Henk J.Th. Goos

Characterization of zebrafish primordial germ cells: Morphology and early distribution of vasa RNA

Research into germ line development is of conceptual and biotechnologic importance. In this study, we used morphology at the level of light and electron microscope to characterize the primordial germ cells (PGCs) of the zebrafish throughout embryonic and larval development. The study was complemented by the detailed analysis of mRNA expression of a putative germ line marker vasa. By morphology alone PGCs were identified at the earliest at the 5‐somite stage in the peripheral endoderm in contact with the yolk syncytial layer. Subsequently, they move from lateral to medial positions into the median mesoderm and from there by means of the dorsal mesentery into the gonadal anlage at day 5 postfertilization (pf), to establish gonads with mesenchymal cells by day 9 pf. Ultrastructural analysis of the 4‐day‐old zebrafish larvae demonstrates the presence of the germ line‐specific structures, nuage, and annulate lamellae. vasa RNA‐positive cells can be followed during zebrafish embryogenesis from the 32‐cell stage onward (Yoon et al., 1997 ). Upon completion of gastrulation, the RNA is exclusively present in the cells of the hypoblast, which as a consequence of convergence and extension movements first arrange themselves in a V‐shaped string‐like conformation to end up, by late somitogenesis, as a string of cells on each side of the midline. We show that the localization of maternal vasa RNA in the ovary changes from cytoplasmic, in the previtellogenic oocytes, to cortical in the vitellogenic oocytes, to concentrate at the boundary of the yolk and cytoplasm in the one cell stage zygote. These results demonstrate that the cortical vasa RNA localization precedes its cleavage furrow‐associated localization in the embryos and is presumably cytoskeleton dependent. vasa RNA localization changes from asymmetric subcellular at the sphere stage, to become entirely cytoplasmic at the dome stage. These data suggest a close resemblance in modes of segregation of the germ plasm in the frog and vasa mRNA in the fish during cleavage stages. Based on the significantly larger size and the stereotype and similar position of morphologically distinct cells, presumed to be PGCs, and their vasa RNA‐positive counterparts, we conclude that vasa RNA‐positive cells are the PGCs and vasa RNA represents a definitive germ line marker in the fish. Dev Dyn 1999;216:153–167.

Evolutionary development of three gonadotropin-releasing hormone (GnRH) systems in vertebrates

Gonadotropin-releasing hormone (GnRH) is the neuropeptide that links the brain to the reproductive system. Most vertebrate species express two forms of GnRH, which differ in amino acid sequence, localization, distribution, and embryological origin. The GnRH system in the ventral forebrain produces a species-specific GnRH form and projects toward the gonadotropic cell in the pituitary. The GnRH neurons of this system originate from the olfactory placode and migrate into the brain during early development. The other GnRH system is localized in a nucleus in the midbrain, where large cells express chicken-GnRH-II, of which the function is still unclear. In modern teleosts, a third GnRH system is present in the terminal nerve, which contains salmon GnRH. The three GnRH systems appear at different times during fish evolution. Besides the two accepted lineages in GnRH evolution (of conserved chicken GnRH-II in the midbrain and of mammalian GnRH or species-specific GnRH in the hypophysiotropic system), we propose a third lineage: of salmon GnRH in the terminal nerve.

Sex steroids and the initiation of puberty in male African catfish (Clarias gariepinus).

The effects of sex steroids on spermatogenesis and testicular androgen secretion were studied in juvenile (spermatogonia present in testes) African catfish. Fish were implanted with Silastic pellets containing 11-ketotestosterone (KT), 11β-hydroxyandrostenedione (OHA), androstenetrione (OA), androstenedione (A), testosterone (T), 5α-dihydrotestosterone (DHT), or estradiol-17β (E2). Control groups received steroid-free pellets. Two weeks later, testis tissue fragments were incubated with African catfish luteinizing hormone (LH) and the amount of OHA secreted in vitro (the main androgen produced by African catfish testes) was quantified. Tissue fragments were then fixed for histological analysis of spermatogenesis. Treatment with KT, OHA, and OA stimulated testicular growth and spermatogenesis (spermatocytes and spermatids were found), whereas T, DHT, A, or E2 had no such effects. All steroids, except for DHT and E2, reduced OHA secretion in the absence and presence of LH to ∼10% of the control values. Previous studies have shown that KT, OHA, and OA have little effect on circulating LH levels in juvenile male African catfish, so that these androgens probably had direct effects on the testis. Inasmuch as OHA, OA, and KT have largely similar effects and because OHA and OA are converted to KT in vivo, we suggest that KT is physiologically the most relevant androgen for the initiation of spermatogenesis in African catfish.The effects of sex steroids on spermatogenesis and testicular androgen secretion were studied in juvenile (spermatogonia present in testes) African catfish. Fish were implanted with Silastic pellets containing 11-ketotestosterone (KT), 11beta-hydroxyandrostenedione (OHA), androstenetrione (OA), androstenedione (A), testosterone (T), 5alpha-dihydrotestosterone (DHT), or estradiol-17beta (E2). Control groups received steroid-free pellets. Two weeks later, testis tissue fragments were incubated with African catfish luteinizing hormone (LH) and the amount of OHA secreted in vitro (the main androgen produced by African catfish testes) was quantified. Tissue fragments were then fixed for histological analysis of spermatogenesis. Treatment with KT, OHA, and OA stimulated testicular growth and spermatogenesis (spermatocytes and spermatids were found), whereas T, DHT, A, or E2 had no such effects. All steroids, except for DHT and E2, reduced OHA secretion in the absence and presence of LH to approximately 10% of the control values. Previous studies have shown that KT, OHA, and OA have little effect on circulating LH levels in juvenile male African catfish, so that these androgens probably had direct effects on the testis. Inasmuch as OHA, OA, and KT have largely similar effects and because OHA and OA are converted to KT in vivo, we suggest that KT is physiologically the most relevant androgen for the initiation of spermatogenesis in African catfish.

The influence of androgen administration on the structure and function of the brain-pituitary-gonad axis of sexually immature platyfish, Xiphophorus maculatus.

SummaryThis report demonstrates that the administration of testosterone (T) or 11-ketotestosterone (11-KT) to sexually immature (8 wks old) male platyfish (Xiphophorus maculatus) of early-and late-maturing genotypes affects the synthesis and/or release of luteinizing hormone-releasing hormone (LHRH), as assessed by immunocytochemical evaluation, increases the number and activity of pituitary gonadotropes, stimulates the production of sperm and, thus, advances the age of sexual maturation over that dictated by the genome. We also show that 11-KT and T affect different LHRH-containing centers in the brain and have differential effects on rate and degree of sexual maturation, regardless of whether the hormones are administered to early or late-maturing genotypes.

Expression and distribution of two gonadotropin-releasing hormones in the catfish brain.

The expression of prepro-catfish GnRH mRNA and prepro-chicken GnRH-II mRNA was investigated by means of in situ hybridization. The differential distribution of cells expressing the respective mRNAs was compared with the distribution of cells immunoreactive for (1) catfish (cf) GnRH and chicken (c) GnRH-II and (2) both GnRH-associated peptides (GAPs). It was found that the prepro-cfGnRH mRNA expressing cells were located in the ventral forebrain, with a similar distribution of the cfGnRH- and cfGAP-immunoreactive perikarya. The prepro-cGnRH-II mRNA expressing cells were exclusively located in the midbrain tegmentum, at the same position as a group of large cGnRH-II- and CIIGAP-immunoreactive perikarya. It was concluded that the peptidergic neurons in the ventral forebrain contain cfGnRH, whereas cGnRH-II perikarya are restricted to the midbrain. The proximal pars distalis of the pituitary, containing the gonadotropin cells, is innervated by fibers immunoreactive for both cfGnRH and cfGAP and originating from the cfGnRH neurons in the ventral forebrain. We could, however, not detect fibers innervating the pituitary that were immunoreactive for cIIGAP.

Testosterone Inhibits 11-Ketotestosterone-Induced Spermatogenesis in African Catfish (Clarias gariepinus)

Abstract Male fish produce 11-ketotestosterone as a potent androgen in addition to testosterone. Previous experiments with juvenile African catfish (Clarias gariepinus) showed that 11-ketotestosterone, but not testosterone, stimulated spermatogenesis, whereas testosterone, but not 11-ketotestosterone, accelerated pituitary gonadotroph development. Here, we investigated the effects of combined treatment with these two types of androgens on pituitary gonadotroph and testis development. Immature fish were implanted for 2 wk with silastic pellets containing 11-ketotestosterone, testosterone, 5α-dihydrotestosterone, or estradiol-17β; cotreatment groups received 11-ketotestosterone in combination with one of the other steroids. Testicular weight and pituitary LH content were higher (two- and fivefold, respectively) in the end control than in the start control group, reflecting the beginning of normal pubertal development. Treatment with testosterone or estradiol-17β further increased the pituitary LH content four- to sixfold above the end control levels. This stimulatory effect on the pituitary LH content was not modulated by cotreatment with 11-ketotestosterone. However, the stimulatory effect of 11-ketotestosterone on testis growth and spermatogenesis was abolished by cotreatment with testosterone, but not by cotreatment with estradiol-17β or 5α-dihydrotestosterone. Also, normal pubertal testis development was inhibited by prolonged (4 wk) treatment with testosterone. The inhibitory effect of testosterone may involve feedback effects on pituitary FSH and/or on FSH receptors in the testis. It appears that the balanced production of two types of androgens, and the control of their biological activities, are critical to the regulation of pubertal development in male African catfish.

Corticosteroids Affect the Testicular Androgen Production in Male Common Carp (Cyprinus carpio L.)

Abstract Our previous experiments to study the effect of stress adaptation on pubertal development in carp showed that repeated temperature stress and prolonged feeding with cortisol-containing food pellets, which mimics the endocrine stress effects, retarded the first waves of spermatogenesis and decreased 11-ketotestosterone (11KT) plasma levels. The objective of the present study was to investigate whether the decrease in plasma 11KT is caused by a direct effect of cortisol on the steroid-producing capacity of the testis or by an indirect effect, such as a decrease in plasma LH. Pubertal and adolescent isogenic male common carp (Cyprinus carpio L.) were fed with either cortisol-containing food pellets or control food pellets over a prolonged period. Our results indicate that cortisol has a direct inhibitory effect on the testicular androgen secretion independent of the LH secretion. Furthermore, the pubertal period is critical to the influence of cortisol regarding testicular androgen secretion, because the effect is no longer observed at adolescence.

Two gonadotropin-releasing hormones from African catfish (Clarias gariepinus)

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the African catfish, Clarias gariepinus, using reverse-phase high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The amino acid sequences of both forms of African catfish GnRH were determined using Edman degradation after digestion with pyroglutamyl aminopeptidase. In addition, both GnRHs were studied by mass spectrometry. The primary structure of African catfish GnRH I is identical to Thai catfish GnRH I, pGlu-His-Trp-Ser-His-Gly-Leu-Asn-Pro-Gly-NH2, and the primary structure of African catfish GnRH II is identical to the widely distributed and highly conserved chicken GnRH II, pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.

A new member of the gonadotropin-releasing hormone family in teleosts: catfish gonadotropin-releasing hormone

Two forms of immunoreactive gonadotropin-releasing hormone (GnRH) were detected in extracts of brain-pituitary tissue from the African catfish, Clarias gariepinus. Catfish I GnRH eluted first from reverse-phase HPLC and was present in larger amounts compared with catfish II GnRH. Chromatographic and immunological studies with four antisera provide evidence that catfish I GnRH is unique compared with identified GnRHs from mammal, chicken, salmon, and lamprey. Catfish II GnRH elutes in the same position as chicken II GnRH and the forms cannot yet be distinguished. GnRHs extracted from female and male catfish tissue appear to be similar in terms of the number of peaks eluted, elution position, quantity, and cross-reactivity with the antisera. The results of the HPLC and radioimmunoassay studies suggest that catfish I GnRH is likely to be 10 amino acids in length, and have an amide at the C terminus similar to the other family members. In addition, catfish I GnRH is probably different in the 5 to 10 amino acid region compared with mammalian GnRH. Finally, catfish I GnRH is likely to have a lysine or arginine residue as it is the most hydrophilic family member. The lack of the salmon form of GnRH and the presence of a unique GnRH form constitute another example of the considerable evolutionary variation that has occurred in the catfish family compared with other teleosts.

Development of three distinct GnRH neuron populations expressing two different GnRH forms in the brain of the African catfish (Clarias gariepinus).

The early development of both the catfish gonadotropin‐releasing hormone (cfGnRH)‐ and the chicken GnRH‐II (cGnRH‐II) system was investigated in African catfish by immunocytochemistry by using antibodies against the GnRH‐associated peptide (GAP) of the respective preprohormones. Weakly cfGnRH‐immunoreactive (ir) neurons and fibers were present at 2 weeks after hatching (ph) but only in the ventral telencephalon and pituitary. Two weeks later, cfGnRH fibers and neurons were also observed in more rostral and in more caudal brain areas, mainly in the preoptic area and hypothalamus. Based on differences in temporal, spatial, and morphologic appearance, two distinct cfGnRH populations were identified in the ventral forebrain: a population innervating the pituitary (ventral forebrain system) and a so‐called terminal nerve (TN) population. DiI tracing studies revealed that the TN population has no neuronal connections with the pituitary. The cGnRH‐II system is present from 2 weeks ph onward in the midbrain tegmentum and only their size and staining intensity increased during development. Based on the comparison of GnRH systems amongst vertebrates, we hypothesize that during fish evolution, three different GnRH systems evolved, each expressing their own molecular form: the cGnRH‐II system in the midbrain, a hypophysiotropic GnRH system in the hypothalamus with a species‐specific GnRH form, and a salmon GnRH‐expressing TN population. This hypothesis is supported by phylogenetic analysis of known GnRH precursor amino acid sequences. We hypothesize, because the African catfish is a less advanced teleost species, that it contains the cfGnRH form both in the ventral forebrain system and in the TN population.J. Comp. Neurol. 437:308–320, 2001.