A study of gonadal organogenesis,and the factors influencing regeneration following surgical castration in Deroceras reticulatum (Pulmonata:Limacidae)

Summary At hatching, the hermaphrodite duct of Deroceras reticulatum consists of a single cell type designated the Gonadal Stem Cell (GSC). Proliferation of the GSC leads to the formation of numerous ductules each of which forms one of the acini of the gonad. The germinal and supporting cells are derived entirely from the GSC. The germ cells differentiate first, followed by the Sertoli and follicle cells. At the early sperm stage of gonadal development the hermaphrodite duct differentiates to function as a seminal vesicle. Once the GSC are committed to this change they lose their regenerative ability. The only remaining GSC are the cells of the acinar epithelium, and these retain their germinal potential until the death of the animal.Regeneration will occur from the hermaphrodite duct provided it is in the immature state, i.e., composed of GSC, and is exposed to the hormonal conditions of a young animal. Nervous connections and the presence of an artery are not necessary for this regeneration. The presence of a functional gonad does not inhibit regeneration.     

Development and early differentiation of gonad in the european eel (Anguilla anguilla [L.], Anguilliformes,Teleostei): A cytological and ultrastructural study

The structure of the gonad of the European eel (Anguilla anguilla [L.]), an “undifferentiated” gonochoristic teleost, was investigated by transmission electron microscopy from 6–8 cm elvers to 22 cm yellow eels with juvenile hermaphroditic gonads. The pear-shaped gonads of 6–8 cm elvers assume, in 12–15 cm eels, a lamellar shape and enlarge by migration of germ cells, which we refer to as primary primordial germ cells. In the gonads of ∼ 16 cm eels, the primary primordial germ cells multiply, giving rise to clusters of germ cells that have ultrastructural characteristics of the primary primordial germ cells but show giant mitochondria, enlarged Golgi complexes, and round bodies not limited by membranes. We refer to these as secondary primordial germ cells. In 16–18 cm eels, syncytial clones of oogonia interconnected by cytoplasmic bridges are also observed. In 18–22-cm-long eels, the gonads contain primordial germ cells, oogonial clones, early oocyte cysts, single oocytes in early growth stages, and primary spermatogonia. Such germ cells are present in the same cross section where they are either intermingled or are in areas of predominantly female germ cells close to areas with predominantly male germ cells. These gonads are juvenile hermaphroditic and should be considered ambisexual because in larger eels they differentiate either into an ovary or into a testis. Somatic cells always envelop the germ cells following their migration into the gonad. These somatic cells first show similar ultrastructural features and then differentiate either into early Sertoli cells investing spermatogonia, or into early follicular (granulosa) cells investing the early previtellogenic oocytes. In eels ∼ 14 cm long, primitive steroid-producing cells also migrate into the gonad. In the ambisexual gonad they differentiate either into immature Leydig cells in the male areas, or into early special cells of the theca in the female areas. Nerve fibers are joined to the steroid-producing cells. Gonad development and differentiation are also associated with structural changes of the connective tissue characterized by the progressive appearance and deposition of collagen fibrils first in the mesogonadium, then in the gonad vascular region, and then in the germinal region. The collagen-rich areas are massive in the male areas and reduced in the female ones. J. Morphol. 231:195–216, 1997. © 1997 Wiley-Liss, Inc.     

Histological and ultrastructural investigation of early gonad development and sex differentiation in Adriatic sturgeon (Acipenser naccarii,Acipenseriformes, Chondrostei)

Gonad development and sex differentiation from embryos to 594‐day‐old individuals were investigated in farmed Acipenser naccarii using light and transmission electron microscopy. The migrating primordial germ cells first appear along the dorsal wall of the body cavity in embryos 1.5 days before hatching. The gonadal ridge, containing a few primary primordial germ cells (PGC‐1) surrounded by enveloping cells, appears in 16‐day‐old larvae. At 60 days, the undifferentiated gonad is lamellar and PGC‐1 multiply, producing PGC‐2. In 105‐day‐old juveniles, a distinct germinal area with advanced PGC‐2 appears on the lateral side near the mesogonium and the first blood vessels are visible. At 180 days, putative ovaries with a notched gonadal epithelium and putative testes with a smooth one appear, together with adipose tissue on the distal side. In 210‐day‐old juveniles, active proliferation of germ cells begins in the putative ovaries, whereas putative testes still contain only a few germ cells. The onset of meiosis and reorganization of stromal tissue occurs in ovaries of 292‐day‐old individuals. Ovaries with developed lamellae enclosing early oocyte clusters and follicles with perinucleolar oocytes occur at 594 days. Meiotic stages are never found, even in anastomozing tubular testes of 594‐day‐old individuals. Steroid producing cells are detected in the undifferentiated gonad and in the differentiated ones of both sexes. Anatomical differentiation of the gonad precedes cytological differentiation and female differentiation largely precedes that of the male. Gonad development and differentiation are also associated with structural changes of connective tissue, viz. collagen‐rich areas are massive in developing testes and reduced in ovaries. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Development of the mammalian gonad: the fate of the supporting cell lineage

Sex determination in mammals is mediated via the supporting cell lineage in the fetal gonad. In the very early stages of gonadal development, the fate of the supporting cell population is critically dependent on the expression of the male-determining gene on the Y chromosome. If this gene is absent or fails to be expressed, or is expressed too late or in too small a number of supporting cells, all supporting cells (XX or XY) differentiate as pre-follicle cells and development proceeds along the female pathway. Supporting cells in which the male-determining gene is expressed in a timely manner differentiate as pre-Sertoli cells; given sufficient such cells, testis cords form and development proceeds in a male direction. If XX supporting cells are also present, a few may be recruited into the pre-Sertoli population and participate in testis cord formation. The subsequent fate of pre-follicle cells depends critically on interaction with the germ cell population in the developing gonad: absence of germ cells may lead to partial masculinization of the gonad, and/or to disappearance of the supporting cell component.     

Evolution and phylogeny of gonad morphology in bony fishes

Gonad morphology at the gross anatomical or histological levelshas long been studied by fisheries biologists to identify annualreproductive cycles and length of breeding season, among othergoals. Comparative surveys across vertebrate taxa have not beendetailed enough, however, to describe fully the differencesand similarities among gonads of bony fishes and other vertebrates,and to use gonad morphology in phylogenetic systematic analyses.An emerging constant among vertebrates is the presence of agerminal epithelium composed of somatic and germ cells in bothmales and females. In females, the germinal epithelium linesthe ovarian lamellae. In males, arrangement of the germinalepithelium into compartments varies among osteichthyans: basaltaxa have an anastomosing tubular testis, whereas derived taxahave a lobular testis. The lobular testis is proposed as a synapomorphyof the Neoteleostei. The annual reproductive cycle is hypothesizedto be the source of morphological variation among testis types.Elongation of germinal compartments during early maturationmay result in a transition from anastomosing tubular to lobulartestes. In all male atherinomorphs surveyed, spermatogonia arerestricted to the distal termini of lobules rather than beingdistributed along the lobule; there is an epithelioid arrangementof Sertoli and germ cells rather than a germinal epithelium.Arrest of the maturation-regression phases is hypothesized tolead to formation of the atherinomorph testis. Atherinomorphsalso have a distinctive egg with fluid, rather than granular,yolk. Variation among germinal epithelia is interpreted in adeveloping phylogenetic framework to understand evolution ofgonad morphology and to propose gonad characters for phylogeneticanalyses.     

Individualisation et organogenèse de la gonade embryonnaire deViviparus viviparus L. (Mollusque Gastéropode Prosobranche à sexes séparés)

Résumé L'origine et l'évolution de la gonade embryonnaire deViviparus viviparus sont étudiées ultrastructuralement. La gonade se forme par migration et multiplication de cellules péricardiques.Plusieurs étapes se succèdent au cours de l'organogenèse gonadique: durant le stade sexuellement indifférencié à un seul type cellulaire, la gonade est constituée de cellules-souches qui paraissent toutes identiques. Le stade suivant ou stade sexuellement indifférencié à deux types cellulaires est caractérisé par la différenciation de cellules germinales et de cellules non germinales. Les cellules non germinales sont ancrées les unes aux autres par des jonctions septées et leurs longs prolongements enveloppent totalement les cellules germinales qui demeurent isolées et n'établissent pas de jonctions avec leurs voisines. La sexualisation de la gonade ne se manifeste qu'après l'organogenèse, vers la fin de la vie embryonnaire ou même après la naissance.

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The individualization and organogenesis of the embryonic gonad in the gonochoric prosobranch molluscViviparus viviparus L.

Summary The origin and evolution of the embryonic gonad ofViviparus viviparus were studied ultrastructurally. The gonad is formed by migration and multiplication of pericardial cells.There are several successive stages during gonad organogenesis: during the sexually undifferentiated stage with one cellular type, the gonad is made up of primordial cells which seem to be identical. The sexually undifferentiated stage with two cell types is characterized by the differentiation of germinal and non-germinal cells. The non-germinal cells are linked to one another by septate junctions and their long extensions completely surround the germinal cells which remain isolated and do not form junctions with their neighbours. The gonad sexualisation does not appear until after organogenesis, towards the end of embryonic development, or even after birth.

     

On the control of germ cell development in Caenorhabditis elegans

After hatching, the germ line progenitor cells in C. elegans begin to divide mitotically; later, some of the germ line cells enter meiosis and differentiate into gametes. In the adult, mitotic germ cells, or stem cells, are found at one end (the distal end) and meiotic cells occupy the rest of the elongate gonad. Removal of two somatic gonadal cells, the distal tip cells, by laser microsurgery has a dramatic effect on germ cell development. In either sex, this operation leads to the arrest of mitosis and the initiation of meiosis in germ cells. The function of the distal tip cell in the intact animal appears to be the inhibition of meiosis (or stimulation of mitosis) in nearby germ cells. During development, this permits growth and, in the adult, it maintains the germ line stem cell population. A change in the position of the distal tip cell in the gonad at an early point in development is correlated with a change in the axial polarity of the germ line tissue. This suggests that the localization of the distal tip cell's inhibitory activity at the distal end of the gonad establishes the axial polarity of the germ line tissue in the intact animal.     

Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells.     

Differentiation of murine premigratory primordial germ cells in culture.

In the mouse embryo, primordial germ cells first appear in the extraembryonic mesoderm and divide rapidly while migrating to the fetal gonad. Shortly after their arrival in the gonad, germ cells sexually differentiate as proliferation ceases. Previous studies have established that primordial germ cells proliferate and migrate in feeder layer culture. To explore cellular regulation of fetal germ cell development, we have used germ cell nuclear antigen 1 (GCNA1), a marker normally expressed only in postmigratory germ cells, to investigate the developmental potency of both pre- and postmigratory cells in this culture system. We found that explanted premigratory germ cells will initiate expression of this marker and are, therefore, capable of undertaking some aspects of gonocyte differentiation without intimate exposure to the fetal gonad. We have also tested whether postmigratory gonocytes are stable in culture. As detected by either alkaline phosphatase or GCNA1, we did not detect long-term survival of either prospermatogonia or oogonia under conditions that support the survival, proliferation, and differentiation of earlier premigratory cells. These observations are consistent with an autonomous cellular mechanism governing the initial stages of gonocyte differentiation, and suggest that differentiation towards gonocytes is accompanied by a change in requirements for cell survival.     

The planarian <Emphasis Type="Italic">nanos</Emphasis>-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A survey of the reproductive biology in Italian branchiopods

A survey of the most important aspects of the reproductive biology of Lepidurus apus lubbocki Brauer, 1873 (Notostraca) is presented. Gametogenesis, germ cell distribution, and somatic tract structure of the male gonad are studied by means of TEM and SEM. The most important data emerging from our observations is the total and widespread degeneration of the sperms. We discuss the real functionality of male germ cells, and the implications on sexuality and reproductive strategies. On the whole, present ultrastructural observations suggest that males of Lepidurus apus lubbocki are not functional. Therefore, we suggest that this form reproduces parthenogenetically as the functional female gonad lacks any associated testicular cells. For comparison, we analyzed the male portion of the hermaphroditic gonad of Triops longicaudatus (LeConte, 1846) from California. In this case functionallity of the testis lobes and sperm were readily apparent.     

The structure of the gonad during natural sex reversal in Monopterus albus (Pisces: Teleostei)

A detailed study on the structure of the gonad of Monopterus albus was made as a basis for analysis of gonadal steroids in this sex-reversing teleost. Two types of males were identifiedand their existence appeared to be a result of the difference in gonadal ontogeny among the individuals in natural populations. The germinal area of the gonad, the gonadal lamellae, exhibited à zoned nature with regard to the location of the female and male germ cells. Observations suggested that the male germ cells originated from gonia pre-existing in the inner zone of the gonadal lamellae before sex reversal. Natural reversal of sex in this protogynous hermaphrodite was found to be usually a postnuptial event and was always accompanied by loss of ovarian tissue and by development of interstitial (Leydig) cells. In the mesogonial region of the gonadal wall, peculiar mesenchyme cells were found, their significance remained uncertain.     

Germ cell nuclear antigen (GCNA1) expression does not require a gonadal environment or steroidogenic factor 1: Examination of GCNA1 in ectopic germ cells and in Ftz-f1 null mice

The germ cell lineage is first recognized as a population of mitotically proliferating primordial germ cells that migrate toward the gonadal ridge. Shortly after arriving at the gonadal ridge, the germ cells begin to initiate a commitment to gamete production in the developing gonad. The mechanisms controlling this transition are poorly understood. We recently reported that a mouse germ cell nuclear antigen 1 (GCNA1) is initially detected in both male and female germ cells as they reach the gonad at 11.5 days postcoitum (dpc). GCNA1 is continually expressed in germ cells through all stages of gametogenesis until the diplotene/dictyate stage of meiosis I. Since GCNA1 expression commences soon after primordial germ cells arrive at the gonadal ridge, we wanted to determine whether the gonadal environment was essential for induction of GCNA1 expression. By examining GCNA1 expression in germ cells that migrate ectopically into the adrenal gland, we determined that both the gonadal and adrenal gland environments allow GCNA1 expression. We also examined GCNA1 expression in Ftz-F1 null mice, which are born lacking gonads and adrenal glands. During embryonic development in the Ftz-F1 null mice, the gonad and most germ cells undergo apoptotic degeneration at about 12.5 dpc. While most of the germ cells undergo apoptosis without expressing GCNA1, a few surviving germs cells, especially outside the involuting gonad clearly express GCNA1. Thus, although the Ftz-F1 gene is essential for gonadal and adrenal development, induction of GCNA1 expression in germ cells does not require Ftz-F1 gene products. The finding that germ cell GCNA1 expression is not restricted to the gonadal environment and is not dependent on the Ftz-F1 gene products suggests that GCNA1 expression may be initiated in the germ cell lineage by autonomous means. Mol. Reprod. Dev. 48:154–158, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of the gonadal germinal epithelium during sex reversal and seasonal testicular cycling in the protogynous swamp eel,Synbranchus marmoratus Bloch 1795 (Teleostei,Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a protogynous, diandric species. During sex reversal, the ovarian germinal epithelium, which forms follicles containing an oocyte and encompassing follicle cells during the female portion of the life cycle, produces numerous invaginations, or acini, into the ovarian stroma. Within the acini, the gonia that formerly produced oocytes become spermatogonia, enter meiosis, and produce sperm. The acini are bounded by the basement membrane of the germinal epithelium. Epithelial cells of the female germinal epithelium, which formerly became follicle (granulosa) cells, now become Sertoli cells in the developing testis. Subsequently, lobules and testicular ducts form. The swamp eel testis has a lobular germinal compartment in both primary and secondary males, although the germinal compartment in testes of secondary males resides within the former ovarian lamellae. The germinal compartment, supported by a basement membrane, is composed of Sertoli and germ cells that give rise to sperm. Histological and immunohistochemical techniques were used to describe the five reproductive classes that were observed to occur during the annual reproductive cycle: regressed, early maturation, mid-maturation, late maturation, and regression. These classes are differentiated by the presence of continuous or discontinuous germinal epithelia and by the types of germ cells present. Synbranchus marmoratus has a permanent germinal epithelium. Differences between the germinal compartment of the testes of primary and secondary males were not observed.     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.     

Induction of meiosis in fetal mouse testis in vitro

Fetal mouse testes and ovaries with their urogenital connections were cultured singly or in pairs on Nuclepore filters. When a testis in which the sex was not yet morphologically detectable was cultured together with older ovaries containing germ cells which were progressing through the meiotic prophase, the male germ cells were triggered to enter meiosis. When older fetal testes in which the testicular cords have developed were cultured together with ovaries of the same age with germ cells in meiosis, the oocytes were prevented from reaching diplotene stage. It was concluded that the fetal male and female gonads secrete diffusable substances which influence germ cell differentiation. The male gonad secretes a "meiosis-preventing substance" (MPS) which can arrest the female germ cells within the meiotic prophase. The female gonad secretes a "meiosis-inducing substance" (MIS) which can trigger the nondifferentiated male germ cells to enter meiosis.     

Meiotic prophase in the left embryonic testis of the Japanese quail (Coturnix coturnix japonica L.)

Spontaneous, transitory feminization of the left embryonic testis occurs in the majority of male Japanese quails from day 11 of incubation until the 2nd day after hatching. The feminized gonad is composed of a testicular part and of an ovarian-like cortical region, which develops outside the tunica albuginea. The cortical region contains numerous germ cells some of which enter meiosis on day 11 (i.e. at the time characteristic of female germ cells) and reach pachytene on day 14. Cortical regions are best developed on day 16 (day of hatching), but regression, accompanied by degeneration of many germ cells, begins shortly afterwards. The mechanism of feminization of the left embryonic testis is discussed.     

Germinal and non-germinal lines in the ovotestis of Helix aspersa: a survey

Summary The study of gonadal organogenesis and differentiation by means of light and electron microscopy suggested the following in Helix aspersa: (1) the distal parts of the acini have components of mesodermal origin, whereas the neck and efferent duct comprise ectodermal elements; (2) a segregation of a germinal line occurs early, during the embryonic life; (3) in juvenile and adult animals, male and female cells arise from a germinal ring located at the base of the acinar neck. Apart from developing oocytes, the epithelium lining the distal region of the acini consists of somatic cells (Sertoli and follicle cells).     

Sexual phenotype of avian chimeric gonads with germinal and stromal cells of opposite genetic sexes

The respective roles of germinal and stromal cells in determining the sexual phenotype of the gonad were analyzed in chimeric gonads obtained by surgical recombination between young avian blastodiscs in ovo. Equivalent territories were exchanged between two blastodisc, in order that the germinal crescent and the gonad territory had a different origin (fig. 3). Embryos used for these experiments carried a sex linked pigment mutation, that made it possible to diagnose the genetic sexes of germ cells and stroma at the time when the gonad was retrieved for examination. On the basis of species, three types of combination were performed: chick germ cells in chick or quail stroma, quail germ cells in chick stroma. In each chimera, the genetic sexes of the two gonadal cell populations could be identical or opposite. However it appeared that the germ cell population was not always homogeneous. In some grafting schemes, ectopic germ cells, located outside the germinal crescent, contributed to the colonization of the experimental gonad. These germ cells were from the same territory as the stroma element of the gonad, i.e., they were of the same species and the same genetic sex. Whatever the case, in 87 chimeras that were studied, the sex phenotype of the gonads always corresponded to the genetic sex of the stroma. Thus the genetic sex of germ cells has no role in the sexual differentiation of the gonadal rudiments.