Connexin 43: its regulatory role in testicular junction dynamics and spermatogenesis

Spermatogenesis is an intensely regulated process of germ cell development which takes place in the seminiferous tubules of the testis. In addition to known endocrine and autocrine/paracrine signaling pathways, there is now strong evidence that direct intercellular communication via gap junction channels and their specific connexins represents an important mechanism in the regulation of spermatogenesis. Another possibility is that connexins may indirectly regulate the spermatogenic process through modulation of tight and adherens junction proteins, further main structural components of the Sertoli-Sertoli junctional complexes at the blood-testis barrier site. The present review is focused on connexin 43 and updates its possible roles and functions in testicular junction dynamics and in the initiation and maintenance of spermatogenesis. In addition, testicular phenotypes of recently generated (1) conventional connexin 43 knockout mice, (2) connexin 43 knockin mice and (3) transgenic mice exhibiting a cell-specific (conditional) connexin 43 knockout will be discussed.     

Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis

Apoptosis appears to have an essential role in the control of germ cell number in testes. During spermatogenesis germ cell deletion has been estimated to result in the loss of up to 75% of the potential number of mature sperm cells. At least three factors seem to determine the onset of apoptosis in male germ cells: (1) lack of hormones, especially gonadotropins and androgens; (2) the specific stage in the spermatogenic cycle; (3) and the developmental stage of the animal. Although male germ cell apoptosis has been well characterized in various animal models, few studies are presently available regarding germ cell apoptosis in the human testis. The first part of this review is focused on germ cell apoptosis in testes of prepubertal boys, with special emphasis on apoptosis in normal and cryptorchid testes. A higher percentage of apoptotic spermatogonia was seen in the cryptorchid testes than in the scrotal testes. The hCG-treatment increased the number of apoptotic spermatogonia. The hCG-treatment-induced apoptosis in spermatogonia had severe long-term consequences in reproductive functions in adulthood. Increased apoptosis after hCG-treatment was associated with subnormal testis volumes, subnormal sperm density and pathologically elevated serum FSH. This finding indicates that increased apoptosis in spermatogonia in prepuberty leads to disruption of testis development. To evaluate the role of apoptosis in human adult testes, apoptosis was induced in seminiferous tubules that were incubated under serum-free conditions in the absence or presence of testosterone. Most frequently apoptosis was identified in spermatocytes. Occasionally some spermatids also showed signs of apoptosis. In short term incubations apoptosis was suppressed by testosterone. Our findings lead to the conclusion that apoptosis is a normal, hormonally controlled phenomenon in the human testis. The role of apoptosis in disorders of spermatogenesis remains to be established.     

Opposite regulation of connexin33 and connexin43 by LPS and IL-1alpha in spermatogenesis

The gap junction proteins, connexins (Cxs), are present in the testis, and among them, Cx43 play an essential role in spermatogenesis. In the present study, we investigated the testicular expression and regulation of another Cx, Cx33, previously described as a negative regulator of gap junction communication. Cx33 mRNA was present in testis and undetectable in heart, liver, ovary, and uterus. In the mature testis, Cx33 was specifically immunolocalized in the basal compartment of the seminiferous tubules, whereas Cx43 was present in both seminiferous tubule and interstitial compartments. During stages IX and X of spermatogenesis, characterized by Sertoli cell phagocytosis of residual bodies, Cx43 was poorly expressed within seminiferous tubules, while Cx33 signal was strong. To evaluate the role of phagocytosis in the control of Cx33 and Cx43 expression, the effect of LPS was analyzed in the Sertoli cell line 42GPA9. We show herein that phagocytosis activation by LPS concomitantly stimulated Cx33 and inhibited Cx43 mRNA levels. These effects appear to have been mediated through IL-1, because the exposure of Sertoli cells to the IL-1 receptor antagonist partly reversed these effects. IL-1 enhanced and reduced, respectively, the levels of Cx33 and Cx43 mRNA in a time- and dose-dependent manner. These data reveal that Cx33 and Cx43 genes are controlled differently within the testis and suggest that these two Cxs may exert opposite and complementary effects on spermatogenesis. Sertoli cell; germ cell proliferation     

Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells

Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).     

Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis

Early in postnatal life the first phase of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This wave of germ cell death is thought to reflect an adjustment of germ cell numbers that can be adequately maintained by Sertoli cells. Caspase 2 is an initiator caspase whose activation has been found to stimulate apoptosis through the mitochondria. The present study investigates if germ cell apoptosis during the first phase of spermatogenesis involves activation of caspase 2. Germ cell apoptosis was found to peak at Postnatal Days (pnds) 15 and 16 in male C57BL/6 mice. Western blot analysis revealed that caspase 2 also increased in the testes at pnd 16. Immunolocalization of total caspase 2 showed staining of germ cells in the periphery of the seminiferous tubules as well as germ cells more centrally located in an area where apoptotic germ cells were observed. Cytoplasmic as well as nuclear staining was observed. Western blot analysis of cytoplasmic and nuclear proteins from pnd 16 testis revealed pro-caspase 2 in both fractions. Further Western blot analysis for caspase 2 detected an increase in the activation of caspase 2 at pnd 16 in proteins isolated from the cytoplasm but not from the nucleus. Proteins isolated from mitochondria from pnd 16 testes revealed an increase in pro-caspase 2 as well as activated caspase 2 corresponding with an increase in cytochrome c in cytoplasmic fractions. Injection of the caspase 2-specific inhibitor z-VDVAD-fmk directly into the testis significantly reduced the observed germ cell apoptosis at pnds 15 and 16. These results suggest that caspase 2 is present in germ cells in the murine testis in early postnatal life and increases in expression in correspondence to the initial wave of germ cell apoptosis. Caspase 2 also localizes to mitochondria, where it is correlated with a release of cytochrome c and germ cell apoptosis. Blockade of caspase 2 activation reduced the number of apoptotic germ cells in the initial wave of germ cell apoptosis, indicating that caspase 2 plays an important role upstream of the mitochondria in germ cell apoptosis during the first phase of spermatogenesis.     

Differential expression of N-Myc downstream regulated gene 2 (NDRG2) in the rat testis during postnatal development

N-Myc downstream regulated gene 2 (NDRG2) is expressed in the testis of adult animals and is involved in cell differentiation and development. However, little is known about the expression pattern of NDRG2 in the testis during postnatal development. Here, we show that NDRG2 is consistently expressed in Leydig cells in the rat testis during postnatal development. However, its expression has also been detected at a high frequency in spermatogenic cells of the seminiferous tubules in young rats but at a much lower frequency in adult rats. Furthermore, high levels of NDRG2 expression have been found in methoxyacetic-acid-induced apoptotic germ cells, particularly at stages X–XIII of the seminiferous epithelium cycle of adult rats. Interestingly, high levels of NDRG2 expression have also been observed in spontaneously apoptotic germ cells in the seminiferous tubules of young and adult rats. Thus, the expression of NDRG2 in germ cells seems to alter during spermatogenesis. These findings suggest that NDRG2 regulates testicular development and spermatogenesis in rats and is involved in the physiological and pathological apoptosis of germ cells. Wu-Gang Hou, Yong Zhao, and Lan Shen contributed equally to this study. This study was supported by the Natural Science Foundation of China (2006: no. 30600340; 2007: no. 30771138; 2008: no. 30871309).     

Influence of hormonal imbalance on the integrity of seminiferous epithelium in the testes of adult rats chronically exposed to letrozole and rats exposed to soya isoflavones during the prenatal period,lactation, and up to sexual maturity

The structural integrity of the germ cells in the seminiferous epithelium and the correct process of spermatogenesis are made possible by proteins that participate in the formation of different types of junctions. This study was performed on samples of the testes of 4 groups (2 experimental and 2 corresponding control) of male Wistar rats. In the first experimental group, the adult rats received letrozole – a nonsteroidal inhibitor of cytochrome P450 aromatase (P450arom). The second experimental group was exposed to soya isoflavones during the prenatal period, lactation, and up to sexual maturity. The aim of this study was to examine the immunoexpression of β-catenin, N-cadherin, occludin, connexin43, annexin V, and advanced glycation end products (AGE) in the seminiferous epithelium of rat testes with chronic estrogen deficiency and of rats exposed to soya isoflavones. Series of sections of the testes were stained using PAS and silver impregnation. Moreover, immunohistochemistry tests were performed. A semi-quantitative determination of protein immunoexpression was performed using Image J. The number of annexin V positive Sertoli cells per tubule were counted manually. Comparisons between the experimental and corresponding control groups were performed using a non-parametric Mann-Whitney U test. The most common alterations were prematurely sloughed germ cells in the lumen of the seminiferous tubules and invaginations of the seminiferous tubules. We observed a lower number of annexin V positive Sertoli cells and a lower expression of N-cadherin and occludin in the seminiferous epithelium of both groups of rats with hormonal imbalances. Moreover, a higher expression of AGE, a lower expression of connexin 43 and a lower amount of reticular fibers in the basal lamina of seminiferous tubules was present in rats treated with letrozole and a higher expression of β-catenin was found in rats exposed to soya isoflavones. The hormonal imbalance between androgens and estrogens resulted in a decreased number of annexin V positive Sertoli cells. This may be associated with a failed clearance of apoptotic germ cells that leads to disturbances in the blood-testis-barrier (BTB) by affecting the expression of junctional proteins in the seminiferous epithelium. Moreover, a decreased level of estrogens was also associated with an increased expression of AGEs and with a changed composition of basal lamina in the seminiferous tubules of rats. These changes could lead to germ cell sloughing and invaginations of the seminiferous tubules.     

Cytokines and junction restructuring events during spermatogenesis in the testis: An emerging concept of regulation

During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli–Sertoli and Sertoli–germ cell interface, which is coupled with germ cell development, such as cell cycle progression, and translocation of the germ cell within the seminiferous epithelium. In the rat testis, restructuring of the blood–testis barrier (BTB) formed between Sertoli cells near the basement membrane and disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa into the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high levels in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells, particularly primary spermatocytes, and Sertoli cells, would induce restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate cytokine-induced junction restructuring, including gap junction and extracellular matrix, and the role of testosterone on junction dynamics in the testis will also be discussed.     

Connexines testiculaires: marqueurs physiopathologiques et cibles potentielles aux toxiques environnementaux

Gap junctions and their constitutive proteins, connexins, are essential in cell homeostasis and are considered as tumour suppressors. The purpose of the present review is to discuss the role of connexins in the testis and their expression in testis physiopathology. Organized in an hexameric arrangement forming a channel that connects cytoplasms of adjacent cells, connexins are implicated in numerous physiological processes such as cell proliferation and differentiation. The balance between cell proliferation/differentiation/apoptosis is prerequisite for limiting anarchic cell proliferation, a major risk of cancer development. Spermatogenesis is a sophisticated model of germ cell proliferation and differentiation in which connexins play an essential role. It is well recognized that alteration of membranous expression of connexins is an early event of germ cell tumoral kinetics and it has been suggested that environmental toxicants such as non-genomic carcinogens, which in most cases impair connexin expression, could be associated with testis tumoral development. The identification of agents capable of regulating the deleterious effects of carcinogens on connexin expression could be today of interest for opening new therapeutic perspectives.     

Apoptosis of fetal testicular cells is regulated by both p53-dependent and independent mechanisms

A portion of fetal germ cells undergoes apoptosis in the physiological context, but the molecular mechanisms of their apoptosis are largely unknown. Because p53 tumor suppressor gene product promotes apoptosis in various types of cells, we have investigated the expression of p53 in fetal gonads and examined the influence of loss of p53 function in fetal gonad cells using mice deficient in the p53 gene. We found that the expression of p53 protein in fetal testis was induced after 15.5 dpc (days post coitum), while the expression was not detected in fetal ovary. The number of apoptotic cells found in the seminiferous tubules of fetal testes was not significantly different between p53-deficient and wild-type mice until 16.5 dpc. At 17.5 dpc, however, more apoptotic cells were observed in wild-type testes than in the p53-deficient mice. In contrast, a similar number of apoptotic cells was found in fetal ovaries throughout these developmental stages. These observations indicate that p53 promotes apoptosis of fetal testicular cells after 16.5 dpc.     

Effects of prenatal exposure to a 900 MHz electromagnetic field on 60-day-old rat testis and epididymal sperm quality

We investigated the effects of exposure in utero to a 900 megahertz (MHz) electromagnetic field (EMF) on 60-day-old rat testis and epididymis. Pregnant rats were divided into control (CG; no treatment) and EMF (EMFG) groups. The EMFG was exposed to 900 MHz EMF for 1 h each day during days 13 ? 21 of pregnancy. Newborn rats were either newborn CG (NCG) or newborn EMF groups (NEMFG). On postnatal day 60, a testis and epididymis were removed from each animal. Epididymal semen quality, and lipid and DNA oxidation levels, apoptotic index and histopathological damage to the testis were compared. We found a higher apoptotic index, greater DNA oxidation levels and lower sperm motility and vitality in the NEMFG compared to controls. Immature germ cells in the seminiferous tubule lumen, and altered seminiferous tubule epithelium and seminiferous tubule structure also were observed in hematoxylin and eosin stained sections of NEMFG testis. Nuclear changes that indicated apoptosis were identified in TUNEL stained sections and large numbers of apoptotic cells were observed in most of the seminiferous tubule epithelium in the NEMFG. Sixty-day-old rat testes exposed to 900 MHz EMF exhibited altered sperm quality and biochemical characteristics.     

Connexin 43 a potential regulator of cell proliferation and apoptosis within the seminiferous epithelium

The gap junction proteins, connexins (Cx), are present in the testis and among them Cx43 play an essential role in spermatogenesis. By using an in vitro proliferation model of germ cells and Sertoli cells, we tempted here to clarify the role of Cx43 in the control of Sertoli and germ cell proliferation and apoptosis. Cx43 was detected in purified preparations of Sertoli cells and spermatogonia and immunolocalized in both cell types identified by vimentin and c-kit, respectively. Inhibition of gap junction coupling by the gap junction inhibitor α-GA significantly enhanced BrdU incorporation in Sertoli cells and reduced the number of activated caspase-3 positive germ cells. Similarly, inhibitory Cx43 and pan-Cx mimetic inhibitory peptides increased proliferation of Sertoli cells and stimulated survival of germ cells. Cx32 mimetic inhibitory peptide also stimulated Sertoli cell proliferation without altering germ cell proliferation and apoptosis. The present results reveal that Cx43 gap junctions between Sertoli cells participate in the control of Sertoli cell proliferation and that Cx43 gap junctions between Sertoli cells and spermatogonia are indirectly involved in germ cell number increase by controlling germ cell survival rather than germ cell proliferation.     

Localisation aberrante de la connexine 43 dans le cancer du testicule

Most cells can communicate directly via gap junction channels. Gap junction intercellular communication (GJIC) participates in the control of cell proliferation. Abnormal expression of connexins (Cx), the constitutive proteins of gap junctions, has been associated with a transformed phenotype. In the seminiferous tubules, connexin Cx43 is predominantly expressed by Sertoli cell and germinal cell membranes. We studied Cx43 expression in four testicular cancers (pure seminoma). Cx43 mRNA and protein characterized by RT PCR and Western blot were found to be similar to controls (normal testes) in each case. However, immunofluorscence study of Cx43 protein indicated a cytoplasmic localization with no membrane expression, excluding the participation of Cx43 in GJIC. The significance of this aberrant localization will be discussed in relation to carcinogenesis.     

Germ cell apoptosis in the testes of normal stallions

Apoptosis in testicular germ cells has been demonstrated in many mammalian species. However, little is known about the stallion (Equus caballus) and rates of apoptosis during spermatogenesis. Morphological and biochemical features of apoptosis reported in other species were used to confirm that the TdT-mediated dUTP Nick end labeling (TUNEL) assay is an acceptable method for identification and quantification of apoptotic germ cells in histological tissue sections from stallion testis. Seminiferous tubules from eight stallions with normal testis size and semen quality were evaluated according to stage of seminiferous epithelium to determine the germ cell types and stages where apoptosis most commonly occurs. Spermatogonia and spermatocytes were the most common germ cell types labeled by the TUNEL assay. A low rate of round and elongated spermatids were labeled by the TUNEL assay. Mean numbers of TUNEL-positive germ cells per 100 Sertoli cell nuclei were highest in stages IV (15.5 +/- 1.0) and V (13.5 +/- 1.1) of the seminiferous epithelial cycle (P < 0.001). An intermediate level of apoptosis was detected in stage VI (P < 0.02). These stages (IV-VI) correspond to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia. Establishing basal levels of germ cell apoptosis is a critical step towards understanding fertility and the role of apoptosis in regulating germ cell numbers during spermatogenesis.     

一种特异性多用途抗人Connexin31抗体的获得与应用

间隙连接蛋白31(Connexin31,Cx31)是间隙连接蛋白(Connexin)家族的一员,目前对于Cx31的功能及其调节方式知之甚少。本实验利用Fmoe固相多肽合成的方法合成Cx31羧基端一个多肽片段(250-266从),经HPLC纯化后偶联到匙孔槭血蓝蛋白,免疫新西兰雄兔后采血检测、并纯化,采用Cx31myc表达蛋白进行Western blotting、细胞免疫荧光染色、免疫沉淀实验,证实得到的抗体为抗间隙连接蛋白31的特异抗体。     

Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by the differentiation of a transient population of germ cells called gonocytes found in the center of the seminiferous tubule. The fate of gonocytes depends upon these cells resuming mitosis and developing the capacity to migrate from the center of the seminiferous tubule to the basement membrane. This process begins approximately Day 3 postpartum in the mouse, and by Day 6 postpartum differentiated type A spermatogonia first appear. It is essential for continual spermatogenesis in adults that some gonocytes differentiate into spermatogonial stem cells, which give rise to all differentiating germ cells in the testis, during this neonatal period. The presence of spermatogonial stem cells in a population of cells can be assessed with the use of the spermatogonial stem cell transplantation technique. Using this assay, we found that germ cells from the testis of Day 0-3 mouse pups can colonize recipient testes but do not proliferate and establish donor-derived spermatogenesis. However, germ cells from testes of Day 4-5 postpartum mice colonize recipient testes and generate large areas of donor-derived spermatogenesis. Likewise, germ cells from Day 10, 12, and 28 postpartum animals and adult animals colonize and establish donor-derived spermatogenesis, but a dramatic reduction in the number of colonies and the extent of colonization occurs from germ cell donors Days 12-28 postpartum that continues in adult donors. These results suggest spermatogonial stem cells are not present or not capable of initiating donor-derived spermatogenesis until Days 3-4 postpartum. The analysis of germ cell development during this time frame of development and spermatogonial stem cell transplantation provides a unique system to investigate the establishment of the stem cell niche within the mouse testis.     

Hereditary defects in both germ cells and the blood-testis barrier system in as-mutant rats: evidence from spermatogonial transplantation and tracer-permeability analysis

The rat mutant allele as is located on chromosome 12. Homozygous (as/as) males show arrested spermatogenesis, mainly at the pachytene spermatocyte stage. It is not clear whether this defective spermatogenesis is caused by a failure in a somatic cell component that supports spermatogenesis or in the germ cell itself. Spermatogonial transplantation was performed to identify the genetically defective site in the as/as testis. In experiment 1, germ cells collected from as/as testes were transplanted into the testes of immunodeficient mice and normal rats. In experiment 2, normal rat germ cells were transplanted into as/as testes. The results of experiment 1 showed arrest of spermatogenesis at the pachytene spermatocyte stage, accompanied by a characteristic morphological feature, i.e., the formation of inclusion-like bodies in the cytoplasm, in both rat and mouse recipients. These results revealed the intrinsic effect of the mutant gene(s) on germ cells. In experiment 2, no restoration of spermatogenesis was detected in the recipient testes despite thorough histological examination. These results suggest that defects in a somatic cell component in as/as testes prevent the donor germ cells from colonizing and regaining their spermatogenetic ability. When the seminiferous epithelium of the as/as testis was examined by electron microscopy, no morphological abnormalities, including the formation of ectoplasmic specializations between adjacent Sertoli cells, were observed in the somatic cell components. However, when cytochrome c was applied as a tracer material, it penetrated the tight junctions between the Sertoli cells, indicating dysfunction of the blood-testis barrier in the as/as testis. The lack of restoration of spermatogenesis in the as/as testis after transplantation of normal germ cells may have been caused by the unfavorable environment in the seminiferous epithelium resulting from the incomplete barrier system between adjoining Sertoli cells. The gene(s) at the as locus may have a role in both germ cell differentiation and the establishment of the blood-testis barrier.