Growth hormone (GH) treatment acts on the endocrine and autocrine/paracrine GH/IGF-axis and on TNF-α expression in bony fish pituitary and immune organs.

There exist indications that the growth hormone (GH)/insulin-like growth factor (IGF) axis may play a role in fish immune regulation, and that interactions occur via tumour necrosis factor (TNF)-α at least in mammals, but no systematic data exist on potential changes in GH, IGF-I, IGF-II, GH receptor (GHR) and TNF-α expression after GH treatment. Thus, we investigated in the Nile tilapia the influence of GH injections by real-time qPCR at different levels of the GH/IGF-axis (brain, pituitary, peripheral organs) with special emphasis on the immune organs head kidney and spleen. Endocrine IGF-I served as positive control for GH treatment efficiency. Basal TNF-α gene expression was detected in all organs investigated with the expression being most pronounced in brain. Two consecutive intraperitoneal injections of bream GH elevated liver IGF-I mRNA and plasma IGF-I concentration. Also liver IGF-II mRNA and TNF-α were increased while the GHR was downregulated. In brain, no change occurred in the expression levels of all genes investigated. GH gene expression was exclusively detected in the pituitary where the GH injections elevated both GH and IGF-I gene expression. In the head kidney, GH upregulated IGF-I mRNA to an even higher extent than liver IGF-I while IGF-II and GHR gene expressions were not affected. Also in the spleen, no change occurred in GHR mRNA, however, IGF-I and IGF-II mRNAs were increased. In correlation, in situ hybridisation showed a markedly higher amount of IGF-I mRNA in head kidney and spleen after GH injection. In both immune tissues, TNF-α gene expression showed a trend to decrease after GH treatment. The stimulation of IGF-I and also partially of IGF-II expression in the fish immune organs by GH indicates a local role of the IGFs in immune organ regulation while the differential changes in TNF-α support the in mammals postulated interactions with the GH/IGF-axis which demand for further investigations.

Elevated amh gene expression in the brain of male tilapia (Oreochromis niloticus) during testis differentiation.

Anti-müllerian hormone (AMH) is expressed in male embryos and represses development of müllerian ducts during testis differentiation in mammals, birds and reptiles. Amh orthologues have been identified in teleosts despite them lacking müllerian ducts. Previously we found sexually dimorphic aromatase activity in tilapia brains before ovarian differentiation. This prompted us to search for further dimorphisms in tilapia brains during sex differentiation and see whether amh is expressed. We cloned the tilapia amh gene and found that it contains 7 exons but no spliced forms. The putative protein presents highest homologies with Amh proteins of pejerrey and medaka as compared to other Perciformes. We analysed amh expression in adult tissues and found elevated levels in testes, ovary and brain. Amh expression was dimorphic with higher levels in XY male brains at 10-15 dpf, when the gonads were still undifferentiated and gonadal amh was not dimorphic. Male brains had 2.7-fold higher amh expression than gonads. Thereafter, amh levels decreased in the brain while they were up-regulated in differentiating testes. Our study indicates that amh is transcribed in male brains already at 10 dpf, suggesting that sexual differentiation may be occurring earlier in tilapia brain than in gonads.

Environmental effects on fish sex determination and differentiation.

Environmental factors affect the sex ratio of many gonochoristic fish species. They can either determine sex or influence sex differentiation. Temperature is the most common environmental cue affecting sex but density, pH and hypoxia have also been shown to influence the sex ratio of fish species from very divergent orders. Differential growth or developmental rate is suggested to influence sex differentiation in sea bass. Studies in most fish species used domestic strains reared under controlled conditions. In tilapia and sea bass, domestic stocks and field-collected populations showed similar patterns of thermosensitivity under controlled conditions. Genetic variability of thermosensitivity is seen between populations but also between families within the same population. Furthermore, in the Nile tilapia progeny testing of wild male breeders has strongly suggested the existence of XX males in 2 different natural populations. Tilapia and Atlantic silverside studies have shown that temperature sensitivity is a heritable trait which can respond to directional (tilapia) or frequency dependent selection. In tilapia, transitional forms within a genetic sex determination (GSD) and environmental sex determination (ESD) continuum seem to exist. Temperature regulates the expression of the ovarian-aromatase cyp19a1 which is consistently inhibited in temperature masculinized gonads. Foxl2 is suppressed before cyp19a1. Recent in vitro studies have shown that foxl2 activates cyp19a1, suggesting that temperature acts directly on foxl2 or further upstream. Dmrt1 up-regulation is correlated with temperature-induced male phenotypes. Temperature through apoptosis or germ cell proliferation could be a critical threshold for male or female sex differentiation.

Tilapia sex determination: Where temperature and genetics meet.

This review deals with the complex sex determining system of Nile tilapia, Oreochromis niloticus, governed by the interactions between a genetic determination and the influence of temperature, shown in both domestic and wild populations. Naturally sex reversed individuals are strongly suggested in two wild populations. This can be due to the masculinising temperatures which some fry encounter during their sex differentiation period when they colonise shallow waters, and/or to the influence of minor genetic factors. Differences regarding a) thermal responsiveness of sex ratios between and within Nile tilapia populations, b) maternal and paternal effects on temperature dependent sex ratios and c) nearly identical results in offspring of repeated matings, demonstrate that thermosensitivity is under genetic control. Selection experiments to increase the thermosensitivity revealed high responses in the high and low sensitive lines. The high-line showed approximately 90% males after 2 generations of selection whereas the weakly sensitive line had 54% males. This is the first evidence that a surplus of males in temperature treated groups can be selected as a quantitative trait. Expression profiles of several genes (Cyp19a, Foxl2, Amh, Sox9a,b) from the gonad and brain were analysed to define temperature action on the sex determining/differentiating cascade in tilapia. The coexistence of GSD and TSD is discussed.

Genetics of sex determination in tilapiine species.

We identified DNA markers linked to sex determining genes in six closely related species of tilapiine fishes. The mode of sex determination differed among species. In Oreochromis karongae and Tilapia mariae the sex-determining locus is on linkage group (LG) 3 and the female is heterogametic (WZ-ZZ system). In O. niloticus and T. zillii the sex-determining locus is on LG1 and the male is heterogametic (XX-XY system). A more complex pattern was observed in O. aureus and O. mossambicus, in which markers on both LG1 and LG3 were associated with sex. We found evidence for sex-linked lethal effects on LG1, as well as interactions between loci in the two linkage groups. Comparison of genetic and physical maps demonstrated a broad region of recombination suppression harboring the sex-determining locus on LG3. Sex-specific recombination suppression was found in the female heterogametic sex. Sequence analysis showed the accumulation of repetitive elements in this region. Phylogenetic analysis suggests that at least two transitions in the mode of sex determination have occurred in this clade. This variation in sex determination mechanisms among closely related species makes tilapias an excellent model system for studying the evolution of sex chromosomes in vertebrates.

Mapping of sox2 and sox14 in tilapia (Oreochromis spp.).

Sox genes encode transcription factors that are involved in a variety of embryonic developmental pathways. Sox2 and Sox14 are located on the same chromosomal arm in several mammalian and bird species and on the basis of comparative maps were suggested as candidate genes for the major sex-determining locus on tilapia LG3. We have sequenced the sox2 and sox14 genes in four tilapia species and mapped them to different chromosomes, LG17 and LG23 respectively. Although excluded as being one of the major sex-determining genes so far mapped in tilapia, sox14 did fall within a QTL region for growth, stress response, embryonic mortality and a minor effect on sex determination.

Temperature effects along the reproductive axis during spawning induction of grass carp (Ctenopharyngodon idella).

For grass carp (Ctenopharyngodon idella) raised in the Ivory Coast (with water temperatures of 26-31 degrees C), induced spawning is obligatory for fry production. However, ovulation rates following hormonal treatment are often low. We hypothesized that high temperatures are an inhibiting factor for the reproductive axis (brain-pituitary-gonad) in these conditions. By in vivo and in vitro experiments, we tried to determine the thermosensitive steps during spawning induction. We compared gonadotropin and maturation-inducing steroid (MIS) profiles during a spawning induction at controlled temperatures of 24 and 28 degrees C in relation to ovulation success. We performed pituitary cell cultures and ovarian fragment incubations at controlled temperatures. The ovulation rate was lower at 28 degrees C (10%) than at 24 degrees C (36%). At the pituitary level, we found only minor thermal impacts on GnRH-stimulated LH release, but our data suggest an increase of the dopaminergic inhibition by high temperatures. The main effects were found at the ovary level, where ovary responsiveness to gonadotropin by MIS synthesis was disturbed, as well as oocyte responsiveness to MIS triggering final maturation, and probably ovulation. These results show the importance of regulating temperature during spawning induction to ensure a high rate of ovulation.

Environment and sex determination in farmed fish.

A plasticity of gonadal sex differentiation was reported in the 1930s following exogenous steroid treatments in fish, but demonstration that environmental factors (temperature, pH, density and social interactions) could influence the sex ratio in gonochoristic species has been relatively recent. In fish, as in reptiles and amphibians displaying environmental sex determination, the main environmental factor influencing sex seems to be temperature (TSD=Temperature Sex Determination). In most thermosensitive species (some Atherinids, Poecilids, Cichlids: tilapias, goldfish, a Siluriform, a flatfishellipsis) male to female ratio increases with temperature and/or ovarian differentiation is induced by low temperatures. Conversely, in some rare species (Dicentrarchus labrax, Ictalurus punctatus), high temperatures may produce female-biased sex ratios and/or low temperatures promote male-biased sex ratios. In the hirame Paralichthys olivaceus, both high and low temperatures induce monosex male populations while intermediate temperatures yield a 1:1 sex ratio (U-shape curve). Fish show particularities in their TSD patterns since mono-sex populations are generally not produced at extreme temperatures, suggesting the existence of strong temperature/genotype interactions. In reptiles, amphibians and fish displaying TSD, temperature treatments must be applied at a critical sensitive period, relatively similar to the hormone sensitive period. In gonochoristic fish, steroid hormones with estrogens in females and 11-oxygenated androgens in males, are probably key physiological steps in the regulation of gonadal sex differentiation. Cytochrome P450-aromatase, enzyme catalysing conversion of androgens to estrogens, seems to be a critical enzyme for ovarian differentiation. Molecular mechanisms of thermosensitivity have been addressed in two species tilapia Oreochromis niloticus and the hirame, where aromatase gene expression is down-regulated by masculinizing temperature treatments. Furthermore, in tilapia the gene expression of 11 beta-hydroxylase (a key enzyme involved in the synthesis of 11-oxygenated androgens) does not appear to be affected by temperature treatments.

Hypothyroidism induces type I iodothyronine deiodinase expression in tilapia liver.

In the current study, the authors examined the effects of experimentally induced hypothyroidism on peripheral thyroid hormone metabolism and growth in two closely related tilapia species: the Nile tilapia (Oreochromis niloticus) and the slower growing black tilapia (Sarotherodon melanotheron). Hypothyroidism, induced by administration of 0.2% methimazole through the food, significantly decreased plasma T(3) and T(4) in both species. This decrease in circulating thyroid hormones was accompanied by an increase in hepatic type II deiodinase (D2) and a decrease in hepatic type III deiodinase (D3). Hepatic type I deiodinase (D1), which is barely expressed in euthyroid tilapia, was significantly upregulated during hypothyroidism. The changes in hepatic D1 and D2 enzyme activity were paralleled by changes in D1 and D2 mRNA levels, indicating pretranslational regulation. Hypothyroidism also resulted in severe growth retardation that was accompanied by an increase in condition factor. Because hyperthyroidism has been shown to decrease the condition factor, these results suggest that thyroid hormones play an essential role in the control of proportional body growth in fish. The authors conclude that (1) hepatic D1 expression is induced by hypothyroidism in tilapia, (2) the changes in hepatic iodothyronine deiodinases during hypothyroidism in tilapia are predominantly regulated at a pretranslational level, and (3) thyroid hormones are involved in the control of proportional body growth in fish.

Search for genes involved in the temperature-induced gonadal sex differentiation in the tilapia, Oreochromis niloticus.

In the tilapia, Oreochromis niloticus, sex is determined by genetic factors (XX/XY) but temperature can also influence the gonadal sex differentiation. Elevated temperatures of 35 degrees C can generate functional male phenotypes if applied before and during sexual differentiation. The genes and mechanisms by which temperature acts on the cascade leading to sex differentiation have been investigated. Two strategies have been followed: 1) Search for novel genes by differential display, and 2) Expression studies of candidate genes. Genetically all-female and all-male progenies were reared at 27 degrees C (natural temperature) and at 35 degrees C (masculinizing treatment) and gonads dissected. Using differential display, we isolated a 300 bp cDNA (MM20C) from temperature-masculinized females. Virtual northern analysis revealed a 1.2 kb transcript in 35 degrees C treated females and males, but hardly any expression in natural females (27 degrees C). Semi-quantitative RT-PCR established a several-fold increase in MM20C expression in 35 degrees C masculinized fry. Elevated expression was observed in natural males (27 degrees C) with higher levels detected in those reared at 35 degrees C. Furthermore, we have analyzed as a candidate gene the P450 11beta-hydroxylase, an important androgen steroidogenic enzyme. Low levels of expression were found in natural males. This coincides with low concentrations of 11 ketotestosterone in the gonads before and during gonadal sex differentiation. Higher expression levels of 11beta-hydroxylase were detected in male gonads at 35 degrees C but levels in phenotypic males were similar to those found for natural females. Previous results reported that expression of aromatase is repressed by masculinizing treatments. Our study demonstrated that masculinizing-temperature can also stimulate the expression of other gene(s).

Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus.

In the tilapia Oreochromis niloticus, sex is determined genetically (GSD), by temperature (TSD) or by temperature/genotype interactions. Functional masculinization can be achieved by applying high rearing temperatures during a critical period of sex differentiation. Estrogens play an important role in female differentiation of non-mammalian vertebrates. The involvement of aromatase, was assessed during the natural (genetic all-females and all-males at 27 degrees C) and temperature-induced sex differentiation of tilapia (genetic all-females at 35 degrees C). Gonads were dissected between 486--702 degree x days. Aromatase gene expression was analyzed by virtual northern and semi-quantitative RT-PCR revealing a strong expression during normal ovarian differentiation concomitant with high levels (465 +/- 137 fg/g) of oestradiol-17 beta (E2-17 beta). This was encountered in gonads after the onset of ovarian differentiation (proliferation of both stromal and germ cells prior to ovarian meiosis). Genetic males exhibited lower levels of aromatase gene expression and E2-17 beta quantities (71 +/- 23 fg/ g). Aromatase enzyme activity in fry heads established a sexual dimorphism in the brain, with high activity in females (377.9 pmol/head/hr) and low activity in males (221.53 pmol/head/hr). Temperature induced the masculinization of genetic females to a different degree in each progeny, but in all cases repression of aromatase expression was encountered. Genetic males at 35 degrees C also exhibited a repression of aromatase expression. Aromatase brain activity decreased by nearly three-fold in the temperature-masculinized females with also a reduction observed in genetic males at 35 degrees C. This suggests that aromatase repression is required in the gonad (and perhaps in the brain) in order to drive differentiation towards testis development. Mol. Reprod. Dev. 59:265-276, 2001.

Endocrine and environmental aspects of sex differentiation in gonochoristic fish.

This paper reviews current knowledge concerning the endocrine and environmental regulation of gonadal sex differentiation in gonochoristic fish. In gonochoristic fish, although potentially active around this period, the hypothalamo-pituitary axis is probably not involved in triggering sex differentiation. Although steroids and steroidogenic enzymes are probably not the initial triggers of sex differentiation, new data, including molecular approaches, have confirmed that they are key physiological steps in the regulation of this process. Environmental factors can strongly influence sex differentiation in gonochoristic fish. The most important environmental determinant of sex would appear to be temperature. Interactions between environmental factors and genotype have been suggested for gonochoristic fish.

Effect of egg deprivation on sex steroids, gonadotropin, prolactin, and growth hormone profiles during the reproductive cycle of the mouthbrooding cichlid fish Oreochromis niloticus.

Various hormones were analyzed during the course of a reproductive cycle in the cichlid fish Oreochromis niloticus: plasma levels of the gonadal steroids 17beta-estradiol (E2), testosterone (T), 17, 20beta-OH progesterone (17,20beta-P), gonadotropin (taGtH), and plasma and pituitary concentrations of prolactin (tiPRL(I) and tiPRL(II)) and growth hormone (tiGH). Two categories of fish were sampled and sacrificed on days 1 and 3 postspawning and at 3-day intervals thereafter: typical incubating females (INC), and nonincubating females (NI), deprived of their eggs just after spawning. Such deprivation is known to suppress maternal behavior and to accelerate ovarian development and especially vitellogenesis, thus shortening the mean interspawning interval. In both groups, variations of the plasma concentrations of E2 and T appeared to depend on ovarian stages, and differences between groups appeared to reflect underlying differences in the kinetics of ovarian development. The observation of noticeable levels of 17,20beta-P in plasma before spawning, when high values of taGtH could also be detected in NI females, suggests the implication of this progestin in the control of final maturation events, as in some other teleosts. Moreover, 17,20beta-P, which was still detected a few days after spawning, but at low concentrations and only in the plasma of INC females, might play a role at the beginning of the reproductive cycle in incubating females (maternal behavior and/or slowing down of ovarian growth). The pituitary and plasma profiles of both tiPRLs isoforms appeared to depend mainly on the kinetics of ovarian development in each group of fish, suggesting a role during the beginning of vitellogenesis. However, the variance of plasma tiPRL(II), which was significantly enhanced during maternal behavior in INC females, also suggests an implication of this hormone in the control of that behavior. Concerning tiGH, comparison of the plasma profiles in INC and NI fish also suggest an influence on the control of maternal behavior, but a main effect of starvation of INC during mouthbrooding cannot be excluded.

Involvement of estrogens in the process of sex differentiation in two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus).

In order to study the physiological implication of sex steroid hormones in gonadal sex differentiation in fish, we first investigated the potential role of estrogens using two fish models: the rainbow trout (Oncorhynchus mykiss) and a tilapia species (Oreochromis niloticus). All experiments were carried out on genetically all-male (XY) and all-female (XX) populations. In vivo treatments with an aromatase inhibitor (ATD, 1,4,6- androstatriene-3-17-dione) result in 100% masculinization of an all-female population in rainbow trout (dosage 50 mg/kg of food) and 75.3% in tilapia (dosage 150 mg/kg of food). In tilapia, the effectiveness of the aromatase inhibition by ATD is demonstrated by the marked decrease of the gonadal aromatase activity in treated animals versus control. No masculinization is obtained following treatment with an estrogen receptor antagonist (tamoxifen) in both species. Aromatase and estrogen receptor gene expression was studied in rainbow trout by semi-quantitative RT-PCR in gonads sampled before, during and after sex-differentiation. Aromatase mRNA is specifically detected in female gonads, 3 weeks before the first sign of histological sex-differentiation, i.e., first female meiosis. Aromatase expression in male gonads is at least a few hundred times less than in female gonads. Estrogen receptor gene is expressed in both male and female gonads at all stages with no dimorphic expression between sexes. Specific aromatase gene expression before ovarian differentiation was also demonstrated using virtual Northern blot, with no expression detected in male differentiating gonads. From these results it can be concluded that estrogen synthesis is crucial for ovarian differentiation, and transcription of the aromatase gene can be proposed as a key step in that process in fish.

Effect of confinement stress on circulating levels of growth hormone and two prolactins in freshwater-adapted tilapia (Oreochromis niloticus).

The aim of the present study was to assess a potential link between confinement stress and prolactin (PRL), the hormone responsible for adaptation to a hypoosmotic environment in freshwater-adapted tilapia (Oreochromis niloticus). The effect of stress on plasma levels of the two tilapia PRL forms, tiPRLI (or tiPRL188) and tiPRLII (or tiPRL177), was examined along with the effects on plasma levels of cortisol and growth hormone (GH). In a preliminary study, various sampling protocols (immediate sampling; sampling one by one; anesthesia at 0.5, 1, 2 ml/liter phenoxyethanol) were tested for their ability to modify basal plasma PRL and cortisol. In fish sampled within 1 min of capture (immediate sampling), no changes in the plasma levels of these hormones were observed, whereas when fish were sampled one at a time, PRL levels did not change but cortisol levels were modified. The immediate sampling protocol was used to study the effects of 1 hr confinement stress, which induced a large increase in plasma cortisol levels as well as increases tiPRLI and tiPRLII levels with kinetics similar to those of cortisol. In contrast, plasma tiGH levels significantly decreased after 1 hr confinement. When this stress situation was removed, plasma cortisol and tiPRL levels decreased and plasma GH levels increased. Two and one-half hours later, values were not significantly different from those measured in control fish. In tilapia exposed to 24 hr confinement stress, similar changes in hormone levels were observed. However, after 24 hr confinement, only cortisol levels were significantly different from those measured in control fish. None of these stress conditions significantly changed plasma chloride levels. Together, these results indicate that both PRL and GH have important roles in the adaptive response of freshwater-adapted tilapia to confinement stress.

Feeding behaviour and food utilisation in tilapia, Oreochromis niloticus: effect of sex ratio and relationship with the endocrine status.

The feeding behaviour of male monosex, female monosex, and mixed groups of Oreochromis niloticus was studied under conditions of self-feeding. Feeding activity was observed almost exclusively during the light period. The food intake pattern was similar whatever the sex ratio, and voluntary food intake (VFI) appeared lower in the male monosex groups than in the others. Male monosex groups displayed higher specific growth rates (SGR) and a lower food conversion ratio than female monosex and mixed groups. The SGR of males was higher in the monosex than in the mixed groups, whereas females of mixed and monosex groups displayed no significant difference in SGR. The efficiency of food utilisation was also analysed: nutrient retention ratios were higher in male monosex than in female monosex and mixed groups. Males displayed a distinctly higher metabolic capacity. Differences in sex-related hormones (11 ketotestosterone = 11-KT, 17beta-Oestradiol = 17beta-E2) and a metabolic hormone (triiodothyronine = T3) were observed between males and females. The hypothesis of an involvement of these hormones in the higher metabolic capacity of males is discussed. The observed differences in feeding behaviour between the different groups also suggest an effect of social interactions on the efficiency of food conversion and thus on the differential growth of males and females.

Consequences of food restriction on short-term growth variation and on plasma circulating hormones in Oreochromis niloticus in relation to sex.

In tilapia, there is a sex-related growth difference between males and females. This study tried to detect any correlation between the somatic growth and the plasma endocrine status. For this, individually marked (Floytags) male and female tilapia (BW 82 +/- 10 g) were either starved or fed on different daily food rations (1, 2, or 3% of the biomass) during 15 days. We have found that specific growth rates (SGR) were positively and significantly related to feeding levels. Growth hormone (GH) plasma levels tended to increase with the decrease in food levels, and thus with the decrease in growth rate. No significant correlation was found between GH levels and SGR. Triiodothyronine (T3) levels in well-fed fish were higher than those in restricted fish (0 and 1%), but no differences in thyroxine (T4) levels were observed. No significant relationship was found between plasma levels of steroid hormones and feeding ration, even though 11-ketotestosterone (11-KT) levels tended to increase with the ration in fed males. SGR were not significantly different between males and females at the same feeding level, but taken as a whole, they were significantly different in favor of males (P < 0.05). There was no important difference in GH levels between the two sexes. Steroid hormones were, in general, higher in males for 11-KT and in females for 17 beta-estradiol (17 beta-E2). Males and females exhibited significant differences in T3 levels (respectively 4.25 +/- 0.18 and 2.71 +/- 0.09 pmol/ml), whatever the food ration, but no significant differences in T4 levels were observed except in the high-ration group. The correlation between T3 levels and SGR was low but stronger in males (r2 = 0.21; n = 90) than in females (r2 = 0.10; n = 105). The slope of the log-log regression of T3 levels with body weight was much lower in females (b = 0.87) than in males (b = 1.31). This relationship suggests the involvement of T3 in tilapia growth and probably in the differential growth between males and females. In both males and females, a significant but low correlation was observed between T3 and 11-KT levels (respectively r2 = 0.12; n = 82 and r2 = 0.08; n = 89), while no correlation was found between the levels of T3 and 17 beta-E2. T3 plasma levels were found to be the most different parameter between males and females. This hormone seemed to be involved in the control of somatic growth, and could explain the differential growth rate between males and females.

The metabolic clearance rate of estradiol-17 beta in rainbow trout, Salmo gairdneri R., estimated by both single injection and constant infusion methods: increase during oocyte maturation.

A dual cannulation of free-swimming rainbow trout is used to estimate the metabolic clearance rate (MCR) of Estradiol-17 beta (E2-17 beta) by both single injection and constant infusion methods. It is shown that E2-17 beta MCR changes significantly during the progress of oogenesis, mainly at the end of the sexual cycle. The same changes in MCR and very similar values are found with both single injection and constant infusion methods: MCR is stable (28.8 ml/hr/kg) from the postovulation period (throughout endogenous vitellogenesis) to early exogenous vitellogenesis. It decreases significantly during advanced exogenous vitellogenesis (18.7 ml/hr/kg) and increases clearly at the onset of oocyte maturation (40.9 ml/hr/kg). A direct relationship between MCR and plasma E2-17 beta occurs: Plasma E2-17 beta levels increase (advanced exogenous vitellogenesis) when MCR decreases. Then estradiol decline takes place at the same time that MCR reaches its highest values (oocyte maturation). An increase in MCR is probably one event required to allow the establishment of an appropriate hormonal environment for oocyte maturation.