Influence of sex and gonadal status of sheep on cortisol secretion in response to ACTH and on cortisol and LH secretion in response to stress: importance of different stressors

There are sex differences in the response to stress and in the influence of stress on reproduction which may be due to gonadal steroids but the nature of these differences and the role of the gonads are not understood. We tested the hypotheses that sex and the presence/absence of gonads (gonadal status) will influence the cortisol response to injection of ACTH, insulin-induced hypoglycaemia and isolation/restraint stress, and that sex and gonadal status will influence the secretion of LH in response to isolation/restraint stress. Four groups of sheep were used in each of three experiments: gonad-intact rams, gonadectomised rams, gonad-intact ewes in the mid-luteal phase of the oestrous cycle and gonadectomised ewes. In Experiment 1 (n=4/group), jugular blood samples were collected every 10 min for 6 h; after 3 h, two animals in each group were injected (i.v.) with ACTH and the remaining two animals were injected (i.v.) with saline. Treatments were reversed 5 days later so that every animal received both treatments. Experiment 2 (n=4/group) used a similar schedule except that insulin was injected (i.v.) instead of ACTH. In Experiment 3 (n=5/group), blood samples were collected every 10 min for 16 h on a control day and again 2 weeks later when, after 8 h of sampling, all sheep were isolated and restrained for 8 h. Plasma cortisol was significantly (P<0.05) elevated following injection of ACTH or insulin and during isolation/restraint stress. There were no significant differences between the sexes in the cortisol response to ACTH. Rams had a greater (P<0.05) cortisol response to insulin-induced hypoglycaemia than ewes while ewes had a greater (P<0.05) cortisol response to isolation/restraint stress than rams. There was no effect of gonadal status on these parameters. Plasma LH was suppressed (P<0.05) in gonadectomised animals during isolation/restraint stress but was not affected in gonad-intact animals, and there were no differences between the sexes. Our results show that the sex that has the greater cortisol response to a stressor depends on the stressor imposed and that these sex differences are likely to be at the level of the hypothalamo-pituitary unit rather than at the adrenal gland. Since there was a sex difference in the cortisol response to isolation/restraint, the lack of a sex difference in the response of LH to this stress suggests that glucocorticoids are unlikely to be a major mediator of the stress-induced suppression of LH secretion.

Peripheral insulin resistance develops in transgenic rats overexpressing phosphoenolpyruvate carboxykinase in the kidney

Aims/hypothesis: To study the secondary consequences of impaired suppression of endogenous glucose production (EGP) we have created a transgenic rat overexpressing the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in the kidney. The aim of this study was to determine whether peripheral insulin resistance develops in these transgenic rats.
Methods: Whole body rate of glucose disappearance (Rd) and endogenous glucose production were measured basally and during a euglycaemic/hyperinsulinaemic clamp in phosphoenolpyruvate carboxykinase transgenic and control rats using [6-3H]-glucose. Glucose uptake into individual tissues was measured in vivo using 2-[1-14C]-deoxyglucose.
Results: Phosphoenolpyruvate carboxykinase transgenic rats were heavier and had increased gonadal and infrarenal fat pad weights. Under basal conditions, endogenous glucose production was similar in phosphoenolpyruvate carboxykinase transgenic and control rats (37.4±1.1 vs 34.6±2.6 µmol/kg/min). Moderate hyperinsulinaemia (810 pmol/l) completely suppressed EGP in control rats (–0.6±5.5 µmol/kg/min, p<0.05) while there was no suppression in phosphoenolpyruvate carboxykinase rats (45.2±7.9 µmol/kg/min). Basal Rd was comparable between PEPCK transgenic and control rats (37.4±1.1 vs 34.6±2.6 µmol/kg/min) but under insulin-stimulated conditions the increase in Rd was greater in control compared to phosphoenolpyruvate carboxykinase transgenic rats indicative of insulin resistance (73.4±11.2 vs 112.0±8.0 µmol/kg/min, p<0.05). Basal glucose uptake was reduced in white and brown adipose tissue, heart and soleus while insulin-stimulated transport was reduced in white and brown adipose tissue, white quadriceps, white gastrocnemius and soleus in phosphoenolpyruvate carboxykinase transgenic compared to control rats. The impairment in both white and brown adipose tissue glucose uptake in phosphoenolpyruvate carboxykinase transgenic rats was associated with a decrease in GLUT4 protein content. In contrast, muscle GLUT4 protein, triglyceride and long-chain acylCoA levels were comparable between PEPCK transgenic and control rats.
Conclusions/interpretation: A primary defect in suppression of EGP caused adipose tissue and muscle insulin resistance.

Co-localization and distribution of corticotrophin-releasing hormone, arginine vasopressin and enkephalin in the paraventricular nucleus of sheep : a sex comparison

The paraventricular nucleus (PVN) is integral to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and contains cells producing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin. We used immunohistochemistry to map these peptides and to resolve the extent of co-localization within PVN cells in intact and gonadectomized male and female sheep. Immunoreactive (ir) CRH, AVP and enkephalin cells were mapped in two rams and two ewes at 180 μm intervals throughout the rostro-caudal extent of the PVN. Similar distributions of AVP-ir cells occurred in both sexes whereas CRH-ir and enkephalin-ir cells extended more rostrally in rams. In groups (n=4) of intact and gonadectomized sheep of both sexes, co-localization and distribution of neuropeptides was influenced by sex and gonadectomy. Males had more AVP and CRH cells than females. Intact animals had more AVP cells than gonadectomized animals. There were no differences between groups in the number or percentage of cells that stained for both CRH and AVP or in the number of cells that stained for both CRH and enkephalin. Differences were observed in the percentage of enkephalin cells that contained CRH with males having a greater percentage of co-localized cells than did females. Differences were also observed in the number and percentage of cells that stained for both enkephalin and AVP; the number of cells that stained for both neuropeptides was greater in males than in females and greater in intact animals than in gonadectomized animals. Differences were observed in the percentage of AVP cells that contained enkephalin, and in the percentage of enkephalin cells that contained AVP with males having a greater percentage of co-localized cells than did females. We conclude that sex and gonadal status affect peptide distribution in the PVN of the sheep which may provide an anatomical basis for sex differences in HPA axis

Control of ovarian development in the yabby (Cherax destructor)

A study under controlled conditions of ovarian development and rematuration in the yabby (Cherax destructot) was undertaken. The purpose of the study was to improve fundamental understanding of the reproductive biology of the species and provide a basis for application to hatchery management in culture. A review was made of the current status of yabby culture in Australia and the present understanding of reproductive biology of decapod Crustacea. The review emphasised factors controlling several aspects of ovarian development, in particular the processes of vitellogenesis. The subsequent study was designed within the context of current hatchery practice and was based on existing knowledge of decapod reproduction, The sexual differentiation of the yabby after hatching was investigated by serial histological sections, and experiments were carried out to investigate the possibility of sex reversal of males. Most of this Investigation was concerned with removing the influence of the androgenic gland in directing male development, with the intent of observing the development of the elementary gonadal tissue into ovary. It was found that in contrast to other crustacean species, the sex of the yabby becomes fixed before the development of external secondary sexual characteristics, and before the androgenic gland can be discerned. Ovarian tissue developed in females at less than 8 weeks after hatching. A preliminary examination was undertaken for feminising parasites in gonadal tissue of a hermaphrodite yabby. Investigation of the ovary after spawning demonstrated that whilst the female was held under constant conditions of temperature and photoperiod, little rematuration occurred. Except for generation of previtellogenic oocytes during the first two days, the gonaciosomatic index remained low for up to 5 months after spawning. If the temperature of the female was reduced to 10°C and maintained constant, the previtellogenic oocytes were partially resorbed over a three week period. Rematuration then commenced, albeit at a low rate because of the reduced temperature, A method for standardising gonadosomatic indices was developed which took into account differences in hepatopancreatic nutrient reserves of individuals and loss of one or more appendages. This part of the study also considered constraints to rematuration and developed a method of accounting for differences in the ability of females to remature after spawning. Experiments were carried out to investigate the effect of crowding and temperature manipulation on initiating ovarian rematuration and to determine the rate of rematuration at 22°C once initiated. The duration of low temperature had no effect on rematuration; an overnight cooling was sufficient to initiate the process, Rematuration to the end of stage 2 vltellogenesis was substantially complete within 10 days. Crowding of females suppressed rematuration, but less than ideal water quality was not found to have any effect. The presence of a male initiated rematuration at a similar rate, but also led to stage 3 vitetlogenesis and spawning. A study was made of the pheromonal influence of the male through water borne factors without success. Rematuration could not be induced in ovigerous females. The literature review indicated that ovarian rematuration was under the control of an ovary stimulating hormone produced by the thoracic nerve ganglia. Attempts were therefore made to stimulate ovarian rematuration by incorporating the thoracic nerve into the diet of females. Attempts were also made to induce the release of ovary stimulating hormone from the thoracic nerve with 5-hydroxytryptamine, and also with octopamine. No effects were found, but a significant difference between the neurophysiology of the yabby and northern hemisphere crayfish was observed, and the implications of this finding are discussed. The study did not produce any conclusive evidence of an ovary stimulating hormone for the yabby. A model of ovarian rematuration which collects the findings of the experimental investigations was developed, and was used to suggest a hatchery broodstock management protocol. This model differs from existing models in that rematuration triggers and nutritional status are considered.

Validation of the aceto-carmine technique for evaluating phenotypic sex in Nile tilapia (Oreochromis niloticus) fry

In this study a method for evaluating phenotypic sex in Nile tilapia was validated. A technique that uses aceto-carmine squash mounts to stain the entire gonadal tissue for microscopic examination (Method 2- squash mounts) was compared with a technique based on traditional histology. Approximately 2600 Nile tilapia fry weighing and measuring, respectively, between 0.25-2.50g and 26-53mm, aged 35 to 60 days after hatch (DAH), were sexed using this methodology. In situ microscopic examination on the gonads was also performed. A reliable sexing using squash mount was possible with fish weighing more than 0.500g, 45-47 DAH. Results from microscopic observation using the aceto-carmine stain coincided 100% with the histological examination. Male gonadal tissue was characterized by the presence of cysts containing spermatogonia and spermatocytes, while females were easily identified by the presence of oocytes at the perinucleolar stage. The technique proved to be efficient not only in terms of evaluating sex proportion in fish but also because it allows immediate evaluation of gonadal sex and demands less time and labour.

Dual-axis hormonal covariation in adolescence and the moderating influence of prior trauma and aversive maternal parenting

Adversity early in life can disrupt the functioning of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes and increase risk for negative health outcomes. The interplay between these axes and the environment is complex, and understanding needs to be advanced by the investigation of the multiple hormonal relationships underlying these processes. The current study examined basal hormonal associations between morning levels of cortisol, testosterone, and dehydroepiandrosterone in a cohort of adolescents (mean age 15.56 years). The moderating influence of childhood adversity was also examined, as indexed by self-reported trauma (at mean age 14.91), and observed maternal aggressive parenting (at mean age 12.41). Between-person regressions revealed significant associations between hormones that were moderated by both measures of adversity. In females, all hormones positively covaried, but also interacted with adversity, such that positive covariation was typically only present when levels of trauma and/or aggressive parenting were low. In males, hormonal associations and interactions were less evident; however, interactions were detected for cortisol-testosterone - positively covarying at high levels of aggressive parenting but negatively covarying at low levels - and DHEA-cortisol - similarly positively covarying at high levels of parental aggression. These results demonstrate associations between adrenal and gonadal hormones and the moderating role of adversity, which is likely driven by feedback mechanisms, or cross-talk, between the axes. These findings suggest that hormonal changes may be the pathway through which early life adversity alters physiology and increases health risks, but does so differentially in the sexes; however further study is necessary to establish causation.

Vasodilatory effects of homologous adrenomedullin 2 and adrenomedullin 5 on isolated blood vessels of two species of eel

In mammals, adrenomedullin (AM) is a potent vasodilator through signalling pathways that involve the endothelium. In teleost fishes, a family of five AMs are present (AM1/4, AM2/3 and AM5) with four homologous AMs (AM1, AM2/3 and AM5) recently cloned from the Japanese eel, Anguilla japonica. Both AM2 and AM5 have been shown to be strong in vivo vasodepressors in eel, but the mechanism of action of homologous AMs on isolated blood vessels has not been examined in teleost fish. In this study, both eel AM2 and AM5 caused a marked vasodilation of the dorsal aorta. However, only AM5 consistently dilated the small gonadal artery in contrast to AM2 that had no effect in most preparations. Neither AM2 nor AM5 had any effect when applied to the first afferent branchial artery; in contrast, eel ANP always caused a large vasodilation of the branchial artery. In the dorsal aorta, indomethacin significantly reduced the AM2 vasodilation, but had no effect on the AM5 vasodilation. In contrast, removal of the endothelium significantly enhanced the AM5 vasodilation only. In the gonadal artery, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) significantly reduced the AM5 vasodilation suggesting a role for soluble guanylyl cyclase in the dilation, but l-NNA and removal of the endothelium had no effect. The results of this study indicate that AM2 and AM5 have distinct vasodilatory effects that may be due to the peptides signalling via different receptors to regulate vascular tone in eel.

Stress and reproduction: central mechanisms and sex differences in non-rodent species

Despite extensive research, the mechanisms by which stress affects reproduction are unknown. Activation of stress systems could potentially influence reproduction at any level of the hypothalamo-pituitary gonadal axis. Nonetheless, the predominant impact is on the secretion of gonadotrophin releasing hormone (GnRH) from the brain and the secretion of the gonadotrophins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), from the gonadotrophs of the anterior pituitary gland. When stress is prolonged, it is likely that secretion of the gonadotrophins will be suppressed but the effects of acute stress or repeated acute stress are not clear. Different stressors activate different pathways for varying durations, and the actions of stress vary with sex and are influenced by the predominance of particular sex steroids in the circulation. The mechanisms by which stress influences reproduction are likely to involve complex interactions between a number of central and peripheral pathways and may be different in males and females. To understand these mechanisms, it is important to determine the stress pathways that are activated by particular stressors and to establish how these pathways affect the secretion and actions of GnRH. Furthermore, there is a need to know how stress influences the feedback actions of gonadal steroids and inhibin.