Adult Seminiferous Tubules Research Articles

Differentiation of fetal sertoli cells in the adult testis.

Sertoli cells proliferate and construct seminiferous tubules during fetal life, then undergo differentiation and maturation in the prepubertal testes. In the adult testes, mature Sertoli cells maintain spermatogonia and support spermatogenesis during the entire lifetime. Although Sertoli-like cells have been derived from iPS cells, they tend to remain immature. To investigate whether Sertoli cells can spontaneously acquire the ability to support spermatogenesis when transferred into the adult testis, we transplanted mouse fetal testicular cells into a Sertoli-depleted adult testis. We found that donor E12.5, E14.5 and E16.5 Sertoli cells colonized adult seminiferous tubules and supported host spermatogenesis 2 months after transplantation, demonstrating that immature fetal Sertoli cells can undergo sufficient maturation in the adult testis to become functional. This technique will be useful to analyze the developmental process of Sertoli cell maturation and to investigate the potential of iPS-derived Sertoli cells to colonize, undergo maturation, and support spermatogenesis within the testis environment.

Morphology of mouse seminiferous tubules.

We developed a technique to analyze the high-resolution three-dimensional (3D) structure of seminiferous tubules. It consists of segmentation of tubules in serial paraffin sections of the testis by marking the basement membrane with periodic acid-Schiff or a fluorescent anti-laminin antibody followed by 3D reconstruction of tubules with high-performance software. Using this method, we analyzed testes from mice at different ages and accurately elucidated the 3D structure of seminiferous tubules, including the number and length of tubules as well as the numbers of connections with the rete testis, branching points, and blind ends. We also developed a technique to identify the precise spermatogenic stage and cellular composition of the seminiferous epithelium. It consists of the combination of lectin histochemistry for acrosomes and immunohistochemistry for specific cell markers visualized with fluorescence. Using this method, we examined seminiferous tubules from normal mice and counted the number of each cell type at each stage, and thereby established a quantitative standard for the cellular composition of the seminiferous epithelium. We then investigated seminiferous epithelia from genetically modified infertile mice deficient in certain cell adhesion molecules and revealed characteristic abnormalities in the cellular composition. We also analyzed the distribution and direction of spermatogenic waves along the length of adult seminiferous tubules as well as the site of the first onset of spermatogenesis in postnatal seminiferous tubules. These methods will be useful for investigating the structure and function of seminiferous tubules in mice and humans under normal and pathological conditions.

SALL4 Expression in Gonocytes and Spermatogonial Clones of Postnatal Mouse Testes

The spermatogenic lineage is established after birth when gonocytes migrate to the basement membrane of seminiferous tubules and give rise to spermatogonial stem cells (SSC). In adults, SSCs reside within the population of undifferentiated spermatogonia (Aundiff) that expands clonally from single cells (Asingle) to form pairs (Apaired) and chains of 4, 8 and 16 Aaligned spermatogonia. Although stem cell activity is thought to reside in the population of Asingle spermatogonia, new research suggests that clone size alone does not define the stem cell pool. The mechanisms that regulate self-renewal and differentiation fate decisions are poorly understood due to limited availability of experimental tools that distinguish the products of those fate decisions. The pluripotency factor SALL4 (sal-like protein 4) is implicated in stem cell maintenance and patterning in many organs during embryonic development, but expression becomes restricted to the gonads after birth. We analyzed the expression of SALL4 in the mouse testis during the first weeks after birth and in adult seminiferous tubules. In newborn mice, the isoform SALL4B is expressed in quiescent gonocytes at postnatal day 0 (PND0) and SALL4A is upregulated at PND7 when gonocytes have colonized the basement membrane and given rise to spermatogonia. During steady-state spermatogenesis in adult testes, SALL4 expression overlapped substantially with PLZF and LIN28 in Asingle, Apaired and Aaligned spermatogonia and therefore appears to be a marker of undifferentiated spermatogonia in mice. In contrast, co-expression of SALL4 with GFRα1 and cKIT identified distinct subpopulations of Aundiff in all clone sizes that might provide clues about SSC regulation. Collectively, these results indicate that 1) SALL4 isoforms are differentially expressed at the initiation of spermatogenesis, 2) SALL4 is expressed in undifferentiated spermatogonia in adult testes and 3) SALL4 co-staining with GFRα1 and cKIT reveals distinct subpopulations of Aundiff spermatogonia that merit further investigation.

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.

Characterization of Mongolian Gerbil Chromatoid Bodies and Their Correlation with Nucleolar Cycle During Spermatogenesis

The aims of the present study were to monitor the nucleolar cycle in Mongolian gerbil spermiogenesis, to verify the relationship between the nucleolar component and chromatoid body (CB) formation and to investigate the function of this cytoplasmic supramolecular structure in spermatogenic cells. Histological sections of adult seminiferous tubules were analysed cytochemically by light microscopy and ultrastructurally by transmission electron microscopy. The results reveal that in early spermatids, the CB was visualized in association with Golgi vesicles indicating that this structure may participate in the acrosome formation process as had been reported in other rodents. In late spermatids, the CB was observed near the axoneme region suggesting that this structure may support spermatozoon tail formation as happens in other species. Chromatoid body was joined with lipid droplets in this same cell type. This observation should be investigated to verify whether CB may be related to steroidal hormone metabolism. In conclusion, our data showed that there is disintegration of primary spermatocyte nucleoli at the beginning of prophase I and a fraction of this nucleolar material migrates to the cytoplasm, where a specific structure is formed, known as the 'chromatoid body', which apparently participates in some parts of the gerbil spermiogenesis process.

Interaction with the mammary microenvironment redirects spermatogenic cell fate in vivo

Previously, we characterized a parity-induced mammary epithelial cell population that possessed the properties of pluripotency and self-renewal upon transplantation. These cells were lineally marked by the expression of beta-galactosidase (LacZ) as a result of mammary-specific activation of a reporter gene through Cre-lox recombination during pregnancy. We used this experimental model to determine whether testicular cells would alter their cell fate upon interaction with the mammary gland microenvironment during pregnancy, lactation, and involution. Adult testicular cells, isolated from seminiferous tubules, were mixed with limiting dilutions of dispersed mammary epithelial cells and injected into epithelium-divested mammary fat pads. The host mice were bred 6-8 weeks later and examined 20-30 days postinvolution. This approach allowed for the growth of mammary tissue from the injected cells and transient activation of the whey acidic protein promoter-Cre gene during pregnancy and lactation, leading to Cre-lox recombination and constitutive expression of LacZ from its promoter. Here we show that cells from adult seminiferous tubules interact with mammary epithelial cells during regeneration of the gland. They adopt mammary epithelial progenitor cell properties, including self-renewal and the production of cell progeny, which differentiate into functional mammary epithelial cells. Our results provide evidence for the ascendancy of the tissue microenvironment over the intrinsic nature of cells from an alternative adult tissue.

Meiotic nucleolar cycle and chromatoid body formation during the rat ( Rattus novergicus) and mouse ( Mus musculus) spermiogenesis

The aims of the present study were to follow the nucleolar cycle in spermiogenesis of the laboratory rodents Rattus novergicus and Mus musculus, to verify the relationship between the nucleolar component and chromatoid body (CB) formation and to investigate the function of this cytoplasmic supramolecular structure in spermatogenic haploid cells. Histological sections of adult seminiferous tubules were analyzed cytochemically by light microscopy and ultrastructural procedures by transmission electron microscopy. The results reveal that in early spermatids, the CB was visualized in association with the Golgi cisterns indicating that this structure may participate in the acrosome formation process. In late spermatids, the CB was observed near the axonema, a fact suggesting that this structure may support the formation of the spermatozoon tail. In conclusion, our data showed that there is disintegration of spermatid nucleoli at the beginning of spermatogenesis and a fraction of this nucleolar material migrates to the cytoplasm, where a specific structure is formed, known as the “chromatoid body”, which, apparently, participates in some parts of the rodent spermiogenesis process.

A Targeted Mutation of Nkd1 Impairs Mouse Spermatogenesis

Nkd1 is an antagonist of the canonical Wnt/beta-catenin signaling pathway. The EF-hand motif of Nkd1 is required for its inhibitory function. Early studies suggested that Nkd1 might play important roles in mouse embryonic development and tumorigenesis. We constructed Nkd1(-/-) mice whose Nkd1 protein lacked the EF-hand and was unable to inhibit Wnt/beta-catenin signaling. The homozygotes were viable and grew normally, but their fertility in males was reduced. In wild-type adult testes, Nkd1 mRNA was expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand caused reductions in the testis weight and sperm count by 30 and 60%, respectively. During testis development, Nkd1 mRNA expression started at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increased in the elongating spermatids, suggesting that the mutant Nkd1 failed to inhibit the Wnt/beta-catenin pathway. These results suggest that deletion of the EF-hand from Nkd1 reduces the number of the elongating spermatids at haploid stage. In contrast, the mutant Nkd1 did not affect intestinal polyposis in Apc(Delta716) mice.

Alternative patterns of transcription and translation of the ribosomal protein L32 mRNA in somatic and spermatogenic cells in mice

The patterns of transcription and translation of the ribosomal protein L32 ( Rpl32) mRNA differ greatly in adult testis and somatic tissues. Northern blots reveal that the levels of Rpl32 mRNA are four- to five-fold higher in prepubertal and adult testes, and purified pachytene spermatocytes and round spermatids than in a variety of nongrowing adult somatic tissues. 5′ RACE demonstrates that transcription in 8-day prepubertal testis, which lacks meiotic and haploid cells, strongly prefers the same start site in the 5′ terminal oligopyrimidine tract (5′ TOP) that is used is somatic cells. The 5′ TOP is a cis element that inhibits translation of many mRNAs in nongrowing somatic cells. Although the sizes of deadenylated Rpl32 mRNAs are indistinguishable in somatic and spermatogenic cells, transcription initiates at 11 sites over a 31-nt segment in adult testis and ∼62% of Rpl32 mRNAs lack a 5′ TOP. In agreement with previous studies, low levels of cycloheximide increase the proportions and sizes of polysomes in absorbance profiles, and increase the proportions and sizes of polysomes translating four 5′ TOP mRNA species including the Rpl32 mRNA in 8-day seminiferous tubules. In contrast, cycloheximide has little or no effect on the absorbance profiles and distribution of Rpl32 mRNA and 5′ TOP mRNAs in adult seminiferous tubules. The failure of cycloheximide to increase the size of polysomes in adult seminiferous tubules implies a block in the pathway by which ribosomes are recruited onto translationally active mRNAs.

Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line.

During normal embryonic development, mammalian germ cells use both cell migration and aggregation to form the primitive sex cords. Germ cells must be able to interact with their environment and each other to accomplish this; however, the molecular basis of early germ cell adhesion is not well characterized. Differential adhesion is also thought to occur in the adult seminiferous tubules, since germ cells move from the periphery to the lumen as they differentiate. In a screen for additional adhesion molecules expressed by the germ line, expression of the homophilic adhesion molecule, Ep-CAM, was identified in embryonic, neonatal and adult germ cells using immunocytochemistry and flow cytometry with an Ep-CAM-specific monoclonal antibody. At embryonic stages, germ cells were found to express Ep-CAM during migration at embryonic day 10.5 and early gonad assembly at embryonic day 12.5. Expression of Ep-CAM was also found on neonatal male and female germ cells. In the adult testis, Ep-CAM was detected only on spermatogonia, and was absent from more differentiated cells. Finally, embryonic stem cells were shown to express this receptor. It is proposed that Ep-CAM plays a role in the development of the germ line and the behaviour of totipotent cells.

Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa.

We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.

Gene expression of mouse M1 and M2 pyruvate kinase isoenzymes correlates with differential poly[A] tract extension of their mRNAs during the development of spermatogenesis

In eukaryotes, different isoenzymes for pyruvate kinase have been characterized. M2-type Pk cDNA from a mouse fetal ovary library was isolated and differential expression for M1 and M2-types during testis development was observed. While the presence of M2 mRNAs decreases throughout the development of spermatogenesis, we deduced that M1 type expression increases in adult testis coinciding with the presence of elongating spermatids in the seminiferous epithelium. Polyadenylation tests showed a concurrent increase in the length of the polyadenylation tail of transcribed M1-type pyruvate kinase mRNAs in prepuberal to adult seminiferous tubules. A similar relationship between poly[A] tail extension and differential increase of gene expression was detected for M1-type mRNA in adult brain and muscle. Length of poly[A] tail of M2-type transcripts is shown to decrease during the development of mouse testis. These results suggest that changes in the length of the poly[A] tail of transcripts are associated with differential expression of both regulated isoenzymes during testicular development.

Adult testicular enlargement induced by neonatal hypothyroidism is accompanied by increased Sertoli and germ cell numbers

Our previous studies have shown that transient neonatal hypothyroidism, induced by treatment with the reversible goitrogen 6-propyl-2-thiouracil (PTU), increases testicular size and daily sperm production in the adult rat by up to 82% and 136%, respectively. The objective of the present study was to examine morphological and functional changes in adult seminiferous tubules associated with PTU-induced increases in testicular size and sperm production. Sprague-Dawley rats were treated with PTU from birth to day 25 or left untreated; for morphometry, all testes were fixed by vascular perfusion at 90 days of age. Although testicular weight was increased 62% in treated rats, gross pathological changes were not evident in these organs, and spermatogenesis appeared morphologically normal. The percent area of testis occupied by seminiferous tubules was equal in control and treated testes, but mean seminiferous tubule diameter and length were increased in the PTU-treated testis. The adult number of Sertoli cells in treated testes was increased by 157%, and the numbers of leptotene spermatocytes and round spermatids were increased 84% and 93%, respectively. These results demonstrate that increases in Sertoli cell numbers result in increased sperm production and support the idea that Sertoli cells are the major regulators of the magnitude of sperm production. Although the round spermatid to Sertoli cell ratio was reduced by nearly 30%, the number of round spermatids per g testis was increased by 14%. This increased efficiency of sperm production was accomplished by an increased density of Sertoli cells along the basement membrane and an increased height of the seminiferous epithelium. Despite the large increase in Sertoli cell numbers in treated rats, Northern blot analysis using Sertoli cell-specific cDNA probes for transferrin and androgen-binding protein indicated that relative steady state levels of mRNAs per Sertoli cell for these two secretory proteins were similar in control and treated rats at 90 days of age.

Biochemical and immunocytochemical analysis of a histone H1 variant from the mouse testis.

An H1 histone variant, H1a, has been isolated and purified from the mouse testis. Biochemical and amino acid analyses indicate its similarity with the rat testis H1a. Specific antibodies against the purified H1a have been generated in rabbits and used to study its tissue and species distribution using protein blotting procedures. We have also used the immunocytochemical technique to determine in situ distribution of H1a in spermatogenic cells and somatic tissues of the mouse. A non-random distribution of H1a has been noted in the nuclei of certain somatic cell types such as Sertoli cells, Leydig cells and neurons. By contrast, hepatocyte nuclei lacked detectable levels of H1a. In adult seminiferous tubules, the early primary spermatocyte nuclei displayed a greater level of immunoreactivity relative to other cell types. Developmental studies indicate its initial expression in the 7-day-old mouse testis concomitant with the appearance of intermediate and type B spermatogonia.

Pertussis toxin enhances follicle-stimulating hormone-stimulated cAMP production in rat seminiferous tubules in a stage-dependent manner

Cyclic (c) AMP production was measured in vitro in dissected pieces of adult rat seminiferous tubules of defined stages of the seminiferous epithelial cycle (VII–VIII and XIV–IV) in the absence and presence of human follicle-stimulating hormone (FSH) (Metrodin, Serono). The basal rate of cAMP production was stage dependent, being about 2-fold higher in stages XIV–IV. FSH stimulated cAMP output in a time- and dose-dependent (stimulation at doses > 3 mg/l) fashion, and also the stimulability of stages XIV–IV (on average 2-fold) was greater than that of stages VII–VIII. When the tubular pieces were incubated in the presence of pertussis toxin (PT, 100 μg/l), the FSH-stimulated cAMP production of stages VII–VIII was enhanced by about 2-fold ( P < 0.01) whereas the basal rate was unaffected. In contrast, neither the basal nor the FSH-stimulated cAMP production of stages XIV-IV were affected by PT. Presence of the G i-protein in both stages studied was demonstrated with PT-induced ADP ribosylation. However, no release of a putative activator of the G i-protein could be demonstrated into spent media of the seminiferous tubules when incubated with freshly separated tubules. It is concluded that the poor FSH stimulability in cAMP production of certain spermatogenic stages of adult seminiferous tubules is at least partly due to endogenous inhibitors acting through the inhibitory G i-protein. This inhibition could be demonstrated in a stage-dependent manner, and was present in stages with the lowest production and least stimulability of cAMP production by FSH.

Apoptosis: A Distinctive Mode of Cell Death that Plays an Opposite Role to Mitosis in Cell Population Kinetics

SUMMARYThe term apoptosis has been coined for a morphologically distinctive mode of cell death, which plays an opposite role to mitosis in controlling the size of animal tissues: it is fundamentally different from the well known phenomenon of necrosis that results from irreversible injury to cells by agents such as toxins and ischaemia.Cells affected by apoptosis condense and bud to produce many membrane‐bounded fragments in which organelles are well preserved. These are phagocytosed and digested by nearby resident tissue cells without associated inflammation; the viable cells merely close ranks. Extensive deletion of cells may thus take place without disorganisation of overall tissue architecture.Apoptosis occurs continuously in healthy animals. It is enhanced in endocrine‐dependent tissues during the reversible shrinkage that follows appropriate hormone withdrawal or administration, and it is also implicated in total and irreversible involution of tissues in normal animals, such as loss of the tadpole tail during metamorphosis and elimination of phylogenetic vestiges during embryonic development.Apoptosis takes place spontaneously and continuously in malignant neoplasms, often grossly retarding their growth, and cytotoxic drug administration and X irradiation enhance apoptosis as well as inhibiting mitosis in neoplasms; tumour regression following therapy with these agents is the result of tipping the balance between the two processes. Hyperthermia enhances apoptosis in foetal tissues and adult seminiferous tubules; its effect on apoptosis in neoplasms needs study. Cell mediated immune attack on cells induces apoptosis, not necrosis; apoptosis may thus sometimes be enhanced in neoplasms by immunological means.The features of apoptosis suggest that it is an active process of cellular self‐destruction rather than a form of cell degeneration. This carries the implication that it is genetically programmed. Further understanding of the regulation of the onset of apoptosis at the molecular level may lead to the discovery of improved methods of tumour treatment.

HYPERPLASIA AND NEOPLASIA OF THE INTERSTITIAL CELLS OF THE TESTICLE

Considerable confusion exists as to the type and terminology of testicular tumors. Many varieties of tumor arise from the complex elements composing the testicle, and identities are further obscured by the possibility of teratoid origin. No attempt will be made to classify tumors of the testicle, but it may be stated that the scope of pure simple tumors of the testicle has been greatly narrowed by the study of teratoma testis and its derivatives. Ewing stated that all the common and nearly all the rarer testicular tumors arise from totipotent sex cells and that the monodermal forms represent one-sided developments of tridermal teratomas. Only rarely, he stated, do the stroma, duct cells, interstitial cells and adult seminiferous tubules give rise to characteristic growths. His view is more extreme than that of most, who separate from the group of teratoma testis and its derivatives the seminoma, or spermatocytoma, as being derived