Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3β-hydroxysteroid dehydrogenase (3β-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3β-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats.
In eukaryotes, diploid cells give rise to haploid cells via meiosis, a program of two cell divisions preceded by one round of DNA replication. Although key molecular components of the meiotic apparatus are highly conserved among eukaryotes, the mechanisms responsible for initiating the meiotic program have diverged substantially among eukaryotes. This raises a related question in animals with two distinct sexes: Within a given species, are similar or different mechanisms of meiotic initiation used in the male and female germ lines? In mammals, this question is underscored by dramatic differences in the timing of meiotic initiation in males and females. Stra8 is a vertebrate-specific, cytoplasmic factor expressed by germ cells in response to retinoic acid. We previously demonstrated that Stra8 gene function is required for meiotic initiation in mouse embryonic ovaries. Here we report that, on an inbred C57BL/6 genetic background, the same factor is also required for meiotic initiation in germ cells of juvenile mouse testes. In juvenile C57BL/6 males lacking Stra8 gene function, the early mitotic development of germ cells appears to be undisturbed. However, these cells then fail to undergo the morphological changes that define meiotic prophase, and they do not display the molecular hallmarks of meiotic chromosome cohesion, synapsis and recombination. We conclude that, in mice, Stra8 regulates meiotic initiation in both spermatogenesis and oogenesis. Taken together with previous observations, our present findings indicate that, in both the male and female germ lines, meiosis is initiated through retinoic acid induction of Stra8.
Spermatogonial stem cells (SSCs or As spermatogonia) in rodents and rams are single cells that can self-renew or form a pair (Apr) that will continue to differentiate and form chains of Aal spermatogonia. Aal spermatogonia differentiate into A1 spermatogonia that after six divisions produce spermatocytes via A2, A3, A4, In, and B spermatogonia. The cell cycle times of each of the generations of A1-B spermatogonia are similar and are about 14% of the duration of the epithelial cycle. In contrast, the cell cycle times of the As,pr,al spermatogonia are highly variable, the minimal cell cycle time being about 30% longer than that of the A1-B spermatogonia. During the epithelial cycle the As,pr,al spermatogonia start to proliferate at about stage X and the Apr and Aal spermatogonia stop dividing around stage II, while the SSCs continue to proliferate until stage VI. There is a feedback regulation between the numbers of A1-B spermatogonia and the length of the proliferative period of the As,pr,al spermatogonia. When the number of A1-B spermatogonia is low, the As,pr,al spermatogonia continue to proliferate longer. The As,pr,al spermatogonia in different epithelial areas produce variable numbers of A1 spermatogonia but always more than needed, subsequently the surplus of A2–A4 will enter apoptosis, ensuring an even distribution of germ cells from In spermatogonia onwards.
All components of the double-stranded DNA break (DSB) repair complex DNA-dependent protein kinase (DNA-PK), including Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs), were found in the radiosensitive spermatogonia. Although p53 induction was unaffected, spermatogonial apoptosis occurred faster in the irradiated DNA-PKcs-deficient scid testis. This finding suggests that spermatogonial DNA-PK functions in DNA damage repair rather than p53 induction. Despite the fact that early spermatocytes lack the Ku proteins, spontaneous apoptosis of these cells occurred in the scid testis. The majority of these apoptotic spermatocytes were found at stage IV of the cycle of the seminiferous epithelium where a meiotic checkpoint has been suggested to exist. Meiotic synapsis and recombination during the early meiotic prophase induce DSBs, which are apparently less accurately repaired in scid spermatocytes that then fail to pass the meiotic checkpoint. The role for DNA-PKcs during the meiotic prophase differs from that in mitotic cells; it is not influenced by ionizing radiation and is independent of the Ku heterodimer.
The response of spermatogonial stem cells in the CBA mouse to 1 MeV fission neutrons was investigated. Spermatogenetic clones arising from surviving stem cells were counted indirectly by determining the fraction of repopulated tubules in testicular sections at four time intervals (3, 5, 8, and 11 weeks) after irradiation with graded doses (50 to 410 rad) of fast neutrons. This fraction, the repopulation index (RI), is proved to be a linear parameter of the number of surviving stem cells when determined between 5 and 11 weeks after irradiation. The mean D/sub 0/ value of the stem cell survival curves in this period amounts to 81 +- 2 rad, pointing to the existence in the mouse testis of a highly radioresistant population of stem cells. Evidence was obtained that age of the animal does not influence stem cell sensitivity once the animal is adult, though clones grow at a slower rate in older animals. Our findings permit an estimate of the number of stem cells present in the normal unirradiated testis. This number amounts to about 1800. The existence of a population of more radiosensitive stem cells cannot be excluded. Stem cell survival after irradiation with 300 kV x rays wasmore » also investigated. The x-ray curve is exponential from 400 rad onward; the value of D/sub 0/ is 242 +- 7 rad, and the quasi-threshold dose D/sub q/ amounts to 165 rad. By comparison with the neutron curve for the same time interval the RBE of 1 MeV fission neutrons was calculated. The RBE decreases from 5.5, at a surviving fraction of stem cells of 0.30, to 4.1 at a fraction of 0.01.« less
Retinoid X receptors (RXRs) are key regulators in retinoid signaling. Knowledge about the effects of 9-cis-retinoic acid (9-cis-RA), the natural ligand for the RXRs, may also provide insight in the functions of RXRs. In this study, the effect of 9-cis-RA on spermatogenesis in vitamin A-deficient (VAD) mice was examined. Administration of 9-cis-RA stimulated the differentiation and subsequent proliferation of the growth-arrested A spermatogonia in the testis of VAD mice. However, compared with all-trans-retinoic acid (ATRA), relatively higher doses of 9-cis-RA were necessary. This could not simply be due to a lower or delayed activity of 9-cis-RA, as simultaneous administration of ATRA and 9-cis-RA did not cause a synergistic effect. Instead, the presence of 9-cis-RA diminished the effect of ATRA by approximately one third. Studies of in vivo transport and metabolism showed that ATRA and 9-cis-RA, after administration to VAD mice, penetrated the testis equally well. However, 9-cis-RA was metabolized much faster than ATRA, and other metabolites were formed. This may account for the above-described differential effects of ATRA and 9-cis-RA on spermatogenesis. Similar to ATRA, 9-cis-RA transiently induced the messenger RNA expression of the nuclear RA receptor RARβ, suggesting a role for this receptor in the effects of retinoids on the differentiation and proliferation of A spermatogonia. In contrast, the messenger RNA expression of the nuclear retinoid receptors RXRα, -β, and -γ was not changed significantly by administration of their ligand, 9-cis-RA. Hence, 9-cis-RA does not seem to exert its effect on spermatogenesis through altered expression of the RXRs.
Abstract Paternal chromatin undergoes extensive structural and epigenetic changes during mammalian spermatogenesis, producing sperm that contain an epigenome optimal for the transition to embryogenesis. Histone modifiers play an important role in this process by encoding specialized regulatory information in the sperm epigenome. Lysine demethylase 6a (KDM6A) promotes gene activation via demethylation of H3K27me3, a developmentally important repressive modification abundant throughout the epigenome of sperm and embryonic stem cells. Despite its developmental importance in pluripotent cells and germ cell progenitors, the function of KDM6A during spermatogenesis has not been described. Here, we show that Kdm6a is transiently expressed in the male germline in late spermatogonia and during the early stages of meiotic entry. Deletion of Kdm6a in the male mouse germline ( Kdm6a cKO) yielded a modest increase in sperm head defects but did not affect fertility or the overall progression of spermatogenesis. However, hundreds of genes were deregulated upon loss of Kdm6a in spermatogenic cells and in an immortalized spermatogonia cell line (GC-1 spg) with a strong bias towards downregulation. Single cell RNA-seq revealed that most of these genes were deregulated in spermatogenic cells at the same stage when Kdm6a is expressed and encode epigenetic factors involved in chromatin organization and modification. A subset of these genes was persistently deregulated in the male germ line across two generations of offspring of Kdm6a cKO males. Our findings highlight KDM6A as a transcriptional activator in the mammalian male germline that is dispensable for spermatogenesis but important for safeguarding gene regulatory state intergenerationally. Author summary Offspring viability and fitness relies upon the development of functional sperm and the integrity of information that they carry. Chromatin is modified and remodeled extensively throughout spermatogenesis to facilitate meiosis, DNA compaction, and to encode gene regulatory information for the next generation. In mice, a paternal germline lacking KDM6A, a histone modifier, yields offspring with reduced lifespans and increased cancer risk. How KDM6A functions in the paternal germline to support offspring health is unknown. Here, we show that Kdm6a expression is limited to a distinct developmental interval when differentiated spermatogonia transition from mitosis to meiosis. During this timepoint, KDM6A acts as a transcriptional activator for hundreds of genes, many of which encode meiotic factors and epigenetic modifiers. Nevertheless, this activity is dispensable for overall spermatogenesis and fertility. Surprisingly, we find a significant overlap in germline transcriptomes of Kdm6a cKO mice and wildtype offspring. We propose that KDM6A encodes gene regulatory information in the male germline that is retained across generations.
ABSTRACT The specialized cell cycle of meiosis transforms diploid germ cells into haploid gametes. In mammals, diploid spermatogenic cells acquire the competence to initiate meiosis in response to retinoic acid. Previous mouse studies revealed that MEIOC interacts with RNA-binding proteins YTHDC2 and RBM46 to repress mitotic genes and promote robust meiotic gene expression in spermatogenic cells that have initiated meiosis. Here, we used the enhanced resolution of scRNA-seq, and bulk RNA-seq of developmentally synchronized spermatogenesis, to define how MEIOC molecularly supports early meiosis in spermatogenic cells. We demonstrate that MEIOC mediates transcriptomic changes before meiotic initiation, earlier than previously appreciated. MEIOC, acting with YTHDC2 and RBM46, destabilizes its mRNA targets, including transcriptional repressors E2f6 and Mga , in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate transcriptional regulator STRA8-MEIOSIN, required for the meiotic G1/S phase transition and meiotic gene expression. We conclude that in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of spermatogenic cells to initiate meiosis. SUMMARY STATEMENT RNA-binding complex MEIOC-YTHDC2-RBM46 destabilizes its mRNA targets, including transcriptional repressors. This activity facilitates the retinoic acid-dependent activation of Meiosin gene expression and transition into meiosis.
