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Developmental expression and characterization of FS39, a testis complementary DNA encoding an intermediate filament-related protein of the sperm fibrous sheath.

Proteins immunologically related to intermediate filaments have been identified in the sperm fibrous sheath but remain uncharacterized. We isolated and characterized a novel intermediate filament-related protein (FS39) localized to the fibrous sheath of the sperm tail. We used Northern blot analysis to establish that FS39 is transcribed predominantly in the testis of mice >18-20 days old. At this age, spermatogenesis has proceeded to the development of the first round haploid spermatids. In situ hybridization revealed that FS39 mRNA is first detectable in late step 3 spermatids, is at its highest level during steps 9 and 10, and diminishes in steps 13 and 14. Western blot analysis identified a single protein of 39 kDa in mouse and rat testis and epididymis, suggesting the protein is conserved in rodents. Indirect immunofluorescence localized FS39 to the fibrous sheath of the sperm tail, and in testis sections expression was detected from step 13 and step 14 spermatids onward, indicating FS39 is under translational control. Southern blot analysis showed FS39 to be a single copy gene, and hybridization to human genomic DNA suggested that a human equivalent gene is present. These results demonstrate that FS39 is transcribed in testis tissue during the haploid phase of spermatogenesis, is present in mature sperm, and codes for a novel 39-kDa intermediate filament-related protein of the fibrous sheath.

Amino Acid Sequence↗

Prolactin actions in the sheep testis: a test of the priming hypothesis.

This study investigated whether prolactin (PRL) plays a priming role in the testis during the nonmating season and thereby facilitates gonadal reactivation. Sexually inactive Soay rams under long days were treated as follows: 1) group C (control) received vehicle, 2) group B received bromocriptine to suppress PRL secretion, 3) group B + PRL received bromocriptine + ovine PRL to reinstate physiological levels of PRL (n = 5/group). Treatments were for 10 wk. The photoperiod was then switched to short days to reactivate the reproductive axis. Testis diameter and sex skin coloration were recorded, and routine blood samples were collected to measure concentrations of FSH, inhibin A, and testosterone (T). At the end of the treatments, blood samples were collected every 10 min for 10 h to monitor LH pulses and the T-response to exogenous LH, and a testis biopsy was collected to assess spermatogenic activity (bromodeoxyuridine [BrDU] method) and expression of PRL receptor (reverse transcription-polymerase chain reaction and immunocytochemistry). There were no significant differences between groups in spermatogenesis (BrDU index) or steroidogenesis (T-response), and no difference in the time taken to achieve full testicular redevelopment under short days. Testis diameter and inhibin A were marginally increased in group B + PRL. Overall, this thorough experiment provides minimal support for the priming hypothesis.

Animals↗

Methylation of CpG dinucleotides alters binding and silences testis-specific transcription directed by the mouse lactate dehydrogenase C promoter.

The mouse lactate dehydrogenase c gene (mldhc) is transcribed only in cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-base pair fragment was able to drive testis-specific transcription in vitro and in transgenic mice. Several testis-specific genes are believed to be regulated at least in part through differential methylation of CpG dinucleotides. We investigated the possibility that transcriptional repression of the mldhc gene is mediated in somatic tissues by hypermethylation of CpG dinucleotides. The CpG dinucleotides within a fragment of the mldhc promoter containing a GC box and tandem activating transcription factor/cAMP-responsive element binding sites are hypermethylated in somatic tissues and hypomethylated in testis. Methylation of the activating transcription factor/cAMP-responsive elements altered the protein binding pattern observed in electrophoretic mobility shift assays using mouse liver but not testis nuclear extract. Furthermore, methylation of an extended mldhc promoter fragment driving lac Z silenced transcription from the promoter in a transient transfection assay. These data suggest that tissue-specific differential methylation plays a role in mldhc silencing in somatic tissues.

Activating Transcription Factors↗

Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules.

The mule (Equus mulus mulus) is a sterile hybrid domestic animal that results from the breeding of a male donkey (Equus asinus) to a female horse (Equus caballus). Usually, spermatogenesis in mules does not advance beyond spermatocytes. In the present study, we performed a comparative and more accurate morphometric and functional investigation of the testis in donkeys and mules. Due to the smaller testis size, lower seminiferous tubule volume density, and fewer germ cells, the total length of seminiferous tubules in mules was significantly smaller than in donkeys. However, the percentage of seminiferous tubules containing germ cells (spermatogonia and spermatocytes) in mules was approximately 95%. The total number of Sertoli cells per testis observed in donkeys and mules was very similar. However, the total number of Leydig cells in mules was approximately 70% lower than in donkeys. At least in part, this difference was probably related to the lower number of germ cells present in mule seminiferous tubules. Although spermatogenesis in mules did not advance beyond secondary spermatocytes/newly formed round spermatids, germ cell associations in the seminiferous epithelium and pachytene spermatocytes nuclear volume in donkeys and mules were similar. The duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Each spermatogenic cycle in donkeys lasted 10.5 days. A similar value was found in mules ( approximately 10.1 days). Considering that the entire spermatogenic process takes approximately 4.5 cycles to be completed, its total duration in donkeys was estimated to last 47.2 days. The results found for mules suggest that the mechanisms involved in the determination of testis structure and function are probably originated from donkeys. Also, the data found for mules suggest that their seminiferous tubules are able to sustain complete spermatogenesis. In this regard, this species is a potential model for transplants of germ cells originated from donkeys and horses or other large animals.

Animals↗

H1t/GC-box and H1t/TE1 element are essential for promoter activity of the testis-specific histone H1t gene.

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue-specific expression of the gene is mediated primarily through elements located within the proximal promoter. Previous work in transgenic animals identified a unique 40-base pair promoter element designated H1t/TE that is essential for spermatocyte-specific expression. The H1t/TE element contains three subelements designated TE2, GC-box, and TE1 based on in vitro footprinting and electrophoretic mobility shift assays. Because GC-box is a consensus site for binding of Sp transcription-factor family members, experiments were performed demonstrating that two Sp family members, Sp1 and Sp3, were present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids. A 95- to 105-kDa form of Sp1 is most abundant in the tissues and cell lines examined, but a 60-kDa form of Sp1 is the most abundant species in spermatocytes and early spermatids. Further examination of Sp1 and Sp3 from adult testis, primary spermatocytes, and early spermatids showed that they can bind to the H1t/TE element. In order to determine the contributions of the subelements to H1t transcription, we mutated each of them in H1t promoter luciferase reporter vectors. Mutation of the GC-box and TE1 subelement reduced expression 77% and 49%, respectively, compared with the wild-type H1t promoter in transient expression assays in a testis GC-2spd cell line that was derived from germinal cells. These studies suggest that Sp transcription factors may be involved in transcription of the H1t gene and the GC-box and the TE1 subelement are required for activation of the H1t promoter.

Animals↗

Adenomatous hyperplasia of the rete testis. A clinicopathologic study of nine cases.

Adenomatous hyperplasia of the rete testis is an uncommon lesion that has recently been described. Nine cases of adenomatous hyperplasia were identified in two institutions from 1980 to 1989. At diagnosis the nine patients ranged in age from 30 to 74 years (mean, 59 years; median, 66 years). Three patients presented with a grossly identifiable solid or cystic testicular hilar mass. In six cases adenomatous hyperplasia was an incidental microscopic finding--five from orchiectomy specimens and one from an autopsy specimen. Microscopically, the hyperplasia consisted of a tubulopapillary epithelial proliferation of rete testis. The lining cells were cuboidal to low columnar and lacked nuclear pleomorphism or mitotic figures. The involvement of the rete testis was predominantly diffuse. In seven cases the seminiferous tubules showed atrophic changes. Ultrastructural and immunohistochemical (keratin, epithelial-membrane antigen: positive; vimentin, muscle-specific actin, desmin, and S-100: negative) studies done on one case showed similar features to those of nonhyperplastic rete testis epithelium. No patient with adenomatous hyperplasia showed local recurrence or metastasis. Possible pathogeneses include hormonal imbalance or stimulatory influence that remains as yet unidentified.

Adenoma↗

Cystic transformation of the rete testis.

In a review of the testicular and epididymal specimens obtained from autopsies (1,798 men) or surgery (518 men), cystic transformation of the rete testis (CTRT) was found in 20 autopsies and 18 surgical specimens. When both testes were studied (autopsies), the lesion was bilateral. Ultrasonography revealed a widened mediastinum testis showing small hypoechoic areas. Arteriography showed thin or irregularly outlined testicular arteries, and the epididymal artery was lacking or appeared stenosed. Simple CTRT (without epithelial alteration) was found in both testes of 17 autopsied patients (all were elderly men) and in eight surgically removed testes from patients with sarcoma, tuberculous orchidoepididymitis, or hematocele. The most frequent epididymal lesion was bilateral efferent duct atrophy. In three patients, the rete testis presented nodular proliferation of calcifying connective tissue. CTRT with columnar transformation of the rete testis epithelium was observed in both testes from three patients with alcoholic cirrhosis, and in 10 surgically removed testes from patients with testicular tumor, cryptorchidism, or nonspecific orchitis. In cirrhotic patients, the efferent ducts appeared atrophied. In patients with testicular tumors, the efferent ducts were infiltrated by carcinoma in situ cells (CISs) and often contained granular material, cell debris, or hyaline globules. In both kinds of CTRT (without or with epithelial metaplasia), the most frequent seminiferous tubule lesions were tubular ectasia, hypospermatogenesis, tubular sclerosis, spermatogonium arrest, and sloughing of immature germ cells (spermatids and spermatocytes). The mechanism leading to CTRT might be mechanic (compression of the epididymis by an epididymal tumor or a spermatic cord tumor, or the result of a long-standing epididymitis or traumatic hemocele); ischemic (autopsied elderly men); hormonal (cirrhotic patients); malformative (cryptorchidism); or unknown (the remaining cases).

Adolescent↗

Image analysis assessment of the abnormal testis biopsy in male infertility.

PURPOSE: We previously demonstrated that testis biopsy image analysis is an effective method for quantifying intratubular spermatogenic cells in the obstructed testis with normal spermatogenesis. As an extension of the initial report, we describe using the quantitative ploidy and morphological characteristics of cells counted with image analysis in abnormal testis biopsies obtained for a male infertility evaluation. MATERIALS AND METHODS: Image analysis using a specifically designed filter was performed on Feulgen stained 5 microns, sections of paraffin embedded testicular tissue. Archival testicular tissue had been obtained using standard biopsy techniques from patients with azoospermia or severe oligospermia. Qualitative classification was based on standard evaluation of hematoxylin and eosin processed tissue. RESULTS: There were 62 biopsies performed in 58 men. Significant differences in the intratubular content of haploid (spermatozoa and spermatids), diploid and tetraploid cells were found among the 5 categories of abnormalities: the Sertoli-cell-only syndrome, spermatocyte arrest, spermatid arrest, hypospermatogenesis and normal spermatogenesis. Moderate variability was found in the proportion of cell types in spermatid arrest and hypospermatogenesis. CONCLUSIONS: Testis biopsy image analysis provides a quantitative method for categorizing abnormalities of intratubular cell content present in male infertility states by using deoxyribonucleic acid content and morphology characteristics. The limitations of the present qualitative analysis system are emphasized by the moderate variability evident within the current categories of spermatid arrest and hypo-spermatogenesis states.

Adult↗

Reconsideration of the lymphatic drainage of the rat testis.

An extensive investigation of the lymphatic drainage from the rat testis was carried out in an effort to detect any characteristics which might be important in the immunological privilege the testis extends to allografts. Three different methods revealed a consistent and very efficient lymphatic drainage involving primarily the iliac and renal lymph nodes and to a lesser extent the external lumbar, para-aortic, and posterior gastric nodes. Within minutes, dye or labeled cells injected into the testis could be found within regional nodes. Node hypertrophy was induced by injecting the testes of F1 hybrids with parental strain lymphoid cells (a regional graft-versus-host reaction). Besides indicating that efficient filtration occurs in these nodes, this also established that they are a hospitable environment for cellular immune reactions. Similarly, the capacity of allogeneic cells injected into the testis to induce production of humoral alloantibodies by recipients confirms that exposure to antigens via this route does not in itself suppress immune responsiveness.

Animals↗

Successful islet/abdominal testis transplantation does not require Leydig cells.

Pancreatic islet allo- and xenografts are not rejected and exhibit long-term beta-cell function if transplanted into the abdominal testis of the diabetic host. Successful transplantation appears dependent on local factors unique to the abdominal testis. Because Leydig cells remain viable in abdominal testes, which also retain high levels of testosterone, the following question was addressed: do Leydig cells and/or their secretory products influence islet transplantability in the successful islet/abdominal testis transplantation model? Streptozotocin-induced diabetic rats (Sprague-Dawley) were injected with 75 mg/kg ethane dimethanesulfonate (EDS) to selectively eliminate Leydig cells prior to or following transplantation with islets isolated from the BBWORdr rat. Subcutaneous silastic tubes packed with estradiol prevented Leydig cell repopulation in the EDS-treated recipient. Grafted diabetic animals, including the EDS-treated rats with serum testosterone at castration levels, became nornoglycemic following islet transplantation and remained so far for up to ten months. Leydig cells were not observed in testes of the EDS- or EDS/estradiol-treated rats, whereas the transplanted islets within these testes appeared structurally normal and highly vascularized. Islets resided within the testicular interstitial compartment and contained alpha-, beta and delta-cells, as identified by electron microscopy. Beta cells were most prominent, contained secretory granules and exhibited a close structural and functional relationship with adjacent intraislet capillaries. We conclude that Leydig cells and Leydig cell secretory products, including testosterone, are not necessary for protecting islets against rejection and they do not play an obligatory role in the success of the islet/abdominal testis transplantation protocol. Leydig cells and Leydig cell secretory products do not promote long-term beta-cell function and are not required for the return to and maintenance of normoglycemia in the grafted diabetic rat.

Animals↗

Prescrotal orchiopexy: an alternative surgical approach for the palpable undescended testis.

PURPOSE: Inguinal exploration has been a standard approach for the management of palpable undescended testis. We performed prescrotal orchiopexy in patients with palpable undescended testes at our institution and we report our results. MATERIALS AND METHODS: We reviewed the charts of patients with palpable undescended testes treated with prescrotal orchiopexy from 1999 to 2002. All children were referred to a university children's hospital and 1 of 2 surgeons performed the procedures. Examination using anesthesia was performed prior to any incision. If the testis was palpable and could be drawn close to the scrotum, prescrotal orchiopexy was performed. Retractile testes were excluded. RESULTS: During this period 291 patients underwent orchiopexy. Prescrotal orchiopexy was performed in 78 patients. Followup was 1 to 36 months (median 6). The overall success rate was 98.8% and the overall complication rate was 4.7%. At 7 months postoperatively 1 patient had a palpable retractile testicle. One patient had wound hematoma and another patient had wound cellulitis. At 31 months of followup 1 patient was considered to have a 25% decrease in testis size. All patients were without clinical evidence of hernia or hydrocele. CONCLUSIONS: Prescrotal orchiopexy is a successful procedure in select patients with a low complication rate. It has the advantage of a single, perfectly cosmetic incision. This approach should be considered an option when performing orchiopexy in a patient with a palpable, mobile undescended testis.

Adolescent↗

Localization of aquaporin-7 in human testis and ejaculated sperm: possible involvement in maintenance of sperm quality.

PURPOSE: We have previously reported the expression of aquaporin-7 (AQP7) in the rat testis and cloned human AQP7 from the testicular cDNA library. However, to our knowledge the spatial expression pattern and biological roles of AQP7 remain to be elucidated in humans. MATERIALS AND METHODS: We investigated AQP7 expression in the human testis and ejaculated sperm from fertile men and from infertile patients. RESULTS: AQP7 was expressed at the tail of spermatids and spermatozoa in the human testis. AQP7 protein was also detected at the middle piece and the anterior tale portion of ejaculated sperm. However, some infertile patients lacked AQP7 expression in ejaculated sperm, although all fertile men expressed AQP7 protein. The motility rate of AQP7 negative sperm was significantly lower than that of AQP7 positive sperm, while the sperm concentration was not different between AQP7 positive and negative subjects. CONCLUSIONS: AQP7 shows a spatial expression pattern in the human testis. AQP7 may be involved in the maintenance of sperm motility. Furthermore, a lack of AQP7 expression in sperm may be an underlying mechanism of male infertility.

Aquaporins↗

Contrasting evolution of expression differences in the testis between species and subspecies of the house mouse.

Regulatory changes in genes involved in reproduction are thought to be prime targets for divergence during speciation, since they are expected to play an important role in sexual selection and sexual conflict. We used microarray analysis of RNA from different wild populations of house mouse subspecies (including Mus m. musculus, Mus m. domesticus, and Mus m. castaneus) and from the sister species Mus spretus to test this assumption. A comparison of expression divergence in brain, liver/kidney, and testis shows a major difference in the evolutionary dynamics of testis-related genes. While the comparison between species confirms an excess in divergence in testis genes, we find that all comparisons between subspecies yield only a very small number of genes with significantly different expression levels in the testis. These results suggest that the early phase of the speciation process may not be driven by regulatory changes in genes that are potential targets of sexual selection, and that the divergence in these genes is only established during a later phase of the speciation process.

Animals↗

A set of testis-specific novel genes collected from a collection of Korean Native Chicken ESTs.

We report a set of testis-specific novel genes by comparing the The Institute for Genomic Research (TIGR) chicken gene index with the 13,132 expressed sequence tag (EST) collection from Korean Native Chicken (KNC). A total of 553 novel transcripts have resulted after comparison of our EST contigs and singlets against the chicken gene index and chicken genome sequences. Interestingly, 76% of the new genes were from testis-specific EST contigs or singlet and analysis of exon/intron structure showed that a significant number of the contigs have intronless coding regions. A large portion of the testis-specific transcripts have intronless coding region indicating that the testis-specific expression pattern is related to a group of single exon genes in the chicken. The chicken EST data that are described in this paper have been submitted to the NCBI dbEST under accession numbers CO759017-CO764274, CO765331-CO768903 and CO769672-CO773972.

Animals↗

Cellular localization of the Mn2+-dependent adenylyl cyclase (AC) in rat testis.

In the present study we have examined the exact cellular localization of the Mn2+-dependent adenylyl cyclase (AC) in the rat testis. Several pieces of evidence indicate that this testis specific enzyme is exclusively localized to haploid cells in the testis: firstly, the appearance of Mn2+-dependent adenylyl cyclase coincides in time with the occurrence of haploid cells (IC) in the rat testis. Secondly, tests of tfm rats and mice, in which little or no haploid cells are found, exhibit negligible Mn2+-dependent AC activity. Finally, separation of various testicular cells by BSA gradient sedimentation (Staput fractionation), revealed that this enzyme activity follows the location of haploid cells; the highest specific AC activities being found in the fractions containing elongated spermatids. Some activity found in the primary spermatocyte fraction (4C cells) can to a large extent be explained by the contamination of haploid cells in this fraction.

Adenylyl Cyclases↗

Effect of low doses of alpha chlorohydrin on the enzymes of glycolytic and phosphogluconate pathways in the rat testis and epididymis.

A biochemical study has been made of the effects of low doses of alpha chlorohydrin on all the glycolytic enzymes and two key enzymes of phosphogluconate pathway i.e. glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconate dehydrogenase (6-PGDH) of rat testis and epididymis. All the glycolytic enzymes of testis and epididymis are decreased after treatment with alpha chlorohydrin. G-6-PDH and 6-PGDH are decreased only in epididymis and not in the testis. LDH, ADH and glucose-6-phosphatase were also studied histochemically to show that the drug affects the glycolytic enzymes of epididymal cells and various testicular cell types of testis. Possible significance of these results is discussed.

Alcohol Dehydrogenase↗

Studies on the scrotal testis in unilateral experimental cryptorchidism in rat and guinea pig.

The effects of experimental unilateral cryptorchidism on the scrotal testis regarding weight, morphology and secretion of tubular fluid and the sertoli-cell specific androgen binding protein (ABP) were studied. In the intact guinea pig testis and epididymis an androgen binding component similar to rat ABP was found. In juvenile and adult rats cryptorchid for 17 and 21 days, respectively, and in guinea pigs cryptorchid for 11 weeks, the scrotal testis seemed unaffected regarding all parameters studied. With reference to previous findings of lowered fertility in unilateral cryptorchidism in man the possible mechanisms by which unilateral cryptorchidism may influence the scrotal testis are discussed.

Age Factors↗

Laminin and type IV collagen in the human testis.

Specimens of normal human testis and biopsies from testes with Sertoli-cell-only syndrome in which the seminiferous tubules had a remarkably thickened lamina propria, were investigated immunohistochemically using specific antibodies against human laminin and human type IV collagen. In the normal testis, both laminin and type IV collagen were localized to the epithelial basement membranes and the peritubular cell layers. In addition, laminin was found in the Sertoli cells. In the pathological testis, structures representing invaginations of the tubular basement membrane were positive for both laminin and type IV collagen. The presence of laminin and type IV collagen in the myoid cell layers, and laminin in the Sertoli cells from both normal and pathological testis and its indication for the secretion of these substances by the myoid and Sertoli cells is discussed.

Biopsy↗