Three-dimensional analysis and in vivo imaging for sperm release and transport in the murine seminiferous tubule.

Spermatozoa released from Sertoli cells must be transported to the epididymis. However, the mechanism of the luminal flow in seminiferous tubules has remained unclear to date. Therefore, in this study, we investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging. Serial 5-µm-thick mouse testicular sections at 50-µm-intervals were prepared and stained by Periodic Acid-Schiff-hematoxylin. After three-dimensional reconstruction of the seminiferous tubules, the localization of the released spermatozoa and the stages observed in the sections were recorded in each reconstructed tubule. Luminal movements in the seminiferous tubules were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system. Spermatozoa without contact to the seminiferous epithelium were not accumulated toward the rete testis. Additionally, such spermatozoa were found on their way not only to the most proximal rete testis but also a more distant rete testis from any stage VIII seminiferous epithelia. In vivo imaging demonstrated that the direction of the flagella of spermatozoa attached to the seminiferous epithelium was repeatedly reversed. The epithelium at the inner curve of the seminiferous tubule was shaken more actively and had fewer spermatozoa attached compared with the epithelium at the outer curve. Our results hence suggest that the luminal flow in the seminiferous tubules is repeatedly reversed and that this physical force helps spermatozoa to be released from Sertoli cells. In brief: Spermatozoa are released from Sertoli cells and flow in the seminiferous tubule to the rete testis. Our results suggest that the luminal flow in the tubules is repeatedly reversed and that this physical force helps spermatozoa release from the Sertoli cells.

Three-dimensional analysis of seminiferous tubules and spermatogenic waves in mice.

The aim of the present study was to reconstruct seminiferous tubules and analyze spermatogenic waves in seminiferous epithelia in developing and adult mice using serial paraffin sections and high-performance three-dimensional (3D) reconstruction software. By labeling the basement membrane of seminiferous tubules with fluorescent immunohistochemistry or periodic acid-Schiff-hematoxylin staining, all seminiferous tubules were reconstructed in 9 testes from 9 different mice, 3 each at 0, 21 and 90 days (adult) postpartum. The 3D structure of seminiferous tubules, including the number and length of tubules as well as the number of connections with the rete testis, branching points and blind ends, was assessed accurately. Although tubules showed marked variations among individual mice, their overall structure was regular and retained from newborn to adult mice. Some seminiferous tubules contained inner portions running distant from the testis surface. In a representative testis at 21 days, the sites at which spermatids initially occurred were examined by labeling acrosomes and were found to be preferentially distributed in the upper and medial portions of the testis close to the rete testis. In a representative adult testis, 76 complete waves with an average length of 16.9 mm were found and their directions were analyzed. The methods used in the present study will be useful for investigating the structure and function of seminiferous tubules in mice and humans under normal and pathological conditions, such as infertility.

Retinoic acid treatment alters germ cell heterogeneity and testicular echotexture in prepubescent ram lambs.

Testicular echotextural attributes are closely associated with spermatogenic development; however, precise characterisation of specific germ cell types is difficult due to tremendous germ cell heterogeneity. Recently, retinoic acid (RA) administration in neonatal mice was found to induce highly synchronised spermatogenesis as adults. A RA-treatment protocol was tested in 17 ram lambs treated with or without RA at 8 weeks of age, with scrotal ultrasonography and blood samples collected until castration 24h or 2.5 weeks later. At 8.2 weeks of age, the nuclear:seminiferous tubule (ST) area was higher in the treated compared with the control group. Serum testosterone concentrations and numerical pixel values (NPVs) of the testicular parenchyma reached a peak at 9 weeks of age in both groups of ram lambs studied. At 10.5 weeks of age, the percentage of ST cross-sections with different germ cells as the most mature germ cell type was lower and the inter-tubular heterogeneity and NPVs were also lower in the treated compared with the control animals. RA manipulation of spermatogenesis in prepubertal ram lambs may provide a suitable model for further investigation of the echotextural characteristics of specific germ cell types and critical developmental events.

Feasibility of high-frequency ultrasound for seminiferous tubule assessment and correlation of B-mode imaging with pathological findings in the testis in azoospermia.

PURPOSE: To determine the feasibility of high-frequency ultrasound (HFUS) for assessing seminiferous tubules and to understand high-resolution B-mode images of the testes in cases of azoospermia. METHODS: We verified how the histopathological images of testicular biopsy specimens can be observed using HFUS images and measurement analysis of seminiferous tubules was performed to 28 testes of 14 cases with azoospermia who underwent preoperative ultrasound and microdissection testicular sperm extraction (micro-TESE). The population consisted of obstructive azoospermia (OA) and non-obstructive azoospermia (NOA), including Sertoli cell-only syndrome (SCOS), and the other pathologies. Statistical verification of differences in seminiferous tubule diameters among preoperative ultrasound examination, ultrasound examination of pathological specimens, and histopathological specimens. We also examined the imagingpathology correlation via a case series presentation, aiming to identify imaging markers of testicular pathology and determine the possibility of predicting each condition. RESULTS: A comparison between HFUS images and histopathology from the same biopsy specimens suggested that ultrasonography could be seen as stereoscopic images due to its significantly greater slice thickness. The diameters of tubules were generally larger in pathological tissues as compared to ultrasonographic findings in OA and SCOS, but not in the other conditions. Comparisons provided insights into the predictability of SCOS and revealed imaging findings such as gaps between tubules and decreased diameter reflective of testicular damage. CONCLUSION: Seminiferous tubules can be observed however the diameter of seminiferous tubules varies in imaging and histopathology depending on the pathology. Imaging findings that reflect testicular damage and the predictability of SCOS were revealed in this study, but further verification is required.

Ultrasonographically determined size of seminiferous tubules predicts sperm retrieval by microdissection testicular sperm extraction in men with nonobstructive azoospermia.

OBJECTIVE: To investigate the value of the ultrasonographically determined size of seminiferous tubules and other conventional parameters for predicting sperm retrieval by microdissection testicular sperm extraction (micro-TESE). DESIGN: Clinical retrospective study. SETTING: Two urological clinics. PATIENT(S): Eight hundred six men with nonobstructive azoospermia. INTERVENTION(S): Micro-TESE. MAIN OUTCOME MEASURE(S): Sperm retrieval. RESULT(S): Sperm retrieval was successful in 240 (29.8%) of the 806 men. In a receiver operating characteristic analysis of sperm retrieval, the area under the curve (AUC) for seminiferous tubules, assessed as 0, 100, 200, 250, or 300 µm, was no less than 0.82 (95% confidence interval [CI] 0.79-0.85). Sensitivity and specificity at a cutoff point of 250 µm were 76.7% and 80.7%, respectively. An AUC of 0.85 (95% CI, 0.81-0.88) was attained in a parsimonious multiple logistic regression model that included age (<30, 30-39, and 40-59 years), low follicle-stimulating hormone (<14 IU/L), history of cryptorchidism, and sex chromosome abnormality in addition to the diameter of seminiferous tubules. CONCLUSION(S): The gray-scale image in testicular ultrasound was shown to be highly predictive of sperm retrieval in micro-TESE in a large series of men with nonobstructive azoospermia.

Heat Stress and Pulsed Unfocused Ultrasound: The Viability of these Physical Approaches for Drug Delivery into Testicular Seminiferous Tubules.

AIM: To investigate the application of Scrotal Heat Stress (SHS) and Pulsed Unfocused Ultrasound (PuFUS) to explore Blood-Testis Barrier (BTB) permeability in adult mice. BACKGROUND: The BTB provides a stable microenvironment and a unique immune barrier for spermatogenesis. Meanwhile, it blocks macromolecular substances access, including therapeutic agents and antibodies, thereby it decreases the therapeutic or immunocontraception effects. OBJECTIVES: To determine the viability of these physical approaches in delivering macromolecular substances into seminiferous tubules. MATERIALS & METHODS: Mice were subjected to receive single SHS intervention at 39°C, 41°C, or 43°C for 30 min. Whereas, mice received the PuFUS intervention at 1.75w/cm2, 1.25w/cm2, and 2.5w/cm2 for 2 min, 5 min, and 10 min, respectively. The Biotin and macromolecular substances (IgG, IgM, and exosomes) were separately injected into the testicular interstitium at different times following SHS or PuFUS interventions, to observe their penetration through BTB into seminiferous tubules. RESULTS: As detected by Biotin tracer, the BTB opening started from day-2 following the SHS and lasted for more than three days, whereas the BTB opening started from 1.5h following PuFUS and lasted up to 24h. Apparent penetration of IgG, IgM, and exosomes into seminiferous tubules was observed after five days of the SHS at 43°C, but none at 39°C, or any conditions tested with PuFUS. CONCLUSION: The current results indicate that SHS at 43°C comparatively has the potential for delivering macromolecular substances into seminiferous tubules, whereas the PuFUS could be a novel, quick, and mild approach to open the BTB. These strategies might be useful for targeted drug delivery into testicular seminiferous tubules. However, further studies are warranted to validate our findings.

Seminiferous tubule molecular imaging for evaluation of male fertility: Seeing is believing.

The proper assessment of male fertility is essential for diagnosing and treating male infertility. Currently, spermiogram and Johnsen testicular biopsy score counts are used to assess male fertility. However, spermiogram is not a suitable option for non-obstructive azoospermia patients, and Johnsen testicular biopsy scores only represent localized and not the overall spermatogenesis. Whole-mount staining was a novel method for evaluating protein expression in the tissue. Thus, we explored its application in human seminiferous tubules. Testicular biopsies from 57 azoospermia patients were categorized as obstructive azoospermia (OA), maturation arrest (MA) and Sertoli-cells only syndrome (SCOS). We performed whole-mount staining of their seminiferous tubules and evaluated the spermatogonial stem cells (SSCs), differentiated spermatogonia (SG), spermatocytes (SPC) and spermatids (SD) with their respective markers (GFRA1, CD117, SYCP3, and PNA) to assess fertility. GFRA1, CD117, SYCP3, and PNA were not expressed in SCOS patients, whereas all of them were detected in OA patients. In MA patients with arrested spermatogenesis at the SPC stage, GFRA1, CD117, and SYCP3, but not PNA were expressed in the seminiferous tubules. In MA patients with arrested spermatogenesis at the spermatogonia stage, only GFRA1 was expressed in the seminiferous tubules. These results were consistent with the Johnsen testicular biopsy score counts except for one patient, where although only Sertoli cells were indicated by the score, SSCs were also detected in the whole-mounts. Collectively, whole-mount staining could be used to analyze the inherent spermatogenesis of seminiferous tubules through staining of germ cells at different stages. It offers a more accurate and promising faster method for assessing male fertility compared with traditional biopsy screening. And it could have potential value for the clinical purpose for male fertility management.

[Doppler color echography in the diagnosis of impotence]. / Eco-color-Doppler nella diagnostica dell'impotenza.

This study reports the experience achieved with duplex and color Doppler ultrasonography in 120 impotent patients. The following morphodynamic parameters of the cavernosal arteries were studies before and after intracorporal injection of a mixture of vasoactive drugs: arterial diameter, wall pulsatility, morphology of the spectral waveform, peak systolic velocity, end diastolic velocity and flow volume. The veno-occlusive mechanism of the corpora cavernosa was studied directly by determination of flow along the deep dorsal vein of the penis and indirectly by serial evaluation of the diastolic flow of the cavernous arteries.

[Testicular artery Doppler flow evaluation in men with degenerative changes of seminiferous tubules]. / Ocena przeplywów naczyniowych w tetnicy jadrowej u mezczyzn ze zmianami zwyrodnieniowymi kanalików kretych jader.

AIM: The aim of this study was to assess the eventual impact of degenerative changes of seminiferous tubules on Doppler flow parameters in testicular vessels. MATERIAL AND METHODS: There were 12 men with degenerative changes of seminiferous tubules (diagnosed by testicular biopsy) in study group and 24 healthy men with normal semen parameters in control group. Patients' age in both groups was respectively 32.22 +/- 5.31 and 29.64 +/- 3.17 (ns). Sonographic biometry of testes volume was performed and the values of qualitative flow indices in terminal section of testicular artery were evaluated. The both groups results were compared with themselves by T-test. Also the values of qualitative flow parameters in testis after biopsy and contralateral one were compared. Additionally the correlation between vascular flow parameters and selected hormone levels (FSH, LH, T, E2 and Prl) was determined. RESULTS: The mean testes volume in the study group was 10.81 +/- 3.77 cm3 and in the control one 14.42 +/- 5.42 cm3 (ns). The values of particular flow indices in both study and control group were respectively: S:D 3.20 +/- 0.95 and 2.92 +/- 0.96 (ns), PI 1.46 +/- 0.46 and 1.23 +/- 0.41 (ns), RI 0.69 +/- 0.09 and 0.66 +/- 0.08 (ns). There were no differences between flow parameters in testis after biopsy and contralateral one. Testosterone was the only hormone correlating with analyzed flow indices (S:D, PI, RI) and the correlation coefficient value was respectively 0.51, 0.49 and 0.52 (p < 0.05). CONCLUSIONS: The significant influence of degenerative changes of seminiferous tubules on vascular flow in testicular artery was not observed. There were no differences between flow parameters in testis after biopsy and contralateral one. The testosterone was the only hormone significantly correlating with analyzed flow indices.

Testicular microlithiasis. A case report.

A 63-year-old man presented with bilateral testicular microlithiasis and right-sided epididymitis, the latter of which responded to antibiotic and anti-inflammatory drugs. Microlithiasis has been associated with nondescent, infection, neoplasia, and other pathologic conditions of the testicles, but there is no evidence that it is a premalignant condition. It is likely to be seen more often with the increasing use of scrotal ultrasonography.

Individual variation related to testicular biometry and semen characteristics in collared peccaries (Tayassu Tajacu Linnaeus, 1758).

The aim of this research was to study the individual variation with regard to the morphometry of the testes evaluated by ultrasonography and semen characteristics and to verify the existence of relationship between these variables in collared peccaries. In addition, the testes of the animals were evaluated by histology in order to determine the proportion occupied by the seminiferous tubules. A total of 52 ejaculates were obtained from ten adult specimens that had been restrained by anesthesia. The testicular measurements (length, height, and width) were performed by ultrasonography, and the testicular volume was calculated according to Lambert's formula. The scrotal circumference was measured by encircling the thickest portion of the testicle with a graduated nylon tape. The semen was collected by electroejaculation. Testicular fragments were analyzed through classic histology for the determination of the area occupied by the seminiferous tubules. The results show a great amount of individual variation with regard to testicular morphometry and semen characteristics. No significant correlations were obtained between testicular measurements and semen characteristics. The histometric analysis revealed that 67.8% of the testes are occupied by seminiferous tubules. Results show that the measurement of testicular dimensions does not serve as an indicator of the quality of semen obtained by electroejaculation in collared peccaries, as there is no correlation between testicular morphometry and semen characteristics in this species that presents large variations among individuals.

Tubular ectasia of the rete testis: a diagnostic dilemma.

Tubular ectasia of the rete testis is a pathologically benign process with complex and varied aetiology. It must be differentiated from neoplastic disease of the testis clinically with patient age, mode of presentation, tumour marker status and the characteristic ultrasound and Doppler study findings. Awareness and diagnosis of this clinical entity can prevent unnecessary surgical intervention in these patients.

Testicular microlithiasis: what does it mean clinically?

OBJECTIVE: To review our current practice of follow-up for boys with testicular microlithiasis (TM), an uncommon condition characterized by calcification within the seminiferous tubules, detected by ultrasonography (US); TM has been associated with both benign and malignant conditions of the testes but the natural history of TM in children remains unclear. PATIENTS AND METHODS: All boys diagnosed with TM over a 14-year period were included in this study. A search of the radiology database was carried out using the keywords 'testicular microlithiasis' and 'testicular calcification'. A retrospective case-note review was then used to determine age at diagnosis, presenting symptoms, indication for testicular US, outcome and follow-up. We also searched Medline/PubMed, using the same keywords for published data on TM from 1970 to 2006. RESULTS: Over the study period 711 testicular scans were taken in 623 patients; seven cases (1.1%) of TM were identified. The mean (range) age at presentation was 12 (7-15) years. The presenting symptoms were testicular pain (three), undescended testes (two), hydrocele (one) and asymptomatic scrotal swelling (one). In five cases the TM was bilateral and in two a solitary kidney was identified. Only one patient had tumour markers measured (beta-human chorionic gonadotrophin and alpha-fetoprotein) and these were within normal limits. On yearly US follow-up, the TM was less prominent in one patient, unchanged in four and two were lost to follow-up. Three patients are currently on yearly US follow-up while two are under the care of adult general surgeons. The analysis of reports published to date indicated that malignancy only develops when TM is associated with other predisposing factors. CONCLUSION: There is no convincing evidence that TM alone is premalignant. However, when it accompanies other potentially premalignant features we recommend annual US follow-up.

Ultrasonic texture and volume of testicles in infertile men.

Ultrasound scanning of the testes and surgical biopsy were performed in 95 infertile men to evaluate the use of ultrasound in male infertility. Ultrasonic testicular volume was calculated using three measurements and the formula of an ellipsoid, and the ultrasonic texture was evaluated and given a score from 1 to 5, indicating increasing degrees of irregularity. The median score was 3 (range 1-5), which was higher than previously found in normal men (median score 2; range 1-5; P < 0.0001). The ultrasonic texture score was lower in testes with a uniform pattern of 100% spermatogenic tubules compared with the rest, both for the right (P < 0.001) and for the left (P < 0.0005) testis. Texture score was correlated with the number of obliterated tubules for both testes (P < 0.001). The mean ultrasonic testicular volume of the right testis was 10.30 ml, and that of the left 10.26 ml. Both were smaller compared with the findings in normal men (P < 0.0001). Ultrasonic testicular volume was negatively correlated with texture score (P < 0.001). A positive correlation between ultrasonic volume and sperm count was seen (P < 0.001). Sperm count was negatively correlated with texture score if calculated together with data from 119 men from the general population (P < 0.001). The study shows that ultrasonic volume and texture are valuable parameters in the evaluation of infertile men.

Tubular ectasia of the testicle: sonographic and MR imaging appearance.

OBJECTIVE: Ectasia of the seminiferous tubules at the level of the mediastinum is a recently recognized benign condition of the testicles. Although it may have typical sonographic features, the condition can at times be difficult to distinguish from tumors on the basis of sonography. We describe the sonographic and MR appearance of this condition in seven men in whom we were able to distinguish tubular ectasia from tumors of the testicles on the basis of the imaging findings. MATERIALS AND METHODS: Ectasia of the seminiferous tubules was histologically proved in a man who had an intratesticular mass seen on sonograms and MR images. We analyzed the sonographic and MR imaging features in this man and in six others who had similar imaging findings but did not have biopsy proof of tubular ectasia. RESULTS: The first patient was a man with bilateral findings who had a unilateral orchiectomy that revealed ectasia of the seminiferous tubules. Spermatocelectomy was performed in another man whose testis was normal on surgical inspection and on subsequent follow-up. Imaging findings have remained unchanged in one man with 3-year imaging follow-up and in three men with clinical follow-up of 8-22 months. One patient was lost to follow-up. At presentation, most of the men were more than 55 years old. All had a scrotal mass typical of a spermatocele on physical examination, with normal testes when the testes could be palpated. The spermatocele was larger than 4 cm in six of 11 involved epididymides. On imaging, in five of the seven patients, the intratesticular process was bilateral, involved the mediastinum testis, began at the periphery adjacent to the spermatocele, and extended for a variable distance within the testis. On sonograms, the lesion was hypoechoic with coarse internal echoes. MR imaging of six of the seven patients showed characteristic findings and allowed the identification of two additional testicles with tubular ectasia that were missed sonographically. Lesions had a homogeneous signal similar to that of the coexisting spermatocele with all pulse sequences. They were hypointense relative to the testis on T1- and proton density-weighted images and, unlike tumors, were not visible on T2-weighted images. CONCLUSION: Our experience suggests that ectasia of the seminiferous tubules can be distinguished from testicular tumor on the basis of characteristic clinical, sonographic, and MR imaging findings. Thus, orchiectomy is unnecessary to establish the diagnosis and to rule out tumor.

Tubular ectasia within the mediastinum testis.

Eleven scrotal sonographic examinations showing a spectrum of findings within the mediastinum testis were collected over a 2 year period. Each case showed numerous small tubular or rounded anechoic structures within the mediastinum testis; often, the findings mimicked a hypoechoic mass. Findings were bilateral in eight of ten patients; one additional patient had only one testis because of orchiectomy. All patients had an associated extratesticular finding, in most cases a spermatocele. Tubular ectasia shares several features with testicular cysts and mechanisms of formation are postulated to be similar to those previously proposed for testicular cysts. Recognizing tubular ectasia is important to avoid unnecessary concern and potential surgery.

Intercellular bridges and apoptosis in clones of male germ cells.

When an As spermatogonium divides to form a pair of Apr spermatogonia the two daughter cells stay interconnected by an intercellular bridge. These cytoplasmic bridges form after every subsequent division leading to large clones of interconnected germ cells. Cohorts of spermatogonia maintain synchronous development throughout spermatogenesis, which has been attributed to the presence of these intercellular bridges. To examine whether apoptotic signals are transduced through the intercellular bridges we studied germ cell apoptosis in whole mounts of seminiferous tubules from non-irradiated and irradiated mouse testes, using whole mount seminiferous tubules and confocal microscopy. This allowed us to use TUNEL staining of apoptotic germ cells and at the same time to study these apoptotic germ cells in their topographical context. Our results show that in response to ionizing radiation single spermatogonia within a clone can undergo apoptosis without affecting their neighboring cells. Additionally, also early spermatocytes were shown to undergo apoptosis individually. Both radiation-induced spermatogonial apoptosis and spontaneous apoptosis of spermatocytes are caused by DNA damage of individual cells. Degeneration of healthy spermatogonia because of regulatory signals, however, follows other death inducing mechanisms, which lead to apoptosis of chains of interconnected spermatogonia.

Tubular ectasia of the rete testis: a potential pitfall in scrotal imaging.

Tubular ectasia of the rete testis (TERT) is a benign entity due to dilation of the tubules of the rete testis. Most of the time it is discovered incidentally on scrotal sonograms and may be misinterpreted as malignant. This article outlines the diagnostic criteria of TERT, its possible causes, its incidence and its potential evolution. Recognizing this entity owing to its characteristic clinical, sonographic and, if necessary, MRI features is important to avoid unnecessary surgery or biopsies.