Andreas Miething

Germ-cell death during prespermatogenesis in the testis of the golden hamster

SummaryDegenerating prespermatogonial germ cells in the testis of the immature golden hamster [aged 14 days post conceptionem (dpc) to 13 days post partum [dpp)] were studied with regard to their morphology and temporal incidence. Judged by their ultrastructural features, these cells clearly take the form of apoptosis and finally are subjected to phagocytosis by neighboring Sertoli cells; only a few germ cells of a presumably incipient, partly variant degenerative morphology cannot, at present, be assigned to the apoptotic mode of cellular death. Degenerating prespermatogonia occur between the 14th dpc and 3rd dpp and again, after an interval in which no such cells are found, from the 9th dpp onwards. This pattern reveals a striking parallelism to the phases of proliferation of these cells, viz., the appearance of M- and T2-prespermatogonia. Both this obvious temporal association of proliferation and degeneration and the classification of prespermatogonial death as apoptosis suggest some developmental significance of the degenerative phenomena investigated.

Physiological purkinje cell death is spatiotemporally organized in the developing mouse cerebellum.

Physiological cell death is crucial for matching defined cellular populations within the central nervous system. Whereas the time course of developmental cell death in the central nervous system is well analyzed, information about its precise spatial patterning is scarce. Yet, the latter one is needed to appraise its contribution to circuit formation and refinement. Here, we document that during normal cerebellar development, dying Purkinje cells were highly localized within the vermal midline and in a lobule specific, parasagittal pattern along the whole mediolateral axis. In addition, single hot spots of cell death localized to the caudal declive and ventral lobule IX within the posterolateral fissure. These hot spots of dying Purkinje cells partly overlapped with gaps within the Purkinje cell layer which supports the classification of different gaps based on histological and molecular criteria, i.e., midline gap, patchy gaps, and raphes. Areas characterized by a high incidence of Purkinje cell death and gaps colocalize with known molecular and functional boundaries within the cerebellar cortex. Physiological cell death can thus be considered to serve as an important regulator of cerebellar histogenesis.

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 γ-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.

Engrailed-2 regulates genes related to vesicle formation and transport in cerebellar Purkinje cells.

Engrailed transcription factors regulate survival, cell fate decisions and axon pathfinding in central neurons. En-2 can also attenuate Purkinje cell (PC) maturation. Here, we use array analysis to scrutinize gene expression in developing PCs overexpressing Engrailed-2 (L7En-2). The majority (70%) of regulated genes was found down-regulated in L7En-2 cerebella, consistent with the known repressive function of Engrailed-2. Differential gene expression, verified by in situ hybridization or Western blotting, was particularly evident during the first postnatal week, when L7En-2 PCs display conspicuous deficits in dendritogenesis. Functional classification revealed clusters of genes linked to vesicle formation and transport. Consistently, Golgi stacks located at the axonal pole of wild type PC somata were rarely detected in L7En-2 PCs. In addition, long continuous stretches of endoplasmic reticulum typically found around the axonal pole of wild type PCs were less frequently observed in transgenic cells. Engrailed-2 might therefore orchestrate PC survival and process formation as a regulator of subcellular organization.

Intercellular bridges between germ cells in the immature golden hamster testis: evidence for clonal and non-clonal mode of proliferation.

SummaryIntercellular bridges of prespermatogonia and of the first A-spermatogonia in the maturing testes of newborn to 17-day-old golden hamsters have been studied by electron microscopy. Incomplete cytokinesis of dividing M- and T2-prespermatogonia and A-spermatogonia produces these bridges, which undergo different developmental fates. Bridges of the first A-spermatogonia are stable beyond subsequent mitoses of these cells; this gradually leads to the formation of bridge-connected groups of synchronously developing germ cells. Thus, the clonal mode of male germ cell proliferation is already established in this period of testis maturation. During mitoses, pre-existing bridges reversibly develop structural modifications, i.e. considerable elongation and formation of a bridge-partitioning complex. In contrast, intercellular bridges of prespermatogonia are mostly severed and become lost during subsequent mitoses of the cells involved; this results in separation of the germ cells and represents a mainly non-clonal mode of M- and T2-prespermatogonial proliferation. Here, too, pre-existing bridges elongate and develop the bridge-partitioning complex during subsequent mitoses of the joined cells, but this is superposed and interrupted by the simultaneous process of disconnection of the bridges.

Multinucleated spermatocytes in the aging human testis: formation, morphology, and degenerative fate.

Summary. The ultrastructural appearance of multinucleated spermatocytes in the aging human testis is documented emphasizing their mode of formation and their degenerative fate. Multinuclearity results from confluence of cell membranes of neighbouring spermatocytes of one clone. Intercellular bridges are not causally involved in this process but are only secondarily detached from the cell membrane and, attaining an intracellular position, subsequently disintegrate. Accompanying or following the process of formation of the multinucleated spermatocytes, these cells undergo degeneration. There are no indications of a regular further development of multinucleated spermatocytes. Though quite rarely found within the aging human seminiferous epithelium, the appearance of these cells represents a germ‐cell loss at the spermatocyte stage.

Alopecia and male infertility in oligotriche mutant mice are caused by a deletion on distal chromosome 9

The recessive mutation oligotriche (olt) affects the coat and male fertility in the mouse. In homozygous (olt/olt) mutants, the coat is sparse, most notably in the inguinal and medial femoral region. In these regions, almost all hair shafts are bent and distorted in their course through the dermis and rarely penetrate the epidermis because the hair cortex is not fully keratinized. During hair follicle morphogenesis, mutant hair follicles exit from anagen one day before those of normal littermates and show a prolongation of the catagen stage. The oligotriche (olt) locus was mapped to distal chromosome 9 within a 5-Mbp interval distal to D9Mit279. Analysis of candidate gene expression revealed that olt/olt mutant mice do not express functional phospholipase C delta 1 (Plcd1) mRNA. This deficiency is the consequence of a 234-kbp deletion involving not only the Plcd1 locus but also the chromosomal segment harboring the genes Vill (villin-like), Dlec1 (deleted in lung and esophageal cancer 1), Acaa1b (acetyl-Coenzyme A acyltransferase 1B, synonym thiolase B), and parts of the genes Ctdspl (carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase-like) and Slc22a14 (solute carrier family 22 member 14). Offspring of olt/olt females, mated with Plcd1−/− knockout males, exhibit coat defects similar to those observed in homozygous olt/olt mutant mice but the spermiogenesis in male offspring is normal. We conclude that the 234-kbp deletion from chromosome 9 harbors a gene involved in spermiogenesis and we propose that the oligotriche mutant be used as a model for the study of the putative tumor suppressor genes Dlec1, Ctdspl, and Vill. We also suggest that the oligotriche locus be named Del(9Ctdspl-Slc22a14)1Pas.

Arrested germ cell divisions in the ageing human testis

Arrested divisions represent a defective type of germ cell division. The cells are irreversibly arrested in a state marked by strongly condensed chromosomes, a dissolved nuclear envelope and a disturbed spindle apparatus. They finally die. The present ultrastructural study of the ageing human seminiferous epithelium is the first to describe such cells in the human testis. The cells usually lie in small groups and show intercellular bridges connecting them. The bridges lack a bridge‐partitioning complex, a structure otherwise characteristic for bridges between dividing germ cells of a clone. Frequently, arrested divisions show signs of cytoplasmic disorganization and severe structural degradation. Centrioles, irregular microtubules, small membranous vesicles and granular electron‐dense material are crowded within the circle of chromosomes. Some cells show numerous small vesicles and larger membranous structures distributed throughout the cytoplasm. The presence of arrested germ cell divisions in ageing men might be connected with local dysfunctions of the Sertoli cell barrier, which are known to appear focally in the ageing human seminiferous epithelium as well.

Primordial germ cells of the rabbit are specifically recognized by a monoclonal antibody labelling the perimitochondrial cytoplasm

Abstract Instrumental for studies investigating the development of germ cells, and especially the separation of the germline in the early embryo, are molecular markers which reliably label germ cells and with which regulative factors of germ cell development may be analyzed. Here, we describe the monoclonal antibody PG-2, which is highly specific for the germ cells of the rabbit embryo and labels the perimitochondrial cytoplasm, as demonstrated by immunogold-silver staining. Identical expression patterns are found in germ cells of either sex from early organogenesis at 10 days post-conception (d.p.c.), when the germ cells leave the hindgut epithelium and settle in the gonadal anlage as primordial germ cells (PGCs), until the time immediately prior to birth (30 d.p.c.), when germ cells are either in their oogonial or prospermatogonial state. The antibody is the first to recognize specifically a cytoplasmic epitope in germ cells of a higher vertebrate and may well recognize the mammalian equivalent of the germ plasm found in inverteb-rates and lower vertebrates. The antibody can be used for early identification of PGCs and may be of help in the elucidation of mammalian germ cell development towards the gonial stages of spermatogenesis and oogenesis.

Proliferative activity of the developing seminiferous epithelium during prespermatogenesis in the golden hamster testis measured by bromodeoxyuridine labeling

Bromodeoxyuridine (BrdU)-labeling was used to study the cell kinetics of the developing seminiferous epithelium in the testes of golden hamsters aged 10.5 to 27.5 days post conception (dpc), i.e., during a period beginning one developmental day before testicular differentiation (11.5 dpc) and extending to the appearance of the first “mature” spermatogonia. Supporting (Sertoli) cells continuously proliferate throughout the period studied. Labeling indices amount to about 30% between the 10.5th and 16.5th dpc, and subsequently decrease to levels below 10% on the 26.5th and 27.5th dpc. Germ cells (prespermatogonia) proliferate between the 10.5th and 15.5th dpc and again, after a period of mitotic quiescence, from the 24.5th dpc onwards. This pattern of prespermatogonial proliferation substantiates and further specifies the successive appearance of M-prespermatogonia (10.5th to 15.5th dpc: proliferating), T1-prespermatogonia (16.5th to 23.5th dpc: quiescent), and T2-prespermatogonia (24.5th to 27.5th dpc: proliferating). Thus, the M-prespermatogonial phase of germ cell proliferation is shown to commence at least 24 h before testicular differentiation. Transitions from M- to T1-phase and from T1-to T2-phase are rather abrupt. Both the latter observation and the comparison with oogonial development in the female at the corresponding time (onset of meiosis) indicate the presence of an underlying control mechanism operative during prespermatogenic development. Due to different nuclear staining patterns, the BrdU-labeling method allows temporal subdivision of the S-phase, thus opening up prospects of more detailed cell-kinetic analyses of the seminiferous epithelium.