Behaviors of nucleus, basal bodies and microtubules during eupyrene and apyrene spermiogenesis in the silkworm, Bombyx mori (Lepidoptera)

Observations with immunostaining for tubulin and electron microscopy revealed that silkworm eupyrene spermiogenesis was characterized by an attachment of the basal body to the nucleus except in the period of movement for unidirectional arrangement. In young eupyrene sperm, a microtubule basket caught the nucleus, which thereafter was transformed elliptically. Microtubules were also observed along the elongated acrosome and mitochondrial derivatives. During apyrene spermiogenesis, however, the basal body was not attached to the nucleus and approached the head cyst cell after the completion of unidirectional arrangement, leaving the round nucleus in the middle of the cell. The presence or absence of the phenomenon in which the basal body attaches to the nucleus seems to be essential in the course of diverse spermatogenesis of the silkworm.     

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.     

Regular and irregular divisions of Lepidoptera spermatocytes as related to the speed of meiotic prophase

Lepidoptera males bear two kinds of meiotic divisions. One is regular (eupyrene) and leads to nucleate, fertilizing spermatozoa. The other (apyrene) shows metaphase I chromosomes clumping together into irregular masses which later split forming daughter cells with unbalanced sets of chromosomes which are eventually extruded from the cells; hence, the spermatids develop into anucleate spermatozoa of unknown function. The apyrene divisions are induced by a haemolymph factor which becomes functional towards pupation. Using incorporation of tritiated thymidine at the premeiotic S-phase as a marker for timing, it was found that the prophase of the apyrene spermatocyte is shorter than that of the eupyrene spermatocyte. It is proposed that meiosis-specific proteins cannot be synthesized during the shortened apyrene prophase and that this is correlated with the irregular chromosome behaviour during the subsequent metaphase-telophase of these spermatocytes.     

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.     

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.     

Application of artificial insemination technique to eupyrene and/or apyrene sperm in Bombyx mori

The silkworm, Bombyx mori, has a dimorphic sperm system. The eupyrene sperm is the sperm to fertilize eggs and the apyrene sperm plays a crucial role for assisting fertilization. Heat-treated (33 degrees C for 96h) Daizo (DH) males, one of the strains in the silkworm, produce only eupyrene sperm, while in triploid males only apyrene sperm are functional. Though both types of males are found to be sterile, double copulation of the two males with a single female greatly increases fertility. Here we examined the fertilizing ability of eupyrene and apyrene sperm by means of an artificial insemination technique previously established in B. mori. Neither the eupyrene sperm collected from DH males, nor the apyrene sperm from triploid males have the ability to fertilize eggs. Artificial insemination with the mixture of eupyrene and apyrene sperm leveled up the frequency of fertilized eggs to more than 80%. When cryopreserved DH sperm (eupyrene sperm) were subjected to the same experiment, more than 95% fertilized eggs were obtained. These results confirmed that apyrene sperm play an important and indispensable role in fertilization in B. mori. Separate collection of functional eupyrene sperm and functional apyrene sperm and success of fertilization by means of the artificial insemination technique are applicable for further studies to elucidate the function of apyrene sperm.     

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.     

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.     

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.     

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.     

Sperm maturation screening and the effect of ecdysone on sperm development of silkworm Bombyx mori

There are two sperm morphs of silkworm, the nucleated spermatozoa (eupyrene) and anucleated spermatozoa (apyrene). Eupyrene sperm cannot complete fertilization successfully without the apyrene sperm. Here a modified rapid and efficient method for sperm identification was developed, after 10 s of fixation in paraformaldehyde and 30 s of 4′6-diamidino-2-phenylindole (DAPI) or propidium Iodide (PI) staining, the sperm bundles can be detected easily using a fluorescence microscope. Sperm maturation process of silkworm from the fifth instar larvae to the adult was described with the above method, the precise time of earliest elongate apyrene bundles was detected on day 2 of pre-pupation, with a ratio of 5% in total sperm bundles, after which the percentage of apyrene sperm bundles increased rapidly and attained a relatively stable ratio of 75% at the end of pupation and nearly 80% after eclosion. Delayed mating leads to apyrene sperm accumulation and damaged fertilization. Previous study showed that ecdysone can increase the frequency of apyrene sperm bundles in vitro. Here 20-hydroxyecdysone (20E) was injected into hemolymph of the 2-d-old fifth instar larvae, the worms entered into mounting period after three days injection, but no apyrene sperm bundles were induced unless day 2 of pre-pupation. Interestingly, maturation of eupyrene sperm bundles were accelerated, and the ratio of eupyrene sperm bundles increased and exhibited a dose-dependent effect after 20E injection, which indicated that the development of eupyrene sperm can be accelerated by ecdysone before pupation of silkworm in vivo. These results will provide new clues for lepidopteran pest control.     

Behavior of centrioles during meiosis in the male silkworm, Bombyx mori (Lepidoptera)

The behavior of centrioles during eupyrene and apyrene meiosis was examined in the silkworm, Bombyx mori , by transmission electron microscopy and indirect immunofluorescence for tubulin. In eupyrene spermatocytes the centrioles, accompanied by axonemes, attached temporarily to the nucleus at diplotene, then detached from the nucleus in diakinesis. After the separation, a beret-shaped structure consisting of a double membrane covered the proximal region of the pair of centrioles. The structure disappeared after breakdown of the nuclear membrane. The centriole, with the axoneme, reattached to the nucleus at telophase I. The process was repeated during meiosis II until the centrioles maintained their nuclear attachment in newly developed spermatids. In stark contrast to their eupyrene counterparts, apyrene spermatocytes were conspicuously devoid of any attachment of the centrioles to the nucleus. These eupyrene-specific and apyrene-specific relationships were consistently and repeatedly found between the nuclear membrane and centrioles, giving rise to suspicion that the behavioral phenomena may be related to differentiation of the dimorphic sperm types.     

The regular divisions of the spermatocytes as related to a meiotic lysine-rich protein fraction

Lepidopteran primary spermatocytes are bipotential leading first to regular (eupyrene) and later to irregular (apyrene) meiotic divisions. The kinetics of the lysine-rich proteins during this dichotomous meiosis was studied using the fluorescent dye sulfoflavine. Throughout the spermatogonial divisions, the chromatin fluoresces while the cytoplasm remains unstained. Reversely, during the meiotic prophase, the cytoplasm fluoresces strongly while the nuclei show only a few weakly fluorescing structures. From premetaphase to telophase the meiotic chromosomes fluoresce strongly again. But during this period, only in the eupyrene cells the cytoplasm remains strongly fluorescent; the fluorescence vanishs in the cytoplasm of the apyrene spermatocytes. Thus, the regular (eupyrene) meiotic divisions and the presence of a lysine-rich protein fraction in the cytoplasm of the dividing spermatocytes of Lepidoptera, are probably related.     

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.     

Apyrene-spermatogenesis-inducing factor is present in the haemolymph of male and female pupae of the codling moth

The Lepidopteran spermatocyte is bipotential producing first eupyrene (nucleate) and later apyrene (anucleate) spermatozoa. It is proposed that this shift in commitment of the spermatocyte from eupyrene to apyrene spermatogenesis is related to an apyrene-spermatogenesis-inducing factor. Using testes transplantations we show that: (1) Apyrene-spermatogenesis-inducing factor becomes active towards pupation since apyrene spermatogenesis appears precociously when the testes of 4th-instar larvae are transplanted into pupae, but not into early 5th-instar larvae, and when testes of diapausing larvae are transplanted into pupae (2) The factor is a haemolymph factor since the experimental testes are transplanted into the thorax, far from their normal location in the abdomen (3) The factor is not sex-determined since both male and female hosts equally induce apyrene spermatogenesis in testes transplanted from diapausing larvae into pupae.     

Double copulation of a female with sterile diploid and polyploid males recovers fertility in Bombyx mori

Silkworm males produce dimorphic sperm, nucleate eupyrene sperm and anucleate apyrene sperm. Apyrene sperm have been speculated to have an assisting role in fertilisation. However, the coexistence of eupyrene and apyrene sperm in the testis and female reproductive organs has made it difficult to define the role of apyrene sperm. Polyploid males are highly sterile. Microscopic observation revealed that the elimination of eupyrene nuclei by peristaltic squeezing caused the sterility of polyploids. Heat-shock applied to pupae of Daizo males (DH) also induced high sterility due to the lack of normal apyrene sperm. When eupyrene sperm of sterile DH males and apyrene sperm of sterile polyploid males were mixed by double copulation, a remarkable increase in fertility of the double-mated females was observed. This finding strongly suggests that the apyrene sperm are indispensable in fertilisation of the silkworm and that polyploid apyrene sperm function as a substitute for diploid sperm. We established an experimental system in which we can separate the two types of sperm for further studies on their functions without chemical and/or mechanical treatments.     

组蛋白H3Ser10磷酸化在家蚕精母细胞减数分裂中的动态分布

【目的】探究组蛋白H3Ser10磷酸化(H3Ser10ph)在家蚕Bombyx mori精母细胞减数分裂中的功能。【方法】解剖并分离家蚕4龄幼虫至蛹期精巢组织,通过丙烯酰胺凝胶包埋制备处于减数分裂不同时期的精巢组织玻片,以免疫荧光标记检测H3Ser10ph抗体在精母细胞减数分裂不同时期的定位特点。【结果】在家蚕有核精子精母细胞减数分裂过程中,组蛋白H3Ser10的磷酸化发生在粗线期染色体的特定位置,双线期H3Ser10ph信号逐渐减弱,至终变期时在染色体上完全检测不到磷酸化信号。随着细胞周期的进行,磷酸化信号又开始逐渐增强,减数第一次分裂中期时达到最高水平。当细胞进入减数第二次分裂前中期时,染色体臂上的H3Ser10ph信号消失,在靠近纺锤体微管的分裂面处有弥散的H3Ser10ph抗体的信号,减数第二次分裂末期,仅剩余非常微弱的H3Ser10ph信号残留于染色体的特定位置。在无核精子精母细胞减数分裂过程中,在中期I至末期I一直在染色体上有较均一的3Ser10ph信号,后期I时纺锤丝微管与赤道面平行。【结论】组蛋白H3Ser10磷酸化与家蚕有核精子和无核精子精母细胞减数分裂中染色质的动态变化相关。     

Effect of fetal bovine serum on the enhancement of in-vitro cultivation of spermatocysts of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae)

Like other Lepidoptera, the silkworm (Bombyx mori) has both nucleated eupyrene and anucleated apyrene sperm that are derived from the same spermatocysts. The former type is responsible for egg fertilization, while the function of the latter is still uncertain. Many hypotheses have been presented concerning the role of the apyrene sperm in mating and fertilization, but none is supported by a convincing experimental approach. The aim of the present study was to enhance the production of apyrene sperm in vitro by using different concentrations of fetal bovine serum (FBS), namely 20%, 30% and 40%, in the culture medium used for cultivating the naked spermatocysts isolated from the silkworm testes at 0 hr, 120 hr, and 192 to approximately 360 hr after the fourth molt. Cultivation of 0-hr spermatocysts was not successful. The development of spermatocysts into eupyrene and apyrene sperm bundles was slightly slower in vitro than in vivo. The overall growth percentage of both eupyrene and apyrene bundles was satisfactory when the spermatocysts were cultivated in TC-100 culture medium containing 30% FBS.     

Precocious reprogramming of eupyrene-apyrene spermatogenesis commitment induced by allatectomy of the penultimate larval instar of the moth Actias selene

A possible causal relationship between the switch-over from eupyrene to apyrene spermatogenesis and pupation in Lepidoptera was examined in Actias selene. Precocious pupation, 11 days earlier than in the controls, was induced by allatectomy on the first day of the penultimate larval instar. The course of the eupyrene spermatogenesis, until nuclear elongation in the spermatid, is not related to pupation. In both allatectomized and control individuals, eupyrene metaphases appear 8 days after ecdysis of the fourth larval instar. Nuclear elongation, however, is triggered in the allatectomized individuals earlier than in the controls, probably by the premature decline of the juvenile hormone titre following allatectomy. Apyrene commitment, on the other hand, is directly related to pupation, as apyrene spermatogenesis begins 2 days after pupation in both control and allatectomized individuals.     

Apyrene sperm from the triploid donors restore fecundity of cryopreserved semen in Bombyx mori

Female moths of Bombyx mori were artificially inseminated with cryopreserved semen. The fertility of inseminated females varied from 0% to 76.9% depending on the strain. Addition of fresh semen from triploid males, which are infertile but whose semen includes intact apyrene sperm, greatly improved fecundity of cryopreserved semen from normal males. Frozen apyrene sperm from the triploid donors also improved the fecundity of females, inseminated with cryopreserved normal semen, but less than fresh semen from triploid males. Fertilization success in B. mori requires the presence of both, intact eupyrene and apyrene sperm. Our results show that eupyrene sperm tolerate the cryopreservation process better than apyrene sperm. Hence, we recommend to add apyrene sperm from the triploid donors as helper sperm routinely to cryopreserved semen in artificial insemination. This may advance the application of cryopreservation as a routine technique to maintain silkworm resources. The technique may also be applicable to other moth and butterfly species which, like B. mori, possess eupyrene and apyrene sperm.