Flow cytometry of human semen: a preliminary study of a non-invasive method for the detection of spermatogenetic defects

BACKGROUND: The pathway of spermatogenesis involves the conversion of diploid stem cells (spermatogonia) to tetraploid primary spermatocytes, followed by meiosis and two cell divisions, first forming diploid secondary spermatocytes and then haploid round spermatids. Differentiation of round spermatids results in spermatozoa containing condensed chromatin. It has long been known that semen from patients with non-obstructive azoospermia or oligospermia contains small numbers of immature germinal cells. In this article, a flow cytometric procedure is described for assessing defects in spermatogenesis by identifying the ploidy of those immature cells. METHODS: Cells in semen samples from 44 infertile patients and 14 controls were stained with propidium iodide, which displays red fluorescence when intercalated between bases in double-stranded DNA. The resulting cell suspension was examined by quantitative flow cytometry, with excitation by laser light (488 nm) and red fluorescence recorded on a logarithmic scale to allow easy differentiation between intensities of tetraploid, diploid and haploid round spermatids, and spermatozoa containing condensed chromatin. RESULTS: The flow cytometric method differentiated between cases of 'Sertoli cell-only' syndrome (complete absence of tetraploid and haploid cells) and cases where spermatogenesis was blocked in meiosis or in spermiogenesis. Flow cytometric histograms from semen samples from normozoospermic, oligozoospermic and azoospermic patients fell into patterns that correlated well with the results obtained from testis histology findings. CONCLUSIONS: The method described may serve as a simple, non-invasive and reliable assay to help clinicians counsel patients with severe male infertility before referring them for testicular surgery to locate spermatozoa for ICSI.     

Misregulation of histone acetylation in Sertoli cell-only syndrome and testicular cancer

In many species, including humans, chromatin remodelling during spermiogenesis is initiated with a marked increase in histone acetylation in elongating spermatids. We have investigated whether this process is disturbed when spermatogenesis is defective or in human testicular tumours. For this purpose, the presence of highly acetylated histone H4 was detected on testicular sections from men with a severe impairment of spermatogenesis of several origins, as well as in different types of testicular tumours. In most tubules devoid of germinal cells (including SCO, Sertoli cell only syndromes) or lacking spermatocytes and spermatids, the Sertoli cells' nuclei showed a global increase in histone H4 acetylation. A similar observation was made in the peritumoral seminiferous tubules of testicular tumour tissues, whenever they were lacking germinal cells, with carcinoma in situ (CIS) cells being hypoacetylated. The global hyperacetylation of elongating spermatids during spermatogenesis could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be disturbed in cases of severe spermatogenesis impairment, as well as in tubes surrounding germ cells in testicular tumours.     

Spermatogenesis in the vasectomized monkey: Quantitative analysis

The seminiferous epithelium in mature vasectomized Macaca fascicularis was examined quantitatively to assess spermatogenesis. Monkeys were bilaterally vasectomized and controls were bilaterally sham operated. At postoperative periods of 10 and 18 months, groups of monkeys were castrated and their testes prepared for morphologic analysis. Diameters were measured in 100 cross sections of seminiferous tubules from each animal. Numbers of spermatogonia (Ad and Ap), preleptotene spermatocytes, pachytene spermatocytes, and step 7 spermatids, relative to Sertoli cell nucleoli, were counted in stage VII tubules. Tubule diameter and germ cell numbers per Sertoli cell nucleoli were not altered by vasectomy. Our study demonstrates quantitatively that spermatogenesis in the monkey is not inhibited up to 18 months following vasectomy.     

Quantitative (stereological) study of the effects of vasectomy on spermatogenesis in rabbits

Using stereological methods, especially the optical disector for unbiased estimation of nuclear number, our recent study demonstrated that long-term (6 or 12 months) vasectomy in the rhesus monkey had no significant effects on spermatogenesis (Peng et al. Reproduction 2002, 124, 847-856). This study aimed to determine the scenario in the rabbit using the same morphometric methodology. Three groups of normal male Japanese white rabbits (aged 4-5 months) were subjected to unilateral vasectomy; 10 days, 6 months and 12 months later both testes and epididymides were removed. Testicular and epididymal methacrylate-embedded sections were obtained for stereology. Vasectomy-induced damage to spermatogenesis was observed, primarily sloughing of spermatogenic cells with a greater reduction in the number of advanced (adluminal) cells. The damage was most severe at 10 days, occurring in all the testes on the vasectomized side and involving sloughing of even type A spermatogonia, the number of which returned to normal at 6 and 12 months. Damage was less severe at 6 and 12 months, being found in half of the testes of the vasectomy side, in which the total numbers of later germ cell types were 24.0-59.1% (spermatocytes) and 0.3-11.6% (spermatids) of control at 6 months, and 20.1-22.1% (spermatocytes) and 0.4-12.0% (spermatids) of control at 12 months. By contrast, Sertoli cell number per testis was unchanged following vasectomy in any group. Epididymis on the vasectomy side, especially at 10 days and 6 months, appeared larger than on the contralateral side, but this difference was not statistically significant, and no sperm granuloma was seen in the epididymis.     

Time-dependent changes in post-mortem testis histopathology in the rat

To clarify the contribution of spontaneous or autolytic post-mortem changes to testis histopathology, the testes of adult rats were examined after animals were left at room temperature for 12, 24, 36, and 48 hours postmortem (n = 2 for all time points except 0 hours postmortem, where n = 3). A progressive decrease in testis weight and seminiferous tubule diameter was observed, as well as detachment of the seminiferous epithelium from the basement membrane. As early as 12 hours postmortem, there was observable clumping and margination of chromatin in Leydig cells, Sertoli cells, spermatogonia, spermatocytes, and step 7-10 spermatids; extensive disintegration of Sertoli cells and residual bodies by 24 hours postmortem; and TUNEL positivity of Leydig cells (by 36 hours postmortem) and step 19 spermatids (at 48 hours postmortem). These findings will aid in ensuring proficient histopathological analysis of testes in toxicity studies.     

Testicular development in cynomolgus monkeys

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

Stage-specific expression of mouse germ cell-less-1 (mGCL-1), and multiple deformations during mgcl-1 deficient spermatogenesis leading to reduced fertility

A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1), is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-1 in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum et al., 2003), postacrosomal sheath, and posterior ring apparently formed. In addition, MN7 and MN13, essential substances for fertilization that are located in sperm heads, were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility.     

Compensatory upregulation of myelin protein zero-like 2 expression in spermatogenic cells in cell adhesion molecule-1-deficient mice

The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells.     

Regulated expression of the ubiquitin protein ligase, E3(Histone)/LASU1/Mule/ARF-BP1/HUWE1, during spermatogenesis.

A ubiquitin protein ligase (E3), E3(Histone)/LASU1 (Mule/ARF-BP1/HUWE1), was recently identified that mediates ubiquitination of core histones, the Mcl-1 anti-apoptotic protein, and the p53 tumor suppressor protein. However, the expression of E3(Histone)/LASU1 remains poorly studied. Because we identified E3(Histone)/LASU1 from the testis, we explored its regulation during spermatogenesis. In the first wave of rat spermatogenesis, E3(Histone)/LASU1 mRNA and protein had peak expression at days 10 and 20, respectively, and decreased with age. Consistent with these findings, immunohistochemistry revealed that E3(Histone)/LASU1 was highly expressed in nuclei from spermatogonia to mid-pachytene spermatocytes. There was no obvious staining in spermatids, when histones are ubiquitinated and degraded. E3(Histone)/LASU1 was also expressed in other tissues. However, except in neuronal cells of the brain, expression was cytoplasmic. Thus, E3(Histone)/LASU1 may play a role in chromatin modification in early germ cells of the testis, but also has functions in other tissues.     

Mutations in the testis-specific NALP14 gene in men suffering from spermatogenic failure

BACKGROUND: Because of the common use of ICSI and the potential genetic aetiology of spermatogenic failure, concern has been raised about transmitting genetic disorders to ICSI offspring. However, to date, in only approximately 15% of all cases of spermatogenic failure, an underlying genetic cause can be identified. We have previously established an association between spermatogenic failure and chromosomal region 11p15. In this study, we set out to explore whether NALP14, a gene recently mapped to 11p15, has a function in spermatogenesis and whether mutations in NALP14 can cause spermatogenic failure. METHODS: We applied two different multiple tissue northern (MTN) blots to determine tissue specificity of NALP14 and performed immunohistochemistry on human testis with anti-NALP14 antiserum. To determine imprinting status of NALP14, we tested the expression pattern of two single-nucleotide polymorphisms (SNPs) in human testis. Finally, we performed a mutation screen of the NALP14 gene in 157 men with azoospermia or severe oligozoospermia by direct sequencing; 158 normospermic men served as controls. RESULTS: NALP14 was, as are the three other genes in 11p15, exclusively expressed in testis. Within the testis, the NALP14 protein was mainly expressed in A dark spermatogonia, mid and late spermatocytes and spermatids. The mutation screen revealed five mutations that occurred only in the patient group. One of these unique mutations introduced an early stop codon in the NALP14 sequence, predicted to result in a severely truncated protein. CONCLUSION: Our data suggest that NALP14 has a function in spermatogenesis and that mutations in this gene might cause spermatogenic failure.     

Germ cell transplantation into X-irradiated monkey testes.

BACKGROUND: An intense debate is ongoing regarding options for fertility protection in oncological patients. Germ cell transplantation has been applied to restore mouse spermatogenesis. Here, an attempt to apply autologous germ cell transplantation to a primate animal model is described. METHODS: Five adult male cynomolgus monkeys were biopsied to retrieve and cryopreserve germ cells before both testes were irradiated (dose 2 Gy). Six weeks later, each monkey received an infusion of its own cell suspension into the right testis, while the left testes were infused with saline. Testis size, sperm counts and serum concentrations of inhibin, FSH and testosterone were analysed weekly for 9 months. Spermatogenic recovery was determined histologically at the end of the study. RESULTS: In four monkeys, the germ cell-infused right testes showed a slight to moderate increase in the rate of regrowth in comparison with the left testes. In two monkeys the right testis proceeded to recover more prominently, resulting in larger right testis volumes and better or full spermatogenic recovery at the study end. Restoration of spermatogenesis occurred as an all-or-nothing event. Inhibin B concentrations increased, while FSH and testosterone concentrations decreased with testicular regrowth. Sperm counts did not recover. CONCLUSIONS: The present study demonstrates the immaturity and complexity of germ cell transplantation as a clinical approach.     

CD147 is required for matrix metalloproteinases-2 production and germ cell migration during spermatogenesis

Spermatogenesis is a highly programmed process that requires the degradation of the extracellular matrix and the remodeling of tight junctions (TJ) to facilitate differentiating germ cell migration. Matrix metalloproteinases (MMPs) are essential in regulating Sertoli cell TJ in the testis. CD147 is known to stimulate the production of MMPs in tumor metastasis and its knockout mice are infertile. However, the functional relationship between CD147 and MMPs in spermatogenesis has not been investigated. In the present study, we examined the expression profile of CD147 and MMPs during mouse testicular development by RT-PCR, western blot and immunofluorescence staining. We also examined CD147 involvement in the production of MMP-2 and the migration of germ cells (GC-1 and GC-2 cells) using CD147 antibody or synthetic microRNA mimics-mediated knockdown. The results showed that CD147 was present at all stages of testicular development from 7 to 56 days post-partum (dpp). CD147 expression was found to increase after 21 days from moderate levels in 7 and 14 days. Of the eight MMPs studied, MMP-2, MMP-7, MMP-9 and MMP-23 were detected to have changes in expression during testicular development, with MMP-2 showing the largest change. CD147 and MMP-2 were co-localized in spermatogonia, spermatocytes and round spermatids in mouse testis, while in human testis, they were co-localized in spermatocytes and round spermatids. MMP-2 expression and migration of GC-1 and GC-2 cells were reduced by interfering with CD147 expression and function in vitro. These data suggest that CD147 regulates migration of spermatogonia and spermatocytes via induction of MMP-2 production during spermatogenesis.     

DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

Changes in Spermatozoa due to Large Doses of Pyridoxine (Vitamin B6)

To investigate the changes of spermatozoa by high doses of vitamin B₆, (B6), the alterations in spermatozoa and testis of rats after the administration of high doses of B6 were evaluated quantitatively and morphometrically. Wistar rats of 11 weeks of age were intraperitoneally injected with 63,125,250 and 500 mg/kg of B6 daily 5 times per week for 6 weeks. Using the spermatozoa taken from the epididymis and ductus deferens, the number, motility and nuclear morphology of spermatozoa were examined. After preparing 7 parameters for the nuclear morphology, the morphometry was performed by an IBAS version 2 (Zeiss) image analysis system. The number of spermatocytes and spermatids in representative stages of spermatogenesis was counted per Sertoli cell histologically. Mild deformation of spermatozoa nuclei occurred in the 63 mg or more exposure groups. In the 125 and 250 mg groups, the decrease in number as well as motility of spermatozoa together with slight decrease of spermatids in late maturation phase (mature spermatids) and the delay in spermia-tion appeared. Phagocytosis of mature spermatids by Sertoli cells was clearly increased in the 250 mg group. The alteration and the decreased number of spermatozoa are suggested to have mainly resulted from alteration of mature spermatids and the increased phagocytosis of mature spermatids by Sertoli cells. Computer-assisted morphometry of spermatozoa nuclei was useful not only to evaluate morphological changes objectively but also to discern them early. Acta Pathol Jpn 42: 861–869, 1992.     

Specific expression of the mRNA for 25 kDA heat-shock protein in the spermatocytes of mouse seminiferous tubules

 The 25 kDa heat-shock protein (Hsp25) is a member of the family of small heat-shock proteins. We investigated the expression and cellular localization of Hsp25 mRNA in the testis of adult and developing mice using Northern blotting and in situ hybridization techniques. In the early postnatal days, i.e., before the onset of spermatogenesis, no Hsp25 mRNA was detected in the testis. At around 10 days postpartum, Hsp25 mRNA began to be expressed in the testis in coincidence with the onset of the first wave of spermatogenesis and increased in amount progressively toward adulthood. Throughout the testis development, the signal for Hsp25 mRNA was localized exclusively to germ cells and was not detected in Sertoli or interstitial cells. The testis of W/Wv mutant mice, which lack the germ cell line, exhibited no Hsp25 mRNA expression. In the testis of normal adult mice, the abundance of Hsp25 mRNA differed among the seminiferous tubules in different stages of spermatogenesis. The most intense signal for Hsp25 mRNA was localized to the spermatocytes at leptotene, zygotene and early pachytene phases, which are present in the tubules of stages I–III and IX–XII. The signal decreased in intensity in the late pachytene and diplotene spermatocytes and was not detected in spermatids. Spermatogonia were also devoid of the signal. These results suggested that Hsp25 plays some specific role in the meiotic prophase of the testicular germ cell. Accepted: 27 Oct 1998     

Spontaneous and age-related testicular findings in beagle dogs

This study was conducted to characterize spontaneous testicular and epididymal microscopic findings in eighty control beagle dogs from toxicity studies. Hypospermatogenesis, characterized by randomly scattered missing spermatids and/or spermatocytes within seminiferous tubules, was observed in 75% of dogs six to seven months of age and declined to fewer than 10% in dogs over eleven months of age. Atrophy/hypoplasia of seminiferous tubules, characterized by subcapsular triangular clusters of tubules containing no germ cells, was observed in 25 to 40% of dogs under twelve months old, decreasing with age to 14 to 17% in dogs twelve to thirty-six months old. Retained spermatids, multinucleate giant cells, intracytoplasmic vacuoles (presumably in Sertoli cells), and swollen spermatocytes were common findings of minimal severity. Six- and seven-month-old dogs had lower testicular weights, less filling of the epididymal tails with sperm, and a two-fold higher incidence of abnormal epididymal content compared to dogs more than eight months of age. Most male beagles were histologically sexually mature by eight to nine months of age. This study confirms published reports that dogs at least ten months of age at necropsy usually are adequate for routine microscopic evaluation of the testes. If evaluation of spermatogenesis is critical, the incidental findings can be minimized by using males over twelve months of age.