DEVELOPMENTAL

154,160-168 (1992)

BIOLOGY

Differential Transcription Spermatogenesis

of Pgk Genes during in the Mouse

JOHN R. MCCARREY,**~ WERNER M. BERG,? STEVE J. PARAGIOUDAKIS,~ PETER L. ZHANG,* DONALD D. DILWORTH,* BRENT L. ARNOLD,$ AND JOHN J. ROSSI+ *Department

of Genetics, Southwest Foundation for Biomedical Research, P.O. Box 28147, San Antonio, Texas 78248; ~Diwision of Reproductive The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 212O5; and $Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, Calgornia 91010

Biology,

Accepted June 10, 1992 We have analyzed the occurrence of transcripts produced from the ubiquitously expressed, X-linked Pgk-1 gene and the testis-specific, autosomal Pgk-2 gene during spermatogenesis in the mouse. We found that tissue specificity, developmental specificity, and cell-type specificity of these mRNAs parallel that previously reported for the two protein isozymes of phosphoglycerate kinase (PGK) encoded by these two genes. This indicates that primary regulation of differential expression of the Pgk genes during spermatogenesis is exerted at the transcriptional level. We first detected Pgk-2 mRNA in preleptotene spermatocytes, indicating that transcription of Pgk-2 is initiated coincident with the onset of meiosis in male germ cells, and then continues to increase in later spermatocytes and postmeiotic round spermatids. This expression initiates prior to an initial decline in Pgk-1 transcript levels observed in pachytene spermatocytes, which apparently follows inactivation of the single X chromosome in spermatogenic cells. However, unlike cessation of Pgk-1 transcription from the inactivated X chromosome in female somatic cells, we show that inactivation of the Pgk-1 locus in spermatogenic cells is not followed by methylation of a key CpG dinucleotide in the promoter region. These results support the idea that specific expression of the Pgk-2 gene in meiotic and postmeiotic spermatogenic cells has evolved to compensate for reduced levels of Pgk-1 gene product caused by transient X-chromosome inactivation in these cells. They further suggest that reinitiation of transcription of the paternal Pgk-1 allele shortly after fertilization is o 1992 facilitated by constitutive hypomethylation in the promoter region of this gene throughout spermatogenesis. Academic

Press, Inc.

INTRODUCTION

Two isozymes of the glycolytic enzyme phosphoglycerate kinase (PGK), each encoded by a separate gene, are differentially expressed during spermatogenesis in the mouse (VandeBerg et ab, 19’7’7;Kramer and Erickson, 1981). The PGK-A isozyme is encoded by the Xlinked &k-l gene and is expressed in premeiotic spermatogenic cells as well as in all somatic cells and in oogenic cells (reviewed by VandeBerg, 1985). Pgk-1 is regulated as a “housekeeping” gene and is transcribed constitutively in all cells harboring one or more transcriptionally active X chromosome. Spermatocytes and spermatids represent the only metabolically active cell types in either sex believed to lack at least one active X chromosome (Lifschytz and Lindsley, 1972). Thus ongoing transcription of the X-linked f’gk-1 gene is not expected in these cells. It is in these same cells, and only in these cells, that the PGK-B isozyme has been detected (VandeBerg et ak, 1977; Kramer and Erickson, 1981). The PGK-B isozyme is encoded by the autosomal Pgk-2 gene, which appar1 To whom 0012-1606192 Copyright All rights

correspondence

should

$5.00

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

be addressed. 160

ently arose as a functional retroposon by reverse transcriptase-mediated processing of a transcript from the Pgk-1 gene (McCarrey and Thomas, 1987; Boer et aL, 1987). Enzymological studies indicate the PGK-A and PGK-B isozymes are structurally and functionally equivalent (Pegoraro and Lee, 1978; Lee et aL, 1980). Studies of tissue distribution of the PGK-B isozyme in marsupials and placental mammals indicate the Pgk-2 gene has evolved an increasingly tissue-specific mode of expression during mammalian evolution (VandeBerg, 1985). Thus it has been proposed that specific expression of the Pgk-2 gene in meiotic and postmeiotic spermatogenie cells has evolved in eutherian mammals to compensate for reduced expression of the Pgk-1 gene in these cells (McCarrey and Thomas, 1987; McCarrey, 1990). Evidence has been reported for both transcriptional (Erickson et al., 1980; Robinson et al., 1989; Gebara and McCarrey, 1992) and post-transcriptional (Gold et al., 1983) regulation of Pgk-2 expression in spermatogenic cells. However, the extent to which each of these mechanisms contributes to regulation of tissue-, developmental stage-, and/or cell-type specificity of Pgk-2 expression has not been completely described.

MCCARREY

ET AL.

Pgk Transcripts

Studies of somatic cells indicate expression of Pgk-1 is regulated solely at the transcriptional level, primarily as a consequence of X chromosome inactivation (XCI) (Singer-Sam et ak, 1990a). XC1 occurs in XX (female) somatic cells as a two-step process, involving initial cessation of transcription from one of the two X chromosomes, followed by stabilization of the inactive state by DNA methylation (Lock et aZ,, 1987). XC1 also occurs in XY (male) spermatogenic cells (reviewed by Handel, 1987) and is presumed to cause a transcriptional silencing of X-linked genes similar to that produced by XC1 in female somatic cells. However, it remains to be determined if the mechanism of XC1 in spermatogenic cells is similar to that which leads to XC1 in somatic cells. The ability to obtain enriched populations of specific spermatogenic cell types, including premeiotic (mitotic) spermatogonia, meiotic spermatocytes, and postmeiotic spermatids (Romrell et a& 1976; Bellve et a& 1977), facilitates the use of molecular techniques to trace changes in the production of specific gene products and/or factors that regulate gene expression throughout spermatogenesis. We have used this approach in the study reported here to characterize the levels of Pgk-1 and Pgk-2 mRNAs and the status of DNA methylation at a key CpG dinucleotide in the Pgk-1 promoter, during spermatogenesis in the mouse. Our results indicate that regulation at the transcriptional level facilitates expression of the Pgk-2 gene at an appropriate stage during spermatogenesis to compensate for diminished production of PGK from the inactivated Pgk-1 gene. These results further indicate that the mechanism of XC1 in spermatogenic cells is different from that in somatic cells in that DNA methylation does not appear to be involved in this process. MATERIALS

AND

METHODS

Cell Separations Spermatogenic cells from CD-l mice (Charles River) were separated by unit gravity sedimentation in a medium (1100 ml) or small (550 ml) Sta-Put chamber (Johns Scientific) according to the methods of Romrell et al. (1976) and Bellve et al. (1977). Populations of primitive type A spermatogonia (purity 2 85%) and Sertoli cells (purity > 90%) were isolated from testes of 6-dayold male mice; type A and B spermatogonia (purities 2 85%) were isolated from testes of &day-old mice; preleptotene, leptotene/zygotene, and early pachytene spermatocytes (purities > 85%) were isolated from testes of l&day-old mice; and late pachytene spermatocytes, round spermatids, and residual bodies (purities > 90%) were isolated from testes of adult (260 days old) mice. For small-scale cell preparations a special “mini”sized (50 ml) Sta-Put chamber was designed (manufac-

in Spernaatogenesis

161

tured by Johns Scientific). The mini Sta-Put chamber was used to purify spermatogenic cells from a single adult mouse testis or from the testes of 5-20 neonatalprebuberal mice, in preparation for analysis of RNA by the reverse transcriptase-polymerase chain reaction (rtPCR). Cells (106-107) in 2 ml of buffer were loaded onto a 50-ml gradient of 2-4s BSA and allowed to sediment for 2.5 hr. Approximately 100 0.5-ml fractions were then collected in microcentrifuge tubes and analyzed for content of specific spermatogenic cell types on the basis of morphology under phase contrast optics. A single adult mouse testis yielded populations of pachytene spermatocytes (2.6 X 10’ cells, 94% purity) from fractions 26-40, round spermatids (2.0 X lo6 cells, 99% purity) from fractions 58-70, and residual bodies (3.6 x lo6 bodies, 98% purity) from fractions 80-92 suitable for preparation of total RNA for subsequent analysis by rtPCR. Similarly, populations enriched to 385% purity for spermatogonia and spermatocytes were recovered from prepuberal and puberal testes, respectively. Testicular and epididymal spermatozoa were isolated as sonication-resistant cells after 3 X 90-set treatments on setting 4 of a sonicator cell disruptor (Heat SystemsUltrasonics, Inc.) in homogenization buffer [0.9% NaCl, 0.05% (v/v) Triton X-100, and 0.01% (w/v) Na azide). Purities were consistently 290% as determined by light microscopy. Preparation

of RNA

For standard Northern blots or rtPCR analysis, total RNA was prepared from complete (decapsulated) testes or pelleted populations enriched for specific spermatogenie cell types by the method of Chirgwin et aZ. (1979). Tissues or cells were homogenized using a Polytron homogenizer (Brinkman) for 3 X 30 set, and the homogenate was then spun through a cushion of 5.7 M cesium chloride at 35,000 rpm for 18 hr at 18°C in an SW 50.1 ultracentrifuge rotor. Poly(A)+ RNA was fractionated from total RNA using a commercially available oligo dT column (Stratagene) according to manufacturer’s directions. Northern

Blots

For standard Northern blots, 30 pg of total RNA or 10 pg of poly(A)+ RNA were electrophoresed in each lane of a 1% denaturing agarose gel containing 2% formaldehyde. RNAs were then blotted to Zetaprobe nylon membranes (Bio-Rad) according to manufacturer’s directions, probed with S2P-labeled DNA fragments specific to the 3’-untranslated regions of the mouse Pgk-1 or Pgk-2genes (Boer et aL, 1987), and analyzed by autoradiography to X-ray film.

162 A

DEVELOPMENTALBIOLOGY S-LIT

Coding

Sequence

3’.UT

z--

The location of primer and probe oligonucleotides for rtPCR of each Pgk gene transcript and the amplification products they produce are schematically represented. The 23%bp Pgk-1 amplification product initiates in the 5’-untranslated region where there is no significant homology with the Pgk-2 gene (McCarrey, 1990) and terminates in the coding sequence. The 300-bp Pgk-2 amplification product initiates in the coding sequence and terminates in the 3’-untranslated region where there is no homology with the Pgk-1 gene (McCarrey, 1990). In each case a third probe oligonucleotide complimentary to a sequence in the interior of the amplified product, but in a nonhomologous region between Pgk-1 and Pgk-2, was used to detect and quantify each amplification product by Southern hybridization. (Drawings not to scale.) (B) The sequences are shown of the oligonucleotides used for primers and probes in rtPCR experiments to detect Pgk gene transcripts. All oligonucleotides were 25 nt in length and are listed in a 5’-3’ orientation.

Reverse Transcriptase-Polymerase

Chain Reaction

rtPCR was used to confirm the initial appearance of

Pgk-2 mRNA and to estimate relative levels of Pgk-1 transcripts in RNA samples from developmentally staged testes or populations of specific spermatogenic cell types. Contaminating genomic DNA was eliminated from each 500-ng sample of RNA as described (Dilworth and McCarrey, 1992). Samples were then subjected to rtPCR amplification either according to the method of Murakawa et al. (1988) or using a kit from PerkinElmer. Synthetic oligonucleotide primers [each 25 nt in length] (Fig. 1) delimiting specific fragments of the Pgk1 and Pgk-2 transcripts were used to initiate amplification. Each amplified Pgk fragment either initiated or terminated in a nonhomologous untranslated region of Pgk-1 or Pgk-2 (Fig. l), such that amplification of only the desired Pgk transcript was ensured. Total RNA was subjected to one round of cDNA synthesis with reverse transcriptase, using either the specific oligonucleotides or random hexamers as primers. This cDNA was then subjected to multiple (20-35) rounds of PCR with the Tag polymerase and the specific oligonucleotide primers. The amplification products were electrophoresed through agarose (LO-1.8%), visualized by ethidium bromide staining and uv illumination, and then

VOLUME 154,1992

vacuum blotted to Zetaprobe membrane and hybridized with another 32P-labeled 25-nt oligonucleotide probe homologous to a region in the middle of each expected amplification product (Fig. l), as described by Murakawa et al. (1988). “No template” and “no reverse transcriptase” controls were run for each experiment to rule out template contamination in reaction components or DNA contamination in RNA samples, respectively, as a source of positive amplification signals. To ensure that representations of relative levels of Pgk-1 transcripts were based on amplification results obtained from a linear range of PCR, an AMBIS radioanalytic imaging system was used to measure hybridization signals on blots of aliquots removed from each rtPCR reaction after various numbers of cycles of PCR (22, 25, 28, or 31). These results (data not shown) showed that amplification was consistently exponential at 22 and 25 cycles of PCR, but then approached saturation after 28 cycles. Thus we chose to use data from 25 cycles of PCR for the Pgk-1 results presented here. To generate a controlled estimate of the relative levels of Pgk-1 mRNA in each population of spermatogenic cells, the products of each 20-~1 reverse transcriptase reaction were divided into two lo-p1 aliquots. One 10-J aliquot was then subjected to 25 cycles of PCR to amplify a 453-bp fragment of the mouse P-actin cDNA using 22-mer primers (upstream primer: 5’-GCGGACTGTTACTGAGCTGCGT-3’;downstreamprimer:5’-GAAGCAATGCTGTCACCTTCCC-3’; based on cDNA sequence from Tokunaga et ah, 1986). The other lo-p1 aliquot was subjected to 25 cycles of PCR to amplify a 237-bp fragment of the mouse Pgk-1 cDNA using the primers described in Fig. 1. Fifteen microliters of the 100 ~1of actin PCR reaction product plus 15 ~1 of the 100 ~1 of Pgk-1 PCR reaction product from each cell type were then run together in a single lane on a 1.8% agarose gel and blotted to Zetaprobe (Bio-Rad) as described (Murakawa et aZ., 1988). This blot was then hybridized with [T-~~P]ATP end-labeled oligonucleotides complimentary to a region in the middle of each amplified product (P-actin probe: 5’-TGCTCCAACCAACTGCTGTCGC-3’; Pgk-1 probe sequence described in Fig. 1) as described (Murakawa et aL, 1988). Hybridization signals were then quantified using the AMBIS system.

Preparation

of DNA

For analysis of DNA methylation, genomic DNA was isolated from primitive type A spermatogonia, pachytene spermatocytes, round spermatids, testicular spermatozoa, caput epididymal spermatozoa, and caudal epididymal spermatozoa as described (Ausubel et al., 1987), with the addition of 0.001% (v/v) P,-mercaptoethanol in the spermatozoa DNA preparations. DNAs were puri-

163

Pgk Transcripts in Spermatcgenesis

of the PGK-B isozyme is regulated at the transcriptional level, we performed a Northern blot analysis on RNA from a variety of tissues from both male and female mice (Fig. 2). A Pgk-2 transcript was detected only in testicular tissue, indicating apparent testis-specific transcription of this gene. Onset and Specificity

: 1

‘.;

/

."',

-1.6

FIG. 2. Tissue specificity of Pgk-2 transcription. Total RNA isolated from the adult mouse tissues noted above each lane was subjected to Northern blot analysis and hybridized with a probe fragment representing the 3’-untranslated region of the mouse Pgk-8 gene. A 1.6-bp transcript was detected in testis, but not in any other tissue tested. M, male; F, female.

tied by phenol-chloroform extraction, ethanol precipitation, and drop dialysis against 0.1X TE [1X TE is 10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. Detection of DNA Methylaticm

by PCR

Analysis of DNA methylation at the CpG dinucleotide in the “H-7” HpaII site of the Pgk-1 promoter was carried out as described by Singer-Sam et al. (1990b). Prior to analysis, genomic DNA was sheared by 10 passages through a 26-guage needle and then digested with XbaI restriction endonuclease to produce appropriate templates for analysis. Twenty two cycles of PCR were then performed on 1 pg XbaI-cut DNA that was also: (1) digested with methylation-sensitive HpaII restriction endonuclease, (2) not further digested, or (3) digested with methylation-insensitive MspI restriction endonuclease. A 25-~1 aliquot of the 100 ~1 of product of each reaction was then electrophoresed in 1.5% agarose and stained with ethidium bromide to visualize amplified fragments.

of Pgk-2 Transcription

We used Northern blot analysis of poly(A)+ RNAs (data not shown) and rtPCR (Fig. 3) to determine the precise age at which Pgk-2 transcription is initiated in the mouse testis. Analysis of testicular RNAs from mice at ages 12-18 days showed that Pgk-2 mRNA is first detectable as early as 12 days, although the signal is relatively faint. A readily apparent signal was detected at 14 days of age and later. This time of onset of expression correlates with the first appearance of spermatocytes as the initial wave of spermatogenesis occurs in these puberal animals (Bellve et aZ., 1977), and suggests that Pgk-2 is expressed specifically in differentiating spermatogenic cells. To test this hypothesis we analyzed RNAs from enriched populations of specific spermatogenie cell types using both Northern blot analysis (Fig. 4) and rtPCR (data not shown). We found no evidence of Pgk-2 mRNA in premeiotic spermatogonia; however, Pgk-2 transcripts were detected in all types of meiotic spermatocytes tested, as well as in postmeiotic round spermatids and in residual cytoplasmic bodies shed from differentiating spermatozoa. Cessation of Pgk-1 Transcription To estimate changes in the relatively low levels of Pgk-1 mRNA in spermatogenic cells, we performed a semiquantitative rtPCR analysis on RNAs from enriched populations of specific spermatogenic cell types (Fig. 5). We compared levels of Pgk-1 mRNA with those of P-actin mRNA, since the latter is encoded by an autosomal gene and is therefore not affected by XCI. We used the values for each mRNA signal from the 6- and

RESULTS

Testis Speci$city

of Pgk-2 Gene Transcription

Testis-specific expression of the Pgk-2 gene has been demonstrated at the protein level by the unique appearance of the PGK-B isozyme in this tissue (VandeBerg et al, 1977). To determine if the testis-specific expression

FIG. 3. Pgk-2 rtPCR: Developmental onset of Pgk-2 transcription in the testis. Total RNA from testes taken from mice at various ages (days of age denoted above each lane) was subjected to rtPCR analysis to detect Pgk-2 mRNA. A 300-bp amplification product was first faintly visible in testicular RNA at 12 days of age and became prominent at 14-18 days of age. (Note: the doublet band is due to separation of single-stranded and double-stranded PCR products.)

164

DEVELOPMENTALBIOLOGY

vOLUME1&&1%?2

Meth&dion

-1.6

FIG. 4. Cell-type specificity of Pgk-2 transcription in the testis. Total RNA isolated from enriched populations of specific spermatogenic cell types was subjected to Northern blot analysis and hybridized with the mouse Pgk-2:3’ probe. No Pgk-2 transcript was detected in premeiotic spermatogonia. However, a 1.6-kb Pgk-2 transcript was detected in all meiotic spermatocyte cell types as well as in postmeiotic round spermatids and residual bodies. Note the increasing intensity of Pgk-2 transcript signal intensity in spermatocytes as meiosis progresses and in postmeiotic round spermatids. Also note the appearance of degraded forms of Pgk-2 RNA (open arrows) in residual bodies in which the Pgk-2 gene is not actively transcribed. 6d gonia, primitive type A spermatogonia; 8d gonia A, type A spermatogonia; 8d gonia B, type B spermatogonia; 18d PreLep, preleptotene spermatocytes; 18d Lep/Zyg, leptotene + zygotene spermatocytes; 18d Path, early pachytenes spermatocytes; Adult Path, late pachytene spermatocytes; Adult R-tid, round spermatids; Adult Res Bod, residual cytoplasmic bodies.

&day Sertoli cell samples to establish a “control somatic cell level” for that mRNA, against which corresponding values from each spermatogenic cell type were normalized (Fig. 5B). Levels of both mRNAs decline during spermatogenesis, since all transcription ceases at approximately step 9 of spermiogenesis (Monesi, 1967). However, to reveal the effects of XCI, which produces cessation of X-linked gene transcription prior to that resulting from subsequent cessation of autosomal gene transcription, we assigned the normalized p-actin figure a value of 100% for each cell type, and then compared the normalized Pgk-1 values to this level (Fig. 50 We found that Pgk-1 transcript levels in primitive type A spermatogonia, type A plus type B spermatogonia, and preleptotene spermatocytes were equivalent to or slightly higher than those in somatic cells, as were levels of p-actin transcript. However, unlike @actin mRNA levels which showed some decrease in late pachytene spermatocytes, but did not drop below 50% of somatic values except in residual bodies, levels of Pgk-1 mRNA showed a slight decrease beginning in leptotene/zygotene spermatocytes and a marked reduction to 50% of somatic values in early pachytene spermatocytes. This decrease continued to less than 20% of somatic values in late pachytene spermatocytes and postmeiotic spermatids.

of Pgk-1

We used PCR amplification of a fragment in the Pgk-1 promoter to determine if the methylation-sensitive restriction endonuclease, HpaII, cleaved at the Pgk-1 H-7 site in DNAs isolated from a variety of premeiotic, meiotic, and postmeiotic spermatogenic cell types and testicular and epididymal spermatozoan cell types. DNAs from male and female liver tissues were run as negative (cleaved fragment from unmethylated, active X chromosome will not be amplified after HpaII cleavage) and positive (uncleaved fragment from methylated, inactive X chromosome will be amplified since methylation inhibits HpII cleavage) controls, respectively. Results are shown in Fig. 6. Following HpoII cleavage (Fig. 6A), no amplified fragment was produced from any of the spermatogenic or spermatozoan DNAs, indicating the H-7 site is unmethylated in all of these cell types, as it is on the active X chromosome in male liver. DNA from female liver did yield a 170-bp amplified fragment, demonstrating the effect of methylation at the H-7 site. In the absence of HpoII cleavage (Fig. 6B), a 170-bp band was produced from the uncleaved template in all DNAs. When these same DNAs were cut with MspI endonuclease (Fig. 6C), which is not sensitive to methylation, none (including female liver DNA) yielded an amplified fragment. The latter result demonstrated that the H-7 site in all of the DNAs could act as a target site for restriction endonuclease cleavage. DISCUSSION

The results demonstrate that differential expression of Pgk-1 and Pgk-2 gene transcripts occurs during spermatogenesis in the mouse in a manner which parallels that previously detected at the protein level for PGK isozymes A and B, respectively (VandeBerg et ah, 1977; Kramer and Erickson, 1981). This indicates that transcriptional regulation of each Pgk gene provides the primary control for differential expression of the Pgk genes during spermatogenesis. The occurrence of posttranscriptional regulation of Pgk-2 gene expression has also been reported (Gold et aL, 1983) and is not discounted by our results. However, it would appear that whereas post-transcriptional regulation may be responsible for some modulation in the developmental stage specificity of expression of the PGK-B isozyme during spermatogenesis, it is transcriptional regulation which determines the tissue and cell type specificity of Pgk-2 gene expression, as well as the cessation of Pgk-1 gene expression during spermatogenesis. Our results show that transcription of the Pgk-2 gene is initiated at the very onset of meiosis. This is in contrast to previous reports suggesting that Pgk-2 transcription is initiated in pachytene spermatocytes (Gold

MC~ARREYETAL. 6dadBdBdP MSSGGLZPPTBM

A

Pgk Transcripts

L E L R R ’

-453 bp -238 bp

B

ActIll

WI

Tv~e

corn

%

net

Somatlc

a

W-1 cDm net

6dS

910 4

1647.5t

8dS 6dG 8dG PL I2 EP LP RT w

1089.2 1467.9 1382.4 1214.2 1348.2 1013 4 600.4 564.1 746

1884.6t 1742.1 1753.6 1916.0 1720.9 936.3 351.2 248.6 22.2

*mean

somatic

acfin

146.7 138.3 121 4 134.6 101.3 60.1 56.4 7.5 value

%

X

Pak-I

Somatic*

%

ActhA

92.4 94.0 92.2

63.0 680 85.0 68.4

502 188 133 ‘1.2

31.3 236 16.0

102.7

500

- 999 8

tmean somatic Pgk-1 value - 1866.1 aAclm cpm net/mean somatic acfin value OPgk-1 cpm net/mean somatic Pgk-1 value “Pgk- 1 % somatic valuelactin % somatic value

C

110

-

Actin

---+--

Pgk-1

mRNA mRNA

[cpm net normalized vs mean somatic

Cell

as % values]

Type

FIG.5. Pgk-1 rtPCR: Cessation of Pgk-1 transcription during spermatogenesis in the testis. (A) Total RNA isolated from cell populations enriched for specific spermatogenic or Sertoli cell types was analyzed by rtPCR to detect Pgk-1 and P-a&in mRNAs as described in the text. After ethidium bromide staining, a 453-bp fl-actin amplification product was detected in all cell types. A 238bp Pgk-1 amplification product was clearly visible in RNAs from Sertoli cells, spermatogonia, and early spermatocytes, but less prevalent in late spermatocytes, round spermatids, or residual bodies. A reverse-positive image of the ethidium bromide-stained agrarose gel is shown. M, 123-bp marker; 6dS, g-day Sertoli cells; 8dS, S-day Sertoli cells; 6dG, primitive type A spermatogonia; 8dG, type A and B spermatogonia; PL, preleptotene spermatocytes; LZ, leptotene plus zygotene spermatocytes; EP, early pachytene spermatoeytes; LP, late pachytene spermatocytes; RT, round spermatids; RB, residual cytoplasmic bodies. (B)

in Spmatogenesis

165

et ah, 1983; Thomas et ab, 1989). Indeed, our results bring into question the previous suggestion that Pgk-2is exemplary of an entire class of genes which initiate transcription in pachytene spermatocytes (Thomas et al, 1989). Rather, it would appear that Pgk-2 is a member of a class of genes, the transcription of which is triggered at or by the onset of meiosis. Other members of this class of genes would likely include the Ldh C, gene (Goldberg, 1990; Millan et ah, 1987), among others (see Hecht, 1992, for review). Thus we would modify the suggestion of Thomas et al. (1989) to predict that there are three (not four) primary classes of genes differentially expressed in spermatogenesis: (1) those which are already expressing or initiate expression in premeiotic spermatogonia (e.g., Pgk-1); (2) those which initiate expression in meiotic spermatocytes (e.g., Pgk-2); and (3) those which initiate expression in postmeiotic spermatids [e.g., protamine 1 (m@) (Kleene et ah, 1985)]. Our rtPCR experiments to detect Pgk-1 transcript levels in spermatogenic cells (Fig. 5) indicate that levels of this X-linked gene transcript initially decrease at about the time of onset of meiosis. These results differ from those reported by Singer-Sam et al. (199Oc), who observed a marked decrease in Pgk-1 transcript levels in type A and B spermatogonia, but are similar to the pattern of Pgk mRNAs determined by Goto et al. (1990) using in situ hybridization on sections of mouse testis. In any case, it is difficult to pinpoint the actual time of XC1 on the basis of reduced levels of an X-linked gene transcript because of the variable introduced by relative stability of RNA transcribed prior to XCI. However, the reduction in Pgk-1 mRNA that we observed during first meiotic prophase correlates with the appearance of cytological indications of XC1 which have been reported previously (reviewed by Handel, 1987). No cytological characteristics of an inactivated X chromosome have been observed in spermatogonia. These results support the suggestions that XC1 is either a prerequisite for (Lifschytz and Lindsley, 1972) or at least a correlate of (Handel, 1987) meiosis in mammalian spermatogenic cells. A Southern blot of the gel shown in A was probed with oligonucleotide probes for each amplification product in separate experiments as described in the text, and the results were analyzed using an AMBIS radioanalytic imaging system. Net counts per minute (cpm) obtained for each spermatogenic cell type were normalized against the mean value for the corresponding gene in the two Sertoli cell samples and expressed as % somatic value. Pgk-1 % somatic values were then norActin). (Note: malized against p-a&in % somatic values (% Pgk-I/% the Pgk-1 oligonucleotide hybridized more efficiently than the actin probe, thus yielding higher cpm values.) (C) Pgk-1 and P-actin rtPCR data from B are plotted to show the relative decrease in levels of Pgk-i mRNA compared to that of P-a&in mRNA during spermatogenesis. Percentage somatic values for each gene were normalized against pactin % somatic values to provide data points in each case.

166

DEVELOPMENTALBIOLOGY

123456789

-170 bp

-170 bp

-170 bp FIG. 6. PCR analysis of methylation at the Pgk-1 H-7 site in spermatogenic cells. Genomic DNAs were prepared from enriched populations of spermatogenic and spermatozoan cell types and analyzed for the presence of methylation at the H-7 site in the promoter region of the Pgk-1 gene as described in the text. Amplification of a 170-bp product indicates an intact template that has not been cleaved at the H-7 site. Lane 1, primitive type A spermatogonia; 2, late pachytene spermatocytes; 3, round spermatids; 4, testicular spermatozoa; 5, caput epididymal spermatozoa; 6, cauda epididymal spermatozoa; 7, adult male liver; 8, adult female liver; 9, 123-bp marker. (A) DNA samples digested with the methylation-sensitive restriction endonuclease, HpaII, yielded an amplification product only in the case of adult female liver (lane 8), which is known to possess an inactive X chromosome with a methylated H-7 site (Singer-Sam et al, 1990b), but not in any of the spermatogenic cell types, indicating a lack of methylation at the H-7 site in these cells. (B) Undigested DNA samples all yielded an amplification product, indicating these DNAs can act as PCR templates if not cleaved at the H-7 site. (C) DNA samples digested with the methylation-insensitive MspI restriction endonuclease failed to yield an amplification product in any case, demonstrating the effect of cleavage at the H-7 site.

Our analysis of methylation at the H-7 HpaII site in the Pgk-1 promoter indicates that, unlike the situation in XX somatic cells, XC1 in spermatogenic cells is neither associated with nor followed by an increase in DNA methylation. The H-7 HpaII site is invariably methylated on the inactive X chromosome and unmethylated on the active X chromosome in somatic cells (SingerSam et aL, 1990b). Although methylation follows the initial inactivation event in somatic cells, it typically occurs within a few days (Lock et al, 1987; Singer-Sam et al, 1990b) after cessation of transcription. Our results suggest that the Pgk-1 promoter region is unmethylated

vOLUME154,1992

in primitive type A spermatogonia prior to any detectable decrease in Pgk-1 transcript level, in spermatocytes as transcript levels begin to decrease, and in round spermatids shortly after transcript levels have decreased. Beyond this, however, our analysis of testicular and epididymal spermatozoan DNAs suggests the Pgk-1 promoter remains unmethylated for at least another 2 weeks as sperm mature, and presumably maintains this state through the time of fertilization and subsequent reactivation of the paternal X chromosome in the early embryo. This represents a notable difference in the process of XC1 in spermatogenic cells compared to that in somatic cells. A similar finding was noted by Venolia et aZ. (1984) who showed that the X-linked Hprt gene appears to remain unmethylated into the postmeiotic stages of spermatogenesis as indicated by the ability of spermatogenie cell DNA to transform Hpr&deficient cells in culture. This does not indicate that the mechanism of initial transcriptional inactivation of Pgk-1 (or other Xlinked loci) is different in spermatogenic and somatic cells. Rather it suggests that the heritable, stabilizing mechanism conferred by DNA methylation is beneficial for terminal maintenance of the inactivated state of the X chromosome in somatic cells, which persists for the lifetime of the individual. However, such a mechanism apparently confers no benefit (and might even be detrimental) to the short-term maintenance of the inactivated state in spermatogenic cells, which appears to last only from the beginning of meiosis until shortly after fertilization. Indeed, the absence of methylation in the Pgk-1 promoter appears to be characteristic of the transient state of XC1 that occurs in germ cells of both sexes, as evidenced by the recent finding of Singer-Sam et al. (1992) that a key HpaII site in the promoter region of the human Pgk-1 gene on the inactive X chromosome is unmethylated in fetal oogonia prior to reactivation of the second X chromosome in fetal oocytes. Recent results indicate that transcription of the paternally derived Pgk-1 allele is initiated as early as the eight-cell stage in the mouse embryo (J. Singer-Sam, personal communication). The absence of methylation in the promoter region of the Pgk-1 allele contributed by the sperm may therefore be an important prerequisite to reinitiation of transcription at this early stage. Such constitutive hypomethylation may also be a general characteristic of CpG island containing housekeeping genes, since a similar lack of methylation has been observed in the Dhfr gene in spermatogenic and spermatozoan cell DNAs (Kafri et ak, 1992). The marked decline we observed in the levels of Pgk-1 mRNA at the onset of meiosis during spermatogenesis suggests these transcripts are relatively labile. Thus transcription of the Pgk-1 gene prior to XC1 during sper-

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ET AL.

Pgk

Transcripts

matogenesis would not appear to be sufficient to provide the stockpile of PGK enzyme required for glycolysis in later spermatogenic cells and in mature spermatozoa after they leave the testis (although this mechanism may be sufficient to provide the required supply of mRNA from certain other X-linked genes which produce more stable transcripts). The expression of the Pgk-2 gene appears to compensate for the lack of or reduction in Pgk-1 mRNA in postmeiotic spermatogenic cells. There is no evidence that the process of XC1 in spermatogenic cells produces any sort of signal to directly initiate transcription of the Pgk-Zgene. Rather, it would appear that the Pgk-2 gene promoter region has evolved so as to specifically respond to transcription factors present in meiotic spermatogenic cells. A 327-bp, tissuespecific enhancer region was identified in the promoter of the human PGK-2 gene on the basis of its capacity to direct appropriate spermatogenic cell-type-specific expression of reporter genes in transgenic mice (Robinson et ah, 1989). Recently a critical 40-bp subregion that appears to bind testis-specific transcription factors has been delineated within this enhancer region in the human PGK-2 gene promoter, and similar sequences have been identified in an analogous location in the mouse Pgk-2 gene promoter (Gebara and McCarrey, 1992). It will be of interest to determine if other genes that initiate expression in spermatocytes are regulated by the same transcription factor(s), thus affecting a coordinate regulation of an entire battery of genes. J.R.M. is very grateful to Dr. Clarke Millette for instruction in the use of the Sta-Put gradient system to isolate enriched populations of spermatogenic cells, and to Dr. Michael McBurney for providing mouse Pgk cDNA clones. This work was supported by grants to J.R.M. from the NIH (HD 23126) and the NSF (DMB-8904741), and to J.J.R. from the NIH (AI 29329) and the Parson’s Foundation. J.R.M. is the recipient of a career development award from the NIH (HD 00829). REFERENCES Ausubel,F. M.,Brent, R., Kingston, R. E., Moore, D.D., Seidman, J. G., Smith, J. A., and Struhl, K. (Eds.) (1987). “Current Protocols in Molecular Biology.” Wiley, New York. Bellve, A. R., Cavicchia, J. C., Millette, C. F., O’Brien, D. A., Bhatnager, Y. M., and Dym, M. (1977). Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J. Cell BioL 74,68-85. Boer, P. H., Adra, C. N., Lau, Y.-F., and McBurney, M. W. (1987). The testis-specific phosphoglycerate kinase gene Pgk-2, is a recruited retroposon. MoL CelL BioL 7,3107-3112. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry l&5294-5299. Dilworth, D. D., and McCarrey, J. R. (1992). Single-step elimination of contaminating DNA prior to rtPCR. PCR: Methods AppL 1,2791,282. Erickson, R. P., Kramer, J. M., Rittenhouse, J., and Salkeld, A. (1980). Quantitation of mRNAs during mouse spermatogenesis: Prota-

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mine-like histone and phosphoglycerate kinase-2 mRNAs increase after meiosis. Proc. NatL Acad Sci USA 77,6086-6090. Gebara, M. M., and McCarrey, J. R. (1992). Protein-DNA interactions associated with the onset of testis-specific expression of the mammalian Pgk-2 gene. Mol. CelL BioL 12,1422-1431. Gold, B., Fujimoto, H., Kramer, J. M., Erickson, R. P., and Hecht, N. B. (1983). Haploid accumulation and translational control of phosphoglycerate kinase-2 messenger RNA during mouse spermatogenesis. Dev. BioL S&392-399. Goldberg, E. (1990). Developmental expression of lactate dehydrogenase isozymes during spermatogenesis. Prog. Clin. Biol. Res. 344, 49-52. Goto, M., Koji, T., Mizuno, K., Tamaru, M., Koikeda, S., Nakane, P. K., Mori, N., Masamune, Y., and Nakanishi, Y. (1990). Transcription switch of two phosphoglycerate kinase genes during spermatogenesis as determined with mouse testis sections in situ. Exp. Cell Res. 186,273-278. Handel, M. A. (1987). Genetic control of spermatogenesis. Results ProbL Cell Difer. 15, l-62. Hecht, N. B. (1992). Gene expression during male germ cell development. In “Cell and Molecular Biology of the Testis. Vol 5. The Testis” (C. Desjardins and L. Ewing, Eds.). Oxford Univ. Press. New York. In press. Kafri, T., Ariel, M., Brandeis, M., Shemer, R., Urven, L., McCarrey, J., Cedar, H., and Razin, A. (1992). Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Den 6,705-714. Kleene, K. C., Distel, R. J., and Hecht, N. B. (1985). Nucleotide sequence of a cDNA clone encoding mouse protamine 1. Biochemistry 24,719-722. Kramer, J. M., and Erickson, R. P. (1981). Developmental program of PGK-1 and PGK-2 isozymes in spermatogenic cells of the mouse: Specific activities and rates of synthesis. Den BioL 87,37-45. Lee, C.-Y., Niesel, D., Pegoraro, B., and Erickson, R. P. (1980). Immunological and structural relatedness of isozymes and genetic variants of 3-phosphoglycerate kinase from the mouse. J. Biol. Chem. 255, 2590-2595. Lifschytz, E., and Lindsley, D. L. (1972). The role of X-chromosome inactivation during spermatogenesis. Proc. NatL Acad Sci. USA 69, 182-186. Lock, L. F., Takagi, N., and Martin, G. R. (1987). Methylation of the Hprt gene on the inactive X occurs after chromosome inactivation. Cell 48, 39-46. McCarrey, J. R. (1990). Molecular evolution of the human Pgk-2 retroposon. Nucleic Acids Res. l&949-955. McCarrey, J. R., and Thomas, K. (1987). Human testis-specific PGK gene lacks introns and possesses characteristics of a processed gene. Nature (London) 326,501-505. Millan, J. L., Driscoll, C. E., LeVan, K. M., and Goldberg, E. (1987). Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence. Proc. Natl. Acad. Sci. USA 84,5311-5315. Monesi, V. (1967). Ribonucleic acid and protein synthesis during differentiation of male germ cells in the mouse. Arch. Anat. Microsc. Morphol. Exp. 56(Suppl. 3/4), 61-74. Murakawa, G. J., Zaia, J. A., Spallone, P. A., Stephens, D. A., Kaplan, B. E., Wallace, R. B., and Rossi, J. J. (1988). Direct detection of HIV-l RNA from AIDS and ARC patient samples. DNA 7,287-295. Pegoraro, B., and Lee, C.-Y. (1978). Purification and characterization of two isozymes of 3-phosphoglycerate kinase from the mouse. Biochim. Biophys. Acta 522,423-433. Robinson, M. O., McCarrey, J. R., and Simon, M. I. (1989). Transcriptional regulatory regions of testis-specific PGK2 defined in transgenie mice. Proc. Natl. Acod. Sci. USA 86,8437-8441. Romrell, L. J., Bellve, A. R., and Fawcett, D. W. (1976). Separation of

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mouse spermatogeniccells by sedimentation velocity. A morphological characterization. Dev. Biol 49,119-131. Singer-Sam, J., Goldstein, L., Dai, A., Gartler, S. M., and Riggs, A. D. (1992). A potentially critical HpoII site of the X chromosome-linked PGKl gene is unmethylated prior to the onset of meiosis of human oogenic cells. Proc. Natl Acad Sci USA 89,1413-1417. Singer-Sam, J., Grant, M., LeBon, J. M., Okuyama, K., Chapman, V., Monk, M., and Riggs, A. D. (1990b). Use of a HpoII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation. Md Cell Bid 10,4987-4989. Singer-Sam, J., Robinson, M. O., Bellve, A. R., Simon, M. I., and Riggs, A. D. (199Oc). Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis. Nucleic Acids Res. 18,1255-1259. Singer-Sam, J., Yang, T. P., Mori, N., Tanguay, R. L., Le Bon, J. M., Flores, J. C., and Riggs, A. D. (1990a). DNA methylation in the 5’

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Differential transcription of Pgk genes during spermatogenesis in the mouse.

We have analyzed the occurrence of transcripts produced from the ubiquitously expressed, X-linked Pgk-1 gene and the testis-specific, autosomal Pgk-2 ...
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