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Original Studies |
Institute of Maternal and Child Research, School of Medicine and Department of Obstetrics and Gynecology (P.K., A.C., O.C., M.V., L.D.), the Human Genetics Program, ICBM, School of Medicine (P.C.), University of Chile, Santiago, Chile; the Department of Obstetrics and Gynecology, University of Valencia (P.C.-C., C.S.), Valencia, Spain; and the Center for Research on Reproduction and Women’s Health and Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine (A.M.), Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Luigi Devoto M.D., University of Chile School of Medicine, P.O. Box 226–3, Santiago, Chile. E-mail: ldevoto{at}machi.med.uchile.cl
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Introduction |
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, and tumor necrosis
factor- (TNF) (1). These polypeptides are essential to the immune
cascade and stimulate or inhibit cell growth depending on their
concentrations and target cells. IL-1ß is known to inhibit
luteinization of cultured murine and porcine granulosa cells (2, 3).
Furthermore, IL-1ß inhibited hCG stimulated-progesterone production
by human granulosa cells cocultured with white blood cells (4). It is
thought that IL-1ß exerts its action through cell surface receptors.
To date two IL-1 receptors (types I and II) have been identified. In
the human, the type I receptor (IL-1RtI) possesses a 213-amino acid
long intracytoplasmic domain, whereas the type II receptor has a short
29-residue intracytoplasmic domain (5). Current information indicates
that IL-1ß signaling occurs exclusively via IL-1RtI (6). Cells
regulated by IL-1ß also produce a IL-1 receptor antagonist (IL-1Ra).
The complete IL-1ß system including genes encoding IL-1ß, IL-1RtI,
and IL-IRa have been detected in the ovary of several species,
including the human (7).
Interestingly, cultured human granulosa and thecal cells did not
express messenger ribonucleic acid (mRNA) for IL-1ß under basal
conditions, but they did express mRNA for IL-1RtI (7). Treatment of
ovarian cell cultures with forskolin induced IL-1ß transcripts, but
only in granulosa cells. In addition, IL-1-like activity has been
detected in human follicular fluid, and the IL-1ß transcript is more
abundant in cells of follicular aspirates than IL-1. An intermediary
role for IL-1 in the ovulatory process has been postulated (5).
The cells comprising the CL have distinct morphological, endocrine, and biochemical properties. Cells present in the CL include small and large luteal cells, fibroblasts, and endothelial and immune cells. These cells change in number, morphology, function, and secretory capability throughout the life span of the CL (8, 9). Cells derived from the CL produce IL-1ß in culture, particularly immune cells (10). However, there is limited information regarding the role of IL-1ß and the cell localization of the IL-I system in the human CL. In the present investigation we examined the ability of IL-1ß to modulate basal and hCG-stimulated progesterone production by human luteal cells. Furthermore, studies were carried out to elucidate the types of luteal cells that express IL-1RtI and IL-1Ra in the human CL.
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Materials and Methods |
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Antibodies
The following mouse monoclonal antibodies (mAb) were gifts from Dr. Maria Rosa Bono (Faculty of Sciences, University of Chile, Santiago, Chile): mAb W6/32 (IgG2a antihuman major histocompatibility complex class I) and mAb GAP8.3 (IgG2a anti-CD45). Dr. Francisco Sánchez-Madrid (The Princess Hospital, Madrid, Spain) provided mAb Bear-I (IgG1 anti-CD 11b) and mAb LIA 3/2 (IgG1 anti-CD 18). Mouse mAb Tuk4 (IgG1 anti-CD14) was purchased from (DAKO Corp., Carpenteria, CA), the labeled F(ab')2 antibody (antimouse IgG) was obtained from Immunotech (Marseille, France), the mouse mAb (antihuman IL-1RtI) was purchased from Genzyme Transgenics Corp. (Cambridge, MA), and rat mAb 4 C1 (IgC2a antihuman IL-1RtI) was obtained from PharMingen (San Diego, CA).
Patients and tissues
CL were obtained from 16 eumenorrheic women, aged 27–38 yr, undergoing laparotomy for tubal sterilization (n = 10) or myomas (n = 6). at the San Borja Arriarán Clinical Hospital, University of Chile, National Health Service (Santiago, Chile). None of the patients had experience infertility or endometriosis and had stopped any hormonal contraception at least 3 months before surgery. The study was approved by the institutional review board of the University of Chile. All patients gave their informed consent for removal of the CL.
After lutectomy, the tissue was transported to the laboratory in sterile saline. The cycle date of each woman was confirmed by endometrial biopsy (11) and classified as early (1–4 days; n = 5), mid (5–9 days; n = 6), or late (10–14 days; n = 5). One piece of each CL was placed in 10% formalin for histological examination (12).
Cell dispersion
Luteal cells were dispersed as previously described (13). Briefly, CL were enzymatically dissociated in culture medium 199/HEPES (25 mmol/L) containing NaHCO3 (26 nmol/L), penicillin (50 IU/mL), collagenase trypsin-free (740 IU/100 mg tissue), and deoxyribonuclease (14 kallikrein inhibitor units/100 mg tissue). After 90 min, luteal cells were washed once with culture medium and twice with Dulbecco’s Ca2+- and Mg2+-free phosphate-buffered saline (PBS) containing ethylenediamine tetraacetate (1 mmol/L). Red blood cells were removed by Histopaque column (density, 1.007). The cell viability was more than 90%, as assessed by the trypan blue exclusion method.
Luteal leukocyte isolation
After luteal dispersion, a fraction of luteal cells was incubated with a panel of mouse mAbs (GAP 8.3, Bear-1, and LIA 3/2) directed to leukocyte-specific molecules as previously described (10). Leukocytes were separated from luteal cells in a magnetic concentrator (DynAl, Chantilly, VA). Subsequent to isolation of luteal leukocytes, the cell viability of luteal cells was greater than 90%, as determined by the trypan exclusion method.
Cell culture
Luteal cells were cultured as previously described (13, 14). Briefly, mixed luteal cells (total luteal cells) and leukocyte-free luteal cells (0.5–1 x105/well) were cultured in serum-free medium and treated with IL-1ß (25 ng/mL) in the absence and presence of hCG (10 IU/mL), for 24 h. To test the specificity of IL-1ß, some cells were treated with anti IL-1RtI mAb in the presence and absence of hCG. Culture medium was collected for determination of progesterone concentrations.
Human granulosa cells were isolated from patients undergoing in vitro fertilization/embryo transfer. The cells were cultured as previously described (19) and used to detect IL-1RtI by indirect immunofluorescence.
Detection of IL-1RtI by flow cytometry
To investigate the protein expression of IL-1RtI on mixed luteal cells, flow cytometric analysis was performed as described previously with minor modifications (10). Briefly, dual color immunofluorescence staining was performed to discriminate the expression level of IL-1RtI on luteal leukocytes and leukocyte-free luteal cells. In the first step, suspensions of mixed luteal cells were simultaneously incubated for double staining with the rat mAb 4C1 (anti IL-1RtI) and the panel of mouse antileukocyte mAbs for 40 min at 4 C, and then washed in PBS. The second step included incubation with biotin-conjugated goat antirat Ig polyclonal antibody for 30 min at 4 C, followed by two washes. In the third step, the cells were incubated with the conjugate streptavidin-phycoerythrin and fluorescein-F(ab)2 anti-mouse Ig for 30 min at 4 C. After two washes, the cells were resuspended at a concentration of 0.6 x 106/mL in PBS and immediately analyzed or stored overnight in PBS containing 0.5% formaldehyde in the dark until analysis. Control samples included assessment of autofluorescence and negative controls (absence of primary mAbs and/or secondary mAbs). No cross-reaction was observed between secondary antibodies, indicating highly specific binding of the mouse or rat Ig.
RNA isolation and Northern analysis
The presence of IL-1RtI mRNA was determined in CL collected throughout the luteal phase. Corpora lutea were kept frozen at -70 C. Total RNA was prepared as described by Chromczynski and Sacchi (15) and was quantified by absorbance at 260 nm. Northern analysis was carried out using Il-1RtI complementary DNA (cDNA) as a probe (Immunex Corp., Seattle, WA). Briefly, 15 µg total RNA were resolved on a 1% formaldehyde-agarose gel, blotted onto a nylon membrane (Schleicher & Schuell, Inc., Nytran), and cross-linked by UV irradiation. The membranes were prehybridized for 1 h at 65 C in a solution containing 0.5 mol/L NaH2PO4 (pH 7), 0.1 mmol/L ethylenediamine tetraacetate, 0.5% BSA, and 7% SDS. Hybridization was carried out overnight at 65 C in the same solution after the addition of the 32P-labeled probe (106 cpm/mL solution). Membranes were washed in 2 x SSC (standard saline citrate)-0.1% SDS for 30 min at room temperature, followed by two washes in 0.1 x SSC-0.1% SDS for 30 min at 60 C. Blots were exposed to x-ray film (reflection NEF-496, DuPont/NEN, Boston, MA), for 72 h.
RT-PCR
The presence of IL-1RtI transcripts in mixed luteal cells and leukocyte-free luteal cells was determined by RT-PCR. Oligonucleotide primers were designed according to previously published sequences of IL-1RtI and IL-1Ra, predicting PCR products of 441 and 511 bp, respectively (16, 17). Primer sequences were as follows: IL-1Rt1: forward, 5'-GCC AAG AGT TCT TTA GGT GCC-3'; reverse, 5'-CTC ACT GCA ACC TCC GTC TC-3'; and IL-1Ra: forward, 5'-CAG AAG ACC TCC TGT CCT ATG AGG-3'; reverse, 5'-GAT GAG CAG GAG GAC CTT CAT-3'. Total RNA (5 µg) was reverse transcribed in a 20-µL reaction containing 10 µmol/L reverse primer, AmpliTaq buffer, 0.2 mmol/L of each deoxy-NTP, 200 U reverse transcriptase (Superscript II, Life Technologies, Inc., Gaithersburg, MD), and 5 U RNasin (Promega Corp., Madison, WI) at 42 C for 60 min. The PCR amplifications were performed by adding 10 µmol/L forward primer, Taq buffer, 0.2 mmol/L of each deoxy-NTP, and 1 U Taq DNA polymerase (Promega Corp.) to the previous reaction mixture in a final volume of 50 µL. The cycling conditions for IL-1RtI were as follows: initial denaturation at 95 C for 5 min, followed by 30 cycles of denaturation at 95 C for 1 min, annealing at 58 C for 1 min, extension at 72 C for 1 min, and a final extension at 72 C for 10 min. Cycling conditions for IL-1Ra were the same, except for the annealing temperature, which was 55 C. Control reactions without cDNA were carried out in parallel and resulted in negative determination. PCR products were analyzed by electrophoresis in 1.25% agarose gel. Purified PCR products were sequenced using the DNA ds Cycle Sequencing System (Life Technologies, Inc.).
Nested PCR for IL-1Ra
IL-1Ra product obtained from RT-PCR was submitted to a second PCR amplification (nested), using the following specific primers: forward, 5'-CAG AAG ACC TCC TGT CCT ATG AGG-3'; and reverse, 5'-GAT GAG CAG GAG GAC CTT CAT-3'. Cycling conditions were the same as described for the first PCR reaction, except for the annealing temperature, which was 60 C. Purified PCR products were sequenced using the DNA ds Cycle Sequencing System (Life Technologies, Inc.).
Immunohistochemical staining procedures
CL were formalin-fixed and paraffin embedded. Five-micron sections were processed for routine hematoxylin-eosin staining and immunohistochemistry as previously described (18). Briefly, the slides were deparaffinized in xylene, followed by rapid rehydration through a graded series of alcohols. For the avidin-biotin-peroxidase method, endogenous peroxidases were blocked with 1% H2O2 in 96% methanol. Nonspecific binding was blocked with horse serum followed by incubation with the primary antibodies: monoclonal mouse antihuman IL-1RtI and Il-1Ra antibody, respectively (Genzyme Corp., Cambridge, MA). Control incubations included deletion of the primary antibody. After rinsing with PBS, sections were incubated with a secondary IgG antibody. Immunoreaction products were visualized by incubating sections with 3'-diaminobenzidine tetrahydrochloride as substrate solution.
Indirect immunofluorescence procedures for lutein and granulosa lutein cells
Lutein or luteinized granulosa cells grown on coverslips were washed twice in prewarmed (37 C) DMEM and twice in prewarmed PBS containing 1.5 mmol/L Ca2+ and fixed in 100% methanol (-20 C for 5 min). Cells were incubated in 10% normal goat serum for 30 min at room temperature and then with 10 µg/mL of the primary antibodies for 2 h at room temperature (18, 19). A fluorescein-conjugated goat antimouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at a 1:200 dilution (30 min at room temperature). Coverslips were mounted on glass slides with Fluoromount G (Fisher Scientific, Malvern, PA) containing 1,4-diazabicyclo-(2,2,2)octane (DABCO, Polysciences, Inc., Warrington, PA) to stabilize fluorescence and were photographed with a Nikon microscope (Melville, NY).
Data analysis
All experiments were conducted in duplicate and repeated at least three times. Experimental data are presented as the mean ± SE. The percent increment in progesterone production was analyzed by two-tailed Student’s t test for comparison between control and treated conditions and between different luteal cell populations. P < 0.05 was considered statistically significant.
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Results |
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illustrates the effect of
hCG (10 IU/mL) on progesterone accumulation by cultured mixed luteal
cells and leukocyte-free luteal cells. The time course of hCG
stimulated-progesterone accumulation was examined in luteal cells of
different ages. Culture medium from mid and late leukocyte-free luteal
cells displayed higher progesterone accumulation compared with that
from mixed luteal cells, respectively (P < 0.05). No
difference was noted in the progesterone accumulation by mixed luteal
and leukocyte-free luteal cells from early CL. These data suggest a
luteal age-dependent interaction among cAMP signaling, luteal
steroidogenic cells, and luteal leukocytes.
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depicts the effect of IL-1ß
(25 ng/mL) in the presence and absence of hCG on progesterone
accumulation by mixed and leukocyte-free early luteal cells. IL-1ß
had no effect on basal progesterone production by either mixed luteal
cells or leukocyte-free luteal cells. In contrast, treatment with
IL-1ß inhibited hCG-stimulated progesterone accumulation in mixed
luteal cells by 60%,(P < 0.05), but did not affect
leukocyte-free luteal cells. This finding suggests that the action of
IL-1ß is leukocyte mediated.
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illustrates that
hCG-stimulated progesterone accumulation by mixed luteal cells was
amplified 2-fold in the presence of IL-1RtI mAb compared with that in
the presence of hCG alone (P < 0.05). Treatment with
IL-1RtI mAb had no effect on basal progesterone accumulation or
hCG-stimulated progesterone accumulation in leukocyte-free luteal cells
(data not shown). These findings suggest that the action of IL-1ß on
steroidogenesis is mediated by the leukocyte IL-1RtI.
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, the upper panel
shows the immunohistochemical identification of IL-1RtI in CL collected
during the midluteal phase. Immunostaining was localized in the
cytoplasmic plasma membrane of luteal cells (Fig. 4A). In control
experiments, no immunostaining was noted when primary antibody was
omitted (Fig. 4B). The middle panel shows
luteinized granulosa cells in primary culture (Fig. 4C, immunofluorescence; Fig. 4D, phase contrast). The cells displayed
numerous cell to cell contacts, and the cell morphology is consistent
with steroidogenic cells. The IL-1RtI protein was visualized in
leukocyte-free granulosa lutein cells by indirect immunofluorescence.
These cells exhibited intense staining for IL-1RtI. However, we noticed
that some of these cells did not stain for IL-1RtI, suggesting that
IL-1RtI is not constitutively present in all granulosa-lutein cells.
The lower panel shows the indirect immunofluorescence for
IL-1RtI in luteal cells. Mixed luteal cells (Fig. 4E) as well as
leukocyte-free luteal cells (Fig. 4F) displayed intense staining for
IL-1RtI. The negative control included the omission of the primary
antibody and showed no staining (Fig. 4G).
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To determine whether the CL is a site of IL-1RtI gene expression
during the luteal phase, Northern blot was performed using IL-1RtI cDNA
as a probe. Figure 5 is an autoradiogram
that illustrates the presence of the IL-1RtI mRNA transcript in CL
(total tissue) of different stages. The cDNA probes yield a major band
of 5.1 kb in the different stages of the CL. Secretory endometrium was
used as a positive control. These data indicate that IL-1RtI mRNA is
expressed during all stages of the luteal phase.
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RT-PCR was used to determine whether IL-1RtI is expressed in both
luteal leukocytes and nonleukocyte luteal cells. Figure 6 depicts a 441-bp PCR product
corresponding to the expected size of IL-1RtI mRNA and was found in
mixed luteal cells, leukocyte-free luteal cells, and luteal leukocytes.
All products obtained were sequenced, confirming individual product
identity. Secretory endometrial cell RNA was used as a positive
control, and peripheral leukocytes that do not express the IL-RtI mRNA
were used as a negative control. In all experiments ß-actin was used
as a control for sample to sample variation.
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Figure 7 shows the
immunohistochemical identification of IL-1Ra from CL collected during
the midluteal phase. The immunostaining was localized in the
cytoplasmic membrane of luteal cells (Fig. 7A). In control experiments,
no immunostaining was noted when primary antibody was omitted (Fig. 7B). The lower panel shows the indirect immunofluorescence
for IL-1Ra in luteal cells. Mixed luteal cells (Fig. 7C) as well as
leukocyte-free luteal cells (Fig. 7D) displayed intense staining for
IL-1Ra. The negative control included the omission of the primary
antibody and shows no staining (Fig. 7E).
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illustrates the RT nested-PCR of
IL-1Ra mRNA in mixed luteal cells. A 551-bp PCR product corresponding
to the expected size of IL-1Ra mRNA was found in mixed luteal cells.
The product obtained was sequenced, confirming product identity.
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We have previously validated by flow cytometry the purity of the
leukocyte-free luteal cell population as greater than 99% (10). In
this study, flow cytometric analysis assessed the level and cellular
distribution of IL-1RtI in the CL. Figure 9 shows the simultaneous detection of
IL-1RtI in luteal leukocytes as well as in leukocyte-free luteal cells.
The upper panel illustrates cells that stain positive for
IL-1RtI. The dots plotted in the upper right
quadrant represent leukocytes, corresponding to 31% of the
positive cells. The dots plotted in the upper left
quadrant represent leukocyte-free luteal cells corresponding 56%
of the positive cells. The signals plotted in the lower left
quadrant represent nonleukocyte luteal cells that did not express
IL-IRt-I, corresponding to 13% of the cells.
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Discussion |
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The pattern of hCG-stimulated progesterone secretion by mixed luteal cells throughout the luteal phase is consistent with previous findings, indicating a progressive decline toward the end of the luteal phase (13). Interestingly, leukocyte-free luteal cells exhibited a greater responsiveness to hCG in term of progesterone secretion, particularly during the late luteal phase. This finding suggests a leukocyte inhibitory effect on hCG-stimulated progesterone production. Consistent with this observation, IL-1ß was able to inhibit hCG-stimulated progesterone production only in mixed luteal cells, indicating a leukocyte-mediating effect.
On the other hand, IL-1ß failed to modify basal progesterone production by mixed or leukocyte-free luteal cells, suggesting that IL-1ß does not exert a direct effect on luteal steroidogenesis. These results point to a leukocyte-produced or released factor(s) that subsequently affects hCG-stimulated progesterone synthesis.
In contrast to its effect during the mid- and late luteal phase,
hCG-stimulated progesterone production was similar in both early mixed
luteal cells and leukocyte-free luteal cells. The lack of effect of
early luteal leukocytes on hCG-stimulated progesterone production is
interesting, possibly suggesting that the population or function of
luteal leukocytes changes during the luteal phase or that the
sensitivity of luteal cells to the leukocyte-derived factor change. The
fact that blood lymphocytes represent the predominant subtypes of
leukocytes in early CL might explain the lack of effect on luteal
steroidogenesis (20). In the monkey, IL-1ß stimulates
PGF2 production by luteal cells isolated only during the
mid and late luteal phase (21). Interestingly, in cultured human
granulosa cells IL-1ß increases cyclooxygenase-2 mRNA levels, which
represents the rate-limiting enzyme in conversion of arachidonic acid
to prostanoids. In addition, IL-1ß up-regulated the receptor for
PGF2 (22). PGF2
inhibits adenyl cyclase activity in luteal cells (1). Our finding that
treatment with IL-1ß inhibited by 60% the hCG-stimulated
progesterone production by mixed early luteal cells and was without
effect on leukocyte-free luteal cells highlights the importance of both
luteal leukocytes and IL-1ß in the modulation of progesterone
production and suggests that PGF2 could drive
the inhibitory action of IL-1ß in luteal cells steroidogenesis.
To understand the specific action of IL-1ß on luteal steroidogenesis,
we examined in vitro the effect of a neutralizing mAb
directed to IL-1RtI on basal and hCG-stimulated progesterone
production. The treatment resulted in a 2.5-fold increase in the
hCG-supported progesterone production, but it was without effect on
basal steroidogenesis. The underlying mechanism controlling the
antigonadotropic activity of IL-1ß in luteal cells cannot be
determined from this study, but several possible scenarios can be
suggested. It has been recently noted that IL-1ß favors the
production/release of PGF2 and nitric oxide by
luteal tissue (21, 22, 23, 24). Both, PGF2 and nitric
oxide inhibit progesterone synthesis at the level of adenyl cyclase
activity and cytochrome P450ssc, respectively. These findings are
consistent with the recent report by Bréard et al.,
who found that IL-1ß diminished hCG-stimulated cAMP generation in
rabbit granulosa cells (25). Collectively, these data support the
idea of a functional role for IL-1ß in human CL regression.
The second part of our study was designed to assess the mRNA expression and cellular localization of IL-1RtI and IL-1Ra within the CL. Immunoreactive IL-1RtI, and gene expression (mRNA) were localized in both somatic cells and immune cells of the gland. This is consistent with our flow cytometric assessment that detected IL-1RtI protein in both cell types. In addition, our data indicate that 56% of cells that stain positively for IL-1RtI are nonimmunological luteal cells. These results are in agreement with those of Piquette et al. (26), who reported that human granulosa-lutein cells collected at the time of oocyte retrieval, and not macrophages, account for the majority of the immunohistochemical staining for IL-1RtI.
Northern blot analysis documented the presence of single mRNA transcript for IL-1RtI (5.1 kb) in the CL. Furthermore, this mRNA transcript is present in CL of different ages. Cellular localization of a fragment of the IL-1RtI transcript was performed by RT-PCR. The 441-bp RT-PCR product corresponded to the amplified IL-1RtI fragment and is localized in leukocyte-free luteal cells and luteal leukocytes. These data indicate that the CL is a site of IL-1RtI gene expression and IL-1ß action.
IL-1 exerts its action through cell surface receptors. A naturally occurring antagonist, IL-1Ra, is also produced by the cells to modulate the actions of IL-1, preventing postreceptor activation (20). Our observations documented the presence of immunoreactive IL-1Ra in histological section of the CL throughout the luteal phase. Furthermore, immunofluorescence procedures identified IL-1Ra in luteal leukocytes and leukocyte-free luteal cells. The IL-1Ra mRNA was only examined in mixed luteal cells. We have previously shown the ability of human luteal cells to produce IL-1ß (9). Taken together these findings indicate the presence of a complete intraluteal IL-1 system in the CL, replete with ligand, receptor, and receptor antagonist.
In summary, this study demonstrated localization of the IL-1 system in both immune and somatic cells of the human CL. In addition, our findings reveal a functional role for IL-1ß in the regulation of luteal steroidogenesis in vitro. The factor(s) that mediates the antigonadotropic action of IL-1ß on hCG-stimulated progesterone secretion is mainly leukocyte dependent. The lack of effect of IL-1ß on progesterone synthesis in leukocyte-free luteal cells suggests a nonsteroidogenic role for IL-1RtI localized in somatic luteal cells.
Finally, these findings highlight the importance of resident macrophages in the regulation of CL development, function, and regression and suggest an interaction between the immune and steroidogenic cells within the corpus luteum.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
Received May 25, 1999.
Revised August 12, 1999.
Accepted August 17, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß
modulation of luteinized human granulosa cell oestrogen and
progesterone biosynthesis. Hum Reprod. 10:3206–3210. receptor
expression by interleukin-1ß in cultures human granulosa-luteal
cells. Endocrinology. 138:3638–3644.This article has been cited by other articles:
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  R. Wu, K. H. Van der Hoek, N. K. Ryan, R. J. Norman, and R. L. Robker Macrophage contributions to ovarian function Hum. Reprod. Update, March 1, 2004; 10(2): 119 - 133. [Abstract] [Full Text] [PDF]
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 F. Miceli, A. Tropea, F. Minici, P. Navarra, A. Lanzone, and R. Apa Interleukin-1{beta} Stimulates Progesterone Production by in Vitro Human Luteal Cells: Evidence of a Mediatory Role of Prostaglandins J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2690 - 2694. [Abstract] [Full Text] [PDF]
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L. Devoto, P. Kohen, R. R. Gonzalez, O. Castro, I. Retamales, M. Vega, P. Carvallo, L. K. Christenson, and J. F. Strauss III Expression of Steroidogenic Acute Regulatory Protein in the Human Corpus Luteum throughout the Luteal Phase J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5633 - 5639. [Abstract] [Full Text] [PDF]
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