The influence of the opioid pentapeptide methionine-enkephalin on seasonal and FSH-induced ovarian recrudescence in the gecko Hemidactylus frenatus.

Although methionine-enkephalin (M-ENK) is implicated in the regulation of reproductive functions in vertebrates, its function in reptiles is little understood. This study aims to elucidate the role of M-ENK on seasonal and follicle stimulating hormone (FSH)-induced ovarian recrudescence in the gecko Hemidactylus frenatus. In the first experiment, administration of 5 µg M-ENK did not affect germinal bed activity or follicular developmental stages I, II, and III (previtellogenic) and IV (vitellogenic), but there were no stage V (vitellogenic) follicles in the ovary. However, there was a significant decrease in the mean numbers of oogonia and primary oocytes in the germinal bed associated with the complete absence of stage IV and V follicles in 25 µg M-ENK-treated lizards in contrast to experimental controls. Furthermore, there was a significant decrease in gonadotropin-releasing hormone - immunoreactive (GnRH-ir) content in the median eminence (ME) and pars distalis (PD) of the pituitary gland and sparse labelling of hypothalamic GnRH-ir neurons in 25 µg M-ENK-treated lizards. In the second experiment, treatment with FSH during the regression phase of the ovarian cycle resulted in the appearance of stage IV and V follicles, in contrast to their absence in the initial controls and treatment controls. However, treatment with 25 µg M-ENK + FSH did not result in the appearance of these follicles, indicating the inhibitory effect of M-ENK on FSH-induced ovarian recrudescence. These findings suggest that M-ENK inhibits the germinal bed and vitellogenic follicular growth in a dose-dependent manner, possibly mediated through the suppression of GnRH release in the ME and PD. In addition, M-ENK may also act at the level of the ovary in the gecko.

The opioid peptide leucine-enkephalin disrupts seasonal and gonadotropin-induced ovarian recrudescence in the gecko Hemidactylus frenatus.

The enkephalins are known to regulate many physiological functions, including reproduction in vertebrates. However, the role of leucine-enkephalin (L-ENK) in the ovarian recrudescence activity of reptiles is not known. In the present study, we studied the influence of L-ENK on seasonal and FSH-induced ovarian recrudescence during the breeding and non-breeding phases of the cycle in the tropical and subtropical gecko Hemidactylus frenatus. In the first experiment, treatment with 5 and 25 µg L-ENK resulted in a dose-dependent inhibitory effect on the hypothalamic gonadotropin-releasing hormone (GnRH) neurons and ovary, as indicated by a significantly decreased percent area of GnRH-immunoreactive (GnRH-ir) fibres in the median eminence and pars distalis of the pituitary gland, concomitant with complete absence of stage V (late vitellogenic) follicles in the ovary compared to those of experimental controls. In the second experiment, administration of FSH to lizards in the regression phase stimulated the recruitment of stage IV and V (vitellogenic) follicles in contrast to their absence in initial controls or treatment controls. However, similar treatment of FSH in combination with 25 µg L-ENK did not result in the development of stage IV or V follicles. Together, these results suggest for the first time that treatment with 5 and 25 µg L-ENK exerts a dose-dependent inhibitory effect on the hypothalamic GnRH release into the median eminence and pituitary gland, leading to the blockade of ovarian recrudescence. These results also suggest a possible direct inhibitory effect of L-ENK at the level of the ovary in the gecko.

Organization of the galaninergic neuronal system in the brain of the gecko Hemidactylus frenatus.

Galanin (GAL) is a 29 amino acid peptide present in the central nervous system (CNS) as well as peripheral tissues in vertebrates. However, the brain distribution pattern of GAL is understudied in reptiles. The aim of this study was to determine the organization of galaninergic neuronal system in the brain of the gecko Hemidactylus frenatus, a tropical and sub-tropical lizard, using rabbit anti-galanin antibody. In the telencephalon, GAL-ir perikarya and fibres were found in the lateral septal nucleus, but only GAL-ir fibres were observed in the striatum, nucleus accumbens, anterior commissure, nucleus centralis amygdalae, dorsal and medial septal nuclei, nucleus of the diagonal band of Broca and in the optic chiasma. In the preoptic region, a cluster of GAL-ir cells and fibres was observed in the periventricular preoptic area and lateral preoptic area. GAL-ir perikarya and fibres were observed in hypothalamic areas such as the supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, periventricular nucleus of the hypothalamus, infundibular recess nucleus and in the median eminence, whereas GAL-ir fibres were present in the pars distalis of the pituitary gland. In the thalamus, GAL-ir fibres were observed in the dorsomedial, dorsolateral, and medial thalamic nuclei. GAL-ir fibres were also detected in mesencephalic areas such as the optic tectum, torus semicircularis, ventral tegmental area and substantia nigra, brain stem as well as the spinal cord. The organization of GAL-ir cells and fibres throughout the gecko brain suggests several neuroendocrine, neuromodulatory and behavioural functions for GAL in lizards. The study provides new insights into the evolutionarily conserved nature of GAL peptide in squamate reptiles and forms a valuable basis for future comparative studies.

Melanin-concentrating hormone interferes with the hypothalamic-pituitary-gonad axis in the Mozambique tilapia.

This study was conducted to elucidate the influence of melanin-concentrating hormone (MCH) along the reproductive-axis in the female tilapia Oreochromis mossambicus. Administration of MCH (4 µg / 0.1 ml saline) for 22 days resulted in significantly lower gonadosomatic index compared to controls. Significant reduction in the mean numbers of follicles at different stages of development such as previtellogenic (stages I-III), vitellogenic (stage IV) and preovulatory (stage V) follicles was observed in MCH-treated fish compared with controls. On the other hand, the rate of atresia was significantly higher in follicles at stages II, III and IV in MCH-treated fish. In addition, in the pituitary gland, sparsely labelled gonadotropin releasing hormone (GnRH)-immunoreactive fibres were observed in MCH-treated fish in contrast to their intense labelling in controls. The serum level of luteinizing hormone (LH) showed significant decrease, but the serum cortisol level rose significantly following MCH treatment compared to those of controls. Collectively, these results indicate for the first time, that MCH treatment blocks follicular development during the ovarian cycle, possibly through the suppression of GnRH-LH axis in fish. The results also indicate that MCH may activate the stress-axis pathway in fish.

The effect of α-MSH treatment on the hypothalamic-pituitary-gonad axis in the cichlid fish Oreochromis mossambicus.

In this investigation, we examined the influence of alpha-melanocyte stimulating hormone (α-MSH), a proopiomelanocortin-derived peptide, along the hypothalamic-pituitary-gonad axis in a cichlid fish Oreochromis mossambicus. Administration of α-MSH (40 µg/0.1 ml saline) for 22 days did not affect the number of stage I (previtellogenic) follicles but caused significant reduction in the mean numbers of previtellogenic (stages II and III), vitellogenic (stage IV) and preovulatory (stage V) follicles compared to those of controls. While the gonadosomatic index was significantly lower, the rate of follicular atresia in stages II, III and IV remained significantly higher in α-MSH-treated fish compared to the controls. Furthermore, the mean percent area of gonadotropin-releasing hormone-immunoreactive (GnRH-ir) fibres and luteinizing hormone-immunoreactive (LH-ir) cells were significantly reduced in the proximal pars distalis  of the pituitary gland in α-MSH-treated fish compared with the controls. Together, our findings suggest for the first time that the treatment of α-MSH blocks the follicular developmental process during the ovarian cycle, possibly through the inhibition of GnRH-LH pathway in teleosts.

Domperidone treatment attenuates stress-induced suppression of reproduction in viviparous mosquitofish Gambusia affinis.

The aim of this study was to determine the effect of stress on reproduction and the possible involvement of dopaminergic systems in the reproductive stress response in the mosquitofish Gambusia affinis. Exposure of fish to aquaculture stressors (four 10 min episodes of stress, each corresponding to a different stressor such as handling, chasing, frequent netting and low water levels), for a period of 30 days caused reduction in the mean numbers of stage I-IV follicles associated with lower number of pregnant females and embryos in most of the developmental stages compared with experimental controls. Besides, increase in the intensity of labelling and the per cent area of tyrosine hydroxylase (TH; a rate-limiting enzyme in the biosynthetic pathway of catecholamines)- immunoreactive (ir) neurons was observed in the preoptic area (POA) and the nucleus preopticus (NPO) regions of the brain concomitant with reduction in the labelling of gonadotropin releasing hormone-immunoreactive (GnRH-ir) fibres in the proximal pars distalis (PPD) of the pituitary gland in stressed fish compared with experimental controls. Treatment of domperidone (DOM) caused an increase in the number of stage II and V follicles and promoted pregnancy rate concomitant with an increase in the number of embryos at various developmental stages compared with those of experimental controls. Similar treatment to stressed fish caused an increase in the number of stages I-V follicles compared with those in stress alone group. The GnRH fibres showed increased immunolabelling in stress + DOM treated fish compared with stress alone fish. On the other hand, TH-immunoreactivity in the POA and the NPO regions was reduced in stress + DOM treated fish compared with stress-alone group. These results suggest that stress inhibits follicular development and subsequent hatching success through the suppression of GnRH and that the inhibition appears to be mediated through dopamine, for the first time in a viviparous fish.

ß-endorphin-induced inhibition of vitellogenic follicular growth in the fish Oreochromis mossambicus: evidence for opioidergic mediation of ovarian stress response.

The opioid peptide ß-endorphin (ß-EP) is known to play modulatory role in vertebrate reproduction. The objective of the present study was to determine the influence of ß-EP and the opioid receptor antagonist naltrexone (NALT) on follicular development in normal, and stressed female tilapia Oreochromis mossambicus. Administration of 4 µg ß-EP, but not 0.5 or 1.5 µg ß-EP daily for 22 days caused a significant reduction in the mean GSI, HSI, and number of Stage IV (vitellogenic) follicles compared to experimental controls during the prespawning phase of the ovarian cycle. Furthermore, the Stage V (vitellogenic) follicles were completely absent during the prespawning phase concomitant with the significantly lower serum levels of estradiol and cortisol in 4 µg ß-EP treated fish compared to experimental controls. On the other hand, exposure of the fish to mild acute stressors for 22 days caused changes in the ovary similar to that of high dose of ß-EP, whereas administration of NALT attenuated these effects. The results reveal that ß-EP exerts inhibitory effect on the vitellogenic follicular growth through the suppression of ovarian steroidogenesis. The study provides evidence for the opioidergic mediation of the ovarian stress response for the first time in fish.

Stress inhibits seasonal and FSH-induced ovarian recrudescence in the lizard,Mabuya carinata.

Stressors (handling, chasing, and noise) applied randomly five times per day for one month to lizards during the recrudescence phase of the ovarian cycle caused a significant reduction in mean number of oocytes and primordial follicles when compared to those of controls. Further, vitellogenic follicles were absent in the ovary of lizards subjected to stressors. Administration of bovine FSH during post-breeding regression phase of the ovarian cycle induced ovarian recrudescence as shown by significant increases in the mean number of oogonia, oocytes, and primordial follicles compared to controls, as well as vitellogenic growth of follicles. However, lizards treated with FSH and exposed to stressors did not exhibit ovarian recrudescence. Furthermore, FSH administration during the post-breeding regression phase caused a significant increase in serum levels of estradiol compared to controls, which was accompanied by significant increases in the relative weight of the liver and oviduct, as well as vitellogenic growth of follicles. Despite administration of FSH to lizards subjected to stressors, there was neither any increase in serum levels of estradiol and weight of the liver nor vitellogenic growth of follicles. The results indicate that repeated application of stressors inhibits vitellogenic growth of follicles by suppression of steroidogenic activity in M. carinata. This is the first report revealing that the ovary does not respond to gonadotrophin treatment under stressful conditions in reptiles.