J. Peute

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.

Annual changes in plasma and liver in relation to vitellogenesis in the female rainbow trout, Salmo gairdneri

As part of a study on the physiological participation of ovarian steroids in the regulation of the synthesis of vitellogenin in the liver of the rainbow trout, Salmo gairdneri, an inventory was made of some relevant variables throughout the annual cycle. The annual reproductive cycle can be divided into four physiological periods: a previtellogenic period (March–April), a period of endogenous vitellogenesis (May–July), a period of exogenous vitellogenesis (August–December), and a period of ovulation and spawning (January–February). Quantitative studies have been carried out of the hepatosomatic and gonadosomatic indices, of the concentrations of total proteins, vitellogenin, free phosphates, and vitellogenin-bound phosphates in the plasma, and of the concentration of total proteins and vitellogenin in the liver. The plasma vitellogenin concentration increased from 0.1 mg/ml in May to 12.9 mg/ml in November, the vitellogenin-bound phosphates were hardly detectable in May, but had increased to 6.1 μmol/ml in December, the hepatosomatic index increased from 12 in March to 30 in December, and the gonadosomatic index increased from 6 in June to 222 in December. The free plasma phosphates varied considerably in the different specimens. The total plasma protein levels had a constant value of about 100 mg/ml throughout the annual cycle, except during previtellogenesis, when the levels had decreased to 60 mg/ml. The total liver protein levels per gram liver gradually decreased from 234 mg in July to 136 mg in January, and increased rapidly again in the following months. Vitellogenin of the type as detected in the plasma, was not found in the liver. During exogenous vitellogenesis, plasma vitellogenins with different degrees of phosphorylation appeared to be present. The ultrastructural examination of the liver indicated large amounts of rough endoplasmic reticulum and a large Golgi system from October to December, whereas glycogen and lipid droplets were found in June and July.

Cyclic changes in the ovary of the rainbow trout, Salmo gairdneri, with special reference to sites of steroidogenesis.

SummaryIn the maturation cycle of ovaries of cultured rainbow trout, three periods can be distinguished: (1) a period of ovulation and previtellogenesis (January–May), (2) a period of exogenous vitellogenesis (May–November/December), and (3) a period of maturation of oocytes (November/December–January). Enzyme cytochemical and electron microscopical data indicate that stroma cells (i.e., interstitial cells and special theca cells) and granulosa cells represent sources of steroids. Steroidogenesis in stroma cells is found throughout the annual cycle, reaching a peak activity in January and February. Weak steroidogenic activity is observed in the granulosa cells of exogenous vitellogenic follicles and young postovulatory follicles. Possible functions of steroids secreted by stroma cells and granulosa cells are discussed.

Morphological and enzyme cytochemical aspects of the testis and vas deferens of the rainbow trout, Salmo gairdneri

SummaryIn the maturation cycle of the testis of cultured rainbow trout, three periods can be distinguished: a period of spermatogonial proliferation (June–September), a period of maturation, spermiogenesis and spermiation (September–January), and a period of full spermiation (January–June). In the third period new primary spermatogonia are formed, and old sperm cells are resorbed. Sperm release occurs seldom, if ever in animals kept in captivity. The presence of 3β-HSD, 3α-HSD and G6PD activities, smooth endoplasmic reticulum and mitochondria with tubular cristae indicates steroidogenesis in Leydig cells. These parameters have been found during the annual cycle; the strongest steroidogenic activity is from January to June, when metabolic activities tend to shift to the pentose phosphate shunt. In June, most steroid synthesizing Leydig cells are abundant, at sites where seminiferous tubules contain primary and secondary spermatogonia. In November, a weak steroidogenic activity has also been demonstrated in Sertoli cells lining post-spermatogonial cysts. Other aggregations of steroid synthesizing cells are present outside the testis in the connective tissue surrounding the epithelium of vas deferens. The epithelium of the vas deferens shows positive reaction for 3α-HSD, G6PD, MD, LD and NADHD throughout the testicular cycle; 3α-HSD and G6PD activities are generally weak. Metabolic activity in this epithelium increases when sperm cells are stored in the lumen of the duct (November–June). Acid phosphatase in the epithelium of the vas deferens is more active during the period when sperm cells are resorbed.

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.

The brain-pituitary-gonadal axis in the rainbow trout, Salmo gairdneri: gonadal hormones and the maturation of gonadotropic cells.

SummaryIntact and castrated juvenile male rainbow trout (Salmo gairdneri) were treated with testosterone and gonadotropic hormone (GTH) to determine the maturational effects of these hormones on the GTH-cells. Electron-microscopic studies of the GTH-cells revealed that GTH and testosterone in intact animals, and testosterone in castrated fish, caused GTH-cell maturation: These cells now displayed the same appearance as GTH-cells in adult trout, including the presence of globules, a well-developed Golgi apparatus and rough endoplasmic reticulum, all of which were absent in GTH-cells of control animals. Animals with stimulated GTH-cells also had an increased GTH content of the pituitary; release of GTH could not be demonstrated. Animals treated with GTH exhibited an accelerated development of the testes, resulting in complete gametogenesis and elevated plasma testosterone levels. These results indicate that exogenous steroids as well as endogenous gonadal steroids can stimulate the full development of GTH-cells and accelerate GTH synthesis. The significance of this stimulating effect of the gonadal hormones with respect to the development of the brain-pituitary-gonadal axis and the onset of puberty is discussed.

Maturational gonadotropin from the African catfish, Clarias gariepinus : purification, characterization, localization, and biological activity

A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.

The seminal vesicle of the African catfish, Clarias gariepinus

SummaryThe seminal vesicle of the African catfish, Clarias gariepinus, consists of 36–44 fingerlike lobes built up of tubules in which a fluid is secreted containing acid polysaccharides, acid-, neutral- and basic proteins, and phospholipids. In this fluid sperm cells are stored. The seminal vesicle fluid immobilizes the sperm cells. After ejaculation, it prolongs the period of sperm activity. The seminal vesicle fluid is secreted by the epithelium lining the tubules. The tubules in the proximal part of the lobes are predominantly lined by a simple cylindrical and those of the distal part by a simple squamous epithelium. These epithelial cells contain enzymes involved in energy-liberating processes, the enzyme activites being proportional to the height of the cells. Interstitial cells between the tubules have enzyme-histochemical and ultrastructural features indicative of steroid biosynthesis. Similar characteristics are found in testicular interstitial cells. The most rostral seminal vesicle lobes and the most caudal testicular efferent tubules form a network of tubules that opens at the point where the paired parts of the sperm ducts fuse with each other. The tubules of most seminal vesicle lobes, however, form a complex system that fuses with the unpaired part of the sperm duct.

Ultrastructure and lipid content of the liver of the zebrafish, Brachydanio rerio, related to vitellogenin synthesis.

SummaryThe female zebrafish is capable of producing mature eggs on the fifth day of each reproductive cycle. During this five-day period the ultrastructure of hepatocytes undergoes several changes. The number of nuclear pores increases rapidly during spawning, followed by a proliferation of RER within 24 h. Two days after spawning, glycogen has disappeared and the liver contains large amounts of lipids. The lipid droplets are closely surrounded by elongated mitochondria. Golgi complexes are abundant, secreting dense bodies. Four days after spawning the hepatocytes tend to regain their pre-spawning appearance. It is suggested that the changes in the hepatocytes, which coincide with special phases of ovarian activity, are related to vitellogenin synthesis. Steroids, especially estradiol-17β, may trigger this process in the liver.

Glycoprotein gonadotropin in the plasma and its cellular origin in the adenohypophysis of sham-operated and ovariectomized rainbow trout, Salmo gairdneri.

SummaryAmong the cells of the pituitary generally believed to produce glycoprotein gonadotropin (GTH) five forms were distinguished, based on the amount and the diameter of granules and globules and the appearance of the rough endoplasmic reticulum. In sham-operated trout so-called “globular” cells predominated, whereas after ovariectomy these were replaced by so-called “cisternal” cells, suggesting that both belong to one GTH-cell type. In addition, ovariectomy caused a strong increase in plasma GTH-levels. This indicates that the transition from globular to cisternal cells is accompanied by extrusion of GTH, and thus points to a storage of GTH in the granules and globules. It is argued that one of the five forms has the morphological characteristics of thyrotropic cells and may not produce glycoprotein GTH.