Michael Friedländer

The eupyrene-apyrene dichotomous spermatogenesis of Lepidoptera: I. The relationship with postembryonic development and the role of the decline in juvenile hormone titer toward pupation

Abstract The relationships between the stages of postembryonic development and the occurrence of eupyrene and apyrene spermatogenesis, and the effects of the decline of the juvenile hormone (J.H.) titer toward pupation in these processes, were studied in the carob moth, Ectomyelois ceratoniae . The accurate timing of the spermatogenetic events was determined daily from the 2nd instar larva to the imago in squashes and electron microscope preparations of testes. Eupyrene spermatids elongate in two phases. In the first, beginning in late 4th instar larva, only flagella elongate, while in the second, beginning in the mid 5th instar larva, both flagella and nuclei elongate. Apyrene spermatogenesis starts just after the beginning of the nuclear elongation of eupyrene spermatids, in the mid 5th instar larva and not in the pupa, as is commonly believed. Using ligatures, topical applications of a J.H. mimic, and testes transplantation, it was found that the nuclear elongation begins in the 5-day-old eupyrene spermatid and cannot be induced earlier; the elongation is inhibited by high titer of the J.H. mimic. Elongation of the flagella, however, is unaffected by fluctuations of the J.H. titer. The onset of the apyrene spermatogenesis, which occurs in the very early 5th instar larva or before, was found to be unrelated to the decline in the J.H. titer toward pupation.

Eupyrene and Apyrene Sperm: Dichotomous Spermatogenesis in Lepidoptera

Publisher Summary This chapter focuses on the dichotomous spermatogenesis in Lepidoptera. Research into how apyrene sperm development occurs and is regulated plays important dividends in understanding of normal spermatogenesis. The existence of apyrene spermatocytes that continue developing in a predictable way to produce spermatozoa after their nuclei become apoptotic presents a very convenient system to study differentiation of eukaryotic cell in the absence of newly synthesized informative RNA. Furthermore, the dichotomous eupyrene–apyrene dichotomy shows cells having the same genetical background but differentiating separately through a series of well characterized morphological markers that are easy to correlate with stages on molecular experimentation. Genetic techniques are poorly developed in the Lepidoptera, but molecular genetic techniques are now very powerful. The chapter gives some indications of where it might be profitable to look for genes controlling apyrene spermatogenesis and look forward to advances in this research area. Lepidopteran insects are important subjects of research in many areas of reproductive ecology, especially those of sperm allocation, sperm competition, and post copulatory sexual selection. The existence of two types of sperm in this group of insects is of fundamental importance to their reproductive biology, and there is no doubt that the evolutionary biology of sperm dichotomy will continue to be of great interest.

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 pu...

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

Abstract 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.

Phylogenetic branching of trichoptera and lepidoptera: An ultrastructural analysis on comparative spermatology

The divergence during the evolution of the sister orders of Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) from their common ancestors is still subject to disagreement. This is partially due to lack of clarity concerning the phylogenetic position of Zeugloptera which is considered to be either a very primitive suborder of Lepidoptera or an order of its own, more archaic than both Trichoptera and Lepidoptera. The ultrastructural analysis on comparative spermatology reported here shows that dichotomous spermatogenesis producing concomitant, normal, anucleate (apyrene) and nucleate (eupyrene) spermatozoa: (1) is generalized in Lepidoptera as it occurs, in addition to higher Lepidoptera, also in the primitive Hepialoidea, (2) is present in Zeugloptera, and (3) is absent from Trichoptera which produce only nucleate spermatozoa. It is concluded that dichotomous spermatogenesis is an evolutionary novelty of Lepidoptera (including Zeugloptera) that was established at the origin of the order, after the divergence of Trichoptera and Lepidoptera.

Intratesticular ecdysteroid titres and the arrest of sperm production during pupal diapause in the tobacco hornworm, Manduca sexta

Abstract Lepidopteran spermatogenesis, which is a dichotomous process producing concomitantly both eupyrene (nucleate) and apyrene (anucleate) spermatozoa, discontinues during diapause. In diapause-averting pupae of Manduca sexta, the intratesticular titre of 20-hydroxyecdysone increases sharply to a post-pupation peak in day-10 pupae, and then drops sharply. In contrast, the titre is persistently low throughout diapause. In diapause-averting pupae, the post-pupation ecdysteroid peak and its abrupt decline, occur shortly before cessation of eupyrene meiosis. Accordingly, eupyrene meiosis continues uninterruptedly in diapausing larvae having experienced neither the peak, nor the fall of the ecdysteroid titre. The advance of apyrene spermatocytes towards meiotic metaphases coincides in diapause-averting pupae with the post-pupation rise in ecdysteroid titre. Accordingly, in diapausing pupae lacking this rise, apyrene spermatocytes stop developing, and lyse, before entering metaphase. The lack of the post-pupation peak of ecdysteroid may be related also to the lysis of eupyrene spermatids in the diapausing larvae. Fluctuations of the intratesticular ecdysteroid content follow the corresponding patterns of fluctuations in haemolymph ecdysteroid content in both diapausing and diapause-averting pupae. These findings do not support the idea that testicular ecdysteroids may play a separate, major role on controlling discontinuity of sperm production during diapause in Manduca.

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

Abstract 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.

Meiotic metaphases are induced by 20-hydroxyecdysone during spermatogenesis of the tobacco hornworm, Manduca sexta

Abstract Spermatocyte progression towards meiotic metaphases is induced in Manduca sexta by the post-wandering peak of 20-hydroxyecdysone occuring at stage day-2 wandering larvae. Abdomens isolated after the peak display meiotic metaphases, while abdomens isolated before the peak show spermatocytes blocked at meiotic prophase. This meiotic block is removed by either implantation of prothoracic glands or injecting 20-hydroxyecdysone into the isolated abdomens. Spermatocytes cannot be induced to undergo metaphases in abdomens isolated before wandering begins. Duration of the unblocking effect of 20-hydroxyecdysone is dose-dependent; the larger the dose injected, the more extended the effect. The unblocking effect may be indirect as in vitro , only testes explanted from day-2 or older wandering larvae bear meiotic metaphases even in the presence of 20-hydroxyecdysone. The 20-hydroxyecdysone titre required for unblocking meiosis is lower than that needed for pupation. Neck ligatures show that cephalic organs are not involved in meiosis unblocking. The effect of 20-hydroxyecdysone in unblocking meiosis differs from its effect on renewal of spermatogenesis in diapausing testes in vitro during which the spermatocytes, failing to undergo metaphases, produce unreduced spermatids.

Tebufenozide (Mimic®), a non-ecdysteroidal ecdysone agonist, induces spermatogenesis reinitiation in isolated abdomens of diapausing codling moth larvae (Cydia pomonella)

Abstract Spermatogenesis discontinues during lepidopteran diapause. To elucidate the mode of action of ecdysteroids on the regulation of this phenomenon, we treated isolated abdomens of diapausing codling moth larvae with the non-ecdysteroidal ecdysone-agonist Mimic® (tebufenozide). Under similar conditions, Mimic®-treated abdomens renewed Spermatogenesis, while corresponding untreated abdomens did not. Apparently, Mimic® induces spermatogenesis reinitiation by attaching to, and saturating, a specific number of receptors causing an “all or none” type of response as: (1) Resumption of spermatogenesis was induced by dosages of Mimic® ranging from 0.25–100 μg in 100% of treated abdomens, while lower dosages induced renewal in only 70% of them. (2) Germ cells developed similarly in all the testes bearing renewed spermatogenesis regardless of the dosage of Mimic® applied to the abdomens. (3) The excessive Mimic® included in the higher doses was innocuous, causing no visible detrimental effects on Spermatogenesis or physiological functions related to abdomen survival. Germ cells were more susceptible than somatic ones to Mimic®, since (1) only part of the abdomens showing spermatogenesis renewal displayed pupal integument or fusion of the testes and (2) all the abdomens showing spermatogenesis renewal displayed apyrene spermatogenesis, a pupal characteristic, regardless of whether the integument was of pupal type or the testes had fused.

Leydig cell differentiation during the reproductive cycle of the seasonal breeder Camelus dromedarius: an ultrastructural analysis.

Spermatogenesis and Leydig cell development in the dromedary were analyzed at the ultrastructural level and correlated with fluctuations of testosterone synthesis during the mating and nonmating seasons. It was found that (1) spermatogenesis and diameter of the seminiferous tubules are dissociated from seasonal fluctuations of testosterone synthesis as they remain similar throughout the year; (2) the volume of the interstitial tissue and the rate of testosterone synthesis are correlated since both increase during the mating season and both diminish during the nonmating season; (3) during the mating season, reduction of the tubular smooth endoplasmic reticulum (SER) and proliferation of condensed SER correspond to the relatively high rate of testosterone synthesis by the 4-ene pathway; (4) during the mating season there is a drastic reduction of the SER and proliferation of myelin figures within the Leydig cells which disrupt at the end of their differentiation. During the nonmating season, testosterone synthesis is probably impaired only at the final stage of differentiation of the Leydig cell.