Mitotic Germ Cells Research Articles

Complete Sertoli cell proliferation induced by follicle-stimulating hormone (FSH) independently of luteinizing hormone activity: evidence from genetic models of isolated FSH action.

Defining the gonadal effects of FSH distinct from those of LH remains difficult. We have characterized and compared the level of Sertoli and germ cell development in three mouse models recently created to isolate FSH activity from LH effects. Two models used LH-deficient hypogonadal (hpg) mice to selectively study either pituitary-independent transgenic (tg) FSH or ligand-independent activated tg FSH receptor (FSHR(+)) expression, and the third model used LH receptor (LHR)-deficient mice to isolate and examine endogenous mouse FSH effects. Stereological evaluation revealed tg-FSH or tg-FSHR(+) activity significantly increased total Sertoli cell numbers per testis in both hpg models relative to control hpg testes. Furthermore, tg-FSH dose-dependently restored hpg Sertoli cells to wild-type (wt) (non-hpg) levels, and LHR-/- testes also exhibited wt Sertoli numbers. Spermatogonial proliferation and meiotic development were enhanced by tg-FSHR(+) or tg-FSH. Despite producing normal Sertoli numbers, isolated tg-FSH activity only increased total spermatogonia and spermatocyte populations to 57 and 44% of wt, which was comparable to spermatogonia and spermatocyte numbers observed in LHR-null testes (45 and 34% of wt). Selective FSH activity initiated round spermatid formation in all three models. However, elongated spermatid formation was detected in tg-FSH and tg-FSHR(+) hpg testes but not in LHR-/- testes, which may reflect even lower intratesticular testosterone levels in LHR-null compared with hpg testes. FSH increased round and elongated spermatid numbers in hpg testes to 16 and 6% of wt without altering intratesticular testosterone levels, but failed to produce spermatozoa demonstrating the inability of FSH to complete spermatogenesis. These findings revealed that full Sertoli cell proliferation can be accomplished by FSH activity without LH requirement, and although postnatal mitotic and meiotic germ cell development can be promoted by FSH alone, LH-mediated effects remain a critical determinant for initiating the full complement of germ cells and final stages of postmeiotic development.

Expression profiling of mammalian male meiosis and gametogenesis identifies novel candidate genes for roles in the regulation of fertility.

We report a comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis, and gametogenesis by using high-density oligonucleotide microarrays and highly enriched cell populations. Among 11,955 rat loci investigated, 1268 were identified as differentially transcribed in germ cells at subsequent developmental stages compared with total testis, somatic Sertoli cells as well as brain and skeletal muscle controls. The loci were organized into four expression clusters that correspond to somatic, mitotic, meiotic, and postmeiotic cell types. This work provides information about expression patterns of approximately 200 genes known to be important during male germ cell development. Approximately 40 of those are included in a group of 121 transcripts for which we report germ cell expression and lack of transcription in three somatic control cell types. Moreover, we demonstrate the testicular expression and transcriptional induction in mitotic, meiotic, and/or postmeiotic germ cells of 293 as yet uncharacterized transcripts, some of which are likely to encode factors involved in spermatogenesis and fertility. This group also contains potential germ cell-specific targets for innovative contraceptives. A graphical display of the data is conveniently accessible through the GermOnline database at http://www.germonline.org.

Molecular mechanisms utilized by alternative c-kit gene products in the control of spermatogonial proliferation and sperm-mediated egg activation.

The c-kit proto-oncogene plays a dual role in the control of male fertility in mice through two alternative gene products: (1). c-kit [the transmembrane tyrosine kinase receptor for stem cell factor (SCF)], which is expressed and functional in differentiating spermatogonia of the postnatal testis, in which c-kit is essential for pre-meiotic proliferation; and (2). tr-kit, an intracellular protein which is specifically accumulated during spermiogenesis through the use of an alternative intronic promoter, and which is able to trigger mouse egg activation when microinjected into the cytoplasm of metaphase II arrested oocytes. Here, we summarize the most recent findings about the molecular pathways through which c-kit regulates cell cycle progression in mitotic germ cells, and those through which sperm-derived tr-kit triggers parthenogenetic completion of meiosis II and pronuclear formation in microinjected mouse eggs.

The emerging phenotype of the testicular carcinoma in situ germ cell

This review summarises the existing knowledge on the phenotype of the carcinoma in situ (CIS) cell. CIS is a common pre‐invasive precursor of testicular germ cell tumours of adolescents and young adults. These tumours display a variety of histological forms. Classical seminoma proliferates along the germ cell lineage, whereas embryonal carcinoma retains embryonic features and readily differentiates into teratomas that resemble various somatic cell lineages. A thorough review of the gene expression in CIS cells in comparison to normal testicular germ cells and overt tumours supports the view that CIS is a common precursor for both tumour types. Impaired cell differentiation resulting in a partial retention of the embryonic features, associated with an increasing genomic instability may be responsible for a remarkable phenotypic heterogeneity of CIS cells. Depending on the degree of differentiation and pluripotency, CIS cells found in adult patients seem to be predestined for further malignant progression into one or the other of the two main types of overt tumours. A new concept of phenotypic continuity of differentiation of germ cells along germinal lineage with a gradual loss of embryonic features based on the analysis of gene expression in all types of germ cells during their ontogeny is presented in this review. The data point out that despite the phenotypic continuum of gene expression, there are two periods of rapid changes of gene expression: first at the transition from primordial germ cells to pre‐spermatogonia, and later during the pubertal switch from the mitotic to meiotic cell division. The persistent expression of embryonic traits in CIS cells, and the high expression of the cell cycle regulators that are typical of mitotic germ cells support our long‐standing hypothesis that CIS cells originate from primordial germ cells or gonocytes and not from germ cells in the adult testis.

Multidrug resistance genes and p-glycoprotein in the testis of the rat, mouse, Guinea pig, and human.

Study of the multidrug resistance phenomenon in tumor cell lines has led to the discovery of the product of the multidrug resistance (MDR) type 1 genes, the plasma membrane P-glycoprotein (P-gp) that functions as an energy-dependent pump for the efflux of diverse anticancer drugs. P-gp was also recently identified in normal epithelial cells with secretory/excretory functions and in the endothelial cells of the capillary blood vessels in the brain and the testis. These endothelial cells are key elements of the blood-brain and blood-testis barriers, respectively. The aim of this study, in the rat, mouse, guinea pig, and human, was to determine whether testicular cells other than the capillary endothelial cells could express MDR type I genes. Immunohistochemistry on testicular sections revealed that P-gp is present in interstitial cells in the mouse, rat, and human testes, in early and late spermatids in guinea pig testis, and in late spermatids in the rat, mouse, and human. Reverse transcription-polymerase chain reaction analysis on isolated mouse, rat, and human cells showed that all somatic testicular cells (Leydig cells, macrophages, peritubular cells, and Sertoli cells) and the cytoplasmic lobes from rat late spermatids expressed MDR type I mRNAs, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. An ontogenesis study in the mouse reveals that type I MDR gene expression begins at 13.5 days postcoitum at the time when the seminiferous cords and the blood vessels appear and are maintained thereafter. Finally, two functional tests on isolated rat cells, the doxorubicin and rhodamine uptake assays, demonstrated that rat testicular macrophages, Leydig cells, peritubular cells, and Sertoli cells displayed a multidrug-resistance activity, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not. Western blot experiments have revealed that a P-gp of 175 kDa is present in the human testis as well as in the rat Leydig cells, testicular macrophages, peritubular cells, and Sertoli cells, but is absent in spermatogonia, spermatocytes, and early spermatids. We conclude that P-gp is involved in the self-protection of the somatic cells and is most probably one of the molecules that confers its functionality to the blood-testis barrier. The absence of expression of MDR type I genes in mitotic and meiotic germ cells probably explains their particular vulnerability to various anticancer drugs. In contrast, expression of the P-gp in the haploid cells most likely reflects the ability of spermatozoa to assume their own antidrug defense.

Mitotic arrest of female germ cells during prenatal oogenesis. A colcemid-like, non-apoptotic cell death.

The sequence of events and a possible reason for germ cell death during oogenesis in the prenatal ovary were studied in rat and mouse embryos. ED 14-22 rat and ED 14-16 mouse embryos were studied using semithin sections for light microscopy and serial ultrathin sections for electron microscopy. In addition, the rat material was 3H-thymidine labelled for historadioautography and cytospin preparations of freshly obtained gonads were immunohistochemically analysed. During the transition from the proliferating oogonial stage to the meiotic prophase about 16% of the postmitotic oocytes do not pass the initial meiotic checkpoint on ED 18/19 in the rat (ED 15/16 in the mouse). These germ cells first show structural signs of mitosis; the diploid number of 'super-condensed' chromosomes are globally formed and are concentrated in the center of the cell. Although the germ cells show all morphological signs of living cells they never have mitotic spindles; the micro-tubulus-organisation-centres (MTOCs) are found peripherally and become concentrated, forming a single centrosomal body (acentriolar MTOC) as detected by immunohistochemistry for the centrosomal protein MPM2 and gamma-tubulin. EM studies show 25 nm tubule-like profiles within the MTOC bodies. The centrioles frequently lie separate from the MTOC material or are not present at all; the germ cells are apparently arrested in a prophase- or metaphase-like stage when they have reached the postmitotic G2/preleptotenal transition and are unable to enter meiosis. Forty-eight to 72 h after the first mitotically arrested germ cells are found, degeneration is seen in these germ cells. This second event, the germ cell death proper, shows neither criteria of apoptosis (cell shrinkage, marginal condensation of chromatin, DNA fragmentation) nor signs of necrosis (cell swelling, pycnosis, inflammation). Both arrested pro- and metaphase-like stages are found with signs of cell death and phagocytosis. The morphological signs of phagocytosis are found in neighbouring pregranulosa cells. The final heterocytotic bodies contain the remnants of the centrosomal (MTOC) material and DAPI-positive DNA material. The pregranulosa cells are mitotically silent during the phase when mitotic arrest and germ cell degeneration is found. The results suggest the presence of a hypothetical 'anti-spindle' factor, which under normal conditions is necessary for induction of meiotic prophase. The structural events of 'arrested mitosis' is reminiscent of those induced by the antimitotic, tubule-degrading drug colcemid. This type of arrest may inhibit meiosis of more than 33% prenatal germ cells and induce their cell death.

Detection of division non-disjunction of meiosis I and II in spermatogenesis of men with idiopathic infertility.

Objective: Increased incidence of germ cell aneuploidy has been noted in men with idiopathic infertility. By using 3-color (chromosome 18, X, Y) fluorescence in situ hybridization (FISH), individual sex-chromosome aneuploidy rate in mitotic and meiotic germ cells could be calculated. However, meiosis is a process of 2 consecutive cell divisions, which leads to the formation of secondary spermatocytes (after first division) and spermatids (after second division). It is not possible to analyze the non-disjunction rate of the individual meiotic division based only on the FISH signals. The secondary spermatocytes and the spermatids are both haploid cells and both have haploid FISH signals.

An abundance of X-linked genes expressed in spermatogonia.

Spermatogonia are the self-renewing, mitotic germ cells of the testis from which sperm arise by means of the differentiation pathway known as spermatogenesis. By contrast with hematopoietic and other mammalian stem-cell populations, which have been subjects of intense molecular genetic investigation, spermatogonia have remained largely unexplored at the molecular level. Here we describe a systematic search for genes expressed in mouse spermatogonia, but not in somatic tissues. We identified 25 genes (19 of which are novel) that are expressed in only male germ cells. Of the 25 genes, 3 are Y-linked and 10 are X-linked. If these genes had been distributed randomly in the genome, one would have expected zero to two of the genes to be X-linked. Our findings indicate that the X chromosome has a predominant role in pre-meiotic stages of mammalian spermatogenesis. We hypothesize that the X chromosome acquired this prominent role in male germ-cell development as it evolved from an ordinary, unspecialized autosome.

Adenosine receptor–adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1 receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2 receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2′,5′-dideoxyadenosine. These data suggest that A1 and A2 adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation. Mol. Reprod. Dev. 58:307–317, 2001. © 2001 Wiley-Liss, Inc.

Adenosine receptor-adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.

Identification and characterization of Caenorhabditis elegans gamma-tubulin in dividing cells and differentiated tissues.

gamma-Tubulin is an essential component of the microtubule-nucleation machinery and therefore plays a crucial role during mitosis. To gain further insights into the function of this protein in the events that take place during embryogenesis and differentiation, we carried out detailed studies on gamma-tubulin during all the developmental stages of Caenorhabditis elegans. We identified the gamma-tubulin gene from this organism and analyzed the localization of the protein by both immunofluorescence and GFP reporter construct. We show that gamma-tubulin association with the centrosome is highly dynamic in mitotic cells, being massively recruited at prophase and released at anatelophase. This accumulation in mitotic centrosomes is dramatic during the first embryonic divisions. We provide the first description of the morphological changes at the centrosome level during the orientation of the mitotic spindle and the flattening of the posterior aster. Loss of function of the gamma-tubulin gene by RNAi induces a strong polyploidization of mitotic germ cells and embryos, but does not affect meiosis and pronuclear migration. In addition, we demonstrate the prominent redistribution of gamma-tubulin in adults at basal bodies of amphid and phasmid neurons, and at the apical membrane of polarized intestinal cells.

Efficiency of spermatogenesis: a comparative approach

Efficiency of spermatogenesis is the estimated number of spermatozoa produced per day per gram of testicular parenchyma. Spermatogenesis is the process of cell division and cell differentiation by which spermatozoa are produced in testes. Efficiency of spermatogenesis is influenced by species differences in the numerical density of germ cell nuclei and in the life span of these cells. Activities of spermatogonia, spermatocytes, and spermatids partition spermatogenesis into three major divisions (spermatocytogenesis, meiosis, and spermiogenesis, respectively). Spermatocytogenesis involves mitotic germ cell division to produce stem cells and primary spermatocytes. Meiosis involves duplication of chromosomes, exchange of genetic material, and two cell divisions that reduce the chromosome number and yield four spermatids. In spermiogenesis, spherical spermatids differentiate into mature spermatids which are released in the lumen of seminiferous tubules as spermatozoa. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various developmental steps throughout spermatogenesis. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest impact occurs during spermatocytogenesis and meiosis. There are species and seasonal influences on the developmental steps in spermatogenesis at which germ cell degeneration occurs. Number of Sertoli cells, amount of smooth endoplasmic reticulum of Leydig cells, and the number of missing generations of germ cells within the spermatogenic stage of the cycle influence efficiency of spermatogenesis. Efficiency of spermatogenesis is influenced to the amount of germ cell degeneration, pubertal development, season of the year, and aging of humans and animals.

Oocyte attrition

During oogenesis, germ cell numbers sharply decrease when meiosis is initiated. There is solid evidence (DNA ladders, in situ detection) that this loss is through apoptosis. Oocyte apoptosis appears to hit mitotic primordial germ cells (PGC), pachytene oocytes and early primordial follicles. The control of oocyte apoptosis is not fully understood, although survival factors (LIF, kit ligand and FGF), as well as death inducing factors (fas ligand, TGFβ), have been identified. Fas ligand binding on oocytic fas may result in caspase 8 activation. Two pathways inducing oocyte apoptosis may then be operating. In the first one, activated caspase 8 will induce activation of executioner caspases. In the second one, activated caspase 8 will trigger the cleavage of the bcl 2 family member Bid, which will act on mitochondria, resulting in cytochrome c release, caspase 9 activation and finally, activation of all executioner caspases. As a consequence of caspase activation, alterations in the cell nucleus (DNAse activation, PARP fragmentation), in the cell cytoskeleton (lamin) and cell metabolism will occur, producing cell death. During folliculogenesis, germ cell loss, owing to oocyte apoptosis, has been postulated within primordial and preantral follicles. Its regulatory mechanisms may be even more complex than those operating in foetal oocytes since additional control factors include EGF/TGFα and bcl 2 (survival) and activin (death inducer). In contrast, oocytes from antral follicles appear to be very unsensitive to death inducing stimuli.

CDNA cloning of a stage-specific gene expressed during HCG-induced spermatogenesis in the Japanese eel.

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)+ RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin betaB subunit, was expressed in the initial phase of spermatogenesis (1-6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames: 3-18 days (two clones), 6-18 days (one clone) and 9-18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full-length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12-15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. It has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG-induced spermatogenesis in the eel.

Stem cell factor and c-kit gene expression and protein localization in the sheep ovary during fetal development.

The aim of this study was to investigate stem cell factor and c-kit gene expression and protein localization in the mesonephros and ovary of sheep fetuses at different days of gestation, using RNA in situ hybridization and immunohistochemical procedures. At days 24 and 26 of gestation, stem cell factor mRNA and protein were present in cells throughout the developing gonad and mesonephros. From day 28 to day 40 of gestation, stem cell factor mRNA and protein became increasingly localized to the cortical region of the ovary, where most germ cells were present as actively proliferating oogonia. From day 40 to day 90 of gestation, stem cell factor mRNA and protein localization were confined mainly to the ovarian cortex. Moreover, within the cortical region, stem cell factor mRNA was low or absent where follicles were first forming and highest in the outer ovarian cortex, where germ cells were undergoing mitosis or the early stages of meiosis. In contrast, stem cell factor protein was present in newly forming follicles, as well as in mitotic and meiotic germ cells, which is consistent with the presence of both membrane-bound and soluble forms of this ligand. However, by day 90 of gestation, both stem cell factor mRNA and protein were observed in the granulosa cells of most (> 90%) primordial follicles. C-kit mRNA and protein were observed from day 24 of gestation in both germ cells and somatic cells but, with increasing gestational age, preferentially in germ cells (for example, pre-meiotic germ cells and both isolated oocytes and follicle-enclosed oocytes). C-kit protein, but not mRNA, was also observed in germ cells that were undergoing meiosis. The results indicate that the cells containing stem cell factor mRNA within the ovary up to day 90 of gestation originated from the gonadal blastema and from cells that migrated from the mesonephros before day 28 of gestation. Since stem cell factor mRNA was absent in both mesonephric cells migrating after day 28 of gestation and in regions where follicles were first forming, it is suggested that these later migrating mesonephric cells are the progenitors of the granulosa cells in the first forming follicles. In conclusion, during follicle formation, c-kit mRNA is localized to germ cells whereas c-kit, together with stem cell factor protein, is localized to both germ cells and somatic cells, consistent with the hypothesis that the presence of this receptor-ligand pair is essential to prevent apoptosis.

Germ-Cell Nondisjunction in Testes Biopsies of Men With Idiopathic Infertility

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.

Concurrent teratogenic and mutagenic action of 2‐methoxyethanol in Drosophila melanogaster larvae resulted in similar phenotypes: Close resemblance to directed mutations

Early third instar larvae of wild-type Drosophila melanogaster were transferred to medium supplemented with 2-methoxyethanol (2-ME) or ethylene glycol monomethyl ether. 2-ME produced terata in adult flies as wing notches and duplications of macrochaetae similar to feeding with methoxyacetic acid (MAA), the oxidation product of 2-ME. Larval feeding with 2-ME also affected the fertility of both females and males. 2-ME or more likely its intermediate oxidation product, methoxyacetaldehyde (MAALD), concurrently generated mutations in the premeiotic stages of the oücytes in the early third instar larvae. The mutagenicity of 2-ME has been confirmed in subsequent small scale experiments. The mutation frequency ranged from 4 × 10–4 to 1 × 10–2. Although terata were not supposed to be heritable, 1.1 to 8.7% of the affected females produced offspring with phenotypic similarity to the female parent. This phenomenon looked like a classical example of inheritance of an acquired character. The question is addressed why a Notch-like phenocopy, generated by larval 2-ME treatment, could bring forth Notch and rudimentary mutants in particular. Administration of 2-ME to larvae, containing the highly active alcohol dehydrogenase variant ADH-71k, exposed the mitotic germ cells and the mitotic somatic cells of the imaginal discs simultaneously to the mutagen MAALD and the teratogen MAA, respectively. The chances for specific gene mutations, though non-adaptive, were likely increased by a feedback mechanism: gene-products that were inhibited or disturbed by the teratogen demanded increased transcription of their encoding genes. Transcribed genes are more susceptible to mutagens. Teratogenesis Carcinog. Mutagen. 19:183–204, 1999. © 1999 Wiley-Liss, Inc.

Concurrent teratogenic and mutagenic action of 2-methoxyethanol inDrosophila melanogaster larvae resulted in similar phenotypes: Close resemblance to directed mutations

Early third instar larvae of wild-type Drosophila melanogaster were transferred to medium supplemented with 2-methoxyethanol (2-ME) or ethylene glycol monomethyl ether. 2-ME produced terata in adult flies as wing notches and duplications of macrochaetae similar to feeding with methoxyacetic acid (MAA), the oxidation product of 2-ME. Larval feeding with 2-ME also affected the fertility of both females and males. 2-ME or more likely its intermediate oxidation product, methoxyacetaldehyde (MAALD), concurrently generated mutations in the premeiotic stages of the oocytes in the early third instar larvae. The mutagenicity of 2-ME has been confirmed in subsequent small scale experiments. The mutation frequency ranged from 4 x 10(-4) to 1 x 10(-2). Although terata were not supposed to be heritable, 1.1 to 8.7% of the affected females produced offspring with phenotypic similarity to the female parent. This phenomenon looked like a classical example of inheritance of an acquired character. The question is addressed why a Notch-like phenocopy, generated by larval 2-ME treatment, could bring forth Notch and rudimentary mutants in particular. Administration of 2-ME to larvae, containing the highly active alcohol dehydrogenase variant ADH-71k, exposed the mitotic germ cells and the mitotic somatic cells of the imaginal discs simultaneously to the mutagen MAALD and the teratogen MAA, respectively. The chances for specific gene mutations, though non-adaptive, were likely increased by a feedback mechanism: gene-products that were inhibited or disturbed by the teratogen demanded increased transcription of their encoding genes. Transcribed genes are more susceptible to mutagens.

XX Germ Cells: The Difference Between an Ovary and a Testis

In mammals, gonadal sex is determined by the action of the testis-determining gene, SRY. In the absence of SRY, the indifferent gonad follows an alternative pathway and develops as an ovary. Both mitotic and meiotic germ cells appear to play an essential role in ensuring ovarian development. Ovaries depleted of germ cells before or after ovarian differentiation has commenced can develop seminiferous cords, suggesting that XX germ cells may inhibit testicular differentiation in the ovary.

Differential Expression of Two γ-Tubulin Isoforms during Gametogenesis and Development inDrosophila

Previous work identified a γ-tubulin gene, γTub23C,inDrosophila(Zhenget al.,1991). We now report identification of a second γ-tubulin gene, γTub37CD.Immunoblot analysis and immunolocalization show that γTub37CD and γTub23C are differentially expressed during gametogenesis and development. During oogenesis, γTub23C was detected at centrosomes and in the cytoplasm of mitotic germ cells, but was not detected in germ cells following completion of mitosis. Conversely, γTub37CD was not detected in proliferating germ cells, but appeared to accumulate in germ cells during egg chamber development. Neither γ-tubulin isoform was detected at the anterior or posterior poles of developing oocytes. During spermatogenesis, only γTub23C was detected at centrosomes, where it showed cell cycle- and differentiation-dependent organization. During the transition into the first meiotic division, γTub23C became organized as a corpuscular focus at centrioles until completion of meiosis II. During postmeiotic spermatid differentiation, γTub23C was detected first as a rod and then as a collar-like structure near the juncture of the nucleus and the elongating flagellum, but was not detected in bundles of mature sperm. The germline-specific CDC25 encoded bytwineis required for organization of γTub23C into corpuscular focus in spermatocytes, but not for separation of centriole pairs in M-phase or postmeiotic organization of γTub23C at centrioles. Following reconstitution of a canonical centrosome at fertilization, only γTub37CD was detected at centrosomes in syncytial embryos, but both γTub37CD and γTub23C were detected at centrosomes in cellularized embryos. Colocalization of these two isoforms suggests that γTub23C and γTub37CD both contain structural features of γ-tubulins essential for localization to centrosomes.