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Menisci help maintain the structural integrity of the knee. However, the poor healing potential of the meniscus following a knee injury can not only end a career in sports but lead to osteoarthritis later in life. Complete understanding of meniscal structure is essential for evaluating its risk for injury and subsequent successful repair. This study used novel approaches to elucidate meniscal architecture. The radial and circumferential collagen fibrils in the meniscus were investigated using novel tissue-preparative techniques for light and electron microscopic studies. The results demonstrate a unique architecture based on differences in the packaging of the fundamental collagen fibrils. For radial arrays, the collagen fibrils are arranged in parallel into ∼10 μm bundles, which associate laterally to form flat sheets of varying dimensions that bifurcate and come together to form a honeycomb network within the body of the meniscus. In contrast, the circumferential arrays display a complex network of collagen fibrils arranged into ∼5 μm bundles. Interestingly, both types of architectural organization of collagen fibrils in meniscus are conserved across mammalian species and are age and sex independent. These findings imply that disruptions in meniscal architecture following an injury contribute to poor prognosis for functional repair.

Infertile men undergoing intracytoplasmic sperm injection have an increased frequency of chromosome abnormalities in their sperm. Men with low sperm concentration (oligozoospermia) have an increased risk of sperm chromosome abnormalities. This study was initiated to determine whether men with severe oligozoospermia (

Recombination between the X and Y chromosomes is limited to the pseudoautosomal region and is necessary for proper segregation of the sex chromosomes during spermatogenesis. Failure of the sex chromosomes to disjoin properly during meiosis can result in individuals with a 47,XXY constitution, and approximately one-half of these result from paternal nondisjunction at meiosis I. Analysis of individuals with paternally derived 47,XXY has shown that the majority are the result of meiosis in which the X and Y chromosomes have failed to recombine. Our studies of sperm have demonstrated that aneuploid 24,XY sperm have a decreased recombination frequency, compared with that of normal sperm. Some studies have indicated a relationship of increased paternal age with 47,XXY offspring and with the production of XY disomic sperm, whereas others have failed to find such relationships. To determine whether there is a relationship between paternal age and recombination in the pseudoautosomal region, single-sperm genotyping was performed to measure the frequency of recombination between a sex-specific locus, STS/STS pseudogene, and a pseudoautosomal locus, DXYS15, in younger men (age ≤30 years) compared with older men (age ≥50 years). A total of 2,329 sperm cells were typed by single-sperm PCR in 20 men who were heterozygous for the DXYS15 locus (1,014 sperm from 10 younger men and 1,315 sperm from 10 older men). The mean recombination frequency was 39.2% in the younger men and 37.8% in the older men. There was no heterogeneity in the frequency of recombination rates. There was no significant difference between the recombination frequencies among the younger men and those among the older men, when analyzed by the clustered binomial Z test ( Z =.69, P =.49). This result suggests that paternal age has no effect on the recombination frequency in the pseudoautosomal region.

Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate molecular and structural changes occurring in four articular cartilage (AC) regions from the knees of anterior cruciate ligament (ACL)-transected rabbits at 3 and 8 weeks post-surgery. Rabbit AC from the lateral and medial femoral condyles (LFC and MFC) as well as from the medial and lateral tibial plateau (MTP and LTP) were processed for histology and for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules (collagen II, aggrecan. biglycan, decorin, fibromodulin, MMP-1, -3, -13, and TIMP-1). While the most severe histological changes were observed in the MTP starting as early as 3 weeks post-ACL transection based on Mankin scores, histological examination demonstrated a progression of osteoarthritic changes in the MFC from 3 to 8 weeks post-surgery. In contrast, very few changes were observed within both the LFC and LTP, and these changes did not worsen with increasing time after surgery. The water content increased significantly in the MFC at 8 weeks post-ACL transection and at both 3 and 8 weeks post-ACL transection in the MTP. Significant decreases in DNA content were observed for the MFC, LTP and MTP at 8 weeks post-ACL transection. Total RNA yields from the MFC and MTP were significantly elevated at 8 weeks post-ACL transection, while in the lateral compartment total RNA was unchanged following ACL transection. Analysis of mRNA levels for a subset of matrix molecules, proteinases and proteinase inhibitors, by RT-PCR demonstrated significant region-specific changes at the mRNA level following ACL transection. These results show that following ACL transection, complex molecular, as well as structural changes occur early in cartilage and that the observed changes are both region-specific and time-dependent.

Four major morphologically distinct classes of cells were identified within the adult rabbit meniscus using antibodies to cytoskeletal proteins. Two classes of cell were present in the fibrocartilage region of the meniscus. These meniscal cells exhibited long cellular processes that extended from the cell body. A third cell type found in the inner hyaline-like region of the meniscus had a rounded form and lacked projections. A fourth cell type with a fusiform shape and no cytoplasmic projections was found along the superficial regions of the meniscus. Using a monoclonal antibody to connexin 43, numerous gap junctions were observed in the fibrocartilage region, whereas none were seen in cells either from the hyaline-like or the superficial zones of the meniscus. The majority of the cells within the meniscus exhibited other specific features such as primary cilia and 2 centrosomes. The placement of the meniscal cell subtypes as well as their morphology and architecture support the supposition that their specific characteristics underlie the ability of the meniscus to respond to different types of environmental mechanical loads.

To account for the increased proportion of paternal nondisjunction in 47,XXY males as compared to other trisomies, it has been suggested that the XY bivalent, with its reduced region of homology, is particularly susceptible to nondisjunction. Molecular studies of liveborn Klinefelter syndrome (47,XXY) individuals have reported an association between the absence of recombination in the pseudoautosomal region and nondisjunction of the XY bivalent. In this study we examined single sperm from a normal 46,XY male to determine if there is any alteration in the recombination frequency of aneuploid disomic 24,XY sperm compared to unisomic sperm (23,X or Y). Two DNA markers STS/STS pseudogene and DXYS15 were typed in sperm from a heterozygous man to determine if recombination had occurred in the pseudoautosomal region. Individual unisomic sperm (23,X or Y) were isolated using a FACStar(Plus) flow cytometer into PCR tubes. To identify disomic 24,XY sperm, 3-colour FISH analysis was performed with probes for chromosomes X,Y and 1. The 24,XY cells were identified using fluorescence microscopy, each disomic sperm was scraped off the slide using a glass needle attached to a micromanipulator and then put into a PCR tube. Hemi-nested PCR analysis of the two markers was performed to determine the frequency of recombination. A total of 329 unisomic sperm and 150 disomic sperm have been typed. The frequency of recombination between the two DNA markers was 38.3% for the unisomic sperm, similar to frequencies previously reported. The 24,XY disomic sperm had an estimated recombination frequency of 25.3%, however, a highly significant decrease compared to the unisomic 23,X or 23,Y sperm (chi(2) = 10.7, P = 0.001). This direct analysis of human sperm indicates that lack of recombination in the pseudoautosomal region is a significant cause of XY nondisjunction and thus Klinefelter syndrome.

Objective To determine the histologic changes that accompany the formation of cell clusters during the early stages of osteoarthritis development in the meniscus, and to characterize the expression phenotype of these cells. Methods Histologic sections of medial menisci from normal and anterior cruciate ligament (ACL)–deficient rabbit knees were immunolabeled with monoclonal antibodies for vimentin to highlight the cytoskeleton of meniscal cells, Ki-67 to identify proliferating cells, and type X collagen to evaluate changes in the cell expression phenotype. Tissue mineralization was assessed by specific staining with alizarin red. Results Following ACL transection, there was an alteration in the normal interconnected network of meniscal cells in the fibrocartilaginous region of the tissue. This led to isolation of islands of cells within the extracellular matrix of the meniscal tissue. These islands of cells displayed 3 different morphologies based on cell composition: 1) stellate cells, 2) stellate as well as round cells, and 3) round cells. Islands composed solely of round cells were more prominent in the latter stages following ACL transection, and the size of these islands increased with time, apparently as the result of cell proliferation. These islands of cells corresponded to the “clusters” previously described in osteoarthritic cartilage. Strong expression of type X collagen colocalized with the deposition of calcium within the meniscal regions enriched with cell clusters. Conclusion Based on the observed changes in cell distribution, morphology, and cell proliferation as well as the previous detection of apoptosis in similar studies of rabbit knee joints, we propose a model for the development of cell clusters in the osteoarthritic meniscus. The morphologic appearance as well as the type X collagen expression phenotype of the meniscal cells forming the clusters is similar to that of hypertrophic chondrocytes. These findings provide a basis for understanding the origin of cell clusters in other joint connective tissues, such as osteoarthritic cartilage.

Hereditary nonpolyposis colon cancer (HNPCC) has been shown to be caused by mutations in the mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2. Recent evidence has demonstrated that mutations in mismatch repair genes disrupt meiosis in mice. A large HNPCC kindred in Newfoundland, Canada, has an hMSH2 mutation-an A--T transversion at the +3 position of the splice-donor site of exon 5. We have studied sperm from men with this hMSH2 mutation, since it is possible that mismatch repair mutations in humans might also have an effect on meiosis and normal segregation of chromosomes. The frequencies of aneuploid and diploid sperm were determined in 10 men with the hMSH2 mutation, by use of multicolor FISH analysis for chromosomes 13, 21, X, and Y. A minimum of 10,000 sperm per man was studied per chromosome probe. Control individuals consisted of men in the same kindred with HNPCC who did not carry the mutation and of other normal men from Newfoundland. A total of 321,663 sperm were analyzed: 200,905 sperm were from men carrying the hMSH2 mutation and 120,758 sperm were from control men. There was a significantly increased frequency of disomy 13, disomy 21, XX, and diploidy in mutation carriers compared with control men. These results suggest that the hMSH2 mutation may affect meiosis in humans.

Sperm chromosome abnormalities were assessed in testicular cancer patients before and after treatment with BEP (bleomycin, etoposide, cisplatin). The frequencies of disomy for chromosomes 1, 12, X, Y and XY were assessed along with diploid frequencies and sex ratios by multicolour fluorescence in situ hybridization (FISH). For each cancer patient, a minimum of 10,000 sperm was assessed for each chromosome probe before and after chemotherapy (CT). Data was analysed "blindly" by coding the slides. A total of 161097 sperm were analyzed, 80,445 before and 80,642 after treatment. The mean disomy frequencies were 0.11% pre-CT vs 0.06% post-CT for chromosome 1, 0.18% vs 0.15% for chromosome 12, 0.10% vs 0.9% for the X chromosome, 0.13% vs 0.10% for the Y chromosome and 0.25% vs 0.20% for XY sperm. There was no significant difference in the frequency of disomy pre-CT vs post-CT for any chromosome except that chromosome 1 demonstrated a significant decrease after CT. The "sex ratios" and frequency of diploid sperm were also not significantly different in pre and post-CT samples with 50.2% X-bearing sperm pre-CT and 50.5% X post-CT and 0.14% diploid sperm pre-CT vs 0.15% diploid sperm post-CT. There was no significant donor heterogeneity among the cancer patients. None of the values in the cancer patients differed significantly from 10 normal control donors. Thus our study suggests that BEP chemotherapy does not increase the risk of numerical chromosomal abnormalities in human sperm.

Sperm karyotype analysis was performed on testicular cancer patients before and after treatment with BEP (bleomycin, etoposide, and cisplatin). A total of 788 sperm chromosome complements was studied, 236 before chemotherapy (CT) and 552 post-CT. There was no significant difference in the total frequency of sperm chromosomal abnormalities pre-CT (10.2%) compared to post-CT (10.7 %). Similarly, there were no significant differences in the frequencies of numerical abnormalities (2.5% pre-CT vs. 2.4% post-CT) or structural abnormalities (6.4% pre-CT vs. 7.4% post-CT). The percentage of X-bearing sperm was also not significantly different before (46.3%) and after CT (50.1%). The results in cancer patients were not significantly different from those in control donors. This study corroborates results from our previous analysis of these same men using multicolor fluorescence in situ hybridization for assessment of aneuploidy for chromosomes 1, 12, X, Y, and XY. Together, these two studies suggest that the sperm of men receiving BEP chemotherapy are not at increased risk of chromosomal abnormalities two or more years after treatment.

Meiotic recombination is essential for the segregation of homologous chromosomes and formation of normal haploid gametes. Decreased recombination is associated with the production of aneuploid sperm in humans. MLH1, a DNA mismatch repair protein, was recently found to mark the sites of recombination in humans. Newly developed immunofluorescence techniques to identify MLH1 foci on synaptonemal complexes (SCs) in pachytene cells from testicular tissue have opened up a new avenue of research on meiotic recombination. Future studies on normal and abnormal recombination in early meiosis will further research in human reproduction and genetics. However, the availability of testicular material will always be a major limiting factor in this kind of study. In order to obtain an adequate number of samples and samples of particular research interest, it is often of benefit to obtain samples from distant regions. Therefore, it is necessary to determine whether the quality of samples and accuracy of MLH1 frequencies change after transporting testicular samples from a distance. In the present study, we examined the recombination frequencies (numbers of MLH1 foci using immunofluorescence techniques) in 6 normal testicular samples. Each sample was split and analyzed in the fresh state and after storage on ice for two days, mimicking overnight courier air transport. The results showed no significant difference in the quality of the SC preparations or in the number of MLH1 foci between these two groups. These results demonstrate that testicular specimens may be shipped on ice without compromising data on chromosome pairing and recombination in early meiosis.

Infertile men undergoing intracytoplasmic sperm injection have an increased frequency of chromosome abnormalities in their sperm. Men with low sperm concentration (oligozoospermia) have an increased risk of sperm chromosome abnormalities. This study was initiated to determine whether men with severe oligozoospermia (10(6) sperm/ml) have a higher frequency of chromosome abnormalities in their sperm compared with men with moderate (1-9 x 10(6) sperm/ml) or mild (10-19 x 10(6) sperm/ml) oligozoospermia. Multicolor fluorescence in situ hybridization analysis was performed using DNA probes specific for chromosomes 13, 21, X, and Y (with chromosome 1 as an autosomal control for the sex chromosomes). Aneuploidy and disomy frequencies were assessed from a total of 603,011 sperm from 30 men: 10 in each of the categories. The mean frequencies of disomy for the patients with mild, moderate, and severe oligozoospermia were 0.17%, 0.24%, and 0.30%, respectively, for chromosome 13 and 0.22%, 0.44%, and 0.58%, respectively, for chromosome 21. For the sex chromosomes, the mean frequencies of disomy for mild, moderate, and severe oligozoospermia were 0.25%, 1.04%, and 0.68%, respectively, for XY, 0.047%, 0.08%, and 0.10%, respectively, for XX, and 0.04%, 0.06%, and 0.09%, respectively, for YY. The frequencies for diploidy also increased from 0.4% for mild to 1.20% for moderate to 1.24% for severe oligozoospermia. There was a significant inverse correlation between the frequency of sperm chromosome abnormalities and the sperm concentration for XY, XX, and YY disomy and diploidy. These results demonstrate that men with severe oligozoospermia have an elevated risk for chromosome abnormalities in their sperm, particularly sex chromosome abnormalities.

Cigarette smoke contains chemi- cals which are capable of inducing aneuploidy in experimental systems. These chemicals have been shown to reach the male reproductive system, increasing oxidative DNA damage in human sperm and lowering semen quality. We have examined the association between smoking and aneuploid sperm by studying 31 Chinese men with similar demographic characteristics and lifestyle factors except for cigarette smoking. None of the men drank alcohol. These men were divided into three groups: nonsmokers (10 men), light smokers (

Human sperm karyotypes can be prepared after fusion of human sperm with Golden hamster oocytes. Most laboratories use one of two methods of sperm capacitation: incubation of freshly-ejaculated sperm in Biggers, Whitten, and Whittingham (BWW) medium for 5-7 h at 37 degrees C or sperm storage in (N-tris [hydroxymethyl]methyl-2-aminoethanesulfonic acid; 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid) (TES)-Tris yolk buffer (TYB) for 1-3 days at 4 degrees C. Since there have been conflicting reports as to whether there is a difference in the frequency of structural chromosomal abnormalities between BWW capacitation and storage in TYB for 2 days, we analyzed a larger number of karyotypes (8974) from 136 donors to determine if there was any difference in the frequency or type of chromosomal abnormalities in sperm treated by fresh BWW capacitation, storage in TYB for 1 day (TYB-1), or storage in TYB for 2 days (TYB-2). There was no difference in the frequency of numerical chromosomal abnormalities or sex ratio in any of the three treatment groups. However, there was a significantly increased frequency of structural chromosomal abnormalities after storage in TYB-1 and TYB-2. There was no difference in the frequency or type of structural chromosomal abnormalities after sperm storage in TYB-1 compared to TYB-2.

Infertile men with azoospermia now have the possibility of fathering children by testicular sperm extraction combined with intracytoplasmic sperm injection. However, there are concerns about the risk of chromosomal abnormalities in their spermatozoa. We have studied aneuploidy frequencies for chromosomes 13, 21, X and Y by multicolour fluorescence in-situ hybridization (FISH) in testicular spermatozoa extracted from three men with non-obstructive azoospermia. The men were 34-37 years of age and had normal follicle-stimulating hormone (FSH) concentrations and normal 46,XY somatic karyotypes. A total of 3324 spermatozoa was analysed. The infertile patients had an elevated frequency of disomy for chromosomes 13, 21, XY disomy compared to controls but none of these reached statistical significance. Also there was no significant difference in the sex ratio or the frequency of diploidy in azoospermic patients compared to normal control donors. This first report on chromosomal aneuploidy in spermatozoa extracted from testes of patients with non-obstructive azoospermia suggests that some azoospermic men do not have a substantially increased risk of chromosomally abnormal spermatozoa.

Sperm chromosomal abnormalities were assessed in testicular cancer patients before, during, and after BEP (bleomycin, etoposide, cisplatin) chemotherapy (CT). Multicolor fluorescence in situ hybridization (FISH) analysis was employed to detect aneuploidy for chromosomes 1, 12, X, and Y, and diploidy. Sperm samples were cryopreserved and coded before analysis to facilitate "blind" analysis. Complete results at all time points was available for only one patient. A total of 60,400 sperm were analyzed: 20,004 before CT, 20,005 during CT, and 20,391 after CT. There was a significant increase in the frequency of 24,XY sperm during (0.33%) and post-CT (0.34%) compared to pre-CT (0.14%). This study suggests that there may be a significantly increased risk of chromosomal abnormalities in sperm of CT patients during and immediately post-CT, similar to that shown in animal models.

Sperm samples from infertile men with oligozoospermia or teratozoospermia were studied by multicolour fluorescence in-situ hybridization (FISH) using DNA probes for chromosomes 13 and 21. A total of 90 809 sperm nuclei from nine infertile men and 182 799 sperm nuclei from 18 control donors were analysed. There was a highly significant increase in the frequency of spermatozoa disomic for chromosome 13 in infertile patients (0.28%) compared to control donors (0.13%) (two-tailed Z statistic P < 0.0001) and for chromosome 21 (0.48% in infertile men versus 0.37% in controls, P < 0.0001). Also there was a significantly increased frequency of diploid spermatozoa in infertile men (0.85%) compared to control donors (0.66%) (P < 0.0001). Our previous studies on these same infertile patients demonstrated increased frequencies of sperm disomy for chromosomes 1 and XY. This suggests that infertile men, who are prime candidates for intracytoplasmic sperm injection, may be at a very small increased risk of aneuploid offspring.

Human sperm chromosomes were studied in a man heterozygous for a pericentric inversion of chromosome (1)(p31q12). Q-banded pronuclear chromosomes were analyzed after in vitro penetration of golden hamster oocytes. A total of 159 sperm were examined: 54% bearing the inverted chromosome 1 and 46% the normal chromosome 1. These frequencies are not significantly different from the theoretical 1∶1 ratio. There were no recombinant sperm with duplications or deficiencies, suggesting that a pairing loop failed to form or that crossing-over was suppressed. The frequency of abnormalities unrelated to the inversion was 5% for numerical, 8.8% for structural, 2.5% for numerical and structural, values not significantly different from control donors studied in our lab. The frequencies of X- and Y-bearing sperm were 46% and 54%, respectively, not significantly different from the expected value of 50%. This is the fifth pericentric inversion studied by human sperm chromosome analysis; recombinant chromosomes have been observed in two of the five cases. Some of the factors associated with an increased risk of recombinant sperm appear to be inversion size greater than 30% of the chromosome and chromosome breakpoints in G-light bands.

A technique for obtaining preparations of human sperm chromosomes was introduced by E. Rudak et al (1978) which is based on the use of hamster oocytes to reactivate human sperm. Detailed descriptions of the methods used in our laboratory have been published elsewhere (Martin, 1983; Martin, 1988). The following procedures currently used in our laboratory are presented below.

Sperm chromosome complements were studied in three men who carried reciprocal translocations. A total of 400 sperm were karyotyped after in vitro penetration of hamster eggs: 217 sperm from t(2;9) (q21;p22), 164 from t(4;6)(q28;p23) and 19 from t(7;14) (q21;q13). All possible 2:2 and 3:1 meiotic segregations were observed for t(2;9) and t(4;6); for t(7;14) only 2:2 segregations were observed. For alternate segregations, the number of normal sperm was not significantly different from the number of sperm carrying a balanced form of the translocation in any of the translocations, as theoretically expected. The percentage of sperm with an unbalanced form of the translocation was 57% for t(2;9), 54% for t(4;6) and 47% for t(7;14). There was no evidence for an interchromosomal effect in any of the translocations since the frequencies of numerical abnormalities (unrelated to the translocation) were within the normal range of control donors. The frequencies of X- and Y-bearing sperm did not differ significantly from 50%. Results from a total of 17 reciprocal translocations studied by sperm chromosomal analysis were reviewed.

Sperm chromosome complements were studied in a man who carried a reciprocal translocation t(12;20)(q24.3;q11). A total of 113 spermatozoa were karyotyped after in-vitro penetration of hamster eggs. 2:2 and 3:1 meiotic segregations were observed with the following frequencies: alternate 47%, adjacent 1 42%, adjacent 2 10%, 3:1 2%. For alternate segregations, the number of normal spermatozoa (25) was not significantly different from the number of spermatozoa carrying a balanced form of the translocation (28), as theoretically expected. The proportion of spermatozoa with an unbalanced form of the translocation was 53%. There was no evidence for an interchromosomal effect since the frequencies of numerical and structural abnormalities (unrelated to the translocation) were within the normal range of control donors.

Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia