Laparoscopic evaluation and treatment of the impalpable testis

Objectives: To evaluate the laparoscopic technique as a diagnostic and therapeutic tool in the management of patients with impalpable testis. Material and Methods: Fifty-nine patients with mean age of 6.3 years underwent laparoscopy to evaluate 85 impalpable testes that were classified as absent, canalicular and intra-abdominal. In the case of testicular absence, the procedure was terminated. In the case of canalicular testis, open inguinal exploration was performed. In intra-abdominal testis, either laparoscopic orchiopexy or orchiectomy was performed. According to the length of the vascular pedicle, orchipexy was performed either with or without vascular ligature. Post-operatively, the treated testes were evaluated according to size and location in the scrotum. Results: Seventeen (20%) of the 85 impalpable testes were diagnosed as absent, 21 (24.7%) as canalicular and 47 (55.3%) as intra-abdominal. Of the canalicular testes, 20 were explored by inguinotomy and one by laparoscopy. All the intra-abdominal testes were treated initially by laparoscopy, four being removed due to atrophy, 31 submitted to vascular ligature and 12 to primary orchipexy. Of those submitted to vascular ligature, 22 underwent a second stage orchipexy, of which 18 laparoscopically and 4 by inguinotomy. Of the 18 testes brought to the scrotum by staged laparoscopic orchipexy, 15 (83.3%) presented normal characteristics in the late follow-up, while of the 12 submitted to primary laparoscopic orchipexy, 8 (66.6%) were normal. There were no perioperative or late complications. Conclusions: Laparoscopy is a minimally invasive procedure with low morbidity that enables precise diagnosis of the impalpable testes. When intra-abdominal testes are found, either immediate laparoscopic orchiectomy, or primary and staged orchipexy are possible, with results equivalent to open procedures, with the advantage of smaller surgical incisions and shorter postoperative recovery.

Morphology of testis and epididymis in an ethanol-drinking rat strain (UChA and UChB)

The objective of the present study was to analyze the prospective alterations of the testis and epididymis in a defined strain of alcoholic rats in order to contribute to our understanding of the effects of chronic alcoholism on reproduction. The testis and epididymis of the animals were submitted to morphological analysis by macroscopy, light microscopy and electron microscopy and to morphometric analysis. The UCh rats showed atrophy of the epithelium and reduction of testis and epididymis weight, liver hypertrophy and fat infiltration and alterations of the hypothalamus-pituitary axis. Ethanol induces changes in the weight and in the epithelium of the testis and epididymis and in the hypothalamus-pituitary axis of the UCh rats.

Cimetidine-induced Leydig cell apoptosis and reduced EG-VEGF (PK-1) immunoexpression in rats: evidence for the testicular vasculature atrophy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Localization of fibronectin in the rat testis : effects of serum and dibutyryl cyclic AMP on fibronectin deposition by peritubular myoid cells in culture

The peritubular zone of the rat testis has an extensive extracellular matrix (ECM). Fibronectin (FN) is distributed primarily in the basal lamina of the seminiferous tubule boundary tissue and is synthesized by peritubular myoid cells. Several extracellular changes are mediated by growth factors and these changes occur at the time of hormone mediated testicular development, particularly in the peritubular zone. The effects of serum or dibutyryl cyclic AMP (cAMP) on FN production by the mesenchymal peritubular myoid cells were evaluated. Rats of various ages (10, 15, 20, 40 and 80 days) were employed for immunofluorescent localization of rat testicular FN in frozen sections. In all age groups tested, FN was primarily present in a broad layer around each seminiferous tubule, and blood vessel, and in variable distribution throughout the interstitial stroma. By day 20 there was no clear distinction in FN staining between the peritubular zone and the interstitial tissue. This indicates an involvement of FN in the ECM developments which occur in the peritubular zone of the testis at this time. The peritubular myoid cells were isolated from 20-22 day old rat testis and cultured on glass coverslips. These cells were grown to confluence with 10% fetal calf serum (FCS) in medium until day 4 and then subcultured to have secondary monocultures maintained with or without serum. By means of immunofluorescence and cytochemistry using avidin-biotin peroxidase complex it was observed that peritubular myoid cells were positive for FN and most of the FN was localized in the perinuclear region. Subcultured peritubular myoid cells maintained for 4 days in medium containing FCS developed an extensive interconnecting FN matrix. In the presence of 0.5 mM cAMP in culture, FN became localized along the filamentous process of peritubular myoid cells and more prominently in the areas of triangulated multi-cell aggregates as well as on the surface of the contracted small spherical cells. The addition of cAMP in the presence of FCS, also caused a noticeable change in the staining pattern; FN was detected along the filamentous process developing into a complex network of cells encased in an extensive matrix. It would appear that the translocation of FN in the cytoplasmic extensions of peritubular myoid cells may be a direct consequence of morphological changes associated with metabolic regulation of cAMP. This may also be related to the puberty associated development of in vivo changes in the ECM produced by peritubular myoid cells.

Tubule detection in testis images using boundary weighting and circular shortest path

In studies of germ cell transplantation, measureing tubule diameters and counting cells from different populations using antibodies as markers are very important. Manual measurement of tubule sizes and cell counts is a tedious and sanity grinding work. In this paper, we propose a new boundary weighting based tubule detection method. We first enhance the linear features of the input image and detect the approximate centers of tubules. Next, a boundary weighting transform is applied to the polar transformed image of each tubule region and a circular shortest path is used for the boundary detection. Then, ellipse fitting is carried out for tubule selection and measurement. The algorithm has been tested on a dataset consisting of 20 images, each having about 20 tubules. Experiments show that the detection results of our algorithm are very close to the results obtained manually. © 2013 IEEE.

Progressive dysgraphia in a case of posterior cortical atrophy

Dysgraphia (agraphia) is a common feature of posterior cortical atrophy (PCA). However, detailed analyses of these spelling and writing impairments are infrequently conducted. LM is a 59-year-old woman with dysgraphia associated with PCA. She presented with a two-year history of decline in her writing and dressmaking skills. A 3D T1-weighted MRI scan confirmed selective bi-parietal atrophy, with relative sparing of the hippocampi and other cortical regions. Analyses of LM's preserved and impaired spelling abilities indicated mild physical letter distortions and a significant spelling deficit characterised by letter substitutions, insertions, omissions, and transpositions that was systematically sensitive to word length while insensitive to real word versus nonword category, word frequency, regularity, imagery, grammatical class and ambiguity. Our findings suggest a primary graphemic buffer disorder underlies LM's spelling errors, possibly originating from disruption to the operation of a fronto-parietal network implicated in verbal working memory.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Determinants of vascular cognitive impairment : White matter lesions, brain atrophy and their neuropsychological correlates

The concept of vascular cognitive impairment (VCI) covers a wide spectrum of cognitive dysfunctions related to cerebrovascular disease. Among the pathophysiological determinants of VCI are cerebral stroke, white matter lesions and brain atrophy, which are known to be important risk factors for dementia. However, the specific mechanisms behind the brain abnormalities and cognitive decline are still poorly understood. The present study investigated the neuropsychological correlates of particular magnetic resonance imaging (MRI) findings, namely, medial temporal lobe atrophy (MTA), white matter hyperintensities (WMH), general cortical atrophy and corpus callosum (CC) atrophy in subjects with cerebrovascular disease. Furthermore, the cognitive profile of subcortical ischaemic vascular disease (SIVD) was examined. This study was conducted as part of two large multidisciplinary study projects, the Helsinki Stroke Aging Memory (SAM) Study and the multinational Leukoaraiosis and Disability (LADIS) Study. The SAM cohort consisted of 486 patients, between 55 and 85 years old, with ischaemic stroke from the Helsinki University Hospital, Helsinki, Finland. The LADIS Study included a mixed sample of subjects (n=639) with age-related WMH, between 65 and 84 years old, gathered from 11 centres around Europe. Both studies included comprehensive clinical and neuropsychological assessments and detailed brain MRI. The relationships between the MRI findings and the neuropsychological test performance were analysed by controlling for relevant confounding factors such as age, education and other coexisting brain lesions. The results revealed that in elderly patients with ischaemic stroke, moderate to severe MTA was specifically related to impairment of memory and visuospatial functions, but mild MTA had no clinical relevance. Instead, WMH were primarily associated with executive deficits and mental slowing. These deficits mediated the relationship between WMH and other, secondary cognitive deficits. Cognitive decline was best predicted by the overall degree of WMH, whereas the independent contribution of regional WMH measures was low. Executive deficits were the most prominent cognitive characteristic in SIVD. Compared to other stroke patients, the patients with SIVD also presented more severe memory deficits, which were related to MTA. The cognitive decline in SIVD occurred independently of depressive symptoms and, relative to healthy control subjects, it was substantial in severity. In stroke patients, general cortical atrophy also turned out to be a strong predictor of cognitive decline in a wide range of cognitive domains. Moreover, in elderly subjects with WMH, overall CC atrophy was related to reduction in mental speed, while anterior CC atrophy was independently associated with frontal lobe-mediated executive functions and attention. The present study provides cross-sectional evidence for the involvement of WMH, MTA, general cortical atrophy and CC atrophy in VCI. The results suggest that there are multifaceted pathophysiological mechanisms behind VCI in the elderly, including both vascular ischaemic lesions and neurodegenerative changes. The different pathological changes are highly interrelated processes and together they may produce cumulative effects on cognitive decline.

Localization of testis-variant histones in rat testis chromatin

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.

Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies

The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double- stranded DNA, thus behaving like a DNA-melting protein.

Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase IIalpha

Topoisomerase II (topo II) is a dyadic enzyme found in all eukaryotic cells. Topo II is involved in a number of cellular processes related to DNA metabolism, including DNA replication, recombination and the maintenance of genomic stability. We discovered a correlation between the development of postnatal testis and increased binding of topo IIalpha to the chromatin fraction. We used this observation to characterize DNA-binding specificity and catalytic properties of purified testis topo IIalpha. The results indicate that topo IIalpha binds a substrate containing the preferred site with greater affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Interestingly, topo IIalpha displayed high-affinity and cooperativity in binding to the scaffold associated region. In contrast to the preferred site, however, high-affinity binding of topo IIalpha to the scaffold-associated region failed to result in enhanced catalytic activity. Intriguingly, competition assays involving scaffold-associated region revealed an additional DNA-binding site within the dyadic topo IIalpha. These results implicate a dual role for topo IIalpha in vivo consistent with the notion that its sequestration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.

Testis-specific histone H1t is truly a testis-specific variant and not a meiotic-specific variant

Immunoblotting studies using highly specific polyclonal anti-histone H1t-IgG, which was extensively characterized by us previously, did not produce a signal with any of the histone H1 subtypes of either 1-day-old or adult rat ovarian nuclei. The absence of histone H1t in ovarian nuclei was also confirmed by indirect immunofluorescence studies. It is concluded, therefore, that histone H1t is truly a testis-specific histone variant and not a meiotic-specific variant.

Interaction of rat testis protein, TP, with nucleosome core particle

Circular dichroism studies have revealed that addition of testis specific protein, TP in vitro, to rat testes nucleosome core particle resulted in a decrease in the compaction of the core particle DNA. This was also corroborated by thermal denaturation analysis. Addition of TP to nucleosome core particle resulted in the conversion of a biphasic transition towards a single phase. However, at the same time there was a 20% reduction in the overall hyperchromicity of core particle DNA at core particle to TP molar ratios of 1:2 and 1:3. These observations along with our earlier report, showing the DNA melting properties of TP, suggest that TP may play an important role in the disassembly process of nucleosome core particle during spermiogenesis.

Hemiorchidectomy leads to dramatic and immediate alterations in pituitary follicle-stimulating hormone secretion and the functional activity of the remaining testis in the adult male bonnet monkey (Macaca radiata)

The aim of the present study was to examine the effect of hemiorchidectomy (HO) on serum FSH, LH, testosterone (T), and inhibin (INH) concentrations as well as on the testicular volume (TV) and on changes in the kinetics of germ cell turnovers in the remaining testis of adult male bonnet monkeys. Blood samples collected at 2200 h at various times before and after HO and testicular biopsies obtained at different periods were subjected to hormone analysis and DNA flow cytometry. Though serum T levels were lowered (p < 0.05) at 12 h after HO, T levels rapidly returned to intact control concentrations by Day 5. While serum LH remained unaltered, serum FSH increased markedly within 2 days of HO and remained significantly (p < 0.05) elevated over the next 90 days. Though serum INH showed a significant decrease (p < 0.05) by 15 min of HO, it returned to approximately 80% of intact levels within one week. The TV of the remaining testis showed maximal increment by Day 30 (p < 0.05) of HO. DNA flow cytometric analysis 24 days after HO showed increases (p < 0.05) in spermatogonia (2C) and primary spermatocytes (4C). These cell types by Day 45 had transformed to round (1C) and elongate (HC) (by 38%, p < 0.001) spermatids. Overall spermatogenesis (conversion of 2C to 1C and HC) showed significant enhancement at Days 110 and 175, suggesting that the spurt in spermatogenic activity is not confined to a single spermatogenic cycle.