Hubert Wartenberg

Development of the early human ovary and role of the mesonephros in the differentiation of the cortex

SummaryThe morphogenesis of the primary gonadal differentiation, of the sexual differentiation and the growth of the ovarian cortex during its early period have been studied on 10 human embryos between 12 and 95 mm CR-length. Semithin sections of glutaraldehyde-OsO4-fixed and plastic-embedded material were used to demonstrate the structural events on a cellular level. The primary gonadal blastema within the genital ridge is formed by two types of somatic cells: cells segregated from the mesonephros and cells of the proliferating coelomic epithelium. The two types of cells show a tendency to intermingle and they enclose the immigrating primordial germ cells. In the female gonad the indifferent period terminates between day 40 and 42 of ovulation age (20 to 23 mm CR-length). Between day 40 and 50 the blastemal content of the indifferent gonad is remodelled and an ovarian cortex differentiates. Cellular strands extending from the primary blastema and strands from the superficial blastemal layer contribute to the formation of the cortex. Within the newly formed medulla, remnants of the disintegrating primary blastema differentiate into medullary cords.Cells of mesonephric origin which invade the growing cortex via the rete blastema interact with cells deriving from the supericial epithelium, and both exert their opposite influence on the germ cells. While female sexual differentiation is characterized by failure of the dark mesonephric cells to completely penetrate the gonadal blastema, the morphogenetic process resulting in the formation of the ovarian cortex shows a strong invasion of the cortex by the dark mesonephric cells. Dark cells advance at the most superficial layer of the cortex and increase in number at the deeper level of the cortex. Onset of oogonial proliferation and meiotic prophase seems to depend on the numerical proportion between the activating dark and the inhibiting light supporting cells.

Morphology of the "spindle-shaped body" in the developing tail of human spermatids.

SummaryAt the beginning of the late cap phase of the normal differentiation of human spermatids, a unique structure, the “spindle shaped body“, occupies part of the future middlepiece. This spindle-shaped body was followed throughout its development, and its functional relationship to the formation of the fibrous sheath is discussed. The spindle-shaped body disappears when the fibrous sheath is completed and the annulus moves down, giving rise to the middle-piece.

Germ cell kinetics during early ovarian differentiation: An analysis of the oogonial cell cycle and the subsequent changes in oocyte development during the onset of meiosis in the rat

The aim of this study was the comparison between the mitoses of oogonia and the initial stages of oocyte meiosis. The structural alterations that the germ cell chromatin undergoes during the oogonial mitosis have been compared with those occurring during the G1‐ and S‐phase just before meiosis. Using plastic embedded 1‐μm sections of fetal rat ovaries (embryonic days = ED 14–20) labeled with 3H‐thymidine and re‐embedded for electron microscopy, a study of the structural conditions of the nuclear chromatin has been combined with a kinetic analysis of the oogonial cell cycle and the transitional period into the meiotic prophase. After ovarian differentiation (ED 14) the oogonia show a non‐clonal, but strong proliferation. On ED 16, proliferation changes to a clonal pattern and decreases during ED 17. A final increase in 3H‐thymidine incorporation on ED 18 characterizes the meiotic S‐phase. On ED 19 the nuclear labeling drops to zero. The mitotic cycle of the oogonia lasts 16.5 hr and can be divided into 11 stages according to the concept of El‐Alfy and Leblond [(1988) Am. J. Anat., 183:45–56] on the basis of the chromatin pattern. The S‐phase (10.0 hours) extends from the telophase‐interphase transition through the interphase to early prophase. The postmitotic G1‐ and S‐phases show a more extensive duration, respectively 10 and 11.5 hours, and differ from their oogonial counterparts by the spherical shape of the nuclei from the very beginning. The chromatin pattern is similar until the end of the S‐phase and lacks any prophase‐like, preleptotenal chromatin condensation before the oocytes exhibit (pre‐) leptotenal structures. Once the germ cell has completed a sequence of clonal mitotic divisions, it irrevocably progresses into meiosis. During an extended postmitotic period, the structural characteristics of meiosis emerge stepwise. Microsc. Res. Tech. 40:377–397, 1998.

Morphological studies on the role of the periductal stroma in the regression of the human male Müllerian duct

SummaryThe regression of the male Müllerian duct has been studied in human embryos and fetuses by means of the semi-thin light microscopic technique and by electron microscopy. After completion of the ducts differentiation during week 7, a periductal stroma is formed by two types of mesenchymal-like cells: light, epitheloid cells originating from the coelomic epithelium and dark, fusiform cells of mesonephric origin. During week 9 these cells condense to a compact cuff in which the light cells occupy the inner core. The duct is entirely sealed by an epitheloid stratum of the periductal stroma. At the same time, the basal lamina thickens up to 300 nm by apposition of extracellular material. During weeks 10 to 12, the inner stromal core is invaded by dark fusiform cells from the peripheral stratum which intermingle with the light cells. The basal lamina dissolves, the epithelio-stromal interface becomes indistinct and finally disappears. During week 13 remnants of the Müllerian duct can be observed. They result from the complete merging of the ductal into the periductal compartment.Müllerian duct regression is divided into two functional steps: First the duct is prevented from growth by the epitheloid cells of the stromal cuff. This process lasts for 2 to 3 weeks. In the second place the basal lamina breaks up under the influence of the dark stromal component. This event launches the regression proper and lasts for another 2 to 3 weeks. Necrosis of cells or programmed cell death does not play a decisive role in the regression of the human Müllerian duct.In the upper, nonregressing part of the duct, light epitheloid cells are scarce and do not seal the duct. A periductal extracellular space is preserved during the entire period and the periductal stroma does not fuse with the ducts epithelium. The epithelio-stromal interface is maintained along this section.

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.

Ultrastructure of fetal ovary including oogenesis

Ultrastructural aspects of the developing human ovary have been the subject of several studies during the last decades. Although most articles deal with the differentiation of the female germ cell (1–18), studies on the role of the somatic blastema and its differentiation into granulosa cells are rare (2,4,6). Even those papers which deal with the ovarian structure of nonhuman mammals concentrate on the cellular characteristics of oogonia (19–22) and premeiotic oocytes (19–26), the structure of intercellular bridges (27–32) and germ cell degeneration (30,33,34), but not on the supporting cell structure (23, 35–39).

Structural Aspects of Gonadal Differentiation in Mammals and Birds

The task of this review is to discuss the morphogenetic mechanisms of gonadal differentiation. The structural events which result in the formation of the genital ridge blastema in the sexually differentiated male or female organ, or in the creation of an ovarian cortex, are difficult to ascertain. Accordingly, several concepts have been elaborated in the past that try to explain gonadal differentiation more or less extensively. Most of these concepts focus on the main aspect of gonadal development: the process of sexual differentiation. Among the different concepts are those conceived from a primarily morphological aspect; others present an entirely functional view.

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.

Simultaneous changes of the perivascular contact area and HIMOT activity in the pineal organ after bilateral adrenalectomy in the rat

SummaryIn the pineal organ of the female rat, proportional changes in the area of contact of pinealocyte processes and glial processes with the perivascular space were found after bilateral adrenalectomy. The contact area was evaluated by measuring the length of pinealocyte and glial cell membranes directly abutting on the basal lamina. In the normal female rat, 40% of the contact area is occupied by pinealocyte and 60% by glial cell processes. Fourteen days after bilateral adrenalectomy, this proportion is reversed. In addition, cell counts demonstrate that more pinealocytes gain access to the pericapillary space due to the experimental conditions.In order to prove whether or not these results indicate an increase of pineal endocrine activity, the melatonin-forming enzyme hydroxyindole-O-methyltransferase (HIOMT, E.C. 2.1.1.4) was assayed. After bilateral adrenalectomy the HIOMT activity was found to significantly increase.