Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatoza by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization. © Wiley-Liss, Inc.     

Acrosome staining and motility characteristics of sterlet spermatozoa after cryopreservation with use of methanol and DMSO

In this study we describe acrosome staining and motility characteristics of fresh and cryopreserved sterlet (Acipenser ruthenus L.) spermatozoa using soybean trypsin inhibitor-Alexa conjugate fluorescent staining and computer-aided sperm analysis (CASA), respectively. Methanol or dimethylsulfoxide (DMSO) were used as cryoprotectants. After cryopreservation a decline in sperm motility characteristics occurred, but no differential effect between cryoprotectant was observed. Cryopreservation caused a significant increase in the percentage of spermatozoa with acrosome stained by SBTI-Alexa for samples cryopreserved using DMSO compared to methanol. These data suggest that the low usefulness of DMSO for cryopreservation of sturgeon spermatozoa is related to its harmful specific effect towards the acrosome, probably by causing its precocious triggering, much before any egg contact.     

Disintegration of spermatozoa in the infundibular sperm-host glands of the fowl

Summary The disintegration of spermatozoa in the infundibular sperm-host glands of the fowl was investigated by electron microscopy. After the 15th day following artificial insemination, secretory granules in the epithelial cells of the sperm-host glands increase in number and size, and subsequently the contents of the granules are released into the glandular lumen, so that the electron density of the lumen increases. At this stage, spermatozoa stored in the glands begin to undergo degenerative changes starting from the head. The heads become distended and chromatin of the nucleus begins to disperse as small masses, simultaneously with the destruction of the acrosome. As the dispersion of chromatin progresses, mitochondria of the middle piece become distended and irregular in shape, and then disintegrate. At the last stage, most of the organelles have disappeared, but the fibrous sheath and axial-filament complex are still identified.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his invaluable advice. The authors also wish to thank Mr. Takayuki Möri for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

Evaluation of rabbit sperm acrosomal integrity and fertilizing ability by use of vital stains.

Three staining procedures to detect sperm acrosome integrity were compared via electron microscopy. Stains were applied to epididymal, freshly ejaculated, in vivo capacitated, and sonicated sperm cells in addition to spermatozoa displaying sequentially removed plasma and outer and inner acrosomal membranes. Sequential membrane removal procedures resulted in removal of plasma membranes from 73% of all sperm cells, removal of plasma and outer acrosomal membranes from 74% of all sperm cells, and removal of plasma and outer and inner acrosomal membranes from 87% of all sperm cells as determined by electron microscopy. Live/dead staining results were not statistically different from subjective microscopic motility evaluations (P less than 0.005) for epididymal, sonicated, freshly ejaculated, and in vivo capacitated sperm samples. All three stains assessed were similarly capable of detecting the acrosome status of freshly ejaculated and of sonicated spermatozoa compared to data obtained by electron microscopy (P = 0.010). However, only the Bryan-Akruk stain afforded data that were closely correlated with data obtained via electron microscopy for all sperm types assessed; the latter included in vivo capacitated spermatozoa and sperm cells rendered free of plasma membranes. Results confirmed an earlier report by successfully effecting sequential removal of rabbit acrosomal membranes and documented use of the Bryan-Akruk acrosomal stain for evaluation of sperm cell populations for fertilizing ability. These findings should prove useful in further investigations of mechanisms involved in achievement of fertilizing ability by rabbit spermatozoa.     

Ultrastructure of trout spermatozoa: Changes after dilution and deep-freezing

Summary Trout spermatozoa taken from the testis, vas deferens and ejaculate are described at the ultrastructural level. The morphology of the spermatozoa head, changes in the middle-piece structure, and the relationship between the centrioles and the flagellum were studied under consideration of their role in the reproduction of this species. Morphological changes observed after dilution of the spermatozoa in freshwater or saline and subsequent freezing deserve attention in connection with certain manipulations used in fish farms and laboratories.     

Ultrastructural changes in rat epididymis after vasectomy

Summary Epididymal biopsies from rats that had undergone unilateral or bilateral vasectomies from one to eight months previously were compared with biopsies from their contralateral side or from normal controls to ascertain what ultrastructural changes had occurred. After vasectomy, spermatozoa appeared to dissolve in the lumen of the caput epididymidis and to be absorbed by the principal cells. About 5 weeks after vasectomy, numerous lamellar accumulations became apparent in the supernuclear region. Their resemblance to lysosomes or residual bodies was confirmed by an acid phosphatase reaction. After 10 weeks, similar lamellar and polymorphic accumulations on the contralateral side of animals with unilateral vasectomies indicated that resorption had also increased on the unligated side.Publication No. 627 of the Oregon Regional Primate Research Center. This study was supported by NIH Grants No. RR-00163 and HD-05969.The author wishes to thank Ms. J. Hren for her excellent technical assistance.     

Electron-microscopic localisation of thiol and disulphide groups by direct monomaleimido-nanogold labelling in the spermatozoa of a marsupial,the tammar wallaby (Macropus eugenii)

This study utilised a commercially available monomaleimido-nanogold reagent to directly label cellular thiol groups (SH) of marsupial (tammar wallaby) spermatozoa before and after reduction of disulphides (S-S) with mercaptoethylamine hydrochloride (MEA). The sperm surface, mitochondrial membranes, axoneme and tail fibres were all labelled with gold particles before MEA treatment and the label intensity was increased after S-S reduction. The acrosomal membranes and matrix of spermatozoa contained no detectable SH prior to MEA treatment. However, after moderate MEA treatment (1 mg/ml) gold label was associated with the acrosomal membrane and invaginated acrosomal membrane within the acrosomal matrix. After exposure to 5 and 10 mg/ml MEA, gold particles heavily labelled the acrosomal matrix. Thus, the acrosomal membranes and matrix of tammar wallaby spermatozoa both contain S-S cross-linked structures, and this may contribute to the unusual stability of the marsupial acrosome. Under all treatment conditions the nucleus remained unlabelled. This is consistent with early studies which indicated that cysteine was absent from the nuclear protamines. The study also demonstrated that monomaleimido-nanogold can be used to resolve SH- and S-S-rich cellular structures directly, in addition to its use to label antibodies and Fab fragments for immunochemical localisation.This study was supported by a grant to J.C.R. from the Australian Research Council. Y.S. was the recipient of an Australian International Development Program Fellowship.     

Ability of chicken spermatozoa to undergo acrosome reaction after liquid storage or cryopreservation

The effects of in vitro storage on the sperm's ability to undergo the acrosome reaction (AR) have never been studied in avian species despite its major importance for reproduction management.The ability of chicken sperm to undergo the AR was measured after liquid storage at 4 °C and after cryopreservation, and its relationship with other semen quality parameters, including viability, mass motility and objective motility parameters measured by computer semen analyser (CASA) was analysed in two different flocks. The percentage of intact acrosome-reacting spermatozoa (IAR) was dramatically decreased by 48 h liquid storage (loss of 2/3 among the spermatozoa initially able to undergo the AR) whereas motility, viability and morphological integrity were reduced by 10-15%. By contrast, cryopreservation did not affect the induction of AR in flock 1 (29% IAR) whereas it was strongly affected in flock 2 (7% IAR). Motility parameters, viability and morphology were considerably altered by freezing in every case (more that 50% loss). Positive correlations were found between the percentage of intact acrosome-reacting spermatozoa and viability, mass motility and many objective motility parameters.Our results showed that the sperm's ability to undergo the AR was much more affected than other sperm functions after storage at 4 °C, while cryopreservation only had an effect in semen with the lowest initial quality. These results raise questions regarding the specific features of chicken sperm biology that must be taken into account in the treatment of semen.     

Induction of acrosome reaction of spermatozoa in <Emphasis Type=Italic>Eriocheir sinensis</Emphasis> by low temperature

The effects of temperatures, durations of treatment, and derivations from spermatophores or spermaries on in vitro acrosome reaction of the spermatozoa in the Chinese mitten crab Eriocheir sinensis were investigated. The results showed that the different temperatures resulted in extremely significant differences (p < 0.01) in the time of beginning acrosome reaction, the time of the maximum percentage of acrosome reaction, and the maximum percentage of acrosome reaction of the spermatozoa from spermatophores; and the low temperature (−20, −80 °C and liquid nitrogen) induced acrosome reaction of more than 90% spermatozoa while 15 and 4 °C didn’t. Similar results occur in the spermatozoa, treated with −80 °C for 15 min, from spermaries but the time of beginning acrosome reaction and the time of the maximum percentage of acrosome reaction were obviously longer than those from spermatophores. In conclusion, low temperature can induce acrosome reaction, which is a novel and efficient operating method of inducing acrosome reaction; the spermatozoa might be affected physiologically to capacitate with chilling. The study may be beneficial to new understandings of mechanism of acrosome reaction and provide the foundational material for artificial fertilization and breeding of this crab and other commercial aquatic crustaceans.     

Ultrastructure of the different zones of the tectorial membrane

The tectorial membrane (t.m.) of mammals, which lies over the organ of Corti, is made up of an agglomerate of protofibrils of varying degrees of hydration. Two types of protofibrils are clearly distinguishable in the mouse t.m. While type-A protofibrils are straight and unbranched (thickness: 110A) demonstrating a periodic structure (period = 70 A), type-B protofibrils are branched and coiled (thickness: 150-200 A). These protofibrils could be systematically ordered according to the different t.m. zones. Type-A protofibrils predominate in the basal layer and in the entire middle zone, where they are interlaced with strongly hydrated type-B protofibrils. Weakly hydrated type-B protofibrils essentially make up the marginal zone (with the marginal net), the covering net, Hensen's stripe and the immediate contact layer with the limbus.     

Ultrastructure of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris and Brachyhypopomus cf. pinnicaudatus (Teleostei: Gymnotiformes)

The ultrastructure of spermiogenic stages and spermatozoa of representatives of two gymnotiform families, Gymnotus cf. anguillaris (Gymnotidae) and Brachyhypopomus cf. pinnicaudatus (Hypopomidae) were studied. Spermiogenesis of both species is characterized by lateral development of the flagellum and formation of a nuclear fossa. Some differences were found between these species, such as whether (B. cf. pinnicaudatus) or not (G. cf. anguillaris) nuclear rotation occurs, permanence of the cytoplasmic channel, and type and localization of the nuclear fossa. In the G. cf. anguillaris spermatozoon the nucleus is spherical with highly condensed chromatin. The nuclear fossa is shallow and lateral and is associated with the centriolar complex through stabilizing fibrils. The midpiece is short, with many vesicles, a cytoplasmic channel, and elongate mitochondria. In the B. cf. pinnicaudatus spermatozoon the ovoid nucleus is elongated lateral and posterior to the centriolar complex, and has highly condensed chromatin. The eccentric nuclear fossa is of the moderate type, and contains the entire centriolar complex. The midpiece is long, with numerous vesicles, elongate mitochondria, and no cytoplasmic channel. In both species the flagella are laterally disposed in relation to the nucleus and comprise of the classical 9+2 axoneme. Most of the characteristics found in the spermatozoa of these two species of Gymnotiformes are shared with species of Characiformes, whereas only a few are also found in Siluriformes. This suggests that Gymnotiformes and Characiformes may be more closely related than previously proposed.     

隐球菌脑膜炎患者治疗后隐球菌超微结构观察

目的研究隐球菌脑膜炎治疗后菌体超微结构的变化,探讨电镜检查在隐球菌活力检测中的应用。方法对8例经过两性霉素B和5-氟胞嘧啶联合治疗8周后,脑脊液中仍然可以查见隐球菌的患者,采用透射电镜对其脑脊液中的隐球菌进行超微结构观察。结果隐球菌菌体结构都显示了明显的变异：菌体大小差异显著,菌体形态变化明显;荚膜结构紊乱,菌体内可见空洞状或多个巨大脂滴,部分菌体胞膜破损,胞浆溢出。结论隐球菌脑膜炎治疗后虽然脑脊液中还存在菌体,但是菌体的超微结构已经发生了重大变化,提示菌体活力降低或死亡。电镜检查可以作为隐球菌活力判定的一种有效手段,提高隐球菌脑膜炎疗效判定的准确性。     

Ultrastructure of STH cells of the pars distalis of castrated male mice after hepatectomy

Summary An electron microscopic study aimed at differentiation between castration gonadotrophs and posthepatectomy STH cells was performed on the pars distalis of the pituitary of castrated male mice, after partial hepatectomy. The ultrastructural features observed permit the distinction of both cell types. In the present experiments some remarkable ultrastructural changes, other than those described in a previous report, have been found in STH cells of hepatectomized mice with or without previous castration. Most of them contained masses of heterogeneous electron density, suggesting fusion of granules. These masses and some secretion granules were observed close to the plasma membrane, apparently in the process of discharging material into the pericapillary space. Granular extrusion was more frequent than normally. An increased number of lysosomes, probably related to the digestion of overproduced secretion material, was evident. The appearance of concentric lamellar formations might be related to an increase in cell movements. STH cells with severe cytoplasmic damage were also found, indicating an increased rate of cell loss.Work carried out with the financial assistance by a grant from the Comisión de Investigación Científica de la Universidad Nacional de La Plata. Thanks are due to the members of the Technical Staff of the Institute for their technical assistance.     

Effect of histone on the nucleolar morphology of cells cultured in vitro

Summary The frequency of various types of nucleoli was investigated in tissue cultures of human embryonal lung and HeLa cells cultured in the presence of calf thymus histone. The nucleolar morphology and the frequency of various nucleolar types were dependent on the concentration of histone in the tissue cultures of the human embryonal lung cells. HeLa cells required longer cultivation with histone to manifest some effect on nucleoli. In both cases, the observed nucleolar changes suggest the depression of nucleolar RNA synthesis.     

Ultrastructure of the age-involuted adult human thymus

Summary Age involuted thymus tissue from a middle aged (33 years) and an old (63 years) man have been examined by electron microscopy and compared with thymus tissue from children. Biopsies had been taken during surgical correction of congenital heart defects.The fine structural architecture of cortex, medulla and connective tissue in the remaining lymphatic islands in the adult thymus investigated was not different to the thymus of children. We were surprised to find vigorous lymphocytopoiesis in the cortical regions and to recognize extended areas of medulla with a cellular composition which obviously provides the same microenvironment for T-cell maturation as the medulla of the non involuted thymus. Our findings are discussed in relation to the increasing arguments that the human thymus serves an immunological function throughout life.This investigation was supported by grants from the Deutsche ForschungsgemeinschaftI express my thanks to Professor Dr. med. Alexander Bernhard (Kiel) for kindly providing the human thymus tissue. The author also appreciates the excellent technical assistance of Mrs. Knauer, Mrs. Parczany, Mrs. Siebke and Mrs. Waluk     

Ultrastructure of possible sites of ultrafiltration in some gastropoda,with particular reference to the auricle of the freshwater prosobranch Viviparus viviparus L.

Summary In the Gastropoda pressure-ultrafiltration of the blood is assumed to be the first step in urine formation. The most probable site of ultrafiltration is the wall of the heart. Since in other animal groups ultrafilters are characterized by a special cell type, the podocyte, the hearts of two pulmonates (Lymnaea stagnalis, Biomphalaria glabrata) and of four prosobranchs (Viviparus viviparus, Bithynia tentaculata, Ampullaria gigas, Littorina littorea) were ultrastructurally investigated, in order to establish whether or not podocytes occur in these structures. It appeared that only in the wall of the auricle of Viviparus podocytes are present. They form a layer underneath the epicardium, the epithelium covering the auricle. It is assumed that in Viviparus ultrafiltration proceeds in the auricle. The possible route of the pro-urine is discussed. The location of the ultrafilters in the other species studied remains still unknown.     

Long link induction between the microtubules of the manchette in intermediate stages of spermiogenesis

Summary Links unusual for their length and variable morphology have been described between manchette microtubules in late stages of rat spermiogenesis. In earlier stages of rat spermiogenesis obvious links longer than 160 Å are rare, the majority being 80 or 120 Å in length. The most easily discernible geometric pattern in cross-sections of assemblies of manchette microtubules in intermediate stages of rat spermiogenesis is that of linear arrays sometimes resulting in long and irregularly folded chains of closely linked microtubules. Colcemid disrupts these arrays and is responsible for the formation of more complex geometric patterns. Six hours after drug administration the manchette is dramatically reduced in length. Sheet-like links of variable dimensions and >160 Å in length interconnect not only microtubules but C-type microtubules as well as other links. These links are similar in morphology to those found in later stages of rat spermiogenesis. It is suggested that the formation of these links may perhaps be dependent upon aspects of microtubule disassembly.Supported by a grant to E. A. MacKinnon by the Medical Research Council of Canada.     

Histological and ultrastructural alterations of rat thyroid gland after short-term treatment with high doses of thyroid hormones

The aim of the present study was to investigate histological alterations of rat thyroid gland after short-term treatment with supraphysiological doses of thyroid hormones. Rats from experimental groups were treated with triiodothyronine (T3) or thyroxine (T4) during five days. In both treated groups, thyrocyte height was reduced and follicular lumens were distended. Progressive involutive changes of the thyroid parenchyma were apparent, including follicular remodeling (fusion) and death of thyrocytes. Morphological changes confirmed by quantitative analysis were more pronounced in the T4-treated group. Our results demonstrate that thyrotoxicosis, whether induced by T3 or T4, leads to different grades of thyroid tissue injury, including some irreversible damages. These changes might be explained at least in part by lack of trophic and cytoprotective effects of the thyroid stimulating hormone. Since the period required for morphophysiological recovery may be unpredictable, findings presented here should be taken into consideration in cases where the thyroid hormones are used as a treatment for thyroid and non-thyroid related conditions.     

The effects of niamid and reserpine on the nerve endings of the pineal gland

Summary Kitten pineal glands were studied cytochemically under normal conditions, after reserpine injection, and after niamid administration. Adrenergic nerve elements were in perivascular spaces while cholinergic terminals were adjacent to pinealocytes, often times in synaptic contact. BA reactions are primarily in dotted vesicles of adrenergic terminals with some reaction in granular vesicles. Positive reaction occurs along neurotubules and membranous structures of adrenergig nerve fibers and terminals indicating membrane-bounded BA's. Niamid increased the number and density of dotted vesicles, and some granular vesicles are increased in density and size. Reserpine produced a loss reaction in dotted vesicles and a loss of vesicle matrix, producing elliptical vesicles. There is loss of reaction of the dotted vesicles, but occasionally, the positive granular reaction remains. Cholinergic terminals demonstrate no changes with either niamid or reserpine. These findings indicate BAs are stored in reserpine sensitive dotted vesicles and membraneous structures. The findings also show that the dotted vesicle matrix is reserpine sensitive and is necessary for storage of the BA's. Possibly biogenic amines cannot be stored or synthesized in terminals unless the matrix of the dotted vesicle is intact.Supported by: HEW Grant No. NS-10326. The University of Texas Medical School at Houston. — Special appreciation to Mrs. Charlotte Smith for her valuable technical assistance. Appreciation to Ciba-Geigy Corporation for supply of Serpasil (reserpine).     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED₁₀₀; ED = effective dose) and a toxic (LD₂₅; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.