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a Department of Physiology, The University of Manitoba, Winnipeg, Manitoba, Canada R3E 3J7
b Departments of Physiology and Obstetrics and Gynaecology, The University of Western Ontario, London, Ontario, Canada N6A 5C1
c Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7400
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are 14 published rodent connexin sequences, with several more in the process of being characterized. Connexins are distinguished by their different sizes: connexin26 (Cx26) is the smallest at 26 kDa, whereas connexin50 (Cx50) is almost twice as large at 50 kDa. All connexins have four membrane-spanning domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-termini. Sequence similarity is concentrated in the transmembrane domains and extracellular loops, whereas most of the sequence and length variation among the connexins resides in their cytoplasmic loop and C-terminal tail regions. It is this diversity that is assumed to account for most of the distinct biophysical, permeability, and regulatory properties that are exhibited by gap junction channels composed of different connexins [1, 2].
Many members of the connexin family could potentially be involved in reproductive processes. In the preimplantation mouse embryo, for example, at least six connexin genes are transcribed, and four of those connexins have been shown to contribute to gap junction assembly beginning with compaction [7–10]. The expression of multiple connexins in preimplantation embryos provides an explanation for the fact that targeted disruption of two of those genes (those encoding Cx43 and Cx31) had no effect on development to the blastocyst stage ([10, 11]; K. Willecke, personal communication).
The ovary is another location where multiple connexins are expressed. Using reverse transcription-polymerase chain reaction, Cx32 and Cx43 mRNAs were identified in both fully grown mouse oocytes and their associated cumulus cells [12]. The presence of both connexins in cumulus cells was confirmed by confocal immunofluorescence microscopy [12]. Expression of Cx32 and Cx43, like intercellular coupling itself, is down-regulated during gonadotropin-induced meiotic maturation [12–15]. Cx40 and Cx45 are also expressed in the rodent ovary: Cx40 is confined to the vascular endothelium whereas Cx45 is coexpressed with Cx43 in some gap junctions of the cumulus granulosa [15]. Connexin37 is also present in mouse ovarian follicles, restricted to the interface between the oocyte and cumulus cells. Targeted disruption of the gene encoding Cx37 resulted in failure of graafian follicle development and lack of ovulation [16].
Gap junctional coupling is also likely to be functionally important in the testis, where there is evidence for the presence of connexins 26, 30, 32, 33, 37, and 43. Whereas Cx37 appears to be restricted to the vascular endothelium, Cx43 is abundant in gap junctions between Leydig, Sertoli, and peritubular cells [17, 18]. It may also couple Sertoli cells with spermatogonia and spermatocytes [19]. A few gap junctions containing Cx26 or Cx32 were noted in the apical regions (spermatids and/or Sertoli cells) of the seminiferous epithelium [17]. Cx33 colocalizes with Cx43 in some Sertoli-Sertoli gap junctions [18]. The spatial distribution of Cx30 in the testis has not been investigated [3].
In the present study, we examined the gonads of fetal and neonatal mice lacking Cx43 to determine whether the absence of this connexin has any consequences for gonadal development (mice lacking Cx43 die soon after birth because of a heart abnormality [11]). We found that in both sexes at the time of birth, the gonads were unusually small. This appears to be caused, at least in part, by a deficiency of germ cells. The germ cell deficiency was traced back as far as Day 11.5 of gestation, implying that it arises before or during the onset of gonadal development. Finally, we used an organ culture technique to show that postnatal folliculogenesis is abnormal in ovaries lacking Cx43. These results make it clear that gap junctional coupling mediated by Cx43 channels plays indispensable roles in germ line development and ovarian folliculogenesis. Some of these results were reported previously in abstract form [20].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mice used in this study were derived from the ES24 line created by Reaume et al. [11] and originally maintained on a C57Bl/6 background; for our experiments they had been bred for two or more generations into the CD1 background. They were maintained and handled in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction. Homozygotes were obtained from heterozygote matings. Pregnant females were anesthetized with CO2 and then killed by cervical dislocation; embryos or fetuses were dissected from the uteri and decapitated. The genotype of each was determined from a skin sample by polymerase chain reaction (PCR) [10]. Embryos and fetuses were washed three times with PBS to remove any adherent tissue coming from the mother, and the forceps were rinsed thoroughly between samples. Each skin sample (2–4 mm2) was digested at 56°C for 8–16 h in 100 µl of solution containing proteinase K (Gibco BRL, Canadian Life Technologies Inc., Burlington, ON, Canada). This solution was prepared by adding 4 µl of proteinase K stock solution (20 mg/ml) to 96 µl proteinase K buffer (10 mM Tris pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.1 mg/ml gelatin, 0.45% Nonidet P-40, 0.45% Tween 20). One microliter of digested skin solution served as template for each PCR reaction; this was combined with 5.1 µl Milli-Q water (Millipore Ltd., Nepean, ON, Canada), placed in a PCR reaction tube, heated to 95°C for 15 min (to inactivate the proteinase K), and then held at 85°C while the remaining reaction ingredients were added ("hot start"). Details of primers and amplification conditions were as described by De Sousa et al. [10]; the reactions to detect the wild-type and targeted alleles were carried out in separate tubes.
Histological Analysis of Gonads
The ovaries and testes from fetuses at 17.5 days postcoitus (dpc), obtained as described above, were fixed in 2% glutaraldehyde and 2% sucrose in 0.1 M phosphate buffer (pH 7.3) for 6 h and then postfixed in 2% OsO4 for 1 h. The organs were dehydrated in increasing concentrations of acetone and then embedded in Epon resin (Marivac Ltd., Halifax, Nova Scotia). The circumference of each organ was measured and the maximum cross-sectional area calculated; the data were examined by two-way ANOVA using Statgraphics software (Manugistics, Inc., Rockville, MD). All the organs were oriented in the same manner in the blocks; if the orientation of one was not optimal, it was not included in the study. Each block was then cut into 0.5-µm serial sections that were transferred to a glass slide on a drop of distilled water. The slides were heated to 70°C to attach the sections. The sections were stained with 0.5% alkaline toluidine blue. Germ cells in the testes were identified by their large, round nuclei, which made them clearly distinguishable from the somatic cells of the seminiferous tubules. Germ cells in the ovaries (primary oocytes) were identified by the presence of meiotic chromosomes in their nuclei and the fact that their nuclei usually appear larger and rounder than those of the surrounding somatic cells.
Immunolocalization
A rat IgM monoclonal antibody (10D9G11; characterized previously [21]), which recognizes the germ cell nuclear antigen (GCNA1) in prospermatogonia of male mice and oogonia and oocytes of female mice from embryonic Day 11.5 onward, was used in this study. We used this antibody to detect germ line cells in the genital ridges and gonads of embryos and fetuses on Days 11.5 and 14.5 of gestation. The embryos and fetuses were obtained from heterozygote matings as described above. Organs were fixed in Bouin's fixative and then embedded in paraffin. Immunocytochemical localization was performed as described previously [21]. Deparaffinized sections were treated with 10D9G11 hybridoma-conditioned medium (undiluted) for 15–30 min at 33°C, followed by an alkaline phosphatase-based avidin-biotin anti-rat detection system (Zymed, South San Francisco, CA). This generates a red fuchsin color reaction that was counterstained lightly with hematoxylin. Sections were photographed with Ektar 25 professional color film (Eastman Kodak, Rochester, NY) using an 80A filter.
Immunofluorescence was used to detect and localize Cx43 in neonatal ovary sections. Before immunostaining, sections were stained with contrast blue (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD). The primary antibody (anti-CT360) was a gift from Dr. Dale Laird (Department of Anatomy and Cell Biology, University of Western Ontario). This rabbit antibody was raised against a synthetic peptide corresponding to residues 360–382 of the C-terminal portion of Cx43 [22]; it was used at a dilution of 1:250. The secondary antibody was fluorescein-conjugated goat anti-rabbit IgG (ICN Canada Ltd., Montréal, PQ, Canada) used at a dilution of 1:50.
Culture of Fetal Ovaries
Ovaries were removed from fetuses at 17.5 dpc and rinsed with PBS; they were then placed in culture in Waymouth MB 752/1 medium (Gibco-BRL) with 10% fetal bovine serum. We used the organ culture system of Eppig and O'Brien [23]. The ovaries were placed in small drops of medium on the polycarbonate membranes (3 µm pore size) of 25-mm cell culture inserts in 6-well plates (Falcon Plastics, Los Angeles, CA). Medium volume below and above the polycarbonate membranes was adjusted so that the ovaries were just wet on their surfaces and were fed continuously from the larger volume of medium below the membranes. The ovaries were cultured for 8 days; medium was changed once on Day 4. On Day 8, the polycarbonate membrane inserts were removed from the wells. The ovaries, which were partially attached to the membranes, were washed once with PBS on the membrane itself (no attempt was made to detach them from the membranes). The portion of each membrane containing the ovary was cut out with fine scissors. The ovaries were fixed in a solution containing 2% glutaraldehyde and 2% sucrose in 0.1 M phosphate buffer (pH 7.3) for 6 h, postfixed in 2% OsO4 for 1 h, and then embedded in epon resin. Each ovary in the embedded block was cut into 0.5-µm serial sections. The sections were transferred onto glass slides and stained with toluidine blue as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Other than the germ cell deficiency, the gonads of 17.5 dpc fetuses did not reveal any obvious morphological defects, raising the possibility that normal gametogenesis might be possible with the few remaining germ cells. In males, gametogenesis begins at puberty when the first wave of germ cells enters meiosis as primary spermatocytes. In female mice, on the other hand, gametogenesis begins around the time of birth with the formation of primordial follicles enclosing primary oocytes. Recently, an organ culture technique was devised [23] that can support the development of neonatal mouse ovaries for a week or more, allowing early stages of folliculogenesis to occur in vitro. We used this technique to determine whether follicles could form and whether their growth could be sustained in the absence of Cx43. Ovaries were removed from fetuses at 17.5 dpc and placed in organ culture; they were then processed for histology after 8 days (primordial, primary, and secondary follicles would be expected to have developed after that length of time in wild-type ovaries [23]). As shown in Figure 5, wild-type or heterozygous ovaries did form follicles after 8 days of culture, with the most advanced follicles becoming multilaminar. In homozygous mutant ovaries, folliculogenesis was clearly retarded or arrested: well-organized multilaminar follicles, comparable to those in wild-type ovaries, were never seen, and the oocytes failed to achieve the size of wild-type or heterozygous oocytes cultured for the same length of time. These results indicate that postnatal folliculogenesis is impaired in mice lacking Cx43.
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Although gap junctions have been identified morphologically within primordial follicles of newborn mice [24], the connexin(s) contributing to those channels has not been identified. In the rat, Cx43 expression in the ovary was first detected after the onset of folliculogenesis [25]. To ascertain whether Cx43 is expressed in neonatal mouse follicles, ovaries from wild-type mice were immunostained using an antibody specific for the C-terminus of this connexin. Gap junction-like (punctate) immunoreactivity in regions of cell apposition was evident throughout the somatic component of the ovaries from postnatal Day 1, when the first primordial follicles had begun to form (Fig. 6). In some cases, Cx43 immunoreactivity could be clearly seen between the pregranulosa cells of developing primordial follicles. There was no clear indication of immunoreactivity within oocytes or along their surfaces. Thus Cx43 is likely present in gap junctions between the somatic cells of ovarian follicles from the onset of folliculogenesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mammalian germ line originates in the embryonic ectoderm of the pregastrula embryo [26, 27]. In the mouse, lineage restriction occurs about the time of gastrulation and involves a founding population of about 45 primordial germ cells (PGCs). PGCs migrate from the extraembryonic region through the hindgut endoderm and then through the dorsal mesentery into the genital ridges [28]. PGCs increase in number during migration and link up to form networks [29]; it is not known whether these germ cell-germ cell contacts involve gap junctions. After entering the genital ridges, which in the mouse occurs on Days 10–11 of gestation, the PGCs continue to proliferate until the sex determination event initiates gonadal differentiation. This begins on embryonic Day 12.5, when there are approximately 25 000 germ cells. In male mice, the germ cells become mitotically quiescent about embryonic Day 14.5 and remain thus until shortly after birth, when mitosis resumes. In the female, all of the germ cells cease mitosis before birth, enter meiotic prophase, and remain in that state until reproductive function begins in the young adult. Folliculogenesis begins with the formation of primordial follicles around the time of birth. Oocytes then start to grow with the postnatal activation of primordial follicles to become primary follicles [30].
The germ cell deficiency in the genital ridges and late-gestation gonads of Gja1-/- fetuses implies that gap junctional coupling via Cx43 channels is a determinant of the migration, proliferation, or survival of PGCs. It is known that migrating as well as postmigratory PGCs require signals from neighboring cells to sustain them; candidate paracrine factors include mast cell growth factor [27, 31–33], tumor necrosis factor 
[34], leukemia inhibitory factor [33, 35, 36], oncostatin M [35, 36], interleukins 11 and 4 [35, 37], ciliary neurotrophic factor [36], pituitary adenylate cyclase-activating polypeptides PACAP-27 and PACAP-38 [38], and basic fibroblast growth factor [29, 34, 36]. One possible explanation for the germ cell deficiency in fetuses lacking Cx43 is that the synthesis and/or secretion of paracrine factors required by PGCs is dependent on gap junctional coupling between the somatic cells with which the PGCs associate, between the PGCs and the somatic cells, or between the PGCs themselves. Whereas Cx43 is expressed in the embryonic ectoderm, where the PGCs originate, and in the extraembryonic and embryonic regions through which they migrate [39], there are at present no data concerning the presence of this connexin in the PGCs themselves.
During the growth of mammalian oocytes, there is continuous coupling with the surrounding granulosa cells via gap junctions [30, 40, 41]. According to morphological evidence, this coupling begins as primordial follicles develop around the time of birth, and expands as folliculogenesis proceeds postnatally [24]. Amino acids, glucose metabolites, and nucleotides are among the molecules known to be transferred to the growing oocyte via gap junctions [41]. In return, the oocyte signals the surrounding granulosa cells to support their proliferation and differentiation, one agent of that signal being a transforming growth factor ß family member, GDF-9 [42]. Our immunolocalization results demonstrated that Cx43 is expressed in the somatic cells of primordial follicles from postnatal Day 1. This correlates with the fact that cultured neonatal Gja1-/- ovaries failed to generate normal secondary follicles, in contrast to cultured heterozygote and wild-type ovaries. Thus gap junctional coupling between granulosa cells via Cx43 channels, along with secreted GDF-9 from the oocyte, may be required to support the proliferation of granulosa cells after primary follicles have formed.
It is interesting to compare the effects of the Gja1 null mutation with those of the Steel-panda allele (Mgf Sl-pan) at the locus encoding mast cell growth factor (MGF). In addition to its role in promoting primordial germ cell migration and proliferation, MGF is also involved in postnatal follicular development. MGF is produced by granulosa cells surrounding growing oocytes while its receptor, c-kit, is expressed on the surface of those oocytes; in both cell types, expression can be detected from the primordial follicle stage onward [43, 44]. There is good experimental evidence that this signaling pathway is required for postnatal oocyte growth and follicular development [45, 46]. The Mgf Sl-pan allele contains a DNA rearrangement upstream of the coding region that severely reduces MGF expression both in fetal and in postnatal ovaries [47, 48]. Mgf Sl-pan/Mgf Sl-pan mice are born with ovaries that are smaller and have 80% fewer oocytes than those of normal mice; those oocytes that are present do not grow beyond 19 µm and remain enclosed within follicles that do not become multilaminar [47]. The germ cell deficiency can be seen in the 11.5 dpc embryo (both male and female) when PGCs have recently arrived in the genital ridges, suggesting that it results from impaired PGC migration [48]. Thus, like the Gja1 null mutation, the Mgf Sl-pan allele reduces embryonic germ cell numbers in both sexes and impairs ovarian follicle development postnatally. This similarity prompts speculation that reduced gap junctional coupling resulting from the loss of Cx43 somehow interferes with signaling between germ line and somatic cells via the MGF/c-kit pathway. Experiments now under way in our laboratory are designed to explore this possibility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 519 661 3827; gkidder{at}physiology.uwo.ca
3 Current address: Subhash C. Juneja, Dept. of Pediatrics and Adolescent Research, Guggenheim-5, Mayo Clinic, Rochester, MN 55905.
Accepted: December 29, 1998.
Received: July 23, 1998.
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