Shorter sperm confer higher competitive fertilization success

Spermatozoa exhibit taxonomically widespread patterns of divergent morphological evolution. However, the adaptive significance of variation in sperm morphology remains unclear. In this study we examine the role of natural variation in sperm length on fertilization success in the dung beetle Onthophagus taurus. We conducted sperm competition trials between males that differed in the length of their sperm and determined the paternity of resulting offspring using amplified fragment length polymorphism (AFLP) markers. We also quantified variation in the size and shape of the female's sperm storage organ to determine whether female morphology influenced the competitiveness of different sperm morphologies. We found that fertilization success was biased toward males with relatively shorter sperm, but that selection on sperm length was dependent on female tract morphology; selection was directional for reduced sperm length across most of the spermathecal size range, but stabilizing in females with the smallest spermathecae. Our data provide empirical support for the theory that sperm competition should favor the evolution of numerous tiny sperm. Moreover, because sperm length is both heritable and genetically correlated with condition, our results are consistent with a process by which females can accrue genetic benefits for their offspring from the incitement of sperm competition and/or cryptic female choice, as proposed by the "sexy sperm" and "good sperm" models for the evolution of polyandry.     

Efficacy of TEPA as a “sperm label” for competitive fertilization studies in the rabbit

In this paper, the conditions necessary to use TEPA [tris (1-aziridinyl)] effectively as a label for spermatozoa in competitive fertilization are established. The fertilizing ability of rabbit spermatozoa treated with 0 and 0.8 mg TEPA/ml was compared at insemination doses of 1, 5, 20, and 40 × 10⁶ spermatozoa. Fertility was assessed by collecting ova from 64 does 48 to 52 h after insemination. TEPA blocked all but 4% of the ova from developing when 1 × 10⁶ spermatozoa were inseminated, but fertility was reduced. When 5 × 10⁶ spermatozoa were inseminated following treatment with 0, 0.6 or 1.2 mg of TEPA/ml, the fertility was 83, 74 and 50% (P<0.05), and the percentage of ova containing more than four blastomeres was 83, 11 and 5% (P<0.05), respectively. The 0.6% TEPA level was selected for a competitive fertilization trial. Equal numbers of sperm from pure Dutch-color and albino sires were combined so that either both types were untreated, only the ‘albino’ semen was treated, only the ‘Dutch’ semen was treated, or both were treated. Does were inseminated with 5 × 10⁶ sperm and allowed to kindle. The litter sizes were 5.6, 3.1, 2.7, and 0 young, and the proportion of Dutch-color progeny was 63, 97, 0 and 0%, respectively, confirming the effectiveness of TEPA as a “label”. Only one of 60 young born resulted from fertilization by a TEPA-treated spermatozoon, demonstrating that few embryos fully escape the TEPA block. Thus, the TEPA concentration and sperm numbers were established to use TEPA effectively as a label for spermatozoa in competitive fertilization studies.     

Decreased sperm survivability in subfertile Delaware roosters as indicated by comparative and competitive fertilization

Duration of fertility following intravaginal and intramagnal insemination of hens with viable spermatozoa from subfertile Delaware roosters was compared with that obtained with spermatozoa from fertile Leghorns and subfertile Wyandotte roosters. In contrast to results with Leghorn and Wyandotte birds, duration of fertility was not increased following intramagnal insemination of spermatozoa from Delaware birds. Competitive fertilization also demonstrated that duration of fertility was less than expected in the spermatozoa from Delaware birds. Heritable subfertility in Wyandotte and Delaware roosters therefore appears to be attributable to distinct sperm defects.     

Turkey sperm mobility influences paternity in the context of competitive fertilization.

We have devised a novel means of investigating competitive fertilization in turkeys, using microsatellite genotyping to identify male parentage. Our results demonstrate that sperm mobility is a mechanism responsible in part for paternity efficiency in turkeys. Sperm mobility is composed of several parameters in which sperm motility is a component. Differences between ejaculates in the number of sperm penetrating into a dense, insert, nontoxic solution were measured and used to classify males into high, average, or low sperm mobility phenotypes. Microsatellite genotyping was used to determine parentage of poults after equal numbers of sperm from 10 males (either high or average phenotype, n = 5, mixed with low phenotype, n = 5) were inseminated simultaneously. In a separate study, the numbers of sperm hydrolyzing the perivitelline layer of eggs were compared between hens inseminated with sperm from high-, average-, or low-phenotype males. Overall, heterospermic inseminations resulted in consistently fewer offspring produced by low-mobility phenotype males. This correlated with physiological data in which semen from the low-mobility males had reduced numbers of sperm at the fertilization site as determined by sperm hole counts in the perivitelline layer of eggs. This is the first illustration of a measurable sperm trait predictive of paternity success in a competitive fertilization trial in turkeys, a species that is predominately reproduced by artificial insemination of multiple-sire pools.     

The relationship between sperm concentration and fertilization in vitro of rabbit ova

Different concentrations of in utero incubated rabbit sperm (1.5 × 10⁴-120 × 10⁴ /ml) were tested to determine whether there is a relationship between sperm concentration and level of fertilization achieved “in vitro” of rabbit ova. While low concentrations (1.5 × 10⁴-4.5 × 10⁴ /ml) resulted in relatively low fertilization (23–36%), those in the range of 13 × 10⁴?120 × 10⁴ /ml gave fertilization rates of 65–83%. Consistently high results were obtained with sperm counts above 40 × 10⁴ /ml. This is in agreement with the concentration of spermatozoa found in vivo in the Fallopian tubes around the time of fertilization (50 × 10⁴ /ml).     

Cat fertilization by mouse sperm injection

Summary Interspecies intracytoplasmic sperm injection has been carried out to understand species-specific differences in oocyte environments and sperm components during fertilization. While sperm aster organization during cat fertilization requires a paternally derived centriole, mouse and hamster fertilization occur within the maternal centrosomal components. To address the questions of where sperm aster assembly occurs and whether complete fertilization is achieved in cat oocytes by interspecies sperm, we studied the fertilization processes of cat oocytes following the injection of cat, mouse, or hamster sperm. Male and female pronuclear formations were not different in the cat oocytes at 6 h following cat, mouse or hamster sperm injection. Microtubule asters were seen in all oocytes following intracytoplasmic injection of cat, mouse or hamster sperm. Immunocytochemical staining with a histone H3-m2K9 antibody revealed that mouse sperm chromatin is incorporated normally with cat egg chromatin, and that the cat eggs fertilized with mouse sperm enter metaphase and become normal 2-cell stage embryos. These results suggest that sperm aster formation is maternally dependent, and that fertilization processes and cleavage occur in a non-species specific manner in cat oocytes.     

Factors affecting the respiration of rabbit sperm measured with an oxygen electrode

The respiration of rabbit sperm was measured by a Clarke type electrode which has two advantages over the conventional Warburg technique, greater sensitivity, and no necessity for a carbon dioxide-free atmosphere. It was not necessary to resaturate the sample chamber of the oxygen monitor with air, down to about 30% desaturation.Rabbit seminal plasma had a measurable oxygen uptake (0.6 μl/hr/ml) but this was much less than for human seminal plasma (4.3 μl/hr/ml). Hoderate dilution of the sperm and storing the semen at 0°C after slow cooling had no effect on oxygen uptake. Unlike those of most other species, rabbit sperm were also little affected by deliberate exposure to cold shock and the respiration before and after rapid cooling to 0°C was about the same. On the other hand very brief exposure of rabbit sperm to 65°C abolished motility and greatly reduced the respiration rate. Bicarbonate (6 mM) stimulated the oxygen uptake of freshly collected samples of rabbit sperm after washing. Increasing the phosphate concentration of the medium to 80 mM did not greatly depress the oxygen uptake.     

In vitro fertilization by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is the latest, and by far the most efficient, variant of micromanipulation-assisted fertilization, whereby a single spermatozoon is selected, aspirated into a microinjection needle and injected to the oocyte cytoplasm. The development of this technique is mainly linked to application in human assisted reproduction for which it enables fertilization with defective spermatozoa that would not otherwise be able to penetrate an oocyte by their proper means. Because ICSI by-passes many steps of the natural fertilization process, it offers an extremely interesting model for the study of basic mechanisms underlying fertilization. This is particularly true for oocyte activation, whose mechanism needs to be revisited in light of the current ICSI research. The massive application of ICSI in human infertility treatment also represents a huge laboratory in which the impact of different genetic and epigenetic anomalies of the male gamete on fertilization and embryonic development can be studied. BioEssays 21:791–801, 1999. © 1999 John Wiley & Sons, Inc.     

Annelid sperm and fertilization biology

This paper provides data on fine particulate organic matter (FPOM) and macroinvertebrates associated to natural and artificial leaf packs in a small woodland stream (Schlaube, Brandenburg). Macroinvertebrate colonisation and the dynamics of FPOM were studied in oven-dried alder leaf packs, air-dried alder leaf packs and packs with artificial leafshaped substrate exposed in the stream during a 68-day period. The importance of FPOM as a potential food source for macroinvertebrates especially in artificial leaf packs was evaluated. Changes in the quantity as well as in the chemical composition of the accumulating FPOM (>63 and <63 μm) was determined using soluble carbohydrates, proteins and chlorophyll a as parameters of the nutritional quality. Mass loss and the chemical changes of alder leaves during the decompositional process were also described. The loss of soluble carbohydrates due to leaching was more rapid in oven-dried alder leaf packs than in air-dried ones. After 3 days of leaf pack exposure weight loss of oven-dried and air-dried leaf packs was nearly comparable, as the similar decay coefficients, k = 0.0228 (oven-dried leaf packs) and k = 0.0214 (air-dried leaf packs), respectively, show. The amount of FPOM per unit leaf area constantly increased in artificial packs, although it remained below that of alder leaf packs at all sampling dates. The nutritional quality of FPOM <63 μm was constantly greater than that of FPOM >63 μm and decreased in both size-fractions with length of exposure. Referring to leaf area the abundance of macroinvertebrates continually increased in all packs till the end of exposure, whereas the numbers in artificial packs remained below that in alder leaf packs. The taxonomic composition of all treatments was very similar with Gammarus pulex being the most abundant taxon in all packs until day 42, while afterwards the caddis fly genus Hydropsyche gained in importance. The amphipod Gammarus pulex in general did not show a preference for air-dried alder leaf packs compared to oven-dried alder leaf and artificial packs. Corresponding dynamics of macroinvertebrate colonisation and FPOM content in artificial packs support the hypothesis that FPOM functions not only as an important food source for macroinvertebrates including gammarideans but also as a control mechanism of macroinvertebrate abundance in stream habitats. Even if the accumulation of FPOM and drifting macroinvertebrates might be influenced by the same abiotic factor (e.g. by reduction in stream velocity inside the packs) it is quite unlikely that only physical properties caused the invertebrates to stay.     

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.     

Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization

Dear Editor, Sperm cooperation has been observed in multiple species(Pizzari and Foster,2008),yet its existence and benefit for reproductive success in mammals remains underexplored.Here,combining tissue-clearing with deep three-dimen-sional (3D) imaging,we demonstrate that postcopulatory mouse sperm congregate into unidirectional sperm cooper-ative clusters at the utero-tubal junction (UTJ),a key physical barrier for passage into the oviduct.Reducing sperm number in male mice by unilateral vasoligation (Uni-Vas) or busulfan-treatment impairs sperm cluster formation and oviduct entry.     

Factors which may affect removal of protamine from sperm DNA during fertilization in the rabbit

In order to derive information about possible mechanisms by which the sperm head is converted into the male pronucleus during fertilization in the rabbit, unfertilized egg homogenate was assayed for two enzyme activities. Protamine was extracted from rabbit sperm, purified, and labelled with [¹⁴C] in an in vitro reaction and used as a probe to assay for a protein kinase which could transfer [³²P]PO₄ from [γ-³²P]ATP onto the substrate. A kinase with a pH optimum of approximately 8.0 to 8.5 is described. Assays for the enzyme glutathione reductase were performed using homogenates from eggs or embryos at three early stages of development. Results suggest that oocytes can oxidize 2.58 × 10^?6 μmol NADPH per minute per oocyte, unfertilized eggs 5.16 × 10^?7 μmol NADPH per minute per ovum, and 20- to 24-hour postcoitus fertilized eggs 2.30 × 10^?6 μmol NADPH per minute per ovum. The relevance of these observations to male pronuclear formation is discussed.