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Regular Article |
a Department of Animal Science, Cornell University, Ithaca, New York 14853
ABSTRACT
Improved methods for culturing spermatogenic cells will facilitate the study of spermatogenesis, treatment of male factor infertility, and genetic modification of the male germ line. The objective of this study was to develop a procedure for achieving male germ cell progression through meiosis in vitro. Testes from 3-day-old bulls were decapsulated and seminiferous tubules were dissociated enzymatically to recover Sertoli and germ cells. Dissociated cells were reaggregated by phytohemagglutinin and encapsulated by calcium alginate, then cultured for up to 14 wk in modified Dulbecco modified Eagle medium/F12 (32°C, 5% CO2 in air). At 2, 5, and 10 wk, cultured cells were examined and evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis for protamine-2 (PRM-2) and transition protein-1 (TP-1) mRNA, expressed specifically in round spermatids. Ploidy was characterized by flow cytometric analysis of DNA content of cultured cells. Only Sertoli cells and gonocytes were observed in seminiferous tubules of 3-day-old testes. By 10 wk of culture, small spherical cells (7–10 µm) were apparent at the margin of cell associations in culture. Following RT-PCR and Northern blot analysis, specific bands corresponding to PRM-2 and TP-1 were detected only in adult testis RNA or after 10 wk of culture. Based on flow cytometry, a haploid population of cells appeared in vitro that was not in 3-day-old bull testis. The novel culture system developed in this study is the first to promote differentiation of gonocytes to presumptive spermatids in vitro based on the expression of spermatid-specific genes.
spermatid, spermatogenesis, testis
INTRODUCTION
Spermatogenesis is the process of germ cell proliferation and differentiation within the seminiferous tubules of the testis that leads to a haploid, free-swimming spermatozoon. Orchestrated in large measure by Sertoli cells, spermatogenesis requires complex endocrine and auto-/paracrine regulation as well as direct cell-to-cell interaction [1–3]. However, precise molecular mechanisms regulating the extensive cross-talk among various somatic and germ cell types remain to be established.
An effective procedure for recapitulating spermatogenesis in vitro would greatly facilitate mechanistic studies of the in vivo process while providing a biological basis for treating selected causes of male infertility and genetically modifying the male germ line. In vitro spermatogenesis (IVS) could be applied to generate developmentally competent haploid spermatids or sperm that could then be used in conjunction with round spermatid injection (ROSI) or intracytoplasmic sperm injection (ICSI) procedures [4, 5] to overcome male factor infertility, especially azoospermia due to maturation arrest [6]. Also, transfection of diploid germ cells in culture followed by IVS would provide a direct approach to genetic modification of the male germ line [7], including targeted gene insertion via homologous recombination.
Devising an IVS culture system that will support germ cell development through meiosis has been especially challenging. Miura et al. [8] reported an organ culture system in which fragments of immature Japanese eel testis, containing only spermatogonia and inactive testicular somatic cells, were maintained and completed spermatogenesis in a chemically defined medium. In rodents, some stage-specific progression of spermatogenesis has been achieved in vitro utilizing organ culture or coculture with immortalized Sertoli cells [9–11].
Mammalian germ cells can be maintained in culture for months during which they retain their full spermatogenic potential [7], but difficulties in establishing conditions for germ cells to proceed to and through meiosis have limited the success of IVS culture systems [2]. Recently, however, meiosis of rat germ cells in culture has been reported using seminiferous tubule segments [11, 12]. By measuring the expression of stage-specific markers phosphoprotein p19 and testis-specific histone TH2B (pachytene spermatocytes) and transition proteins TP-1 and TP-2 (round spermatids) as a function of days in culture, Hue et al. [12] demonstrated an increase in the round spermatid to spermatocyte ratio during a 3-wk culture. The increased ratio corresponded to an increase in haploid cells in culture based on ploidy analysis. Retention of germ cell-Sertoli cell associations during tissue dissociation was considered to be a critical feature of this culture system. Tesarik et al. [6] recently reported a live human birth following ROSI using spermatids recovered after in vitro differentiation of primary spermatocytes. However, complete IVS from gonocytes through spermatocytogenesis, meiosis, and spermiogenesis has not been reported previously for mammalian species.
The primary objective of this study was to determine if dissociated cells of the neonatal bovine testis, both fresh and frozen-thawed, could be reassembled to recapitulate spermatogenesis during long-term culture. Testis cells from dissociated seminiferous tubules were reaggregated and encapsulated in calcium alginate to promote and sustain interaction between germ cells and Sertoli cells without limiting permeability to media components. Results indicate that these conditions promoted germ cell differentiation from gonocytes to presumptive round spermatids.
MATERIALS AND METHODS
Testis Dissociation
The tunica albuginea was manually decapsulated from 3-day-old bull testes, and 2 g of the exposed parenchyma were cut into pieces and washed for 20 min in Ca++, Mg++-free PBS (Sigma Chemical Co., St. Louis, MO). Tissue was dissociated in 50 ml of enzyme solution A containing 0.5 mg/ml collagenase (Type A, Sigma), 10 µg/ml DNase I (Sigma), 1 µg/ml soybean trypsin inhibitor (Gibco BRL, Grand Island, NY), and 1 mg/ml hyaluronidase (Sigma) in Ca++, Mg++-free PBS, and incubated for 30 min at ambient temperature (
25°C). Dissociated cells and tubule fragments were centrifuged at 400 x g and decanted to remove peritubular cells, then the material was incubated with enzyme solution B (same as solution A with collagenase increased to 5 mg/ml) for 30 min. The material was centrifuged at 600 x g for 10 min.
After centrifugation, the supernatant was removed and dissociated cells were washed twice in Dulbecco modified Eagle medium/F12 (DMEM/F12) medium (Gibco), then placed immediately into culture or frozen and stored in liquid nitrogen. For freezing, testicular cells were suspended in DMEM/F12 at a concentration of 1 x 109 cells/ml. Freezing medium (10% dimethyl sulfoxide, 20% bovine calf serum [BCS] in DMEM/F12) was added slowly to a volume equal to the original cell suspension, and mixed. Aliquots of the final cell suspension (1.0 ml per cryovial) were held at -70°C for at least 12 h, and then stored in liquid N2. Swirling in a 32°C water bath thawed cells, and DMEM/F12 was added slowly to five times the initial volume [13]. The thawed cell suspension was centrifuged at 600 x g for 10 min, and the cell pellet was resuspended in DMEM/F12 for reaggregation.
Calcium Alginate Encapsulation and In Vitro Culture
Washed, dissociated cells from fresh testis or frozen-thawed cell suspensions were resuspended to 1 x 109 cells/ml in DMEM/F12 medium containing 0.5% BCS (Hyclone, Logan, UT) and 100 µg/ml phytohemagglutinin (Sigma). After incubation for 10 min at 32°C, reaggregated cells were centrifuged, the supernatant was removed, and 1.0% sodium alginate (Sigma) in 0.9% NaCl was added without disrupting the pellet. After further incubation for 10 min at 32°C, the aggregated, alginate-treated cells were extruded into 1.0 ml sodium alginate solution in a petri dish using a fire-polished Pasteur pipette (200 µm outside diameter [o.d.]). Extruded strands of alginate-treated cells were drawn with a column of 1.0% sodium alginate into the tip of a 9'' Pasteur pipette (1.0 mm o.d.) and solidified by transferring into 1.5% CaCl2 in 0.9% NaCl. Alginate-encapsulated cells were then transferred to culture medium.
Culture medium consisted of modified Hepes-buffered DMEM/F12 medium supplemented with 10 µg/ml insulin-transferrin-selenium solution (Gibco), 10-4 M vitamin C (Sigma), 10 µg/ml vitamin E (Sigma), 3.3 x 10-7 M retinoic acid (Sigma), 3.3 x 10-7 M retinol (Sigma), 1 mM pyruvate (Sigma), 2.5 x 10-5 U bovine FSH (Sioux, Sioux Center, IA), 10-7 M testosterone (Sigma), 1x antibiotic-antimycotic solution (Gibco), 10% BCS [11]. Alginate-encapsulated cell aggregates were transferred to 1.0 ml of culture medium in a 24-well dish (Falcon, Becton-Dickinson, Lincoln Park, NJ) and cultured for up to 14 wk at 32°C in a humidified atmosphere of 5% CO2 in air. Approximately 80% of culture medium was replaced on alternate days. The cell culture protocol was repeated on three separate cell preparations from two bulls.
Histological Examination
Tissue from 3-day-old bull testis was fixed in Bouin fluid, embedded in paraffin wax, and sectioned at 5 µm using standard histological procedures for examination of starting material. Sections were then stained with hematoxylin and eosin. Cultured, encapsulated cells were recovered and examined before and after dissociation using an Olympus inverted microscope with phase-contrast and Hoffman illumination.
RNA Preparation and Analysis Using Reverse Transcription-Polymerase Chain Reaction and Northern Blot Analysis
The mRNA expression of protamine-2 (PRM-2) and TP-1 was assessed in cultured cells from neonatal bull testes using reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis. At 0 (3-day-old testis), 2, 5, and 10 wk of culture, total RNA of cultured cells was extracted by the Trizol method (Gibco).
Reverse transcription was carried out using 200 ng of total RNA, 5 mM MgCl2, 1 U of RNase inhibitor, and 0.2 U of DNase I at 37°C for 30 min, then 42°C for 1 h after adding 1 mM dNTP, 2.5 µM random hexamers, and 2.5 U murine leukemia virus reverse transcriptase (Roche, Branchburg, NJ) [14]. The PCR for PRM-2 (5'-AGAGGCCGCTACCACTACAGACACA-3', 5'-ACTTAGAGCTGCCTTCCGCATCTCC-3'; 140 bp) [15], TP-1 (5'-GGAAGAGCAGCCTGAAGAGT-3', 5'-TCAGGTCTCCTTGGCAGTCC-3', 150 base pairs [bp]) [16] and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 5'-ACGGCACAGTCAAGGCAGAG-3', 5'-GTGATGGCGTGGACAGTGGT-3'; 379 bp, from GenBank U85042) was performed in a 20-µl reaction containing 10 mM Tris-HCl (pH 8.3), 2 mM MgCl2, 50 mM KCl, 0.25 mM dNTP, 3–5 pmol of each primer, and 1.25 U of Taq DNA polymerase (Gibco). The PCR was initiated with denaturation at 94°C for 5 min followed by 40 cycles of 30 sec at 94°C, 30 sec at 65°C, and 30 sec at 72°C. A final extension for 10 min at 72°C completed the PCR; then PCR products were separated by 2% agarose gel electrophoresis and verified by automated nucleotide sequencing. Negative controls included mock reverse transcription without RNA or PCR with distilled deionized water. Testicular cells from adult testis were used as positive control.
Northern blot analysis was performed by the protocol of Wang and Johnson [17]. Briefly, 15 µg of total RNA per sample was electrophoresed in denaturing 1.5% agarose, 2.2 M formaldehyde gels in 1x morpholinepropanesulfonic acid. Gels were blotted onto nitrocellulose membrane (Genescreen Plus; New England Nuclear, Boston, MA). 32P-Labeled probes were prepared using random labeling kit (Stratagene, La Jolla, CA). Hybridization was performed at 42°C in a solution of 1% SDS (Gibco), 50% formamide (Sigma), 2 mg/ml herring sperm DNA (Gibco), 5x Denhardt reagent, and 5x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA [pH 7.7]). Following hybridization, the membrane was washed at a final stringency of 65°C in 0.1x saline-sodium citrate (0.15 M NaCl and 0.015 M sodium citrate) and exposed for appropriate times using Kodak X-OMAT autoradiographic film.
Flow Cytometric Analysis of DNA Content
Flow cytometry was carried out to differentiate germ cell populations based on nuclear DNA content. Encapsulated cells were isolated mechanically after 10 and 14 wk of culture, washed in Ca++, Mg++-free PBS, and permeabilized with 0.1% Triton-X (Sigma) for 30 min. Testicular cells from adult and 3-day-old bulls were prepared as controls with and without haploid germ cells, respectively. Cell nuclei were stained with propidium iodide (Sigma) at 50 µg/ml, and single color analysis for DNA ploidy was completed on 2 x 104 cells/sample using a Becton Dickinson FACS Calibur flow cytometer.
RESULTS
Morphological and Histological Analysis of Encapsulated Cells Cultured In Vitro from Fresh and Frozen-Thawed Testicular Cells of 3-Day-Old Bulls
Parenchyma of 3-day-old bull testis was characterized by seminiferous tubules containing only immature Sertoli cells and gonocytes (Fig. 1A). The integrity and dimensions of the extruded calcium alginate-containing aggregated testis cells did not change (Fig. 1B), and intercellular association during culture was effectively maintained. By 10 wk of culture, small spherical cells (7–10 µm) were apparent at the margins of encapsulated cells in culture (Fig. 1C). When encapsulated cells were dissociated, a distribution in cell size from 7 to 20 µm was observed. Cell morphology was generally spherical with apparently intact plasma membranes based on the distinct margins of individual cells (Fig. 1D). Cell morphology and integrity was similar for testis cells cultured after freezing and thawing.
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Messenger RNA Expression of PRM-2 and TP-1 Genes in Encapsulated Cell Mass Cultured In Vitro
Expression of PRM-2 and TP-1 by the encapsulated cells cultured for 2, 5, and 10 wk was analyzed by RT-PCR. Testicular tissue samples from an adult and a 3-day-old bull were used as positive and negative controls, respectively, and GAPDH was included as an internal control for gene expression. Specific bands corresponding to mRNA of PRM-2 and TP-1 were detected only in samples cultured for 10 wk and in adult testis, indicating the presence of spermatids (Fig. 2A). The detected PCR products were verified by automated nucleotide sequencing and used as templates for probes for Northern blot analysis. Frozen-thawed testis cells were also competent to develop in vitro, as similar patterns for PRM-2 and TP-1 expression were observed following culture of frozen-thawed germ cells (Fig. 3).
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To confirm RT-PCR results, RNA isolated from testicular and in vitro-cultured tissue was analyzed by Northern blot hybridization with 32P-labeled probes for PRM-2 and TP-1. Transcripts of 0.47 and 0.37 kilobases (kb) were detected using probes for PRM-2 and TP-1, respectively, in samples cultured for 10 wk and in adult testis (Fig. 2, B and C).
Flow Cytometric Analysis of DNA Content in Cultured Cells
DNA content of individual cells was analyzed by flow cytometry after staining with propidium iodide. A major population of presumptive diploid cells based on fluorescence intensity of dispersed adult testis cells and washed, ejaculated bull sperm was detected with a relative fluorescence intensity of 440 (Fig. 4). A slight shift in the distribution of cellular DNA content was detected by 10 wk of culture and a subpopulation of testis cells with haploid DNA (relative fluorescence intensity 220) was detected in cell associations cultured after 14 wk in vitro and in adult testis but not in 3-day-old bull testis (Fig. 4). The leading peak is attributed to fragmented cells based on microscopic observation. The distribution of cells cultured for 14 wk in vitro had a similar fluorescence intensity pattern to those from adult testis.
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DISCUSSION
The complete recapitulation of spermatogenesis in vitro remains an elusive goal in reproductive biology. While germ cell viability and functionality can be maintained for extended periods in culture [7], only limited differentiation of spermatogenic cells has been achieved in vitro [2]. Several reports in recent years have demonstrated that germ cells cultured in association with Sertoli cells can progress through discrete steps in the sequence of spermatogenesis during short-term culture, including initiation or completion of meiosis and initiation of spermiogenesis [9, 12, 18, 19]. However, long-term culture conditions that result in the progression of undifferentiated mammalian gonocytes through spermatocytogenesis and meiosis have not been reported.
The complex Sertoli-germ cell interactions required for the orchestration of spermatogenesis in vivo [3], including intercellular junctions and endocrine and paracrine regulation, appear to be critical to the in vitro process also, as culture systems utilizing tubule fragments [12] or homogenates [19] stimulate germ cell differentiation more successfully than other approaches. A potential limitation to this approach is the permeability barrier to critical medium constituents created by peritubular cells and basement membrane components that persist in tubule fragments. To avoid this potential constraint, peritubular cells were removed and seminiferous tubules were completely dissociated enzymatically [20].
However, direct association among testis cells (germ cells and Sertoli cells) can be reestablished by aggregation with lectins such as concanavalin A [21]. In the present study, dissociated testis cells were first aggregated by phytohemagglutinin followed by encapsulation with calcium alginate [22, 23] before being transferred to culture medium (Fig. 1B). Sodium alginate is a polysaccharide that forms a hydrogel when complexed with calcium and is used widely for culturing and transplanting cells, tissues, and embryos because it is nontoxic, highly permeable, biodegradable, and provides three-dimensional support [24]. Though larger in diameter than endogenous seminiferous tubules (200 versus 50 µm diameter, respectively), alginate-encapsulated cell aggregates approximated the cell density and linear arrangement of native tubules (Fig. 1, B and C). Extruded alginate strands containing aggregated testis cells were delicate but patency and close intercellular associations were maintained throughout 14 wk of in vitro culture (Fig. 1, B and C). Whether lectin-mediated aggregation and alginate encapsulation facilitated cell adhesion and the formation of specific junctional complexes observed in vivo remains to be determined.
Despite the low number of testis cells present in the long-term cultures, evidence for the progression of gonocytes to presumptive round spermatids was supported by expression of PRM-2 and TP-1 between mRNA 5 and 10 wk of culture (35–70 days). Expression of these genes has been shown to be spermatid-specific in a variety of species [25–28]. Hue et al. [12] also used stage-specific markers to describe changes in the spermatocyte and spermatid population in the postpartum rat testis between 18 and 60 days and in pubertal rat testes cultured for 20 days. The appearance of spermatid-specific markers in the rat testis was coincident with the appearance of haploid germ cells. These results must be interpreted with caution, however, as evidence for spermatid development without the completion of meiosis has been presented for the mouse [29] and Drosophila [30]. Germ cell development in the present culture system has not yet been assessed for cell-specific morphological markers other than cell size.
Little change in distribution of cellular DNA content was observed in neonatal bull testis cells cultured for 10 wk (Fig. 4) when PRM-2 was first detected. However, a peak in cells containing haploid DNA content was apparent following 14 wk of culture, suggesting that the number of spermatids present after 10 wk of culture was adequate to detect PRM-2/TP-1 expression by RT-PCR and Northern blotting but too low to be resolved by flow cytometric analysis. This observation is consistent with the low number of cells in culture corresponding in size to round spermatids (7–10 µm, Fig. 1D) and the relatively weak signal detected on Northern blots (Fig. 2, B and C). A cell peak with 4C DNA content that would provide evidence for spermatocytes in meiotic prophase is not observed. This would lead to the conclusion that meiosis is not occurring if observed only for cultured cells. However, absence of a 4C DNA peak appears to be due to dissolution of cells following permeabilization and dissociation with detergent, as the peak is absent from treated but not untreated adult testis cells.
Under the present culture conditions, physiological constraints to the initiation of spermatogenesis at puberty somehow were circumvented. The timing of presumptive spermatid appearance in culture was delayed compared to the progression and duration of bovine spermatogenesis in vivo [31, 32] but highly accelerated relative to the onset of puberty as sperm are not formed in vivo until around 6 mo of age [33]. Presence of testosterone and other medium components increased permeability to these components, and the intercellular associations that developed due to in vitro conditions were apparently adequate to stimulate Sertoli cell function and consequently germ cell proliferation and differentiation.
The delayed detection of haploid cells by flow cytometry compared to the initial expression of spermatid-specific mRNAs suggests that improving culture conditions or longer-term culture of neonatal testis cells may be required to increase spermatid production and initiate spermiogenesis. Additionally, initiating cultures with seminiferous tubule cells from peripuberal or adult testes using the present system of aggregation and encapsulation could enhance the appearance of and postmeiotic changes to spermatids. However, identification of appropriate probes for evaluating the progression of spermatogenesis in vitro will be required when starting with sexually mature animals to confirm that advanced germ cells did not arise simply by limited differentiation of precursor cells.
Differentiation of frozen-thawed testis cells following aggregation and encapsulation was similar to that observed for fresh, unfrozen cells as demonstrated previously following spermatogonial transplantation [34]. General appearance of cell cultures from frozen-thawed preparations and expression of stage-specific markers during culture indicated that both germ cells and Sertoli cells survived the relatively simple freezing and thawing procedures and were competent to divide and differentiate. Developmental competence of cryopreserved germ cells has important practical ramifications for the application of IVS technology.
While recapitulating spermatogenesis in vitro will greatly facilitate mechanistic studies of the in vivo process, the primary impetus for developing an effective IVS procedure has been to generate developmentally competent haploid spermatids or sperm to be used in conjunction with ICSI or ROSI procedures in treating selected causes of male infertility [4–6]. The present study demonstrates that dissociation, reaggregation, and encapsulation of Sertoli cells and germ cells can improve long-term culture conditions such that germ cell differentiation can be achieved when starting with an infantile testis. These conditions could not only enhance current approaches to treating male factor infertility but may provide the option of preserving testicular tissue for future use from young male patients anticipating the loss of gonadal function due to chemotherapy or other treatments.
Applications of procedures presented here can also be extended to genetic modification of the germ line of domestic and laboratory animals and conservation efforts for threatened and endangered species where male fertility is an issue. Further refinement of culture conditions and characterization of Sertoli and germ cells produced in vitro are imperative to determine whether presumptive spermatids are produced by normal progression through primary spermatocytes and meiosis or an abberent differentiation during spermatocytogenesis
ACKNOWLEDGMENTS
We thank Dr. Rodman Getchell for providing flow cytometric analysis of testis cells and Dr. Patricia Johnson and Seung-Jun Yoo for excellent technical support.
FOOTNOTES
First decision: 5 January 2001.
1 This research was supported by Genex Cooperative, Inc./CRL, Shawano, WI, and the New York State Center for Advanced Technology, Cornell University, Ithaca, NY. 
2 Correspondence: John E. Parks, Department of Animal Science, Cornell
University, 201 Morrison Hall, Ithaca, NY 14853. FAX: 607 255 9829;
jep5{at}cornell.edu
Accepted: April 23, 2001.
Received: November 29, 2000.
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