Mammalian Spermatids Research Articles

TH2BS11ph histone mark is enriched in the unsynapsed axes of the XY body and predominantly associates with H3K4me3-containing genomic regions in mammalian spermatocytes

BackgroundTH2B is a major histone variant that replaces about 80–85% of somatic H2B in mammalian spermatocytes and spermatids. The post-translational modifications (PTMs) on TH2B have been well characterised in spermatocytes and spermatids. However, the biological function(s) of these PTMs on TH2B have not been deciphered in great detail. In our attempt to decipher the unique function(s) of histone variant TH2B, we detected the modification in the N-terminal tail, Serine 11 phosphorylation on TH2B (TH2BS11ph) in spermatocytes.ResultsThe current study is aimed at understanding the function of the TH2BS11ph modification in the context of processes that occur during meiotic prophase I. Immunofluorescence studies with the highly specific antibodies revealed that TH2BS11ph histone mark is enriched in the unsynapsed axes of the sex body and is associated with XY body-associated proteins like Scp3, γH2AX, pATM, ATR, etc. Genome-wide occupancy studies as determined by ChIP sequencing experiments in P20 C57BL6 mouse testicular cells revealed that TH2BS11ph is enriched in X and Y chromosomes confirming the immunofluorescence staining pattern in the pachytene spermatocytes. Apart from the localisation of this modification in the XY body, TH2BS11ph is majorly associated with H3K4me3-containing genomic regions like gene promoters, etc. These data were also found to corroborate with the ChIP sequencing data of TH2BS11ph histone mark carried out in P12 C57BL6 mouse testicular cells, wherein we found the predominant localisation of this modification at H3K4me3-containing genomic regions. Mass spectrometry analysis of proteins that associate with TH2BS11ph-containing mononucleosomes revealed key proteins linked with the functions of XY body, pericentric heterochromatin and transcription.ConclusionsTH2BS11ph modification is densely localised in the unsynapsed axes of the XY body of the pachytene spermatocyte. By ChIP sequencing studies in mouse P12 and P20 testicular cells, we demonstrate that TH2BS11ph is predominantly associated with H3K4me3 positive genomic regions like gene promoters, etc. We propose that TH2BS11ph modification could act alone or in concert with other histone modifications to recruit the appropriate transcription or XY body recombination protein machinery at specific genomic loci.

Nuclear envelope remodelling during human spermiogenesis involves somatic B-type lamins and a spermatid-specific B3 lamin isoform.

The nuclear lamina (NL) is a filamentous protein meshwork, composed essentially of lamins, situated between the inner nuclear membrane and the chromatin. There is mounting evidence that the NL plays a role in spermatid differentiation during spermiogenesis. The mouse spermatid NL is composed of the ubiquitous lamin B1 and the spermatid-specific lamin B3, an N-terminally truncated isoform of lamin B2. However, nothing is known about the NL in human spermatids. We therefore investigated the expression pattern and localization of A-type lamins (A, C and C2) and B-type lamins (B1, B2 and B3) during human spermiogenesis. Here, we show that a lamin B3 transcript is present in human spermatids and that B-type lamins are the only lamins detectable in human spermatids. We determine that, as shown for their mouse counterparts, human lamin B3, but not lamin B2, induces strong nuclear deformation, when ectopically expressed in HeLa cells. Co-immunofluorescence revealed that, in human spermatids, B-type lamins are present at the nuclear periphery, except in the region covered by the acrosome, and that as the spermatid matures the B-type lamins recede towards the posterior pole. Only lamin B1 remains detectable on 33-47% of ejaculated spermatozoa. On spermatozoa selected for normal head density, however, this fell to <6%, suggesting that loss of the NL signal may be linked to complete sperm nucleus compaction. The similarities revealed between lamin expression during human and rodent spermiogenesis, strengthen evidence that the NL and lamin B3 have conserved functions during the intense remodelling of the mammalian spermatid nucleus.

H3K79 methylation directly precedes the histone-to-protamine transition in mammalian spermatids and is sensitive to bacterial infections.

In both mammalian and Drosophila spermatids, the completely histone-based chromatin structure is reorganized to a largely protamine-based structure. During this histone-to-protamine switch, transition proteins are expressed, for example TNP1 and TNP2 in mammals and Tpl94D in Drosophila. Recently, we demonstrated that in Drosophila spermatids, H3K79 methylation accompanies histone H4 hyperacetylation during chromatin reorganization. Preceding the histone-to-protamine transition, the H3K79 methyltransferase Grappa is expressed, and the predominant isoform bears a C-terminal extension. Here, we show that isoforms of the Grappa-equivalent protein in humans, rats and mice, that is DOT1L, have a C-terminal extension. In mice, the transcript of this isoform was enriched in the post-meiotic stages of spermatogenesis. In human and mice spermatids, di- and tri-methylated H3K79 temporally overlapped with hyperacetylated H4 and thus accompanied chromatin reorganization. In rat spermatids, trimethylated H3K79 directly preceded transition protein loading on chromatin. We analysed the impact of bacterial infections on spermatid chromatin using a uropathogenic Escherichia coli-elicited epididymo-orchitis rat model and showed that these infections caused aberrant spermatid chromatin. Bacterial infections led to premature emergence of trimethylated H3K79 and hyperacetylated H4. Trimethylated H3K79 and hyperacetylated H4 simultaneously occurred with transition protein TNP1, which was never observed in spermatids of mock-infected rats. Upon bacterial infection, only histone-based spermatid chromatin showed abnormalities, whereas protamine-compacted chromatin seemed to be unaffected. Our results indicated that H3K79 methylation is a histone modification conserved in Drosophila, mouse, rat and human spermatids and may be a prerequisite for proper chromatin reorganization.

Identification of Potential Regulatory Elements in the 5′ and 3′ UTRs of 12 Translationally Regulated mRNAs in Mammalian Spermatids by Comparative Genomics

To facilitate identifying translational control elements by studies of mutations in transgenic mice, a database of orthologous 5' and 3' ends of 12 messenger RNA (mRNA) species from 13 to 23 mammals that undergo delayed translational activation in spermatids was constructed for the Acev2, Akap3, Akap4v2, Gapdhs, Odf1, Prm1, Prm2, Prm3, Smcp, Spata18, Tnp1, and Tnp2 mRNAs. This database, available here, was searched for conserved sequences in conserved positions and known translational control elements. Numerous potential mRNA-specific elements were identified, including upstream open reading frames, conserved sequences upstream and downstream of the poly(A) signal, and noncanonical and multiple poly(A) signals. RNA electrophoresis mobility shift assays demonstrate that Y-box proteins bind 30 of the 36 permutations of the degenerate Y-box recognition sequence (YRS), [UAC][CA]CA[UC]C[ACU], and this information was used to identify hundreds of YRSs in the untranslated region (UTR) database. Collectively, these findings suggest that the distal ends of both UTRs are particularly well conserved, implying that translation of each mRNA is regulated by mechanisms involving the poly(A) binding protein and the closed loop. In addition, the 5' flanking regions of all 12 genes have conserved, gene-specific sequences and configurations of elements that resemble the binding site of the testis-specific isoform of cyclic AMP response element modulator, and all 12 genes lack retrogene paralogues, demonstrating the efficacy of mechanisms that limit the proliferation of retroposons in the male germ line. This study illustrates the power of comparative genomics in identifying novel hypothetical regulatory elements for analysis with biochemical and in vivo genetic approaches.

Male Germ Cell Apoptosis and Epigenetic Histone Modification Induced by Tripterygium wilfordii Hook F

Multiglycosides of Tripterygium wilfordii Hook f (GTW), a Chinese herb-derived medicine used as a remedy for rheumatoid arthritis, are considered to be a reversible anti-fertility drug affecting the mammalian spermatids. However, the mechanism behind this effect is still unknown. To study the possible mechanism behind the impact of GTW on spermatogenesis, we administered 4 groups of 4-week-old male mice with different doses of GTW. We found a dose-dependent decrease in the number of germ cells after 40 days of GTW treatment, and an increase in apoptotic cells from the low-dose to the high-dose group. During this same period the dimethylated level of histone H3 lysine 9 (H3K9me2) in GTW-treated testes germ cells declined. Additionally, spermatogonial stem cells (SSCs) from 6-day-old mice were isolated to evaluate the possible effect of GTW or triptolide on development of SSCs. We found a significantly higher incidence of apoptosis and lower dimethylation level of H3K9me2 in the SSCs of GTW or triptolide treatment than in controls. Thus, these data suggest that the GTW-induced apoptosis might be responsible for the fertility impairment in mice. This damage could be traced back to the early stages of spermatogenesis. GTW also affected the epigenetic modification of H3K9 in spermatogenesis. Molecular dynamics simulation suggested that triptolide and dimethylated or trimethylated H3K9 might have similar interaction mechanisms with EED (embryonic ectoderm development). These candidate activation mechanisms provide the first glimpse into the pathway of GTW-induced gonad toxicity, which is crucial for further research and clinical application.

Maybe repressed mRNAs are not stored in the chromatoid body in mammalian spermatids

The chromatoid body is a dynamic organelle that is thought to coordinate the cytoplasmic regulation of mRNA translation and degradation in mammalian spermatids. The chromatoid body is also postulated to function in repression of mRNA translation by sequestering dormant mRNAs where they are inaccessible to the translational apparatus. This review finds no convincing evidence that dormant mRNAs are localized exclusively in the chromatoid body. This discrepancy can be explained by two hypotheses. First, experimental artifacts, possibly related to peculiarities of the structure and function of the chromatoid body, preclude obtaining an accurate indication of mRNA localization. Second, mRNA is not stored in the chromatoid body, because, like perinuclear P granules in Caenorhabditis elegans, the chromatoid body functions as a center for mRNP remodeling and export to other cytoplasmic sites.

Electron microscopy analysis of histone acetylation and DNA strand breaks in mouse elongating spermatids using a dual labelling approach

Chromatin remodelling steps in mammalian spermatids include posttranslational modifications of histones and DNA fragmentation. Histone H4 hyperacetylation (AcH4) establishes a chromatin state that facilitates DNA repair in somatic cells. So we sought to determine whether a similar link exists in spermatids by combining immunogold labelling with detection of DNA strand breaks, making use of gold particles of different sizes. DNA strand breaks were not detected in the vicinity of AcH4 chromatin, suggesting that this modified histone may not be involved in the aetiology of DNA fragmentation and repair in spermatids. The AcH4 reactivity, however, indicates that chromatin remodelling is distributed throughout the nucleus.

A novel requirement in mammalian spermatid differentiation for the DAZ-family protein Boule

Reproduction is required for the survival of all animals, yet few reproductive genes have been shown to have a conserved requirement for fertility across the animal kingdom. Remarkably, the RNA binding protein BOULE, the oldest member of the DAZ (Deleted in AZoospermia) family of genes, appears to have maintained its conserved functional motif and spermatogenic expression from insects to humans. Boule mutations lead to a pachytene meiotic arrest before metaphase in Drosophila males and C. elegans females, and human BOULE can restore meiosis in the fly testis, suggesting a conserved meiotic function of human BOULE. However, the physiological function of BOULE in mammals is not yet known. We generated Boule knockout mice and found it to be required only for spermatogenesis, as in Drosophila. Interestingly, meiosis completed normally in the absence of Boule, and haploid round spermatids were readily detected. However, round spermatids did not progress beyond step 6, revealing a novel role for Boule in spermiogenesis, the differentiation of round spermatids into mature spermatozoa. Expression of key regulators of spermiogenesis was unaffected in Boule(-/-) mice, suggesting that Boule regulates germ-cell differentiation through a novel pathway.

Inactivation of the Mouse Adenylyl Cyclase 3 Gene Disrupts Male Fertility and Spermatozoon Function

Mammalian spermatids and spermatozoa express functional G protein-coupled receptors. However, bicarbonate-regulated soluble adenylyl cyclase (AC), the major AC present in these cells, is not directly coupled to G proteins. To understand how G protein-coupled receptors signal in spermatozoa, we investigated whether a conventional transmembrane cyclase is present and biologically active in these cells. Here, we provide evidence for expression of type 3 AC (AC3) in male germ cells and describe the effects of disruption of the AC3 gene on fertility and function of mouse spermatozoa. As previously reported in rat, AC3 mRNA is expressed in mouse testes and localized, together with soluble AC mRNA, mainly in postmeiotic germ cells. AC3 protein was detected by immunolocalization in round and elongating spermatids in a region corresponding to the developing acrosome and was retained in the mature spermatozoa of the epididymis. Forskolin caused a small increase in cAMP production in mouse spermatozoa, but this increase could not be detected in the AC3(-/-) mice. Inactivation of the AC3 gene did not have overt effects on spermatogenesis; however, AC3(-/-) males were subfertile with only three litters generated by 11 males over a period of 6 months. When used in in vitro fertilization, spermatozoa from these AC3(-/-) mice produced few embryos, but their fertilizing ability was restored after removal of the zona pellucida. Despite an apparently normal structure, these spermatozoa had decreased motility and showed an increase in spontaneous acrosome reactions. These data support the hypothesis that AC3 is required for normal spermatid or spermatozoa function and male fertility.

The cancer/testis antigen CAGE-1 is a component of the acrosome of spermatids and spermatozoa

Cancer/testis antigens (CTAs) are characterized by their restricted expression pattern. In normal individuals their expression is largely restricted to the testis. In the case of cancer patients, CTA expression has also been frequently observed in the tumoral cells. CTAs are considered to be promising targets for immunotherapy. However, almost nothing is known about the properties defined by the vast majority of CTAs. Here, we have investigated the expression pattern and localization of the CTA CAGE-1 during mouse spermatogenesis. We show that protein CAGE-1 is 849 amino acids long. Analysis of the first spermatogenic wave of pubertal mice by RT-PCR and immunoblotting showed that CAGE-1 is predominantly expressed during postmeiotic stages. CAGE-1 localizes to the acrosomal matrix and acrosomal granule, as demonstrated by immunocytochemistry at the light and electron microscopic level. Taken together, our results allowed to define protein CAGE-1 as a novel component of the acrosome of mammalian spermatids and spermatozoa.

Transcriptional Regulation via ACTive Transport

Molecules called kinesins are critical for the movement of chromosomes, organelles, and messenger RNAs molecules within cells. Macho et al. now show that the testis-specific kinesin KIF17b can function in the transport of the coactivator ACT and affect transcriptional activity. ACT is a coactivator of CREM-mediated transcription in mammalian spermatids. KIF17b acts as a dose-dependent inhibitor of ACT-dependent transcription by excluding ACT from the nucleus. This work begins to address the molecular mechanism by which germ-cell gene expression is regulated. B. Macho, S. Brancorsini, G. M. Fimia, M. Setou, N. Hirokawa, P. Sassone-Corsi, CREM-dependent transcription in male germ cells controlled by a kinesin. Science 298 , 2388-2390 (2002). [Abstract] [Full Text]

Characterization of POMT2, a novel member of the PMT protein O-mannosyltransferase family specifically localized to the acrosome of mammalian spermatids.

Over the past few years it has emerged that O-mannosyl glycans are not restricted to yeasts and fungi but are also present in higher eukaryotes, including humans. They play a substantial role in the onset of muscular dystrophy and neuronal migration disorders, like muscle-eye-brain disease. Protein O-mannosyltransferase genes (PMTs) are evolutionarily conserved from yeast to human; however, little is known about these enzymes in higher eukaryotes. In this study, we cloned the first PMT2 subfamily members from human (hPOMT2), mouse (mPomt2), and Drosophila (DmPOMT2). A detailed characterization of the mammalian POMT2, with emphasis on mouse Pomt2, shows that mammalian POMT2 is predominantly expressed in testis tissue. Due to differential transcription initiation of the mPomt2 gene, two distinct mRNA species that vary in length are formed. The shorter transcript is present in all somatic tissues examined. Expression of the corresponding hPOMT2 cDNA in mammalian cells identified POMT2 as an integral membrane protein of the endoplasmic reticulum with an apparent molecular weight of 83 kDa. The longer mPomt2 transcript is restricted to testis and encodes a testis-specific mPOMT2 protein isoform. Using in situ hybridization and immunolocalization, we demonstrate that in testis tissue mPOMT2 localizes to maturing spermatids and is abundant within the acrosome, a sperm-specific organelle essential for fertilization. Our data suggest a novel and specific role for the putative protein O-mannosyltransferase POMT2 in the maturation and/or function of sperm in mammals.

Targeting and fusion proteins during mammalian spermiogenesis.

Regulated exocytosis is controlled by internal and external signals. The molecular machinery controlling the sorting from the newly synthesized vesicles from the Golgi apparatus to the plasma membrane play a key role in the regulation of both the number and spatial location of the vesicles. In this context the mammalian acrosome is a unique vesicle since it is the only secretory vesicle attached to the nucleus. In this work we have studied the membrane trafficking between the Golgi apparatus and the acrosome during mammalian spermiogenesis. During bovine spermiogenesis, Golgi antigens (mannosidase II) were detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgiacrosome flow may be relatively unselective, with Golgi residents retrieved before spermination is complete. Surprisingly, rab7, a protein involved in lysosome/endosome trafficking was also found associated with the acrosomal vesicle during mouse spermiogenesis. Our results suggest that the acrosome biogenesis is associated with membrane flow from both the Golgi apparatus and the endosome/lysosome system in mammalian spermatids.

Rat testis motor proteins associated with spermatid translocation (dynein) and spermatid flagella (kinesin-II).

In this study, we report sites in the seminiferous epithelium of the rat testis that are immunoreactive with antibodies to the intermediate chain of cytoplasmic dynein and kinesin II. The study was done to determine whether or not microtubule-dependent motor proteins are present in Sertoli cell regions involved with spermatid translocation. Sections and epithelial fragments of perfusion-fixed rat testis were probed with an antibody (clone 74.1) to the intermediate chain of cytoplasmic dynein (IC74) and to kinesin-II. Labeling with the antibody to cytoplasmic dynein was dramatically evident in Sertoli cell regions surrounding apical crypts containing attached spermatids and known to contain unique intercellular attachment plaques. The antibody to kinesin II reacted only with spermatid tails. The levels of cytoplasmic dynein visible on immunoblots of supernatants collected from spermatid/junction complexes treated with an actin-severing enzyme (gelsolin) were greater than those of controls, indicating that at least some of the dynein may have been associated with Sertoli cell junction plaques attached to spermatids. Results are consistent with the conclusion that an isoform of cytoplasmic dynein may be responsible for the apical translocation of elongate spermatids that occurs before sperm release. Also, this is the first report of kinesin-II in mammalian spermatid tails.

Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins.

Lamina-associated polypeptide 2 (LAP2) and the thymopoietins (TPs) are a family of proteins described in somatic cells of mammals, which are derived by alternative splicing from a single gene. For one of the members of the family (LAP2 = TPbeta) it has been shown that this integral membrane protein locates to the inner membrane of the nuclear envelope, and that it binds to chromatin and B-type lamins. In the present study, we observed that during the third phase of spermatogenesis (i.e. spermiogenesis), TP-labelling shifted progressively to one half of the nuclear periphery in round spermatids. In the elongating spermatid the signal then becomes restricted to one spot located at the posterior (centriolar) pole of the nucleus. Changes in localization are accompanied by the disappearance, first of TPgamma, and later on of LAP2/TPbeta. TPalpha is the only member of the family detectable in the mature sperm. Concomitantly, lamin B1, the only nuclear lamina protein known to be expressed in mammalian spermatids, showed a similar behaviour, i.e. shifted progressively to the centriolar pole of spermatid nuclei before it became undetectable in fully differentiated mature sperms. These results are the first demonstration that expression and localization patterns of TPs are coordinately and differentially regulated with lamins during a differentiation process.

Specific binding of a 47-kilodalton protein to the 3' untranslated region of rat transition protein 2 messenger ribonucleic acid.

The synthesis of transition protein (TNP) 2, one of the predominant nuclear proteins of mammalian spermatids, was shown to be posttranscriptionally regulated, by storing the untranslated mRNA for about 3-5 days in the cytoplasm of differentiating spermatids. It has been proposed that binding of a cytoplasmic protein to a conserved motif of 8 nucleotides (nt) in the 3' untranslated region (3'UTR) of TNP2 mRNA is involved in this translational control mechanism. In this report, we show that deletion or variation of the conserved 8-nt motif (GCCAT-CAC) in rat TNP2-3'UTR abolishes the capacity of the in vitro-transcribed RNA to reconstitute specific RNA-protein complexes in RNA bandshift assays. Using Northwestern analysis, we identified specific binding to the TNP2-3'UTR of four proteins of 45, 47, 49, and 60 kDa, all of which are stage-specifically regulated in male germ cell differentiation. Deletion of the 8-nt motif in the 3'UTR specifically prevented binding of the 47-kDa protein, the interaction of which is thought to be mediated by the RNA secondary structure. Analysis of the RNA secondary structure revealed that the 8-nt motif is an essential element of a specific stem-loop structure that is predicted for rat wild-type TNP2-3'UTR. Therefore we assume that the 47-kDa protein plays an important role in specific RNA-protein complex formation of rat TNP2-3'UTR that may be a central event in the translational control of rat TNP2 mRNA.

Transcription and masking of mRNA in germ cells: involvement of Y-box proteins.

Gametogenesis is directed by various specialized genetic mechanisms which, to a considerable extent, apply to the production of both eggs and sperm and have been conserved across a wide spectrum of eukaryotic organisms. Two key aspects which are discussed here are: germ-cell-specific gene transcription; and translational repression (masking) of mRNA accumulated in oocytes and spermatocytes/spermatids. Together, these two processes conspire to deliver often large amounts of essential proteins at the appropriate stages of development. It is perhaps not surprising that recent evidence points to a functional link between transcription activation and translation repression, both processes being determined in the nucleus and involving common components. One set of components which has been studied recently are members of the Y-box family of regulatory proteins. Most information of the involvement of Y-box proteins in germ cell development comes from studies on amphibian oocytes and mammalian spermatids. In these cells, Y-box proteins have been detected as major components of both maternal and paternal mRNP particles and have been shown to be instrumental in the masking process. Y-box proteins are also implicated in the regulation of several germ-cell-specific genes. Possible connections between these processes are discussed.

Biogenesis of the Posterior-Tail Plasma Membrane Domain of the Mammalian Spermatozoon: Targeting and Lateral Redistribution of the Posterior-Tail Domain-Specific Transmembrane Protein CE9 during Spermiogenesis

We used immunoperoxidase histochemistry and confocal immunofluorescence microscopy to examine the events involved in the compartmentalization of CE9 to the posterior-tail plasma membrane domain during spermatogenesis in the rat. We identified two major episodes of spermatogenesis during which CE9 appeared to accumulate in relatively large amounts intracellularly within elements of the secretory pathway. The first episode encompassed cells from preleptotene through early pachytene primary spermatocytes and was evident as intense intracellular labeling of the endoplasmic reticulum and the Golgi complex. The second episode encompassed spermatids in steps 8-12 of spermiogenesis and was evident as intense intracellular labeling of the Golgi complex and smaller vesicular structures observed within the cytoplasm of the spermatid. Between these two episodes, CE9 was detected in considerably reduced amounts. Although present within the Golgi complex and the acrosomic system throughout much of the first half of spermiogenesis, CE9 was not detected on the tail of the spermatid until steps 8-9 of spermiogenesis. Although detected initially in relatively small amounts along the entire length of the tail beginning at steps 8-9, there was no evidence for the presence of relatively large amounts of CE9 on the tail or anywhere else on the surface of the spermatid until after step 11 of spermiogenesis. Between step 11 and steps 13-14 of spermiogenesis, CE9 was observed to accumulate in relatively large amounts on the whole tail coincident with its apparent loss from the Golgi complex. CE9 was observed to then undergo further compartmentalization to the posterior-tail domain sometime between steps 13-14 of spermiogenesis and spermiation. Our results suggest that CE9 is synthesized and enters the secretory pathway throughout much of spermatogenesis, but that the site of accumulation of CE9 varies considerably as a function of development. With respect to the biogenesis of the posterior-tail plasma membrane domain, our results suggest that CE9 is targeted from the Golgi complex to the plasma membrane of the whole tail during mid to late spermiogenesis and then redistributes laterally into the posterior-tail domain coincident with the caudal migration of the annulus late in spermiogenesis. This proposed pathway has a number of important implications for the logistical capabilities of the mammalian spermatids.

Identification of a functional cyclic adenosine 3',5'-monophosphate response element in the 5'-flanking region of the gene for transition protein 1 (TP1), a basic chromosomal protein of mammalian spermatids.

Transition protein 1 (TP1) is a small basic chromosomal protein that appears in mammalian spermatids during the period of chromatin condensation. The gene for TP1 from several species contains an apparent cAMP response element (CRE) in the immediate 5'-flanking region. The recent identification of high expression of the novel CRE-activating protein (CREM tau) in advanced testicular germ cells provided a stimulus to ask whether or not the TP1 CRE is functional. To this end we show both by gel retardation and by footprint assays that TP1 CRE forms specific bound complexes with proteins in whole testis nuclear extracts and that these complexes involve CREM as evidenced by recognition by a specific antibody. In addition, the TP1 CRE forms specific bound complexes with bacterially expressed CREM tau. Finally, the TPI CRE conveys protein kinase A-dependent induction to a linked chloramphenicol acetyl transferase gene when transfected into JEG-3 cells. Accordingly, TP1 is a good candidate for a testis-specific gene subject to CREM tau regulation.

Ultrastructure of goat testes: centriolar adjunct in spermiogenesis.

During the morphological study of goat spermatogenesis, cross and oblique sections of the neck region (connecting piece) of the spermatid were closely studied in conjunction with the shaping of the sperm head; the centriolar adjunct (CA) briefly appeared as a fragile organelle in spermiogenesis. As is the case of other mammalian spermatids, the CA expanded about 1 microns in length from the distant end of the proximal centriole (PC). The prolongation of this structure began in an early stage of the cap phase in spermiogenesis, and it usually oriented itself parallel to the flat plane of the flattening spermatid head. In other words, the CA appears to regulate the flattening direction of the spermatid head. Thus, the expanding direction of this structure showed a declination of about 25 degrees C toward the ninth microtubule in the axoneme of the sperm tail. After regulating the head shape, this accessory apparatus quickly disappeared during the maturation phase. If this observation is correct and not an artifact of sampling procedures, the basic mechanisms of sperm motility might be elucidated.