Germ Cell Migration,Proliferation and Differentiation during Gonadal Morphogenesis in All‐Female Japanese Flounder (Paralichthys Olivaceus)

In this study, all genetic female (XX) broods of Japanese flounder were produced artificially by mating the females with sex‐reversed males. The proliferation and migration of primordial germ cells (PGCs), formation of ovary and oogenesis were described in detail. After hatching, around 20 individual PGCs migrated from the lateral to the dorsal of trunk region. At 15 days posthatching (dph), a part of PGCs were covered by a single layer somatic cells and formed the genital ridge. By 22 dph, the elongated gonadal primordia appeared under the ventral kidney, where the PGCs were totally enclosed by somatic cells. During the process of migration, PGCs were presumed to be mitotically inactive. From 63 to 73 dph, somatic cells rearrangement resulted in the formation of a narrow crevice, which became deeper and formed ovarian lumen. However, at 52 dph, dramatic mitotic proliferation of germ cell occurred and germline nest formed before the appearance of ovarian lumen. The onset of intensive germ cell proliferation and appearance of cell nests could be accepted as a criterion of initial ovarian differentiation. Then germ cells and somatic epithelial cells were gradually delimited by basement membrane and formed the germinal epithelium. In this period, results from in situ hybridization revealed that the early forkhead box L2 (pofoxl2) was expressed in somatic cells and oocytes in primary growth, which indicated the prefollicle cells formed. Then oogonia or oocytes, follicle cells, basement membrane, and theca cells composed a follicle complex. Finally, oocytes underwent meiosis and developed into to mature eggs. Anat Rec, 301:727–741, 2018. © 2017 Wiley Periodicals, Inc.     

The Basement Membrane and the Sex Establishment in the Juvenile Hermaphroditism During Gonadal Differentiation of the Gymnocorymbus ternetzi (Teleostei: Characiformes: Characidae)

Although there are several studies on morphogenesis in Teleostei, until now there is no research describing the role of the basement membrane in the establishment of the germinal epithelium during gonadal differentiation in Characiformes. In attempt to study these events that result in the formation of ovarian and testicular structures, gonads of Gymnocorymbus ternetzi were prepared for light microscopy. During gonadal development in G. ternetzi, all individuals first developed ovarian tissue. The undifferentiated gonad was formed by somatic cells (SC) and primordial germ cells (PGCs). After successive mitosis, the PGCs became oogonia, which entered into meiosis originating oocytes. An interstitial tissue developed. In half of the individuals, presumptive female, prefollicle cells synthesized a basement membrane around oocyte forming a follicle. Along the ventral region of the ovary, the tissue invaginated to form the ovigerous lamellae, bordered by the germinal epithelium. Stroma developed and the follicle complexes were formed. The gonadal aromatase was detected in interstitial cells in the early steps of the gonadal differentiation in both sexes. In another half of the individuals, presumptive male, there was no synthesis of basement membrane. The interstitium was invaded by numerous granulocytes. Pre‐Leydig cells proliferated. Apoptotic oocytes were observed and afterward degenerated. Spermatogonia appeared near the degenerating oocytes and associated to SCs, forming testicular tubules. Germinal epithelium developed and the basement membrane was synthesized. Concomitantly, there was decrease of the gonadal aromatase and increase in the 3β‐HSD enzyme expression. Thus, the testis was organized on an ovary previously developed, constituting an indirect gonochoristic differentiation. Anat Rec, 298:1984–2010, 2015. © 2015 Wiley Periodicals, Inc.     

Ultrastructural analysis of folliculogenesis in the ovary of the yellow spotted stingray, Urolophus jamaicensis.

The ovary of the yellow spotted ray, Urolophus jamaicensis, is embedded in the epigonal gland, a lymphomyeloid organ. The covering of the ovary is composed of a germinal epithelium that is cuboidal and dome-shaped with microvilli. Adjacent cells have elaborate intercellular folds that create dilated intercellular spaces. In previtellogenic follicles, the follicle cells are simple cuboidal and contain modest amounts of synthetic or transport organelles. As vitellogenesis proceeds, the epithelium becomes multilaminar. Follicle cells are columnar as yolk precursors are transported from the maternal circulation, through the follicle cell cytoplasm, to the oocyte. Large, round cells occur in the follicle wall that contain lipid-like substances. These cells decrease in size and number as folliculogenesis proceeds and eventually disappear prior to ovulation. Columnar follicular cells and the oocyte have cellular extensions that impinge upon the zona pellucida. Transosomes are follicle cell extensions that indent the oocyte membrane. Tips of transosmes become enclosed by a layer of oocyte plasmalemma. The tips of transosomes pinch off and become resident in the ooplasm. Dense staining material occurs on the inner surface of the transosome membrane derived from the follicle cell. In Other animals, this material has been described as ribosome-like. This study is the first to document the presence of transosomes in a group other than Aves or reptiles. Follicle cells are supported by an extremely thick basal lamina. Subjacent to the lamina is the vascularized theca with fibroblasts embedded in a collagenous network. There is no differentiation into definitive theca interna and externa. In vitellogenic eggs, extensive inward folding of the follicular epithelium occur thereby generating more surface area for the transport of yolk precursors to the oocyte. Atretic follicles are common.     

Immunolocalization of Fas and Fas ligand in the ovaries of women with polycystic ovary syndrome: relationship to apoptosis

Both Fas (APO-1, CD95), an apoptosis-inducing receptor, and its ligand, Fas ligand (FasL, CD95L), have been localized to the ovary. Granulosa cell apoptosis occurs in antral follicular atresia. In polycystic ovary syndrome (PCOS), antral follicles accumulate with some atretic features. The ovarian expression of Fas and FasL was examined in PCOS by immunohistochemistry and correlated with immunodetection of apoptotic cells. Fas immunostaining was present in pre-antral follicle oocytes, some primary and secondary pre-antral follicle granulosa cells, and both granulosa and theca of antral follicles. Thecal staining persisted with advancing atresia, while granulosa staining declined. In antral follicles, abundant Fas-positive cells co-localized with scattered nuclei immunopositive for apoptosis. Ovarian vascular myocytes were strongly Fas-immunopositive. FasL immunostaining was present in pre-antral follicles in oocytes and variably in granulosa. In antral follicles, granulosa and thecal FasL staining increased with advancing atresia. Normal control ovaries showed follicular Fas and FasL staining patterns similar to those in PCOS, but vascular staining was less prominent. In one healthy follicle, Fas immunostaining was seen in the oocyte and weakly in mural granulosa and theca interna. The results suggest that in PCOS, an alteration in Fas-mediated apoptosis, does not cause abnormal folliculogenesis, but may promote ovarian vascular remodelling.     

Immunohistochemical localization of the bone morphogenetic protein receptors in the porcine ovary

The bone morphogenetic protein (BMP) family is emerging as playing a crucial role in regulating normal follicle growth and determining ovulation rate. BMPs exert their effects via BMP receptors (BMPR-IA, -IB and -II). However, there is a paucity of information relating to the expression of the BMPRs within the ovary of large polyovular species such as the pig. Furthermore, there is a lack of information on the expression of BMPRs by fetal ovaries of any species. The purpose of this study was to investigate temporal and spatial expression of the BMPRs in the porcine ovary, at different developmental stages. Immunohistochemistry for BMPR-IA, BMPR-IB and BMPR-II was performed using sections from paraffin wax-embedded ovaries, obtained from fetal (n = 15), prepubertal (n = 3) and cycling postpubertal (n = 4) pigs. Results confirmed the presence of all three receptors in the fetal egg nests and in the granulosa cell layer of follicles ranging from primordial to late antral stages. Immunostaining was also observed in oocytes, theca layer, corpus luteum and ovarian surface epithelium. The expression of BMPRs by fetal ovaries may be related to follicle formation, whereas expression in pre- and post-pubertal animals indicates BMPs are involved in regulating porcine ovarian follicle growth.     

雌激素受体β在小鼠及猪卵巢中表达的比较研究

目的 研究雌激素受体β(ERβ)在小鼠和猪卵巢中的表达差异. 方法通过免疫组织化学的方法,研究ERβ在小鼠及猪卵巢中不同类型细胞中的表达量和细胞内的定位情况. 结果在小鼠卵巢中,ERβ在黄体和各级生长卵泡的卵母细胞质中均有大量表达,在各级卵泡的颗粒细胞和膜细胞中表达量很少,生殖上皮和间质细胞中没有检测到表达信号.猪卵巢中ERβ强表达于各级卵泡的膜细胞中,各种直径的腔前卵泡的颗粒细胞中也有表达,但有腔卵泡颗粒细胞有的表达,有的则未见表达,间质细胞和生殖上皮中无明显表达. 结论 ERβ在小鼠和猪卵巢中的表达和定位差异很大.表明ERβ在小鼠和猪卵巢中的作用也可能不同.     

PCNA in the ovary of zebrafish (Brachydanio rerio, Ham.-Buch.)

PCNA, a nuclear protein intimately involved in DNA synthesis, has been localized immunohistochemically in ovaries of zebrafish (Brachydanio rerio, Ham.-Buch.). Positively stained cells were detected in the follicle epithelium of all stages of growing oocytes and degenerating oocytes. Furthermore, PCNA was found to be present in nuclei of oogonia and oocytes in meiotic prophase. Follicle epithelium showed an increasing number of PCNA-positive cells during egg cell development with a very low index of <0.5% at the beginning and a maximum of >7% in stage IV oocytes that are rapidly incorporating yolk protein. In this phase, follicle epithelium often showed groups of stained cells in limited zones of proliferation. In degenerating oocytes, over 60% of the former follicle cells were positive for PCNA. Nuclei of oogonia were always positive and the nucleus of stage III oocytes showed strong positivity. Stage III is the stage in which amplification of rRNA genes occurs and numerous nucleoli apear in the peripheral caryoplasm. It is concluded that PCNA expression is related to amplification of rDNA.     

A qualitative and quantitative study of rete ovarii development in the fetal rat: Correlation with the onset of meiosis and follicle cell appearance

Development of the rete ovarii and its contribution to the cells of the ovary were examined in fetal rats. Histochemical and autoradiographic techniques were used for the observations between days 15 and 21 of gestation. The data presented indicate that the rete system contributes somatic cells to the ovary before birth. The basement membrane which surrounds the cuboidal epithelium of the mesonephric tubules immediately adjacent to the ovary becomes discontinuous on day 15 of gestation. The mesonephric epithelial cells in this region form a knot or clump of pleiomorphic cells, with no apparent tubular organization, and this clump later becomes surrounded by a basement membrane. On day 17 of gestation the newly established fetal rete ovarii is comprised of three regions; (1) the extraovarian mesonephric tubules (ER), (2) intraovarian cords of flattened epithelioid cells which surround the oogonia (IR), and (3) a knot or clump of cells connecting the ER and IR regions (CR). The entire rete system is enclosed by a continuous basement membrane as defined by Periodic Acid-Schiff Reagent techniques. Autoradiographic and quantitative analyses demonstrate that the ER tubule cells proliferate and are incorporated into the other regions of the rete system. These processes begin on day 17 and continue until at least day 21 of gestation. The role these mesonephric tubule cells may play in the regulation of meiosis and their early contribution to the presumptive granulosa cell population is discussed.     

Distribution patterns of TGF-alpha, laminin and fibronectin and their relationship with folliculogenesis in rat ovary

Many growth factors are considered to be involved in regulatory functions in the ovary. Specific factors mediate local cell-cell interactions in relation to follicle development. As a result of the complexity of the estrous cycles in experimental models, it is not easy to determine the role of a growth factor such as transforming growth factor alpha (TGF-alpha) in the system. Moreover, little is known about possible interactions of TGF-alpha and laminin and fibronectin in basement membranes during estrous cycles in relation to follicle development. Therefore, the present study was designed to investigate distribution patterns of TGF-alpha, laminin and fibronectin and their possible roles during follicle maturation in normal rat ovary. Ovaries were obtained from 6 adult virgin female rats and fixed in buffered neutral formalin. TGF-alpha, laminin, and fibronectin distribution patterns were evaluated using 5-7-microm-thick serial sections using the immunoperoxidase method. It was found that TGF-alpha was predominantly localised in nuclei of oocytes. Varying amounts of TGF-alpha were found in granulosa cells and interstitial thecal cells which form follicles. In addition, laminin and fibronectin were found predominantly in vascular walls, outer layers of granulosa cells and basement membranes of cuboidal/columnar surface epithelium of rat ovary. Therefore, we suggest that TGF-alpha is involved in follicular maturation. Moreover, because laminin was found to be present in between parenchymal follicle cell layers, we suggest that they were attached to supportive stromal cells by fibronectin. As TGF-alpha is associated with follicles and their relationship with the extracellular matrix, TGF-alpha may also induce formation of basement membranes which contains laminin and fibronectin components.     

Chronic cholestasis is associated with hypogonadism and premature ovarian failure in adult rats (cholestasis causes ovarian hypogonadism)

Hypogonadism is a well-known complication in males with chronic liver diseases. However, the consequences of chronic hepatopathies on female reproductive capacities have received relatively little attention. The present study evaluates the effect of chronic obstructive jaundice on the ovary of adult cycling rats. Estrous cyclicity was monitored to check the functional status of the hypothalamic–pituitary–ovarian axis. Ovarian changes were assessed using histomorphometric, immunohistochemical, and ultrastructural techniques. Chronic cholestasis was associated with estrous cycle irregularities, diminished ovarian weight, primordial follicle loss, atretic follicle prevalence, marked stromal fibrosis, and diminished immunoexpression of proliferation marker and estrogen receptors, in addition to many ultrastructural alterations in theca, granulosa cells, and oocytes of antral follicles. The results establish that chronic cholestasis causes hypogonadism and premature ovarian insufficiency in adult cycling female rats.     

Immunohistochemical localization of superoxide dismutase in the human ovary.

To elucidate the physiological function of superoxide dismutase (SOD) in the ovary, we examined the immunohistochemical distribution of CuZn-SOD in the human ovary. We also measured the CuZn-SOD concentration in human follicular fluid by enzyme-linked immunosorbent assay (ELISA). The germinal epithelium and tunica albuginea showed no or weak immunoreactivity. No or weak staining activity was also observed in the non-antral follicle. Once the follicle began to form the antral cavity, theca interna cells began to show intensive immunostaining of SOD, as compared with no staining in the granulosa and theca externa cells. In the gestational corpus luteum, theca and granulosa lutein cells showed intensive and moderate staining activity, respectively. The concentration of CuZn-SOD was 0.222 +/- 0.186 ng/mg protein (mean +/- SD) in the preovulatory follicular fluid. In the present study, the immunohistochemical distribution of SOD was confirmed in the human ovary for the first time. Taking into consideration the fact that SOD catalyses the dismutation reaction of superoxide anion radicals, the present results suggest that theca interna cells play an important role in the protection of the developing oocyte from oxygen radicals by acting as a blood-follicular barrier during follicle maturation.     

Morphology of the ovary of Caiman crocodilus (Crocodylia: Alligatoridae).

We describe the ovarian histology and characterize the folliculogenesis of adult females of Caiman crocodilus in order to compare them with other species of Reptilia. The gonad has a thin irregular cortex and a well-developed reticular stroma, composed of vascularized chordae, drained by large lymphatic vessels and separated by extensive lacunae. Simple cuboidal to squamous epithelium, dense connective tissue with numerous elastin fibers, and smooth muscle constitute the ovarian wall. This morphology is similar to that of other Crocodylia, Aves and Testudines. Germinal nests are distributed in the ovarian cortex, some of them with oogonia. Oocytes leave the germinal nests with a single layered cuboidal granulosa, remaining simple during vitellogenesis, as in other Archosauria and Testudines and different from Squamata. As the oocyte grows, the theca is formed by numerous fibroblasts, collagen fibers, and smooth muscle fibers, becoming very thick, highly vascularized and developing muscle bands at advanced vitellogenesis. Folliculogenesis shares morphological features with other species of Reptilia. The corpus luteum is similar to that described for Archosauria; during luteolysis, the theca shrinks and the amount of luteinic cells decreases. The relationship of the corpus luteum to vitellogenesis and egg maintenance is unclear in Crocodylia. Follicular regression in Caiman crocodilus corresponds to an invasive atresia type. Histological features seen in the ovary of Caiman crocodilus are similar to those described in Alligator mississippiensis indicating that these features could be shared among Crocodylia. Also several of these morphological characteristics are observed in Aves suggesting a similar reproductive functional morphology in Archosauria.     

Germline cell formation and gonad regeneration in solitary and colonial ascidians

The morphology of ascidian gonad is very similar among species. The testis consists of variable number of testicular follicles; the ovary consists of ovarian tubes that are thickened forming the germinal epithelium with stem cells for female germ cells with the exception of botryllid ascidians. Peculiar accessory cells that would be germline in origin accompany the oocytes. Using vasa homologues as a molecular marker, germline precursor cells can be traced back to the embryonic posterior‐most blastomeres and are found in the tail of tailbud embryo in some solitary and colonial ascidians. In Ciona, they are subsequently located in the larval tail, while in colonial botryllid ascidians vasa‐expressing cells become obscure in the tail. Recent evidence suggests that ascidian germ cells can regenerate from cells other than embryonic germline. An ensemble of the embryonic stringency of germ cell lineage and the postembryonic flexibility of gonad formation is discussed. Developmental Dynamics 240:299–308, 2011. © 2011 Wiley‐Liss, Inc.     

Oogenesis in the pentastomidRaillietiella aegypti (Cephalobaenida)

In the pentastomidRaillietiella aegypti the structure of the genital apparatus and of oogonia, previtellogenic and vitellogenic oocytes was studied by light and electron microscopy. The ovarian wall consists of a single-layered epithelium covered by a basement membrane of varying thickness. The ovary is characterized by numerous, lateral, sac-like evaginations in which the oocytes lie.Centrioles indicate mitotic activities in oogonia. In previtellogenic oocytes the ooplasm is poor in organelles, containing only a few mitochondria and rough endoplasmic reticulum, but many free ribosomes. In vitellogenic oocytes there is a rapid multiplication of mitochondria and a proliferation of the rough endoplasmic reticulum. The cytoplasm also contains cortical vacuoles, yolk spheres, dictyosomes and pinocytotic vesicles. The mitochondria lie in one big cluster and combine to the Balbiani body (=yolk nucleus). Two types of yolk spheres differing in morphological details are formed in the oocytes ofR. aegypti.     

Oogonia and oocytes degeneration and the nutritive macrophages in the process of the development of the ovary in embryos of the domestic pig (Sus scrofa dom. L.)

Summary In the development the embryonic ovary numerous oogonia and oocytes degenerate. A histochemical study was carried out on the degenerated oogonia and oocytes in different stages of differentiation and development of the ovary in the pig embryos. In the oogonia the degenerative changes include chiefly the chromatin in interphasic and mitotic nuclei. The most numerous degenerations of the nuclei are in meiotic prophase. The degenerative process involves the changes in nuclear volume. Both, the large swollen nuclei, with chromosomes displaced and clustered at one pole, and also the small pycnotic nuclei are observed. Different kinds of oocytes degenerations are discussed.Special attention was paid to the gonadal cord cells and their role in nutrition of gonocytes and in the follicle formation. Some of the cord cells are transformed into macrophages, which ingeste degenerated oocytes. The supposed nutritive functions of these macrophages (nutritive macrophages) are discussed.     

The Mussel Mytilus galloprovincialis in the Bay of Naples: New Insights on Oogenic Cycle and Its Hormonal Control

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17β-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3β-HSD, 17β-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039–1049, 2019. © 2019 Wiley Periodicals, Inc.     

Smad4蛋白及mRNA在卵巢不同发育阶段的表达

目的：探讨Smad4在不同发育阶段大鼠卵巢中蛋白及mRNA的表达。 方法： 选择不同发育时期大鼠卵巢，运用免疫组化方法检测卵巢中Smad4蛋白表达，并进行图像分析；采用半定量逆转录聚合酶链反应(RT-PCR)方法检测Smad4 mRNA在卵巢中的表达。 结果： 免疫组化结果显示Smad4主要表达在各级卵泡中，在卵巢发育早期，Smad4主要在原始卵泡和窦前卵泡中表达；随着卵巢的发育成熟，Smad4在窦状及成熟卵泡颗粒细胞和卵泡膜细胞的表达与间质细胞比较无显著差异（P>0.05）。Smad4在卵泡中的表达强度也发生了变化：随着卵泡的发育，Smad4在窦状及成熟卵泡卵母细胞的表达与窦前卵泡卵母细胞比较明显减弱（P<0.05，P<0.01）；在卵泡膜细胞的表达逐渐增强（P<0.01），而在各级卵泡颗粒细胞中的表达无显著差异（P>0.05）。RT-PCR结果显示各阶段卵巢均有mRNA的表达，从第3周起Smad4 mRNA的表达明显增强，与生后1 d比较差异显著（P<0.05）。 结论： 卵巢内存在Smad4，提示TGF-β家族对卵泡发育的调节很可能是通过Smad信号转导模式实现的。     

Localization of Smad4 in the ovary of the European hedgehog (Erinaceus europaeus L.)

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor β (TGFβ) superfamily, TGFβ, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor (GDF) are present in the ovaries of many animals. In the present study, we investigated the immunolocalization of Smad4, a signaling molecule of the TGFβ superfamily, during folliculogenesis in the ovary of the European hedgehog (Erinaceus europaeus L., 1758). Immunolocalization studies revealed that Smad4 was widely seen in the ovary, mainly in the follicle, though its location and staining intensity varied with the different stages of the developing follicle. In the primordial follicles and early growing follicles, Smad4 protein was mainly localized in the cytoplasm of the oocyte with a half-moon staining pattern. In the pre-antral follicles, Smad4 protein was mainly located in the granulosa cells, theca cells and diffusely distributed in the interstitial cells surrounding the follicle. In the corpora lutea, the immunostaining for Smad4 was very intense. These results suggested that Smad signal transduction may play an important role in folliculogenesis and conceivably may participate in subsequent pregnancy.     

"Waste" follicular aspirate from fertility treatment--a potential source of human germline stem cells?

Fertility clinics worldwide routinely produce a large volume of 'waste' follicular aspirate, which is potentially an abundant source of immature ovarian follicles. Current attempts to cultivate these further in vitro to yield viable mature oocytes for fertility treatment have not yet achieved much success. Instead, recent lines of evidence have emerged that are suggestive of a potential stem cell niche within such immature ovarian follicles. The recent discovery of follicular renewal and putative germ-line stem cells within the postnatal mammalian ovary shook the foundations of reproductive biology by challenging the established dogma that mammalian females lose the capacity for germ cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. More intriguingly, another recent study in the Drosophila model provided compelling evidence that somatic progenies (nurse cells) of germ-line stem cells had the ability to revert back to the stem-cell-like state. This introduces the exciting possibility that within the mammalian ovarian follicle, similar somatic progenies of germ-line stem cells may also possess a greater intrinsic ability to revert back into functional stem cells. If this is the case, then a favored candidate would be the cumulus/granulosa of immature ovarian follicles, since such cells are true homologues of nurse cells found within the Drosophila ovary. The successful elucidation of a human germ-line stem cell niche within immature ovarian follicles is likely to have huge ramifications in stem cell biology and regenerative medicine.     

Surface epithelium of the fetal guinea-pig ovary: a light and electron microscopic study.

The surface epithelium in fetal guinea-pig ovaries was examined from the time of early sexual differentiation, about 34-days, until approximately ten days before birth. At day 34 the epithelium varied greatly in appearance as seen in the light microscope and possessed a superficial layer of flattened or culoidal cells. By day 58 the epithelium had changed into one layer of regularly arranged columnar cells. During the same period the number of germinal cells decreased. Connections between the germinal cords and the surface epithelium were observed from day 34, being especially numerous and broad at days 34 and 42 and decreasing in number and size from day 46 onwards. The basement membrane beneath the epithelium gradually increased in thickness. In the electron microscope two types of somatic cells could be distinguished. One type formed a superifical single layer connected by junctional complexes and exhibited intracellular bundles 60 A microfilaments running straight through the apical part of the cell, attached to junctional complexes on either side. These bundles were found frequently between days 34 and 42, but were rarely seen after the forth-sixth day. Microvillous projections into the coelomic cavity were especially numerous from day 34 to day 46. The other type of somatic cells lay in close proximity to the germinal cells: microfilaments or junctional complexes were not observed. The subepithelial basement lamina was continuous with that surrounding the connections and the germinal cords.