Investigation of assisted fertilization and biology of reproduction by sperm microinjection

Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that under-lie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed.     

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.     

不同取卵时间对兔ICSI胚胎体外发育的影响

目的探讨不同取卵时间对兔ICSI胚胎体外发育的影响。方法采用Piezo操作系统对实验兔进行辅助体外受精。结果hCG注射后14、16、18h取卵,ICSI后的受精率分别为82.2％、75.9％和0.0％,对受精卵进行体外发育培养,桑椹胚的发育率分别为72.9％、70.0％、0.0％,囊胚的发育率分别为62.2％、53.3％、0.0％。14h和16h之间受精率、桑椹胚率、囊胚率差异不显著（P〉0.05）,但是14h采卵比16h要好;18h和14h、16h之间差异显著（P〈0.05）。结论不同取卵时间影响实验兔的ICSI体外受精率及胚胎的体外发育率,hCG注射后14h取卵最有利于兔ICSI胚胎的发育。     

猪胞浆内单精子注射研究进展

胞浆内单精子注射将体外受精和显微操作技术相结合,已成功应用于哺乳动物受精机理研究,家畜性控胚胎生产,动物转基因等领域。综述了猪胞浆内单精子注射的主要影响因素,包括卵母细胞体外成熟和预处理、精子的选择和处理、注射卵的人工激活以及操作技术的改进,为进一步提高猪胞浆内单精子注射的效率以及该技术的应用提供参考。     

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.     

Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperm injection

To evaluate the potential for fertilization by sperm injection into fish eggs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological changes of gametes on the possible performance of intracytoplasmic sperm injection (ICSI), four different combinations of injection conditions were conducted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval stages, and another 3 (2%) developed normally and hatched. The highest fertilization rate (blastodisc formation) was achieved by injection of activated spermatozoa into nonactivated eggs (35%). Injections were most effective when performed within the first hour after egg collection. Flow cytometric analysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Our study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.     

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.     

猪卵母细胞的体外受精及多精受精

对用于猪体外受精(IVF)的研究方法和技术,如传统的液滴IVF、透明带下注射精子受精(SUZI)、卵母细胞质内单精注射受精(ICSI)及细管IVF等进行了简述。与其它动物相比,进行猪卵的体外受精研究,多精受精现象特别明显。大量的研究表明,猪卵的多精受精不但与其品种特性有关,而且与卵母细胞成熟的程度、透明带的异常、受精时获能精子的浓度、输卵管分泌物、受精液蛋白添加成分、NaHCO3浓度、咖啡因、pH值以及温度等因素密切相关。     

Fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection

We demonstrated normal fertilization processes (as determined by pronuclear formation, pronuclear apposition and syngamy) in porcine oocytes either following intracytoplasmic spermatozoon (ICSI) or isolated sperm head injection. Microtubule organization and chromatin configuration were investigated in these oocytes during the first cell cycle. Following ICSI, the microtubular aster was organized from the neck of the spermatozoon and filled the whole cytoplasm. These male-derived microtubules appear to move both pronuclei to the center of oocytes. These cytoskeletal changes are analogous to those seen following conventional fertilization. In contrast, following isolated sperm head injection, the sperm aster was not seen. Instead, the microtubule matrix was organized from the cortex and then filled the whole cytoplasm in all cases in normally fertilized oocytes following injection (n = 35). This organization is similar to what has been shown in the parthenogenetically activated oocytes. Chromosome analysis revealed that the oocytes injected with isolated sperm heads were fertilized normally. At 7 days following injection, the incidence of blastocoele formation following ICSI (38%) and isolated sperm head injection (22%) was higher than that following sham injection (2%). These results suggested that successful fertilization and preimplantation development occurred in porcine oocytes following either ICSI or isolated sperm head injection. Our results also indicated that fertilization processes can occur by self-assembled microtubules within cytoplasm in the absence of a sperm centrosome. Mol. Reprod. Dev. 51:436–444, 1998. © 1998 Wiley-Liss, Inc.     

Cross species fertilization and development investigated by cat sperm injection into mouse oocytes

The use of intracytoplasmic sperm injection (ICSI) in model animals is a powerful approach for the study of species-specific fertilization processes and multiploidy embryogenesis. In this study, we examined the fertilization process in mouse oocytes following injection of a single mouse or cat sperm, two mouse spermatozoa or mouse and cat spermatozoa. These treatments did not affect histone H3K9 acetylation or methylation, although the pattern of DNA methylation differed following the injection of cat sperm. Immunocytochemical staining revealed that sperm chromatin was normally incorporated with female mouse chromatin following any of the four injection scenarios. Furthermore, metaphase was successfully entered to reach a normal two-cell stage, and cell division could even persist to produce blastocyst stage embryos. In addition, both mouse and cat Pou5l and Nanog mRNA were expressed in the hybrid embryos. These results suggest that, although epigenetic modification of DNA is affected by the sperm injection treatment, fertilization and cleavage occur in a non-species-specific manner. In addition, despite abnormal division of the chromosomes, intra- and inter-species ICSI produced embryos that could develop into blastocysts.     

Combined analysis of endometrial thickness and pattern in predicting outcome of in vitro fertilization and embryo transfer: a retrospective cohort study

Objective  

To evaluate the combined effect of endometrial thickness and pattern on clinical outcome in patients undergoing in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET).     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.     

Clinical experience with flow cytometric separation of human X- and Y-chromosome bearing sperm

Numerous methods to separate human X- and Y-bearing sperm have been reported with unconfirmed separation after DNA analysis and inconsistent birth results. Successful flow cytometric separation of sperm resulting in alteration of the sex ratio of young born has been demonstrated in several animal species. Flow cytometric separation of human X- and Y-bearing sperm (MicroSort) has been confirmed after DNA analysis by fluorescence in situ hybridization (FISH). Pregnancies and births have resulted from the use of MicroSort after intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).     

Fertilization of oocytes and birth of normal pups following intracytoplasmic injection with spermatids in mastomys (Praomys coucha)

The mastomys is a small laboratory rodent that is native to Africa. Although it has been used for research concerning reproductive biology, in vitro fertilization (IVF) and intracytoplasmic sperm injection are very difficult in mastomys because of technical problems, such as inadequate sperm capacitation and large sperm heads. The present study was undertaken to examine whether mastomys spermatids could be used to fertilize oocytes in vitro using a microinsemination technique, because spermatids are more easily injected than mature spermatozoa into oocytes. Most mastomys oocytes (80%-90%) survived intracytoplasmic injection with either round or elongated spermatids. Round spermatids had little oocyte-activating capacity, similar to those of mice and rats, and exogenous stimuli were needed for normal fertilization. Treatment with an electric pulse in the presence of 50 microM Ca2+ followed by culture in 10 mM SrCl2 led to successful oocyte activation. After injection of round spermatids into preactivated oocytes, 93% of oocytes were normally fertilized (male and female pronuclei formed), and 100% of cultured oocytes developed to the 2-cell stage. However, none reached term after transfer into recipient females. Elongated spermatids, which correspond to steps 9-11 in rats, activated oocytes on injection without additional activation treatment. After embryo transfer, five offspring (6% per transfer) developed to term. These results indicate that microinsemination with spermatids is a feasible alternative in animal species that are refractory to IVF and sperm injection and that using later-stage spermatids may lead to increased production of viable embryos that can develop into normal offspring.     

SNAREs in mammalian sperm: possible implications for fertilization

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.     

Early-cleavage is a reliable predictor for embryo implantation in the GnRH agonist protocols but not in the GnRH antagonist protocols

Background  

To test if early-cleavage was a strong predictor of pregnancy in patients receiving either a GnRH agonist long protocol or a GnRH antagonist protocol for in-vitro fertilization treatment (IVF) and intracytoplasmic sperm injection (ICSI).     

Complete fertilization failure in ICSI

Abstract  Fertilization failure is one of the causes of infertility that becomes evident only after in vitro fertilization (TVF) and intracytoplasmic sperm injection (ICSI) have been attempted. Although the frequency of incidence of fertilization failure is low, if fertilization failure is encountered, medical treatment is usually stopped and serious psychological damage may occur to the patient.
While fertilization failure in IVF can be dealt with using ICSI, there is no treatment for fertilization failure in ICSI. At present, clinical investigations are being conducted to evaluate oocyte activation in combination with ICSI to cope with fertilization failure of ICSI.     

Current trends in evaluation of sperm function: in vitro selection and manipulation of male gametes for assisted conception

With increasing medical utilization of assisted reproductive technology (ART), scientists and clinicians have been able to study extensively multiple cell functions operating synchronously and flawlessly during the events preceding, before and after fertilization. Critical evaluation of the functional status of spermatozoa for in vitro techniques such as sperm-mucus interaction, acrosome reaction status, sperm-zona pellucida binding and penetration tests, hyaluronic acid binding assay, and computer assisted semen analysis etc. can direct a male partner of an infertile couple to more aggressive forms of treatments. In vitro selection of functionally competent sperm cells is a pre-requisite for successful outcome in in vitro fertilization or in intracytoplasmic sperm injection (ICSI). Direct injections of acrosome-intact spermatozoa into oocyte during ICSI bypassing the normal events of sperm oocyte interaction and fusion events have raised concerns with regard to fertilization abnormalities and genetic issues. The present communication briefly reviews the sperm function tests with emphasis on its correlation with fertility outcome, and the currently employed sperm selection and manipulation procedures which may have implications in assisted conception programs.     

The role of Ca2+ in oocyte activation during In Vitro fertilization: Insights into potential therapies for rescuing failed fertilization

At fertilization the mature mammalian oocyte is activated to begin development by a sperm-induced series of increases in the cytosolic free Ca²⁺ concentration. These so called Ca²⁺ oscillations, or repetitive Ca²⁺ spikes, are also seen after intracytoplasmic sperm injection (ICSI) and are primarily triggered by a sperm protein called phospholipase Czeta (PLCζ). Whilst ICSI is generally an effective way to fertilizing human oocytes, there are cases where oocyte activation fails to occur after sperm injection. Many such cases appear to be associated with a PLCζ deficiency. Some IVF clinics are now attempting to rescue such cases of failed fertilization by using artificial means of oocyte activation such as the application of Ca²⁺ ionophores. This review presents the scientific background for these therapies and also considers ways to improve artificial oocyte activation after failed fertilization.     

Triploidy--the breakdown of monogamy between sperm and egg

The advent of assisted reproductive technology (ART) has taught us a great deal about human fertilization patterns. Thirty years of experience with IVF and cultivation of early embryos has provided a unique view into the mechanisms of normal and aberrant human fertilization. Here we review the different types of triploidy following conventional in vitro fertilization and intracytoplasmic sperm injection, as well as the mechanisms giving rise to digynic and dispermic fertilization. Additionally, the role of the centrosome in triploidy, the genetic analysis of triploid embryos and the potential for therapeutic enucleation are explored. Lastly, we review our own clinical experience with human fertilization patterns following > 20,000 treatment cycles of assisted reproduction.