| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Departments of Physiology,
b Obstetrics and Gynaecology,
c Paediatrics, The University of Western Ontario, London, Ontario, Canada N6A 5C1
d Child Health Research Institute, London, Ontario, Canada N6C 2V5
ABSTRACT
Connexin43 (Cx43), a gap junction protein encoded by the Gja1 gene, is expressed in several cell types of the testis. Cx43 gap junctions couple Sertoli cells with each other, Leydig cells with each other, and spermatogonia/spermatocytes with Sertoli cells. To investigate the role of this communication pathway in spermatogenesis, we studied postnatal testis development in mice lacking Cx43. Because such mice die shortly after birth, it was necessary to graft testes from null mutant fetuses under the kidney capsules of adult males for up to 3 wk. Grafted wild-type testes were used as controls. In our initial experiments with wild-type testes, histological examination indicated that the development of grafted testes kept pace with that of nongrafted testes in terms of the onset of meiosis, but this development required the presence of the host gonads. When excised grafts were stimulated in vitro with cAMP or LH, there was no significant difference in androgen production between null mutant and wild-type testes, indicating that the absence of Cx43 had not compromised steroidogenesis. Previous research has shown that Cx43 null mutant neonates have a germ cell deficiency that arises during fetal life, and our analysis of grafted testes demonstrated that this deficiency persists postnatally, giving rise to a "Sertoli cell only" phenotype. These results indicate that intercellular communication via Cx43 channels is required for postnatal expansion of the male germ line.
developmental biology, gametogenesis, Leydig cells, Sertoli cells, spermatogenesis, testis, testosterone
INTRODUCTION
Spermatogenesis is a complex, multistage process that is coordinated by both paracrine and endocrine signals. These signals are required to support the development of four or five layers of germ cells in progressive stages of mitosis, meiosis, and spermiogenesis within each region of the seminiferous tubule. Another avenue of intercellular signalling for coordination of spermatogenesis is potentially provided by gap junctions. Gap junctions are arrays of intercellular membrane channels that allow cells to communicate directly with one another. The channels are formed from proteins called connexins (reviewed in [1]). Six connexins oligomerize to make a connexon hemichannel in one cell membrane, which then docks with a connexon in the adjacent cell membrane forming an intercellular channel that allows the passage of small molecules (<1 kDa) from cell to cell. The connexins are encoded by a multigene family, and the expression of several different connexins has been documented in rodent testes [2]. Morphological studies have identified gap junctions between Sertoli cells, between Sertoli cells and spermatogonia/spermatocytes, between cells of the peritubular layer, and between Leydig cells of the interstitial compartment [2–7].
There are several lines of evidence indicating that gap junctional coupling may be essential for normal testicular function. Male germ cells must be in contact with Sertoli cells to develop in vivo or in vitro [8]. FSH is a regulator of male germ cell development, yet Sertoli cells are the only cells in the testis that possess FSH receptors (reviewed in [9]). The effects of FSH on germ cell development must therefore be exerted indirectly, and one possible pathway is gap junctional communication between Sertoli cells and germ cells. Regulation of testosterone secretion may involve communication between adjacent Leydig cells [10]. The fact that Leydig cells are coupled via gap junctions suggests that the steroidogenic response of the testis to gonadotropins could also be modulated by this signaling pathway.
The most abundant connexin identified in the testis to date is connexin43 (Cx43). Electrophysiological and immunochemical data suggest that Cx43 is the predominant, if not the only, connexin contributing to functional gap junctions in Leydig cells [4, 11]. In contrast, Cx43 is just one of several connexins expressed, at least at the mRNA level, in Sertoli cells, germ cells, and peritubular cells [2], with expression being highest in Sertoli cells [7]. Given the widespread distribution of Cx43 in the testis, we hypothesized that spermatogenesis and/or testosterone production is compromised in the absence of this connexin.
To evaluate this hypothesis, we studied postnatal testis development in mice lacking Cx43. We used mice in which the neoR gene had replaced most of the second exon of the Gja1 gene [12]. Because the entire coding sequence of Cx43 resides within the second exon, the replacement results in a null mutation [12, 13]. The homozygous mutant mice die shortly after birth because of a severe heart abnormality; therefore, it was necessary to graft fetal testes into adult hosts [14, 15] to allow the mutant testes to develop postnatally. The initial experiments in this study were aimed at ensuring that testes removed from late gestation fetuses would continue to develop under an adult mouse kidney capsule. Another aspect of the Cx43 null mutant phenotype is a pronounced germ cell deficiency affecting both sexes; however, neonatal testes do contain some identifiable germ cells [13]. After determining optimal grafting conditions, our goals were to ascertain whether the germ cell deficiency of the mutant fetuses could be overcome by postnatal spermatogonial proliferation and whether the mutation affected testosterone production by Leydig cells.
MATERIALS AND METHODS
Mice
Mice heterozygous for the mutant allele (Gja1+/Gja1-) on the CD1 background were bred and maintained in the Department of Animal Care and Veterinary Services at the University of Western Ontario. Heterozygotes were mated to produce the homozygous mutant fetal mice used in grafting experiments. The morning a vaginal plug was detected was termed Day 0.5 of gestation. Wild-type fetuses from the same dams were used as controls. Mice used as graft recipients were 18–20 g Prkdcscid/Prkdcscid adult males (C.B-17/IcrHsd-scid; Harlan Sprague Dawley, Indianapolis, IN). These mice were bred and maintained in the barrier facility at the Robarts Research Institute (London, ON, Canada). All mice used in this study 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.
Collection of Fetal Testes
Pregnant mice were anesthetized with CO2 and killed by cervical dislocation on Day 17.5 of gestation. The uterine horns were removed through an abdominal incision, and each fetus was carefully extracted from its individual amniotic sac and then killed immediately by decapitation. Approximately 5 mm of tail was clipped from each fetus, washed in detergent solution, and kept for genotyping. The testes of each fetus were removed and placed in 0.5 ml of Medium 199, pH 7.4 (Life Technologies, Burlington, ON, Canada) in a 35- x 10-mm Falcon petri dish, and nongonadal tissue was removed. Fetal testes were maintained on Millicell culture plate inserts with Isopore polycarbonate membrane, 3.0-µm pore size (Millipore Canada Ltd., Nepean, ON, Canada) over Medium 199 at 37°C for 2 days until grafting.
Genotyping
The genotype of each fetus was determined by polymerase chain reaction (PCR) techniques. Tail clippings were digested with 6 µl of a solution containing 1.13 mg/ml proteinase K in 50 mM KCl, 50 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, and 0.5% Tween 20. The digestion was carried out overnight at 58°C. The digest was then diluted 1:5 with water and heated at 95°C for 15 min to inactivate the proteinase K. One microliter of each digest was used for each PCR, which utilized one of three sets of primers. For the Gja1 wild-type allele the primers were 5'-CCCCACTCTCACCTATGTCTCC-3' and 5'-ACTTTTGCCGCCTAGCTATCCC-3', and they generated a 519-base pair (bp) amplicon. For the Neo (disrupted) allele, the primers were 5'-CTTGGGTGGAGAGGCTATTC-3' and 5'-AGGTGAGATGACAGGAGATC-3', generating a 280-bp amplicon. A third set of primers was used as a positive control for each PCR; these primers were designed to amplify a 206-bp segment of the T-cell receptor gene. The sequences were 5'-CAAATGTTGCTTGTCTGGTG-3' and 5'-GTCAGTCGAGTGCACAGTTT-3'. Each PCR mixture contained either the Neo or the Gja1 primers plus the positive control primers (0.2 µM each) in 20 mM Tris-HCl, pH 8.4, containing 1.5 mM MgCl2, 0.2 mM dNTPs, 50 mM KCl, and 0.125 units of Platinum Taq polymerase (Life Technologies) in a 10-µl reaction volume. Cycling conditions were 3-min soak at 94°C, 94°C for 30 sec, 64°C for 30 sec (with the temperature of this step dropping 0.5°C per cycle to eventually reach 58°C), followed by 72°C for 30 sec. The last (37th) cycle was followed by a 7-min soak at 72°C. PCR products were separated on a 3% agarose gel at 100 V for 30 min.
Grafting Procedure
Male SCID mice to be used for graft recipients were removed from their microisolator cages in a sterilized biological safety cabinet where surgery was carried out. Each mouse was injected i.p. with 0.04 ml/10 g body weight of an anesthetic solution consisting of 2.5% ketamine hydrochloride (Ketaset; Ayerst Veterinary Laboratories, Guelph, ON, Canada) and 0.2% xylazine (Rompun; Bayer, Etobicoke, ON, Canada). After 10 min, a second half-dose was given, which allowed the mouse to remain unconscious for approximately 20 min by which time the surgery was complete. Vaseline was placed over the eyes of each unconscious mouse to prevent drying. In those recipients to be castrated before grafting, the abdominal area and dorsal right side were shaved and wiped with iodine. A lower abdominal medial incision, approximately 1 cm wide, exposed the recipient's testes. The blood vessels supplying the testes were clamped with forceps for at least 8 sec to collapse the vessels, preventing excess bleeding. Each testis was removed with scissors and the associated fat pad was replaced. The incisions were closed with two or three sutures each. For all recipients, the right kidney was exteriorized through a small dorsolateral incision. A puncture was made in the kidney capsule with a 3-mm-diameter glass rod. Keeping the kidney capsule moist with sterile water, the fetal testes were inserted through the puncture. After the kidney was replaced in the body cavity, the peritoneal opening was closed with sutures, and the skin was closed with wound clips. Buprenorphine analgesic (0.3 mg/ml; Rickitt and Colman Products, Hull, England) diluted 1:1000 with sterile water was injected i.p. at 0.018 ml/g. Each mouse remained on a 37°C heating pad after surgery for approximately 1 h until consciousness was fully regained. To retrieve the grafts, host mice were anesthetized with CO2 and killed by cervical dislocation. The surgical incision was opened and the graft was carefully cut from the kidney and placed immediately in PBS. The graft was peeled away from the kidney tissue and any excess kidney capsule was removed with 30-gauge needles. The grafts were then placed into Bouin fixative for histological examination or into Medium 199 for assessment of capacity to produce androgen.
Histology
Recovered grafts were fixed in Bouin fixative overnight and then transferred to 70% ethanol until needed. The testes were embedded in paraffin and sectioned at a thickness of 5 µm. For routine histological examination, sections were stained with hematoxylin and eosin.
Immunocytochemistry
To identify germ cells, sections from grafted testes were immunostained with a rat monoclonal antibody (10D9G11; George C. Enders, University of Kansas Medical Center, Kansas City, KS) against germ cell nuclear antigen (GCNA1). This same antibody had been used by us previously to characterize the germ cell deficiency in fetal gonads lacking Cx43 [13]. A mouse monoclonal antibody against proliferating cell nuclear antigen (PCNA) (Sigma Chemical Co., St. Louis, MO) was used to specifically label cells undergoing mitotic division. For both antibodies, immunostaining was preceded by treatment of hydrated slides with blocking solution, 2% BSA (Life Technologies) in PBS, for 45 min. Anti-GCNA1 or anti-PCNA primary antibody (100 µl) was placed on each slide for 1 h at room temperature. Anti-GCNA1 was used as undiluted hybridoma supernatant, and anti-PCNA was diluted 1:300 in blocking solution. Slides were then rinsed once with PBS for 5 min before applying secondary antibody for 1 h. The secondary antibody for GCNA-immunostained sections was horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (ICN Canada Ltd., Montréal, PQ, Canada) diluted 1:250 in blocking solution, and the secondary antibody for PCNA-immunostained sections was a goat anti-mouse HRP-conjugated IgG (Caltag Laboratories, Burlingame, CA) diluted 1:5000 in blocking solution. All slides were then rinsed once in PBS for 10–30 min. Germ cells or proliferating cells were visualized using 100 µl of colorimetric peroxidase detection substrate (True Blue Peroxidase; Kirkegaard & Perry Laboratories, Gaithersburg, MD). True Blue was left on the slides for 10 min and then rinsed away with water. GCNA1-immunostained slides were dehydrated as above and mounted in permount. The number of germ cells per seminiferous tubule was determined by counting GCNA1-positive cells in 40 tubules from each genotype or developmental age. PCNA-immunostained slides were covered with a coverslip and examined immediately.
The distribution of Cx43-containing gap junctions in the grafted testes was investigated using a rabbit polyclonal antibody (CT360; Stephen Lye, Mount Sinai Hospital, Toronto, ON, Canada) raised against a synthetic peptide corresponding to residues 360–382 of the C-terminal portion of Cx43. Hydrated slides were blocked with 2% BSA in PBS for 45 min. The CT360 primary antibody was diluted 1:250 in blocking solution and applied to slides for 1 h. After rinsing for 5 min, a goat anti-rabbit fluorescent secondary antibody (ICN Canada) diluted 1:50 in blocking solution was applied for 1 h and rinsed for 10–30 min in PBS. A drop of antifade solution was added to each slide before applying coverslips.
Detection of Apoptotic Cell Death
The frequency of apoptosis in grafted testes was assessed using the in situ TUNEL reaction kit from Roche Diagnostics (Laval, PQ, Canada). Sections were dewaxed and rehydrated as described above and treated for 20 min at room temperature with proteinase K (20 µg/ml in 10 mM Tris/HCl; Life Technologies). They were then permeabilized on ice for 2 min in 0.1% Triton X-100/0.1% sodium citrate followed by two rinses with PBS. The TUNEL staining reaction was carried out according to the manufacturer's instructions. Sections treated with DNase I (1 µg/ml, 10 min at room temperature) to introduce DNA strand breaks served as positive controls, whereas sections treated with the TUNEL mix without the terminal deoxynucleotidyl transferase served as negative controls. The fluorescent signal from the incorporated fluorescein-dUTP was converted into a colorimetric signal using a peroxidase-conjugated sheep anti-fluorescein antibody (Roche Diagnostics). The peroxidase reaction was visualized with the True Blue system.
Measurement of Testosterone Production
The capacity of grafted testes to produce androgens in vitro was tested 3 wk after the transplantation procedure. Testes were harvested and freed from attached renal tissue, the tunica albuginea was removed, and the testes were placed immediately in Medium 199 at 37°C. The medium was sampled after 1 h, and this first sample was subjected to RIA to determine the amount of androgen that had accumulated prior to the onset of the test procedure. These data were subtracted from the output measured after a further 4 h in the presence or absence of LH. One testis from each fetal donor served as a control and was incubated in 1.0 ml of fresh Medium 199 for 4 h. The remaining gonad from the same fetus was incubated for the same length of time in the presence of ovine LH (400 pg/ml NIH-oLH26, a concentration that elicits robust androgen production in vitro). At the end of the incubation period, the culture medium was collected and centrifuged at 2500 x g for 10 min, and the supernatant was frozen for later estimation of androgen content by a standard RIA procedure [16].
Electron Microscopy
Ovaries were removed from host mice after 3 wk, cut into small pieces, and fixed for 1 h at room temperature in 1.6% glutaraldehyde in 0.1 M phosphate buffer containing 2% (w/v) sucrose (pH 7.3). Pieces were postfixed with 1% OsO4 in phosphate buffer for 1 h at 4°C and then stained with 1% aqueous uranyl acetate overnight at 4°C. Samples were dehydrated by passing them through an ascending series of ethanols and then infiltrated with LR White resin (London Resin Company Ltd., Reading, U.K.) overnight. Specimens were embedded in blocks of fresh LR White and polymerized at 60°C for 24 h in gelatin capsules (size 00; Polysciences, Warrington, PA). Thin sections (60–70 nm) were counterstained with 2.5% uranyl acetate and Reynold lead citrate before being viewed with a Phillips 201 electron microscope at 60 kV.
RESULTS
Fetal Testes Can Develop in an Adult Kidney
We had previously used a kidney grafting procedure to study postnatal folliculogenesis in ovaries lacking Cx43 [15]. In those experiments, the host ovaries were removed at the time of grafting, an alteration known to enhance the developmental competence of the graft [17]. In the present study, our first experiments were aimed at determining whether grafted fetal testes could develop under an adult kidney capsule and whether the presence or absence of the host testes affected the outcome. Grafted testes did develop in the adult host kidneys, but the best outcomes occurred when the host testes were left intact (Fig. 1). Wild-type grafts developing in intact hosts (Fig. 1B) more closely resembled testes of the same age that had developed in situ (Fig. 1A), with more cells (spermatocytes) growing towards the lumen of the seminiferous tubules, as compared with grafts developing in gonadectomized hosts (Fig. 1C). This was true for different periods of postnatal growth up to 3 wk (the longest period we observed). Heterozygote testes were indistinguishable from wild-type testes. To ensure that the grafting environment did not alter Cx43 expression, sections were labeled with a Cx43-specific antibody (Fig. 2). Grafted and nongrafted testes showed similar Cx43 expression patterns, with most immunostaining in the basal region of the tubules as reported previously [6, 7].
|
|
Germ Cell Deficiency of Gja1-/Gja1- Testes Persists Postnatally
Wild-type and mutant testes were examined histologically at Gestational Day 17.5 (before grafting) and after 1, 2, and 3 wk of postnatal development in the kidneys (Fig. 3, hematoxylin and eosin staining; Fig. 4, GCNA1 staining). Although the seminiferous tubules of wild-type (Gja1+/Gja1+) and null mutant (Gja1-/Gja1-) grafts appeared morphologically similar at Day 17.5 (Fig. 3, A and B) and after 1 wk of postnatal development (Fig. 3, C and D), after 3 wk of postnatal development the mutant tubules were clearly different, with many fewer cells and vacuous lumena (compare Fig. 3E with Fig. 3F). Labeling with the anti-GCNA1 germ cell-specific antibody (Fig. 4) revealed that this difference arises because the mutant seminiferous tubules are deficient in germ cells at all postnatal ages. The number of germ cells per seminiferous tubule was determined for grafted wild-type and mutant testes and for nongrafted wild-type testes of comparable age (Fig. 5). The mutant testes had at least 90% fewer germ cells. Although germ cell numbers increased during postnatal development in the wild-type testes (whether developing in situ or in kidney grafts), those numbers remained extremely low and did not increase in the mutant grafts.
|
|
|
As a test of cell proliferation in the mutant seminiferous tubules, sections were immunostained for PCNA, an accessory protein of DNA polymerase 
that is a marker for cells proceeding through S phase [18]. When sections of grafted testes were labeled with an antibody that recognizes PCNA, proliferating cells could be identified in both wild-type and mutant seminiferous tubules (Fig. 6). The number of PCNA-positive cells in the mutant tubules was severely reduced by 3 wk after birth, when Sertoli cell proliferation is known to decline [19]. Thus, cell proliferation in the testis can still occur when Cx43 is absent but eventually reaches a very low level in the mutant testes because of the paucity of germ cells. Immunolabeling of contiguous sections with either anti-GCNA1 or anti-PCNA (Fig. 7) allowed us to confirm that some of the PCNA-positive cells in the mutant testes were germ cells.
|
|
During the first wave of spermatogenesis in juvenile mice, spermatogonial proliferation is opposed by a high rate of apoptotic cell death that reaches a peak 3 wk after birth [20]. We used the in situ TUNEL reaction to look for changes in apoptotic cell death frequency in mutant testes developing in kidney grafts. TUNEL staining detected DNA strand breaks in virtually all cells in DNase-treated sections of wild-type seminiferous tubules (Fig. 8A), but no such staining was observed in negative controls, where the terminal deoxynucleotidyl transferase enzyme was omitted from the reaction (Fig. 8B). The TUNEL reaction applied to sections of grafted testes revealed very few apoptotic cells in either wild-type (Fig. 8, C and E) or mutant (Fig. 8, D and F) seminiferous tubules of any postnatal age up to 3 wk. This finding indicates that the absence of Cx43 did not influence the frequency of spermatogonial apoptosis but suggests that that apoptosis was suppressed in the grafted testes.
|
Androgen Production Persists in Gja1-/Gja1- Testes
When the pituitary hormone, LH, binds to its receptor on Leydig cells, cAMP is generated to stimulate testosterone synthesis (reviewed in [21]). A variety of second messengers, including cAMP, are known to pass through Cx43 gap junctions [22–24]. We therefore tested grafted mutant testes to determine whether their ability to produce androgens in response to LH had been compromised. Grafts were removed from adult kidneys after 3 wk and placed in culture medium with or without LH for 4 h, and the medium was then harvested for RIA. There was no difference in androgen production between Gja1+/Gja1+ and Gja1-/Gja1- testes under these conditions (Fig. 9). Correspondingly, we could not detect any morphological differences between wild-type and mutant Leydig cells when examined in situ with the electron microscope (Fig. 10). The mutant Leydig cells exhibited numerous lipid droplets and mitochondria and had copious smooth endoplasmic reticulum, all features of cells undergoing steroidogenesis. These results indicate that steroidogenesis persists in Leydig cells lacking Cx43.
|
|
DISCUSSION
One of the potential pitfalls with genetic ablation experiments in mice is the real possibility that the null mutation created will cause unexpected embryonic lethality, preventing study of the adult physiological process under investigation. In our case, the lethality caused by the cardiac defect of Cx43-deficient fetuses [12] precluded analysis of the effects of the mutation on fertility. The organ grafting procedure that we employed has, however, allowed us to evaluate the state of gametogenesis in both sexes. In females, the absence of Cx43 results in failure of the granulosa cell population to expand after birth, resulting in arrest of ovarian folliculogenesis in the primary or early secondary follicle stage [15]. In males, as shown in the present study, the germ cell population of the testis fails to expand postnatally in the absence of Cx43. The results obtained from both sexes imply a role for Cx43 in supporting cell proliferation and/or survival. The fact that both ovaries and testes from null mutant fetuses can continue to develop in kidney grafts, albeit with specific deficiencies, confirms the general health of tissues taken from such fetuses. Thus, the kidney grafting method employed here should be useful for studying other organ systems in lethally mutant mouse models.
When we began the present study, we first established that development of the grafted testes was closer to normal, i.e., more like that of testes developing in situ, when the host testes were left intact. This finding is in contrast to the situation in the female, where the development of ovary grafts is improved when the host is ovarectomized [17]. The reasons for this difference are unclear at present, although sex differences in the temporal pattern of gonadal development and/or dependence on gonadotrophic hormones could be involved.
In both sexes, the germ cells develop in an avascular environment. Gap junctions provide a means for efficient transport of nutrients to cells located away from the blood supply and provide a conduit for sharing of second messengers downstream of ligand binding to cell surface receptors. Given that spermatogenesis is a synchronized and spatially patterned process of cell proliferation and differentiation, gap junctional intercellular coupling would be expected to play an important role. Our results demonstrate that Cx43, likely the most abundant gap junction protein in the testis, plays a critical role because it is required for postnatal expansion of the germ cell population. In the mouse, germ cells (spermatogonia and spermatocytes) are coupled with Sertoli cells via gap junctions containing Cx43 [7], although it is possible that other connexins also are present. Hence the failure of the Cx43-deficient germ cell population to expand could reflect reduced support from the Sertoli cells. However, because Sertoli cells are also coupled with one another via Cx43 channels, the defect may also or alternatively involve a functional deficit in the Sertoli cells themselves (although their own ability to proliferate was apparently not compromised). In any case, the defect in germ line expansion in the mutant testes is not the result of a total failure of spermatogonia to proliferate, because some germ cells identified by GCNA1 immunostaining were also PCNA positive. Neither is this defect due to an elevated frequency of apoptosis. It could be that the rate of proliferation of the few remaining spermatogonia in the null mutant testes is low enough to be balanced by cell death, even at the low rate of apoptosis seen in the grafts, preventing recovery from the germ cell deficiency that exists at birth.
The "Sertoli cell only" phenotype resulting from the absence of Cx43 closely resembles the situation in genetically modified mice in which either Cx32 or Cx40 has replaced Cx43 [25]. In both of these "knockin" lines, the replacement of Cx43 with another connexin did not result in neonatal lethality, allowing the mutant mice to survive to adulthood. Thus Cx32 and Cx40 are able to function in place of Cx43 in the developing heart but not in the testis. Not only is gap junctional coupling as mediated by Cx43 necessary for postnatal testis development and spermatogenesis, but this connexin must play some role that is at least partially unique in that it cannot be fulfilled by two other connexins.
An unexpected finding in this study was that the frequency of TUNEL-positive cells in grafted testes, regardless of genotype, was much lower than expected for juvenile testes [20]. In adult rodents, the frequency of apoptotic cell death in seminiferous tubules is generally low but can increase in response to metabolic stress such as that caused by ischemia/reperfusion [26, 27]. Therefore, some condition pertaining to the kidney graft situation caused a reduction in apoptosis frequency to a level typical of adult testes. One explanation might be that the higher testosterone levels of the adult hosts suppressed apoptosis [20].
Given that Cx43 is the only connexin known to be present in Leydig cell gap junctions [3, 4, 11], it was also surprising that androgen production persisted in the mutant testes. This finding indicates, at least under the maximal LH stimulation of our in vitro assay system, that testosterone production is not dependent on Cx43 gap junctions. A future research priority will be to determine whether the responsiveness of Leydig cells to suboptimal LH levels is compromised by the mutation. However, Cx43 expression may be dependent on testosterone; treatment with hCG, which stimulated testosterone production by Leydig cells in vitro as well as in vivo, caused a reduction in Cx43 expression in both contexts [28]. LH signaling may negatively regulate gap junctional coupling in Leydig cells, in which case loss or reduction of that coupling would not compromise steroidogenesis but may in fact enhance it. Further experimentation is needed to explore this possibility.
ACKNOWLEDGMENTS
We thank Cheryl Ackert for assisting with the first kidney grafts, Jacqui Bocking for help with the TUNEL staining, Stephen Lye for supplying Cx43 antibody, Va
ek Pitelka for assistance with the RIAs, and Mike Risley for help in interpreting the histology of the grafted testes.
FOOTNOTES
First decision: 16 February 2001.
1 This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to G.M.K.) and the Medical Research Council of Canada (to D.K.P.). 
2 Correspondence: Gerald M. Kidder, Department of Physiology, The University
of Western Ontario, London, ON, Canada N6A 5C1. FAX: 519 661 3827; gerald.kidder{at}med.uwo.ca
Accepted: April 20, 2001.
Received: January 19, 2001.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  S. Mhaouty-Kodja, A. Lozach, R. Habert, M. Tanneux, C. Guigon, S. Brailly-Tabard, J.-P. Maltier, and C. Legrand-Maltier Fertility and spermatogenesis are altered in {alpha}1b-adrenergic receptor knockout male mice J. Endocrinol., November 1, 2007; 195(2): 281 - 292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Brehm, M. Zeiler, C. Ruttinger, K. Herde, M. Kibschull, E. Winterhager, K. Willecke, F. Guillou, C. Lecureuil, K. Steger, et al. A Sertoli Cell-Specific Knockout of Connexin43 Prevents Initiation of Spermatogenesis Am. J. Pathol., July 1, 2007; 171(1): 19 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sridharan, L. Simon, D. D. Meling, D. G. Cyr, D. E. Gutstein, G. I. Fishman, F. Guillou, and P. S. Cooke Proliferation of Adult Sertoli Cells Following Conditional Knockout of the Gap Junctional Protein GJA1 (Connexin 43) in Mice Biol Reprod, May 1, 2007; 76(5): 804 - 812. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. de Montgolfier, J. Dufresne, M. Letourneau, J. J. Nagler, A. Fournier, C. Audet, and D. G. Cyr The Expression of Multiple Connexins Throughout Spermatogenesis in the Rainbow Trout Testis Suggests a Role for Complex Intercellular Communication Biol Reprod, January 1, 2007; 76(1): 2 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. N Kahiri, M W. Khalil, F. Tekpetey, and G. M Kidder Leydig cell function in mice lacking connexin43. Reproduction, October 1, 2006; 132(4): 607 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. B. Francis and C. W. Lo Primordial germ cell deficiency in the connexin 43 knockout mouse arises from apoptosis associated with abnormal p53 activation Development, September 1, 2006; 133(17): 3451 - 3460. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Fiorini, X. Decrouy, N. Defamie, D. Segretain, and G. Pointis Opposite regulation of connexin33 and connexin43 by LPS and IL-1{alpha} in spermatogenesis Am J Physiol Cell Physiol, March 1, 2006; 290(3): C733 - C740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Flenniken, L. R. Osborne, N. Anderson, N. Ciliberti, C. Fleming, J. E. I. Gittens, X.-Q. Gong, L. B. Kelsey, C. Lounsbury, L. Moreno, et al. A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia Development, October 1, 2005; 132(19): 4375 - 4386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Liu, K. P. Lehmann, M. Sar, S. S. Young, and K. W. Gaido Gene Expression Profiling Following In Utero Exposure to Phthalate Esters Reveals New Gene Targets in the Etiology of Testicular Dysgenesis Biol Reprod, July 1, 2005; 73(1): 180 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Aravindakshan and D. G. Cyr Nonylphenol Alters Connexin 43 Levels and Connexin 43 Phosphorylation Via an Inhibition of the p38-Mitogen-Activated Protein Kinase Pathway Biol Reprod, May 1, 2005; 72(5): 1232 - 1240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Fischer, R. Brehm, L. Konrad, S. Hartmann, S. Kliesch, R. M. Bohle, and M. Bergmann Connexin 33: A Rodent-Specific Member of the Gap Junction Protein Family? J Androl, January 1, 2005; 26(1): 75 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. I. Gittens, K. J. Barr, B. C. Vanderhyden, and G. M. Kidder Interplay between paracrine signaling and gap junctional communication in ovarian follicles J. Cell Sci., January 1, 2005; 118(1): 113 - 122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Fiorini, B. Mograbi, L. Cronier, I. Bourget, X. Decrouy, M. Nebout, B. Ferrua, A. Malassine, M. Samson, P. Fenichel, et al. Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration J. Cell Sci., September 15, 2004; 117(20): 4665 - 4672. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Aravindakshan, M. Gregory, D. J. Marcogliese, M. Fournier, and D. G. Cyr Consumption of Xenoestrogen-Contaminated Fish during Lactation Alters Adult Male Reproductive Function Toxicol. Sci., September 1, 2004; 81(1): 179 - 189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. SAEZ, V. M. BERTHOUD, M. C. BRANES, A. D. MARTINEZ, and E. C. BEYER Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions Physiol Rev, October 1, 2003; 83(4): 1359 - 1400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Rodemer, T.-P. Thai, B. Brugger, T. Kaercher, H. Werner, K.-A. Nave, F. Wieland, K. Gorgas, and W. W. Just Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice Hum. Mol. Genet., August 1, 2003; 12(15): 1881 - 1895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Schlatt, A. Honaramooz, M. Boiani, H. R. Scholer, and I. Dobrinski Progeny from Sperm Obtained after Ectopic Grafting of Neonatal Mouse Testes Biol Reprod, June 1, 2003; 68(6): 2331 - 2335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. I. Gittens, A. A. Mhawi, D. Lidington, Y. Ouellette, and G. M. Kidder Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis Am J Physiol Cell Physiol, April 1, 2003; 284(4): C880 - C887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. St-Pierre, J. Dufresne, A. A. Rooney, and D. G. Cyr Neonatal Hypothyroidism Alters the Localization of Gap Junctional Protein Connexin 43 in the Testis and Messenger RNA Levels in the Epididymis of the Rat Biol Reprod, April 1, 2003; 68(4): 1232 - 1240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Risley, I. P. Tan, and J. Farrell Gap Junctions with Varied Permeability Properties Establish Cell-Type Specific Communication Pathways in the Rat Seminiferous Epithelium Biol Reprod, September 1, 2002; 67(3): 945 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.D. Houghton, K.J. Barr, G. Walter, H.-D. Gabriel, R. Grummer, O. Traub, H.J. Leese, E. Winterhager, and G.M. Kidder Functional Significance of Gap Junctional Coupling in Preimplantation Development Biol Reprod, May 1, 2002; 66(5): 1403 - 1412. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||
