In vitro fertilization with flow-cytometrically-sorted bovine sperm

An attractive feature of IVF is that fewer sexed sperm are needed than for artificial insemination. However, sperm sexed by flow cytometry/cell sorting are probably pre-capacitated, necessitating modifications to standard IVF systems for optimal success. With current procedures, the percentages of oocytes fertilized with sorted and unsorted frozen bovine sperm are similar, and events during the first cell cycle are timed similarly for sorted and unsorted sperm. However, in most cases, blastocyst production with sorted sperm was approximately 70% of controls produced with unsorted sperm. In some early studies, there appeared to be an unexplained delay of about half a day in blastocyst development. Nevertheless, some dozens of apparently normal calves, pre-sexed with 90% accuracy, have resulted from frozen embryos produced via IVF with sexed sperm. IVF also has proven useful as a bioassay for improving sperm-sorting procedures such as determining potential detrimental effects of laser power. It is likely that use of IVF in cattle breeding programs will increase considerably when sexed, frozen sperm become commercially available.     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.     

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.     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

Investigations of motility and fertilization potential in thawed cryopreserved mouse sperm from cold-stored epididymides

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.     

In vitro fertilization of two species of deer mouse eggs by homologous or heterologous sperm and penetration of laboratory mouse eggs by deer mouse sperm

Newly ovulated eggs from immature deer mice (Peromyscus maniculatus and P. polionotus) and mature laboratory mice (Mus musculus) treated with PMSG and HCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis of mature males. The time required for capacitation of deer mouse sperm in culture was estimated to be about two to five hours based on the dispersal of sperm agglutination and increase of sperm motility. The rate of sperm penetration through the zona pellucida of deer mouse eggs by homologous or heterologous sperm was relatively high (72-91%) but that of laboratory mouse eggs by deer mouse sperm was low (20-21%). After penetration through the zona pellucida, a high proportion of deer mouse eggs (79-93%) were fertilized by homologous or heterologous deer mouse sperm but no laboratory mouse eggs were fertilized by sperm of two species of deer mice. The zona pellucida was dissolved in a higher proportion of laboratory mouse eggs cultured with P. maniculatus (45%) than with P. polionotus sperm (3.4%), but this did not happen by incubation of deer mouse eggs with homologous or heterologous sperm. It seems that there is little difference in sperm penetration and fertilization between these two closely related species of deer mice but the reactions between the mouse eggs and deer mouse sperm are quite different.     

Reliable recovery of inbred mouse lines using cryopreserved spermatozoa

Since the mouse has become the most detailed model system to investigate the genetics and pathogenesis of human diseases, large numbers of new mouse strains have and continue to be produced. In nearly all animal facilities, the maintenance of breeding colonies is limited and mouse strains have to be archived in an efficient way. This study was undertaken to test the reliability of recovering mouse lines by use of cryopreserved spermatozoa from individual male mice. In contrast to many studies, spermatozoa and oocytes were derived from the same genetic background. 30 C3HeB/FeJ males belonging to three different categories (wild-type, F1-generation of ENU-treated males, and defined mutants) were recovered by producing at least 20 offspring from each donor. Independent of the experimental group, every single male was successfully recovered. Archiving mouse strains by cryopreservation of spermatozoa may, therefore, offer a reliable way to preserve genetically valuable mouse strains and provides an efficient management strategy for animal facilities. Received: 15 February 1999 / Accepted: 19 April 1999     

In vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs.

In vitro fertilization of rat and mouse eggs by ejaculated or epididymal spermatozoa in chemically defined media was studied. Penetration rates by ejaculated sperm was very low (0 to 8%) in the rat, but 11 to 41% of eggs were penetrated by ejaculated sperm in the mouse. The optimal concentration of sperm for in vitro fertilization appears to be similar whether ejaculated or epididymal sperm were used. The time of sperm penetration in the mouse eggs, however, was delayed for one-half to one hour when ejaculated sperm were used. The importance of sodium pyruvate, sodium lactate and glucose in the medium containing bovine serum albumin for in vitro fertilization of rat eggs was examined. When rat eggs in cumulus clot were exposed to epididymal sperm preincubated for five hours, the presence of sodium pyruvate, sodium lactate and glucose was found to play an important role. When exposed to non-incubated epididymal sperm sodium pyruvate could be omitted without much decline of the fertilization rate. When the denuded eggs were exposed to non-incubated sperm, penetration rates were very low (0 and 5%) in the absence of pyruvate. It appears that although lactate, pyruvate and glucose are all important for in vitro fertilization of rat eggs, pyruvate can be supplied by the follicular cells surrounding the eggs.     

Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.     

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.     

Interactions of aged gametes: In vitro fertilization using in vitro-aged sperm and in vivo-aged ova in the mouse

A study of varying combinations of in vitro-aged sperm and in vivo-aged ova at 3 hr intervals from 0–24 hr resulted in failures at different steps of the fertilization process during in vitro fertilization of mouse ova. Significant decreases caused by sperm aging, ova aging, and sperm × ova aging interaction were found in sperm penetration. Pronuclear formation was not affected by sperm aging and was enhanced by ova aging, and there was a significant effect of sperm × ova aging interaction. Sperm aging significantly influenced the prometaphase stage of the fertilization process. Therefore, it is suggested that the detrimental fertilization effects resulting from aging gametes are due to different mechanisms in sperm and ova, that these mechanisms are affected at different times, and that they affect different steps in the fertilization process.     

Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro

Mouse sperm were incubated in medium with or without 24 mM lactate and assessed for (1) motility characteristics including hyperactivation—a computer-assisted motion analysis system was used; (2) capacitation—a chlortetracycline fluorescent dye binding assay was used; and (3) ability to penetrate oocytes. Lactate affected all aspects of motility and delayed the rates of both hyperactivation and capacitation. When a concentration of 8 × 10³ sperm/ml was used for insemination in vitro, sperm preincubated 60–90 minutes in medium with lactate prior to insemination in lactate-free medium fertilized fewer oocytes than did sperm preincubated in lactate-free medium. Use of a calcium-sensitive electrode demonstrated that lactate chelated appreciable amounts of calcium in the medium. Capacitation was assayed in sperm incubated 60 minutes in medium with various concentrations of lactate or CaCl₂. When medium containing lactate was compared to medium without lactate but having a similar level of free calcium, the level of capacitation of sperm incubated with lactate was less than half that of sperm incubated without lactate. These results demonstrate that including 24 mM lactate in the medium can have detrimental effects on mouse sperm hyperactivation and capacitation. The detrimental effects on capacitation are partly but not completely due to the chelation of calcium by lactate.     

Successful fertilization and hatching of four European cyprinid species using cryopreserved sperm

In this study we tried to develop a uniform method of sperm cryopreservation for four cyprinid fish species indigenous to Hungarian waters: the roach (Rutilus rutilus L.), the bream (Abramis brama L.), the silver bream (Blicca bjoerkna L.) and the barbel (Barbus barbus L.). The sperm was frozen in liquid nitrogen vapor in the presence of five extenders (350 mm fructose, 30 mm Tris, pH 8.0; 350 mm glucose, 30 mm Tris, pH 8.0; 300 mm sucrose, 30 mm Tris, pH 8.0; 200 mm KCl, 30 mm Tris, pH 8.0 and modified Kurokura's extender) and two cryoprotectants: 10% methanol (MeOH) and 10% dimethyl‐sulfoxide. The highest post‐thaw motility (roach: 77 ± 6%, bream: 77 ± 6%, silver bream: 67 ± 5%, barbel: 75 ± 6%), fertilization (roach: 84 ± 4%, bream: 83 ± 2%, silver bream: 63 ± 2%, barbel: 70 ± 4%) and hatching (roach: 74 ± 2%, bream: 67 ± 6%, silver bream: 54 ± 2%, barbel: 61 ± 4%) rates were found when either fructose or glucose extenders were used in combination with MeOH as cryoprotectant for all four investigated species. Strong correlations were found between post‐thaw motility of the sperm and fertilization or hatching rates, which indicates that motility can be used to predict fertilization success in these species.     

In vitro fertilization of hamster and human oocytes by microinjection of human sperm

In this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influcnce on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 × 10⁶ sperm/ ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.     

Inhibition of in vitro fertilization by mouse anti-mouse sperm sera and preliminary antigen identification

Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.     

Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa

Spermatozoa from cauda epididymis of mature mice were suspended in preservation solution (Dulbecco's PBS containing raffinose in combination with glycerol, DMSO or skim milk as freezing protective agents). The suspension was frozen by the dry ice-alcohol method and preserved for 1-120 days in liquid nitrogen (-196 degrees C). Highest sperm viability after thawing was obtained with a combination of 10% raffinose and 5% glycerol or with a combination of 10% raffinose and 10% DMSO. These frozen thawed sperm were found to have fertilizing capacity when used for in vitro fertilization. The 2-cell embryos obtained through the above procedures developed into normal pups at a high rate when transferred into the oviducts of pseudopregnant female mice.