Endocrine and paracrine regulation of zebrafish spermatogenesis: the Sertoli cell perspective

Spermatogonial stem cells (SSCs) either self-renew or differentiate into spermatogonia that further develop into spermatozoa. Self-renewal occurs when residing in a specific micro-environment (niche) While displacement from the niche would tip the signalling balance towards differentiation. Considering the cystic type of spermatogenesis in fish, the SSC candidates are single type A undifferentiated (Aund) spermatogonia, enveloped by mostly one niche-forming Sertoli cell. When going through a self-renewal cell cycle, the resulting new single type Aund spermatogonium would have to recruit another Sertoli cell to expand the niche space, while a differentiating germ cell cyle would result in a pair of spermatogonia that remain in contact with their cyst-forming Sertoli cells. In zebrafish, thyroid hormone stimulates the proliferation of Sertoli cells and of type Aund spermatogonia, involving Igf3, a new member of the Igf family. In cystic spermatogenesis, type A und spermatogonia usually do not leave the niche, so that supposedly the signalling in the niche changes when switching from self-renewal to differentiation. Recombinant zebrafish (rz) Fsh down-regulated Sertoli cell anti-müllerian hormone (amh) mRNA levels, and rzAmh inhibited differentiation of type A und spermatogonia as well as Fsh-stimulated steroidogenesis. Thus, for Fsh to efficiently stimulate testis functions, Amh bioactivity should be dampened. We also discovered that Fsh increased Sertoli cell Igf3 gene and protein expression; rzIgf3 stimulated spermatogonial proliferation and Fsh-stimulated spermatogenesis was significantly impaired by inhibiting Igf receptor signaling. We propose that in zebrafish, Fsh is the major regulator of testis functions and, supported by other endocrine systems (e.g. thyroid hormone), regulates Leydig cell steroidogenesis as well as Sertoli cell number and growth factor production to promote spermatogenesis.

Endocrine and paracrine regulation of zebrafish spermatogenesis: the Sertoli cell perspective

Spermatogonial stem cells (SSCs) either self-renew or differentiate into spermatogonia that further develop into spermatozoa. Self-renewal occurs when residing in a specific micro-environment (niche) While displacement from the niche would tip the signalling balance towards differentiation. Considering the cystic type of spermatogenesis in fish, the SSC candidates are single type A undifferentiated (Aund) spermatogonia, enveloped by mostly one niche-forming Sertoli cell. When going through a self-renewal cell cycle, the resulting new single type Aund spermatogonium would have to recruit another Sertoli cell to expand the niche space, while a differentiating germ cell cyle would result in a pair of spermatogonia that remain in contact with their cyst-forming Sertoli cells. In zebrafish, thyroid hormone stimulates the proliferation of Sertoli cells and of type Aund spermatogonia, involving Igf3, a new member of the Igf family. In cystic spermatogenesis, type A und spermatogonia usually do not leave the niche, so that supposedly the signalling in the niche changes when switching from self-renewal to differentiation. Recombinant zebrafish (rz) Fsh down-regulated Sertoli cell anti-müllerian hormone (amh) mRNA levels, and rzAmh inhibited differentiation of type A und spermatogonia as well as Fsh-stimulated steroidogenesis. Thus, for Fsh to efficiently stimulate testis functions, Amh bioactivity should be dampened. We also discovered that Fsh increased Sertoli cell Igf3 gene and protein expression; rzIgf3 stimulated spermatogonial proliferation and Fsh-stimulated spermatogenesis was significantly impaired by inhibiting Igf receptor signaling. We propose that in zebrafish, Fsh is the major regulator of testis functions and, supported by other endocrine systems (e.g. thyroid hormone), regulates Leydig cell steroidogenesis as well as Sertoli cell number and growth factor production to promote spermatogenesis.(AU)

Sertoli cell and spermatogenic efficiencies in Pêga Donkey (Equus asinus)

The donkey (Equus asinus) is a domesticated species from the Equidae family that is economically important and very well adapted to the arid regions of South America, Africa and Asia. However, except for a few studies in the literature related to testis structure and function in donkeys, to our knowledge there is no report regarding spermatogenic and Sertoli cell efficiencies in this equid species. Therefore, in the present study our main aims were to study these important parameters in Pêga donkeys. For this purpose, five sexually mature donkeys that had their testis perfused-fixed with buffered glutaraldehyde and routinely prepared for histological and stereological evaluationswere investigated during the equine breeding period, which is from September to February in the South Hemisphere. The results found showed that, similar to most mammalian species already investigated, from the theoretical expected number, expressive germ cell loss occurred during the spermatogonial (~75%) and meiotic (~25%) phases of spermatogenesis, in such a way that, from each 10 spermatozoa expected to be formed from initial type A differentiated spermatogonia, only around 2 would be formed. However, mainly due to the higher Sertoli cell efficiency found for Pêga donkeys (~15 spermatids per Sertoli cell), spermatogenic efficiency (daily sperm production per testis gram) observed for this species (~42 million) is the highest observed for the domestic mammals already investigated, being ~110% higher than the value described in the literature forstallions.

Sertoli cell and spermatogenic efficiencies in Pêga Donkey (Equus asinus)

The donkey (Equus asinus) is a domesticated species from the Equidae family that is economically important and very well adapted to the arid regions of South America, Africa and Asia. However, except for a few studies in the literature related to testis structure and function in donkeys, to our knowledge there is no report regarding spermatogenic and Sertoli cell efficiencies in this equid species. Therefore, in the present study our main aims were to study these important parameters in Pêga donkeys. For this purpose, five sexually mature donkeys that had their testis perfused-fixed with buffered glutaraldehyde and routinely prepared for histological and stereological evaluationswere investigated during the equine breeding period, which is from September to February in the South Hemisphere. The results found showed that, similar to most mammalian species already investigated, from the theoretical expected number, expressive germ cell loss occurred during the spermatogonial (~75%) and meiotic (~25%) phases of spermatogenesis, in such a way that, from each 10 spermatozoa expected to be formed from initial type A differentiated spermatogonia, only around 2 would be formed. However, mainly due to the higher Sertoli cell efficiency found for Pêga donkeys (~15 spermatids per Sertoli cell), spermatogenic efficiency (daily sperm production per testis gram) observed for this species (~42 million) is the highest observed for the domestic mammals already investigated, being ~110% higher than the value described in the literature forstallions.(AU)

Effects of mild calorie restriction on reproductive parameters of pubertal and sexually mature male Wistar rats

Calorie restriction (CR) has been reported to have pro-longevity effects in a myriad of species. However, a deleterious effect of CR on reproduction is also observed, though seemingly negligible if CR is very mild. Full or partial suppression of reproduction by CR is worthy of concern when envisioning the use of this methodology to prolong lifespan in humans. Our aim was to investigate the effects of mild CR on several reproductive parameters, such as the onset of puberty, fertility, reproductive organ bi ometry, testis morphometry and testosterone and cholesterol plasma levels. Therefore, male Wistar rats were subjected to 30%CR from age 24 days until the ages of 50 and 150 days. Age-matched animals received food ad libitum (AL) and served as controls. The day of onset of balano-preputial separation, indicative of puberty, showed no difference between diet groups. In relation to the fertility parameters evaluated, considering mainly the number of pups per female, there was a trend for better performance in CR rats. Body weight, as well as the absolute and relative weights of the epididymal fat pad, was reduced by CR at both ages investigated, whereas testosterone and cholesterol plasma levels were not changed. The weight of ventral prostate, also the tubular diameter and luminal size were smaller in CR rats at age 50 days, but seemed to recover later since no differences were observed at age 150 days. In conclusion, our results suggested that 30%CR does not compromise reproduction in male Wistar rats.

Effects of mild calorie restriction on reproductive parameters of pubertal and sexually mature male Wistar rats

Calorie restriction (CR) has been reported to have pro-longevity effects in a myriad of species. However, a deleterious effect of CR on reproduction is also observed, though seemingly negligible if CR is very mild. Full or partial suppression of reproduction by CR is worthy of concern when envisioning the use of this methodology to prolong lifespan in humans. Our aim was to investigate the effects of mild CR on several reproductive parameters, such as the onset of puberty, fertility, reproductive organ bi ometry, testis morphometry and testosterone and cholesterol plasma levels. Therefore, male Wistar rats were subjected to 30%CR from age 24 days until the ages of 50 and 150 days. Age-matched animals received food ad libitum (AL) and served as controls. The day of onset of balano-preputial separation, indicative of puberty, showed no difference between diet groups. In relation to the fertility parameters evaluated, considering mainly the number of pups per female, there was a trend for better performance in CR rats. Body weight, as well as the absolute and relative weights of the epididymal fat pad, was reduced by CR at both ages investigated, whereas testosterone and cholesterol plasma levels were not changed. The weight of ventral prostate, also the tubular diameter and luminal size were smaller in CR rats at age 50 days, but seemed to recover later since no differences were observed at age 150 days. In conclusion, our results suggested that 30%CR does not compromise reproduction in male Wistar rats.(AU)

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 vari ous organisms, even when started later in life. Despite benefits for health and lifespan, CR’s negative impact on reproduct ion 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 dele terious 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 or gan 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 libitu m (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 analyze d plasma parameters or weight of androgen - dependent organs. More over, CR did not interfere with expression of the assessed testicular genes, or most of the hepatic genes, but it did cause an increas e in the levels of peroxisome proliferator - activated receptor gamma ( Pparg ) and mouse sulfo transferase ( mSTa ); and a decre ase 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 improv ing health and longevity, even when started later in life.

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 vari ous organisms, even when started later in life. Despite benefits for health and lifespan, CRs negative impact on reproduct ion 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 dele terious 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 or gan 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 libitu m (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 analyze d plasma parameters or weight of androgen - dependent organs. More over, CR did not interfere with expression of the assessed testicular genes, or most of the hepatic genes, but it did cause an increas e in the levels of peroxisome proliferator - activated receptor gamma ( Pparg ) and mouse sulfo transferase ( mSTa ); and a decre ase 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 improv ing health and longevity, even when started later in life.(AU)

Expression of GFP in transgenic tilapia under the control of the medaka B-actin promoter: establishment of a model system for germ cell transplantation

The Nile tilapia (Oreochromis niloticus) stands out as one of the most important fresh water edible fish, possessing remarkable characteristics that make it desirable for both commercial culture and as a laboratory model. For the utilization of tilapia in germ cell transplantation experiments, appropriate cell markers are required to evaluate the colonization behavior of donor-derived germ cells in recipient gonads. Here we report the production of a medaka β-actin/EGFP transgenic tilapia strain expressing the green fluorescent protein (GFP) reporter gene in several tissues including germ cells in testis and ovary. Fluorescent observations in F2 generation transgenic individuals showed GFP positive cells along the body axis in pre-hatched embryos, while in hatching embryos the GFP gene was strongly expressed in the area surrounding the gills, operculum and in the cephalic region. In early larvae, fluorescent cells were scattered throughout the body, forming aggregations around the dorsal-cephalic and mouth areas. At 38 days post-fertilization, juvenile fish expressed the GFP homogeneously in the whole body. GFP fluorescence was also observed in caudal fins, muscle, and in several internal organs (gills, heart, testes, and ovaries) in 140 and 240 day F2 and F3 individuals. Immunohistochemistry using a monoclonal anti-GFP antibody in juvenile and adult gonads showed that both mitotic and meiotic germ cells were labeled with GFP. The utilization of this transgenic line in a germ cell transplantation system could offer a fast and reliable screening of donor-derived transgenic offspring, as well as accurate tracing of donor-derived cell colonization in the recipient gonad by means of immunohistochemistry using GFP antibodies. In the future, germ cell transplantation using Nile tilapia also could help to preserve the genetic resources of threatened cichlids, through cryopreservation and interspecies transplantation of germ cells from endangered cichlids into O. niloticus recipients.(AU)

Expression of GFP in transgenic tilapia under the control of the medaka B-actin promoter: establishment of a model system for germ cell transplantation

The Nile tilapia (Oreochromis niloticus) stands out as one of the most important fresh water edible fish, possessing remarkable characteristics that make it desirable for both commercial culture and as a laboratory model. For the utilization of tilapia in germ cell transplantation experiments, appropriate cell markers are required to evaluate the colonization behavior of donor-derived germ cells in recipient gonads. Here we report the production of a medaka β-actin/EGFP transgenic tilapia strain expressing the green fluorescent protein (GFP) reporter gene in several tissues including germ cells in testis and ovary. Fluorescent observations in F2 generation transgenic individuals showed GFP positive cells along the body axis in pre-hatched embryos, while in hatching embryos the GFP gene was strongly expressed in the area surrounding the gills, operculum and in the cephalic region. In early larvae, fluorescent cells were scattered throughout the body, forming aggregations around the dorsal-cephalic and mouth areas. At 38 days post-fertilization, juvenile fish expressed the GFP homogeneously in the whole body. GFP fluorescence was also observed in caudal fins, muscle, and in several internal organs (gills, heart, testes, and ovaries) in 140 and 240 day F2 and F3 individuals. Immunohistochemistry using a monoclonal anti-GFP antibody in juvenile and adult gonads showed that both mitotic and meiotic germ cells were labeled with GFP. The utilization of this transgenic line in a germ cell transplantation system could offer a fast and reliable screening of donor-derived transgenic offspring, as well as accurate tracing of donor-derived cell colonization in the recipient gonad by means of immunohistochemistry using GFP antibodies. In the future, germ cell transplantation using Nile tilapia also could help to preserve the genetic resources of threatened cichlids, through cryopreservation and interspecies transplantation of germ cells from endangered cichlids into O. niloticus recipients.