| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reproductive Technology |
The Institute for Biogenesis Research,2 Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822
Department of Cell Biology and Anatomical Sciences,3 The Sophie Davis School of Biomedical Education/The City University of New York Medical School, New York, New York 10031
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/
kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.
assisted reproductive technology, early development, fertilization, oocyte development, sperm motility and transport
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Two issues have stimulated this pursuit: whether a built-in genetic program accounts for the progression of spermatogonial stem cells into meiotic and postmeiotic phases of differentiation and whether somatic cells (Sertoli cell and peritubular myoid cells) can modulate the hypothetical spermatogenic genetic clock. XY-bearing primordial germ cells, the ancestors of the spermatogonial stem cell lineage, provide a partial answer to these two issues. When primordial germ cells home in the gonadal ridge, they proliferate and initiate meiosis; meiosis is aborted when Sertoli cells differentiate and organize testicular cords [2]. In the fetal testis, germ cells cease proliferation and differentiation until puberty, when uninterrupted spermatogenesis becomes established. In contrast, XX germ cells, surrounded by follicular cells, advance into meiotic prophase I and arrest in diplotene as primary oocytes. The gonadal ridge environment does not appear critical for meiosis because XX- and XY-bearing germ cells can complete meiosis in ectopic sites, such as the female or male adrenal gland, and reaggregate in an embryonic lung organ culture system [3]. A corollary from these observations is that meiosis appears to be cell signaling and gonadal ridge independent but contingent to a still undefined germinal cell built-in clock modulated by either Sertoli cells or follicular cells. In this context, experimental evidence has been presented that supports the temporal cycling capacity of the Sertoli cell lineage in the embryonic, postnatal, and adult testis [4]. In addition, it has been proposed that Sertoli cells cultured on an extracellular matrix achieve an undefined differentiation status, which may contribute to spermatogenesis in vitro [5].
The recapitulation in vitro of the uninterrupted progression of spermatogonial stem cells into mature sperm capable of producing offspring still remains to be shown. Morphologic and molecular parameters have been used to monitor the differentiation of spermatogenic cells in vitro. Two major in vitro approaches have been pursued: 1) the coculture of mammalian spermatogenic cells with Sertoli cells [6–9] and 2) the creation of immortalized murine primordial germ cells [10–12]. It was initially reported that a spermatogenic stem cell line, produced by cotransfection of male germ cell lines with the simian virus 40 large tumor antigen and a mutant of p53, was able to generate meiotic and postmeiotic cells [13]. This report was not confirmed. A telomerase-immortalized mouse type A spermatogonial cell has been recently reported to give rise to spermatocytes and spermatids in the presence of stem cell factor [14]. Previous work from several laboratories has shown that spermatids can develop from spermatogenic cell precursors in coculture with Sertoli cells [7–9]. In vitro-developed round spermatids display typical vacuolated mitochondria, chromatoid bodies, and flagella [15]. More recently, the coculture of mouse spermatids that display one to four flagella with an asynchronous beating pattern and spermatid propelling capacity has been reported [16]. An important issue has been to determine whether in vitro-developed uniflagellated or multiflagellated spermatids can generate normal and fertile offspring. Herein, we show that preexisting spermatocytes in coculture with Sertoli cells can produce haploid spermatids, which can generate normal offspring following nuclear injection into mature mouse oocytes. We also show that breeding of offspring thus generated can produce normal offspring.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B6D2F1 mice (C57BL/6 X DBA/2) were used to prepare Sertoli-spermatogenic cell cocultures and also as oocyte donors. Surrogate females of spermatid-injected oocytes were CD1 females mated with vasectomized males of the same strain. All animals were maintained in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and the Animal Facility of The City University of New York Medical School/City College of New York (CUNY/CCNY) and those prepared by the Committee on the Care and Use of Laboratory Animals (National Institutes of Health). The protocol for animal handling and treatment was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii and CUNY/CCNY.
Culture Media
Serum-free, chemically defined medium (designated TKM) was used for coculturing mouse Sertoli cells with spermatogenic cells [16, 17]. For oocyte handling and embryo culture, activated oocytes were cultured in a bicarbonate-buffered CZB medium with 5.5 mM glucose [18, 19] at 37°C under 5% CO2 in air. Oocyte manipulation was performed in Hepes-buffered CZB with 20 mM Hepes and 5 mM NaHCO3 [20] at room temperature in air.
Preparation of Sertoli-Spermatogenic Cell Cocultures
Testes of prepuberal male mice (13–18 days old) were used to prepare Sertoli-spermatogenic cell cocultures. Testicular samples were fixed in Bouin solution and embedded in paraffin according to standard procedures to assess in hematoxylin-eosin-stained sections the population of spermatogenic cells. Cocultures were prepared according to an already reported protocol [6, 17, 21] and maintained in TKM (Eagle Minimum Essential Medium, EMEM; supplemented with 5 µg/ml of insulin; 5 µg/ml of transferrin; 500 ng of follicle-stimulating hormone [NIH-oFSH-S16]; 133 µIU/ml of growth hormone [human, recombinant; Eli Lilly, Indianapolis, IN]; 10 ng/ml of epidermal growth factor; 10 µg/ml of insulin-like growth factor 1 [Upstate Biotechnology, Lake Placid, NY]; 0.5 µM retinol [Sigma, St. Louis MO]; 0.1 µM each of testosterone and dihydrotestosterone [Calbiochem, La Jolla, CA]; 4 mM glutamine; 1 mM sodium pyruvate; 0.1 mM nonessential amino acids; and antibiotics [100 U/ml of penicillin and 100 µg/ml of streptomycin]). Samples were plated in plastic culture dishes (35 mm in diameter) at high cell density (approximately 3 x 106 cells/ml) to maintain Sertoli cells in a contact-inhibited state and incubated at 34°C in a humidified air-CO2 atmosphere. Samples were also plated on glass coverslips placed in plastic culture dishes for indirect immunofluorescence.
Microscopic Examination of Sertoli-Spermatogenic Cell Cocultures and Criteria for the Identification of Round Spermatids
Cocultures prepared in plastic dishes and glass coverslips were examined at 1- to 2-day intervals using an inverted microscope with Hoffman modulation contrast optics. Hoechst 33342 DNA staining was used to evaluate the number of cultured spermatogenic cells in clusters. Two complementary procedures were used to identify spermatogenic cell types in the cocultures: videomicroscopy and indirect immunofluorescence. For videomicroscopy, real-time cell and axoneme movements were recorded with a video camera (Optronics, Bolton, MA) connected to a digital recorder. The sample was maintained at 34°C using an automatic temperature stage controller (Warner Instruments, Hamden, CT). Spermatogonial cells were recognized by their pulsatile movement, and spermatocytes displayed a characteristic nuclear clockwise-counterclockwise rotation [17, 21]. A substantial number (70%) of spermatids developed one to four flagella, each with characteristic wavelike movement [16]. When no flagella were detected, spermatids were recognized with Hoffman optics by a characteristic centrally located nuclear dense mass [20]. The number of flagellated and nonflagellated spermatids varies with the age of the mouse donor and coculture time. In general, the spermatid average is approximately 25% and 35% of the total spermatogenic cell population in 6-day-old cocultures prepared from 15- and 18-day-old donors, respectively. Anti-phosphorylated histone H2AX monoclonal antibody (working dilution: 1:50; Upstate Biotechnology) or anti-
-tubulin monoclonal antibody (working dilution: 1:100; Sigma) followed by anti-mouse IgG conjugated with fluorescein isothiocyanate (working dilution: 1:200; Jackson Immunoresearch Laboratories, West Grove, PA) were used to determine the temporal appearance of the XY bivalent in primary spermatocytes (leptotene to diakinesis) and axoneme in round spermatids, respectively. Controls included omission and dilution of the primary antibody.
Round Spermatid Nuclear Injection into In Vivo-Matured Oocytes
At the end of the coculture (from 5 to 13 days after plating), spermatogenic cells, including round spermatids, were gently detached from the dish using a pipette. Media with cells were transferred to a 60-mm dish, kept on ice, and covered with mineral oil (Squibb & Sons, Princeton, NJ). Many of the collected spermatids displayed a single nucleus; others were multinucleated due to the formation of symplasts containing two to four nuclei within a common cytoplasm. Single-nucleated and multinucleated spermatids were transferred by means of a large-boar pipette (about 20 µm in outer diameter) into 50 µl of Hepes-CZB under mineral oil in a large microsurgical operation plastic dish (95 x 8 mm) and kept there for up to 5 min. Cells were then transferred to a 50-µl drop of Hepes-CZB containing 12% (wt/vol) polyvinylpyrrolidone (360 kDa; ICN Biochemicals, Costa Mesa, CA) and kept there for less than 5 min. Single spermatids were repeatedly aspirated and ejected from an injection pipette (8-µm inner diameter) until its plasma membrane was removed by shearing forces. The plasma membrane of multinucleated spermatids was ruptured in the same way. A single nucleus, surrounded by cytoplasm, was injected into in vivo-matured oocytes as previously reported [20]. Oocytes were activated by placing them for 5–6 h in Ca2+-free CZB containing 10 mM SrCl2. Those oocytes with two distinct polar bodies by the end of SrCl2 treatment were recorded as activated. Activated oocytes were further cultured in CZB medium under 5% CO2 in air.
Chromosome Analysis of Spermatid-Injected Oocytes
To examine the chromosomes of spermatid-injected oocytes, some nuclei of cultured round spermatids were injected into mature oocytes. These injected-oocytes were activated by SrCl2 for 5–6 h and then cultured for 13–14 h in CZB medium containing 0.01µg/ml of vinblastine to arrest them at the metaphase of the first mitotic division. They were fixed and air-dried for chromosome examination [22]. We did not discriminate two groups of chromosomes from male and female pronuclei. We considered that the injected cell was a haploid spermatid when a zygote had both the second polar body and 40 metaphase chromosomes. Cells with more than 40 chromosomes were considered injected with either spermatocytes or spermatogonia.
Embryo Transfer and Raising Pups
Two-cell zygotes (24 h after onset of oocytes activation) were transferred into oviducts of pseudopregnant surrogate females. Females were euthanized on Day 19.5 of generation to determine the number of implantation sites and live offspring. Live offspring were raised by lactating foster mothers. Individual pups were weighted and sexed at birth. These pups were allowed to sexually mature. Then they were mated between them or with normal males and females to test their fertility.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Testes from 13- to 18-day-old mice were used to prepare the cocultures to ensure that no preexisting spermatids were present in the samples at the time of plating (verified by histologic examination of representative samples). The most advanced spermatogenic cells in the testes of 13-day-old mice were early pachytene spermatocytes (Fig. 1A). Middle-to-late pachytene spermatocytes were detected in the testes of 15-day-old mice (Fig. 1B). Diplotene-diakinesis (primary spermatocytes) and secondary spermatocytes (meiosis II) in a few seminiferous tubules were seen in testes of 18-day-old mice (Fig. 1C). The gradual development of a seminiferous tubular lumen was visualized from Days 13 to 18 (Fig. 1A through C). Round spermatids are known to appear in mouse testis by Day 20 after birth [23]. Five days after plating, clusters of interconnected spermatogenic cells (mainly spermatogonial and primary spermatocyte progenies) were seen attached to Sertoli cell surfaces (Fig. 1D and E). Solitary spermatogenic cells were also observed. The larger cell aggregates consisted of approximately 300–1000 overlapping spermatogenic cells as determined by Hoechst 33342 nuclear staining (Fig. 1E). During daily renewal of the culture medium, many spermatogenic cells loosely associated with Sertoli cell surfaces were removed from the dish. Many cells underwent programmed cell death characterized by the formation and release of apoptotic bodies and cessation of the typical cell movements [21].
|
A valuable parameter for distinguishing spermatogonial from primary spermatocyte cell progenies was monitoring the immunoreactive patterns of phosphorylated histone H2AX (-H2AX) (Fig. 2A), a marker for DNA double-strand breaks, which occur during homologous synapsis at meiosis and are highly expressed in testis [24]. Figure 2A shows a Sertoli-spermatogenic cell coculture prepared from a 16-day-old mouse. After 72 h of coculture, the nuclei of spermatogenic cells, but not Sertoli cells, are H2AX immunoreactive (Fig. 2B through E). The XY bivalent appeared as a distinct fluorescent mass at the nuclear periphery of the pachytene spermatocyte progeny (denoted by a box in Fig. 2A and D through E). At high magnification, the fluorescent mass displayed the negative image of the unstained cores of the X and Y chromosomes and their pairing region (Fig. 2F and G). The condensed, highly fluorescent XY bivalent was detected in neither spermatogonia nuclei (denoted by an oval in Fig. 2A through C) nor leptotene-early zygotene spermatocytes (not shown).
|
When spermatogenic cells from 15- to 18-day-old males were cultured for 7–10 days, we detected by Hoffman modulation contrast a number of round cells with a spherical nucleus containing a characteristic dense mass (Fig. 3A and B). Many of these cells displayed one to two hairlike appendages, each with the repetitive wavelike motion of typical flagella. In some instances, four flagella were seen emerging from a single cell at the same cellular pole. When more than one flagellum was present per cell, the motion of each flagellum was asynchronous with respect to that of the other. Examples of flagellated spermatids attached to Sertoli cell surfaces and free flagellated spermatid propelled by four asynchronous beating flagella can be seen at www.sci.ccny.cuny.edu/kier.
|
The axoneme is known to initiate its assembly during step 4 of spermiogenesis, preceding the development of the manchette, which assembles at step 8 and disassembles by step 14 [25]. The presence of an axoneme was confirmed by the linear -tubulin immunoreactive pattern along each filamentous projection, ending in a coiled tip (Fig. 3C through H). -Tubulin immunostaining did not detect the presence of a manchette in the in vitro-developed round spermatids, thus suggesting that the round spermatids corresponded to developmental steps preceding step 8 of spermiogenesis. We have not made attempts to monitor the development of the acrosome (presumably at the Golgi phase in the cocultured round spermatids).
When spermatogenic cells from 13-day-old mice were cocultured for 10–12 days, less than 1% of the cells judged as round spermatids (by the presence of a centrally located nuclear dense mass) displayed flagella. Cells considered to be spermatids by their nuclear features were isolated and their nuclei injected individually into mature oocytes to examine their chromosomal complement. All of the injected nuclei derived from cocultured cells prepared from 15- to 18-day-old mice had flagella, whereas most of the injected nuclei from 13-day-old donor mice had no flagella. Although only 15% of the cells from 13-day-old mice were haploid, approximately 75% of the flagellated cells from 15- to 20-day-old mice proved to be haploid (Table 1).
|
Most oocytes with successful round spermatid injection developed into 2-cell zygotes. In comparison with in vivo-developed spermatids (control), cultured round spermatids were inferior in their ability to contribute to zygote's preimplantation or postmplantation development (Table 2). Nevertheless, it is significant to point out that female and male offspring all developed into fertile adults with normal litter size (data not shown). Histologic examination of their testes and epididymis (Fig. 4A and B) and ovaries (not shown) revealed no sign of gametogenic abnormalities.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The observation of uniflagellated and multiflagellated, motile round spermatids extends our earlier observations (and the observations of others [7, 8]) that meiosis can be completed in vitro [13] and generate haploid spermatids capable of developing a motile flagellum [15, 16]. It is important to emphasize that the sole presence of flagellated cells in Sertoli-spermatogenic cell cocultures does not signify that these cells are spermatids. In fact, any cell with a centriole-derived basal body is capable of generating an axoneme [27]. The demonstration that approximately 75% of the flagellated and approximately 15% of the nonflagellated cells developed in coculture with Sertoli cells proved to be haploid and that most of the in vivo-matured oocytes with successful nuclear injection developed into two-cell stage zygotes indicates both the spermatid nature and fertilizing potential of these flagellated cells. When compared with freshly collected round spermatids from adult testis, cultured round spermatids were less competent in contributing to zygote development. Nevertheless, female and male offspring produced by nuclear injection all developed into fertile adults with normal litter size.
Two aspects of this work deserve further discussion. The first aspect is whether the presence of Sertoli cells is required for the differentiation of spermatogenic cells in vitro. The preservation of the Sertoli-spermatogenic cell partnership has been regarded as critical in fostering the in vivo and in vitro differentiation of spermatogenic cells [6, 8, 9]. In vivo, the spatial distribution of spermatogenic cells in testicular cords and the timing of the spermatogenic cycle after puberty appear to be controlled by Sertoli cell cyclical patterns of gene expression [4, 28]. When Sertoli cell expression of the Steel (c-kit ligand) gene in the infertile Steel/Steeldickie mutant mouse is restored by adenovirus-mediated gene delivery in vivo, partial spermatogenesis (up to round spermatids) was observed [29]. Round spermatids collected from these mice produced adenoviral, DNA-free, fertile offspring after intracytoplasmic injection into oocytes [29]. In organ and cell culture, the entry into meiosis of male germinal cells does not require inducing signals but can be inhibited by the differentiation status of Sertoli cells [3]. Although these and other observations support a role of the Sertoli cell lineage during normal spermatogenesis, it has been reported that a transformed spermatogonial stem cell can divide and differentiate in the absence of supportive cells into single spermatogonia and spermatocytes (not linked by intercellular bridges as in the intact testis) and nonflagellated spermatids [14]. The fertilization potential of presumptive nonflagellated spermatids derived from transformed spermatogonial stem cell precursors [14] awaits demonstration. Although we are confident that most flagellated cells developed in cocultures of Sertoli cells and spermatogenic cell precursors from 15- to 18-day-old mice are spermatids with full developmental potential, we are not certain whether all flagellated and nonflagellated cells found in the cocultures prepared from 13-day-old mice are spermatids with developmental possibilities.
The second aspect of this work relates to the connotation of motile flagella in round spermatids cocultured with Sertoli cells. Rapid flagellar growth in fractionated mouse spermatids (in the absence of Sertoli cells) was first reported by Gerton and Millette [30]. The in vitro development of motile flagella from rat spermatids cocultured with Sertoli cells was also documented [15]. Motile flagella developed in vitro consist of the characteristic nine plus two microtubular organization but lack of outer dense fibers [15, 30]. Other authors have also reported flagellar growth in vitro,[31–33] but typical wavelike flagellar movement was not observed. Mouse round spermatids cocultured with Sertoli cells can generate one to four flagella, all emerging from the same cellular pole and displaying actively propagating bending waves [16]. The generation of multiple motile flagella by cultured round spermatids demonstrates an in vitro origin in contrast with the uniflagellated spermatids developed in vivo. In addition, multiple flagella indicate that spermatid centrosomes, each consisting of a centriolar pair, have duplicated and migrated to the same cellular pole. In vivo, developing spermatids contain only one centrosome, with the distal centriole giving raise to the axoneme and the proximal centriole developing a complex head-to-tail coupling apparatus [25]. The observation of multiple flagella suggests that a mechanism may operate in vivo to prevent centrosome duplication and thus ensure that just one axoneme develops. Details of the currently unknown mechanism of intracellular centrosomal polarity in round spermatids can now be explored in the in vitro-developed round spermatids. Furthermore, flagellated round spermatids developed from preexisting mouse primary spermatocytes mimic the biflagellated green alga Chlamydomonas, providing experimental opportunities for examining the mechanism of intraflagellar transport [34] involving motor proteins, raft proteins, and cargo proteins participating in anterograde (toward the tip of the axoneme) and retrograde direction (toward the cytoplasmic body of the spermatid) during axonemal growth.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
4 Correspondence: Abraham L. Kierszenbaum, Department of Cell Biology and Anatomical Sciences, CUNY Medical School, 138th St. and Convent Ave., Harris Hall, Suite 306, New York, NY 10031. FAX: 212 650 6812; kier{at}med.cuny.edu
Received: 30 December 2002.
First decision: 1 February 2003.
Accepted: 21 February 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  R. Sa, R. Neves, S. Fernandes, C. Alves, F. Carvalho, J. Silva, N. Cremades, I. Malheiro, A. Barros, and M. Sousa Cytological and Expression Studies and Quantitative Analysis of the Temporal and Stage-Specific Effects of Follicle-Stimulating Hormone and Testosterone During Cocultures of the Normal Human Seminiferous Epithelium Biol Reprod, November 1, 2008; 79(5): 962 - 975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kita, T. Watanabe, K. Ohsaka, H. Hayashi, Y. Kubota, Y. Nagashima, I. Aoki, H. Taniguchi, T. Noce, K. Inoue, et al. Production of Functional Spermatids from Mouse Germline Stem Cells in Ectopically Reconstituted Seminiferous Tubules Biol Reprod, February 1, 2007; 76(2): 211 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Suganuma, P. Pelczar, J. F. Spetz, B. Hohn, R. Yanagimachi, and S. Moisyadi Tn5 Transposase-Mediated Mouse Transgenesis Biol Reprod, December 1, 2005; 73(6): 1157 - 1163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sofikitis, E. Pappas, A. Kawatani, D. Baltogiannis, D. Loutradis, N. Kanakas, D. Giannakis, F. Dimitriadis, K. Tsoukanelis, I. Georgiou, et al. Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment Hum. Reprod. Update, May 1, 2005; 11(3): 229 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Vigier, M Weiss, M H Perrard, M Godet, and P Durand The effects of FSH and of testosterone on the completion of meiosis and the very early steps of spermiogenesis of the rat: an in vitro study J. Mol. Endocrinol., December 1, 2004; 33(3): 729 - 742. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hong, T. Liu, H. Zhao, H. Xu, W. Wang, R. Liu, T. Chen, J. Deng, and J. Gui Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro PNAS, May 25, 2004; 101(21): 8011 - 8016. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Marcon and G. Boissonneault Transient DNA Strand Breaks During Mouse and Human Spermiogenesis:New Insights in Stage Specificity and Link to Chromatin Remodeling Biol Reprod, April 1, 2004; 70(4): 910 - 918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Godet, A. Damestoy, S. Mouradian, B. B. Rudkin, and P. Durand Key Role for Cyclin-Dependent Kinases in the First and Second Meiotic Divisions of Rat Spermatocytes Biol Reprod, April 1, 2004; 70(4): 1147 - 1152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kurita, S. M. Burgess, and N. Sakai Transgenic zebrafish produced by retroviral infection of in vitro-cultured sperm PNAS, February 3, 2004; 101(5): 1263 - 1267. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
