cellular localization of the mrnas of the somatic and testis-specific cytochromes c during...

MOLECULAR REPRODUCTION A N D DEVELOPMENT 34:196205 (1993)

Cellular Localization of the mRNAs of the Somatic and Testis-Specific Cytochromes c During Spermatogenesis in the Rat CARLOS R. MORALES,' LAURA E. HAKE,' AND NORMAN B. HECHT' 'Department of Anatomy, McGill University, Montreal, Quebec, Canada and 2Department of Biology, Tufts University, Medford, Massachusetts

ABSTRACT During mammalian spermatogenesis, two forms of cytochrome c, cytochromes c, and ct, are present in male germ cells. During meiosis, cytochrome ct begins to replace cytochrome c,. At least four size classes of cytochrome c, mRNA are expressed in all somatic cells and in early stages of male germ cells. In addition, a cytochrome c, transcript of 1.7 kB has been detected in rodent testes and is abundant in post meiotic male germ cells. Here we utilize "in situ" hybridization to define the cellular sites where the four ubiquitous cytochrome c, transcripts, the 1.7 kB cytochrome c, transcripts, and the testis-specific cytochrome c, transcripts are expressed in the rat. Low levels of cytochrome c, mRNAs are detected in Leydig cells, myoepithelial cells, Sertoli cells, all types of spermatogonia, and during meiotic prophase. The 1.7 kB cytochrome c, mRNA is first detected in late stages of meiotic prophase and reaches its highest levels in steps 1 to 9 spermatids. No cytochrome c, mRNAs are detected in spermatids between steps 10 to 19. Low levels of cytochrome c, mRNAs, initially detected in zygotene spermatocytes, reach maximal levels in round spermatids. For all three probes the majority of the silver grains are localized randomly throughout the cytoplasm, suggesting that neither the translating nor non-translating (the 1.7 kB mRNA) forms of cytochrome c, mRNA nor the cytochrome ct mRNAs are sequestered during spermatogenesis. The absence of cytochrome c, or c, mRNAs in steps 10-19 spermatids suggest that the cytochrome c, protein does not tu rn over rapidly in late stage male germ cells. 0 1993 Wiley-Liss, Inc.

Key Words: Mitochondria, Isozymes, Male Germ Cells

INTRODUCTION The mammalian testis contains two distinct isozymes

of cytochrome c, cytochrome c, and cytochrome c, (Hen- nig, 1977; Goldberg et al., 1977; Pettigrew and Moore, 1987). Cytochrome c, is a highly conserved protein encoded by a multipseudogene family in mammals which is ubiquitously expressed in all somatic tissues (Scar- pulla et al., 1981, 1986; Scarpulla 1984; Limbach and Wu, 1985; Wu and Limbach, 1985). In the mouse cytochrome c, is encoded by four different sizes of mRNA of about 1.3, 1.1, and 0.5-0.7 kB. These transcripts are widely expressed in all somatic cell types and in the

0 1993 WILEY-LISS, INC.

testis have been detected in germ cells ranging from type A spermatogonia to round spermatids (Hake et al., 1990; Hecht, 1986, 1992). The 1.7 kB cytochrome c, mRNA appears to be predominantly, if not solely, expressed in the testis and is most abundant in isolated populations of round spermatids. In the testis, cytochrome c, protein has been localized by immunofluorescence to interstitial cells, Sertoli cells, and to early stages of germ cells (Goldberg et al., 1977). As spermatogenesis proceeds the cytochrome c, protein is re- placed by the testis specific cytochrome, cytochrome c,.

Cytochrome ct is a single copy gene which is widely expressed in the testes of many animals (Virbasius and Scarpulla 1988). In the mouse and rat the amino acid sequence of cytochrome c, differs from that of cytochrome c, in 14 and 15 of 104 amino acids, respec- tively. Although the amino acid sequences of the rat and mouse cytochrome c, proteins are identical, the cytochrome ct proteins differ in four amino acids between the two closely related species. Cytochrome c, protein has been detected by immunofluorescence in primary spermatocytes and in subsequent stages of the differentiating male germ cells of the mouse (Goldberg et al., 1977). Northern blot analysis has shown cytochrome ct mRNAs to be present in RNA preparations from isolated populations of meiotic and post-meiotic mouse germ cells (Hake et al., 1990).

To more precisely define the cellular sites where cytochrome c, and cytochrome c, gene expression occurs in the mammalian testis, we have performed in situ hybridization with three different probes for the cytochromes c. We present here data showing the cellular localization of the ubiquitous cytochrome c, mRNAs, the 1.7 kB testicular cytochrome c, transcript, and the mRNAs encoding the testis-specific cytochrome c,.

MATERIALS AND METHODS Processing of Testicular Tissue

Adult male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of Nembutal, and the

Received June 18, 1992; accepted August 6 , 1992. Address reprint requests to Norman B. Hecht, Department of Biology, Tufts University, Medford, MA 02155.

CELLULAR LOCALIZATION OF mRNAs 197

Fig. 1. Cross section of rat testis hybridized in situ with a [“HI- labeled coding region probe for cytochrome c, RNAs and visualized by radioautography. Silver grains (some of them indicated with black circles) overlay Sertoli cells (S), type A spermatogonia (A), pachytene spermatocytes (P), and step 1 round spermatids. Elongated spermatids (E, separated by black broken line) are unreactive. White open circles indicate some of the numerous osmiophilic lipid inclusions present in the cytoplasm of the step 15 elongated spermatids which are not silver

testes were fixed by perfusion through the abdominal aorta with 4% paraformaldehyde, 2% glutaraldehyde, and 3% dextran in 0.05 M phosphate buffer (pH 7.4) for 10 min. Following perfusion, the testes were removed and immersed in the same fixative for 3 h a t 4°C.

The tissues were cut into small blocks of similar sizes, embedded in 4% agar and chopped with a Vibro- tome into 75 pm thick frontal sections. Groups of six tissue sections of approximately 2 x 3 mm were col- lected in autoclaved vials and washed three times in RNAase-free 0.05 M phosphate buffer (pH 7.4) a t 4°C. Glycine (1 M) was added to the buffer t o neutralize aldehyde groups.

In Situ Hybridization With Specific Probes Prehybridization and hybridization procedures were

performed as described in Morales et al. (1991). Briefly,

grains. x 800. Fig. 2. Cross section of rat testis hybridized in situ with a [‘HI-

labeled coding region probe for cytochrome c, RNAs and visualized by radioautography. Most of the silver grains overlay Sertoli cells (S), preleptotene spermatocytes (PL), pachytene spermatocytes (PI, and step 7 round spermatids (R). Step 19 elongated spermatids (El, which are separated from the round spermatids by a black broken line, are unreactive. X800.

the Vibrotome slices were transferred from the phosphate buffer to the prehybridization buffer containing 4 x SSC (standard sodium citrate) and I X Denhardt’s solution for 1 h at room temperature with gentle agita- tion. The sections (six per vial) were then immersed in hybridization buffer containing 1 ml 8 x SSC, 1 ml deionized formamide, 100 p120x Sarcosyl, 200 p11.2 M phosphate buffer, 20 p1 100 x Denhardt’s solution, and 150 ng of [3H]-labelled probe. The specific activities of the probes were 2.4-3.1 x lo7 cpmipg for the cyt c, and the cyt ct antisense RNA probes, 4.3 x l o 7 cpmlpg for the 1.7 cyt c, antisense RNA probe and 2.4-3.5 x lo7 cpmipg for the control sense RNA probes. After hybridization overnight a t 35”C, the sections were rinsed se- quentially a t 40°C in 4 x SSC, l x SSC, and 0 . 1 ~ SSC €or 1.5 h. Following the washes, the sections were immersed in potassium ferrocyanide-reduced osmium for

198 C.R. MORALE$ ET AL.

Fig. 3. Cross section of rat testis hybridized in situ with a ["HI- labeled probe specific to the cytochrome c,. 1.7 kB RNA and visualized by radioautography. A weak reactivity overlies the meiotic spermatocytes (arrowheads) which are separated by white broken lines from the rest of the seminiferous epithelium. ~ 8 0 0 .

Fig. 4. Cross section of rat testis hybridized in situ with a ['HI-

labeled probe specific to the cytochrome c, 1.7 kB RNA and visualized by radioautography. A strong radioautographic reaction is observed over the step 5 round spermatids (R), which have been separated by white and black broken lines from the rest of the seminiferous epithelium. S, Sertoli cells; B, type B spermatogonia; P, pachytene spermatocytes; E, step 17 elongated spermatids. x800.

15 min and quickly dehydrated in 50, 70, 90, 95, and 100% ethanol and propylene oxide, and embedded in Epon.

Preparation of Cytochrome c, and Cytochrome c, Probes

[3H]-labelled probes corresponding to sense and antisense transcripts of the cytochrome subclones were generated from pGEM clones linearized in the poly- linker using the Promega in vitro transcription kit with either [3H]ATP or [3H1UTP (specific activity >40 Cii mmol, Amersham) and the protocol of the supplier. The probes corresponding to the ubiquitous cytochrome c, transcripts contained 45 nt of 5' untranslated region (UTR), the entire coding region and 94 nt of the 3' UTR

(Hake et al., 1990). The cytochrome ct probes contained 110 n t of 5' UTR, the entire coding region and 142 n t of 3' UTR. The sense and antisense RNA probes corresponding to the 1.7 kB cytochrome c, mRNA were generated from a genomic clone containing 500 n t of the 5' UTR unique to this transcript (Hake and Hecht, in press). After transcription, DNA templates were removed by DNAase 1 digestion and free nucleotides were removed with a Sephadex G-50 column.

Radioautography and Quantitation of Epon Sections

Epon embedded testicular tissues were trimmed and cut in an ultramicrotome. Semithin sections (1 pm thick) were prestained with ironlhematoxylin and then


Figs. 54. Comparison between cross sections of rat testis hybridized in situ with the [3H1-labeled coding region probe for cytochrome c, (Fig. 5) and the probe specific to the 5'UTR ofthe 1.7 kB cytochrome c, (Figs. 6 and 7). Note that in Fig. 5 the silver grains overlay the cells in the basal compartment of the seminiferous epithelium as well as the cells of the adluminal compartment composed of pachytene spermatocytes (P) and step 9 spermatids (arrows). Residual bodies (RBI are

devoid of hybridization. S, Sertoli cells. In Figs. 6 and 7 the radioautographic reaction appears to be restricted to the round spermatids separated by white broken lines from the rest of the seminiferous epithelium. S, Sertoli cells; A, type A spermatogonia; B, type B spermatogonia; P, pachytene spermatocytes; E, elongated spermatids. x 800.

200 C.R. MORALES ET AL.

Figs. 8 and 9. Comparison between cross sections of rat testis hybridized in situ with the ['HI labeled antisense 1.7 kB specific cytochrome c, probe (Fig. 8 ) and a control sense 1.7 kB specific cytochrome c, RNA probe (Fig. 9). While the antisense RNA probe (Fig 8 ) generates a radioautographic reaction in round spermatids (demarcated by broken white line), the sense RNA probe does not (Fig. 9). X 800.

coated with a Kodak NTB-2 nuclear emulsion (Kopriwa and Leblond, 1962; Kopriwa, 1973). After 7,14, and 21 days of exposure, they were developed with Kodak

To compare the number of silver grains generated by the coding region cyt c, probe and the 1.7 cyt c, probe, a quantitative analysis was conducted on areas of the seminiferous epithelium containing steps 1 and 9 spermatids. These areas were first measured with a MOP-3 instrument (Reichert-Jung, Austria) on micrographs ( x 800) of radioautographs developed 21 days after the in situ hybridization. Subsequently, the number of silver grains overlying such areas was counted and expressed as number of grains per pm2. Corrections were made for the differences in specific activities of each probe.

D-170.

RESULTS To compare the sites of transcription for the two iso-

forms of cytochrome c in the seminiferous epithelium and particularly in the germ cells, three specific L3H]- labeled RNA probes were used for in situ hybridization with rat testicular tissues. Since maximum preservation of the tissue and excellent morphology are essen- tial for cellular RNA localization analyses, chopped testicular sections (75 pm thick) were fixed by perfusion

and following in situ hybridization, the tissues were post-fixed in potassium ferrocyanide-reduced osmium and embedded in Epon.

Cellular Localization of All Cytochrome c, mRNAs

Using an antisense RNA that hybridized to the coding regions of the four classes of cytochrome c, mRNA detected in rodent testes, a weak hybridization signal was seen in somatic cells of the testis, including Leydig cells, myoepithelial cells and Sertoli cells, and in all types of spermatogonia (type A, intermediate, and B spermatogonia). The radioautographic reaction became slightly stronger in early spermatocytes (preleptotene, leptotene, and zygotene stages), increased in early, mid, and late pachytene spermatocytes and became very strong in round spermatids, up to and including step 9 of spermiogenesis. No radioautographic reaction above background was seen in spermatids between steps 10 and 19 (Figs. 1 ,2 ,5 , 14).

Cellular Localization of the 1.7 kB Cytochrome c, mRNAs

Although the 1.7 kB cytochrome c, mRNA contains the coding region present in all cytochrome c, mRNAs, it also contains a novel 5' untranslated region (Hake


Figs. 10-13. Cross sections of rat testis hybridized in situ with a [3H1-labeled cytochrome ct RNA probe and visualized by radioautogra- pby. Note that the radioautographic reaction is strong in pachytene spermatocytes (PI and round spermatids (R) demarcated by white bro-

ken lines. Reaction is negligible (background level) or absent in elongated spermatids (El. Cells in the basal compartment (below the lower white broken lines) are also unreactive. S, Sertoli cells; A, type A spermatogonia; PL, preleptotene spermatocytes. X800.


TABLE 1. Number of Silver Grainsilo0 pm2 Detected in the Cytoplasm of Round Spermatids Following

Hybridization of Testicular Sections With Probes for the Cvtochromes c,

S tep 1 Step 9 Probe detecting spermatids spermatids

The coding regions of 10 2 3 12 t 3

The 1.7 kB cytochrome c, mRNA cytochrome c, rnRNAs

8 2 2 12 2 4

and Hecht, in press) and is predominantly detected in post-meiotic germ cells (Hake et al., 1990). Using an antisense RNA probe specific to the 5' untranslated region of the 1.7 kB cytochrome c, transcript, a hybridization signal slightly above background was first detected in pachytene spermatocytes. Increased levels of hybridization were seen in the diplotene and dividing spermatocytes present in stages XI11 and XIV, respec- tively, of the cycle of the seminiferous epithelium. The strongest hybridizations were seen in steps 1 to 9 spermatids. No radioautographic reactions above back-

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ground were observed in steps 10 to 19 spermatids (Figs. 3,4,6-9,151.

Cellular Localization of Cytochrome ct mRNAs Using an antisense RNA specific for the coding re-

gion of the testis-specific cytochrome, low levels of cytochrome c, mRNAs were first detected in zygotene spermatocytes. An increased level of hybridization was seen during spermatocyte development and spermiogenesis, reaching a maximum reaction in round spermatids (Figs. 10-13, 16). As seen with the two probes that hybridized t o the 1.7 kB and ubiquitous cytochrome c mRNAs, the cytochrome ct probe did not detect silver grains in elongated spermatids.

Control Hybridizations Sense RNA probes that were transcribed from the

same pGEM vectors as antisense probes but from the opposite strand did not yield any radioautographic reactions, indicating that the hybridizations observed with the antisense probes were specific (Fig. 9).

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A

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9 10 12 14 18 19 Fig. 14. Diagrammatic representation of somatic cytochrome graphic reaction became slightly stronger in early spermatocytes (pre-

leptotene, leptotene, and zygotene), continued to increase in early, mid, and late pachytene spermatocytes and became very strong in round spermatids up to and including step 9 spermatids. The radioautographic reaction was negligible or absent in late spermatids at steps 10-19.

mRNA expression during spermatogenesis in the rat demonstrated by in situ hybridization with the cytochrome c, coding region RNA probe. Black dots indicate the cells that express these mRNAs and the density of the dots represents the concentration of mRNAs in germinal cells. This probe generated a weak reaction in all types of spermatogonia (type A, intermediate, and type B spermatogonia). The radioauto-

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CELLULAR LOCALIZATION OF mRNAs

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Fig. 15. Diagrammatic representation of testicular cytochrome mRNA expression during spermatogenesis in the rat demonstrated by in situ hybridization with the 1.7 kB cytochrome c, RNA probe. Black dots indicate the cells that express these mRNAs and the density of the dots represents the concentration of their mRNAs. This probe gener-

Quantitation of Cytochrome c, mRNA Levels To determine the proportion of the hybridization sig-

nal detected by the cytochrome c, coding region probe that is attributable to the 1.7 kB cytochrome c, mRNA, a quantitative comparison was made of the number of silver grains generated by the cytochrome c, coding region probe and the 1.7 kB cytochrome c, probe. Areas of round spermatids a t steps 1 and 9 were measured with a MOP-3 instrument (Reichert-Jung, Austria) and the number of silver grains overlying each area was counted and expressed as number of grains per pm2. A correction factor was used for any differences in specific activities of the probes. The results showed that the number of silver grains was remarkably similar for both probes at the same steps of spermatid development (Table 1, Figs. 14-16), suggesting the 1.7 kB cytochrome c, transcript is the source of some of the meiotic cytochrome c, mRNA and most of the post-meiotic cytochrome c, mRNA.

DISCUSSION These in situ hybridization studies extend our earlier

analyses of cytochrome c gene expression during sper-

9 10 12 14

203

19

ated a weak positive reaction in diplotene and dividing spermatocytes. The radioautographic reaction was higher in all round spermatids (steps 1-91, but no radioautographic reaction was observed in late spermatids (steps 10-19).

matogenesis (Hake et al., 1990). We have previously shown by Northern blot analysis of RNA samples pre- pared from isolated populations of mouse male germ cells that cytochrome c, mRNAs can be detected in germ cells from type A spermatogonia to round spermatids. Here we extend these observations to a second species of rodent, the rat, and confirm that little, if any, of the three ubiquitously expressed cytochrome c, mRNAs is present in spermatids.

Previous studies have revealed that cytochrome c, is encoded by a t least four size classes of mRNAs in all rodent tissues. In addition, a fifth cytochrome c, mRNA of 1.7 kB has been detected in the testis. Northern blots with RNAs isolated from individual populations of germ cells have suggested the 1.7 kB transcript is predominantly present in the round spermatids of the mouse. The in situ hybridizations establish the post- meiotic expression of the 1.7 kB cytochrome c, RNA in the rat.

In situ hybridization with a RNA probe specific for the 1.7 kB cytochrome c, mRNA reveals that this transcript is first detected in the rat towards the end of meiosis and, as previously shown by Northern blots of mouse testicular RNAs, is predominantly found in


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Fig. 16. Diagrammatic representation of testicular cytochrome mRNA expression during spermatogenesis in the rat demonstrated by in situ hybridization with the cytochrome ct RNA probe. Black dots indicate the cells that express these mRNAs and the density of the dots represents the concentration of mRNA in germinal cells. This probe

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9 10 12 14 18 19

round spermatids. Genomic cloning studies and RNAase protection assays have revealed that the 1.7 kB mRNA results from cytochrome c, transcription from an alternative upstream promoter of the cytochrome c, gene (Hake and Hecht, in press). Analysis of the translational status of the somatic cytochrome c mRNAs revealed that the 1.7 kB transcript, unlike the 1.3, 1.1, and 0.5-0.7 kB transcripts, is almost solely present in the non-polysomal fraction (Hake et al., 1990).

In situ hybridization establishes that the probe specific to the 1.7 kB cytochrome c, mRNA hybridized ex- clusively to mRNAs found in late stages of spermatocytes and in round spermatids (steps 1-9). Since both cytochrome c, probes generated a similar grain density with respect to silver grain quantity over the round spermatids, most of these hybridization signals are from the 1.7 transcript. These results suggest that during spermatogenesis the four smaller cytochrome c, transcripts are primarily present in spermatogonia and spermatocytes, whereas the 1.7 kB mRNA is the pre- dominant cytochrome c, mRNA in round spermatids.

generated a weak radioautographic reaction that started in zygotene spermatocytes and gradually increased during spermatocyte development to reach a maximum reaction in round spermatids (steps 1-91, This probe did not hybridize to RNA in elongated spermatids.

The decline in amount of the four smaller polysomal cytochrome c, mRNAs during spermiogenesis has been previously shown in Northern blots (Hake et al., 1990).

The majority of the silver grains were localized randomly throughout the cytoplasm for both of the cytochrome c, and the cytochrome ct probes, suggesting that neither the translating cytochromes c, and c, mRNAs nor the non-translating 1.7 kB cytochrome c, mRNAs are sequestered in male germ cells.

The methodological approach utilized in this study is similar to our recent investigation where we used in situ hybridization to determine the cellular localization of transition protein 1 and protamine 1 transcripts in the rat testis (Morales et al., 1991). The technique con- sists of hybridizing [3H]-labeled RNA probes to testicular sections obtained with a tissue chopper immediately after perfusion and before embedding. This procedure usually avoids degradation of RNAs, since it bypasses a series of steps unavoidable during paraffin embedding, such as deparaffinization, hydration, treatment with 0.2 N HC1, and proteolytic treatment with proteinase K. Cellular morphology is also well maintained by a


post-fixation step with ferrocyanide-reduced osmium and subsequent embedding in plastic resins. In contrast to the use of frozen sections, a more tedious procedure that often results in loss of the sections, the procedure we have used yields superior tissue preservation and resolution and also permits visualization at the EM level (Morales e t al., 1991).

During male germ cell development, the testis-specific cytochrome c, cytochrome ct, begins to replace cytochrome c, in germ cell mitochondria during meiosis. Our in situ hybridization data is consistent with this finding, since significant levels of cytochrome ct mRNAs are detected in early stages of meiotic germ cells in the rat with mRNA levels rising in round spermatids. Surprisingly little, if any, cytochrome C, mRNA was detected in steps 10 to 19 spermatids, suggesting that cytochrome ct is either a very long lived protein in the mitochondria of spermatids and spermatozoa or a pool of cytochrome C, apoprotein exists in the cytoplasm of the elongating spermatids. The absence of cytoplasmic ribosomes or any cytoplasmic protein synthesis in spermatozoa indicates that the cytochrome ct, a nuclear encoded protein of the spermatozoan mitochondria, must remain functional during at least the life of the spermatozoon (Alcivar et al., 1989). Although we detect the highest levels of the 1.7 kB cytochrome c, mRNAs in round spermatids at the time of cytochrome c, synthesis, polysomal gradient analysis has shown most of the 1.7 kB mRNA to be in the non-polysomal fraction, indicating that the 1.7 kB may be a non-functional RNA (Hake and Hecht, in press). It is likely that the 1.7 kB cytochrome c, mRNA is evidence of a mechanism leading to the down regulation of cytochrome c, during spermatogenesis.

ACKNOWLEDGMENTS This research was supported by a MRC grant (to

C.R.M.)andNIHgrantHD 11878 (t0N.B.H.). C.R.M. is a fellow of the Fonds de la Recherche en Sante du Que- bec. The authors thank Polly Bradley for her excellent secretarial assistance.

REFERENCES Alcivar AA, Hake L, Millette CF, Transler JM, Hecht NB (1989):

Mitochondria1 gene expression in mouse male germ cells. Dev Biol 135263-271.

Goldberg E, Sberna D, Wheat TE, Urbanski GJ, Margoliash E (1977): Cytochrome c: Immunofluorescent localization of the testis-specific form. Science 196:1010-1012.

Hake LE, Alcivar AA, Hecht NB (1990): Changes in mRNA length accompany tranlational regulation of the somatic and testis specific cytochrome c genes during spermatogenesis in the mouse. Develop- ment 110:249-257.

Hake LE, Hecht NB: Utilization of an alternative transcription initia- tion site of somatic cytochrome c in the mouse produces a testis- specific cytochrome c mRNA. J Biol Chem (in press).

Hecht NB (1986): Regulation of gene expression during mammalian spermatogenesis. In Rossant J, Pedersen R (eds): “Experimental Approaches to Embryonic Development.” Cambridge, Cambridge University Press. pp. 151-193.

Hecht NB: Gene expression during male germ cell development. In Ewing L, Desjardins C (eds): “Cell and Molecular Biology of the Testes” New York: Oxford University Press (in press).

Hennig B (1975). Change of cytochrome c structure during development of the mouse. Eur J Biochem 55:167-183.

Kopriwa BM (1973): A reliable, standardized method for ultrastruc- tural electron microscope radioautography. Histochemistry 37:l-17

Kopriwa BM, Leblond CP (1962) Improvements in the coating tech- niques of radioautography. J Histochem Cytochem 10:269-284.

Limbach KJ, Wu R (1985): Characterization of a mouse somatic cytochrome c gene and three cytochrome c pseudogenes. Nucl Acids Res 2:617-630.

Morales CR, Kwon YK, Hecht NB (1991): Cytoplasmic localization during storage and translation of the mRNAs of transition protein 1 and protamine 1, two translationally regulated transcripts of the mammalian testis. J Cell Sci 100:119-131.

Pettigrew GW, Moore GR (1987): “Cytochromes c: Biological Aspects.” (Rich A, ed.) New York: Springer-Verlag.

Scarpulla RC (1984): Processed pseudogenes for rat cytochrome c are preferentially derived from one of three alternate mRNAs. Mol Cell Biol4:2279-2288.

Scarpulla RC, Agne KM, Wu R (1981): Isolation and structure of a rat cytochrome c gene. J Biol Chem 256:6480-6486.

Scarpulla RC, Kilar MC, Scarpulla KM (1986): Coordinate induction of multiple cytochrome c mRNAs in response of thyroid hormone. J Biol Chem 261:4660-4662.

Virbasius JV and Scarpulla RC (1988): Structure and expression of rodent genes encoding the testis-specific cytochrome c. J Biol Chem

Wu R, Limbach KJ (1985): Characterization of a mouse somatic cytochrome c gene and three cytochrome c pseudogenes. Nucl Acids Res 13:617-630.

263:6791-6796.

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