Subzonal microinjection of mouse spermatozoa: insufficient sperm motility might induce phagocytosis

Acrosome-reacted CB6F1 mouse spermatozoa with slight flagellar motility were microinjected under the zona pellucida of CB6F1 mouse oocytes. Electron microscopy revealed the presence of swollen and decondensed sperm heads in the oocyte cytoplasm. Sixty-one percent of the microinjected oocytes reached a morphologically apparent two-cell stage, but chromosomal analysis demonstrated only haploid chromosomal complements in all cases. The exposure of microinjected oocytes to suspensions of spermatozoa of mice homozygous for a 2,4 reciprocal translocation resulted in normal fertilization and embryonic development with a maternally as well as a paternally derived haploid genome. Identical results were obtained with oocytes microinjected with medium and subjected to in vitro fertilization thereafter. Thus it can be suggested that the microinjected spermatozoa with insufficient flagellar motility are incorporated into the oocyte cytoplasm by phagocytosis. These spermatozoa do not induce a polyspermy block but induce the oocyte to parthenogenetic development.     

体外受精-胚胎移植中完全受精失败的原因分析

受精是生命起源至关重要的一个步骤。在辅助生殖的过程中,完全受精失败发生具有其复杂性和不可预见性。受精失败常伴随着一些胞间调控机制异常,其中,可能阻滞在与精子穿越卵冠丘复合体、精子-透明带结合/穿透、精子-卵膜结合、卵子激活、精子去浓缩或原核形成等任一阶段。通过卵胞浆内单精子注射可以避免大部分受精失败现象,但某些患者仍无法成功受精,即使采用辅助人工激活也无法完全避免其发生。对于在辅助生殖过程中完全受精失败患者,结合其卵子成熟情况、精子质量及相关检测结果,在后续周期调整临床方案可有效避免受精失败的再次发生。     

Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization

The mammalian zona pellucida (ZP) is an extracellular glycoprotein structure formed around growing oocytes, ovulated eggs and preimplantation embryos. The specific functions of ZP are highly determined by its morphological structure. Studies on cat oocytes during maturation and after fertilization were undertaken, using routine transmission (TEM) and scanning electron microscopy (SEM). Two basic ZP layers – outer with rough spongy appearance and inner with smaller fenestrations and smooth fibrous network – were visible. Deposits, secreted by oviductal cells formed new layer, the so called oviductal ZP. After fertilization outer ZP showed rougher meshed network due to fusion between filaments as a consequence from sperm penetration while the inner was smoother with melted appearance. The presented data on the SEM and TEM characteristics of cat oocytes, together with our previous studies on carbohydrate distribution suggest that during oocyte maturation and fertilization ZP undergoes structural and functional rearrangements related to sperm binding and penetration.     

ENU‐induced mutant allele of Dnah1, ferf1, causes abnormal sperm behavior and fertilization failure in mice

Given attention to both contraception and treatment of infertility, there is a need to identify genes and sequence variants required for mammalian fertility. Recent unbiased mutagenesis strategies have expanded horizons of genetic control of reproduction. Here we show that male mice homozygous for the ethyl‐nitroso‐urea‐induced ferf1 (fertilization failure 1) mutation are infertile, producing apparently normal sperm that does not fertilize oocytes in standard fertilization in vitro fertilization assays. The ferf1 mutation is a single‐base change in the Dnah1 gene, encoding an axoneme‐associated dynein heavy chain, and previously associated with male infertility in both mice and humans. This missense mutation causes a single‐amino‐acid change in the DNAH1 protein in ferf1 mutant mice that leads to abnormal sperm clumping, aberrant sperm motility, and the inability of sperm to penetrate the oocyte's zona pellucida; however, the ferf1 mutant sperm is competent to fertilize zona‐free oocytes. Taken together, the various mutations affecting the DNAH1 protein in both mouse and human produce a diversity of phenotypes with both subtle and considerable differences. Thus, future identification of the interacting partners of DNAH1 might lead to understanding its unique function among the sperm dyneins.     

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.     

Birth of domestic cat kittens of predetermined sex after transfer of embryos produced by in vitro fertilization of oocytes with flow-sorted sperm

Our goals were to: (1) determine if domestic cat sperm could be sorted to high purity by flow cytometry after overnight shipment of cooled samples; (2) evaluate the efficiency with which sorted sperm could be used to generate cat embryos in vitro; and (3) determine if live kittens of predetermined sex could be produced after transfer of embryos derived by IVF using sorted sperm. Semen samples (n = 5) from one male were extended in electrolyte-free solution and shipped overnight at 4 °C to the sorting facility. Samples were adjusted to 75 × 10⁶ sperm/mL and stained with Hoechst 33342. After 1 h at 34.5 °C, samples were adjusted to 50 × 10⁶ sperm/mL with 4% egg yolk TALP + 0.002% food dye and sorted by high-speed flow cytometry. Later resort analysis confirmed purities of 94% and 83% for X- and Y-chromosome bearing sperm, respectively. Sorted sperm were centrifuged, re-suspended in TEST yolk buffer and shipped overnight to the IVF laboratory. After IVF of in vivo matured oocytes with X-chromosome bearing sperm, cleavage frequency was 62% (54/87). After IVF of IVM oocytes with control, X- or Y-chromosome bearing sperm, the incidence of cleavage was 42% (48/115), 33% (40/120), and 35% (52/150), respectively, and blastocyst development was 53% (21/40), 50% (11/22), and 55% (23/42), respectively (P > 0.05). On Day 2, 45 embryos produced by IVF of in vivo matured oocytes with X-chromosome bearing sperm were transferred to the oviduct of four Day 1 recipients, three of which subsequently delivered litters of one, four, and seven female kittens, respectively. In conclusion, we confirmed that sperm sorting technology can be applied to domestic cats and established that kittens of predetermined sex can be produced.     

Development of androgenetic mouse embryos produced by in vitro fertilization of enucleated oocytes

Enucleated mouse oocytes were successfully fertilized in vitro, and the resultant androgenetic eggs developed to the blastocyst stage. The proportion of enucleated oocytes fertilized in vitro was high (87–99%) at sperm concentrations ranging from 10–100 × 10⁴/ml. At high sperm concentrations (100–1,000 × 10⁴), 35–45% of the fertilized eggs resulted in heterozygous bispermic androgenones. The proportion of hemizygous haploid and heterozygous diploid androgenones developing to blastocysts was 11% and 43%, respectively. Hemizygous diploidization, however, showed no positive effect on development. These results clearly show that the procedure reported here is efficient and reliable for the production of androgenetic eggs. © 1993 Wiley-Liss, Inc.     

Wheat germ agglutinin treatment of follicle cell-free mouse oocytes inhibits fertilization

Controversy exists whether treatment of follicle cell-free oocytes with wheat germ agglutinin (WGA) prevents fertilization. Lack of inhibition in one case has led to the suggestion that acrosin may not be a zona lysin. To re-examine the effect of the WGA, the zona pellucida of follicle cell-free mouse oocytes was made more resistant to proteinase digestion by treatment with 10 or 50 μg/ml WGA. Such WGA-treated oocytes showed decreased fertilizability when washed to remove excess WGA and incubated with capacitated spermatozoa. Oocyte cleavage was used as an end point, because a large number of spermatozoa adhered to the eggs after WGA treatment, making observation of sperm penetration and pronucleus formation unreliable. Resistance to proteinase digestion increased, and the fertilizability decreased with the higher amount of WGA. The action of WGA was most likely not mediated by a direct effect on sperm motility, sperm acrosin activity, sperm binding to the zona pellucida, or oocyte cleavage. WGA did not affect the acrosome reaction of guinea pig spermatozoa. These data show that WGA treatment of follicle cell-free mouse oocytes results in decreased fertilizability, possibly by rendering the zona pellucida more resistant to sperm proteinase digestion.     

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6–42.2%) and blastocyst formation (2.9–8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.     

Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (10⁵ and 10⁶/mL) and oocytes were incubated with 10⁶ TCID₅₀/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (10⁵/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 10⁶/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (10⁵/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (10⁶ sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF.     

Time and process of sperm penetration into cumulus-free mouse eggs fertilized in vitro

Sperm penetration through the zona pellucida and fusion of the sperm head with the vitellus were observed continuously and filmed under phase optics in cumulus-free living mouse eggs inseminated in vitro with capacitated epididymal sperm. Most spermatozoa penetrated the zona pellucida, traversed the perivitelline space, and fused with the vitellus at an angle nearly perpendicular to the surface. The mean duration required for sperm to penetrate the zona pellucida was 20 minutes with a range of 15–26 minutes. Sperm traversed the perivitelline space in less than one second. The initial contact of sperm with the vitellus generally took place at the tip of the sperm head. When the tip of the sperm head contacted the vitellus there was an immediate reduction in the rate of flagellation, followed by the gradual sinking of the sperm head into the vitellus.     

Characterization of mouse-hamster-human cross-reacting antioocyte monoclonal antibodies produced by intrasplenic immunization of mice with 12 zona-free mouse oocytes

Several intrasplenic immunizations with batches of ～15 or ～30 zona-free, unfertilized mouse oocytes resulted in 200–300 hybrids, respectively, among which about 20 positive clones were selected from each fusion between splenic plasma cells and SP2/0 myeloma cells. When nonimmunized splenic plasma cells were used, only one antibody, showing weak immunoreaction, was obtained from ～370 hybrids collected from 2 fusions. From one immunization with a total of 12 zona-free, unfertilized mouse oocytes, 15 positive clones were selected for further study. Eleven of these 15 antibodies reacted with antigens only in unfertilized oocytes but not in fertilized, pronuclear stage oocytes. Three antibodies, which recognized antigens in paraffin-embedded oocyte sections, did not label growing ovarian oocytes, indicating that the antibodies were specific to ovulated, unfertilized oocytes. These antibodies did not detect any antigen epitopes in the panel of tissues examined. The molecular weight of one antigen, corresponding to a IgM antibody that is present both in ooplasma and zona pellucida, was ～116 kDa. Cross-reactivity to blots of unfertilized zona-free hamster oocytes was demonstrated by 6 antibodies and to unfertilized human oocytes by 7 antibodies. Three antibodies cross-reacted with both hamster and human oocytes. The study indicates that the intrasplenic immunization is an appropriate means of raising antibodies against unfertilized, zona-free mouse oocytes and that the method applied offers an easy way to select antibodies against human oocytes for functional studies. © 1994 Wiley-Liss, Inc.     

Developmental capacity of rat embryos produced by in vivo or in vitro fertilization

This work compares the ability of rat zygotes fertilized in vitro or in vivo to develop into viable embryos. All oocytes were from adult cyclic females. After the first cleavage, the zygotes were transferred to oviducts of pseudopregnant recipients. Their fate was examined on day 13 at laparotomy and again on day 20. Ninety-five of 146 in vivo fertilized zygotes developed into normal sized 13-day fetuses and 72 (55%) to apparently normal near-term fetuses. Forty-six of 135 in vitro fertilized zygotes developed up to day 13, and 30 (24%) developed to term. It appears that the probability that in vitro fertilized rat zygotes will develop into viable embryos is about half the chance of in vivo fertilized zygotes. Since the two types of zygotes were morphologically identical, the morphological appearance of the two-cell stage is not an adequate criterion for judging developmental potential.     

Bovine oocyte development following different oocyte maturation and sperm capacitation procedures

Various procedures have been reported for successful in vitro maturation and in vitro fertilization (IVM/IVF) of bovine follicular oocytes. Direct comparisons of these different recommended procedures have been rare. In this research, involving a total of 5,128 oocytes, a series of experiments were conducted to compare oocyte maturation, fertilization, and development in vitro with 2 maturation systems (with or without added hormones) and 3 types of sperm treatment procedures. Oocytes were collected from ovarian antral follicles (2–7 mm in diameter) within 3 hr after slaughter of cows or heifers. Those with intact or at least 4 layers of cumulus cells were selected for IVM/IVF. Oocytes were incubated for 22 hr in either Medium 199 with 7.5% fetal calf serum (M199 + FCS) alone or M199 + FCS with added hormones (M199 + FCS + H; oFSH 0.5 μg/ml, oLH 5.0 μg/ml, and E₂ 1.0 μg/ml) at 39°C in 5% CO₂ and 95% air. For IVF, frozen-thawed sperm were treated with either 0.1 μM calcium ionophore A23187 (A23187) for 1 min, or 10 or 100 μg/ml heparin (H10 or H100) for 15 min. Our results demonstrated the following: (1) both M199 + FCS and M199 + FCS + H supported maturation development to the metaphase II stage (90–95%, P > 0.05); (2) when oocytes were matured in M199 + FCS without added hormones, A23187 sperm treatment was superior to H10 or H100 treatment for fertilization and blastocyst development of the inseminated oocytes (P < 0.05); (3) when oocytes were matured in M199 + FCS + H, A23187 treated sperm again produced a higher fertilization rate than the H10 group (P < 0.05), but the development to the blastocyst stage was similar among all 3 sperm treatment groups (P > 0.05); (4) direct comparison of the 2 maturation systems with A23187 treated sperm resulted in no difference in all criteria measured; however, (5) when compared retrospectively, beneficial effects of added hormones are evident for blastocyst development (but not for fertilization) when sperm were treated with heparin procedures. © 1993 Wiley-Liss, Inc.     

Effects of ultrashort gamete co-incubation time on porcine in vitro fertilization

A reduction in co-incubation time has been suggested as an alternative method to reduce polyspermic fertilization. The aim of this study was to evaluate the effect of short periods of gamete co-incubation during pig in vitro fertilization. A total of 2833 in vitro matured oocytes were inseminated with thawed spermatozoa and coincubated for 0.25, 1, 2, 3, 7, 10 min and 6 h. The oocytes from the 0.25–10 min groups were washed three times in modified Tris-buffered medium (mTBM) medium to remove spermatozoa not bound to the zona and transferred to the same medium (containing no spermatozoa) until 6 h of co-incubation time were completed. After 6 h, presumptive zygotes from each group were cultured in NCSU-23 medium for 12–15 h to assess fertilization parameters. After each period of co-incubation, 45–50 oocytes from each group were stained with Hoechst-33342 and the number of spermatozoa bound to the zona was counted. Although the number of zona bound spermatozoa increased (p < 0.05) with the co-incubation time, no increase was observed in penetration rates among groups from 2 min to 6 h of co-incubation time (ranging from 53.5 ± 2.8 to 61.3 ± 2.6%). Similarly, the efficiency of fertilization reached a maximum for the 2 min of co-incubation group with values ranging between 32.3 ± 2.4 and 41.9 ± 2.5%. The reduction of co-incubation time did not affect the monospermy rate (range: 71.3 ± 3.4–80.2 ± 3.8%) and the mean number of spermatozoa/oocyte (range: 1.2 ± 0.4–1.4 ± 0.5). These results show that, under our in vitro conditions, high penetration rate can be obtained with co-incubation times as short as 2 min, although monospermy could not be improved using this strategy.     

Possible involvement of CD81 in acrosome reaction of sperm in mice

Tetraspanin CD81 is closely homologous in amino acid sequence with CD9. CD9 is well known to be involved in sperm-egg fusion, and CD81 has also been reported to be involved in membrane fusion events. However, the function of CD81 as well as that of CD9 in membrane fusion remains unclear. Here, we report that disruption of the mouse CD81 gene led to a reduction in the fecundity of female mice, and CD81-/- eggs had impaired ability to fuse with sperm. Furthermore, we demonstrated that when CD81-/- eggs were incubated with sperm, some of the sperm that penetrated into the perivitelline space of CD81-/- eggs had not yet undergone the acrosome reaction, indicating that the impaired fusibility of CD81-/- eggs may be in part caused by failure of the acrosome reaction of sperm. In addition, we showed that CD81 was highly expressed in granulosa cells, somatic cells that surround oocytes. Our observations suggest that there is an interaction between sperm and CD81 on somatic cells surrounding eggs before the direct interaction of sperm and eggs. Our results may provide new clues for clarifying the cellular mechanism of the acrosome reaction, which is required for sperm-egg fusion.     

In vitro fertilization in nonhuman primates

Ovulation induction, sperm capacitation, and fertilization have been studied for over 50 years in nonhuman primates but it has only been in the past 20 years that extensive studies on sizeable numbers of embryos have been carried out. Of over 200 species of nonhuman primates only a few have been studied and the majority of the findings come from studies of the squirrel monkey, baboon, rhesus, and cynomolgus macaque. Nevertheless, the fertilization process appears to be similar to that identified in other mammals and in man.     

Binding of sperm proacrosin/beta-acrosin to zona pellucida glycoproteins is sulfate and stereodependent. Synthesis of a novel fertilization inhibitor

Specific binding of spermatozoa to the zona pellucida that surrounds mammalian eggs is a key step in the fertilization process. However, the sperm proteins that recognise zona pellucida receptors remain contentious despite longstanding research efforts to identify them. Here we present evidence that proacrosin, a tissue-specific protein found within the acrosomal vesicle of all mammalian spermatozoa, is a multifunctional protein that mediates binding of acrosome-reacted spermatozoa to zona glycoproteins via a stereospecific polysulfate recognition mechanism. Using sulfated versus non-sulfated forms of chemically defined compounds in binding assays employing native proteins in their normal cellular location or conjugated to FluoSpheres, we have attempted to identify the sulfation "code" required for recognition. Results show that protein conformation is important for specificity and that at least 2 sulfate groups are required to cross-link spatially separated docking sites on proacrosin. The consistently most effective inhibitory compounds were suramin and quercetin-3beta-d-glucoside sulfate. The results support our hypothesis that proacrosin is one of several proteins in the acrosomal matrix that retain acrosome reacted spermatozoa on the zona surface prior to penetration. They also establish, as a proof-of-principle, the feasibility of synthesising sulfated compounds of high specificity as antifertility agents for human or animal use.