Spermatogenesis in the testes of diapause and non-diapause pupae of the sweet potato hornworm, Agrius convolvuli (L.) (Lepidoptera: Sphingidae)

Dichotomous spermatogenesis was examined in relation to diapause in the sweet potato hornworm, Agrius convolvuli. In non-diapause individuals, eupyrene metaphase began during the fifth larval instar and eupyrene spermatids appeared in wandering larvae. Bundles of mature sperm were found after pupation. Apyrene spermatocytes also appeared during the fifth larval instar, but meiotic divisions occurred irregularly and their nuclei were discarded from the cells during spermiogenesis. Morphometric analyses of flagellar axonemes showed a variable sperm number in apyrene bundles. The variation ranging from 125 to 256 sperm per bundle indicated abnormal divisions or the elimination of apyrene spermatocytes. In diapause-induced hornworms, spermatogenesis progressed similarly during the larval stages. The cessation of spermatogenesis during diapause is characterized by 1) secondary spermatocytes and sperm bundles degenerating gradually as the diapause period lengthens, and 2) spermatogonia or primary spermatocytes appearing throughout diapause. A TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay revealed that DNA fragmentation occurred in the nuclei of secondary spermatocytes and early spermatids. Aggregates of heterochromatin along the nuclear membrane indicated the onset of apoptosis, and condensed chromatin was confirmed by electron microscopy to be the apoptotic body. These results show that the degenerative changes in spermatogenic cells during pupal diapause were controlled by apoptosis.     

Effects of Environmental Conditions and Aging on Eupyrene Sperm Movement in Male Adults of Polygonia c-aureum (Lepidoptera: Nymphalidae)

The effects of temperature, photoperiod and aging on eupyrene sperm movement from the testis to the duplex in Polygonia c-aureum male adults were examined in relation to seasonal form and imaginal diapause. In males of both summer and autumn forms obtained under long-day and short-day conditions, respectively, the number of eupyrene sperm bundles in the duplex increased linearly with age during the early stage of adult life at 21 degrees C, and no marked difference was observed between the two seasonal forms. Photoperiod during the adult stage did not influence the rate of sperm movement in autumn forms with diapause. The lower the temperature, the smaller the number of sperm bundles moved from the testis to the duplex. Sperm movement did not appear to occur during a period of chilling at 5 degrees C. Males of the autumn form which were pre-incubated for 30 days and chilled for 60 days failed to resume rapid sperm movement at the final incubation temperature of 21 degrees C. Those which were pre-incubated only for 15 days at 21 degrees C had much fewer sperm bundles in the duplex after chilling than those pre-incubated for 45 days. These results suggest the possibilities that eupyrene sperm movement is suppressed strongly during and after overwintering in the field, and that the eupyrene sperm to be used for reproduction in spring are transferred from the testis to the duplex before overwintering.     

Control of spermatogenesis resumption in post-diapausing larvae of the codling moth

The lepidopteran primary spermatocytes produce first eupyrene (nucleated) and later apyrene (anucleated) spermatozoa. The shift to apyrene commitment of the spermatocytes is related to an apyrene-spermatogenesis-inducing factor (ASIF) becoming active towards pupation. During diapause, the primary spermatocytes lyse and spermatogenesis ceases. The renewal of the dichotomous spermatogenesis in the testes of post-diapausing, last-instar larvae of the codling moth was studied in vivo and in vitro. In vivo, the post-diapausing larvae resume the two types of spermatogenesis. Since ASIF activity is related to pupation, the earliest apyrene spermatids appear one day before pupation, as in non-diapausing larvae. In vitro, renewal of spermatogenesis occurs if 20-hydroxy-ecdysone is added to the medium, but only eupyrene spermatids occur since the testes are explanted before ASIF activity has started. These spermatids are unreduced and develop directly from primary spermatocytes which do not undergo meiotic divisions. Moreover, only flagella develop in these spermatids and the nuclei remain spherical. Post-diapause resumption of spermatogenesis is thus a complex process in which meiosis-blocking and meiosis-deblocking factors, ecdysteroids, and the ASIF play regulative roles.     

In vitro cultivation of spermatocysts to matured sperm in the silkworm Bombyx mori

Bombyx spermatogonia are bipotential, producing nucleate eupyrene sperm and anucleate apyrene sperm. An in vitro cultivation of spermatocysts of Bombyx mori from spermatocytes to matured sperm was established. The present experiment made clear that: (i) spermatocysts must be isolated; (ii) constant shaking at 45 r.p.m. was necessary; and (iii) the addition of Bombyx hemolymph (BH) was indispensable for successful cultivation. In the absence of BH, spermatogenesis proceeded normally for 2 or 3 days and, thereafter, spermatocytes and sperm bundles began to degenerate. The best results for normal eupyrene spermatogenesis were obtained when culture medium containing BH of the corresponding stage was used in every exchange of the medium at 72 h intervals. None or only a small number of apyrene sperm bundles was produced by this culture system when spermatocysts from larval testes were used, although eupyrene spermatogenesis proceeded normally to form matured, or squeezed, sperm bundles.     

Inhibition of Spermiogenesis of Eupyrene Spermatozoa by Elevated,Sublethal Temperatures During Pupal-Adult Development of the Bertha Armyworm,Mamestra con figurata Wlk. (Lepidoptera,Noctuidae)

Summary

Eupyrene spermiogenesis and spermatozoa production in Mamestra configurata Walker were inhibited by temperatures >24° C during a “critical period” of about 7 days during the first half of post-diapause pupal-adult development. The critical period coincided with the appearance of early-stage eupyrene spermatids. If temperatures >24° C occurred during the critical period, eupyrene spermatozoa production was reduced, the result of irreversible autolysis of the early- and mid-stage eupyrene spermatids. Each day of exposure to 25 or 27.5° C during the critical period reduced the number of eupyrene cysts with spermatozoa on average by 12 to 14%. There were no observable effects on eupyrene spermiogenesis and spermatozoa production of exposing post-diapausing pupae to 25 or 27.5° C before or after the critical period. Apyrene spermatogenesis was not affected by 25 or 27.5° C.

Exposure of pupae to 25°or 27.5° C during diapause had no apparent effect on eupyrene spermiogenesis and spermatozoa production. Meiosis usually was not detected among the eupyrene cysts with lalje primary spermatocytes until after diapause, indicating that meiosis is suppressed during diapause. By providing a meiotic block, diapause governs the time of onset of the temperature-sensitive critical period of spermiogenesis. In nature, male pupae of M. configurata in diapause are protected from sterilizing temperatures in the soil during August and post-diapause developing pupae pass through the critical period of spermiogenesis in spring (April to June) when soil temperatures throughout the range of M. configurata in Canada are well below 25° C. The distribution of M. configurata in the northern region of the temperate zone may in part be attributable to the sensitivity of spermatogenesis to relatively mild temperatures.     

Abnormal spermatogenesis at low temperatures in the Japanese red-bellied newt,Cynops pyrrhogaster: possible biological significance of the cessation of spermatocytogenesis

In newt testis, spermatocytes never appear during winter, because secondary spermatogonia die by apoptosis just before meiosis. In the current study, we examined the effect of low temperatures on spermatogenesis. Incubation of newts at low temperatures (8, 12, 15 degrees C) induced defects in spermatogenesis in a temperature-dependent manner. At 8 degrees C, multinucleated giant cells (MGCs) were observed in spermatocytes and spermatogenesis never proceeded beyond meiosis. Although spermatocytes completed meiotic divisions at 12 degrees C, severe cell death was observed in the spermatids. At 15 degrees C both normal and abnormal spermiogenesis were observed. Under these conditions, impaired meiotic synapsis/recombination and down-regulation of the expression of the DMC1 protein, which play pivotal roles in meiotic pairing in eukaryotes, were also observed. Furthermore, to examine the quality of the sperm produced at low temperature for supporting development, artificial insemination was performed. The eggs inseminated with spermatozoa derived from newts kept at 15 degrees C demonstrated a restricted developmental capacity, even though these spermatozoa had an equal capacity for carrying out fertilization to those kept at 22 degrees C. These results suggest that meiosis at low temperatures cause the production of abnormal spermatozoa. Conservation and the significance of this phenomenon in poikilothermic vertebrates living in the temperate zones are also discussed.     

Significance of peristaltic squeezing of sperm bundles in the silkworm, Bombyx mori: elimination of irregular eupyrene sperm nuclei of the triploid

Silkworm (Lepidoptera) males produce dimorphic sperm: nucleate eupyrene sperm and anucleate apyrene sperm. The eupyrene sperm are ordinary sperm to fertilise the eggs, while the function of apyrene sperm remains uncertain. After meiosis, 256 sperm cells are enclosed by a layer of cyst cells, forming a sperm bundle. We have previously documented that the nucleus of eupyrene sperm anchors to the head cyst cell, which locates at the anterior apex of the bundle, by an acrosome tubule-basal body assembly. Neither the basal body attachment to the nucleus nor the acrosome is seen in apyrene sperm, and the nuclei remain in the middle region of the bundle. Peristaltic squeezing starts from the anterior of the bundles in both types of sperm, and cytoplasmic debris of the eupyrene sperm, and both the nuclei and debris of apyrene sperm, are eliminated at the final stage of spermatogenesis. Since the irregularity of meiotic division in apyrene sperm is known, we used triploid silkworm males that show irregular meiotic division even in eupyrene spermatocytes and are highly sterile. The irregular nuclei of the triploid are discarded by the peristaltic squeezing just as those of the apyrene sperm. Transmission electron microscopic observations disclose the abnormality in the acrosome tubule and in the connection to the basal body. The peristaltic squeezing of sperm bundles in the silkworm appears to be the final control mechanism to eliminate irregular nuclei before they enter female reproductive organs.     

Behavior of mitochondria during eupyrene and apyrene spermatogenesis in the silkworm,Bombyx mori (Lepidoptera), investigated by fluorescence in situ hybridization and electron microscopy

Summary Changes in the morphology and quantity of mitochondria and mitochondrial DNA during eupyrene and apyrene spermatogenesis in the silkworm were examined by electron microscopy and by fluorescence in situ hybridization with a 2 kb silkworm mitochondrial DNA clone (pBmMtE2). In the eupyrene spermatogenesis, the spermatocytes at early prophase I contained only a small amount of cytoplasm and showed a rather faint signal. As the cells grew larger in the later prophase I, the signal grew stronger. In the eupyrene spermatids, an especially strong signal was evident in the nebenkerns, in which all the cell's mitochondria were aggregated, and the strong fluorescence was maintained in mitochondrial derivatives. On the other hand, the apyrene cells were markedly smaller throughout spermatogenesis, showing much fainter signals for mitochondrial DNA than the eupyrene. Electron microscopy disclosed considerable differences in the behavior of mitochondria between the apýrene and the eupyrene cells. The observed qualitative and/or quantitative differences in the mitochondria may have some physiological bearing on the spermatogenesis of the two types of sperm.Abbreviations FISH fluorescence in situ hybridization - FITC fluorescein isothiocyanate - kb kilo base pair - PI propidium iodide - PBS phosphate-buffered saline     

Glucose and ecdysteroid increase apyrene sperm production in in vitro cultivation of spermatocysts of Bombyx mori

Two types of sperm, nucleate eupyrene and anucleate apyrene, occur in the silkworm as in other lepidopteran species. Hormones and other substances have been assumed to play important roles in sperm dimorphism. We established an in vitro cultivation system for silkworm spermatocytes, and found that apyrene sperm are not produced when spermatocytes from larval testes are cultivated, though eupyrene spermatocytes develop normally into mature sperm. Based on the fact that ecdysteroid titers increase rapidly and peak 1 day after spinning, and that the amount of glycogen reaches its peak 1 day before the spinning stage, we studied the effects of adding glucose and/or 20-hydroxyecdysone to the culture medium. The experiments disclosed a significant additive effect of both substances on apyrene sperm production.     

Control of the eupyrene-apyrene sperm dimorphism in Lepidoptera

Lepidoptera males bear concomitantly nucleate (eupyrene) and anucleate (apyrene) spermatozoa. Both kinds of spermatozoa reach the spermatheca of inseminated females but only the eupyrene ones fertilize the eggs. The functions of the apyrene spermatozoa are still uncertain. Eupyrene spermatogenesis is regular and highly sensitive to genetic and experimental manipulations while apyrene spermatogenesis is irregular and withstands these manipulations. Both kinds of spermatozoa derive from the same kind of bipotential spermatocytes. The shift of spermatocyte commitment from eupyrene to apyrene spermatogenesis is induced by a haemolymph factor that becomes active just before or after pupation, depending on species. Accordingly, eupyrene spermatogenesis starts during larval instars and stops after pupation while apyrene spermatogenesis begins just before or after pupation, depending on the species, and persists in the imago. The shift is related to shortening of meiotic prophases and blocking synthesis of a meiotic lysine-rich protein fraction in apyrene cells. From spermatogonia proliferation to early spermatocytes, spermatogenesis is a quasi-independent process. Afterwards, it becomes discontinuous and is punctuated by predetermined stations. Progress to a subsequent station is an 'all or none' phenomenon, regulated by cues linked to fluctuations of the main morphogenetic hormones titers. In absence of a particular cue, the cells stop advancing towards the next station and eventually degenerate.     

Peristaltic squeezing of sperm bundles at the late stage of spermatogenesis in the silkworm, Bombyx mori

Silkworm (Lepidoptera) males produce dimorphic sperm, nucleate eupyrene sperm, and anucleate apyrene sperm. The eupyrene sperm is the ordinary sperm fertilizing eggs, while the function of the apyrene sperm, which are about four times as numerous as the eupyrene sperm, is still uncertain. We found the peristaltic phenomenon at the very late stage of spermatogenesis. Peristalsis occurs in both eupyrene and apyrene sperm bundles. Through peristaltic action, cytoplasm of the eupyrene sperm and both cytoplasm and nuclei of the apyrene sperm are discarded from the posterior end of the sperm bundles. Peristaltic squeezing seems to be a process to eliminate the irregular nuclei of apyrene sperm while preserving the nuclei of eupyrene sperm.     

Spermatogenesis in Hydra oligactis. II. How temperature controls the reciprocity of sexual and asexual reproduction

Hydra oligactis undergo two mutually exclusive modes of reproduction: at warm temperatures (18-22 degrees C) animals reproduce asexually by budding, while at cold temperatures (10-12 degrees C) gamete differentiation occurs. Using a monoclonal antibody which is specific for cells of the sperm lineage, it was discovered that under conditions where sperm differentiation does not occur (18-22 degrees C), cells continually enter the sperm pathway but progression down the pathway is prematurely halted, effectively blocking the production of sperm. To elucidate the mechanism by which completion of sperm differentiation is controlled, the cell cycle times of interstitial cells entering the sperm pathway at both the restrictive (18 degrees C) and permissive (10 degrees C) temperatures were examined. It was envisaged that at the restrictive temperature the cell cycle times of committed cells would lengthen as they proceeded down the pathway, leading to dilution and eventual loss of cells at later stages of sperm differentiation. This did not occur. Although cells of the sperm lineage were found overall to divide more slowly at 18 degrees C than at 10 degrees C, at both temperatures the cell cycle times shortened as cells proceeded further down the pathway, making a dilution mechanism untenable. The effect of high temperature on the survival of cells was then tested by subjecting animals to a heat shock. Within 12 hr of the increase in temperature, the total number of sperm lineage interstitial cells dropped 10-fold while the total numbers of epithelial and somatic interstitial cells remained virtually unchanged. A distinct consequence of this cell loss was the disappearance of cells furthest down the sperm pathway. It is proposed that as cells move down the sperm pathway, they become increasingly sensitive to high temperature which adversely affects their survival; the higher the temperature, the earlier in the pathway cells die. The lethal effect is abolished by lowering the temperature, allowing sperm differentiation to continue to completion. The possible adaptive advantages of temperature controlling gametogenesis are discussed.     

BmPMFBP1 regulates the development of eupyrene sperm in the silkworm,Bombyx mori

Sperm deliver the male complement of DNA to the ovum, and thus play a key role in sexual reproduction. Accordingly, spermatogenesis has outstanding significance in fields as disparate as infertility treatments and pest-control, making it a broadly interesting and important focus for molecular genetics research in a wide range of species. Here we investigate spermatogenesis in the model lepidopteran insect Bombyx mori (silkworm moth), with particular focus on the gene PMFBP1 (polyamine modulated factor 1 binding protein 1). In humans and mouse, PMFBP1 is essential for spermatogenesis, and mutations of this gene are associated with acephalic spermatozoa, which cause infertility. We identified a B. mori gene labeled as “PMFBP1” in GenBank’s RefSeq database and sought to assess its role in spermatogenesis. Like in mammals, the silkworm version of this gene (BmPMFBP1) is specifically expressed in testes. We subsequently generated BmPMFBP1 mutants using a transgenic CRISPR/Cas9 system. Mutant males were sterile while the fertility of mutant females was comparable to wildtype females. In B. mori, spermatogenesis yields two types of sperm, the nucleated fertile eupyrene sperm, and anucleated unfertile apyrene sperm. Mutant males produced abnormal eupyrene sperm bundles but normal apyrene sperm bundles. For eupyrene sperm, nuclei were mislocated and disordered inside the bundles. We also found the BmPMFBP1 deficiency blocked the release of eupyrene sperm bundles from testes to ejaculatory seminalis. We found no obvious abnormalities in the production of apyrene sperm in mutant males, and double-matings with apyrene-deficient sex-lethal mutants rescued the ΔBmPMFBP1 infertility phenotype. These results indicate BmPMFBP1 functions only in eupyrene spermatogenesis, and highlight that distinct genes underlie the development of the two sperm morphs commonly found in Lepidoptera. Bioinformatic analyses suggest PMFBP1 may have evolved independently in lepidoptera and mammals, and that despite the shared name, are likely not homologous genes.     

Effect of thermoperiod on diapause intensity in Pyrrhocoris apterus (Heteroptera Pyrrhocoridae)

The intensity of adult diapause in Pyrrhocoris apterus was measured in two series of experiments as the duration of pre-oviposition period at a constant temperature of 25 degrees C after transfer from short (12L:12D) to long day conditions (18L:6D). Higher diapause intensity was induced with a thermoperiod than at constant temperatures. After the induction throughout larval instars 3-5 and during 4 weeks of adult life at short days and a thermoperiod of 25/15 degrees C the pre-oviposition period was 30+/-4 and 26+/-3 days. After induction at constant 25 degrees C the pre-oviposition period was 22+/-3 and 23+/-4 days, while after induction at constant 20 degrees C it was 17+/-4 and 19+/-4 days. Induction at a lower constant temperature of 20 degrees C was thus followed by a less intense diapause than the induction at a higher constant temperature of 25 degrees C. These counterintuitive results are discussed. The oxygen consumption rate measured at experimental temperatures prior to transfer from short to long days was higher at thermoperiodic conditions than at constant temperatures and it was similar at constant 20 and 25 degrees C. Thus, the oxygen consumption rate measured prior to the transfer was highest (indication of the least intense diapause) in the insects that showed later, after the transfer to long days, the longest pre-oviposition period (indication of the most intense diapause). Within the first two days after transfer to constant 25 degrees C, oxygen consumption rate measured at 25 degrees C decreased in the thermoperiodic insects, while it transiently increased in insects from constant 20 degrees C. Two days and later after the transfer, oxygen consumption rate was similar in all groups. Cold hardiness was not correlated with diapause intensity. The low lethal temperature in diapausing insects was correlated with the night temperature during diapause induction.     

Roles of actin networks in peristaltic squeezing of sperm bundles in Bombyx mori

Two types of sperm are produced in the silkworm, Bombyx mori. Nucleate eupyrene sperm is an ordinary sperm that contributes to fertilization, while anucleate apyrene sperm is considered to play important roles in assisting eupyrene sperm. At the very late stage of spermatogenesis, a phenomenon called "peristaltic squeezing" occurs in both types of sperm, whereby cytoplasm of the eupyrene and nuclei of the apyrene sperm are discarded from the posterior end, forming matured sperm. In this study, rhodamine-phalloidin staining for actin was applied to sperm bundles. Before the start of peristaltic squeezing, actin filament networks are spread on the cyst cells and constrictions by the networks appear in several places of the bundles. Actin particles, which are later recognized as circlets, are localized within the bundles. Squeezing action by the networks occurs from the anterior region and transfers toward the posterior, eliminating cytoplasm together with circlets from the posterior end. It seems that actin filaments contribute to the peristaltic squeezing of the sperm bundles in Bombyx mori.     

Identification of the starting point for spermatogenesis resumption in the post-diapause development of the sweet potato hornworm, Agrius convolvuli L

The resumption of spermatogenesis in post-diapause development was examined in the sweet potato hornworm (Agrius convolvuli) with in vivo bromodeoxyuridine (BrdU) incorporation experiments used to determine the starting point. Diapausing pupae were “overwintered” by chilling at 10 °C for over 4 months, after which they initiated post-diapause development by transferring the pupae to 25 °C with a 12-h light/12-h dark photoperiod. The testes of living, post-diapause pupae were injected with BrdU, which is incorporated into newly synthesized DNA strands. During the first 2 days after diapause termination, the nuclei of spermatogonia and spermatocytes failed to label with BrdU. However, on day 3 of post-diapause pupae (PDP3), labeling studies showed that cell proliferation was initiated by spermatogonia, but not by spermatocytes. In both hemolymph and testes, ecdysteroid concentrations rose gradually, reaching 0.3 μg/ml hemolymph at PDP3. These results led to the following three conclusions. The spermatogonial cell division is highly suppressed during diapause. After a long-term diapause, spermatogenesis resumes in the spermatogonia but not in the spermatocytes of diapause-terminated pupae. Cell division begins in advance of peak ecdysteroid concentrations. The latter result indicates that in post-diapause development, high concentrations of the hormone are not required to initiate spermatogonial proliferation.     

Consequences of diapause on post‐diapause development,reproductive physiology and population growth of Chilo partellus (Swinhoe)

We investigate the effects of diapause on post‐diapause development, reproductive physiology and population growth of Chilo partellus (Swinhoe) (Crambidae: Lepidoptera). Aestivating and hibernating larvae of C. partellus are exposed to diapause terminating conditions (consisting of an LD 12 : 12 h photocycle at 27 ± 1 °C and 65 ± 5% relative humidity with a fresh diet) to terminate the diapause and observations are made on percentage pupation, pupal duration and weight, adult reproductive performance and population growth parameters. We find that the diapause in C. partellus significantly reduces the percentage pupation and weights of pupae, ultimately lowering the weight and reproductive performance of adults. Reduced weights of adult females are found to be directly associated with a lower number of egg cells in ovaries. Nevertheless, the reproductive performance of C. partellus males is also found to be greatly affected in the diapause (hibernation and aestivation) experiencing population in terms of the deposition of a lower number of spermatophores and eupyrene sperm in the reproductive tracts of females compared with the nondiapausing population. The results of the present study clearly indicate that a reduction in longevity, fecundity and egg viability, as well as a reduced rate of deposition of spermatophores and eupyrene sperm, in a diapause experiencing population of C. partellus ultimately leads to a reduction in population growth parameters, thus having implications for bio‐ecology and population dynamics under a changing climatic scenario.     

THE EUPYRENE-APYRENE DICHOTOMOUS SPERMATOGENESIS OF LEPIDOPTERA. ORGAN CULTURE STUDY ON THE TIMING OF APYRENE COMMITMENT IN THE CODLING MOTH

Lepidopteran spermatogenesis is dichotomous, producing eupyrene (nucleated) and apyrene (anucleated) spermatozoa. The eupyrene precedes the apyrene spermatogenesis. The timing of the switchover from eupyrene to apyrene spermatogenesis was determined by cultivating testes of accurately aged codling moth larvae in a medium containing mammalian serum but neither hemolymph nor insect hormones. In cultures, eupyrene spermatogenesis occurred in testes dissected from either 4th or 5th instar larvae, probably due to macromolecular factor-like activity of the serum of the medium. But apyrene spermatogenesis occurred only in testes explanted during or after the fourth day of the 5th instar larva. It is concluded that: (1) An apyrene spermatogenesis inducing factor (ASIF) becomes active on the fourth day of the 5th instar larva in addition to the already existing macromolecular factor. (2) Primary spermatocytes can develop into either eupyrene or apyrene spermatozoa. (3) The apyrene spermatogenesis commitment and pupal commitment of other tissues coincide about the fourth day of the 5th instar larva.     

Temperature-dependent activation of ERK/MAPK in yolk cells and its role in embryonic diapause termination in the silkworm Bombyx mori

The silkworm Bombyx mori requires 2-3 months of low temperature (5 degrees C) to terminate embryonic diapause. The molecular mechanisms, however, are unknown. Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) is temperature-dependently activated in the yolk cells of diapausing eggs after 45 days at 5 degrees C, coincident with the acquisition of developmental competence of the embryos at 25 degrees C. Yolk cell granulation and dissociation also begin in diapause eggs incubated at 5 degrees C for 45 days. We used dechorionated egg culture as a model system of diapause termination and observed that both yolk cell dissociation and embryonic development are inhibited by MAPK-ERK kinase (MEK) inhibitor U0126. Therefore, we suggest that ERK in yolk cells has a role in regulating changes in yolk morphology and termination of embryonic diapause in B. mori.     

Meiotic chromosome missegregation during apyrene meiosis in the gypsy moth,Lymantria dispar,is preceded by an aberrant prophase I

The gypsy moth, Lymantria dispar, produces two structurally and genetically distinct types of spermatozoa. The eupyrene spermatozoa are genetically haploid and structurally typical. The apyrene spermatozoa are anucleate and structurally different from eupyrene spermatozoa. To understand further the events contributing to meiotic chromosome missegregation in apyrene spermatocytes, we examined the progression of meiosis in these cells with respect to their eupyrene counterparts. Chromosomal bouquet formation and fusion of nucleolar organizing regions are disrupted in apyrene nuclei. In addition, the chromatin of apyrene nuclei is prematurely and extremely condensed compared with that of eupyrene nuclei. An antibody to the conserved synaptonemal complex protein 3 (SCP3) labeled eupyrene pachytene chromosomes, but not apyrene pachytene chromosomes. In addition, apyrene meiotic spindles are missing a subset of microtubules, which likely include kinetochore microtubules. Because the condensation behavior of meiotic chromatin in apyrene spermatocytes deviates from that of eupyrene spermatocytes, we examined the appearance and distribution of the phosphorylated form of histone H3, but no significant differences in histone H3 phosphorylation were found between apyrene and eupyrene spermatocytes. We argue that because a pachytene checkpoint is not initiated in apyrene spermatocytes, this system may provide a way to understand better the underlying biochemical connections between pairing, recombination, synapsis, kinetochore assembly and segregation of chromosomes during meiosis in a higher eukaryote.