Electron microscopy analysis of histone acetylation and DNA strand breaks in mouse elongating spermatids using a dual labelling approach

Chromatin remodelling steps in mammalian spermatids include post‐translational modifications of histones and DNA fragmentation. Histone H4 hyperacetylation (AcH4) establishes a chromatin state that facilitates DNA repair in somatic cells. So we sought to determine whether a similar link exists in spermatids by combining immunogold labelling with detection of DNA strand breaks, making use of gold particles of different sizes. DNA strand breaks were not detected in the vicinity of AcH4 chromatin, suggesting that this modified histone may not be involved in the aetiology of DNA fragmentation and repair in spermatids. The AcH4 reactivity, however, indicates that chromatin remodelling is distributed throughout the nucleus.     

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.     

Changes Induced to Bull Spermatids and Testicular Spermatozoa by a Single Peritesticular Injection of Ethylene Dibromide

Peritesticular injection of ethylene dibromide to bulls resulted in misshaping, abnormal chromatin condensation and decrease of UV-DNA and UV-protein contents of elongating spermatids.     

Effects of lithium carbonate on rat seminiferous tubules: an ultrastructural study

Lithium salts are commonly used for treatment of bipolar disorder but prolonged treatment with therapeutic doses induces substantial toxic effects. In the present study we examined the effects of lithium carbonate on the ultrastructure of rat seminiferous tubules. Rats were exposed to lithium carbonate at doses of 35 mg/kg/day for 21 days. After lithium treatment, the tunica propria widened and folded together with convolutions of the basement membrane, myoid cells and lymphatic endothelium. In the seminiferous epithelium loss of germ cell attachment and appearance of expanded intercellular spaces between spermatogenic cells were observed. Early stages of spermatogenic cells showed nuclear protrusions or swellings because of an extensive enlargement of the outer nuclear membrane. Round spermatids exhibited abnormally shaped acrosomes and dilation of the subacrosomal space. Many abnormal, degenerated late spermatids with random orientation were seen towards the basal and adluminal compartments of the seminiferous epithelium. In addition, spermatids exhibited alteration in F-actin bundle ectoplasmic specialization and contained many mitochondria-associated granular bodies.     

Ultrastructural cytochemistry of epithelial gland-like component in synovial sarcoma

In order to analyze the mucoid substance in the epithelial component of synovial sarcoma, electron microscopic and cytochemical studies were made on three of these neoplasms. The mucoid substances in the glandular lumen were intensely stained with ruthenium red (RR), appearing as granular, fibrillar and amorphous structures. RR staining of proteoglycans was diminished after treatment with chondroitinase AC or ABC, and was partially diminished by exposure to streptomyces hyaluronidase. Trypsin treatment did not affect RR staining of proteoglycans in the lumen. On thin sections stained with periodic acid-thiocarbo-hydrazide-silver proteinate (PA-TCH-SP), deposits of reaction product were observed on the mucoid substances within the lumen, and were localized in the Golgi complex, including the rough endoplasmic reticulum, small vesicle and lysosome-like dense body. Trypsin digestion decreased the stain intensity of PA-TCH-SP. These results indicate that the lumen of the gland-like component contains glycoproteins as well as proteoglycans mainly consisting of chondroitin sulfate and hyaluronic acid, and suggest that GERL (Novikoff) is closely related to production, storage and transport of glycoproteins in the cytoplasm of tumor cells.     

Acute lymphoblastic leukaemia in rats induces pathomorphological changes in germ cells

Summary.  Concern about the reproductive potential of long-term survivors of acute lymphoblastic leukaemia (ALL) prompted an investigation into the impact of the disease on spermatogenic cells. Using rats as a model, histological, immunocytochemical and electron microscopic analysis was applied to investigate changes in the seminiferous epithelium. In rats transplanted with leukaemic cells at early puberty, degenerate primary spermatocytes and spermatids were prevalent within stage VIII tubules. Electron microscopically, step 8 spermatids showed acrosomal abnormalities and nuclear contour distortion. In the distorted step 9 spermatids, the microtubules of the manchette were abnormally oriented or deficient. Antitubulin antibody staining was reduced in elongating spermatids in the group transplanted with leukaemic cells at early puberty but was not observed in the older leukaemic group. Step 13 spermatids showed extracted chromatin and degenerate step 19 spermatids were occasionally found. Similar but less severe changes were seen in the group of rats transplanted with leukaemic cells at late puberty. We conclude that germinal cell damage induced by ALL is dependent on the developmental maturity of the seminiferous epithelium. The present findings are of particular importance when interpreting the impact of anticancer chemotherapeutics on germinal cells in patients with ALL.     

Effect of vasectomy on sperm nuclear chromatin condensation in the rabbit

Histone-to-protamine exchange in haploid spermatids is known to play a central role for male fertility. The present study investigates, for the first time, the effects of vasectomy on the expression of protamines in the rabbit. During normal spermatogenesis, protamine-1 and protamine-2 mRNA were expressed from step 5 round spermatids to step 11 elongated spermatids. In unilaterally vasectomized animals, control testes revealed normal spermatogenesis with normal protamine expression, while vasectomized testes exhibited both normal spermatogenesis and spermatogenic arrest. Some testes with normal spermatogenesis revealed delayed expression of both protamine-1 and protamine-2. Furthermore, multinucleated round spermatids were a regular finding in these testes. In both treated and untreated animals, a higher percentage of spermatozoa from the cauda epididymis had highly condensed chromatin when compared with those from the testis. The percentage of spermatozoa with highly condensed chromatin from testes and epididymides from the vasectomized side of treated animals remained unchanged from controls. As the integrity of nuclear chromatin is important for oocyte fertilization, especially in intracytoplasmic sperm injection (ICSI), where most of the natural selection mechanisms are bypassed, our data add valuable information for the treatment of infertility by ICSI, showing that vasectomy may affect nuclear chromatin integrity of testicular spermatids but not epididymal spermatozoa. Microsurgical epididymal sperm aspiration (MESA), therefore, may be superior to testicular sperm extraction (TESE) in vasectomized patients.     

Isolation and characterization of a novel cDNA encoding a DNA-binding protein (Hils1) specifically expressed in testicular haploid germ cells

A cDNA encoding a protein homologous with histone H1 has been cloned from a haploid germ cell specific cDNA library. Deduced amino acid sequence (170 amino acids) showed 40% identity with histone H1 globular domain. Messenger RNA of the gene was observed exclusively in the testis, and was accumulated after post-natal day 23. Western blotting analysis showed that the protein encoded by this gene is about 19 kDa in molecular weight, and it was exclusively recovered from the nuclei of testicular germ cells. Immunohistochemical analysis showed that the protein was localized to the nuclei of round and elongating spermatids, consistent with the results of immunoblot analysis. Thus, the gene product was named Hils1 (histone H1 like protein in spermatids 1). In vitro DNA-binding experiments using DNA-cellulose mini-columns showed that Hils1 was able to bind to both double and single stranded-DNAs in a non-sequence-specific manner. These findings suggest that Hils1 may play an important role in the structural changes of spermatid nuclei, such as nuclear condensation, and gene regulation of haploid germ cell differentiation.     

Immunolocalization assessment of metastasis-associated protein 1 in human and mouse mature testes and its association with spermatogenesis

Aim： To investigate the stage-specific localization of metastasis-associated protein 1 （MTA1） during spermatogenesis in adult human and mouse testis. Methods： The immunolocalization of MTA1 was studied by immunohistochemistry and Western blot analysis. The distribution pattern of MTA1 in mouse testis was confirmed by using quantitative analysis of purified spermatogenic cells. Results： The specificity of polyclonal antibody was confirmed by Western blot analysis. MTA1 was found expressed in the nucleus of germ cells, except elongate spermatids, and in the cytoplasm of Sertoli cells; Leydig cells did not show any specific reactivity. MTA1 possessed different distribution patterns in the two species： in humans, the most intensive staining was found in the nucleus of round spermatids and of primary spermatocytes while in mice, the most intense MTA 1 staining was in the nucleus of leptotene, zygotene and pachytene spermatocytes. In both species the staining exhibited a cyclic pattern. Conclusion： The present communication initially provides new evidence for the potential role of MTA1 in mature testis. In addition, its distinctive expression in germ cells suggests a regulatory role of the peptide during spermatogenesis.     

Identification of differentiation antigens in mouse testicular germ cells recognized by monoclonal antibody TRA 55

We have isolated a monoclonal antibody (mAb) TRA 55, which recognizes mouse testicular germ cells from mid-pachytene spermatocytes to the early stages of haploid spermatids during differentiation. Immunohistochemical analysis produced strong positive staining of the nuclei and faint staining in the cytoplasm of germ cells. At meiotic division, when the nuclear membrane disappeared, a specific positive signal could be observed on metaphase chromosomes. When germ cells produced haploid spermatids, antigenicity became suddenly weak and soon disappeared. TRA 55 did not react with testicular somatic cells, such as Sertoli cells or Leydig cells. Western blot analysis of the whole testis showed four positive bands with molecular weights of 43, 46, 49 and 55 kDa. Three bands of 43, 49 and 55 kDa, and a single band of 46 kDa were recovered in cytoplasmic and nuclear fractions of testicular germ cells, respectively. Chronological changes in the Western blot pattern indicated that these antigens became detectable in the testis at the age of 10 days. Furthermore, all antigens were resistant to periodate treatment, suggesting that the epitope was in an amino acid rather than a sugar moiety. These antigen molecules may play important roles in the differentiation of germ cells at the later stages of meiotic prophase and meiotic division in the mouse testis.     

胀亡初级精母细胞的形态观察及测量

目的研究患者精液中胀亡初级精母细胞的各种形态及变化规律。方法采用瑞-姬氏染色,于光镜油镜下对生精细胞及胀亡生精细胞进行观察、摄像及测量。结果1．初级精母细胞与胀亡初级精母细胞(无核胀出型)胞体直径与胞核直径的比值明显缩小；2．胀亡初级精母细胞可表现出一系列的形态学变化：(1)细胞核肿胀明显,几乎占满整个胞体；(2)细胞核肿胀伴细胞核染色质溶解；(3)细胞核膨胀,使胞核胀出于胞体外：(4)由于胞核膨胀,使胞膜上胀出泡状物；(5)部分胀亡细胞核不膨胀,但胞质内含大量空泡,使胞体胀大；(6)细胞核肿胀、溶解,并出现细胞膜破坏、崩解。结论应用光学显微镜在瑞-姬氏染色下检查胀亡生精细胞,其细胞结构清晰、可靠,该方法简单、实用,并完全可以用于临床患者的检验和研究,是理想的检测胀亡生精细胞的方法。     

Nucleolar vacuoles observed in rat oocytes

Recently Merveille and al (1984) using light microscope have observed intranucleolar cavities in rat ovocytes from large antral follicles. They have showed that the frequency of these vacuoles increases when follicular growth is stimulated by gonadotropins. In this paper, the ultrastructure of the nucleolar cavities has been studied. Two types of cavities are visible in these nucleoli: 1. nucleolar "interstices" present at the periphery of the nucleoli in remnants of the granular component and of the dense fibrillar components; 2. nucleolar "vacuoles" which are located in the homogeneous substance forming the greatest part of the nucleolus. The nucleolar vacuoli generally are clear-cut and spherical. The density of their content is similar to the nucleoplasm but they don't communicate to the nucleoplasm. By means of cytochemical to detect Ag-NOR proteins (Ploton and al [1983]) and basic proteins (Sheridan and Barnett [1984]), dense fibrillar component of the nucleolus but no basic proteins may be seen in the wall of the cavity. Moreover no evidence of relation between the presence of the vacuoles and the process of follicular atresia has been found.     

Improved Staining Method for Differentiating Immature Germ Cells from White Blood Cells in Human Seminal Fluid

A new staining method for differentiating WBCs from immature germ cells in seminal fluid has been studied. It is a combination of Bryan's sperm stain, which particulary stains the acrosomal cap of the spermatozoa and the spermatid, and Leishman's blood stain which stains the WBCs in the same way as found in blood smears. The peroxidase positive granules in the cytoplasm of the PMN leukocytes are seen clearly. Thus, it is possible to differentiate PMN leukocytes from non-separated spermatids when they are present in a common cytoplasm. The staining of acrosomal cap permits differentiation between spermatids and lymphocytes.     

Characterization of membrane occupation and recognition nexus repeat containing 3, meiosis expressed gene 1 binding partner,in mouse male germ cells

Mammalian spermatogenesis is a well-organized process of cell development and differentiation. Meiosis expressed gene 1 (MEIG1) plays an essential role in the regulation of spermiogenesis. To explore potential mechanisms of MEIG1''s action, a yeast two-hybrid screen was conducted, and several potential binding partners were identified; one of them was membrane occupation and recognition nexus repeat containing 3 (MORN3). MORN3 mRNA is only abundant in mouse testis. In the testis, Morn3 mRNA is highly expressed in the spermiogenesis stage. Specific anti-MORN3 polyclonal antibody was generated against N-terminus of the full-length MORN3 protein, and MORN3 expression and localization was examined in vitro and in vivo. In transfected Chinese hamster ovary cells, the antibody specifically crossed-reacted the full-length MORN3 protein, and immunofluorescence staining revealed that MORN3 was localized throughout the cytoplasm. Among multiple mouse tissues, about 25 kDa protein, was identified only in the testis. The protein was highly expressed after day 20 of birth. Immunofluorescence staining on mixed testicular cells isolated from adult wild-type mice demonstrated that MORN3 was expressed in the acrosome in germ cells throughout spermiogenesis. The protein was also present in the manchette of elongating spermatids. The total MORN3 expression and acrosome localization were not changed in the Meig 1-deficient mice. However, its expression in manchette was dramatically reduced in the mutant mice. Our studies suggest that MORN3 is another regulator for spermatogenesis, probably together with MEIG1.     

Comprehensive evaluation of CDX2 in invasive cervical adenocarcinomas: immunopositivity in the absence of overt colorectal morphology

CDX2 is a member of the caudal-related homeobox gene family that is expressed during the normal development of the intestinal tract. In addition to staining adenocarcinomas of the alimentary system, studies have demonstrated CDX2 positivity in a percentage of ovarian mucinous and endometrioid tumors, carcinoids, and some adenocarcinomas of other sites such as the urinary bladder, prostate, lung, and pancreas. However, CDX2 immunostaining in cervical adenocarcinomas has not been examined in detail with comparison to important clinicopathologic characteristics including histopathologic subtype, tumor stage, and patient follow-up. In this study of 81 invasive cervical adenocarcinomas, 24 of the cases (30%) demonstrated nuclear positivity. Ten of the 15 (67%) endometrioid tumors had positive nuclear staining, compared with 7 of the 33 (21%) endocervical "usual-type" carcinomas, and 7 of the 33 (21%) remaining subtypes (adenosquamous, glassy cell, clear cell, serous, villoglandular, enteric). The frequency of nuclear staining for the endometrioid subtype was significantly different compared with that for the endocervical and other subtypes (P=0.003). Some cases showed granular cytoplasmic staining with or without corresponding nuclear positivity. Positive nuclear or cytoplasmic staining for CDX2 did not correlate with disease stage or patient outcome. Our results indicate that cervical adenocarcinomas can show nuclear immunopositivity for CDX2 even in the absence of overt morphologic features of colorectal differentiation. The frequency and pattern of CDX2 staining in the more common histologic subtypes of cervical adenocarcinoma (endocervical usual-type and endometrioid) is parallel to that which is seen for adenocarcinomas of the upper gastrointestinal tract and pancreaticobiliary system.     

Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis

Histone-to-protamine exchange in haploid spermatids is preceded by hyperacetylation of core histones resulting in decreased DNA-histone interaction. During normal spermatogenesis, immunohistochemistry with a polyclonal antihyperacetylated histone H4 antibody displayed a strong signal in nuclei of elongating spermatids and, in addition, spermatogonia. Quantitative analysis revealed 98.2 +/- 1.1% of immunopositive spermatids. The percentage of positive spermatids was significantly reduced in infertile men exhibiting at least qualitatively normal spermatogenesis (scores 10-8, 93.1 +/- 6.6%) and impaired spermatogenesis (scores 7-1, 74.9 +/- 23.4%). In seminiferous tubules showing spermatogenic arrest at the level of round spermatids, only 59.5 +/- 16.5% of spermatids were immunopositive for hyperacetylated histone H4. These data demonstrate that the decrease of histone acetylation in spermatids associated with impaired spermatogenesis corresponds with the well known reduction of protamine expression in these cells and confirms the essential role of histone hyperacetylation for correct histone-to-protamine exchange. In seminiferous tubules exhibiting round spermatid maturation arrest, there was an additional signal in nuclei of spermatocytes, suggesting that premature hyperacetylation of histone H4 may result in precocious histone-to-protamine exchange followed by infertility. This is in accordance with data from transgenic mice, where it has been demonstrated that premature expression of protamine-1 results in precocious chromatin condensation followed by sterility.     

Calcium localized in juxtanuclear granules of epiphyseal chondrocytes with a dilute glyoxal bis(2-hydroxyanil) solution

The glyoxal bis(2-hydroxyanil) (GBHA) method for staining labile calcium deposits in chondrocytes was modified. Fresh blocks of epiphyseal cartilage stained in this dilute GBHA solution revealed calcium in the juxtanuclear Golgi vesicles, each approximately 1μ in diameter. Prolonged staining revealed previously described 0.5μ granules throughout the cytoplasm which tended to mask the juxtanuclear reaction. The red GBHA reaction product was believed to be a chelate of calcium since it was removable with EGTA, and stable in Na₂CO₃ and KCN solution. When the tissue blocks stained with the dilute GBHA solution were restained with the original concentrated GBHA solution, refractile bodies containing red GBHA-positive granules were revealed at the periphery of the hypertrophied chondrocytes. The presence of calcium in the juxtanuclear Golgi vesicles and in the pericellular refractile bodies may indicate that the Golgi is involved in the transport of minerals to the matrix.     

Characterisation of a subpopulation of sperm with massive nuclear damage,as recognised with the sperm chromatin dispersion test

Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7‐dibrom‐4‐hydroxy‐mercury‐fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two‐dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double‐ and single‐strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele.