Insights into differentiation and function of the transition region between the seminiferous tubule and rete testis.

Seminiferous tubules physically connect to the rete testis through short segments called the transition region (TR). During fetal development, this specialized junction is considered the initial site where testis cords begin to form and to grow in length well beyond birth and into adulthood and form convoluted tubular cores. Mitotic activity of the Sertoli cell, the somatic cell of the epithelium, ceases before puberty, but modified Sertoli cells in the TR remain immature and capable of proliferation. This review presents what is known about this specialized region of the testis, with an emphasis on the morphological, molecular and physiological features, which support the hypothesis that this short region of epithelial transition serves as a specialized niche for undifferentiated Sertoli cells and spermatogonial stem cells. Also, the region is populated by an elevated number of immune cells, suggesting an important activity in monitoring and responding to any leakage of autoantigens, as sperm enter the rete testis. Several structure/function characteristics of the transition region are discussed and compared across species.

Duration of spermatogenesis and identification of spermatogonial stem cell markers in a Neotropical catfish, Jundiá (Rhamdia quelen).

Spermatogenesis is a process driven by stem cell, where germ cell cycle is under the control of a specific genotype species. Considering that Jundiá (Rhamdia quelen) is a Neotropical catfish with great economical importance and useful experimental model, little information is available on basic aspects of its reproductive biology, especially on spermatogenesis. As a result, this study aimed to characterize the male germ cells, estimate the duration of spermatogenesis and evaluate the expression of selected stem cell genes in Jundiá testis. Similar to other fish species, our results showed a remarkable decrease of germ cell nuclear volume during Jundiá spermatogenesis, particularly from type A undifferentiated to late type B spermatogonia and from diplotene to late spermatids. Using a S-phase marker, bromodeoxyuridine (BrdU), the combined duration of meiotic and spermiogenic phases in this species was estimated in approximately 7 days. This is considered very short when compared to mammals, where spermatogenesis last from 30 to 74 days. Selected stem cell genes were partially sequenced and characterized in Jundiá testis. Expression analysis showed higher plzf and pou5f3 mRNA levels in the cell fractions enriched by type A undifferentiated spermatogonia. These results were further confirmed by in situ hybridization that showed strong signal of plzf and pou5f3 mRNA in type A undifferentiated spermatogonia. Altogether, these information will expand our knowledge of the reproductive biology of this species, contributing to improve its production and management, and also for biotechnological applications, such as germ cell transplantation.

Characterization of spermatogonial cells and niche in the scorpion mud turtle (Kinosternon scorpioides).

Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male's life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.

Spermatogenesis in a neotropical marsupial species, Philander frenatus (Olfers, 1818).

Despite the singular morphology of the male genital system and the different reproductive strategies of marsupials, little emphasis has been given to the testis morphology and spermatogenic kinetics in this mammalian order. The present study aimed to investigate the testis function and the duration of spermatogenesis in the southeastern four-eyed opossum, Philander frenatus. Testes of six adult males were routinely processed for histological and stereological analyses. In order to determine the duration of spermatogenesis, intratesticular injections of tritiated thymidine were performed 1h, 13days and 21days before the sacrifice. Based on the development of the acrosomic system, ten stages of the seminiferous epithelium cycle were characterized. The mean body and testis weights for the P. frenatus were respectively 326±20g and 0.4±0.05g, providing a gonadosomatic index of 0.3±0.02%. The most advanced germ cell types labeled at 1h, 13days and 21days after thymidine injections were, respectively, preleptotene spermatocytes at stage IV, pachytene spermatocytes at stage IV and diplotene spermatocytes at stage IX. Based on the stages frequencies and the most advanced labeled germ cells, each spermatogenic cycle and the entire spermatogenic process lasted respectively 13.5±0.5 and 60.9±2.4days. When compared to the vast majority of eutherian mammals already investigated, these data indicate that the Philander frenatus presents a relatively long duration of spermatogenesis.

Antagonistic regulation of spermatogonial differentiation in zebrafish (Danio rerio) by Igf3 and Amh.

Fsh-mediated regulation of zebrafish spermatogenesis includes modulating the expression of testicular growth factors. Here, we study if and how two Sertoli cell-derived Fsh-responsive growth factors, anti-Müllerian hormone (Amh; inhibiting steroidogenesis and germ cell differentiation) and insulin-like growth factor 3 (Igf3; stimulating germ cell differentiation), cooperate in regulating spermatogonial development. In dose response and time course experiments with primary testis tissue cultures, Fsh up-regulated igf3 transcript levels and down-regulated amh transcript levels; igf3 transcript levels were more rapidly up-regulated and responded to lower Fsh concentrations than were required to decrease amh mRNA levels. Quantification of immunoreactive Amh and Igf3 on testis sections showed that Fsh increased slightly Igf3 staining but decreased clearly Amh staining. Studying the direct interaction of the two growth factors showed that Amh compromised Igf3-stimulated proliferation of type A (both undifferentiated [Aund] and differentiating [Adiff]) spermatogonia. Also the proliferation of those Sertoli cells associated with Aund spermatogonia was reduced by Amh. To gain more insight into how Amh inhibits germ cell development, we examined Amh-induced changes in testicular gene expression by RNA sequencing. The majority (69%) of the differentially expressed genes was down-regulated by Amh, including several stimulators of spermatogenesis, such as igf3 and steroidogenesis-related genes. At the same time, Amh increased the expression of inhibitory signals, such as inha and id3, or facilitated prostaglandin E2 (PGE2) signaling. Evaluating one of the potentially inhibitory signals, we indeed found in tissue culture experiments that PGE2 promoted the accumulation of Aund at the expense of Adiff and B spermatogonia. Our data suggest that an important aspect of Fsh bioactivity in stimulating spermatogenesis is implemented by restricting the different inhibitory effects of Amh and by counterbalancing them with stimulatory signals, such as Igf3.

Sertoli cells are capable of proliferation into adulthood in the transition region between the seminiferous tubules and the rete testis in Wistar rats.

Sertoli cells (SCs) play a crucial role in testis differentiation, development and function, determining the magnitude of sperm production in sexually mature animals. For over 40 years, it has been considered that these key testis somatic cells stop dividing during early pre-pubertal phase, between around 10 to 20 days after birth respectively in mice and rats, being after that under physiological conditions a stable and terminally differentiated population. However, evidences from the literature are challenging this dogma. In the present study, using several important functional markers (Ki-67, BrdU, p27, GATA-4, Androgen Receptor), we investigated the SC differentiation status in 36 days old and adult Wistar rats, focusing mainly in the transition region (TR) between the seminiferous tubules (ST) and the rete testis. Our results showed that SCs in TR remain undifferentiated for a longer period and, although at a lesser degree, even in adult rats proliferating SCs were observed in this region. Therefore, these findings suggest that, different from the other ST regions investigated, SCs residing in the TR exhibit a distinct functional phenotype. These undifferentiated SCs may compose a subpopulation of SC progenitors that reside in a specific microenvironment capable of growing the ST length if needed from this particular testis region. Moreover, our findings demonstrate an important aspect of testis function in mammals and opens new venues for other experimental approaches to the investigation of SC physiology, spermatogenesis progression and testis growth. Besides that, the TR may represent an important site for pathophysiological investigations and cellular interactions in the testis.

Successful xenogeneic germ cell transplantation from Jundia catfish (Rhamdia quelen) into adult Nile tilapia (Oreochromis niloticus) testes.

Fish germ cell transplantation presents several important potential applications for aquaculture, including the preservation of germplasm from endangered fish species with high genetic and commercial values. Using this technique in studies developed in our laboratory with adult male Nile tilapias (Oreochromis niloticus), all the necessary procedures were successfully established, allowing the production of functional sperm and healthy progeny approximately 2months after allogeneic transplantation. In the present study, we evaluated the viability of the adult Nile tilapia testis to generate sperm after xenogeneic transplant of germ cells from sexually mature Jundia catfish (Rhamdia quelen) that belong to a different taxonomic order. Therefore, in order to investigate at different time-periods post-transplantation, the presence and development of donor PKH26 labeled catfish germ cells were followed in the tilapia seminiferous tubules. From 7 to 20days post-transplantation, only PKH26 labeled spermatogonia were observed, whereas spermatocytes at different stages of development were found at 70days. Germ cell transplantation success and progression of spermatogenesis were indicated by the presence of labeled PKH26 spermatids and sperm on days 90 and 120 post-transplantation, respectively. Confirming the presence of the catfish genetic material in the tilapia testis, all recipient tilapias evaluated (n=8) showed the genetic markers evaluated. Therefore, we demonstrated for the first time that the adult Nile tilapia testis offers the functional conditions for development of spermatogenesis with sperm production from a fish species belonging to a different order, which provides an important new venue for aquaculture advancement.

The Sertoli cell: one hundred fifty years of beauty and plasticity.

It has been one and a half centuries since Enrico Sertoli published the seminal discovery of the testicular 'nurse cell', not only a key cell in the testis, but indeed one of the most amazing cells in the vertebrate body. In this review, we begin by examining the three phases of morphological research that have occurred in the study of Sertoli cells, because microscopic anatomy was essentially the only scientific discipline available for about the first 75 years after the discovery. Biochemistry and molecular biology then changed all of biological sciences, including our understanding of the functions of Sertoli cells. Immunology and stem cell biology were not even topics of science in 1865, but they have now become major issues in our appreciation of Sertoli cell's role in spermatogenesis. We end with the universal importance and plasticity of function by comparing Sertoli cells in fish, amphibians, and mammals. In these various classes of vertebrates, Sertoli cells have quite different modes of proliferation and epithelial maintenance, cystic vs. tubular formation, yet accomplish essentially the same function but in strikingly different ways.

INSL3 stimulates spermatogonial differentiation in testis of adult zebrafish (Danio rerio).

INSL3 (insulin-like peptide 3) is a relaxin peptide family member expressed by Leydig cells in the vertebrate testis. In mammals, INSL3 mediates testicular descent during embryogenesis but information on its function in adults is limited. In fish, the testes remain in the body cavity, although the insl3 gene is still expressed, suggesting yet undiscovered, evolutionary older functions. Anti-Müllerian hormone (Amh), in addition to inhibiting spermatogonial differentiation and androgen release, inhibits the Fsh (follicle-stimulating hormone)-induced increase in insl3 transcript levels in zebrafish testis. Therefore, the two growth factors might have antagonistic effects. We examine human INSL3 (hINSL3) effects on zebrafish germ cell proliferation/differentiation and androgen release by using a testis tissue culture system. hINSL3 increases the proliferation of type A undifferentiated (Aund) but not of type A differentiating (Adiff) spermatogonia, while reducing the proliferation of Sertoli cells associated with proliferating Aund. Since the area occupied by Aund decreases and that of Adiff increases, we conclude that hINSL3 recruits Aund into differentiation; this is supported by the hINSL3-induced down-regulation of nanos2 transcript levels, a marker of single Aund spermatogonia in zebrafish and other vertebrates. Pulse-chase experiments with a mitosis marker also indicate that hINSL3 promotes spermatogonial differentiation. However, hINSL3 does not modulate basal or Fsh-stimulated androgen release or growth factor transcript levels, including those of amh. Thus, hINSL3 seems to recruit Aund spermatogonia into differentiation, potentially mediating an Fsh effect on spermatogenesis.

Functionalized nanomaterials: are they effective to perform gene delivery to difficult-to-transfect cells with no cytotoxicity?

Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death.

Thyroid hormone stimulates the proliferation of Sertoli cells and single type A spermatogonia in adult zebrafish (Danio rerio) testis.

Thyroid hormones participate in regulating growth and homeostatic processes in vertebrates, including development and adult functioning of the reproductive system. Here we report a new stimulatory role of thyroid hormone on the proliferation of Sertoli cells (SCs) and single, type A undifferentiated spermatogonia (A(und)) in adult zebrafish testes. A role for T3 in zebrafish testis is suggested by in situ hybridization studies, which localized thyroid receptor α (thrα) in SCs and the ß (thrß) mRNA in Sertoli and Leydig cells. Using a primary zebrafish testis tissue culture system, the effect of T3 on steroid release, spermatogenesis, and the expression of selected genes was evaluated. Basal steroid release and Leydig cell gene expression did not change in response to T3. However, in the presence of FSH, T3 potentiated gonadotropin-stimulated androgen release as well as androgen receptor (ar) and 17α-hydroxylase/17,20 lyase (cyp17a1) gene expression. Moreover, T3 alone stimulated the proliferation of both SCs and A(und), potentially resulting in newly formed spermatogonial cysts. Additional tissue culture studies demonstrated that Igf3, a new, gonad-specific member of the IGF family, mediated the stimulatory effect of T3 on the proliferation of A(und) and SCs. Finally, T3 induced changes in connexin 43 mRNA levels in the testis, a known T3-responsive gene. Taken together, our studies suggest that T3 expands the population of SCs and A(und) involving Igf signaling and potentiates gonadotropin-stimulated testicular androgen production as well as androgen sensitivity.

Germ cell transplantation as a potential biotechnological approach to fish reproduction.

Although the use of germ cell transplantation has been relatively well established in mammals, the technique has only been adapted for use in fish after entering the 2000s. During the last decade, several different approaches have been developed for germ cell transplantation in fish using recipients of various ages and life stages, such as blastula-stage embryos, newly hatched larvae and sexually mature specimens. As germ cells can develop into live organisms through maturation and fertilization processes, germ cell transplantation in fish has opened up new avenues of research in reproductive biotechnology and aquaculture. For instance, the use of xenotransplantation in fish has lead to advances in the conservation of endangered species and the production of commercially valuable fish using surrogated recipients. Further, this could also facilitate the engineering of transgenic fish. However, as is the case with mammals, knowledge regarding the basic biology and physiology of germline stem cells in fish remains incomplete, imposing a considerable limitation on the application of germ cell transplantation in fish. Furthering our understanding of germline stem cells would contribute significantly to advances regarding germ cell transplantation in fish.

Effects of inducible nitric oxide synthase (iNOS) deficiency in mice on Sertoli cell proliferation and perinatal testis development.

Nitric oxide (NO) plays crucial roles in several physiological and pathological conditions. The iNOS isoform produces high levels of NO independent of intracellular calcium and, in the testis, which is expressed in Sertoli (SC), Leydig (LC) and germ cells. The testicular roles of NO are unclear, but it can inhibit LC testosterone production. Our aim was to evaluate the effects of iNOS deficiency on testis development in mice from late fetal life through early puberty. Therefore, testes from wild type (C57BCL/6) and iNOS(-/-) mice (B6.129P2- Nos2(tm1Lau) /J) were sampled at various ages between e18.5 and Pnd20 and evaluated by histological and stereological analyses; proliferating cells were labelled with (3)H-thymidine. At all ages, testis weight and anogenital index, a measure of fetal androgen exposure, were greater in iNOS-deficient mice than in wild type mice. At all ages after birth, iNOS-deficient mice exhibited increased (p < 0.05) SC number per testis, and this was accounted for by a higher SC proliferation index (p < 0.05) in iNOS-deficient mice, especially on Pnd1 and Pnd5. Similarly, LC number per testis was higher (p < 0.05) in iNOS(-/-) mice than in wild type at all post-natal ages. Highly positive and significant correlations were observed between the proliferation index for SC, LC and peritubular myoid cells on e18.5 and post-natally. Although lumen formation was slightly advanced in iNOS(-/-) mice, no obvious other effects on pubertal testis development were observed. These results imply that NO may normally constrain testis somatic cell development, especially SC, perhaps by limiting testosterone production. Removal of this constraint results in normal, but larger, testes with greater sperm production. Our data pinpoint the window of iNOS (NO) action on SC proliferation and raise the possibility that experimental manipulation of NO in early post-natal life could be used to enhance SC proliferation if this was deficient for any reason.

Effects of adult onset mild calorie restriction on weight of reproductive organs, plasma parameters and gene expression in male mice.

Calorie restriction (CR) extends lifespan and delays onset of age-related diseases in various organisms, even when started later in life. Despite benefits for health and lifespan, CR's negative impact on reproduction is documented in some animals. Studies employing approximately 40% CR detected a delay in sexual maturation and impairment of fertility, which were combined with extension of the reproductive period. In contrast, mild CR (10-20%) is apparently not deleterious to reproduction. Hence, we hypothesized that mild CR started at 8 months of age would prolong reproductive capabilities and improve health parameters of male mice. To test this hypothesis, we assessed the effects of 10 and 20% CR on reproductive organ weights, selected plasma parameters and hepatic/testicular gene expression in normal male mice of heterogeneous genetic background. Starting at 8 months of age (adult), mice were assigned to 3 regimen groups: 10% CR (n = 8), 20% CR (n = 9) or ad libitum (AL; n = 8). Four months of CR were sufficient to reduce glycemia in a non-fasted protocol. Mild CR initiated in adulthood did not significantly impact final body weight, most of the analyzed plasma parameters or weight of androgen-dependent organs. Moreover, CR did not interfere with expression of the assessed testicular genes, or most of the hepatic genes, but it did cause an increase in the levels of peroxisome proliferator-activated receptor gamma (Pparg) and mouse sulfotransferase (mSTa); and a decrease in glucose-6-phosphatase-α (G6pc) mRNA, which might signify improvement of body condition. The important finding of our study was that a mild CR regimen, as low as 10 and 20%, was sufficient to impair glycemia in a non-fasted state, and also the levels of plasma IGF-1, corroborating the concept that mild CR has the potential for improving health and longevity, even when started later in life.

Androgen receptor signalling in peritubular myoid cells is essential for normal differentiation and function of adult Leydig cells.

Testosterone synthesis depends on normal Leydig cell (LC) development, but the mechanisms controlling this development remain unclear. We recently demonstrated that androgen receptor (AR) ablation from a proportion of testicular peritubular myoid cells (PTM-ARKO) did not affect LC number, but resulted in compensated LC failure. The current study extends these investigations, demonstrating that PTM AR signalling is important for normal development, ultrastructure and function of adult LCs. Notably, mRNAs for LC markers [e.g. steroidogenic factor 1 (Nr5a1), insulin-like growth factor (Igf-1) and insulin-like factor 3 (Insl3)] were significantly reduced in adult PTM-ARKOs, but not all LCs were similarly affected. Two LC sub-populations were identified, one apparently 'normal' sub-population that expressed adult LC markers and steroidogenic enzymes as in controls, and another 'abnormal' sub-population that had arrested development and only weakly expressed INSL3, luteinizing hormone receptor, and several steroidogenic enzymes. Furthermore, unlike 'normal' LCs in PTM-ARKOs, the 'abnormal' LCs did not involute as expected in response to exogenous testosterone. Differential function of these LC sub-populations is likely to mean that the 'normal' LCs work harder to compensate for the 'abnormal' LCs to maintain normal serum testosterone. These findings reveal new paracrine mechanisms underlying adult LC development, which can be further investigated using PTM-ARKOs.

Sertoli cell numbers and spermatogenic efficiency are increased in inducible nitric oxide synthase mutant mice.

Nitric oxide (NO) is produced via oxidation of l-arginine by nitric oxide synthases (NOSs), and is known as inducible (iNOS), neuronal, endothelial or testis-specific. Suggesting important functions for NOS in the normal rat and mouse testis, iNOS is reported to be constitutively expressed in Leydig cells (LC), Sertoli cells (SC) and germ cells. In our study, we sought to provide further insights into the roles of iNOS in the adult mouse testis using iNOS(-/-) mice. Perfusion-fixed testes from wild type (WT) and iNOS(-/-) mice were used for histological and stereological evaluations. Some of the mice had been injected with (3) H-thymidine to label proliferating cells and to determine the duration of spermatogenesis that was unaffected in iNOS(-/-) mice. Both LC nuclear volume and individual cell size were significantly decreased in iNOS(-/-) mice, but the total number of LC per testis was increased (p < 0.05) by approximately 16%. The number of SC per testis was strikingly increased (approximately twofold) in iNOS(-/-) mice, and testis weight and DSP per gram of testis (spermatogenic efficiency) were similarly increased. The anogenital distance was also significantly increased in iNOS(-/-) mice, and this key endpoint suggests that the augmentation observed for the SC number may be related to increased foetal T-exposure during the masculinization programming window. Compared with WT testes, the numbers of spermatocytes and spermatids and SC per tubule cross sections were significantly increased in iNOS(-/-) mice. Except for stages V-VI and VII-VIII, iNOS(-/-) mice exhibited approximately 3.5-fold fewer apoptotic germ cells than in WT mice. Taken together, our results provide new evidence that iNOS plays an important role in numerical and functional regulation of key somatic cells in the testis, which in turn impacts on germ cells and their survival and thus on daily sperm production.

Spermatogenic efficiency in the spiny rat, Trinomys moojeni (Rodentia: Echimyidae).

The spiny rat (Trinomys moojeni) is a rodent found in the Atlantic Forest, which is considered one of the most diverse and threatened biomes in the world. Knowledge on reproductive biology and physiology is critical to conservation and species management, allowing the prevention of extinction and the use of males in natural and artificial reproduction programs. The main objectives of the present study were to investigate the testis structure as well as spermatogenic and Sertoli cell efficiency in the spiny rat captured in the Caraça Natural Reserve, a fragment of the Atlantic Forest located in the State of Minas Gerais, Brazil. Ten sexually mature spiny rats were analyzed. Intraperitoneal injections of tritiated thymidine were administered to estimate duration of spermatogenesis. The testes were perfused-fixed in buffered glutaraldehyde and routinely processed for histological and morphometric analyses as well as the characterization of the stages of seminiferous epithelium cycle. Volume density (%) of seminiferous tubules and Leydig cells were 97 +/- 0.3 and 0.3 +/- 0.02, respectively. The duration of one spermatogenic cycle and total duration of spermatogenesis were 8.6 +/- 0.1 and 38.5 +/- 0.5 days, respectively. Due to the very great volume density of the seminiferous tubules, short duration of spermatogenesis, tubule length per gram of testis (approximately 40 m), great Sertoli cell efficiency (approximately 15 spermatids per Sertoli cell) and large number of Sertoli cells per testis gram (53 million), spermatogenic efficiency in the spiny rat (82 million) is by far the greatest of the mammalian species investigated thus far.

Testis stereology, seminiferous epithelium cycle length, and daily sperm production in the ocelot (Leopardus pardalis).

Similar to most wild felids, the ocelot (Leopardus pardalis) is an endangered species. However, knowledge regarding reproductive biology of the ocelot is very limited. Germ cell transplantation is an effective technique for investigating spermatogenesis and stem cell biology in mammals, and the morphologic characterization of germ cells and knowledge of cycle length are potential tools for tracking the development of transplanted germ cells. Our goal was to investigate basic aspects related to testis structure, particularly spermatogenesis, in the ocelot. Four adult males were used. After unilateral orchiectomy, testis samples were routinely prepared for histologic, stereologic, and autoradiographic analyses. Testis weight and the gonadosomatic index were 11+/-0.6g and 0.16+/-0.01%, respectively, whereas the volume density of seminiferous tubules and Leydig cells was 83.2+/-1.6% and 9.8+/-1.5%. Based on the acrosomic system, eight stages of spermatogenesis were characterized, and germ cell morphology was very similar to that of domestic cats. Each spermatogenic cycle lasted 12.5+/-0.4 d, and the entire spermatogenic process lasted 56.3+/-1.9 d. Individual Leydig cell volume was 2522mum(3), whereas the number of Leydig and Sertoli cells per gram of testis was 38+/-5x10(6) and 46+/-3x10(6). Approximately 4.5 spermatids were found per Sertoli cell, whereas daily sperm production per gram of testis was 18.3+/-1x10(6), slightly higher than values reported for other felids. The knowledge obtained in this study could be very useful to the preservation of the ocelot using domestic cat testes to generate and propagate the ocelot genome.

Duration of spermatogenesis in the bullfrog (Lithobates catesbeianus).

The bullfrog (Lithobates catesbeianus) has substantial economic importance and has also been used as an experimental model for biological studies in the fields of pharmacology, medicine, and reproductive biology, especially studies addressing gametogenesis. However, there is a lack of comprehensive information in the literature regarding testis structure and function in this amphibian. The main objective of the current study was to estimate the duration of the various phases of spermatogenesis in this vertebrate. Sixteen sexually mature bullfrogs received an intracoelomic administration of tritiated thymidine. Testes were analyzed at various times between 1h and 33 d after administration to detect the most advanced germ cell types labeled at each interval, as well as labeled preleptotene spermatocytes, which presumably originated from spermatogonial stem cells. The duration of the spermatogonial, spermatocytic, and spermiogenic phases of spermatogenesis in the bullfrog were approximately 18, 14, and 8 d, respectively. Thus, the total duration of the spermatogenesis process from early spermatogonia through to spermatozoa was 40 d in this species, similar to that of most previously investigated mammalian species. To our knowledge, this is the first reliable report on the duration of the full spermatogenic process in any amphibian species. These findings will be very useful for tracking the pace of germ cells in studies involving spermatogonial transplantation in lower vertebrates.

Cell junctions in fish seminiferous epithelium.

Similar to mammals, in fish the cellular interactions between Sertoli cells (SC) and germ cells (GC) in the seminiferous epithelium have important structural and functional roles. In this review, we give a brief summary of these interactions, in particular those on the cell junctions. Despite the scarcity of detailed empirical data, it appears that both basic types of adhesive junctions (actin- and intermediate filaments-related) are present between SC. However, the actin-related multifunctional junction known as the "ectoplasmic specialization" is seemingly present only in some cartilaginous fish. Conversely, SC in other fish species are joined by actin-related junctions similar to typical zonulae or puncta adherens found in other epithelia. Adhesive junctions are also found between SC and GC and between GC and GC, and due to their particular characteristics these junctions are known as "desmosome-like junctions". In terms of intercellular communication, connexins and gap junctions have been shown to occur between SC in fish, and they may be involved in the coordination of the synchronous development of GC within the cysts. It is also possible that gap junctions may form an interconnected network between SC and GC within a cyst. Concerning the SC barrier, tight junctions between fish SC apparently form a functional barrier only in cysts containing haploid GC, and different from mammals, meiotic GC in fish are not shielded from the vascular system. In summary, although still not well investigated, cell-cell interactions in the seminiferous epithelium of fish seem to be crucial for GC development, and their disturbance, for example by changing environmental conditions, will probably affect GC survival and fertility.