Purification and characterization of nuclear basic proteins of human sperm.

Highly purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein. Microheterogeneity observed in some of these protamines originates from slight variations in their amino acid composition as well as from post-synthetic modifications. Two of these protamines previously considered as two different proteins are in fact the same protein with different degrees of phosphorylation. All these protamines and intermediate basic proteins are characterized by high amounts of arginine and cysteine. Three of the protamines and all five intermediate basic proteins are also histidine-rich.

A high frequency of Y chromosome deletions in males with nonidiopathic infertility.

Microdeletions of the long arm of the human Y chromosome are associated with spermatogenic failure and have been used to define three regions of Yq (AZFa, AZFb, and AZFc) that are recurrently deleted in infertile males. In a blind study we screened 131 infertile males (46 idiopathic and 85 nonidiopathic) for Y chromosome microdeletions. Nineteen percent of idiopathic males, with an apparently normal 46,XY chromosome complement had microdeletions of either the AZFa, AZFb, or AZFc region. There was no strict correlation between the extent or location of the deletion and the phenotype. The AZFb deletions did not include the active RBM gene. Significantly, a high frequency of microdeletions (7%) was found in patients with known causes of infertility and a 46,XY chromosome complement. These included deletions of the AZFb and AZFc regions, with no significant difference in the location or extent of the deletion compared with the former group. It is recommended that all males with reduced or absence sperm counts seeking assisted reproductive technologies be screened for deletions of the Y chromosome.

A genetic study of angiotensin I-converting enzyme levels in human semen.

The plasma level of angiotensin I-converting enzyme (ACE) has been shown to be under genetic control. An insertion/deletion polymorphism in the ACE gene is associated with differences in the level of ACE in the plasma and inside T-lymphocytes. An ACE isoform is present in large amounts in spermatozoa and is expressed under an alternative, germ cell-specific promoter, whereas ACE present in the seminal fluid is the somatic form of ACE. We have investigated the effect associated with the I/D polymorphism on the level of ACE in seminal fluid and in spermatozoa. No differences in the level of ACE measured in the seminal fluid or in the spermatozoa were associated with the ACE I/D genotypes. We conclude that the modulation of expression associated with the I/D polymorphism is restricted to the somatic ACE promoter. These results also suggest that if one allele modulating the expression of ACE was under positive selection pressure, it was not through an effect on the semen concentration of ACE.

Another case of trisomy 4 with double minute chromosomes in acute non-lymphocytic leukemia.

A further case of trisomy 4 with double minute chromosomes in acute non-lymphocytic leukemia is reported. The non-random association between these two cytogenetic abnormalities is reinforced. A possible relation with environmental exposure is discussed.

Acute myelomonocytic leukemia in a XYY man.

A case of acute myeloid leukemia (M4) in a 29-year-old male with a 47,XYY karyotype is reported. This aneuploidy was found in both bone marrow cells and mitogen-stimulated lymphocytes. Monosomy 7 correlated with myelodysplastic features. The possible role of XYY in increasing the risk of leukemia is discussed.

Human sperm mitochondrial function related to motility: a flow and image cytometric assessment.

Current evaluation of male fertility, routinely estimated by sperm count, motility, and morphology, provides only crude information about the fertility state of individuals. Both flow and image cytometry were applied to mitochondrial activity and sperm motility respectively. Sperm samples from fertile donors were concomitantly measured for Rhodamine 123 (Rh123) uptake (an estimation of mitochondrial activity), percentage of dead cells, and motility characteristics, such as percentage of motility, curvilinear velocity, and amplitude of lateral head displacement. These measurements were done under experimental conditions known to modulate sperm motility (temperature and time course survival in a capacitating medium). Bimodal distributions were found for Rh123 uptake. Flow cytometry-derived parameters were essentially time-dependent whereas motility characteristics were primarily temperature-dependent. Correlations were found between various flow cytometry-derived parameters and motility characteristics. Most of the correlations were obtained after a 24 h incubation in a capacitating medium. The most significant correlation in every experimental condition concerned the percentage of motile spermatozoa and the Rh123 uptakes. The drop in motility observed after a 24 h incubation was paralleled by a markedly lower drop in mitochondrial activity. The data suggest that these two complementary techniques represent an improvement in basic and/or clinical assessment of the functional spermatozoa status.

The nuclear status of human sperm cells by TEM image cytometry: nuclear shape and chromatin texture in semen samples from fertile and infertile men.

Changes in nuclear size, shape, and chromatin texture during spermiogenesis and epididymal transport of human sperm were recently analyzed using transmission electron microscopy (TEM) image cytometry followed by multivariate statistical analysis of data. In the present study, this same methodology was used to investigate the nuclear morphology of spermatozoa in semen samples from fertile and infertile men. Analysis was carried out on a large series of micrographs of sections of sperm nuclei from a donor group with proven fertility and from a patient group with a mean infertility duration of 10 years with no obvious male or female infertility factors (only a slight decrease in the proportion of sperm heads with normal morphology was noted in routine semen tests). For the patient group, it was found that nuclei had a significantly less flattened shape (i.e., increased roundness as a consequence of increased thickness and decreased length). Furthermore, significant differences between donor and patient groups were found for most parameters of chromatin texture. In the patient group, chromatin was less condensed, and there was more homogeneous distribution of the different degrees of chromatin condensation. In addition, the organization of chromatin condensation and distribution along the major axis of the nucleus was found to be significantly different in the two groups. Stepwise linear discriminant analysis indicated a good classification rate of only 66% for nuclei of patients when using the eight major nuclear parameters, thus indicating the striking heterogeneity of nuclear morphology for both patient and donor groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The nuclear status of human sperm cells.

In the last decade, and in particular since the development of in vitro fertilization techniques, the nuclear status of human sperm cells has shown to be a key parameter in the assessment of male fertility. The shape and condensed state of the mature sperm nucleus are determined by structural and functional events that occur during spermiogenesis. This paper reviews essential findings on re-organization of the nucleus during sperm differentiation and maturation, and reports recent data on the architecture, biochemical composition and stability of the nucleus in human ejaculated spermatozoa. Different methods used to evaluate nuclear maturity in relation to male fertility are critically appraised.

Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa.

The distribution of actin in hamster sperm cells was studied during spermiogenesis, epididymal transit, in vitro capacitation and acrosome reaction by immunogold procedures using a polyclonal and two monoclonal antiactin antibodies. A predominant actin labeling (F-actin) was detected in the subacrosomal space of spermatids. Actin labeling was also observed under the plasma membrane of intercellular bridges and along the outer acrosomal membrane. In late spermatids there was both F-actin depolymerization and a loss of actin immunolabeling, thus suggesting a dispersion of G-actin monomers. No obvious labeling was evidenced in residual bodies. This pattern was observed with the three antiactin probes. In contrast, an actin labeling reappeared over the fibrous sheath of the flagellum in epididymal spermatozoa but only when the polyclonal antibody was used. Only one single actin reactive band was detected by immunoblotting of sperm extracts. Since the sperm tails were NBD phallacidin negative they were considered to contain either G-actin or actin oligomers rather than bundles of actin filaments. It is suggested that G-actin originating in the head of late spermatids was redistributed to the flagellum of epidymal spermatozoa. No further changes were noted after capacitation and acrosome reaction thus indicating no apparent effect on actin polymerization and distribution.

[The testis in a 46, XX male. Apropos of an ultrastructural examination]. / Le testicule chez l'homme 46 XX. A propos d'une observation ultrastructurale.

The ultrastructure of a testicular biopsy was carried out in a man with 46 XX chromosomes who had a masculine phenotype and in whom the hormone levels showed that the interstitial tissue of the gland was functioning well. Observations showed that the exocrine appearance of the testis was comparable to that described in Klinefelter's syndrome, namely germinal aplasia with invasion by Sertoli cells. On the other hand the interstitial glandular tissue showed morphological characteristics associated with normal androgen activity, which is unusual to find in Klinefelter's syndrome. Lipid inclusions, pigmentation and Reinke cristalloids were found. The cytogenetic techniques that happened to be used did not manage to show up even a small fragment of the Y chromosome. Various theories have been considered to explain the constitution of such a phenotype with this chromosome abnormality.

Identification of transcripts by macroarrays, RT-PCR and in situ hybridization in human ejaculate spermatozoa.

Round spermatids contain high levels of extremely varied mRNAs that are synthesized either throughout early spermatogenesis or during spermiogenesis from the haploid genome. Concomitantly, with major changes in the chromatin organization, arrest of transcription occurs at midspermiogenesis. However, previous investigations using RT-PCR have revealed the persistence of numerous and different transcripts in ejaculated spermatozoa. In the present study, a step-by-step analysis by means of macroarray hybridization, RT-PCR and in situ hybridization was performed to identify more accurately the different mRNA species found in the human ejaculated spermatozoa. The data showed an extended pattern of various transcripts encoding a diverse range of proteins involved in signal transduction and cell proliferation. For the first time, they demonstrated that mRNAs coding for the transcription factors NFkappaB, HOX2A, ICSBP, protein kinase JNK2, growth factor HBEGF and receptors RXRbeta and ErbB3 accumulate within the sperm nucleus. The origin and fate of the sperm transcripts remain subject to discussion.

Ultrastructural abnormalities of human spermatozoa.

The observation of sperm cells in the scanning and electron microscope reveals that malformed spermatozoa show either a single anomaly of each of their structural components--acrosome, nucleus, axoneme and accessory structures--or a combination of these anomalies. A defined semen profile can be established when the majority of ejaculated spermatozoa present a predominantly single anomaly or the same combination of associated anomalies. Only in these cases can there be a relationship between morphological defects and impairment of the fertilizing ability. The validity of the ultrastructural examination of sperm cells depends upon the data obtained by light microscopy, and quantification of the frequencies of the ultrastructural alterations is necessary to define clearly the limits of abnormality.

The cellular biology of mammalian spermatids: a review.

During spermiogenesis, young spermatids undergo complex morphological, biochemical and physiological changes that result in the formation of highly polarized flagellated spermatozoa. Many of the changes that occur during this time are essential for the production of fertile sperm. Spermiogenesis includes modifications of the nucleus and perinuclear organelles (perinuclear theca, manchette), formation of the acrosomic system originating from the Golgi apparatus, assembly of the tail structures, topographical arrangement of the cell surface and cytoplasmic reorganization the final phase of which results in release of spermatozoa into the lumen of seminiferous tubules. A number of genes, including the protamine genes, are transcribed in haploid round spermatids. Furthermore, sequential expression of a few protooncogenes takes place during spermiogenesis. Both nuclear and cytoplasmic proteins are synthesized by spermatids. Numerous findings show clear functional relationships between late spermatids and Sertoli cells. Sertoli cell products may directly affect late spermatid development and metabolism. In turn, late spermatids may regulate Sertoli cell function, presumably via residual bodies. The ability for spermatids to modulate the functional activity of Sertoli cells is demonstrated by recent data on cyclin-protein-2, beta nerve growth factor and cytokines that are involved in gonadal cell-cell interaction.

Use of the acridine orange staining on smears of human spermatozoa after heat-treatment: evaluation of the chromatin condensation.

A simple, rapid procedure was described to evaluate the degree of chromatin compactness in human spermatozoa. Smears of fixed spermatozoa were heat-treated and stained with acridine orange. Smears from the same sperm samples were also stained with acidic aniline blue. Three groups of semen could be distinguished according to the percentage of red sperm heads observed under fluorescence microscope. 1) Semens with spermatozoa whose chromatin appeared normal before and after heat-treatment. 2) Semens with spermatozoa whose chromatin appeared normal before and abnormal after heat-treatment. 3) Semens with spermatozoa whose chromatin appeared abnormal before and after heat-treatment. The positive correlation between the percentages of red heads and the percentages of blue stained heads suggests that modifications in the biochemical composition of the basic protein component associated with DNA are responsible for the denaturation process.

Quantitative ultrastructural analysis of the principal cells in the human epididymis.

A morphometric study of the principal epithelial cells of the epididymis has been carried out in six young adult men. Samples were taken in the head, body and tail of the epididymis and processed for ultrastructural study. The cytoplasm was subdivided into four zones. Their section areas were measured by planimetry and the volume density of cytoplasmic organelles assessed using the point-counting method. The general organization of the principal cells of the epididymis was identical along the duct. Quantitative analysis revealed that cells of the head contained significantly larger amounts of coated-vesicles and of Golgi saccules than the body and the tail. The functional implications of these results is important for the understanding of receptor-mediated endocytosis and protein synthesis in the different parts of the human epididymal duct.

Computerized sperm motility and application of sperm cryopreservation.

An objective method for measuring sperm motion characteristics was developed on an Intellect 100 Quantel Image Analysis System suitable for various image cytometric applications. It provided overall analysis of percent motility (% MS) as well as individual and mean measurements of motion characteristics, including vigor characteristics such as curvilinear velocity (Vc), straight line velocity (Vsl), and trajectory pattern characteristics, that is, progressiveness ratio (PR) and amplitude of lateral head displacement (Alh). Evaluation of the method for reproducibility and accuracy showed reliable measurements of these parameters measured on a minimum of 70 motile sperm, sufficient to describe adequately the sperm population. A study was performed comparing motion characteristics of 30 semen samples falling in a normal range before and after cryopreservation in cryoprotector medium (CM). A mean motility rate of recovery (MRR) of 45% was obtained. Only sperm count and concentration in motile forms among initial semen variables correlated weakly with MRR. Velocity recovery rate (VRR) approached 1 with a marked variability among ejaculates. Distribution profile of Vc was highly modified by freezing in CM: spermatozoa that were initially fast and progressive were the most resistant to cryoaggression. PR and Alh values were little affected by freezing in CM. The tolerance of various samples from a given patient was highly variable for % MS and Alh and less variable for Vc and PR. This illustrates the difficulty in predicting the effect of freezing on motility characteristics and, therefore, of extrapolating from semen variables the ability of frozen-thawed samples to fertilize.

Autoradiographic investigation of sperm transit through the male mouse genital tract after tritiated thymidine incorporation.

The transit of spermatozoa in the genital tract of the male mouse was investigated by quantitative light microscopic autoradiography after intraperitoneal injection of tritiated thymidine. Transit duration in the caput and the corpus of the epididymis was shown to be 3 days; the total duration of transit in the genital tract was 5 days. These findings indicate that the time required for the transit of spermatozoa in the epididymal caput and corpus was comparable to that calculated in other mammals studied. However, the duration of sperm storage in the epididymal cauda appeared to be shorter than that previously reported for rodents.

[Structural anomalies in disorders human of sperm motility in 60 cases of asthenospermia]. / Les anomalies de structure dans les troubles de la mobilité du spermatozoïde humain a propos de 60 cas d'asthénospermie.

The morphological study of sperm cells was carried out in sixty cases of proven asthenospermia. In fifteen of the cases, structural abnormalities, related to impaired cell mobility, have been shown, namely when spermatozoa, which were immobile in a large majority, presented short tails or characteristic ultrastructural abnormalities of the axonemal complex.