Defective Histone Transition during Spermiogenesis in Heterozygous SEGREGATION DISTORTER Males of DROSOPHILA MELANOGASTER

Males of Drosophila melanogaster that are heterozygous for the segregation distorter (SD) chromosome produce a gross excess of SD-bearing offspring because most of the non-SD-bearing sperm are dysfunctional. These dysfunctional sperm exhibit abnormalities in chromatin condensation and compaction during spermiogenesis. Use of the fluorescent dye sulfoflavine, which is specific for basic proteins, has now revealed that the dysfunctional sperm are also defective in the normal transition from somatic to spermatid-specific histones.     

Temperature Sensitivity of Negative Segregation Distortion in Drosophila Melanogaster

Previous work has shown that the direction of segregation distortion in the SD (Segregation Distorter) system in Drosophila melanogaster can sometimes be reversed, but this was found only with rather weak distorters and the effect was not large. The present study reports large negative segregation distortion in a strong distorter, SD-72 chromosome. In the presence of a specific X chromosome, supp-X(SD), the proportion, k, of SD-72 chromosomes recovered from the SD-72/cn bw males ranges from 0.99 at 20° to 0.11 at 28.5°, whereas with a standard-X chromosome, k ranges from 0.99 to 0.95 for the same temperature range. The temperature-sensitive period is during spermiogenesis. Using a mating system in which the sperm supply is nearly exhausted, it was shown that the negative distortion at high temperatures is due to an absolute reduction in the number of SD-72 chromosomes and an absolute increase in the number of cn bw chromosomes recovered. After adjusting for non-SD-related temperature effects, the amount of decrease in the number of SD-72 progeny is nearly the same as the amount of increase in the number of cn bw progeny, suggesting that the dysfunction switches from a spermatid carrying one homolog to one carrying the other. Negative distortion requires a radical revision of current hypotheses for the mechanism of segregation distortion and a possible modification of the current model is suggested, based on differential recovery of dysfunction in the two homologs during spermiogenesis.     

Environmental effects on Segregation-distorter in Drosophila melanogaster: Irradiation of SD-72 at the onset of spermatogenesis

To ascertain the effect of irradiation at the onset of spermatogenesis on Segregation-distorter, irradiation treatments were administered to very late third instar male SD larvae. Part of the experimental and control groups were stored. The first four daily broods from unstored irradiated males exhibited depressed k values and aberration products. The percentage of aberrations observed per sperm batch is inversely correlated with the k values exhibited. The SD mechanism appeared to be most sensitive to irradiation prior to or at Prophase I. Using aberration products as an index, stored males transfer few sperm which were in meiotic or premeiotic stages at the time of treatment. The induction of these aberrations affected the action of the SD mechanism very little. The SD-bearing exceptional chromosomes were 0.9 of the total exceptions recovered. Lack of mating activity depressed the drive of SD in both irradiated and control groups. Prolongation of spermiogenesis may allow recovery of SD+ gametes which were rendered temporarily dysfunctional.This work was completed at Michigan State University during the tenure of a National Institutes of Health Fellowship and partially supported by a grant to Dr. Armon F. Yanders from the U.S. Atomic Energy Commission (Contract AT (11-1) 1933)     

Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system

Segregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD⁺ chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD⁺ chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD⁺ sperm elimination remain unclear. Here we show that SD/SD⁺ males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD⁺ spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD⁺ genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD⁺ chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization.     

On the Components of Segregation Distortion in DROSOPHILA MELANOGASTER. II. Deletion Mapping and Dosage Analysis of the SD Locus

Segregation distorter (SD) chromosomes are preferentially transmitted to offspring from heterozygous SD/SD⁺ males owing to the induced dysfunction of the SD⁺-bearing sperm. This phenomenon involves at least two major loci: the Sd locus whose presence is necessary for distortion to occur and the Rsp locus which acts as the site of Sd action. Several additional loci on SD chromosomes enhance distortion.—In a previous study deletions were used to map the Sd locus and to determine some of its properties. We have extended this analysis with the isolation and characterization of 14 new deletions in the Sd region. From our results we conclude (1) SD chromosomes contain a single Sd locus located in region 37D2-6 of the salivary gland chromosome map. Deletion of this locus in any of three SD chromosomes now studied results in complete loss of ability to distort a sensitive chromosome; (2) the reduced male fecundity observed in many homozygous SD or SD_i/SD_j combinations is due at least in part to the action of the Sd locus. The fecundity of these males can be substantially increased by deletion of one Sd locus. Thus, it is the presence of two doses of Sd rather than the absence of Sd⁺ that produces the lowered male fecundity in SD homozygotes; (3) Sd behaves as a neomorph, whereas Sd⁺, if it exists at all, is amorphic with respect to segregation distortion; (4) these results support a model in which the Sd product is made in limiting amounts and the interaction of this product with the Rsp locus causes sperm dysfunction. The Sd product appears to act preferentially at Rsp^s (sensitive-Responder) but may also act at Rspⁱ (insensitive-Responder).     

Additive Effects of Multiple SEGREGATION DISTORTER ( SD) Chromosomes on Sperm Dysfunction in DROSOPHILA MELANOGASTER

A portion of the Segregation distorter (SD) chromosome, including both the Sd and E(SD) loci, has been moved by insertional translocation from SD Roma into Y^L . This Dp(2;Y)SD chromosome shows a negligible reduction in its ability to cause dysfunction of Rsp ^s-bearing sperm when compared to the parent SD chromosome, suggesting that SD can still act effectively, even when removed from its normal second chromosome milieu, and that its activity level does not depend on pairing with a normal autosomal homologue. Male genotypes have been constructed using this Dp(2;Y)SD along with a standard SD chromosome (either SD Roma or R( SD-36)-1^bw) and a third chromosome suppressor of SD (TM6) in all possible three-way combinations. The observed level of SD-mediated dysfunction in each case is most compatible with a model that assumes that all SD elements act additively (in terms of M, the probit transformation of the probability of sperm dysfunction), rather than multiplicatively. The additive action of SD elements contrasts with the independent response to SD activity exhibited by multiple Rsp^s copies.     

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small ''bag'' called the spermatheca. Later on, hermaphrodites continually produce oocytes¹. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for >50% of the total cells in a typical adult worm². Therefore, isolating sperm from males is easier than from that of hermaphrodites.Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome³. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population³.Males can be easily distinguished from hermaphrodites by observing the tail morphology⁴. Hermaphrodite''s tail is pointed, whereas male tail is rounded with mating structures.Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids².Males are directly dissected on a small drop of ''Sperm Medium''. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes⁵, and Nelson⁶ previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process⁷. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.Download video file.^{(33M, flv)}     

The relationship between first and second chromosome segregation ratios from Drosophila melanogaster males bearing Segregation Distorter

Summary Drosophila melanogaster males heterozygous for the second chromosome locus Segregation Distorter preferentially transmit this chromosome to their progeny due to a dysfunctioning of SD ⁺-bearing sperm. SD males with a normal sex chromosome constitution produce more females than males among SD ⁺ progeny. This report shows that this unequal recovery of sexes is enhanced from XY/Y; SD/SD ⁺ males and enhanced still further from XY/O; SD/SD ⁺ males. It is argued that the probability that a SD ⁺-bearing sperm will dysfunction is related to its sex chromsome complement, with the relative probabilities of dysfunction ranked O> Y> X> XY. It is shown that a modified probit analysis accounts for the relationship between sex ratio and second chromosome segregation frequency for all paternal genotypes. Finally, SD/SD ⁺ males show no increase in sex chromosome nondisjunction with respect to a control.R. E. Denell was supported by U.S.P.H.S. Training Grant No. GM00337 and by a U.S.P.H.S. Postdoctoral Fellowship; George L. Gabor Miklos was supported by A.E.C. Contract No. AT (04-3)-34 PA150.     

Further Characterization of Genetic Elements Associated with the Segregation Distorter Phenomenon in DROSOPHILA MELANOGASTER

Segregation Distorter, SD, associated with the second chromosome of Drosophila melanogaster, is known to cause sperm bearing the non-SD homologue to dysfunction in heterozygous males. In earlier studies, using different, independently derived, SD chromosomes, three major loci were identified as contributing to the distortion of segregation ratios in males. In this study the genetic components of the SD-5 chromosome have been the subjects of further investigation, and our findings offer the following information. Crossover analysis confirms the mapping of the Sd locus to a position distal to but closely linked with the genetic marker pr. Spontaneous and radiation-induced recombinational analyses and deficiency studies provide firm support to the notion that the Rsp (Responder) locus lies within the proximal heterochromatin of chromosome 2, between the genetic markers lt and rl and most likely in the heterochromatin of the right arm. The major focus of this study, however, has been on providing a better definition of the genetic properties of the Enhancer of SD [E(SD)]. Our findings place this locus within the region of the two most proximal essential genes in the heterochromatin of the left arm of chromosome 2. Moreover, our analysis reveals a probable association of the E(SD) locus with a meiotic drive independent of that caused by Sd.     

Chromosomal Control of Fertility in DROSOPHILA MELANOGASTER . I. Rescue of T(Y;A)/bb Male Sterility by Chromosome Rearrangement

Analysis of X-ray-induced deletions in the Segregation Distorter (SD) chromosome, SD-5, revealed that this chromosome had a gene proximal to lt in the centric heterochromatin of 2L that strongly enhanced the meiotic drive caused by the SD chromosome. This Enhancer of Segregation Distortion [E(SD)] locus had not been characterized in earlier studies of SD chromosomes because it cannot be readily separated by recombination from the Responder (Rsp) locus in the proximal heterochromatin of 2R.—To determine whether E(SD) is a general component of all SD chromosomes and to examine further its effects on distortion, we produced deletions of E(SD) in three additional SD chromosomes. Analysis of these deletions leads to the following conclusions: (1) along with Sd and Rsp, E(SD) is common to all SD chromosomes; (2) the E(SD) allele on each SD chromosome enhances distortion by the same amount, which indicates that allelic variation at the E(SD) locus is not responsible for the different drive strengths seen among SD chromosomes; (3) E(SD) causes very little or no distortion by itself in the absence of Sd; (4) E(SD), like Sd, acts in a dosage-dependent manner; (5) E(SD) exerts its effect in cis or trans to Sd; and (6) if E(SD)⁺ exists, its function is not related to SD.     

Investigations on the polymorphism of sperm diaphorase in man

Summary The polymorphism of sperm diaphorase (SD) was investigated in 141 unrelated persons from Hessen, Germany, by high voltage thin-layer agarose gel electrophoresis (Age) and thin-layer isoelectric focusing on polyacrylamide gel (Pagif). In addition to the three known common phenotypes SD 1, 2-1, and 2, two further phenotypes with the preliminary designation SD 3-1 and SD 3-2 were discovered. This polymorphism can thus be explained in terms of three alleles, SD¹, SD², and SD³ segregating at an autosomal locus. The allele frequencies calculated from the five different phenotypes SD 1, 2, 2-1, 3-1, and 3-2 are: SD¹=0.7553, SD²=0.2234, and SD³=0.0213. As we also found SD activity in female reproductive tract tissues (ovaries, oviducts, uterus), the term gonadal diaphorase (GD) appears to be applicable.     

On the Components of Segregation Distortion in DROSOPHILA MELANOGASTER

The segregation distorter (SD) complex is a naturally occurring meiotic drive system with the property that males heterozygous for an SD-bearing chromosome 2 and an SD⁺-bearing homolog transmit the SD-bearing chromosome almost exclusively. This distorted segregation is the consequence of an induced dysfunction of those sperm that receive the SD⁺ homolog. From previous studies, two loci have been implicated in this phenomenon: the Sd locus which is required to produce distortion, and the Responder (Rsp) locus that is the site at which Sd acts. There are two allelic alternatives of Rsp—sensitive (Rsp^sens) and insensitive (Rsp^ins); a chromosome carrying Rsp^ins is not distorted by SD. In the present study, the function and location of each of these elements was examined by a genetic and cytological characterization of X-ray-induced mutations at each locus. The results indicate the following: (1) the Rsp locus is located in the proximal heterochromatin of 2R; (2) a deletion for the Rsp locus renders a chromosome insensitive to distortion; (3) the Sd locus is located to the left of pr (2-54.5), in the region from 37D2-D7 to 38A6-B2 of the salivary chromosome map; (4) an SD chromosome deleted for Sd loses its ability to distort; (5) there is another important component of the SD system, E(SD), in or near the proximal heterochromatin of 2L, that behaves as a strong enhancer of distortion. The results of these studies allow a reinterpretation of results from earlier analyses of the SD system and serve to limit the possible mechanisms to account for segregation distortion.     

Segregation distorter in D. melanogaster males: An effect of female genotype on recovery

Summary Segregation Distorter (SD) is a gene affecting sperm recovery in Drosophila melanogaster. In a cross SD/SD ⁺ x SD⁺, the proportion of SD/SD ⁺ zygotes recovered (k) is larger than the 0.50 expected. Previous investigations have shown that the relative recovery of SD- and SD ⁺-bearing sperm is determined at or before the meiotic divisions. Evidence is presented here that k is influenced by the genotype of the female parent. It is suggested that the SD gene product causes a difference in the enzymatic or structural complement of the SD- and SD ⁺-bearing sperm which largely determines their relative functionality. The effect of the female genotype on recovery is interpreted as an interaction between this physiological difference and the environment of the female reproductive tract.Adapted from a dissertation presented by the senior author in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported by PHS Training Grant No. GM 00337 and PHS Research Grant No. GM 12334 from the National Institute of General Medical Sciences.     

Live for the moment—Adaptations in the male genital system of a sexually cannibalistic spider (Theridiidae, Araneae)

Monogyny in spiders culminates in extreme traits, like dramatic male self-sacrifice and emasculation of the male by the female during copulation. Here we show that monogynous males can be highly adapted for this fatal sexual behaviour. Dwarf males of the one-palped theridiid spider Tidarren argo, which are cannibalised immediately after the insertion of their single copulatory organ, stop spermiogenesis when reaching adulthood. Their testes atrophy, which might economise the energy expenditures of these males. We also found that the amount of seminal fluid produced is stored in an enlarged seminal vesicle until the single sperm induction takes place. The volume of the seminal vesicle is similar to the sperm droplet taken up into the copulatory organ (palpal organ). Sperm uptake takes much longer than in related species most likely due to the large amount of seminal fluid. As shown by histological observations males are able to fill one of the paired female sperm storage organs during copulation thereby presumably impeding subsequent charging by rival males.     

CAENORHABDITIS ELEGANS Fertilization-Defective Mutants with Abnormal Sperm

Seven new fertilization-defective mutants of C. elegans have been isolated and characterized; six are temperature sensitive, one is absolute and all are autosomal recessive. One mutation is in a previously described gene, while the other six define six new fer genes that appear to code for sperm-specific functions necessary for normal fertilization. In all fer mutants, both males and hermaphrodites accumulate sperm in near normal numbers. In hermaphrodites, mutant sperm contact the oocytes, but fail to fertilize them. Instead, the sperm are swept into the uterus by the passing oocytes and are expelled when oocytes are laid. Males of two fer mutants do not transfer sperm during copulation, but the other mutant males transfer sperm that fail to move to the spermatheca. Spermatozoa from fer-1 and fer-4 mutants are motility-defective in vitro as well as in vivo, and their pseudopods have an altered morphology. The period of development during which mutant hermaphrodites are temperature sensitive for fertility overlaps the time of sperm development. Some mutants are temperature sensitive throughout the entire period, and others are temperature sensitive during or just prior to spermiogenesis. In fer-4/+ and fer-7/+ males, the fertility of the mutation-bearing sperm is diminished, reducing the transmission ratio. This implies some post-meiotic expression of these genes.—This set of mutants provides a variety of functional and structural alterations in nematode sperm that should help identify and analyze gene products involved in sperm morphogenesis and motility.     

Sperm of Doradidae (Teleostei: Siluriformes)

Spermatic characteristics were studied in 10 species representing several distinct groups within the catfish family Doradidae. Interestingly, different types of spermatogenesis, spermiogenesis and spermatozoa are correlated with intrafamilial groups previously proposed for Doradidae. Semi-cystic spermatogenesis, modified Type III spermiogenesis, and biflagellate sperm appear to be unique within Doradidae to the subfamily Astrodoradinae. Other doradid species have sperm with a single flagellum, cystic spermatogenesis, and spermiogenesis of Type I (Pterodoras granulosus, Rhinodoras dorbignyi), Type I modified (Oxydoras kneri), or Type III (Trachydoras paraguayensis). Doradids have an external mode of fertilization, and share a few spermatic characteristics, such as cystic spermatogenesis, Type I spermiogenesis and uniflagellate sperm, with its sister group Auchenipteridae, a family exhibiting sperm modifications associated with insemination and internal fertilization. Semi-cystic spermatogenesis and biflagellate spermatozoa are also found in Aspredinidae, and corroborate recent proposals that Aspredinidae and Doradoidea (Doradidae + Auchenipteridae) are sister groups and that Astrodoradinae occupies a basal position within Doradidae. The co-occurrence in various catfish families of semi-cystic spermatogenesis and either biflagellate spermatozoa (Aspredinidae, Cetopsidae, Doradidae, Malapturidae, Nematogenyidae) or uniflagellate sperm with two axonemes (Ariidae) reinforces the suggestion that such characteristics are correlated. Semi-cystic spermatogenesis and biflagellate sperm may represent ancestral conditions for Loricarioidei and Siluroidei of Siluriformes as they occur in putatively basal members of each suborder, Nematogenyidae and Cetopsidae, respectively. However, if semi-cystic spermatogenesis and biflagellate sperm are ancestral for Siluriformes, cystic spermatogenesis and uniflagellate sperm have arisen independently in multiple lineages including Diplomystidae, sister group to Siluroidei.     

X-Linked Elements Associated with Negative Segregation Distortion in the Sd System of Drosophila Melanogaster

Three elements, M(1), M(2) and M(3), found in a special X chromosome, supp-X(SD), modify the degree and direction of segregation distortion in the SD system of Drosophila melanogaster. The first element, M(1), is located between the y and the cv loci, probably close to the y locus. The second element, M(2), is located near the cv locus and the third element, M(3), is located between the y and the car loci. The M(1) element appears to cause a relatively small amount of reduction in the rate of recovery of the SD-72, but not the cn bw, chromosome from SD-72/ cn bw males, when raised at 27.5°. The M(2) and the M(3) elements cause considerable decrease in the recovery rate of the SD-72 chromosome, whereas they increase the recovery rate of the cn bw chromosome. The amount of decrease is nearly the same as the amount of increase for each element. Some type of ``switch' mechanism in the directions of distortion is suggested for each of these two elements and their effects appear to be approximately additive.