Lectin binding sites in human seminiferous epithelium, in CIS cells and in seminomas

The distribution of glycoconjugates in germ cells during spermatogenic differentiation, in carcinoma-in-situ (CIS) cells and in seminoma were studied by lectin histochemistry. The results show that human germ cells are rich in carbohydrate-containing compounds with specific alterations in the expression of glucosyl moieties during germ cell development. CIS cells reveal different lectin binding sites from spermatogenic cells, but the distribution of glycosubstances in CIS cells is similar to that of seminoma cells which supports the suggestion of the malignant nature of CIS germ cells.     

Recognising the Sertoli-cell-only (SCO) syndrome: a case study

Total Sertoli-cell-only (SCO) syndrome is often confused with a focal SCO picture, in which testicular illness caused damage to seminiferous tubules and compromised the Sertoli cell range of maturation and functions, but from which still some spermatozoa can be retrieved for assisted reproductive techniques. Here, a possibly new SCO syndrome phenotype is reported exhibiting complete lack of germ cells despite normal architecture of the seminiferous tubules with presence of mature Sertoli cells and normal Leydig cells in the intertubular tissue. Sertoli cells are immunonegative for the prepubertal differentiation markers cytokeratin-18, anti-Muellerian hormone and M2A antigen, but reveal a positive signal for the gap junctional protein connexin 43 known to be expressed in Sertoli cells with an adult type of differentiation. The complete lack of germ cells in combination with fully differentiated adult-type Sertoli cells in this case is in contradiction with known SCO subtypes and with the current hypothesis of reciprocal regulation of Sertoli and germ cell differentiation.     

Lectin-Binding Sites in Testis of Men with Acrosomeless Round-Headed Spermatozoa/Testikuläre Lektinbindungsstellen bei Männern mit akrosomenlosen Rundkopfspermatozoen

We report herein about lectin histochemistry of seminiferous epithelia in two infertile men with exlusely acrosomeless round-headed spermatozoa. FITC-conjugated lectins (ConA, PNA, RCA II, WGA) have been employed on tissue sections of Bouin fixed testicular biopsies. RCA II gave a dot-like fluorescence of the acrosomal region and WGA gave a cap-like acrosomal fluorescence of spermatids. PNA-a marker of acrosomal differentiation-failed to stain spermatids. The binding of ConA to germ cells was not influenced by this syndrome. In conclusion, the syndrome of acrosomeless round-headed spermatozoa is associated with selective perturbations of testicular lectin-binding sites. They might contribute of the inability of sperm cells to adhere to and penetrate into ova.     

Lectin-Binding Sites in Normal Human Testis/Lektinbindungsstellen normaler humaner Hoden

Nine fluorescein isothiocyanate (FITC)-labelled lectins have been used to investigate the distribution of glycoconjugates in unfixed frozen and Bouin-fixed sections of normal human testis. Interstitial cells and lamina propria of seminiferous tubuli were stained by PNA, HPA, RCA II, SBA, ConA, and WGA indicating an abundance of the following glycoconjugates: N-GlcNAc, N-GalNAc, Gal, and Man. The germinative cells were stained cytoplasmatically by ConA (Alpha-D-Man/-Glc). Sertoli cells showed the same pattern with ConA. Early spermatids fixed PNA and RCA II in the acrosomal region. Elongated spermatids fixed WGA on their acrosomes and fainty on the flagellae too indicating abundance of N-GlcNAc residues. The findings argue for differentiation-related modifications of lectin-binding sites on germinative cells and the usefulness of Bouin-fixed samples for lectin histochemistry.     

Differentiation of murine male germ cells to spermatozoa in a soft agar culture system

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system (SACS), which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS). The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa, Dazl, OCT-4, C-Kit, GFR-α-1, CD9 and α-6-integrin), meiotic cells (LDH, Crem-1 and Boule) and post-meiotic cells (Protamine-1, Acrosin and SP-10). Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.     

Major differences between the testis and epididymis in the induction of granulomas in response to extravasated germ cells. I. A light microscopical study in mice

A spermatic granuloma is a chronic inflammatory lesion which surrounds extravasated spermatozoa. Clinically, the lesion develops in the interstitial spaces of the epididymis and vas deferens, and only exceptionally in the testis itself. In the present study, murine testes and epididymides were injured using a needle and the histological appearances of these organs was then compared. Traumatic injury induced extravasation of germ cells in both testes and epididymides. A few days later, spermatic granulomas consistently formed in the epididymides, however, such lesions were not induced in the testes. To examine the possibility that epididymal spermatozoa have inherently greater ability to form spermatic granulomas than do testicular germ cells, isolated epididymal spermatozoa or testicular germ cells were locally injected into the testes and epididymides of recipient mice. Spermatic granulomas readily formed in the epididymides after local injection of either epididymal spermatozoa or testicular germ cells. In contrast, such lesions did not form in the testes even when epididymal spermatozoa were injected. Therefore, this study suggests that the microenvironment of the testicular interstitium, rather than the extravasated components from the ruptured seminiferous tubules, is the main factor determining the limited formation of spermatic granulomas in the testis.     

Quantification of seminal germ cells in azoospermia: correlations with testicular histology and TESE outcome

In the treatment of male infertility by intra-cytoplasmic injection of spermatozoa (ICSI) extracted from testicular tissue (TESE), the high incidence of negative TESE outcome calls for non-invasive prognostic methods. Literature suggests that seminal haploid germ cell detection could be one. For this purpose, a multi-parametric stringent flow cytometric method was applied to 50 TESE patients for the quantification of ejaculated germ cells. Cells from 50 ejaculates were identified and quantified as spermatozoa (HC, highly condensed), round spermatids (1N), primary spermatocytes (SPC) (4N) or diploid cells (2N, including somatic and non-testicular cells) by their DNA and mitochondria staining and laser scatter characteristics, and compared with testicular biopsy histology and TESE outcome. Whereas 96% of patients displayed a diploid peak in the distribution histograms, the HC, 1N and 4N peaks were absent from the majority of samples. In 13 ejaculates, either a HC or 1N or 4N peak, or a combination of these, was discernible. Although seminal germ cell numbers bore no overall association with elongated spermatids (ES) in histology or spermatozoa retrieval in TESE outcome, 4N cells per ejaculate were correlated with the percentage of tubule sections showing SPC as the most advanced germ cells. The incidence of HC peaks was higher in patients showing some ES in histology or sperm retrieval than in the sperm-negative groups. In groups with suspected obstruction showing nearly full spermatogenesis and maximal sperm retrieval, there was no incidence of a HC peak. Germ cell peaks were associated with germ cell degeneration noted in testicular histology. In conclusion, seminal germ cells cannot provide good prognosis for TESE, although their presence could indicate the spermatogenic activity in the testis.     

Immunomorphological aspects of the tubuli recti and the surrounding interstitium in normal mice

The tubuli recti (TR) are immunologically special, because the lymphocytes preferably accumulate around them during the course of T-cell dependent testicular autoimmunity in mice. This finding implies that the testicular interstitium around the TR is where autoreactive lymphocytes can gain access to autoimmunogenic germ cell antigens. In the present study, the histoarchitecture of the TR was minutely examined in normal mice. Three-dimensional analysis showed that 14-16 TR appeared to be connected to the rete testis (RT). Electron microscopical analysis revealed that the epithelial cells in the TR formed protruding cytoplasmic strings, with active endocytosis of degenerated spermatozoa, and exhibited three different morphological characteristics. Furthermore, a few macrophages were found to have penetrated into the TR. Immunohistochemical image analysis revealed that more macrophages specifically accumulated around the TR than the RT or seminiferous tubules. These findings indicate that macrophages preferentially accumulate around the TR, where contact between germ cell antigens and macrophages may occur under normal and pathological conditions.     

A germ cell-specific nuclear antigen recognized by a monoclonal antibody raised against mouse testicular germ cells

A monoclonal antibody (mAb TRA104) raised against mouse testicular germ cells was able to recognize the nuclei of testicular germ cells at all the stages of differentiation from embryonic gonocytes to spermatids and did not react with any somatic cells. The antigen recognized by mAb TRA 104 was exclusively present in testicular extracts. The molecular weights and isoelectric point (pI) of the antigens determined by Western blotting analysis were 60–110 kDa and 7.2, respectively. This antigen(s) is referred to as a germ cell-specific nuclear antigen(s) (GENA) since GENA was first detected specifically in the genital ridge at around 12 days of gestation by Western blotting analysis. In the testis, the expression increased gradually until adulthood whereas it was lost in the ovary by postpartum day 5. Thus, GENA is a molecule(s) exclusively present in the nuclei of germ cells and may be a useful marker with which to study the mechanism of germ cell development and differentiation at the molecular level.     

Identification and characterization of an antigen recognized by monoclonal antibody TRA 54 in mouse epididymis and vas deferens

Spermatozoa in testicular fluid are known to have weak forward motility and cannot fertilize eggs. The epididymis is known to participate in sperm maturation leading fertilization, but little is known about the specific epididymal molecules involved in the modification of sperm. In this study, we characterized the new pattern of expression of an antigen previously identified in testicular germ cells by monoclonal antibody (mAb) TRA 54. This antigen is expressed in epididymal and vas deferens epithelial cells in mice older than 24 days but not during younger developmental stages. Evaluation by immunohistochemistry shows that antigen expression is limited to the cytoplasm of a specific cell population of epithelia along the epididymal regions and vas deferens of adult mice. The molecules synthesized and released by epididymal and vas deferens epithelia into their lumen seem to bind on spermatozoa moving down through the ducts. Immunoblot analysis showed that the molecules recognized by mAb TRA 54 in testis and epididymis were similar and share a common epitope involving carbohydrate domains. Interestingly, the antigens identified in epididymal and vas deferens epithelial cells were expressed independently of testicular germ cells and are produced in an androgen-dependent manner. Finally, the molecules recognized by mAb TRA 54 seem to play an important role in spermatogenesis, as well as in epididymal function related to spermatozoa maturation and ability to fertilize.     

Distribution of glycoconjugates in normal rat ventral prostate and their use as markers of androgen-controlled secretory function in culture

The distribution of glycoconjugates in uncultured and cultured rat ventral prostate was studied by using eight fluorescent lectins. The prostate pieces were cultured in defined medium with or without testosterone for 1-14 days. Each lectin revealed a characteristic binding pattern. Con A, LCA, WGA, and RCA I stained both epithelial and interstitial components. SBA and PNA were specific for the epithelium: SBA stained the region of the Golgi complex; PNA showed the brightest fluorescence in the apical part of the cells representing the region of secretory granules. In culture without testosterone the epithelial cells gradually lost their fluorescence, whereas the stromal fluorescence increased. The basement membrane was disorganized. With testosterone the integrity of the epithelium and stroma was maintained, and the staining pattern of the lectins was in the main similar as in vivo. However, at 14 days a change in the staining pattern of apical cytoplasm with PNA was noted, indicating that in long-term cultures, in addition to testosterone, other hormones and growth factors are necessary to complete especially the last stages of the secretory process in the epithelial cells.     

Lectin Histochemical Investigations of the Distal Gut of Chicks with Special Emphasis on the Follicle‐associated Epithelium

Carbohydrates on epithelial cell surfaces play an important role as attachment sites for different microorganisms like bacteria, viruses and protozoa. To obtain more information about the distribution of carbohydrates on the luminal surface along the intestine, lectin histochemical studies on different gut segments of chicks of different age groups were carried out using a panel of 13 lectins with specificities for Man, Glc, Gal, GalNAc, GlcNAc or GlcNAc oligosaccharides and Sia. Furthermore, we tried to find out whether previously reported specificities of certain lectins for M cells (membranous or multifold cells) in the bursa of Fabricius (BF) can be observed also on M cells of the intestine. As a result we were able to demonstrate binding of all lectins employed in these studies in all investigated gut segments. In some cases, the application of the same lectin led to varying staining intensities of the same histological structures in different age‐groups (e.g. staining of the brush border with WGA, LEA, MAA or Conarva) or different gut segments (e.g. staining of goblet cells with CMA II, LEA and MPA). Hence, terminal carbohydrate residues of glycoconjugates on the intestinal epithelium vary depending on age and organ site. As glycoconjugates can act as attachment sites for microorganisms, these differences in the distribution of sugar residues may be one explanation for the site‐specificity of certain pathogens. Furthermore, the binding of lectins to the follicle‐associated epithelium (FAE) of the BF differs from that to the FAE of the intestine again stressing the site specificity of lectin binding. Thus, up to now no universal M‐cell marker along the chicken intestine exists.     

Bilateral prepubertal testicular biopsies predict significance of cryptorchidism-associated mixed testicular atrophy, and allow assessment of fertility

INTRODUCTION: Mixed atrophy of the testis (MAT), a frequent finding in biopsies of formerly cryptorchid and/or infertile patients, is defined as the synchronous occurrence of both seminiferous tubules containing germ cells and Sertoli cell only-tubules in variable proportions. In tubules containing germ cells, different types of abnormalities in spermatogenesis may be seen. The presence of adult spermatids in the biopsy, even in small numbers, correlates with successful spermatozoa retrieval for "in vitro" fertilization techniques. Currently, it is unknown whether precursor lesions of MAT can be identified in cryptorchid patients during childhood. MATERIAL AND METHODS: Eighteen formerly cryptorchid adults who had undergone testicular biopsies in childhood had a repeat testicular biopsy to evaluate infertility. In prepubertal biopsies, abnormalities of the testicular parenchyma were classified into types I (slight alterations), II (marked germinal hypoplasia), and III (severe germinal hypoplasia). In postpubertal biopsies, the percentage of tubules containing germ cells and Sertoli cell only-tubules were estimated, as well as the presence of complete spermatogenesis. Abnormalities in spermatogenesis were classified into lesions of the adluminal or basal compartments of seminiferous tubules. RESULTS: Comparison between prepubertal and postpubertal biopsies revealed that most specimens developing from type III lesions presented with incomplete spermatogenesis (P<0.0001) and more severe lesions of the germinal epithelium (P=0.049). DISCUSSION: Type III lesions correlated with MAT characteristics that confer a worse prognosis for in vitro fertilization. Thus, MAT characteristics may be predicted in prepubertal cryptorchid patients, allowing a fertility prognosis. The pathogenesis of these lesions, and their possible inclusion into the spectrum of the testicular dysgenesis syndrome, are discussed.     

Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.     

Lectin affinity of the seminiferous epithelium in healthy and cryptorchid post-pubertal boars

The present study describes the sugar content of the seminiferous epithelium, using lectin histochemistry, in healthy boars and in boars with unilateral and bilateral abdominal cryptorchidism. In healthy boars the apical cytoplasm of Sertoli cells exhibited abundant glucosyl (Con A and WGA lectins), galactosyl (HPA, DBA, SBA and PNA lectins), and fucosyl (AAA lectin) residues. Spermatogonia and spermatocytes contained abundant glucosyl (Con A and WGA lectins) and fucosyl (AAA lectin) residues. In spermatids, galactosyl (SBA and PNA lectins) and glucosyl (Con A and WGA lectins) residues increased progressively throughout spermiogenesis, and fucosyl (AAA lectin) residues decreased. As compared with healthy boars, the scrotal testis of unilateral cryptorchid boars showed decreased amounts of fucosyl (AAA lectin) and galactosyl (HPA and DBA lectins) residues on the Sertoli cell apical cytoplasm; spermatocytes exhibited higher content of glucosyl (Con A lectin) residues and spermatids showed altered nature of glucosyl (Con A and WGA lectins) and galactosyl (SBA and PNA lectins) complexes. In abdominal testes of unilateral and bilateral cryptorchid boars, immature Sertoli cells and spermatogonia showed decreased fucosyl (AAA lectin), and increased glucosyl (Con A and WGA lectins) and galactosyl (SBA and PNA lectins) contents. These results suggest that the seminiferous epithelium of healthy boars has polarized activity with the apical compartment implicated in germ cell-Sertoli cell adhesion and interaction, in transport of ions, substrates and fluids, and in acrosomal differentiation. In scrotal testes, unilateral abdominal cryptorchidism could lead to defective germ cell-Sertoli cell adhesion, impaired acrosomal differentiation and increased ionic transport in the apical compartment of the seminiferous epithelium. Unilateral and bilateral cryptorchidism could induce increased ionic transport and membrane permeability in the seminiferous epithelium of abdominal testes.     

Isoforms of angiotensin I-converting enzyme in the development and differentiation of human testis and epididymis

Angiotensin I-converting enzyme (ACE; CD143, Kininase II, EC 3.4.15.1) is known to be crucial for male fertility in animal models. We therefore studied its testicular (tACE) and somatic (sACE) isoforms in foetal and adult human testis and epididymis using monoclonal antibodies and cRNA probes. During spermatogenesis, tACE was found only in differentiating germ cells and was the only isoform within the seminiferous tubules of adult men. Although tACE mRNA was present in spermatocytes, tACE protein was initially found in post-meiotic step 3 spermatids and increased markedly during further differentiation. The enzyme was strictly confined to the adluminal membrane site of elongating spermatids and was localized at the neck and midpiece region of released and ejaculated spermatozoa. In contrast, sACE was expressed heterogeneously in Leydig cells and endothelial cells of the testicular interstitium, and homogeneously along the luminal surface of epithelial cells lining the ductuli efferents, corpus and cauda of epididymis, and vas deferens. The cell- and site-restricted pattern of sACE corresponded to that found in foetal tissues except an additional and transient expression of sACE in foetal germ cells and foetal Sertoli cells. Our study documents for the first time in humans the regulation and unique cellular distribution of ACE isoforms during the ontogenesis of the lower male genital tract.     

Osteotesticular protein tyrosine phosphatase expression in rodent testis

PURPOSE: In the last decade the novel receptor-like protein tyrosine phosphatase was identified and termed osteotesticular tyrosine phosphatase (OST-PTP) due to its restricted expression in bone and testis. OST-PTP expression is regulated during osteoblast differentiation and it shows stage specific expression in the testis. Confined OST-PTP expression in the basal compartment of the seminiferous tubules suggests that this protein may be a good candidate for a rodent germ stem cell marker. To test this hypothesis we determined the exact pattern of OST-PTP expression in the rodent testis. MATERIALS AND METHODS: Adult mouse and rat paraffinized testis sections were subjected to in situ hybridization using riboprobes against the receptor and catalytic domains of the protein OST-PTP. RESULTS: OST-PTP testicular expression in rodents is not confined to the spermatogonia, as previously suggested, but is also present in Sertoli cells in a stage independent pattern. This finding excludes the hypothesis that OST-PTP is a germ stem cell identification marker in rodents. In addition, we report the identification of a testicular OST-PTP isoform lacking part of a catalytic domain that is widely expressed during spermatogenesis in all cell types. CONCLUSIONS: This finding implies tight control over OST-PTP expression in the testis, which in turn suggests an important role for OST-PTP and its isoform in male germ cell differentiation.     

Identification of differentiation antigens in mouse testicular germ cells recognized by monoclonal antibody TRA 55

We have isolated a monoclonal antibody (mAb) TRA 55, which recognizes mouse testicular germ cells from mid-pachytene spermatocytes to the early stages of haploid spermatids during differentiation. Immunohistochemical analysis produced strong positive staining of the nuclei and faint staining in the cytoplasm of germ cells. At meiotic division, when the nuclear membrane disappeared, a specific positive signal could be observed on metaphase chromosomes. When germ cells produced haploid spermatids, antigenicity became suddenly weak and soon disappeared. TRA 55 did not react with testicular somatic cells, such as Sertoli cells or Leydig cells. Western blot analysis of the whole testis showed four positive bands with molecular weights of 43, 46, 49 and 55 kDa. Three bands of 43, 49 and 55 kDa, and a single band of 46 kDa were recovered in cytoplasmic and nuclear fractions of testicular germ cells, respectively. Chronological changes in the Western blot pattern indicated that these antigens became detectable in the testis at the age of 10 days. Furthermore, all antigens were resistant to periodate treatment, suggesting that the epitope was in an amino acid rather than a sugar moiety. These antigen molecules may play important roles in the differentiation of germ cells at the later stages of meiotic prophase and meiotic division in the mouse testis.     

新生小鼠睾丸组织和作为异种移植物的人类胎儿睾丸组织在免疫缺陷小鼠中的发育情况(英文)

目的:采用同种和异种睾丸组织移植的方法,研究新生小鼠睾丸组织及人类未成熟睾丸组织异种移植物在免疫缺陷小鼠体内发育不同时期生精细胞的组成和基因表达情况中生精细胞的发育情况。方法:以免疫缺陷小鼠为受体,新生小鼠睾丸组织和人类未成熟睾丸组织为供体,分别进行同种和异种移植。通过对移植物的组织形态学观察和分子生物学检测,对各个时期同种移植物中的生精细胞组成及其特异性基因的表达情况进行评估并与末受损小鼠的情况相比较;对人睾丸组织异种移植物的存活及其生精细胞在异体异位的发育情况进行探讨。结果:新生小鼠睾丸组织在成年雄性去势免疫缺陷小鼠体内的发育状况在移植开始的一个阶段与在体睾丸组织的发育情况基本相同,各级生精细胞的出现及其基因表达均与在体睾丸组织中相类似,而移植7-8星期后生精小管发生退化现象。人未成熟睾丸组织在受体中存活并且进一步生长;组织学观察还发现,生精细胞的发育速度与在体相比具有加速的倾向。结论:新生小鼠睾丸组织同种移植物的发育与在体情况基本相同,而人类未成熟睾丸组织异种移植物的发育与正常生理状态相比较呈现出加速的倾向。     

Use of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm injection programmes for the treatment of 47,XXY non-mosaic Klinefelter men

We evaluated the role of the sensitive quantitative telomerase assay (SQTA) in the management of men with non-mosaic Klinefelter's syndrome (KS). Diagnostic testicular biopsy (DTB) was performed in 24 men with KS. A part of the DTB was stained and the remaining fragment was processed for the SQTA. After 3-18 months, a therapeutic testicular biopsy (TTB) was performed in the same testicle and the recovered specimens were processed to identify spermatozoa. Men with a SQTA outcome equal to 0.00 Units microg-1 protein (n = 7) demonstrated therapeutic testicular biopsy material that was negative for spermatogenic cells. In five men with a SQTA outcome of 8.11-38.03 Units microg-1, the most advanced germ cell was the spermatogonium/primary spermatocyte. In the remaining 12 men, the most advanced spermatogenic cell in the TTB was the spermatozoon. In these men, the SQTA outcome was equal to 25.76-92.68 Units microg-1 protein. Using 39.00 Units microg-1 protein as a cut-off value, the accuracy of the SQTA in identifying men positive for spermatozoa was 91.6%. It appears that the SQTA has a role for identifying non-mosaic KS men who have testicular spermatozoa.