Spermiogenesis defects in human: detection of transition proteins in semen from some infertile men.

Semen samples from 60 infertile men were examined by flow cytometry following propidium iodide staining. Of these, 23 samples contained young haploid cells. Transition proteins (TP1 and/or TP2) were detected in 12 of these, using immunohistochemical staining. The presence of TPs in spermatids in semen indicates inhibition in the differentiation pathway from round spermatids to spermatozoa. Cells of this type were found in semen from patients with nonobstructive azoospermia, severe to extreme cases of oligozoospermia, asthenozoospermia and teratozoospermia.

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.

[Evaluation of sperm samples from infertile men by flow cytometry].

INTRODUCTION: Human spermatogenesis begins at adolescence and continues throughout life. This process includes morphologic, cytologic and biological changes, leading to the formation of mature spermatozoa. Male infertility may be caused by several reasons, including oligozoospermia at variable degrees and complete absence of mature spermatozoa. Routine spermatogram, measuring sperm counts, motility and morphology, might not provide complete information in the evaluation of these cases. This study is aimed to evaluate the possible use of flow cytometry in the identification of different sperm cell populations in sperm samples obtained from infertile men, and in determining the different cell types in various groups of infertile men. MATERIALS AND METHODS: Sperm samples from normal and infertile men (the latter were azoospermic or oligoteratozoospermic OTA) underwent flow cytometry analysis, after preparation with TNE buffer and staining with Propidium Iodide. The separation of germinal cells into different populations, according to their DNA content and chromatin condensation, was evaluated. The WINMDI (http://fac.-scripps.edu, J. Trotter) software was used for data analysis. RESULTS: Flow cytometric analysis enabled identification of several cell populations in sperm samples, including haploid, diploid and tetraploid cells. Certain cellular distribution patterns were observed in sperm samples from infertile men: mature haploid cells, diploid cells, domination of tetraploid or non-mature haploid cells, and combination of these patterns. These patterns appeared in a statistically different manner among fertile and infertile men; the median value of mature haploid cells was higher in normal men (91%, compared to 85% in the OTA group and 0% in the azoospermic men), while the median value of diploid and tetraploid cells was higher in azoospermic men (72% and 8.5% respectively, compared to only 1% and 0% in normal men). CONCLUSIONS: These findings suggest that flow cytometry of sperm samples may serve as a non-invasive tool for investigations of male infertility and for identification of appropriate candidates for interventional treatment.

Evidence for biological rhythm in spermatogenesis in the pubertal hamster (Mesocricetus auratus): a flow cytometric study.

The number of cells in the S-phase fraction of the cell cycle reflects proliferative activity. Using flow cytometry histograms and the Phoenix M+ cell cycle program, the percent of cells in the S-phase fraction was measured in single cell suspensions prepared from testes of hamsters of different ages. A cyclical pattern with a period of 9 days, superimposed on another rhythm with a 38 day period was observed (p < 0.01) during hamster maturation and it disappeared after the second spermatogenic wave, where the S phase values reached a plateau. It was concluded that maturing animals passed through a stage in which testicular biological rhythm was involved. Therefore it was concluded that it takes approximately two spermatogenic waves before the proliferation rate in the testis reached a steady state.

Evaluation of damage to the testicular cells of golden hamsters caused by experimental cryptorchidism using flow cytometry and confocal microscopy.

Artificial unilateral cryptorchidism was performed in golden hamsters which were then held for different periods of time. The non-operated side was used as a control. At various times from 4 to 15 days, hamsters were killed, testes were removed and weighed, single cell suspensions were prepared for flow cytometry analysis and seminiferous tubules were fixed for confocal microscopy. Using DNA staining by propidium iodide or acridine orange followed by flow cytometry analysis, a marked decrease in the haploid condensed cell fraction was detected at the beginning stages of experimental cryptorchidism. In correlation with flow cytometry results, spermiogenic arrest at stages IX and X of seminiferous epithelium was detected in these animals by confocal microscopy and there were no mature forms of haploid cells in the cryptorchid testis. In the testis with more severe damage, there were almost no haploid cells in the seminiferous tubules of cryptorchid animals. In addition, a significant decrease in tetraploid cell fraction and an increase in S-phase fraction was obtained in severe cases. This may be explained by cell arrest before entrance into meiosis. Destruction of tubule structure and cell arrangement were also observed by confocal microscopy in such cases. In conclusion, flow cytometry, combined with confocal analysis, added useful information about spermatogenesis disturbances in cryptorchid testis and it may be used as diagnostic tools in other cases of spermatogenic disorders.

Meiosis in the golden hamster: a confocal microscopy and flow cytometric analysis.

In this study, confocal microscopy and flow-cytometry were utilized to follow meiosis in hamster spermatogenesis. Confocal microscopy was used as an analytical tool to observe spermatocytes inside the tubules following meiotic progression consecutively at defined spermatogenic stages. To study spermatocyte differentiation, the structure of the synaptonemal complex was studied in detail at various stages of hamster spermatogenesis using the antibody against SC3 (the protein of axial/lateral element). The synaptonemal complex was observed from the leptotene stage until the first meiotic division with maximal staining in mid-pachytene spermatocytes, suggesting a role for SC3 at this postrecombinational stage. In addition, 3-dimensional (3D) images of synaptonemal complex were observed, providing information about spatial distribution of the chromosomes within the nuclei of spermatocytes at different stages of meiosis. Changes in spermatocyte sizes and DNA condensation allowed assessment of meiosis by flow cytometry. Changes in chromatin condensation at different stages of hamster meiosis were followed, revealing decondensation from early to late pachytene stages. The analysis also allowed a comparing of chromatin status of mitotic and meiotic chromosomes, confirming the less compact structure of the latter, possibly connected to increased transcriptional activity during meiosis.

Procarbazine effects on spermatogenesis in golden hamster: a flow cytometric evaluation.

The response of hamster testis to the administration of 450mg/kg procarbazine (PCB) over a period of 4 weeks was evaluated. Flow cytometry was used to investigate changes in cell populations in testicular single cell suspensions and to correlate these changes with those observed in histological sections. PCB caused significant decrease in testicular and epididymal weight and a drastic reduction in haploid cells and spermatogenic arrest, demonstrating variation among the test animals. The results obtained confirm previous observations concerning detrimental effects of PCB upon spermatogenesis in species such as the rat and mouse, though its effect on hamster testis is milder and does not include the germinal stem cells. The histological evaluation of the testis showed a good correlation with flow cytometric evaluation, emphasizing the usefulness of this method in providing quantitative and rapid results.

Spermatogenesis in the golden hamster: the role of c-kit.

c-kit is related to the family of transmembrane tyrosine kinase receptors. Mutations in genes for either c-kit or its ligand, Steel factor, result in infertility, but the role of c-kit/SCF system in spermatogenesis is not well understood. In this study Western blot analysis together with confocal microscopy were used to follow c-kit expression in hamsters during the first spermatogenic wave in mature animals and in old age. Three antibodies raised against different domains of c-kit were tested on Western Blot. Confocal microscopy was performed after incubation of fixed seminiferous tubules with tested antibodies followed by binding of FITC-labeled secondary antibody. Longitudinal sections of seminiferous tubule were observed by confocal microscopy to determine in which stages of spermatogenesis and in which cell types c-kit was found. C-kit bands of 80,140, and 150 kDa were observed on Western blot, indicating that c-kit is a name related to several proteins sharing some common domains. Only the band of 150 kDa correlated with positive staining of c-kit in tubules using confocal microscopy. We term this protein c-kit150T (150 kDa, testis). We demonstrated that c-kit150T appeared in differentiating hamster spermatogonia at stages VII-VIII of adult spermatogenesis and at day 13-14 during the first spermatogenic wave. It remained attached to the cell until late pachytene. This suggests that c-kit may play a role in preparing the germinal cells to enter meiosis. In order to evaluate the effect of aging on the number of germ cells, B2 spermatogonia/Sertoli cell ratio was calculated in the group of young animals (5-7 months) compared to this ratio in older ones (20-26 months). A significant decrease (P < 0.01) in the number of B2 spermatogonia in the group of old hamsters as compared to young ones was seen. The calculated value for the B2 spermatogonia/Sertoli cell ratio was 5.6 +/- 0.7 in young animals and 3.8 +/- 1.2 in the 20-26 months ones. In addition, decrease in the intensity of staining for c-kit was detected in the old hamsters. These may be the reasons for subfertility in old age and in other cases of testicular disorders.

Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study.

DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double-stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process.

Spermatogenesis in the golden hamster during the first spermatogenic wave: a flow cytometric analysis.

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36-38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88-90%) in the hamster occurred in the testis and only 10-12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205-211, 2000.

A comparative study of spermatozoal chromatin using acridine orange staining and flow cytometry.

Spermatozoa obtained from fish (Clarias gariepinus), human (Homo sapiens), turkeys (Meleagris gallapova), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), and monkeys (Macaca fascicularis) were stained with acridine orange before measuring fluorescence by flow cytometry. These mature sperm from various species produced different intensities of fluorescence while displaying similar ratios of red/green fluorescence. Comparison of the green fluorescence values for the various species showed the sequence (descending order of fluorescence values) human, turkey, monkey, hamster, rat and fish. The DNA complement (as base pairs in the haploid genome) of the various species did not increase in direct proportion to the fluorescence values. This suggests that the DNA was not equally accessible to the dye in the different species tested. The similarity in ratios of red/green fluorescence suggests that the structure of DNA in the chromatin is similar in the different species but abnormal 'satellite' populations of cells that show higher red/green fluorescence ratios than the parent population have been found in sperm samples from monkeys and from some infertile men. Their high red fluorescence intensities were not caused by RNA because treatment with RNAse did not alter the red fluorescence. It is possible that these cells contain larger amounts of denatured (single stranded) DNA.

In-vitro human spermatozoa nuclear decondensation assessed by flow cytometry.

The process of sperm chromatin decondensation occurs when a spermatozoon enters an ovum. Protamine disulphide bonds are reduced to SH and the polycationic protamines combine with the polyanionic egg protein, nucleoplasmin, thus being stripped from DNA which then combines with histones. Defective chromatin decondensation will thus prevent further development of the male pronucleus. In this study human sperm samples were incubated in vitro at 28 degrees C (using a medium in which the polyanion, heparin, substitutes for nucleoplasmin and beta-mercaptoethanol for egg glutathione) for 10, 20 and 30 min before stopping the reaction with formalin (to 3.6%). The DNA of the fixed cells was stained with Acridine Orange by a one-step method and subjected to flow cytometry and data analysis, in which a zone characteristic of condensed chromatin is outlined on red-green fluorescence contour plots. After 20 min of incubation 97% of the control spermatozoa that were in the mature window (WIN M) had decondensed and moved out of this region. Defects in sperm decondensation were seen in four semen samples of the 20 that were tested. In cases where spermatozoa fail to produce a fertilized egg the cause may lie with defective chromatin quality, including failure of the sperm chromatin to decondense. The method described here is a simple procedure for detecting sperm samples containing such defective cells.

Multiple actions of a hybrid PACAP antagonist: neuronal cell killing and inhibition of sperm motility.

Pituitary stimulating adenylate cyclase (PACAP) is a major regulatory peptide with two active molecular forms: PACAP-27 and PACAP-38. Both molecular forms promote neuronal survival and protect against neurotoxicity. Based on our previous hybrid peptide strategy in designing vasoactive intestinal peptide (VIP) antagonists, novel PACAP analogues were synthesized (neurotensin6-11 PACAP7-27 and neurotensin6-11 PACAP7-38). In addition to the hybrid modification, the methionine in position 17 was replaced by norleucine (Nle). Treatment of rat cerebral cortical cultures for five days with the putative PACAP antagonists (1 nM) resulted in a 35-45% reduction in neuronal cell counts as compared to controls. Neuronal cell death was already obtained at picomolar concentrations for the neurotensin6-11 PACAP7-27 antagonist with 70% death at 10(-8) M. Co-administration of the PACAP hybrid analogue with picomolar amounts of PACAP-27 or Nle17-PACAP-27 attenuated the reduction in neuronal cell counts. While the protective effects of both analogues exhibited a peak at 1 pM concentrations, the Nle-containing agonist displayed a broader range of active concentrations (10(-12)M-10(-9) M). The putative PACAP antagonist also inhibited sperm motility (golden hamster) in a dose-dependent manner as assessed in vitro. Complete inhibition was observed at 10 microM, suggesting a role for PACAP in sperm motility and sexual function. Thus, previous findings of a large number of PACAP and PACAP receptors in the nervous system and the reproductive system are now correlated with a function in neuronal survival and sperm motility. The structure-activity studies suggest that the methionine in position 17 and the first six amino acids are important in the determination of PACAP activity, knowledge that may facilitate PACAP-based drug design.

Epididymal maturation of chromatin in spermatozoa from control monkeys (Macaca fascicularis) and those treated with Cetrorelix, a gonadotropin-releasing hormone antagonist.

In order to examine changes in sperm chromatin upon epididymal maturation in the macaque epididymis (Macaca fascicularis), spermatozoa were obtained from six regions of the duct and examined for the state of their chromatin condensation by flow cytometry after staining with acridine orange. To see whether changes were affected by androgens, tissue was obtained from five monkeys treated with the gonadotropin releasing hormone (GnRH) antagonist Cetrorelix. Spermatozoa were recovered from treated and control animals after 16 days (at hemicastration) and another 9 days of treatment. Chromatin condensation of epididymal spermatozoa from controls displayed an increase upon maturation. After 16 days of GnRH-antagonist treatment, spermatozoa in the caput epididymidis displayed greater fluorescence than those from controls, but this was reduced during epididymal transit to values found in the distal epididymal regions of the controls. It is concluded that epididymal chromatin condensation 1) is normal in GnRH-antagonist-treated monkeys as long as sperm are being produced and 2) can compensate for poor testis function so that spermatozoa with normal states of chromatin condensation are found in the distal cauda epididymidis and probably the ejaculate.

Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining.

The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.

Effects of pivalic acid and sodium pivalate on L-carnitine concentrations in the cauda epididymidis and on male fertility in the hamster.

In this study, administration of pivalic acid or its sodium salt was found to decrease the L-carnitine concentration in the epididymal lumen of the hamster; it also tested whether this decrease affected sperm cell motility, chromatin structure, or fertilizing capacity. Provision of pivalic acid or its sodium salt (20 mM or 40 mM) in the drinking water of mature male golden hamsters for 30 days reduced (by 72%, 75%, and 83% in three experiments) the L-carnitine concentration of the cauda epididymidis but did not inhibit sperm chromatin condensation, as assessed by flow cytometry. The treatments did not alter the location of motile sperm in the epididymidis nor did they appreciably affect the motility of sperm obtained from the distal cauda epididymidis. The numbers and percentage of ova that reached the 2-cell stage 36-40 h after uterine insemination with spermatozoa from control and treated hamsters served as a measure of sperm fertility. Treatment with pivalic acid or sodium pivalate did not render male hamsters infertile although it appeared to reduce the fertilizing ability of their spermatozoa. These results suggest that the high concentration of L-carnitine present in the lumen of the cauda epididymidis is not required for maturation of sperm chromatin or development of sperm motility.

Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry.

Inasmuch as caput epididymal and even testicular spermatozoa are now being used to generate pregnancies by direct injection into the oocyte, differences in the chromatin of spermatozoa from proximal and distal locations in the epididymis were studied. Acridine Orange staining was used to investigate chromatin structure in human spermatozoa which had left the testis and were undergoing maturation in the epididymis. Measurement of green and red fluorescence intensities of human spermatozoa by flow cytometry demonstrated a decrease in binding of Acridine Orange to DNA as the spermatozoa traversed the epididymis. Using spermatozoa from the cauda epididymis as the standard, the percentages of spermatozoa from the efferent duct, proximal corpus epididymis, midcorpus epididymis, distal corpus epididymis, proximal cauda epididymis and distal cauda epididymis that had matured with regard to chromatin condensation were 28 +/- 5, 39 +/- 3, 49 +/- 5, 64 +/- 5, 69 +/- 6 and 74 +/- 4% respectively. It may be concluded that eggs fertilized by ejaculated spermatozoa receive a more highly condensed form of chromatin than that received by eggs inseminated with proximal epididymal or testicular spermatozoa.