Active immunization against gonadotrophin-releasing hormone in Chinese male pigs: effects of dose on antibody titer, hormone levels and sexual development

The objective of this study was to determine the optimal dose of a GnRH vaccine for immunocastration of Chinese male pigs, based on immune, endocrine and testicular responses. Forty-two crossbred (Chinese Yanan x Large White) male pigs were randomly assigned to one of the five treatments as follows: (I) 0 microg (control, n=8); (II) 10 microg (n=8); (III) 62.5 microg (n=8); (IV) 125 microg (n=8); (V) 250 microg (n=10), D-Lys6-GnRH tandem dimer (TDK) peptide equivalent of conjugate (TDK-OVA), using Specol as the adjuvant. Pigs were immunized at 13 and 21 weeks of age and were slaughtered at 31 weeks of age. Blood samples for antibody titer and hormone assays were collected at 13, 21, 24 and 31 weeks of age. At these time-points, testis size was also measured. At slaughter, testis weight was recorded and fat samples were collected for androstenone assay. Four animals, one out of each immunized group, responded poorly to the immunization (non-responders). At slaughter, serum testosterone and LH levels, fat androstenone levels and testis size/weight of these non-responders were similar to those in control animals. Antibody titers of non-responders were substantially lower (P<0.05) than in other immunized pigs. For the animals that responded well to the immunization (immunocastrated pigs), serum testosterone and LH levels, fat androstenone levels and testis size or weight were reduced (P<0.05) as compared to either controls or non-responders, at all doses tested. There was a significant effect of dose of TDK-OVA on antibody titers. The overall mean antibody titers in the 62.5 or 125 microg dose group (53.6 and 50.5% binding, respectively) were significantly higher than in the 10 or 250 microg group (39.2 and 40.24% binding, respectively). At slaughter, there was a significant dose effect on testis size or weight and on serum testosterone levels, but there was no dose effect on serum LH levels and fat androstenone levels. Testis size or weight in the 10 microg group was reduced to a lesser extent (P<0.05) than in the three higher dose groups. At slaughter, in comparison to controls, mean testis size of immunocastrated pigs in treatments II-V was reduced to 55, 21, 33 and 25%, respectively, whereas testis weight was reduced to 39, 12, 18 and 14%, respectively. Reduction of testis size and/or weight is important for visual assessment of castration at the slaughterline, therefore, it is concluded that a dose of 10 microg peptide is not suitable. We conclude that, within the dose-range studied, the 62.5 microg dose is optimal for future GnRH immunization studies or future practical use in immunocastration of Chinese male pigs.     

Evolutionary aspects of gonadotropin-releasing hormone and its receptor

Summary 1. Gonadotropin-releasing hormone (GnRH) was originally isolated as a hypothalamic peptide hormone that regulates the reproductive system by stimulating the release of gonadotropins from the anterior pituitary. However, during evolution the peptide was subject to gene duplication and structural changes, and multiple molecular forms have evolved.2. Eight variants of GnRH are known, and at least two different forms are expressed in species from all vertebrate classes: chicken GnRH II and a second, unique, GnRH isoform.3. The peptide has been recruited during evolution for diverse regulatory functions: as a neurotransmitter in the central and sympathetic nervous systems, as a paracrine regulator in the gonads and placenta, and as an autocrine regulator in tumor cells.4. Evidence suggests that in most species the early-evolved and highly conserved chicken GnRH II has a neurotransmitter function, while the second form, which varies across classes, has a physiologic role in regulating gonadotropin release.5. We review here evolutionary aspects of the family of GnRH peptides and their receptors.     

Monoamine- and peptide-containing elements in the nemertine digestive tract

Detailed comparative-histological data on localization and morphological peculiarities of catecholamine-, serotonin-, neurotensin-and FMRFamide-containing elements in the nemertine digestive system are presented for the first time. Using fluorescent histochemistry and immunohistochemistry, pharynx, esophagus, and midgut were studied in five species from three genera of the White Sea nemertines. Described in various nemertine species are intra-and subepithelial cells of the open and closed type, containing biologically active substances. Their processes are distributed in basi-and subepithelial nerve plexuses and can make contact with lumen of the digestive tract. Species-specificity in localization of the cells containing certain biologically active substances is noticed both along the length of the tract and with respect to its epithelial layer. Peculiarities and common regularities in distribution are discussed, as well as a possible function of monoamine-and peptide-containing elements in the digestive tract of the studied nemertines and other invertebrates.     

Neurochemistry of the gustatory subgemmal plexus

Nerve fibers present in the basal plexus of the vallate papilla of the rat tongue were analyzed using cytochemical, immunocytochemical and ultrastructural methods to investigate whether the subgemmal plexus is subdivided into neurochemical compartments and to provide a clear definition of the reciprocal spatial relationships between nitrergic, peptidergic and acetylesterase positive structures. Several neuronal fibers were detected under the chemoreceptorial epithelium. Some of these fibers were in contact with the taste buds and in some cases neuronal projections were also present between the buds or inside them; some others fibers were present below this layer but in a more peripheral area. Antibodies against CGRP, SP and CCK stained fibers just below the chemoreceptorial epithelium, whereas fibers more distally located were immunolabeled by anti VIP, NOS-1 and NF-200 antibodies. Some double staining experiments were conducted using confocal microscopy. Other sections were processed cytochemically for AChE and subsequently for NADPH-d in colocalization experiments. All the data obtained using these techniques confirmed the results obtained with single immunostaining, as did the ultrastructural results. In conclusion, the present work demonstrates that the subgemmal plexus is a bilayered structure, suggesting that the complex relationship between the two layers plays a pivotal role in taste and in the control of processes ancillary to taste, such as control of vascular or secretory mechanisms.     

Estrus induction in white rhinoceros (Ceratotherium simum)

The estrous cycle length in the white rhinoceros (Ceratotherium simum) is either 4 or 10 wk. The cause(s) for this variation as well as the poor fertility rate in captivity remains under debate in this species. Most captive adult white rhinoceros undergo long anovulatory periods without luteal activity which are considered a major reason for their low reproductive rate. In this study, the synthetic progestin chlormadinone acetate (CMA) was tested in combination with hCG or the GnRH analogue deslorelin for its efficiency to induce ovulation in fourteen females without luteal activity and in three, regular cycling females. HCG (N = 12), injectable GnRH analogue (N = 8) and GnRH analogue implants (N = 15) were given to induce ovulation after CMA treatment. Treatment success was determined using both transrectal ultrasonography and progesterone metabolite EIA analysis. A preovulatory sized follicle (3.5 ± 0.1 cm) or a corpus luteum (5.1 ± 0.7) was present on the ovary one day after induction in 93.1% of the treatments. Despite this high rate of ovarian response, ovulation rate differed between the study groups. The ovulation rate for hCG, injectable GnRH analogue and GnRH analogue implants was 66.7%, 62.5% and 93.3%, respectively. Ovulation rate in cyclic females treated with GnRH implants was 100% (6/6) compared with 89% (8/9) in females without luteal activity receiving the same treatment. The length of the estrous cycle when induced with hCG was 4 wk (85.7%). The estrous cycle when induced with GnRH analogue was predominantly 10 wk long. Two females without luteal activity treated with GnRH became pregnant. In conclusion, CMA in combination with GnRH analogue implants was highly effective to induce ovulation in white rhinoceroses and thus can contribute to efforts aimed at increasing natural mating and reproductive rates in the captive white rhinoceros population.     

Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

Organization of the orthogon – main and minor nerve cords

The central nervous system of flatworms has been regarded as comprised of the bilobed brain, the longitudinal cords and the connecting transverse commissures forming a so called orthogon. The peripheral nervous system comprises the submuscular and subepidermal plexuses. As a confusion in the terminology of the longitudinal nerve cords has prevailed, two concepts have been introduced, the main nerve cords (MCs) and the minor cords. The MCs have been defined as the pair of longitudinal nerve cords that (1) start with strongest roots in the brain, (2) consist of wide fibre bundles and (3) are associated with more neurons (particularly amninergic marker neurons) than the other cords. Longitudinal nerve cords in other positions are thinner and have less pronounced contact with the brain. They have collectively been named minor cords. Support for the special status of the MCs has been obtained from studies of the neuroanatomy of Catenulida, Macrostomida, Proseriata, Tricladida and Lecithoepitheliata and of parasitic flatworms. Using the above mentioned criteria for the MCs and the results of recent studies, we present the following hypothese: The MCs in all flatworms correspond to each other and have a common origin. This revised version was published online in July 2006 with corrections to the Cover Date.     

LH responses to single doses of exogenous GnRH in the Cape mole rat (Georychus capensis): the pituitary potential for opportunistic breeding

The Cape mole rat Georychus capensis is a solitary mole rat that inhabits the winter rainfall region of the Western Cape Province of South Africa. Circulating basal concentrations of luteinizing hormone (LH) were found to be significantly higher in the breeding season in both sexes. During both the breeding and non-breeding season, administration of exogenous gonadotropin-releasing hormone (GnRH) increased circulatory LH levels. The magnitude of the LH response to an overdose of exogenous GnRH both in and out of the breeding season in males and females was not significantly different. Typically, seasonally breeding species exhibit a down-regulation of the pituitary and reproductive functions out of the breeding season. It appears that there is no down-regulation of GnRH receptors at the level of the pituitary out of the breeding season, because the pituitary responds to an exogenous GnRH challenge equally both in and out of the breeding period. The Cape mole rat exhibits the potential for opportunistic breeding out of the breeding period, provided that environmental factors are favourable. This finding questions whether this mole rat is actually a seasonal breeder or whether reproduction is hindered by the ecological constraint of the lack of opportunities to burrow and find mates at certain times of the year.