Primary cilia in the developing pig testis

In vertebrates, a variety of cell types generate a primary cilium. Cilia are implicated in determination and differentiation of a wide variety of organs and during embryonic development. However, there is little information on the presence or function of primary cilia in the mammalian testis. Therefore, the objective of this study was to characterize expression of primary cilia in the developing pig testis. Testicular tissue from pigs at 2–10 weeks of age was analyzed for primary cilia by immunocytochemistry. Expression of primary cilia was also analyzed in testicular tissue formed de novo from a single cell suspension ectopically grafted into a mouse host. Functionality of primary cilia was monitored based on cilia elongation after exposure to lithium. Analysis showed that the primary cilium is present in testis cords as well as in the interstitium of the developing pig testis. Germ cells did not express primary cilia. However, we identified Sertoli cells as one of the somatic cell types that produce a primary cilium within the developing testis. Primary cilium expression was reduced from the second to the third week of pig testis development in situ and during de novo morphogenesis of testis tissue from a single cell suspension after xenotransplantation. In vitro, primary cilia were elongated in response to lithium treatment. These results indicate that primary cilia on Sertoli cells may function during testicular development. De novo morphogenesis of testis tissue from single cell suspensions may provide an accessible platform to study and manipulate expression and function of primary cilia.     

Pre-sertoli specific gene expression profiling reveals differential expression of Ppt1 and Brd3 genes within the mouse genital ridge at the time of sex determination

In mammals, testis determination is initiated when the SRY gene is expressed in pre-Sertoli cells of the undifferentiated genital ridge. SRY directs the differentiation of these cells into Sertoli cells and initiates the testis differentiation pathway via currently ill-defined mechanisms. Because Sertoli cells are the first somatic cells to differentiate within the developing testis, it is likely that the signals for orchestrating testis determination are expressed within pre-Sertoli cells. We have previously generated a transgenic mouse line that expresses green fluorescent protein under the control of the pig SRY promoter, thus marking pre-Sertoli cells via fluorescence. We have now used suppression-subtractive hybridization (SSH) to construct a normalized cDNA library derived from fluorescence-activated cell sorting (FACS) purified pre-Sertoli cells taken from 12.0 to 12.5 days postcoitum (dpc) fetal transgenic mouse testes. A total of 35 candidate cDNAs for known genes were identified. Detection of Sf1, a gene known for its role in sex determination as well as Vanin-1, Vcp1, Sparc, and Aldh3a1, four genes previously identified in differential screens as gene overexpressed in developing testis compared with ovary, support the biological validity of our experimental model. Whole-mount in situ hybridization was performed on the 35 candidate genes for qualitative differential expression between male and female genital ridges; six were upregulated in the testis and one was upregulated in the ovary. The expression pattern of two genes, Ppt1 and Brd3, were examined in further detail. We conclude that combining transgenically marked fluorescent cell populations with differential expression screening is useful for cell expression profiling in developmental systems such as sex determination and differentiation.     

Germ cell development in the XXY mouse: evidence that X chromosome reactivation is independent of sexual differentiation

Prior to entry into meiosis, XX germ cells in the fetal ovary undergo X chromosome reactivation. The signal for reactivation is thought to emanate from the genital ridge, but it is unclear whether it is specific to the developing ovary. To determine whether the signals are present in the developing testis as well as the ovary, we examined the expression of X-linked genes in germ cells from XXY male mice. To facilitate this analysis, we generated XXY and XX fetuses carrying X chromosomes that were differentially marked and subject to nonrandom inactivation. This pattern of nonrandom inactivation was maintained in somatic cells but, in XX as well as XXY fetuses, both parental alleles were expressed in germ cell-enriched cell populations. Because testis differentiation is temporally and morphologically normal in the XXY testis and because all germ cells embark upon a male pathway of development, these results provide compelling evidence that X chromosome reactivation in fetal germ cells is independent of the somatic events of sexual differentiation. Proper X chromosome dosage is essential for the normal fertility of male mammals, and abnormalities in germ cell development are apparent in the XXY testis within several days of X reactivation. Studies of exceptional germ cells that survive in the postnatal XXY testis demonstrated that surviving germ cells are exclusively XY and result from rare nondisjunctional events that give rise to clones of XY cells.     

Mesenchymal cell precursors of peritubular smooth muscle cells of the mouse testis can be identified by the presence of the p75 neurotrophin receptor

In the mouse embryo, at approximately 11.5 days postcoitum (dpc), cells migrate from the mesonephros into the developing testis to contribute to the somatic population of the interstitial compartment (i.e., peritubular myoid cells, Leydig cells, and endothelial cells). Studies from this laboratory have shown that the interstitial population of mesenchymal cells in fetal and newborn mouse testis express the p75 neurotrophin receptor (p75NTR, formerly known as the low-affinity nerve growth factor receptor); part of the cell population progressively congregates around testis cords, later to be replaced by contractile peritubular myoid cells, which express smooth muscle cell markers. In the present study, we show that the migrating cells and the p75NTR-expressing cells are the same population. We also show that the neurotrophin receptor is a useful endogenous marker to follow cell migration within the urogenital ridge and to identify and isolate mesenchymal precursors of myoid cells. A time-course immunolocalization study of the location of p75NTR-bearing cells within the urogenital ridge of mouse embryos between 10.5 and 12.5 dpc showed that the interstitium of the fetal testis was progressively occupied by p75NTR+ cells. The progressive increase of p75NTR expression within the developing testis was confirmed by immunoblot analysis of proteins isolated from the fetal gonads. Organ cultures of isolated testes or testis-mesonephros grafts confirmed that p75NTR+ cells do not appear in the testis unless a mesonephros is attached to it. Cells bearing the p75NTR receptor, purified from 12.5-dpc male mouse mesonephroi by immunomagnetic sorting, were able to differentiate in vitro into myoid cells. Immunofluorescence analysis of postnatal testis sections confirmed the presence around the tubules of cells coexpressing p75NTR and alpha-smooth muscle actin. The ability to identify and purify precursors of myoid cells may be of considerable help for studying the mechanisms regulating their differentiation.     

Aspects of germinal cyst and sperm development in Poecilia latipinna (Teleostei: Poeciliidae).

The structure of the testis of Poecilia latipinna is described with particular reference to Sertoli cell-germ cell relationships during development and maturation of the germinal cyst. The cyst develops when primary spermatocytes become surrounded by a single layer of Sertoli cells at the testis periphery. As spermatogenesis and then spermiogenesis proceed, the cyst moves centrally in the testis toward the ducts comprising the vasa efferentia. In addition to being a structural part of the germinal cyst, the Sertoli cells phagocytize residual bodies cast off by developing spermatids and form an association with mature bodies cast off by developing spermatids and form an association with mature sperm, which resembles that observed in mammals, before the sperm are released into the vasa efferentia as a spermatozeugmata. The results of this investigation are discussed in view of what is known concerning testis structure in other teleosts and similarities between cell functions in teleosts and mammals. It is concluded that teleost Sertoli cells, teleost lobule boundary cells and mammalian Sertoli cells are homologous.     

Sertoli cell vacuolization and abnormal germ cell adhesion in mice deficient in an inositol polyphosphate 5-phosphatase

The dynamic nature of cellular interactions during differentiation of germ cells and their translocation from the basement membrane to the lumen of the seminiferous tubules requires the existence of complex and well-regulated cellular adhesion mechanisms in the testis. Successful migration of the developing germ cells is characterized by dynamic breakage and reformation of cadherin-containing adherens junctions between the germ cells and Sertoli cells, the polarized somatic cells of the testis that support and nourish the developing gametes. Here, we demonstrate the accumulation of abnormally swollen, actin-coated, endosome-like structures that contain intact adherens junctions and stain positive for N-cadherin and beta-catenin in the Sertoli cell cytosol of mice deficient in Inpp5b, an inositol polyphosphate 5-phosphatase. Simultaneous to the formation of these abnormal structures, developing germ cells are prematurely released from the seminiferous epithelium and sloughed into the epididymis. Our results demonstrate a role for Inpp5b in the regulation of cell adhesion in the testis and in the formation of junctional complexes with neighboring cells, and they emphasize the important and essential role of phosphoinositides in spermatogenesis.     

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75^NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.     

精索静脉曲张对青春期大鼠睾丸中VEGF和Flt-1蛋白表达的影响

目的研究血管内皮生长因子(VEGF)及其受体Flt-1蛋白在实验性左侧精索静脉曲张(ELV)大鼠睾丸中的表达和定位,探讨它们在精索静脉曲张(VC)致男性不育中的作用。方法建立青春期大鼠ELV模型,采用免疫组化法检测VEGF及Flt-1在ELV4周、8周组及相应对照组大鼠睾丸中的表达变化。结果 VEGF和Flt-1蛋白在大鼠睾丸中定位具有细胞特异性。VEGF蛋白表达于生精细胞、精子细胞发育中的顶体、Sertoli和Leydig细胞胞质内;Flt-1表达于精子细胞发育中的顶体及Leydig细胞胞质中。ELV4周组睾丸中VEGF蛋白的表达显著增加(P<0.01),8周时其表达量下降(P<0.01);ELV4周组与8周组睾丸中Flt-1蛋白的表达均比相应对照组下降(P<0.01),ELV8周组比4周组显著减少(P<0.01)。结论 ELV可影响青春期大鼠睾丸中VEGF和Flt-1蛋白的表达量,可能会影响精子的发生、发育,因而该变化可能是VC引起男性不育的原因之一。     

The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals

The stem cell niches at the apex of Drosophila ovaries and testes have been viewed as distinct in two major respects. While both contain germline stem cells, the testis niche also contains "cyst progenitor" stem cells, which divide to produce somatic cells that encase developing germ cells. Moreover, while both niches utilize BMP signaling, the testis niche requires a key JAK/STAT signal. We now show, by lineage marking, that the ovarian niche also contains a second type of stem cell. These "escort stem cells" morphologically resemble testis cyst progenitor cells and their daughters encase developing cysts before undergoing apoptosis at the time of follicle formation. In addition, we show that JAK/STAT signaling also plays a critical role in ovarian niche function, and acts within escort cells. These observations reveal striking similarities in the stem cell niches of male and female gonads, and suggest that they are largely governed by common mechanisms.     

Postnatal development of lectin-binding pattern in the rat testis and epididymis

Seven rhodamine-conjugated lectins were utilized to study the distribution of glycoproteins in the developing rat testis and epididymis. In the testis a clear developmental pattern was found in Leydig cells and the cell boundaries between Sertoli and spermatogenic cells, as well as during acrosome formation. Some of the first degenerating meiotic cells and the apical extensions of the Sertoli cells at the time of spermiation also displayed a characteristic lectin binding. The epididymal differentiation was characterized by an increasing lectin binding of the subapical Golgi zone and apical surface, and intratubular secretion prior to the arrival of sperm. After the accumulation of tubular secretion and sperm some epithelial cells were transformed into narrow (initial segment) and light cells (distal caput, cauda) with a strong affinity for some lectins. These cells appeared to be responsible for the absorption and digestion of tubular material derived from the testis and of surplus secretion and/or sperm structures.     

Morphological Studies on the Testis of Adults of Moniliformis (Acanthocephala)

The morphology of the testis in young adult males of Moniliformis dubius developing in the rat has been studied with the aid of light and electron microscopes. In one-day-old male worms, the testis is organised as two zones. One zone consists of individual germ-line cells, while the other consists of a supporting syncytium which embeds the cells and forms the boundary of the testis. The surface of the testis is covered by a fibrous non-cytoplasmic coat. In seven-day-old male worms, the syncytium has lost its compact form, breaking down into multinucleate units connected by cytoplasmic processes and apparently forming a loose syncytial network throughout the testis. The germ cells are now randomly distributed and are surrounded by wide spaces among the segments of the supporting syncytium. The fibrous coat, lined internally by an irregular layer of the syncytium, forms the testis envelope. This basic structure is maintained in the testis of 14-day-old and sexually mature male worms.     

Cytoplasmic filaments in fetal and neonatal pig testis

Leydig cells in developing fetal pig testis contained during the fetal regressive phase large accumulations of intermediate filaments. Before and after this period these filaments were arranged in a criss-cross fashion. In the pig as well as in the dog testis these filaments have been characterized as vimentin. Within the vimentin aggregates occasionally a weak positive actin reaction was seen in pig but not in dog Leydig cells. Microfilaments were hardly observed. Most Sertoli cells contained a layer of actin microfilaments close to the basal cell membrane. In the lower cell compartment and around the nucleus (intermediate) vimentin filaments could be observed in a criss-cross configuration.     

Ameboid cells in spermatogenic cysts of caecilian testis

Sertoli cells constitute a permanent feature of the testis lobules in caecilians irrespective of the functional state of the testis. The developing germ cells are intimately associated with the Sertoli cells, which are adherent to the basal lamina, until spermiation. There are irregularly shaped cells in the cores of the testis lobules that interact with germ cells at the face opposite to their attachment with Sertoli cells. These irregularly shaped (ameboid) cells first appear in the lumen of the cysts containing primary spermatocytes and are continually present until spermiation. We did not observe any cytoplasmic continuity between a Sertoli cell and an ameboid cell. Both light microscopic and TEM observations reveal a phagocytic role for the ameboid cells: they scavenge the residual bodies shed by spermatozoa. Organization of the ameboid cells is grossly different from that of the spermatogenic and Sertoli cells. They appear to develop from the epithelium at the juncture of the collecting ductule with the testis lobule.     

The reproductive cycle of yellowfin bream, Acanthopagms australis (Günther), with particular reference to protandrous sex inversion

Yellowfin bream, Acanthopagrus australis , of all age classes were collected from Moreton Bay, Australia. The species possessed typical sparid ovotestes in which the testis and ovary occur in separate zones. During the spawning period (June-August) juveniles, functional males and functional females could be distinguished by the macroscopic appearance of the gonad. The sex ratio of males to females decreases with age, indicating protandrous sex inversion.
Histological and structural study of the ovotestis showed all fish have previtellogenic cells in the ovarian zone but only juvenile and male fish have developing spermatogenic cells in the testis. Most juveniles become functional males by the age of two years but a small proportion of juveniles develop directly into functional females (primary females). Protandrous sex inversion commences after the spawning period when male fish appear with spermatozoa and no other spermatogenic cells in the testis. During the period November-January male fish with no spermatogenic cells are common and a reduction in size of the testis occurs so that by March-April the ovotestis becomes structurally and histologically similar to the female ovotestis. Some fish remain functional males during their whole life-history (primary males). In functional females vitellogenic cells are present in the ovary only during the spawning period and the testis remains very small in size.     

Spatiotemporal patterns of expression of neurotrophins and neurotrophin receptors in mice suggest functional roles in testicular and epididymal morphogenesis.

Several reports have established that the action of neurotrophins is not restricted to the nervous system but can affect a broad range of non-neuronal cells. Nerve growth factor (NGF) is present in adult testis and has been suggested as a potential regulator of meiosis in rat seminiferous epithelium. Here we present an extensive immunohistochemical study on neurotrophins and their receptors (p75 and trk) in the developing mouse testis and epididymis, and in fetal human testis. During the early steps of testicular and epididymal organization in the mouse, strong p75 immunoreactivity is detectable in the gonadal ridge in the mesenchyme that is excluded from the evolving testicular cords, and in the mesenchymal cells of the mesonephros. Later in organogenesis, most of the p75-positive interstitial cells of the testis coexpress neurotrophin-3 (NT-3) and the truncated trk B receptor in a developmentally regulated pattern. Our Western blot data confirm the expression of these molecules. These findings suggest that neurotrophin receptors play a role in early inductive events during critical periods of testicular and epididymal development. During fetal and postnatal histogenesis, an increasing number of NT-3- and p75-positive mesenchymal cells start to express alpha-smooth muscle isoactin, suggesting a role for the so-called neurotrophic system in the differentiation of testicular myoid cells and epididymal smooth muscle cells. In the testis of an 18-wk gestational-age human fetus, immunohistochemical analysis has shown intense immunoreactivity of mesenchymal cells to antibodies for neurotrophin receptors p75, trk A, and trk C, and their ligands NGF and NT-3. In addition, we found that in the human fetal testis, the interstitial cells that are differentiating into peritubular myoid cells are associated with a dense network of nerve fibers. Our data suggest that neurotrophins and their receptors are involved in a multifunctional system that regulates cell differentiation and innervation in the developing testis and epididymis.     

Identification of novel markers of mouse fetal ovary development

In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process.     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Two distinct origins for Leydig cell progenitors in the fetal testis

During the differentiation of the mammalian embryonic testis, two compartments are defined: the testis cords and the interstitium. The testis cords give rise to the adult seminiferous tubules, whereas steroidogenic Leydig cells and other less well characterized cell types differentiate in the interstitium (the space between testis cords). Although the process of testis cord formation is essential for male development, it is not entirely understood. It has been viewed as a Sertoli-cell driven process, but growing evidence suggests that interstitial cells play an essential role during testis formation. However, little is known about the origin of the interstitium or the molecular and cellular diversity within this early stromal compartment. To better understand the process of mammalian gonad differentiation, we have undertaken an analysis of developing interstitial/stromal cells in the early mouse testis and ovary. We have discovered molecular heterogeneity in the interstitium and have characterized new markers of distinct cell types in the gonad: MAFB, C-MAF, and VCAM1. Our results show that at least two distinct progenitor lineages give rise to the interstitial/stromal compartment of the gonad: the coelomic epithelium and specialized cells along the gonad–mesonephros border. We demonstrate that both these populations give rise to interstitial precursors that can differentiate into fetal Leydig cells. Our analysis also reveals that perivascular cells migrate into the gonad from the mesonephric border along with endothelial cells and that these vessel-associated cells likely represent an interstitial precursor lineage. This study highlights the cellular diversity of the interstitial cell population and suggests that complex cell–cell interactions among cells in the interstitium are involved in testis morphogenesis.