Nuclear Vacuoles Research Articles

Bladder Cancer Therapy Related Histopathologic Changes

Various forms of modern therapy of urinary bladder cancer are associated with characteristic changes. Surgical therapy related morphology alterations usually do not present significant differential diagnostic problems. Postoperative spindle cell nodule may resemble a spindle cell sarcoma, but the fact that it arises within 120 days following surgery makes the diagnostic interpretation less cumbersome. Changes induced by BCG include epithelial denudation of the bladder, small superficial granulomas and chronic inflammation. Other forms of immunotherapy are associated with formation of lymphatic follicles or diffuse lymphocytic infiltrates. Radiation induced changes may appear many years after therapy and include atypia, bizarre appearance and multinucleation of urothelial cells. In the late phase, stroma is particularly affected. It is important not to misdiagnose radiation induced changes for CIS or invasive nested carcinoma. Chemotherapy-induced atypia may be recognized cytologically and histologically by the characteristic changes of the nuclear/cytoplasmic ratio, multiple nuclei, nuclear vacuolization, and architectural disturbances in the bladder epithelium. Photodynamic therapy induces sharply demarcated necrosis in the short post-therapy course. Laser treatment destroys the tumor, and may induce atypia in adjacent endothelial cells. Gene therapy-related changes have not been well defined yet.

Sperm characteristics and ultrastructure of testes of rats after long-term treatment with the methanol subfraction of Carica papaya seeds.

The contraceptive efficacy of Carica papaya seeds after short-term evaluation has been well established. We have examined the safety and mechanism of contraception in rats after long-term treatment with the methanol subfraction (MSF) of C. papaya seeds. The test substance was administered orally to the male albino rats (n = 40) at 50 mg per kg body weight each day for 360 days. Control animals (n = 40) received olive oil as a vehicle. Recovery was assessed up to 120 days after treatment withdrawal. Sperm parameters, serum testosterone levels, fertility, histology and ultrastructure of the testis, haematology and serum clinical chemistry were evaluated to establish the safety and efficacy of the test substance. Safety of long-term treatment was evidenced by unaltered health status, organ weight, haematology and clinical chemistry, and by an increase in body weight. The mechanism of contraception was shown by reduction in nuclear and cytoplasmic volume, normal nuclear characteristics and vacuolization in the cytoplasmic organelles of the Sertoli cells, as well as nuclear degeneration in spermatocytes and spermatids indicating disturbed spermatogenesis. Leydig cells were normal. Initial effects were observed in Sertoli cells at 60 days of treatment. Spermatocytes and spermatids were affected after 120-240 days of treatment. A significant decline in sperm count and viability, total inhibition of sperm motility, increased numbers of sperm abnormalities, normal serum testosterone levels and 100% sterility were evident after 60 days of treatment. All the altered parameters, including percent fertility, were restored to control level 120 days after treatment withdrawal. It is concluded that the MSF is safe for long-term treatment and the mechanism of contraception is shown by its effect on spermatid differentiation in the testis, possibly mediated by the Sertoli cell factors.

Ultrastructural observations on nucleolar changes during mouse spermiogenesis.

When applying a new silver staining technique on developing mouse spermatids, it could be shown that strong argyrophilia in the "padlock" like nucleolus of very early spermatids is confined to its fibrillar and granular component, whereas the fibrillar centre is devoid of silver. During further steps the granular component disappears together with the fibrillar centre. The last remaining nucleolar part, the silver positive fibrillar component disintegrates at the beginning of nuclear elongation. Instead of it clusters of coiled fibers bordered with granules of approximately 40-60 nm in diameter, both silver positive, appear in the nucleoplasm. As chromatin condensation proceeds, these silver positive structures, now intimately attached to the centrally occurring focus of condensing chromatin decrease more and more in size and density. In later stages accumulations of silver positive material will appear in the posterior region of the nucleus, will leave it, and stays as silver positive "juxtanuclear body" in the nuclear pocket formed by the redundant nuclear envelope. As spermatid development continues, the "juxtanuclear body" disappears together with the nuclear pocket. The small silver positive fibrous clusters disintegrate too so that the mature sperm only contains the space in which they formerly existed, now called "nuclear vacuole". A possible connection between silver staining pattern and RNA synthesis is discussed.

On the origin of nuclear vacuoles in spermatozoa a fine structural and cytochemical study in mice.

Morphological and cytochemical changes in nucleoli of spermatids of the mouse during spermiogenesis were examined. Thus, it could be shown that the nucleolar derivatives of the elongation-phase, clusters of coiled fibers that are bordered with spheroidal bodies, show comparable chemical properties as the precursor structure, the fibrillar component, which is known for its transcriptional activity. With progressing development, these nucleolar derivatives continually decrease in size and density, preserving their chemical properties. They persist far beyond into the maturation-phase and will decrease at the time chromatin condensation appears finished. There only remain the spaces in which they formerly existed, now called nuclear vacuoles. A possible function of nuclear vacuoles with respect to chromatin decondensation during the process of fertilization is mentioned.

Antitumor effects of polysorbate-80 coated gemcitabine polybutylcyanoacrylate nanoparticles in vitro and its pharmacodynamics in vivo on C6 glioma cells of a brain tumor model

Polybutylcyanoacrylate (PBCA) nanoparticles coated with polysorbate-80 have been extensively studied for delivery of drugs into the animal models; however, 1% polysorbate-80 coated gemcitabine PBCA nanoparticles (GCTB-PBCA-NPs) remain unknown. In this study, we investigated the inhibitory effects of brain targeted 1% polysorbate-80 coated GCTB-PBCA-NPs on C6 glioma cells in vitro and in vivo. GCTB-PBCA-NPs were prepared by emulsion polymerization and freeze drying. C6 glioma cells treated with 1% polysorbate-80 coated GCTB-PBCA-NPs showed poor growth with less cell density and increased detachment. Cell morphology was also greatly altered with nuclear vacuoles, ruptured cells and dead cells. Meanwhile, by flow cytometry, the numbers of cells treated with 1% polysorbate-80 coated GCTB-PBCA-NPs showed increase in G0/G1 phase and decreased in the S phase ( P < 0.01) compared with the blank control. CCK-8 assay also showed that GCTB could significantly inhibited cell proliferation in a concentration dependent manner. Finally, various preparations were injected (90 mg preparation per kg body weight) into the brain tumor model, which was produced after inoculating C6 glioma cells into Sprague Dawley (SD) rats for 14 days, it was shown that 1% polysorbate-80 coated GCTB-PBCA-NPs could significantly extend the survival time compared with the saline control ( P < 0.05). Taken together, 1% polysorbate-80 coated GCTB-PBCA-NPs can effectively inhibit the growth of C6 glioma cells in vitro and enhance antitumor activity on brain tumor in vivo.

Semen Characteristics in a Sub-Fertile Arabian Stallion with Idiopathic Teratospermia

A 5-year-old Arabian stallion was managed for breeding with fresh/extended semen during a period of 8 months with a resulting per cycle pregnancy rate of 26.3%. The stallion was in good health and no abnormalities of the reproductive tract were observed. Evaluation of several ejaculates revealed that sperm production and semen quality were mostly unchanged during the period of evaluation, that sperm production was normal and that semen quality was extremely poor. The most prevalent sperm defects were abnormal heads and mid-pieces. Most abnormal heads were microcephalic and/or tapered and considerable variation in sperm head dimensions within and among ejaculates was observed. A unique defect characterized by swollen/roughened mid-piece caused by accumulation of cytoplasmic-like material and abnormal mitochondrial sheath was observed. Nuclear vacuoles, acrosome defects, and teratoids were also prevalent and most sperm presented multiple abnormalities. The absence of any clear cause or any signs of testicular degeneration, combined with normal sperm production, and constant abnormal sperm production suggest an inherent, congenital disturbance of spermatogenesis as the cause of teratospermia in this case.

Ultrastructure and cytochemistry of vitellogenesis in Wenyonia virilis Woodland, 1923 (Cestoda, Caryophyllidea)

AbstractVitellogenesis in Wenyonia virilis was examined by transmission electron microscopy (TEM), including the cytochemical detection of glycogen at the ultrastructural level with the periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) technique. Mature vitelline follicles have cells in various stages of development, progressing from immature cells of gonial type near the periphery of the follicle to maturing and mature vitellocytes towards the centre. Maturation is characterized by: (1) increase in cell volume; (2) increase in nuclear surface area restoring the N/C (nucleo-cytoplasmic) ratio; (3) nucleolar transformation; (4) extensive development of parallel cisternae of GER, the shell-protein producing units; (5) development of Golgi complexes, engaged in shell-granule/shell-globule formation and packaging; (6) synthesis and storage of glycogen in the cytoplasm; (7) simultaneous, independent formation and storage of intranuclear glycogen; (8) continuous fusion of small shell-granules into larger shell-globules and fusion of these into large shell-globule clusters with a progressive increase in the number and size of the latter; and (9) disintegration of GER in the medial layer of vitellocyte cytoplasm, degenerative changes and accumulation of glycogen and shell-globule clusters within the cytoplasm. The functional significance of numerous shell-globule clusters and the relatively small amount of nuclear and cytoplasmic glycogen is analysed. Unlike vitellogenesis of other caryophyllids, the nuclear glycogen of mature vitellocytes in W. virilis is randomly dispersed in the nucleoplasm and never forms a high central accumulation, the so-called “nuclear vacuole”. The nutritive function of vitellocytes appears greatly reduced in W. virilis, a fact perhaps related to the intrauterine development of the early embryos. The ultrastructure of vitellogenesis in W. virilis is compared with that in other lower cestodes, both monozoic and polyzoic. Conclusions concerning interrelationships of the vitellogenesis pattern of the ultrastructural cytochemistry of mature vitellocytes of W. virilis to intrauterine embryonation, absence of uterine glands and an extensive uterus characteristic for this species, are drawn and discussed.

Qualité et sélection du spermatozoïde

Selection of a live and morphologically normal spermatozoa is a fundamental stage in ICSI success because of its potential effects on early and late embryo development. In addition to the routine tests such as the spermogram and the spermocytogram, a number of tests have been developed for this purpose : the hyaluronic acid test, which measures the rate of DNA fragmentation using TUNEL (% of fragmented DNA) or electrophoresis separation of SPZ, SPZ evaluation using the FISH method, MSOME (motile sperm organelle morphology examination) techniques, et IMSI (intracytoplasmic morphologically selected sperm injection), which can evaluate the nuclear vacuoles, etc.

Human Sperm Ultrastructures and Fertility

Abstract Human sperm has functional domains on the head and tail (flagellum). Since functional molecules are organized into the substructures of these domains, sperm with good appearance are expected to be fertile. Thus, it is of interest to see how sperm fine morphologies relate to fertility. The sperm head is divided into the acrosome and postacrosome regions. The acrosome region is further divided into the anterior acrosome for the acrosome reaction and posterior acrosome (equatorial segment) for gamete membrane fusion. The postacrosome region is involved in egg activation. In addition, the human sperm head has varieties of nuclear vacuoles. High-resolution light microscopy, which has recently been used in IMSI (intracytoplasmic morphologically selected sperm injection), is helpful in identifying vacuoles. However, IMSI is still insufficient for analysis of the contents of vacuoles; electron microscopy and evaluation tests for sperm DNA damage are more helpful for profound analysis of the nature of nuc...

Effects of scrotal insulation in Nellore bulls ( Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30–36-month-old, 500–550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1–5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased ( P < 0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 ± 0.6 and 17.0 ± 0.8, respectively; mean ± S.E.M.) was higher ( P < 0.05) in agreement with the incidence of nuclear vacuoles (14.0 ± 5.0 and 12.3 ± 2.3) and abnormal chromatin (24.4 ± 7.2 and 30.8 ± 2.8). Although the frequency of cleaved oocytes decreased only on 21 d ( P < 0.05), blastocyst rates were lower ( P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage ( R 2 = 0.38, 0.48, and 0.30, respectively) and blastocyst rates ( R 2 = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates ( R 2 = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, Bandicota indica

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.

Ultrastructural findings in semen samples of infertile men infected with Chlamydia trachomatis and mycoplasmas

The aim of this study was to describe the ultrastructural features observed in semen samples of men with infertility and subfertility of unknown cause infected with Chlamydia trachomatis and mycoplasmas. The findings observed by ultrastructure included destruction or persistence of bacteria in leukocytes, phagocytosis of spermatozoa by leukocytes, and structural damage of spermatozoa.

Sertoli Cell Morphological Alterations in Albino Rats Treated with Etoposide during Prepubertal Phase

Sertoli cells are very important to spermatogenesis homeostasis because they control germ cell proliferation, differentiation, and death. Damages to Sertoli cells cause germ cell death and affect fertility. Etoposide is a potent chemotherapeutic drug largely used against a variety of cancers. However, this drug also kills normal cells, especially those undergoing rapid proliferation. In the testis, etoposide acts predominantly on intermediate and type B spermatogonia. Etoposide was shown to permanently alter Sertoli cell function when administered to prepubertal rats. Based on this, we decided to investigate whether etoposide can affect Sertoli cell morphology. For this, 25-day-old rats were treated with etoposide during 8 consecutive days and killed at 32, 45, 64, 127, and 180 days old. Testes were fixed in Bouin's liquid or in a mixture of 2.5% glutaraldehyde and 2% formaldehyde for analysis under light and electron microscopes, respectively. Sertoli cells showed morphological alterations such as the presence of chromatin clumps close to the nuclear membrane, nucleus displacement, and cytoplasmic vacuolization. Some Sertoli cells also showed nuclear and cytoplasmic degenerative characteristics, suggesting that etoposide causes severe damages to Sertoli cell.

High-power microscopy for selecting spermatozoa for ICSI by physiological status

Sperm selection for intracytoplasmic sperm injection (ICSI), based on standard morphology, can fail to select normal cells, and actual methods to evaluate their physiological status do not allow their later use for ICSI. Some authors have demonstrated that sperm selection based on high-magnification morphology is associated with a better ICSI outcome, above all in subjects with severe testicular failure. In this study there was an evaluation of mitochondrial function, chromatin structure and sperm aneuploidies on whole sperm samples from 30 subjects: 10 normozoospermic controls and 20 patients that were severely oligozoospermic due to testicular damage or partial obstruction of the seminal ducts. All severely oligozoospermic patients showed worse mitochondrial function and chromatin status, while sperm aneuploidies were significantly increased only in those subjects with severe testicular damage ( P < 0.001). In the latter patients the analysis of a single spermatozoon, performed after morphological selection by high-magnification microscopy, showed significantly better mitochondrial function, chromatin status and aneuploidy rate than observed in unselected cells (all P < 0.001). Interestingly, these parameters were further improved when nuclear vacuoles were lacking. These results suggest a strong relationship between high-magnification morphology and the status of spermatozoa, and they may explain the better results of ICSI obtained using spermatozoa selected by high-magnification microscopy.

What’s the signification of nuclear vacuoles in human sperm?

What’s the signification of nuclear vacuoles in human sperm?

Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles

Spermatozoa selection at high magnification before intracytoplasmic sperm injection seems to be positively associated with pregnancy rates after day 3 embryo transfers. The aim was to demonstrate an association between the presence of vacuoles in sperm nuclei and the competence of embryos to develop to day 5. Grading of spermatozoa at ×6000–×12,500 magnification: grade I, no vacuoles; grade II, ≥2 small vacuoles; grade III, ≥1 large vacuole; grade IV, large vacuoles with other abnormalities. The outcome of embryo development in a group of 25 patients after sibling oocyte injection with the four different grades of spermatozoa showed no significant difference in embryo quality up to day 3. However, the occurrence of blastocyst formation was 56.3 and 61.4% with grade I and II spermatozoa respectively, compared with 5.1% with grade III and 0% with grade IV respectively ( P < 0.001). Spermatozoa selection at high magnification using Nomarski interference contrast is useful to identify more precisely the size and the number of nuclear vacuoles that greatly exert a negative effect on embryo development to the blastocyst stage. These observations confirm previous studies pointing to possible ‘early and late paternal effects’, both of which may have an impact on early embryonic development.

Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at ×8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher ( P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher ( P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.

Les indications de l’IMSI

ICSI failures (essentially fertilization and implantation failures) can be due to the quality of the oocytes, the spermatozoon injected and its manipulation, uterine factors and paternal factors, such as DNA fragmentation of the spermatozoa. Pre-selection of the spermatozoon is the crucial phase for ensuring successful ICSI. The characteristics of the sperm must be checked under the microscope at a magnification of x 6600 as certain abnormalities, in particular nuclear vacuoles, cannot be seen at x 300. In our centre, we conducted a study on 72 patients which showed that with more than 30% of fragmented DNA, neither implantation or birth was achieved using ICSI. Performing IMSI on the same patients allowed us to obtain implantation rates of 17.4% with 30 to 40 % of fragmented DNA and of 33.3% above 40%. Births were achieved in 17.4% and 28.6% of cases respectively. IMSI has the advantage of allowing extremely careful selection of the spermatozoon microinjected, so that it is of normal shape, with the least vacuoles possible and a low DNA fragmentation rate. It is, however, a complicated technique that cannot be routinely performed.

Hypothermia-related testicular toxicity of reserpine in mice

Local cooling of the testis was reported to cause testicular abnormality, but there is no information regarding the effect of whole body cooling, such as hypothermia on spermatogenesis. We investigated whether hypothermia would cause testicular toxicity in mice. Male mice were administered with a single intraperitoneal dose of 100 mg/kg of reserpine. The rectal temperature decreased to 29 °C 6 h post dosing and thereafter sustained at lower values (24−25 °C) until 96 h post dosing. The histopathological examination of the testis showed nuclear vacuolation of round spermatids at stages I−V in mice examined 24, 48, 72 and 96 h post dosing. The lesions were more severe in the groups examined 72 and 96 h post dosing than in the groups examined 24 and 48 h post dosing. These results suggested that hypothermia condition at lower than 30 °C and sustained for more than 18 h could induce nuclear vacuolation of round spermatids at stages I−V in the testis. In order to demonstrate that the testicular lesion in the reserpine-treated mice was induced by hypothermia, mice were given a single intraperitoneal dose of 100 mg/kg of reserpine, and each dosed group was housed under different environmental conditions for 72 h; one consisted of housing the mice individually at room temperature, and the other consisted of housing the mice individually under heated condition. In the mice administered with reserpine and housed at room temperature, hypothermia below 30 °C with a minimal of 26 °C was observed for 66 h. In contrast, the mice administered with reserpine and housed under heated condition maintained the rectal temperature of 33−36 °C. Nuclear vacuolation of round spermatids at stages I−V in the testis was observed in reserpine-treated mice maintained at room temperature, but not in reserpine-treated mice kept under heated condition. These data strongly indicated that nuclear vacuolation of round spermatids at stages I−V observed in reserpine-treated mice was related to hypothermia.

Detection of the intranuclear microsporidium Nucleospora salmonis in naturally and experimentally exposed Chinook salmon Oncorhynchus tshawytscha by in situ hybridization

Nucleospora salmonis, an intranuclear microsporidian parasite of salmonid fish, is often difficult to observe in histological sections or wet mount preparations from lightly infected tissues because of its small size and location within the nuclei of lymphoblast-type cells. Diagnosis of infections by conventional light microscopy is directly dependent upon distinguishing different stages of the parasite from host cell nuclear material or vacuoles. To assist detection of stages of the parasite in tissues of its primary host, the Chinook salmon (Oncorhynchus tshawytscha), we developed a nonradioactive in situ hybridization (ISH) method. The new method was then used to detect N. salmonis among Chinook salmon after both natural and experimental exposures to the parasite. Probes derived from the small subunit ribosomal DNA (ssu-rDNA) sequence of the microsporidium were labeled with digoxigenin deoxyuridine triphosphate (DIG-dUTP) and hybridized to parasite DNA present in infected tissues. The ISH procedure effectively identified merogonic and spore stages of N. salmonis in paraffin-embedded tissues of clinically and subclinically infected fish. A Nucleospora-like microsporidium was also detected by ISH in tissues of a nonsalmonid fish, the English sole (Pleuronectes vetulus), using probes designed to a region of the ssu-rDNA of N. salmonis.