Fluorescent in situ hybridization (FISH) on paraffin-embedded placental tissues as an adjunct for understanding the etiology of early spontaneous abortion.

OBJECTIVES: An investigation of first-trimester spontaneous abortions (SAs) for those cases in which karyotype is not available was designed to test the efficiency of fluorescence in situ hybridization (FISH) on paraffin-embedded tissues combined with pathological examination for understanding the etiology of SAs. METHODS: Pathological examination of 202 placental tissues from SAs was performed. FISH analysis was then carried out on paraffin-embedded tissue sections from the same abortion products with probes specific for chromosomes 13, 16, 18, 21, X, Y. RESULTS: FISH could be achieved in 196 cases (97%). After pathological analysis alone, the etiology of SAs was evoked in 40 cases. The suspected diagnosis was confirmed by FISH in 26 cases (13.2%). After combined pathological and FISH analysis, the etiology of SAs was identified in 83 from the 196 cases (42.3%) with the probe set used. CONCLUSION: The present study demonstrates the value of FISH on paraffin-embedded tissues as an adjunct for understanding the etiology of SAs for those cases in which karyotype is not available. Combination of pathological and FISH analysis increases the yield of diagnosis by a factor of 3.2. The results also demonstrate that predictions of the karyotype from pathological examination should be avoided.

Changes in protamine 1 distribution in human sperm nucleus during in vitro sperm-oocyte interaction: an immunoelectron microscopic study.

OBJECTIVE: To determine whether the process of sperm nuclear destabilization would begin before sperm-oocyte fusion in humans. DESIGN: Changes in the distribution of human protamine 1 were investigated in human spermatozoa from the ejaculate, in spermatozoa selected by swim-up or Percoll techniques, and in spermatozoa bound to zona pellucida (ZP) from oocytes that failed to fertilize in an IVF program. SETTING: Center for Infertility and Assisted Reproductive Technology, and university departments. PATIENT(S): Fifteen couples undergoing an IVF program. INTERVENTION(S): Women underwent a similar superovulation induction protocol that consisted of GnRH agonist associated with hMG. MAIN OUTCOME MEASURE(S): Comparative immunoelectron microscopic study of sperm nucleus labeling with an anti-human protamine 1 specific protamine monoclonal antibody. RESULT(S): After selection by swim-up or by Percoll, spermatozoa show a significantly lower nuclear labeling than in the ejaculate. After binding to the ZP, labeling increases, more in spermatozoa selected by swim-up than by Percoll, but, after Percoll selection, labeling in zona-bound spermatozoa is lower than in the ejaculate. CONCLUSION(S): In humans sperm binding to the ZP induces differences in the accessibility of the anti-human protamine 1 antibody, which are consistent with structural rearrangements of the DNA-nucleoproteins complex. These modifications must be a prelude to sperm decondensation, protamines replacement by histones, and subsequent reactivation of the sperm genome in the oocyte.

Nuclear maturity of human spermatozoa selected by swim-up or by Percoll gradient centrifugation procedures.

OBJECTIVE: To investigate the relationship between sperm preparation techniques and nuclear maturity, as evidenced by the electrophoretic profiles of sperm nuclear proteins. DESIGN: Analysis of sperm nuclear quality in sperm populations used for IVF. SETTING: Center for infertility and assisted reproductive technology and university departments. PATIENTS: Twenty-seven men undergoing an infertility work-up. MAIN OUTCOME MEASURES: Comparative electrophoretic investigation of nucleoproteins extracted from spermatozoa selected by swim-up or Percoll techniques. RESULTS: Nuclear maturity level is improved after the two methods of selection but is more improved after Percoll. In the two groups, selected spermatozoa contain less histones. Moreover, Percoll gradients appeared to enrich for spermatozoa with less intermediate proteins and more mature nucleoproteins of P2 family than swim-up spermatozoa. CONCLUSION: Percoll may offer advantages in terms of the quality of the selected spermatozoa that may influence the outcome of assisted conception techniques.

Diamine oxidase activity and biochemical markers in human seminal plasma.

Diamine oxidase (DAO), an enzyme which degrades polyamines, is present at a very high level in human seminal plasma and is assumed to come mainly from the prostate. The possible relationships between DAO activity and biochemical markers of accessory sex glands were evaluated in 139 men in barren marriages. Four groups were formed: normozoospermic (n = 41), asthenozoospermic (n = 29), oligoasthenozoospermic (n = 35) and azoospermic (n = 34). DAO activity was the highest in the asthenozoospermic group and was significantly different from that in the azoospermic one. For all specimens, a positive correlation was demonstrated between DAO activity and the prostatic markers citric acid and acid phosphatase. However, DAO activity was correlated with citric acid only in the oligoasthenozoospermic and the azoospermic groups. Acid phosphatase and citric acid were linked in all groups. These results implicate the DAO enzyme in changes in sperm metabolism leading to a loss of motility and suggest that DAO comes partly from the upper part of the genital tract (testis and/or epididymis), in addition to the prostatic gland secretion, accounting for the absence of correlation with prostatic markers in normozoospermic and asthenozoospermic groups.

Ultrastructural and immunocytochemical study of P1 protamine localization in human testis.

Two monoclonal antibodies (MAbs) directed against human protamine P1 were realized. Anti-P1 specificity was assessed by western-blot and confirmed by ELISA. Monoclonal antibody 97-3 was selected. Protamine P1 was specifically demonstrated in human testis by immunoelectron microscopy, using 97-3 MAb and an indirect post-embedding immunogold technique. Our results clearly demonstrated the precise time of appearance of P1 protamine in the nuclei of human spermatids. P1 first appeared in the nucleus of step 5 spermatids and its concentration was increased in steps 6-8 spermatids, cytoplasm was not labelled.

Decondensation of human sperm nuclei and HP1 protamine degradation from normospermia and asthenospermia in Xenopus egg extracts.

The process of human sperm decondensation has been studied in vitro in cytoplasmic extracts prepared from unfertilized Xenopus laevis eggs. The chromatin decondensation-recondensation cycle was divided into four stages according to chromatin appearance. Spermatozoa from normospermia and asthenospermia were evaluated according to their capacity to reach these stages, and their DNA integrity was assessed by acridine orange (AO) staining. We observed a significant difference between normospermia and asthenozoospermia in the ability to achieve the cycle of chromatin decondensation-recondensation. These results correlated with AO staining. The role of human protamine 1 degradation in the decondensation process was evaluated by immunostaining. It was found not to be a prerequisite for the earlier stage of chromatin decondensation and it was not implied in the latest stages of pronuclear development.

Depletion in nuclear spermine during human spermatogenesis, a natural process of cell differentiation.

Polyamines (PA), polycations present in all mammalian cells, are essential for cell proliferation and differentiation. In vitro, PA are known to bind to DNA with a high affinity. In vivo, the intimate association of endogenous PA with highly condensed chromatin has been reported. During spermatogenesis, when processes of cell proliferation and differentiation take place, the potential role of polyamines has not been studied in depth. We report here the PA levels measured in human spermatogenic cell nuclei at different stages of differentiation. Cell populations (spermatocytes and round, elongating, or elongated spermatids) were obtained after submitting human testes to a trypsin-deoxyribonuclease digestion, then to a centrifugal elutriation and Percoll gradient centrifugation. A significant and progressive nuclear spermine level decrease was observed from primary spermatocytes to elongated spermatids. This release of spermine from nuclei was concomitant with three major events in mammalian spermiogenesis: the reduction of DNA transcription activity, the replacement of histone proteins by protamines, and the compaction of chromatin. This is the first report arguing a release of nuclear spermine during an in vivo physiological cell differentiation process.

Preparation of spermatogenic cell populations at specific stages of differentiation in the human.

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.

Anomalous distribution of nuclear basic proteins in round-headed human spermatozoa.

An electrophoretic analysis of nuclear proteins has been carried out in normal and in round-headed human spermatozoa. Results revealed an anomalous distribution of nuclear basic proteins in round-headed spermatozoa. They contained more histones and especially more intermediate proteins and less protamines than normal spermatozoa.

Development of elastic-system fibers in human vocal cords.

The morphogenesis of elastic fibers in human, fetal and adult vocal cords was studied by light microscopy and transmission electron microscopy. The elastic system includes elastic, elaunin and oxytalan fibers at different stages. The development of elastic-system fibers in human vocal cord is characterized by every stage of maturation, whether normal, stifled or accelerated, according to areas.

Electrophoretic characteristics of nuclear proteins from human spermatozoa.

A comparative electrophoretic analysis of nuclear proteins was investigated in ejaculated human semen. The results confirm the biochemical heterogeneity of nucleoproteins in sperm with normal routine parameters and demonstrate the same heterogeneity in semen with defective routine parameters: nucleoproteins comprise histones, intermediate proteins, and protamines in the two groups. Individual qualitative and quantitative differences are observed within and between the two groups. The results allow a better knowledge of nuclear characteristics of ejaculated human spermatozoa but it does not permit the establishment of a relationship between biochemical heterogeneity of sperm nucleus and decreased fertility.

Human spermatozoal nuclear maturity in normozoospermia and asthenozoospermia.

Ejaculated human spermatozoa were studied to assess their nuclear maturity. After SDS or SDS-EDTA treatment, asthenozoospermic semen had a lower resistance to decondensation than normozoospermic semen and contained more stained immature nuclei after aniline blue staining. It showed a higher uptake of ethidium bromide, specific for DNA. There was no difference in the binding of 14C iodoacetamide in the two groups. Therefore, asthenozoospermic semen could be characterized by its relative nuclear immaturity.

Effect of duration of abstinence on maturity of human spermatozoa nucleus.

Human sperm are a heterogeneous population, particularly with respect to their morphology, motility, and degree of nuclear maturity. The characteristics of human sperm and the degree of nuclear condensation with variable sexual abstinence times (long, 7 days; short, 12 h) have been studied. Long abstinence led to an increase in the number of sperm and a decrease in their motility, but their morphology remained unchanged. The DNA-protein complex demonstrated by ethidium bromide uptake was unchanged, but there was a significant increase in nuclear stability upon treatment with SDS. The duration of abstinence hardly affected the degree of nuclear condensation or stability of human sperm. The heterogeneity observed is essentially of testicular origin.

PIP: Spermatogenesis is a constant process; however, periods of abstinence do affect the number of sperm stored in the tail of the epididymis and the vas deferens as well as the percentage of stable nuclei. Volunteers either abstained for a short period of 1 ejaculation/day or a longer period of 1 ejaculation/7 days. Longer periods of abstinence showed an increase in sperm volume, concentration, and total count, as well as decreased sperm motility. Sperm morphology, however, showed no change. Longer abstinence also produces an increase in the percentage of stable nuclei, as shown when 50 mcl sperm was combined in solutions of 0.5 ml 1% sodium dodecyl sulphate and the same solution with 0.5 ml 1% ethidium bromide. Finally, shorter periods of abstinence were not shown to increase homogeneity of sperm populations, the composition of which is determined by testicular origin.

Effect of proteinase inhibitors on rabbit uterine ultrastructure.

Rabbit uterine epithelium was examined by electron microscopy after treatment with two proteinase inhibitors, aprotinin and antipain, administered as a single injection. Both compounds had similar effects, those of antipain being slighter. After treatment, uterine epithelium showed an increased number of very enlarged cells. Lateral cell membranes were often brocken off; basal cell membrane and basement membrane integrities are impaired. The possible interference of the system proteinase-proteinase inhibitors with the implantation process was discussed.

[Hydrosalpinx and sterility. Value of studying tubal microbiopsies using scanning electron microscopy]. / Hydrosalpinx et stérilité. Intérêt de l'étude de microbiopsies tubaires en microscopie électronique à balayage.

Microbiopsies were taken from the tubes of 13 women who were sterile with hydrosalpinges. They were studied using scanning electron microscopy. It was possible to point out several degrees in the evolution of these lesions of the tubal epithelium. These lesions occurred in the fimbrial portion, the ampulla and the isthmus of the tubes. The value of this investigation in diagnosis and prognosis in cases of sterility due to hydrosalpinx is discussed.

The hedgehog uterus. A comparative transmission electron-microscopic study.

The ultrastructure of the luminal epithelium of the hedgehog uterus is described on the basis of material taken from 11 animals in three different hormonal situations: castrated, active and hibernating animals. The whole uterine epithelium is composed of microvillous cells. Its appearance is very similar in ovariectomized and hibernating animals. It differs from that observed in active animals where the epithelium is taller, microvilli are more numerous and longer, and where nuclei and cytoplasm display a very active ultrastructural appearance. The results now available indicate that ultrastructural changes occurring within the cells are certainly correlated with plasma sex steroid hormone concentrations. The present paper also reports the regular occurrence of nuclear bodies in uterine cells.

[Oral epithelium during tooth eruption in the rat: a scanning electron microscopic study]. / L'épithélium buccal au cours de l'éruption dentaire chez le rat: étude au microscope électronique à balayage.

The evolution of the ultrastructural aspect of the oral epithelium during the pre- and posteruptive periods of rat molars was investigated using a scanning electron microscope. The eruption is preceded by a rupture of the superficial layers of the oral epithelium with a desquamation following concentrical circles. As the tooth erupts, the epithelial attachment is generated from ameloblasts.

Dynamic changes in plasma luteinizing hormone and testosterone after stress in the male rat. Influence of adrenalectomy.

In 60-day old intact male rats, stress imposed by a strange environment increased the levels of plasma LH and testosterone. Adrenalectomy, performed at 50 days of age, decreased plasma level of testosterone in basal conditions. However, without affecting the plasma level of LH significantly, stress increased plasma testosterone, albeit to a lesser extent, in the adrenalectomized rats. Stimulation of the testicular secretion by the high level of ACTH seems to be the most likely explanation for the observed testosterone peak in the adrenalectomized rat.

Implantation in the rabbit: ultrastructural features of nuclei involved in symplasm formation.

Mitoses, direct division or cell fusion are observed during symplasm formation in the rabbit uterus epithelium. These phenomenon are examined through transmission electron-microscopic studies. Two types of direct division are described: (1) nuclear scission after folding of the membrane and (2) nuclear scission by invagination of the membrane. The existence of amitoses has long been contested in mammals; the present study tends to show that direct division is not an accidental karyorrhexis but is organized at the cellular level.

Scanning electron microscopic study of hedgehog uteri.

Ultrastructural studies of hedgehog uteri (Erinaceus europaeus L.) have been made using animals in anestrus, in estrus and in estrus after sojourn of a week with a male. In estrus and anestrus the uterine epithelium is homogeneous, regularly interrupted by orifices of glands. It is composed of microvillous cells only. Microvilli decrease in number and length in anestrus. A new type of cell, a ciliated cell, appears after copulation. Probable correlation of ultrastructural aspects of endometrium with hormonal situation is discussed.