Comparison of intracytoplasmic sperm injection for inbred and hybrid mice

We compared the results of intracytoplasmic sperm injection (ICSI) that leads to full term development of hybrid (B6C3F1 and B6D2F1) and inbred (C57BL/6) mouse embryos. Although fertilization and pre-implantation development of C57BL/6 eggs were similar to those of F1 hybrid eggs, post-implantation development of the embryos from C57BL/6 females was significantly poorer than those of the eggs from hybrid females. Reciprocal crosses of C57BL/6 and B6C3F1 gametes revealed that the low rate of post-implantation development of C57BL/6 embryos was due to oocyte factor(s), rather than the sperm factor.     

Efficient production of intersubspecific hybrid mice and embryonic stem cells by intracytoplasmic sperm injection

Recently, mice and embryonic stem (ES) cells with allelic polymorphisms have been used extensively in the field of genetics and developmental biology. In this study, we examined whether intersubspecific hybrid mice and ES cells with these genotypes can be efficiently produced by intracytoplasmic sperm injection (ICSI). Frozen-thawed spermatozoa from wild-derived strains, JF1 (Mus musculus molossinus), MSM (M. m. molossinus), HMI (M. m. castaneus), and SWN (M. m. spp.), were directly injected into mature oocytes from laboratory mice ([C57BL/6 x DBA2]F1; M. m. domesticus). The in vitro and in vivo developmental capacity of F1 embryos was not significantly different among the groups (P > 0.05), and term offspring were efficiently obtained in all groups (27%-34% of transferred embryos). However, the mean body and placental weights of the offspring differed significantly with genotype (P < 5 x 10(-10)), with the HMI hybrid greatest in both body and placental weights. In an application study using these F1 offspring, we analyzed their mitochondrial DNA using intersubspecific polymorphisms and found the consistent disappearance of sperm mitochondrial DNA in the F1 progeny. In a second series of experiments, we generated F1 blastocysts by injecting MSM spermatozoa into C57BL/6 oocytes and used them to generate hybrid ES cell lines. The ES cell lines were established at a high efficiency (9 lines from 20 blastocysts) and their allelic polymorphisms were confirmed. Thus, ICSI using cryopreserved spermatozoa allows the efficient and immediate production of a number of F1 hybrid mice and ES cell lines, which can be used for polymorphic analysis of mouse genetics.     

Production of mice through intracytoplasmic injection of sperm or spermatogenic cells

Summary Direct injection of spermatozoa or spermatogenic cells into oocytes bypasses many normal fertilization processes, yet it is a powerful tool to analyze basic principles/mechanism underlying normal fertilization and prefertilization processes. Birth of normal, fertile mouse offspring following injection of round spermatid nuclei into oocytes suggests that all the postmeiotic modifications in male gametes evolved as processes solely dedicated for the delivery of their nuclei into the eggs. Birth of normal mouse offspring following injection of infertile spermatozoa with grossly misshaped heads indicates that structurally abnormal spermatozoa are not necessarily genomically abnormal. The spermatozoa with disrupted plasma membranes are generally considered dead, but they are capable of producing normal offspring by injection, suggesting that cell death and nucleus death are not synonymous at least for the spermatozoa.     

Intracytoplasmic sperm injection effects in infertile azh mutant mice

Several reports in the literature describe men with infertility resulting from abnormal sperm head shape or decapitation defects of their spermatozoa. These defects are similar to those shown for the spermatozoa from azh (abnormal spermatozoon head shape) mice. The present study examines the efficiency and effects of intracytoplasmic sperm injection (ICSI) in successive generations of azh mice generated with this method. Three successive generations of azh mice were produced with ICSI. In all three ICSI series, more than 80% of 2-cell embryos were obtained, and more than 35% of embryos transferred gave rise to normal live offspring. In addition, ICSI was used to cross homozygous azh/azh males with homozygous azh/azh females, and live offspring were obtained. The ICSI-derived males were tested for their fecundity and abnormalities of sperm morphology. Spermatozoa from ICSI-derived azh/+ males did not show any impairment of fecundity in in vitro fertilization. These spermatozoa successfully fertilized oocytes from both C57BL/6 and B6D2F1 females, with fertilization rates ranging from 70%- 92%. The proportion of morphologically normal spermatozoa was similar in azh/+ males from three successive generations of ICSI (57.8%, 54.8%, and 49.0%, respectively), and no differences were noted when comparing ICSI-derived males with males derived by mating (57.6%) and with wild-type controls (61.6%). Detailed analysis differentiating between specific types of anomalies of sperm morphology did not reveal significant differences among the examined groups. The results of the present study demonstrate that ICSI does not enhance the azh mutation phenotype in the offspring and brings no risks when applied continuously. Moreover, serial (successive generations) ICSI is highly efficient in maintaining valuable mice with fertility problems.     

Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection

The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI.     

Cryopreservation of bovine blastocysts obtained by intracytoplasmic sperm injection

The freezability and survivability of zona-intact and zona-free (hatched) bovine blastocysts obtained by intracytoplasmic sperm injection (ICSI) were assessed. Day 7 or 8 blastocysts were cryopreserved by slow freezing using 1.5 M glycerol and 0.2 M sucrose. Embryos were exposed to solutions in a 2-step procedure at room temperature and frozen in a programmed cell freezer. Blastocysts that re-expanded within 6 h of post-thaw culture were considered viable. The cleavage, morula and blastocyst development rates after ICSI were 52.4 (131/250), 39.7 (52/131), and 24.4% (32/131), respectively. Blastocyst stage embryos were randomly divided into 2 groups. The first group of embryos was frozen with their zonae intact, while the second group was allowed to hatch from their zonae during the additional 18 h culture, after which they were frozen. The data showed that more Group 2 blastocysts (14/16, 87.5%) than Group 1 (12/16; 75.0%; P<0.05) survived, and more zona-free bovine blastocysts frozen with glycerol as the cryoprotective agent (CPA) than zona-intact blastocysts after slow freezing retained their viability.     

The treatment of obstructive azoospermia by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) allows the treatment of virtually every type of male infertility. Unlike in vitro fertilization (IVF), its success does not depend on sperm concentration, motility or morphology and most of the physical barriers to fertilisation are by-passes. Since ICSI does not require strongly motile sperm, its use has now been expanded to incorporate immature sperm from the testes and epididymides. Successful fertilisation, pregnancies and healthy babies have all been reported. However, concerns about the safety of ICSI remain due to its short clinical history and the lack of testing on animal models. Male fertility potential for assisted reproduction by ICSI cannot be measured by conventional parameters. Sperm DNA integrity is increasingly recognised as a more useful indicator. Studies have shown that sperm with higher levels of DNA damage have lower fertilisation rates after IVF and ICSI. Sperm with DNA damage above a certain threshold are associated with a longer time to conceive in otherwise apparently fertile couples and a higher miscarriage rate. DNA damage has been shown to be associated with impaired embryo cleavage. Our group has shown that sperm DNA from testicular sperm is less fragmented than that from epididymal sperm and suggest its preferred use in ICSI. In addition to nuclear (n) DNA we also assessed the quality of mitochondrial (mt) DNA from testicular sperm from men with obstructive azoospermia undergoing ICSI. We observed that couples achieving a pregnancy had both less mtDNA deletions and less nDNA fragmentation. We found inverse relationships between pregnancy and sperm mtDNA deletion numbers, size and nDNA fragmentation. No relationships were observed with fertilisation rates. With this knowledge, we designed an algorithm for the prediction of pregnancy based on the quality of sperm nDNA and mtDNA. Each year 40,000 men have a vasectomy in the UK but every year 2500 request a reversal to begin a second family. For such men, vasectomy reversal has recently been replaced in part by testicular biopsy via fine-needle testicular sperm aspiration (TESA) or percutaneous epididymal sperm aspiration (PESA) performed at an outpatient clinic and subsequently used in ICSI. Since these were previously fertile men it has been assumed that they had ‘fertile’ sperm. However the assited conception success rates of these mens partners has not been assessed until recently. We have shown a significant reduction in the clinical pregnancy rates in the partners of men who had had a vasectomy ≥10yrs previously. There is also evidence to suggest that spermatogenesis is significantly impaired in vasectomised men. Marked decreases in spermatocytes, spermatids and spermatozoa have been observed. We have found this to be associated with concomitant increases in apoptotic markers, such as Fas, FasL and Bax. The quality of the remaining sperm is also compromised. Sperm DNA from vasectomized men shows substantial damage which increases with time after surgery. This new use of ICSI will be discussed.     

Normal reproductive development of offspring derived by intracytoplasmic injection of porcine sperm grown in host mice

For establishment of gonadal xenografting, it is essential to clarify whether offspring derived from gametes grown in host mice harboring xenografts have normal reproductive development. This study examined the secretory profiles of gonadal hormones in relation to sexual maturation or ovarian cyclicity in pigs generated by intracytoplasmic sperm injection using xenogeneic sperm (Xeno-ICSI pigs, four males and one female). We also assessed the developmental activity of gametes obtained from these pigs using in vitro culture systems, or by mating with conventionally produced (conventional) pigs. During the growth of male Xeno-ICSI pigs, serum inhibin and testosterone concentrations were generally within ranges for those hormones in conventional pigs. Histologically, there were no differences in the growth and differentiation of seminiferous tubules between Xeno-ICSI and conventional pigs. Parameters of semen quality, including volume, pH, sperm concentration, and the percentage of motile sperm were not different from those in conventional pigs. Among the Xeno-ICSI pigs, individual differences were noted in the ability of sperm to penetrate oocytes and to produce blastocysts. However, oocytes after in vitro fertilization using these sperm developed into blastocysts containing more than 31 cells. One conventional sow delivered 12 piglets after being mated with a male Xeno-ICSI pig. During growth of the female Xeno-ICSI pig, serum progesterone concentrations had a sudden increase at 41 wk of age, suggesting CL formation. After puberty, this animal showed cyclic changes in the serum concentrations of progesterone and inhibin, and delivered 10 piglets after AI using fresh sperm obtained from a conventional boar. In conclusion, these findings demonstrated that both male and female Xeno-ICSI pigs had normal reproductive abilities.     

Normal spermatogenesis and fertility in Ddi1 (DNA damage inducible 1) mutant mice

The ubiquitin proteins play important role in proteasomal degradation and their balanced action is essential for the crucial process of spermatogenesis. The disruption of various ubiquitinating proteins in mice revealed defective spermatogenesis, thus inferring their important function in spermatogenesis. However, the role of some testis-specific ubiquitinating proteins still needs to be discovered. This study was planned to study the in vivo function of testis-specific and evolutionarily conserved ubiquitin shuttle gene, Ddi1 (DNA damage inducible 1). Ddi1 knockout mice were generated by CRISPR/Cas9 technology and we found that Ddi1 knockout mice were fertile without obvious alterations in reproductive parameters, such as sperm number and morphology. Histological examination of testicular tissues manifested compact seminiferous tubule structure along with all type of germ cells in the knockout mice. Moreover, cytological studies of spermatocytes did not exhibit any noteworthy difference in the progression of prophase I which endorse the fact that Ddi1 has not any vital function during meiosis. Overall, these findings suggested that Ddi1 is not critical for mouse fertility under normal laboratory conditions. The outcome of this study will help researchers to avoid overlap that will not only save their resources but also concentrate their focus on indispensable genes in spermatogenesis and fertility.     

Comparison of mice born after intracytoplasmic sperm injection with in vitro fertilization and natural mating

The procedures of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are routinely used in modern medicine to overcome infertility and, in animal husbandry, to propagate lines with compromised fertility. However, there remains concern that manual selection and injection of whole sperm into oocytes could contribute to pre- and postnatal developmental defects. To address this, we have used gene expression profiling and immunophenotyping to characterize offspring generated by these procedures. We used gametes from glutathione peroxidase 1 knockout (Gpx1-/-) mice as a sensitized screen responsive to oxidative stress from artificial reproduction technologies (ART). There were no differences between IVF and ICSI derived offspring in gene expression patterns, and minor differences in hematopoietic parameters. Furthermore there were only minor differences between these IVF and ICSI pups and those derived from natural mating. These data demonstrate for the first time in that there is no significant phenotypic affects of ICSI when compared to IVF and we identified a relatively minor influence of the artificial fertilization methods on phenotype of offspring compared with natural mating. These observations would support the use of ICSI for derivation of mutant mouse lines and may be of some importance for the use of this technique in human ART.     

Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation

The niche is considered to play an important role in stem cell biology. Sertoli cells are the only somatic cells in the seminiferous tubule that closely interact with germ cells to create a favorable environment for spermatogenesis. However, little is known about how Sertoli cells develop to form the male germ line niche. We report here that Sertoli cells recovered and dissociated from testes of donor male mice can be microinjected into recipient testes, form mature seminiferous tubule structures, and support spermatogenesis. Sertoli cells from perinatal donors had a dramatically greater capacity for generating seminiferous tubules than those from adult donors. Furthermore, transplantation of wild-type Sertoli cells into infertile Steel/Steel(dickie) testes created a permissive testicular microenvironment for generating spermatogenesis and spermatozoa. Thus, our results demonstrate that the male germ line stem cell niche can be transferred between animals. In addition, the technique provides a novel tool with which to analyze spermatogenesis and might provide a mechanism for correcting fertility in males suffering from supporting cell defects.     

Birth defects in infants conceived by intracytoplasmic sperm injection: an alternative interpretation.

OBJECTIVE: To test the hypothesis that liveborn infants conceived by intracytoplasmic sperm injection are at an increased risk of having a major birth defect. DESIGN: Reclassification of the birth defects reported in infants born after intracytoplasmic sperm injection in Belgium and comparison with prevalence estimated in Western Australian population by means of same classification system. SETTING AND SUBJECTS: 420 liveborn infants who were conceived after intracytoplasmic sperm injection in Belgium and 100,454 liveborn infants in Western Australia delivered during the same period. MAIN OUTCOME MEASURES: Estimates of birth prevalence of birth defects and comparisons of odds ratios between cohort conceived after intracytoplasmic sperm injection and Western Australian infants. RESULTS: Infants born after intracytoplasmic sperm injection were twice as likely as Western Australian infants to have a major birth defect (odds ratio 2.03 (95% confidence interval 1.40 to 2.93); P = 0.0002) and nearly 50% more likely to have a minor defect (1.49 (0.48 to 4.66); P = 0.49). Secondary data-led analyses, to be interpreted with caution, found an excess of major cardiovascular defects (odds ratio 3.99), genitourinary defects (1.33), and gastrointestinal defects (1.84), in particular cleft palate (5.11) and diaphragmatic hernia (7.73). CONCLUSIONS: These results do not confirm the apparently reassuring results published by the Belgian researchers of intracytoplasmic sperm injection. Further research is clearly required. Meanwhile, doctors practising intracytoplasmic sperm injection should bear this alternative interpretation in mind when they counsel couples and obtain informed consent for the procedure.     

Influence of testosterone substitution on sperm suppression by LHRH agonists

To investigate the effect of LHRH (GnRH) agonists on sperm suppression, we studied the effect of a depot preparation of D-Trp6 LHRH in 10 normal men for 30 weeks. In addition, to determine the role of androgenic substitution on sperm suppression, the volunteers were divided into two groups: group 1 (n = 5) received a low dose T substitution (125 mg of T enanthate every month), while group 2 (n = 5) received a normal T substitution (120 mg of T undecanoate every day). Four men became azoospermic in group 1 and none in group 2. Moreover, administration of additional T injections in 1 volunteer of group 1 resulted in the reappearance of spermatozoa in the ejaculate. Return to the low dose therapy produced azoospermia. These results suggest that testosterone supplementation supports spermatogenesis.     

Incubation of sperm heads impairs fertilization and early embryo development following intracytoplasmic sperm injection (ICSI) by decreasing oocyte activation in mice

When intracytoplasmic sperm injection (ICSI) is performed in mice, isolation of sperm heads is usually performed prior to injections in order to increase the efficiency of the procedure. Consequently, the isolated sperm heads undergo an inevitable incubation in vitro. However, little is known about the effects of this incubation step on fertilization and embryo development following ICSI. When we incubated sperm heads at 37 °C, there was a significant time-dependent decrease in fertilization and blastocyst formation. Moreover, the DNA integrity of the sperm heads was maintained over 12 h incubation. Using assisted oocyte activation, these defects in fertilization and embryo development were rescued. Taken together, incubation of sperm heads following isolation can affect the oocyte-activating capacity of sperm thereby compromising fertilization and embryo development associated with ICSI.     

Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been applied successfully in the treatment of male infertility in humans and in fertilization research in mice. However, the technique has had limited success in producing offspring in other species including the rabbit. The aim of this research was to test the in vitro and in vivo developmental of rabbit oocytes after ICSI. Sperm used for ICSI were collected from mature Dutch Belted buck and washed 2-3 times with PBS +0.1% polyvinyl alcohol (PVA) and then mixed with 10% polyvinyl pyrrolidone (PVP) prior to microinjection. Oocytes were collected from superovulated does 14-15 hr after hCG injection and were fertilized by microinjection of a single sperm into the ooplasm of each oocyte without additional activation treatment. After ICSI, the presumed zygotes were either cultured in KSOM +0.3% BSA for 4 days or transferred into oviducts of recipient does at the pronuclear or 2-cell stage. A high percentage of fertilization (78%, n = 114) and blastocyst development (39%) was obtained after ICSI. Control oocytes, receiving a sham injection, exhibited a lower activation rate (31%, n = 51) and were unable to develop to the blastocyst stage, suggesting that the blastocysts developed following ICSI were derived from successful fertilization rather than parthenogenetic development. A total of 113 embryos were transferred to six recipient does. Two recipients became pregnant and delivered seven live young. Our results demonstrated that rabbit oocytes can be successfully fertilized and activated by ICSI and can result in the birth of live offspring.     

Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken

Transplantation of male germ cells into sterilized recipients has been widely used in mammals for conventional breeding and transgenesis purposes. This study presents a workable approach for germ cell transplantation between male chickens. Testicular cells from adult and prepubertal donors were dispersed and transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. In comparison to males transplanted with testicular cell preparations from adult donors, in which the first ejaculates with sperm were recovered about 5 wk after transfer, a substantial interval (about 10 wk) was necessary to obtain ejaculates after the transfer of testicular cells from prepubertal donors. However, in both cases, recipient males produced ejaculates capable of fertilizing ova and producing progeny expressing donor genes.     

Low temperature storage of rhesus monkey spermatozoa and fertility evaluation by intracytoplasmic injection

The objective was to develop a sperm freezing procedure suitable for use in the propagation of valuable founder animals by assisted reproductive technologies. Here, we report a comparison of processing methods by measuring the motility of fresh and frozen-thawed rhesus monkey spermatozoa and fertility via intracytoplasmic spermatozoa injection (ICSI) of sibling oocytes. Washed spermatozoa were frozen in straws or in pellets using different cryoprotective media and processed post-thaw with or without a density gradient centrifugation step. Among the four study series, motility post-thaw was improved with density gradient centrifugation (17-24% versus 75%, P<0.01) achieving levels similar to fresh spermatozoa. Spermatozoa injected oocytes (total n=377) were co-cultured on BRL cells and observed for fertilization and development. With spermatozoa frozen in straws in liquid nitrogen vapors, the fertilization rate after ICSI was lower than with fresh spermatozoa (40-44% versus 77-86%, P<0.05), even with the Percoll-enriched fraction that exhibited robust motility. In contrast, somewhat slower freezing of spermatozoa in pellets on dry ice supported fertilization rates (73%) that were similar to the fresh counterpart. Developmental rates of fertilized eggs were similar in all experiments. A total of 106 embryo transfers has resulted in the first primate born after ICSI with F/T ejaculated spermatozoa plus 22 other infants to date. Additionally, a 3-4 h incubation after thawing improved the fertilization rate with spermatozoa from a male with poor post-thaw recovery of sperm motility. In conclusion, an acceptable fertilization rate after ICSI with motile, frozen-thawed primate spermatozoa was observed comparable to that obtained with fresh spermatozoa allowing small quantities of competent spermatozoa to be used with ICSI to facilitate propagation of desirable primate genotypes.     

Effect of testosterone oenanthate on spermatogenesis and serum testosterone concentrations in adult mice

Administration of 80 or 160 micrograms testosterone oenanthate s.c. three times per week for 8 or 12 weeks reduced testis weight and increased seminal vesicle weight in mice. Radioimmunoassay indicated that treatment increased serum testosterone concentrations. Treatment with testosterone oenanthate decreased the number of step 16 and step 7 spermatids, pachytene spermatocytes and type A spermatogonia, and particularly reduced the proportion of step 7 spermatids which matured to form step 16 spermatids.     

Normospermic versus teratospermic domestic cat sperm chromatin integrity evaluated by flow cytometry and intracytoplasmic sperm injection

Teratospermia (>60% of morphologically abnormal spermatozoa) is well documented in felids. Even morphologically normal spermatozoa from teratospermic ejaculates have reduced ability to undergo tyrosine phosphorylation, acrosome react, and bind and penetrate oocytes compared with normospermic (<40% abnormal spermatozoa) counterparts. However, it is unknown whether fertilization deficiencies originate at a nuclear level. This study examined whether fertilization failure also was attributable to abnormal sperm chromatin, using the sperm chromatin structure assay (SCSA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Aliquots of unprocessed and swim-up-processed (to isolate morphologically normal spermatozoa) spermatozoa from teratospermic and normospermic domestic cats were analyzed by the flow cytometric SCSA. Swim-up-processed sperm were incubated with in vivo-matured oocytes or used for ICSI. Teratospermic ejaculates expressed more (P < 0.05) chromatin heterogeneity (abnormal chromatin structure) than their normospermic counterparts, both in unprocessed and swim-up-processed samples. Fertilization success in vitro was higher (P < 0.05) from normo- compared with teratospermic inseminates. Similar (P > 0.05) proportions of oocytes fertilized after ICSI using spermatozoa from normo- and teratospermic cats. Results reveal that teratospermia in the cat is expressed at the nuclear level as increased sperm chromatin heterogeneity, but ICSI showed that this does not apparently affect fertilization rates if the zona pellucida and oolemma can be bypassed.