In-vitro differentiation of germ cells from frozen testicular biopsy specimens

In some men with germ cell maturation arrest, spermatogenesis can be resumed during in-vitro culture of testicular biopsy samples. In this study, we examined whether similar differentiation events can be induced in cultured germ cells from cryopreserved testicular biopsy specimens. Fresh and cryopreserved aliquots of the same testicular biopsy samples were cultured in medium supplemented with FSH and testosterone. After 24 and 48 h of culture, the progression of spermatogenesis and the percentage of Sertoli cells with DNA damage, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), were evaluated. Spermatogenesis progressed in a similar way in fresh and cryopreserved aliquots over the first 24 h of culture. However, in contrast to fresh aliquots, no additional progress of spermatogenesis was detected between the 24 and 48 h time points. The percentage of TUNEL-positive Sertoli cells in fresh aliquots showed only a moderate increase after 24 h of culture, whereas most Sertoli cells from cryopreserved aliquots became TUNEL-positive during the same culture period. These data show that limited progression of spermatogenesis can be achieved by culturing cryopreserved testicular biopsy specimens for 24 h, but no additional benefit can be expected from prolonging the culture beyond this time point.     

Mitotic activity of Sertoli cells in adult human testis: an immunohistochemical study to characterize Sertoli cells in testicular cords from patients showing testicular dysgenesis syndrome

During puberty, normal somatic Sertoli cells undergo dramatic morphological changes due to the differentiation of immature pre-Sertoli cells in functionally active adult Sertoli cells. Sertoli cell maturation is accompanied with loss of their mitotic activity before onset of spermatogenesis and loss of pre-pubertal and occurrence of adult immunohistochemical Sertoli cell differentiation markers. Testes of infertile adult patients often exhibit numerous histological signs of testicular dysgenesis syndrome (TDS) such as microliths, Sertoli cell only (SCO) tubules, tubules containing carcinoma in situ and immature seminiferous tubules (Sertoli cell nodules). Sertoli cell tumours, however, are very rare neoplasms possibly due to the fact that the mechanism and temporal origin of neoplastic Sertoli cells underlying Sertoli cell tumourigenesis still remain unknown. To clarify the state of Sertoli cell differentiation in both immature seminiferous tubules of adult patients with TDS and Sertoli cell tumour, we compared the expression of the Sertoli cell differentiation markers vimentin, inhibin-α, anti-Muellerian-hormone, cytokeratin 18, M2A-antigen, androgen receptor and connexin43 with that of SCO tubules with hyperplasia. In addition, we demonstrated for the first time the existence of proliferating Sertoli cells by Ki67- and PCNA-immunostaining in Sertoli cell nodules of the adult human testis. Our data indicate that mitotically active Sertoli cells in Sertoli cell nodules will be arrested prior to puberty and, contrary to dogma, do not represent foetal or neonatal cells. Since all markers in Sertoli cell nodules revealed a staining pattern identical to that in neoplastic Sertoli cells, but different to that in Sertoli cells of SCO tubules with hyperplasia, it may be speculated that Sertoli cell tumours in adult men may originate from Sertoli cell nodules.     

LPS-induced transient testicular dysfunction accompanied by apoptosis of testicular germ cells in mice

The aim of this study was to clarify effects of inflammation on spermatogenesis in LPS-administered mice. ICR mice were treated by intraperitoneal injection for 7 days with either physiological saline (control) or 0.1 mg lipopolysaccharide (LPS)/kg body weight/day. Control mice were killed at 24 h after the last injection and the LPS-treated group after 24 h or 1, 3, or 5 weeks. Sperm concentration and motility in the cauda epididymis were examined as well as immunohistochemical localization of Fas and FasL and germ cell apoptosis. Sperm concentration and motility markedly fluctuated in LPS-treated mice. Increase of apoptotic cells was common in all post-LPS treatment groups, with a peak at 24 h after LPS injection. In contrast to the lack of Fas immunoreactivity in control testes, LPS-treated groups demonstrated Fas in many germ cells, especially in spermatocytes and spermatids. Immunoreactivity for FasL, on the other hand, was positive for some Sertoli cells, Leydig cells, and germ cells in both control and LPS-treated groups at all time points. The results suggest that the Fas/FasL system mediates apoptosis of germ cells in LPS-treated mice testes. LPS-administered mice thus provide a good experimental model for the study of transient disruption of spermatogenesis.     

Detection of molecular variability in rice tungro bacilliform viruses from India using polymerase chain reaction-restriction fragment length polymorphism

Rice tungro bacilliform virus (RTBV) with rice tungro spherical virus (RTSV) causes the destructive tungro disease of rice. In order to ascertain the molecular variability of RTBV in India, primers were designed to amplify a polymorphic DNA fragment of the virus. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis on a number of field isolates indicated mixed infections and molecular heterogeneity in the viral genome.     

Respiratory pattern in midline-lesioned brainstems and hemibrainstems from adult turtles

Discrete midline lesions uncouple left and right respiratory motor output in mammals, but not in frogs and lampreys. To address this question in reptiles, isolated adult turtle brainstems were cut along the midline while recording respiratory motor output (bursts of action potentials) on left and right hypoglossal (XII) nerves. XII motor bursts were synchronized as long as a small portion of the midline was still intact. When turtle brainstems were completely cut along the midline and separated into hemibrainstems, XII motor bursts were produced that could be abolished by mu-opioid receptor (MOR) activation or exposure to high pH (7.80) solution. Also, 13/57 hemibrainstems expressed episodic discharge (>1.75 bursts/episode). To test whether crossed connections were necessary to express a long-lasting increase in burst frequency (i.e., frequency plasticity), phenylbiguanide (PBG, 5-HT₃ receptor agonist, 20 μM) was bath-applied to hemibrainstems. Although PBG significantly increased burst frequency by 0.43 ± 0.10 bursts/min after 60 min, no frequency plasticity was observed because burst frequency returned to near baseline levels after a 2-h washout. Thus, crossed connections in turtle brainstems synchronize respiratory motor output and are not required for normal respiratory pattern formation, but are required for PBG-dependent frequency plasticity.     

Terminal transferase positive cells in testicular biopsy specimens from boys with acute lymphoblastic leukaemia.

Thirty nine testicular biopsy samples from 37 boys with acute lymphoblastic leukaemia were examined histologically and analysed for the presence of terminal deoxynucleotidyl transferase (TdT) positive cells. Immunological and histological findings correlated in 35 samples. Thirty boys with histologically and immunologically negative biopsy specimens stopped treatment, two subsequently relapsed in the testis, and five relapsed in the marrow. This examination of samples for TdT positive cells did not improve the precision of early diagnosis of testicular relapse.     

Quantification of immunocompetent cells in testicular germ cell tumours

The immunocompetent cells present in the different histological patterns of 43 testicular germ cell tumours were evaluated. CD3 + and CD45RO + (UCHL1 +) T lymphocytes, CD68 + and MAC 387 + macrophages, CD20 + (L26 +) B lymphocytes, and kappa and lambda + plasma cells were counted. The number of immunocompetent cells per mm² of tumour tissue, excluding the necrotic areas, was evaluated. Microscopic fields were randomly selected by two observers. In order to guarantee randomization each surface was divided into parts, numbered through a lattice, and some fields were chosen via a random numbers table. This procedure yielded significantly different counts from those obtained on subjective selection. The number of T-lymphocytes and macrophages was higher in seminomas than in the non-seminomatous testicular germ cell tumours ( P < 0.05) Embryonal carcinomas had more T-lymphocytes than immature teratomas. No significant differences were found among testicular germ cell tumours with regards to the B-lymphocytes, with the exception of the high number of B-lymphocytes in mature teratomas. Kappa + and lambda + plasma cells were few in the testicular germ cell tumours. Randomization in the quantification of immunocompetent cells in testicular germ cell tumours is a good means for evaluation of immune response in all the tumour mass, not only in the areas with the most intense inflammatory cell infiltrate, and permits comparison of testicular germ cell tumours with other malignant tumours. Study of immunocompetent cells in every histological type of testicular germ cell tumour is useful in comparing them with other extra-testicular germ cell tumours.     

Quantification of immunocompetent cells in testicular germ cell tumours

The immunocompetent cells present in the different histological patterns of 43 testicular germ cell tumours were evaluated. CD3 + and CD45RO + (UCHL1 +) T lymphocytes, CD68 + and MAC 387 + macrophages, CD20 + (L26 +) B lymphocytes, and kappa and lambda + plasma cells were counted. The number of immunocompetent cells per mm² of tumour tissue, excluding the necrotic areas, was evaluated. Microscopic fields were randomly selected by two observers. In order to guarantee randomization each surface was divided into parts, numbered through a lattice, and some fields were chosen via a random numbers table. This procedure yielded significantly different counts from those obtained on subjective selection. The number of T-lymphocytes and macrophages was higher in seminomas than in the non-seminomatous testicular germ cell tumours (P < 0.05) Embryonal carcinomas had more T-lymphocytes than immature teratomas. No significant differences were found among testicular germ cell tumours with regards to the B-lymphocytes, with the exception of the high number of B-lymphocytes in mature teratomas. Kappa + and lambda + plasma cells were few in the testicular germ cell tumours. Randomization in the quantification of immunocompetent cells in testicular germ cell tumours is a good means for evaluation of immune response in all the tumour mass, not only in the areas with the most intense inflammatory cell infiltrate, and permits comparison of testicular germ cell tumours with other malignant tumours. Study of immunocompetent cells in every histological type of testicular germ cell tumour is useful in comparing them with other extra-testicular germ cell tumours.     

The significance of giant cells in human testicular seminomas

Summary Results of cell kinetic analyses on transurethrally obtained material from urinary bladder are compared with parallel immunohistochemical tests on carcinoembryonic antigen (CEA) and tissue polypeptide antigen (TPA), performed on the same material. Labelling index increases from 1.4% in slight to 20% in marked urothelial atypia. CEA reaction in slight atypia is slight or moderate, slight, moderate or distinct in atypia, and moderate to distinct in carcinoma in situ. TPA always shows moderate to distinct reactions. Cell kinetically, urothelial carcinomas mas yield similar gradations. They were positive for CEA in 70% and for TPA in 100%. In G0 and GI carcinomas, negative and slightly positive reactions predominate, poorly differentiated lesions yield predominantly distinct reactions. In all grades, TPA ranges from slight to distinctly positive. As in cell kinetic analyses, there is a relationship between differentiation grade and stage for CEA expression. This does not apply for TPA.The results permit us to draw conclusions on the different biological and histogenetical behavior of urothelial carcinomas. There are undoubtedly differences in the behavior of papillary-exophytical and solid invasive carcinomas in terms of both cell kinetics and immunohistochemistry.Dedicated to Professor Dr. Dr. h.c. mult. W. Doerr on the occasion of his 70th birthdaySupported by Hoyer GmbH and Company 4040 Neuss/W. Germany     

The fine structure of testicular interstitial cells in mice

The testicular interstitial cells of mice contain an abundant agranular endoplasmic reticulum occurring as a network of interconnected tubules. Unusual features of the reticulum are occasional extensive whorls of concentric membranes and the occurrence of bundles of parallel double-walled tubules. The mitochondria have tubular cristae and are occasionally very large. In contrast to the interstitial cells of other species that have been described, the mouse interstitial cells show a segregation of the cytoplasm into areas of dense agranular reticulum and other areas where the agranular reticulum is relatively sparse. The latter areas contain scattered cistenae of granular reticulum, many free ribosomes in clusters, mitochondria, lipid droplets and small granules. The mitochondria and lipid droplets are often encircled by cisternae of the reticulum. Biochemical evidence from other laboratories, taken with the present results, indicates that the membranes of the endoplasmic reticulum in mouse interstitial cells are the site of the enzymes that mediate the synthesis of testosterone from progesterone. There is also an indication that cholesterol biosynthesis is associated with the agranular reticulum. The membranes of the agranular reticulum may also serve as a reservoir for the storage of cholesterol.     

Antibodies against testicular germinal cells in lepromatous leprosy.

Testicular germinal cell antibodies were found in 44 of 59 patients with lepromatous leprosy and in 4 of 10 patients with tuberculoid disease. A similar pattern was found in 12 of 262 controls and normal subjects. The antibody was found to be of the IgG class and 40 of 49 of these antibodies were shown to be complement fixing. Spermatozoal antibodies were detected in 12 patients, but no ovarian antibodies were found in any specimen. There was no close correlation between erythema nodosum leprosum and testicular antibodies. It was found that the characteristic of the testicular antibody in leprosy was its ability to be absorbed by Mycobacterium BCG suspension suggesting that this is another antibody induced by infection. A similar fluorescent pattern was seen in some patients who did not have leprosy, but in these cases, it could not be abolished with BCG. It is concluded that autoimmunity may be 1 of the factors involved in the pathogenesis of orchitis in leprosy.     

Granular transformation of Sertoli cells in testicular disorders.

In order to study the granular transformation of Sertoli cells the following testicular specimens were reviewed: 58 postmortem biopsies from 21 children and 37 young adult males with normal histologic pattern; 165 biopsies from prepubertal cryptorchid testes; 38 biopsies and 18 surgical specimens from postpubertal-cryptorchid testes; bilateral biopsies from eight men with Del Castillo's syndrome, 14 men with retractile testes, and five men with obstructive azospermia; 17 bilateral and seven unilateral biopsies from 24 men with varicocele; seven unilateral biopsies plus five surgical specimens from 12 men with male pseudohermaphroditism; one biopsy and one surgical specimen from two men with macroorchidism; and the autopsy specimens from 28 adult men with acquired immunodeficiency syndrome (AIDS). Sertoli cells with eosinophilic granular cytoplasm were found in the testes of one prepubertal and four postpubertal cryptorchid males, two males with Del Castillo's syndrome, two males with retractile testes, four males with varicocele, two male pseudohermaphrodites, two males with macroorchidism, and one male with AIDS and interstitial orchitis. Histochemical and ultrastructural examination of granular Sertoli cells revealed that these cells accumulate secondary lysosomes and show scant cytoplasmic organelles. In the males with varicocele or retractile testes, these lysosomes were probably heterolysosomes that had degraded the germ cells and testicular fluid accumulated in the lumen of the ectatic seminiferous tubules of these testes. A similar mechanism is also probable in the male with interstitial orchitis that had caused germ cell destruction. In the other cases, in which the tubules showed reduced lumen and severe germ cell depletion, the abundant lysosomes are probably cytolysosomes. The development of these cytolysosomes might be related to the Sertoli cell dysgenesis present in these testes.     

Force responses to rapid length changes in single intact cells from frog heart.

1. Force transients in response to step perturbations in length were recorded in intact atrial cells from frog heart at various temperatures (6-15 degrees C). Length changes of various sizes and in either direction, complete in 0.5 ms, were applied to single myocytes near slack length (initial sarcomere length 2.1-2.2 microns) just before the peak of an isometric twitch. The frequency response of the force transducers used was 2-4 kHz in Ringer solution. 2. An early quick force recovery phase was clearly observed after the elastic force response to the length step and before the start of much slower recovery processes. The quick recovery phase became progressively faster with larger shortening steps and was almost as fast as that originally described in intact frog skeletal muscle fibres (rate constants above 1000 s-1 in large releases at 10 degrees C). 3. The force-extension relation determined at the end of the length change (T1 curve) indicates that an instantaneous shortening of 0.5-0.6% of the initial cell length (L0) brings the force to zero. The force--extension relation determined at the end of the quick recovery phase (T2 curve) showed that the early recovery leads to an almost complete restoration of the original force with small stretches and releases (up to 0.3% L0) and that it becomes negligible in shortening steps of about 1.4% L0. 4. The results suggest that the mechanical properties of attached cross-bridges and the rate of transitions between attached cross-bridge states are approximately the same in frog atrial cells and fast skeletal muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of telomere length in cells from pleural effusion: Asbestos exposure causes telomere shortening in pleural mesothelial cells

We estimated the telomere lengths of neoplastic and non‐neoplastic mesothelial cells and examined their correlation with asbestos exposure and the expression of markers of mesothelial malignancy. Cell blocks of pleural effusion obtained from 35 cases of non‐neoplastic disease (NN), 12 cases of malignant mesothelioma (MM) and 12 cases of carcinomatous effusion due to lung adenocarcinoma (LA) were examined. Fifteen of the 35 NN cases had pleural plaques (NNpp+) suggestive of asbestos exposure, and the other 20 cases had no pleural plaques (NNpp‐). Telomere length was measured using the tissue quantitative fluorescence in situ hybridization method, and expressed as normalized telomere‐to‐centromere ratio. NN cases had significantly longer telomeres than MM (P < 0.001) and LA (P < 0.001) cases. Telomeres in NNpp+ cases were slightly shorter than those of NNpp‐ cases (P = 0.047). MM and LA showed almost the same telomere length. NN cases with shorter telomeres tended to show aberrant expression of epithelial membrane antigen (EMA), CD146, glucose transporter 1 (GLUT1) and IGF‐II messenger RNA‐binding protein 3 (IMP3). These results suggest that telomere shortening and subsequent genetic instability play an important role in the development of MM. Measurement of telomere length of cells in pleural effusion might be helpful for earlier detection of MM.     

Telomere length in subpopulations of human hematopoietic cells

In order to test the hypothesis that the telomere length in human hematopoietic cells correlates with their proliferative potential, we analyzed the telomere length in highly purified subpopulations of bone marrow cells. Cells were sorted on the basis of CD34 and CD38 cell surface markers, and two samples were additionally sorted on the basis of Hoechst 33342 dye efflux allowing isolation of side population (SP) cells. The telomere length in limiting numbers of sorted cells was analyzed using a newly developed fluorescence in situ hybridization (flow-FISH) method in which hybridization of telomere probe in cells of interest is measured relative to control cells in the same tube. In all seven bone marrow samples analyzed, the telomere length in CD34(+)CD38(-) cells was longer than in CD34(+)CD38(+) cells from the same donor (p < 0.02). Results with sorted SP cells were less clear: the telomere fluorescence in these cells was very heterogeneous, and a reproducible difference in telomere length relative to CD34(+)CD38(-) cells could not be observed. We conclude that the telomere length in subpopulations of hematopoietic cells does appear to be correlated with the known proliferative potential of such cells and that further characterization of cells on the basis of telomere length is warranted for enrichment of very rare precursors of hematopoietic and other tissues.     

Chlorphentermine-induced lipidosislike ultrastructural alterations in testicular Leydig and Sertoli cells

Prolonged administration of the anorectic drug chlorphentermine to rats, mice, and rabbits causes the formation of abnormal cytoplasmic inclusions in Leydig and Sertoli cells. The abnormal inclusions display either concentrically lamellated patterns with a 45 Å periodicity, or a crystalloid structure. These alterations correspond to chlorphentermine-induced changes previously observed in other tissues; they are interpreted as the result of a drug-induced phospholipidosis.     

Heterosexual activity and cycle length variability: effect of gynecological maturity.

Previous studies linking heterosexual activity to women's menstrual cycle variability have failed to take into account the effects of gynecological maturity. One hundred thirty-two women, all at least seven years postmenarche and not using birth control pills, completed daily records of their cycles and their heterosexual behavior. Data from women classified as sexually celibate or as regularly sexually active (having sex at least once per week in every nonmenstruating week) replicated previous findings while controlling for gynecological maturity: Women classified as celibate had more variable cycles than women who engaged regularly in heterosexual activity. An interaction between gynecological maturity and sexual status was also found, precluding a comparison involving women who were sexually active on an irregular basis. The interaction revealed that increased gynecological maturity is associated with less variable cycles in the sexually sporadic women, but is not associated with cycle variability in either celibate or sexually regular women. Possible biological mechanisms for these findings and their implications are discussed.