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Reproductive Technology; |
Department of Physiology and Cell Biology,4 University of Nevada School of Medicine, Reno, Nevada 89557
Institute for Biogenesis Research,5 University of Hawaii School of Medicine, Honolulu, Hawaii 96822
ABSTRACT
We have developed a method that effectively removes all of the perinuclear materials of a mouse sperm head, including the acrosome, plasma membrane, perinuclear theca, and nuclear envelope. By injection of a single purified sperm head into a metaphase II mouse oocyte followed by activation with strontium chloride, 93% of the zygotes developed into two-cell embryos. Although only
17% of the transferred two-cell embryos were born alive, all live pups developed into adults, and they appeared to be normal in reproduction and behavior. We detected RNA species, including mRNAs and miRNAs from the purified sperm heads. Our data demonstrate that pure membrane-free sperm heads are sufficient to produce normal offspring through intracytoplasmic sperm injection and that at least part of the RNA molecules are deeply embedded in the sperm nucleus.
assisted reproductive technology, early development, embryo, fertility, fertilization, intracytoplasmic sperm injection, in vitro fertilization, small RNAs, sperm
Intracytoplasmic sperm injection (ICSI) has been widely used as a treatment for severe male factor infertility and idiopathic fertilization failure in fertility clinics [1]. In basic biomedical research, ICSI provides an excellent tool for studying the cellular and molecular mechanisms of fertilization and early embryonic development [2]. Previous studies have shown that healthy offspring can be produced through ICSI as long as the genetic integrity of the injected sperm is intact [2, 3]. In natural fertilization, a spermatozoon looses its acrosome before passing through the zona pellucida and perivitelline space. When an intact spermatozoon is injected into an oocyte, the acrosome also is introduced into the oocyte. This may have adverse effects on oocyte survival and activation, and on the early development of the embryo [4–6]. When both the acrosome and plasma membrane are removed prior to sperm injection, the onset of oocyte activation is more rapid, and the embryonic development is improved [7]. Perinuclear materials contain oocyte-activating factors, and the oocyte cannot be activated if a spermatozoon without or with defective perinuclear materials is injected [6, 8, 9]. However, such an oocyte can be activated either by electric shock or by chemical treatment [8–12].
RNA can be obtained from washed sperm or from sperm heads freed from their tails and necks by detergent treatment [13–16]. These RNA species had been regarded as carryover products originating from the testis during spermatogenesis. Although some testis-derived mRNAs can be detected in the fertilized eggs [17], no direct evidence shows that these sperm-borne RNA species have a role in oocyte activation and embryonic development. A recent study reports that sperm from male mice carrying a Kit mutation can deliver the mRNA transcripts derived from the mutant Kit allele into the oocytes during fertilization, causing the offspring to display white spot phenotype [18]. This study suggests that mRNAs carried by sperm into the oocytes may affect the phenotype of the offspring.
In the present study, we report that the injection of a membrane-free, pure sperm nucleus into an oocyte can produce normal and healthy offspring in mice and that both mRNAs and miRNAs can be detected in these pure sperm nuclei.
All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. The medium used for culturing oocytes after ICSI was a bicarbonate-buffered Chatot, Ziomet, and Bavister medium (CZB) supplemented with 5.56 mM D-glucose and 4 mg/ml BSA. The medium used for oocyte collection and ICSI was a modified CZB with 20 mM Hepes-Na, 5 mM NaHCOB3, and 0.1 mg/ml polyvinyl alcohol (cold water soluble) instead of BSA. Chatot, Ziomet, and Bavister medium was used under 5% CO2 in air, and Hepes-buffered CZB solution (Hepes-CZB) was used under 100% air. The pH of these media was 7.4. The reason for our using CZB instead of the currently popular KSOM medium is that 10 mM SrCl2, which we used to activate mouse oocytes, tends to precipitate in the KSOM medium. As long as oocytes and embryos are from hybrid mice, we have not observed any differences between these two media in their ability to support preimplantation development of embryos.
Adult B6D2F1 (C57BL/6 x DBA/2) hybrid mice were used to collect oocytes and spermatozoa. Adult random-bred ICR (albino) female mice mated with vasectomized males of the same strain were used as embryo recipients. C57BL/6 x DBA/2 and ICR mice were generated and maintained as breeder colonies at the University of Hawaii (Honolulu, HI). All procedures described within were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Hawaii and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Cauda epididymal spermatozoa were allowed to disperse into CZB medium. The spermatozoa then were washed once with CZB medium by centrifugation. Sperm pellets were resuspended in a medium (pH 7.8) consisting of 250 mM NaCl, 100 mM Tris-HCl, 10 mM EGTA, 0.2% (w/v) lysolecithin (LL; L-
-phosphatidylcholine; Avanti Polar Lipids Inc., Alabaster, AL), and 0.1% (w/v) pronase (Calbiochem, La Jolla, CA). The sperm were incubated at 37°C for 30 min, followed by centrifugation. The pellets were processed for electron microscopy [19], RNA isolation, or ICSI.
Intracytoplasmic Sperm Injection
Intracytoplasmic sperm injection was carried out as described [8, 20], with some modifications. An aliquot (25 µl) of sperm suspension was mixed thoroughly with 50 µl Hepes-CZB containing 8%–12% (w/v) polyvinylpyrrolidone (Mr 360 000). A drop of this suspension was transferred under paraffin oil (Squibb & Sons, Princeton, NJ) in a plastic dish (100 x 100 mm) previously placed on the stage of an inverted microscope equipped with a micromanipulation system. Two types of spermatozoa were injected into oocytes: 1) a sperm head isolated from the tail by applying a single or a few Piezo pulses to the neck region, and 2) a membrane-free pure sperm head after treatment with LL + pronase. Approximately 15 oocytes in a group were injected within 5 min. Intracytoplasmic sperm injection was completed within 2 h after collection of oocytes from the oviduct.
Culture and Transfer of ICSI Embryos to Surrogate Mothers
Intracytoplasmic sperm injection oocytes were cultured in CZB medium at 37°C under 5% CO2 in air. Two-cell embryos developed from ICSI oocytes were transferred into oviducts of pseudopregnant CD1 (albino) females that had been mated during the previous night with vasectomized males of the same strain. Surrogate females were killed on Day 19 of pregnancy, and their uteri were examined for the presence of live fetuses. Live fetuses, if present, were collected by cesarean delivery and raised by lactating CD1 foster mothers.
Total RNA containing the small RNA fractions were isolated using mirVana miRNA isolation kit according to the manufacturer's instructions (Ambion Inc., Austin, TX). Briefly, the frozen LL + pronase-treated sperm heads were washed with PBS four to six times before the lysis buffer was added. The sperm pellet was resuspended in 10 vol lysis/binding buffer, followed by vigorous vortex for 2–5 min. A 1:10 volume of miRNA homogenate additive was added and incubated on ice for 10 min. Total RNA was extracted by adding an equal volume of acid-phenol:chloroform. Small RNA was extracted from the total RNA using a filter cartridge with 100 µl preheated (95°C) elution solution. The concentration of small RNA was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE).
PCR-Based Detection of mRNAs and Small RNAs
Total RNAs extracted from treated or intact sperm were further treated with a DNA-free kit (Ambion) to remove potential DNA contamination. First-strand cDNAs were synthesized from the DNA-free total RNAs using 400 units of superscript III reverse transcriptase (Invitrogen) with random primers (Invitrogen) at 50°C for 60 min in the presence of 1 µg total RNA in a 40-µl reaction volume. RNase H (1 µl; Promega) was added to the reaction and incubated at 37°C for 20 min for RNA removal. The cDNAs were purified using a PCR purification kit (Qiagen, Valencia, CA) in 200 µl elution buffer. Polymerase chain reaction detection of 23 testis-specific mRNAs was performed, and Gapdh was used as a loading control. After 40 cycles of amplification, the PCR products were run on 2% agarose gels to visualize the amplicons. Primer sequences for these 23 testis-specific mRNAs are listed in Table 1, and these primers were designed such that each pair encompasses at least one intron. Preparation of small RNA cDNA (srcDNA) libraries and the semiquantitative PCR were performed as previously described [21]. Primer sequences for the 28 testis-specific or testis-preferential miRNAs can be found in Table 2. Two ubiquitously expressed miRNAs, let-7a and let-7d [21–25], were used as loading controls.
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Complete Removal of Nonnuclear Materials from Mouse Spermatozoa
When mouse spermatozoa from the caudal epididymis were treated with LL and pronase and then washed thoroughly with PBS, only sperm heads were observed (Fig. 1). To further verify the complete removal of all of the membranous structures from the sperm heads, electron microscopy was performed on the treated and washed sperm. After the LL + pronase treatment, all sperm had lost their tails, necks, acrosomes, plasma membranes, and nuclear envelopes (Fig. 2, C and D). In contrast, the untreated control sperm had all of them (Fig. 2, A and B). Therefore, after LL + pronase treatment, we obtained sperm heads completely free of all membranous structures. These pure sperm nuclei then were used for the ICSI and RNA analyses reported below.
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Development of Mouse Oocytes Injected with Pure Sperm Nuclei
When single membrane-free, pure sperm nuclei were injected into oocytes, 93% of ICSI-surviving oocytes developed into two-cell embryos after SrCl2-induced oocyte activation (Table 3). This rate is comparable to that of the control group, in which single sperm heads without the treatment were injected (87%). The two-cell embryos were transferred into the oviducts of surrogate mothers. The percentages of the transferred two-cell embryos that developed to full term and were born live were 58% for the membrane-intact (control) group and 17% for the membrane-free (treated) group (Table 3). A total of 29 of the 30 live-born pups derived from oocytes injected with the pure sperm nuclei grew into adults and all looked grossly normal. We randomly selected two males and four females and bred with wild-type females and males, respectively. All produced normal-looking offspring with normal litter size and normal litter interval (data not shown). These data suggest that injection of a pure sperm nucleus into an oocyte can produce normal fertile offspring.
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The Membrane-Free, Pure Sperm Nuclei Contain Both mRNAs and miRNAs
The electron microscopic examination confirmed that the LL+ pronase-treated sperm heads were free of membranous structures, representing essentially pure sperm nuclei (Fig. 2, C and D). To avoid contamination from the disassociated membrane structures from the supernatant, we further washed the treated sperm heads until no membranous structures were seen under the high-power (100x objective in oil) light microscope. We used a differential precipitation method and isolated two RNA fractions, which were enriched in mRNAs and miRNAs, respectively. For both the treated and control groups, total RNA and miRNA fractions were isolated from sperm collected from the caudal epididymides of three adult mice. The average yields of total RNA and small RNA fractions from treated sperm were 498 ± 27 ng per million caudal epididymal sperm and 97 ± 38 ng per million caudal epididymal sperm, compared with those from untreated sperm at 2140 ± 94 ng per million caudal epididymal sperm and 397 ± 48 ng per million caudal epididymal sperm, respectively.
The total RNA was reversed transcribed using random primers, and resultant cDNAs were used for detecting 23 testis-specific genes by PCR. The 23 testis-specific genes were identified by in silico screening of the Unigene collection (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unigene) for the expressed sequence tag clusters that were derived exclusively from the testis and also by our expression profiling analyses using multiple mouse tissues and 11 purified spermatogenic cell types (Yan, unpublished data). Among all of the 23 testis-specific genes tested, all were detected in the intact sperm. A total of 17 of the 23 mRNAs were detected in the pure sperm nuclei, and the levels of 3 of the 17 mRNAs appeared to be lower than those in the intact sperm (Fig. 3). Since the treated sperm heads are free of acrosome and perinuclear materials, it is likely that these mRNAs are from the sperm nuclei. Moreover, some mRNA species are likely to be located outside of the nucleus, given that more mRNAs can be detected in the intact sperm with membranous structures. Using a PCR-based small RNA detection method [21], we examined the levels of 28 testis-specific or testis-preferential miRNAs [23] in membrane-free sperm nuclei and in intact sperm. All 28 miRNAs analyzed were detected in the intact sperm, whereas 14 of 28 were detected at lower levels in the membrane-free pure sperm nuclei (Fig. 4). These data demonstrate that the membrane-free sperm nuclei contain small RNA as well.
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Previous work has shown that injection of single sperm heads lacking acrosome and plasma membrane into oocytes results in a higher percentage of live-born normal offspring at term compared with the injection of intact sperm heads [7, 26]. Simultaneous removal of acrosome and plasma membrane can be achieved by brief exposure of sperm to either Triton X-100 or LL. Lysolecithin is preferred because it is an endogenous product of sperm derived from the hydrolysis of membrane phospholipids by phospholipase A [27, 28]. In the present study, LL was used in conjunction with pronase to remove the acrosomal membrane, nuclear envelope, and other perinuclear materials. A 30-min incubation completely removed all membrane structures and perinuclear materials, as verified by electron microscopy (Fig. 2, C and D). Using these membrane-free sperm nuclei, we performed experiments and intended to answer two longstanding questions: Can injection of a membrane-free, pure sperm nucleus into an oocyte produce live-born normal offspring? Can RNA species be detected in these pure sperm nuclei? Our results provide positive answers to both of these questions.
A Sperm Nucleus with Intact Genomic Integrity Is the Only Prerequisite for Producing Healthy and Normal Offspring in Mice
This study showed that the purified mouse sperm nucleus is capable of participating in normal development of offspring, except that it lacks the ability to activate an oocyte. This is due to the fact that the pure mouse sperm nucleus does not contain the sperm-borne oocyte-activating factor, which normally resides in the perinuclear materials [8, 9]. Proper chemical reagents or physical conditions can substitute for sperm-borne oocyte-activating factor. Strontium, which was used in this study, is one example of a chemical oocyte activator. The sperm centrosome, which plays a crucial role in the development of the sperm aster, which brings the male and female pronuclei to the center of the zygote in many animals, is not essential in the mouse [29], and perhaps in other common laboratory rodents.
It was somewhat unexpected that the percentage of live offspring born after injection of pure sperm nuclei was significantly lower than the percentage following injection of membrane-intact sperm heads (17% vs. 58%; Table 3). It has been shown that treatment of sperm with LL alone does not decrease, but instead increases the percentage of live normal offspring at birth [7]. It is likely that the decrease in live birth rate following injection of pure sperm nuclei was due to the damage to the sperm genome done during the treatment with pronase. The 30-min pronase treatment completely removed the perinuclear materials, and it may also have digested a significant amount of protamines, and possibly some other sperm nuclear proteins, since pronase has a broad specificity. Other possibilities that cannot be excluded include the following: 1) the pronase treatment may have removed some extranuclear components that may be important for embryonic development; and 2) the treatment may have damaged the RNAs contained within the sperm nuclei (see below), and this damage may have caused the defect in embryonic development if these RNAs indeed play a role. Nevertheless, complete removal of the acrosome, plasma membrane, and perinuclear materials from sperm heads results in pure sperm nuclei, and injection of a pure sperm nucleus into oocytes followed by SrCl2 activation can produce live offspring that can develop into adults with normal behavior and normal reproductive capacity. This study further demonstrates that in mice, a single sperm nucleus with intact genomic integrity is the only prerequisite for obtaining a healthy normal offspring through ICSI.
The Sperm Nucleus Contains Various RNA Species
Sperm-borne RNAs have been reported by numerous studies [30–32]. However, many investigators believe that these RNAs are testicular transcripts carried over by residual cytoplasm and cytoplasmic droplets, or are trapped within the plasma membrane and perinuclear materials of sperm during cytoplasm removal and spermiation. It has been suggested that these "leftover" testicular transcripts do not have a direct role in development. This notion has changed within the past few years, because several studies have convincingly demonstrated that not only are the sperm-borne mRNAs detected in fertilized eggs [17], but these transcripts have the potential to generate phenotypes in the offspring as well [18]. Although these studies do not provide concrete evidence that the sperm-borne mRNAs have a direct role in fertilization or embryonic development, it is clear that sperm do contain RNAs, and these transcripts can be delivered into the oocyte during fertilization and can produce phenotypes. However, the precise location of the RNA within the sperm remains a mystery. Several studies have shown that RNA is still detectable after mild treatment of sperm with detergents (e.g., Triton X-100 or Tween-20) to remove tails and other membranous structures attached to the sperm [13–16]. A series of studies [2, 4, 5, 7] showing enhanced fertilization, embryonic development, and high rate of live-born normal offspring using acrosome-free, plasma-free sperm for ICSI suggests that the sperm-borne RNA species must be hidden within the perinuclear materials or even deeply embedded in the sperm nucleus if these sperm-borne RNA species are indeed important for fertilization and/or embryonic development. In the present study, we attempted to isolate both large RNA (e.g., mRNAs) and small RNA (e.g., miRNAs, piRNAs, etc.) species from pure sperm nuclei, and both populations of RNA were consistently obtained from these membrane-free, pure sperm nuclei. It is highly unlikely that these RNA species were derived from contaminating membranous structures from the supernatants, because after the LL + pronase treatment the pellets were thoroughly washed a minimum of four times and the electron microscopy observation did not show membranous structures. However, the fact that the membrane-free sperm nuclei contain much less RNAs than untreated intact sperm suggests that, indeed, many RNAs are located outside the sperm nucleus and are probably carried by the residual cytoplasm, cytoplasmic droplets, and other membranous structures attached to the intact sperm. The different amount of RNAs carried by the intact sperm or pure sperm nuclei may account for the absence of many RNA species that are normally detected in the intact sperm. On the other hand, the mRNAs and miRNAs detected in pure sperm nuclei may represent the truly functional population of sperm-borne RNAs if they do have a role in fertilization and/or early embryonic development. Our data here suggest that the sperm nucleus contains numerous RNA species, including mRNAs and miRNAs. Our findings are consistent with those in several previous reports, which demonstrate that RNAs are a component of the plant spermatozoid nucleus [33] and are visualized as an enzyme-gold complex of RNase and colloidal gold in rat and human sperm nuclei [34]. The function of these RNAs remains an interesting topic for future studies.
In summary, the present study demonstrates that a single sperm nucleus is sufficient to produce a healthy offspring using ICSI, and the sperm nucleus contains mRNAs and miRNAs, which may have a role in fertilization and embryonic development.
ACKNOWLEDGMENTS
The authors thank Drs. Larisa Baryshnikova and Christopher von Bartheld and Mrs. Hiroko Yanagimachi for excellent assistance in the electron microscopic imaging, and David Young and Jason Michaels for editing the text.
FOOTNOTES
1Supported by a start-up fund from the University of Nevada Reno to W.Y. 
Correspondence: 2Wei Yan, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Anderson Biomedical Science Bldg. 105C/111, 1664 North Virginia St., MS 352, Reno, NV 89557. FAX: 775 784 6903; e-mail: wyan{at}medicine.nevada.edu
Correspondence: 3Ryuzo Yanagimachi, Institute for Biogenesis Research, University of Hawaii School of Medicine, 1960 East-West Rd., Honolulu, HI 96822. FAX: 808 956 7316; e-mail: yana{at}hawaii.edu
Received: 6 December 2007.
First decision: 31 December 2007.
Accepted: 15 January 2008.
REFERENCES
This article has been cited by other articles:
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  R. Grant-Downton, S. Hafidh, D. Twell, and H. G. Dickinson Small RNA Pathways Are Present and Functional in the Angiosperm Male Gametophyte Mol Plant, May 1, 2009; 2(3): 500 - 512. [Abstract] [Full Text] [PDF]
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 C. Li, E. Mizutani, T. Ono, and T. Wakayama Production of normal mice from spermatozoa denatured with high alkali treatment before ICSI Reproduction, May 1, 2009; 137(5): 779 - 792. [Abstract] [Full Text] [PDF]
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