Successful Maturation Research Articles

A role for N-glycanase in the cytosolic turnover of glycoproteins.

Successful maturation determines the intracellular fate of secretory and membrane proteins in the endoplasmic reticulum (ER). Failure of proteins to fold or assemble properly can lead to their retention in the ER and redirects them to the cytosol for degradation by the proteasome. Proteasome inhibitors can yield deglycosylated cytoplasmic intermediates that are the result of an N-glycanase activity, believed to act prior to destruction of these substrates by the proteasome. A gene encoding a yeast peptide:N-glycanase, PNG1, has been cloned, but this N-glycanase and its mammalian homolog were reported to be incapable of deglycosylating full-length glycoproteins. We show that both the yeast PNG1 enzyme and its mammalian homolog display N-glycanase activity towards intact glycoproteins. As substrates, cytosolic PNGase activity prefers proteins containing high-mannose over those bearing complex type oligosaccharides. Importantly, PNG1 discriminates between non-native and folded glycoproteins, consistent with a role for N-glycanase in cytoplasmic turnover of glycoproteins.

27. Analysis of a SAGE (serial analysis of gene expression) catalogue of human granulosa cells

Mammalian ovarian follicles are composed of an oocyte surrounded by granulosa (or follicle) cells. Granulosa cells are essential for successful oocyte maturation and ovulation. A gene expression profile, or transcriptome, provides a catalogue of the genes expressed by a cell or tissue and the levels of individual transcript expression. In the case of the granulosa cell, its transcriptome should allow insight into granulosa cell function and potentially lead to markers of follicle quality for use in human assisted reproduction such as in vitro fertilization (IVF). We used SAGE (serial analysis of gene expression) as a technique to determine the transcriptome of granulosa cells. SAGE relies on the assumption that a 14base sequence whose position is defined in relation to the 3'-end of any transcript uniquely identifies the gene from which the transcript originated (1). Human granulosa cells were obtained from Otago Fertility Services, Dunedin, New Zealand from 4 adult females, at the time of IVF treatment. After purification, total RNA (5.96 μg) was extracted from these cells using the Qiagen RNeasy kit. The SAGE library was constructed with the Invitrogen I-SAGE kit. Briefly, SAGEtags were extracted from concatemerized ditag sequences using SAGE2000 software (Invitrogen, version B). In total 59 clones were sequenced, yielding 1339 SAGEtags. Selected human SAGE libraries (normal ovary, heart, liver, lung, pancreas and white blood cell) were downloaded from the NIH Sagemap website (ftp.ncbi.nih.gov/pub/sage/seq/) and their relative SAGEtag abundances were compared to that of the human granulosa SAGE library. It was found that human granulosa cells had a unique pattern of gene expression and a number of tags were found that had high abundance in the human granulosa SAGE library, but were absent from other libraries (for example, hydroxysteroid (11-beta) dehydrogenase I and scavenger receptor class B member 1). Those genes that were common to many cell and tissue libraries tended to be structural and house-keeping genes (for example gamma actin). PCR techniques were used to independently validate the SAGE catalogue. (1) Velculescu, V. E., Zhang, L., Vogelstein, B., Kinzler, K. W. (1995). Serial analysis of gene expression. Science 270, 484– 487.

Murine embryonic stem cell in vitro differentiation: applications to the study of vascular development.

The present review summarizes knowledge accumulated during the last decade concerning in vitro endothelial differentiation from embryonic stem (ES) cells. There is now growing evidence that ES cells may provide a powerful model system to determine the cellular and molecular mechanisms of vascular development. ES cells differentiate into the endothelial lineage by successive maturation steps recapitulating in vivo events observed in the embryo. Further maturation of ES-derived embryoid bodies either in three dimensional gels or in confrontation cultures with tumor spheroids can also provide a model of physiological or tumoral angiogenesis. The data obtained from experimental in vitro differentiation of genetically modified mouse ES cells highlight the potential and the complementarity of this model system to in vivo gene knock out studies. We also consider and discuss some of the potential applications of ES cell technology in vascular biology for future directions in basic research and medicine, by manipulation of differentiation and the generation of cell populations for analysis and transplantation for therapeutic use.

Developmental stages of myeloid dendritic cells in mouse bone marrow.

The lineage relationship of dendritic cells (DC) with other hematopoietic cell types has been studied extensively, resulting in the identification of different bone marrow (BM) progenitors that give rise to distinct DC types. However, the identity of the different maturation stages of DC precursors in the BM remains unclear. In this study we define the in vivo developmental steps of the myeloid DC lineage in mouse BM. To this end, BM cells were separated according to their expression of CD31 (ER-MP12), Ly-6C (ER-MP20) and ER-MP58 antigens, and stimulated to develop into myeloid DC, using granulocyte macrophage colony stimulating factor as a specific growth factor. DC developed from three BM subpopulations: ER-MP12(hi)/20(-) (early blast cells), ER-MP12(+)/20(+) (myeloid blasts) and ER-MP12(-)/20(hi) (monocytes). The kinetic and phenotypic features of DC developing in vitro indicate that the three populations represent successive maturation stages of myeloid DC precursors. Within the earliest ER-MP12(hi)/20(-) population, DC precursors exclusively occurred in the myeloid-restricted ER-MP58(hi) subset. By using switch cultures, we show that these BM precursor subpopulations, when stimulated to develop into macrophages using macrophage colony stimulating factor, retain the ability to develop into myeloid DC until advanced stages of maturation. Together, these findings support a common ER-MP12/20-defined differentiation pathway for both macrophages and myeloid DC throughout their BM development.

Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes

Major histocompatibility complex (MHC) class I and II molecules are highly polymorphic proteins that bind and present foreign peptides to the clonally distributed alphabeta receptors (TCR) of T lymphocytes. As a population, the immature T lymphocytes generated in the thymus express a very diverse set of TCR specificities. A process of positive selection filters this broad repertoire to optimize peripheral T cells for antigen recognition in the context of available MHC products. Only those precursor T cells whose TCRs generate an adequate but not excessive signalling response to self-peptides bound to the expressed MHC proteins undergo successful maturation. Here we show that post-thymic self-recognition facilitates the antigen reactivity of mature T cells. Both experimental and physiological interruption of T-cell contact with self-peptide MHC ligands leads to a rapid decline in signalling and response sensitivity to foreign stimuli. Because the adaptive immune system must be recruited early in an infectious process when antigen is limiting, these findings suggest that positive selection ensures predictable T-cell recognition of available self-ligands, which in turn promotes efficient responses to pathogens.

Replacement of fresh dietary components by a dry feed for successful maturation of male Litopenaeus vannamei (Boone) broodstock

Replacement of fresh dietary components by a dry feed for successful maturation of male <i>Litopenaeus vannamei</i> (Boone) broodstock

Prediction of wrist arteriovenous fistula maturation with preoperative vein mapping with ultrasonography

Objective: The purpose of this study was to determine whether the preoperative minimal cephalic vein size in the forearm was predictive of successful wrist fistula maturation to a functional hemodialysis access. Methods: Forty-four consecutive patients underwent evaluation before surgery with ultrasound scan imaging to map the entire cephalic vein in preparation for the construction of an arteriovenous fistula at the wrist. Measurements of the vein diameter were obtained from the ultrasound scan images at eight representative sites. Patients were clinically followed to determine maturation of the fistula to provide a functional hemodialysis access. The smallest diameter of the cephalic vein then was used as a preoperative predictor of fistula maturation. Results: Successful maturation of the arteriovenous fistula was achieved in 22 of the procedures (50%). Cephalic veins with a minimal diameter of 2.0 mm or less were used for anastamosis in 19 patients (43%), and three of these procedures (16%) led to a functional access site. The remaining 25 patients (57%) had minimal cephalic vein diameters greater than 2.0 mm, producing a successful maturation in 19 of the fistula creations (76%). A significantly higher rate of successful fistula maturation in those patients with a preoperative minimal cephalic vein size greater than 2.0 mm was realized ( P = .0002, χ 2 test, with Yates correction for continuity). Conclusion: In patients with a minimal cephalic vein size of 2.0 mm or less, a procedure other than wrist fistula should be considered for optimization of dialysis access. (J Vasc Surg 2002;36:460-3.)

Salvage of poorly developed arteriovenous fistulae with percutaneous ligation of accessory veins

Many arteriovenous (AV) fistulae fail to achieve an adequate blood flow or size for successful cannulation because of accessory veins. We describe a simple technique to ligate accessory veins that does not require a surgical incision. In this retrospective study, 17 end-stage renal disease patients underwent ligation of accessory veins of poorly developed AV fistulae. There were 14 men and 3 women, and their average age was 50 ± 13 years. There were 14 radiocephalic and 3 brachiocephalic fistulae. After identifying accessory veins with a fistulogram, two nonabsorbable 2-0 polypropylene (Prolene) sutures were placed percutaneously around each accessory vein in proximity to the AV fistula. Successful ligation was confirmed with a repeat fistulogram. This procedure was undertaken after 4 ± 3 months following surgical placement. Successful maturation was defined as adequate blood flow to support effective hemodialysis and adequate caliber to allow for repeated cannulation with a 15G or 16G needle. Of 17 AV fistulae, 15 (88%) successfully matured 1.7 ± 1 month (range, 0.3 to 6 months) after the procedure. The average number of accessory veins ligated was 1.7 ± 0.8 (range, 1 to 3). All AV fistulae that matured after ligation of accessory veins were functioning at 44.5 ± 12 weeks after first use. A technique for salvaging nonmaturing AV fistulae not requiring surgical cutdown for ligation of accessory veins is described. AV fistulae mature quickly after ligation of accessory veins. This is a rapid and safe procedure that can increase the prevalence of AV fistulae. © 2002 by the National Kidney Foundation, Inc.

In vitro culture and in vitro maturation of mouse preantral follicles with recombinant gonadotropins

Objective: To develop an effective method for in vitro maturation of preantral follicles isolated from mice ovarian tissue. Design: Isolated preantral follicles were randomly allocated to designed experimental groups for study. Setting: University-based research lab. Patient(s): Healthy, normal mice. Intervention(s): Superovulation with pregnant mare serum gonadotropin and hCG. Main Outcome Measure(s): Morphological changes and E 2 production were assessed. Result(s): To obtain competent oocytes, preantral follicles must be cultured with medium containing insulin and recombinant gonadotropins (i.e., recombinant FSH and recombinant LH), with a change of medium daily. A high initial recombinant LH or recombinant FSH facilitates E 2 secretion, enhances granulosa cell outgrowth, and has earlier antral formation. However, prolonged culture in high-recombinant LH or recombinant FSH triggers early differentiation and luteinization of granulosa cells, which results in low metaphase II oocyte and blastocyst formation. Conclusion(s): We have developed a culture system that allows the successful maturation of preantral follicles in vitro. The matured follicles are a physiologically functional unit that not only secrete E 2 but also generate competent oocytes. In a special condition, 90% of the cultured follicles survived, 53.5% of them produced MII oocytes, and 50% of the derived MII oocytes were fertilized and reached the blastocyst stage after culture in vitro.

Self-recognition and the regulation of CD4+ T cell survival.

CD4+ T cells differentiate in the thymus from committed precursors to mature naive cells ready for peripheral circulation. Successful maturation depends on adequate but not excessive signaling upon T cell receptor (TCR) engagement of self-peptide/MHC class II molecule ligands present in the thymic environment. Persistent TCR signaling throughout development from the CD4+CD8+ to the CD4+ state is required for completion of the developmental process. Recent work has suggested that a continuation of this signaling is essential for sustained survival of CD4+ T cells once they leave the thymus but our studies suggest otherwise. Although we found clear evidence for active TCR signaling involving recognition of self-ligands in peripheral lymphoid tissues, we did not see a substantial effect of loss of such signaling on the life-time of naive CD4+ T cells. Based on a careful review of the literature, we conclude that essentially all previous claims that MHC class II recognition plays a significant role in the survival of CD4+ T cells can be reinterpreted as an effect of self-recognition on proliferation in lymphopenic environments, maintaining population numbers without a marked effect on individual cell viability. We propose a possible explanation for why, in contrast, the viability of naive CD8+ T cells appears to show such self-MHC dependence and suggest that a primary function of self-recognition by T cells may be to enhance responses to foreign antigen.

Gene trap analysis of germ cell signaling to Sertoli cells: NGF-TrkA mediated induction of Fra1 and Fos by post-meiotic germ cells.

Analysis of complex signalisation networks involving distinct cell types is required to understand most developmental processes. Differentiation of male germ cells in adult mammals involves such a cross-talk between Sertoli cells, the somatic component which supports and controls germinal differentiation, and germ cells at their successive maturation stages. We developed a gene trapping strategy to identify genes, which, in Sertoli cells, are either up- or down-regulated by signals emitted by the germinal component. A library of approximately 2,000 clones was constituted from colonies independently selected from the Sertoli line 15P-1 by growth in drug-containing medium after random integration of a promoter-less (beta)geo transgene (neo(r)-lacZ fusion), which will be expressed as a fusion transcript from a 'trapped' cellular promoter, different in each clone. A first screen conducted on 700 events identified six clones in which beta-galactosidase activity was increased and one in which it was repressed upon addition of germ cells. The targeted loci were identified by cloning and sequencing the genomic region 5' of the insert. One of them was identified as the gene encoding Fra1, a component of the AP1 transcription regulatory complex. Accumulation of Fra1 mRNA was induced, both in 15P-1 and in freshly explanted Sertoli cells, by addition of either round spermatids or nerve growth factor (NGF). The effect of NGF was mediated by the TrkA receptor and the ERK1-ERK2 kinase kinase pathway. Fos and Fra1 transcription were induced within the first hour after addition of the neurotrophin, but, unlike what is observed after serum induction in the same cells, a second wave of transcription of Fra1, but not of Fos, started 16 hours later and peaked at higher levels at about 20 hours. These results suggest that AP1 activation may be an important relay in the Sertoli-germ cell cross-talk, and validate the gene trapping approach as a tool for the identification of target genes in cell culture systems.

Relation between gender and vascular access complications in hemodialysis patients

Native arteriovenous (AV) fistulae for hemodialysis vascular access are believed to be associated with fewer complications than synthetic polytetrafluoroethylene (PTFE) grafts. We conducted a study among patients in the Dialysis Morbidity and Mortality Study to compare risk factors for complications of AV fistulae and PTFE grafts in men and women and to examine the effect of age on vascular access complications. We analyzed data from 833 incident patients with end-stage renal disease who had a PTFE graft (n = 621) or AV fistula (n = 212) in use 1 month after starting hemodialysis therapy. Follow-up using inpatient and outpatient Medicare administrative data identified a 1.8-times greater risk for a subsequent vascular access procedure for PTFE grafts (0.71 procedures/access-year) than for AV fistulae (0.39 procedures/access-year). Men with grafts and women with grafts or fistulae had a greater risk for a first subsequent access procedure than did men with fistulae (0.79, 0.65, and 0.59 versus 0.33 procedures/access-year, respectively). After adjustment for age, race, presence of diabetes mellitus, and history of smoking, peripheral vascular disease, and cardiovascular disease, use of a PTFE graft compared with an AV fistula was associated with a greater risk for a first subsequent procedure in men (relative hazard, 2.2; 95% confidence interval [CI], 1.6 to 2.9), but not in women (relative hazard, 1.0; 95% CI, 0.7 to 1.4). The excess risk associated with a PTFE graft compared with an AV fistula was limited to men in the lower three quartiles of age (ie, ≤72 years). These data raise concern that the potential benefits of AV fistulae over PTFE grafts are not realized in women and older men. A better understanding of the determinants of successful access maturation and maintenance in these groups is needed.

The susceptibility of Lymnaea fuscus to experimental infection with Fasciola hepatica.

Three experiments on the infection of Lymnaea fuscus with Fasciola hepatica were carried out to determine if successful infections and maturation of the parasite were dependent on the size of snails at miracidial exposure. The first experiment was performed using 1-4-mm-high snails from 2 populations of L. fuscus and 1 population of Lymnaea palustris. In these snails each subjected to a single bimiracidial exposure, the prevalence of F. hepatica infection at day 35 postexposure ranged from 20.3% to 46.2% in snails measuring 1 mm in height at exposure; it was lower in the 2-mm snails and was 0 in higher size classes. The second experiment was performed by subjecting 1- and 4-mm L. fuscus to 1, 2, and 3 bimiracidial exposures. The prevalence of F. hepatica infection at day 35 postexposure was maximum in the 1-mm snails exposed once to miracidia and decreased with increasing number of exposures. The results were negative in 4-mm snails. Cercarial shedding of F. hepatica was studied in the third experiment using 1- and 2-mm L. fuscus each subjected to a single bimiracidial exposure. The total number of cercariae released from these snails was less than 50. From these results, it can be concluded that L. fuscus showed a partial resistance to F. hepatica infection due to snail age.

Branchipodopsis species — specialists of ephemeral rock pools

The anostracan Branchipodopsis genus is widespread throughout southern Africa and is the second most speciose anostracan taxon in this sub-continent. Branchipodopsis species are particularly dominant in small short-lived and clear rock pools, to the vagaries of which they are extremely well adapted. Such rock pools were studied in the Drakensberg region, in the eastern Free State and in south-eastern Botswana. Common features of Branchipodopsis-inhabited rock pools are the transparency of the water and the very low conductivity values (generally below 50μScm−1, often less than 10μScm−1). These shallow (usually less than 50cm) water bodies with limited buffering capacity show major fluctuations in pH values (often between about pH 4 and 11), depending on the time of the day and the stage in the hydrocycle. Rock pools also closely follow ambient air temperatures with resulting fluctuations of values between 10 and 40°C. Depending on local climatic conditions, small rock pools are usually short-lived and have several wet/dry cycles during one rainy season. Branchipodopsis species are the record-holders in this race against time with maturation usually being reached within the first week after inundation. Broods of resting eggs are generally small (less than 80 eggs) but are produced almost daily. Often (in 30% of cases in south-eastern Botswana), erratic rainfall does not allow sufficient time for successful maturation and reproduction. As a safe-guarding mechanism in B. wolfi in south-eastern Botswana, only some of the eggs hatch on each occasion, depending on environmental conditions, thus extending the chances for successful recruitment. While short-range dispersal of floating eggs by overflows is common, long-range dispersal (e.g. by wind) seems to be rare and to be restricted to shallow pools with little vegetation. Limited effective dispersal may explain the high number of endemic species (e.g. four in the Drakensberg region) and the large morphological variation in some widespread species such as B. wolfi.

Egg maturation and parthenogenetic recovery of diploidy in the scorpionLiocheles australasiae (Fabricius) (Scorpions, Ischnuridae)

In the scorpion Liocheles australasiae, egg maturation and parthenogenetic recoveries of chromosome number and nuclear DNA content were examined by histological, karyological observations and quantitative measurements of DNA. The primary oocyte becomes mature through two successive maturation divisions. The first maturation division takes place in the primary oocyte to produce a secondary oocyte and a first polar body. The second maturation division soon occurs in the secondary oocyte, in which the nucleus is divided into a mature egg nucleus and a second polar body nucleus, not followed by cytoplasmic fission. The first polar body, in one case, was successively divided into two second polar bodies; in the other case it was not divided. In either case, these polar bodies remained attached to the early embryo. The fate of these polar bodies during further embryogenesis were studied. In the karyological analysis, the chromosome number was divided into two groups, one from 27-32, the other was 54-64. The former was presumably the metaphase chromosome number at the meiotic division; the latter was presumably the metaphase chromosome number at the mitotic division. DNA content in the diploid nucleus of the primary oocyte, doubled before the maturation divisions, was reduced through the maturation divisions by one-half in the nuclei of the secondary oocyte and the first polar body and by one-fourth in the nuclei of the egg and the second polar bodies. The first reduction of DNA content corresponded to halving the number of the chromosomes in the first maturation division and the second to the nuclear division in the secondary oocyte. These reductions represent a common process of egg maturation, except the final production of the mature egg with two haploid nuclei, an egg nucleus, and a second polar body nucleus. These two nuclei, which were formed apart in the mature egg, drew near to fuse into a zygote nucleus. The chromosome number and nuclear DNA content were doubled in the zygote and each blastomere in embryos, supporting the hypothesis that the egg nucleus fuses with the second polar body nucleus and this conjugation initiates subsequent embryonic development.

Control of Carpel and Fruit Development in Arabidopsis

The fruit is a highly specialized plant organ that occurs in diverse forms among the angiosperms. Fruits of Arabidopsis thaliana, which are typical of the > 3000 species of Brassicaceae, develop from a gynoecium that consists of two fused carpels. The mature gynoecium of Arabidopsis is composed of an apical stigma, a short style, and a basal ovary that contains the developing ovules. After the ovules are fertilized, the fruit elongates and differentiates a number of distinct cell types, allowing for the successful maturation and the eventual dispersal of the seeds. Although the processes involved in carpel and fruit morphogenesis are not well understood, recent studies have identified a large number of mutants that display abnormal gynoecium and fruit development. The detailed phenotypic description of these mutants together with recent cloning of many of these genes has begun to shed light on this interesting and complex developmental process. Here we review the growing collection of Arabidopsis genes known to control the initiation and development of the gynoecium and resulting fruit.

Functional Early Endosomes Are Required for Maturation of Major Histocompatibility Complex Class II Molecules in Human B Lymphoblastoid Cells

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.

Effect of boron deprivation on reproductive parameters inXenopus laevis

Low boron (−B), boric acid supplemented (+B), or traditional beef liver and lung (BLL) diets were administered to adult male and female Xenopus laevis for either 28 d or 120 d to evaluate reproductive status. The boron content was the only difference between the −B and +B diets. To evaluate further the potential impact of boron deprivation on adult reproduction, a detailed evaluation of ovary and testis morphology and weight, oocyte fecundity and maturation, and sperm count and dysmorphology was conducted. Adult females were depleted of boron for 120 d and subsequently superovulated to flush mature eggs from the ovaries. Following an additional 45-d oocyte maturation period in which the frogs were maintained on their respective diets, the ovaries were removed and oocytes evaluated. Adults administered the −B diet had distinctly atrophied ovaries and testes when compared to adults administered either the +B or BLL diet. Females administered the −B diet had a much greater proportion of immature oocytes in stages 1 and 2 (ca. −B = 87% vs. +B = 23% following the 120-d regimen) than females fed the +B diet, which had a greater proportion of mature oocytes in stages 5 and 6 (mature) (ca. −B = 3% vs. +B = 45% following the 120-d regimen). Co-culture of progesterone with normal stage 1 and 2 oocytes to stages 5 and 6 oocytes in vitro allowed for the successful maturation of stages 1 and 2 oocytes from the +B or BLL diet-fed females, but not from the −B females, which did not respond to hormone treatment. The adverse effects of low dietary boron in the female Xenopus were noted following both depletion periods; however, the effects were more dramatic following 120 d of boron depletion. Reductions in sperm counts and increases in sperm dysmorphology rates were noted in males fed the −B diet when compared to males fed either the BLL or the +B diet. These results on reproduction, development, and maturation further indicate the nutritional essentiality of boron in Xenopus. J. Trace Elem. Exp. Med. 12:187–204, 1999. © 1999 Wiley-Liss, Inc.

Teratisms in living and fossil megaspores of the Lycopsida: tetrad arrangement and exine ontogeny

Abnormal megaspore tetrad configurations fromSelaginella laevigataand ofLagenicula crassiaculeata(Lower Carboniferous) have been studied by SEM. These abnormalities reflect the variation expected in the presumed evolution of the seed-plants from free-sporing heterosporous ancestors. In addition, they indicate a possible role for the aborted members of a tetrad in the successful development and maturation of the functional spore(s). Aborted members of tetrads also reveal the later stages of the developmental sequence occurring in spore wall construction and highlight the interplay of sporopollenin production, its colloidal flocculation and polymerization, and distortion caused by protoplast expansion. Possible causes of abortion in both living and extinct lycopsid megaspores are discussed.

Teratisms in living and fossil megaspores of the Lycopsida: tetrad arrangement and exine ontogeny

Abnormal megaspore tetrad configurations fromSelaginella laevigataand ofLagenicula crassiaculeata(Lower Carboniferous) have been studied by SEM. These abnormalities reflect the variation expected in the presumed evolution of the seed-plants from free-sporing heterosporous ancestors. In addition, they indicate a possible role for the aborted members of a tetrad in the successful development and maturation of the functional spore(s). Aborted members of tetrads also reveal the later stages of the developmental sequence occurring in spore wall construction and highlight the interplay of sporopollenin production, its colloidal flocculation and polymerization, and distortion caused by protoplast expansion. Possible causes of abortion in both living and extinct lycopsid megaspores are discussed.