Segregation products of male mice doubly heterozygous for the RB(6.16) and RB(16.17) translocations: Influence of sperm karyotype on fertilizing competence under varying mating frequencies

The meiotic segregants of male mice heterozygous for Rb(6.16)24Lub and Rb(16.17)7Bnr were viewed, for the first time, at first cleavage metaphase. Chromosomes were analyzed after G-banding, C-banding, and karyotyping. To study sperm aging effects, chromosomes of 202 one-cell zygotes derived from males mating at intervals of approximately 3,14, and 21 days were examined. At least 89.6% of sperm-derived complements were products of 2:2 segregation; at most, a possible 6.4% were 3:1 segregants. The six expected types of 2:2 segregants, both balanced and unbalanced, were equifrequent in the total zygote population derived from sperm of all ages. When the data were analyzed according to mating frequency, the 3-day sperm population considered most likely to be fresh showed a deficiency of the segregant nullisomic for chromosome 6 and disomic for chromosome 17, when compared with the reciprocal segregant (P < 0.025) as well as to all other 2:2 segregants (P < 0.05). However, these sperm fertilized in greater numbers (P < 0.01) than their reciprocal segregant (disomic for 6 and nullisomic for 17) in the 14-day sperm population. While sperm with chromosomal abnormalities are capable of fertilization, the competence of segregants nullisomic for 6 and disomic for 17 apparently depends on the prior storage period in the male. Further, the results suggest that the effect of aneuploidy on sperm function is dependent on the specific chromosome(s) involved.     

Evidence for differential maturation of reciprocal sperm segregants in the murine Rb(6.16) translocation heterozygote.

The fertilizing ability of unaged sperm and those aged experimentally in the cauda by surgically ligating the corpus epididymis in males carrying the Rb(6.16) translocation was studied. Chromosomally normal females were inseminated with unaged sperm delivered by males mating at 3-day intervals, and aged sperm were studied after matings on 6-14 postoperative days. The sperm chromosome complement was analyzed in first-cleavage metaphase zygotes after sequential G- and C-banding of the chromosomes. Of 283 metaphasic zygotes in the control group, 183 (or 64.7%) were analyzed and showed a ratio of 2.7:1 for chromosomally normal and balanced segregants of the translocation, deviating significantly (P less than 0.001) from the expected 1:1. The ratio of X- to Y-bearing sperm also deviated from expected (P less than 0.01) mostly due to a significant deficiency (P less than 0.05) of balanced sperm that were X-bearing. Fertilized oocytes were recovered from matings of 10 males on days 6-8 postoperatively, and, of 139 metaphasic one-cell zygotes, 101 (or 72.3%) were analyzed. These showed a Mendelian ratio of 1:1 for normal and balanced segregants. The sex ratio in the aged group (57Y:41X) also showed no deviation from 1:1. The results, which reveal significant physiological distortions for both the segregation and the sex ratios in males heterozygous for the Rb(6.16) translocation, suggest that differential maturation of the translocation-bearing sperm and the chromosomally normal reciprocal exists. The findings further support the concept that sperm chromosomal complement affects their maturation and function, and that factors on chromosome 6 and the X or Y chromosome additively affect sperm function.     

The murine Rb(6.16) translocation: alterations in the proportion of alternate sperm segregants effecting fertilization in vitro and in vivo

Summary The segregation products of the mouse Rb(6.16)24 Lub male translocation carrier were analyzed at first cleavage metaphase to determine whether the proportion of normal, balanced, and unbalanced sperm segregants differ in fertilizations occurring in vivo and in vitro. From 34 males, the sperm genomes in 268 firstcleavage mouse embryos were analyzed cytogenetically: 137 and 131 following in vivo and in vitro fertilization, respectively. Both systems demonstrated a preponderance of alternate (67.2% and 54.2%) as compared to adjacent segregation (10.2% and 13.7% as estimated). A contingency table showed that the distribution of reciprocal alternate segregants differed significantly between the two fertilization environments (x ²=20.64, P<0.0005). Whereas chromosomally normal sperm were 3.6 times more likely than the balanced reciprocals to fertilize in vivo (78.3% normal:21.7% balanced), 11 ratios were recovered following in vitro fertilization (43.7% normal: 56.3% balanced). The data also showed an excess of Y-bearing sperm with the translocation in both in vivo and in vitro fertilization groups. In the latter these segregants were 3 times more likely than X-bearing ones to effect fertilization. These data suggest a phenotypic disadvantage of translocation-X-bearing sperm, possibly mediated through altered haploid gene expression on chromosome 6 and gene expression on the Y. The results show clear evidence for prezygotic selection in vivo and indicate that the environment in which fertilization occurs significantly affects the transmission frequency of this specific translocation.     

Estimates of sperm sex chromosome disomy and diploidy rates in a 47,XXY/46,XY mosaic Klinefelter patient

A 47,XXY/46,XY male was investigated for the incidence of aneuploidy in sperm sex chromosomes using a three-colour X/Y/18 fluorescence in situ hybridisation (FISH) protocol. A total of 1701 sperm nuclei were analysed. The ratio of X-bearing to Y-bearing sperm did not differ from the expected 1 : 1 ratio although there were more 23,Y sperm than 23,X sperm (844 vs 795). There was a significantly increased proportion of disomy XY and XX sperm compared with normal controls (0.41% vs 0.10%, P < 0.001 and 0.29% vs 0.04%, P < 0.01). However, the incidence of YY sperm was similar to the controls (0.06% vs 0.02%). The diploidy rate was also significantly increased (1.7% vs 0.13%, P < 0.0001), as was disomy 18 (0.71% vs 0.01%) and 25,XXY (0.47% vs 0%). The results support the hypothesis that some 47,XXY cells are able to undergo meiosis and produce mature spermatozoa. Patients with mosaic Klinefelter syndrome with severe oligozoospermia have significantly elevated incidences of disomy XY and XX sperm and may be at a slightly increased risk of producing 47,XXX and 47,XXY offspring. Additionally, they may be at risk of producing offspring with autosomal trisomies. Hence, patients with Klinefelter mosaicism scheduled for intracytoplasmic sperm injection intervention should first undergo FISH analysis of their sperm to determine their risk. Received: 16 November 1998 / Accepted: 16 February 1999     

Sperm chromosome analysis of two males heterozygous for a t(2;17)(q35;p13) and t(3;8)(p13;p21) reciprocal translocation

Summary Sperm chromosome complements from two males, one heterozygous for the reciprocal translocation t(2;17)(q35;p13) (n = 18) and one for t(3;8) (p13;p21) (n = 73), were analyzed. Only 2:2 segregations were observed with t(2;17): alternate, 56%; adjacent-I, 33%; adjacent-II, 11%. Both 2:2 and 3:1 meiotic segregations occurred in t(3;8): alternate, 34.2%; adjacent-I, 43.8%; adjacent-II, 20.5% and 3:1, 1.4%. A significant excess of chromosomally normal versus balanced sperm complements was observed with both translocation heterozygotes. The frequencies of other chromosome aberrations unrelated to the translocations were 16.7% for t(2;17) and 8.2% for t(3;8). The ratio of X-bearing to Y-bearing sperm was not different from the theoretically expected ratio of 1:1.     

Sex ratio distortion in bovine sperm correlates to recombination in the pseudoautosomal region

A total of 2122 single sperm from 35 bulls belonging to six different paternal half-sib groups were analysed with respect to two markers in the bovine pseudoautosomal region (PAR) and sex-specific loci on the X and Y chromosomes, respectively. A segregation ratio significantly different from 1:1 was observed in a test over all families, with a higher proportion of X-bearing gametes (53.5%). The analysis of recombination conducted separately for X- and Y-bearing sperm showed that X-bearing sperm cells possess highly significant individual and between-family variability in recombination rate, whereas Y-bearing sperm show linkage homogeneity. To test whether the two phenomena are related, different logistic regression models were fitted to the data. The results show that sex ratio significantly correlates with changes in recombination rate among X-bearing but not among Y-bearing sperm. Different hypotheses to explain these observations are discussed.     

Sex ratio in normal and disomic sperm: evidence that the extra chromosome 21 preferentially segregates with the Y chromosome.

In humans, deviations from a 1:1 male:female ratio have been identified in both chromosomally normal and trisomic live births: among normal newborns there is a slight excess of males, among trisomy 18 live borns a large excess of females, and among trisomy 21 live borns an excess of males. These differences could arise from differential production of or fertilization by Y- or X-bearing sperm or from selection against male or female conceptions. To examine the proportion of Y- and X-bearing sperm in normal sperm and in sperm disomic for chromosomes 18 or 21, we used three-color FISH (to the X and Y and either chromosome 18 or chromosome 21) to analyze >300,000 sperm from 24 men. In apparently normal sperm, the sex ratio was nearly 1:1 (148,074 Y-bearing to 148,657 X-bearing sperm), and the value was not affected by the age of the donor. Certain of the donors, however, had significant excesses of Y- or X-bearing sperm. In disomy 18 sperm, there were virtually identical numbers of Y- and X-bearing sperm; thus, the excess of females in trisomy 18 presumably is due to selection against male trisomic conceptions. In contrast, we observed 69 Y-bearing and 44 X-bearing sperm disomic for chromosome 21. This is consistent with previous molecular studies, which have identified an excess of males among paternally derived cases of trisomy 21, and suggests that some of the excess of males among Down syndrome individuals is attributable to a nondisjunctional mechanism in which the extra chromosome 21 preferentially segregates with the Y chromosome.     

Chromosome aberrations in 450 sperm complements from eight controls and lack of increase after chemotherapy in two patients

Summary Four hundred fifty sperm complements from eight controls were analyzed. A conservative estimate of aneuploidy was 1.8% with a hyperhaploid rate of 0.9% (4/450). The overall frequency of structural aberrations was 8.9% (40/450). The proportion of X-bearing (47.5%) and Y-bearing (52.5%) sperm did not differ significantly. Sperm complements were analyzed from a cancer patient 9 months after poly chemotherapy (n = 63) and from a patient being treated with Imurek (azathioprine) (n = 30). There was no significant increase in the incidence of numerical and structural chromosome aberrations in the sperm of either patient. The percentages of X-bearing and Y-bearing sperm were not significantly different from the expected 50%.     

Reproductive costs of mating with a sibling male: sperm depletion and multiple mating in Cephalonomia hyalinipennis

Polygynous parasitoid males may be limited by the amount of sperm they can transmit to females, which in turn may become sperm limited. In this study, I tested the effect of male mating history on copula duration, female fecundity, and offspring sex ratio, and the likelihood that females will have multiple mates, in the gregarious parasitoid Cephalonomia hyalinipennis Ashmead (Hymenoptera: Bethylidae: Epyrinae), a likely candidate for sperm depletion due to its local mate competition system. Males were eager to mate with the seven females presented in rapid succession. Copula duration did not differ with male mating history, but latency before a first mating was significantly longer than before consecutive matings. Male mating history had no bearing on female fecundity (number of offspring), but significantly influenced offspring sex ratio. The last female to mate with a given male produced significantly more male offspring than the first one, and eventually became sperm depleted. In contrast, the offspring sex ratio of first‐mated females was female biased, denoting a high degree of sex allocation control. Once‐mated females, whether sperm‐depleted or not, accepted a second mating after a period of oviposition. Sperm‐depleted females resumed production of fertilized eggs after a second mating. Young, recently mated females also accepted a second mating, but extended in‐copula courtship was observed. Carrying out multiple matings in this species thus seems to reduce the cost of being constrained to produce only haploid males after accepting copulation with a sperm‐depleted male. I discuss the reproductive fitness costs that females experience when mating solely with their sibling males and the reproductive fitness gain of males that persist in mating, even when almost sperm‐depleted. Behavioural observations support the hypothesis that females monitor their sperm stock. It is concluded that C. hyalinipennis is a species with a partial local mating system.     

The segregation of a translocation t(1;4) in two male carriers heterozygous for the translocation

Summary We examined the meiotic segregation pattern of a t(1;4)(p36.2;q31.3) reciprocal translocation in two male cousins heterozygous for the translocation. The wife of subject 1 had four recognized spontaneous abortions and two carrier daughters, and the wife of subject 2 had three recognized spontaneous abortions and no liveborn children. The results showed that subject 1 had an imbalance rate of 54% and subject 2 had an imbalance rate of 61% with respect to the translocation. This was not statistically different (P = 0.3174) and the 95% confidence intervals overlapped for each segregation type. The sex ratio of X- and Y-bearing sperm was not statistically different than the expected 50%. The rate of structural abnormalities was 11.3% in subject 1 and 17.8% in subject 2. Both of these values were above the range of control subjects in our lab, but only subject 2's value fell outside the 95% confidence interval for the control population.     

Sperm Storage and Use After Multiple Mating in Dinarmus basalis (Hymenoptera: Pteromalidae)

Multiple mating and its effects on the sex ratio in Dinarmus basalis (Hymenoptera: Chalcidoidea, Pteromalidae), an ectoparasitoid of Callosobruchus maculatus (Coleoptera: Bruchidae), were investigated under controlled conditions. Once-mated females suffer a sperm depletion about 21 days after mating and thereafter are constrained to produce only haploid males. On the other hand, three-times-mated females store more sperm in their spermathecae (335 vs 147) and produce daughters during the major part of their reproductive life. Consequently, once-mated females showed a male-biased life time sex ratio (mean = 0.31) as opposed to three-times-mated females (mean = 0.63). Females can copulate only at the beginning of their reproductive life, and multiple mating must occur before egg-laying activity. This behavioral strategy could be an avoidance of consanguinity in a promiscuous environment. This ectoparasitoid species reveals a reproductive strategy which promotes polyandry and a very high sperm efficiency.     

Double Infection,Recombination, and Segregation of Two R Factors,R 100 and Rts1, and Their Possible Bearing on the Genetic Structure of R 100

Applying the observation by Yokota et al (1969) that a cell doubly harboring an R factor (R100) and a temperature sensitive R factor (Rts1) produces segregant R factors with various resistance patterns, a total of 271 segregant R factors were obtained. There were 163 resistant to (sul, str, kan), 39 resistant to (sul, str, cml, kan), 62 resistant to (sul, str, tet, kan) and finally 7 resistant to (tet, kan). More than 90% of the former 3 segregants were fi⁺ and the remainder, including all of the (tet, kan) segregants, were fi^?. Some fi^? segregants with the former 3 resistance patterns and all of the (tet, kan) segregants were nontransmissible. All of these segregants were still temperature sensitive. Based upon the results of three experiments; (a) the growth at 43 C to observe linked loss of the kan gene and the genes derived from R 100, (b) a conjugal analysis of the relevant resistant markers, and (c) a transductional analysis of these same markers, several conclusions were made. The 2 R factors both consisting of a circle were supposed to have recombined to form a larger circle which then further resulted in the final formation of smaller circles. The possible bearing of these observations and conclusions on the genetic structure of R 100 was discussed.     

Sperm competition in the melon fly,Bactrocera cucurbitae (Diptera: Tephritidae): Effects of sperm “longevity” on sperm precedence

Sperm competition inBactrocera cucurbitae was studied by double matings of one female with normal and sterile males, with different intervals between the first and the second matings and with or without allowing oviposition after the first or the second mating. When the interval was less than 4 days, the last-male sperm precedence,P ₂ , was not different from 0.5, but as the interval was prolonged,P ₂ was higher than 0.5. There was no significant difference between treatments in which females were allowed to oviposit after the first mating and only after the second mating. The reason for the higherP ₂ when the interval was long was therefore attributed not to sperm usage for egg fertilization during the two matings but, possibly, to sperm mortality. ThatP ₂ was 0.5 for shorter intervals suggests that particular sperm replacement mechanisms such as removal and inactivation are absent in B. cucurbitae. Our study is the first to demonstrate a significant effect of short sperm longevity on the last-male sperm precedence.     

Sperm (ejaculate) competition in Drosophila melanogaster, and the reproductive value of females to males in relation to female age and mating status

• 1 In double mating experiments with Drosophila melanogaster in which one male had been irradiated, it was confirmed that sperm displacement is extensive, i.e. the second male to mate displaces most of the previously-stored sperm.
• 2 The predominance of the second ejaculate over the first increases with the interval between the two matings, from about P₂= 0.83 (second mating on the first day after the first mating) to about P₂= 0.99 (interval between mating = 14 days) where P₂ is the proportion of offspring fathered by the second male.
• 3 A more accurate method for calculating P₂ values is developed for experiments in which sperm are ‘labelled’ by irradiation treatment (equation 1).
• 4 Observations of the reducing egg production of the female throughout life were also obtained. A model is examined which incorporates both the sperm competition and egg production data to predict the reproductive value to a male of a mating with a given type of female, varying in age and mating status. The relative value (in terms of probable numbers of progeny gained) of a mating with a virgin or 4 day post-mating female is about twice that of a 14 day post-mating female, mainly because of the fecundity difference.
• 5 Some evolutionary aspects of sperm competition and multiple mating in insects are reviewed and discussed.

     

Direct chromosomal analysis of human spermatozoa: preliminary results from 18 normal men.

Chromosomal analysis of 240 spermatozoa from 18 normal men was performed using in vitro fertilization of zona-free golden hamster eggs. The frequency of chromosome abnormalities in this population was 9.2% (22/240). Of the abnormal complements, 18 were aneuploid (13 hyperploid and five hypoploid) and four had a chromosome break. The sex ratio of Y-bearing to X-bearing sperm was .68. The frequency and type of sperm chromosome abnormalities is compared with those seen in spontaneous abortions.     

Sex‐specific repeatabilities and effects of relatedness and mating status on copulation duration in an acridid grasshopper

In species with direct sperm transfer, copulation duration is a crucial trait that may affect male and female reproductive success and that may vary with the quality of the mating partner. Furthermore, traits such as copulation duration represent the outcome of behavioral interactions between the sexes, for which it is important—but often difficult—to determine which sex is in phenotypic control. Using a double‐mating protocol, we compared copulation durations between (1) virgin and nonvirgin and (2) sibling and nonsibling mating pairs in rufous grasshoppers Gomphocerippus rufus. Nonvirgin copulations took on average approximately 30% longer than virgin copulations, whereas relatedness of mating partners was not a significant predictor of copulation duration. Longer nonvirgin copulations may represent a male adaptation to sperm competition if longer copulations allow more sperm to be transferred or function as postinsemination mate guarding. The absence of differences between pairs with different degrees of relatedness suggests no precopulatory or preinsemination inbreeding avoidance mechanism has evolved in this species, perhaps because there is no inbreeding depression in this species, or because inbreeding avoidance occurs after copulation. Controlling for the effects of male and female mating status (virgin vs. nonvirgin) and relatedness (sibling vs. nonsibling), we found significant repeatabilities (R) in copulation duration for males (R = 0.33; 95% CI: 0.09–0.55) but not for females (R = 0.09; 95% CI: 0.00–0.30). Thus, copulation durations of males more strongly represent a nontransient trait expressed in a consistent manner with different mating partners, suggesting that some aspect of the male phenotype may determine copulation duration in this species. However, overlapping confidence intervals for our sex‐specific repeatability estimates indicate that higher sampling effort is required for conclusive evidence.     

No Plastic Responses to Experimental Manipulation of Sperm Competition per se in a Free‐Living Flatworm

In the absence of sperm competition evolutionary theory predicts low mating rates and low ejaculate expenditure per mating, and sex allocation theory for simultaneous hermaphrodites predicts a strongly female‐biased sex allocation. In the presence of sperm competition a shift towards a more male‐biased sex allocation and a higher ejaculate expenditure are predicted. The free‐living flatworm Macrostomum lignano has been shown to respond plastically in mating rate, testis size, and sperm transfer to manipulation of the social group size, a proxy of the strength of sperm competition. However, manipulation of social group size may manipulate not only sperm competition, but also other factors, such as food supply and metabolite concentration. In this study we therefore manipulated sperm competition per se by repeatedly exposing individuals to partners that have either mated with rivals or not, while keeping the social group size constant. Our results suggest that M. lignano does not have the ability to detect sperm competition per se, as worms experimentally exposed to the presence or absence of sperm competition did not differ in sex allocation, sperm transfer or mating behavior. A response to our manipulation would have required individual recognition, the ability to detect self‐referencing tags, or tags or traces left by rivals on or in the mating partners. We first discuss the possibility that highly efficient sperm displacement may have decreased the difference between the treatment groups and then propose three alternative cues that may allow M. lignano to respond plastically to the social group size manipulation used in earlier studies: assessment of the mating rate, chemical cues, or tactile cues.     

Meiotic Nondisjunction and Aneuploids in Intersyngenic Hybrids of PARAMECIUM CAUDATUM

Artificially induced intersyngenic crosses in Paramecium caudatum can produce viable and fertile hybrids. When F₁ hybrids of double E mating type (Mt¹/Mt³ or Mt¹²/Mt³) were crossed with mating type O (mt/mt), aberrant segregants of double E and single O type were produced. This segregation was not explained by ordinary equal or unequal crossing over. Breeding analyses of these segregants by using linkage between Mt and cnrA (a behavioral mutant) revealed that they were produced by meiotic nondisjunction of bivalent chromosomes carrying Mt genes, and thus the double E and single O segregants were aneuploids: trisomics ( Mt¹/Mt³/mt or Mt ¹²/Mt³/mt) and monosomics (mt), respectively. An aberrant segregant was also obtained for another locus, tnd 2, independent of both Mt and cnrA, suggesting the occurrence of meiotic nondisjunction throughout hybrid genomes. These aneuploids will be useful for genetic study in this species. The occurrence of meiotic nondisjunction in the intersyngenic hybrids also suggests that syngens of P. caudatum have been reproductively isolated for long enough to develop chromosomal incompatibility in their meiotic process.     

Variable Female Mating Positions and Offspring Sex Ratio in the Spider Pityohyphantes phrygianus (Araneae: Linyphiidae)

Chromosomal sex determination and male heterogamety have been thought to seriously impede direct sex ratio control. However, in Pityohyphantes phrygianus, a solitary sheetweb spider with a skewed sex ratio, earlier experimental studies suggested that there are options for female control of offspring sex ratio, if females change their position during the normal mating sequence. Here we show that under natural conditions there is considerable between-female variation in positions, especially after termination of mating. Computer simulations of the orientation of female inner genitalia suggest that sperm are placed in different storage sites depending on the positions adopted. This means that a specific position after mating might potentially influence offspring sex ratio. The variance in offspring sex ratio among females in earlier experiments was binomially distributed, which leads us to conclude that females control the mean sex ratio but do not exercise direct control of the sex of individual offspring.     

Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting

Intact, viable X and Y chromosome-bearing sperm populations of the rabbit were separated according to DNA content with a flow cytometer/cell sorter. Reanalysis for DNA of an aliquot from each sorted population showed purities of 86% for X-bearing sperm and 81% for Y-bearing sperm populations. Sorted sperm were surgically inseminated into the uterus of rabbits. From does inseminated with sorted X-bearing sperm, 94% of the offspring born were females. From does inseminated with sorted Y-bearing sperm from the same ejaculates, 81% of the offspring were males. The probability of the phenotypic sex ratios differing from 50:50 were p less than 0.0003 for X-sorted sperm and p less than 0.004 for Y-sorted sperm. Thus, the phenotypic sex ratio at birth was accurately predicted from the flow-cytometrically measured proportion of X- and Y-bearing sperm used for insemination.