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Analysis of cell-type-specific gene expression during mouse spermatogenesis.

In rodents, changes in gene expression during spermatogenesis can be monitored by sampling testis from each day during postnatal development. However, changes in gene expression at the tissue level can reflect changes in the concentration of an mRNA in a specific cell type, changes in volume of specific cells, or changes in the cell-type composition. This reflects the cellularity of the tissue. Here we have combined techniques that assess the expression profiles of genes at the whole-tissue level, differential display and DNA array, and, at the level of cellularity, in situ hybridization. Combining results from these techniques allows determination of the cell-type-specific gene-expression patterns of many genes during spermatogenesis. Differential display was used to determine expression profiles with high sensitivity and independent of prior knowledge of the sequence, whereas DNA arrays quickly assess the expression profiles of all the genes. This identified three groups of gene-expression profiles. The major group corresponds to genes that are upregulated in spermatocytes during either the mid- or late- pachytene phase of spermatogenesis (stages VII-XI). This pachytene cluster was gradually extinguished in the later spermatid stages but was followed by another cluster of genes expressed in spermatids. Finally, a group of genes was downregulated during spermatogenesis and probably expressed in nongerm cells. We believe that expression of most genes can be described by a combination of these cell-type-specific expression patterns.

Animals↗

Differentiation of smooth muscle cells in the fetal rat testis and ovary: localization of alkaline phosphatase, smooth muscle myosin, F-actin, and desmin.

The histochemical demonstration of alkaline phosphatase (AP) activity and localization of smooth muscle myosin (SMM), F-actin, and desmin were carried out on frozen sections of testes and ovaries from 15-day-old fetal to newborn rats. The presence of immunocytochemically localized SMM and desmin was confirmed by Western blot analysis of proteins from isolated gonads. The development of smooth muscle cells was predominant in the testis. The first SMM-positive cells with an increasing intensity for F-actin and desmin appeared in the testicular tunica albuginea and around the testicular cords by the age of 16 days. A continuous layer of SMM- and F-actin-positive (but not uniformly desmin-positive) myoid cells was detected in the newborn testis. In the early gonads and in the newborn ovary, a majority of the interstitial cells expressed desmin, indicating that, in undifferentiated tissues, non-myogenic cells may also express desmin. During fetal development, male and female gonocytes showed a decrease in F-actin content but retained their high AP activity. In the cortex of the newborn rat ovary, the observed high AP activity and the presence of desmin may be associated with the postnatal histogenesis of the follicles. The presence of SMM-containing cells in the hilus of the ovary may be required for the demarcation of the ovary from the mesonephros by the constriction of the mesovarium. The occurrence of SMM-positive cells predominantly in male fetuses suggests that the development of the contractile cells in the fetal testis may be induced by testicular androgens.

Actins↗

Tsp57: a novel gene induced during a specific stage of spermatogenesis.

Recently, we described the identification of a novel protein, nuclear receptor-associated protein 80 (RAP80), which is highly expressed in spermatocytes and appears to have a role in regulating gene expression. To identify proteins interacting with this protein, we performed yeast two-hybrid screening using full-length RAP80 as bait. This screen identified one in-frame clone encoding a novel testis-specific protein (Tsp), referred to as Tsp57. Tsp57 encodes a basic protein with a mass of 56.8 kDa. The amino acid sequence of Tsp57 is highly conserved (87%) between mouse and human. The mouse and human Tsp57 genes map to chromosomes 9A1 and 11q21, respectively. Northern blot analysis showed that the expression of Tsp57 mRNA was highly restricted to the testis and temporally regulated during testicular development. Tsp57 mRNA was greatly induced between Day 21 and Day 25 of postnatal testicular development. In situ hybridization analysis demonstrated that the hybridization signal for Tsp57 mRNA was strongest in sections of seminiferous tubules at stages VI-VIII of spermatogenesis, consistent with the conclusion that Tsp57 is most highly expressed in round spermatids. Study of Tsp57 expression in several purified subpopulations of spermatogenic cells confirmed maximum levels of expression in round spermatids. Consistently, Tsp57 expression was absent in testes from vitamin A-deficient mice, which do not have any round spermatids, and was reduced in RARalpha null mice, which have lowered numbers of round spermatids in their testes. These results indicate the possibility that Tsp57 protein plays a role in the postmeiotic phase of germ cell differentiation. Tsp57 contains two putative nuclear localization signals: NLS1 and NLS2. Examination of the cellular localization showed that the green fluorescent protein-Tsp57 fusion protein localized to both cytoplasm and nucleus. After deletion of NLS1 but not NLS2, Tsp57 localized solely to the cytoplasm, indicating a role for NLS1 in the nuclear localization of Tsp57. The localization suggests a nuclear function for Tsp57. Pull-down analysis demonstrated that Tsp57 and RAP80 form a complex in intact cells.

Amino Acid Sequence↗

Rete testis hyperplasia with hyaline globule formation. A lesion simulating yolk sac tumor.

The presence of eosinophilic, hyaline globules in association with epithelial hyperplasia was noted in the rete testis of three patients with germ cell tumors. In the more florid examples, this proliferation formed a solid and microcystic pattern that, in association with the hyaline globules, mimicked a yolk sac tumor component. However, the bland cytologic features of the cells and the conformation to the configuration of the rete testis were keys to its reactive nature. A subsequent review of 48 testicular specimens containing well-defined areas of the rete testis showed hyaline globule formation in the rete testis or tubuli recti in 16 of 27 germ cell tumors, one of five other testicular tumors (four stromal tumors and one plasmacytoma), and none of 16 nonneoplastic cases. Many of the cases that had hyaline globules also showed epithelial hyperplasia. Further analysis demonstrated an incidence of rete testis invasion by neoplasm in cases that had hyaline globules, with or without epithelial hyperplasia, that was significantly higher (p less than 0.01) than that seen in neoplastic cases lacking hyaline globules. We concluded that this pseudoneoplastic reaction developed secondary to invasion of the rete testis by tumor. Immunostains supported the nonneoplastic nature of the proliferative lesions and indicated that the globules represented various proteins that had been absorbed from the lumen of the rete testis by the epithelial-lining cells but not successfully secreted.

Albumins↗

Cryptorchidism: fowler-stephens procedure or autotransplantation-a new experimental model.

OBJECTIVES: To investigate the effect of different operative procedures on testicular development in cryptorchidism. METHODS: One hundred ten postpubertal Wistar-WU rats were divided into four groups. Group 1 (35 rats) underwent microsurgical autotransplantation of the right testis with end-to-end anastomosis of the testicular vessels. Group 2 (35 rats) underwent the Fowler-Stephens maneuver with intra-abdominal fixation of the testis. To determine the influence of temperature on testicular development, group 3 (35 rats) underwent the Fowler-Stephens maneuver with scrotal replacement of the testis. Group 4 (5 rats) was a control group without operative treatment. To assess the early and late changes of testicular ischemia and reperfusion, groups 1 to 3 were divided into seven subgroups (5 rats each) with defined postoperative intervals (0.5 hour, 2 hours, 4 hours, 8 hours, 16 hours, 14 days, and 60 days). Testicular development, sperm density, pH value, tissue lactate concentration, and tubular diameter were the parameters used to determine success or failure. RESULTS: After autotransplantation, testicular development exhibited an atrophy rate of less than 50% and a significant decrease in groups 2 and 3 after 60 days. The sperm density was significantly different between groups 2 and 4 (control). The pH value was significantly different after 2 hours in groups 2 and 3. The tissue lactate concentration revealed no significant differences. The tubular diameters were significantly diminished in group 2 after 60 days. CONCLUSIONS: The smaller atrophy rate of the testes suggests that we successfully established a model in the rat to compare the operative outcome after autotransplantation and the Fowler-Stephens procedure in cryptorchidism. The significant difference in the tubular diameters for the intra-abdominally placed testes might be caused by the exposure of the testis to a higher temperature.

Anastomosis, Surgical↗

Testicular dysgenesis does not affect expression of anti-müllerian hormone by Sertoli cells in premeiotic seminiferous tubules.

Anti-Müllerian hormone (AMH) immunoreactivity was studied on paraffin sections obtained from archival testicular biopsies of 29 children with intersex disorders and of 22 controls. Strong AMH immunoreactivity was observed in Sertoli cell cytoplasm from 8 fetal weeks until puberty. During pubertal maturation, in both normal and intersex patients, AMH expression was present in premeiotic seminiferous tubules, but was no longer detected in neighboring tubules with meiotic development. AMH immunostaining was abolished in the testis of one patient with persistent Müllerian ducts due to a mutation of the AMH gene, but was conserved in the testes of two patients with mutations of the AMH receptor gene. Testicular dysgenesis usually results in sexual ambiguity, with low testosterone and AMH serum levels and persistence of Müllerian derivatives. AMH immunoreactivity was conserved in premeiotic seminiferous tubules of dysgenetic testes, and also in sex-cord cells of a gonadoblastoma. In patients with asymmetric gonadal differentiation, the streak gonad was AMH-negative. In conclusion, secretion of AMH is a constitutive feature of the immature Sertoli cell and its expression is altered only by mutations of the AMH gene, but not by gonadal dysgenesis. The degree of regression of Müllerian ducts and serum AMH levels reflect the number, not the functional value, of Sertoli cells present in the immature testis.

Adolescent↗

Cytological evaluation of spermatogenesis and organization of the germinal epithelium in the male slider turtle, Trachemys scripta.

The germ cell development in the slider turtle (Trachemys scripta) testis was investigated by viewing the histology of the seminiferous epithelium in plastic sections with a light microscope. Germ cell morphologies in the slider turtle testis were similar to the morphologies of other vertebrate germ cell types. However, the slider turtle seminiferous epithelium contained germ cells that progress through spermatogenesis in a temporal rather than a spatial pattern, resulting in a single spermatogenic event that climaxed with one massive sperm release in November. Mature sperm then are stored within the epididymis until breeding commences in the following spring. The germ cell development strategy in the slider turtle is different from that of other amniotes and is more reminiscent of the developmental strategy found in the anamniotic testis. This temporal progression of germ cells through spermatogenesis within a tubular testis represents a transitional model that may be evolutionarily significant.

Animals↗

Immunoexpression of the steroidogenic enzymes 3-beta hydroxysteroid dehydrogenase and 17 alpha-hydroxylase, C17,20 lyase and the receptor for luteinizing hormone (LH) in the fetal rat testis suggests that the onset of Leydig cell steroid production is independent of LH action.

The production of testosterone in the adult testis is mainly regulated by LH. Testosterone is essential for normal development of the male fetus. The regulation of testosterone production in the fetal testis is less clear than in the adult testis, and there are indications that at least the onset of androgen production in the fetal testis takes place in an LH-independent way. The aim of the present study was to compare the onset of synthesis and pattern of expression of LH receptors and two important steroidogenic enzymes, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17alpha-hydroxylase/17,20-lyase (P450c17), in fetal rat gonads. Whole fetuses (13.5 and 14.0 postcoitum (p.c.), testes, and ovaries dissected from fetuses on Days 14.5 p.c. to 20.5 p.c. and testes obtained on Days 3, 5, and 7 postpartum were fixed in Bouin's solution and processed for immunocytochemistry. In all samples of fetal testis, 3beta-HSD was detected on Day 14.5 p.c., and immunoexpression of P450c17 appeared one day later on Day 15.5 p.c. Thereafter, immunoexpression of both enzymes remained intense throughout gestation and postnatally. In contrast, immunoexpression of LH receptors was detectable only from Day 16.5, when a few weakly positive cells were present, but it became more intense one and two days later. Similar to the immunostaining for 3beta-HSD and P450c17, immunostaining for LH receptor remained strong throughout gestation and during the first 7 days of postnatal life. No immunoexpression of any of the three proteins studied was detected in the fetal ovary at any age. These data show very early immunoexpression of 3beta-HSD, which was detected one day before the reported start of steroid production by the fetal Leydig cells. In contrast, immunoexpression of LH receptors was evident only after immunoexpression of both the steroidogenic enzymes studied had became apparent and after the reported start of steroidogenesis, which is consistent with the theory that onset of steroidogenesis in the fetal rat testis is an LH-independent process.

3-Hydroxysteroid Dehydrogenases↗

Vasectomy and vasovasostomy. I. Timing of histologic changes in immature and mature dog testis after vasectomy.

The effects of vasectomy on the development and maintenance of spermatogenesis was studied using immature and mature dog testes. Bilateral vasectomy in immature dogs delayed the development of advanced spermatocytes, spermatids, and spermatozoa for about 3 months postsurgery. Spermatogenesis appeared to be recovered to control levels by 4 months postvasectomy. Spermatogenesis in mature dogs was also altered after bilateral vasectomy. Decreased numbers of advanced spermatocytes as well as maturation arrest was observed by 3 weeks postvasectomy. Seminiferous tubule cell layers quickly decreased to one to three layers as the lumina became occluded with sloughed cells by 3 to 6 weeks postvasectomy. Recovery in terms of the numbers of spermatocytes, spermatids, and spermatozoa was evident by 13 weeks postvasectomy, although occasional dog testes did not recover and appeared to be more sensitive to vasectomy-induced damage. It thus appears that vasectomy temporarily inhibits both the development and maintenance of spermatogenesis in immature as well as mature dog testes. Spermatogenesis does recover but may be maintained at somewhat lower levels after vasectomy. Changes are rapid in onset and take several weeks to be reversed. Some dog testes appear to be more sensitive to the damage and it may be irreversible in these testes.

Animals↗

The in vivo roles of müllerian-inhibiting substance.

The fetal testis functions as the sex differentiator by imposing a masculine pattern of development upon a genetic program that is inherently female. Two hormones produced by the fetal testis mediate the differentiation of the müllerian and Wolffian ducts (Figs. 1 and 4). MIS actively inhibits the development of the müllerian ducts, and testosterone induces the differentiation of the Wolffian ducts. The absence of these two hormones during fetal development in the female (the hormonal equivalent of no testes) permits müllerian duct differentiation and does not induce Wolffian duct development. The in vivo outcomes of ectopic MIS exposure or MIS deficiency illustrate the balance required to coordinately differentiate and cause regression of the respective male and female genital ducts. The observations made in the MIS-deficient mice demonstrate that codevelopment of both genital duct systems interferes with normal development of both systems and ultimately interferes with reproduction and fertility. Thus, reproduction and fertility in mammals appear to be most efficient if only one type of genital duct system develops. The phenotypes of the MIS-overexpressing transgenic mice and the MIS-deficient mice are similar yet different. Some of the explanations that might reconcile these differences probably lie with the receptor for MIS. Since the MIS-overexpressing transgenic mice are exposed to pharmacological levels of MIS during development, it seems possible that this may lead to productive interactions with other related receptors. Candidate genes have been isolated for the MIS receptor that are membrane-bound serine/threonine kinases (Baarends et al., 1994; di Clemente et al., 1994) similar to those cloned for the TGF-beta (Lin et al., 1992) and activin (Mathews and Vale, 1991) type II receptors. Interestingly, expression of these putative MIS receptor genes is localized by in situ hybridization to the mesenchymal cells adjacent to the müllerian ducts, suggesting that MIS most likely alters the surrounding mesenchyme to elicit müllerian duct regression. Experiments are underway to isolate the mouse MIS receptor gene to thereby generate MIS receptor-deficient mice and to compare the phenotype with the MIS gain-of-function and loss-of-function animals. Isolation of the human MIS receptor gene will facilitate the identification of human PMDS patients with normal levels of MIS that have mutations in the MIS receptor gene. Finally, studies of the MIS receptor gene will open up avenues for the molecular characterization of signal transduction pathways that mediate müllerian duct regression and Leydig cell proliferation control.

Androgen-Insensitivity Syndrome↗

Radiation associated malignancies of the esophagus.

This report documents the experience of the authors with two patients who had received thoracic radiation for disseminated teratocarcinomas of the testis, survived their malignancies, and subsequently developed squamous cell carcinomas of the esophagus. To the authors' knowledge, only 11 other cases of esophageal malignancies arising in patients who had received previous mediastinal irradiation have been reported in the world literature. With increasingly successful mediastinal radiation for malignant disease resulting in prolonged patient survival, an increasing number of such patients with subsequent esophageal malignancies can be anticipated. Close follow-up of patients receiving radiation therapy to periesophageal tissues is recommended, and prompt evaluation of any symptoms of esophageal dysfunction is indicated.

Adult↗

Impaired spermatogenic ability of testicular germ cells in mice deficient in the LIM-kinase 2 gene.

LIM-Kinase (LIMK), including LIMK1 and LIMK2, is the only known catalytic protein among LIM-family molecules. It is well known that LIMK phosphorylates and inactivates cofilin, an actin-depolymerizing factor regulating actin reorganization, while in vivo functions have remained to be elucidated. In the present study, we generated Limk2 gene-deficient mice in which three LIMK2 isoforms were disrupted in a Cre-mediated fashion. Impaired cofilin phosphorylation was clearly observed in Limk2-/- fibroblasts stimulated with bradykinin or lysophosphatidic acid, thereby suggesting that Cdc42 or Rho-dependent LIMK activation did not occur. However, Limk2-/- mice did not exhibit embryonic lethality or any phenotypic abnormalities in postnatal growth and development, except for spermatogenesis in the testis. The testes of Limk2-/- mice were smaller in size and partial degeneration of spermatogenic cells in the seminiferous tubules was apparent in association with increased apoptosis. In addition, the viability of Limk2-/- spermatogenic cells, when cultured under stressed conditions, was diminished. Furthermore, the potential for germ cells to differentiate in a regenerative state was severely impaired in Limk2-/- testis. Experimental hyperthermia induced impairment of ADF/cofilin phosphorylation and the formation of intranuclear cofilin inclusions in mutant germ cells. Based on these findings, we propose that LIMK2, especially the testis-specific isoform tLIMK2, plays an important role in proper progression of spermatogenesis by regulation of cofilin activity and/or localization in germ cells.

Actin Depolymerizing Factors↗

Structural characterization of the mouse Hfh4 gene, a developmentally regulated forkhead family member.

Hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) is a forkhead/winged-helix transcription factor family member that has a unique temporal and spatial pattern of gene expression in the developing and adult lung, choroid plexus, testis, and oviduct. To characterize HFH-4 further, mouse genomic clones were isolated and analyzed. The Hfh4 gene is encoded on a 5.5-kb region located on the distal end of mouse chromosome 11 and consists of two exons and one intron. Unlike most forkhead genes, the DNA binding domain is divided between two exons, and the intron position corresponds precisely to the site of gene translocations involving two known human forkhead homologues. Multiple putative transcription start sites are identified in a G+C-rich sequence that does not contain TATA or CAAT boxes. Within 2.1 kb of 5' flanking sequence are three identical E boxes and multiple putative transcription factor binding sites. Transfection of plasmids containing Hfh4 5' flanking sequence linked to a reporter gene results in promoter activity in lung epithelial cells but not in epithelial-like fibrosarcoma cells, suggesting that this 5' flanking sequence can function as a promoter with the proper cell-type specificity.

Amino Acid Sequence↗

Early testicular development in sex reversed mice.

A comparison is made of two sets of data bearing on the development of testes in XX, Sxr mice. Although Spoljar and Drews (1978) reported no significant difference in the growth rates of XX and XY testes, nevertheless their data suggest that such a difference may exist. This confirms earlier work by Mittwoch and Buehr (1973), who found the testicular volumes of XX embryos aged 15 and 16 days to be below those of XY embryos.

Animals↗

The ultrastructure of the interstitial cells in human fetal ovaries.

Steroid-active interstitial cells (stromal lutein cells) in human fetal ovaries of 12, 15 and 22 weeks of gestation were studied by means of electron microscope. The large cells, which are predominantly situated in the vicinity of capillaries, show high structural similarity to Leydig cells (type A) of the fetal testis. Both are characterized by extensively development of smooth endoplasmic reticulum, accumulation of lipids, and numerous mitochondria of the tubular type containing osmiophilic inclusions. Whether the interstitial cell system of the fetal ovary has any specific function in developmental physiology of the genital system cannot be decided on the basis of the present results. Its differentiation may only indicate adequate response of competent mesenchymal cells to gonadotropic stimulation.

Endoplasmic Reticulum↗

D2-40 functions as an effective chondroid marker distinguishing true chondroid tumors from chordoma.

Chordomas and low-grade chondrosarcomas of the central nervous system share many histological features, generating, at times, considerable diagnostic difficulty and, not infrequently, requiring immunohistochemical analysis for appropriate classification. While both chordomas and chondrosarcomas stain positively for S100, only chordomas typically express epithelial antigens like cytokeratins and epithelial membrane antigen. Positive or negative staining with these latter two markers currently represents the only immunohistochemical technique that effectively distinguishes chordomas from chondrosarcomas. A marker that is reliably positive in chondrosarcomas and negative in chordomas has, to date, not been reported. D2-40 is a monoclonal antibody initially developed against M2A, a fetal testis-related antigen now known as podoplanin (aggrus), which has been found to stain a diverse collection of both benign and malignant tissues. In this study, we systematically investigated D2-40 immunoreactivity in a series of 22 chordomas, 20 chondrosarcomas, and 12 enchondromas, in conjunction with cytokeratin and S100 immunostaining. We found that D2-40 robustly and reliably immunostains low-grade chondroid neoplasms (100% of enchondromas and 94% of grades I and II chondrosarcomas), but not chordomas. By contrast, we observed generally strong and diffuse cytokeratin positivity in all cases of chordoma, but not in cases of enchondroma or low-grade chondrosarcoma. Thus, we show that D2-40 behaves as a chondroid marker differentiating true chondroid neoplasms from chordoma. We also demonstrate D2-40 immunoreactivity in two cases of chordoid meningioma and, in doing so, tentatively provide a means to distinguish this tumor from chordoma.

Adult↗

Germ cell transplantation: a potential treatment of severe testicular failure.

Although the process of spermatogenesis is relatively efficient and resistant to damage, male infertility can result from exposure to toxic agents such as chemotherapeutic regimes, radiation, or occupational exposures to chemicals. Other types of infertility may result from migratory defects or poor survival of primordial germ cells during development, abnormal repopulation of the tubules by spermatogonia during development, or low cellularity of the testis (hypospermatogenesis). Presently, there are no effective therapies available to treat these patients. Recent studies in animal models have demonstrated that isolated testicular germ cells collected from testes may be transplanted into sterile recipient mice to regenerate spermatogenesis. This technology will have widespread applications in efforts to manipulate the genome and produce transgenic offspring, to improve agricultural species, to enhance sperm production in endangered species, to improve our understanding of the control mechanisms regulating spermatogenesis, and to treat male infertility.

Animals↗