Testicular Blood Research Articles

P–095 Outcomes and predictive factors of successful salvage microdissection testicular sperm extraction (mTESE) after failed TESE in men with non-obstructive azoospermia: results from a multicenter study

Abstract Study question We assessed the outcome and predictors of successful salvage microdissection testicular sperm extraction (mTESE) in non-obstructive azoospermia (NOA) men previously submitted to unfruitful classic (cTESE). Summary answer The sperm retrieval rate at salvage mTESE was almost 50%. Hypospermatogenesis and low FSH values were associated with positive outcomes at salvage mTESE What is known already In men with NOA testicular sperm can be retrieved using cTESE in approximately 50% of cases. mTESE has been proposed as a salvage treatment option for men with a previously failed TESE, but data are scarce. Study design, size, duration Multicenter, cross-sectional study. Complete data from 61 NOA men who underwent mTESE after a failed cTESE between 01/2014 and 10/2020, at 6 tertiary referral centers in Italy were analysed. Participants/materials, setting, methods All men underwent testicular ultrasound, hormonal and genetic blood testing. Histopathological diagnosis from TESE was collected in every man. Semen analyses were based on the 2010 WHO reference criteria. mTESE was performed according to the technique of Schlegel et al. (1999). Descriptive statistics and logistic regression models were used to investigate potential predictors of positive sperm retrieval (SR+) after salvage mTESE. Main results and the role of chance Overall, median (IQR) age and testicular volume were 35 (31–38) years and 10 (6–15) ml, respectively. Baseline serum FSH and total testosterone levels were 17.1 (8.6–30.4) mUI/mL and 4.7 (3.5–6.4) ng/mL, respectively. Sertoli-cell-only (SCO) syndrome, maturation arrest (MA) and hypospermatogenesis were found in 24 (39.3%), 21 (34.4%) and 16 (26.2%) men after cTESE, respectively. Spermatozoa were retrieved in 30 (49.2%) men at salvage mTESE. Patients with a diagnosis of hypospermatogenesis had a higher rate of SR + [12/16 (75%)] than those with MA [12/21 (57.1%)] and SCOS [6/24 (25%)] after salvage mTESE (p < 0.01), which was bilateral in 36 (59%) cases. FSH was higher [16.5 (8–22) vs. 8.9 (5–13) mUI/mL, p < 0.01] in SR- patients compared to SR+. No difference in clinical characteristics was found between patients with SR+ and SR- at salvage mTESE. There were no significant complications after mTESE. Multivariable logistic regression analysis showed that hypospermatogenesis (OR 9.7; p < 0.01) and low FSH levels (OR 0.9, p < 0.001) were independent predictors of SR+ after salvage mTESE, after accounting for age. Limitations, reasons for caution Despite we analysed one of the largest series of salvage mTESE, the samples size is too small to draw general conclusions. Because of the multicenter nature of the study we cannot rely on standardization of surgical techniques for TESE. Wider implications of the findings: This is one of the larger studies on salvage mTESE. The selection of patients for salvage mTESE is of critical importance. Trial registration number na

ПАХОВЫЕ ГРЫЖИ У ПАЦИЕНТОВ ПОЖИЛОГО ВОЗРАСТА. НОВЫЙ МЕТОД АТЕНЗИОННОЙ ГЕРНИОПЛАСТИКИ

Aim of the study. To develop a new method of tension-free inguinal hernioplasty in elderly patients, which will expand the arsenal of effective open methods of surgical treatment of inguinal hernias and improve the results of their treatment. Material and methods. A total of 172 elderly patients with inguinal hernias were treated. Hernioplasty according to the Lichtenstein method was performed in 83 patients, and according to the developed method – in 89 patients. The testicular volume and blood flow in a.testicularis were determined by ultrasound before surgery, in the early postoperative period, and 1 year after surgery. Results. The developed method of hernioplasty has the following advantages: 1) it permits to reduce significantly the contact area of the elements of the spermatic cord with a mesh implant; 2) the back wall of the inguinal canal is strengthened not only by the mesh implant, but the aponeurosis of the oblique abdominal muscle; 3) a window in a mesh implant corresponds to the diameter of the elements of the spermatic cord, 4) no decrease in testicular volume at the site of the operative intervention is observed in the late postoperative period. Conclusion. The proposed method of hernioplasty is simple to use and surpasses the well-known Lichtenstein method in a number of parameters.

Testicular Morphological and Ultrasonographic Characterization of Male Gray Brocket Deers (Mazama gouazoubira) in Different Reproductive Status

Background: Gray brocket deer (Mazama gouazoubira) populations have been declining due to human intervention. Yet, only a few studies have assessed ultrasonographic testicular characteristics in cervids. Considering the relevance of monitoring testicular size, blood flow, and parenchyma, the present study aims to establish baseline information on scrotal circumference, testicular volume, and spectral Doppler parameters, to describe differences among adult male gray brocket deer in different reproductive status, and to correlate ultrasound parameters with testes size measurements.Materials, Methods & Results: Six adult male gray brocket deers were used in the study. Scrotal circumference and testicular volume were measured. B mode ultrasound images of testes (longitudinal and cross-sectional views) and epididymes were subjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel standard deviation (PSD). Using spectral Doppler, supratesticular artery blood flow velocities (peak systolic velocity - PSV, end diastolic velocity - EDV, time-average maximum velocity - TAMAX, resistivity - RI and pulsatility indices - PI) were obtained. Semen was analyzed through total motility, vigor, and concentration tests. Three animals were normospermic (F+ group) and three were oligo/azoospermic (F- group). Groups were compared using were compared using a one-way ANOVA or Kruskal-Wallis followed by Student-Newman-Keuls (SNK) test. Ultrasound parameters were correlated to testes size parameters using Pearson’s correlation for parametric variables and Spearman’s correlation for non-parametric variables. F+ group presented significantly higher scrotal circumference (14.57 ± 1.19 cm), testicular volume (26.18 ± 4.94 cm3), and testes cross-sectional NPV (69.88 ± 24.00) and PSD (10.78 ± 3.42) than group F- (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). No significant differences were observed between the groups regarding the spectral Doppler ultrasound parameters. Significant correlations were observed between scrotal circumference and longitudinal (r = 0.76) and cross-sectional testes NPV (r = 0.89), and testicular volume was correlated with longitudinal (r = 0.78) and cross-sectional testes NPV (r = 0.91) and with cross-sectional testes PSD (ρ = 0.82).Discussion: Increased testicular echogenicity (higher NPV) has been positively associated with improved testicular growth, cell population expansion, inner and outer seminiferous tubules diameter, spermatids percentages and testis weight. In addition, more heterogenous testes (higher PSD) were associated with higher sperm output. It was suggested that the animals in group F- had compromised testicular development and spermatogenesis. The correlation observed between testes NPV and scrotal circumference was proposed to be associated with seminiferous tubules impairment. The F- group showed lower testicular volume, NPVs and PSDs in cross-sectional testicular images, suggesting higher protein levels and lower lipid contents were present in their parenchyma, influencing in testicular echogenicity and echotexture. No differences in spectral Doppler parameters were observed between the two groups. Also observed in stallions. However, PSV, EDV, TAMAX could be potential infertility indicators in other mammalians. These different results may be due to different locations of the evaluated vessel, species and techniques, age, ambient temperature, pathological conditions, and anaesthesia. Thus, it is suggested that scrotal circumference, testicular volume, and testes NPV are good indicators of male reproductive health in gray brocket deer and may help with better male selection in the species.

Impact of lunar phase at birth on testicular morphology, semen characteristics and blood chemistry of male goats

ABSTRACT The aim was to investigate the possible effects of different lunar phases at birth on the pre-pubertal changes in the body weights, blood parameters and hormonal profile of male goats. Based on the birth date, 33 male Nubian kids (3 months of age) were selected to represent the new (NL), full (FL) and old (OL) phases of the lunar cycle. The FL group exhibited the signs of puberty earlier than the NL and OL groups (P=0.011). At the age of puberty, bucks in the FL group had significantly higher ejaculate volume (P=0.010), sperm cell concentration (P=0.004), mass motility (P=0.016) and the percentage of live spermatozoa (P=0.008) as compared to the NL and OL groups. Additionally, male goats in the FL group showed significantly higher serum testosterone levels at week 25, 26, 27 and 28 of age compared with the NL and OL groups (P=0.002, 0.013, 0.038 and 0.004, respectively). In conclusion, the growing males in the FL group reached the puberty earlier than the NL and OL groups. Moreover, this study may help to maximize the semen quality and develop more effective AI strategies in the modern goat production systems.

Penoscrotal edema: a case report and literature review

BackgroundPenoscrotal edema is typically caused by lymphatic obstruction, which can have both primary and secondary causes. Studies describing congenital penoscrotal edema are rare. Surgery can be divided into two types: The first approach involves extensive removal of diseased tissue and tissue reconstruction. The second approach is removal of the lesions and creating additional lymphatic vascular anastomoses.Case presentationWe present a case report of a 15-year-old patient with recurrent penoscrotal edema and swelling of both lower extremities. The literature were also reviewed to provide additional information. Physical examination revealed slow lymphatic reflux of the lower extremities and no obvious abnormalities in testicular morphology, bilaterally, or blood supply. Surgery was performed by excising the affected skin and subcutaneous tissue and the flaps was cut in the middle in Y shape to cover the penis and scrotum. Postoperative follow-up revealed wound integrity and patient satisfaction with the outcome.ConclusionExcision and reconstructive surgery are the primary treatments for penoscrotal edema. The majority of reported patients undergoing excision and reconstruction achieved satisfactory reshaping and improved their life quality.

151 Associations of Testicular Size and Arterial Blood Flow with Sexual Development and Semen Characteristics in Dorper Rams Raised in a Subtropical Climate

Spectral Doppler (velocimetric) indices are important indicators of blood perfusion and physiological status of internal organs. Doppler ultrasound is used in andrology to assess the blood flow in the testicular arteries, aiding in the diagnosis of scrotal disorders and abnormal spermatogenesis. It was proposed that blood flow indices measured in the testicular artery, namely, blood flow resistance and pulsatility index, were significant predictors of semen quality in clinically healthy dogs. There is a paucity of studies on the associations of testicular morphology and blood flow dynamics with spermatogenesis in growing rams. Forty-five Dorper rams (24 rams aged 8 to 11 months and 21 rams aged 12 to 24 months) were examined using a portable ultrasound scanner connectecd to a linear-array 7.5-MHz transducer. Measurements of the testes were taken with built-in electronic calipers on images containing the largest cross-sectional area of each testicle in 3 planes (coronal, sagittal, and transverse). Colour and spectral Doppler scans were performed immediately after scrotal ultrasonography to detect the testicular arteries and record the following velocimetric indices: peak systolic velocity (PSV, cm/s), end-diastolic velocity (EDV, cm/s), resistive index (RI = [PSV – EDV)/PSV], and pulsatility index [PI = (SPV – EDV)/mean velocity] of the blood vessels. The length (9.7 ± 0.3 v. 9.0 ± 0.2 cm; mean ± SD) of the testes and scrotal circumference (33.3 ± 0.5 v. 31.8 ± 0.4 cm) were greater (P < 0.05) in sexually mature rams than in peri-pubertal rams. There were no differences (P > 0.05) between the 2 age groups of Dorper rams in mean values for blood flow indices measured in testicular arteries. Semen was collected by ejaculation into the artificial vagina from 38% (8/21) of post-pubertal rams and 21% (5/24) of peri-pubertal animals (Chi-squared test; P < 0.05). Semen volume was negatively correlated with PI of the testicular arteries (r = –0.57, P = 0.04). In summary, the attainment of sexual maturity in Dorper rams maintained in a subtropical climate was associated with significant elongation of testes but not with the changes in testicular volume and blood perfusion. Testicular artery PI was a significant predictor of semen volume in peri-pubertal and sexually mature rams. Based on this and several previous studies, B-mode ultrasonography combined with echotextural analyses of the epididymides and accessory sex glands and serum testosterone measurements provide more useful information on the progression of sexual maturation and present semen quality in rams than the ultrasonographic assessment of testicular dimensions and an array of blood flow indices.

Can germ cell neoplasia in situ be diagnosed by measuring serum levels of microRNA371a-3p?

PurposeDiagnosing germ cell neoplasia in situ (GCNis) can detect germ cell tumours (GCTs) at the pre-invasive stage. To date, testicular biopsy with the potential of surgical complications is the only way of safely diagnosing GCNis. Recently, microRNAs (miRs) 371-3, and miR 367 were shown to be valuable serum biomarkers of GCTs. We explored the usefulness of these candidate miRs as a marker for GCNis.Methods27 patients with GCNis and no concomitant GCT were enrolled. All patients underwent measuring serum levels of miR-371a-3p and miR-367-3p before treatment, 11 had repeat measurement after treatment, 2 also had testicular vein blood examinations. Serum levels were measured by quantitative PCR. In addition, four orchiectomy specimens of patients with GCT were examined immunohistochemically and by in situ hybridization (ISH) with a probe specific for miR-371a-3p to look for the presence of this miR in GCNis cells.ResultsThe median serum level of miR-371a-3p was significantly higher in patients with GCNis than in controls, miR-367 levels were not elevated. Overall, 14 patients (51.9%) had elevated serum levels of miR-371a-3p. The highest levels were found in patients with bilateral GCNis. Levels in testicular vein serum were elevated in both of the cases. After treatment, all elevated levels dropped to normal. In two orchiectomy specimens, miR-371a-3p was detected by ISH in GCNis cells.ConclusionsMeasuring miR-371a-3p serum levels can replace control biopsies after treatment of GCNis. In addition, the test can guide clinical decision making regarding the need of testicular biopsy in cases suspicious of GCNis.

Modulation of Testicular and Whole Blood Trace Element Concentrations in Conjunction with Testosterone Release Following Kisspeptin Administration in Male Rabbits (Oryctolagus cuniculus)

The present study investigated the role of kisspeptin-10 on reproductively significant trace elements in relation to testosterone release in male rabbits, Oryctolagus cuniculus. Groups of rabbits were exposed to single 1 μg kisspeptin dose (i.v., saphenous vein), while simultaneous groups were pretreated with a kisspeptin antagonist, peptide-234 (50 μg) 20 min before administering kisspeptin. Sequential blood sampling was done through marginal ear vein puncture at staggered time intervals: 0, 0.5, 1, 2, 4, and 24 h to determine serum testosterone. Testes and whole blood were collected at 4 and 24 h post dosage to determine trace element concentrations through atomic absorption spectrophotometry. In testes, zinc (Zn), manganese (Mn), and Fe concentrations showed significant increases at 24 h, while copper (Cu) concentration was found elevated at 4 and 24 h both (P < 0.001). In whole blood, Zn and Cu concentrations were significantly elevated at 4 and 24 h, while Mn and cobalt (Co) concentrations showed increases only at 24 h (P < 0.001). Blood iron concentration was not altered in the blood. In contrast, no change occurred in testicular Co, and chromium or nickel concentrations in either testes or blood. Compared to control and predose groups, serum testosterone levels increased gradually and peaked at 2 h (P < 0.001) post kisspeptin treatment but declined thereafter. Pretreatment with antagonist abolished all increases in trace elements and testosterone concentrations. The present study provides first evidence that reproduction- and fertility-related peptide "kisspeptin" modulates testicular and blood trace elements and that this action is likely GPR54-dependent.

Relationships of serum thyroid hormones and follicle-stimulating hormone concentrations to Sertoli cell differentiation during the first wave of spermatogenesis in euthyroid ram lambs

The main purpose of this study was to determine if temporal relationships exist between serum concentrations of free fractions of thyroxin (fT4) and triiodothyronine (fT3), follicle-stimulating hormone (FSH) levels, and Sertoli cell differentiation in euthyroid ram lamb testes. Additionally, testicular thyroid hormone (TH) receptors (TRs) were identified using immunohistochemistry and Western blot analysis. Weekly testicular biopsies and jugular blood samples were collected from 12 ram lambs over the 9 weeks of study. Hormone concentrations and the numbers of dividing Sertoli cells per seminiferous tubule (ST) area were analyzed relative to chronological age of animals and the two distinctive stages of Sertoli cell differentiation: (a) tight junction/ST lumen formation and (b) the onset of support mechanisms for the development of multiple germ cell types (presence of primary spermatocytes in >95% STs). Circulating FSH concentrations increased (p<0.05) immediately after first detection of ST lumen and reached a nadir (p<0.05) just prior to the end of the first wave of spermatogenesis. A decline in both fT4 and fT3 levels (p<0.05) occurred after Sertoli cells had formed the ST lumen and began supporting germ cell differentiation. There was a positive correlation between the numbers of proliferating Sertoli cells and serum fT4 (r=0.51, p<0.001) and fT3 (r=0.52, p<0.001) concentrations. TRs were expressed throughout the study period; however, prior to the formation of ST lumen, two isoforms were detected while only one TR isoform was present by the end of the first wave of spermatogenesis. Overall, the exit of Sertoli cells from the cell cycle that presages their final differentiation begins when THs and FSH levels are high, suggesting a permissive role of these hormones in the maturation of STs in prepubertal ram lambs.

Doxorubicin-induced vascular toxicity: Targeting potential pathways to reduce procoagulant activity.

e13080 Background: In a former study in mice using the gonads as an end-organ prototype, we have characterized by real-time intravital imaging an acute deleterious effect of doxorubicin (DXR) on the gonadal vasculature, manifested by a reduction in blood flow and disintegration of the vessel wall. We hypothesized this pattern may represent the formation of microthrombi. We aimed to further characterize the effect of DXR on platelets’ function and to use potentially protectants to reduce DXR acute effect on the blood flow. Methods: 100 µg/mouse 24 hours and 1 hour prior to DXR treatment (8 mg/kg), or with eptifibatide (integrilin,75µg/mouse) 90min prior to DXR treatment. Testicular arterial blood flow was examined in real-time by pulse wave Doppler ultrasound. Platelet adhesion to confluent endothelial cells (EC) was evaluated following exposure of EC to DXR (100 µM) for 4h followed by exposure to whole blood under defined shear rates. Fixed platelets were immunostained by anti- CD41a antibody. DXR effect on platelet adhesion was determined by pre-incubation of platelet rich plasma for 15min with increasing concentrations of DXR and induction of aggregation by ADP. For in vivo study, mice were injected with either LMWH (Enoxaparin; Clexane). Results: There was a significant 3.6-fold increase in platelet adhesion to DXR-exposed EC (p&lt;0.002) reflecting the toxic effect of DXR on EC. Yet, significant DXR- dose dependent decrease in platelet aggregation was observed reaching up to 40% inhibition at 100 µM (p&lt;0.001). Testicular arterial blood flow was preserved as a result of pre-treatment with LMWH or eptifibatide prior to DXR (P&lt;0.01). Conclusions: DXR-induced acute vascular toxicity may trigger the coagulation pathway while enhancing platelet adhesion yet inhibiting massive aggregation, which result in compromised blood flow due to microthrombi formation. Anti-platelet/anti-coagulant agents appear to be effective in reducing the detrimental effect of DXR on the vasculature.

The Effects of Supplemental Vitamin E on Reproductive Development of Prepubertal Karya Male Lambs

The objective of this study was to determine the effect of dietary vitamin E supplementation on testicular and behavioural development, and histological appearance of testes in Karya male lambs. Thirteen lambs weaned at 2.5 months of age were used in the experiment. Experiment was carried out over a period of 70 days and all lambs were fed ad libitum on concentrate and 100 g/day clover hay per lamb. In addition, the experimental group (n=6) received a supplement of 45 mg/day vitamin E (dl-α-tocopherol acetate) per lamb. Testicular measurements and blood testosterone concentrations were recorded every second week. Behavioural tests were carried out four times per animal at three days intervals during the last 12 days of the experiment. After slaughtering, testicular tissue samples of all animals were collected and evaluated for the histological appearances. Except for mount attempts (P<0.05), sexual behaviour and testicular growth characteristics of the lambs were similar. The mounting parameters, testosterone concentrations and testes and epididymides weights were similar between the two groups. However, the mean of mounting weight was found lower (P<0.05) with Vitamin E lambs compared to control (28.78±3.48 vs. 33.98±3.64 kg, respectively). The area of seminiferous tubules was statistically different (P<0.001) between the two groups. However, there were no significant differences between the two groups for most of the reproductive characters. These findings suggest that supplementation of Vitamin E during early prepubertal age of Karya male lambs does not have considerable effects on the reproductive characteristics.

Prognostic Value of the Resistance Index in the Undescended Testis Relative to Patient's Age and Time-Point of Surgery

Purpose The undescended testis is one of the most common disease in paediatric urology. The aim of the present study was to analyse the testicular parenchymal blood flow as measured by the resistance index (RI) in relation to the patient's age and potential changes in RI following surgery. Material and Methods From 04/07 – 03/09 we prospectively studied 100 boys with uni- or bilateral undescended testes aged 1 to 14 using a high resolution linear ultrasound scanner. The RI was determined with simultaneous detection of B-mode and colour-flow Doppler before and 6 weeks after surgery. The obtained indices were compared with those of the descended contralateral testis. Results A total of 143 undescended testes (43 bilateral, 57 unilateral) and 57 descended contralateral testes were examined. In both groups the mean RI was 0,55 irrespecitve of the patient's age. Similarly, we did not observe any significant changes in RI following surgical correction of the undescended testis. Conclusions In contrast to progressive histo-pathological changes in untreated undescended testis over time, the time-point of surgery appears to have no relevant impact on the testicular parenchymal blood perfusion as measured by the RI. Therefore, the RI failed to show a predictive value with regard to both diagnosis and therapy of the undescended testis.

Desenvolvimento testicular, espermatogênese e concentrações hormonais em touros Angus

Este estudo foi realizado com a finalidade de avaliar a evolução das secreções hormonais e do epitélio seminífero em touros da raça Angus de 10 a 38 semanas de idade. Foram castrados 1 a 5 animais em intervalos de quatro semanas (total de 25 touros) para coleta de amostras do parênquima testicular e do plasma sanguíneo. As variáveis relacionadas ao crescimento testicular, aos aspectos quantitativos da espermatogênese e aos níveis hormonais foram transformadas em logaritmo e avaliadas por meio de análise de variância. O diâmetro dos testículos e túbulos seminíferos e o peso testicular apresentaram variações mais acentuadas após 26 semanas de idade. A porcentagem do parênquima testicular ocupado pelos túbulos seminíferos aumentou de 49,3 para 75,2% durante o experimento. A maioria dos túbulos (&gt;90%) apresentou-se com células de Sertoli somente entre 10 e 14 semanas, mas na 18ª (13,8±1,7%) e 22ª semanas (19±1%), o número de túbulos com gonócitos e espermatogônias aumentou em relação às semanas iniciais. Espermatogônias intermediárias e B predominaram na 26ª semana (24,5±8,2%) e a porcentagem de túbulos com espermatócitos foi mais elevada na 30ª semana (42,3±9,9%). Espermátides arredondadas foram detectadas partir da 26ª semana e, na 38ª semana, 62,3±1,5% dos túbulos seminíferos continham espermátides alongadas ou maduras. As variações mais acentuadas no crescimento testicular e, principalmente, no peso testicular após as 26 semanas coincidiram com o estabelecimento da meiose, com as alterações morfológicas do núcleo e nucléolo das células de Sertoli (indicativos do processo de diferenciação das mesmas), com os níveis reduzidos de androstenediona e os incrementos significativos de testosterona e estradiol 17beta. As associações entre o crescimento testicular e os níveis de FSH e LH na circulação periférica foram menos evidentes.

Lignin-Stimulated Reduction of Oxidative DNA Lesions in Testicular Cells and Lymphocytes of Sprague-Dawley Rats In Vitro and Ex Vivo

: Lignin biopolymers constitute 30% of plant biomass and belong to the most abundant organic polymers on earth. We showed previously that this important component of dietary fiber exhibited a protective effect against the overall DNA damage induced by H2O2 or N-methyl-N′-nitro-N-nitrosoguanidine in hamster lung cells and human foreskin cells cultured in vitro. The objective of the present work was to examine DNA-protective effects of lignin in rat testicular cells and rat peripheral blood lymphocytes using in vitro and ex vivo experiments. H2O2 and visible light-excited methylene blue (MB) were used as DNA-damaging agents. Testicular cells were chosen because the germinal epithelium of testes is one of the most proliferately active tissues potentially susceptible to DNA-damaging effects. As a second target peripheral blood lymphocytes were chosen because dietary lignin or its metabolites circulate in the animal organism probably through the blood system. For the in vitro experiments, isolated cells were preincubated with lignin for 2 h before treatment with one of the oxidative agents. In ex vivo experiments, the cells were exposed to H2O2 or visible light-excited MB after isolation from rats fed either a common diet or a lignin-supplemented diet. The water-soluble, sulfur-free lignin used in experiments was obtained by fractionation of hardwood hydrolysate. The level of direct single-strand DNA breaks in H2O2-treated cells was measured by the classical comet assay, and the level of oxidative DNA lesions in visible light-treated cells was measured by a modified comet assay. We found that lignin reduced DNA lesions induced by H2O2 or visible light-excited MB both in vitro and ex vivo. The major conclusion of our study is that lignin polymer obtained by fractionation of hardwood hydrolysate manifested a specific type of antimutagenic effect.

Hormones: what the testis really sees.

Various barriers in the testis may prevent hormones from readily reaching the cells they are supposed to stimulate, especially the hydrophilic hormones from the pituitary. For example, LH must pass through or between the endothelial cells lining the blood vessels to reach the surface of the Leydig cells, and FSH has the additional barrier of the peritubular myoid cells before it reaches the Sertoli cells. The specialised junctions between pairs of Sertoli cells would severely restrict the passage of peptides from blood to the luminal fluid and therefore to the cells inside this barrier, such as the later spermatocytes and spermatids. There is evidence in the literature that radioactively labelled LH does not pass readily into the testis from the blood, and the concentration of native LH in the interstitial extracellular fluid surrounding the Leydig cells in rats is only about one-fifth of that in blood plasma. Furthermore, after injection with LHRH, there are large rises in LH in the blood within 15 min, at which time the Leydig cells have already responded by increasing their content of testosterone, but with no significant change in the concentration of LH in the interstitial extracellular fluid. Either the Leydig cells respond to very small changes in LH, or the testicular endothelial cells in some way mediate the response of the Leydig cells to LH, for which there is now some evidence from co-cultures of endothelial and Leydig cells. The lipophilic steroid hormones, such as testosterone, which are produced by the Leydig cells, have actions within the seminiferous tubules in the testis but also in other parts of the body. They should pass more readily through cells than the hydrophilic peptides; however, the concentration of testosterone in the fluid inside the seminiferous tubules is less than in the interstitial extracellular fluid in the testis, especially after stimulation by LH released after injection of LHRH and despite the presence inside the tubules of high concentrations of an androgen-binding protein. The concentration of testosterone in testicular venous blood does not rise to the same extent as that in the interstitial extracellular fluid, suggesting that there may also be some restriction to movement of the steroid across the endothelium. There is a very poor correlation between the concentrations of testosterone in fluids from the various compartments of the testis and in peripheral blood plasma. Determination of the testosterone concentration in the whole testis is also probably of little predictive value, because the high concentrations of lipid in the Leydig cells would tend to concentrate testosterone there, and hormones inside these cells are unlikely to have any direct effect on other cells in the testis. The best predictor of testosterone concentrations around cells in the testis is the level of testosterone in testicular venous blood, the collection of which for testosterone analysis is a reasonably simple procedure in experimental animals and should be substituted for tissue sampling. There seems to be no simple way of determining the concentrations of peptide hormones in the vicinity of the testicular cells.

Circulating concentrations of dimeric inhibin A and B in the male rhesus monkey (Macaca mulatta).

The purpose of this study was to determine the relative concentrations of inhibin A and B in peripheral serum of the adult male rhesus monkey and to examine the testicular contribution to these circulating forms of inhibin. In addition, inhibin B concentrations were also determined in peripheral sera of neonatal and juvenile males and in spermatic vein blood of adults. Immunoradiometric assays specific for the measurement of inhibin A and B were used. These assays also provided an opportunity to reexamine the physiological significance of a replacement infusion of recombinant human (rh)-inhibin A previously employed to study the role of this hormone in regulating FSH secretion in the monkey. In intact adults, the mean (+/-SE) serum concentration of inhibin B was 1008 +/- 184 pg/mL. In contrast, circulating inhibin A concentrations were very low (< 46 pg/mL). Inhibin B was consistently detected in neonatal monkey serum (275 +/- 57 pg/mL), and concentrations of this inhibin dimer increased throughout postnatal development, reaching maximum values in adulthood. Circulating inhibin A concentrations in neonatal and juvenile monkeys were undetectable (< 7 pg/mL). Both forms of inhibin were generally undetectable in castrate sera. The ratio of inhibin B concentrations in testicular venous blood to those in the peripheral circulation was 1.4:1. These findings indicate that, in the male monkey, inhibin B is the principal form of circulating dimeric inhibin, and that this hormone is derived exclusively from the testis. The elevated levels of circulating inhibin B in the juvenile male monkey suggest that, during this phase of development, testicular inhibin B secretion is relatively gonadotropin independent. Additionally, we found that the concentration of circulating inhibin A in castrate animals that had earlier received an iv infusion of rh-inhibin A (832 ng/h/kg BW) was 9881 +/- 2135 pg/mL, indicating that this mode of inhibin replacement may not have been entirely physiological.

Testicular microvascular blood flow: alteration after Leydig cell eradication and ischemia but not experimental varicocele.

Testicular microvascular blood flow is known to exhibit vasomotion, or pulsatile flow. In the present study we have used laser-Doppler flowmetry to study microvascular blood flow in the rat testis and to examine the response of the microvasculature to pharmacological stimulation and pathophysiological conditions. With a mean microvascular flow rate of 13.3 +/- 1.7 perfusion units (PU), the mean cycle amplitude was 3.4 +/- 0.6 PU, and the cycle frequency was 10.3 +/- 0.8 cycles per minute. Blood flow parameters did not differ between left and right testes, between scrotal testes and testes in a 35 degree C glass testicle receptacle, or among testicular regions. Perifusion of seminiferous tubules and their vasculature with 0.1 microgram/microliter epinephrine significantly reduced microvascular blood flow and eliminated vasomotion. Elimination of Leydig cells and intratesticular testosterone also eliminated vasomotion but did not significantly alter mean blood flow rates. Thirty days after imposition of experimental left varicocele (ELV) there were no significant changes in microvascular blood flow parameters. Testicular torsion of sufficient degree and duration to destroy spermatogenesis did not induce a change in mean microvascular blood flow rate 24 hours after repair of torsion, but testicular vasomotion was eliminated in the majority of animals. We conclude that microvascular flow is altered in some testicular pathologies and not in others. The mechanisms underlying changes in microvascular blood flow are at present unknown.

Leydig cell function after experimental testicular torsion despite loss of spermatogenesis.

Little is known about specific testicular cell responses to periods of testicular torsion. In particular, the steroidogenic capacity of Leydig cells in the post-torsion testis is unknown. Male Sprague-Dawley rats (450-550 g) underwent no torsion (control) or a 720 degrees unilateral testicular torsion for either 0 (sham), or 1 or 2 Such torsions have previously been shown to cause progressive damage to the rat testis. One, 15, or 30 days after torsion repair all animals (n = 5-10/group) were prepared for testicular venipuncture and intravenous infusion of ovine luteinizing hormone (LH) via the femoral vein. Testicular venous blood was collected directly from the surface of the testis both 5 minutes prior to and 90 minutes after infusion of predetermined ED50 (0.1 microgram) or ED100 (0.5 microgram) doses of LH. Testicular venous serum (TVS) was assayed for testosterone (T) by radioimmunoassay. Control animal TVS T concentrations before LH infusion and 90 minutes after ED50 and ED100 LH stimulation were 103 +/- 25, 621 +/- 103, and 1,055 +/- 140 ng/ml, respectively. Testes having experienced a 1-hour torsion did not have a significantly (P < 0.05) reduced capacity to respond to ED50 and ED100 stimulation at either 15 and 30 days after the torsion. Testes having experienced a 2-hour torsion did have significantly reduced (P < 0.05) ED50 responses at both 1 hour and 30 days after torsion repair. More remarkably, significant steroidogenic reserve was still present in testes after torsion, which in previous studies had been shown to have permanent loss of spermatogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of 19-hydroxyandrostenedione and 19-hydroxytestosterone by porcine Leydig cells in vitro and in vivo

19-Hydroxytestosterone and 19-hydroxyandrostenedione have been identified as secretory products of the testes in the mature male domestic pig. Their isolation and identification were made by reverse-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry (CGC-MS) of extracts from testicular vein blood and media of incubations with Leydig cells. Blood was collected from veins on the surface of the testes of anaesthetized boars. Collagenase-dispersed Percoll-purified cells (> 90% pure) were incubated (20 x 10(6) cells/flask) with androstenedione (8.75 mumol/l) or [3H]androstenedione (5 x 10(6) c.p.m.) for < 60 min. Steroids were recovered from plasma or media by solid-phase extraction and the unconjugated fractions chromatographed isocratically in two solvent systems (acetonitrile:water, 37:63 (v/v) and methanol:water, 70:30 (v/v)) before CGC-MS analysis. 19-Hydroxytestosterone was present in greater quantities than 19-hydroxyandrostenedione in testicular vein blood; it was also seen as a quantitatively significant metabolite of unlabelled and radioactive androstenedione in the incubation studies. The demonstration of the secretion of 19-hydroxyandrogens from porcine testes thus raises questions concerning the physiological significance of a testicular, rather than an adrenal, secretion of these compounds.

Evaluation of the role of germ cells in regulating the route of secretion of immunoactive inhibin from the rat testis.

During normal sexual maturation of the male rat there is a progressive change in the route of secretion of inhibin by the Sertoli cell, from a predominantly basal route of secretion in prepuberty to a predominantly apical route of secretion in adulthood. This change may be monitored by comparing the levels of inhibin in testicular (TV), spermatic and peripheral (PV) venous blood and the levels in testicular interstitial fluid (IF). This study has assessed the role of germ cells in effecting this change by assessing (a) the effect of total germ cell depletion by X-irradiation of the males in utero, and (b) the effect of selective germ cell depletion in adulthood using the testicular toxicant, methoxyacetic acid (MAA). Female rats were X-irradiated on day 20 of gestation to produce male offspring whose testes were germ-cell deficient. Blood and IF samples were collected from groups of these offspring and age-matched controls at 35 and 100 days of age. In blood and IF samples, inhibin concentrations were significantly higher at 35 days of age than at 100 days. The absence of germ cells in X-irradiated animals did not affect the age-related fall in inhibin levels, nor the change in the predominant route of secretion of inhibin from the testis into blood. Testosterone was almost undetectable in 35-day-old controls, but was raised significantly by 100 days of age. In X-irradiated animals, testosterone levels were increased significantly at 35 days of age, and the levels in most samples were increased even more substantially by 100 days of age. However, PV levels of testosterone in 100-day-old X-irradiated animals were significantly lower than in controls. LH and FSH levels were raised in X-irradiated animals compared with their age-matched controls, but FSH levels in X-irradiated animals still fell with age, as in the controls. The role of specific germ cell types in regulating the route of secretion of inhibin from the normal adult testis was studied after depletion (80-100%) of pachytene and later spermatocytes by a single oral administration of MAA (650 mg/kg) to adult rats. At 3 days after MAA treatment, coincident with the loss of pachytene spermatocytes, plasma inhibin levels were increased significantly in blood and IF samples, and this was associated with a dramatic change in the route of secretion of inhibin from the testis, with increased secretion of this peptide via the base of the Sertoli cell into IF and TV blood.(ABSTRACT TRUNCATED AT 400 WORDS)