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PubMed1999

Round spermatid-specific transcription of the mouse SP-10 gene is mediated by a 294-base pair proximal promoter.

Spermiogenesis is the terminal phase of male germ cell differentiation during which haploid spermatids engage in coordinate expression of a number of testis-specific genes, including those specifying acrosomal proteins. To begin to understand the transcriptional regulation during acrosomal biogenesis, we initiated promoter analysis of the gene encoding the acrosomal protein SP-10. SP-10 was previously shown to be transcribed within Golgi-phase round spermatids in the human. The present study characterizes SP-10 gene expression during spermiogenesis in the mouse and identifies regions of the mouse SP-10 (mSP-10) promoter that are capable of driving round spermatid-specific transcription in vivo. Expression of mSP-10 mRNA was initiated in early round spermatids coincident with acrosomal biogenesis and was terminated prior to nuclear elongation. The core promoter of mSP-10 lacked a TATA box but contained a canonical initiator (Inr) element surrounding the transcription start site. Using transgenic mice, we showed that the -408 to +28-base pair (bp) or the -266 to +28-bp mSP-10 5' flanking region is sufficient to direct round spermatid-specific expression of a green fluorescent protein reporter gene. On the other hand, the -91 to +28-bp mSP-10 gene fragment lacked promoter activity in vivo. This is the first functional characterization of a testis-specific gene promoter active in early round spermatids.

Acrosome↗
PubMedDate not supplied

Temporal coincidence of the appearance of elongated spermatids and of histochemical reaction of 11 beta-hydroxysteroid dehydrogenase in rat Leydig cells.

The serial semithin sections of the testes of perfused rats (age: 25-41 postnatal days) were evaluated microscopically on each alternative day for the morphology of spermatogenetic cells and especially for spermatid differentiation. The histochemical reaction for non-specific esterases, 3 alpha-hydroxysteroid-dehydrogenase (HSDH), 3 beta-HSDH, 11 beta-HSDH, 17 beta-HSDH were performed in cryostat sections of corresponding rat testes (age: 25-55 days). Eight different types of germ cell associations (designated as A-H) appeared from day 25 to day 41. Seven steps of differentiation of spermatids were characterized. The turning point of spermatid maturation was observed on day 35. Prior to this day, mainly spermatids step 1 were identified. On day 35 spermatids steps 1-5 were present. The reactions of non-specific esterases, 3 beta-HSDH, 17 beta-HSDH and 3 alpha-HSDH were observed in the Leydig cells of all testes. Only from day 35 onwards could 11 beta-HSDH be demonstrated. The temporal sequence of the differentiation of spermatids and the appearance of histochemical activity of 11 beta-HSDH in the Leydig cells seem to be closely correlated. An additional observation concerning the presence of germ cell debris was made in the seminiferous tubules during the postnatal development. Large amounts of debris were observed only till day 33.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2004

In vitro maturation of fresh and frozen-thawed mouse round spermatids.

Both initiation and maintenance of spermatogenesis are hormonally regulated by follicle stimulating hormone (rFSH) and testosterone. Co-culture systems also have important roles in the maintenance of spermatogenic cells. In this study, the effects of FSH and testosterone, co-culture system with Vero cells and co-culture supplemented with the hormones for maturation of frozen-thawed spermatids were determined. Testicular cells were suspended from the testis of National Medical Research Institute (NMRI) male mice and divided into two parts. The first aliquot of suspension was allocated for using as fresh and the rest was quickly cryopreserved. The frozen specimens were thawed and washed using Dulbecco modified Eagle's minimum essential medium (DMEM) medium. The fresh specimens were cultured in four groups: control (cultured on DMEM with 10% FBS), hormone (cultured on a medium supplemented with rFSH and testosterone), co-culture (cultured on Vero cells) and co-culture + hormone (cultured on Vero cells combined with rFSH and testosterone). The frozen-thawed specimens were cultured accordingly. The number of spermatids was recorded daily and the survival rates of each group were evaluated using Trypan blue test. The results showed that the number of the elongating spermatids was increased during the first day of the culture of fresh hormone, co-culture and co-culture + hormone groups. Viability rates of all kinds of the spermatid reduced during the 96 h of culturing. Our findings showed that the addition of hormone could support cell viability better than the co-culture. They also confirmed that the fresh round spermatid cells can progress into elongating and elongated spermatid only within the first 2 days of the culture in hormone, co-culture and co-culture + hormone groups. In the frozen-thawed specimens no extra significant increase in the number of cells was observed.

Androgens↗
PubMed2006

Loss of TSLC1 causes male infertility due to a defect at the spermatid stage of spermatogenesis.

Tumor suppressor of lung cancer 1 (TSLC1), also known as SgIGSF, IGSF4, and SynCAM, is strongly expressed in spermatogenic cells undergoing the early and late phases of spermatogenesis (spermatogonia to zygotene spermatocytes and elongating spermatids to spermiation). Using embryonic stem cell technology to generate a null mutation of Tslc1 in mice, we found that Tslc1 null male mice were infertile. Tslc1 null adult testes showed that spermatogenesis had arrested at the spermatid stage, with degenerating and apoptotic spermatids sloughing off into the lumen. In adult mice, Tslc1 null round spermatids showed evidence of normal differentiation (an acrosomal cap and F-actin polarization indistinguishable from that of wild-type spermatids); however, the surviving spermatozoa were immature, malformed, found at very low levels in the epididymis, and rarely motile. Analysis of the first wave of spermatogenesis in Tslc1 null mice showed a delay in maturation by day 22 and degeneration of round spermatids by day 28. Expression profiling of the testes revealed that Tslc1 null mice showed increases in the expression levels of genes involved in apoptosis, adhesion, and the cytoskeleton. Taken together, these data show that Tslc1 is essential for normal spermatogenesis in mice.

Animals↗
PubMed1989

Expression of replication-dependent histone genes in avian spermatids involves an alternate pathway of mRNA 3'-end formation.

In somatic cells the expression of replication-dependent histone genes is coupled to the S phase of the cell cycle. However, we have found a number of novel H2a, H2b, and H3 poly(A)+ RNA species in avian haploid round spermatids. The spermatid-specific H2a and H2b 0.8-kilobase RNAs are transcribed from a subset of the replication-dependent H2a and H2b gene families. Two cDNAs derived from the spermatid-specific H2b transcripts were isolated and sequenced. The structures of these cDNAs reveal that the spermatid-specific RNAs are identical to the 0.5-kilobase poly(A)- H2b mRNAs expressed in proliferating somatic cells, except for the addition of poly(A) at the 3' ends. The site of poly(A) addition in the spermatid-specific RNAs is located 26 to 28 nucleotides 3' of the poly(A)- H2b mRNA terminus. Thus, the hairpin structures and purine-rich elements required for the U7 small nuclear ribonucleoprotein-mediated cleavage reaction that generates the 3' ends of poly(A)- H2b mRNAs are not utilized in spermatids and are retained in the poly(A)+ H2b RNAs.

Amino Acid Sequence↗
PubMed1986

Diploid spermatids: a manifestation of spermatogenic impairment in XOSxr and T31H/+ male mice.

Microdensitometry of Feulgen-stained spermatogenic cell preparations from six XOSxr and four T31H/+ male mice revealed large numbers of spermatids with a diploid amount of DNA. In XOSxr mice, where the diploid spermatids usually constituted the majority of the spermatid population, there were large differences from mouse to mouse in the proportion of diploid spermatids (range, 35%-94%). There were large losses of both haploid and diploid spermatids during spermiogenesis, and the later condensed spermatids of both types were grossly misshapen. It is suggested that the omission of one meiotic division, and the other spermiogenic anomalies may be manifestations of a meiotic "quality control" mechanism that destroys the products of those pachytene spermatocytes which have unsynapsed or incompletely synapsed chromosomes.

Animals↗
PubMed2001

The hsp60B gene of Drosophila melanogaster is essential for the spermatid individualization process.

The 60-kDa heat shock protein family (Hsp60) is found in prokaryotes, mitochondria, and chloroplasts. The Hsp60 proteins promote proper protein folding by preventing aggregation. In Drosophila melanogaster, the hsp60 gene is essential for a variety of developmental processes, beginning at early embryogenesis. In this study we show that an additional member of the Drosophila hsp60 gene family, hsp60B, is essential in male fertility. In males homozygous for a mutation of the hsp60B gene, developmental processes appeared normal throughout most of spermatogenesis, including spermatocyte growth, meiosis, and spermatid elongation. At these stages, mitochondria also displayed a differentiation process similar to wild-types. However, we found that the mutation disrupted a late stage of spermatogenesis, the spermatid individualization process. In this process, the individualization complex is assembled at spermatid nuclear heads, traverses along spermatid tails, and generates membranes for each of the spermatids in a cyst. Our analysis further shows that the individualization complex in sterile males displayed abnormal morphology as it was traveling along the spermatid tails. The Drosophila Hsp60 proteins are believed to be exclusively localized in the mitochondria. Our observation that the hsp60B mutation displayed no apparent defect in mitochondrial differentiation during spermatogenesis suggests that the Hsp60B protein may operate in a nonmitochondrial location.

Alleles↗
PubMed1990

Effect of glutathione depletion on the cytotoxicity of xenobiotics and induction of single-strand DNA breaks by ionizing radiation in isolated hamster round spermatids.

The role of glutathione (GSH) in cellular protection mechanisms in round spermatids from hamsters was studied. Isolated spermatids were largely depleted of GSH by treating the cells for 2 h with the GSH conjugating agent diethyl maleate (DEM). This treatment resulted in a 90% decrease of the cellular GSH content, but did not affect the ATP content. Exposure of isolated spermatids to cumene hydroperoxide (CHP), a compound which is detoxicated by the GSH redox cycle, showed that the cytotoxicity of the peroxide was markedly potentiated by GSH depletion of the cells. The cytotoxicity was reflected by the cellular ATP content. A decrease of the ATP content of the GSH-depleted spermatids was observed at 5-6-fold lower CHP concentrations, as compared to control cells. An increased cytotoxicity in GSH-depleted cells was also observed using 1-chloro-2,4-dinitrobenzene (CDNB), which is a reactive compound that is detoxicated by glutathione conjugation. The induction of single-strand DNA breaks by gamma radiation was 3-5-fold higher in GSH-depleted spermatids as compared to control cells. This radiation-induced damage was estimated under hypoxic conditions (500 p.p.m. O2 in N2). GSH depletion did not affect the repair of single-strand DNA breaks following the irradiation. The present results indicate that cellular GSH has an important function in the defence mechanisms of round spermatids against peroxides, electrophilic xenobiotics and radiation-induced DNA damage.

Adenosine Triphosphate↗
PubMed2004

Failure to assemble the peri-nuclear structures in GOPC deficient spermatids as found in round-headed spermatozoa.

Deletion of the GOPC gene encoding mouse GOPC (Golgi-associated PDZ- and coiled-coil motif-containing protein) causes infertile round-headed spermatozoa, which have acrosome-less round heads and deformed tails (Yao et al, 2002). This study investigated how GOPC deficient spermatids fail to assemble the peri-nuclear structures in round-headed spermatozoa during spermiogenesis in GOPC knockout mouse testes. In step 1-8 spermatids, Golgi-derived proacrosomal vesicles that are transported to the perinuclear region formed acrosome-like vesicles of various sizes, called pseudoacrosomes. The marginal ring of the acroplaxome, which is generally formed between the descending edge of a developing acrosome and nuclear envelope in a wild spermatid, was poorly formed between the pseudoacrosome and nuclear envelope. In step 9-11 elongating spermatids, a majority of pseudoacrosomes were detached from the nucleus and disappeared from the perinuclear region by spermiation. Concomitantly, several failures occurred on the nucleus, manchette, postacrosomal sheath (perinuclear theca), and posterior ring. Ectoplasmic specializations were poorly formed, and did not always associate with developing spermatids. Consequently, spermatid nuclear elongation to form round-headed spermatozoa developed was impaired. In addition to these sequential failures, the posterior ring deficiency was attributed to the tail deformation destined to occur during epididymal maturation as reported in an accompanying paper (Suzuki-Toyota et al, 2004 in this issue), its eventual phenotype being reminiscent of the round-headed spermatozoa of human infertile globozoospermia.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Stage-specific expression of the Atce1/Tisp40alpha isoform of CREB3L4 in mouse spermatids.

The maturation of haploid spermatids into spermatozoa relies on the timely production of proteins required for spermatid differentiation. The mammalian CREB3L4 (cAMP responsive element binding protein 3-like 4) gene encodes a bZIP transcription factor that associates with the membrane of the endoplasmic reticulum. CREB3L4 is presumed to play an important role in protein maturation via its involvement in the cellular response to endoplasmic reticulum stress. In mice, the Creb3l4 gene gives rise to 2 distinct classes of mRNAs through the use of alternate promoters. Transcripts that initiate upstream of the first coding exon encode a 370-amino acid (aa) protein designated Tisp40beta, whereas transcripts that initiate downstream of the first coding exon encode Atce1/Tisp40alpha, a truncated (315-aa) form of Tisp40beta. In the mouse testis, Creb3l4 transcripts are known to be expressed exclusively in postmeiotic spermatids but the presence of CREB3L4 protein in spermatids has not been formally demonstrated. We produced an antibody directed against the carboxy terminus of mouse CREB3L4 and used it in immunostaining experiments to document that CREB3L4 protein accumulates in post-meiotic spermatids in a stage-specific manner. Moreover, we show that Atce1/Tisp40alpha is the major form of CREB3L4 in mouse testis. These findings suggest that testis-specific isoforms of Creb3l4 could play an important role in spermatid differentiation.

Animals↗
PubMed1989

Endocytosis in spermatids during spermiogenesis of the mouse.

In the course of spermiogenesis in the mouse, spermatid cytoplasm contains numerous membrane pits, vesicles and membranous tubules which are frequently anastomosed. Pale and dense multivesicular bodies (MVB) and secondary lysosome-like structures are also present in the cytoplasm. In order to study the pathway of non-specific adsorptive endocytosis in spermatids, cationic ferritin (CF) was directly microinjected into the lumen of seminiferous tubules, and added to germinal cell culture. Tissue and cultures were fixed at various time intervals after injection. Two-5 hr after microinjection of tracer, CF was found simultaneously in vesicles, tubules, MVB and in lysosome-like bodies present in spermatids at all steps of spermiogenesis. Various membranous components of the Golgi medulla, and the innermost transsaccule of the Golgi cortex were labelled simultaneously. In primary cultures of spermatids, the vesicles contained the marker 5 min after its deposition; 10 min after deposition, CF was evident in tubules; at 30 min, CF was present in pale MVB; at 1 hr, the dense MVB and lysosome-like bodies were labelled. Finally, at 2 hr 30 min, vesicles and tubules of the Golgi medulla contained CF grains. Apparently spermatids are very active cells in the process of adsorptive endocytosis throughout spermiogenesis. Endocytosis in spermatids is probably one of the mechanisms involved in the uptake of material used to build up spermatozoa components. The strong labelling of the Golgi region probably point to its role in recycling endocytosed membranes.

Animals↗
PubMed1982

[Morphology of spermatids in elderly subjects].

This study has been performed with the aim of clarifying whether spermatid differentiation still proceeds regularly in advanced age. Specimens have been observed of testicular tissue from 108 patients orchidectomized because of prostatic carcinoma and before initiation of hormone therapy. In 30 subjects, 65 to 85 years old, mature spermatids have been found in the semithin sectioned germinal epithelium. The testicular tissue of these 30 patients has been investigated by means of transmission electron microscope. In the studied material most of the spermatids appear malformed. In patients less than 75 years old, about 70% of all spermatids are abnormal, in older patients nearly 90%. The malformations concern all cellular components, but can be seen most frequently in the acrosome and in the nucleus. There are isolated malformations, e.g. of the acrosome; however also combined malformations, e.g. the acrosome, the nucleus and the tail can be observed. In addition to these disturbances of spermatid differentiation, even disturbances of spermiation can be found, presumably caused by a misled function of the Sertoli cells. In these cases, a spermatid loses its contact with the Sertoli cell before final maturation.

Acrosome↗
PubMed1981

Stimulation of protein synthesis in round spermatids from rat testes by lactate.

Lactate markedly increased the rate of [3H]leucine incorporation into the protein of isolated round spermatids (steps 1-8) from rat testes. Four kinds of hexoses, glucose, fructose, galactose, and monnose, also stimulated [3H]leucine incorporation, but to much lesser extents than lactate. Ribose had no effect. The glucose-induced stimulation of protein synthesis was entirely suppressed by iodoacetate and NaF, whereas iodoacetate and NaF were without effect on the lactate-induced increase in protein synthesis. Lactate stimulated both protein synthesis and ATP production in the spermatids. However, both of these stimulatory effects of lactate were completely blocked by DNP and rotenone. Rotenone entirely blocked oxygen consumption, as expected, whilst DNP enhanced it additively with lactate. Moreover, lactate was without influence on either transport of alpha-[3H]AIB into spermatids or incorporation of [3H]leucine into protein of a cell-free system of spermatids. These findings suggest that lactate may increase the protein synthesis of spermatids in the same fashion as glucose, and that the effect of lactate in increasing the level of ATP during incubation in vitro may be a major factor in the mechanism of stimulation of protein synthesis in the spermatids.

Adenosine Triphosphate↗
PubMed1994

Seasonal changes in spermatogenesis and ultrastructure of developing spermatids in the Japanese rat snake, Elaphe climacophora.

Seasonal changes in spermatogenesis and the ultrastructure of developing spermatids in the Japanese rat snake, Elaphe climacophora, were observed by light and transmission electron microscopy. Spermatogenesis in this species began in August, continued through October and ceased in November. No spermatozoa were found during other periods. Japanese rat snakes mate from May to July, and spermatogenesis occurs after the mating season. Therefore, according to Girons' classification (1982), the reproduction of this species was classified as a postnuptial type. Although the morphological changes in developing spermatids during spermiogenesis in these snakes fundamentally resembled those in mammals, some unique features were detected. The most prominent characteristic of the developing spermatids was lipid-like structures. These structures first appeared in early round spermatids, gradually increased in size and number, and, finally, aligned around the nucleus of mature spermatids. Although spermatozoa with lipid-like structures were released from the epithelium, stored spermatozoa in vas deferenses had none of these structures. Lipid-like structures that apparently separated from spermatozoa were scattered in the vas deferens. Prominent elongation of mitochondria was also remarkable in elongated spermatids.

Acrosome↗
PubMed2003

Phosphorylation of rat spermatidal protein TP2 by sperm-specific protein kinase A and modulation of its transport into the haploid nucleus.

Transition protein 2 (TP2), which is expressed during stages 12-15 of mammalian spermiogenesis, has been shown to undergo phosphorylation immediately after its synthesis. We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA). We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA). Reverse transcription-PCR analysis indicated that Cs-PKA was present in the haploid spermatids and absent from premeiotic germ cells. The rat Cs-PKA transcript was amplified and sequenced using the isoform-specific primers. The sequence of rat Cs-PKA at the N terminus differs from mouse and human by one amino acid. Western blot analysis using specific anti-Calpha1 antibodies revealed that Calpha1-PKA is absent in haploid spermatid cytosol. We have also established an in vitro nuclear transport assay for the haploid round spermatids. Using this assay, we have found that the cytoplasmic factors and ATP are absolutely essential for translocation of TP2 into the nucleus. Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus.

Active Transport, Cell Nucleus↗
PubMed2003

[Advances in microinsemination with spermatid].

Men with non-obstructive azoospermia(NOA) can now be treated by using intra-oocyte round spermatid injection(ROSI) or elongated spermatid injection(ELSI). Spermatids can be retrieved from semen or from testis biopsy specimens. But the rates of fertilization and pregnancy with spermatids have been disappointing. Many problems limiting success rate and hindering a wide application of this technique still remain unresolved, including the incomplete maturation of spermatid nuclear, oocyte activation and identification of a live spermatid.

Azoospermia↗
PubMed1999

Cadmium concentrations in the testes, sperm, and spermatids of mice subjected to long-term cadmium chloride exposure.

BACKGROUND: Exposures to cadmium have been reported to reduce male fertility and there are several hypotheses that suggest how reduced male fertility may result from incorporation of cadmium into sperm chromatin. The purpose of this study was to determine whether mice subjected to long-term intraperitoneal cadmium exposure incorporated cadmium into their sperm chromatin. METHODS: Male mice were exposed to 0.1 mg/kg body weight cadmium in the form of CdCl2 via intraperitoneal injection once per week for 4, 10, 26, and 52 weeks and then sacrificed. The cadmium contents of the liver, testes, pooled sperm, and pooled spermatids from dosed and control animals were determined by atomic absorption spectroscopy. Cadmium and zinc contents in individual sperm and spermatid heads were determined by particle-induced x-ray emission. RESULTS: Atomic absorption spectroscopy revealed that although cadmium accumulated in the liver and testes, cadmium was not detected in pooled sperm or spermatid samples down to minimum detectable limits of 0.02 microg/g dry weight. Particle-induced x-ray emission analyses did not show the presence of cadmium in any sperm or spermatid head down to minimum detectable limits of 15 microg/g dry weight. Particle-induced x-ray emission analyses also demonstrated that phosphorus, sulfur, and zinc concentrations in individual sperm and spermatid heads were not altered by exposure to CdCl2. CONCLUSIONS: Because cadmium was not incorporated into sperm chromatin at levels above 0.02 microg/g dry weight, the data cast doubt on hypotheses that suggest that reduced male fertility may result from incorporation of cadmium into sperm chromatin.

Animals↗
PubMed1996

Micronuclei induced in round spermatids of mice after stem-cell treatment with chloral hydrate: evaluations with centromeric DNA probes and kinetochore antibodies.

The chromosomal effects of chloral hydrate (CH) on germ cells of male mice were investigated using two methods to detect and characterize spermatid micronuclei (SMN); (a) anti-kinetochore immunofluorescence (SMN-CREST) and (b) multicolor fluorescence in situ hybridization with DNA probes for centromeric DNA and repetitive sequences on chromosome X (SMN-FISH). B6C3F1 mice received single intraperitoneal (i.p.) injections of 82.7, 165.4, or 413.5 mg/kg and round spermatids were sampled at three time intervals representing cells treated in late meiosis, early meiosis, or as spermatogonial stem cells. No increases in the frequencies of SMN were detected for cells treated during meiosis using either SMN-CREST or SMN-FISH methods. After spermatogonial stem-cell treatment, however, elevated frequencies of SMN were detected by both methods. With SMN-FISH, dose trends were observed both in the frequencies of spermatids containing micronuclei and in the frequency of spermatids carrying centromeric label. These findings corroborate the recent report by Allen and colleagues [Allen JW et al.(1994): Mutat. Res. 323:81-88] that CH treatment of spermatogenic stem cells induced SMN. Furthermore, our findings suggest that chromosomal malsegregation or loss may occur in spermatids long after CH treatment of stem cells. Further studies are needed to understand the mechanism of action of the CH effect on stem cells and to determine whether similar effects are induced in human males treated with CH.

Anesthetics, Intravenous↗