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c INRA PRMD, 37380 Nouzilly, France
d INSERM-INRA U 418, Hôpital Debrousse, 69322 Lyon Cedex 05, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The differentiation of pachytene spermatocytes into round spermatids results in a change in the ploidy of the cells (4C 
1C) and is associated with modifications in the expression of many genes in the germ cells [17, 18]. Among them, phosphoprotein p19 (p19) and the testis-specific histone TH2B are mainly expressed in pachytene spermatocytes [13, 19, 20], whereas transition proteins TP1 and TP2 appear specific to haploid cells [13, 21, 22]. Moreover, in the rat [23, 24], as in many other species, there are important developmental changes in the spermatogenic process during sexual maturation leading to the sequential occurrence of the different categories of germ cells. For instance, in rats, primary spermatocytes are first apparent on Day 15 postpartum while round spermatids are first apparent on Days 25–26.
In the present study, we determined first the relative changes in the mRNA levels of p19, TH2B, TP1, and TP2 in the testis of young rats between 18 and 60 days postpartum and related those results to the sequential appearance of the different types of spermatogenic cells during this period. Our other aim was to reproduce the changes observed in vivo in a culture system using whole cell population of seminiferous tubules from 23- to 25-day-old rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley or Wistar rats of different ages were used in these experiments. Animals were killed by decapitation, and their testes were quickly removed; the testes were either immersed in solution D [25], fixed in Bouin's fixative, or collected in Ham's F-12/Dulbecco's Modified Eagle's medium (F12/DMEM, 1:1) (Gibco BRL Life Technologies, Cergy-Pontoise, France). In some experiments, rats were injected with 50 mg/kg 5-bromo-2'-deoxyuridine (BrdU; Sigma, La Verpillière, France) 9 days before they were killed, to label testicular cells undergoing DNA synthesis.
Preparation and Coculture of Somatic and Germinal Cells from Seminiferous Tubule Segments
The tunica albuginea of testes was removed and the tissues were digested at 32°C in F12/DMEM (1:1) containing 0.9 mg/ml collagenase (Serva; Boehringer Ingelheim Bioproducts, Gagny, France), 2 µg/ml lima bean trypsin inhibitor, and 10 µg/ml DNase (both from Sigma) for 10 min under gentle agitation. Seminiferous tubules were harvested by low-speed centrifugation, washed twice with F12/DMEM mixture without enzymes, and then cut into small fragments with two lancets. Tubular fragments were digested for 5–10 min as above, collected and washed, and then gently resuspended in culture medium, avoiding, as far as possible, disruption of the interactions between Sertoli cells and spermatogenic cells. The culture medium consisted of 15 mM Hepes-buffered F12/DMEM supplemented with antibiotics, 1.2 g/L NaHCO3, 10 µg/ml insulin, 10 µg/ml transferrin, 10-4 M vitamin C, 10 µg/ml vitamin E, 10-7 M testosterone, 3.3 x 10-7 M retinoic acid, 3.3 x 10-7 M retinol, 10-3 M pyruvate (all from Sigma), and 50 ng/ml NIH (Bethesda, MD) porcine FSH (lot #AFP 5600; obtained through NIDDK, Rockville, MD).
Cell samples were seeded (Day 0) at about 106 cells/cm2 in bicameral chambers (Falcon, Elvetec, Vénissieux, France) in culture medium supplemented as above and containing 0.2% fetal calf serum. Incubation was carried out at 32°C in a water-saturated atmosphere of 95% air:5% CO2. The medium was removed 36 h later and replaced by serum-free culture medium. The medium in the basal compartment was then changed every other day.
At selected days of culture, cells were detached from culture dishes by trypsinization. An aliquot of the cell suspension was used to determine the number of cells and to assess cell viability by trypan blue exclusion. Another aliquot was deposited on a glass slide, air dried, and fixed in absolute ethanol/acetic acid (3:1 v:v); the cells were then subjected to Feulgen staining (see below). Cells from other wells were used for RNA extraction (see below).
RNA Extraction and Analysis
Total RNA from whole testis or from cultured cells was extracted by the method of Chomczynski and Sacchi [25], and sequences corresponding to p19 and TP1 mRNAs on the one hand and to TH2B and TP2 on the other hand were coamplified by reverse transcription-polymerase chain reaction (RT-PCR) in the presence of [
-33P]dATP (ICN, Orsay, France or Isotopchim, Ganagobie, France) as follows.
First-strand cDNA synthesis. Reactions were carried out using 1 µg of total RNA in a total volume of 20 µl containing 200 U Moloney murine leukemia virus reverse transcriptase (Gibco BRL) in the reaction mixture 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM of each dNTP, and 20 U RNAsin (both from Promega, Charbonnières, France) and the specific primers for either p19 and TP1 or TH2B and TP2 mRNAs (25 pmol each; Eurogentec, Nantes, France). Initially, the total RNA was denatured at 70°C for 10 min and chilled on ice. First-strand cDNAs were obtained after 1 h at 37°C. The samples were then treated at 100°C for 5 min to terminate the RT.
PCR.
Coamplification reactions were carried out on 50 ng reverse-transcribed RNAs in a final volume of 30 µl containing 0.3 U Goldstar polymerase (Eurogentec), 75 mM Tris-HCl (pH 9), 20 mM (NH4)2SO4, 0.01% Tween 20, 1 mM MgCl2, 2 µM dNTPs, 1 µCi [-33P]dATP (3000 Ci/mmol), and 25 pmol each of the 5' and 3' sequence-specific primers for p19 and TP1 or TH2B and TP2. The samples were overlaid with mineral oil and denatured at 94°C for 3 min. Amplification was performed in sequential cycles at 94°C for 1 min, 59°C (for p19-TP1) or 57°C (for TH2B-TP2) for 1 min, and 72°C for 1 min. After the last cycle, all samples were incubated for an additional 10 min at 72°C. The exponential phase of the PCR was determined between 12 and 22 cycles. The absence of contaminants was routinely checked by RT-PCR assays of negative control samples in which the RNA samples were replaced with sterile water or reverse transcriptase was not added.
Oligonucleotides used for RT and amplification. Oligonucleotides used in the RT reactions were chosen in the 3' end of the mRNAs and were specific to each mRNA. Information about these primers is given in Table 1. All oligonucleotides used in the PCR contained 52% G-C, to avoid differential yields of the amplified products, and had calculated melting temperatures of 63°C or above as determined by TM OLIGO (Citi 2, Paris, France). They were selected in order to generate products of sizes different enough to be easily separated by electrophoresis on agarose gels. The sequence of these primers, their location on the mRNA, and the size of the expected amplified products obtained with these primers are given in Table 2.
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Analysis of amplified products. Aliquots of 10 µl of each PCR sample were electrophoresed in parallel with size markers on 3% Metaphor agarose (FMC, Tebu, France) gels. The gels were stained with 0.5 µg/ml ethidium bromide in Tris-borate EDTA buffer; the bands were cut from the gels under UV illumination and then melted in distilled water at 100°C. Scintillation counting was performed in 3 ml of Emulsifier Scintillator Plus (Packard, Rungis, France). These results were plotted on a semi-log scale against the number of cycles of PCR. The ratios of p19- to TP1- and TH2B- to TP2-related PCR products were calculated in the zone where the curves for each coamplified mRNA were parallel straight lines.
The values of the ratios obtained were identical at least for amplification of cDNAs synthesized from 25 to 100 ng of total RNA. The interassay coefficient of variation of the method was 11.7% under the conditions of the present studies, i.e., when different PCRs were performed from the same batch of reverse-transcribed RNA. The coefficient of variation between the different wells of the PCR apparatus used in the present studies (DNA thermal cycler 480, Perkin Elmer [Saint-Quentin-en-Yvelines, France]; or crocodile II, Appligen [Illkirch, France]) was less than 15%.
In order to assess the specificity of the method, the PCR products were sequenced with an automated DNA sequencer (ABI prism 377; Perkin Elmer) and a dye terminator kit (Perkin Elmer). The percentages of homology with the expected sequence were 94% for p19, 95% for TP1, 98% for TH2B, and 96% for TP2.
Cytological Methods
Testis imprints. A freshly dissected testis was cut in half, and an exposed surface was allowed gently to touch a chemically clean, dust-free glass microscope slide. From a cut surface, 8–10 such imprints were made on each of two slides.
Feulgen staining. The cells from testis imprints or those detached from culture wells were stained by the Feulgen reaction method as described previously [26], and their ploidy was determined by image analysis using the Samba 2005 Image Analysis System (Alcatel TITN, Meylan, France). On every day studied, 500 to 1000 cells were counted.
Cytochemical studies on histological sections. Testes were fixed in Bouin's fluid for 12–24 h and then embedded in paraffin; 2- or 8-µm thin sections of testes were deparaffinized and rehydrated. Then they were stained by the periodic acid-Schiff reaction or incubated with a monoclonal mouse anti-BrdU antiserum (Dako, Trappes, France) as described elsewhere [13].
Immunocytochemical studies on cultured cells. Cells were rinsed with PBS and fixed with Bouin's fixative for 20 min at room temperature. After five washes with PBS, two immunocytochemical reactions were successively performed. First, the cells were incubated with the mouse monoclonal antibody MN7, which recognizes a 90-kDa protein present in the acrosome from step 2 to 18 (a kind gift of Professor K. Toshimori [27]); second, the cells were incubated with an anti-BrdU antibody (see above) as described in detail elsewhere [13].
Electron microscopy. Cultured cells were fixed in bicameral chambers for 1 h at room temperature and then during at least 24 h at 4°C. The fixative solution contained 4% glutaraldehyde in 0.1 M buffered sodium cacodylate (pH 7.4). Postfixation was performed with 2% OsO4 and 1.5% ferricyanide in 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature for 1.5 h. Fixed cells were mechanically scraped from the support, then dehydrated by three successive 5-min immersions in gradual ethanol concentrations (70%, 90%, 100%), and subsequently submitted to progressive impregnations in epon resin by three successive 1.5-h incubations in epon-epoxypropane (1:1; 1:2; 2:1, v:v) and pure epon, overnight at 4°C. Polymerization was done at 60°C for 48 h. Observations of ultrathin sections were performed with a Philips CM10 electron microscope at 80 kV.
Analysis of the Data
The numerical data are presented with their SEM. Each in vitro experiment was repeated at least three times, and selected data are presented herein. Data shown in Figures 1 and 4 were examined by one-way ANOVA followed by the Newman-Keuls test. Data shown in Figure 3, a–c, were examined by one-way ANOVA. Then the significance of the evolution during the culture of the p19:TP1 and TH2B:TP2 ratios, or of the amount of radioactivity incorporated into each mRNA-related PCR product, was assessed by testing the significance of the linearity of the regression line obtained by relating the value of the ratio or the amount of radioactivity to the day of the culture [28]. Paired Student's t-test was used in all the other cases. When required, values were logarithmically transformed before testing.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the second set of experiments, we looked for the changes in "pachytene-specific" (i.e., p19 and TH2B) and "spermatid-specific" (TP1 and TP2) mRNAs in testes of Sprague-Dawley rats between Days 18 and 60 of postnatal life. The quantitative measurement of a specific mRNA is rather difficult in such a heterogeneous organ as the testis, in which the proportions of the various populations of somatic and germ cells vary with its development. Hence, we chose to monitor the changes in the value of the ratio between one mRNA specific to pachytene spermatocytes and one mRNA specific to round spermatids. Therefore, p19 and TP1 mRNAs on the one hand and TH2B and TP2 mRNAs on the other hand were coamplified by RT-PCR (see Materials and Methods) to obtain the values of the p19:TP1 and TH2B:TP2 ratios at selected days of life (Fig. 1). The mean value of the p19:TP1 ratio was about 2 on Days 18 and 20 (1.90 ± 0.13); it decreased steadily until Day 40 and remained at this low level until Day 60 (0.17 ± 0.02, p < 0.05 vs. Days 18–20). As for the TH2B:TP2 ratio, a value could not be calculated before Day 32, since no amplification of TP2 mRNA was obtained under our experimental conditions. However, this ratio decreased dramatically between Day 32 (9.4 ± 1.6) and Day 40 (1.0 ± 0.1) and remained low until Day 60 (p < 0.05 vs. Day 32).
Next, our aim was to set up a culture system allowing us to reproduce, at least in part, the changes observed in vivo. For that purpose, small tubular segments from 23- to 25-day-old Wistar rats testes were seeded in bicameral chambers and cultured in a chemically defined medium (see Materials and Methods).
At this age, 59% of tubule sections were at stages IV–VIII and 32% were at stages XII–XIV (not shown). In addition, rats were injected with BrdU 9 days before they were killed in order to obtain labeled pachytene spermatocytes, mainly of stages IV–VI, and to monitor the fate of these in vitro.
During the first days of culture, cells migrated away from the tubules and spread out on the insert surface so that at the end of the first week, the shape of the tubules had disappeared. Germinal cells were in close association with Sertoli cells or placed on their surface, either alone or most often in clusters (data not shown). When tubule segments were seeded at about 1 x 106 cells per insert, the total number of cells decreased quickly during the first week of culture and then remained roughly stable (4 x 105 cells per insert) until the end of the experiment. The proportion of viable cells, as assessed by trypan blue exclusion, remained close to 70% throughout the culture period (Fig. 2). The p19:TP1 and TH2B:TP2 ratios (Fig. 3a) decreased 4-fold (p < 0.05) and 9-fold (p < 0.001), respectively, between Day 0 and Day 9 of culture, down to values of 1.31 ± 0.11 for p19:TP1 and 1.85 ± 0.18 for TH2B:TP2. Then both ratios remained low until the end of the experiment. In order to determine which term(s) of the ratios was (were) responsible for the changes observed during the culture, the amount of radioactivity incorporated in each PCR product within the exponential phase of the reaction, at every day of culture studied, was expressed as a percentage of the radioactivity incorporated in the corresponding product on Day 0 of the experiment [13] (Fig 3, b and c). An almost linear decrease (p < 0.001) was observed between Day 0 and Day 21 for TH2B mRNA-related PCR products. Moreover, at the end of the culture, the amount of radioactivity in the p19 band was 30 ± 3% of that on Day 0 (p < 0.05); it was 3 ± 1% for TH2B (p < 0.02). By contrast, for both TP1 and TP2 mRNAs, the amount of radioactivity incorporated increased progressively (both p < 0.02) to become 2- to 4-fold higher on Days 9–16 than on Day 0; thereafter it decreased until the end of the culture (p < 0.001 and p < 0.02, respectively) to 90 ± 8% and 40 ± 1% of the values on Day 0 for TP1 and TP2, respectively.
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Ploidy analysis of the cells (Fig. 4) showed that during the first 10 days of culture there was a rather parallel decrease in the 2C and 4C populations (both p < 0.05); thereafter, there was a dramatic decrease in the number of 4C cells (p < 0.05), while the number of 2C cells remained fairly constant. Very importantly, whereas no 1C cell could be observed on Days 0 and 1, a 1C population was present in the culture from Day 3 onward; it increased between Day 3 and Day 9 (p < 0.01) and then remained unchanged between Day 9 and Day 21.
These latter results were confirmed by cytological analysis of the culture (Fig. 5). On Day 1, both BrdU-labeled and nonlabeled pachytene spermatocytes were present, but no spermatid was observed. On Day 5, the most advanced BrdU-labeled germ cells were secondary spermatocytes; rare round spermatids were observed that did not react with the anti-BrdU antibody. However, on Day 7, both BrdU-labeled secondary spermatocytes and labeled round spermatids were present in the culture, the latter being most often in quadruplets. Fewer pachytene spermatocytes were observed on Day 12 than before, whereas many labeled secondary spermatocytes and quadruplets of labeled spermatids were present. At the end of the culture, labeled primary and secondary spermatocytes were rare, but quadruplets of BrdU-labeled spermatids, not reacting with the MN7 antibody, were still present; also, unlabeled secondary spermatocytes in a rather high number were observed (a more quantitative cytological study has been published elsewhere [29]). The ultrastructural aspect of a pachytene spermatocyte and of two secondary spermatocytes on Day 2 of culture, and of a young round spermatid on Day 7 of culture, is shown in Figure 5, e and f, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The quantitative measurement of several mRNA species in such heterogeneous samples as testes from pubertal rats of different ages, or cocultures of both somatic and germ cells from seminiferous tubules, is rather difficult. It may require large amounts of cells and/or sophisticated and/or time-consuming methodologies [30–32]. Indeed, the proportions of the various populations of both somatic and germinal cells change in the developing rat testis, and the rate of survival and differentiation of cultured somatic and germinal cells may vary throughout the culture period. However, in an attempt to validate the in vitro culture system described herein, it was necessary to be able to compare gene expression by cultured germinal cells to that of those cells in vivo. Hence we chose to monitor the changes in the value of the ratios p19:TP1 and TH2B:TP2 mRNA-related PCR products and not to perform an absolute quantification of these mRNAs. In that way, "contaminating" RNA from somatic testicular cells or from other types of germinal cells not expressing these genes (spermatogonia) should not interfere in the assay. It may be noted, however, that both the p19:TP1 and TH2B:TP2 ratios were somewhat lower in extracts of whole testes of pubertal rats than on Day 0 of culture of seminiferous tubule segments from rats of corresponding age. This is most likely explained by the fact that the time course of evolution of these ratios in vivo was followed using Sprague-Dawley rats, whereas all in vitro experiments used Wistar rats. Indeed, we have observed in several instances that for our colonies of rats, Sprague-Dawley animals exhibit lower p19:TP1 and TH2B:TP2 ratios, at the same age, than Wistar rats (unpublished results). Another advantage of such a method is its rather high sensitivity for detecting a small number of round spermatids. Indeed, since during the meiotic process the expression of pachytene spermatocyte-specific genes decreases whereas the expression of round spermatid-specific genes increases, both the numerator and the denominator of the ratios change in an anti-parallel way when meiosis is progressing.
The decrease in the total number of cells per culture well during the first week of the experiments could be explained by a rather similar loss of both somatic and germinal cells. Assuming that about 60% of the seeded 2C cells were somatic cells [24], the changes in the number of the 2C on the one hand and the 1C plus 4C cells on the other hand, together with the number of cells lost during that period, fit with such a hypothesis and with results from others [9]; nevertheless, the remaining cells exhibited a rather good level of cell viability throughout the 3-wk period of culture.
The decrease in the values of the p19:TP1 and TH2B:TP2 ratios could be related to the appearance of the 1C cell population, which happened after several days of culture, and/or to an enhanced expression of TP1 and TP2 by pachytene spermatocytes (see below). However, a 3- to 4-fold decrease in the value of the p19:TP1 ratio was observed within 9 days in vitro, which fits with the amplitude of the decrease of this ratio that occurred in whole testes of rats between 25 and 34 days of life (i.e., at the time of appearance of round spermatids in vivo). Moreover, it should be noted that the importance of the 1C population became maximal on Day 9 of culture, whereas maximal values for TP1 and TP2 mRNA-related PCR products were observed later. This fits with the fact that TP1 [33] and TP2 (unpublished results) gene expression is high only from steps 6–7 of spermiogenesis, i.e., 4–5 days after completion of meiosis [2]. Very importantly, from Day 9 of culture onward, germinal cells from seminiferous tubules contained relative amounts of the TP1- and TP2-related mRNAs that were 2- to 4-fold higher than on Day 0 of the experiment, whereas the 4C cell population decreased dramatically throughout the culture period. Taken together, these results suggest strongly that TP1 and TP2 mRNAs measured beyond 9 days of culture were mostly mRNAs newly synthesized by round spermatids that had differentiated recently in vitro. That the increase in the 1C cell population was rather small was not unexpected, since the conversion of primary spermatocytes to spermatids in vivo is almost 10-fold lower in 30-day-old rats than in adult animals [24]. The decrease in the levels of TP1 and TP2 mRNAs at the very end of the culture might indicate that the present culture conditions could not complete the requirement for spermiogenesis. Indeed, this decrease was apparently not related to important changes either in cell number or in cell viability.
The fact that TP2 mRNA could not be detected in whole testes of Sprague-Dawley rats before 32 days of life, but was amplified from tubule segments of 23- to 25-day-old Wistar rats, might be explained by dilution of this germ cell-specific RNA, by RNAs from the intertubular connective tissue, and/or by some difference between these two strains of animals (see above). Moreover, since both TP1 and TP2 mRNAs could be amplified in RNA extracts of seminiferous tubules devoid of round spermatids, it is very likely that TP1 and TP2 genes are already transcribed, but at a very low level, before completion of meiosis.
The occurrence of the two meiotic divisions under the present conditions of culture was further substantiated by labeling pachytene spermatocytes in vivo with BrdU and then monitoring their fate in vitro. In that way, BrdU-labeled secondary spermatocytes were observed on Day 5 of culture, whereas BrdU-labeled round spermatids were present from Day 7 onward. Since the labeled pachytene spermatocytes seeded were mainly from stages IV to VI, it appears that the duration of this step of the meiotic process was rather similar in vitro and in vivo [2, 23, 34].
To our knowledge, this is the first description of a culture system that allows, over a 3-wk period, the functional differentiation of pachytene spermatocytes from stages IV to VI into round spermatids. The viability of these spermatids was assessed not only by morphological methods but also by monitoring their ability to transcribe spermatid-specific genes until the end of the culture period. Hence, the present culture system should be helpful in establishing the role of paracrine factors that are only believed to be involved in germ cell development (reviews in [4, 5]), either by adding them to the culture medium or by preventing their expression or blocking their action in vitro with use of antisense oligonucleotides or specific antibodies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Ph. Durand, INSERM-INRA U418, Hôpital Debrousse, 29 Rue Soeur Bouvier, 69322 LYON Cedex 05, France. FAX: (33) 4.78.25.61.68; durand{at}lyon151.inserm.fr
Accepted: March 25, 1998.
Received: October 31, 1997.
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