ABSTRACT

The ovulatory process in mammals involves gross physiological events in the ovary that cause transient deterioration of the ovarian connective tissue and rupture of the apical walls of mature follicles. This gonadotrophin-induced process has features similar to an acute inflammatory reaction that affects most of the ovary. The present study reveals that the ovulatory events include induction of mRNA for pancreatitis-associated protein-III (PAP-III). Immature Wistar rats were primed with 10 IU equine chorionic gonadotrophin (eCG) s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG, s.c.. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for RT-PCR differential display to detect PAP-III gene expression in the stimulated ovarian tissue. Northern blotting showed that transcription was significantly higher at 4-12 h after the ovaries had been stimulated by hCG. In situ hybridisation indicated that PAP-III mRNA expression was limited mainly to the hilar region of the ovarian stroma, with most of the signal emanating from endothelial cells that lined the inner walls of blood vessels, and from small secondary follicles. Treatment of the animals with ovulation-blocking doses of indomethacin (an inhibitor of prostanoid synthesis) or epostane (an inhibitor of progesterone synthesis) revealed that ovarian transcription of PAP-III mRNA was moderately dependent on ovarian progesterone synthesis. In conclusion, the present evidence of an increase in PAP-III gene expression in gonadotrophin-stimulated ovaries provides further evidence that the ovulatory process is comparable to an inflammatory reaction.
INTRODUCTION

Pancreatitis-associated protein (PAP) was discovered in 1984 as a secretory product of the pancreas following the experimental induction of acute inflammation in the rat pancreas (Keim et al. 1984, Keim et al. 1994). Two years later, a homologous rat protein called peptide 23 was identified as a secretory product from cultured hypophyseal somatotropes that had been stimulated by GHRH (Yokoyo & Friesen, 1986). Subsequently, other homologs of PAP were found in a variety of tissues including the gastric pyloric gland (Yamamoto et al. 1992), the small intestines (Chakraborty et al. 1995a, Iovana et al. 1993), and the uterus (Chakraborty et al. 1995b). Collectively, these related proteins are products of the C-type lectin supergene family (Katsumata et al. 1995). They are especially abundant in the inflamed pancreas, in parts of the gastrointestinal tract, and in GHRH-stimulated somatotropes, yet their physiological role remains unknown (Chakraborty et al. 1995a, Frigero et al. 1993, Bowers 1995).

In the present study, we have characterized the temporal and spatial patterns of expression of PAP-III mRNA in the ovaries of gonadotrophin-primed immature rats during the ovulatory process. Expression of mRNA for this protein was detected by RT-PCR differential display of RNA extracts from rat ovaries at specific intervals following the injection of human chorionic gonadotrophin (hCG) to induce ovulation in the experimental animals. The temporal pattern of PAP-III mRNA expression during the peri-ovulatory period was characterized by Northern blot analysis. The spatial distribution of ovarian PAP-III mRNA was determined by in situ hybridisation. In addition, the effects of inhibition of ovarian prostaglandin (PG) and progesterone (P₄) synthesis on PAP-III mRNA production were assessed. The results reveal a significant increase in expression of the PAP-III gene in certain areas of the ovarian stroma, in secondary follicles, and rarely in limited regions of mature ovarian follicles in response to an ovulatory dose of hCG, a homolog of LH.
MATERIALS AND METHODS

Animals and Treatments In Vivo

Immature Wistar rats were induced to superovulate by equine chorionic gonadotrophin (eCG) and hCG treatment as described previously (Espey et al. 2000a). In this experimental model, the ovaries begin to ovulate at 12 h after hCG, and ovulation is essentially complete by 16 h after hCG (Tanaka et al. 1989). Ovarian RNA was extracted at the peri-ovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hCG. At each interval, the ovaries from 10 animals were pooled for extraction of the total RNA. These nucleic acid extracts were used for differential display and for Northern blotting. In parallel groups of animals, indomethacin and epostane were injected s.c., also as described previously (Espey et al. 2000a). These anti-ovulatory agents were administered at 3 h after hCG in doses of 1.0 mg and 5.0 mg, respectively, because these are the intervals after hCG that these agents are known to effectively block ovarian PG and P₄ (Espey et al. 1990, Tanaka et al. 1992). The ovarian RNA was extracted from the treated animals at 8 h after hCG, and Northern analyses were performed to determine whether blockage of ovarian PG and/or P4 synthesis affected the transcription of ovarian Pap-III. The ovulation rate in the various experimental animals was determined by a procedure that also has been described previously (Espey et al. 2000a). For the determination of ovulation rate and the extraction of ovarian RNA, rats were killed by exposure to CO₂. The animals were acquired, retained, and used in compliance with the NIH Guide and with the approval of the institutional animal care review committee.

Differential Display Protocol to Detect PAP-III mRNA Transcription

The steps of the differential display were carried out as described previously (Espey et al. 2000a). In brief, RNA was extracted by a standard guanidine isothiocyanate/cesium chloride procedure from ovaries taken at the six different peri-ovulatory intervals identified above. RT-PCR was performed using primers from an RNAimage Kit (G508, GenHunter Corporation, Nashville, TN). The primer set that yielded a differentially expressed fragment of cDNA for PAP-III was comprised of the poly-T primer 5'-HTTTTTTTTTG-3' and the random primer 5'-HGCAAGTT-3' (i.e., primer set G62), with "H" representing the attachment of a HindIII restriction site to the primers.

Northern Analysis Protocol to Confirm Differential Expression of PAP-III mRNA

After extraction and re-amplification of the differentially expressed cDNA, a standard Northern analysis was performed to confirm that the expression of the parent mRNA for PAP-III increased significantly during the ovulatory process. This unique cDNA fragment was subcloned using a pCR-TRAP Cloning System (P404, GenHunter), and cloning colonies containing the PAP-III cDNA were identified by secondary Northern analyses. The Northern blots included the same six peri-ovulatory intervals indicated above. The different blots were all prepared from the same pool of RNA that was extracted from ten ovaries at each of the designated intervals following hCG administration to the animals.

Sequencing Protocol for the PAP-III cDNA Fragment

Samples of the cloning colonies that contained PAP-III cDNA were cloned further in LB medium containing 10 mg tetracycline/1.0 ml medium. The harvested plasmid DNA was purified using a Wizard Minipreps DNA Purification System (A750, Promega). Manual sequencing of the cDNA was performed using a Sequenase Version 2.0 DNA Sequencing Kit (US70140, Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The nucleic acid sequence was analyzed by a BLASTn search of the database server at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov).

In Situ Hybridization Protocol for Assessment of Spatial Distribution of PAP-III mRNA

In situ hybridisation was performed as described previously (Espey et al. 2000a). Ovaries were removed from three individual rats at each of the six peri-ovulatory intervals described above and were fixed overnight in 4% paraformaldehyde. The ovarian sections were routinely hybridized with sense probe, as an internal control to detect any background signal.

Statistical Analysis

The intensity of the signals from the Northern blots was analysed by the NIH-image densitometry program, as described previously (Espey et al. 2000a). Numerical data are presented as means ± SEM, based on data from three Northern blots that were prepared from the RNA extracts of pools of ovaries from 10 animals at each time point. The significance of the differences among the six principal time points of 0, 2, 4, 8, 12, and 24 h after hCG was determined by the Duncan's multiple range test after a completely randomised one-way analysis of variance of the means of the groups. The probability value used as the cutoff between "significant" and "not significant" was P = 0.05.
RESULTS

Differential Display of PAP-III cDNA During the Ovulatory Process

Following RT-PCR, the sub-populations of radioactively labeled cDNAs that were generated from RNA extracts at each of the six stages of the peri-ovulatory period were separated from one another by electrophoresis on polyacrylamide gels. The autoradiograph of these PAGE results revealed a faint, but differentially expressed, cDNA band that was more intense at 4, 8, and 12 h after hCG, and was minimal at 0 and 24 h into the ovulatory process (Figure 1). Sizing on an agarose minigel revealed that this differentially displayed cDNA was approximately 260 bp in length (including both primers). The most intense cDNA band (i.e., the band in the 8-h lane) was excised from the acrylamide gel and re-amplified for use as a probe in Northern analysis.

Northern Analysis of PAP-III mRNA Expression During Ovulation

The Northerns revealed a temporal pattern of expression of mRNA (eventually identified as PAP-III mRNA) during ovulation that was comparable to the expression of cDNA on the differential display autoradiograph (Figure 2). In order to compare the intensity of the signals from the Northern blots with other data on gene expression during ovulation, the intensity of the signal from the 8-h lane was arbitrarily set at 100%, and the densities at the other times during the peri-ovulatory period were expressed as fractions of that maximum. Accordingly, the NIH-image program was used to digitised all of the bands on three Northerns, and the ratio of the density of each experimental band to its corresponding b-actin control band was calculated for each lane. Means (± SEM) of the signal densities at 0, 2, 4, 8, 12, and 24 h after hCG were 2.9% ± 2.9%, 9.1% ± 2.8%, 33.7% ± 4.7%, 100%, 51.2% ± 7.3%, and 19.3% ± 4.8%, respectively. Thus, PAP-III gene expression increased approximately 34-fold within 8 h after initiation of the ovulatory process by injecting hCG into the animals. Subsequently, at 24 h after hCG (i.e., during early luteal development), PAP-III gene expression declined to a level that was close to the level of the 0-h control value.

Sequence of the cDNA Fragments for PAP-III

After the hCG-induced expression of the PAP-III gene was confirmed by Northern analysis, the cDNA fragment of this gene was subcloned and sequenced. The NCBI accession number for the fragment is #AF159102. This cDNA is essentially identical to a segment of a PAP-III gene reported for the Wistar rat (NCBI accession #L20869, (Frigerio et al. 1993) and the Sprague-Dawley rat (NCBI accession #U09193, (Dusetti et al. 1995). It is also homologous to the regenerating-III (reg-III) gene in the mouse (NCBI accession #D63362, (Narushima et al. 1997).

Effects of Indomethacin and Epostane on PAP-III Gene Expression

For this experiment, Northern blots were prepared from RNA that was extracted from control ovaries at 0 and 8 h into the ovulatory process, or that was extracted from experimental ovaries taken at 8 h after hCG from rats that had been treated 5 h earlier with an ovulation-inhibiting dose of either epostane, or indomethacin. These experimental intervals were selected in order to compare the present data with related studies using epostane and indomethacin (Espey et al. 2000a, Espey et al. 2000b). The signal density (normalised against the b-actin control) of the 8-h control lane was arbitrarily set at 100% (Figure 3). There was minimal expression of PAP-III mRNA at 0 h, but substantial expression at 8 h. In animals treated with the anti-ovulatory agent indomethacin, which blocks PG synthesis (Espey et al. 1991), the signal density was 74.2% ± 14.1%, but this mean was not statistically different from the 8-h control value. In animals treated with the anti-ovulatory agent epostane, which blocks P₄ synthesis (Espey et al. 1990), the signal density of 48.2% ± 7.3% was moderately, but significantly (P £ 0.05), lower than the 8-h control value. In contrast, the ovaries from a parallel group of gonadotrophin-treated animals that were administered (by a procedure described previously (Espey et al. 2000b)) 10 mg of exogenous progesterone prior to the epostane injection exhibited a recovery of the ovarian PAP-III mRNA level to 101.5% ± 10.0% of the 8-h control value (Figure 3). The ovulation rates in parallel groups of animals that were treated with indomethacin and epostane, alone, were significantly inhibited, but there was recovery of the ovulation rate in the epostane-treated group that received P₄ (Figure 3).

Localisation of Ovarian PAP-III mRNA Expression by In Situ Hybridisation

In situ hybridisation confirmed the temporal pattern of PAP-III mRNA expression that was observed in the differential display autoradiographs and in the Northern blots. There was minimal signal from the 0-h control ovaries, an increasing signal up to 8 h after hCG treatment, and a declining signal by 24 h after injecting the animals with hCG (Figure 4). Hybridisation was localised primarily in stromal tissue in the vicinity of the ovarian hilus and to a much lesser extent in thecal tissue between some of the larger follicles. There was not a distinct pattern of distribution of PAP-III mRNA in the stromal tissue. Closer examination of the in situ data showed that most of the signal was emitted from presumptive endothelial cells of ovarian blood vessels and from a limited number of secondary follicles that were visible in the ovarian sections (Figure 5). Small, concentrated areas of PAP-III mRNA signal could also be detected sporadically along the antral border of the granulosa layer of follicles that had grown to a mature size (Figure 6).
DISCUSSION

PAP is a term that is commonly (but not consistently) used to connote members of a closely related group of lectin-type proteins/genes that have substantial homology with PAP-I, which was initially detected in the inflamed pancreas of rats 6 h after the experimental induction of pancreatitis (Keim et al. 1984). The segment of PAP mRNA that has been identified in the present differential display study is identical to PAP-III, which was first discovered 8 years ago during screening of a rat jejunal cDNA library (Figerio et al. 1993). The complete mRNA sequence of PAP-III is 773 nucleotides, with a single open-reading frame of 174 codons (Figerio et al. 1993). The sequence is 66% homologous to PAP-I and 63% homologous to PAP-2. It is also homologous to the so-called ‘regeneration protein’ (reg) found in regenerating islets of Langerhans of the rat pancreas (Narushima et al. 1997, Terazono et al. 1988). Other, related members of the lectin superfamily include pancreatic stone protein (PSP) (Chakraborty et al. 1995a), pancreatic thread protein (PTP) (Katsumata et al. 1995), and hepatocarcinoma-intestine-pancreas (HIP) protein (Bartoli et al. 1998).

The principal cellular origins of PAP proteins have not been completely defined. In the inflamed pancreas, they reportedly are produced in cells of the islets (Katsumata et al. 1995, Terazono et al. 1988), or in acinar cells close to the islets (Chakraborty et al. 1995a). In the anterior pituitary of GHRH-treated rats, or in treated cell cultures, they are in somatotropes (Yokoyo & Friesen 1986, Yamamoto et al. 1992, Chakraborty et al. 1995a, Bowers 1995, Chakraborty et al. 1995c). In the small intestines, PAP proteins/mRNAs are constitutively expressed in the columnar epithelial cells of the ileum, jejunum and duodenum (Frigerio et al. 1993, Masciotra et al. 1995). Also, they have been identified in goblet cells and Paneth cells of the jejunum (Masciotra et al. 1995). (Paneth cells are immune cells in the intestinal mucosa that contain bright eosinophilic granules with a variety of antimicrobial proteins (Ouellette & Selsted 1996).) In the uterus of oestrogen-stimulated pregnant rats, peptide23/PAP mRNA was localised in the luminal epithelial cells of the endometrium (Chakraborty et al. 1995b). In contrast, in the present study, PAP-III mRNA was conspicuous in luminal endothelial cells in parts of the ovarian vasculature. In addition, the in situ signal for this message was localised in the epithelial (granulosa) cells of secondary follicles and in a few presumptive anomalies of folliculogenesis that were rarely observed along the antral surface of the granulosa layer of large tertiary follicles. These limited structures in the granulosa layer might be related to the Call-Exner bodies, or to ‘microfollicles,’ which represent aberrant entities that occasionally become entrapped in growing follicles without having any known deleterious effect (Gosden et al. 1989, Meng et al. 1994, Rodgers et al. 2000).

Although it is not clear whether the various PAP proteins have a common function, at least a few distinct characteristics of these members of the lectin superfamily have been established. First, it is evident that the experimental induction of acute inflammation in the pancreas results in more than a 300-fold increase in PAP mRNA within 12 h (Iovanna et al. 1991). Under such acute conditions PAP comprises about 5% of the proteins secreted from the pancreas. However, this protein reportedly has a half-life of only about 4.8 min (Chakraborty et al. 1995c), and its secretion returns to undetectable levels when the pancreas has totally recovered (Iovanna et al. 1991). It should be noted that, in contrast to many of the other proteins secreted by the pancreas, PAP does not have any proteolytic activity (Heller et al. 1999). Second, GHRH induces the expression of PAP (i.e., peptide 23) by hypophyseal somatotropes, whereas somatostatin inhibits such secretion (Yokoyo & Friesen 1986, Bowers 1995, Bartoli et al. 1998). However, even though the secretion of pituitary PAP is affected by the same factors that regulate growth hormone secretion, this protein is not homologous to GH (Katsumata et al. 1995). These observations have led to the suggestion that PAP may be involved in cell proliferation, since GHRH is known to stimulate somatotrope hyperplasia in vitro (Katsumata et al. 1995, Bowers 1995). Third, in quite a different pattern from the above examples, PAP is expressed continuously by the small intestines (but not by the colon) (Chakraborty et al. 1995a, Frigerio et al. 1993, Maschiotra et al. 1995). However, as in other tissues, the function of PAP in the gastrointestinal tract has not been established. Fourth, the role of PAP in luminal epithelial cells of the uterus has not been established. This substance is not produced by the uteri of immature rats, and its secretion in adult rats is limited mainly to the later stages of pregnancy, with maximum expression on day 12 of gestation (Chakraborty et al. 1995b).

In the report of Chakraborty et al. (1995b) on PAP expression in the uterus, the investigators concluded that b-oestradiol rather than P₄ is the major regulator of expression of this gene. Their conclusion was based in part on evidence that, during the sexual cycle of the rat, uterine PAP was maximal on the day of oestrus and minimal on the day of dioestrus. However, the study did not include an actual analysis of circulating steroids. Instead, the investigators relied on an earlier research paper that analysed steroid levels during the oestrus cycle of the rat (Smith et al. 1975). In support of their hypothesis that b-oestradiol rather than P₄ promotes PAP secretion, they cited that plasma b-oestradiol is highest and P₄ is lowest on the day of oestrus (Chakraborty et al. 1995b). However, close examination of the cited reference indicates that b-oestradiol is merely at baseline level on the day of oestrus, while P₄ is declining, yet still significantly elevated, on the morning of oestrus (Smith et al. 1975). Clarification of this incongruity is necessary to interpret the present data on ovarian PAP-III expression, because ovarian PAP mRNA increased in response to an ovulatory dose of gonadotrophin in a pattern that was parallel to the well known ovulatory increase in ovarian P₄ and concomitant with the well known pause in ovarian b-oestradiol synthesis (Espey et al. 1991, Espey et al. 1990). Therefore, the present evidence that ovarian PAP-III mRNA is at least partially influenced by ovarian P₄ production is an observation that might not be contradictory to the reported effect of steroids on PAP expression in the uterus.

The background information in the preceding paragraph is only marginally helpful in deciphering the significance of PAP-III mRNA expression in the gonadotrophin-stimulated ovary. It is evident from the current Northern data that ovarian PAP expression is transient—increasing only at the time when the mature follicles are undergoing an acute inflammatory reaction that disintegrates the follicles and causes them to rupture (Espey et al. 2000b, Espey & Lipner 1994). Also, it is evident from the present in situ hybridisation data that most of the PAP was outside the larger ovarian follicles and spatially distributed in a pattern that suggested this agent may not be directly involved in the events that lead to degradation and rupture of the follicle wall. Instead, it would appear that the increase in ovarian PAP might be a response to, rather than a cause of, the physiological changes that occur in ovulatory follicles. In this regard, it would appear relevant that PAP has been proposed as a protective agent in leukocyte-mediated lung injury (Heller et al. 1999), and it has been considered as a potential component of a defense mechanism against infection (Frigerio et al. 1993). Therefore, it is possible that ovarian PAP-III mRNA is expressed in conjunction with a protective response to the hyperemia, exudation, proteolysis, and inflammation that characterise the physiological process of ovulation (Espey & Lipner, 1994). Such a hypothesis would be congruent with what is known about PAP expression in the inflamed pancreas. On the other hand, such an explanation would be difficult to correlate with PAP expression in the anterior pituitary, unless it could be shown that this hypophyseal gland responds to stimulation by GHRH in a manner that is at least mildly comparable to the inflammatory reaction in gonadotrophin-stimulated ovaries. In another direction, the present results do not rule out the possibility that ovarian PAP expression is associated in some way with the reported infiltration of neutrophils and macrophages into the ovary at the time of ovulation (Brannstrom et al. 1994). In any event, the existing information collectively suggests that the increase in ovarian PAP-III mRNA could be a protective response to the degradative events of the ovulatory process. Therefore, the tentative hypothesis arising from this study is that ovarian PAP expression might function in some way to reduce trauma to the ovarian vasculature and to the developing secondary follicles during the acute inflammatory reaction that is characteristic of ovulation.

ACKNOWLEDGEMENTS

We appreciate the reliable assistance of Claire Lo in performing the in situ hybridisation work. This study was supported by NSF Grant #9870793 (LLE) and NIH Grant HD16229 (JSR).
REFERENCES

Bartoli C, Baeza N, Figarella C, Pellegrini I & Figarella-Branger D 1998 Expression of peptide-23/pancreatitis-associated protein and reg genes in human pituitary and adenomas: Comparison with other fetal and adult human tissues. Journal of Clinical Endocrinology and Metabolism 83 4041-4046

Bowers CY 1995 Editorial: A new pituitary hormone. Endocrinology 136 1329-1331.

Brannstrom M, Pascoe V, Norman RJ & McClure N 1994 Localization of leukocyte subsets in the follicle wall and in the corpus luteum throughout the human menstrual cycle. Fertility and Sterility 61 488-495.

Chakraborty C, Katsumata N, Myal Y, Schroedter IC, Brazeau P, Murphy LJ, Shiu RP & Friesen HG 1995a Age-related changes in peptide-23/pancreatitis-associated protein and pancreatic stone protein/reg gene expression in the rat and regulation by growth hormone-releasing hormone. Endocrinology 136 1843-1849.

Chakraborty C, Vrontakis M, Molnar P, Schroedter IC, Katsumata N, Murphy LJ, Shiu RP & Friesen HG 1995b Expression of pituitary peptide 23 in the rat uterus: regulation by estradiol. Molecular and Cellular Endocrinology 108 149-154.

Chakraborty C, Sharma S, Katsumata N, Murphy LJ, Schroedter IC, Robertson MC, Shiu RP & Friesen HG 1995c Plasma clearance, tissue uptake and expression of pituitary peptide 23/pancreatitis-associated protein in the rat. Journal of Endocrinology 145 461-469.

Dusetti NJ, Frigerio J-M, Szpirer C, Dagorn J-C & Iovanna JL 1995 Cloning, expression and chromosomal localization of the rat pancreatitis-associated protein III gene. Biochemical

Journal 307 9-16.

Espey LL, Adams RF, Tanaka N & Okamura H 1990 Effects of epostane on ovarian levels of progesterone, 17b-estradiol, prostaglandin E₂, and prostaglandin F₂_a during ovulation in the gonadotropin-primed immature rat. Endocrinology 127 259-263.

Espey LL, Tanaka N, Adams RF & Okamura H 1991 Ovarian hydroxyeicosatetraenoic acids compared with prostanoids and steroids during ovulation in rats. American Journal of Physiology 260 E163-E169.

Espey LL & Lipner H 1994 Ovulation. In The Physiology of Reproduction, pp 725-780. New York: Raven Press.

Espey LL, Yoshioka S, Russell D, Ujioka T, Vladu B, Skelsey M, Fujii S, Okamura H & Richards JS 2000a Characterization of ovarian carbonyl reductase gene expression during ovulation in the gonadotropin-primed immature rat. Biology of Reproduction 62 390-397.

Espey LL, Yoshioka S, Russell DL, Robker RL, Fujii S & Richards JS 2000b Ovarian expression of a disintegrin and metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat. Biology of Reproduction 62 1090-1095.

Frigerio J-M, Dusetti NJ, Garrido P, Dagorn J-C & Iovanna JL 1993 The pancreatitis associated protein III (PAP III), a new member of the PAP gene family. Biochimica et Biophysica Acta 1216 329-331.

Gosden RG, Brown N & Grant K 1989 Ultrastructural and histochemical investigations of Call-Exner bodies in rabbit Graafian follicles. Journal of Reproduction and Fertility 85 519-526.

Heller A, Fiedler F, Schmeck J, Luck V, Iovanna J & Koch T 1999 Pancreatitis-associated protein protects the lung from leukocyte-induced injury. Anesthesiology 91 1408-1414.

Iovanna J, Orelle B, Keim V & Dagorn JC 1991 Messenger RNA sequence and expression of rat pancreatitis-associated protein, a lectin-related protein overexpressed during acute experimental pancreatitis. Journal of Biological Chemistry 266 24664-24669.

Iovanna JL, Keim V, Bosshard A, Orelle B, Frigerio JM, Dusetti N & Dagorn JC 1993 PAP, a pancreatic secretory protein induced during acute pancreatitis, is expressed in rat intestine. American Journal of Physiology 265 G611-G618.

Katsumata N, Chakraborty C, Myal Y, Schroedter IC, Murphy LJ, Shiu RPC & Friesen HG 1995 Molecular cloning and expression of peptide 23, a growth hormone-releasing hormone-inducible pituitary protein. Endocrinology 136 1332-1339.

Keim V, Rohr G, Stöckert HG & Haberich FJ 1984 An additional secretory protein in the rat pancreas. Digestion 29 242-249.

Keim V, Iovanna JL & Dagorn JC 1994 The acute phase reaction of the exocrine pancreas. Gene expression and synthesis of pancreatitis-associated proteins. Digestion 55 65-72.

Masciotra L, Lechene de la Porte P, Frigerio JM, Dusetti NJ, Dagorn JC & Iovanna JL 1995 Immunocytochemical localization of pancreatitis-associated protein in human small intestine. Digestive Diseases and Sciences 40 519-524.

Meng N, Nakashima N, Nagasaka T, Fukatsu T, Nara Y, Yoshida K, Kawaguchi T & Takeuchi J 1994 Immunohistochemical characterization of extracellular matrix components of granulosa cell tumor of ovary. Pathology International 44 205-212.

Narushima Y, Unno M, Nakagawara K, Mori M, Miyashita H, Suzuki Y, Noguchi N, Takasawa S, Kumagai T, Yonekura H & Okamoto H 1997 Structure, chromosomal localization and expression of mouse genes encoding type III Reg, RegIIIa, RegIIIb, RegIIIg. Gene 185 159-168.

Ouellette AJ & Selsted ME 1996 Paneth cell defensins: endogenous peptide components of intestinal host defense. FASEB Journal 10 1280-1289.

Rodgers RJ, Irving-Rodgers HF & van Wezel IL 2000 Extracellular matrix in ovarian follicles. Molecular and Cellular Endocrinology 163 73-79.

Smith MS, Freeman ME & Neill JD 1975 The control of progesterone secretion during the estrus cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 96 219-226.

Tanaka N, Espey LL & Okamura H 1989 Increase in ovarian blood volume during ovulation in the gonadotropin-primed immature rat. Biology of Reproduction 40 762-768.

Tanaka N, Espey LL, Stacy S & Okamura H 1992 Epostane and indomethacin actions on ovarian kallikrein and plasminogen activator activities during ovulation in the gonadotropin-primed immature rat. Biology of Reproduction 46 665-670.

Terazono K, Yamamoto H, Takasawa S, Shiga K, Yonemura Y, Tochino Y & Okamoto H 1988 A novel gene activated in regenerating islets. Journal of Biological Chemistry 263 2111-2114.

Yamamoto T, Katsumata N, Tachibana K, Friesen HG & Nagy JI 1992 Distribution of a novel peptide in the anterior pituitary, gastric pyloric gland, and pancreatic islets of rat. Journal of Histochemistry and Cytochemistry 40 221-229.

Yokoyo S & Friesen HG 1986 Human growth hormone (GH)-releasing factor stimulates and somatostatin inhibits the release of rat GH variants. Endocrinology 119 2097-2105.
FIGURE LEGENDS

FIG. 1. Autoradiograph of differentially displayed PAP-III cDNA (arrows). Note that the cDNA bands representing PAP-III are mainly visible in the 4-, 8-, and 12-h lanes.

FIG. 2. Intensity of Northern blot signals at the six intervals of the peri-ovulatory period following hCG administration to the rats. The signal density at 8 h was arbitrarily set at 100%, and the other points on the graph represent the mean values from three Northerns prepared from pooled extracts of RNA. Bands taken from a representative Northern blot with PAP-III cDNA, along with the b-actin control, is shown below the linear graph. Note that the greatest intensity is at 8 h after hCG.

FIG. 3. Comparison of the % of signal from Northern blots containing RNA extracted at 8 h after hCG from animals that were also treated with either 1 mg of indomethacin (Indo) or 5 mg of epostane (Epo) administered at 3 h after hCG. Bar graphs are based on NIH-image analyses of three Northerns that were probed with PAP-III cDNA. An additional group of rats received epostane plus progesterone (E + P) as described in the Materials and Methods. The signal from the 8-h control lane (Ctrl) was arbitrarily set at 100% OD. In parallel groups of rats, the ovulation rate was determined at 24 h after hCG.

FIG. 4. Change in intensity of the in situ hybridisation signal during the six peri-ovulatory intervals following hCG administration. Light-field micrographs on the left show the histology of ovarian sections stained with hematoxylin and eosin (H & E), while the dark-field micrographs of the same sections show the localisation of PAP-III mRNA as detected by hybridisation of a ³⁵S-labeled anti-sense probe derived from the PAP-III cDNA.

FIG. 5. Closer view of the distribution of the PAP-III probe in the ovary. Arrows pointing to the left identify secondary follicles that emitted relatively strong signal. Arrows pointing to the right single out a large blood vessel that was outlined by the PAP-III signal.

FIG. 6. Another high magnification view of the distribution of PAP-III mRNA in the ovary. Most of the signal in the field is emitted from presumptive small blood vessels in the stroma of this ovary. The arrow pointing downward in the lower right corner of the field marks a longitudinal segment of a larger blood vessel. Arrows pointing to the left identify isolated spots along the antral surface of the granulosa that emitted relatively strong signal. Notice that the granulosa in the large follicle in the upper left corner of the field is void of any comparable signal.