Circular dichroism studies of ethidium bromide binding to the isolated nucleolus

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.     

Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure

The aim of the study was to determine whether a prior diagnostic testicle biopsy can predict success or failure of testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI) in patients with non-obstructive azoospermia caused by testicular failure, and what is the minimum threshold of sperm production in the testis which must be surpassed for spermatozoa to reach the ejaculate. Forty-five patients with non-obstructive azoospermia caused by testicular failure underwent diagnostic testicle biopsy prior to a planned future TESE-ICSI procedure. The diagnostic testicle biopsy was analysed quantitatively, and correlated with the quantitative findings of spermatogenesis in patients with normal spermatogenesis, as well as with the results of subsequent attempts at TESE-ICSI. Men with non-obstructive azoospermia caused by germinal failure had a mean of 0-6 mature spermatids/seminiferous tubule seen on a diagnostic testicle biopsy, compared to 17-35 mature spermatids/tubule in men with normal spermatogenesis and obstructive azoospermia. These findings were the same for all types of testicular failure whether Sertoli cell only, maturation arrest, cryptorchidism, or post-chemotherapy azoospermia. Twenty-two of 26 men with mature spermatids found in the prior testis biopsy had successful retrieval of spermatozoa for ICSI, 12 of their partners became pregnant, and are either ongoing or delivered. The study suggests that 4-6 mature spermatids/tubule must be present in the testis biopsy for any spermatozoa to reach the ejaculate. More than half of azoospermic patients with germinal failure have minute foci of spermatogenesis which are insufficient to produce spermatozoa in the ejaculate. Prior diagnostic testicle biopsy analysed quantitatively (for the presence of mature spermatids) can predict subsequent success or failure with TESE-ICSI. Incomplete testicular failure may involve a sparse multi-focal distribution of spermatogenesis throughout the entire testicle, rather than a regional distribution. Therefore, it is possible that massive testicular sampling from many different regions of the testes may not be necessary for successful TESE-ICSI.     

Correlation of testicular pathology and sperm extraction in azoospermic men with ejaculated spermatids detected by immunofluorescent localization

Limiting testicular biopsy for intracytoplasmic sperm injection (ICSI) to those with a high chance of having testicular spermatozoa has not been possible because of the poor predictive value of current clinical and laboratory methods. In order to predict testicular pathology and sperm extraction, we characterised the semen of 28 men with azoospermia due to gonadal failure in terms of the presence of spermatids using an immunological method. The results were compared with the assessment of testicular biopsies by histology and the extraction of spermatozoa into culture medium. Washed cellular elements in the ejaculate were smeared on microscope slides and fixed in 100% methanol, before incubation with acrosome-specific monoclonal antibody (18.6), fluorescein isothiocyanate-labelled anti-mouse goat IgG, and examination by epifluorescent microscopy. Semen from men with oligozoospermia and obstructive azoospermia served as positive and negative controls, respectively. Twelve patients who had positive immunofluorescence (one or more spermatids present) had spermatozoa retrieved from their testes (five hypospermatogenesis, seven focal spermatogenesis), and 16 patients with negative immunofluorescence (spermatids absent) had apparent Sertoli cell-only syndrome (12) or maturation arrest histological pattern (four). However, four patients with apparent Sertoli cell-only syndrome had testicular spermatozoa present after extraction from the biopsy. Plasma follicle stimulating hormone concentration and testicular volume did not predict retrieval of seminal spermatids or testicular spermatozoa. We conclude that the immunofluorescent localization of one or more spermatids in the ejaculate can be used to predict the likelihood of obtaining testicular spermatozoa for ICSI. However, in some patients with Sertoli cell-only syndrome, spermatozoa could still be recovered in the absence of apparent seminal spermatids.     

RNA synthesis in spermatocytes and spermatids and preservation of meiotic RNA during spermiogenesis in the mouse

The rate of RNA synthesis at different stages of spermatogenesis in the mouse, and the preservation of RNA from the diploid to the haploid phase of spermatogenesis were studied in homogeneous germ cell fractions separated by velocity sedimentation at unit gravity. The uridine pool expansion method was used for determining the rate of RNA synthesis: seminiferous tubules were labelled in culture with increasing concentrations of [3H]-uridine and the incorporated radioactivity was estimated in cell fractions separated by velocity sedimentation. The results indicate that before nuclear elongation, round spermatids (steps 1 to 8 of spermiogenesis) synthesize RNA at the same rate per DNA content as middle-late pachytene spermatocytes. The preservation of RNA molecules synthesized in meiosis was investigated by labelling pachytene spermatocytes with T3H]uridine in vivo and collecting samples of germ cells at definite stages of spermatogenesis at various time intervals thereafter. The results show that a considerable proportion the RNA synthesized during the pachytene stage is preserved through spermatid development until late spermiogenesis.     

A simple and objective approach to identifying human round spermatids

Although round spermatids have been studied extensively using staining techniques and electron microscopy, little information is available about their appearance in living conditions. We describe a method of collecting and identifying round spermatids from ejaculates and testicular biopsies. The validity of the selection procedure was confirmed by fluorescence in-situ hybridization. Based on cell size, morphological characteristics of nucleus and cytoplasm, and on the nucleus/cytoplasm ratio, we harvested a population of cells that was 84% haploid. This procedure can be applied to select spermatids for clinical or research purposes.     

A prospective study of multiple needle biopsies versus a single open biopsy for testicular sperm extraction in men with non-obstructive azoospermia

To identify the predictive factors for testicular sperm extraction (TESE) and to understand the pathology associated with TESE, we carried out a prospective study in 40 consecutive men with azoospermia due to primary gonadal failure. The main outcome measure was the retrieval of at least one testicular spermatozoon. Endocrine and biophysical profiles, testicular histology, Johnsen score and testicular spermatids were used as predictors of sperm extraction. Spermatogenesis was quantified with the Johnsen score. A variable pattern of spermatogenesis was common, being present in 20 (50%) patients. Visualisation of testicular spermatids on testicular histology showed a strong association with TESE (P < 0.0001). Statistically significant differences were detected in plasma follicle stimulating hormone (FSH) and testicular volume between patients who had hypospermatogenesis and Sertoli cell-only or maturation arrest. There were no significant differences in Johnsen score, biophysical and endocrine profiles between the groups with successful and failed TESE. However, a statistically significant trend occurred with changes in histological pattern [chi2 for trend, P = 0.001; Pearson's coefficient (r) = 0.6], Johnsen score (P = 0.022; r = 0.5), testicular volume (P = 0.01; r = 0.5) and plasma FSH concentrations (P = 0.044; r = 0.4), albeit to a limited degree. Difference in the interpretation of histological patterns with different assessors was observed. The type of occupation or risk factors for azoospermia showed no association with testicular pathology or TESE. Variable histological patterns in different tubules in the same individual may explain the poor correlation of TESE with endocrine and biophysical profiles, Johnsen score and histological pattern. Differences in the amount of tissue used for TESE and histopathology, and misinterpretation of testicular histology rather than failure to quantify spermatogenesis may explain the poor correlation between histological patterns and TESE. Testicular spermatids predicted TESE. However, considerable overlap in values means that no single variable can provide a perfect discrimination between the groups with successful and failed TESE.     

Reinitiation of spermatogonial mitotic differentiation in inactive old BDF1 mouse seminiferous tubules transplanted to W/Wv mouse testis

The seminiferous epithelia of old mice (33 mo of age) are composed of spermatogonia and Sertoli cells. Histochemical examination using the anti-c-kit monoclonal antibody demonstrated that the number of differentiating type A spermatogonia decreases with age. To elucidate the differential activity of old mouse spermatogonia, we transplanted extremely thin seminiferous epithelia of old BDF, mice into W/Wv mouse testes and examined whether or not they could reinitiate differentiation. Artificially cryptorchid mice were used as the control. At 2 wk after transplantation, spermatocytes and round spermatids were detected in transplanted seminiferous tubules of the control, whereas the most advanced spermatogenic cells in those of old mice were spermatocytes. At 4 wk after transplantation, although elongated spermatids were detected in transplanted tubules of the control, haploid cells (spermatids) were still undetectable in those derived from old mice. Thus, meiosis was never restored, although spermatogonia of old mice can reinitiate differentiation into spermatocytes under suitable testicular conditions. Since it has been reported in several mammalian species that age-related changes in the testicular microenvironment lead to the gerontal cessation of spermatogenesis, the present results suggest that both a defective extratubular environment and a defective intratubular environment may cause the cessation of spermatogenesis in old BDF, mice.     

Characterization of domains in mice of calnexin-t, a putative molecular chaperone required in sperm fertility, with use of glutathione S-transferase-fusion proteins

Calnexin-t (calmegin) is a male germ cell-specific variant of calnexin, a membrane bound-molecular chaperone in the endoplasmic reticulum (ER). Although it is temporally expressed during spermatogenesis, it has recently been shown to be highly involved in sperm fertility. To investigate the biochemical states of calnexin-t during spermatogenesis, we produced a series of glutathione S-transferase-fusion proteins with several specific coding domains of calnexin-t. Immunostaining and 45Ca2+ overlay assays clearly showed that the internal proline-rich repeat region has Ca2+-binding ability and contains an epitope recognized by monoclonal antibody 1C9. Western blot analysis of protein extracts from the testes of 10-, 18-, 26-, and 60-day-old mice revealed only a single 101-kDa protein during testicular development by 1C9. Anti-C, a cytoplasmic domain-specific antibody generated by immunization with recombinant protein, produced the same results, indicating that the 101-kDa form of calnexin-t is prevalent at all stages of spermatogenesis expressing calnexin-t. In paraffin sections of mouse testis, Anti-C stained spermatocytes and spermatids intensely, whereas 1C9 stained spermatocytes only slightly but spermatids intensely, suggesting that the affinity of 1C9 for its epitope is lower in pachytene spermatocytes than in spermatids. Acid phosphatase treatment of the 101-kDa form generated a 93-kDa band that in turn could be recovered to the 101-kDa form by incubation with HeLa cell S100 fraction, indicating that the 101-kDa form is a phosphorylated type of calnexin-t. The sites of phosphorylation were shown to be restricted to the cytoplasmic domain. Our results suggest that the structure of the ER luminal domain of calnexin-t is likely to differ in middle pachytene versus haploid germ cell phases. In addition, the cytoplasmic domain of calnexin-t was shown to be highly phosphorylated immediately after protein synthesis and constitutively phosphorylated during spermatogenesis.     

Differences in ethidium bromide and 4'-6-diamidino-2-phenylindole staining profiles with regard to DNA fragmentation during apoptosis

To simply and directly evaluate DNA fragmentation during apoptosis induced in mouse cultured hepatocytes by an anti-Fas antibody, we examined the fluorescence intensity in cell nuclei stained with ethidium bromide and 4'-6-diamidino-2-phenylindole by optiphoto fluorescence microscopy. The intensity of the former staining for the nuclear DNA of apoptotic cells was clearly decreased compared to that of non-apoptotic cells, whereas no difference in the fluorescence intensity for the latter stain between the apoptotic and non-apoptotic groups was observed. Thus, the use of optiphoto fluorescence microscopy, in conjunction with both stains, constitutes a useful tool for the evaluation of apoptotic DNA fragmentation.     

Assessment of testicular cytology by fine-needle aspiration and the imprint technique: are they reliable diagnostic modalities?

OBJECTIVE: To investigate whether testicular cytology may be considered diagnostic in the evaluation of infertile men. PATIENTS AND METHODS: Specimens of testicular tissue obtained either surgically (imprint smear) or through fine-needle aspiration (FNA) were used as a source of cytological smears; 58 testes from 24 men with azoospermia or severe oligospermia and from five men with advanced prostate cancer were evaluated cytologically and compared with the histological diagnosis. RESULTS: FNA caused no apparent trauma. The results from FNA smears generally agreed with the histological findings but four patients with no spermatozoa in the FNA smears were diagnosed histologically as hypospermatogenic and two others judged histologically as having Sertoli-cell-only (SCO) syndrome and spermatogenic arrest had detectable spermatozoa in their FNA smears. There was complete agreement between the results of imprint smears and histological findings in those patients with SCO syndrome and spermatogenic arrest. There were no evident differences in sperm counts between hypospermatogenesis and normal spermatogenesis on the imprint slides, but FNA smears detected this difference. CONCLUSION: FNA of the testis is a relatively non-invasive and reproducible technique for evaluating qualitative and quantitative cytology. However, it is insufficient for diagnosing some testicular pathologies. Imprint smears supplement the histological diagnosis, especially if the histological slides are stained unsatisfactorily.     

A comparison between open and percutaneous needle biopsies in men with azoospermia

Open testicular biopsy is a classic method of investigation in men with azoospermia. Recently, percutaneous needle biopsy of the testis has been used in attempts to obtain material for histopathological diagnosis in such cases and to retrieve spermatozoa for intracytoplasmic sperm injection (ICSI). To determine whether a 19 gauge (G) and a 21G butterfly needle could be used for percutaneous aspiration of testicular tissue to determine the presence of mature spermatids and assess spermatogenesis, 10 patients (16 testes) and 12 patients (17 testes) underwent 19G or 21G needle biopsy respectively, immediately followed by open testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with open biopsy. With the 19G needle, in the 14 cases where material was obtained there was full agreement with open biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13 biopsies yielding material were predictive in this respect. Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. But for a detailed histopathological diagnosis, however, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable.     

Expression of Escherichia coli TehA gives resistance to antiseptics and disinfectants similar to that conferred by multidrug resistance efflux pumps

The genes tehAB located at 32.3 min on the Escherichia coli chromosome were initially identified by their ability to mediate resistance to potassium tellurite (128 micrograms of K2TeO3 per ml) when overexpressed with a high-copy-number plasmid. The genes encode an integral membrane protein (TehA) of 36 kDa with 10 putative transmembrane segments and a second protein (TehB) of 23 kDa. Overexpression of TehAB results in hypersensitivity to dequalinium CI and methyl viologen (paraquat). Expression of TehA alone gives similar hypersensitivity. Overexpression of TehA gave resistance to tetraphenylarsonium CI, ethidium bromide, crystal violet and proflavin. The efflux of ethidium, measured by fluorescence quenching, revealed that TehA transported ethidium at twice the control rate and 10% of the rate of the highly resistant efflux transporter Emr Eco. Addition of tellurite had no effect on ethidium transport. In addition to the ethidium transport assay, a proflavin fluorescence assay which was approximately 200-fold more sensitive was also used. TehA was also found to have proflavin efflux activity. The addition of TeO32- to the proflavin transport assay on TehA caused a 20% increase in transport rate. Both ethidium and proflavin transport were found to be energy dependent.     

稀有金属甲氨喋呤配合物与小牛胸腺DNA相互作用的研究

用溴化乙锭为探针研究了稀有金属甲氨喋呤(methotrexate MTX)配合物与小牛胸腺DNA的作用机制，探讨了其作用模式，即甲氨喋呤与DNA是非嵌插结合，稀有金属甲氨喋呤配合物与DNA之间的作用为静电方式和一定的嵌入方式。并求得稀有金属甲氨喋呤配合物与DNA的结合常数。     

Assessment of testicular function in experimental varicocele rats by phosphorus-31 magnetic resonance spectroscopy

To assess testicular function in experimental varicocele rats, we used 31P magnetic resonance (MR) spectroscopy and compared MR spectroscopic parameters with flow cytometric DNA analysis. In vivo 31P MR spectroscopy and flow cytometric DNA analysis of testes were performed in 10 sham-operated and 9 induced varicocele rats. Although the testicular phosphomonoester (PM)/ATP ratio did not differ between sham-operated and induced varicocele rats, the phosphodiester (PD)/ATP ratio was significantly reduced and the inorganic phosphate (Pi)/ATP ratio was significantly increased in induced varicocele rats. There was no difference between the right and left sides. DNA flow cytometry showed a decrease in the percentage of haploid cells in induced varicocele rats, regardless of the side. This study indicates that in vivo 31P MR spectroscopy provides valuable information for assessment of testicular function.     

Use of immature germ cells for the treatment of male infertility

Both animal experimentation data and preliminary clinical experience converge to suggest that normal progeny can be obtained by fertilizing oocytes with spermatids, the youngest male germ cells to have a set of haploid chromosomes. Spermatids can be obtained from the ejaculate of many patients with non-obstructive azoospermia. The use of ejaculated spermatids in the treatment of non-obstructive azoospermia is thus to be considered as an alternative to that of testicular spermatozoa. Fertilization with ejaculated spermatids makes it possible to avoid the potential adverse consequences of extensive testicular biopsy and may thus become the treatment of first choice. The recourse to testicular spermatids represents a treatment of last chance if no spermatids can be recovered either from the ejaculate and no spermatozoa from the testis.     

Clinical aspects associated with Sertoli-cell-only histology

OBJECTIVE: To assess the clinical features found in infertile men in whom the histological diagnosis of Sertoli-cell-only (SCO) was made on testicular biopsy. PATIENTS AND METHODS: A retrospective review was carried out of the seminal fluid analysis, testis size and follicle-stimulating hormone (FSH) levels of 72 men who had bilateral testicular biopsies due to infertility when one (30) or both (42) of bilateral testicular biopsies showed tubules containing only Sertoli cells. In a subgroup of 15 men, the biopsies were re-examined to correlate the morphological features with the plasma FSH level. RESULTS: When both biopsies showed bilateral SCO the patient had azoospermia (86%) or oligozoospermia (14%); the testicular size was normal in 36% and the FSH level was normal (43%), raised (21%) or grossly elevated (more than twice normal, 36%). When one biopsy showed SCO, the opposite testis showed appearances which varied from grossly impaired spermatogenesis to almost normal spermatogenesis. The clinical findings were also very variable. CONCLUSIONS: The clinical features associated with the histological diagnosis of SCO are extremely variable. Biopsy evidence of bilateral SCO cannot be relied upon to indicate a total absence of spermatogenesis in the testes.     

Direct physical measurements on substituted agarose gels: evidence for intercalation of gel-bound ethidium into transfer RNA

The cation of the salt ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) has been covalently linked to an agarose matrix through an intermediate 3,3'-diaminodipropylaminosuccinyl spacer arm. Partition binding and visible absorption spectral measurements on the gel were used to monitor the binding of transfer RNA to the covalently bound ethidium group. Direct fluorescence measurements of the formation of the gel-bound complex indicate that this binding involves the intercalation of the ethidium groups into the tRNA molecule. Dissociation of the ethidium-tRNA complex was monitored as a function of sodium chloride concentration by both direct solution spectral measurement of the released tRNA and by fluorescence quenching measurements of the dissociation of the intercalation complex. The derivatized gel has been shown to be capable of the fractionation of tRNA species by elution with a positive salt gradient under column flow conditions.     

Stereological evaluation of human spermatogenesis after suppression by testosterone treatment: heterogeneous pattern of spermatogenic impairment

Testosterone (T) treatment suppresses gonadotropin levels in normal men and is a promising reversible contraceptive that induces azoospermia in approximately 70% of subjects and oligospermia in the remainder; however, the basis of this variable response is unclear. This study aimed to investigate this reported variable response by examining the spermatogenic process and quantitating germ cell number in men after T-induced gonadotropin withdrawal. Ten normal fertile men (31-46 yr), already planning to undergo vasectomy, either received T enanthate (200 mg, i.m., weekly) for 19-24 weeks (n = 5; TE group) or proceeded directly to surgery (n = 5; controls), at which time a unilateral testicular biopsy was taken, and germ cell numbers were estimated using the optical disector stereological method. In response to TE treatment, serum T levels rose 2-fold, and FSH/LH levels became undetectable. Sperm counts fell to azoospermia in 4 men and to 21 million/mL in the fifth man. The mean number of type A spermatogonia per 100 Sertoli cells was unchanged, but type B spermatogonia fell markedly to 10% of the control values, and later germ cell types decreased to 11-18% of the control values. The pattern of germ cell suppression varied widely and showed no relationship with sperm count or the time to azoospermia. Despite the presence of elongated spermatids (1.4-20% of the control), four men remained azoospermic. Two TE subjects with similar early germ cell complements and elongated spermatid numbers had sperm counts of zero and 21 million/mL; the latter man demonstrated marked variability in germ cell numbers between adjacent tubules. We conclude that 1) the principal spermatogenic lesion in TE-treated men is the marked (90%) inhibition of type A-->B spermatogonial maturation. Other sites are also affected, particularly the release and/or survival of elongated spermatids during transit; and 2) a steady state in germ cell number may not be established even after 4-5 months of TE treatment. The findings suggest that TE treatment does not adequately or consistently withdraw hormonal support for spermatogenesis, leading to variable between- and within-individual patterns of germ cell suppression.     

Serum inhibin B as a marker of spermatogenesis

Inhibin B is produced by Sertoli cells, provides negative feedback on FSH secretion, and may prove to be an important marker for the functioning of seminiferous tubules. The purpose of the present study was to examine the relationship between the spermatogenic function of the testis of subfertile men and the plasma concentrations of inhibin B and FSH. These parameters were estimated in a group of 218 subfertile men. Serum inhibin B levels were closely correlated with the serum FSH levels (r = -0.78, P < 0.001), confirming the role of inhibin B as feedback signal for FSH production. The spermatogenic function of the testis was evaluated by determining testicular volume and total sperm count. Inhibin B levels were significantly correlated with the total sperm count and testicular volume (r = 0.54 and r = 0.63, respectively; P < 0.001). Testicular biopsies were obtained in 22 of these men. Inhibin B was significantly correlated with the biopsy score (r = 0.76, P < 0.001). Receiver operating characteristic analysis revealed a diagnostic accuracy of 95% for differentiating competent from impaired spermatogenesis for inhibin B, whereas for FSH, a value of 80% was found. We conclude that inhibin B is the best available endocrine marker of spermatogenesis in subfertile men.     

The effect of mercuric ions on the excited states of DNA-intercalated ethidium bromide

The first excited triplet state of DNA-intercalated ethidium bromide is produced with a quantum yield of 0.01 +/- 0.002 on irradiation at 532 nm. A difference extinction coefficient of 1.5 +/- 0.2 x 10(3) m2 mol-1 is measured for the triplet state at 380 nm. Mercuric ions quench the first excited singlet state of DNA-intercalated ethidium bromide via induced spin orbit coupling to give an increased yield of ethidium triplet states. The same mercuric ion that quenches the singlet state then quenches the triplet state, via the same mechanism, with a rate constant of ca 3.5 x 10(3) s-1. An upper limit for the rate of detachment of Hg2+ from its binding site in DNA may be fixed at ca 10(3) s-1.