In Situ Localization of Messenger Ribonucleic Acid for an Oviduct ...

BIOLOGY OF REPRODUCTION 55, 1107-1118 (1996)

In Situ Localization of Messenger Ribonucleic Acid for an Oviduct-Specific Glycoprotein during Various Hormonal Conditions in the Golden Hamster Hiromi Komiya, Tomoko Onuma, Masahiko Hiroi, and Yoshihiko Araki2 Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata-City 990-23, Japan ABSTRACT The oviductal epithelium secretes specific glycoproteins that associate with the egg after ovulation. Several published reports including our preliminary studies have suggested that ovarian steroids regulate the secretion of oviduct-specific glycoproteins in several mammalian species. The objective of this study, using golden hamsters, was to analyze the hormonal effects on gene expression of these molecules more precisely during various hormonal conditions (estrous cycle, ontogeny, pregnancy, GnRH analogue treatment, and ovariectomy) by in situ hybridization. The message for the hamster oviduct-specific glycoprotein (HOGP) was detected by a digoxigenin-labeled single-strand specific DNA probe inparaffin sections. Data from these studies show the following. 1) In the oviduct, the signal was detected in both the perinuclear region and the basal region in the ampulla but was predominantly detected in the basal region inthe isthmus. 2) The signal intensity was high in the ampulla compared with the isthmus. 3) During a normal estrous cycle, the message level was significantly altered between the estrous and diestrous stages in the ampulla but not in the isthmus. In addition, the signal did not disappear at any stage in either the isthmus or ampulla. 4) The HOGP message was first observed from around 14 days of age and then decreased in parallel with serum estradiol levels during aging. 5) The signal was also observed in the oviductal epithelium of pregnant animals at term and of postpartum animals. 6) When we treated the animals with TAP144-SR (GnRH analogue) or performed an ovariectomy, which caused diminution of serum estradiol and progesterone levels, the message of HOGP was significantly decreased. Moreover, the message expression was greatly induced after estradiol administration to GnRH analogue-treated animals, whereas a high level of serum progesterone slightly inhibited HOGP message expression. These results suggest that elevation of the serum estradiol/progesterone level affects the HOGP gene expression in the ampulla. However, a high serum estradiol level did not induce the gene expression rapidly, suggesting that an adequate serum hormonal level over a given period of time may be important for the HOGP gene expression. INTRODUCTION In general, the fertilization process involves a series of complex interactions between complementary molecules present on the surface of the gametes. In mammals, the zona pellucida (ZP), the extracellular glycocalyx surrounding the oocyte, is believed to mediate the relative species specificity of sperm binding, blocking of polyspermy, and protection of the growing embryo from fertilization to imAccepted July 1, 1996. Received May 2, 1996. 'This work was supported by Grants-in-Aid for General Scientific Research, No. 07671764 and 05404055, and a Grant-in-Aid for Scientific Research, International Scientific Research Program, No. 07044220 from the Ministry of Education, Science and Culture, Japan. 2Correspondence: Yoshihiko Araki, Department of Obstetrics & Gynecology, Yamagata University School of Medicine, 2-2-2 lida-Nishi, Yamagata-City 990-23, Japan. FAX: 81236285396; e-mail: [email protected]. yamagata-u.ac.jp

plantation [1]. Several sperm proteins have been reported to serve as ZP receptors during the fertilization process (for review, see Wassarman [2], Miller and Shur [3], Ramarao et al. [4], and Aitken [5]). However, in most species, the interaction between the receptor(s) on the sperm plasma membrane and the complementary ligand(s) on the ZP has not been fully understood. Since the organ in which the ZP shows its multiple physiological functions is the oviduct, it is generally believed that oviductal luminal fluid provides a beneficial microenvironment for fertilization and/or early embryonal development. It has been reported that epithelial cells of the oviduct secrete specific glycoproteins into the oviductal luminal fluid (mouse [6, 7], hamster [8-10], rabbit [11, 12], pig [1315], sheep [16, 17], cow [18-20], baboon [21-23], and human [24, 25]). In some species, these molecules have been characterized as ZP-and/or perivitelline space-associated glycoproteins [6-10, 13, 16, 26-28]. It has also been reported that oviduct-specific glycoproteins are associated with the sperm surface, and it has been suggested that they may be involved in some sperm functions, including sperm capacitation, the acrosome reaction, or binding to the ZP [29-32]. However, the biochemical and molecular characterization of the molecules and their physiological significance has not been elucidated. Previous studies from this laboratory have reported the molecular characterization of mouse [33] and hamster [34] oviduct-specific glycoproteins. When their N-terminal amino acid sequence was compared with sequences of oviductspecific glycoproteins from other mammalian species (baboon [35], bovine [36], human [37], sheep [38], and pig [39]), it was found that these molecules are highly conserved and share identity with a protein of chitinase family [40]. These results strongly suggest that a significant degree of homology exists among oviduct-specific glycoproteins of various mammalian species. It is generally accepted that the mammalian oviduct has a secretory role during the estradiol (E2)-dominated phase of the reproductive cycle [41]. The secretion of oviductspecific glycoproteins has also been reported as E2-dependent in some species (human [24, 42], baboon [22, 23, 35], pig [43], cow [20], and sheep [44, 45]). Despite these results on the secretion of oviductal proteins, little or no information is available on the gene expression of the oviduct-specific glycoprotein during various hormonal conditions in rodents. Since rodents have an incomplete estrous cycle (they lack a spontaneous luteal phase, and the corpora lutea of the estrous cycle are short-lived and nonfunctional) [46], the secretion of the molecule may be somewhat different from that in the cattle or primates. The purpose of the studies presented in this paper was to investigate the hormonal effects on gene expression of the oviduct-specific glycoprotein in the Syrian golden hamster (Mesocricetus auratus). This animal has proven to be an excellent model for this type of study since the estrous cycle in this species is short (4 days), relatively stable, and well characterized.

1107

KOMIYA ET AL.

1108 1)Control

Day ·-I 42

1

63

1 sam oil(=1)fday TAP-144SR

TAP-t44-SR

I

TAP-144SR

2) E2 treatment o 1+

TAP-14R

21

42

f+~

I

TAP-144SR

63

IE2:

.Op

y for 14dayI

TAP-144SR

3) E2+P4 treatment o

TAP.144-R

21

T

TAP.1444R

63

42

t

E2: 1.0glday for 14days P4:O.hg/day fr 14days

TAP.144SR

FIG. 1. Experimental design for hormone administration to GnRH analogue (TAP-144-SR)-treated animals. Sesame oil (200 ild) alone or containing either E2 or E2 +P 4 was injected into animals over 14 days as described in Materials and Methods. Arrows indicate the time of TAP144-SR administration to the animal.

into four experimental groups by various hormonal conditions. The estrous cycles were determined by the following criteria: Day 1 (estrus), the day on which postestrus discharge was found in the vagina; Day 2 (diestrus Day 1); Day 3 (diestrus Day 2); and Day 4 (proestrus). Some animals were killed by excess ether inhalation at 1100 h on each day of the estrous cycle. For the ontogeny experiments, immature hamsters (8 days-4 wk old) and aged hamsters (18 mo old) were used. For the study in pregnant animals, pregnant hamsters at term (around Day 15 after mating) were used. For the ovariectomy study, young female hamsters (4 wk old) were either ovariectomized or sham-ovariectomized by the dorsal route for 60 days. Animals were treated with the GnRH analogue leuprolide acetate (TAP-144-SR) using 4 mg/kg. TAP-144-SR was injected s.c. three times at 3-wk intervals into female hamsters (4 wk old at the first injection). The GnRH analogue-treated animals received injections of E2 (1.0 Lg/day) only, E2 (1.0 pug/day) and P4 (0.5 mg/day), or sesame oil (containing benzyl benzoate) only for 14 days. Both E2 and P4 were dissolved in benzyl benzoate and administered s.c. in a volume of 200 l1I sesame oil. The experimental design for hormonal administration is summarized in Figure 1. Preparation of Serum for Hormonal Assays

In this paper, we report the tissue localization and quantitative analysis, by in situ hybridization, of the hamster oviduct-specific glycoprotein (HOGP) mRNA in various hormonal conditions including hormone replacement. MATERIALS AND METHODS Chemicals Restriction endonucleases, digoxigenin (DIG)- 11-dUTP, alkaline phosphatase-conjugated sheep anti-DIG Fab fragments (anti-DIG-AP), nitroblue tetrazolium (NBT), and a DIG DNA labeling kit were obtained from Boehringer Mannheim (Indianapolis, IN). Disodium 3-(4-methoxyspiro-4-yl) phenyl phosphate (CSPD) was from Tropix (Bedford, MA). Taq DNA polymerase was purchased from Stratagene (La Jolla, CA). Paraformaldehyde was from Kanto Chemical Co., Inc. (Tokyo, Japan). Low-gelling-temperature agarose (Sea Plaque agarose) was purchased from FMC Bioproducts (Rockland, ME). Positive-charged nylon membranes (Hybond N +) were from Amersham (Buckinghamshire, UK). Formamide and diethylpyrocarbonate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Leuprolide acetate (TAP-144-SR) was obtained from Takeda Chemical Industries, Ltd. (Osaka, Japan). Estradiol-1713 (E2 ), progesterone (P4), benzyl benzoate, and sesame oil were obtained from Nacalai tesque, Inc. (Kyoto, Japan). Ultrapure chemicals were from Sigma Chemical Co. (St. Louis, MO) and Bio-Rad Laboratories (Hercules, CA). All other chemicals were obtained commercially and were of the highest purity available. Animals and Experimental Design Female and male golden hamsters were purchased from Japan SLC Inc. (Hamamatsu, Japan) and given free access to food and water. The female hamsters were 5-6 wk old on the day of arrival. They were maintained and bred at the Animal Center in our University under 12L:12D conditions. For the study of HOGP expression during the normal estrous cycle, female animals (8-10 wk old) were divided

The serum E2 and P4 levels were confirmed by cardiocentesis. Blood collected by cardiac puncture was allowed to clot at room temperature for 30 min and then centrifuged at 1000 x g for 30 min. The serum was stored at -70 0 C until used. Concentration of E 2 was measured by enzyme immunoassay (DELFIA Estradiol Kit; Pharmacia K.K., Tokyo, Japan). Serum P4 level was measured by a direct RIA using reagents obtained from Diagnostic Products Corp. (Los Angeles, CA). Specific Probe Preparation Preparation of the DIG-labeled antisense DNA probe prepared by the asymmetrical polymerase chain reaction (PCR) method [47] was described previously [33]. Briefly, a pBluescript SK(-) vector cDNA containing the deletion mutant clone of a HOGP cDNA (HRD-8) [34] was digested with Pvu II. The Pvu II fragments were separated by 0.75% low-gelling-temperature agarose gel electrophoresis, and the band containing HRD-8 cDNA was excised from the gel. Using this deleted-cDNA fragment as the template, the DIG-labeled antisense probe (HOGP probe) was produced by the asymmetrical PCR method as follows: the template cDNA (20-30 ng) was amplified with 50 pI1 of PCR reaction mixture containing 10 mM Tris-HCI (pH 8.3), 50 mM KC1, 1.5 mM MgC12, 0.01% gelatin, 100 mM dATP, 100 mM dCTP, 100 mM dGTP, 65 mM dTTP, 35 mM DIGdUTP, and 1.25 IU of Taq polymerase. The 5'-specific oligonucleotide primer (sense side) corresponded to 579-602 of pBluescript SK(-), and the 3'-specific oligonucleotide primer (antisense side) corresponded to 831-817 of pBluescript SK(-), respectively. The concentrations of primers used in this PCR were set to 2 pmol (5'-specific primer) and 100 pmol (3'-specific primer). The sample was subjected to 45 cycles of PCR, denaturing at 94°C for 1 min, annealing at 55°C for 2 min, and extending at 72°C for 1 min. To prepare the DIG-labeled sense probe (sense HOGP probe) for the control experiments, the concentrations of primers were changed to 100 pmol (5'-specific primer) and 2 pmol (3'-specific primer).

LOCALIZATION OF A HAMSTER OVIDUCT-SPECIFIC GLYCOPROTEIN mRNA Northern Blot Analysis Total oviductal RNA from each estrous cycle of the hamsters was prepared by the method of Sambrook et al. [48] as described previously [34]. The total RNAs separated by 1.2% agarose gel (containing 40 mM 3-[N-morpholino] propanesulfonic acid [pH 7.2]/0.5 mM EDTA/6% formaldehyde/5 mM sodium citrate) electrophoresis were transferred to a Hybond N+ membrane by capillary blotting. After blotting, the RNAs were cross-linked to the membrane by exposure to UV light; then the membrane was incubated in the hybridization buffer (modified Church's buffer) [34, 49] containing 0.25 mM Na 2HPO 4 (pH 7.2)/1 mM EDTA/20% SDS/0.5% casein for 1 h at 65°C. After incubation, DIG-labeled single-strand DNA probe was added to a final concentration of 10 ng/ml, and hybridization was performed for 12-15 h at 65°C. After hybridization, the membrane was washed three times in 20 mM Na2 HPO4 (pH 7.2) containing 1 mM EDTA/1% SDS (washing buffer A) for 20 min at 65C and then transferred into washing buffer B (100 mM maleic acid [pH 8.0] containing 3 M NaCl, 0.3% Tween 20) and incubated with shaking for 5 min at room temperature. Nonspecific binding sites were blocked by blocking buffer (washing buffer B containing 0.5% casein), and the membrane was incubated with antiDIG-AP for 30 min. At the end of the reaction, the membrane was washed at least four times with washing buffer B and then incubated for 5 min in substrate buffer (100 mM Tris-HCl [pH 9.5] containing 100 mM NaCl/50 mM MgC1 2) for equilibration. The equilibrated membrane was transferred in the substrate buffer containing 0.24 mM CSPD as fluorescent substrate. The amount of product was visualized by exposure to x-ray film (Fuji New RX; Fuji Photo Film Co. Ltd., Kanagawa, Japan) as described previously [34]. Light Microscopic Study For analyzing the cyclic changes in oviductal epithelium, oviductal tissue from various hormonal conditions was observed by light microscopy. The tissue was fixed immediately with 4% paraformaldehyde in PBS (pH 7.4) at 4°C for 10 h and embedded in paraffin wax. The embedded tissue was sliced into sections (3 ,um thick) and stained with hematoxylin-eosin. Ciliated cells were counted by scanning 300 cells per block, and the percentage of ciliation was determined from three different blocks of ampulla or isthmus. In Situ Hybridization

The procedure for in situ hybridization has been previously described [33]. Briefly, serial cross sections (3 Ixm thick) from tissue for hematoxylin-eosin staining (see above) were deparaffinized and treated with 0.2 N HC1 for 20 min at room temperature and then with proteinase K (10 ixg/ml) for 15 min at 37°C. After postfixation with PBS (pH 7.4) containing 4% paraformaldehyde, these sections were dehydrated. Hybridization was carried out for 12-16 h in 5-strength saline-sodium phosphate/EDTA buffer (SSPE) (single-strength SSPE: 150 mM NaCl, 10 mM NaH 2PO 4, 1 mM EDTA, pH 7.4), containing 5-strength

Denhardt's solution, 0.5% SDS, 100 Ixg/ml denatured salmon sperm DNA, 50% formamide, and 500 ng/ml singlestrand DIG-labeled DNA probe prepared by the asymmetrical PCR method as described above. After hybridization, the sections were washed five times with 50% formamide

1109

in double-strength SSC (single-strength SSC: 150 mM sodium chloride, 15 mM sodium citrate) at 37°C for 1 h, twice with double strength SSC at 37°C for 15 min, and once with half-strength SSC at room temperature for 5 min. The sections were allowed to react with anti-DIG-AP diluted 1: 500 in blocking solution for 1 h at room temperature. After two washes with 100 mM Tris-HC1 (pH 7.5) and 150 mM NaCl, the sections were transferred into 100 mM Tris-HCl (pH 9.5), 50 mM MgCI 2, and 100 mM NaCl. The visualization was performed by use of 0.41 mM NBT and 0.38 mM 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for 1 h to overnight in a darkroom. To confirm the specificity of the HOGP probe to the oviduct-specific glycoprotein mRNA signal, parallel experiments were carried out using the sense HOGP probe as a control. Some sections were hybridized with the HOGP probe in the presence of an excess amount of either homologous or nonhomologous (salmon sperm DNA) unlabeled DNA as additional control experiments. The tissue specificity of the HOGP probe was also evaluated by reactivity to the various hamster tissues (oviduct, uterus, ovary, liver, spleen, kidney, lung, heart, and stomach). The number of in situ hybridization positive cells was determined by scanning 300 cells per block, and the percentage of such cells was determined from three different blocks of ampulla or isthmus per hamster. All experiments represented in the same figure were performed by identical conditions. Densitometry The results from either Northern blot or in situ hybridization were photographed, and the print from each experiment was scanned using a Microtek Image Scanner (ScanMaker IIXE). The resulting image was digitized into an Apple Power Macintosh 8100 computer, and the relative density was traced using the NIH image program (version 1.33f) as previously described [50, 51]. Statistics Data obtained from densitometric scanning, cell counts, or serum hormonal levels are indicated as the mean SEM. Statistical analysis of the results was performed using Dunnett's test. Differences were regarded as significant at p < 0.05. RESULTS Specificity of the HOGP Probe for In Situ Hybridization The HOGP probe strongly hybridized with the oviductal epithelial cells from mature hamsters at estrus (Day 1) (Fig. 2A). To confirm the specificity of the HOGP probe, various control experiments were performed. First, the probe showed no detectable positive signal with other tissues examined (liver, uterus, ovary, spleen, stomach, kidney, lung, heart, and brain [data not shown]). Second, when a tissue section of the oviduct was hybridized with the sense HOGP probe (Fig. 2B) or with the HOGP probe in the presence of an excess amount of homologous unlabeled DNA (Fig. 2C), the HOGP mRNA signal was abolished. On the other hand, the HOGP mRNA signal was not significantly altered when the tissue was hybridized with the HOGP probe in the presence of an excess amount of nonhomologous (salmon sperm) DNA (Fig. 2D). Moreover, random-labeled pBluescript SK(-) DNA did not hybridize with mature hamster oviduct (data not shown). These results demonstrate

KOMIYA ET AL.

1110 FIG. 2. Specificity of the HOGP probe. The ampulla (in mature female hamster oviduct at estrus) section was stained with HOGP probe (A); the serial adjacent sections were hybridized with HOGP sense probe (B), with HOGP probe in the presence of unlabeled HOGP-cDNA (C), or an excess amount of unlabeled salmon sperm DNA (D). L, lumen; S, stroma. x350.

that the HOGP probe used in this study was specific for detection of HOGP gene expression. HOGP Gene Expression during the Estrous Cycle Cyclic changes in the oviductal epithelium during the estrous cycle. It is well known that the epithelium of the oviduct is composed of two cell types, ciliated cells and nonciliated (secretory) cells. It is also well known that the epithelium shows cyclic change in morphology during the menstrual cycle or estrous cycle in several mammalian species [41, 44, 52-56]. According to our previous study concerning the mouse homologue of HOGP mRNA expression, the message was observed only in the nonciliated cells in the oviductal epithelium [33]. In preliminary studies, we checked the morphological changes in the epithelial cells by hematoxylin-eosin staining. In the ampulla, as summarized in Table 1, the mean percentage of ciliated cells was highest during diestrus (Days 2 and 3), decreased slightly during proestrus (Day 4), and reached its lowest level during estrus (Day 1). At diestrus, the serum P4 level was significantly low, while the serum E2 level increased. The serum P4 level was significantly high at estrus (Day 1), whereas serum E2 level showed its highest level at proestrus

(Day 4) and decreased drastically during estrus (Day 1). These data indicate that, in the hamster, the ampullary epithelium shows a cyclic change in the constitution of ciliated and nonciliated cells during the estrous cycle. On the other hand, light microscopic studies showed that, in the isthmus, most epithelial cells (> 95%) were nonciliated, and the number of ciliated cells was very low and did not significantly change through the estrous cycle (data not shown). Thus, we conclude that a cyclic change in the morphology of the oviductal epithelium of the isthmus is not as marked as that of the ampulla during the estrous cycle. Northern blot analysis. Using the HOGP probe, Northern blot analysis was performed on the total RNA extracted from the oviduct at each stage of estrous cycle. As shown in Figure 3, a 2.5-kb HOGP message was detected at all stages of the estrous cycle. The results obtained from the densitometric study revealed that the HOGP mRNA intensity and the patterns of the bands were not significantly changed throughout the estrous cycle (data not shown). These results suggest that the differences in the level of HOGP mRNA expression during the normal estrous cycle seen by in situ hybridization (see below) could not be quantified by Northern blot analysis.

TABLE 1. Intact hamsters: percentage ciliation and in situ hybridization (ISH) positive cells in the ampulla of the oviductal epithelium in cycling animals. Item measured % Ciliated cells % ISH positive cells Expression ratio* Serum E2 level (pg/ml) Serum P4 level (ng/ml)

Day 1 (n = 5) 9.9 -+4.0 91.1 + 4.0 1.01 28.2 + 8.7 11.4 + 1.5

Day 2 (n = 4)

Day 3 (n = 6)

Day 4 (n = 4)

29.4 + 4.13 51.4 + 8.0a 0.76 67.5 + 21.5a 2.3 + 1.2a

28.4 + 9.1a 76.1 + 3.9 a 1.04 91.8 + 12.4a 2.1 0.8a

20.4 + 9.3 79.6 + 12.0 1.00 114.5 19.3a 7.2 + 2.3a

* Expression ratio = mean % ISH positive cells/mean % nonciliated cells (100 minus mean % ciliated cells). p < 0.01, compared with the value of Day 1.

LOCALIZATION OF A HAMSTER OVIDUCT-SPECIFIC GLYCOPROTEIN mRNA

In situ hybridization in the ampulla. By in situ hybridization experiments with the HOGP probe, HOGP mRNA was observed within the oviductal epithelium throughout the estrous cycle, as shown in Figure 4. Figure 4A indicates the patterns of HOGP mRNA expression in the ampullary region during estrus. In this region, the oviductal epithelium consisted of a single layer of cuboidal cells, and most of the cells were stained at estrus (Day 1). The localization of HOGP mRNA was more dominant in the perinuclear region than in the basal region. On Day 2, the oviductal epithelial cells became more columnar in shape, and the signal-positive cells seemed to be dispersed. The signal intensity was decreased compared with that of Day 1. The oviductal epithelium on Day 3 remained columnar and cell height tall, similar to Day 2 . The mean percentage of in situ hybridization positive cells was increased in comparison with that on Day 2 (Table 1), and the signal appeared to be relatively intense (Fig. 4A). On Day 4, the cuboidal cells and the columnar cells were dispersed. The signal intensity of HOGP mRNA appeared to be dense, changing to the pattern of Day 1. Since pretreatment with HCI or proteinase K before in situ hybridization has an effect on cell morphology, hematoxylin-eosin staining was performed to confirm the cell types in the oviductal epithelium during the estrous cycle using serial sections as described previously [33]. Because the epithelium of the oviduct is composed of two cell types, ciliated cells and nonciliated (secretory) cells, the percentage of nonciliated cells in the oviductal epithelium can be calculated by subtracting the percentage of ciliated cells (Table 1) from 100; using these derived percentages, the expression ratio of in situ hybridization positive cells to nonciliated cells (Table 1) indicates that not all nonciliated cells expressed the HOGP message, especially on Day 2, when ciliated cells of the oviductal epithelium have no message for HOGP. These data were quantitatively analyzed by densitometry as shown in Figure 5. These results show that although HOGP mRNA never disappeared in the ampulla throughout the estrous cycle, the signal intensity of the message was significantly altered (p < 0.05), especially between the estrous stage and the diestrous stage. In situ hybridization in the isthmus. The shape of the epithelial cells in the isthmus and the staining pattern did not change throughout the estrous cycle (Fig. 4B). The shape of the cell was columnar, and the cell height was tall in all stages of the cycle. Unlike the pattern in the ampulla, the localization of the HOGP mRNA signal was restricted to the basal region. Throughout the estrous cycle, 95% of the cells were HOGP message-positive, a value similar to that of nonciliated cells in the isthmus. The relative intensity of the signal was also analyzed by densitometry. Data show that the signal intensity was significantly lower in the isthmus compared to the ampulla, and no significant difference in the message expression was observed through the estrous cycle in the isthmus (Fig. 5). HOGP Gene Expression in Ontogeny The animals used in these studies consisted of immature hamsters (8, 11, 14, 21, and 28 days old) and older (18 mo old) hamsters. Serum E2 and P4 levels in these animals are

shown in Table 2. As expected, we could not detect any signal in the oviductal epithelium from the 8-day-old immature hamsters (Fig. 6A). Although there appeared to be individual differences, a faintly positive signal could be observed in the perinuclear region in 11-day-old animals (Fig.

1111

FIG. 3. HOGP gene expression during the estrous cycle: Northern blot analysis. HOGP RNAs were isolated from hamster oviducts during the estrous cycle. A) Aliquots containing 5 g of total oviductal RNA were resolved on a 1.2% formaldehyde agarose gel. After electrophoresis, the RNAs were transblotted to a nylon membrane, and the hybridized signal was detected using the DIG-labeled HOGP probe. Lane 1, Day 1 (estrus); 2, Day 2 (diestrus Day 1); 3, Day 3 (diestrus Day 2); 4, Day 4 (proestrus). B) Ethidium bromide staining demonstrates quantity and quality of total RNA.

6B). The HOGP mRNA was detected in all 14-day-old animals (Fig. 6C). At this age, most oviductal epithelial cells showed a positive reaction. In 21-day-old animals, the cellular localization of the message and the tissue localization of in situ hybridization positive cells were similar to that observed in 14-day-old animals. The intensity of the signal increased with age until the animals became sexual mature. However, in the 18-mo-old hamsters, the height of the oviductal epithelial cells was lower, and cells were cuboidal in shape, and the intensity of the signal was significantly weaker than that of the 28-day-old hamsters (Fig. 6F). HOGP Gene Expression in Pregnancy The HOGP mRNA signal was detected by in situ hybridization in pregnant hamsters on Day 15 after mating (Fig. 7). The oviductal epithelium of the ampulla consisted of columnar cells. The signal intensity was as high as that of the mature hamsters (at the estrous stage; Fig. 7, A and B). The message was predominantly localized in the basal regions, with weak localization in perinuclear regions (Fig. 7B). In the isthmus of the oviduct of pregnant hamsters, the HOGP mRNA was localized mainly in the basal region of epithelial cells (data not shown). In postpartum animals, the serum E2 level is known to suddenly decrease after delivery. With withdrawal of serum E2, the shape of the oviductal epithelium in both ampulla and isthmus became cuboidal by the 1 th postpartum day. In these animals, the HOGP mRNA signal spread throughout the cytoplasm of

1112

KOMIYA ET AL.

FIG. 4. HOGP gene expression during the estrous cycle: in situ hybridization. The ampullary (A)and isthmus (B) regions of the oviduct during the estrous cycle. D1, 2, 3, and 4 refer to days of the estrous cycle. L, lumen. S, stroma. x220.

the epithelium and the signal intensity was decreased as compared to that in the pregnant hamsters (Fig. 7C). Changes in HOGP Gene Expression in Relation to EP4, Levels Effects of ovariectomy. In situ hybridization patterns for ovariectomized animals are shown in Figure 8. Sixty days after ovariectomy, the reproductive tract from these animals significantly changed its shape and appeared atrophic. The oviductal epithelium became short and cuboidal, and it appeared atrophied in both the ampulla and isthmus (serum E2 : 19.20 + 4.77 pg/ml, P4 : 3.13 + 0.77 ng/ml; n = 4). The levels of both E2 and P4 showed values similar to that

from menopausal women [57]. In this condition, the HOGP mRNA was significantly suppressed, and the signal was dispersed and faintly observed in the perinuclear region of the oviductal epithelium (Fig. 8B). Effects of GnRH analogue treatment and hormonal administration. Figure 9 shows changes in the HOGP mRNA expression after treatment with GnRH analogue (TAP-144SR). As in the ovariectomy study, administration of the cyclic GnRH analogue caused a decrease in serum E2 level (serum E2: 28.62 + 10.24 pg/ml, P4: 3.63 2.14 ng/ml; n = 5). As the hypoestrogenic state continued, the entire oviductal epithelium of the ampulla and isthmus appeared atrophied, and the HOGP mRNA signal was significantly


1113

gene expression, the elevation of the serum P4 level in itself is not strongly effective in suppressing HOGP gene expression. DISCUSSION

'U0 4)

Day 1

Day 2

Day 3

Day4

*: p < 0.05 ( vs. the ampulla expression of Day 1) FIG. 5. Quantitative trends of HOGP mRNA expression during the estrous cycle. The level of HOGP mRNA intensity in the epithelium of the oviduct was measured using the NIH image program as described in Materials and Methods. Densitometric scanning was carried out in five different areas in the oviductal epithelium at each estrous stage. The relative signal intensity for HOGP mRNA in ampulla (solid bar) or isthmus (open bar) is shown with standard deviation. The mean intensity on Day 1 of the estrous cycle in ampulla was expressed as 100%. * p < 0.05 (vs. the mean intensity on Day 1 of the estrous cycle in ampulla).

lower in the ampullary region (Figs. 9B and 10B). However, the signal never completely disappeared regardless of the long-term hypoestrogenic state, a finding similar to the situation in the ovariectomized animals described above. The signal was faintly localized in the perinuclear region. At 12 wk after the final treatment with GnRH analogue, the HOGP mRNA signal was restored within the epithelium of the oviduct (Fig. 9C). In this condition, the oviductal epithelium was columnar and tall, the serum hormonal levels returned to those of cyclic changes, and the estrous cycle of animals was normalized. To confirm the hormonal dependency of HOGP gene expression, E2 treatment was given to GnRH analogue treated-hypoestrogenic animals. Moreover, since P4 levels rise after a short period of E2 secretion during the natural estrous cycle in the hamster, combined E 2 +P4 treatment was also given to the GnRH analogue treated-hypoestrogenic animals (Figs. 1 and 10). After E2 treatment, the oviductal epithelium became columnar and tall in shape (serum E2: 58.03 ± 14.25 pg/ml, P4: 4.87 + 2.20 ng/ml; n = 6). Most of epithelial cells were in situ hybridization positive, and the tissue localization of the positive cells were similar to that in estrus (Day 1). The intensity of staining was restored and reached the level before GnRH analogue treatment (Fig. 10C). On the other hand, treatment with both E2 and P4 for 14 days partially decreased the expression of HOGP mRNA, and the localization of the HOGP mRNA signal was restricted mostly 10.09 pg/ml, P4: to the basal region (serum E2: 55.47 17.57 ± 7.04 ng/ml; n = 4; Fig. 10D). These results suggest that although a low serum E2 level suppresses HOGP

The present study has demonstrated, for the first time, the changes in tissue/cellular localization of HOGP mRNA under various hormonal conditions in the golden hamster, using a specific single-stranded DNA probe. Although Northern blot analysis did not show a significant difference in the level of message expression during the estrous cycle (Fig. 3), in situ hybridization studies clearly showed a specific message expression pattern on each day of the cycle, especially in the ampullary region of the oviduct (Fig. 4). On the basis of Northern blot data and in situ hybridization studies, the total quantity of HOGP message does not change drastically; however, the signal distribution in the message-positive cells in the ampulla appears to be altered during the estrous cycle. Parallel studies by hematoxylineosin-stained tissue sections suggested that quantitative differences of the HOGP gene expression within the oviductal ampullary epithelium (Fig. 5) were not accompanied by changes in ciliation of the oviductal epithelial cells, as summarized in Table 1. Subsequently, we investigated the expression pattern of HOGP message during ontogeny and pregnancy. Data in Figures 6 and 7 suggest that the elevation of serum E2 level was necessary for the HOGP gene expression; however, the HOGP mRNA was dispersed within the oviductal epithelium and weakly observed in the perinuclear region when the serum E2 level was low. This result was supported by data obtained from studies of ovariectomized and GnRH analogue-treated animals, which showed diminution in both serum E2 and P4 levels (Figs. 8 and 9). On the other hand, in the pregnant animals, which showed both high serum E2 and P4 levels, the cellular HOGP mRNA localization was predominantly in the basal rather than in the perinuclear region. In addition, results of administration of E2 and P4 to GnRH analogue-treated animals presented in Figure 10 also suggest the importance of serum E2 and P4 levels for HOGP gene expression. Taken together, these results lead us to conclude that the expression of the HOGP gene is regulated by both serum E2 and P4 . Unlike baboon oviduct-specific glycoprotein gene expression, which is strongly suppressed by P4 [35], elevation in serum P4 is not strongly effective in the suppression of HOGP gene expression in the hamster oviduct, although it caused a change in localization of HOGP mRNA, as shown in Figure 10D. It should be noted that HOGP gene expression shows hormonal dependency (Table 1); however, there is at least a 24-h lag between the elevation of serum E2 level and the expression of the message. In the hamster, a previous study demonstrated that serum E2 increased from Day 10 to Day 18, and this was followed by a drop between Days 18 and 21 [58]. The present study confirms this trend of serum E2 levels during ontogeny (Table 2). However, the

TABLE 2. Hormonal levels during ontogeny in hamster. Age Hormone Serum E2 (pg/ml) Serum P4 (ng/ml)

8 days (n = 5) 6.17 ± 3.14 0.63 ± 0.04

11 days (n = 5) 11.38 ± 9.33 1.21 0.44

14 days (n = 5)

21 days (n = 5)

28 days (n = 5)

18 months (n = 3)

18.20 ± 7.43 2.04 ± 1.16

12.54 + 4.95 6.45 ± 1.41b

49.28 ± 15.26 a 15.30 + 5.30 a

14.01 ± 1.97 7.24 ± 4.01b

a p < 0.01, b p < 0.05, compared with the value of 8-day-old animals.

1114

KOMIYA ET AL.

FIG. 6. HOGP gene expression during ontogeny. The oviduct was obtained from immature (8 days-4 wk old) or aged hamsters (18 mo old): (A) 8 days old, (B)11 days old, (C) 14 days old, (D) 21 days old, (E)28 days old, or (F) 18 mo old. x125.

intensity of HOGP mRNA continued to increase during Days 11-28 after birth (Fig. 6). Since this result indicated that elevation of the serum E2 level does not affect HOGP gene expression rapidly, it is important to have an adequate serum E2 level over a given period of time. In addition, the message never disappears completely during the estrous cycle, in aged animals with low levels of E2 , or even during pregnancy. The human homologue of the HOGP may be more E2 -dependent because its synthesis is suppressed immediately after delivery [24, 37], presumably because of rapidly decreasing serum E2 levels after delivery. This observation is consistent with the trend shown in the present study. To date, it is generally considered that the oviduct-specific glycoprotein may have a role(s) in the fertilization FIG. 7. HOGP gene expression in the pregnant animal. Ampullary epithelium (A) on Day 1 of a normal estrous cycle (positive control), (B)in pregnancy at term (E 2, 255.80 pg/ml; P4, 24.08 ng/ml); or (C) postpartum 11 days (E 2, 16.88 pg/ml; P4, 8.09 ng/ml). L, lumen; S, stroma. x200.

process and/or early development of the embryo because these molecules associate with the egg and/or spermatozoa [6-10, 13, 16, 26-32, 59]. It is noteworthy that although HOGP message expression seems to be relatively high in the epithelium of the ampulla at the time of fertilization (Day 1 of the estrous cycle), no significant hormonal dependency of the gene expression was observed in the isthmus (Figs. 4 and 5). In addition, the message remains strongly even during pregnancy. This fact suggests that, at least in the hamster, the oviduct-specific glycoprotein may have a role not only in fertilization or early developmental processes but also in some other reproductive processes. Further study will be needed to obtain definitive evidence on this possibility. In the last decade, oviduct-specific glycoproteins have


1115

FIG. 8. HOGP gene expression in ovariectomized hamster. Ampullary epithelium (A) on Day 1 of a normal estrous cycle (positive control) or (B)in an ovariectomized hamster. L, lumen; S, stroma. x215.

been identified in several mammalian species, including the hamster (for review, see Malette et al. [60]). Some of these glycoproteins (sheep, bovine, porcine, baboon, and human) have been characterized as E2 -dependent secretory proteins on the basis of data obtained from native oviductal tissue, fluid, or cell culture systems analyzed by one- or two-dimensional SDS PAGE [20, 22-24, 35, 42-45]. In addition, mRNA expression of oviduct-specific glycoproteins of these animals (except the pig) detected by Northern blot demonstrated correlation with E2 levels [35-38]. Recently, using in situ hybridization, Murray and DeSouza reported that mRNA encoding an estrogen-dependent oviduct secretory protein in the sheep is localized in the apical tips and basal compartments of fimbria and ampulla epithelial cells, implying translation at unique cytoplasmic foci [61]. On the other hand, morphological changes in the oviductal epithelial cells during various hormonal conditions have been reported in some mammalian species [52, 54-56, 62-65].

These studies reported that ciliogenesis of oviductal epithelial cells was E2 -dependent in primates and carnivores but not in cattle. In hamsters, our present data obtained from light-microscopic studies show that the number of ciliated cells in the ampulla changed during the estrous cycle (Table 1). Therefore, ciliogenesis of the hamster oviductal epithelium appears to be controlled by ovarian steroids. However, the significant decrease in signal intensity observed on Day 2 (Figs. 4A and 5) does not appear to be due to the change in ciliogenesis in the ampullary oviductal epithelium, since our preliminary data using electron microscopic in situ hybridization suggest that HOGP messages, as well as the message of the mouse homologue of HOGP [33], are observed only within nonciliated secretory-type cells (Komiya et al., manuscript in preparation). Using a specific monoclonal antibody to HOGP as the probe, an immunohistochemical study reported that this antibody strongly reacted with the isthmus but not with the FIG. 9. Effects of GnRH analogue (TAP144-SR) treatment. TAP-1 44-SR was injected as described in Figure 1. Ampullary epithelium (A)on Day 1 of a normal estrous cycle (positive control), (B)in TAP-144-SRtreated animals, or (C)on Day 4 of a normal estrous cycle after 12 wk from the final TAP-1 44-SR treatment. L, lumen; S, stroma. x240.

1116

KOMIYA ET AL.

FIG. 10. Effect of GnRH analogue (TAP144-SR) treatment: hormone replacement. TAP-1 44-SR, E2, and P4 were injected as described in Figure 1. Ampullary epithelium (A)on Day 1 of a normal estrous cycle (positive control), (B)in TAP-1 44-SR-treated animals, (C) in TAP-1 44-SR-treated animals followed by E2injection, or (D) in TAP-144-SR-treated animals followed by E2+P4 injection. L, lumen; S, stroma. x180.

ampulla of the oviduct [8]. A possible explanation for this discrepancy in the expression patterns shown in the immunohistochemical study and the in situ hybridization study may be as follows. Since the secretory activity of the HOGP in the ampulla may be greater than in the isthmus, the HOGP message is strongly detected by in situ hybridization but not observed strongly by immunohistochemistry. Alternatively, since the antigen determinant of this monoclonal antibody is a carbohydrate moiety [8], the immunohistochemical staining patterns may reflect the presence of the sugar moiety but not the peptide backbone of the glycoprotein within the oviductal epithelium. In other studies, immunohistochemical staining methods identified oviduct-specific glycoproteins mostly in the ampullary region in some other species (mouse, pig, sheep) of the oviduct [44, 59, 66]. Since Northern blot studies using HOGP or mouse oviduct-specific glycoprotein cDNA reported that mRNA expression was extremely restricted in the oviduct [33, 34], the cDNA probe is more suitable for the detection of the molecule than the monoclonal antibody to HOGP [8]. At present, we do not have direct evidence indicating whether or not this molecule is an essential factor for the fertilization process. In order to examine details of the physiological function(s) of the oviduct-specific glycoprotein more precisely, we are using the mouse system to analyze genomic DNA, which includes the promoter region. This approach will also give us important information concerning the molecular mechanism for transcription of the oviduct-specific glycoprotein gene. Since we have already reported molecular characterization of a mouse oviductspecific glycoprotein as a homologue of HOGP [33, 34], it will be interesting to compare the data from the mouse system with the data presented in this paper in order to understand more clearly the physiological function(s) of a molecule widely observed in the mammalian oviduct.

ACKNOWLEDGMENTS We are grateful to Drs. Marie-Claire Orgebin-Crist, Daulat Ram P Tulsiani, Majorie D. Skudlarek, and Ms. Catherine A. Chayko (Vanderbilt University School of Medicine) for their helpful discussion and a critical reading of the manuscript.

REFERENCES 1. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, Vol. 1. New York, NY: Raven Press; 1994: 189-317. 2. Wassarman PM. Cell surface carbohydrate and mammalian fertilization. In: Fukuda M (ed.), Cell Surface Carbohydrate and Development. Boston, MA: CRC Press; 1992: 215-238. 3. Miller DJ, Shur BD. Molecular basis of fertilization in the mouse. In: Lennarz WJ (ed.), Seminar in Developmental Biology, vol.5. London, UK: Academic Press; 1994: 255-264. 4. Ramarao CS, Myles DG, Primakoff P. Multiple roles for PH-20 and fertilin in sperm-egg interaction. In: Lennarz WJ (ed.), Seminar in Developmental Biology, Vol. 5. London, UK: Academic Press; 1994: 265-271. 5. Aitken RJ. The complexities of conception. Science 1995; 269:3940. 6. Kapur RP, Johnson LV. An oviductal fluid glycoprotein associated with ovulated mouse ova and early embryos. Dev Biol 1985; 112:8993. 7. Kapur RP, Johnson LV. Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos. J Exp Zool 1986; 238:249-260. 8. Araki Y, Kurata S, Oikawa T, Yamashita T, Hiroi M, Naiki M, Sendo E A monoclonal antibody reacting with the zona pellucida of oviductal egg but not with that of the ovarian egg of the golden hamster. J Reprod Immunol 1987; 11:193-208. 9. Leveille MC, Roberts KD, Chevalier S, Chapdelaine A, Bleau G. Uptake of an oviductal antigen by the hamster zona pellucida. Biol Reprod 1987; 36:227-238. 10. Oikawa T, Sendai Y, Kurata S, Yanagimachi R. A glycoprotein of oviductal origin alters biochemical properties of the zona pellucida of hamster egg. Gamete Res 1988; 19:113-122. 11. Oliphant G, Ross PR. Demonstration of production and isolation of three sulfated glycoproteins from the rabbit oviduct. Biol Reprod 1982; 26:537-544.

LOCALIZATION OF A HAMSTER OVIDUCT-SPECIFIC GLYCOPROTEIN mRNA 12. Oliphant G, Reynolds AB, Smith PF, Ross PR, Marta JS. Immunocytochemical localization and determination of hormone-induced synthesis of the sulfated oviductal glycoproteins. Biol Reprod 1984; 31: 165-174. 13. Brown CR, Cheng WKT. Changes in composition of the porcine zona pellucida during development of the oocyte to the 2- to 4-cell embryo. J Embryol Morphol 1986; 92:183-191. 14. Hedrick JL, Wardrip NJ, Berger T. Differences in the macromolecular composition of the zona pellucida isolated from pig oocytes, eggs and zygotes. J Exp Zool 1987; 241:257-262. 15. Buhi WC, Vallet JL, Bazer FW. De novo synthesis and release of polypeptides from cyclic and early pregnant porcine oviductal tissue in explant culture. J Exp Zool 1989; 252:79-88. 16. Sutton R, Nancarrow CD, Wallace ALC, Rigby NW. Identification of an oestrus-associated glycoprotein in oviducal fluid of the sheep. J Reprod Fertil 1984; 72:415-422. 17. Gandolfi F, Brevini TAL, Richardson L, Brown CR, Moor RM. Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. Development 1989; 106: 303-312. 18. Joshi MS. Isolation, cell culture and immunocytochemical characterization of oviduct epithelial cells of the cow. J Reprod Fertil 1988; 83:249-261. 19. Malayer LR, Hansen PJ, Buhi WC. Secretion of proteins by cultured bovine oviducts collected from estrus through early diestrus. J Exp Zool 1988; 248:345-353. 20. Boice ML, Geisert RD, Blair RM, Verhage HG. Identification and characterization of bovine oviductal glycoproteins synthesized at estrus. Biol Reprod 1990; 43:457-465. 21. Fazleabas AT, Verhage HG. The detection of oviduct-specific proteins in the baboon (Papio anubis). Biol Reprod 1986; 35:455-462. 22. Verhage HG, Fazleabas AT. The in vitro synthesis of estrogen-dependent proteins by the baboon (Papio anubis) oviduct. Endocrinology 1988; 123:552-558. 23. Verhage HG, Boice ML, Mavrogianis P, Donnelly K, Fazleabas AT. Immunological characterization and immunocytochemical localization of oviduct-specific glycoproteins in the baboon (Papio anubis). Endocrinology 1989; 124:2464-2472. 24. Verhage HG, Fazleabas AT, Donnelly K. The in vitro synthesis and release of proteins by the human oviduct. Endocrinology 1988; 122: 1639-1645. 25. Wagh PV, Lippes J. Human oviductal fluid proteins. III. Identification and partial purification. Fertil Steril 1989; 51:81-88. 26. Kan FWK, St-Jacques S, Bleau G. Immunoelectron microscopic localization of an oviductal antigen in hamster zona pellucida by use of a monoclonal antibody. J Histochem Cytochem 1988; 36:1441-1447. 27. Wegner CC, Killian GJ. Origin of oestrus-associated glycoproteins in bovine oviductal fluid. J Reprod Fertil 1992; 95:841-854. 28. Boice ML, McCarthy TJ, Mavrogianis PA, Fazleabas AT, Verhage HG. Localization of oviductal glycoproteins within the zona pellucida and perivitelline space of ovulated ova and early embryos in baboons (Papio anubis). Biol Reprod 1990; 43:340-346. 29. Lippes J, Wagh PV. Human oviductal fluid (hOF) proteins. IV. Evidence for hOF proteins binding to human sperm. Fertil Steril 1989; 51:89-94. 30. McNutt T, Rogowski L, Vasilatos-Younken R, Killian G. Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation. Mol Reprod Dev 1992; 33:313-323. 31. King R, Killian G. Purification of bovine estrus-associated protein and localization of binding on sperm. Biol Reprod 1994; 51:34-42. 32. Kimura H, Matsuda J, Ogura A, Asano T, Naiki M. Affinity binding of hamster oviductin to spermatozoa and its influence on in vitro fertilization. Mol Reprod Dev 1994; 39:322-327. 33. Sendai Y, Komiya H, Suzuki K, Onuma T, Kikuchi M, Hoshi H, Araki Y. Molecular cloning and characterization of a mouse oviduct-specific glycoprotein. Biol Reprod 1995; 53:285-294. 34. Suzuki K, Sendai Y, Onuma T, Hoshi H, Hiroi M, Araki Y. Molecular characterization of a hamster oviduct-specific glycoprotein. Biol Reprod 1995; 53:345-354. 35. Donnelly KM, Fazleabas AT, Verhage HG, Mavrogianis PA, Jaffe RC. Cloning of a recombinant complementary DNA to a baboon (Papio anubis) estradiol-dependent oviduct-specific glycoprotein. Mol Endocrinol 1991; 5:356-364. 36. Sendai Y, Abe H, Kikuchi M, Satoh T, Hoshi H. Purification and molecular cloning of bovine oviduct-specific glycoprotein. Biol Reprod 1994; 50:927-934. 37. Arias EB, Verhage HG, Jaffe RC. Complementary deoxyribonucleic

38.

39.

40.

41.

42.

43.

44.

45. 46.

47.

48.

49. 50. 51.

52.

53.

54. 55.

56.

57.

58.

59.

1117

acid cloning and molecular characterization of an estrogen-dependent human oviduct glycoprotein. Biol Reprod 1994; 51:685-694. DeSouza MM, Murray MK. An estrogen-dependent secretory protein, which shares identity with chitinase, is expressed in a temporal and regional specific manner in the sheep oviduct at the time of fertilization and embryo development. Endocrinology 1995; 136:2485-2496. Buhi WC, Alvarez IM, Choi I, Simmen FA. A recombinant cDNA to a porcine oviductal estrogen-dependent secretory protein (POESP). Biol Reprod 1994; 50(suppl 1):165 (abstract). Hakala BE, White C, Recklies AD. Human cartilage gp-39, a major secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J Biol Chem 1993; 268:25803-25810. Brenner RM, Slayden OD. Cyclic changes in the primate oviduct and endometrium. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, Vol. 1. New York, NY: Raven Press; 1994: 541-569. Rapisarda JJ, Mavrogianis PA, O'Day-Bowman MB, Fazleabas AT, Verhage HG. Immunological characterization and immunocytochemical localization of an oviduct-specific glycoprotein in the human. J Clinical Endocrinol & Metab 1993; 76:1483-1488. Buhi WC, Ashworth CJ, Bazer FW, Alvarez IM. In vitro synthesis of oviductal secretory proteins by estrogen-treated ovariectomized gilts. J Exp Zool 1989; 262:426-435. Murray MK. Biosynthesis and immunocytochemical localization of an estrogen-dependent glycoprotein and associated morphological alteration in the sheep ampulla oviduct. Biol Reprod 1992; 47:889-902. Murray MK. An estrogen-dependent glycoprotein is-synthesized and released from the oviduct in a temporal- and region-specific manner during early pregnancy in the ewe. Biol Reprod 1993; 48:446-453. Terkel J. Neuroendocrine processes in the establishment of pregnancy and pseudopregnancy in rats. Psychoneuroendocrinology 1988; 13:528. Gyllenstein UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 1988; 85:76527656. Sambrook J, Fritsch EF, Maniatis T Extraction, purification, and analysis of messenger RNA from eukaryotic cells. In: Molecular Cloning-A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989. Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci USA 1984; 81:1991-1995. Araki Y, Orgebin-Crist M-C, Tulsiani DRP Qualitative characterization of oligosaccharide chains present on the rat zona pellucida glycoconjugates. Biol Reprod 1992; 46:912-919. Tulsiani DRP, Skudlarek MD, Araki Y, Orgebin-Crist M-C. Purification and characterization of two forms of -D-galactosidase from rat epididymal luminal fluid: evidence for their role in the modification of sperm plasma membrane glycoprotein(s). Biochem J 1995; 305: 41-50. Verhage HG, Bariether ML, Jaffe RC, Akbar M. Cyclic changes in ciliation, secretion, and cell height of the oviduct epithelium in women. Am J Anat 1979; 156:505-522. Odor DL, Gaddum-Rosse P, Rumery RE, Blandau RJ. Cyclic variations in the oviductal ciliated cells during the menstrual cycle and after estrogen treatment in the pig-tailed monkey, Macaca nemestrina. Anat Rec 1980; 198:35-57. Bariether ML, Verhage HG. Control of the secretory cell in cat oviduct by estradiol and progesterone. Am J Anat 1981; 162:107-118. Odor DL, Gaddum-Rosse P, Rumery RE. Secretory cells of the oviduct of the pig-tailed monkey, Macaca nemestrina, during the menstrual cycle and after estrogen treatment. Am J Anat 1983; 166:149172. Verhage HG, Mavrogianis PA, Boice ML, Li W, Fazleabas AT. Oviduct epithelium of the baboon: hormonal control and the immunogold localization of oviduct-specific glycoproteins. Am J Anat 1990; 187: 81-90. Judd HL, Judd GE, Lucas WE, Yen SSC. Endocrine function of the postmenopausal ovary: concentration of androgens and estrogens in ovarian and peripheral vein blood. J Clin Endocrinol & Metab 1974; 39:1020-1024. Vomachka AJ, Greenwald GS. The development of gonadotropin and steroid hormone patterns in male and female hamsters from birth to puberty. Endocrinology 1988; 105:960-966. Kapur RP, Johnson LV. Ultrastructural evidence that the specialized regions of the murine oviduct contribute a glycoprotein to the extracellular matrix of mouse oocytes. Anat Rec 1979; 221:720-729.

1118

KOMIYA ET AL.

60. Malette B, Paquette Y, Merlen Y, Bleau G. Oviductin possess chitinase- and mucin-like domains: a lead in the search for the biological function of these oviduct-specific ZP-associating glycoproteins. Mol Reprod Dev 1995; 41:384-397. 61. Murray MK, DeSouza MM. Messenger RNA encoding an estrogendependent oviduct secretory protein in the sheep is localized in the apical tips and basal compartments of fimbria and ampulla epithelial cells implying translation at unique cytoplasmic foci. Mol Reprod Dev 1995; 42:268-283. 62. Brenner RM, Resko JA, West NB. Cyclic changes in oviductal morphology and residual cytoplasmic estradiol binding capacity induced by sequential estradiol-progesterone treatment of spayed Rhesus monkeys. Endocrinology 1974; 95:1094-1104.

63. Verhage HG, Murray MK, Boomsma RA, Rehfeldt PA, Jaffe RC. The postovulatory cat oviduct and uterus: correlation of morphological features with progesterone receptor levels. Anat Rec 1984; 208:521531. 64. Sawyer HR, Olson PN, Gorell TA. Effects of progesterone on the oviductal epithelium in estrogen-primed prepubertal beagles: light and electron microscopic observations. Am J Anat 1984; 169:75-87. 65. Murray MK. Epithelial lining of the sheep ampulla oviduct undergoes pregnancy-associated morphological changes in secretory status and cell height. Biol Reprod 1995; 53:653-663. 66. Buhi WC, Alvarez IM, Sudhipong V, Dones-Smith MM. Identification and characterization of de novo-synthesized porcine oviductal secretory proteins. Biol Reprod 1990; 43:929-938.