Cytochrome Ct Research Articles

Expression of mitochondrial marker proteins during spermatogenesis.

Spermatogenesis is a highly complex, hormonally regulated cytodifferentiation process finally leading to the production of spermatozoa. In addition to other events germ cell differentiation is characterized by a gradual structural modification of many organelles including mitochondria which play a unique role. The morphological and functional development of germ cell mitochondria is a reflection of the permanent change in the testicular microenvironment which occurs when the germ cells are slowly moving from the base of the seminiferous epithelium to the lumen. Concomitant with the structural changes, several mitochondrial proteins are known to be expressed and synthesized during distinct phases of the organelle's development. This review pays particular attention to these transiently expressed mitochondrial proteins such as hsp60, Lon protease, sulphydryl oxidase and cytochrome ct. Furthermore, the biological function of this stepwise gene activation during mitochondrial and germ cell development is discussed.

In male mouse germ cells, copper-zinc superoxide dismutase utilizes alternative promoters that produce multiple transcripts with different translation potential.

Copper-zinc superoxide dismutase (SOD-1) is an enzyme that is widely expressed in eukaryotic cells and performs a vital role in protecting cells against free radical damage. In mouse testis, three different sizes of SOD-1 mRNAs of about 0.73, 0.80, and 0.93 kilobases (kb) are detected. The 0.73-kb mRNA is found in early stages of male germ cells and in all somatic tissues. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase H digestions and Northern blot analyses reveal that the three SOD-1 mRNAs are derived from two transcripts, a ubiquitously expressed transcript and a post-meiotic transcript, which differ by 114-120 nucleotides. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5'-untranslated region. Using a probe derived from the 5'-untranslated region of the 0.93-kb SOD-1 mRNA, we have established that it originates from an alternative upstream promoter contiguous with the somatic SOD-1 promoter. Polysomal gradient analysis of the three mouse testis SOD-1 mRNAs reveals that the 0.93-kb SOD-1 mRNA is primarily non-polysomal, while the 0.80- and 0.73-kb SOD-1 mRNAs are mostly polysome associated. A faster migrating form of the 0.93-kb SOD-1 mRNA is present on polysomes as a result of partial deadenylation. In a cell-free translation system, the 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-1 mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-1 mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-1 mRNAs undergo adenylation changes, and one of the post-meiotic SOD-1 mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form.

Immortalized germ cells undergo meiosis in vitro.

Establishing mammalian germ-cell lines capable of differentiation in vitro would greatly facilitate the study of gametogenesis and the meiotic process that is so fundamental for reproduction and the maintenance of genetic diversity of the species. We have established two germ-cell lines [GC-2spd(ts) and GC-3spc(ts)] by cotransfecting primary mouse testicular germ cells with the simian virus 40 large tumor antigen gene and the gene coding for a temperature-sensitive mutant of p53. Both cell lines express the germ cell-specific lactate dehydrogenase C4 isozyme and cytochrome ct isoform. At the permissive temperature of 37 degrees C, the GC-2spd(ts) line generates cells with a haploid DNA content and morphologic and biochemical features of round spermatids, including the appearance of an acrosomic granule. The identification of a flagellar axoneme when these cells are cultured at 32 degrees C further indicates that these cells correspond to the early spermatid stages of spermiogenesis.

Heterogeneity in the 5' untranslated region of mouse cytochrome cT mRNAs leads to altered translational status of the mRNAs.

Previous studies have shown that the differential regulation of mouse somatic cytochrome c (cyt cS) and testicular cytochrome c (cyt cT) during spermatogenesis is accompanied by changes in mRNA length [Hake et al. (1990) Development, 110, 249-257]. When analyzed by polysomal gradient sedimentation, cytochrome cT sediments in two broad size classes: non-polysomal mRNAs are about 0.6 to 0.75 kb and polysomal mRNAs range from 0.7 to 0.9 kb. Both classes of mRNAs shorten to about 0.5 kb following deadenylation. Oligonucleotide-directed cleavage of the cytochrome cT RNAs by RNase H reveals that the size heterogeneity of cytochrome cT mRNAs resides in the 5' untranslated regions (UTRs). Ribonuclease protection assays reveal that multiple cytochrome cT mRNAs are transcribed from six different transcriptional start sites spanning a region of 59 nucleotides in the 5'UTR from +1 to +59. Transcripts derived from the first and second transcriptional initiation sites are not loaded onto polysomes as efficiently as those transcripts initiated from the other start sites. Each of the longer mRNAs has an upstream open reading frame, which starts at +8 and ends at +136 in the 5'UTR of the cytochrome cT transcript. Computer analysis suggests that the lengthened 5'UTR sequences allow additional hairpin structures to be formed. Since the upstream open reading frame and the additional stem loop structure are absent in the 5' UTRs of the cytochrome cT mRNAs initiated from the four downstream start sites, we suggest that these sequences in the two longest cytochrome cT transcripts hinder their loading onto polysomes.

Immunoelectron Microscopic Localization of Testicular and Somatic Cytochromes c in the Seminiferous Epithelium of the Rat1

Somatic and testis-specific cytochromes c were localized ultrastructurally in the seminiferous epithelium by immunocytochemistry using monospecific antibodies. Cytochrome cS was lost from the mitochondria as spermatogenesis advanced, while there was a relative increase in cytochrome cT during the zygotene-to-pachytene transition; this was in agreement with other studies that have suggested activation of the cytochrome cT gene during prophase of the first meiotic division. Cytochrome cT was highly concentrated in mitochondria that were being degraded within cytoplasmic lobes of spermatids and in residual bodies that were phagocytized by Sertoli cells. The two isoforms were found to coexist within the same mitochondrion during the transitional period from cytochrome cS to cytochrome cT predominance. In addition, both cytochromes c were present in the chromatoid bodies of spermatocytes and round spermatids; this suggests that the chromatoid body may be involved in the storage of these isozymes and possibly in their differential expression within germ cell mitochondria. Apocytochrome c was concentrated in mitochondria and chromatoid bodies of the germ cells and also scattered in the cytoplasm. The presence of the holoprotein and apoprotein immunoprobes within the chromatoid bodies of spermatocytes and spermatids was an interesting observation that raises questions regarding the precise location of the synthesis of cytochromes c in spermatogenic cells.

Cellular localization of the mRNAs of the somatic and testis‐specific cytochromes c during spermatogenesis in the rat

During mammalian spermatogenesis, two forms of cytochrome c, cytochromes cs and ct, are present in male germ cells. During meiosis, cytochrome ct begins to replace cytochrome cs. At least four size classes of cytochrome cs mRNA are expressed in all somatic cells and in early stages of male germ cells. In addition, a cytochrome cs 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 cs transcripts, the 1.7 kB cytochrome cs transcripts, and the testis-specific cytochrome ct transcripts are expressed in the rat. Low levels of cytochrome cs mRNAs are detected in Leydig cells, myoepithelial cells, Sertoli cells, all types of spermatogonia, and during meiotic prophase. The 1.7 kB cytochrome cs mRNA is first detected in late stages of meiotic prophase and reaches its highest levels in steps 1 to 9 spermatids. No cytochrome cs mRNAs are detected in spermatids between steps 10 to 19. Low levels of cytochrome ct 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 cs mRNA nor the cytochrome ct mRNAs are sequestered during spermatogenesis. The absence of cytochrome cs or ct mRNAs in steps 10-19 spermatids suggest that the cytochrome ct protein does not turn over rapidly in late stage male germ cells.

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.

Stage-dependent appearance of sulfhydryl oxidase during spermatogenesis in the testis of rat and hamster

Sulfhydryl oxidase (SOx), an enzyme that catalyzes the oxidation of sulfhydryl compounds, appears in the spermatogenic cells of rat and hamster testes in a stage-dependent manner. It first appears in pachytene spermatocytes at stage I in both the animal species studied. SOx immunoreactivity is associated with mitochondria of these cells. The fate of such mitochondria is species-dependent. In rat, the immunoreactive mitochondria aggregate during maturation phase and are retained in the residual bodies. Spermatozoa free of SOx are released into the lumen. On the other hand, in hamster, the immunoreactive mitochondria arrange themselves around the midpiece of spermatozoa. In such a case, residual bodies lack SOx. The appearance of SOx coincides with the appearance of LDH-X in the spermatogenic cells. Like many other proteins such as LDH-X, RSA-1 and cytochrome ct, SOx provides yet another example of differential gene activation associated with a developmental process of gametes.

Effects on fertility of rabbits after active immunization with rat testicular cytochrome ct.

A Male rabbit was immunized with rat testicular cytochrome (Cyt) ct and mated with normal, unimmunized females. The matings resulted in abnormal pregnancies: no offspring or stillborn or undersized liveborn offspring weighing 25-30 gm each. Another unusual observation was that fur-pulling behavior, normally exhibited by pregnant female rabbits at the end of the gestational period, was absent in all of these pregnancies. Therefore, immunization of a normal rabbit with testicular cyt ct appeared to interfere with physiological and behavioral aspects of pregnancy in normal female rabbits. The immunological basis of these findings remains to be determined.

Analyses of Stage-Specific Multiple Forms of Lactate Dehydrogenase and of Cytochrome c during Spermatogenesis in the Mouse

Spermatogenesis is a programmed developmental process characterized by the inactivation of certain genes and the activation of other, testis-specific genes. Synthesis of unique gene products such as LDH-C4 and cytochrome ct occurs only at precise stage of germ cell formation. The developmental sequences of gene activation for these proteins was observed by immunohistochemical procedures. LDH-C4 is first detectable during mid-pachytene of the primary spermatocyte. The C subunits appear to be uniformly distributed throughout the cytoplasm of the spermatocyte. The mid-pachytene stage also marks the first appearance of cytochrome ct. The association of this electron transport protein with spermatocyte mitochondria is reflected in a granular fluorescence of specific antibody-treated sections of testis. Neither the C subunit of LDH, nor cytochrome ct appear in leptotene or early pachytene primary spermatocytes. These analyses indicate that there is stage-specific protein synthesis in the primary spermatocyte which is characterized by differential activation of the LDH-C locus and of the gene coding for cytochrome ct.