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Biology of Reproduction 60, 522-526 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Sequential Expression of Zona Pellucida Protein Genes during the Oogenesis of Domestic Cats

Katarina Jewgenow1,a, and Jörns Fickela

a Institute for Zoo Biology and Wildlife Research, D-10252 Berlin, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of the present study was to determine the earliest developmental stage of zona pellucida (ZP) protein gene expression during oogenesis in domestic cats (Felis catus) by means of immunohistochemical and molecular biological methods.

Semi-thin sections (1 µm) from domestic cat ovaries were treated with anti-cat ZP serum raised in guinea pig, and then incubated with silver-labeled anti-guinea pig IgG. To distinguish between the three ZP proteins, total RNA was extracted from freshly isolated cat primordial, primary, and secondary follicles as well as from cumulus-oocyte complexes (COCs) and subjected to reverse transcription (RT). The generated cDNAs were used for polymerase chain reaction (PCR) with specific feline ZPA, ZPB, and ZPC gene primers. All amplified products were sequenced to confirm their identity. Neither ZP mRNAs nor ZP proteins were detectable in primordial and early primary follicles. The immunohistological approach indicated the expression of ZP proteins in some of the primary follicles as well as in secondary follicles and COCs. Follow-up by RT-PCR revealed that only one ZP (ZPB) was expressed in growing primary follicles (70–80 µm), whereas all three ZP mRNAs were detectable in secondary follicles and COCs. We therefore assume a sequential synthesis of zona proteins in the cat ovary.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
About 1500 preantral follicles can be recovered from a single cat ovary by mechanical dissection [1]. If these ova could be grown in vitro, they would be a potentially rich source of genetic material, especially from members of rare or endangered cat species. However, we still have comparatively little understanding of gamete development within the cat ovary during the growth phase from resting to antral follicles. Better knowledge of oogenesis is necessary for an understanding not only of the cellular and molecular bases of fertility, but for introducing effective long-term culture systems for "oocyte rescue" in order to retain genetic diversity in rare species.

The comparative biology approach has already been very fruitful in gaining knowledge for oogenesis research, using the mouse as a model species. During oogenesis, mammalian oocytes become more complex in their cytoplasmic organization. They become competent to resume meiosis and are able to undergo fertilization and cleavage division [2]. These changes depend on the production of new gene products and organelles during oogenesis as well as on the modification and redistribution of existing ones. One of the most conspicuous changes during oogenesis is the secretion of the zona pellucida (ZP), which is the extracellular coat surrounding the mammalian oocyte. This structure is involved in species-specific sperm-egg binding, in the induction of the acrosome reaction, and in the post-fertilization blockage of polyspermy [3]. Although the mouse is an elegant model for studying oogenesis in vivo and in vitro, questions remain as to whether or not this model accurately reflects the mechanisms of oocyte development in other species. The few data available regarding ZP protein gene expression in other mammalian species reflect differences among mammals and between the three zona proteins (for review, see [4]).

The aim of the present study was to determine the earliest developmental stage of ZP gene transcription and protein synthesis during oogenesis 1) by detection of mRNAs of the three ZP protein genes in primordial, primary, secondary, and tertiary follicles; and 2) by immunohistochemical detection of ZP proteins with specific anti-cat ZP antibodies. The purpose was to develop a tool permitting the qualitative analysis of mRNA in isolated cat preantral follicles as well as the monitoring of gene expression of an oogenesis marker (ZP synthesis) to accompany our in vitro studies in cats [5, 6], since ZP genes are not only a potential paradigm for studying mechanisms of oocyte-specific gene expression but also a useful marker for oocyte growth and differentiation [7].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All chemicals were obtained from Sigma (Sigma Chemie GmbH, Deisenhofen, Germany) unless stated otherwise and were of the highest purity available. Ovaries were obtained from 14 domestic cats ovario-hysterectomized at local veterinary clinics. The exact age and reproductive history of each donor was almost always unknown; however, the majority of donors were 12–24 mo old and believed to be nulliparous. After excision, ovaries were placed into PBS at 4°C and were processed within 4 h.

Collection of Cumulus-Oocyte Complexes (COCs) and Preantral Follicles

After being washed, ovaries were minced in 10 ml PBS using a scalpel blade. COCs were released from antral follicles by repeated slicing of the ovarian cortex. All COCs exhibiting uniform, dark-pigmented ooplasm and an intact cumulus cell layer (Fig. 1D) were chosen, transferred into a 0.5-ml tube containing 100 µl PBS, and snap-frozen at -70°C. The dissection procedure for preantral follicles has been described elsewhere [1]. In brief, ovaries were carefully pressed through a cell dissociation sieve (60 mesh; Sigma) together with 20 ml PBS. The resulting cell suspension was passed through a series of nylon sieves (100 µm, 70 µm, 40 µm cell strainers; Falcon, Becton Dickinson Labware, Franklin Lakes, NJ). Every cell strainer was rinsed in 10 ml PBS, allowing the recovery of all follicles with a diameter exceeding the appropriate mesh size. The cell suspension flushed from the 40-µm sieve consisted of primordial and early primary follicles with one layer of granulosa cells (Fig. 1A). The suspension was washed and adjusted to 500 follicles per 100 µl PBS; each 100 µl suspension was transferred into a 1.5-ml tube. In the same way, growing preantral follicles with diameters of 70–80 µm (Fig. 1B) as well as those larger than 100 µm (Fig. 1C) were obtained by flushing the corresponding cell strainers. Calibrated glass pipettes were used to separate them from cells and tissue shreds, which were flushed from the respective sieves as well. The growing follicles that were obtained (70–80 µm, n = 50; > 100 µm, n = 20) were transferred into 1.5-ml tubes containing 100 µl PBS. Follicle aliquots were immediately snap-frozen at -70°C and stored until further use. Three repeats with 4 cats per repeat were performed.



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  		FIG. 1. Isolated preantral follicles (A–C) from domestic cat ovaries, and an intact feline cumulus oocyte-complex (D). A) Primordial follicles and early primary follicle with unilaminar layer of granulosa cells (40–50 µm). Diameter of oocytes is about 30–40 µm. B) Growing primary follicles (70–80 µm) with one or two layers of granulosa cells and oocytes of 50-µm diameter. C) Secondary preantral follicles (> 100 µm) with diameter and oocytes of 90–100-µm diameter. Bar = 75 µm.

Reverse Transcription (RT) Polymerase Chain Reaction (PCR) of Zona Pellucida mRNA

Total RNA from preantral follicles and COCs was extracted as described by Chomczynski and Sacchi [8]. RNA concentrations were determined photometrically. For RT reactions, 20 ng, 40 ng, 80 ng, 100 ng, and 200 ng total RNA (in 3 µl) were used. The RT reaction (final volume 20 µl) consisted of 40 units Maloney murine leukemia virus-reverse transcriptase (Perkin-Elmer, Irvine, CA), 2 units ribonuclease (RNase) inhibitor (Perkin Elmer), 5 mM MgCl2, 4 mM deoxy (d) NTPs (each) (rTth; Boehringer Mannheim, Mannheim, Germany), 20 pmol oligo(dT)12–18 primers or random hexamers (parallel assay) (Pharmacia LKB, Piscataway, NJ) in 50 mM KCl, and 10 mM Tris pH 8.3. Incubation was as follows: 30 min at 37°C, 15 min at 42°C, 5 min at 99°C, and 5 min at 0°C. The concentrations of all components as well as incubation times had been optimized previously. In parallel we performed RT reactions using DNA-polymerase from Thermophilus spec. (Boehringer Mannheim) at 70°C according to the manufacturer's instructions. Ten microliters of each RT reaction (cDNA) was subsequently used for a 50-µl PCR containing 1.25 units Taq polymerase (Perkin Elmer), 2 mM MgCl2, 400 µM dNTP, 10 pmol each primer, 50 mM KCl, and 10 mM Tris pH 8.3. After denaturation for 3 min at 95°C, amplification was for 35 cycles at 94°C, 30 sec; 50°C, 45 sec; and 72°C, 50 sec, with a final elongation phase of 5 min at 72°C. PCR conditions for ZP matrices were optimized using cDNA of COCs. Amplification products were cloned and subsequently identified by dideoxy sequencing [9].

Primer sequences were determined using OLIGO software (V.5.0; National Biosciences Inc., Plymouth, MN) according to Harris et al. [10], referring to the longest cDNA as ZPA and to the shortest as ZPC. The following primers were used: felZPAforw 5'-GGGAAGTTAAAATCTGTGAGC-3' (nt 841–861); felZPArev 5'-TGGTTTTGTCTGGTGACTG-3' (nt 1147–1164); felZPBforw 5'-GGTGGCAGCTAGAGATGTGAG-3' (nt 882–902); felZPBrev 5'-CGTTCTGTGGCGGATAGAGAC-3' (nt 1153–1173); felZPCforw 5'-CAGGCCGAAGTCCACAC-3' (nt 589–605); and felZPCrev 5'-TCATGTTTCTGGGGTCATTAG-3' (nt 806–826). An internal control PCR was performed with all cDNAs to amplify a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene fragment with the following primers: GAPDHforw 5'-TGCACCACCAACTGCTTAGC-3' (nt 478–597 of the rat GAPDH cDNA) and GAPDHrev 5'-CAAGAAGGTGGTGAAGCAGG-3' (nt 839–858) [11]. All RTs and PCRs were performed 3 times, each time with pooled RNA from the respective cell preparations of 4 cats.

Immunohistological Detection of ZP Proteins in Cat Ovary Sections

Ovaries from two domestic cats were fixed with 3% glutaraldehyde immediately after hysterectomy and embedded in Unicryl (British BioCell International, Cardiff, UK). Semi-thin sections (1 µm) were cut and subsequently incubated in 50 mM glycine in PBS for 15 min and in PBS with 5% BSA, 0.1% gelatin, and 1% normal goat serum for 30 min. After being washed in PBS with 0.1% BSA (3 x 5 min), the slides were treated with anti-cat ZP serum (1:80, raised in guinea pig [12]) for 24 h at 4°C, incubated with gold-conjugated goat anti-guinea pig antibody (GAGp GP-US; BioTrend Chemikalien GmbH, Köln, Germany; 1:50) for 4 h, and fixed in 2% glutaraldehyde for 10 min. The sections were enhanced in R-Gent (BioTrend) for 30 min, counterstained in methylene blue, and embedded in Entellan (Merck, Darmstadt, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The antiserum against zona pellucida proteins of domestic cats reacted very specifically with extracellular zona proteins on semi-thin ovarian tissue sections (Fig. 2, A and B). Using immunohistochemistry, ZP antigens were not detectable intracellularly either in oocytes or in granulosa cells. All oocytes from secondary and antral follicles were surrounded by a thick zona pellucida (Fig. 2A). About 30% of follicles with a unilaminar layer of granulosa cells (60 out of 208 follicles on 4 slides) were characterized by a partial coating of ZP antigens in the perivitelline space (Fig. 2B). Because of the limited number of follicles analyzed, this relationship does not reflect the real quantitative distribution of primordial and primary follicles in the cat ovary.



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  		FIG. 2. Semi-thin section of plastic-embedded domestic cat ovaries with immunohistological labeling of zona pellucida proteins. A) Oocyte of an antral follicle. Insert represents the control to the immunostaining, achieved by omitting the primary antibody. B) Early growing follicle with one and more layers of granulosa cells expressing a complete (1) and a partial coating of ZP antigens in the perivitelline space (2). Bar = 10 µm.

Extensive RT-PCRs with specific primers for the feline ZPA, ZPB, and ZPC gene did not detect any ZP signal in primordial and early primary follicles (40–50 µm; Fig. 3, lanes 1–3), although up to 1 µg (in 100 ng steps) of the respective cDNA was used in the PCR. These results were independent from the kind of primers used in the RT reaction. GAPDH PCR with 20 ng cDNA amplified the expected fragment (Fig. 3, lanes 4, 8, 12, 16). The use of DNA-polymerase from Thermophilus spec. in the RT-PCR at 70°C to avoid possible cDNA synthesis problems due to potential secondary RNA structures did not change the outcome, nor did a preamplification step with 10 cycles before the "regular" PCR. In growing primary follicles (70–80 µm; Fig. 3, lanes 5–7), only ZPB gene expression was detectable (Fig. 3, lane 6), whereas in secondary follicles (> 100 µm; Fig. 3, lanes 9–11) and COCs (Fig. 3, lanes 13–15), all three ZP gene transcripts were detectable. Sequence comparison of those PCR fragments with the respective sequences published by Harris et al. [10] confirmed their identity with the zona pellucida gene sequences. Additionally, sequencing revealed a point mutation within the ZPB gene fragment at position 978 (transition T->C, Fig. 4A). The same transition T->C was detected within the ZPC gene fragment at position 783 (Fig. 4B). Neither mutation altered the amino acid sequence.



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  		FIG. 3. Gel electrophoresis of zona pellucida PCR products from follicles of different developmental stages (10 µl of each PCR, PCR with 200 ng cDNA, cDNA primed with oligo(dT)12–18). POF, primordial folllicles; PF, growing primary follicles; SF, secondary follicles. The arrow (lane 6) indicates the earliest detectable PCR product. M, molecular weight marker, 100-bp ladder.



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  		FIG. 4. A) Nucleotide and deduced amino acid sequence of a part of the 249-bp felis catus ZPB gene PCR fragment (w/o primers); lower sequence (*) according to Harris et al. [10]; the underlined C at position 978 indicates a transition (T->C). B) Nucleotide and deduced amino acid sequence of a part of the 199-bp felis catus ZPC gene PCR fragment (w/o primers); lower sequence (*) according to Harris et al. [10]; the underlined C at position 783 indicates a transition (T->C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This appears to be the first study investigating the starting point of the expression of all three zona pellucida genes during oogenesis in a nonrodent animal by RT of mRNA obtained from isolated follicles. Since it was not our objective to differentiate between oocytes and granulosa cells, we used whole follicles as functional units of oogenesis. It is still debated whether the zona components originate from oocytes, from granulosa cells, or from both, and whether or not the appearance of ZP is specific to a particular stage of follicular development. In mice, oocytes alone are capable of producing all zona proteins; in other species, granulosa cells also contribute to the synthesis of zona components. Immunohistochemical studies and in situ hybridization experiments led to ZP-positive staining of granulosa cells in secondary follicles and follicles at more advanced stages [13–15]. Because of our cell isolation method, disintegration of follicle structures would have led to damage of the oocytes. However, the separation of follicles according to their size allows a detailed analysis of mRNA species using the highly sensitive RT-PCR method [16], therefore providing an additional marker to determine the developmental stage of the follicles.

Oocytes in the mid-growth stage produce approximately ten times more RNA (particularly ribosomal RNA) than most somatic cells [17]. The mature mouse egg contains a total of 0.3–0.55 ng RNA, approximately 65% being rRNA, 20% 5S RNA and tRNA, and 15% heterogeneous RNA, the last fraction containing about 8% poly(A)+ RNA (mostly mRNA), which is potentially available for translation into protein [17]. Isolation and analysis of genes involved in oogenesis present a major challenge for research since they usually require the construction of cDNA libraries from ovaries and follicles. The RT-PCR results presented show the possibility of obtaining a sufficient amount of intact mRNA, which could be used for cDNA libraries of particular stages of oogenesis. On the basis of the sequential ZP expression pattern that we demonstrated, a specific and growth-phase-dependent yield of follicles at different developmental stages is possible. Using a similar methodological approach, it was shown that the human FSH receptor is expressed in growing preantral follicles but not in dormant primordial follicles [16]. Analysis of gene expression in isolated preantral follicles, which were classified according to their morphology and their size, is a powerful approach in humans and other animals in which the developmental stage of oocytes is impossible to determine by the prepubertal age of ovary donors.

The GAPDH gene transcript was detectable in all follicle classes, showing that all cells of all developmental stages were actively synthesizing mRNA. The GAPDH gene is controlled mostly by energy requirements and is always expressed constitutively, whereas for the transcription of ZP genes in cat ovaries, follicles from the resting pool (40–50 µm) have to be recruited to growing primary follicles (> 70 µm).

The first detectable gene was ZPB, whose transcripts were present in growing follicles with one or two layers of granulosa cells (70–80 µm), specifying the results of our immunohistochemical ZP-detection. Therefore, not only transcription of the ZPB gene, but also translation is evident in growing primary follicles. The two other zona proteins are expressed in the cat only after development to the next stage. The sequential ZP expression pattern described here raises the question as to what might be responsible for such a maturation-dependent "switch on" of the zona pellucida gene expression. We believe that the transcription is probably triggered by growth factors or developmentally induced transcription factors. Even though such a scenario is very likely, it remains to be elucidated. This sequential transcription and, presumably, synthesis differ entirely from the expression pattern observed in mice. In mice, ZP1 (ZPB) transcripts were detected only after growth initiation of the oocytes [7], whereas the mouse ZP2 (ZPA) gene was already being transcribed before birth in primary oocytes [17] and in resting follicles. ZP2 (ZPA) and ZP3 (ZPC) proteins are coordinately expressed in mouse oocytes during their 3 weeks of growth phase [7]. It has been shown that the elimination of either ZP2 (ZPA) or ZP3 (ZPC) synthesis by the use of either antisense oligonucleotides or gene knockouts prevents zona pellucida assembly [18–20]. During ovulation, the level of ZP3 (ZPC) mRNA declines dramatically until it becomes undetectable in fertilized eggs [21]. For mouse oocytes surrounded by two or more layers of cumulus cells, an increase in ZP3 (ZPC) transcripts was demonstrated in in vitro cultures, with peak levels at 48 h after commencement of cultivation [22].

Little is known about the pattern of ZP expression in other species. Most data are available from contraceptive and immunohistological studies. In some species, active immunization with zona proteins results in oophoritis, with a progressive depletion of the resting oocyte pool, whereas in other species a reversible infertility is documented [23]. Contraceptive effects or ovarian dysfunction mainly depends on the zona protein from which the immunogen was derived as well as on the target animal immunized. Studies in monkeys (Macaca fascicularis) immunized with recombinant rabbit ZP proteins showed that antibodies against a recombinant 75-kDa protein (ZPA family) were responsible for ovarian pathology, while antibodies against a recombinant 55-kDa protein (ZPB family) accounted for infertility effects without any ovarian pathology. This might be due to the fact that the ZPA protein is expressed earlier in follicular development than is ZPB [23]. Using in situ hybridization to analyze the pattern of expression of zona pellucida glycoproteins in cynomolgus monkey (Macaca fascicularis) ovaries, Martinez et al. [14] found that ZPA and ZPC were expressed as early as in primordial (ZPC) and primary (ZPA) follicles. In contrast to our results, ZPB was not detected before the secondary follicle stage. Immunohistochemical studies of rabbit, marmoset, rhesus monkey, and human ovaries showed the presence of ZPC on primordial follicles [13], although those results are nonconclusive since absence of cross-reactivity [24] to the other ZPs was not shown. On the other hand, in squirrel monkeys, reversible ovarian dysfunction in terms of both ovarian steroid secretion and oocyte production was found after immunization with ZPC from the porcine zona pellucida, leading to the conclusion that only advanced stages of folliculogenesis were affected by such treatment, whereas the pool of resting oocytes remains uninfluenced [25]. In rabbit ovaries, the 55-kDa protein (ZPB family) is localized within primordial oocytes and ovarian follicles of more advanced stages, whereas the 45-kDa (ZPC family) and the 75-kDa (ZPA family) proteins are lacking primordial follicles [26]. The disparity among species may be caused by different methods and ZP antibodies used, but surely indicates a species specificity in ZP synthesis during oogenesis. Since the properties of mammalian ZP vary in different species [27], it is very likely that the time frames and sites of ZP protein synthesis also vary [26].

Because of its major role in the fertilization processes (interaction with sperm, induction of acrosome reaction, control of polyspermy), the ZP is an attractive target for contraceptive vaccination. The ZP possesses a strong immunological heterospecificity [28]. Active immunization with various forms of ZP glycoproteins leads to a blockage in fertility that can be either reversible or irreversible. Irreversible infertility is potentially useful in controlling populations of animals such as dogs, horses (mares), and stray cats. Analysis of such irreversibility reveals that intervention does not occur at the level of sperm-egg interaction but is due to the disruption of normal ovarian oogenesis [29]. This is acceptable, sometimes even desirable, for the development of animal immunocontraceptives, but must be avoided for human purposes or for controlling reproduction of rare animals in zoos. Therefore, it is important to determine which ZP proteins do not alter ovarian follicular development [23] but do elicit antibodies that interfere with sperm binding only.

In conclusion, we demonstrated the sequential synthesis of zona proteins in the cat ovary. ZPB is synthesized and already excreted in growing follicles with one layer of granulosa cells (primary follicles). The sequential expression of cat zona proteins is of practical relevance for designing an immunocontraceptive for felid species. ZPB antigens are suited for controlling the feral cat population. The induced infertility would be irreversible, since the destruction of early follicles would exhaust the resting oocyte pool. In the case of rare felid species in which infertility must be reversible (e.g., in controlled zoo populations), the two other zona proteins (ZPA, ZPC) are suitable contraceptive vaccinogens. In the latter case, application of a crude protein mixture (e.g., porcine zonae pellucidae vaccine [30]) is not recommended.


   ACKNOWLEDGMENTS
 
The authors thank Susanne Auls and Doris Fichte for technical assistance. We also acknowledge John E. Cooper for his advice and for improving the English.


   FOOTNOTES
 
1 Correspondence: K. Jewgenow, Institute for Zoo Biology and Wildlife Research, PF 601103, D-10252 Berlin, Germany. FAX: 49 030 5126104; jewgenow{at}izw-berlin.de Back

Accepted: September 28, 1998.

Received: May 19, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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