Division Of Spermatocytes Research Articles

Histological organization of roe deer testis throughout the seasonal cycle: variable and constant components of tubular and interstitial compartment.

Seasonally regulated breeding in roe deer, Capreolus capreolus, is associated with significant changes in testis mass, structure and function. This study has quantified seasonal changes of morphometric parameters and cellular composition in roe deer testis parenchyma. Tissue samples were collected bimonthly during a complete annual cycle. Morphometric parameters of seminiferous tubules were measured and the number of different cell types was counted using a computer-aided image-analyzing system. A scheme of eight tubular epithelium stages for active spermatogenesis was devised according to the spermatid development. Stage I is characterized by the occurrence of new round spermatids, stage IV by spermiation and stage VIII by the meiotic division of spermatocytes. The average diameter of seminiferous tubules varied between 88.4+/-3.6 micro m (February) and 216.8+/-9.2 micro m (June). Also numbers of spermatogonia, spermatocytes and spermatids per tubule cross-section showed considerable seasonal changes. In December and February the germinative epithelium mainly consists of Sertoli cells and spermatogonia. In February, the first differentiated spermatogonia enter meiosis, and in April even spermatids occasionally occur, which reach their highest numbers during the rut in August. Both the expansion and the proportion of tubular and interstitial compartment change seasonally and result in differing cell densities. Assuming numerically constant populations of Sertoli cells and interstitial cells during the entire year, the hypothetical cell numbers per mm(2) of the tubular and interstitial areas were calculated for the seasonally variable total areas of tissue cross-sections. The concordance of these theoretical values with measured cell densities provided evidence that the total numbers of Sertoli cells, as well as interstitial cells, remain really constant throughout the seasonal cycle. The exact quantification of variable and constant components provides basic data for characterization of cell type and stage-specific processes of spermatogenesis.

Germ cell apoptosis in the testes of normal stallions

Apoptosis in testicular germ cells has been demonstrated in many mammalian species. However, little is known about the stallion ( Equus caballus) and rates of apoptosis during spermatogenesis. Morphological and biochemical features of apoptosis reported in other species were used to confirm that the TdT-mediated dUTP Nick end labeling (TUNEL) assay is an acceptable method for identification and quantification of apoptotic germ cells in histological tissue sections from stallion testis. Seminiferous tubules from eight stallions with normal testis size and semen quality were evaluated according to stage of seminiferous epithelium to determine the germ cell types and stages where apoptosis most commonly occurs. Spermatogonia and spermatocytes were the most common germ cell types labeled by the TUNEL assay. A low rate of round and elongated spermatids were labeled by the TUNEL assay. Mean numbers of TUNEL-positive germ cells per 100 Sertoli cell nuclei were highest in stages IV (15.5±1.0) and V (13.5±1.1) of the seminiferous epithelial cycle ( P<0.001). An intermediate level of apoptosis was detected in stage VI ( P<0.02). These stages (IV–VI) correspond to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia. Establishing basal levels of germ cell apoptosis is a critical step towards understanding fertility and the role of apoptosis in regulating germ cell numbers during spermatogenesis.

Phosphorylation of high-mobility group protein A2 by Nek2 kinase during the first meiotic division in mouse spermatocytes.

The mitogen-activated protein kinase (MAPK) pathway is required for maintaining the chromatin condensed during the two meiotic divisions and to avoid a second round of DNA duplication. However, molecular targets of the MAPK pathway on chromatin have not yet been identified. Here, we show that the architectural chromatin protein HMGA2 is highly expressed in male meiotic cells. Furthermore, Nek2, a serine-threonine kinase activated by the MAPK pathway in mouse pachytene spermatocytes, directly interacts with HMGA2 in vitro and in mouse spermatocytes. The interaction does not depend on the activity of Nek2 and seems constitutive. On progression from pachytene to metaphase, Nek2 is activated and HMGA2 is phosphorylated in an MAPK-dependent manner. We also show that Nek2 phosphorylates in vitro HMGA2 and that this phosphorylation decreases the affinity of HMGA2 for DNA and might favor its release from the chromatin. Indeed, we find that most HMGA2 associates with chromatin in mouse pachytene spermatocytes, whereas it is excluded from the chromatin upon the G2/M progression. Because hmga2-/- mice are sterile and show a dramatic impairment of spermatogenesis, it is possible that the functional interaction between HMGA2 and Nek2 plays a crucial role in the correct process of chromatin condensation in meiosis.

DEFINITIVE EXPRESSION OF C-MOS IN LATE MEIOTIC PROPHASE LEADS TO PHOSPHORYLATION OF A 34 KDA PROTEIN IN CULTURED RAT SPERMATOCYTES

To investigate the role of c-mos in rat spermatogenesis, expression of c-mos, MAP kinase kinase (MAPKK), MAP kinase (MAPK), cdc2 and protein kinase A (PKA) by spermatogenic cell culture of 14 day-old rats was examined. MAPKK and PKA expressions were constitutive, whereas the expression of MAPK and cdc2 in spermatogonia initially decreased, but later increased on meiotic maturation of spermatocytes. c-mos expression was definitive of late meiotic prophase. c-mos immunoprecipitates prepared from the c-mos-enriched fraction (pI9.0–9.6) could form complex(es) in the cultured spermatogenic cell lysates.In vitro phosphorylation of the c-mos immune complexes revealed a 34 kDa protein that was phosphorylated at serine and threonine residues as a target of the c-mos signal. Its pI value was 4.4–4.5, and cdc2 was not detected, making it different from cdc2 (p34). These results suggest that the phosphorylation of the 34kDa protein by the c-mos signal may play a crucial role in the meiotic division of rat spermatocytes.

Effects of anti-myosin drugs on anaphase chromosome movement and cytokinesis in crane-fly primary spermatocytes.

To investigate whether myosin is involved in crane-fly primary spermatocyte division, we studied the effects of myosin inhibitors on chromosome movement and on cytokinesis. With respect to chromosome movement, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) added during autosomal anaphase reversibly perturbed the movements of all autosomes: autosomes stopped, slowed, or moved backwards during treatment. BDM added before anaphase onset altered chromosome movement less than when BDM was added during anaphase: chromosome movements only rarely were stopped. They often were normal initially and then, if altered at all, were slowed. To confirm that the effects of BDM were due to myosin inhibition, we treated cells with ML-7, a drug that inhibits myosin light chain kinase (MLCK), an enzyme necessary to activate myosin. ML-7 affected anaphase movement only when added in early prometaphase: this treatment prevented chromosome attachment to the spindle. We treated cells with H-7 as a control for possible non-myosin effects of ML-7. H-7, which has a lower affinity than ML-7 for MLCK but a higher affinity than ML-7 for other potential targets, had no effect. These data confirm that the BDM effect is on myosin and indicate that the myosin used for chromosome movement is activated near the start of prometaphase. With respect to cytokinesis, BDM did not block furrow initiation but did block subsequent contraction of the contractile ring. When BDM was added after initiation of the furrow, the contractile ring either stalled or relaxed. ML-7 blocked contractile ring contraction when added at all stages after autosomal anaphase onset, including when added during cytokinesis. H-7 had no effect. These results confirm that the effects of BDM are on myosin and indicate that the myosin used for cytokinesis is activated starting from autosomal anaphase and continuing throughout cytokinesis.

Morphological characterization of the seminiferous cycle in the goat ( Capra hircus): A histological and ultrastructural study

The cycle of spermatogenesis/seminiferous cycle was investigated in the goat testis using both light and electron microscopy techniques. Using the various cell associations and the accompanying changes in spermatid shape and location, the cycle was divided into eight (8) successive stages. The cycle began with the accomplishment of spermiation (stage 1) and ended with apical migration and close attachment of late maturation phase spermatids at the Sertoli cell apex accompanied by adluminal retention of residual bodies with dense staining inclusions (stage 8). The early stages of the cycle (stages 1-4) were therefore characterized by the presence of only one generation of spermatids, the second one appearing only after the division of secondary spermatocytes in stage 4. Consequently, stages 5-8 had two generations of spermatids; Golgi or cap phase as well as maturation phase spermatids. Although stages 5 to 7 appeared as distinct entities, stages 6 and 7 were rather short-lived and considered as continuations of stage 5. Therefore, the 8 stages of the cycle in the goat were further condensed into 6 main divisions. The duration of each stage was estimated by the frequency of occurrence in sections. Among these, stage 1 had the highest frequency (34%) followed by stages 5-7 (27%). Stages 8 and 4 had the shortest frequency (up to 9%) while stages 2 and 3 had 13% and 12% respectively. These results indicate that, like most domestic species, goats have a cycle of 8 stages with 6 main divisions, the longest being stage 1.

Meiotic arrest and germ cell apoptosis in androgen-binding protein transgenic mice.

The fundamental role of androgen-binding protein (ABP) in spermatogenesis remains obscure after nearly 25 yr since its first characterization. In the present investigation, we used a transgenic mouse model that overexpresses rat ABP to examine the potential involvement of this protein in the regulation of processes occurring during spermatogenesis. Specifically, homozygous or heterozygous transgenic mice were analyzed in terms of spermatogenic progression, DNA fragmentation pattern, and germinal cell ploidy status. All animals homozygous for transgenic ABP exhibited an increased accumulation of primary spermatocytes and cells at metaphase with abnormal morphology and localization within the seminiferous epithelium. Analysis of DNA fragmentation by in situ techniques and agarose gel electrophoresis provided evidence for an increased occurrence of apoptosis in the transgenic animals, principally involving pachytene spermatocytes and cells at metaphase. Flow cytometric analysis of the DNA content of isolated germ cells revealed a reduction in the number of haploid cells, an increase in the number of tetraploid cells, and the appearance of a hypotetraploid cell population, consistent with degenerating primary spermatocytes. In mice heterozygous for the transgene, the effects were less prominent, and the degree to which spermatogenesis was compromised correlated with the levels of ABP messenger RNA in individual animals. The present results are interpreted to suggest that ABP can act as a modulator of spermatogenesis by regulating completion of the first meiotic division of primary spermatocytes.

Spermatogenese beim Menschen: Grundlagenforschung und Klinik

Histological evaluation of human spermatogenesis suffers from the hazy border line between normal and pathological germ cell development. This border line needs better definition for histological fertility diagnosis and the early detection of germ cell tumors. Testicular biopsies from more than 2,900 patients with fertility disturbances and more than 1,900 patients with testicular tumors were investigated by means of semithin sectioning, different immunocytochemical methods and transmission electron microscopy. Cellular systems of the human testes possess a degree of autonomy from the body. Their morphological and functional heterogeneity reveals characteristics of cells that are not terminally differentiated. In the testis of an adult, fertile man not only the proliferation of spermatogonia, maturation divisions of spermatocytes and differentiation of spermatids take place, but also abortive germ cells, as well as apoptotic and degenerative cells appear. Disturbances of spermatogenesis are defined by the evaluation of quantity and quality of germ cell alterations. Compensatory and non compensatory defects of spermatogenesis may be distinguished. Deficiency of spermatogonial cell types, multilayered spermatogonia, megalospermatocytes, malformed spermatids and single tumor cells in the face of sufficient development of mature spermatids are considered compensatory defects of spermatogenesis. Dominating malformed germ cells or tumor cells accompanied by an arrest or lack of spermatogenesis, however, represent non-compensatory defects of spermatogenesis. In addition, normal organization and function of the microvasculature, Leydig cells and compartmentalizing cells in the intertubular space are prerequisites for spermatogenesis. The neuroendocrine function of Leydig cells may be responsible for regulating the blood flow rate and the permeability to hormones and nutritive substances. Finally, for patients a successful definition of the border line between normal and pathological events of germ cell development may be essential for early detection of germ cell tumors. Therefore, anatomical sciences not only contribute to basic research, advanced diagnostics and therapeutic concepts related to diseases of the male gonad, but also to the improvement of assisted reproduction.

Expression of mitotic cyclin B1 is not confined to proliferating cells in the rat testis.

Spermatogenesis is a precisely controlled and timed process comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, and the maturation and differentiation of haploid spermatids. Cell proliferation is controlled by genes involved in the regulation of the cell cycle. Among the principal regulatory proteins are cyclins, which are categorized according to their appearance during the cell cycle. B-type cyclins are mitotic cyclins and function at the G2/M transition of the cell cycle. We have investigated the expression and regulation of cyclin B1 during rat spermatogenesis. Rat cyclin B1 was isolated from a testis cDNA library and further used as a probe to detect mRNA expression. Northern blot hybridization of testis mRNA revealed the presence of a single 1.7-kilobase transcript. In situ hybridization showed stage-specific expression during spermatogenesis with highest expression found in late pachytene spermatocytes and early round spermatids. This pattern was confirmed in fractions of isolated germ cells. Immunocytochemistry displayed highest protein levels in round spermatids. Depletion of gonadotropins did not change the quantitative and qualitative expression pattern of cyclin B1. Therefore, the signals triggering the onset of cyclin B1 expression seem not to originate from the pituitary-gonadal endocrine axis and might therefore be paracrine factors originating within the germinal epithelium. Our observations suggest that cyclin B1 plays a hitherto unknown role in spermatid maturation in addition to its known function in dividing cells.

Blastocyst formation following intracytoplasmic injection of in-vitro derived spermatids into bovine oocytes.

Various types of male gametes, such as spermatozoa, spermatids and spermatids obtained after the in-vitro division of secondary spermatocytes, were injected into the in-vitro matured bovine oocytes in order to examine the possibility of utilizing in-vitro derived spermatids for intracytoplasmic injection. There were no significant differences in the development of bovine oocytes injected with various types of male gametes (cleavage rate, 31.0-37.8%; morula rate, 11.1-18.2%; blastocyst rate, 4.4-1.4%). Similarly, 22.0% of sham-injected oocytes (injection of medium plus polyvinylpyrrolidone) cleaved parthenogenetically but none of them developed to morula stage. There were no significant differences in embryo quality (assessed by cell number) of blastocysts obtained by the injections of various types of male gametes (124.0 +/- 9.6-152.5 +/- 15.5). This study demonstrates that various types of spermatogenic cells can be used for intracytoplasmic injection of oocytes to produce viable embryos.

Ultrastructural aspects of spermatogenesis in Metamicrocotyla macracantha (Alexander, 1954) Koratha, 1955 (Monogenea; Microcotylidae).

Ultrastructural aspects of spermatogenesis, spermiogenesis and of the mature spermatozoon of a microcotylid monogenean Metamicrocotyla macracantha parasite from Mugil liza, are described. The irregularly-shaped spermatogonia divides by successive mitoses, forming the primary spermatocytes, identified by the presence of synaptonemal complexes in their nuclei. The spermatids formed by meiotic cell divisions of the secondary spermatocytes, differentiate into a mature spermatozoon. Cross sections of the head and the middle region of mature spermatozoa show the nucleus with strong condensed chromatin, the mitochondria with short cristae, peripheral microtubules and two axonemes with a 9 + 1 pattern, confirming the characteristics of this genus.

Study on the mammalian spermatogenic pathway

The mammalian spermatogenic pathway is a complex process that involves the mitotic proliferation of spermatogonia, the meiotic division of spermatocytes, chromosomal condensation, the production of sperm-specific proteins, and the morphogenic differentiation of spermatids to mature sperm. Elucidating the molecular basis of mammalian spermatogenesis is of great importance, not only because spermatogenesis is a fundamental biological event in the organism but because understanding of this mechanism could lead to the development of new therapeutics to overcome certain genetic diseases. Numerous cytological studies have described, in detail, the dramatic morphological changes of the differentiating spermatogenic cells, however, the molecular basis for many of these steps has yet to be elucidated. What has hampered the progress of such research is the lack of suitable experimental systems. Establishing cultured cell lines of testicular cells has been attempted for many times without much success. We have studied the molecular basis for the mammalian spermatogenic pathway, making use of two unique systems; cell-free transcription reactions in nuclear extracts of animal organs and primary culture of rat testicular cells. Expression of a variety of genes is activated or inactivated as the spermatogenic pathway progresses. The isozyme for glycolytic enzyme phosphoglycerate kinase (PGK) changes in spermatogenic cells from somatic-type PGK-1 to sperm-type PGK-2. We showed that the PGK isozyme switch occurs at the mRNA level during the pachytene spermatocyte stage, suggesting that Pgk-1 transcription ceases and Pgk-2 transcription is instead induced at this stage. The mechanism of the Pgk transcription switch was then investigated by cell-free transcription and DNA transfection experiments. We found that the activation of Pgk-2 transcription in the testis is caused mainly by an Ets family transcription factor named TAP-1, and that the same protein is likely to be involved in the inactivation of the Pgk-1 gene during spermatogenesis. Silencer-like negative regulatory DNA sequences and proteins bound to them seemed to be responsible for the repression of Pgk-1 transcription in the somatic tissues. Throughout the spermatogenic pathway, spermatogenic cells remain in close contact with each other and with somatic Sertoli cells in the seminiferous tubules. Although a number of reports have suggested that Sertoli cells play an important role in spermatogenesis, little is known about the mechanism by which these cells influence spermatogenic cell differentiation.4+ f1p4

Changes of aspartate transcarbamylase activity in the gonad of Sepia officinalis L. during the sexual cycle

1. 1. Aspartate transcarbamylase activity was measured in the testis and the ovary throughout the sexual cycle of Sepia officinalis. 2. 2. Peaks of ATCase activity were correlated with spermatocyte division during sexual maturation and reproduction in males, and with follicle cell divisions during previtellogenesis in females. 3. 3. Thus ATCase appeared to be a good index for phases of gonadal cell multiplication in both sexes and is proposed as a bioassay to study the mitogenic factor from the optic gland.

An ?axis-like? material in the centromeric region of metaphase-I chromosomes from mouse spermatocytes

This study reports the persistence of axis-like structures in the centromeric region of both homologues during the metaphase-I and anaphase-I stages of meiotic division of mouse spermatocytes. A novel type of silver 'argentaffin' technique (NH4-Ag) is employed. This technique includes the treatment of glutaraldehyde-fixed tissues with dilute ammonium hydroxide followed by a reduction of aldehyde groups with sodium borohydride. Staining is accomplished with ammoniacal silver nitrate in darkness followed by sulfite washing. The lateral elements of synaptonemal complexes and the single chromosomal axes of diplotene spermatocytes show a prominent reactivity with this technique. The pattern of very small grains over condensed chromatin is uniform and gives only a light opacity to the electron beam. The presence of an axis-like structure is seen in every centromeric end of meiotic chromosomes at metaphase I and anaphase I. The chromatin (heterochromatin) that surrounds the centromeric filament and some material distributed in irregular linear arrays along some of the homologues also showed a higher electron opacity than the bulk of deoxyribonucleoprotein. While the former is related to C+ heterochromatin, the latter could represent dispersed material of diplotene axes. It is suggested that the disposal of axial material is differentially delayed at the centromeric regions. The present evidence supports the hypothesis that axial fragments or lateral-element segments persisting at these regions contribute to the cohesiveness of centromeres of sister chromatids during normal disjunction.

Meiotic chromosome structure. Kinetochores and chromatid cores in standard and B chromosomes of Arcyptera fusca (Orthoptera) revealed by silver staining

The behaviour of two chromosome structures in silver-stained chromosomes was analyzed through the first meiotic division in spermatocytes of the acridoid species Arcyptera fusca. Results showed that at diakinesis kinetochores and chromatid cores are individualized while they associate in bivalents of metaphase I; only kinetochores and distal core spots associate in the sex chromosome. Metaphase I is characterized by morphological and localization changes of both kinetochores and cores which define the onset of anaphase I. These changes analyzed in both autosomes and in the sex chromosome allow us to distinguish among three different substages in metaphase I spermatocytes. B chromosomes may be present as univalents, bivalents, or trivalents. Metaphase I B univalents are characterized by separated cores except at their distal ends and individualized and flat sister kinetochores. At anaphase I sister kinetochores of lagging B chromatids remain connected through a silver-stained strand. The behaviour of cores and kinetochores of B bivalents is identical with that found in the autosomal bivalents. The differences in the morphology of kinetochores of every chromosome shown by B trivalents at metaphase I may be related to the balanced forces acting on the multivalent. The results show dramatic changes in chromosome organization of bivalents during metaphase I. These changes suggest that chromatid cores are not involved in the maintenance of bivalents. Moreover, the changes in morphology of kinetochores are independent of the stage of meiosis but correlate with the kind of division (amphitelic-syntelic) that chromosomes undergo.

The relationship between nuclear envelope-derived annulate lamellae and the asymmetric division of primary spermatocytes in aphids

The structure of dividing primary spermatocytes of Amphorophora tuberculata (Aphididae, Hemiptera) as determined by electron microscopy and serial sectioning is described. The developmental stages examined extend from late prophase I to late telophase I. We looked for any asymmetric organization that could be causally linked to the differences in chromatin behaviour between the two daughter nuclei towards the end of meiosis I of this species. In late prophase I, evaginations of the nuclear envelope in the vicinity of two neigh-bouring centrosomes develop into closed cytoplasmic compartments with a dense content. The compartments open in prometaphase I and come to lie together with fragments of the nuclear envelope within the spindle area. Since nuclear pores are preserved in the membranes, intraspindle annulate lamellae have formed. These and material of presumed nuclear origin associated with them are asymmetrically distributed within the cell. Although dispersed at stages beyond prometaphase I, the material may be largely incorporated into one of the two daughter cells and thus be decisive for further development. Some annulate lamellae form a cap at the chromosome surface opposite to the neighbouring centrosomes in prometaphase I. These membranes may prevent interaction between spindle microtubules and chromosomes until a bipolar spindle forms in metaphase I. At this stage, both the banana-shaped autosomal bivalent and the X univalent occupy the equatorial plane. This is strange, because the X univalent has microtubular connections with one spindle pole and would be expected to migrate towards that pole. Possibly, the kinetochore of the X chromosome is inactive, and remains so in anaphase I, when the X univalent remains located between the two autosomal half-bivalents.

Inhibition of meiotic divisions of rat spermatocytes in vitro by polycyclic aromatic hydrocarbons

The toxic effects of polycyclic aromatic hydrocarbons (PAH) on spermatogenic cells undergoing meiotic division were investigated in vitro. Toxicity was assayed as alterations in cell nucleus morphology and cell survival and by DNA flow cytometry. Benzo[ a]pyrene (BP) and 7,12-dimethylbenz[ a]anthracene (DMBA) inhibited the progression of spermatocytes through meiotic division and were highly cytotoxic at concentrations higher than 1 μM. These results were obtained upon addition of a drug-metabolizing system, indicating that the seminiferous tubules lack the enzymes required for the initiation o PAH metabolism. The spindle poisons, e.g., vincristine and Colcemid, a group of direct-acting agents, affected spermatogenesis during meiotic division in a manner similar to that observed with PAH. In contrast, adriamycin did not inhibit meiotic division, although it did induce the formation of meiotic micronuclei as a result of chromosome breakage. It is concluded that low concentrations, i.e., 0.1 μM of PAH, strongly inhibit meiotic division, presumably after metabolic activation to reactive molecules functionally resembling direct-acting alkylating agents. High concentrations of PAH are cytotoxic.

Sheathed nuclear division in primary spermatocytes of Orgyia antiqua (Lepidoptera, Insecta)

The restructuring of spermatocytes during the first meiotic division is examined in the moth Orgyia antiqua (Lymantriidae, Lepidoptera) using transmission electron microscopy. Particular emphasis was placed on the behaviour of the perispindle membrane system. These membranes develop from layers of smooth endoplasmic reticulum wrapped around the prophase I nucleus and are retained until early telophase I. The original nuclear envelope is dissolved in metaphase I. Polar fenestrae in the perispindle membrane stacks are filled with numerous irregular membrane elements. The formation of new nuclear envelopes around the daughter nuclei takes place inside the perispindle membrane system. Finally, the membrane stacks rupture concomitantly with spindle elongation in late telophase I. Thus, division of primary spermatocytes in Orgyia antiqua has a surprising degree of similarity with the so-called closed mitosis. This mode of division is typical for many protozoa, algae and fungi. In the pertinent cells, the original nuclear envelope persists around the spindle area during nuclear division. In order to distinguish the closed mitosis from the situation in Orgyia antiqua spermatocytes, the term ‘sheathed nuclear division’ is suggested for the latter.

Centromeric dots in crane-fly spermatocytes: meiotic maturation and malorientation.

During the first meiotic division in crane-fly spermatocytes, the two homologs of a metaphase bivalent each bear two sister kinetochores oriented toward the same pole. We have previously reported treatments that increase the percentage of metaphase bivalents in which one or both homologs have bipolar malorientations: kinetochore microtubules extending from a homolog toward both poles. The maloriented homologs lag at anaphase. Treatments that induce this behavior include: (a) recoverey from exposure to low temperatures or Colcemid or Nocodazole concentrations that prevent spindle formation but allow nuclear membrane breakdown, and (b) exposure to 6 degrees C, a temperature that permits spindle assembly but slows progression through meiosis. Giemsa staining methods reveal two 0.5 micron diameter dots at the centromeric region of each metaphase homolog; these often are more separated in maloriented homologs. This investigation was undertaken to assess whether this separation precedes the establishment of bipolar malorientation, and hence may be a cause of it, or is only a consequence of forces resulting from bipolar malorientation. Analysis showed that, in untreated cells, the average center-to-center distance between sister centromeric dots increases during the course of meiosis I. After the above-mentioned treatments, center-to-center distances similar to those normally seen in untreated half-bivalents at anaphase I were seen in bivalents, both after and before nuclear membrane breakdown. Longer exposure to temperatures that arrested meiosis increased the degree of dot separation. Based on our data, we conclude that normal orientation during the first meiotic division is aided by the close apposition of centromeric dots, and that a time-dependent maturation occurs causing centromeric dots to separate for the second meiotic division and facilitating orientation of sister kinetochores to opposite poles. If centromeric maturation occurs either prior to or during early stages of the first meiotic division, then it may contribute to persisting bipolar malorientation.

The infrastructure and Reversibility of Testicular Atrophy Induced by Ethylene Glycol Monomethyl Ether (EGME) in the Rat

Inhalation exposure to 300 ppm ethylene glycol monomethyl ether (EGME) for 3 days produced degenerative changes in spermatocytes of pachytene and meiotic division at spermatogenic stage XIV in rats. However, a wide range of germ cell types including spermatogonia was affected and the stage-specific damage was not discernible after 2 weeks exposure to 300 ppm EGME. The stage-specific damage was related to exposure concentration-time course. In early stages, degenerating spermatocytes showed nuclear chromatin clumping around synaptonemal complexes, cytoplasmic vesiculation with electron-dense material deposition, and disruption of the plasma membrane. Chromosomal microtubules in the meiotic division of spermatocytes were discontinued with deposition of electron-dense chromatin material. Sertoli cells showed cytoplasmic vacuolization, contact loss to germ cells, and cytoplasmic processes fragmentation with disrupted microtubules. Degenerative pachytene or meiotic spermatocytes were associated with disrupted Sertoli-germ cell relationship, chromosomal microtubules, and synaptonemal complexes. Spermatid degeneration and giant cell formation were observed after spermatocyte degeneration. Spermatid degeneration appeared to be a secondary change resulting from disrupted Sertoli-to-germ cell association. After 14 days post-exposure (PE) following 2 weeks exposure, some tubules were lined with regenerating spermatocytes with or without round spermatids. By 42 days PE, many tubules regained normal germinal epithelium, but some tubules were still atrophic even after 84 days PE. Reversibility of testicular atrophy was inversely proportional to severity of damaged stem cells.