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.

Attachment behaviors, depression, and anxiety in nonoffending mothers of child sexual abuse victims.

The purpose of this study was to examine the psychological well-being and attachment behavior of nonoffending mothers of child sexual abuse victims (CSAVs). This topic is significant because it is the mothers who most often provide support for young child victims. Two sets of data on maternal depression, state and trait anxiety, and Ainsworth's maternal attachment behaviors were analyzed. First, 38 mothers of CSAVs were compared based on the presence or absence of maternal history of abuse. Second, from the original 38 mothers of CSAVs, 27 mothers were compared to a matched group of mothers of nonabused children. Children in both data sets were 6 to 48 months. In the first data set, there were no significant differences in depression, anxiety, and attachment behaviors based on mothers' personal history of abuse. However, in the second data set, mothers of CSAVs had heightened levels of depression and anxiety and diminished maternal attachment behaviors.

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.

Screening families with young children for child maltreatment potential.

The incidence of child maltreatment in the United States is increasing. According to the third National Incidence Study of Child Abuse and Neglect (NIS-3), the number of abused and neglected children doubled between the years 1986 and 1993 to 2.8 million (Sedlak & Broadhurst, 1996). This same study found that the number of children who were seriously injured quadrupled during this period of time to nearly 570,000. Conservative estimates suggest that 10% to 20% of children under 12 years of age experience physical or sexual abuse or neglect. In over 80% of these circumstances, the perpetrator is a family member. Risk indicators have been identified that significantly increase the likelihood of abuse or neglect of children. Interventions that reduce these risk factors have been found to be associated with a decreased incidence of abuse and neglect (Andrews, 1994; Barber-Madden, Cohn, & Scholesser, 1988; Garbarino, 1986; Helfer, 1987). However, existing risk assessment or intervention guides for use with families of children under the age of 3 years are not concise enough for use in a busy primary care setting. For this reason, the Parenting Maltreatment Risk and Intervention Protocol was designed to aid in the identification of families with children under the age of 3 who are at risk for child abuse and neglect and to guide initial intervention, referral, and follow-up care.

Childhood social predictors of adolescent antisocial behavior: gender differences in predictive accuracy and efficacy.

This study examined the ability of several childhood, school-based, social variables to correctly classify antisocial adolescents. Children (N = 314; 163 boys, 151 girls) in the 3rd-5th grade were assessed on academic and social variables (i.e., peer rejection, aggression, withdrawal, and low prosocial behavior) and followed forward for 6-7 years until the 9th and 10th grade. Adolescent antisocial outcomes included a consensus measure formed from diagnostic interviews, contact with juvenile authorities, adolescent self-report, and mother's report. The gender-differential predictive accuracy and efficacy of the early predictor domains to a consensus measure of antisocial behavior were compared with the same estimates found for adolescent self-report of antisocial behavior. Both gender and criterion-method differences were found. For girls, regardless of the measure of antisocial behavior, early academic problems were the strongest predictors of future problems. For boys' self-reported antisocial outcomes, peer rejection was the strongest independent predictor. For consensus-reported antisocial outcomes, both early fighting-anger and withdrawn behavior displayed equally strong predictive relations. For boys, the combination of early fighting-anger and disruptive and withdrawn behavior was the strongest set of predictors for the consensus measure of antisocial functioning. Predictive accuracy and efficacy estimates are discussed in terms of predictive strength as well as the cost-benefit of misidentification.

The development of alcohol and other substance use: a gender study of family and peer context.

OBJECTIVE: This study examined early school-based academic and social variables with concurrent family conflict in predicting adolescent alcohol and other drug use. METHOD: 365 children were assessed initially in grades 2-4 on academic-related and social behavior variables using teacher ratings and rankings, peer nominations and ratings and direct observation of playground and classroom behavior. They were reassessed in grades 9-10, using interviews and questionnaires to determine the initiation and sequence of their use of alcohol, tobacco, marijuana and other hard drugs. RESULTS: In three sets of analyses, independently for males and females, lifetime abstainers were compared with adolescents who had used (1) only alcohol; (2) alcohol and tobacco; (3) alcohol, tobacco and marijuana; and (4) all three plus other hard drugs. The drug classifications represent a normative-deviant continuum of adolescent drug use. Constructs were developed for early academic and social predictors as well as concurrent family conflict. The results showed more wide-ranging academic and social difficulties during elementary school for children falling at the more deviant end of the drug use continuum. For girls, the concurrent home environment appeared to moderate the effect of early academic and social variables. CONCLUSIONS: Substance use established by ages 14-15 can be predicted by academic and social behavior displayed at ages 7-9. This suggests that prevention efforts for alcohol and other drugs may be more effective if directed at earlier antecedent behaviors rather than those that are concurrent with substance use.

Performance of third-year primary-care-track students in an integrated curriculum at Case Western Reserve University.

In 1994, Case Western Reserve University School of Medicine established a Primary Care Track (PCT) with an integrated curriculum as part of The Robert Wood Johnson Foundation's Generalist Physician Initiative. This study compared the performance of the first cohort of students to participate in the PCT third year with that of their classmates and determined student attitudes toward their experiences. The performances of 24 PCT and 81 traditional students on the Medical School Admissions Test (MCAT) and the United States Medical Licensure Examination (USMLE) Step 1 and 2 were compared using analysis of variance. Grades on the six core clerkships were compared using chi-square analysis. Performances of the PCT students and a subset of traditional students on the generalist school's objective structured clinical exam (OSCE) were compared using multivariate analysis. The students reported their perceptions on a questionnaire. The traditional students had significantly higher scores on the physical science section of the MCAT and on the USMLE Step 1, but at the end of year three, their USMLE Step 2 scores did not differ. Grade distributions in the core clerkships did not differ, except in psychiatry, where the PCT students received honors significantly more often. The PCT students had a lower mean score on the internal medicine National Board of Medicine Examiners shelf exam but performed better on the generalist OSCE exam. A majority of PCT students reported that they would choose the integrated third year again and recommend it to others.

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.