Changes in lectin binding pattern of gonads of developing mice

Changes in lectin binding of developing fetal mouse testes and ovaries were examined by light and electron microscopy, with much attention paid particularly to those in carbohydrates of germ cells. Characteristic binding patterns were observed with three lectins (BPA, GS-I, and GS-II) in the germ cells and the somatic cells during the process of testicular and ovarian development. GS-I and BPA, which showed similar binding patterns, preferentially bound to the plasma membrane and small dense bodies (SDB) of germ cells in both testes and ovaries during the 12th to 14th day post coitum (p.c.). In the fetal testes on day 16 p.c., the reaction with both GS-I and BPA completely disappeared. While, in the ovaries, a weak reaction with these lectins was retained as it was in germ cells until the 16th day p.c. The reaction with GS-II was restricted to Sertoli cells in the fetal testes during the 12th to 14th day p.c., and thereafter disappeared on day 16 p.c. The distribution of GS-II binding sites was in agreement with that of the glycogen granules. No positive staining with GS-II was seen in the ovaries throughout their development. These results indicate that certain glycoconjugates containing D-galactose and N-acetyl-D-galactosamine residues are expressed on the cell surface and in the SDB of germ cells during the period of the 12th to 14th day p.c., and that striking changes in function as well as in structure may take place in both germ cells and somatic cells during the 14th to 16th day p.c. in association with testicular and ovarian development.     

Changes in lectin binding pattern of gonalds of developing mice

Summary Changes in lectin binding of developing fetal mouse testes and ovaries were examined by light and electron microscopy, with much attention paid particularly to those in carbohydrates of germ cells. Characteristic binding patterns were observed with three lectins (BPA, GS-I, and GS-II) in the germ cells and the somatic cells during the process of testicular and ovarian development. GS-I and BPA, which showed similar binding patterns, preferentially bound to the plasma membrane and small dense bodies (SDB) of germ cells in both testes and ovaries during the 12th to 14th day post coitum (p.c.). In the fetal testes on day 16 p.c., the reaction with both GS-I and BPA completely disappeared. While, in the ovaries, a weak reaction with these lectins was retained as it was in germ cells until the 16th day p.c. The reaction with GS-II was restricted to Sertoli cells in the fetal testes during the 12th to 14th day p.c., and thereafter disappeared on day 16 p.c. The distribution of GS-II binding sites was in agreement with that of the glycogen granules. No positive staining with GS-II was seen in the ovaries throughout their development. These results indicate that certain glycoconjugates containing d-galactose and N-acetyl-d-galactosamine residues are expressed on the cell surface and in the SDB of germ cells during the period of the 12th to 14th day p.c., and that striking changes in function as well as in structure may take place in both germ cells and somatic cells during the 14th to 16th day p.c. in association with testicular and ovarjan development.     

Stem cell factor and c-kit gene expression and protein localization in the sheep ovary during fetal development

The aim of this study was to investigate stem cell factor and c-kit gene expression and protein localization in the mesonephros and ovary of sheep fetuses at different days of gestation, using RNA in situ hybridization and immunohistochemical procedures. At days 24 and 26 of gestation, stem cell factor mRNA and protein were present in cells throughout the developing gonad and mesonephros. From day 28 to day 40 of gestation, stem cell factor mRNA and protein became increasingly localized to the cortical region of the ovary, where most germ cells were present as actively proliferating oogonia. From day 40 to day 90 of gestation, stem cell factor mRNA and protein localization were confined mainly to the ovarian cortex. Moreover, within the cortical region, stem cell factor mRNA was low or absent where follicles were first forming and highest in the outer ovarian cortex, where germ cells were undergoing mitosis or the early stages of meiosis. In contrast, stem cell factor protein was present in newly forming follicles, as well as in mitotic and meiotic germ cells, which is consistent with the presence of both membrane-bound and soluble forms of this ligand. However, by day 90 of gestation, both stem cell factor mRNA and protein were observed in the granulosa cells of most (> 90%) primordial follicles. C-kit mRNA and protein were observed from day 24 of gestation in both germ cells and somatic cells but, with increasing gestational age, preferentially in germ cells (for example, pre-meiotic germ cells and both isolated oocytes and follicle-enclosed oocytes). C-kit protein, but not mRNA, was also observed in germ cells that were undergoing meiosis. The results indicate that the cells containing stem cell factor mRNA within the ovary up to day 90 of gestation originated from the gonadal blastema and from cells that migrated from the mesonephros before day 28 of gestation. Since stem cell factor mRNA was absent in both mesonephric cells migrating after day 28 of gestation and in regions where follicles were first forming, it is suggested that these later migrating mesonephric cells are the progenitors of the granulosa cells in the first forming follicles. In conclusion, during follicle formation, c-kit mRNA is localized to germ cells whereas c-kit, together with stem cell factor protein, is localized to both germ cells and somatic cells, consistent with the hypothesis that the presence of this receptor-ligand pair is essential to prevent apoptosis.     

Localization of Forssman glycolipid and GM1 ganglioside intracellularly and on the surface of germ cells during fetal testicular and ovarian development of mice

Changes in the expression pattern and intracellular localization of Forssman glycolipid (FA) and GM1 ganglioside (GM1) in fetal mouse gonads were examined during germ cell differentiation by immunofluorescence microscopy and immunoelectron microscopy. In male germ cells from the 12th to 14th day p.c., anti-FA binding was localized in granular structures aggregated on one side of the cytoplasm and/or in the plasma membrane. On day 16 p.c., some germ cells still showed patch-like positive reactions in the plasma membrane, but by day 18 p.c., positive reactions for FA had completely disappeared. The female germ cells showed granular bindings of anti-FA scattered throughout their cytoplasm during the 13th to 16th day p.c., although the positive reactions in female germ cells on day 12 p.c. tended to be found in one side of cytoplasm and/or plasma membrane similar to those in male germ cells from 12th to 14th day p.c. On day 18 p.c., positive reactions remained in the plasma membrane of some germ cells, but these positive reactions disappeared before birth. Immunoelectron microscopic observation showed that the sites of anti-FA bindings were equivalent to the "small dense bodies" (SDB) and the Golgi lamellae both in male and female germ cells. On the other hand, GM1 was not detected in male germ cells at any time during fetal testicular development, whereas an anti-GM1 reaction was detected in the plasma membrane of female germ cells from the 16th to 18th day p.c. (oocytes in the first meiotic prophase).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural aspects of gonadal morphogenesis in Bufo bufo (Amphibia Anura) 1. Sex differentiation

The morphogenesis of gonads in Bufo bufo tadpoles was studied, and ultrastructural differences between sexes were identified. All specimens analyzed initially developed gonads made up of a peripheral fertile layer (cortex) surrounding a small primary cavity. Subsequently a central layer of somatic cells (medulla) developed. Both layers were separated by two uninterrupted basal laminae between which a vestige of the primary cavity persisted. During female differentiation, the peripheral layer continued to be the fertile layer. In males, the central layer blended into the peripheral layer and the basal laminae disappeared. The somatic cells of the central layer came into direct contact with the germ cells; this did not occur in females. Testicular differentiation continued with the migration of germ cells towards the center of the gonad. The somatic elements surrounding the germ cells appeared to play an active role in their transfer to the center of the gonad. The peripheral layer shrank and became sterile. Two basal laminae then re-formed to separate the fertile central layer from the peripheral sterile one. Germ cells have always been thought to perform a passive role in sex differentiation in amphibians. Following the generally accepted "symmetric model", the mechanism of gonad development is symmetrical, with cortical somatic cells determining ovarian differentiation and medullary somatic cells determining testicular differentiation. In contrast, we found that sex differentiation follows an "asymmetric" pattern in which germ cells tend primarily toward a female differentiation and male differentiation depends on a secondary interaction between germ cells and medullary somatic cells.     

Localization of forssman glycolipid and GM1 ganglioside intracellularly and on the surface of germ cells during fetal testicular and ovarian development of mice

Summary Changes in the expression pattern and intracellular localization of Forssman glycolipid (FA) and GM1 ganglioside (GM1) in fetal mouse gonads were examined during germ cell differentiation by immunofluorescence microscopy and immunoelectron microscopy.In male germ cells from the 12th to 14th day p.c., anti-FA binding was localized in granular structures aggregated on one side of the cytoplasm and/or in the plasma membrane. On day 16 p.c., some germ cells still showed patch-like positive reactions in the plasma membrane, but by day 18 p.c., positive reactions for FA had completely disappeared. The female germ cells showed granular bindings of anti-FA scattered throughout their cytoplasm during the 13th to 16th day p.c., although the positive reactions in female germ cells on day 12 p.c. tended to be found in one side of cytoplasm and/or plasma membrane similar to those in male germ cells from 12th to 14th day p.c. On day 18 p.c., positive reactions remained in the plasma membrane of some germ cells, but these positive reactions disappeared before birth. Immunoelectron microscopic observation showed that the sites of anti-FA bindings were equivalent to the small dense bodies (SDB) and the Golgi lamellae both in male and female germ cells. On the other hand, GM1 was not detected in male germ cells at any time during fetal testicular development, whereas an anti-GM1 reaction was detected in the plasma membrane of female germ cells from the 16th to 18th day p.c. (oocytes in the first meiotic prophase).Therefore, these results indicate that kinetic translocation of FA between the plasma membrane and the SDB and Golgi lamellae takes place during germ cell differentiation in fetal gonads. Moreover, the ganglioside composition containing GM1 seems to change in association with the first meiotic prophase.     

Differential expression of acidic cytokeratins 18 and 19 during sexual differentiation of the rat gonad.

The expression of cytokeratins (CKs) 8, 18 and 19 was analyzed in male and female rat gonads from the undifferentiated stage (12.5 days of gestation) until two weeks after birth by indirect immunofluorescence, using specific monoclonal antibodies anti-CK 8 (LE41), anti-CK 19 (LP2K) and anti-CK 18 (LE65 and RGE53). In the undifferentiated blastema, the somatic cells were stained for CK 8 and CK 19, whereas no detectable immunoreactivity for CK 18 was obtained. The same staining CK pattern was observed in ovaries, in the somatic cells of ovigerous cords and in primary follicles. The staining was progressively decreasing in growing follicles after one week after birth. At the onset of testicular differentiation, when the first Sertoli cells differentiate in the gonad of 13.5-day old male fetuses, positive staining for CK 18 became evident, in addition to CK 8 and CK 19 expression. In the following days, CK 8, CK 18 and CK 19 were detected in Sertoli cells in the differentiating seminiferous cords, but progressively the reactivity for CK 19 decreased and was no longer observed after 18.5-19.5 days of gestation. In all cases, CKs were found to be coexpressed with vimentin, and germ cells were negative for both vimentin and CKs. The results reported here show first, that CKs are expressed before sexual differentiation in gonadal blastema in which no epithelial organization is observed, and second, that there is a CK 18/CK 19 shift in expression during morphogenesis of the testis which is not observed in the differentiating ovary. Future studies will have to determine whether these differences in CK expression are due to epitope-masking phenomena or to the regulation of CK synthesis.     

Germ cell deficiency causes testis cord differentiation in reconstituted mouse fetal ovaries

Sex-reversal in fetal ovaries was studied by using a dissociation-reconstitution technique. Gonads of 12.5 gestation-day male and female mouse fetuses were dissociated into single cells. To eliminate germ cells, the dissociated cells were cultured for 14 h, and then somatic cells attached to culture dishes were harvested and aggregated by gyratory culture for 24 h. The aggregates were then transplanted into ovarian bursa in ovary-ectomized nude mice. The recovered explants were examined histologically. Male somatic cells developed into testes containing Sertoli cells, Leidig cells, and tunica albuginea. Female somatic cells formed testis cords and differentiated into Sertoli cells, but they did not differentiate into other testis components or ovarian tissues. However, aggregates consisting of both female and male somatic cells differentiated into well-developed testes containing Leidig cells and tunica albuginea as well as Sertoli cells. Enzyme marker analysis showed significant contributions of female cells in these organized testes. In contrast, aggregates containing both female germ cells and somatic cells developed into ovaries and did not differentiate into any testicular tissues. The results indicate that female somatic cells in fetal gonads at 12.5 gestation day have the potency to form testis cords and differentiate into Sertoli cells. The subsequent steps in testis development require the contributions of male cells. The present study also suggests that testicular differentiation is independent of germ cells but ovarian development involves the interaction between germ cells and somatic cells.     

Autonomous regulation of sex-specific developmental programming in mouse fetal germ cells

In mice, unique events regulating epigenetic programming (e.g., genomic imprinting) and replication state (mitosis versus meiosis) occur during fetal germ cell development. To determine whether these processes are autonomously programmed in fetal germ cells or are dependent upon ongoing instructive interactions with surrounding gonadal somatic cells, we isolated male and female germ cells at 13.5 days postcoitum (dpc) and maintained them in culture for 6 days, either alone or in the presence of feeder cells or gonadal somatic cells. We examined allele-specific DNA methylation in the imprinted H19 and Snrpn genes, and we also determined whether these cells remained mitotic or entered meiosis. Our results show that isolated male germ cells are able to establish a characteristic "paternal" methylation pattern at imprinted genes in the absence of any support from somatic cells. On the other hand, cultured female germ cells maintain a hypomethylated status at these loci, characteristic of the normal "maternal" methylation pattern in endogenous female germ cells before birth. Further, the surviving female germ cells entered first meiotic prophase and reached the pachytene stage, whereas male germ cells entered mitotic arrest. These results indicate that mechanisms controlling both epigenetic programming and replication state are autonomously regulated in fetal germ cells that have been exposed to the genital ridge prior to 13.5 dpc.     

The control of the differentiation of female embryonic germ cells in the bird

The left gonad from female chick embryos at 4–12 days of incubation was cultured in vitro as pieces of intact gonad, pieces of isolated cortex, and groups of pure germ cells. All cultures were maintained for a time equal to 17 days in ovo. At the end of the culture period, a cytological and quantitative study was made on the germ cells.The results show that some germ cells in pieces of intact 6-day gonad and pieces of 6-day cortex complete their normal developmental sequence and enter zygotene. This shows that the factors that control the differentiation of the germ cells reside in the cortex of the gonad and their expression does not depend upon the pituitary and the medullary estrogens after 6 days of incubation.Germ cells that are cultured as isolated cells do not attach to the tissue culture substrate, do not divide mitotically, and do not enter zygotene. Evidence is presented that suggests 12-day germ cells do enter zygotene when cultured with pieces of 12-day cortex. These data suggest the differentiation of the female germ cells is regulated by the somatic cells of the cortex.     

Adenohypophysis regulates cell proliferation in the gonads of the developing chick embryo

The aim of the present study was to evaluate the effect of hypophysectomy on cell proliferation in the left ovary and the left testis of 8- to 14-day-old chick embryos. Hypophysectomy was performed by the partial decapitation technique. At 44-46 h of incubation, chick embryo heads were sectioned at the mesencephalic level and the prosencephalic region removed. Embryos were further incubated until 8-14 days of development. Cell division was evaluated by bromodeoxyuridine (BrdU) incorporation and by counting the total number of somatic and germ cells in the gonads. The ovary displayed an exponential increase in the number of somatic and germ cells and a higher rate of BrdU incorporation compared to the testis. BrdU incorporation was reduced in the ovary of hypophysectomized embryos at 9-14 days of incubation, while in the testis, the reduction was significant at 14 days of development. Changes in the total number of somatic and germ cells further suggest that the absence of hypophysis affects the growth of the ovary earlier than the growth of the testis. Reduction in the number of somatic and germ cells after hypophysectomy in the ovary was reversed by a hypophyseal graft on the chorioallantoic membrane. The adenohypophysis regulates, probably through gonadotropic hormones, proliferation of somatic and germ cells in the gonads during chick embryo development.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Immunohistochemical study of caspase-3-expressing cells within the pancreas of non-obese diabetic mice during cyclophosphamide-accelerated diabetes

During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.     

Localization of activated caspase-3-positive and apoptotic cells in the developing tooth germ of the mouse lower first molar

This study examined the immunohistochemical detection of activated caspase-3, and its association with apoptosis, during tooth morphogenesis of the mouse lower first molar. The distribution of cells positive for caspase-3 closely corresponded with the localization of the terminal deoxynucleotidyl transferase-mediated deoxyuridine-5-triphosphate-biotin nick end labelling (TUNEL)-positive apoptotic cells through the developmental course of tooth germs from embryo day 12 (E12) to E19, thus showing that the apoptosis occurring in the developing odontogenic tissue was induced by the activation of the caspase family. The specific distribution pattern of apoptotic cells in the developing odontogenic epithelial tissue from the initiation (E12) of tooth germ to the completion of tooth crown morphology (E19) also suggests that apoptotic events are related not only to a deletion of functionally suspended cells, but also participate in initiation and the completion of tooth morphogenesis. Electron microscopic examination revealed that apoptotic cells were present in the primary enamel knot, and these apoptotic cells were phagocytized by neighbouring odontogenic epithelial cells, thus indicating the prompt disposal of any dead cells by epithelial cells.     

Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-beta-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome ct and lactate dehydrogenase-C4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.     

The effect of retinoic acid on the meiosis in the chick embryo (Gallus gallus domesticus)

In this study it was shown that the injection of retinoic acid (RA) into incubated eggs on day 9 or 14 induced entry the males germ cells into preleptotene stage of prophase I on day 17, which are absent in the control embryos. At the same time the meiosis marker SCP3 was detected in the germ cells. Which was also absent at control embryos. On day 19 in male embryos the number of male germ cells at the stage preleptoteny increased, but there were no germ cells in the following stages of the prophase of meiosis. In 20-day-old chicks meiotic germ cells were absent. Thus, white it is shown that the influence of RA on the developing chicken embryos induces the entry of germ cells into preleptotene stage of prophase I meiosis. However, further meiotic transformations don't occur. Thus RA is only one of many factors providing meiotic cell division.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

Presence and localization of molecules related to the cholinergic system in developing rat testis

The presence of cholinergic molecules was recently found in male gametes of different animal species; very little is known from this point of view about the somatic component of the gonad. In the present study, a contribution is given to the localization of some cholinergic-like molecules, such as acetylcholinesterase (AChE), nicotinic acetylcholine and muscarinic acetylcoline (nAchR and mAChR, respectively) receptors during postnatal development of the testis. Maturation stages were checked by use of histochemical stainings, such as DAPI for nuclear shape changes, and PSA binding to reveal acrosomal maturation. The distribution of cholinergic-like molecules, revealed by immunocytochemical methods in both gametes and somatic cells, varied with development. Generally, during early stages, molecules immunologically related to AChE, and to mAChR's were mainly found in the interstitial and peritubular compartment, while, during maturation, they were found in Sertoli cells and in differentiating germ cells. nAChR's were not found in the somatic compartment, except in the blood vessel walls, and were distributed in mature germ cells, mainly in the flagella. The presence of cholinergic molecules in somatic as well as germ cells could play a role in cell-to-cell communications affecting testicular cell differentiation and function.