 ng/mL; P <
0.05; data not shown; see Fig. 4) or progesterone (10 or 100 ng/mL;
P < 0.05; data not shown) levels on day 1 or 4.
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. Low doses (0.1 and 1 ng/mL) of r-hFSH did not
enhance VEGF levels, whereas the 10 and 100 ng/mL doses markedly
increased (P < 0.05) VEGF concentrations. Progesterone
levels increased (P < 0.05) slightly in response to 1
ng/mL r-hFSH, whereas the 10 and 100 ng/mL doses elicited a large
increase (P < 0.05). NLGC exhibited similar
dose-response curves for VEGF and progesterone production on day 1 of
culture, with the only difference being that the relative stimulation
by r-hFSH was lower (data not shown) than that on day 4. In contrast,
LGC production of VEGF and progesterone on days 1 and 4 only increased
(P < 0.05) in response to the highest (100 ng/mL) dose
of r-hFSH (data not shown; see Fig. 4).
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Figure 7 depicts the results of crystal violet assays
that give an index (i.e. absorbance) of NLGC and LGC number
on days 1 and 4 of culture. Absorbance values on day 1 were lower
(P < 0.05) for NLGC than LGC, suggesting less
adherence of NLGC to the fibronectin-coated slides. The absorbance
(i.e. cell number) for NLGC did not change from day 1 to day
4 of culture for any treatment group. The absorbance for LGC cultured
in control medium alone also did not differ between days 1 and 4. In
contrast, LGC cultured in the presence of 100 ng/mL gonadotropins
(i.e. r-hFSH, r-hLH, and hCG) had a 1.5-fold greater
absorbance (i.e. cell number) compared to the control
value.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-fold higher) within 4 h of hCG treatment
(16). Collectively, the data suggest an important role for the
gonadotropin surge in mammalian species to stimulate an early increase
in VEGF production, which may be important for follicle rupture and/or
luteinization. Our studies also demonstrated that a low dose (100 IU) was as effective as the standard ovulatory bolus (1000 IU) of r-hCG in enhancing VEGF production by granulosa cells. The data support other observations by this laboratory (21) that a 10-fold lower dose of hCG is sufficient to initiate follicular processes associated with oocyte maturation (e.g. reinitiation of meiosis) and luteinization of the granulosa cells (i.e. increased progesterone production). Thus, the levels of LH/CG required to stimulate many periovulatory events, including VEGF production by LGCs, may be less than the surge levels achieved in natural or IVF-related cycles. However, monkeys receiving 100 IU r-hCG had shorter luteal phases than animals receiving a standard ovulatory dose (21). It is postulated that luteal phase defects (e.g. a short luteal phase) can result from inadequate vascularization of the corpus luteum (11). It is possible that the lower hCG dose did not fully luteinize the cells, or only a subset of cells within the follicle(s) luteinized. Indeed, the pronounced rise in VEGF production by cells from the low (100 IU) group in response to in vitro hCG treatment was similar to the large increase observed with NLGC and contrasts to the minimal response observed with luteinized granulosa cells collected after the standard (1000-IU) bolus. Moreover, NLGC lost their ability to produce VEGF during culture if gonadotropins were not present, whereas LGC cultured in the absence of gonadotropins maintained elevated VEGF levels. These studies suggest an initial dependence on increased, if not surge (21), levels of LH/CG for the establishment of appreciable VEGF synthesis that is lost (i.e. independent of gonadotropins) after luteinization of the granulosa cells.
This study is also the first to show that hCG and hLH directly
stimulate VEGF production and secretion by primate granulosa cells. The
large increase (5-fold) in VEGF synthesis by NLGC after in
vitro hCG treatment was previously observed for progesterone
production (29) and is probably due to gonadotropin binding to
unoccupied LH receptors induced by the follicular stimulation protocol.
In contrast, hCG or hLH treatment was not as effective in increasing
VEGF synthesis by LGC; this attenuated response can be attributed to
the higher basal VEGF levels, LH/CG receptor desensitization, or the
lack of unoccupied receptors after in vivo exposure to a
large bolus of hCG. The latter is supported by the results of study 1,
in which a more pronounced response to in vitro hCG
treatment was observed with cells collected from the low hCG (100 IU)
and FSH treatment groups, than from the high (1000 IU) hCG treatment
group. Human (30) and bovine (31) granulosa cells cultured in the
presence of hCG/LH also increased VEGF mRNA expression. In contrast,
rat granulosa cells cultured with LH did not respond with increased
VEGF mRNA expression, even though in vivo hCG treatment was
a highly effective inducer of VEGF mRNA (16). Interestingly, these
researchers observed that decreasing oxygen tension elevated VEGF mRNA
expression by rat granulosa cells (32). This lack of an LH/CG effect
may be intrinsic to the immature rat model (i.e. the
granulosa cells may not reach the appropriate maturational state to
respond to LH) (16), or it may indicate a species difference with
regard to VEGF regulation or action within the ovary.
Recombinant hFSH, either administered in vivo as an ovulatory bolus (2500 IU) or added to cultures in high (10–100 ng/mL) doses also stimulated VEGF production by granulosa cells. This laboratory reported recently that a bolus of r-hFSH was as effective as the standard bolus of hCG in eliciting periovulatory events (e.g. reinitiation of oocyte meiosis and luteinization of granulosa cells) during IVF-related cycles in monkeys (33). These observations are consistent with the concept arising from rodent models (34, 35) that the midcycle FSH surge can play a role with (or substitute for) the LH surge to initiate periovulatory events, including VEGF production by granulosa cells. Our data appear to contrast somewhat with those reported by Neulen et al. (30) of no effect of a single dose (1 IU/mL) of FSH on VEGF mRNA expression by human luteinized granulosa cells from women. However, we observed a much greater response to FSH by NLGC than LGC in macaques. This difference could be related to measurement of VEGF mRNA vs. protein or to the presence of nonluteinized cells within the LGC preparation pooled from macaque follicles.
Earlier studies of angiogenesis in the ovary focused on bFGF, as this factor plays a critical role in a variety of tissues and is present in the developing corpus luteum (10, 32). However, bFGF concentrations in medium from NLGC and LGC cultures in our study were extremely low and did not increase after either in vivo or in vitro gonadotropin treatment. In fact, in vitro LH treatment caused a significant decline in bFGF levels in culture medium. The apparent lack of bFGF production, however, could be due to our inability to measure macaque bFGF that may have remained associated with the cells and extracellular matrix. Our evidence of VEGF secretion is particularly relevant because in vitro angiogenesis (chemotactic and proliferative) assays indicate that conditioned medium from cultures of follicular cells and luteal cells contains a releasable growth factor (10). Nevertheless, luteinized cells may produce several angiogenic factors, as immunoneutralization studies demonstrated that antibodies to VEGF blocked 65% of the angiogenic activity within luteal cell-conditioned medium (36), whereas bFGF antibodies eliminated 25% of the angiogenic activity (10, 36).
Four isoforms of VEGF arise from a single gene through alternative splicing of the VEGF mRNA (11). Although the VEGF ELISA used in our study recognizes all isoforms, we presume that the secreted forms of VEGF (i.e. VEGF 121 and VEGF 165) (11) account for the majority produced by macaque granulosa cells. However, additional studies will be required to elucidate the VEGF isoforms or related molecules (11) produced by granulosa/luteal cells and their regulation by gonadotropin and other factors.
Progesterone production by the NLGC was highly correlated with VEGF production throughout the culture period. It is possible that progesterone acts locally via steroid receptor activation to mediate hCG/LH-stimulated VEGF production; this pathway is necessary for ovulation and luteinization in both rodent (37) and primate (38) species. In the rat, blockade of progesterone production by administration of aminoglutethimide, a P450 side-chain cleavage inhibitor, also prevented VEGF mRNA expression (39). Retinal pigment epithelial cells also produce greater levels of VEGF after exposure to progesterone (40). Alternatively, we cannot rule out that VEGF affects progesterone synthesis or progesterone receptor expression in granulosa cells during the periovulatory interval.
After the onset of the ovulatory gonadotropin surge, the follicular microvasculature becomes highly permeable as a result of increased endothelial fenestrations as well as intracellular gaps between endothelial cells (3, 5). Previous studies in a variety of species linked the ovulatory gonadotropin stimulus with the production of inflammatory mediators (e.g. histamine, eicosanoids, platelet-activating factor, and bradykinin) that were postulated to mediate the increase in capillary permeability (41). VEGF should be included as a possible candidate, as a bolus of hCG increases VEGF secretion, and this factor is 5,000- to 50,000-fold more potent than histamine in increasing cell permeability (42, 43). The increased VEGF production by LGC is also consistent with a role for VEGF in the angiogenic process within the corpus luteum. Additional studies are needed to elucidate the physiological role(s) of VEGF in the processes of follicular maturation, ovulation, and development of the corpus luteum.
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Acknowledgments |
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Footnotes |
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Received March 10, 1997.
Revised April 30, 1997.
Accepted May 12, 1997.
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R Gomez, I Lima, C Simon, and A Pellicer Administration of low-dose LH induces ovulation and prevents vascular hyperpermeability and vascular endothelial growth factor expression in superovulated rats Reproduction, April 1, 2004; 127(4): 483 - 489. [Abstract] [Full Text] [PDF]
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J. Balasch, M. Guimera, O. Martinez-Pasarell, J. Ros, J. A. Vanrell, and W. Jimenez Adrenomedullin and vascular endothelial growth factor production by follicular fluid macrophages and granulosa cells Hum. Reprod., April 1, 2004; 19(4): 808 - 814. [Abstract] [Full Text] [PDF]
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, where MSE is the mean
square error from ANOVA of all data in a treatment group) were
determined for control (17 pg/mL and 6 ng/mL), r-hFSH (593 and 405),
r-hLH (145 and 359), and hCG (138 and 349) treatment groups. *, Daily
means for VEGF and progesterone production by control cells were
different (P < 0.05) from those for production by
the r-hFSH-, r-hLH-, and hCG-treated cells across all days. r-hFSH-,
r-hLH-, and hCG-treated cells exhibited similar VEGF and progesterone
levels. a–d, Means within a treatment group (e.g.
r-hFSH) with unlike superscripts are different (P
< 0.05) across days.
