Rüdiger W. Schulz

Spermatogenesis in fish.

Spermatogenesis is a developmental process during which a small number of diploid spermatogonial stem cells produce a large number of highly differentiated spermatozoa carrying a haploid, recombined genome. We characterise morphologically the different germ cell stages with particular attention for the spermatogonial generations, including the stem cells and their specific capacity to colonise a recipients testis after transplantation. We propose a nomenclature for fish germ cells to improve the comparability among different teleost fish but also to higher vertebrates. Survival and development of germ cells depends on their continuous and close contact to Sertoli cells, and we review their multiple roles in the cystic mode of spermatogenesis seen in fish. We then discuss gene expression patterns associated with testis maturation. The endocrine system of vertebrates has evolved as master control system over spermatogenesis. In fish, both pituitary gonadotropins LH and FSH stimulate gonadal sex steroid hormone production directly by activating Leydig cells. Information is reviewed on the effects of progestin, androgens, and estrogens on global testicular gene expression patterns (microarray analysis), and on the molecular mechanisms by which steroids regulate specific candidate genes (identified by subtractive hybridization approaches) during early stages of testis maturation. Moreover, progestin and androgen effects on spermiation and milt hydration are discussed. Sex steroids mainly act via receptors expressed by Sertoli cells. One type of response is that Sertoli cells change growth factor expression, which subsequently modulates germ cell proliferation/differentiation via mechanisms yet to be characterised. Finally, we review data on germ cell autonomous processes, mainly derived from loss-of-function mutant fish lines, before identifying a number of focus areas for future research activities.

Control of puberty in farmed fish

Puberty comprises the transition from an immature juvenile to a mature adult state of the reproductive system, i.e. the individual becomes capable of reproducing sexually for the first time, which implies functional competence of the brain-pituitary-gonad (BPG) axis. Early puberty is a major problem in many farmed fish species due to negative effects on growth performance, flesh composition, external appearance, behaviour, health, welfare and survival, as well as possible genetic impact on wild populations. Late puberty can also be a problem for broodstock management in some species, while some species completely fail to enter puberty under farming conditions. Age and size at puberty varies between and within species and strains, and are modulated by genetic and environmental factors. Puberty onset is controlled by activation of the BPG axis, and a range of internal and external factors are hypothesised to stimulate and/or modulate this activation such as growth, adiposity, feed intake, photoperiod, temperature and social factors. For example, there is a positive correlation between rapid growth and early puberty in fish. Age at puberty can be controlled by selective breeding or control of photoperiod, feeding or temperature. Monosex stocks can exploit sex dimorphic growth patterns and sterility can be achieved by triploidisation. However, all these techniques have limitations under commercial farming conditions. Further knowledge is needed on both basic and applied aspects of puberty control to refine existing methods and to develop new methods that are efficient in terms of production and acceptable in terms of fish welfare and sustainability.

Spermatogenesis and its endocrine regulation

Three major phases compose spermatogenesis: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and spermiogenesis, the restructuring of spermatids into flagellated spermatozoa. The process is fuelled by stem cells that, when dividing, either self-renew or produce spermatogonia that are committed to proliferation, meiosis, and spermiogenesis. During all phases, germ cells are in close contact with and require the structural and functional support of Sertoli cells. In contrast to germ cells, these somatic cells express receptors for sex steroids and follicle-stimulating hormone (FSH), the most important hormones that regulate spermatogenesis. A typical Sertoli cell response to an endocrine stimulus would be to change the release of a growth factor that would then mediate the hormones effect to the germ cells. Recent studies in the Japanese eel have shown, for example, that in the absence of gonadotropin Sertoli cells produce a growth factor (an orthologue of anti-Müllerian hormone) that restricts stem cell divisions to the self-renewal pathway; also estrogens stimulate stem cell renewal divisions but not spermatogonial proliferation. Gonadotropin or 11-ketotestosterone (11-KT) stimulation, however, induces spermatogonial proliferation, which is in part mimicked by another Sertoli cell-derived growth factor (activin B). Since FSH (besides luteinizing hormone, LH) stimulates steroidogenesis in fish, and since FSH is the only gonadotropin detected in the plasma of sexually immature salmonids, increased FSH signalling may be sufficient to initiate spermatogenesis by activating both Sertoli cell functions and 11-KT production. Another important androgen is testosterone (T), which seems to act via feedback mechanisms that can compromise FSH-dependent signalling or steroidogenesis. The testicular production of T and 11-KT therefore needs to be balanced adequately. Further research is required to elucidate in what way(s) 11-KT stimulates later stages of development, such as entry into meiosis and spermiogenesis. At this period, LH becomes increasingly important for the regulation of androgen production. Results from mammalian models suggest that during the later phases, the control of germ cell apoptosis via Sertoli cell factors is an important regulatory mechanism. In many species, sperm cells cannot fertilize eggs until having passed a maturation process known as capacitation, which includes the acquisition of motility. Progestins that are produced under the influence of LH appear to play an important role in this context, which involves the control of the composition of the seminal plasma (e.g., pH values).

Discrepancy Between Molecular Structure and Ligand Selectivity of a Testicular Follicle-Stimulating Hormone Receptor of the African Catfish (Clarias gariepinus)

Abstract A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.

Histological and Stereological Evaluation of Zebrafish (Danio rerio) Spermatogenesis with an Emphasis on Spermatogonial Generations

Abstract The zebrafish has become an important vertebrate model for basic and biomedical research, including the research field of the biology of reproduction. However, very few morphological and stereological data are available regarding zebrafish testis structure and spermatogenesis. In this careful histomorphometric evaluation of the testis, we studied spermatogonial cells using molecular markers, determined the combined duration of meiotic and spermiogenic phases, and examined the formation of the Sertoli cell barrier (tight junctions). We found at least nine spermatogonial generations and propose a morphology-based nomenclature for spermatogonial generations that is compatible with the one used in higher vertebrates. The number of germ cells per cyst increased dramatically (1 to ∼1360 cells) from undifferentiated spermatogonia type A to early spermatids. The combined duration of meiotic and spermiogenic phases is approximately 6 days, one of the shorter periods among the teleost fish investigated to date. The number of Sertoli cells per cyst increased 9-fold during the maturational cycle of spermatogenic cysts and stabilized in the meiotic phase at a ratio of approximately 100 early spermatids per Sertoli cell (Sertoli cell efficiency). Similarly to mammals, Sertoli cell proliferation ceased in the meiotic phase, coinciding with the formation of tight junctions between Sertoli cells. Hence, the events taking place during puberty in the germinal epithelium of mammals seem to recapitulate the “life history” of each individual spermatogenic cyst in zebrafish.

Gonadotropins, their receptors, and the regulation of testicular functions in fish

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.

Serum levels of 11-oxotestosterone in male and 17β-estradiol in female rainbow trout (Salmo gairdneri) during the first reproductive cycle

From September 1980 to August 1981 the serum levels of 11-oxotestosterone (11-O-T) in 231 male and of 17 beta-estradiol (E2) in 207 female rainbow trout were measured radioimmunologically, and compared with the development stages of the gonads. Using histological criteria, testicular development was divided into seven stages, and ovarian development into four stages. In males, 11-O-T levels remained below 6 ng/ml until the appearance of spermatozoa. Maximum values (126 ng/ml) were observed in spermiating males. In females, E2 levels were below 2 ng/ml during the period of slow oocyte growth. Maximum values (average of 31 ng/ml) were observed during exogenous vitellogenesis.

11-Oxygenated androgens in female teleosts: prevalence, abundance, and life history implications.

Although 11-ketotestosterone (11-KT) has been found in blood of females of several diadromous fish species, the importance, abundance, and prevalence of this and related 11-oxygenated androgens in females have not been investigated. To address this issue and to determine whether the differences among androgen profiles relate to specific life history strategies, particularly diadromous migrations, fish (males and females) of around 30 species were sampled and 5 androgens were measured by radioimmunoassay. Levels of 17beta-estradiol and cortisol were also determined to evaluate ovarian and interrenal activity at the time of sampling. Testosterone (T) was the predominant androgen in most sexually recrudescent females. Only in female eel and sturgeon were 11-oxygenated androgens present in levels as high as, or higher than, those of T, although substantial amounts were also found in blood of mullet and salmonids. 11-KT was generally the most abundant 11-oxyandrogen, levels being higher than those of 11beta-hydroxytestosterone or 11beta-hydroxyandrostenedione. It is concluded that 11-oxygenated androgens are quantitatively minor steroids in most female fish. There was no convincing evidence to support the notion that the presence of 11-oxygenated androgens in blood is an adaptation specific to migratory fishes.

Seasonal changes in and effect of castration/androgen replacement on the plasma levels of five androgens in the male three-spined stickleback, Gasterosteus aculeatus L

The seasonal changes in plasma levels of the androgens 11-ketotestosterone (OT), testosterone (T), 11 beta-hydroxytestosterone (OHT), 11-ketoandrostenedione (OA), and 11 beta-hydroxyandrostenedione (OHA) were measured in the male three-spined stickleback (Gasterosteus aculeatus L). OT was the dominant plasma androgen in the breeding season in summer and was the only androgen that peaked during this period. The levels of OT correlated closely with the development of male secondary sexual characters and reproductive behavior. T and OHT were low in all seasons, whereas OHA and OA displayed the highest levels in early winter. During the postbreeding period, the time of active spermatogenesis, all measured steroids were low. Castration resulted in an almost complete loss of plasma OT and reduced T, whereas OHT, OHA, and OA were not reliably influenced. Androstenedione implants in castrated fish increased plasma T and OA implants increased plasma OT, suggesting a nontesticular site of conversion.

Functional characterization and expression analysis of the androgen receptor in zebrafish (Danio rerio) testis

The biological activity of androgens, important for male sexual differentiation and development, is mediated by the androgen receptor (AR) that binds to specific DNA recognition sites regulating the transcription of androgen target genes. We investigated androgen production by adult zebrafish testis tissue, and identified 11beta-hydroxyandrostenedione, 11-ketoandrostenedione (OA), and 11-ketotestosterone (11-KT) as main products, and hence potential ligands, for the zebrafish Ar. These androgens were then included in the pharmacological characterization of the zebrafish Ar. The zebrafish Ar responded well in terms of binding and transactivation to synthetic androgens as well as to testosterone and 11-KT, and reasonably well to OA and androstenedione. In situ hybridization analysis of zebrafish testis revealed that ar mRNA expression was detected in the subpopulation of Sertoli cells contacting early spermatogonia.