Stem cell and niche development in the postnatal rat testis

Adult tissue stem cells self-renew and differentiate in a way that exactly meets the biological demand of the dependent tissue. We evaluated spermatogonial stem cell (SSC) activity in the developing rat testis and the quality and accessibility of the stem cell niche in wild type, and two busulfan-treated models of rat pup recipient testes using an SSC transplantation technique as a functional assay. While our results revealed a 69-fold increase in stem cell activity during rat testis development from neonate to adult, only moderate changes in SSC concentration were observed, and stem cells from neonate, pup, and adult donor testes produce spermatogenic colonies of similar size. Analysis of the stem cell niche in recipient rat testes demonstrated that pup testes support high levels of donor stem cell engraftment when endogenous germ cells are removed or compromised by busulfan treatment. Fertility was established when rat pup donor testis cells were transplanted into fetal- or pup-busulfan-treated recipient rat pup testes, and the donor genotype was transmitted to subsequent generations. These results provide insight into stem cell/niche interactions in the rat testis and demonstrate that techniques originally developed in mice can be extended to other species for regenerative medicine and germline modification.     

An electron microscopic study of cultured rat testicular fragments

Summary Fragments of testicular tissue of 26-day-old rats were grown as organ cultures during one to six weeks. Electron microscopic studies showed that these tissues can be maintained in vitro for prolonged periods of time, although the most differentiated elements (Leydig and spermatic cells other than spermatogonia) fail to continue their development and degenerate rather rapidly. The connective tissue structures preserve their usual architecture, but the basal membrane of the tubules appears extremely folded and detached from the epithelium. After four weeks in culture, spermatogonia without differentiating are still present, and among them the presence of a more primitive type is noted. Sertoli cells are well preserved and ultrastructurally they present the characteristics of the adult type. The possibility exists that differentiation of these two lines of cells may be achieved in vitro if the factors necessary for their growth and differentiation are recognized and incorporated in the culture system.     

The pituitary cleft of the rat: An electron microscopic study

The epithelium of the pituitary cleft and its colloid have been studied at the ultrastructural level in normal, adrenalectomized, castrated, dehydrated and lactating adult rats. Both control and experimental animals showed cells in different stages of degradation inside the cleft and mixed with the colloid. These exfoliated cells seems to have originated from the cleft's epithelium or the adjacent granular cells.In adrenalectomized and castrated rats the rostral epithelium of the cleft had large intercellular spaces that in some instances appeared to open directly into the cleft. Occasionally the adenohypophysial canaliculi of the adrenalectomized rats were also seen connecting with the cleft. In lactating and castrated animals dark cells appeared more frequently in the rostral and caudal epithelium of the cleft.The results obtained suggest that the colloid is originated at least in part by involuted pituitary cells. There is also evidence that the follicular cavities of the Pars distalis and the cleft are continuous structures.     

An electron microscopic study of a 'Sertoli-like' cell in the testis of a barnacle, Balanus

The organization of a 'Sertoli-like' cell (SLC) is described in the testis of a barnacle, Balanus eburneus. Underlying a relatively thick basal lamina are 'sheet-like' cytoplasmic extensions that develop from the base of the SLC and almost completely enclose the germ cells. Junctional structures are frequently observed between the SLCs and between an SLC and a developing germ cell. Portions of the SLC extend deep into the testis and subdivide into branches. Developing sperm are seen within inpocketings of the plasmalemma of the branches. Noteworthy among the SLC's organelles are large, well-developed mitochondria, an abundant smooth endoplasmic reticulum and masses of glycogen particles. Intercellular bridges are present between germ cells which are at the same stage of development. In some respects the organization of the SLC in B. eburneus is suggestive of that in the Sertoli cell in the testis of vertebrates.     

Differentiation of the golden hamster oviduct epithelial cells during postnatal development: an electron microscopic study

The ultrastructural changes in the process of differentiation of the epithelial cells of the golden hamster oviduct during postnatal development were investigated by means of electron microscopy. In the epithelium of the ampulla of the neonatal oviducts, no differentiated ciliated cells or secretory cells were identified. In these undifferentiated cells, free ribosomes were well developed, but rough endoplasmic reticulum (RER) and the Golgi apparatus were undeveloped. Cells undergoing ciliogenesis were first identified at 3.5 days after birth, and some ciliated cells appeared at 4.5 days. In the nonciliated cells, marked changes in the organelles were observed at this time. Subsequently, some nonciliated cells containing well-developed RER and Golgi apparatus were observed at 9.5 to 10.5 days after birth, and a few mature secretory cells were observed at 10.5 days. An increase in secretory granules occurred in the secretory cells at 12.5-15.5 days after birth. Many fully mature ciliated and secretory cells were observed at 15.5 days after birth. After 20.5 days after birth, the epithelium was identical with that of the adult golden hamster. Quantitative data indicated that the differentiation of ciliated cells began earlier and took place over a more extended period of time than did that of the secretory cells in the golden hamster oviduct during postnatal development.     

A light and electron microscopic study of the pre- and postnatal development and secretory differentiation of the pars tuberalis of the rat hypophysis

Summary The development of the pars tuberalis was studied in the rat fetus from 13 days of gestation to 6 weeks after birth. After the closure of Rathke's pouch, the pars tuberalis anlage is clearly distinguishable from the anlagen of the partes intermedia and distalis. It comprises the entire basal portion of the adenohypophysial anlage; the limit between the anlagen of the pars tuberalis and the pars distalis is defined by Atwell's recess, i.e. the pathway taken by the hypophysial vessels coming from the vascular plexus of the median eminence.At 14 days the pars tuberalis cells are characterized by the presence of glycogen which persists in the adult. Their secretory differentiation (elaboration of granules with a diameter of 100–120 nm) is obvious at 15 days of gestation. It therefore, clearly precedes that of the other hypophysial cell types. Its functional differentiation takes place well before its adhesion to the primary vascular plexus of the portal system. Cystic formations appear just before birth in the pars tuberalis, much later than those of the pars distalis.These observations on the development of the pars tuberalis, together with previous observations on the adult PT in various species, showing that the specific glandular cells of the pars tuberalis are cytologically different from all known adenohypophysial cell types, seem to indicate a specific endocrine function of this lobe.     

Immunolocalization of cytochrome P450 aromatase in rat testis during postnatal development

Aromatization of androgens into estrogens in rat testis is catalyzed by the microsomal enzyme cytochrome P450 aromatase. In this work, aromatase cellular site was investigated in prepuberal, peripuberal and postpuberal testis, from 10-, 21- and 60-day-old rats respectively. Paraffin-embedded testis sections were processed for P450arom immunostaining using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. Next, biotinylated anti-rabbit IgG was applied, followed by ABC/HRP/complex amplification with diaminobenzidine as chromogen. Prepuberal testis sections showed a strong immunoreactivity of aromatase in Sertoli cell cytoplasm while interstitial cells were immunonegative. In peripuberal testis sections, cytoplasmic immunoreaction was weak in Sertoli cells, but it was strong in spermatocytes and sporadic in Leydig cells. Postpuberal testis sections displayed a moderate aromatase immunoexpression in spermatocytes while a strong immunostaining was observed in round and elongated spermatids, as well as in Leydig cells. These results indicate a different age-dependence of aromatase localization in rat testicular cells during gonadal development. In particular, inside the seminiferous tubules, the aromatization site moves from Sertoli cells to late germ cells, suggesting a proliferative role of aromatase in prepuberal testis and its subsequent involvement in meiotic and post-meiotic germ cell maturation.     

5alpha-reductase isoenzymes 1 and 2 in the rat testis during postnatal development

The pubertal initiation of spermatogenesis is reliant on androgens, and during this time, 5alpha-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5alpha-reductase (5alphaR) isoenzymes (5alphaR1 and 5alphaR2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5alphaR isoenzymes and their relationship to the production of 5alpha-reduced androgens in the postnatal rat testis. Both 5alphaR1 and 5alphaR2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5alphaR1 and 5alphaR2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60-160 days of age. The developmental pattern of both 5alphaR isoenzyme activity levels was mirrored by the testicular production of 5alpha-reduced metabolites. Although 5alphaR1 was greater than 5alphaR2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5alpha-reduced androgen biosynthesis. The peak in 5alphaR isoenzymes and 5alpha-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5alpha-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5alphaR inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5alphaR activity during puberty by 75%-86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5alpha-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5alphaR inhibitors. We conclude that both 5alphaR1 and 5alphaR2 isoenzymes are involved in the peak of 5alpha-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.     

Smad4蛋白在不同龄大鼠睾丸的定位研究

探讨转化生长因子－β超家族肽类的细胞内信号转导分子Smad4蛋白在发育不同阶段大鼠睾丸的表达与分布。分别选用出生后3、7、14、28天以及成年大鼠,应用免疫组织化学ABC法结合葡萄糖氧化酶－DAB－硫酸镍铵增强技术及蛋白质免疫印迹技术,检测Smad4蛋白在大鼠睾丸的表达、定位和发育变化,并通过图像分析技术对免疫组织化学结果进行统计学分析。结果显示,发育各个阶段的间质细胞中都有较强的表达,免疫阳性产物位于细胞质内,而各级生精细胞则无阳性反应,且随着大鼠睾丸发育阶段的变化而蛋白表达量逐渐增多,为TGF－β超家族成员在精子发生和发育过程中的分子机理提供了直接证据。     

An electron microscopic study of the human infundibulum

Summary For a limited period during the oogenesis of Protopterus, blebs of the perinuclear cistern contain, in addition to other inclusions, a special kind of microtubular elements. Most of these blebs face parts of multiple nucleolar bodies that extend toward and make contact with the inner nuclear membrane. The microtubular lumen contains a finely dispersed material of moderate electron density which seems to be in contact with this nucleolar material.Aside from these intracisternal structures there are, within both the perinuclear cytoplasm and the nucleoplasm, similar microtubular arrays without apparent connection with the nuclear envelope. These are either enclosed by membranes derived from those of the envelope or unconfined, having escaped through breaks in their respective bounding membranes. Extracisternal tubules are presumed to have passed their period of putative functional activity and to be undergoing a process of regression and subsequent disintegration.Among possible roles attributable to the intracisternal microtubular apparatus are the following: (1) It may serve for the transport of special nucleolar components to the cytoplasm, possibly to be incorporated in the matrix of developing perinuclear mitochondria; (2) it may provide openings in the nuclear membranes for the direct passage of particulate elements between nucleus and cytoplasm; (3) it may be instrumental in the breakdown of parts of the nuclear envelope prior to its restitution during the subsequent phase of oogenesis.Supported by grants NB-00840 and NB-05219 from the U.S.P.H.S.     

An electron microscopic study of bud development in Saccharomycodes ludwigii and Saccharomyces cerevisiae

Summary Examination of sectioned cells fixed in KMnO₄ has shown that the wall of the first bud of a cell of Saccharomycodes ludwigii arises as an extension of the main wall of the parent, while in subsequent buds it develops by extension of the half-septum remaining at a previous detachment scar. Septa are formed by the deposition of wall material on each side of an electron transparent plate which develops centripetally. Structural changes occur in the marginal region of the septum prior to rupture of the main wall and the separation of cells at the surface of the septum-plate. The broken walls remain as annular rings around the scars following the successive development of buds at both apices of the cell.In Saccharomyces cerevisiae the bud wall arises as a direct extension of the parent wall or as an extension of an additional inner layer developed locally.The two types of bud origin are compared in the two yeasts and a comparison is also made with the development of buds, fission cells, conidia and germ tubes in other organisms.