The ability to develop an activity that transfers histones onto sperm chromatin is acquired with meiotic competence during oocyte growth.

Following fertilization, the oocyte remodels the sperm chromatin into the male pronucleus. As a component of this process, during meiotic maturation, oocytes develop an activity that transfers histones onto sperm DNA. To further characterize this activity, we tested whether oocytes at different stages of growth could, upon entry into metaphase of maturation, transfer histones onto sperm DNA, as judged by chromatin morphology and immunocytochemistry. Meiotically competent growing oocytes, which spontaneously enter metaphase upon culture, transferred histones onto sperm chromatin, whereas incompetent oocytes did not, even when treated with okadaic acid to induce germinal vesicle breakdown (GVBD) and chromosome condensation. When incompetent oocytes were cultured until they acquired the ability to undergo GVBD, only a small proportion also developed histone-transfer activity during maturation. However, this proportion significantly increased when the oocytes were cultured as granulosa-oocyte complexes. The failure of histone-transfer activity to develop in incompetent oocytes treated with okadaic acid was not linked to low H1 kinase activity nor rescued by injected histones. Because competent, but not incompetent, oocytes produce natural calcium oscillations, incompetent oocytes were exposed to SrCl2. One-third of treated oocytes produced at least one Ca2+ oscillation and, following insemination, the same proportion transferred histones onto sperm DNA. Histone transfer did not occur in oocytes pretreated with the Ca2+ chelator, BAPTA-AM. These results indicate that the ability to develop histone-transfer activity is acquired by growing oocytes near the time of meiotic competence, that it is separable from this event, and that it may be regulated through a Ca2+-dependent process.     

Liposome‐mediated DNA transfer to chicken sperm cells

Abstract

The efficacy of using liposomes to transfer DNA to chicken sperm cells was investigated. Liposomes were prepared from dilauroyl (12:0) phosphatidylcholine (DLPC), dimyristoyl (14:0) phosphatidyl choline (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (EYPC) or lipids extracted from sperm cell membranes. The efficiency of trapping of DNA into the liposomes, transfer of the DNA from the liposomes to the sperm cells and the effect of the liposomes on the fertilizing ability of the sperm cells were determined. Increasing the concentration of lipid in the liposome preparations increased the trapping efficiency of DNA into liposomes but lowered the transfer of DNA to sperm. Including stearylamine (SA) in the liposomes increased the incorporation of DNA into the liposomes and the DNA transfer to sperm cells, while including lauroyllysophosphatidylcholine (LPC) along with SA resulted in the highest transfer efficiency from liposomes to sperm. The transfer of DNA from liposomes to sperm cells was lowered by increasing the number of sperm cells, while decreasing the number of sperm cells lowered the fertility. The sperm cells remained fertile after exposure to low levels of DPPC or lipofectin reagent or to high levels of SA and LPC. The best conditions for liposome‐mediated gene transfer to chicken sperm cells are thus using either lipofectin reagent at .006 to .06 μmol/ml and 5 × 10⁷ sperm or with DPPC liposomes comprised of 10 μmol/ml total lipid including 5 mol% SA and 20 mol% LPC with 2.5 × 10⁸ sperm cells. The use of liposomes to enhance the transfer of DNA to sperm cells may make the use of sperm cells as gene transfer vectors possible.     

Superovulation Using the Combined Administration of Inhibin Antiserum and Equine Chorionic Gonadotropin Increases the Number of Ovulated Oocytes in C57BL/6 Female Mice

Superovulation is a reproductive technique generally used to produce genetically engineered mice. Superovulation in mice involves the administration of equine chorionic gonadotropin (eCG) to promote follicle growth and then that of human chorionic gonadotropin (hCG) to induce ovulation. Previously, some published studies reported that inhibin antiserum (IAS) increased the number of ovulated oocytes in ddY and wild-derived strains of mice. However, the effect of IAS on the C57BL/6 strain, which is the most widely used inbred strain for the production of genetically engineered mice, has not been investigated. In addition, the combined effect of IAS and eCG (IASe) on the number of ovulated oocytes in superovulation treatment has not been examined. In this study, we examined the effect of IAS and eCG on the number of ovulated oocytes in immature female mice of the C57BL/6 strain in superovulation treatment. Furthermore, we evaluated the quality of obtained oocytes produced by superovulation using IASe by in vitro fertilization (IVF) with sperm from C57BL/6 or genetically engineered mice. The developmental ability of fresh or cryopreserved embryos was examined by embryo transfer. The administration of IAS or eCG had a similar effect on the number of ovulated oocytes in C57BL/6 female mice. The number of ovulated oocytes increased to about 3-fold by the administration of IASe than by the administration of IAS or eCG alone. Oocytes derived from superovulation using IASe normally developed into 2-cell embryos by IVF using sperm from C57BL/6 mice. Fresh or cryopreserved 2-cell embryos produced by IVF between oocytes of C57BL/6 mice and sperm from genetically engineered mice normally developed into live pups following embryo transfer. In summary, a novel technique of superovulation using IASe is extremely useful for producing a great number of oocytes and offspring from genetically engineered mice.     

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.     

DNA uptake in swine sperm: Effect of plasmid topology and methyl‐beta‐cyclodextrin‐mediated cholesterol depletion

Sperm‐mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still undergoing development and optimization to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real‐time PCR‐based assay to assess the absolute number of exogenous plasmids internalized into the spermatozoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found that DNA internalization into the nucleus of porcine sperm cells is better under specific methyl‐β‐cyclodextrin (MCD)‐treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA. Mol. Reprod. Dev. 79: 853–860, 2012. © 2012 Wiley Periodicals, Inc.     

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.     

Intrabursal transfer of spermatozoa (ITS): a new route for artificial insemination of mice.

Artificial insemination (AI) by direct injection of epididymal spermatozoa into the reproductive tract of females is simpler and more convenient than in vitro fertilization (IVF) and subsequent transfer of fertilized eggs to recipient oviducts for simultaneous acquisition of a large number of pups. Introduction of epididymal spermatozoa into oviducts via the oviductal wall or via vaginal and intrauterine routes is currently the most commonly used method for AI in mice. In this study, we explored another route for AI of the mouse and found that transfer of spermatozoa into a space near the infundibulum between the ovary and ovarian bursa enables in vivo fertilization of ovulated oocytes at the ampulla. When 1 microL of a sperm suspension containing 1 x 10(4) spermatozoa freshly isolated from B6C3F1 males was intrabursally injected into superovulated B6C3F1 females on E (embryonic day) 0.4 (10:00 AM), 5 of 7 females yielded 2-cell embryos with rates of efficiency ranging from 4 to 21% (11% on average), which were much lower than those (91% on average) for embryos obtained by natural mating. All the 2-cell embryos derived from injection of sperm developed in vitro to hatched blastocysts. Similar results were obtained from injection of 1 microL of sperm suspension containing 1 x 10(3) spermatozoa, although in vivo fertilizing ability was slightly improved (28% on average). When 1 microL of sperm suspension containing 1 x 10(4) spermatozoa was injected intrabursally into superovulated females that had been mated with vasectomized males, 6 of 10 mice (60%) yielded 19 normal mid-gestational fetuses with an average litter size of 3.2, which was much lower than that (14.5) for embryos obtained by natural mating. Although the present findings appear to be preliminary, this technique, based on the intrabursal transfer of spermatozoa, will be of practical use for AI in mice, particularly for transgenic and mutant mice that are often difficult to breed.     

Injection of sperm heads into immature rat oocytes

When sperm heads are injected into rat oocytes at the germinal vesicle stage the sperm heads remain intact until the germinal vesicle breaks down. Then they decondense but do not form pronuclei. This observation agrees with the results reported for in vitro fertilization of immature oocytes from rats, mice and hamsters.     

In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation.

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and beta-mercaptoethanol. Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro-developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center.     

利用卵胞浆精子注射（ICSI）技术生产转基因小鼠

在掌握小鼠ICSI技术的基础上,进行了ICSI技术生产转基因小鼠的研究。来自成年KM小鼠附睾尾的精子,使用未加抗冻剂的HEPES-CZB溶液,在液氮中冻融1次后,用于本实验。解冻精子与DNA混匀1min后,精子头被显微注射到B6D2F1小鼠成熟卵母细胞质中。精子头与pEGFP-N1环状DNA共注射生产的ICSI受精卵,在CZB溶液中培养至囊胚期时,39.1%(9/23)的囊胚表达GFP基因。精子注射后6h,直接移植ICSI受精卵后,7只妊娠受体一共产仔30只,效率为23.8%(30/126)。Southernblot分析其中16只小鼠发现,3只(18.8%)转基因小鼠同时整合了GFP和Neomycin基因,它们全部来自精子和线性DNA混合的实验组(阳性效率为33.3%,3/9),相反,精子与环状质粒DNA共注射生产的7只ICSI后代中,没有检测到外源基因。转基因小鼠整合的外源基因能够传递给它们后代。结果说明,利用ICSI技术可以高效地生产转基因小鼠,宿主基因组可能更容易整合线性化的外源基因。     

Liposomes fuse with sperm cells and induce activation by delivery of impermeant agents

Sperm cell activation is a critical step in fertilization. To directly investigate the cell signaling events leading to sperm activation it is necessary to deliver membrane impermeant agents into the cytoplasm. In this study, the use of liposomes as possible agent-loading vectors was examined using (1) the octadecylrhodamine B (R18) and NBD phosphatidylethanolamine (NBD DHPE)/rhodamine phosphatidylethanolamine (rhod DHPE) fusion assays in bulk samples, (2) membrane transfer of fluorescence from liposome membranes labeled with R18 and rhodamine-tagged phosphatidylethanolamine (TRITC DHPE), and (3) lumenal transfer of impermeant calcium ions from liposomes to sperm cells, a process that stimulated sperm cell activation. Intermediate-sized unilamellar liposomes (98.17+/-15.34 nm) were prepared by the detergent-removal technique using sodium cholate as the detergent and a phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) lipid composition. In the R18 fusion assays, self-quenching increased logarithmically with increasing concentrations of R18 in the liposome membranes; addition of unlabeled sperm to R18-labeled liposomes lead to a rapid release of self-quenching. In the NBD DHPE/rhod DHPE resonance energy transfer (RET) fusion assay, RET was rapidly reduced under similar conditions. In addition, individual sperm became fluorescent when TRITC DHPE-labeled liposomes were incubated with unlabeled sperm cells. Incubation of sperm cells with empty liposomes did not significantly affect sperm cell activation and did not alter cell morphology. However, incubation with Ca (10 mM)-loaded liposomes resulted in a time-dependent increase in sperm cell activation (7.5-fold over controls after 15 min). We conclude that liposomes can be used for direct loading of membrane-impermeant agents into sea squirt sperm cell cytoplasm, and that delivery occurs via fusion and content intermixing.     

An essential role for functional telomeres in mouse germ cells during fertilization and early development

Late generations of telomerase-null (TR(-/-)) mice exhibit progressive defects in highly proliferative tissues and organs and decreased fertility, ultimately leading to sterility. To determine effects of telomerase deficiency on germ cells, we investigated the cleavage and preimplantation development of embryos derived from both in vivo and in vitro fertilization of TR(-/-) or wild-type (TR(+/+)) sperm with either TR(-/-) or TR(+/+) oocytes. Consistently, fertilization of TR(-/-) oocytes with either TR(+/+) or TR(-/-) sperm, and TR(-/-) sperm with TR(+/+) oocytes, resulted in aberrant cleavage and development, in contrast to the normal cleavage and development of TR(+/+) oocytes fertilized by TR(+/+) sperm. Many (>50%) of the fertilized TR(-/-) eggs developed only one pronucleus, coincident with increased incidence of cytofragmentation, in contrast to the normal formation of two pronuclei and equal cleavage of wild-type embryos. These results suggest that both TR(-/-) sperm and oocytes contribute to defective fertilization and cleavage. We further found that a subset (7-9%) of telomeres was undetectable at the ends of some metaphase I chromosomes from TR(-/-) spermatocytes and oocytes, indicating that meiotic germ cells lacking telomerase ultimately resulted in telomere shortening and loss. Dysfunction of meiotic telomeres may contribute to aberrant fertilization of gametes and lead to abnormal cleavage of embryos, implying an important role of functional telomeres for germ cells undergoing fertilization and early cleavage development.     

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.     

Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice.

We compared liposomes and empty viral capsids for their use as vehicles for DNA transfer into cells and animals. DNA binding capacity was high for liposomes, but DNase I protection of DNA bound to liposomes was only moderate in comparison to DNA incorporated into viral capsids. Cellular uptake of radiolabeled and physiologically active DNA was also compared. For animal studies we chose an endogenous retroposon as target gene. To identify recombinational events we replaced a part of this gene with an artificial sequence not present in the mouse genome. The recombination rate for DNA fragments transfected in Polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DNA transfer methods, mediated by positively charged liposomes (DOTMA) and by empty Polyoma viral capsids.     

Effects of antisperm antibodies on early cleavage of fertilized ova.

Early cleavage of zygotes was abnormal after oocytes were fertilized with sperm cells from one of 64 infertile couples studied in our human in vitro fertilization and embryo development procedure. The abnormal cleavage, which did not affect fertilization, was not due to polyspermy or parthenogenetic activation of oocytes, but to antisperm antibodies present on the sperm cells. These antibodies did not react with zona-intact or zona-free human oocytes, although they reacted with the acrosomal (strongly) and tail (weakly) regions of human sperm. The antibodies recognized a double band comprising two glycoproteins of 14 +/- 3 and 18 +/- 3 kDa and a protein band of 22 +/- 3 kDa on the Western blot of lithium diiodosalicylate-solubilized human sperm extract. Antisera were raised in rabbits against the double band (14 +/- 3- and 18 +/- 3-kDa antigens) and the 22 +/- 3-kDa protein. Passive transfer of affinity-purified human or rabbit immunoglobulins directed against the double band (14 +/- 3- and 18 +/- 3-kDa antigens), but not those directed against the 22 +/- 3-kDa protein, caused a significant inhibition of early cleavage of oocytes without affecting pronuclear formation in mice. These results suggest that the sperm surface antigens of 14 +/- 3 and 18 +/- 3 kDa may provide an extranuclear signal to oocytes to divide in mice and humans. These antigens may also find clinical applications in the management of immunoinfertility and in the development of an antisperm contraceptive vaccine for humans.     

Detection and expression of hepatitis B virus X gene in one and two-cell embryos from golden hamster oocytes in vitro fertilized with human spermatozoa carrying HBV DNA

The main objectives of this study were to introduce motile human sperm carrying Hepatitis B virus (HBV) DNA to golden hamster oocytes in a co-culture environment and to detect the replication and expression of the HBx gene in early embryonic cells. Zona-free hamster oocytes were inseminated with human sperm carrying pBR322-HBV DNA plasmid using the in vitro fertilization (IVF) technique. Both the one- and two-cell stages of early embryonic development were studied. PCR, RT-PCR, and Dot hybridization were performed to observe the HBx gene and its expression in these stages. "Fluorescence in situ hybridization" (FISH) was carried out to confirm the integration of HBV into the pronucleus, nucleus, and the chromosomes of embryos. The results showed that we have the ability to obtain a fertilization rate of 80%. RT-PCR showed that the HBx gene could be expressed in both one- and two-cell stages of embryonic development. The data suggested the possibility of sperm as a vector for the vertical transmission of HBV DNA to the next generation.     

Detection of transgene in progeny at different developmental stages following testis-mediated gene transfer

We recently reported that exogenous DNA injected into testis as a liposome complex can be transferred into the egg via sperm by natural mating and integrated in the genome (testis-mediated gene transfer: TMGT). Here, we studied the efficiency of each of the several liposomes in associating foreign DNA with sperm, the expression of an introduced gene in early embryos, and the presence of the DNA in fetuses and pups at different ages. The CMV/beta-actin/EGFP fusion gene, encapsulated with different liposomes, was injected into rat testis, and spermatozoa in the cauda epididymis were obtained 1, 4, and 14 days after injection. We tested each of the 8 liposomes, and found that only 2, DMRIE-C and SuperFect, led to the detection of foreign DNA on all of the days examined, with relatively higher ratios of rats having positive sperm. By means of TMGT using either of those two liposomes, more than 80% of morula-stage embryos expressed EGFP, as observed by fluorescence microscopy. Then we detected introduced DNA in the progeny by PCR and Southern dot blot, and found that the ratio of animals carrying the foreign DNA decreased as they developed, and that only a part of postpartum progeny were foreign-DNA-positive with high incidence of mosaicism. These results suggest that, although, the success rate is still limited, foreign DNA could be integrated into the genome of the progeny by TMGT at least under specific experimental conditions, the efficiency of which depends largely on the characteristics of the liposome. The results also suggest that TMGT could be applicable to fetal gene therapy as well as to the generation of transgenic animals.     

Characterization of glycolysis and pentose phosphate pathway activity during sperm entry into the mouse oocyte

Studying the events that occur during gamete fusion and sperm decondensation in the oocyte remains difficult because sperm-oocyte fusion and subsequent sperm decondensation represent a short part of the fertilization process, and their exact timing is difficult to determine. There is therefore a need for greater understanding of the events that occur during this period. The main purpose of this study was to examine the metabolic aspects of this time frame by characterizing glucose metabolism (glycolytic and pentose phosphate pathway [PPP] activities) during sperm fusion and decondensation into zona-free oocytes in mice. The metabolism of glucose through both glycolysis and the PPP was measured in ovulated MII oocytes, free of cumulus cells, and the levels of glucose metabolized were found to be low. Upon sperm entry, both glycolytic and PPP activity increased substantially. To determine whether this elevation in glucose metabolism was part of the activation process, the metabolism of parthenogenetically activated oocytes was measured, and no increase in metabolism was observed. The characterization of glucose metabolism during sperm fusion and decondensation into the oocyte, and comparison to parthenogenetically activated oocytes, showed that the fertilizing sperm is responsible for an increase in both glycolytic and PPP activity during fusion and/or decondensation. The significance of this observation during the fertilization process and for the developing embryo is as yet unclear and warrants further investigation.