Role of the spermatogenic–Sertoli cell interaction through cell adhesion molecule-1 (CADM1) in spermatogenesis

Endocrine and local secretory factors have long been known to be required for spermatogenesis. Evidence has been accumulating in recent years indicating that direct contact between spermatogenic and Sertoli cells is also required for spermatogenesis. Cell adhesion molecules of various types have been found in the mammalian testis that are expressed in spermatogenic and/or Sertoli cells and involved in homophilic and/or heterophilic binding. We have cloned a novel cell adhesion molecule, cell adhesion molecule-1 (CADM1), also known as immunoglobulin superfamily 4A or spermatogenic immunoglobulin superfamily, from the mouse testis. CADM1 belongs to the immunoglobulin superfamily and is composed of three immunoglobulin-like domains, a transmembrane domain, and a short intracellular domain. In the seminiferous epithelium, CADM1 is expressed in intermediate spermatogonia through to early pachytene spermatocytes as well as in elongating spermatids—but not in round spermatids, mature spermatozoa, or Sertoli cells. One of the heterophilic binding partners of CADM1 has proven to be a poliovirus receptor, another member of the immunoglobulin superfamily that is expressed in Sertoli cells. Knockout mice for CADM1 develop male infertility due to defective spermatogenesis. These findings suggest that cell adhesion molecules between spermatogenic and Sertoli cells play essential roles in spermatogenesis.     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

The role of angiotensin-(1–7) receptor Mas in spermatogenesis in mice and rats

Evidence regarding the components of the renin–angiotensin (Ang) system suggests that this system plays an important role in male reproduction. However, there are few data available in the literature on the effects of Ang-(1–7) on the male reproductive system. The present study investigated the effects of the genetic deletion and chronic blockage of Ang-(1–7) receptor Mas on spermatogenesis and male fertility. The localization of Mas in mouse and rat testes was determined by binding assays and immunofluorescence, whereas the testis structure and spermatogenic process were morphologically and stereologically analysed by light microscopy. Ang-(1–7) binding and immunofluorescence revealed the presence of Mas in the testes of mice and rats. Although the total numbers of Sertoli and Leydig cells per testis and Leydig cell size were similar in both wild-type and Mas -deficient mice, Mas ^−/– animals exhibited a significant reduction in testis weight and a greater volume of apoptotic cells, giant cells and vacuoles in the seminiferous epithelium. In both mice and rats, an increased number of apoptotic cells were found during meiosis. Due to disturbed spermatogenesis, daily sperm production was markedly reduced in Mas ^−/– mice. Moreover, chronic infusion of A-779 [an Ang-(1–7) antagonist] in rats significantly increased the total number of apoptotic cells and primary spermatocytes in particular stages of spermatogenesis. Taken together, these findings strongly suggest that Ang-(1–7) receptor Mas plays an important role in the regulation of spermatogenesis.     

DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

Atypical development of Sertoli cells and impairment of spermatogenesis in the hypogonadal (hpg) mouse

Testes of hypogonadal (hpg) mice show arrested postnatal development due to congenital deficiencies of gonadotrophin-releasing hormone (GnRH) and gonadotrophin synthesis and secretion. Follicle-stimulating hormone (FSH), androgen or oestrogen treatment restore qualitatively normal spermatogenesis in hpg testes. Understanding the cellular and molecular changes accompanying hormone-induced spermatogenesis in hpg mice requires detailed morphological analyses of the germ cells and Sertoli cells in the untreated hpg testis. We compared seminiferous epithelial cytology in adult hpg, immature and adult wild-type mice using unbiased optical disector-based stereology, immunolocalization of Sertoli cell microtubules (MT), espin (a component of the blood-testis barrier), markers of Sertoli cell maturity (p27(kip1) and WT-1), and electron microscopy. Hpg testes had marked reductions in weight, seminiferous cord volume and length, and severe spermatogenic impairment with germ cells per testis < 1% of adult wild-type testes. Sertoli cell nuclei expressed WT-1 in hpg testes, but often were centrally located, similar to 9-14-day-old wild-type testes, and they expressed p27(kip1), indicating that hpg Sertoli cells were post-mitotic. Hpg testes had significantly (P < 0.05) reduced Sertoli cells per testis (0.56 million) compared with 10-day wild-type (1.15 million) and adult wild-type testes (2.06 million). Immunofluorescence labelling of normal adult Sertoli cells showed supranuclear MT columns and basally located espin, but these features were absent in 10-day-old and hpg Sertoli cells. Hpg Sertoli cells showed pleomorphic nuclear ultrastructure with mature-type nucleoli, similar to normal adult-type Sertoli cells, but hpg Sertoli cells exhibited incomplete tight junctions that lacked ectoplasmic specializations. We conclude that in hpg mice, chronic gonadotrophin insufficiency restrains Sertoli cell proliferation and maturation, forming pseudo-adult-type Sertoli cells that are incapable of supporting germ cell proliferation and maturation.     

Stage-specific expression of mouse germ cell-less-1 (mGCL-1), and multiple deformations during mgcl-1 deficient spermatogenesis leading to reduced fertility

A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1), is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-1 in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum et al., 2003), postacrosomal sheath, and posterior ring apparently formed. In addition, MN7 and MN13, essential substances for fertilization that are located in sperm heads, were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility.     

Chronic chromium exposure-induced changes in testicular histoarchitecture are associated with oxidative stress: study in a non-human primate (Macaca radiata Geoffroy)

BACKGROUND: Reproductive toxicity of chromium is in dispute despite positive findings in rodents. Recently we reported epididymal toxicity of hexavalent chromium (CrVI) in bonnet monkeys and in this paper we report its testicular toxicity. METHODS: Adult monkeys (Macaca radiata) were given drinking water containing CrVI (100, 200, 400 p.p.m.) for 6 months and testes were removed for ultrastructural and biochemical analyses. RESULTS: CrVI treatment disrupted spermatogenesis, leading to accumulation of prematurely released spermatocytes, spermatids and uni- and multinucleate giant cells in the lumen of seminiferous tubules. Transmission electron microscopy revealed granulation of chromatin and vacuolation between acrosomal cap and manchette microtubules of elongated spermatids and in the Golgi area of round spermatids. Pachytene spermatocytes had fragmented chromatin and swollen mitochondria with collapsed cristae. Spermatocytes and spermatogonia in the basal compartment were unaffected. Macrophages containing phagocytosed sperm and dense inclusions in Sertoli cells were seen. Specific activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase and concentrations of the non-enzymatic antioxidants glutathione, vitamins A, C and E decreased, while concentrations of H(2)O(2) and hydroxyl radicals increased in the testis of chromium-treated monkeys. Withdrawal of chromium treatment for 6 months normalized spermatogenesis and the status of pro- and antioxidants in the testis. CONCLUSIONS: CrVI disrupts spermatogenesis by inducing free radical toxicity, and supplementation of antioxidant vitamins may be beneficial to the affected subjects.     

Testis structure in the sys (symplastic spermatids) mouse

Testes of mice with the recessive insertional mutation termed symplastic spermatids (sys) were assessed for structural and developmental abnormalities. Homozygous (sys/sys) males are infertile due to an abnormality in spermatogenesis leading to azoospermia. The major interruption to spermatogenesis occurs when the intercellular bridges that connect round spermatids open prematurely resulting in the formation of symplasts. Symplasts contain as many as 285 nuclei. Development of spermatids within symplasts is arrested just before, or just after, elongation of the spermatid nuclei begins. Symplasts degenerate and appear to be phagocytized by Sertoli cells and by intratubular macrophages. In addition, degeneration of young round spermatids and also spermatocytes occasionally is observed. Spermatocyte degeneration is substantial in some tubules and leaves them depleted of cells other than basal compartment cells. Sertoli cell abnormalities are prominent and include intracellular vacuolation, absence of apical processes surrounding round spermatids, degeneration, and occasional sloughing. Although reduplication and infolding of the basal lamina is also seen, this does not appear as a common phenomenon. The sys phenotype is first manifest in animals between 19 days and 22 days of age. Considerable variability is seen in testis histology of prepubertal animals; some display degenerating pachytene spermatocytes and virtually no Sertoli cell vacuoles, while others display vacuoles without apparent elevated numbers of degenerating spermatocytes. Although this study has not revealed the primary cell type(s) affected by the insertional inactivation event, it is possible that the abnormalities in the Sertoli cells are responsible for germ cell degeneration as it is generally recognized that deficits in the Sertoli cell can result in major germ cell abnormalities but not vice versa.     

Changes in the distribution of microtubules and intermediate filaments in mammalian Sertoli cells during spermatogenesis

We have studied the distribution of microtubules and intermediate filaments in mammalian Sertoli cells during spermatogenesis. The arrangement of microtubules was determined, by indirect immunofluorescence, in ground squirrel testes that were 1) fixed, mechanically fragmented, and attached to polylysine-coated slides, and 2) fixed, embedded in polyethylene glycol, and sectioned. Intermediate filament patterns were determined, also by indirect immunofluorescence, in sections of unfixed rat testis. Results from these studies were confirmed and extended using electron microscopy. Microtubules first become evident in lateral processes that embrace round spermatids. When spermatids elongate and become situated in apical crypts of Sertoli cells, the microtubules become oriented parallel to the long axis of Sertoli cells and surround the crypts. As spermatids mature and acquire a saucer shape, apical microtubules progressively concentrate in Sertoli cell regions adjacent to the acrosome and eventually form discrete C-shaped structures that disappear during spermiation. Intermediate filaments in rat Sertoli cells are centered around the nucleus. From perinuclear regions, filaments extend toward desmosome-like junctions with early spermatogenic cells and into the apical cytoplasm where they have a transient association with crypts containing elongate spermatids. Filaments amongst crypts are most evident in early stages of the spermatogenic cycle when apical crypts are situated deep within the epithelium. They become less evident and eventually disappear as spermatids assume a more apical position. Our fluorescence studies and ultrastructural analyses indicate that the association of intermediate filaments with crypts is specific to regions adjacent to the dorsal or convex aspect of spermatid heads. In these regions, approximately 8 to 12 uniformly aligned filaments are intimately associated with actin filaments in ectoplasmic specializations surrounding the crypts. We conclude that, like actin, the distribution of microtubules and intermediate filaments changes in Sertoli cells during spermatogenesis. The distribution of microtubules correlates with the irregular columnar shape of Sertoli cells. We suspect that the apically situated intermediate filaments may play a role in anchoring or positioning Sertoli cell crypts deep within the epithelium during the early stages of the spermatogenic cycle.     

The expression pattern of PARP-1 and PARP-2 in the developing and adult mouse testis

Although the importance of the PARP family members in the adult testis has already been acknowledged, their expression in the developing testis has not been addressed. We performed immunohistochemistry by using PARP-1 and PARP-2 antibodies on the developing mouse testis at embryonic day (E) 15.5, E17.5, postnatal day (PN) 0, PN3, PN9, PN20 and adult. Our results showed that at embryonic and early postnatal days, the expression of PARP-1 was in the nuclei of gonocytes and spermatogonia. PARP-1 was positive in interstitial cells with nuclear localization at all studied ages. At embryonic and early postnatal days, the expression of PARP-2 was in the cytoplasm of gonocytes and spermatogonia. During the progress of spermatogenesis, PARP-2 was localized in the cytoplasm of pre-leptotene spermatocytes on PN9, in the cytoplasm of pachytene spermatocytes on PN15 and in the cytoplasm of round spermatids on PN20. In the adult, PARP-2 staining can still be observed in the cytoplasm of spermatogonia, but to a much lesser degree than in the round and elongating spermatids. For all the studied ages, PARP-2 was positive in Sertoli cells and interstitial cells with cytoplasmic localization. Our results indicate that PARP proteins are present in germ and somatic cells during testis development in mice.     

Chromosome aberrations and spermatogenic disorders in mice with Robertsonian translocation (11; 13)

Objective: To determine the diagnostic features of Robertsonian (Rob) translocation (11; 13) in mice and the mechanisms underlying the effect on spermatogenesis and reproductive decline. Methods: A Rob translocation (11; 13) mouse model was established by cross-breeding, and confirmed by chromosome analysis. Chromosome aberrations and translocation patterns were identified in mice with Rob translocation (11; 13) by fluorescence in situ hybridization (FISH). Spermatogenic disorders were investigated at different stages of spermatogenesis. Immunofluorescent analysis was performed on sections of testis and epididymis specimens during spermatogenic meiosis. The weight of the testes and reproductive decline were recorded. Results: The crossed Rob translocation (11; 13) mouse has 39 chromosomes, including a fusion chromosome (included chromosomes 11 and 13) using dual color FISH. There was no difference in the distribution pattern of SYCP3 and γH2AX in spermatocytes between Rob translocation and wild-type mice; however, round haploid spermatids presented characteristic morphologic changes of apoptosis and the number of haploid spermatids was decreased. Furthermore, the immature germ cells were released into the epididymis and the number of mature sperm was reduced. Conclusions: Chromosome aberrations and spermatogenic disorders may result from apoptosis of round haploid spermatids and a reduced number of mature sperm in Rob translocation (11; 13) mice. Abnormal sperm and reduced number of sperm may be one of the main reasons for reproductive decline and male infertility in Rob translocation (11; 13) mice.     

Presence of synaptonemal complex protein 1 transversal filament-like protein in human primary spermatocytes

The synaptonemal complex (SC) is involved in the pairing of chromosomes during meiosis. We found that antibodies raised against a protein component (P1) of the mouse synaptonemal complex, mouse SCP1, also identified the SC in human primary spermatocytes. Biopsies from 18 men presented with infertility were evaluated by light-field microscopy and grouped into five categories: normal spermatogenesis, Sertoli cell-only syndrome, meiotic disturbances, spermiogenic (i.e. differentiation) disturbances, and other combined disturbances. In all the normal subjects the SCP1 antibody distinctly stained the synaptonemal complexes of primary spermatocytes, whereas Sertoli cells, spermatogonia or spermatids were never stained. In three of the groups, which had germ cells but showed spermatogenic disturbances, the staining was similar to that seen in normal subjects. In sharp contrast to this, in sections from men with Sertoli cell-only syndrome no specific staining was seen. This study demonstrates that a SCP1-related protein is also conserved in the synaptonemal complex in meiotic cells from man. Further studies will reveal to what extent the absence or the non-functionality of SCP1 contributes to male infertility.      

CD147 is required for matrix metalloproteinases-2 production and germ cell migration during spermatogenesis

Spermatogenesis is a highly programmed process that requires the degradation of the extracellular matrix and the remodeling of tight junctions (TJ) to facilitate differentiating germ cell migration. Matrix metalloproteinases (MMPs) are essential in regulating Sertoli cell TJ in the testis. CD147 is known to stimulate the production of MMPs in tumor metastasis and its knockout mice are infertile. However, the functional relationship between CD147 and MMPs in spermatogenesis has not been investigated. In the present study, we examined the expression profile of CD147 and MMPs during mouse testicular development by RT-PCR, western blot and immunofluorescence staining. We also examined CD147 involvement in the production of MMP-2 and the migration of germ cells (GC-1 and GC-2 cells) using CD147 antibody or synthetic microRNA mimics-mediated knockdown. The results showed that CD147 was present at all stages of testicular development from 7 to 56 days post-partum (dpp). CD147 expression was found to increase after 21 days from moderate levels in 7 and 14 days. Of the eight MMPs studied, MMP-2, MMP-7, MMP-9 and MMP-23 were detected to have changes in expression during testicular development, with MMP-2 showing the largest change. CD147 and MMP-2 were co-localized in spermatogonia, spermatocytes and round spermatids in mouse testis, while in human testis, they were co-localized in spermatocytes and round spermatids. MMP-2 expression and migration of GC-1 and GC-2 cells were reduced by interfering with CD147 expression and function in vitro. These data suggest that CD147 regulates migration of spermatogonia and spermatocytes via induction of MMP-2 production during spermatogenesis.     

Specific expression of the mRNA for 25 kDA heat-shock protein in the spermatocytes of mouse seminiferous tubules

 The 25 kDa heat-shock protein (Hsp25) is a member of the family of small heat-shock proteins. We investigated the expression and cellular localization of Hsp25 mRNA in the testis of adult and developing mice using Northern blotting and in situ hybridization techniques. In the early postnatal days, i.e., before the onset of spermatogenesis, no Hsp25 mRNA was detected in the testis. At around 10 days postpartum, Hsp25 mRNA began to be expressed in the testis in coincidence with the onset of the first wave of spermatogenesis and increased in amount progressively toward adulthood. Throughout the testis development, the signal for Hsp25 mRNA was localized exclusively to germ cells and was not detected in Sertoli or interstitial cells. The testis of W/Wv mutant mice, which lack the germ cell line, exhibited no Hsp25 mRNA expression. In the testis of normal adult mice, the abundance of Hsp25 mRNA differed among the seminiferous tubules in different stages of spermatogenesis. The most intense signal for Hsp25 mRNA was localized to the spermatocytes at leptotene, zygotene and early pachytene phases, which are present in the tubules of stages I–III and IX–XII. The signal decreased in intensity in the late pachytene and diplotene spermatocytes and was not detected in spermatids. Spermatogonia were also devoid of the signal. These results suggested that Hsp25 plays some specific role in the meiotic prophase of the testicular germ cell. Accepted: 27 Oct 1998     

Testis of the lizard Mabuya carinata: a light microscopic and ultrastructural seasonal study

Histomorphology and ultrastructure of the testis during breeding and nonbreeding phases of the reproductive cycle of the lizard Mabuya carinata are studied. Observations of the ultrastructural features of the testis during breeding and nonbreeding phases of the reproductive cycle reveal a prenuptial type of spermatogenesis and a clearcut discontinuous spermatogenic cycle. Seminiferous tubules are enlarged and there is active spermatogenesis as shown by the presence of all the stages of spermatogenesis (spermatogonia to spermatids) and spermatozoa during the breeding phase (November). During the nonbreeding phase (April) only spermatogonia and Sertoli cells are seen in the shrunken seminiferous tubules. Leydig cells and Sertoli cells show distinct changes in the morphological appearance with hypertrophy of the cells in breeding phase and atrophy of the cells in the nonbreeding phase of the reproductive cycle. The present study suggests that Sertoli cells and Leydig cells functions are synchronous in the lizard M. carinata.