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Autoradiographical localization of oxytocin-binding sites in the guinea-pig ovary at different stages of the oestrous cycle

L. Zhang, J. J. Dreifuss, M. Dubois-Dauphin, and E. Tribollet

ABSTRACT

The discovery that oxytocin is synthesized and stored in corpora lutea of ruminants has fostered a renewed interest in the possible roles of oxytocin in ovarian function. In the present study we describe the distribution of binding sites for oxytocin in the guinea-pig ovary. Sections were reacted with a radioiodinated oxytocin antagonist (125I-labelled OTA) to yield autoradiograms on film. Specific binding sites for oxytocin were defined as those which bound 0·05 nmol 125I-labelled OTA/l and where 1 μmol non-radioactive oxytocin/1 displaced the radioactivity. 125I-Labelled OTA consistently labelled the ovarian stroma and the theca interna, but not the corpora lutea, the granulosa cells or the theca externa. The amount of 125I-labelled OTA bound to ovarian stroma and theca interna was high in animals killed during dioestrus, and low during and shortly after oestrus. These data suggest that the binding sites are regulated by steroid hormone levels and that in the guinea-pig ovary oxytocin could exert a role in follicular steroidogenesis, maturation or ovulation rather than in luteal function. Oxytocin-binding sites were also shown in the uterus but their numbers varied only slightly during the cycle.

Journal of Endocrinology (1991) 131, 421–426

DOI:
https://doi.org/10.1677/joe.0.1310421
Volume 131: Issue 3,
Pages:
421–NP
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Developmental expression of the prolactin receptor gene in rat gonads

F-P Zhang, A Rannikko, J Toppari, A Bartke, and I Huhtaniemi

Abstract

The prolactin receptor (PRLR) is a member of the cytokine/prolactin/GH receptor family, and it is widely expressed in various mammalian tissues. Expression of the two different forms of PRLR, differing in the length of their cytoplasmic domains, was studied in rat gonads during fetal and postnatal development. The two forms of PRLR mRNA were analyzed by reverse transcription (RT)-PCR using primer pairs specific for the different forms. The specificity of the cDNA species generated by RT-PCR was verified by Southern hybridization using nested 32P-labeled oligonucleotides. The results indicated that both forms of PRLR mRNA are expressed in the rat testis and ovary, which is in agreement with previous reports. The onset of expression of the two PRLR forms occurs on day 14·5 of fetal life in rat testis. In the ovary, the long form of PRLR mRNA appears 1 day before the short form, i.e. these forms begin to be expressed on fetal days 14·5 and 15·5 respectively. In situ hybridization with antisense cRNA probes specific to each form of the PRLR mRNAs demonstrated specific hybridization of both forms, localized in Leydig cells from day 18·5 of fetal life and at the postnatal ages studied. Compared with our previous findings concerning the ontogeny of LH receptor gene expression, PRLR gene expression starts earlier in development and exhibits no sexual dimorphism. The presence of two forms of PRLR mRNA in the fetal gonads suggest that they might play differential roles in gonadal development and function.

Journal of Endocrinology (1995) 147, 497–505

DOI:
https://doi.org/10.1677/joe.0.1470497
Volume 147: Issue 3,
Pages:
497–505 (9 total)
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The Effect of GH-Releasing Peptide-2 (GHRP-2 or KP 102) on GH Secretion from Primary Cultured Ovine Pituitary Cells Can be Abolished by a Specific GH-Releasing Factor (GRF) Receptor Antagonist

Danxing Wu, Chen Chen, Kazuo Katoh, Jin Zhang, and Iain J. Clarke

Abstract

A newly synthesised GH-releasing peptide, KP 102 (also named GHRP-2), was studied in an in vitro perifusion system of primary cultured ovine anterior pituitary cells. Application of KP 102 to the perifusion medium caused a dose-dependent increase in GH secretion. Dose-response relationships indicated that KP 102 had similar potency to GRF and was 10-fold more potent than earlier generations of GH-releasing peptide (GHRP-6 and GHRP-1) tested in same system. The response to a second application of KP 102 given within 1 h of initial application was significantly lower than the response to the first application. When KP 102 (or GRF) was applied first and then GRF (or KP 102) given 1 h later, the second response was not attenuated. When GRF and KP 102 were coadministered, an additive effect on release of GH was obtained. The effect of maximal dose of KP 102 (100nM) on GH release was totally abolished by [Ac-Tyr1, d-Arg2] GRF 1-29 (1μM) which is believed to be a specific antagonist for the GRF receptor. Blockade of Ca2+ channels by Cd2+ (2mM) diminished the basal GH secretion and abolished the increase in GH release in response to KP 102 (100nM). These data suggest that the action of KP 102 is blocked by a GRF receptor antagonist and therefore acts through a different receptor to that employed by earlier generations of GH-releasing peptides. GH release in response to KP 102 involves an increase in Ca2+ influx and there is no cross-desensitization between KP 102 and GRF responses.

DOI:
https://doi.org/10.1677/joe.0.140R009
Volume 140: Issue 2,
Pages:
R9–R13
Free access

Responses to the cannabinoid receptor-1 antagonist, AM251, are more robust with age and with high-fat feeding

M K Judge, Y Zhang, and P J Scarpace

Endocannabinoids (EC) are involved in regulating energy homeostasis, particularly in promoting hyperphagia and the consumption of a palatable diet. We have previously shown that rats given a high-fat (HF) diet display a transient hyperphagia that is normalized by a process partially dependent on leptin. We now propose that the induction of this hyperphagia is mediated, at least partially, by the EC signaling system. Obesity, including diet-induced and age-related, is associated with dysregulation of the EC system, and obese rodent models are hypersensitive to a cannabinoid-1 (CB1) receptor antagonist. This suggests that aged rats will be more responsive to the anorectic effects of a CB1 receptor antagonist. To test this, we examined the responsiveness to CB1 receptor antagonist, AM251, in young and aged rats during two experimental paradigms. First, we administered AM251 simultaneously with the introduction of an HF diet. Second, AM251 treatment began after the establishment of diet-induced obesity. Responses were measured by changes in body weight and composition, calorie intake, serum leptin, and biochemical indicators. The results demonstrated three key findings. 1) CB1 receptor activity contributes to the hyperphagia seen with the introduction of an HF diet. 2) Increased AM251 sensitivity and efficacy is increased with age and HF feeding, with the greatest responsiveness observed in HF-fed, aged rats. 3) AM251 sensitivity is elevated to a greater extent with HF diet than with established obesity. Thus, both age and an HF diet are associated with enhanced anorectic responses to AM251, but the underlying mechanism of these responses remains speculative.

DOI:
https://doi.org/10.1677/JOE-09-0210
Volume 203: Issue 2,
Pages:
281–290 (10 total)
Restricted access

The effects of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on intracellular adenosine 3′,5′-monophosphate (cAMP) levels and GH secretion in ovine and rat somatotrophs

D Wu, C Chen, J Zhang, C Y Bowers, and I J Clarke

Abstract

The mechanism of action of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on GH release was investigated in ovine and rat pituitary cells in vitro. In partially purified sheep somatotrophs, GHRP-2 and GH-releasing factor (GRF) increased intracellular cyclic AMP (cAMP) concentrations and caused GH release in a dose-dependent manner; GHRP-6 did not increase cAMP levels. An additive effect of maximal doses of GRF and GHRP-2 was observed in both cAMP and GH levels whereas combined GHRP-6 and GHRP-2 at maximal doses produced an additive effect on GH release only. Pretreatment of the cells with MDL 12,330A, an adenylyl cyclase inhibitor, prevented cAMP accumulation and the subsequent release of GH that was caused by either GHRP-2 or GRF. The cAMP antagonist, Rp-cAMP also blocked GH release in response to GHRP-2 and GRF. The cAMP antagonist did not prevent the effect of GHRP-6 on GH secretion whereas MDL 12,330A partially reduced the effect. An antagonist for the GRF receptor, [Ac-Tyr1,d-Arg2]-GRF 1–29, significantly diminished the effect of GHRP-2 and GRF on cAMP accumulation and GH release, but did not affect GH release induced by GHRP-6. Somatostatin prevented cAMP accumulation and GH release responses to GHRP-2, GRF and GHRP-6. Ca2+ channel blockade did not affect the cAMP increase in response to GHRP-2 or GRF but totally prevented GH release in response to GHRP-2, GRF and GHRP-6. These results indicated that GHRP-2 acts on ovine pituitary somatotrophs to increase cAMP concentration in a manner similar to that of GRF; this occurs even during the blockade of Ca2+ influx. GHRP-6 caused GH release without an increase in intracellular cAMP levels. GH release in response to all three secretagogues was reduced by somatostatin and was dependent upon the influx of extracellular Ca2+. The additive effect of GHRP-2 and GRF or GHRP-6 suggested that the three peptides may act on different receptors. In rat pituitary cell cultures, GHRP-6 had no effect on cAMP levels, but potentiated the effect of GRF on cAMP accumulation. The synergistic effect of GRF and GHRP-6 on cAMP accumulation did not occur in sheep somatotrophs. Whereas GHRP-2 caused cAMP accumulation in sheep somatotrophs, it did not do so in rat pituitary cells. These data indicate species differences in the response of pituitary somatotrophs to the GHRPs and this is probably due to different subtypes of GHRP receptor in rat or sheep.

Journal of Endocrinology (1996) 148, 197–205

DOI:
https://doi.org/10.1677/joe.0.1480197
Volume 148: Issue 2,
Pages:
197–205 (9 total)
Free access

Impaired cardiac contractile function in ventricular myocytes from leptin-deficient ob/ob obese mice

F Dong, X Zhang, X Yang, L B Esberg, H Yang, Z Zhang, B Culver, and J Ren

The level of the obese gene product leptin is often positively correlated with body weight, supporting the notion that hyperleptinemia contributes to obesity-associated cardiac dysfunction. However, a link between leptin levels and cardiac function has not been elucidated. This study was designed to examine the role of leptin deficiency (resulting from a point mutation of the leptin gene) in cardiomyocyte contractile function. Mechanical properties and intracellular Ca2 + transients were evaluated in ventricular myocytes from lean control and leptin-deficient ob/ob obese mice at 12 weeks of age. Cardiac ultrastructure was evaluated using transmission electron microscopy. ob/ob mice were overtly obese, hyperinsulinemic, hypertriglycemic, hypoleptinemic and euglycemic. Ultrastructural examination revealed swelling and disorganization of cristae in mitochondria from ob/ob mouse ventricular tissues. Cardiomyocytes from ob/ob mice displayed reduced expression of the leptin receptor Ob-R, larger cross-sectional area, decreased peak shortening and maximal velocity of shortening/relengthening, and prolonged relengthening but not shortening duration compared with lean counterparts. Consistent with mechanical characteristics, myocytes from ob/ob mice displayed reduced intracellular Ca2 + release upon electrical stimulus associated with a slowed intracellular Ca2 + decay rate. Interestingly, the contractile aberrations seen in ob/ob myocytes were significantly improved by in vitro leptin incubation. Contractile dysfunction was not seen in age- and gender-matched high fat-induced obese mice. These results suggested that leptin deficiency contributes to cardiac contractile dysfunction characterized by both systolic and diastolic dysfunction, impaired intracellular Ca2 + hemostasis and ultrastructural derangement in ventricular myocytes.

DOI:
https://doi.org/10.1677/joe.1.06241
Volume 188: Issue 1,
Pages:
25–36 (12 total)
Free access

Both stress experience and age determine the impairment or enhancement effect of stress on spatial memory retrieval

Y Yang, J Cao, W Xiong, J Zhang, Q Zhou, H Wei, C Liang, J Deng, T Li, S Yang, and L Xu

It has been documented that stress or glucocorticoids have conflicting effects on memory under different conditions. However, it is not fully understood why stress can either impair or enhance memory. Here, we have examined the performance of six age groups of Wistar rats in a water maze spatial task to evaluate the effects of stress under different conditions. We found that the impairment or enhancement effect of an 'elevated platform' (EP) stress on memory was dependent on previous stress experience and on age. EP stress impaired memory retrieval in water maze naive animals, but enhanced rather than impaired memory retrieval in young water maze stress-experienced animals. Furthermore, exogenously applied corticosterone or foot shock stress before water maze training prevented the impairment of memory retrieval that should be induced by treatment with corticosterone or foot shock before the 'probe trial'. Again, memory retrieval was enhanced in young animals under these conditions, and this enhancement can be prevented by the glucocorticoid receptor antagonist RU 38486. Thus, glucocorticoid receptor activation not only induced impairment of memory but also increased the capacity of young animals to overcome a later stress. The present findings suggest that the effect of stress on memory can be switched from impairment to enhancement dependent on both stress experience and age.

DOI:
https://doi.org/10.1677/joe.0.1780045
Volume 178: Issue 1,
Pages:
45–54 (10 total)
Restricted access

Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium

A Rannikko, T-L Penttilä, F-P Zhang, J Toppari, M Parvinen, and I Huhtaniemi

Abstract

Stage-specific expression of the FSH receptor (FSHR) gene in the rat seminiferous epithelium was studied. Using transillumination-assisted microdissection for sample preparation and Northern hybridization for analysis of total RNA, we first reassessed the stage specificity of the FSHR gene expression in the adult rat testis. Sixfold higher FSHR mRNA levels were found in stages XIII–I compared with stage VI of the seminiferous epithelial cycle, which had the lowest signal level (P<0·01). The other stages had intermediate signal levels. In situ hybridization showed distribution of grains which confirmed the data obtained by Northern analysis. Prepubertal stage-specific FSHR gene expression was studied using in situ hybridization. Stage specificity could first be demonstrated at the age of 16 days when the average grain counts in stages I–IV were threefold higher than in stages VI–VII (P<0·01). The present data are in agreement with earlier findings on stage-specific FSH binding and FSHR gene expression using both microdissected and stage-synchronized seminiferous tubules. The onset of stage-specific FSHR gene expression is concomitant with maturation of the Sertoli cell population and completion of the first generation of spermatocytes. This supports the hypothesis that spermatogonia and spermatocytes may be involved in the regulation of FSHR gene expression.

Journal of Endocrinology (1996) 151, 29–35

DOI:
https://doi.org/10.1677/joe.0.1510029
Volume 151: Issue 1,
Pages:
29–35 (7 total)
Free access

POMC overexpression in the ventral tegmental area ameliorates dietary obesity

Lourdes M Andino, Daniel J Ryder, Alexandra Shapiro, Michael K Matheny, Yi Zhang, Melanie K Judge, K Y Cheng, Nihal Tümer, and Philip J Scarpace

The activation of proopiomelanocortin (POMC) neurons in different regions of the brain, including the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the solitary tract curtails feeding and attenuates body weight. In this study, we compared the effects of delivery of a recombinant adeno-associated viral (rAAV) construct encoding POMC to the ARC with delivery to the ventral tegmental area (VTA). F344×Brown Norway rats were high-fat (HF) fed for 14 days after which self-complementary rAAV constructs expressing either green fluorescent protein or the POMC gene were injected using coordinates targeting either the VTA or the ARC. Corresponding increased POMC levels were found at the predicted injection sites and subsequent α-melanocyte-stimulating hormone levels were observed. Food intake and body weight were measured for 4 months. Although caloric intake was unaltered by POMC overexpression, weight gain was tempered with POMC overexpression in either the VTA or the ARC compared with controls. There were parallel decreases in adipose tissue reserves. In addition, levels of oxygen consumption and brown adipose tissue uncoupling protein 1 were significantly elevated with POMC treatment in the VTA. Interestingly, tyrosine hydroxylase levels were increased in both the ARC and VTA with POMC overexpression in either the ARC or the VTA. In conclusion, these data indicate a role for POMC overexpression within the VTA reward center to combat HF-induced obesity.

DOI:
https://doi.org/10.1530/JOE-10-0418
Volume 210: Issue 2,
Pages:
199–207 (9 total)
Free access

Functional characterization of hyperpolarization-activated cyclic nucleotide-gated channels in rat pancreatic β cells

Yi Zhang, Yunfeng Liu, Jihong Qu, Alexandre Hardy, Nina Zhang, Jingyu Diao, Paul J Strijbos, Robert Tsushima, Richard B Robinson, Herbert Y Gaisano, Qinghua Wang, and Michael B Wheeler

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate pacemaker activity in some cardiac cells and neurons. In the present study, we have identified the presence of HCN channels in pancreatic β-cells. We then examined the functional characterization of these channels in β-cells via modulating HCN channel activity genetically and pharmacologically. Voltage-clamp experiments showed that over-expression of HCN2 in rat β-cells significantly increased HCN current (I h), whereas expression of dominant-negative HCN2 (HCN2-AYA) completely suppressed endogenous I h. Compared to control β-cells, over-expression of I h increased insulin secretion at 2.8 mmol/l glucose. However, suppression of I h did not affect insulin secretion at both 2.8 and 11.1 mmol/l glucose. Current-clamp measurements revealed that HCN2 over-expression significantly reduced β-cell membrane input resistance (R in), and resulted in a less-hyperpolarizing membrane response to the currents injected into the cell. Conversely, dominant negative HCN2-AYA expression led to a substantial increase of R in, which was associated with a more hyperpolarizing membrane response to the currents injected. Remarkably, under low extracellular potassium conditions (2.5 mmol/l K+), suppression of I h resulted in increased membrane hyperpolarization and decreased insulin secretion. We conclude that I h in β-cells possess the potential to modulate β-cell membrane potential and insulin secretion under hypokalemic conditions.

DOI:
https://doi.org/10.1677/JOE-09-0068
Volume 203: Issue 1,
Pages:
45–53 (9 total)