Electron microscopy in the investigation of asthenozoospermia.

Asthenozoospermia, defined as low sperm motility, is a significant cause of subfertility in men. Its origins are diverse and in some instances cannot be ascertained. However, severely reduced motility can often be associated with abnormalities in the structure of the sperm tails, which can only be detected by transmission electron microscopy (TEM). In this respect, TEM is an important adjunct to the traditional methods of semen analysis. This review examines the development of the current state of knowledge of sperm tail abnormalities. These may be genetic in origin, or they may be acquired as a result of extrinsic factors. At present, consistent molecular markers are not available to characterise many of the genetic defects. However, TEM can distinguish specific defects of genetic origin and the non-specific structural anomalies that are typical of an acquired condition. It can also differentiate sperm structural anomalies from necrospermia, or sperm death, which is another significant cause of asthenozoospermia. In this modern era of assisted reproduction, it is possible in some instances to circumvent the problems of sperm immotility and to achieve fertilisation and pregnancy using intracytoplasmic sperm injection (ICSI). However, because of the possible genetic origin of asthenozoospermia, many scientists working in the field of infertility believe that it is of the utmost importance to investigate the causes of asthenozoospermia. This review considers the continuing relevance of TEM to the evaluation of sperm tail abnormalities in the context of current reproductive techniques.

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women.

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.

Ultrastructure of the early human feto-maternal interface co-cultured in vitro.

BACKGROUND: The study was designed to investigate the ultrastructural features of the early human feto-maternal interface when generated by in-vitro co-culture, and compare these with findings reported previously from human pregnancies. METHODS: Placental villi and decidua parietalis tissues from 8-12 week pregnancies were co-cultured in vitro over a 4-day period. The co-incubations were ended at 24 h intervals and processed for electron microscopical studies, and for immunocytochemistry using anti-cytokeratin antibody (CAM 5.2) for trophoblast. RESULTS: Loss of the syncytium at points of contact with the decidual stroma, cytotrophoblast column formation, differentiation and invasion of extravillous trophoblast (EVT) cells into the decidual stroma over the 4-day period of co-culture were observed. Cellular components, such as actin filaments, microtubules, glycogen granules and lamellipodic processes found in EVT cells were consistent with active cellular locomotion. CONCLUSIONS: These ultrastructural studies emphasize the usefulness of this model in investigating the formation of the feto-maternal interface of human pregnancy. The recruitment of cytotrophoblast to the syncytium by a process involving fusion of the intervening plasma membranes, and the migration of EVT cells causing little or no damage to the surrounding decidual cells, resemble in-vivo data.

Mechanical isolation and in vitro growth of preantral and small antral human follicles.

OBJECTIVE: To develop a procedure for isolating small human follicles and to determine their growth requirements. DESIGN: Preantral and early antral follicles were isolated manually, allocated randomly to experimental groups, and cultured for a few weeks. SETTING: Patients giving informed consent in hospitals. PATIENT(S): Women undergoing laparotomy or oophorectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicular size, E2, histology. RESULT(S): Human FSH (at a dose of 1.5 U/mL) induced antral growth of follicles, and the addition of human LH (2.5 ng/mL) to human FSH stimulated growth and antral development. Histologic studies showed that most of the early antral follicles did not contain an oocyte and already had begun to undergo atresia before culturing. Levels of E2 increased in the incubation medium as the follicles increased in size, but those levels were significantly greater when the follicles contained oocytes. CONCLUSION(S): It is possible to grow small human follicles after they have been isolated manually. To develop successfully, they require a low concentration of human LH in addition to human FSH. The rate of atresia between the preantral and early antral stages in vivo is very high; therefore, it is worthwhile to develop techniques for isolating and culturing the follicles before the antral stages.

The endometrial nucleolar channel system as an indicator of progestin potency in HRT.

A quantitative assessment has been made of nucleolar channel systems (NCS) in the endometrial glands of postmenopausal women receiving hormone replacement therapy. The women were taking conjugated equine oestrogen and one of five progestins. The number of NCS induced was related to the dose of progestin administered. The minimum doses of progestin inducing a comparable response to premenopausal secretory phase endometria were found to be 1-2.5 mg norethindrone, 150 micrograms norgestrel and 20 mg dydrogesterone. Progesterone and medroxyprogesterone acetate were inadequate at the doses tested. The results show that the quantification of endometrial gland NCS would be a useful addition to the biochemical and morphological assessments made of any new progestin treatment.

Two techniques for electron opaque staining of elastic fibres using tannic acid in fresh and formalin fixed tissue.

Two electron microscopic staining techniques, one using tannic acid-glutaraldehyde as a fixative, and the other using tannic acid-uranyl acetate solution as a stain on ultra-thin sections of glutaraldehyde fixed material, were directly compared for elastic fibre staining on several human and animal tissues. Various concentrations of tannic acid were compared using both techniques. The two techniques were also compared on formalin fixed tissues. The use of tannic acid-uranyl acetate solution as a stain on processed tissue is by far the more consistent technique and achieves equally good results on glutaraldehyde or formalin fixed tissue. It is suggested that the use of the term tannic acid technique/method should be reserved for this particular method to achieve a meaningful interpretation of results in scientific papers.

The inhibitory activity of a peptide derivative against the growth of simian immunodeficiency virus in C8166 cells.

The peptide derivative Ro 31-8959 is a potent and selective inhibitor of the aspartic proteinases encoded by HIV-1 and HIV-2 and it arrests the growth of both viruses in cell culture. We have demonstrated similar effects against the simian immunodeficiency virus SIVmac251 in the human T-cell line, C8166 (ED50 = 6nM) with a therapeutic index of 4,500. The antiviral activity of Ro 31-8959 was 250 and 22 times greater than that of ddI and ddC, respectively. The mode of action was confirmed by accumulation of the polyprotein p55 with concomitant reduction of the cleavage product, p27, and by the production of immature virions.

Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties.

We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non-metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non-saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer.

A survey of the ultrastructural defects associated with absent or impaired human sperm motility.

The sperm tails of 400 patients having absent or impaired sperm motility were examined by electron microscopy. A wide variety of fine-structural defects were observed although all of the patients fell into clearly defined groups. Total or partial dynein arm deficiency was observed in 12 patients (3%). Ninety-one patients (23%) had sperm with a spectrum of fine-structural defects, whereas 90 patients (23%) were necrospermic. Subjects with low motility, but with at least a few tails of normal structure, had a 5% pregnancy rate, whereas those patients with similar overall motility, but in whom no normal sperm were seen, produced no pregnancies. The results confirm the importance of making an electron microscopical examination of the sperm of patients with asthenozoospermia.

Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short-term culture.

Trophoblastic cells, of at least 95 per cent purity by immunofluorescence and morphological criteria, were obtained from human term placenta by a simple trypsinisation method without the additional purification steps or complex culture conditions used by others. The differentiation of these cells was followed over four days in culture by fluorescence immunocytochemistry, by scanning and transmission electron microscopy and by light microscopy. The results support the idea that the isolated cells are cytotrophoblast and that these differentiate during this time into cells with characteristics of villous syncytiotrophoblast. This process involved first the formation of a multicellular layer of mononucleated cells, then the development of a syncytium of multinucleated cells and, not necessarily concurrently, functional differentiation. This may be a useful model for the study of syncytiotrophoblast function.

Loss of cytomegalovirus infectivity after treatment with castanospermine or related plant alkaloids correlates with aberrant glycoprotein synthesis.

Many plants contain polyhydroxyalkaloids which are potent inhibitors of glucosidases, enzymes involved in oligosaccharide trimming. These are important in determining the final configuration of specific glycoproteins. Human cytomegalovirus (CMV) encodes a number of glycoproteins, some of which ultimately reside in the outer envelope of the mature virion and are important for virus infectivity. Treatment with three polyhydroxyalkaloids, castanospermine (CAST), deoxynojirimycin (DNJ) and 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) blocked the growth of infectious virus, as determined by yield reduction and plaque reduction assays. However, in the presence of CAST, CMV infected cells continued to shed virions into the extracellular medium, as determined by electron microscopy. Envelope glycoproteins of virions produced after treatment with CAST (2.5 mM) were immunoprecipitated with a monoclonal antibody (F5) specific for the gcI family of glycoproteins. Analysis by PAGE-SDS showed an absence of gcI complex 2 (gp52 disulphide-linked to gp130) with a proportional increase in gcI complex 1 (gp52 disulphide-linked to gp95). The results indicated that gp130 alone, or linked to gp52, was important for CMV infectivity. As well as being potential targets for antiviral agents against CMV, inhibitors of glycoprotein trimming reactions may define components of the virion surface important for infectivity.

Iatrogenic epithelial change caused by endotracheal intubation of neonates.

Tracheae of 20 neonates were obtained at postmortem examination. All neonates had been intubated for between 4 h and 105 days. The tracheal epithelial lining was examined by both light and scanning electron microscopy. Five tracheae which had never been intubated served as controls. All of these showed ciliation throughout their length. Those neonates who had been intubated showed epithelial change which ranged from a simple deciliation to a full stratified squamous epithelium. The degree of change was broadly related to the duration of intubation.

Fine structure of cells infected with human cytomegalovirus after treatment with 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

Infection with human cytomegalovirus (CMV) is characterized by cytological changes which are readily visualized by electron microscopy using ultrathin sections of infected cells. Treatment of such cells with 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), a potent inhibitor of CMV, is effective when initiated at early or late times after infection and the response to such treatment has been studied by fine structural analysis. Inhibition of viral DNA synthesis by DHPG treatment (50 microM) late in virus infection resulted in a cessation of virus growth accompanied by a lack of development and possible regression in skein-like intranuclear inclusions together with a depletion in cytoplasmic dense bodies. Such changes were accompanied by the appearance of nuclear dense bodies. These were also present when virus growth was reduced (5 microM-DHPG) rather than completely inhibited (50 microM-DHPG) by treatment initiated from the time of infection. The nuclear bodies were predominantly of a reticular type structure after the early treatment but mainly of a homogeneous form when virus growth was interrupted at late times. Their presence appeared to be connected with the ability of infected cells to initiate the synthesis of late proteins and their morphology may relate to the extent of such protein synthesis. Unlike cytoplasmic dense bodies, provisional findings on the characterization of the nuclear bodies suggested that the 69K matrix protein was not present in abundance.

Human cytomegalovirus infections in vitro after treatment with arildone.

Arildone (WIN 38020), a broad spectrum antiviral, aryl-beta-diketone (4-[6-(2-chloro-4-methoxy)phenoxyl]hexyl-3,5-heptanedione), blocks the replication of human cytomegalovirus at a stage prior to the synthesis of virus-specific DNA. Inhibitory action was demonstrated against a number of virus isolates from neonates and immune-compromised patients. Intranuclear sites of virus replication, highlighted by DNA-staining methods or immunofluorescence, were absent after Arildone treatment and corresponded with the lack of ultrastructural changes associated with productive infection. The abundance of early antigens in cells treated with Arildone was evidence for expression of the viral genome and this was confirmed by detection of immediate-early viral proteins in the presence of the drug. The results suggest that Arildone prevents the replication of human cytomegalovirus at a stage after virion uncoating but prior to viral DNA synthesis.