Effect of vascular endothelial growth factor and testis tissue culture on spermatogenesis in bovine ectopic testis tissue xenografts

Bovine ectopic testis tissue grafting is a technique that can be used to study bovine spermatogenesis and for the production of germ cells for a variety of applications. Approximately 10% of seminiferous tubule cross sections in testis grafts contain spermatids, providing a unique tool to investigate what regulates germ cell differentiation. We hypothesized that manipulation of testis tissue grafts would increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation. To test this hypothesis, bovine testis tissue was treated with vascular endothelial growth factor (VEGF) at the time of grafting or explant cultured for 1 wk prior to grafting. For the VEGF experiment, 8-wk donor tissue and graft sites were treated with 1 microg of VEGF in order to increase angiogenesis at the graft site. For the testis tissue culture experiment, 4-wk-old donor testis was cultured for 1 wk prior to grafting to stimulate spermatogonial stem cell proliferation. Testis tissue grafts were removed from the mice 24 wk after grafting. VEGF treatment increased graft weight and the percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal. Cultured testis tissue grafts were smaller and had fewer seminiferous tubules per graft. However, there was no difference in the percentage of seminiferous tubule cross sections that contained any germ cell type between groups. These data indicate for the first time that bovine testis tissue can be manipulated to better support germ cell differentiation in grafted tissue.     

The ultrastructure of mouse testicular interstitial tissue containing plutonium-239 and its significance in explaining the observed distribution of plutonium in the testis.

The technique of autoradiography with Araldite-embedded sections was used to study the distribution of 239Pu in mouse testis at various times post-injection. Adjacent sections were examined with both the light microscope and electron microscope. The autoradiographs showed that from 1 week to 3 months postinjection, most 239Pu in located in interstitial tissue. The major change in distribution observed was that the early diffuse deposit in interstitial tissue is concentrated in macrophages with increasing time post-injection. This is a real change of distribution as the amount of 239Pu in mouse testis remains constant from 1 week to 3 months post-injection. Study of the ultrastructure of interstitial tissue indicated that the accumulation of 239Pu in macrophages may be brought about in two ways. First, there may be phagocytosis of dead cells containing 239Pu. Second, 239Pu may follow the transfer of waste products of hormone synthesis from Leydig cells into macrophages. The significance of these observations is discussed with regard to the deposition of 239Pu in human testis.     

Grafting period and donor age affect the potential for spermatogenesis in bovine ectopic testis xenografts

Bovine testis tissue xenografts contain elongating spermatids 6 mo after grafting. The percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal varies depending on donor age and rarely exceeds 10%. These data indicate significant changes are occurring to bovine testicular cells during the first weeks of life. The objective of this research was to xenograft testis tissue from multiple ages of bull calves for 24 or 36 wk in order to gain a better understanding of early bovine testis development. Testis tissue from 1-, 2-, 4-, and 8-wk-old calves was grafted onto the backs of castrated immunodeficient mice. Testis tissue from all donor ages grew, differentiated, and produced testosterone and elongating spermatids. Testis tissue grafts from 1- and 8-wk-old calves had elongating spermatids in greater than 5.5% of seminiferous tubule cross sections at the time of graft removal regardless of grafting period. Four-week-old donor tissue never had more than 5.2% of seminiferous tubule cross sections with elongating spermatids. Extending the grafting period from 24 to 36 wk resulted in an increase in the percentage of seminiferous tubule cross sections with elongating spermatids from 2% to 10% in 2-wk donor tissue. These data demonstrate that both donor age and grafting period may be important factors regulating the maturation of bovine testis xenografts, indicating that intrinsic differences exist within testis tissue at these donor ages. These data provide the framework for further study of bovine spermatogenesis using ectopic testis xenografting.     

Detection of cell proliferation in pig testis and intestine sections using monoclonal anti-bromodeoxyuridine antibody and immunogold silver staining

Summary For the first time a monoclonal antibody against 5-bromodeoxyuridine was used to detect cell proliferation in pig testis and intestine sections. The influence of several parameters such as mode of injection, addition of thymidine biosynthesis inhibitor, tissue fixation, hydrolysis and revelation was examined. The technique of choice consisted in intravenously injecting the animals with 50 mg/kg BUdR added to 10 mg/kg FUdR 2 h before tissue collection and Bouin fixation; hydrolysis of sections was performed by HCl 4N: Ethanol 70° (1:1 v/v); revelation of BUdR was made by a secondary antibody linked to colloidal gold particles, followed by a silver enhancement step. The data were superior when compared to those obtained by direct immunofluorescence and by the PAP technique. The described method is convenient and sensitive, provides an intense nuclear labelling without background and allows simultaneous examination of histology. The advantages over the technique using tritiated thymidine are particularly obvious when fast screening of numerous samples is required or when new experimental protocols are developping.     

Detection of cell proliferation in pig testis and intestine sections using monoclonal anti-bromodeoxyuridine antibody and immunogold silver staining

For the first time a monoclonal antibody against 5-bromodeoxyuridine was used to detect cell proliferation in pig testis and intestine sections. The influence of several parameters such as mode of injection, addition of thymidine biosynthesis inhibitor, tissue fixation, hydrolysis and revelation was examined. The technique of choice consisted in intravenously injecting the animals with 50 mg/kg BUdR added to 10 mg/kg FUdR 2 h before tissue collection and Bouin fixation; hydrolysis of sections was performed by HC1 4N: Ethanol 70 degrees (1:1 v/v); revelation of BUdR was made by a secondary antibody linked to colloidal gold particles, followed by a silver enhancement step. The data were superior when compared to those obtained by direct immunofluorescence and by the PAP technique. The described method is convenient and sensitive, provides an intense nuclear labelling without background and allows simultaneous examination of histology. The advantages over the technique using tritiated thymidine are particularly obvious when fast screening of numerous samples is required or when new experimental protocols are developing.     

Localization of a proteolytic enzyme within the efferent and deferent duct epithelial cells of the turkey (Meleagris gallopavo) using immunohistochemistry

Turkey seminal plasma contains a serine protease found to be distinct from the spermatozoal acrosin. However, the origin and biological roles of this enzyme are unknown. Our experimental objective was to identify the cellular source of this protease within the male reproductive tract. The enzyme was isolated from seminal plasma using benzamidine-Sepharose 6B chromatography. A synthetic substrate, Nalpha-benzoyl-DL-arginine p-nitroanilide, was used to detect fractions containing the enzyme. The affinity chromatography technique yielded a 150-fold increase in amidase activity. Analysis of the protease by SDS-PAGE revealed two protein bands with relative molecular masses of 37 000 and 61 000. Proteolytic activity was detected within the smaller band as evidenced by casein digestion. Further analysis of the purified protein revealed that the smaller protein band was glycosylated. To determine the cellular source of the protease, a panel of mouse monoclonal antibodies was then developed against the purified protease, and used in immunohistochemistry. Frozen tissue sections from the liver, testis, epididymal region, and deferent duct were fixed in 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) (octylphenoxy)polyethoxyethanol followed by routine immunohistochemistry procedures. Monoclonal antibodies did not bind to tissue sections from either the liver or testis, or to blood plasma proteins. Both the distal portion of the efferent duct and the deferent duct were immunoreactive. We concluded that the protease found in turkey seminal plasma is concentrated to the distal efferent duct and the deferent duct epithelial cells.     

High level of endostatin in epididymal epithelium: protection against primary malignancies in this organ?

Rete testis and epididymis are rare locations for primary tumors or metastasis. Assuming that this may be related to expression level of angiogenic inhibitors, we focused our study on the expression pattern of collagen 18/endostatin. In situ hybridization and immunohistochemistry for collagen 18 and endostatin were carried out on sections of human rete testis and epididymis as well as on epididymal adenoma and human testicular tissue with or without carcinoma in situ (CIS). In situ hybridization revealed strong expression of collagen 18 mRNA in rete testis, efferent ducts and epididymal duct. Immunostaining showed collagen 18 in epithelium and basement membrane as well as in blood vessels of rete testis. Further, in both efferent ducts and epididymal duct, collagen 18 was mainly localized in the basement membrane of these ducts and of the blood vessel wall. Endostatin immunostaining was localized in the epithelium of rete testis, efferent ducts and epididymal duct. This pattern of endostatin staining was absent in epididymal adenoma tissue while tumor associated blood vessels exhibited strong endostatin staining. No endostatin staining was detectable in normal germinal epithelium and CIS cells while Leydig cells exhibited strong endostatin staining. High endostatin expression in epididymis may protect this organ against tumor development. Gene therapeutic strategies providing high expression of endostatin in normal epithelia may be useful to prevent tumor development.     

Monoclonal antibody localization of sperm surface antigen secreted by the epididymis of the baboon (Papio cynocephalus)

A monoclonal antibody (BSA6) was generated against an antigenic determinant secreted by the epididymis of the baboon and present on the acrosomal surface of the spermatozoa. This determinant was first secreted by the principal cells of the proximal corpus region, as determined by fluorescent microscopy performed on Bouin-fixed epididymal tissue sections. The secretory product subsequently bound on the lateral acrosomal surfaces in the distal corpus region, but became uniformly distributed over the acrosomal region in the cauda epididymidis. The antigenic determinant had a molecular weight of 82,000 (western blot technique). The testis, caput and other somatic tissues were devoid of the antigen, indicating the restriction of the antigen to spermatozoa and epithelial cells of the corpus epididymidis. Examination of similar tissue from immature baboons indicated that the secretion of this antigen was age-dependent, secretion beginning at about 4 years of age.     

Quantitative determination of hyaluronidase in tissue sections

Summary A method for the determination of hyaluronidase in histological sections is described. This method is based on incubation of tissue sections in a medium containing hyaluronic acid as substrate. Depolymerisation of the substrate during incubation as well as the total nitrogen content are measured in the same section. The comparison of these two values gives information concerning the hyaluronidase activity. This assay was tested in experiments with rat testis. It was also found that our procedure is sensitive enough for the estimation of hyaluronidase activity in sections from kidney. From these experiments with kidney sections it can be concluded that: 1. The pH optimum for renal hyaluronidase (3.5) differs from that in the testis (4.7). 2. In the renal cortex more hyaluronidase activity was detected than in the medulla.Department of Transplantology.Department of Histology and Embryology.     

Immunolocalization of cytochrome P450 aromatase in rat testis during postnatal development

Aromatization of androgens into estrogens in rat testis is catalyzed by the microsomal enzyme cytochrome P450 aromatase. In this work, aromatase cellular site was investigated in prepuberal, peripuberal and postpuberal testis, from 10-, 21- and 60-day-old rats respectively. Paraffin-embedded testis sections were processed for P450arom immunostaining using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. Next, biotinylated anti-rabbit IgG was applied, followed by ABC/HRP/complex amplification with diaminobenzidine as chromogen. Prepuberal testis sections showed a strong immunoreactivity of aromatase in Sertoli cell cytoplasm while interstitial cells were immunonegative. In peripuberal testis sections, cytoplasmic immunoreaction was weak in Sertoli cells, but it was strong in spermatocytes and sporadic in Leydig cells. Postpuberal testis sections displayed a moderate aromatase immunoexpression in spermatocytes while a strong immunostaining was observed in round and elongated spermatids, as well as in Leydig cells. These results indicate a different age-dependence of aromatase localization in rat testicular cells during gonadal development. In particular, inside the seminiferous tubules, the aromatization site moves from Sertoli cells to late germ cells, suggesting a proliferative role of aromatase in prepuberal testis and its subsequent involvement in meiotic and post-meiotic germ cell maturation.     

Combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining

Summary A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step — Incubation of the tissue sections with the monoclonal primary antibodies; Second step — biotinylated anti-rat or anti-mouse IgG; Third step — monoclonal PAP complex; Fourth step — ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step — the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4-and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells. Stronger staining intensity was obtained for desmin-like immunoreactivity (LI) (within myofibroblasts of the lamina propria and muscle cells of the blood vessels)-and vimentin-LI (within Sertoli cells, myofibroblasts of the lamina propria and fibroblasts of the interstitium) of the human testis. The full combination (6 step-reaction) permits the detection of smallest quantities of an antigen in sections of different tissues using highly diluted primary antibodies. The technique represents a further alternative among the immunocytochemical amplification methods.     

Bisulfite genomic sequencing of microdissected cells

Mapping of methylation patterns in CpG islands has become an important tool for understanding tissue-specific gene expression in both normal and pathological situations. However, the inherent cellular heterogeneity of any given tissues can affect the outcome and interpretation of molecular studies. In order to analyse genomic DNA methylation on a pure cell population from tissue sample, we have developed a simple technique of single-cell microdissection from cryostat sections which can be combined with bisulfite-mediated sequencing of 5-methylcytosine. We report here our results on the methylation status of the androgen receptor gene studied by bisulfite genomic sequencing on purified cells isolated from human testis.     

Evaluation of the safety and efficacy of testicular biopsies in llamas

Evaluation of the reproductive function of Lama glama is generally considered to be a challenging task due to the difficulty of obtaining representative semen samples. One method that has been proposed for evaluation of testicular function in these animals is histologic examination of testicular needle biopsies. This study was undertaken to examine the safety and efficacy of using needle biopsies to assess testicular function in this species. One randomly selected testicle from each of 16 sexually mature llamas was biopsied with a 14-gauge self-firing biopsy instrument. The llamas were evaluated over a 6-week period with thermography for temperature changes of the scrotum. At the end of the 6-week trial, the llamas were castrated and sections of each testis were fixed in Bouin's solution for histologic examination. Immediately prior to castration, an additional biopsy was taken from each testis to compare the tissue obtained via biopsy with sections from the corresponding testis obtained after castration. A qualitative grading scale was used to compare the seminiferous tubules from each testis. No difference was found between the biopsied and the nonbiopsied testes (P = 0.69). The percentage of normal tubules between the biopsied and the nonbiopsied sides also did not differ (P = 0.70). Furthermore, the percentage of normal seminiferous tubules did not differ between the needle biopsy samples and the corresponding tissue samples obtained at castration (P = 0.48). The number of round seminiferous tubules counted in each biopsy section ranged from 3 to 67. There was no significant difference in the thermographic images of the scrotum between the biopsied and the nonbiopsied testes. This study supports testicular biopsies as a safe and useful procedure in the evaluation of testicular function.     

Sudan Black and Osmic Acid as Staining Agents for Testicular Interstitial Cells

Two standard cytological techniques have heen modified to stain specifically the interstitial cells of the testis. In Method 1, the tissue is fixed in Zenker-formol or Regaud's fluid for several hours or overnight and subsequently postchromed in 3% K₂Cr₂O₇ for 72 hr at 37°C. After paraffin embedding, sections are cut at 5μ, dewaxed, brought down to 70% alcohol and stained in an unfiltered saturated solution of Sudan black in 70% alcohol for 10-30 min. Sections are washed briefly in 70% alcohol to remove all excess dye, differentiated, if necessary, in 50% alcohol, downgraded to water and mounted in Farrants' medium or glycerol jelly. Interstitial cells: deep blue black; remainder of testicular tissue: light blue. Method 2 is essentially the Champy-Kull technique but specific staining for mitochondria is omitted and the sections are downgraded to water; then they are mounted in Farrants' medium or glycerol jelly without further treatment. In this way osmicated lipoids are preserved. Interstitial cells: conspicuous due to the variable number of black granules in their cytoplasm; the remainder of the tissue: yellow.     

Localization of transferrin and transferrin receptors in rat testes

One of the major proteins secreted by rat Sertoli cells in culture is a transferrin-like protein (Skinner and Griswold, 1980). The purpose of this study was to quantitate the amount of testicular transferrin in fluids isolated from the testis by the use of a radioimmunoassay and to determine the location of transferrin and transferrin receptors in the testis by indirect immunofluorescence. Seminiferous tubule fluid, rete testis fluid, and testicular lymph were collected from rat testes and were found to contain 141 micrograms, 47 micrograms and 3.7 mg transferrin per ml of fluid, respectively. Serum was found to contain 3.7 mg/ml transferrin. Paraffin sections of rat testis were incubated with rabbit anti-rat transferrin, biotinylated goat anti-rabbit and fluorescein-conjugated avidin. Immunoreactive transferrin was thus localized on the proacrosome and nuclear cap of developing spermatids. Late spermatids showed transferrin over the entire region of the head but mature testicular spermatozoa exhibited little fluorescence. The interstitial tissue between seminiferous tubules fluoresced brightly, indicating a large amount of transferrin in this area. By pretreating sections with rat transferrin, the receptor for the protein was localized on and in spermatocytes and early round spermatids. Dividing germ cells were brightly fluorescent.     

The effects of tissue sample size and media on short-term hypothermic preservation of porcine testis tissue

The objective of this study was to develop effective strategies for hypothermic preservation of immature porcine testis tissue to maintain structural integrity and cell viability. In Experiment 1, testes from 1-week-old piglets were used to study the effects of tissue sample size (as intact testes or fragments of 100-or 30 mg) and the use of one of 9 different media on hypothermic preservation of the testis tissue for 6 days. The examined media included: Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM), Leibovitz L15 (L15), L15 with fetal bovine serum (FBS, at 10%, 20% or 50%), HypoThermosol solution-FRS (HTS), Ham’s F12, and Media 199. On days 0, 3, and 6, testis tissues were digested to compare the cell survival rates. Tissue sections were also semi-quantitatively assessed to determine the efficiency of different preservation strategies. There was no effect of testis sample size (P > 0.05), but cell survival rates of testis cells isolated from preserved testis tissues changed depending on the media and day (P < 0.05). Testis tissue within HTS did not show morphological changes after 6 days. In Experiment 2, two of the top performing media (20% FBS-L15 and HTS) were selected for immunocytochemical detection of gonocytes. Proportions of gonocytes (%) in isolated testis cells, however, did not differ between the two media on days 0, 3, or 6. These results show that testis tissue can be maintained for 3 days at 4°C with high cell survival rate, and tissue morphology can be preserved for at least 6 days in HTS.     

Spermatogenesis in ferret testis xenografts: a new model

Testis xenografting is both a promising tool to study spermatogenesis and a means to preserve the genetic information and reproductive potential of prepubertal male animals. The present study was conducted to evaluate this technique using testis tissue from domestic ferrets, an important biomedical model and a model for the conservation of small carnivore species. Fresh testis tissue from 8-wk-old ferrets was implanted ectopically under the skin on the backs of castrated nude mice and subsequently evaluated for testosterone production and establishment of spermatogenesis at 10, 20, 25, and 30 wk after xenografting. A total of 40% of fresh ferret xenografts were harvested. Seminal vesicles were collected from the recipient mice and weighed as an assay for bioactive testosterone. The weights of seminal vesicles from the mice showed no significant difference from those of uncastrated, control nude mice, indicating that the xenografts were producing physiologically relevant amounts of testosterone. The ferret testis xenografts produced differentiating germ cells and sperm at the same time as did testis from age-matched control ferrets. These data demonstrate the ability of Mustelidae testicular tissue to establish spermatogenesis in nude mice after testis xenografting.     

Application of laser-capture microdissection to analysis of gene expression in the testis

The isolation and molecular analysis of highly purified cell populations from complex, heterogeneous tissues has been a challenge for many years. Spermatogenesis in the testis is a particularly difficult process to study given the unique multiple cellular associations within the seminiferous epithelium, making the isolation of specific cell types difficult. Laser-capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. This technology has enhanced our ability to directly examine gene expression in enriched testicular cell populations by routine methods of gene expression analysis, such as real-time RT-PCR, differential display, and gene microarrays. The application of LCM has however introduced methodological hurdles that have not been encountered with more conventional molecular analyses of whole tissue. In particular, tissue handling (i.e. fixation, storage, and staining), consumables (e.g. slide choice), staining reagents (conventional H&E vs. fluorescence), extraction methods, and downstream applications have all required re-optimisation to facilitate differential gene expression analysis using the small amounts of material obtained using LCM. This review will discuss three critical issues that are essential for successful procurement of cells from testicular tissue sections; tissue morphology, capture success, and maintenance of molecular integrity. The importance of these issues will be discussed with specific reference to the two most commonly used LCM systems; the Arcturus PixCell IIe and PALM systems. The rat testis will be used as a model, and emphasis will be placed on issues of tissue handling, processing, and staining methods, including the application of fluorescence techniques to assist in the identification of cells of interest for the purposes of mRNA expression analysis.     

Immunolocalization of androgen receptors in testicular cells of prepubertal and pubertal pigs

In the testis, androgen receptors are known to mediate autocrine and paracrine effects of androgens on Leydig cell function and spermatogenesis. The pig presents some unusual features with regard to the synthesis of testosterone and estrogens in the male gonads. In testes from prepubertal males, testosterone level was lower than in testes from adult boars, while estrogen secretion was relatively high and comparable to that of mature porcine gonad. Immunolocalization of androgen receptors and intensity of immunohistochemical staining was age-dependent. In testis sections from adult boars, androgen receptors were found in nuclei of all somatic cells such as Leydig cells, Sertoli cells, and peritubular-myoid cells, whereas in sections from immature pigs only in the Leydig cell cytoplasm showed positive immunoreaction for androgen receptors. In control tissue sections incubated with omission of the primary antibody, no positive staining was observed. Detection of the androgen receptors in testicular cells of the pig is important for understanding of their central role in mediating androgen action.