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.

Thermal manipulation and GnRHa therapy applied to the reproduction of lambari-do-rabo-amarelo, Astyanax altiparanae females (Characiformes: Characidae) during the non-breeding season.

Lambari-do-rabo-amarelo Astyanax altiparanae in the wild reproduce during spring and summer, but females undergo vitellogenesis throughout the year, including the non-spawning winter period when water temperatures are low. The present study investigated the physiological role of temperature modulation on the hypothalamus-pituitary-gonads axis of lambari during winter, as well as the effects of gonadotropin releasing hormone agonist (GnRHa) therapy. Captive females were exposed to two different temperatures (20 °C and 27 °C) and were injected weekly with GnRHa for 21 days during winter (Control, CTR; Low dose; LD and high dose of GnRHa, HD). At the end of the 21-days period gonadosomatic index (GSI), oocyte stage of development and theoretical fecundity were evaluated, together with plasma levels of 17ß-estradiol (E2). Gene expression of the two pituitary gonadotropins follicle-stimulating hormone (fshß) and luteinizing hormone (lhß), as well as hepatic vitellogenin-A (vtgA) expression were also analyzed. At the end of the experimental period, females from the six different experimental conditions were induced to spawn using human chorionic gonadotropin (hCG). Spawning performance parameters and plasma levels of the maturation inducing steroid (MIS) were analyzed. Gene expression of fshß did not change with temperature manipulation, but females exposed to 27 °C and supplemented with a HD of GnRHa exhibited an increased fshß gene expression, associated with higher E2 levels. The higher water temperature alone was able to increase E2 levels. At both water temperatures GnRHa injections induced a decrease in E2 levels. GnRHa injected females had a lower vtgA gene expression levels at 20 °C. Even with differences in the gene expression of gonadotropins among the various temperature/GnRHa treatments, GSI and oocyte diameter did not change, but GnRHa enhanced the number of vitellogenic oocytes at 20 °C. The reproductive performance of lambari induced to spawn with hCG was better after the combined treatment with GnRHa and summer temperature.

Ovarian differentiation and development in cachara Pseudoplatystoma fasciatum.

One thousand five hundred cachara or tiger shovelnose catfish Pseudoplatystoma fasciatum, obtained from induced reproduction, were used to determine the onset of ovarian differentiation and development and to record the main characteristics of this process. Samples were collected from 0 to 240 days post-fertilization (dpf) and the results classified into stages I-XII. Ovarian formation was histologically detected for the first time when juveniles measured mean ± s.d. 51·5 ± 8·3 mm total length (LT ) at 39-45 dpf (stages I-V), with intense somatic cell proliferation originating in the ovarian cavity. Both LT and age of fish had a positive correlation (P < 0·001) with ovarian differentiation, but LT showed a greater correlation (r(2) = 0·95) than age (r(2) = 0·85), especially during the initial stages of development. From stages VI to VII, the ovarian cavity was enlarged and undifferentiated oogonia were present. At stage VIII, small projections formed in the ovarian stroma towards the ventral region of the gonad (future ovarian lamellae) and the basal membrane and differentiated oogonia nests could be seen. At stages IX and X, the germ cells entered meiosis and folliculogenesis was completed by stages XI and XII, which can be considered late in comparison to other Siluriformes. This study has demonstrated that ovarian differentiation in P. fasciatum begins with an intense proliferation of squamous epithelial cells (somatic cells) during the early stages of development and that sex inversion protocols could, thus, be applied successfully before this period. Furthermore, the results have demonstrated that both size and age can influence gonad differentiation and development in this species.

Testes of Astyanax altiparanae: the Sertoli cell functions in a semicystic spermatogenesis.

The Astyanax altiparanae (lambari) is a South American freshwater fish belonging to the family Characidae. Although some authors have described reproductive aspects of this species, this is the first study about the morphology of the testes throughout the annual reproductive cycle of A. altiparanae. Fish spermatogenesis differs from that in mammals as it occurs in cysts whose borders are defined by cytoplasmic processes of Sertoli cells, thus creating a favorable environment for spermatogenesis. The functions commonly attributed to fish Sertoli cells were investigated using stereological, light and electron microscopy in A. altiparanae. Results showed that when the Sertoli cells of A. altiparanae are in contact with germ cells, they plan a support function that culminates in the production of spermatozoa. After releasing spermatozoa, modified Sertoli cells form the duct epithelium, transform into secretory cells and release a secretion into the duct lumen where spermatids and sperm are located. Thus, the present study revealed important aspects of the testes of A. altiparanae, and propose a sequence of functions played by the Sertoli cells in this species.

The administration of exogenous prostaglandin may improve ovulation in pacu (Piaractus mesopotamicus).

Based on the reports of unsuccessful ovulation in pacu (Piaractus mesopotamicus) by fish farmers and researchers undertaking artificial reproduction programs, we evaluated the use of prostaglandin F (PGF) to improve pacu ovulation. This study was conducted during two spawning seasons (2009/2010 and 2010/2011) with two samplings in the first season and one sampling in the second season. A total of 45 females was sampled in this study. The control group was injected with carp pituitary extract (crude extract, 6 mg/kg), and the treatment group received PGF (2 mL per fish in the 2009/2010 season and 5 mL per fish in the 2010/2011 season) in addition to the crude extract. In both seasons, 100% (N = 4, 2009/2010 first sampling; N = 5, 2009/2010 second sampling; and N = 3, 2010/2011) of the PGF-treated fish spawned. In contrast, 53.0% (N = 9) and 83.3% (N = 10) of the control fish spawned in the first and second samplings of the 2009/2010 season, respectively, and only 25.0% (N = 1) spawned in the 2010/2011 season. Fecundity, fertility, and hatching rates did not differ (P > 0.05) between the treated and control fish. Based on oocyte volume frequency analysis, ovaries of the control fish had more (P < 0.05) vitellogenic oocytes with germinal vesicle breakdown that remained unovulated after spawning, whereas more (P < 0.05) of previtellogenic oocytes were present in the ovaries of the PGF-treated fish. In conclusion, administration of exogenous prostaglandin may improve the outcome of hormonally induced spawning in tropical migratory fish.

Spawning failure in Brycon amazonicus may be associated with ovulation and not with final oocyte maturation / A falha na desova de fêmeas de Brycon amazonicus pode estar relacionada com ovulação e não com a indução a maturação final dos ovócitos

Avaliaram-se os possíveis mecanismos envolvidos com a falha na desova de matrinxãs (Brycon amazonicus), submetidas à indução hormonal por extrato bruto de hipófise de carpa. Para tal, após a extrusão, os ovários foram coletados e analisados histomorfometricamente. Nas fêmeas que não desovaram (FNDs), a maioria dos ovócitos vitelogênicos remanescentes nos ovários atingiu a maturação final, apresentando quebra de vesícula germinativa, mas não foram ovulados (NOs). Consequentemente, estas fêmeas apresentaram frequências mais baixas de folículos pós ovulatórios (5%) quando comparadas com a que desovou (FD) (23%). Com relação aos NOs, os valores se inverteram e a frequência destes nas FNDs (21%) foi maior do que na FD (3%). Estes dados indicam que as falhas na desova desta espécie estão provavelmente relacionadas com a ovulação, uma vez que a maturação final dos ovócitos ocorre de forma similar tanto nas FNDs como na FD. Os dados sugerem que as substâncias que promovem a ovulação, como as prostaglandinas, podem aumentar o sucesso de desova em peixes reofílicos.

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.

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish.

Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology-transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species.

Occurrence and significance of ovarian and follicular regression in cachara Pseudoplatystoma fasciatum (Linnaeus, 1766): a histology approach

The processes of ovarian regression and follicular atresia which reproduction was not induced by hormone in confined cachara, Pseudoplatystoma fasciatum, were investigated. The macro and microscopic characteristics (oocytes diameter and histology) of the ovaries were described every 20 days, in four stages: initial regression (Rg I = first 20 days), intermediate regression (Rg II = from 21st to 40th day), final regression (Rg III = from 41st to 80th day) and the recovering stage, called resting II (R II = from 81st to 150th day). The experiment was conducted from late January (summer - longer days) to May (autumn - shorter days). In the beginning, A0 samples showed oocyte diameters ranging from 437.5 to 1,187.5mm, suggesting that oocytes were in perinucleolar, at final maturation and atretic phases. After 150 days, the diameters reached the lowest values and a ruptured zona radiata, as well as the nearly complete reabsorption of the yolk could be visualized. At the same time, a sharp decrease in the mean values of the gonadosomatic index (GSI), water temperature, photophase and rainfall was observed. The gradual involution of this long process was dynamic and complex, affecting the spawning success (fertilization, eclosion and larvae survival rates) and, consequently, the whole productive system.

Estudaram-se os processos de regressão ovariana e atresia folicular em cachara, Pseudoplatystoma fasciatum, mantida em cativeiro, na reprodução não induzida por hormônios. As características macro e microscópicas (diâmetro dos ovócitos e histologia) dos ovários foram descritas a cada 20 dias, em quatro estádios: na regressão inicial (Rg I - os primeiros 20 dias), na regressão intermediária (Rg II - do 21° ao 40° dia), na regressão final (Rg III - do 41° ao 80° dia) e na fase de recuperação ou de repouso II (R II - do 81° ao 150° dia). O experimento foi realizado do final de janeiro (verão-dias longos) a maio (outono-dias curtos). No início do experimento, as amostras apresentaram ovócitos com diâmetros que variaram de 437,5 a 1.187,5mm, sugerindo encontrarem-se nas fases perinucleolar, de maturação final e atrésicos. Aos 150 dias, os diâmetros atingiram os menores valores e pôde-se visualizar a zona radiata rompida e o vitelo reabsorvido. Concomitantemente, houve diminuição abrupta dos valores médios do índice gonadossomático, da temperatura da água, das horas de luz e de chuva. A involução gradual do longo processo foi dinâmica e complexa, afetando o êxito da desova (taxas de fertilização, de eclosão e de sobrevivência de larvas) e, conseqüentemente, o sistema produtivo.

The reproductive cycle of male catfish Pseudoplatystoma fasciatum (Teleostei, Pimelodidae) revealed by changes of the germinal epithelium. An approach addressed to aquaculture.

The aims of the present study were to analyze the gonadal structure of Pseudoplatystoma fasciatum males during their annual cycle to enhance understanding of their reproductive biology and to improve the hormonally induced reproduction and culture of this species in hatcheries. We adopted the recently proposed method that establishes reproductive classes that are based on variations of the germinal epithelium within the year. Five reproductive classes were established: maturation (early, middle, and late), regression and recrudescence. Our observations revealed that in the spawning season P. fasciatum testes display two main functions: sperm production and sperm storage. We also concluded that the analysis of the variation of germinal epithelium was satisfactory when applied to this freshwater catfish and should be adopted for other fish species.