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.     

Heterotopic transplantation as a model to study the regulation of spermatogenesis; some histomorphological considerations about sperm decline in man

A novel animal model (syngeneic neonatal testicular graft transplanted under the skin of the outer ear in adult inbred Fischer rats that had been castrated, hypophysectomised, and/or subjected to hormonal replacement therapy) was developed to study regulation of spermatogenesis and Sertoli cell number. Given that Sertoli cell number and testicular size are important in determining spermatozoan production rates, this model was first used to study Sertoli cell proliferation, testicular size, and establishment of germ cells. The specific objectives were to determine the developmental pattern of Sertoli cell numbers in transplanted testes and the effect of number of testes transplanted, sex of hosts, pituitary hormonal removal, and replacement on Sertoli cell number, hormonal status of the host, and establishment of germ cells. A few tubules had complete spermatogenesis at 90 days posttransplantation, indicating that Sertoli cells in some of these tubules were functional. Leydig cell structure appeared to be normal, but the density of these interstitial cells was greater than that in testes of intact rats. Although the weight of the seminal vesicles and prostate were maintained in the castrated host with transplants, both serum FSH and LH concentrations were higher than intact control rats. Leukocytic infiltration of testes was not observed in intact rats or in rats receiving neonatal testes. Although transplanted testes showed a delay in reaching the plateau value for Sertoli cell number per testis and although the value reached was lower than in intact testes, the developmental pattern of Sertoli cell proliferation (early division of cells followed by stabilized number of cells) in transplanted testes was similar to that in intact rats. Hypophysectomy reduced the growth of testicular grafts, and hormonal replacement via retransplantation to pituitary intact hosts enhances Sertoli cell proliferation and testicular growth. When two on four testes were transplanted into castrated males or ovariectomized female hosts for 65 days, there was no difference in the graft weights or Sertoli cell numbers between sexes of hosts. Four transplanted testes per rat produced more total testicular parenchyma and a greater number of Sertoli cells per testis than did two testes regardless of sex of the host. Transplantation of six or eight testes produced more total Sertoli cells/host than that found in testes of intact rats. Using hormonal therapy in hypophysectomized hosts, the testicular parenchymal weight was greater for pituitary-intact hosts and FSH-LH combination than the control media. There was no statistically significant difference among the media control, LH, FSH, and GH. This testicular transplant model has shown that the period of Sertoli cell proliferation can be delayed by hypophysectomy, that Sertoli cell number can be influenced by endogenous and exogenous hormones, and that a major component in regulation of testicular size is at the level of the testis. Hence, this model should facilitate study of experimental endocrine manipulation control and potential experimental intervention to increase Sertoli cell number, testicular size, and spermatogenesis. Regarding human sperm count decline in recent years, there appears to be no significant decline in Sertoli cell number or spermatogenic potential in a group of North American men. However, there was a significant decline in volume/man of Leydig cells and volume/man of Leydig cell cytoplasm.     

Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels

To elucidate the role of germ cells in the regulation of inhibin B secretion, serum inhibin B levels in prepubertal boys and adult men whom had a concurrent testicular biopsy showing either normal or impaired testicular function were compared. In addition, by immunohistochemistry the cellular localization of the two subunits of inhibin B (alpha and betaB) were examined in adult testicular tissue with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only tubules (SCO) as well as in normal testicular tissue from an infant and a prepubertal boy. Adult men with testicular biopsy showing normal spermatogenesis (n=8) or spermatogenic arrest (n=5) had median inhibin B levels of 148 pg/mL (range, 37-463 pg/mL) and 68 pg/mL (range, 29-186 pg/mL), respectively, corresponding to normal or near-normal levels of our reference population (165 and 31-443 pg/mL; n=358). Men with SCO (n=9) had undetectable or barely detectable (n=1) serum levels of inhibin B. In contrast to adults, prepubertal boys with SCO (n=12) all had measurable serum inhibin B levels that corresponded to our previously determined normal range in healthy prepubertal boys (n=114). However, in postpubertal samples from the same SCO boys, inhibin B levels were undetectable as in the adult SCO men. Intense inhibin alpha-subunit immunostaining was evident in Sertoli cells in both prepubertal and adult testes. In the prepubertal testis, positive immunostaining for the betaB-subunit was observed in Sertoli cells. In the adult testis, intense immunostaining for the betaB-subunit was evident in germ cells from the pachytene spermatocyte to early spermatid stages and to a lesser degree in Leydig cells, but not in Sertoli cells or other stages of germ cells. Thus, surprisingly, in adult men the two subunits constituting inhibin B were expressed by different cell types. We speculate that during puberty Sertoli cell maturation induces a change in inhibin subunit expression. Thus, immature Sertoli cells express both alpha and betaB inhibin subunits, whereas fully differentiated Sertoli cells only express the alpha-subunit. The correlation in adult men between serum inhibin B levels and spermatogenesis may be due to the fact that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells, including the stages from pachytene spermatocytes to early spermatids.     

A comparative study of the glycolipids of human, bird and fish testes and of human sperm

The glycolipids of human testis and sperm have been compared. Both adult testis and the sperm exhibited remarkably complex, but generally similar, patterns of glycolipids. In particular, both contained appreciable amounts of the sulfogalactosylmonoalkylmonoacylglycerol, recently shown to be the principal glycolipid of the testis and sperm of a number of animals. In contrast, immature (prebuteral) human testis did not contain this compound. To extend knowledge on the possible distribution of sulfogalactosylmonoalkylmonoacylglycerol in the testes of other chordates, we have also analysed the glycolipids of the testes of a number of birds and fish. None of the testes from these species contained the above compound. Instead, sulfogalactosylceramide was found to be a major glycolipid of the testis of mature fowl, duck and skate-fish and sulfogalactosylglucosylceramide of the testis of mature salmon and trout. Immature duck testis contained only a trace of sulfogalactosylceramide. These studies reveal intriguing differences between the sulfatides of various chordates, lend support to the concept that sulfatides increase markedly in testis at a specific stage of spermatogenesis and suggest an important role for sulfatides in testicular and spermatozoal function.     

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.     

Susceptibility of alpine ibex to conjunctivitis caused by inoculation of a sheep-strain of Mycoplasma conjunctivae

The genetic expression of insulin-like growth factor II (IGF-II) mRNA was studied in healthy adult testes and in hypoplastic testes of polled Murciano-Granadina goats by means of in situ hybridization. A positive reaction in spermatogonia, pachytene spermatocytes and a few peritubular myoid cells was observed using the ovine antisense oligonucleotide in healthy testes. The hypoplastic testes displayed a loss of germinal epithelium and a slight thickening of the basement membranes. A limited number of immature germinal cells displayed a lesser hybridization reaction, while the expression of IGF-II mRNA observed in the peritubular myoid cells was similar to that seen in healthy testes. In hypoplastic testes, IGF-II mRNA expression within germinal cells decreased with increasing hypoplasia within the seminiferous epithelium and there was no hybridization within the tubules in cases of severely disrupted spermatogenesis. These results suggest that testicular hypoplasia is associated with changes in the expression of IGF-II mRNA.     

A study of the fluctuating factors in the seminiferous epithelial cycle in mice with special attention to statistical studies on diurnal fluctuations and on variations due to testes and testis loci in the relative frequencies of the stages

The influence of the testis and loci within testes on diurnal variations and fluctuations in the seminiferous epithelial cycle was investigated in 30 50-60 day old mice. Histological examination of the seminiferous tubules revealed a typical cellular arrangement for each stage of spermatogenesis in virtually all of the specimens. No apparent diurnal fluctuations were observed in the relative frequency of the various stages between specimens obtained at 6 different times of sacrifice or among the uniform periods over a 24-hour period. However, a significant (p less than .05) difference in the frequencies of the stages was observed between testes among individuals and loci within testes.     

Bezafibrate down-regulates fibrinogen biosynthesis in human hepatoma HepG2 cells

Cellular distribution of HIV-1 proviral DNA has been studied, by in situ PCR hybridization, in the testes of infected men who died at various stages of the disease. In seropositive asymptomatic subjects, HIV-1 proviral DNA was present in the nuclei of germ cells at all stages of their differentiation. The presence of provirus did not induce germ cell damage, was associated with normal spermatogenesis, and was not accompanied by morphologic signs of immune response. The observed HIV hybridization pattern of germ cells suggests clonal infection. Mechanisms responsible for HIV penetration in testicular germ cells remain to be clarified; however, the possibility of a direct infection of the germ cells by cell-free virus is suggested. In the testes of AIDS-deceased men, histologic features of hypoplasia with arrested spermatogenesis were evident, and few infected spermatogonia and spermatocytes were observed. The whole of these data demonstrates that the testis is a site of early viral localization that fails to elicit an immunological response, and that HIV-seropositive men produce infected spermatozoa that are released in the genital tract.     

Patterns of EEG alpha during word processing and relations to recall

The following relations were derived from large-scale testing of feed protein quality and its bearings on testicular function in growing Wistar rats: testicle weights and live weights r = 0.77 for juvenile animals and r = 0.36 for adult animals; nuclear volumes of Leydig's cells to testicle weights r = 0.84 for juvenile animals and r = 0.43 for adult animals; testosterone in testicular tissue, ng/g, to testicle weights r = 0.55 for juvenile animals and r = 0.80 for adult animals; testosterone per live weight, ng/100 g, to nuclear volumes of Leydig's cells r = 0.76 for juvenile animals and r = 0.46 for adult animals. Critical thresholds above which complete spermatogenesis is always ensured may be established for testosterone (50+/-5 ng/100 g live weight) and for the nuclear volume of Leydig's cells (30 mum3). Another conclusions drawn from the above results is that high testosterone levels in the testes of adult animals were not attributable to high testosterone biogenesis of Leydig's cells, as might have been assumed from high activities of that kind recordable from some nuclear volumes.     

Muscle metabolism of professional athletes using 31P-spectroscopy

PURPOSE: Cocaine abuse has reached epidemic proportions in the United States. Our recent study has shown that cocaine has adverse action on spermatogenesis and fertility in male rats. The indirect action of cocaine occurs by blocking the reuptake of neurotransmitter, which causes local vasoconstriction. In this study we evaluate blood flow to the testes after subcutaneous injection of cocaine to male rats. METHODS: Male Sprague-Dawley rats were divided into two main groups. The treatment group received subcutaneous cocaine (30 mg/kg body weight) and the control animals received normal saline. Xenon-133 wash out experiments were carried out on testes at 5, 10, 15, 20, 30, 45, 60, 90 min and 4.5 hours after injection of cocaine or normal saline. Statistical analysis was done by SPSS V. 7.S for windows. P < 0.05 was considered statistically significant. RESULTS: There was a reduction in testicular blood flow after cocaine administration to male rats. This vasoconstrictor effect was most pronounced at 15 min after injection of cocaine and persisted up to 60 min. At 90 min, the early restitution of blood flow to ischemic tissue occurred. There was a significant increase in testicular blood flow in cocaine-treated groups than in the control group during restitution phases at 90 min. At 4.5 hours, there was no difference in blood flow in both groups. CONCLUSION: This study demonstrates that cocaine, when given subcutaneously at a 30 mg/kg body weight dose, results in prolonged vasoconstriction of the blood vessels to the testes. Adverse effects of cocaine on the testes may be in part due to ischemic and postischemic reperfusion injury to the organ.     

Inhibition of nucleolar protein nucleolin by electroporation with anti-nucleolin antibodies results in an increase of the nucleolar size

We produced an antibody that recognized only early stages of spermatogonia in Japanese eel testis. This antibody (anti-spermatogonia-specific antigen-1, anti-SGSA-1) recognized a band of about 38 kDa in Western blot analysis of extracts from eel testis. This antigen was observed by immunohistochemistry only in type-A and early type-B spermatogonia and could not be seen in the late type-B spermatogonia, which appeared after the initiation of spermatogenesis by a single injection of human chorionic gonadotropin. Immunoreactive SGSA-1 was absent in spermatocytes, spermatids, spermatozoa, Sertoli cells, and interstitial Leydig cells. Similarly, this antigen was also detected only in type-A/primary spermatogonia in the testes of two species of teleosts, medaka (Oryzias latipes) and tilapia (Oreochromis niloticus), as well as a toad (Xenopus laevis). These results imply that the disappearance of SGSA-1 in late type-B/secondary spermatogonia is a critical step in the progression of spermatogenesis, and indicate that anti-SGSA-1 is a useful marker for analysis of the molecular mechanism controlling the differentiation of spermatogonia in lower vertebrates.     

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.     

Deoxyribonucleic acid histogram of testes in primary transsexualism

The influence of oestrogen on spermatogenesis was evaluated by comparing its effect on the testes of primary male transsexuals and those who had undergone hormone therapy for prostatic carcinoma. Eight primary transsexuals were studied. They had been diagnosed on clinical and psychiatric evidence and had been on oestrogen therapy for several years since puberty. Histological sections of testicular tissue obtained at reassignment surgery from 8 phenotypic male transsexuals (aged 24-32 years) with an XY chromosome complement were studied by light microscopy. The formalin-embedded specimens were analysed by flow cytometry for deoxyribonucleic acid (DNA) histograms. Both the histology and DNA histograms revealed a pattern of maturation arrest in 12 of 16 testes in which the diploid cell compartment occupied most of the spermatogenetic element, followed by tetraploid and monoploid cells. Two testes showed impaired spermatogenesis and 2 were normal. The DNA histograms and pathology were also evaluated in 20 testes after 3 to 8 years of hormone therapy in patients with advanced prostatic carcinoma (aged 60-78 years). No maturation arrest was found in these patients. Sixteen of them had a pattern of fibrosis and atrophy and 4 had impaired spermatogenesis. It was concluded that oestrogen influenced spermatogenesis and affected the maturation of spermatogonia mostly during puberty.     

Effects of zinc on intact and castrated cockerels

1. Fourteen-week-old intact and castrated cockerels (White Leghorn) were injected intramuscularly with zinc (100 micrograms/kg body weight) as zinc ammonium sulphate solution once daily for 4 weeks and the effects on testes, pituitary and adrenal glands investigated histologically. 2. In intact cocks zinc decreased testes weight significantly, inhibited spermatogenesis and disturbed testicular hormone production. There was an increase in pituitary gonadotrophic cell activity, perhaps an indication of feed-back response to low concentration of testicular hormone. Cortical and medullary cells of the adrenal glands showed signs of activation. 3. No discernible effects of zinc injection on pituitary and adrenal cells of castrates were observed. This may reflect a decrease in pituitary and adrenal responsiveness to repeated stimulation with zinc. 4. The results indicate that the effectiveness of a given dose of zinc was dependent on the physiological condition of the cockerels and the effective site of zinc action was centred in the testes.     

Chromatin condensation behaviour of the Y chromosome in the human testis. I. Evidence for decondensation of distal Yq in germ cells prior to puberty with a switch to Sertoli cells in adults

The condensation behaviour of the human Y chromosome in germ cells and Sertoli cells of pre- and post-pubertal testes was followed by fluorescence in situ hybridisation using probes for three different regions of the Y chromosome. Patterns of expansion or contraction of signal are taken to reflect degrees of condensation of the related Y chromatin and hence its potential for genetic activity. For probe pHY2.1, which labels the distal non-fluorescent and fluorescent heterochromatin of the Y chromosome (Yq12), an expanded signal seen in gonocytes of the prepubertal testis is superseded by a condensed signal seen in adult germ cells at all but the zygotene stage of meiotic prophase when meiotic pairing takes place. In contrast, Sertoli cells show a condensed signal pre-pubertally but a greatly expanded signal in the adult testis. A totally condensed pHY2.1 signal is found in a chromosomally normal man with Sertoli-cell-only syndrome. It is hypothesised that control over at least some facets of spermatogenesis may not, in the adult, be autonomous to the germ cells, but rather may emanate from the Sertoli cells. Chromatin expansion at zygotene could, however, be important for pairing and crossing over in the XY bivalent, successful synapsis ensuring survival of spermatocytes into the post-meiotic stages.     

The value of laparoscopy in the diagnosis and treatment of non-palpable testicular cryptorchism

Laparoscopy is useful in both the diagnosis and the management of impalpable testes. Intra-abdominal testicles can be removed laparoscopically if atrophic or can be partly devascularized by spermatic vessel clipping if apparently normal. Assessment of testicular revascularization would be desirable prior to subsequent orchidopexy. A second-stage vasal-based orchidopexy can then be performed once adequate testicular reperfusion via the deferential pedicle is believed to have occurred. We have used both diagnostic and therapeutic laparoscopy in the management of 103 non-palpable testes over a period of 6 years. Open procedures following laparoscopy included 57 orchidopexies, 11 orchiectomies and one microvascular testicular autotransplant. Thirteen laparoscopic interventions were performed: 5 orchiectomies for atrophic testes and 8 testicular vessel clippings followed by 6 second stage open inguinal orchidopexies. Color Doppler duplex ultrasonography was not found to be reliable for assessment of testicular revascularization following spermatic vessel clipping. There were 3 complications which were all related to puncture with the Veress needle.     

Expression of oestrogen receptor beta (ER beta) occurs in multiple cell types, including some germ cells, in the rat testis

The identification of a second oestrogen receptor (beta) has prompted a re-evaluation of the potential sites of action of oestrogens. The aim of the present study was to characterize immunoexpression of ER beta expression in the testis to complement earlier data which had demonstrated that expression of ER alpha is confined to testicular interstitial Leydig cells. In all testes studied, including those from both fetal (day 20.5 p.c.) and adult rats, ER beta was found to be expressed in multiple cell types. Sertoli cell nuclei were immunopositive at all ages. In adult testes expression in Sertoli cells was not stage dependent and was unaffected by ablation of Leydig cells. In fetal testes ER beta was also expressed in peritubular cells, fetal Leydig cells and gonocytes. In the pubertal and adult testis ER beta was detected in the nuclei of spermatogonia and most pachytene spermatocytes. Weak immunopositive staining was present in the cytoplasm of spermatocytes undergoing the second meiotic division. In conclusion the widespread expression of ER beta in the testis is consistent with a role for oestrogens in modulating spermatogenesis, and hence fertility, in the male.     

Doppler ultrasound of the testis in azoospermic subjects as a parameter of testicular function

Azoospermia frequently represents the end-point of different pathological conditions that cause important quantitative and qualitative alterations of both spermatogenesis and testicular structure, including intratesticular blood vessels. In this study we performed colour Doppler ultrasound of the testis in 12 azoospermic subjects affected by primary testicular pathology (four bilateral post-orchitis, four postradiotheraphy for cancer, four post-traumatic) aged 28.2+/-3.3 (mean+/-SD) years, in six subjects affected by obstructive azoospermia aged 29.7+/-2.4 years and in 20 age-matched fertile subjects (aged 28.6+/-2.5 years). The analysis of intratesticular vessels per organ was quantified using a semiquantitative score: category 0, no vessels visible; category 1, between one and three intratesticular vessels visible; and category 2, more than three vessels visible. In obstructive azoospermic patients and in fertile subjects there were always more than three intratesticular vessels. No intratesticular vessels were detected in eight testes (33.3%) and fewer than three vessels in 16 testes (66.6%) in subjects affected by primary testicular pathology. In azoospermic subjects the testicular structure of the testis was evaluated by diagnostic fine needle aspiration cytology (FNAC) performed in the middle portion of the testis. In non-obstructive azoospermic patients this procedure showed the presence of only Sertoli cells in all cases. When detectable vessels were present, a new aspiration was performed in these areas. In 12 out of 16 cases, spermatogenetic cells including mature spermatozoa, were found when the FNAC was performed in testicular regions showing the presence of blood vessels. These results indicate that colour Doppler sonography of the testis may be useful in the differential diagnosis of azoospermia and suggest the evaluation of the intratesticular blood vessel distribution before performing any method to retrieve intratesticular spermatozoa for intracytoplasmic sperm injection.