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.     

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.     

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.     

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.     

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.     

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.     

Movement of spermatozoa in the reproductive tract of adult male Spodoptera litura: daily rhythm of sperm descent and the effect of light regime on male reproduction

Sperm production and movement from the fused testes into the male reproductive tract of the common cutworm Spodoptera litura were studied in insects maintained in a 12 h:12 h light dark (LD) regime. Two types of sperm bundles, eupyrene (nucleated) and apyrene (anucleate) were present in the adult testes. Eupyrene bundles constituted about 25% of the total. Descent of spermatozoa from the testes into the upper vas deferens (UVD) first occurred about 24-30 h before adult eclosion. On entering the reproductive tract, eupyrene spermatozoa remained in bundles while apyrene bundles became dissociated before they reached the UVD. Downward movement of both eupyrene and apyrene spermatozoa within the male tract occurred in a daily rhythm. Sperm descent from the testes into the UVD occurred during the early scotophase, followed by their further descent into the seminal vesicle (SV) during the photophase. Spermatozoa remained in the SV for only a short duration, whence sperm quickly passed through the lower vas deferens into the duplex, which acted as the main sperm storage organ until mating was initiated. During mating 80% of sperm left the duplex, but mating did not influence the number of sperm bundles that subsequently descended into the duplex or the rate of their descent. There was no evidence of sperm reflux. Rearing in constant light (LL) and in constant dark (DD) reduced the number of eupyrene sperm present in the testes of adults that emerged in LL and DD compared to controls (LD), although there was no significant effect on the number of apyrene sperm in the testes. The rhythmic pattern of sperm descent was suppressed in both LL and DD regimes, and the number of sperm in the duplex was adversely affected, with a marked impact in LL reared insects. Male longevity, mating behaviour, oviposition and fertility were found to be more severely affected in LL than in DD.     

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.     

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.     

Effects of 20-hydroxyecdysone on sperm release from the testes of the Mediterranean flour moth,Anagasta kuehniella (Zeller)

Abdominal injection of 1 μg aqueous 20-hydroxyecdysone into Anagasta kuehniella, anytime prior to the initiation of sperm release from the testes, prevents the impending release of eupyrene sperm bundles. Apyrene sperm release is not prevented and there is complete recovery of eupyrene release by the following cycle 24 hr later. If 20-hydroxyecdysone is administered on consecutive days, no eupyrene bundles are released and although apryene sperm release continues, it diminishes with time. The effect of 20-hydroxyecdysone in preventing eupyrene release is dose dependent. Administration of decreasing 20-hydroxyecdysone dosages results in increasing numbers of eupyrene bundles released. When a single injection of 20-hydroxyecdysone is administered to isolated abdomens, recovery time of eupyrene sperm release is slower than in whole moths and total recovery is not seen even by 5 days after administration. Apyrene sperm release is also affected to a greater extent than in whole moths, and in some cases, no apyrene release was detected at all. Treatment with 20-hydroxyecdysone prevents cupyrene bundles from passing through the testicular basilar membrane into the vasa efferentia, thus causing a build up of bundles near the basilar membrane but no disintegration of these eupyrene sperm bundles.     

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.     

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.     

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.     

Induction of motility of apyrene spermatozoa and dissociation of Eupyrene sperm bundles of the silkmoth,Bombyx mori,by initiatorin and trypsin

Summary

The activator, or inducer of motility, of apyrene spermatozoa of the silkmoth, Bombyx mori, was shown to be present in the posterior glandula (g.) prostatica. The activity of this factor in this gland was lost by boiling at 100°C for 10 min. Bundles of eupyrene sperm were dissociated by treatment with either a g. prostatica homogenate or trypsin, and their dissociation was concentration-dependent. On the contrary, for activation of apyrene sperm, the optimal trypsin concentration was 0.45–1.80 μg/ml and activation decreased at higher and lower trypsin concentrations. Microscopic observation showed that the dissociation rate of eupyrene sperm bundles by the g. prostatica homogenate corresponded to that by 0.45–9.0 μg/ml of trypsin. An autolysate of the g. prostatica and digests of seminal fluid with the g. prostatica homogenate or trypsin did not activate apyrene sperm. Of 11 endopeptidase inhibitors tested, antipain, leupeptin, TLCK, TPCK and PMSF strongly inhibited sperm activation by the g. prostatica homogenate, suggesting that the activator is an endopeptidase of the wine protease type. The 6 exopeptidase inhibitors tested did not inhibit activation of apyrene spermatozoa and the dissociation of eupyrene sperm bundles are both caused by the same factor, initiatorin, an endopeptidase of the serine protease type present in the prostatic secretion.     

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.     

Effects of sublethal dose of chlorfluazuron on testicular development and spermatogenesis in the common cutworm, Spodoptera litura

This paper describes the physiological mechanism of action of chlorfluazuron on testicular development and spermatogenesis when sublethal doses (LD₁₀: 1.00 ng/larva or LD₃₀: 3.75 ng/larva) are applied topically to the cuticle of newly moulted fifth instars of the common cutworm Spodoptera litura (F.) (Lepidoptera, Noctuidae). These doses disrupt the growth and development of testes by decreasing the volume and weight of testes and thickness of testes sheath as compared with that of the controls. Sublethal doses of chlorfluazuron also significantly reduce the protein content of the testis, but do not affect the carbohydrate and lipid contents in newly emerged treated males when measured in μg/mg of testis as compared with that of the controls. Additionally, such doses disrupt spermatogenesis by reducing the number and size of eupyrene and apyrene sperm bundles in the testis. Very few or no eupyrene sperm bundles are observed in vas deferens of pre‐ and newly moulted adults compared with controls. This result shows that the transfer of sperm bundles from testes to vas deferens is delayed in treated males. The effects of chlorfluazuron on testicular development and spermatogenesis is thought to be one of the factors responsible for the reduction in fecundity, fertility and hatchability caused by sublethal doses of chlorfluazuron.     

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     

Effect of constant light on male sterility in the cotton leafworm Spodoptera littoralis

Females of the cotton leafworm (Spodoptera littoralis) reared in long day conditions (LD 16:8 h) and mated to males kept throughout the whole period of development in continuous light (LL) oviposit very small numbers of mostly sterile eggs. It was found that in control males reared from the first larval instar in long day conditions there was a large accumulation of euperene sperm bundles in their testes on day 1 after imaginal moult. On day 10 of adult life the number of the sperm bundles was very small. In males kept from the first instar in continuous light there was also high number of sperm bundles on day 1 after imaginal moult but it did not decrease significantly on day 10 as was observed in controls. Transfer of different developmental stages of S. littoralis from long day conditions to continuous light resulted in a big difference in the density of eupyrene sperm bundles in their testes. In control insects reared through the whole of their development in long day conditions there was a significant decrease in the density of eupyrene sperm bundles on day 10 of adult life. By contrast, in males in continuous light, regardless of their developmental stage when transferred, there was either no change in density of sperm bundles in day 10 adults or there was a significant increase in comparison with day 1 adults. The highest density of eupyrene sperm bundles was observed in day 10 adults when they were transferred to continuous light shortly before moulting to the last instar (as day 4 larvae in the last stadium or day 1 pupae). Generally, the density of eupyrene sperm bundles on day 10 of adult development was about 2–2.5 times higher in males in continuous light then those under long day conditions. The results presented here indicate that the last larval instar and the pupa are the stages most sensitive to constant light treatment, which greatly reduces the amount of eupyrene sperm bundles released from the testes.     

Motility and dynamics of eupyrene and apyrene sperm in the spermatheca of the monandrous swallowtail butterfly Byasa alcinous

Lepidopteran males produce two sperm types: nucleated eupyrene sperm and non‐nucleated apyrene sperm. Although apyrene sperm are infertile, both sperm types migrate from the spermatophore to the spermathecal after copulation. As a dominant adaptive explanation for migration of apyrene sperm in polyandrous species, the cheap filler hypothesis suggests that the presence of a large number of motile apyrene sperm in the spermatheca reduces female receptivity to re‐mating. However, apyrene sperm are also produced in males of the monandrous swallowtail butterfly Byasa alcinous Klug. To identify the role of apyrene sperm in these males, the present study examines the number of spermatozoa produced and transferred and the dynamics and motility of spermatozoa in the spermatheca for each type of sperm. Apyrene sperm represents approximatey 89% of the sperm produced and transferred, which is comparable to polyandrous species. Two‐day‐old males transfer approximately 17 000 eupyrene and 230 000 apyrene spermatozoa to a spermatophore; approximately 5000 eupyrene and 47 000 apyrene spermatozoa arrive at the spermatheca. Eight days after copulation, most eupyrene spermatozoa remain in the spermatheca and a quarter of them are still active. However, the number of apyrene spermatozoa decreases and those remaining lose their motility after the arriving at the spermatheca. Consequently, 8 days after copulation, no motile apyrene sperm are found. The high proportion of apyrene sperm in the spermatophore, as well as in sperm migration, suggests that the production and migration of apyrene sperm is not simply an evolutionary vestigial trait. The possible functions of apyrene sperm in monandrous species are discussed.