Human Testicular Biopsies Research Articles

Proteomic Analysis of Testis Biopsies in Men Treated with Injectable Testosterone Undecanoate Alone or in Combination with Oral Levonorgestrel as Potential Male Contraceptive

Treatment with injectable testosterone undecanoate (TU) alone or in combination with oral levonorgestrel (LNG) resulted in marked decreases in sperm concentrations. In this study, we used proteomic analyses to examine the cellular/molecular events occurring in the human testis after TU or TU + LNG treatment. We conducted a global proteomic analysis of the human testicular biopsies before and at 2 weeks after TU alone or TU + LNG treatment. Proteins showing significant changes in expression were identified and analyzed. As a result, 17 and 46 protein spots were found with significant differential expression after the treatment with TU alone and TU + LNG, respectively. TU treatment changed the expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), proteasome inhibitor PI31 subunit (PSMF1), and superoxide dismutase [Mn] mitochondrial precursor (SOD2). These proteins inhibit "assembly", induce cell death, and promote compensatory "cell survival" in the testis. After TU + LNG treatment, "proliferation/cell survival" and "apoptosis/death" were the predominant responses in the testis. TU + LNG treatment inhibited the expression of Prolyl 4-hydroxylase beta subunit (P4HB) and Annexin A2 (Annexin II). These proteins are involved in apoptosis and cell proliferation, respectively. TU + LNG treatment also enhanced the expression of SOD2 and Parvalbumin alpha (Pvalb). These two proteins may protect testicular cells against apoptosis/death and promote cell survival. In conclusion, TU and TU + LNG treatments suppress spermatogenesis through different pathways by changing the expression of different proteins. hnRNP K, PSMF1, SOD2, P4HB, Annexin II, and Pvalb, are key proteins that may be early molecular targets responsible for spermatogenesis suppression induced by hormone treatment.

Male Gamete Empowerment

The degree of spermatogenesis varies among infertile men from a barely compromised sperm number in the ejaculate to their complete absence-the condition known as azoospermia. The scarcity of gametes often represents a major hindrance to overcoming spermatogenic failure through the use of assisted fertilization techniques. This induced us to attempt replication of the spermatozoon by "male genome cloning." We then investigated whether it is possible to induce human spermatogenesis in host testes by transplanting germ cells obtained from human testicular biopsy specimens into mouse seminiferous tubules. After plating the sorted germ cells on a feeder layer, one putative spermatogonial stem cell colony proliferated up to day 9 of culture. The production of male gametes by ooplasmic somatic cell haploidization has been tested in our laboratory. Attempts to induce neogametogenesis from embryonic stem cells has yielded mouse offspring following fertilization with sperm-like cells.

A Prostaglandin D2 system in the human testis

As shown recently, cyclooxygenase 2 (COX2), the inducible key enzyme for the prostaglandin (PG) biosynthetic pathway, is abundantly present in interstitial cells of testes of men suffering from different forms of impaired spermatogenesis and sub- or infertility, but it is absent in human testes with normal spermatogenesis. Although the spectrum of the downstream products of COX2 action in testis, namely PGs, and their effects are not known, our results show that Prostaglandin D2 (PGD2) likely plays a role. We describe (a) PGD2 synthetases, as well as receptors for PGD2 (DP) in testicular interstitial cells of men suffering from spermatogenic damage and infertility, and report that (b) PGD2 is produced by and can affect Leydig cells of an animal model, which expresses testicular COX2 and DP.

Adult human testicular tissue with impaired spermatogenesis survives as ectopic xenograft

To provide a laboratory model to evaluate survival and maintenance of adult human spermatogenesis from men with a variety of pathological conditions. Research study (IRB-approved) of transplantation by xenografting of human testicular biopsies in immunodeficient mice. A total of 8 testicular biopsies, obtained from four men diagnosed with obstructive azoospermia and one man with maturation arrest, and multiple testicular samples from two testes (orchiectomy specimen) of a patient undergoing a sex reversal operation after prolonged hormonal suppression, were used for the experiments. The specimens were grafted into castrated, six-week old male, immunodeficient ICR SCID mice. At the time of grafting a piece of testicular tissue was also fixed for histological exam and to serve as a reference for graft development. The remaining fragments of testis tissue were inserted subcutaneously into the dorsal skin and analyzed at different time intervals (between 1 and 19 weeks). At the time of analysis, the following were documented: a) histology; b) measurement of the diameter of the seminiferous tubules; c) maintenance or development of spermatogenesis. The xenografts from each donor survived and all established blood supply; however, grafts from donors with complete spermatogenesis at the time of grafting showed consistently massive degenerative changes of the germinal epithelium and progressive decline and disappearance of spermatogenic activity. On the other hand, from 2 and up to 19 weeks, all the testis grafts of patients exhibiting absent spermatogenesis maintained their tubular morphology and showed almost no degenerative changes. These pioneering observations suggest that: a) adult human testis grafts from patients with complete spermatogenesis undergo progressive degenerative changes in xenografts; b) testis grafts of patients with less advanced spermatogenesis (histologically comparable to prepubertal boys) are more likely to survive. These findings provide strong support to the hypothesis that, as already demonstrated in domestic animal species, xenografting could be used to initiate and maintain spermatogenesis in grafts obtained from prepubertal boys prior to gonadotoxic treatments.

Testicular pain following vasectomy: a review of postvasectomy pain syndrome.

Testicular pain following vasectomy: a review of postvasectomy pain syndrome.

Growth, Development, Reproduction, Physiological and Behavioural Studies on Living Organisms, Human Adults and Children Exposed to Radiation from Video Displays

Various living organisms, human workers and children were tested for any biological action resulting from exposure to radiation from video display terminals (VDTs). VDTs were powered by a 50-Hz alternating volt age of 220 V. Measured electric and magnetic fields were 13 V/M and 50 nT, respectively. Living organisms were maintained under their normal breeding conditions and control values were obtained before switching on the VDT. Various effects related to the irradiation time were demonstrated, i.e. growth delay in algae and Drosophila, a body weight deficiency in rats, abnormal peaks of mor tality in Daphnia and Drosophila, teratological effects in chick embryos and behavioural disturbances in rats. The embryonic and neonatal periods showed a high sensitiv ity to the VDT radiation. In humans, after 4 h of working in front of a VDT screen, an increase in tiredness and a decrease in the resistance of the immune system were observed in workers. In prepubertal children, 20 min of exposure were sufficient to induce neuropsychological disturbances; pre-pubertal young people appear to be particularly sensitive to the effect of the radiation. ln human testicular biopsies cultured in vitro for 24 h in front of a VDT screen, mitotic and meiotic disturbances, the appearance of degeneration in some aspects of the cells and significant disorganisation of the seminiferous tubules were demonstrated and related to modification of the metabolism of the sample. An experimental appa ratus has been developed and tested that aims to pre vent the harm from VDT radiation. Known commercially as the emf-Bioshield®, it ensures effective protection against harmful biological effects of VDT radiation.

O-064. Enrichment of round spermatogenetic cells from human testicular biopsies

O-064. Enrichment of round spermatogenetic cells from human testicular biopsies

Chromosome in situ suppression hybridisation in human male meiosis.

Chromosome in situ suppression hybridisation with biotinylated whole chromosome libraries permits the unequivocable identification of specific human somatic chromosomes in numerous situations. We have now used this so called 'chromosome painting' technique in meiotically dividing cells, isolated from human testicular biopsy. It is shown that the method allows identification of target homologues, bivalents, and sister chromatids throughout the relevant stages of meiosis. Thus, a more accurate study of meiosis per se is now available to increase our understanding of such processes as first meiotic synapsis of homologues and chiasma formation/meiotic crossing over, which are still outstanding biological enigmas. The new technology also makes it possible, for the first time, (1) to obtain direct numerical data in first meiotic non-disjunction for individual chromosomes, and (2) to quantify segregation in male carriers of structural rearrangements. We exemplify the use of the chromosome painting technique for a first meiotic segregation analysis of an insertional translocation carrier.

Epidermal Growth Factor Receptors (EGFR) Localization in Human Testis

An indirect immunofluorescence technique was used to detect and localize epidermal growth factor receptors (EGFR) in human testicular biopsies in different testicular diseases. Monolateral biopsies from twelve infertile subjects were studied from qualitative/quantitative points of view and were examined by immunofluorescence study with anti-EGFR monoclonal antibody. Two different patterns of EGFR expression were observed: a very weak presence of EGFR was detectable on Sertoli cells, peritubular basal structures, and interstitial compartment in testicular biopsies showing a normal spermatogenic status; and an important increase in EGFR expression was observed on the cytoplasm of Sertoli cells and on peritubular basal structures in biopsies exhibiting various degrees of hypospermatogenesis and in a case of Sertoli cells only syndrome. Germ cells did not show EGFR immunolocalization. EGFR seems to be present on different testicular target cells. EGFR expression increases on peritubular and Sertoli cells in the presence of significant tubular damage.

Pattern of compartmentation in human seminiferous tubules showing dislocation of spermatogonia.

The pattern of compartmentation of the seminiferous epithelium was investigated, using a lanthanum tracer technique, in human testicular biopsies of adult infertile men (age 27 to 44 years), where dislocation of spermatogonia from the basal lamina occurred. Spermatogonia type A and B were found in a two- or three-layered arrangement, in aberrant locations throughout the seminiferous epithelium, and in intratubular positions associated with fragments of Sertoli cell cytoplasm. Tracer impregnation was found around spermatogonia in a multilayered arrangement, indicating the extension of the basal compartment in a luminal direction. Single spermatogonia within the second or third layer of the seminiferous epithelium were regularly found to be surrounded by tracer. The junctional complex between the lateral membranes of adjacent Sertoli cells was devoid of tight junctions. Tracer penetration around spermatogonia in a more luminal position was prevented by intact Sertoli cell junctional complexes; tracer was also absent from intraluminal located spermatogonia associated with cytoplasmic fragments of Sertoli cells. The luminal extension of the basal compartment associated with the dislocation of spermatogonia clearly differs from the pattern of compartmentation during the movement of primary spermatocytes within undisturbed epithelium. There is a strong incidence of elevated serum levels of follicle-stimulating hormone (greater than 7 U/l), indicating a suppression of Sertoli cell function; this may be the cause for the dislocation of spermatogonia and the changes of compartmentation.

Mechanism of germ cell degeneration in human and guinea pig testes

The mechanism of germ cell degeneration in the contralateral testes of men with varicocele or torsion of the spermatic cord has not yet been fully elucidated. The major cause of such a gap in our knowledge is due to the lack of a suitable animal model. We used guinea pigs as an experimental animal model, to evaluate the suitability of this animal species to study the testicular pathology.Unilateral torsion of the spermatic cord was surgically induced in 36 animals. Groups of six animals were sacrificed at intervals of 4,8,12,16,20 and 24 months. Pieces of tissues were obtained from the contralateral testis of each animal. Human testicular biopsies were obtained from patients with either torsion of the spermatic cord or varicocele. Tissue specimens were fixed, dehydrated and embedded in epon following the usual procedure.

Immunostaining of transferrin and transferrin receptor in human seminiferous tubules

Transferrin (TF) and transferrin receptor (TFr) were studied in human testicular biopsy specimens with the use of immunostaining techniques. A polyclonal antibody to human TF (obtained in goat), a murine monoclonal antibody (B3/25) to human TFr, and antisera antigoat IgG and antimouse IgG, both labeled with peroxidase, were used. In seminiferous tubules of subjects with normal spermatogenesis, TF was found mainly in Sertoli cells and, in lesser amounts (probably related to the presence of receptor-TF complexes), in spermatocytes and early spermatids. TFrs were found only in spermatocytes and early spermatids. In patients with spermatogenetic disorders, TF was always found in Sertoli cells, whereas TFrs were found in spermatocytes only when they were present. These results seem to demonstrate that in human seminiferous tubules, Sertoli cells are devoted to the production and/or storage of TF, whereas spermatocytes and early spermatids use TF.

Germ cell quantitation in human testicular biopsy.

Quantitative analysis of human seminiferous epithelium was carried out using an improved method of glutaraldehyde and osmium fixation with plastic embedding. Part of each biopsy specimen was fixed in Bouin's fixative and embedded in paraffin for comparison. Epon embedded tissue had very little artifactual damage compared with paraffin embedded tissue sections. The germ cell to Sertoli cell ratios were determined by counting the various germ cells per "unit" tubular area. Data obtained by this method reflect a remarkable stability of Sertoli cell number and germ cell-Sertoli cell ratios both between biopsies from different individuals and between biopsies from right and left testes from the same individual. Agreement between the present results and those of earlier studies based on paraffin embedded testicular specimens supports the validity of this method of germ cell quantitation of human testicular biopsy samples.

Quantitative evaluation of spontaneous and radiation-induced polyploidisation processes in human and murine testes.

Flow cytometric analysis of human and murine testicular tissue was performed in order to determine whether cells exist with a DNA content differing from the expected categories 1 c, 2 c and 4 c, and to estimate the frequencies of diploid elongated spermatids. About 1.5 per cent of the murine testicular cells had an 8 c DNA content. In human testis, no 8 c cells were detected. A slight increase of 8 c cells was observed in the murine testis, following irradiation. An average of 1.8 per cent of the elongated spermatids in 10 control mice were diploid. In 12 human testicular biopsies, an average of 4.7 per cent of diploid elongated spermatids was observed among all elongated spermatids in mice. Acute or split-dose exposure, with 15 Gy roentgen rays, to spermatocytes increased the spontaneously occurring level of diploid elongated spermatids 25-fold.

Demonstration of thiamine pyrophosphatase in human germ cells and sertoli cells, a histochemical study

The distribution of thiamine pyrophosphatase (TPPase) was studied in the human testicular biopsy tissue with the help of a modified histochemical gel method. The spermatogonia showed a large round strongly positive reaction zone in the supranuclear region. The pachytene spermatocytes exhibited a large semicircular TPPase reaction zone around the nucleus directing towards the lumen. The early round spermatids were characterized by a small, round, and weak supranuclear reaction zone. The elongated late spermatids were devoid of TPPase activity. The Sertoli cells possessed a "streamer" like formation of strong TPPase activity spreading from basal to apical portion.

Aspiration biopsy of testis: another method for histologic examination

The most important method for evaluating the pathogenesis of male infertility is open testicular biopsy. Herein we describe a method of aspiration biopsy of testis for histologic examination. Sexually mature dogs and rats treated with chemotherapeutic agents and ionizing radiation were followed with periodic testicular aspiration biopsy during and after treatment. The histologic findings from the aspiration biopsy compare with the results of routine histologic examinations in assessing spermatogenetic activity and delineating pathologic changes. The puncture in the experimental animals was performed under general anesthesia. In human patients testicular biopsy could be done under local anesthesia in an outpatient clinic. The procedure would be less painful, minimally invasive, and more cost-effective.

Lymphocyte infiltration in human testicular biopsies.

Testicular biopsies from 166 men with a clinical diagnosis of azoospermia or oligozoospermia were analysed for the presence of leucocytic infiltrations. In 8 cases (4.8%) a pronounced focal or general leucocytic infiltration was found. In all cases the vast majority of the cells were lymphocytes and their presence was not correlated to the histopathological diagnosis. Usually the lymphocytes were within the interstitial tissue, but in one case the lymphocytes were migrating through the wall of the seminiferous tubules. The spermatogenesis was most disturbed in the tubules located beneath the lymphocytic infiltrations. The changes observed were morphologically very similar to those described in experimental autoimmune orchitis and therefore it may be suggested, that immunological processes might mediate the deleterious effects of different factors on the testis more often than has been suspected.

153. Incubation study of 3H-pregnenolone with human bilateral testicular biopsy samples

153. Incubation study of 3H-pregnenolone with human bilateral testicular biopsy samples

In vitro differentiation of diakinesis figures in human testis.

Fragments of tissue from human testicular biopsies were incubated at 31°C in medium supplemented with 10% w/v foetal calf serum and enriched with 4 mM L-glutamine. The tissue was supported during growth at the gas/medium interface. Autoradiography was used to follow the progress of differentiating spermatogonia. Activity of the testicular epithelium was observed throughout 21 days of culture. Many diakinesis figures were observed between 10–14 days growth in culture.

Synaptosomal complexes and associated structures in microspread human spermatocytes.

Human spermatocytes processed with a modified microspreading technique which involves the use of sodium dodecyl-sulphate (SDS) have been used to construct synaptonemal complex (SC) karyotypes. Twenty two pachytene spermatocytes were selected for length quantitation. The mean values of relative lengths and centromeric indexes of each SC agree closely with values obtained by three-dimensional reconstructions (Holm and Rasmussen, 1977), except for SCs #4--5, 6--7 and 19--20. Absolute lengths are consistently longer in spreads (10.7% longer than in sections, on average). The mean total length of the SC complement is 258.7 micrometer. Six morphological types of XY pairs have been described. On the basis of the relationships between the XY pair, nucleolar development and autosome behavior, these six XY types are assumed to develop in succession. Type O XY pairs occur during late zygotene, types I and II XY pairs occur during early to mid-pachytene, and types III, IV and V occur during later pachytene substages. Alignment of the X and Y axes is observed at late zygotene, and formation of the SC occurs in relation with type I XY pairs. Progressive desynapsis occurs in types II and III. Splitting and fusion of the X and Y axes attain a maximum in types IV and V. The breakdown of the dense bodies associated with the X and Y axes occurs during stage V. --Bar-like structures, having a mean length of 2,100 A are associated with SCs in all the pachytene substages defined by the XY types. The average number of bars per nucleus is 46.2 (SD = 8.4, N = 20), and the average SC length per bar is 5.57 micrometer. The distribution along the SCs of 923 bars shows that near-termini locations are preferred (SC length per bar, 2.98 micrometer) and centromere regions are avoided (SC length per bar, 16.9 micrometer). --On the basis of these data, bars are similar to recombination nodules described in other organisms. The availability of a standard SC karyotype for microspreads and a temporal sequence given by the XY pair provide a basis for rapid screening of chromosome aberrations in human testicular biopsies.