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Endocrinology Vol. 143, No. 5 1833-1844
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

The Regulated Expression of the Pregnancy-Associated Plasma Protein-A in the Rodent Ovary: A Proposed Role in the Development of Dominant Follicles and of Corpora Lutea

Ariel Hourvitz, Akira Kuwahara, Jon D. Hennebold, Adriano B. Tavares, Hiroaki Negishi, Taek Hoo Lee, Gregory F. Erickson and Eli Y. Adashi

Division of Reproductive Sciences (A.H., A.K., J.D.H., A.B.T., H.N., E.Y.A.), Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah 84112; and Department of Reproductive Medicine (T.H.L., G.F.E.), University of California, San Diego, La Jolla, California 92093

Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Huntsman Cancer Institute, 2000 Circle of Hope, Room 5221, Salt Lake City, Utah 84112. E-mail: . eadashi{at}hsc.utah.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Compelling evidence exists displaying that the intrafollicular IGF-I system constitutes an obligatory mediator of FSH action in the murine ovary. Within this system, the ovarian IGF binding protein-4-directed protease (IGFBP-4ase) may have a critical role. Human IGFBP-4ase has been proved identical to the previously well-characterized pregnancy-associated plasma protein-A (PAPP-A). This communication reports the cloning and sequencing of the mouse PAPP-A cDNA as well as its expression and cellular localization in the mouse ovary. PAPP-A mRNA was undetectable in ovaries of untreated immature 25-d-old mice. Treatment with PMSG led to a marked time-dependent increase in PAPP-A expression in well-defined subsets of granulosa cells and follicles. Specifically, PAPP-A expression was detectable exclusively in centrifugally residing membrana granulosa cells of antral follicles during a 3- to 36-h period post PMSG. PAPP-A expression then fell to nondetectable levels in dominant preovulatory follicles at 48 h post PMSG. Treatment of PMSG-primed mice with human CG caused a rapid reinduction of PAPP-A expression in granulosa cells of dominant follicles and was sustained at relatively high levels throughout the ovulation and luteinization. These results suggest a role for gonadotropin-stimulated PAPP-A gene expression in the physiologic processes of dominant follicle development, ovulation, and luteogenesis in the mammalian ovary. The early onset and extended duration of gonadotropin-dependent PAPP-A expression in granulosa cells may serve to degrade the antigonadotropin IGFBP-4. Accordingly, successful antral follicle development, ovulation, and corpus luteum formation may be contingent on an IGFBP-4-deplete/PAPP-A-replete circumstance, hence resulting in an IGF-I-replete intrafollicular microenvironment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE EXISTS SUBSTANTIAL evidence to the effect that the IGF family plays a central role in determining FSH action in murine and human ovaries (1, 2, 3). In the murine, there exists a complete intrafollicular IGF-I system (4, 5), replete with a ligand (IGF-I), a receptor (type I), IGF-binding proteins (IGFBPs 4 and 5), and IGFBP-4- and -5-directed endopeptidases (IGFBP-4ase and IGFBP-5ase) (6, 7). The essential ovarian function of IGF-I in the mouse has been documented by the observation that female null igf-1 mutants are infertile, owing to the failure of FSH to stimulate dominant follicle formation (8, 9). Direct in vitro evidence that bioavailable (unbound) intrafollicular IGF-I is required for FSH action has also been furnished in cultures of rat granulosa cells (10, 11). As such, a concept has emerged according to which IGF-I may constitute an obligatory determinant of FSH responsiveness and thus of dominant follicle growth and development (12).

IGFBP-4 is a potent inhibitor of IGF-I action in all tissues studied, including the granulosa cell (13). The regulation of IGFBP-4 activity in the intrafollicular microenvironment seems to be critically important for the very ability of FSH to promote dominant follicle formation (7, 14, 15).

A biochemically defined protease(s) capable of degrading IGFBP-4 has been reported in a number of different cell types (16, 17, 18, 19, 20, 21), including the rodent granulosa cell (7). The physiological relevance of IGFBP-4ase is supported by the fact that proteolysis of IGFBP-4 results in increased bioavailability of IGFs. It is tempting to speculate that granulosa cell-derived IGFBP-4ase may, indirectly, play a pivotal role in regulating the FSH-dependent development of the healthy follicle. Until recently, despite considerable efforts, the molecular identity of the IGFBP-4ase was unknown. However, studies of cultured human fibroblasts, of osteoblastic cells, and of luteinized granulosa cells have identified an IGFBP-4-specific protease (IGFBP-4ase) as a protein previously known as pregnancy-associated plasma protein-A (PAPP-A) (22, 23). More recently, the recombinant form of PAPP-A was definitely shown to display specific IGFBP-4 proteolytic activity (24). Evidence of identity between the IGFBP-4ase activity of human ovarian follicular fluid and PAPP-A has also been recently furnished (25). PAPP-A is a member of the metzincin family of metalloproteinases, one characterized by a molecular mass of about 200 kDa.

The aim of the present study was to identify the mouse counterpart of the human PAPP-A cDNA and to analyze its ovarian expression, hormonal regulation, and cellular localization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In vivo protocols
Two sets of C57BL/6J mice, purchased from The Jackson Laboratory (Bar Harbor, ME), were employed for these experiments: immature female mice and randomly cycling mature female mice. The mice were managed in compliance with NIH guidelines for the care and use of animals.

Immature mice.
At 25 d of life, mice (n = 42; 3 mice for each time point) were injected, ip, with 10 IU PMSG (Sigma, St. Louis, MO) in 0.1 ml saline. Thereafter, mice were killed 2, 4, 8, 12, 16, 24, 36, and 48 h after PMSG injection. Forty-eight hours post PMSG, some PMSG-primed mice (n = 18) were injected with 10 IU human CG (hCG) (Sigma) in 0.1 ml saline. In turn, hCG-treated mice were killed 3, 6, 9, 12, 24, and 48 h after injection. All mice were killed by CO2 asphyxiation, and the ovaries were removed and either frozen or paraformaldehyde-fixed until used.

Mature mice.
Adult randomly cycling female mice (10 wk of age) were exposed to a photoperiod of 14 h light, 10 h dark, with lights on at 0600 h. Estrous cycles were monitored by daily examination of vaginal cytology, and only those animals exhibiting at least two consecutive 4- to 5-d ovulatory cycles were used. Mice were killed at the indicated stages of the cycle, and the ovaries were removed and paraformaldehyde-fixed until used.

Total RNA isolation
Total RNA was isolated using an RNAeasy Kit (QIAGEN, Valencia, CA) according to the manufacturer’s directions. The resultant RNA was quantified by absorbance at A260.

Sequencing of rapid amplification of cDNA ends (RACE)-derived partial mouse PAPP-A cDNAs
To secure the full-length sequence of the mouse PAPP-A cDNA, we have undertaken rapid amplification (RACE) of both the 5' and 3' ends of the relevant partial cDNAs (26). Pooled ovarian material from PMSG-primed immature mice (n = 12) was obtained 3, 6, 9, and 12 h post hCG administration. Total ovarian RNA was extracted as described above. The latter (100 µg total RNA), in turn, was used to isolate Poly A+ RNA by means of an oligo-dT magnetic bead system (PolyATract mRNA Isolation System, Promega Corp., Madison, WI). The resultant Poly A+ RNA was used as a template to synthesize two separate populations of full-length single-strand cDNAs by means of the SmartRACE cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Briefly, the switching mechanism at the 5' end of the RNA transcript (SMART) sequence was incorporated into both the 5' and 3' RACE cDNA during RT, thus making it possible to concurrently prime both RACE PCR reactions with SMART as well as with PAPP-A-specific primers. The gene-specific primers used for PCR were antisense (5'-TGTCGGATACAATCTCTCGCTGGGTTC-3'), and sense (5'-CCTCAGCCAGCCCTTCTACCACAGC-3'). A touchdown (27) PCR protocol was used: 5 cycles at 94 C for 50 sec, 68 C for 3 min; followed by 5 cycles at 94 C for 50 sec, 66 C for 50 sec, and 72 C for 3 min; and finally, 30 cycles at 94 C for 50 sec, 65 C for 50 sec, and 72 C for 3 min. Amplicons were analyzed on a 1.2% agarose gel stained with ethidium bromide, recovered using the QIAquick PCR purification kit (QIAGEN), and sequenced (both strands).

Northern blot analysis
Formaldehyde-denatured RNA [20 µg total RNA/lane and 0.24–9.5 kb RNA ladder (Life Technologies, Inc., Gaithersburg, MD)] was separated on denaturing 1% agarose-formaldehyde gels and transferred to nylon membranes (Magna Graph, MSI, Westboro, MA) (28). The above nylon membranes were prehybridized for 2–6 h at 42 C in 5x SSPE, 50% formamide, 5x Denhardt’s, 0.25% SDS, and 100 µg/ml denatured salmon sperm DNA. A partial mouse PAPP-A cDNA (553 bp), a PCR product (GenBank AF 258461; nucleotides 1599–2151) generated from mouse ovaries using PAPP-A-specific primers (F: 5'-TGAGTCTCTGACCATTTGGGTGAC-3', B: 5'-TTGTCGGCAGAAAAGGGAGCAG-3'), was labeled with 5 µCi {32P} deoxy-CTP using the random-hexanucleotide-primed second-strand synthesis method (rediPrime II; Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). The resultant probe was denatured for 5 min in a boiling water bath, transferred to ice, and hybridized overnight at 42 C with the above mentioned nylon membranes, using the above prehybridization solution. Thereafter, membranes were sequentially washed three times for 5 min at room temperature with 5x saline sodium citrate (SSC) and 0.5% SDS, followed by two washes for 15 min at 60 C with 1x SSC and 0.75% SDS. The blots were ultimately rinsed with 4x SSC. To quantify the extent of hybridization, the membranes under study were exposed to a Phosphor Screen (Molecular Imager System; Bio-Rad Laboratories, Inc., Hercules, CA), and the resultant digitized data were analyzed with Molecular Analyst software (Bio-Rad Laboratories, Inc.). The membranes were then stripped by heating to 95 C in 0.2x SSC/0.5% SDS and reprobed with a {32P}-labeled PCR product corresponding to the mouse ß-actin cDNA, to correct for possible variation in RNA loading and/or transfer. Each experiment was carried out at least three times with three different sets of animals, in an effort to minimize possible errors introduced by a given individual experiment.

Semiquantitative RT-PCR
First-strand cDNA was synthesized from total ovarian RNA. Briefly, 1 µg total RNA and 0.5 µg oligo(dT)12–18 (Amersham Pharmacia Biotech, Uppsala, Sweden) were mixed in diethyl ester pyrocarbonic acid-treated water, to a final vol of 30 µl, heated to 70 C for 2 min, and the reaction finally quenched on ice for 2 min. RT reactions were carried out using final concentrations of 50 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 75 mM KCl, 1 mM deoxynucleotide triphosphates, 37 U RNAguard Ribonuclease Inhibitor from human placenta (Amersham Pharmacia Biotech), 10 mM dithiothreitol, 0.1 mM each of deoxynucleotide triphosphates (dNTPs), 0.1 mM oligo(dT)12–18, and 400 U Moloney murine leukemia virus reverse transcriptase (M-MLV Reverse transcriptase, Life Technologies, Inc.). This mixture was incubated at 37 C for 1 h and inactivated at 70 C (10 min).

Different tissue cDNAs were purchased from Origene Technologies (Rockville, MD; Rapid-Scan Gene Expression Panel).

Serial dilutions of cDNAs corresponding to the different experimental time points were used for PCR amplification. Included were a primer set for ß-actin (0.5 µM each; forward primer, 5'-CCCCATTGAACATGGCATTGTTAC-3'; reverse primer, 5'-TTGATGTCACGCACGATTTCC-3') in a 25-µl reaction vol with 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1%Triton X-100 (Promega Corp.), 2.5 mM MgCl2, 400 µM each dNTP, and 0.625 U Taq DNA Polymerase (Promega Corp.). The concentrations used ensured that the reaction could be quantified within the log phase of the amplification reaction. PCR was performed for 27 cycles (initial denaturation at 94 C for 3 min, then 27 cycles at 94 C for 1 min, 59 C for 1 min, 72 C for 1 min, and a final incubation at 72 C for 7 min). The reaction mix (7 µl) was run on a 1.5% agarose gel stained with ethidium bromide and quantified using UV imaging (Gel Doc 1000, Bio-Rad Laboratories Inc.) and Molecular Analyst software (Bio-Rad Laboratories, Inc.). These same conditions were used for PCR reactions designed to quantify the relative levels of the ovarian expression of PAPP-A, except that the PCR reaction was carried out for 33 cycles, the primer set being: forward primer: 5'-CTCTTTCACGCCCAATCAAGTC-3'; reverse primer-5'GATGAGCACCTGGAGATTGATGC-3'. Signals corresponding to ovarian PAPP-A expression were normalized, relative to ß-actin, for each sample. Experimental replication of each time point was performed in triplicate for all three sets.

In situ hybridization
In situ hybridization was performed as previously described (29), with minor modifications. Mouse ovaries were obtained from two sets of animals: immature gonadotropin-primed (at the indicated time points) and adult randomly cycling (at the indicated phase of the estrous cycle). To compare the expression intensity between tissue sections under the same experimental conditions, ovarian sections from three different mice, at each time point studied, were placed on the same slide to ensure reproducibility. These experiments were repeated at least three times. Freshly-dissected ovaries were immediately fixed in 4% paraformaldehyde in PBS, overnight, at 40 C. Paraffin-embedded tissues were sectioned at 10 µm and mounted onto poly-L-lysine-coated slides. Sections were deparaffinized, rehydrated, rinsed with diethyl ester pyrocarbonic acid water, digested with proteinase K, and acetylated. A partial mouse PAPP-A cDNA (553 bp), a PCR product (GenBank AF 258461; nucleotides 1599–2151), generated from mouse ovaries with the same PAPP-A-specific primers used in the Northern Blot analysis, was cloned into pGEM-T Easy Vector (Promega Corp.). Tissues were hybridized for 16 h at 60 C within 100 µl of a hybridization solution containing 1 x 106 cpm of a labeled mouse PAPP-A antisense or sense probe. At the conclusion of the hybridization phase, sections were washed, treated with ribonuclease (RNase A for 30 min, at 37 C), gradually desalted (2x SSC, 1x SSC, and 0.5x SSC), incubated with 0.1x SSC at 75 C for 40 min, dehydrated, and exposed to Hyperfilm-ß max (Amersham Pharmacia Biotech, Arlington Heights, IL) for 3 d. Thereafter, the ovarian sections were defatted, dehydrated, coated with NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), and exposed for 2 wk at 4 C. The autoradiograms were developed, fixed, and counterstained with hematoxylin.

Detection of apoptosis
DNA fragmentation was detected in adjacent serial ovarian sections by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method, using the ApopTag Plus in situ Apoptosis Detection kit (Intergen, Purchase, NY).

Statistical analysis
Each experiment was carried out at least three times, with 3–4 mice at each time point. Data points are presented as mean ± SE. Statistical significance (Fisher’s protected least significance difference) was determined by ANOVA to assess differences between mean densities, adjusted for the average background density. All analyses were performed using Statview for Macintosh (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and sequencing of the mouse PAPP-A cDNA
To clone the mouse PAPP-A cDNA, six sets of primer pairs were prepared, based on the nucleotide sequence of human PAPP-A (GenBank X68280). Substantial homology between the species was assumed. Three of the six sets of primers: 1) F: 5'-TCGAGCACTTCAGTCTGTGGAAG-3', B: 5'-AGAGTCGGGGTCAAAGCAAGTC-3'; 2) F: 5'-CGGTTCAACTTTGATGGTGGAGAG-3', B: 5'-ATTCTGGCGACTTGATTGGGCGTG-3'; and 3) F: 5'-TGAGTCTCTGACCATTTGGGTGAC-3', B: 5'-AACTGAAGCTCACACGTACCGC-3' yielded amplicons corresponding to partial mouse ovarian PAPP-A cDNAs (668, 524, and 1137 bp, respectively). Another partial mouse PAPP-A cDNA fragment (618 bp) was generated using primers corresponding to the above mouse cDNA sequences: (F: 5'-CTCTTTCACGCCCAATCAAGTC-3', B: 5'-GCATCAATCTCCAGGTGCTCATC-3'). Both strands of the resultant four cDNAs were sequenced, using gene-specific primers, by the ABI 377 automated DNA sequencer of the Huntsman Cancer Institute at the University of Utah Health Sciences Center.

The derived nucleotide sequences of the four different partial cDNA sequences of mouse PAPP-A were aligned using the CLUSTALW program, yielding a composite sequence of 2759 bp. Screening of the mouse ovarian cDNA library with a mouse PAPP-A amplicon (GenBank AF 258461; nucleotides 1599–2151) failed to produce positively hybridizing clones. RACE technology yielded a 3' fragment (1815 bp), which was isolated and sequenced. In contrast, no 5' product could be generated. Alignment of the former composite cDNA with the 3' RACE fragment yielded a cDNA of 4505 bp.

The mouse PAPP-A cDNA: homology with human PAPP-A
A BLASTn search of the nonredundant database of the National Center for Biotechnology Information against the partial mouse PAPP-A sequence (GenBank AF 258461) revealed substantial homology with the human PAPP-A gene (GenBank NM 002581). The nucleotide sequences of the partial mouse cDNA and of the corresponding partial human cDNA proved 88% identical. At the amino acid level, the partial mouse and human cDNAs shared 89% identity, a figure further increased to 93% when conservative amino acid substitutions were included (similarity) (Fig. 1Go).



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  		Figure 1. The amino acid sequence of the human and mouse PAPP-A. The partial amino acid sequence of the mouse PAPP-A (GenBank AF258461) was aligned with the complete human PAPP-A amino acid sequence (GenBank X68280). Black shaded residues are identical to the consensus sequence. Gray shaded residues are similar to the consensus sequence. The numbers at the top indicate the residue position.

 
Ovarian PAPP-A mRNA expression during a simulated ovarian cycle in immature mice
To analyze the expression of PAPP-A transcripts during a simulated ovarian cycle, mouse ovarian mRNA samples were subjected to Northern blot analysis using a mouse PAPP-A cDNA probe (GenBank AF 258461, nucleotides 1155–1817). Fig. 2AGo displays ovarian PAPP-A transcripts, approximately 13 kb in size, a size similar to that detected by Northern blot analysis of human placental mRNA and cultured human fibroblasts. However, a minor band of 8.5 kb, previously reported for the above-mentioned mRNA, could not be detected (22, 30, 31). Studied 48 h post PMSG injection, ovarian PAPP-A gene expression remained at control levels. However, 3 h after hCG administration, a statistically significant (P < 0.05) 2.9-fold increase in ovarian PAPP-A expression was noted. To further quantify ovarian PAPP-A gene expression during a simulated ovarian cycle, semiquantitative RT-PCR was undertaken using oligonucleotide primer pairs corresponding to the mouse PAPP-A cDNA. The results of this analysis, normalized for ß-actin expression, confirmed and extended the ovarian expression patterns previously noted on Northern blot analysis. Specifically, note was made of a significant (P < 0.05) increase in ovarian PAPP-A expression 24 h post PMSG injection (4.8-fold increase over control), a decrease to control levels 48 h post PMSG injection, and a significant (P < 0.05) increase 6 h after hCG injection (3.4-fold increase over control levels). These results indicate that PAPP-A transcripts are subject to hormonal regulation during the folliculogenic (preovulatory), ovulatory, and postovulatory phases of the ovarian cycle (Fig. 2Go, B and C).



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  		Figure 2. Regulated ovarian expression of mouse PAPP-A mRNA during a simulated estrous cycle. A, Northern blotting. Total ovarian RNA (20 µg) from superovulated C57BL/6 mice, obtained at the indicated time points, was electrophoresed and transferred to a nylon membrane, and the membrane was probed with a partial mouse PAPP-A cDNA probe as described under Materials and Methods. The panel shown reflects a representative experiment out of a total of three independent experiments (0.24- to 9.5-kb RNA ladder) (Life Technologies, Inc.). B and C, Semiquantitative RT- PCR. Ovarian cDNAs, corresponding to different time points during a simulated ovarian cycle, were analyzed as described under Materials and Methods. Seven microliters of the resultant PCR product were run on a 1.5% agarose gel stained with ethidium bromide and quantified by UV imaging and Molecular Analyst. Each sample was analyzed in triplicate. The panel shown reflects a representative experiment out of a total of three independent experiments. PAPP-A expression was normalized, relative to ß-actin. Results are expressed as fold activation over control, representing the mean ± SEM of three independent experiments.

 
PAPP-A gene expression in different mouse tissues: tissue specificity studies
Northern blot analysis with a commercial RNA-impregnated membrane failed to detect any of the 12 tissues tested (i.e. mouse brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, muscle, lung, testis, and skin), data not shown. To further study PAPP-A gene expression, 24 commercially-available cDNAs, representing a broad range of tissues, were subjected to PCR analysis. As shown (Fig. 3Go), this more sensitive technology revealed the testicular, kidney, breast, and embryonic cDNAs to be most abundant. PAPP-A transcripts could also be detected in the brain, heart, spleen, skin, muscle, gastrointestinal tract, ovary, prostate, and uterus. PAPP-A transcripts were not detected in the thymus, liver, lung, and adrenal.



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  		Figure 3. PAPP-A expression in different mouse tissues. RT- PCR. cDNAs corresponding to 24 mouse different tissues (Rapid-Scan Gene Expression Panel) were analyzed as described under Materials and Methods. Ten microliters of the resultant PCR product were run on a 1.5% agarose gel and stained with ethidium bromide. The panel reflects a representative experiment out of a total of two independent experiments. E, Embryo; Small Int., small intestine.

 
Cellular localization of PAPP-A transcripts in PMSG-primed (preovulatory) immature mouse ovaries
To identify the cellular sites of expression of PAPP-A transcripts in immature PMSG-primed ovaries, use was made of in situ hybridization. A control sense probe was associated with a very low nonspecific background signal in all sections examined (data not shown). No ovarian PAPP-A expression could be detected in the untreated state, thereby confirming the aforementioned Northern blot and RT-PCR results (Fig. 4AGo). In vivo treatment with 10 IU PMSG produced a time-dependent increase in PAPP-A mRNA (Fig. 4Go, B–E), the time course of induction paralleling that documented in the above-mentioned RT-PCR and Northern blot experiments. As shown in Fig. 4BGo, a positive (albeit weak) signal for PAPP-A transcripts was observed as early as 3 h after the administration of PMSG, a signal restricted to granulosa cells of a few follicles. The signal corresponding to granulosa cell PAPP-A transcripts rose to apparent peak levels 12 h post PMSG injection, high levels of expression being maintained through the 24 and 36 h post PMSG time points (Fig. 4Go, C–E). Most, if not all, of the PAPP-A mRNA was expressed in the granulosa cells of large antral follicles (Fig. 4Go, C and D). Interestingly, 48 h post PMSG, ovarian PAPP-A mRNA was greatly reduced, to a point wherein no specific hybridization signal was apparent in Graafian preovulatory follicles (Fig. 4FGo), in keeping with the Northern and RT-PCR data.



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  		Figure 4. In situ hybridization analysis of ovarian PAPP-A mRNA in immature PMSG-treated mice. Dark-field photomicrographs depicting the distribution 35S-labeled probe (white grains). A, Untreated control displays little or no labeling. B, PMSG (3 h) reveals a weak positive signal in some granulosa cells in a few follicles. C, PMSG (12 h) discloses a strong signal in granulosa cells of some antral follicles. PMSG (24 h) (D) and PMSG (36 h) (E) display a similar strong labeling pattern. F, PMSG (48 h) labeling was markedly reduced. All photographs were taken at 4x magnification.

 
Cellular localization of PAPP-A mRNA in PMSG-primed/hCG-triggered (ovulatory and postovulatory) mouse ovaries
PMSG-primed/hCG-triggered ovaries displayed intense labeling of PAPP-A transcripts at all time points tested. The signal localized to granulosa cells of preovulatory follicles and to corpora lutea (Fig. 5Go). Time course studies revealed ovarian PAPP-A mRNA expression to rise from undetectable levels at the time of hCG injection (48 h post PMSG: see Fig. 4FGo) to maximal levels, within 3 h after treatment with hCG (Fig. 5AGo). This level of expression was sustained (in granulosa cells of preovulatory follicles) when assessed 6 h and 12 h post hCG stimulation (Fig. 5Go, B and C). After ovulation, the newly-formed corpora lutea (24 and 48 h post hCG time points) displayed distinct labeling (Fig. 5Go, D and E). The signal seemed most prominent in a subset of the large cells, which exhibited spherical nuclei containing one or two large nucleoli (Fig. 6Go). The structure of these cells is typical of granulosa-lutein cells. An occasional cell within the theca, stroma, and surface epithelium also seemed to be labeled.



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  		Figure 5. Comparison of the distribution of the PAPP-A mRNA in ovaries of immature 25-d-old PMSG-primed hCG-treated mice and normal adult cycling animals. Dark-field photomicrographs: white dots represent silver grains in the autoradiographic emulsion. A, hCG (3 h) reveals a strong hybridization signal in granulosa cells of growing antral (pre-Graafian) follicles; B, hCG (6 h); C, hCG (12 h); D, hCG (24 h) discloses distinct labeling in some luteal cells; E, hCG (48 h); F, adult ovary at proestrus (2000 h). All photographs were taken at 4x magnification.

 


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  		Figure 6. Analysis of the cellular localization of PAPP-A mRNA in the corpus luteum. Photomicrographs of a PMSG-primed ovary after 48 h of hCG treatment (Fig. 5EGo). Low magnification (20x) bright-field (A) and dark-field (B) photomicrographs of the same section of a corpus luteum (CL) after hybridization with PAPP-A antisense probe. The hybridizing signals are present in a subset of granulosa-lutein cells. C, Higher magnification (40x) bright-field photograph of inset shown in panels A and B. The hybridizing signal (black silver grains) occurs in a subset of large lutein cells containing one or two nucleoli (arrowheads). No hybridizing signal was detected in the endothelial cells ({therefore}).

 
To further address the question of physiological relevance, the expression pattern of PAPP-A in the ovaries of adult randomly cycling mice was also examined. As shown in Fig. 5FGo, a pattern of ovarian hybridization similar to that noted in immature ovaries was apparent in antral follicles and in corpora lutea of adult randomly cycling mice.

As shown in Fig. 7Go, heterogeneity was noted in labeling intensity among granulosa cells of PMSG-primed and PMSG-primed/hCG-triggered antral follicles. The message encoding PAPP-A localized exclusively to the centrifugally residing membrana granulosa cells of developing antral follicles. No detectable signal was noted for the inner periantral (i.e. granulosa cells adjacent to the antrum) and cumulus granulosa cells (Fig. 7Go, A and B). It is noteworthy that most, but not all, of the centrifugally residing membrana granulosa cells displayed PAPP-A labeling. This cell-specific pattern of PAPP-A expression was noted in developing antral follicles of all PMSG-primed ovaries at the 3, 12, 24, and 36 h time points (Fig. 7Go, A–D).



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  		Figure 7. Analysis of the cellular localization of PAPP-A mRNA in sections of gonadotropin-stimulated immature mouse ovaries. Bright-field (A, C, E, and G) and dark-field photomicrographs of the same sections (B, D, F, and H). A and B, PMSG (24 h): *, Healthy follicles; AF, atretic follicle; arrowheads, preantral follicles; magnification, 10x. C and D, PMSG (24 h), high power: MGC, Membrana granulosa cells; EGG, oocyte; SE, surface epithelium; PGC, periantral granulosa cells; CGC, cumulus granulosa cells; T, theca; PAF, preantral follicle; GSE, germinal surface epithelium; magnification, x20. E and F, hCG (3 h): POF, preovulatory follicle; magnification, x10. G and H, hCG (3 h): Panels C and G, High power of a preovulatory follicle; magnification, x20.

 
Given an ovulatory dose of hCG, a similar cell-specific pattern of PAPP-A mRNA expression was apparent in healthy follicles (Fig. 7Go, E–H). The PAPP-A mRNA was highly expressed in centrifugally located membrana granulosa cells but proved very low or undetectable in periantral and cumulus granulosa cells (Fig. 7Go, E and F). This cell-specific pattern of expression was seen in all hCG-stimulated healthy follicles throughout the entire ovulatory sequence, e.g. oocyte meiotic maturation, cumulus expansion, and ovulation.

TUNEL analysis
To evaluate the atretic nature of the follicles with PAPP-A hybridization signals, we carried out an apoptosis analysis of adjacent serial sections at each time point in the PMSG/hCG experiment. TUNEL analysis revealed that all PAPP-A-positive follicles were healthy (i.e. apoptosis-free). No detectable PAPP-A expression was seen in atretic (i.e. apoptotic, Fig. 8Go) or healthy preantral (e.g. primordial, primary, or preantral secondary follicles; Fig. 7Go, A–H) in any of the ovaries examined.



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  		Figure 8. TUNEL analysis for PAPP-A in sections of gonadotropin-stimulated immature ovaries. Analysis of in situ hybridization for PAPP-A and apoptosis in adjacent serial sections of mouse ovaries after 24 h of PMSG-treatment (panels A and C). Dark-field photomicrographs hybridized with the antisense PAPP-A probe. White dots represent silver grains in the autoradiographic emulsion. A, Low power, 4x; C, Higher power of inset shown in panel A, 20x. Panels B and D, TUNEL analysis. The 3'OH ends of fragmented DNA are stained brown by exogenously added TdT and digoxigenin-labeled deoxyuridine 5-triphosphate. B, Low power, 4x. D, Higher power (20x) of inset shown in panel B. Atretic antral follicle with apoptotic granulosa cells (arrowheads). Healthy antral follicle (right), showing no apoptosis. Note the atretic and healthy follicles are PAPP-A-negative and -positive, respectively (compare panels C and D).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present manuscript documents, for the first time, the gonadotropin-dependent regulation and pattern of expression of the PAPP-A gene in the murine ovary. Specifically, we observed that after FSH- and LH (hCG)-stimulation, the only ovarian cells expressing detectable PAPP-A mRNA are the centrifugally located membrana granulosa cells of healthy growing antral as well as ovulating follicles and the granulosa lutein cells of corpora lutea. This is compelling evidence that PAPP-A gene expression in the ovary is a specific consequence of gonadotropin-stimulation and that, when expressed, PAPP-A is detected in the membrana granulosa cells of dominant follicles and in the granulosa-lutein cells of corpora lutea.

Cloning efforts yielded only a partial mouse PAPP-A cDNA. In spite of intense efforts, the 5' region of the PAPP-A cDNA could not be delineated. A partial explanation for this apparent failure might be the exceptionally high GC content (90%), which characterized this region of the (human PAPP-A) cDNA (30). The partial protein sequence of mouse PAPP-A comprised 1367 amino acids, the full human open reading frame consisting of 1627 amino acids. The Protein Family Database of Alignments and hidden Markov Models (HMM’s) (Pfam; Internet address: http://www.sangerac.uk/software/pfam) and the Prosite Database of Families and Domains (Internet address-http://www.expasy.ch/prosite) domain-searching tools revealed a neutral zinc metallopeptidase, a zinc-binding region signature (TMIHEIGHSL, amino acids 299–308), three sequences similar to the Notch (Delta-Serrate-Lag-2) domain found in the Notch family of transmembrane homeotic genes such as lin-12 and GLP-1 (32), and five stretches homologous to the complement control protein (CCP) modules. The CCP modules (also known as short consensus repeats, SCRs, or SUSHI repeats), each containing approximately 60 amino acid residues, have been identified in many proteins germane to the mammalian complement system and to selectins, a group of cell adhesion molecules that mediate the localization of circulating leukocytes (33, 34). Interestingly, the complement protein C1s was recently found to be IGFBP-5ase, synthesized and secreted by fibroblast and smooth muscle cells (35). The functions of the Notch and CCP domains in PAPP-A remain to be elucidated.

The finding that PAPP-A gene expression is gonadotropin-dependent, under in vivo circumstances, identifies FSH and LH/hCG as important regulatory molecules in the control of ovarian PAPP-A gene activity. Preliminary in vitro studies, entailing cultured rat granulosa cells, revealed the induction of PAPP-A transcription to be FSH-dependent (Hourvitz et al., unpublished data). As such, the regulated expression of PAPP-A in the healthy antral follicle might be expected to play a role in follicular growth and maturation after selection. The preceding statement is based on the following. Granulosa cell-derived IGFBPs have been reported to be selectively expressed in atretic (but not healthy antral) follicles. IGFBP-4 has been shown to act as an antigonadotropin by virtue of its IGF-I-sequestering property (15, 36, 37). Moreover, the expression of IGFBP-4 transcripts in granulosa cells precedes the appearance of morphological signs of atresia. It was thus suggested that IGFBP-4 could serve as a marker of incipient follicular atresia in the mouse ovary (38). Given that intraovarian IGF-I is obligatory for FSH action and that IGFBP-4 is a potent inhibitor of this process, it seems reasonable to propose that the regulated expression of PAPP-A may serve the enhancement of gonadotropin action through the degradation of IGFBP-4. It might be speculated that PAPP-A is another player in the intrafollicular positive feedback loop. Induced by FSH (as is the type I IGF receptor), PAPP-A could increase bioavailable IGF-I, thereby enhancing FSH action, while increasing type I IGF receptor representation brought about by FSH.

Our finding of PAPP-A gene expression in mouse granulosa cells of healthy growing antral, but not atretic follicles, is compatible with previous studies wherein IGFBP-4ase bioactivity was assessed in human follicular fluid. Specifically, Chandrasekher et al. (16) reported IGFBP-4ase activity in the follicular fluid of estrogen-dominant (healthy), but not androgen-dominant (atretic), human follicles. Conover et al. (25) confirmed and extended these findings by demonstrating that anti-PAPP-A antibodies are capable of neutralizing IGFBP-4ase activity in human follicular fluid. This new finding raised the possibility that follicular PAPP-A and IGFBP-4ase are one and the same, as previously reported for the human fibroblast (22). In addition to this, more recently, Overgaard et al. (24) were able to display data revealing that human recombinant PAPP-A expressed from mammalian cells specifically cleaves IGFBP-4 in an IGF-dependent way. This recombinant protein behaves antigenically and functionally like the native PAPP-A. Independently, Sjoberg et al. (39) have documented PAPP-A immunoreactivity in both human follicular fluid and corpora lutea. More recently, we have observed that PAPP-A transcripts are localized to the granulosa cells of dominant human follicles (40); meanwhile, Conover et al. (23) demonstrated the proteolytic activity of PAPP-A on IGFBP-4, using conditioned media from cultured human luteinizing-granulosa cells obtained from estrogen-dominant follicles.

It is clear, from our in situ hybridization data, that the major site of PAPP-A gene expression in the murine ovary (during the follicular phase of the cycle) is the centrifugally-residing membrana granulosa cell of the growing healthy antral follicle. The identification of PAPP-A mRNA only in healthy follicles supports the notion that gonadotropin-induced PAPP-A gene activity represents an early marker of follicles selected to enter the growth trajectory. The absence of detectable PAPP-A mRNA in the granulosa cells of healthy preantral and atretic antral follicles suggests that the PAPP-A gene is not transcribed in these follicles, despite exposure to PMSG. The molecular mechanisms underlying the apparent absence of PAPP-A mRNA in atretic follicles remain to be elucidated. This question relates to the more fundamental issue of ovarian follicular selection, i.e. why do only a few follicles develop in response to gonadotropic stimulation, when all follicles are presumably comparably exposed. The latter presumption may, in fact, be erroneous. It was previously shown (41) that exposure to FSH may not be equal among follicles, because selective FSH receptor and IGF-I expression significantly amplifies FSH-receptor gene expression and, presumably, FSH action only in a subset of follicles. FSH in turn, induces IGF-I receptor expression (42, 43), thereby effecting an intrafollicular positive feedback loop. It has been previously speculated (43) that granulosa-cell IGF-I expression (possibly responsible for selective follicular responsiveness to FSH) is stimulated by an oocyte-derived signal.

Overgaard et al. (44) screened for the expression of the human PAPP-A gene in fifty different human tissues, using dot blotting. Whereas the placenta displayed a very high signal, the only other tissue displaying a PAPP-A signal above background was the kidney. Using sensitive semiquantitative RT-PCR, the authors were able to document the expression of PAPP-A in 13 tissues analyzed, including endometrium, myometrium, ovary, breast, prostate, colon, and kidney. The most abundant PAPP-A signal was seen in the term placenta (250- to 3000-fold higher than in the other tissues). We, in turn, were unable to detect a PAPP-A signal in any of the 12 tissues tested using Northern Blot analysis. These results are in agreement with previous human studies wherein Northern blots of poly(A+) RNA from heart, lung, skeletal muscle, brain, liver, kidney, and pancreas failed to detect PAPP-A mRNA. However, the use of Northern blotting for large mRNAs, such as PAPP-A, is technically difficult and is characterized by relatively low sensitivity. Using the more sensitive method of RT-PCR, we were able to document the expression of PAPP-A in most of the tissues tested, including the brain, the heart, spleen, skin, muscle, gastrointestinal tract, ovary, prostate, and uterus. The highest signals were noted in the testis, kidney, breast, and embryo at different stages of development.

The PAPP-A mRNA signal proved intense in subtypes of corpora lutea cells in both superovulated immature mice and randomly cycling adult mice. However, given that the preovulatory signal is selectively expressed in the membrana granulosa of preovulatory follicles, together with expression pattern of PAPP-A in the corpus luteum, it is likely that the luteal signal is localized to some granulosa-lutein cells. The identification of PAPP-A transcripts in the corpus luteum suggests that PAPP-A may be physiologically relevant to the function of this important postovulatory structure.

Our data document PAPP-A to be expressed in a highly-restricted fashion in healthy growing antral follicles. It is intriguing that the hybridization signal in healthy follicles was limited to centrifugally residing membrana granulosa cells. Although the molecular mechanism responsible for this cell-specific pattern of PAPP-A gene expression is unknown, it might involve a regulatory gradient of centrifugal oocytic signals. Classical work has established the concept of granulosa cell heterogeneity in response to FSH stimulation (45, 46). Viewed in conjunction with previous in vivo and in vitro studies (47), our data fit the hypothesis that morphogens emanating from the oocyte may play a role in regulating differential PAPP-A gene activity in the membrana granulosa cells (46). It will be interesting to investigate whether oocytic signals such as growth differentiation factor-9 and growth differentiation factor-9B/BMP-15 are involved.

In summary, using the murine ovary as a model, we demonstrated the membrana granulosa cells of healthy antral follicles and the granulosa-lutein cells of the corpus luteum as the only ovarian sites of PAPP-A/IGFBP-4ase mRNA expression. This selective pattern of expression points to a role for PAPP-A in the organized growth, function, and survival of these two fundamentally important tissues, the healthy follicle and the corpus luteum (Fig. 9Go). Although the various biological effects of PAPP-A in the murine ovary remain to be established, it represents a novel and potentially important gene, with respect to the dominant follicle, the ovulatory process, and the corpus luteum.



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  		Figure 9. Schematic illustration of PAPP-A expression during follicular growth and luteinization.

 


   Footnotes
 
This work was supported, in part, by NIH Research Grants RO1-HD-30288 and RO1-HD-37845 (to E.Y.A.), American Physician Fellowship Award (to A.H.), CAPES/Brazil BEX 1007/99-8 (to A.B.T.), CNPq/Brazil 870.313/97-5 (to A.B.T.), and by cooperative agreement U54HD 12303 (to G.F.E.) as part of the Specialized Cooperative Centers Program in Reproduction Research.

Abbreviations: CCP, Complement control protein; hCG, human CG; IGFBP, IGF binding protein; IGFBP-4ase, IGFBP-4-directed protease; PAPP-A, pregnancy-associated plasma protein-A; RACE, rapid amplification of cDNA ends; SMART, switching mechanism at the 5' end of the RNA transcript; SSC, saline sodium citrate; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling.

Received November 5, 2001.

Accepted for publication January 11, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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