Distribution of GnRH and Kisspeptin Immunoreactivity in the Female Llama Hypothalamus

Llamas are induced non-reflex ovulators, which ovulate in response to the hormonal stimulus of the male protein beta-nerve growth factor (β-NGF) that is present in the seminal plasma; this response is dependent on the preovulatory gonadotrophin-releasing hormone (GnRH) release from the hypothalamus. GnRH neurones are vital for reproduction, as these provide the input that controls the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. However, in spontaneous ovulators, the activity of GnRH cells is regulated by kisspeptin neurones that relay the oestrogen signal arising from the periphery. Here, we investigated the organisation of GnRH and kisspeptin systems in the hypothalamus of receptive adult female llamas. We found that GnRH cells exhibiting different shapes were distributed throughout the ventral forebrain and some of these were located in proximity to blood vessels; sections of the mediobasal hypothalamus (MBH) displayed the highest number of cells. GnRH fibres were observed in both the organum vasculosum laminae terminalis (OVLT) and median eminence (ME). We also detected abundant kisspeptin fibres in the MBH and ME; kisspeptin cells were found in the arcuate nucleus (ARC), but not in rostral areas of the hypothalamus. Quantitative analysis of GnRH and kisspeptin fibres in the ME revealed a higher innervation density of kisspeptin than of GnRH fibres. The physiological significance of the anatomical findings reported here for the ovulatory mechanism in llamas is still to be determined.

Effect of multiday exercise on serum hormones and metabolic substrate concentrations in racing sled dogs

Prolonged submaximal exercise relies on the steady delivery of oxidizable substrates to the working muscle, with the sources of those substrates either stored reserves or food absorbed from the gastrointestinal tract during exercise. Fat oxidation could be advantageous for this type of exercise because of potentially greater reserves, but recent studies suggest that athletic dogs remain highly dependent on carbohydrate to fuel exercise despite ingesting a high fat diet. The purpose of this study was to characterise the pattern of exercise-induced hormone and substrate concentrations as they relate to carbohydrate and fat metabolism during prolonged submaximal exercise in dogs. Two studies (a 10-dog pilot study and a subsequent primary study using 54 Alaskan sled dogs) were conducted with the dogs running 160 km/day for 4 or 5 days. Blood samples were obtained within 60 min of cessation of daily exercise and in the second study within 30 min of the start of the next day of exercise. Samples were analysed for key hormones and substrates. Results demonstrated the development of a strong hormonal stimulus for glycogenolysis/gluconeogenesis that coincided with sparing and replenishment of muscle glycogen. The stimulus for glycogenolysis/gluconeogenesis tended to diminish during rest periods in the early stages of the exercise challenge, but remained increased during later rest periods and for several days after the conclusion of exercise. These data support the hypothesis that in the face of a high-fat diet, ultra-endurance racing sled dogs rely on large amounts of hepatic glucose output to support prolonged submaximal exercise.

Nucleoplasmic Reticulum Formation in Human Endometrial Cells is Steroid Hormone Responsive and Recruits Nascent Components

The nuclei of cells may exhibit invaginations of the nuclear envelope under a variety of conditions. These invaginations form a branched network termed the nucleoplasmic reticulum (NR), which may be found in cells in pathological and physiological conditions. While an extensive NR is a hallmark of cellular senescence and shows associations with some cancers, very little is known about the formation of NR in physiological conditions, despite the presence of extensive nuclear invaginations in some cell types such as endometrial cells. Here we show that in these cells the NR is formed in response to reproductive hormones. We demonstrate that oestrogen and progesterone are sufficient to induce NR formation and that this process is reversible without cell division upon removal of the hormonal stimulus. Nascent lamins and phospholipids are incorporated into the invaginations suggesting that there is a dedicated machinery for its formation. The induction of NR in endometrial cells offers a new model to study NR formation and function in physiological conditions.

SGK regulates pH increase and cyclin B–Cdk1 activation to resume meiosis in starfish ovarian oocytes

Tight regulation of intracellular pH (pHi) is essential for biological processes. Fully grown oocytes, having a large nucleus called the germinal vesicle, arrest at meiotic prophase I. Upon hormonal stimulus, oocytes resume meiosis to become fertilizable. At this time, the pHi increases via Na+/H+ exchanger activity, although the regulation and function of this change remain obscure. Here, we show that in starfish oocytes, serum- and glucocorticoid-regulated kinase (SGK) is activated via PI3K/TORC2/PDK1 signaling after hormonal stimulus and that SGK is required for this pHi increase and cyclin B–Cdk1 activation. When we clamped the pHi at 6.7, corresponding to the pHi of unstimulated ovarian oocytes, hormonal stimulation induced cyclin B–Cdk1 activation; thereafter, oocytes failed in actin-dependent chromosome transport and spindle assembly after germinal vesicle breakdown. Thus, this SGK-dependent pHi increase is likely a prerequisite for these events in ovarian oocytes. We propose a model that SGK drives meiotic resumption via concomitant regulation of the pHi and cell cycle machinery.

SGK regulates pH increase and cyclin B-Cdk1 activation to resume meiosis in starfish ovarian oocytes

Tight regulation of intracellular pH (pHi) is essential for biological processes. Fully-grown oocytes, having a large nucleus called the germinal vesicle, arrest at meiotic prophase-I. Upon hormonal stimulus, oocytes resume meiosis to acquire fertilizability. At this time, pHi increases through Na+/H+ exchanger activity. However, regulation and function of this change remains obscure. Here we show that in starfish oocytes, serum- and glucocorticoid-regulated kinase (SGK) is activated by the PI3K/TORC2/PDK1 signaling after hormonal stimulus, and is required for the pHi increase and cyclin B–Cdk1 activation. Furthermore, when we clamped pHi at 6.7, corresponding to the pHi of unstimulated ovarian oocytes, hormonal stimulus normally induced cyclin B–Cdk1 activation; thereafter, oocytes initiated germinal vesicle breakdown (GVBD), but failed to complete it. Thus, SGK-dependent pHi increase is likely prerequisite for completion of GVBD in ovarian oocytes. We propose a model that SGK drives meiotic resumption through concomitant regulation of pHi and the cell-cycle machinery.

Protein phosphatases in pancreatic islets

The prevalence of diabetes is increasing rapidly worldwide. A cardinal feature of most forms of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing β-cells, impaired glucose-sensitive insulin secretion from the β-cell, or a combination thereof, the reasons for which largely remain elusive. Reversible phosphorylation is an important and versatile mechanism for regulating the biological activity of many intracellular proteins, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to the stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating the phosphorylation of proteins involved in the insulin secretory process by the β-cell have been extensively investigated. However, far less is known about the role and regulation of protein dephosphorylation by various protein phosphatases. Herein, we review extant data implicating serine/threonine and tyrosine phosphatases in various aspects of healthy and diabetic islet biology, ranging from control of hormonal stimulus–secretion coupling to mitogenesis and apoptosis.

Small-Molecule Hormones: Molecular Mechanisms of Action

Small-molecule hormones play crucial roles in the development and in the maintenance of an adult mammalian organism. On the molecular level, they regulate a plethora of biological pathways. Part of their actions depends on their transcription-regulating properties, exerted by highly specific nuclear receptors which are hormone-dependent transcription factors. Nuclear hormone receptors interact with coactivators, corepressors, basal transcription factors, and other transcription factors in order to modulate the activity of target genes in a manner that is dependent on tissue, age and developmental and pathophysiological states. The biological effect of this mechanism becomes apparent not earlier than 30–60 minutes after hormonal stimulus. In addition, small-molecule hormones modify the function of the cell by a number of nongenomic mechanisms, involving interaction with proteins localized in the plasma membrane, in the cytoplasm, as well as with proteins localized in other cellular membranes and in nonnuclear cellular compartments. The identity of such proteins is still under investigation; however, it seems that extranuclear fractions of nuclear hormone receptors commonly serve this function. A direct interaction of small-molecule hormones with membrane phospholipids and with mRNA is also postulated. In these mechanisms, the reaction to hormonal stimulus appears within seconds or minutes.

Involvement of G protein and purines in Rhinella arenarum oocyte maturation

SummaryWe investigated the participation of Gαi protein and of intracellular cAMP levels on spontaneous and progesterone-mediated maturation in Rhinella arenarum fully grown follicles and denuded oocytes.Although progesterone is the established maturation inducer in amphibians, Rhinella arenarum oocytes obtained during the reproductive period (competent oocytes) resume meiosis with no need for an exogenous hormonal stimulus if deprived of their enveloping follicular cells, a phenomenon called spontaneous maturation. In amphibian oocytes, numerous signalling mechanisms have been involved in the rapid, non-genomic, membrane effects of progesterone, but most of these are not fully understood.The data presented here demonstrate that activation of the Gαi protein by Mas-7 induced maturation in non-competent oocytes and also an increase in GVBD (germinal vesicle breakdown) in competent oocytes. Similar results were obtained with intact follicles independent of the season. The activation of adenylyl cyclase (AC) by forskolin seems to inhibit both spontaneous and progesterone-induced GVBD. In addition, the high intracellular levels of cAMP caused by activation of AC by forskolin treatment or addition of db-cAMP inhibited maturation that had been induced by Mas-7 and in a dose-dependent manner. Treatment with H-89, a protein kinase A (PKA) inhibitor, was able to trigger GVBD in a dose-dependent manner in non-competent oocytes and increased the percentages of GVBD in oocytes competent to mature spontaneously. The results obtained with whole follicles and denuded oocytes were similar, which suggested that effects on AC and PKA were not mediated by follicle cells. The fact that Mas-7 was able to induce maturation in non-competent oocytes in a similar manner to progesterone and to increase spontaneous maturation suggests that Gαi activation could be an important step in meiosis resumption. Thus, the decrease in cAMP as a result of the regulation of the G proteins on AC and the inactivation of PKA by H-89 could contribute to the activation of MPF (maturation promoting factor) and induce maturation of the oocytes of Rhinella arenarum.

Signal Protein-Derived Peptides as Functional Probes and Regulators of Intracellular Signaling

The functionally important regions of signal proteins participating in their specific interaction and responsible for transduction of hormonal signal into cell are rather short in length, having, as a rule, 8 to 20 amino acid residues. Synthetic peptides corresponding to these regions are able to mimic the activated form of full-size signal protein and to trigger signaling cascades in the absence of hormonal stimulus. They modulate protein-protein interaction and influence the activity of signal proteins followed by changes in their regulatory and catalytic sites. The present review is devoted to the achievements and perspectives of the study of signal protein-derived peptides and to their application as selective and effective regulators of hormonal signaling systems in vitro and in vivo. Attention is focused on the structure, biological activity, and molecular mechanisms of action of peptides, derivatives of the receptors, G protein α subunits, and the enzymes generating second messengers.

Clathrin is essential for meiotic spindle function in oocytes

In the mammalian ovary, oocytes are arrested at prophase of meiosis I until a hormonal stimulus triggers resumption of meiosis. During the subsequent meiotic maturation process, which includes completion of the first meiotic division and formation of the second metaphase spindle, oocytes acquire competence for fertilization. Recently, it was shown that clathrin, a cytosolic protein complex originally defined for its role in intracellular membrane traffic, is also involved in the stabilization of kinetochore fibers in mitotic spindles of dividing somatic cells. However, whether clathrin has a similar function in meiotic spindles in oocytes has not been investigated previously. Our results show that endogenous clathrin associates with the meiotic spindles in oocytes. To study the function of clathrin during meiotic maturation, we microinjected green fluorescent protein-tagged C-terminal and N-terminal dominant-negative clathrin protein constructs into isolated porcine oocytes prior toin vitromaturation. Both protein constructs associated with meiotic spindles similar to endogenous clathrin, but induced misalignment and clumping of chromosomes, occurrence of cytoplasmic chromatin and failure of polar body extrusion. These data demonstrate that clathrin plays a crucial role in meiotic spindle function in maturing oocytes, possibly through spindle stabilization.