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Biology of Reproduction 64, 310-316 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Effects of Testosterone on Production of Perivitelline Membrane Glycoprotein ZPC by Granulosa Cells of Japanese Quail (Coturnix japonica)1

Jianzhi Pana,b, Tomohiro Sasanamia, Yoshinori Konoc, Tsukasa Matsudac, and Makoto Mori2,a,b

a Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan b The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan c Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

ABSTRACT

Avian perivitelline membrane, an investment homologous to the zona pellucida of mammalian oocytes, is composed of at least two glycoproteins. Previous studies have indicated that one of the components, a glycoprotein homologous to mammalian ZPC, is produced and secreted by the granulosa cells of developing follicles of the chicken ovary. In the present study, we evaluated the expression and regulation of ZPC in Japanese quail (Coturnix japonica) granulosa cells both in vivo and in vitro. Western blot analysis of the SDS-solubilized granulosa layer using anti-quail ZPC antiserum showed that the amount of ZPC increased in parallel with follicular development. Northern blot analysis of total RNA using cDNA of quail ZPC showed that the increase in mRNA expression was also correlated with follicular development. To investigate the regulation of ZPC production, the granulosa cells were cultured in a medium containing steroid hormones such as progesterone, estradiol-17ß, or testosterone. By measuring ZPC protein and mRNA with Western and Northern blot analyses, respectively, we found that addition of testosterone maintained ZPC contents in the culture of the granulosa cells, and that ZPC mRNA expression was high in the culture with testosterone compared to the control. These results suggest that testosterone stimulates ZPC protein production at the gene transcription level.

developmental biology, follicle, follicular development, gene regulation, granulosa cells, oocyte development

INTRODUCTION

Mature oocytes of vertebrates are surrounded by an extracellular matrix generally termed the egg envelope, although different names have been adopted for different classes [1]. For most mammalian species, an egg envelope called the zona pellucida (ZP) comprises three glycoproteins: ZPA, ZPB, and ZPC [2]. These components are assembled into a three-dimensional matrix around the oocyte and serve several functions in the process of fertilization, including species-specific sperm-egg binding, induction of the acrosome reaction, and prevention of polyspermy [3]. The seminal work of Bleil and Wassarman [4–6] during the 1980s established that ZPC is a major component responsible for the initial sperm-egg recognition in mouse oocyte. In amphibians, the majority of the ligand activity involved in sperm binding is also derived from ZPC [7, 8].

The avian egg is a complex of an oocyte and extracellular matrices. At the time of ovulation, the oocyte is surrounded by the perivitelline membrane, which is called the inner layer of the vitelline membrane after laying [9]. The vitelline membrane consists of three layers: an inner layer, an outer layer, and a continuous membrane in between [10]. The outer layer adheres to the inner layer after ovulation in the infundibulum part of the oviduct [9]. The inner layer is a three-dimensional network of fibers and a structure similar to that observed between the granulosa cells and the oocyte in follicles before ovulation [11].

Two glycoproteins were identified as components of the inner layer of the vitelline membrane in the avian egg: 33- and 175-kDa glycoproteins in quail [12], and 32- and 183-kDa glycoproteins in hens [13]. We have found that antiserum raised against the 33-kDa glycoprotein of a quail egg can recognize a protein with a molecular mass of 35 kDa in the perivitelline membrane in preovulatory follicles [14]. Modification of the 35-kDa protein to 33 kDa has been demonstrated by the deletion of 26 amino acids from the N-terminus, with oviductin-like protease secreted from the infundibulum part of the oviduct [15]. The 35-kDa protein band had also been detected in the lysate of granulosa cells, indicating that the origin of the 35-kDa protein is the granulosa cells of the preovulatory follicle.

The cDNA encoding for this protein was cloned (GenBank accession no. D89097 for chicken and AB012606 for quail), and a high degree of homology of its nucleotide sequence and primary polypeptide structure with those of mammalian, amphibian, and fish ZPC homologues was noted [16]. Hence, it was suggested that this protein be called ZPC. Although a common ancestor might be suggested for the ZPC homologues of various species, the expression of ZPC appears to be regulated differently in different species. Clear evidence shows that ZPC is synthesized by the oocytes themselves in mice [17] and in Xenopus sp. [18], whereas the granulosa cells in the follicles also participate in the formation of ZPC in rabbits [19] and in cynomolgus monkeys [20]. On the other hand, a glycoprotein homologous to ZPC in chorion, the fish egg envelope, is produced in the liver and transported to the ovary by the blood circulation, like vitellogenin [21, 22].

To our knowledge, the regulation of mammalian ZPC production in oocytes has not been studied, but the gene expression of a fish ZPC homologue in the liver has been found to be regulated by estrogens [23–25]. Moreover, down-regulation of a ZPC homologue by human chorionic gonadotropin and 11-ketotestosterone has been reported in the testes of Japanese eel [26].

Although the physiological importance of avian ZPC for fertilization suggests it to be the candidate for a "sperm receptor" in quail [27], the regulation of ZPC production in granulosa cells during the ovulation cycle remains obscure. To investigate the regulatory mechanism of ZPC production, we have studied the expression of ZPC during the ovulation cycle by Western and Northern blot analyses. Moreover, we cultured the granulosa cells with various steroid hormones to study the endocrine control of ZPC production.

MATERIALS AND METHODS

Animals and Tissue Preparation

Female Japanese quail (Coturnix japonica), 15–30 wk of age, were individually caged under a photoperiod of 14L:10D (lights-on at 0500 h) and provided with water and a commercial diet (Tokai Kigyo, Toyohashi, Japan) ad libitum. Almost all birds lay eggs regularly at the same time every day, ranging from 1500 to 1800 h. The exact time of oviposition was recorded automatically for each bird to the nearest 5 min, and ovulation was assumed to occur 15–30 min after oviposition of the previously ovulated oocyte [28].

Ovaries from at least three birds were removed after cervical dislocation. Follicles from each ovary were dissected, cleaned of residual connective tissue, and placed in a physiological saline. The granulosa layer was isolated as a sheet of granulosa cells sandwiched between the perivitelline membrane and the basement membrane as previously described [29].

Cell Culture

To isolate the granulosa cells, the granulosa layers were dispersed by incubation at 37°C for 10 min with 500 U/ml of collagenase (Type I; Sigma, St. Louis, MO) in Hanks' balanced salt solution (BSS) as previously described [30]. After having been washed repeatedly with Hanks' BSS, the cell suspension was diluted with RPMI-1640 medium (Gibco/Life Sciences, Gaithersburg, MD) to a final concentration of 1 x 107 viable cells/ml. The density and viability of the cell preparations were determined using a hemocytometer and the trypan blue exclusion technique [31]. The viability of the cell preparations was greater than 95%. Aliquots (1 x 106 cells) of each cell suspension were pipetted into 12- x 75-mm polystyrene tubes (Becton Dickinson, Franklin Lakes, NJ), and the final volume of each tube was brought to 0.5 ml with the medium. When steroid hormone was added to the medium, the ethanol concentration never exceeded 0.1%. Cells were then incubated as suspension cultures for up to 48 h at 39°C while being shaken constantly in a humidified atmosphere of 5% CO2 and 95% air.

Western Blotting

The granulosa layers were solubilized in 1% SDS buffered at pH 6.8 with 70 mM Tris-HCl (TBS), followed by centrifugation at 14 500 x g for 15 min. Next, SDS-PAGE was performed as described previously [32] using 12% and 5% polyacrylamide for resolving and stacking gels, respectively. Protein content of the samples was determined according to the method described by Lowry et al. [33] using BSA as a standard. For Western blotting, gels were equilibrated in a transfer buffer (25 mM Tris, 40 mM aminocaproic acid, and 20% methanol) for 15 min at room temperature. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon, Millipore, Bedford, MA) using a semidry transfer system at 1 mA/cm2 of the gel for 1 h [34]. The membrane was blocked for 2 h with 0.1% gelatin in saline buffered at pH 7.4 with 10 mM TBS to reduce the nonspecific background. The PVDF membranes were washed three times for 30 min in 0.1% Tween-20 in TBS (TBS-T) and then incubated overnight at room temperature with anti-quail ZPC antiserum [14] diluted with TBS (1:2000). The membranes were again washed three times with TBS-T and three times with PBS for 10 min each wash, then reacted with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Cappel, Durham, NC) diluted with PBS (1:1000) for 2.5 h at room temperature. Washing with PBS was repeated three times for 10 min each time. Finally, the membrane was reacted with 3,3'-diaminobenzidine tetrahydrochloride (6 mg per 10 ml) buffered at pH 7.6 with 50 mM TBS containing 0.03% hydrogen peroxide and 0.03% NiCl2.

Northern Blotting

For Northern blotting, total RNA was isolated from granulosa layers or cultured granulosa cells by Isogen (Nippon Gene, Toyama, Japan), which utilizes a modification of the acid guanidinium thiocyanate-phenol-chloroform extraction method [35]. The RNA concentration and purity were determined by absorbance at 260 and 280 nm. Total RNA was denatured at 65°C for 15 min in a formaldehyde-containing buffer and subjected to electrophoresis in 1% agarose and 2.2 M formaldehyde gels. After electrophoresis, the gels were stained with ethidium bromide, and RNA was transferred to a Hybond-N+ nylon membrane (Amersham Life Science, Bucks, England) by overnight capillary blotting in a 20x saline-sodium phosphate-EDTA buffer (SSPE; 1x SSPE is 150 mM NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA [pH 7.4]). Membranes were baked at 80°C for 2 h, followed by ultraviolet cross-linking. Transcript size was estimated by comparison with 0.2–10 kilobase (kb) RNA size standards (Novagen, Madison, WI). Equal loading of RNA onto gels was verified by ethidium bromide staining of 18S and 28S bands of ribosomal RNA, because ß-actin mRNA, a common housekeeping gene, is not expressed at consistent levels during follicular development in chicken [36].

The blots were prehybridized at 42°C for 3 h with 20 µg/ml of calf thymus DNA in 50% formamide, 5x SSPE, and 5x Denhardt's solution (1x Denhardt's solution is 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, and 0.02% BSA).

The blots were hybridized with a 32P-labeled cDNA probe at 42°C for 16–20 h in the same solution. The probe used was quail ZPC cDNA (778-base HincII restriction fragment, position 88–866 in quail ZPC cDNA; GenBank accession no. AB012606). The radiolabeled cDNA probe was generated by random primer labeling of 50 ng of cDNA with {alpha}-32P-deoxycytidine 5'-triphosphate (1.85 MBq/reaction) using a kit (Redi Prime II random prime labeling system; Amersham). After hybridization, the membrane was washed twice with 2x SSPE and 0.1% SDS for 10 min each time at room temperature, followed by two washes in 1x SSPE and 0.1% SDS and two washes in 0.1x SSPE and 0.1% SDS for 15 min each time at 65°C. Membranes were exposed to autoradiographic film at -80°C for 1–5 days.

The amounts of ZPC mRNA transcript were analyzed by densitometric scanning of the autoradiographs and were normalized against the values obtained for ribosomal RNA.

3H-Leucine Incorporation into ZPC

After the culture of granulosa cells, the medium was removed, and the cells were washed with Hanks' BSS and incubated with 0.5 ml (0.57 MBq) of 3H-leucine (2.55 TBq/mmol; Amersham) in leucine-free RPMI-1640 medium. After incubation, the medium was collected, and the cells were washed twice with ice-cold PBS and then lysed with 0.5 ml of RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM TBS [pH 7.5]). The medium or the cell lysate (100 µl) was incubated for 1 h at 4°C with 25 µl of anti-ZPC antiserum (1:10 dilution). The immune complex was precipitated by centrifugation at 10 000 x g for 1 min after having been incubated for 30 min at 4°C with 25 µl of Immunoprecipitin (Staphylococcus aureus Cowan I; Gibco BRL, Gaithersburg, MD). The precipitates were washed twice with ice-cold PBS and solubilized in 50 µl of Laemmli's sample buffer by heating at 95°C for 5 min. The supernatants, after centrifugation at 10 000 x g for 1 min, were measured for their radioactivity by a liquid scintillation counter.

Statistical Analysis

All experiments were repeated from two to five times. Data were analyzed for significant differences using ANOVA, and means were compared using Duncan's multiple range test. A P value of less than 0.05 denoted a statistically significant difference.

RESULTS

ZPC Contents and mRNA Expression During Follicular Development

Western blotting of the SDS-solubilized granulosa layer obtained from the yellow follicles at defined points in time before ovulation is shown in Figure 1. Antiserum to quail ZPC [14] detected a 35-kDa immunoreactive product in the granulosa layer. Intensity of the immunostaining appeared to increase progressively during follicular development. The results of densitometric analysis of the immunostaining, expressed as an arbitrary unit per follicle, also revealed the increase in ZPC protein during follicular development (Fig. 1).



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  		FIG. 1. Increase in ZPC protein in the granulosa layer during follicular development. Aliquots (3 µg protein per lane) of the SDS-solubilized granulosa layer (granulosa cells + perivitelline membrane + basement membrane) of preovulatory follicles obtained at varying points in time before ovulation were separated on SDS-PAGE, and immunoreactive protein was detected using anti-quail ZPC antiserum. The signals were quantified and expressed per follicle, based on the total protein of the granulosa layer derived from one follicle. Representative of three independent experiments

Northern blotting of the total RNA extracted from various tissues, including the granulosa layer, theca layer, oviduct, liver, kidney, spleen, and several parts of digestive tracts, showed that only the granulosa layer contained the mRNA transcript of ZPC at approximately 1.4 kb (data not shown). The RNA extracted from the granulosa layers of various sizes of follicles were compared by Northern blotting, and the levels of ZPC mRNA transcript increased as the follicle developed to the largest preovulatory stage (Fig. 2).



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  		FIG. 2. Northern blot analysis of ZPC mRNA in granulosa layers of the largest (F1) to the fourth-largest (F4) follicles and the small, yellow follicles (SYF). Each lane contained 2.6 µg of total RNA. The size of the respective mRNA is indicated on the right in kilobases

Effects of Steroid Hormones on ZPC Production In Vitro

To investigate the effects of various steroid hormones on ZPC production, the granulosa cells obtained from the largest and the second-largest follicles were cultured for 48 h with increasing concentrations of progesterone, estradiol-17ß, or testosterone, and the media during 0–24 and 24–48 h of culture were subjected to Western blotting. Although no significant differences were found in the contents of ZPC in the culture medium during 0–24 h (data not shown), the addition of 100 nM and 1 µM testosterone caused an increase in the ZPC contents in the medium during 24–48 h (Fig. 3). This effect was observed in the granulosa cells of the largest and the second-largest follicles. Other hormones caused no significant increase, but the addition of progesterone to the culture of the granulosa cells of the largest follicle caused a decrease in ZPC content.



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  		FIG. 3. Effects of various steroid hormones on ZPC protein production. Granulosa cells of the largest (F1) and the second-largest (F2) follicles were cultured with 10, 100, or 1000 nM progesterone (P), estradiol-17ß (E2), or testosterone (T) for 48 h. The ZPC protein in the medium of 24–48 h of culture (8 µl of culture medium per lane) was detected using anti-quail ZPC antiserum. Immunoblots shown here are representative of at least three experiments

To examine the effect of testosterone more closely, the culture media were recovered every 6 h and subjected to Western blotting. As shown in Figure 4, the ZPC contents of the media were high during the initial 12 h of the culture, and the effect of testosterone was not obvious. Intensity of immunostaining in the media of the control culture faded by 30 h to an almost nondetectable level, whereas that of the culture with testosterone remained for up to 30 h. Consequently, the effect of testosterone became prominent after 18 h of culture in the largest follicle and after 12 h in the second-largest follicle.



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  		FIG. 4. Effect of testosterone on ZPC protein contents in the medium. Granulosa cells of the largest (F1) and the second-largest (F2) follicles were cultured with (T) or without (C) 1 µM testosterone for 30 h. The culture medium was changed and recovered every 6 h, and the ZPC protein was detected using anti-quail ZPC antiserum (8 µl of culture medium per lane)

Effect of Testosterone on 3H-Leucine Incorporation into ZPC

To verify that the effect of testosterone observed after 18 or 12 h (in the cells of the largest and the second-largest follicles, respectively) of culture was due to synthesis of the ZPC protein, the granulosa cells cultured for 24 h with or without 1 µM testosterone were incubated with 3H-leucine for 6 h, and the radioactivity immunoprecipitated with anti-quail ZPC antiserum was measured. As shown in Figure 5A, the radioactivity incorporated into ZPC was high in the cells cultured with testosterone. The incorporation of radioactivity into ZPC was linear for up to 8 h of the incubation (Fig. 5B).



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  		FIG. 5. A) Effect of testosterone on 3H-leucine incorporation into the immunoprecipitated ZPC protein. After 24 h of culture of the granulosa cells of the largest (F1) and the second-largest (F2) follicles with (T) or without (C) 1 µM testosterone, the cells were further incubated with 3H-leucine for 6 h. The incorporation of radioactivity into ZPC was measured by immunoprecipitation. Values are the mean ± SEM of triplicate experiments. **P < 0.01 compared with corresponding control (C). B) Time course of 3H-leucine incorporation into the immunoprecipitated ZPC protein. Granulosa cells were pooled from the three largest follicles. Values are the mean ± SEM of triplicate experiments

Effect of Testosterone on ZPC mRNA Expression

The effect of 1 µM testosterone on ZPC mRNA expression in vitro was investigated by Northern blotting. The granulosa cells were cultured with or without 1 µM testosterone for 24 h, and the total RNA was subjected to Northern blotting. As shown in Figure 6, mRNA levels increased when granulosa cells from the largest and the second-largest follicles were cultured with testosterone.



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  		FIG. 6. Representative Northern blot analysis of ZPC mRNA in granulosa cells of the largest (F1) and the second-largest (F2) follicles after 24 h of culture with (T) or without (C) 1 µM testosterone. Each lane contained 12.3 µg of total RNA

DISCUSSION

As reported previously in studies of hens [16, 37], quail granulosa cells produce ZPC, which is one of the components of the perivitelline membrane, and the ZPC content in follicles increases during the final stage of follicular development. The present study is, to our knowledge, the first to report that ZPC production is stimulated by testosterone in avian granulosa cells.

Little is known about the pattern and regulation of ZPC expression in other species. Immunohistochemical studies of rabbit, marmoset, rhesus monkey, and human ovaries have shown the presence of ZPC as early as in primordial follicles [38]. The in situ hybridization studies of cynomolgus monkey (Macaca fascicularis) ovaries also showed that ZPC was expressed in primordial follicles [39]. In the present study, we did not investigate the presence of ZPC in the prehierarchal follicles, but the ZPC contents expressed per unit of protein in the granulosa layer or per follicle were at undetectable levels in the small, yellow follicles. An abrupt increase both in ZPC protein and ZPC mRNA was observed only during the final 48 h of follicular development. In bovine ovaries, the follicle cells, especially the cumulus and corona radiata cells, contribute to ZPC mRNA synthesis to an increasing extent during follicular development, whereas during the early phases of development, the mRNA encoding ZPC is predominantly localized in the oocyte [40].

Such disparity among species indicates a species specificity in ZPC synthesis during follicular development. Because the properties of the egg envelope vary in different species [41], the ontogeny and sites of ZPC protein synthesis also are likely to vary. In mice, clear evidence shows that glycoproteins of the zona pellucida are synthesized exclusively by the oocyte itself [17], and the ZPC transcript has not been detected in granulosa cells [42]. In dogs and pigs, however, follicle cells take part in zona protein synthesis [43, 44]. Lee and Dunbar [19] have also demonstrated the presence of mRNA encoding the 55-kDa protein (ZPB family) in cultured rabbit granulosa cells.

Our study showed that the ZPC contents of the media gradually decreased in the control culture, whereas those in the culture with testosterone remained at significant levels. Moreover, intensity of ZPC mRNA was prominent when the granulosa cells were cultured with testosterone. To date, studies on the hormonal regulation of ZPC production have been limited. The immunohistochemical study of bovine follicles suggested that synthesis of ZPC in follicle cells was influenced by the medium used [40]. A cumulus-oocyte complex matured in vitro in a medium supplemented with FSH, LH, and heat-inactivated estrus cow serum showed high amounts of ZPC as well as enhanced frequencies of in vitro fertilization and blastocyst development. In contrast, a cumulus-oocyte complex matured in a medium without these supplements revealed reduced ZPC synthesis.

In female birds, androgen is synthesized by the theca cells of medium-sized follicles [45]. The peak concentration of testosterone in serum occurs 6 to 10 h before ovulation [46], and follicular venous plasma of the fourth- to second-largest follicles contain a significantly higher concentration of testosterone than that in peripheral plasma [47]. Secretion of androgen is regarded as the result of excess substrate, which cannot be metabolized to estrogen by aromatase, and the role of androgen in avian female reproduction has been unclear. However, the granulosa cells in chicken follicles express an androgen receptor [48], indicating that androgens may act on the follicular development via a paracrine and/or an autocrine action.

Johnson et al. [49] reported that testosterone, in coordination with estradiol-17ß, suppressed LH-stimulated progesterone production in hen granulosa cells, and Lee and Bahr [50, 51] showed that testosterone acted as a competitive inhibitor of cytochrome P450scc. It should be noted, however, that they observed the effects of testosterone on LH-stimulated progesterone production during short-term incubation. In contrast, Phillips et al. [52] reported that testosterone caused a significant increase in progesterone production during 48 h of culture. We also demonstrated that testosterone stimulated progesterone production during 66 h of culture of quail granulosa cells [53]. Thus, the role of testosterone in steroidogenesis is inconsistent and depends on the experimental conditions. Although ovulation can occur without any preovulatory increase in plasma testosterone [54], testosterone treatment induces ovulation in vitro, and administration of anti-testosterone antiserum in vivo effectively blocks ovulation [55]. Moreover, testosterone suppresses the activity of the plasminogen activator, a serine protease implicated in the process of follicular rupture [56]. Thus, testosterone is regarded as an important sex hormone in female birds. The present study has explored another important role of testosterone in avian follicular development.

In contrast to the effect of testosterone, addition of progesterone seemed to cause a decreased intensity of the immunoreactive band of ZPC protein in the culture media. Although we have not pursued the effect of progesterone on ZPC production in the present study, the response to progesterone might vary during the ovulation cycle, because the effect of progesterone was prominent in the granulosa cells of the largest follicle.

We have not investigated the precise mechanism of ZPC secretion from cells and the formation of fibrous molecules outside the cells. We speculate that ZPC secreted from granulosa cells is directly integrated into the perivitelline membrane in the space between oocyte and granulosa cells, because ZPC could not be detected in the serum. However, in our in vitro system, ZPC was in soluble form in the culture medium. This might be due to the disruption of the morphology of the granulosa cells and the oocyte. It is quite interesting in this regard that the soluble form of ZPC is present in bovine follicular fluid [40]. Efforts are currently underway to investigate the topology of ZPC secretion, in which selective secretion of ZPC to the apical surface of the granulosa cells forms the perivitelline membrane.

In conclusion, we have demonstrated that the addition of testosterone maintains ZPC contents in the culture of granulosa cells, and that ZPC mRNA expression is high in the culture with testosterone compared to the control. These results suggest that testosterone stimulates ZPC protein production at the gene transcription level.

FOOTNOTES

First decision: 6 July 2000.

1 Supported by Grants-in-Aid for Scientific Research (09660300 and 11660280 to M.M.) from the Ministry of Education, Science, Sports and Culture of Japan. Back

2 Correspondence: Makoto Mori, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. FAX: 81 54 238 4866; acmmori{at}agr.shizuoka.ac.jp Back

Accepted: August 28, 2000.

Received: June 8, 2000.

REFERENCES

  1. Dumont JN, Brummett AR. Egg envelopes in vertebrates. In: Browder LW (ed.), Developmental Biology: A Comprehensive Synthesis, vol. 1. New York: Plenum Press; 1985: 235–288.
  2. Harris JD, Hibler DW, Fontenot GK, Hsu KT, Yurewicz WC, Sacco AG. Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. DNA Seq 1994; 4:361–393.[Medline]
  3. McLeskey SB, Dowds CR, Carballada R, White RR, Saling PM. Molecules involved in mammalian sperm-egg interaction. Int Rev Cytol 1998; 177:57–113.[Medline]
  4. Bleil JD, Wassarman PM. Mammalian sperm-egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 1980; 20:873–882.[CrossRef][Medline]
  5. Bleil JD, Wassarman PM. Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm. J Cell Biol 1986; 102:1363–1371.[Abstract/Free Full Text]
  6. Bleil JD, Wassarman PM. Galactose at the nonreducing terminus of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein ZP3 is essential for the glycoprotein's sperm receptor activity. Proc Natl Acad Sci U S A 1988; 85:6778–6782.[Abstract/Free Full Text]
  7. Tain J, Gong H, Thomsen GH, Lennarz WJ. Gamete interactions in Xenopus laevis>: identification of sperm binding glycoproteins in the egg vitelline envelope. J Cell Biol 1997; 136:1099–1108.[Abstract/Free Full Text]
  8. Vo LH, Hedrick JL. Independent and hetero-oligomeric-dependent sperm binding to egg envelope glycoprotein ZPC in Xenopus laevis. Biol Reprod 2000; 62:766–774.[Abstract/Free Full Text]
  9. Bain JM, Hall JM. Observations on the development and structure of the vitelline membrane of the hen's egg: an electron microscope study. Aust J Biol Sci 1969; 22:653–665.[Medline]
  10. Bellairs R, Harkness M, Harkness RD. The vitelline membrane of the hen's egg: a chemical and electron microscopical study. J Ultrastruct Res 1963; 8:339–359.[CrossRef]
  11. Wyburn GM, Aitken RNC, Johnston HS. The ultrastructure of the zona radiata of the ovarian follicle of the domestic fowl. J Anat 1965; 99:469–484.[Medline]
  12. Mori M, Masuda N. Proteins of the vitelline membrane of quail (Coturnix coturnix japonica) eggs. Poult Sci 1993; 72:1566–1572.[Medline]
  13. Kido S, Doi Y. Separation and properties of the inner and outer layers of the vitelline membrane of hen's eggs. Poult Sci 1988; 67:476–486.
  14. Kuroki M, Mori M. Origin of 33 kDa protein of vitelline membrane of quail eggs: immunological studies. Dev Growth Differ 1995; 37:545–550.[CrossRef]
  15. Pan J, Sasanami T, Nakajima S, Kido S, Doi Y, Mori M. Characterization of progressive changes in ZPC of the vitelline membrane of quail oocyte following oviductal transport. Mol Reprod Dev 2000; 55:175–181.[CrossRef][Medline]
  16. Takeuchi Y, Nishimura K, Aoki N, Adachi T, Sato C, Kitajima K, Matsuda T. A 42-kDa glycoprotein from chicken egg-envelope, an avian homolog of the ZPC family glycoproteins in mammalian zona pellucida. Its first identification, cDNA cloning and granulosa cell-specific expression. Eur J Biochem 1999; 260:736–742.[Medline]
  17. Bleil JD, Wassarman PM. Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro. Proc Natl Acad Sci U S A 1980; 77:1029–1033.[Abstract/Free Full Text]
  18. Yamaguchi S, Hedrick JL, Katagiri C. The synthesis and localization of envelope glycoproteins in oocytes of Xenopus laevis using immunocytochemical methods. Dev Growth Differ 1989; 31:85–94.[CrossRef]
  19. Lee VH, Dunbar BS. Developmental expression of the rabbit 55-kDa zona pellucida protein and messenger RNA in ovarian follicles. Dev Biol 1993; 155:371–382.[CrossRef][Medline]
  20. Martinez ML, Fontenot GK, Harris JD. The expression and localization of zona pellucida glycoproteins and mRNA in cynomolgus monkeys (Macaca fascicularis). J Reprod Fertil Suppl 1996; 50:35–41.[Medline]
  21. Hamazaki T, Iuchi I, Yamagami K. A spawning female-specific substance reactive to anti-chorion (egg-envelope) glycoprotein antibody in the teleost, Oryzias latipas. J Exp Zool 1985; 235:269–279.[CrossRef]
  22. Hamazaki TS, Nagahama Y, Iuchi I, Yamagami K. A glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes. Dev Biol 1989; 133:101–110.[CrossRef][Medline]
  23. Hyllner SJ, Oppen-Bernsten DO, Helvik JK, Walther BT, Haux C. Oestradiol-17ß induces the major vitelline envelope proteins in both sexes in teleosts. J Endocrinol 1991; 131:229–236.[Abstract/Free Full Text]
  24. Murata K, Iuchi I, Yamagami K. Synchronous production of the low- and high-molecular-weight precursors of the egg envelope subunits, in response to estrogen administration in the teleost fish, Oryzias latipes. Gen Comp Endocrinol 1994; 95:232–239.[CrossRef][Medline]
  25. Larsson DGJ, Hyllner SJ, Haux C. Induction of vitelline envelope proteins by estradiol-17ß in 10 teleost species. Gem Comp Endocrinol 1994; 96:445–450.
  26. Miura T, Kudo N, Miura C, Yamauchi K, Nagahama Y. Two testicular cDNA clones suppressed by gonadotropin stimulation exhibit ZP2- and ZP3-like structures in Japanese eel. Mol Reprod Dev 1998; 51:235–242.[CrossRef][Medline]
  27. Mori M, Huang JS, Kuroki M. Evidence suggesting that the 33 kDa glycoprotein mediates sperm binding of quail perivitelline layer. Zygote 1998; 6:112.
  28. Woodard AE, Mather FB. The timing of ovulation, movement of the ovum through the oviduct, pigmentation and shell deposition in Japanese quail (Coturnix coturnix japonica). Poult Sci 1964; 43:1427–1432.
  29. Gilbert AB, Evans AJ, Perry MM, Davidson MH. A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus). J Reprod Fertil 1977; 50:179–181.[Abstract/Free Full Text]
  30. Mori M, Kantou T. Changes in progesterone production in granulosa cells during the ovulatory cycle of the Japanese quail (Coturnix coturnix japonica). Gen Comp Endocrinol 1987; 68:57–63.[CrossRef][Medline]
  31. Roberts RD, Sharp PJ, Burt DW, Goddard C. Insulin-like growth factor-I in the ovary of the laying hen: gene expression and biological actions on granulosa and thecal cells. Gen Comp Endocrinol 1994; 93:327–336.[CrossRef][Medline]
  32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685.[CrossRef][Medline]
  33. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265–275.[Free Full Text]
  34. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 1987; 262:10035–10038.[Abstract/Free Full Text]
  35. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156–159.[Medline]
  36. You S, Bridgham JT, Foster DN, Johnson AL. Characterization of the chicken follicle-stimulating hormone receptor (cFSH-R) complementary deoxyribonucleic acid, and expression of cFSH-R messenger ribonucleic acid in the ovary. Biol Reprod 1996; 55:1055–1062.[Abstract]
  37. Waclawek M, Foisner R, Nimpf J, Schneider WJ. The chicken homologue of zona pellucida protein-3 is synthesized by granulosa cells. Biol Reprod 1998; 59:1230–1239.[Abstract/Free Full Text]
  38. Grootenhuis AJ, Philipsen HLA, de Breet-Grijsbach JTM, van Duinm M. Immunocytochemical localisation of ZP3 in primordial follicles of rabbit, marmoset, rhesus monkey and human ovaries using antibodies against human ZP3. J Reprod Fertil 1996; 50:43–54.
  39. Martinez LL, Fontenot GK, Harris JD. The expression of zona pellucida glycoproteins and mRNA in cynomolgus monkey (Macaca fascicularis). J Reprod Fertil 1996; 50:35–41.
  40. Kolle S, Sinowatz F, Boie G, Palma G. Differential expression of ZPC in the bovine ovary, oocyte, and embryo. Mol Reprod Dev 1998; 49:435–443.[CrossRef][Medline]
  41. Dunbar BS, Avery S, Lee V, Prasad S, Schwahn D, Schwoebel E, Skinner S, Wilkins B. The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology, and developmental expression. Reprod Fertil Dev 1994; 4:59–76.
  42. Philpott CC, Ringuette MJ, Dean J. Oocyte-specific expression and developmental regulation of ZP3, the sperm receptor of the mouse zona pellucida. Dev Biol 1987; 121:568–575.[CrossRef][Medline]
  43. Nickson DA, Boyd JS, Eckersall PD, Ferguson JM, Harvey MJ, Renton JP. Molecular biological methods for monitoring maturation and in vitro fertilization in bitches. J Reprod Fertil 1993; 47:231–240.[CrossRef]
  44. Kolle S, Sinowatz F, Boie G, Totzauer I, Amselgruber W, Plendl J. Localization of the mRNA encoding the zona protein ZP3{alpha} in the porcine ovary, oocyte, and embryo by nonradioactive in situ hybridization. Histochem J 1996; 28:441–447.[CrossRef][Medline]
  45. Etches RJ, Duke CE. Progesterone, androstenedione and oestradiol content of theca and granulosa tissues of the 4 largest ovarian follicles during the ovulatory cycle of the hen (Gallus domesticus). J Endocrinol 1984; 103:71–76.[Abstract/Free Full Text]
  46. Johnson Al, van Tienhoven. Plasma concentrations of six steroids and LH during the ovulatory cycle of the hen, gallus domesticus. Biol Reprod 1980; 23:386–393.[Abstract]
  47. Etches BJ, Croze F, Duke E. Plasma concentrations of luteinizing hormone, progesterone, testosterone and estradiol in follicular and peripheral venous plasma during the ovulation cycle of the hen. Adv Physiol Sci 1981; 33:89–98.
  48. Yoshimura Y, Chang C, Okamoto T, Tamura T. Immunolocalization of androgen receptor in small, preovulatory, and postovulatory follicles of laying hens. Gen Comp Endocrinol 1993; 97:81–89.
  49. Johnson PA, Green C, Lee HT, Bahr JM. Inhibition of progesterone secretion from granulosa cells by estradiol and androgens in the domestic hen. Endocrinology 1988; 123:473–477.[Abstract/Free Full Text]
  50. Lee HT, Bahr JM. Inhibitory site of androgens and estradiol in progesterone biosynthesis in granulosa cells of the domestic hen. Endocrinology 1989; 125:760–765.[Abstract/Free Full Text]
  51. Lee HT, Bahr JM. Inhibition of the activities of P450 cholesterol side-chain cleavage and 3ß-hydroxysteroid dehydrogenase and the amount of P450 cholesterol side-chain cleavage by testosterone and estradiol-17ß in hen granulosa cells. Endocrinology 1990; 126:779–786.[Abstract/Free Full Text]
  52. Phillips A, Scanes CG, Hahn DW. Effect of androgens and gonadotropins on progesterone secretion of chicken granulosa cells. Comp Biochem Physiol 1985; 81A:847–852.
  53. Sasanami T, Mori M. Effects of oestradiol-17ß and testosterone on progesterone production in the cultured granulosa cells of Japanese quail. Br Poult Sci 1999; 40:536–540.[CrossRef][Medline]
  54. Johnson AL, van Tienhoven A. Effects of aminoglutethimide on luteinizing hormone and steroid secretion, and ovulation in the hen, Gallus domesticus. Endocrinology 1984; 114:2276–2283.[Abstract/Free Full Text]
  55. Tanaka K, Inoue T. Role of steroid hormones and catecholamines in the process of ovulation in the domestic fowl. In: Wada M, Ishii S, Scanes CG (eds.), Endocrinology of Birds: Molecular to Behavioral. Berlin: Springer-Verlag; 1990: 59–68.
  56. Tilly JL, Johnson AL. Presence and hormonal control of plasminogen activator in granulosa cells of the domestic hen. Biol Reprod 1987; 37:1156–1164.[Abstract]



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