Loss of bmp15 function in the seasonal spawner Atlantic salmon results in ovulatory failure.

Bone morphogenetic protein 15 (BMP15) is an oocyte-specific growth factor important for successful female reproduction in mammals. While mutations in BMP15/Bmp15 cause ovulatory deficiency and/or infertility in certain mammalian species, loss of bmp15 in zebrafish, a continuous spawner and the only bmp15 knockout model in fish to date, results in complete arrest of follicle development and later female-to-male sex reversal, preventing to examine effects on ovulation/fertilization. Here, we used Atlantic salmon, a seasonal spawner, and generated bmp15 mutants to investigate ovarian development and fertility. Histological and morphometric analyses revealed that in biallelic frameshift (bmp15 fs/fs) mutant ovaries, folliculogenesis started earlier, resulting in an advanced development compared to wild-type (WT) controls, accompanied by a weaker expression of the (early) oocyte-specific factor figla. This precocious ovarian development was followed in bmp15 fs/fs females by enhanced follicle atresia during vitellogenic stages. Although genes involved in steroid synthesis and signaling (star, cyp11b, cyp17a1 and esr1) were dramatically higher in late vitellogenic bmp15 fs/fs mutant ovaries, estradiol-17ß plasma levels were lower than in WT counterparts, potentially reflecting compensatory changes at the level of ovarian gene expression. At spawning, bmp15 fs/fs females displayed lower gonado-somatic index values and reduced oocyte diameter, and the majority (71.4%), showed mature non-ovulating ovaries with a high degree of atresia. The remaining (28.6%) females spawned eggs but they either could not be fertilized or, upon fertilization, showed severe malformations and embryonic mortality. Our results show that Bmp15 is required for proper follicle recruitment and growth and later ovulatory success in Atlantic salmon, providing an alternative candidate target to induce sterility in farmed salmon. Moreover, since loss of bmp15 in salmon, in contrast to zebrafish, does not result in female-to-male sex change, this is the first mutant model in fish allowing further investigations on Bmp15-mediated functions in the ovulatory period.

Endocrine and local signaling interact to regulate spermatogenesis in zebrafish: follicle-stimulating hormone, retinoic acid and androgens.

Retinoic acid (RA) is crucial for mammalian spermatogonia differentiation, and stimulates Stra8 expression, a gene required for meiosis. Certain fish species, including zebrafish, have lost the stra8 gene. While RA still seems important for spermatogenesis in fish, it is not known which stage(s) respond to RA or whether its effects are integrated into the endocrine regulation of spermatogenesis. In zebrafish, RA promoted spermatogonia differentiation, supported androgen-stimulated meiosis, and reduced spermatocyte and spermatid apoptosis. Follicle-stimulating hormone (Fsh) stimulated RA production. Expressing a dominant-negative RA receptor variant in germ cells clearly disturbed spermatogenesis but meiosis and spermiogenesis still took place, although sperm quality was low in 6-month-old adults. This condition also activated Leydig cells. Three months later, spermatogenesis apparently had recovered, but doubling of testis weight demonstrated hypertrophy, apoptosis/DNA damage among spermatids was high and sperm quality remained low. We conclude that RA signaling is important for zebrafish spermatogenesis but is not of crucial relevance. As Fsh stimulates androgen and RA production, germ cell-mediated, RA-dependent reduction of Leydig cell activity may form a hitherto unknown intratesticular negative-feedback loop.

Development of supermale and all-male Atlantic salmon to research the vgll3 allele - puberty link.

BACKGROUND: Farmed Atlantic salmon are one of the most economically significant global aquaculture products. Early sexual maturation of farmed males represents a significant challenge to this industry and has been linked with the vgll3 genotype. However, tools to aid research of this topic, such as all-male and clonal fish, are still lacking. The present 6-year study examined if all-male production is possible in Atlantic salmon, a species with heteromorphic sex chromosomes (males being XY, females XX), and if all-male fish can be applied to further explore the vgll3 contribution on the likelihood of early maturation. RESULTS: Estrogen treatment of mixed sex yolk sac larvae gave rise to one sexually mature hermaphrodite with a male genotype (XY) that was used to produce both self-fertilized offspring and androgenetic double haploid (dh) offspring following egg activation with UV treated sperm and pressure shock to block the first mitotic division. There were YY supermales among both offspring types, which were crossed with dh females. Between 1 and 8% of the putative all-male offspring from the eight crosses with self-fertilized supermales were found to have ovaries, and 95% of these phenotypic females were also genetically female. None of the offspring from the one dh supermale cross had ovaries. When assessing the general contribution of the vgll3 locus on the likelihood of early post-smolt sexual maturation (jacking) in the all-male populations we found individuals that were homozygous for the early maturing genotype (97%) were more likely to enter puberty than individuals that were homozygous for the late maturing genotype (26%). However, the likelihood of jacking within individuals with an early/late heterozygous genotype was higher when the early allele came from the dam (94%) compared to the sire (45%). CONCLUSIONS: The present results show that supermale Atlantic salmon are viable and fertile and can be used as a research tool to study important aspects of sexual maturation, such as to further explore the sex dependent parental genetic contribution to age at puberty in Atlantic salmon. In addition, we report the production of viable double haploid supermale fish.

The initiation of puberty in Atlantic salmon brings about large changes in testicular gene expression that are modulated by the energy status.

BACKGROUND: When puberty starts before males reach harvest size, animal welfare and sustainability issues occur in Atlantic salmon (Salmo salar) aquaculture. Hallmarks of male puberty are an increased proliferation activity in the testis and elevated androgen production. Examining transcriptional changes in salmon testis during the transition from immature to maturing testes may help understanding the regulation of puberty, potentially leading to procedures to modulate its start. Since differences in body weight influence, via unknown mechanisms, the chances for entering puberty, we used two feed rations to create body weight differences. RESULTS: Maturing testes were characterized by an elevated proliferation activity of Sertoli cells and of single undifferentiated spermatogonia. Pituitary gene expression data suggest increased Gnrh receptor and gonadotropin gene expression, potentially responsible for the elevated circulating androgen levels in maturing fish. Transcriptional changes in maturing testes included a broad variety of signaling systems (e.g. Tgfß, Wnt, insulin/Igf, nuclear receptors), but also, activation of metabolic pathways such as anaerobic metabolism and protection against ROS. Feed restriction lowered the incidence of puberty. In males maturing despite feed restriction, plasma androgen levels were higher than in maturing fish receiving the full ration. A group of 449 genes that were up-regulated in maturing fully fed fish, was up-regulated more prominently in testis from fish maturing under caloric restriction. Moreover, 421 genes were specifically up-regulated in testes from fish maturing under caloric restriction, including carbon metabolism genes, a pathway relevant for nucleotide biosynthesis and for placing epigenetic marks. CONCLUSIONS: Undifferentiated spermatogonia and Sertoli cell populations increased at the beginning of puberty, which was associated with the up-regulation of metabolic pathways (e.g. anaerobic and ROS pathways) known from other stem cell systems. The higher androgen levels in males maturing under caloric restriction may be responsible for the stronger up-regulation of a common set of (449) maturation-associated genes, and the specific up-regulation of another set of (421) genes. The latter opened regulatory and/or metabolic options for initiating puberty despite feed restriction. As a means to reduce the incidence of male puberty in salmon, however, caloric restriction seems unsuitable.

Correction to: Entry into puberty is reflected in changes in hormone production but not in testicular receptor expression in Atlantic salmon (Salmo salar).

Following publication of the original article [1], the authors would like to apologize for an error in Fig. 5e, the correct graph is presented below and shows the significant increase in pituitary mRNA levels of fshb in recruited males in the SGA stage.

Entry into puberty is reflected in changes in hormone production but not in testicular receptor expression in Atlantic salmon (Salmo salar).

BACKGROUND: Puberty in male Atlantic salmon in aquaculture can start as early as after the first winter in seawater, stunts growth and entails welfare problems due to the maturation-associated loss of osmoregulation capacity in seawater. A better understanding of the regulation of puberty is the basis for developing improved cultivation approaches that avoid these problems. Our aim here was to identify morphological and molecular markers signaling the initiation of, and potential involvement in, testis maturation. METHODS: In the first experiment, we monitored for the first time in large Atlantic salmon males several reproductive parameters during 17 months including the first reproductive cycle. Since testicular growth accelerated after the Winter solstice, we focused in the second experiment on the 5 months following the winter solstice, exposing fish from February 1 onwards to the natural photoperiod (NL) or to continuous additional light (LL). RESULTS: In the first experiment, testis weight, plasma androgens and pituitary gonadotropin transcript levels increased with the appearance of type B spermatogonia in the testis, but testicular transcript levels for gonadotropin or androgen receptors did not change while being clearly detectable. In the second experiment, all males kept under NL had been recruited into puberty until June. However, recruitment into puberty was blocked in ~ 40% of the males exposed to LL. The first morphological sign of recruitment was an increased proliferation activity of single spermatogonia and Sertoli cells. Irrespective of the photoperiod, this early sign of testis maturation was accompanied by elevated pituitary gnrhr4 and fshb and testicular igf3 transcript levels as well as increased plasma androgen levels. The transition into puberty occurred again with stable testicular gonadotropin and androgen receptor transcript levels. CONCLUSIONS: The sensitivity to reproductive hormones is already established before puberty starts and up-regulation of testicular hormone receptor expression is not required to facilitate entry into puberty. The increased availability of receptor ligands, on the other hand, may result from an up-regulation of pituitary Gnrh receptor expression, eventually activating testicular growth factor and sex steroid release and driving germ and Sertoli cell proliferation and differentiation.

Regulation of spermatogonial development by Fsh: The complementary roles of locally produced Igf and Wnt signaling molecules in adult zebrafish testis.

Spermatogenesis is a cellular developmental process characterized by the coordinated proliferation and differentiation activities of somatic and germ cells in order to produce a large number of spermatozoa, the cellular basis of male fertility. Somatic cells in the testis, such as Leydig, peritubular myoid and Sertoli cells, provide structural and metabolic support and contribute to the regulatory microenvironment required for proper germ cell survival and development. The pituitary follicle-stimulating hormone (Fsh) is a major endocrine regulator of vertebrate spermatogenesis, targeting somatic cell functions in the testes. In fish, Fsh regulates Leydig and Sertoli cell functions, such as sex steroid and growth factor production, processes that also control the development of spermatogonia, the germ cell stages at the basis of the spermatogenic process. Here, we summarize recent advances in our understanding of mechanisms used by Fsh to regulate the development of spermatogonia. This involves discussing the roles of insulin-like growth factor (Igf) 3 and canonical and non-canonical Wnt signaling pathways. We will also discuss how these locally active regulatory systems interact to maintain testis tissue homeostasis.

Termination of puberty in out-of-season male Atlantic salmon smolts.

Environmental conditions are known to contribute to the phenotypic plasticity in the age of sexual maturation of Atlantic salmon (Salmo salar). Here, we report on an observation of out-of-season male Atlantic salmon initiating puberty as pre-smolts (jacks) but failing to complete maturation as post-smolts. Jacks were identified based on elevated plasma 11-ketotestosterone (range, 3-12 ng/ml) and the occurrence of type B spermatogonia in January 2017. However, these males failed to show running milt as post-smolts at the expected time in May 2017. Subsequently, 6 out of the 21 (32%) suspected "terminated jacks" went on to become grilse, whereas only 1 of the 22 (5%) males that showed no signs of initiating puberty in January became grilse in December 2017. Therefore, "terminated" jacks were more likely to mature as grilse than the males that remained immature. Why these pubertal pre-smolt males did not complete maturation is unclear but could be related to the transfer of fish from conditions of warm water and long days, risk factors for early maturation, to conditions of cold water and short days, which are expected to delay the age of maturation. We provide a description of the conditions under which male Atlantic salmon appear to have terminated the process of sexual maturation.

Effects of re-stripping on the seminal characteristics of pacu (Piaractus mesopotamicus) during the breeding season.

Seminal characteristics in teleost fish with an annual reproductive period, such as pacu (Piaractus mesopotamicus), may vary during the breeding season. The sperm formed before the beginning of the spawning period may be stored for a long time, causing damage to the cells. Therefore, re-stripping may be an important way to eliminate the "old" and allow for the collection of "new" spermatozoids. In this study, we analyzed the seminal characteristics of hormonally induced pacu at the beginning, middle and end of the breeding season, and we analyzed samples from re-stripped males (stripped first at the beginning, re-stripped in the middle, and re-stripped again at the end of the season) during two breeding seasons. The sperm density, ionic composition, pH, and osmolality were similar among the groups. The semen volume, seminal plasma protein concentration and incidence of morphologically anomalous sperm increased over time. In addition, some parameters that are associated with good-quality semen decreased, such as sperm motility, viability and DNA integrity. Moreover, we observed a positive association among motility, viability and DNA integrity for sperm with elevated 11-ketotestosterone, but there was no such association for fshb or lhb mRNA levels in the pituitary. The semen that was obtained earlier (at the beginning) or from re-stripped males exhibited better characteristics than the other samples collected. In conclusion, collecting semen from pacu at the end of breeding season should be avoided; it is preferable to strip early and then re-strip later in the season, and this approach may be used for diverse aquaculture purposes.

Androgens directly stimulate spermatogonial differentiation in juvenile Atlantic salmon (Salmo salar).

We studied the effects of androgens on early stages of spermatogenesis along with androgen receptor binding characteristics and the expression of selected testicular and pituitary genes. To this end, immature Atlantic salmon postsmolts received testosterone (T), adrenosterone (OA, which is converted in vivo into 11-ketotestosterone, 11-KT) or a combination of the two androgens (T+OA). Treatment with OA and T elevated the plasma levels of 11-KT and T, respectively, and co-injection of OA with T lead to high 11-KT levels but prevented plasma T levels to reach the levels observed after injecting T alone. Clear stimulatory effects were recorded as regards pituitary lhb and gnrhr4 transcript levels in fish receiving T, and to a lesser extent in fish receiving OA (but for the lhb transcript only). The two androgen receptors (Ara1 and Ara2) we cloned bound T and 11-KT and responded to these androgens in a similar way. Both androgens down-regulated testicular amh and increased igf3 transcript levels after 1 week of treatment, but effects on growth factor gene expression required sustained androgen stimulation and faded out in the groups with the decreasing T plasma levels. In fish exhibiting a sustained elevation of 11-KT plasma levels (OA and T+OA groups) for 2 weeks, the number of differentiating spermatogonia had increased while the number of undifferentiated spermatogonia decreased. Previous work showed that circulating gonadotropin levels did not increase following androgen treatments of gonad-intact immature male salmonids. Taken together, androgen treatment of immature males modulated testicular growth factor expression that, when sustained for 2 weeks, stimulated differentiation, but not self-renewal, of undifferentiated type A spermatogonia.

Loss of Fshr Prevents Testicular Maturation in Atlantic Salmon (Salmo salar L.).

Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr-/-) or in-frame mutations (fshrif/if). Nearly all wt males matured while all fshr-/- males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr-/- males compared with maturing males. More than half of the fshrif/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.

Androgen feedback effects on LH and FSH, and photoperiodic control of reproduction in male three-spined sticklebacks, Gasterosteus aculeatus.

Sexual maturation in the stickleback is controlled by photoperiod. The aim of this study was to find out whether changes in feedback effects exerted by sex steroids could mediate the photoperiodic effect, which is regarded to be of an all-or-nothing character. To that end, males were castrated and treated with different doses of testosterone (T) and in one experiment also with the aromatase inhibitor fadrozole (AI) and kept under different photoperiods. In control fish, long day (LD 16:8) stimulated maturation, associated with more hypertrophied kidneys (a secondary sexual character) and higher levels of pituitary lhb and fshb mRNA than under short day conditions (LD 8:16). Under LD 8:16, low doses of T suppressed both lhb and fshb mRNA levels. However, with the use of high doses of T and/or longer photoperiods the inhibitory effects on lhb and fshb mRNA levels became less clear or instead positive effects were observed. Under intermediate photoperiod conditions, the negative feedback effect of a low dose of T on fshb was more prominent with shorter photoperiods, whereas no such shift was observed for lhb mRNA. The inhibitory effect of the low dose of T on lhb mRNA levels under LD 8:16 was abolished by AI, whereas the stimulatory effect of the high dose of T was not. The negative feedback effects were more marked under short days than under long days, whereas positive feedback effects were more marked under long days. The suppression of both fshb and lhb mRNA levels by low androgen levels, especially under short days, may inhibit maturation completely unless a rise of androgens above threshold levels would allow complete maturation.

A progestin (17α,20ß-dihydroxy-4-pregnen-3-one) stimulates early stages of spermatogenesis in zebrafish.

Recently, evidence has been provided for multiple regulatory functions of progestins during the late mitotic and meiotic phases of spermatogenesis in teleost fish. For example, our previous studies suggested that 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), potentially via Sertoli cells that express the progesterone receptor (pgr) gene, can contribute to the regulation of zebrafish spermatogenesis. To further our understanding of the function of DHP at early spermatogenetic stages, we investigated in the present study the expression of genes reflecting Sertoli cell function and spermatogenic development in adult zebrafish testis after DHP treatment in tissue culture. Moreover, using an in vivo model of estrogen-mediated down-regulation of androgen production to interrupt adult spermatogenesis, we studied the effects of DHP on estrogen-interrupted spermatogenesis. In this model, DHP treatment doubled the testis weight, and all differentiating germ cell types, such as type B spermatogonia and primary spermatocytes, were abundantly present and incorporated the DNA-synthesis marker (BrdU). Accordingly, transcript levels of germ cell marker genes were up-regulated. Moreover, transcripts of two Sertoli cell-derived genes anti-müllerian hormone (amh) and gonadal soma-derived growth factor (gsdf) were up-regulated, as were three genes of the insulin-like growth factor signaling system, insulin-like growth factor 2b (igf2b), insulin-like growth factor 3 (igf3) and insulin-like growth factor 1b receptor (igf1rb). We further analyzed the relationship between these genes and DHP treatment using a primary zebrafish testis tissue culture system. In the presence of DHP, only igf1rb mRNA levels showed a significant increase among the somatic genes tested, and germ cell marker transcripts were again up-regulated. Taken together, our results show that DHP treatment induced the proliferation of early spermatogonia, their differentiation into late spermatogonia and spermatocytes as well as expression of marker genes for these germ cell stages. DHP-mediated stimulation of spermatogenesis and hence growth of spermatogenic cysts and the associated increase in Sertoli cell number may in part explain the elevated expression of Sertoli cell genes, but our data also suggest an up-regulation of the activity of the Igf signaling system.

Pituitary gonadotropin and ovarian gonadotropin receptor transcript levels: seasonal and photoperiod-induced changes in the reproductive physiology of female Atlantic salmon (Salmo salar).

In female Atlantic salmon kept at normal light conditions, pituitary follicle-stimulating hormone beta (fshb) transcript levels were transiently elevated one year before spawning, re-increased in February, and remained high during spawning in November and in post-ovulatory fish in December. The first increase in plasma 17b-estradiol (E2), testosterone (T) and gonadosomatic index (GSI) was recorded in January; E2 rose up to one month prior to ovulation, while T and GSI kept increasing until ovulation. Pituitary luteinizing hormone beta (lhb) transcript levels peaked at the time of ovulation. Except for transient changes before and after ovulation, ovarian follicle stimulating hormone receptor (fshr) transcript amounts were relatively stable at a high level. By contrast, luteinizing hormone receptor (lhcgr) transcript levels started out low and increased in parallel to GSI and plasma E2 levels. Exposure to continuous light (LL) induced a bimodal response where maturation was accelerated or arrested. The LL-arrested females showed previtellogenic oil droplet stage follicles or primary yolk follicles only, and fshb and E2 plasma levels collapsed while fshr increased. The LL-accelerated females showed elevated lhb transcript levels and slightly elevated E2 levels during early vitellogenesis, and significantly elevated lhcgr E2 and GSI levels in late vitellogenesis. We conclude that Fsh-dependent signaling stimulates recruitment into and the sustained development through vitellogenesis. Up-regulation of lhcgr gene expression during vitellogenesis may reflect an estrogenic effect, while elevated fshr gene expression following ovulation or during LL-induced arrestment may be associated with ovarian tissue remodeling processes.

Sex differentiation in Atlantic cod (Gadus morhua L.): morphological and gene expression studies.

BACKGROUND: In differentiated gonochoristic species, a bipotential gonad develops into an ovary or testis during sex differentiation. Knowledge about this process is necessary to improve methods for masculinizing genetically female Atlantic cod for the subsequent purpose of producing all-female populations. METHODS: Gonads were examined histologically in juveniles from 14 to 39 mm total body length (TL). Number and size of germ cells were determined in a subset of the samples. Relevant genes were cloned, and mRNA levels determined by qPCR of amh, cyp19a1a; dax1 (nr0b2); shp (nr0b2a) and sox9b in a mixed-sex and an all-female population ranging from 12-49 mm TL. RESULTS: Individuals between 14-20 mm TL could be separated in two subgroups based on gonad size and germ cell number. Ovarian cavity formation was observed in some individuals from 18-20 mm TL. The mixed sex population displayed bimodal expression patterns as regards cyp19a1a (starting at 12 mm TL) and amh (starting at 20 mm TL) mRNA levels. After approximately 30 mm TL, cyp19a1a and amh displayed a gradual increase in both sexes. No apparent, sex-dependent expression patterns were found for dax1, shp or sox9b transcripts. However, shp levels were high until the larvae reached around 35 mm TL and then dropped to low levels, while dax1 remained low until 35 mm TL, and then increased sharply. CONCLUSIONS: The morphological sex differentiation in females commenced between 14-20 mm TL, and ovarian cavities were evident by 18-20 mm TL. Testis development occurred later, and was morphologically evident after 30 mm TL. This pattern was corroborated with sexually dimorphic expression patterns of cyp19a1a from 12-13 mm TL, and a male-specific increase in amh from 20 mm TL.

Cloning, pharmacological characterization and expression analysis of Atlantic cod (Gadus morhua, L.) nuclear progesterone receptor.

To better understand the role(s) of progesterone in fish spermatogenesis, we cloned the nuclear progesterone receptor (Pgr) of Atlantic cod. The open-reading frame of the cod pgr consists of 2076 bp, coding for a 691-amino acids-long protein that shows the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that the cod Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency in activating the receptor. During ontogenesis, the pgr mRNA was undetectable in embryo's 24 h after fertilization, but became detectable 4 days after fertilization. During the larval stage, the expression levels increased steadily with the development of the larvae. In adult fish, pgr was predominantly expressed in gonads of both sexes. During the onset of puberty, testicular pgr transcript levels started to increase during rapid spermatogonial proliferation, and peaked when spermiation started. In situ hybridization studies using testis tissue during the rapid growth phase containing all germ cell stages indicated that in cod, pgr mRNA is predominantly located in Sertoli cells that are in contact with proliferating spermatogonia. Taken together, our data suggests that the Pgr is involved in mediating progestagen stimulation of the mitotic expansion of spermatogonia, and in processes associated with the spermiation/spawning period in Atlantic cod.

Sertoli cell proliferation in the adult testis is induced by unilateral gonadectomy in African catfish.

Survival and development of male germ cells depends on their close contact with Sertoli cells. In the cystic spermatogenesis found in fish, one germ cell clone, initially a single undifferentiated spermatogonium type A, is enclosed by and accompanied through spermatogenesis by a group of Sertoli cells. Previous work showed that after forming such spermatogenic cysts, Sertoli cells proliferated mainly during the mitotic expansion of the spermatogonial clone in the cyst. Here, we used unilateral gonadectomy (ULG) as experimental model to study Sertoli cell proliferation at the start of cyst development in adult African catfish testis. Four days after surgery, we observed a particularly strong increase in the number of mitotic Sertoli cells along with a significant increase in the number of mitotic single type A spermatogonia. Proliferation of pairs of spermatogonia or of larger germ cell clones, however, did not change. At the same time, pituitary transcript levels of the three gonadotropin-subunits (cga, glycoprotein hormones, alpha polypeptide; fshb, follicle stimulating hormone, beta polypeptide; lhb, luteinizing hormone, beta polypeptide) were not different between sham-operated and ULG males. However, expression of the gonadotropin-releasing hormone receptor gene gnrhr1 was significantly reduced after ULG, and Lh plasma levels were slightly elevated. In the testis remaining after ULG, Fsh receptor (fshr) mRNA levels increased significantly but luteinizing hormone/choriogonadotropin receptor (lhcgr) mRNA levels did not change. Circulating androgen levels did not differ between groups, but testicular androgen release increased significantly 2- to 3-fold after ULG. Considering the strong steroidogenic potency of Fsh and the expression of the fshr gene by Leydig cells in catfish, we explain the absence of an effect of ULG on circulating androgen levels by an Fshr-mediated, compensatory increase in the steroid production of the remaining testis, perhaps supported in addition by the increased Lh plasma levels. Since Fsh is a major stimulator of mammalian Sertoli cell proliferation, we propose that ULG-induced activation of the Fsh signalling system also promoted Sertoli cell proliferation and - possibly as a consequence of that - proliferation of single type A spermatogonia, providing the basis for an increased spermatogenic capacity.

Role of the Melanocortin System in Gonadal Steroidogenesis of Zebrafish.

In teleost, as in other vertebrates, stress affects reproduction. A key component of the stress response is the pituitary secretion of the adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. In zebrafish, Mc2r was identified in male and female gonads, while ACTH has been shown to have a physiological role in modulating reproductive activity. In this study, the hypothesis that other melanocortins may also affect how the zebrafish gonadal function is explored, specifically steroid biosynthesis, given the presence of members of the melanocortin signaling system in zebrafish gonads. Using cell culture, expression analysis, and cellular localization of gene expression, our new observations demonstrated that melanocortin receptors, accessory proteins, antagonists, and agonists are expressed in both the ovary and testis of zebrafish (n = 4 each sex). Moreover, melanocortin peptides modulate both basal and gonadotropin-stimulated steroid release from zebrafish gonads (n = 15 for males and n = 50 for females). In situ hybridization in ovaries (n = 3) of zebrafish showed mc1r and mc4r in follicular cells and adjacent to cortical alveoli in the ooplasm of previtellogenic and vitellogenic oocytes. In zebrafish testes (n = 3), mc4r and mc1r were detected exclusively in germ cells, specifically in spermatogonia and spermatocytes. Our results suggest that melanocortins are, directly or indirectly, involved in the endocrine control of vitellogenesis in females, through modulation of estradiol synthesis via autocrine or paracrine actions in zebrafish ovaries. Adult zebrafish testes were sensitive to low doses of ACTH, eliciting testosterone production, which indicates a potential role of this peptide as a paracrine regulator of testicular function.

Pituitary Gonadotropin Gene Expression During Induced Onset of Postsmolt Maturation in Male Atlantic Salmon: In Vivo and Tissue Culture Studies.

Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.