The GK rat beta-cell: a prototype for the diseased human beta-cell in type 2 diabetes?

Increasing evidence indicates that decreased functional beta-cell mass is the hallmark of type 2 diabetes (T2D) mellitus. Nowadays, the debate focuses on the possible mechanisms responsible for abnormal islet microenvironment, decreased beta-cell number, impaired beta-cell function, and their multifactorial aetiologies. This review is aimed to illustrate to what extend the Goto-Kakizaki rat, one of the best characterized animal models of spontaneous T2D, has proved be a valuable tool offering sufficient commonalities to study these aspects. We propose that the defective beta-cell mass and function in the GK model reflect the complex interactions of multiple pathogenic players: (i) several independent loci containing genes responsible for some diabetic traits (but not decreased beta-cell mass); (ii) gestational metabolic impairment inducing an epigenetic programming of the pancreas (decreased beta-cell neogenesis and/or proliferation) which is transmitted to the next generation; and (iii) loss of beta-cell differentiation due to chronic exposure to hyperglycemia/hyperlipidemia, inflammatory mediators, oxidative stress and to perturbed islet microarchitecture.

Type 2 diabetes - a matter of failing beta-cell neogenesis? Clues from the GK rat model.

Now that reduction in beta-cell mass has been clearly established in humans with type 2 diabetes mellitus (T2D), the debate focuses on the possible mechanisms responsible for decreased beta-cell number. Appropriate inbred rodent models are essential tools for this purpose. The information available from the Goto-Kakizaki (GK) rat, one of the best characterized animal models of spontaneous T2D, is reviewed in such a perspective. We propose that the defective beta-cell mass in the GK model reflects mostly a persistently decreased beta-cell neogenesis. The data discussed in this review are consistent with the notion that poor proliferation and/or survival of the endocrine precursor cells during GK foetal life will result in a decreased pool of endocrine precursors in the pancreas, and hence an impaired capacity of beta-cell neogenesis (either primary in the foetus or compensatory in the newborn and the adult). As we also demonstrated that beta-cell neogenesis can be pharmacologically reactivated in the GK model, our work supports, on a more prospective basis, the concept that facilitation of T2D treatment may be obtained through beta-cell mass expansion after stimulation of beta-cell regeneration/neogenesis in diabetic patients.

Defective IGF2 and IGF1R protein production in embryonic pancreas precedes beta cell mass anomaly in the Goto-Kakizaki rat model of type 2 diabetes.

AIMS/HYPOTHESIS: The Goto-Kakizaki (GK) rat is a spontaneous model of type 2 diabetes. Defective beta cell mass detectable in late fetal age precedes the onset of hyperglycaemia. Our hypothesis was that an embryonic IGF production deficiency might be involved in beta cell mass anomaly in the diabetic GK rat. To test this, we evaluated during pancreatic organogenesis: (1) the beta cell development in GK rats on embryonic day (E) 13.5 and E18.5; (2) IGF2 and IGF1 receptor (IGF1R) pancreatic protein production on E13.5 and E18.5; (3) the in vitro development of GK pancreatic rudiment on E13.5; and (4) the in vitro effect of IGF2 addition on beta cell mass. MATERIALS AND METHODS: Beta cell quantitative analyses were determined by immunohistochemistry and morphometry. IGF2 and IGF1R pancreatic protein production was evaluated using western blot analyses. Dorsal pancreatic rudiments were dissected on E13.5, separated from surrounding mesenchyme and cultured for 7 days without or with recombinant IGF2. RESULTS: While beta cell mass was already decreased on E18.5, the differentiation of the first beta cells was in fact normal in E13.5 GK pancreas. Moreover, defective IGF2 and IGF1R protein production was detected in GK pancreatic rudiment as early as E13.5. The isolated GK pancreatic rudiment as maintained in vitro mimics the GK beta cell deficiency observed in vivo. This last approach enabled us to show that GK beta cells were fully responsive to IGF2 as far as their net growth is concerned. CONCLUSIONS/INTERPRETATION: In diabetic GK rat, defective IGF2 and IGF1R protein production in embryonic pancreas precedes beta cell mass anomaly. IGF2 supplementation expands the pool of beta cells.

Undernutrition does not alter the activation of beta-cell neogenesis and replication in adult rats after partial pancreatectomy.

In previous work, we demonstrated that a 65% protein calorie food restriction started during the third trimester of gestation in rats caused a reduced beta-cell mass at 4 days of life that persisted until adult age. In this study with adult undernourished (U) rats, we investigated 1) whether undernutrition affects the beta-cell growth potential and both beta-cell proliferation and differentiation and 2) the implication of the IGFs, highly responsive to nutritional status, in these processes. To this end, we used the 90% pancreatectomy (Px) procedure in U and control (C) adult rats. The results show that, on day 2 after Px, beta-cell replication was significantly higher in C rats, whereas the beta-cell neogenesis was markedly increased in U/Px rats. Both the serum levels of IGF-I and the liver IGF-I mRNA expression were reduced in adult U rats before and after Px compared with C rats. Pancreatic IGF-I mRNA expression was reduced in U animals on day 0. However, on day 2 after Px, the increase of pancreatic IGF-I mRNA expression was significantly higher in U rats than in C rats. These data suggest that beta-cells still have the capacity to regenerate in the adult U rats, with a higher efficiency than C rats on day 2, and that both beta-cell neogenesis and beta-cell replication are stimulated. The increased pancreatic IGF-I mRNA may be instrumental in these processes.

Protein-caloric food restriction affects insulin-like growth factor system in fetal Wistar rat.

We have previously shown that fetuses from protein-caloric undernourished pregnant rats (35% of control diet during the last week of pregnancy) at 21.5 d post coitum exhibit increased beta-cell mass. This alteration is correlated with increased insulinemia and total pancreatic insulin content, a pattern similar to that reported in infants of mild diabetic mothers. In this work, we investigated in undernourished fetuses: 1) whether availability of growth factors such as insulin, GH, and IGFs and their binding proteins (IGFBPs) could be implicated in this alteration, and 2) the beta-cell mitogenic response to IGFs in vitro. The results show that maternal undernutrition increases pancreatic IGF-I expression and islet IGF-I receptor content in undernourished fetuses, whereas hepatic IGF-I expression and serum IGF-I levels were decreased. No changes were observed in serum IGF-II, and its expression was diminished in undernourished pancreases and unchanged in the liver, compared with control fetuses. Serum levels and liver and pancreatic mRNA expression of IGFBP-1 were found to be normal in undernourished fetuses, whereas the serum concentration and abundance of IGFBP-2 mRNA in pancreas were increased. Finally, the beta-cell mitogenic response to IGFs in vitro was significantly increased in undernourished fetal islets, compared with controls. In conclusion, in undernourished fetuses the increased beta-cell mass can be related to the stimulation of replicative beta-cell response due to locally increased pancreatic IGF-I mRNA; this effect is perhaps potentiated or favored by the enhanced islet IGF-I receptor content and pancreatic IGFBP-2 gene expression.

beta-cell function and viability in the spontaneously diabetic GK rat: information from the GK/Par colony.

The GK rat model of type 2 diabetes is especially convenient to dissect the pathogenic mechanism necessary for the emergence of overt diabetes because all adult rats obtained in our department (GK/Par colony) to date have stable basal mild hyperglycemia and because overt diabetes is preceded by a period of normoglycemia, ranging from birth to weaning. The purpose of this article is to sum up the information so far available related to the biology of the beta-cell in the GK/Par rat. In terms of beta-cell function, there is no major intrinsic secretory defect in the prediabetic GK/Par beta-cell, and the lack of beta-cell reactivity to glucose (which reflects multiple intracellular abnormalities), as seen during the adult period when the GK/Par rats are overtly diabetic, represents an acquired defect (perhaps glucotoxicity). In terms of beta-cell population, the earliest alteration so far detected in the GK/Par rat targets the size of the beta-cell population. Several convergent data suggest that the permanently reduced beta-cell mass in the GK/Par rat reflects a limitation of beta-cell neogenesis during early fetal life, and it is conceivable that some genes among the set involved in GK diabetes belong to the subset of genes controlling early beta-cell development.

Glucose metabolism and beta-cell mass in adult offspring of rats protein and/or energy restricted during the last week of pregnancy.

An association between low birth weight and later impaired glucose tolerance was recently demonstrated in several human populations. Although fetal malnutrition is probably involved, the biological bases of such a relationship are not yet clear, and animal studies on the matter are scarce. The present study was aimed to identify, in adult (8-wk) female offspring, the effects of reduced protein and/or energy intake strictly limited to the last week of pregnancy. Thus we have tested three protocols of gestational malnutrition: a low-protein isocaloric diet (5 instead of 15%), with pair feeding to the mothers receiving the control diet; a restricted diet (50% of the control diet); and a low-protein restricted diet (50% of low-protein diet). Only the low-protein diet protocols, independent of total energy intake, led to a lower birth weight. The adult offspring female rats in the three deprived groups exhibited no decrease in body weight and no major impairment in glucose tolerance, glucose utilization, or glucose production (basal state and hyperinsulinemic clamp studies). However, pancreatic insulin content and beta-cell mass were significantly decreased in the low-protein isocaloric diet group compared with the two energy-restricted groups. Such impairment of beta-cell mass development induced by protein deficiency limited to the last part of intrauterine life could represent a situation predisposing to impaired glucose tolerance.

Effect of gliclazide treatment on insulin secretion and beta-cell mass in non-insulin dependent diabetic Goto-Kakisaki rats.

The Goto-Kakisaki rat is a genetic non-overweight model of non-insulin-dependent diabetes mellitus. Adult Goto-Kakisaki rats exhibit a mild basal hyperglycaemia (11 mmol/l) with impaired glucose tolerance, elevated basal plasma insulin level, a failure of insulin release in response to glucose together with a 50% depletion of the total pancreatic beta-cell mass and insulin stores. We have examined the effects of long-term (4 weeks) gliclazide treatment on the severity of diabetes in adult male Goto-Kakisaki rats (10-12 weeks of age). Gliclazide was administered orally (10 mg/kg per day). Gliclazide-treated Goto-Kakisaki rats were evaluated against Wistar and untreated Goto-Kakisaki rats. In the gliclazide-treated Goto-Kakisaki rats, basal plasma glucose levels declined progressively reaching 8 mmol/l as a mean at the end of treatment, and their basal insulin levels decreased to values similar to those in non-diabetic Wistar rats. Despite their total pancreatic beta-cell remaining unaffected, their pancreatic insulin stores were twice increased, with a similar improvement of the insulin content per individual beta-cell. Furthermore, the glucose-stimulated insulin release as evaluated in vivo during an intravenous glucose tolerance-test was significantly improved (twice increased) in the gliclazide-treated Goto-Kakisaki rats. This was correlated with a modest but significant enhancement of the early phase of insulin release in vitro (isolated perfused pancreas), in response to glucose. However, the overall insulin response in vitro remained clearly defective with no reappearance of the late phase of insulin release. The in vitro response to arginine (which was basically amplified in the Goto-Kakisaki model) or to gliclazide were kept unchanged after the gliclazide treatment. In conclusion, chronic gliclazide does not exert any beta-cytotrophic effect, but improves beta-cell function in the adult Goto-Kakisaki rat as far as it lowers basal insulin release, increases beta-cell insulin stores, and increases the glucose-induced insulin release.

Continuous glucose monitoring in the free-moving rat.

The aim of this work was to set up an experimental model of glycemic fluctuations for assessing in the conscious freely moving rat, the performance of a continuous glucose-monitoring system, using a pocket-calculator-size electronic control unit and a miniaturized subcutaneous glucose sensor. The well-known triphasic glycemic pattern following streptozotocin injection (initial peak and secondary hypoglycemia preceding the establishment of permanent hyperglycemia) was used as a way to obtain spontaneous changes in blood glucose level over a wide concentration range. This report demonstrates that streptozotocin injection produced highly reproducible changes in the current generated by the sensor: an initial peak and a secondary nadir, during which blood sampling provided the evidence of hyperglycemia associated with immunoreactive hypoinsulinemia, and of hypoglycemia associated with hyperinsulinemia, respectively. This reproducible experimental model should be valuable for the assessment of a continuous glucose-monitoring system.

Impaired pancreatic beta cell function in the fetal GK rat. Impact of diabetic inheritance.

The Goto-Kakisaki (GK) rat is a genetic model of non-insulin-dependent diabetes. At 21.5 d of age we found that GK fetuses had an increased plasma glucose concentration, a decreased plasma insulin level, and a reduced pancreatic beta cell mass. To investigate the beta cell function during fetal life we used a hyperglycemic clamp protocol applied to the mothers, which allowed us to obtain a steady-state hyperglycemia in the corresponding fetuses. At variance, with Wistar (W) fetuses, plasma insulin concentration in GK fetuses did not rise in response to hyperglycemia. In contrast, GK fetal pancreas released insulin in response to glucose in vitro to the same extent as W fetal pancreas. Such a discrepancy between the in vivo and in vitro results suggests that the lack of pancreatic reactivity to glucose as seen in vivo is extrinsic to the fetal GK beta cell. Finally, the importance of gestational hyperglycemia was investigated by performing crosses between GK and W rats. Fetuses issued from crosses between W mother and GK father or GK mother and W father had a beta cell mass close to normal values and were still able to increase their plasma insulin levels in response to hyperglycemia in vivo. Our data suggest that hyperglycemia in utero does not influence the severity of the decrease of the beta cell mass or the lack of the insulin secretory response to glucose in the fetal GK rat. Moreover they indicate that conjunction of GK genes originating from both parents is necessary in order for these defects to be fully expressed.

Impaired phosphoinositide metabolism in glucose-incompetent islets of neonatally streptozotocin-diabetic rats.

The effects of nutrient and neurotransmitter stimuli on insulin release, loss of phosphoinositides (PI), and production of inositol phosphates (InsP) were investigated in islets from neonatally streptozotocin-injected (nSTZ) rats. In islets from nSTZ rats, insulin secretory responses to 16.7 mM D-glucose and 10.0 mM D-glyceraldehyde were reduced compared with controls. Contents in phosphatidylinositol 4-monophosphate [PtdIns(4)P] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], but not in phosphatidylinositol, were diminished. Glucose effects on breakdown of PtdIns(4)P and PtdIns(4,5)P2 and on total InsP accumulation were both reduced. D-Glucose was unable to increase the levels of both inositol trisphosphate isomers, Ins(1,3,4)P3 and Ins(1,4,5)P3. Glyceraldehyde also failed to promote InsP formation. By contrast, the ability of 1.0 mM carbachol or 300 nM cholecystokinin to stimulate insulin secretion and InsP generation was still observed. Thus a disturbed coupling between nutrient recognition and activation of phospholipase C, possibly together with a shortage of available polyphosphoinositides, could be responsible for the altered islet PI turnover in the nSTZ rats. It is proposed that such defects may contribute to the impairment of glucose-stimulated insulin secretion in this model of non-insulin-dependent diabetes mellitus.

Use of a subcutaneous glucose sensor to detect decreases in glucose concentration prior to observation in blood.

The development of a hypoglycemic alarm system using a subcutaneous glucose sensor implies that a decrease in blood glucose is rapidly followed by a decrease in the signal generated by the sensor. In a first set of experiments the linearity and the kinetics of the response of sensors implanted in the subcutaneous tissue of normal rats were investigated during a progressive increase in plasma glucose concentration: the sensitivities determined between 5 and 10 mM and between 10 and 15 mM were not significantly different, and a 5-10 min delay in the sensor's response was observed. In a second set of experiments, performed in diabetic rats, the kinetics of the decrease in subcutaneous glucose concentration following insulin administration was monitored during a decrease in plasma glucose level, from 15 to 3 mmol/L. During the 20 first min following insulin administration, the sensor monitored glucose concentration in subcutaneous tissue with no lag time. Subsequently, the decrease in the estimation of subcutaneous glucose concentration preceded that of plasma glucose. This phenomenon was not observed when the same sensors were investigated in vitro during a similar decrease in glucose concentration and may be due to a mechanism occurring in vivo, such as the effect of insulin on glucose transfer from the interstitial space to the cells surrounding the sensor. It reinforces the interest of the use of implantable glucose sensors as a part of a hypoglycemic alarm.

Decreased glucose-induced cAMP and insulin release in islets of diabetic rats: reversal by IBMX, glucagon, GIP.

The first aim of the study was to investigate the possibility that a defect on the islet adenosine 3',5'-cyclic monophosphate (cAMP) production could be involved in the failure of the glucose-induced insulin secretion in the neonatal streptozotocin diabetic rats. Exposure to glucose concentration that induced a rise of the cAMP content in the control islets did not elicit any significant increase in cAMP in diabetic islets. Forskolin, isobutyl methylxanthine (IBMX), glucagon, or pertussis toxin amplified the cAMP accumulation and the insulin release to the same extent in both types of islets. Somatostatin, prostaglandin E2, UK-14304, or galanin inhibited cAMP accumulation and insulin release to the same extent in both types of islets. Our second purpose was to investigate whether the use of activators of adenylate cyclase could restore the beta-cell competence to glucose in diabetic rats. The addition of IBMX, glucagon, or gastric inhibitory polypeptide (GIP) to perifused islets of diabetic rats amplified their insulin response to glucose, and a clear biphasic pattern of the release was regained. In conclusion, although there is no major alteration of the functionality of the adenylate cyclase in the beta-cells of the diabetic rats, we have identified a defective glucose-induced cAMP generation that could be explained by a block in the step(s) linking glucose metabolism and activation of adenylate cyclase.

Modification of the sensitivity of glucose sensor implanted into subcutaneous tissue.

The mechanism of reducing the glucose sensitivity of sensors implanted into the subcutaneous tissue of the normal rat was evaluated (n = 10) by comparing sensitivities observed in vitro and in vivo. In vivo sensitivity was significantly lower than that observed in vitro before implantation (p < 0.005). Most interestingly, in vitro sensitivity immediately after explanation did not differ from that in vivo and increased progressively during rinsing (p < 0.02 after 30 min). These results demonstrate that the reduction of in vivo sensitivity was not due to a local factor or factors but to a reversible alteration of the glucose sensor characteristics induced in vivo by some local factor(s). This suggests that modifications of the outer sensor membrane, the nature of which remains to be determined, may prevent this effect and resolve the problem.

Mitochondrial deoxyribonucleic acid content is specifically decreased in adult, but not fetal, pancreatic islets of the Goto-Kakizaki rat, a genetic model of noninsulin-dependent diabetes.

Considerable interest has recently been focused on the putative role of mutations in the mitochondrial genome for the development of noninsulin-dependent diabetes. The Goto-Kakizaki (GK) rat, a genetic model of defective insulin secretion and hyperglycemia, is characterized by partial maternal inheritance. Because the mitochondrial genome is known to be maternally transmitted, the aim of this study was to investigate whether the GK syndrome can be explained in terms of alterations of the mitochondrial DNA (mtDNA). For this purpose, pancreatic islets were isolated from adult and fetal control Wistar and diabetic GK rats. Using electron microscopy, the ultrastructural morphology of beta-cell mitochondria was analyzed in control and GK islets. It was found that the beta-cells of adult GK rats had a significantly smaller mitochondrial volume and an increased number of mitochondria per unit tissue volume as compared with the beta-cells of corresponding control islets. Moreover, mtDNA and mtRNA were isolated from the islets and, as a control tissue, from liver, and subsequently analyzed using Southern and Northern blot techniques. No major deletions or restriction fragment polymorphism could be detected in mtDNA from both GK liver and GK islets. The mtDNA sequence of the transfer RNAleu(UUS) gene was identical in both strains of rats. mtDNA contents of fetal GK islets and fetal GK liver were not different from those of fetal Wistar rats. However, adult GK islets contained markedly less mtDNA than the corresponding control islets, contrary to the mtDNA contents of adult liver, which were similar in the two strains. The lower islet mtDNA contents were paralleled by a decreased content of islet mtRNA (12S ribosomal RNA and cytochrome b messenger RNA). Islet insulin messenger RNA contents were similar in GK and Wistar rats. In conclusion, our results do not support a role of a genetic defect in mtDNA as a cause of the GK syndrome. Instead, mtDNA damage may occur specifically in islet cells as a consequence of the disturbed metabolic environment of the adult GK rat. It is speculated that a long-lasting metabolic dysfunction may induce mtDNA damage and/or inhibition of mtDNA replication leading to a gradual and late decrease in the mitochondrial volume fraction and subsequently an impaired capacity for oxidative metabolism.

A method for obtaining monodispersed cells from isolated porcine islets of Langerhans.

Micro or macroencapsulation of islets of Langerhans have been proposed as a bioartificial pancreas. Encapsulation of dispersed single cells instead of porcine islets should improve the oxygenation of encapsulated tissue. The aim of this work was, therefore, to develop techniques for dissociating porcine islets and test cell viability and function. After islet isolation and purification, islets were dispersed into single cells with collagenase and DNAse in either an extracellular type ionic solution or a UW solution. After culture, islets or cells were perfused with Krebs buffer. Two consecutive stimulations from 2.8 mM to 20 mM glucose were performed. Viability of cells (trypan blue) was higher than 85% after dispersion in ES or UW solutions. Islets or dispersed cells responded similarly to both stimulations with a return to basal rate between stimulations. No difference was found between cell function cultured during 18 hours or 6 days. However, islet function was improved by a long period of culture. In conclusion, this study demonstrates that dissociated cells performed as well as native islets up to six days culture.

Reduction of acetaminophen interference in glucose sensors by a composite Nafion membrane: demonstration in rats and man.

Amperometric glucose sensors typically monitor the production of hydrogen peroxide generated in the course of the enzymatic oxidation of glucose. At the applied potential necessary to oxidize the peroxide produced, other species are also electroactive and contribute to the signal. Interference of ascorbate or urate has been effectively eliminated, but that resulting from the widely used analgesic acetaminophen is not. The aim of this work was to reduce this interference, which was found to be possible by introducing a membrane constructed of Nafion. We compared the in vitro sensitivity to acetaminophen of five Nafion sensors with that of five non-Nafion sensors with identical glucose sensitivity (2.0 +/- 0.4 vs 1.9 +/- 0.1 nA.mmol-1.l-1, NS): sensitivity to acetaminophen was 12.2 +/- 2.7 vs 30.8 +/- 6.3 nA.mmol-1.l-1, respectively (p < 0.05). These sensors were tested in rats by implanting in each animal one Nafion and one non-Nafion sensors. The in vivo sensitivity to glucose was similar (0.33 +/- 0.09 vs 0.30 +/- 0.05 nA.mmol-1.l-1, NS). The current generated by an acetaminophen infusion (plasma acetaminophen plateau = 140 +/- 10 mumol/l) was much decreased in the case of the Nafion sensor: 0.5 +/- 0.3 vs 2.0 +/- 0.7 nA, p < 0.05). Five Nafion sensors were implanted in the subcutaneous tissue of normal human volunteers who were given on oral dose of 500 mg acetaminophen.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one on steroidogenesis in gonads of rat fetuses.

1. The effect of 5 alpha-reductase inhibitor 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA) on the fetal rat steroidogenesis was examined. 18- and 20-day-old fetal ovaries were incubated in the presence of dcAMP (1 mM) and spironolactone (10 mM) and progesterone production was assessed with and without 4-MA (0.01-50 microM). 2. High concentrations of 4-MA significantly decreased the production of progesterone. 3. Similar inhibition of the testosterone synthesis was observed in 16-day-old fetal testes explanted either in the control medium (M199) or in the presence of LH (100 ng/ml) when treated with 4-MA. The effect was rapidly reversible in both gonads.

Ontogenesis of the in vitro response of rat testis to gonadotropin-releasing hormone.

The age-related evolution of the in vitro effects of a gonadotropin releasing hormone agonist ([D-Trp6]-GnRH) on the secretion of testosterone by the testis, cultured during 3 days on a Millipore filter floating on M199 medium, was studied during the perinatal period in the rat. The basal and luteinizing hormone (LH)-stimulated secretions by testes explanted on fetal day 14.5 were unaffected by the agonist. With fetal testes explanted on days 16.5 and 18.5 post-conception, the agonist inhibited, in a concentration-dependent manner, both basal and LH-stimulated secretions from the second or the third day of treatment onwards. With fetal and neonatal testes explanted on days 20.5, 21.5 and 31.5 post-conception, the GnRH agonist also had a long-term inhibitory effect on LH-stimulated secretion, but increased basal secretion. This stimulatory effect was already observed after 4 h of culture, and was maintained for 3 days. These results suggest that, during fetal development, the cellular mechanisms involved in the negative testicular response to GnRH are differentiated 3-5 days before those involved in the positive response. Lastly, after 3 days of preculture in hormone-free medium, fetal testes explanted on day 14.5 displayed long-term GnRH agonist inhibition of in vitro basal secretion of testosterone. This observation points out a spontaneous differentiation of the negative responsiveness to GnRH in the cultured fetal testis.

Responsiveness of rat ovaries to dcAMP in the perinatal period: evidence for an inhibitory influence in vivo.

The progesterone production by rat ovaries from 18-day-old fetuses to 6-day-old neonates was measured in vitro in the presence of dibutyryl cAMP (dcAMP, 1 mM). A pronounced decline was observed at the end of fetal life. The 5 alpha-reductase activity did not seem sufficient to explain this decrease. Preculture of the ovaries for 48 h in the basal medium enhanced responsiveness to the nucleotide. Addition of spironolactone, an inhibitor of 17 alpha-hydroxylase to dcAMP did not modify this evolution. 3 beta-hydroxysteroid dehydrogenase activity, detectable in fetal ovaries in the absence of dcAMP was also increased after preculture. In the presence of spironolactone and trilostane, the pregnenolone production showed the same evolution as progesterone and was also enhanced after culture. These results suggest the existence of inhibitory factor(s) present in vivo at the end of fetal life.