Sperm morphology and arrangement along the male reproductive tract of the butterfly Euptoieta hegesia (Insecta: Lepidoptera)

Summary

The present study was undertaken to describe the morphological and organizational modifications that occur in apyrene and eupyrene spermatozoa along the male adult reproductive tract of the butterfly, Euptoieta hegesia. Testis, vas deferens, vesicula seminalis and ductus ejaculatorius were studied by transmission electron microscopy. In the testis, both sperm types are organized into cysts; apyrene sperm are devoid of extracellular structures while eupyrene ones have lacinate and reticular appendages. In the testis basal region, both sperm pass through an epithelial barrier and lose their cystic envelope. The eupyrene morphological and organizational modifications are more drastic than the apyrene ones. From the vas deferens to the ductus ejaculatorius, apyrene sperm are dispersed in the lumen and acquire several concentric layers that are formed by the folding of their abundant cell membrane. The apyrene distribution observed here suggests that their functions include eupyrene transportation. Eupyrene sperm, however, remain aggregated along the tract. In the vas deferens, they are covered by a filamentous material that develops into a homogeneous matrix surrounding the spermatozoa coat in the vesicula seminalis and the ductus ejaculatorius. Eupyrene sperm undergo complex morphological changes that include the loss of lacinate appendages and the formation of a dense and heterogeneous extracellular coat. The formation of the matrix and the coat in eupyrene extratesticular sperm is related to the loss of lacinate appendages. These changes are in general extracellular and are probably important for sperm maturation.     

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.     

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.     

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.     

Morphology of the male reproductive system and sperm ultrastructure of Leucoptera coffeella (Lepidoptera: Lyonetiidae)

The male reproductive tract of Leucoptera coffeella was processed for light and transmission electron microscopy. In the testis, the eupyrene cells are arranged in individual cysts, while the apyrene cysts form aggregates, never observed in other Lepidoptera. Both cysts contain 128 spermatozoa, which differ from the typical pattern. In the seminal vesicle, both types of spermatozoa are dispersed in the lumen, also different from other Lepidoptera. The apyrene spermatozoa are similar to those observed for other Lepidoptera. They present an anterior region covered by a dense cap and the flagellum is composed of a 9 + 9 + 2 axoneme and two mitochondrial derivatives. The eupyrene spermatozoa, however, differ from the typical pattern for Lepidoptera. Their anterior region contains a nucleus, an acrosome and a peculiar arc of eight accessory microtubules connected to the plasma membrane by dense bridges. In the nucleus–flagellum region, the ninth accessory microtubule is assembled between both mitochondrial derivatives, to participate in the axoneme. The flagellum comprises a 9 + 9 + 2 axoneme and two mitochondrial derivatives with paracrystalline cores. External to the plasma membrane and close to the accessory microtubules, there are tufts of an amorphous material, suggesting reduced lacinate appendages, while the reticular ones are absent. The reduction of lacinate appendages and the absence of sperm bundles in the seminal vesicle support the concept that the appendages of other Lepidoptera could be associated with the eupyrene aggregations. The characters ‘number of spermatozoa per cyst’ and ‘absence of bundles’ should be considered plesiomorphic, supporting the position of this taxon in the base of the Ditrysia.     

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.     

Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000     

Ultrastructure and function of long and short sperm in Cicadidae (Hemiptera)

The cicada, Graptopsaltria nigrofuscata, produces two distinct sizes of sperm, as determined by either nuclear volume of early spermatids or nuclear length of mature sperm. Between both sperm, there is no difference in location of the acrosome and flagellum during spermiogenesis. The acrosome is covered by an anteacrosomal bleb, which is inserted in a common mass, spermatodesm, derived from cyst cells. Both kinds of sperm linked to the spermatodesm form sperm bundles, respectively. During copulation, the sperm bundles are transported from the vesicula seminalis of the male to the bursa copulatrix of the female. Morphometric analyses of the nuclear length revealed that the two kinds of sperm reach the bursa copulatrix in the same condition as that found in the vesicula seminalis. Once transferred inside the latter, the sperm bundles disintegrated to individual sperm within a few hours, and the tail components, such as the axoneme and mitochondrial derivatives, become separated from each other over time. The tail completely splits from the sperm nucleus 24 h after copulation. Fertile sperm accumulate in the spermatheca, the final storage organ, where only long sperm survived for any length of time. Fertilized eggs examined by vital staining contain only sperm with long nuclei.     

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.     

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.     

Effects of larval exposure to sublethal concentrations of the ecdysteroid agonists RH-5849 and tebufenozide (RH-5992) on male reproductive physiology in Spodoptera litura

Sublethal concentrations of the bisacylhydrazine moulting hormone agonists, RH-5849, and tebufenozide (RH-5992) were fed to sixth (final) instar larvae of Spodoptera litura. Both RH-5849 and tebufenozide adversely affected the mating success of S. litura when the surviving treated males were crossed with normal females. The ecdysone agonists decreased the longevity of treated males and of untreated females when crossed with treated males. The number of eggs laid by untreated females mated to treated males was decreased, and the fertility (percentage of hatching success) of the resulting eggs was reduced. These effects on male reproductive success were at least in part explained by a reduction in the number of sperm transferred during mating. The adverse effects of tebufenozide on male reproductive function were qualitatively the same as those of RH-5849, but tebufenozide was active at lower concentrations. To understand the reason for these adverse effects on male reproduction, we investigated the effects of the insecticides on male reproductive physiology. Male reproductive tract development and testicular volume of resulting adult moths were adversely affected by sublethal larval exposure to the ecdysone agonists. Dose-dependent reductions occurred in the production of eupyrene and apyrene spermatozoa in the adult testes, and in the number of spermatozoa released from the testes into the male reproductive tract. The descent into the male tract of both eupyrene and apyrene sperm was found to start at the normal stage of development in both normal and treated insects, but the daily rhythm of sperm descent was subsequently disturbed in the insecticide-treated moths. This affected the numbers of sperm in the upper vas deferens (UVD), seminal vesicle (SV), and duplex (duplex). Injections of RH-5849 given to pharate adult or newly emerged adult S. litura also caused drastic reduction in the number of sperm in the upper regions of the male tract, when measured 24 h after injection. The possible importance of pest population reduction through the sublethal anti-reproductive effects of insecticides is 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.     

Impact of male mating history on the temporal sperm dynamics of Choristoneura rosaceana and C. fumiferana females

In the oblique-banded leafroller, Choristoneura rosaceana, and the spruce budworm, C. fumiferana, male reproductive performance decreases with consecutive matings. While the onset time of mating did not vary, the time spent mating was longer in mated than in virgin males. Furthermore, a decline observed in the spermatophore mass with successive matings was associated with a concomitant decline in its apyrene and eupyrene spermatozoa content. In the hours following mating, spermatozoa migrate from the spermatophore, located in the bursa copulatrix, to the spermatheca. Regardless of the male's previous mating history, the number of apyrene sperm dropped rapidly in the days following mating whereas the number of eupyrene spermatozoa declined gradually. As the temporal pattern of sperm movement was similar in all treatments, females mated with previously-mated males would suffer from sperm shortage sooner than those mated with virgins. Large C. rosaceana females stored more apyrene spermatozoa in their spermatheca than small ones, irrespective of the time after mating or male mating history, while only large females mated with once-mated males received more apyrene sperm and accessory gland secretions than small ones mated with virgin or twice-mated males. The results obtained in this study are discussed in relation with their potential impact on the reproductive success of both sexes.     

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.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

Circadian rhythm of sperm release in the gypsy moth,Lymantria dispar: ultrastructural study of transepithelial penetration of sperm bundles

Release of mature bundles of spermatozoa from the testis into the vas deferens is a critical but poorly understood step in male insect reproduction. In moths, the release of sperm bundles is controlled by a circadian clock which imposes a temporal gate on the daily exit of bundles through the terminal epithelium-a layer of specialized epithelial cells separating testis follicles from the vas deferens. The sequence of cellular events associated with the daily cycle of sperm release was investigated by scanning and transmission electron microscopy. In the hours preceding sperm release, there is a solid barrier between the testis and the vas deferens formed by the interdigitation of cytoplasmic processes of adjacent terminal epithelial cells. At the beginning of the sperm release cycle, sperm bundles protrude through this barrier while the terminal epithelial cells change their shape and position relative to the bundles. Subsequently, the cyst cells enveloping the sperm bundles break down and spermatozoa move out of the testis through the exit channels formed between the epithelial cells. Afterwards, cyst cell remnants and other cellular debris are released into the vas deferens lumen, and the epithelial barrier is reconstructed due to phagocytic activity of its cells. These data provide a foundation on which to build an understanding of the cellular mechanisms of clock-controlled sperm release in insects.     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.     

The structural mechanism of trypsin-induced intrinsic motility in Manduca sexta spermatozoa in vitro

Lepidopteran males produce eupyrene (nucleate) and apyrene (anucleate) spermatozoa, but in the female only eupyrene spermatozoa leave the spermatheca and fertilize the eggs. Both kinds of spermatozoa lack intrinsic motility in the male genital duct. They become motile in the spermatophore, in a process involving proteases from the male duct. In vitro, trypsin induces immotile spermatozoa to become motile. We studied the changes spermatozoa of Manduca sexta undergo during trypsin-induced motility and found that (a) they mimick rather closely those occurring in vivo during normal sperm maturation in genital ducts and (b) they are time- and dose-dependent. As in vivo, they comprise, successively, (a) disappearance of an extracellular matrix that maintains the integrity of eupyrene bundles in the seminal vesicle, (b) dispersion of the eupyrene bundles and intermingling of eupyrene and apyrene spermatozoa and (c) "hatching" of eupyrene spermatozoa from individual enclosing envelopes that are formed in the seminal vesicle. "Hatching" may not directly be related to motility since eupyrene spermatozoa become motile before "hatching" and motile apyrene spermatozoa never "hatch". Rather "hatching" may be related to the capacitation of eupyrene spermatozoa to either leave the spermatheca or fertilize the eggs, or both, as neither apyrene spermatozoa, nor those eupyrene spermatozoa that fail to "hatch", leave the spermatheca.     

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.     

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.