Genetic parameters of biokinematic variables of the trot in Spanish Purebred horses under experimental treadmill conditions.

The purpose of this study was to estimate the genetic parameters of biokinematic variables in Spanish Purebred (SPB) horses in order to select those of sufficient interest to be measured in the pre-selection of the animals for possible inclusion in the breeding programme. Kinematic analysis of 130 SPB horses 4.6+/-1.5 years old were recorded at the trot (4m/s) on a treadmill. Genetic parameters were estimated using VCE software and a bivariate mixed animal model including age and stud as fixed effects and animal additive genetic effect and residual error as random effects. In general, heritabilities were high (0.33-0.88). The angular variables presented the lowest heritabilities, whereas the maximum height of the fore-hoof and the duration of swing phase in the hindlimb gave the highest scores. Genetic correlations were also very high, so it was possible to reduce the number of breeding programme characteristics to stride duration, hindlimb swing phase duration, range of stifle and elbow angles, minimal angle of carpus, and minimal retraction-protraction angle of the hindlimb.

Modulation of granulosa cell deoxyribonucleic acid synthesis and differentiation by activin.

FSH stimulates follicular growth through inducing granulosa cell proliferation. Our working hypothesis is that mitogenesis is facilitated by a locally produced growth/differentiation factor(s) that modulates FSH action in developing granulosa cells. The present study was undertaken to examine the effect of the related gonadal peptides activin and inhibin on granulosa cell proliferation. Monolayer cultures of granulosa cells isolated from preantral/early antral follicles in immature rat ovaries were established by incubation overnight in serum-free medium 199. Treatment was initiated with serum-free medium containing recombinant human (rh) activin and/or rh-inhibin in the presence or absence of FSH. After incubation for 18 h, medium was collected for progesterone determination (as a marker of cell differentiation), and the cell monolayers were incubated for 2 h in the presence of [3H]thymidine to measure DNA synthesis. Activin dose dependently (1-100 ng/ml) stimulated DNA synthesis (minimal effective dose, 1 ng/ml), whereas inhibin or FSH alone was without effect. When activin (1 ng/ml), but not inhibin, was present with FSH, the gonadotropin caused dose-dependent increases in [3H]DNA synthesis over a wide range of FSH concentrations (1-100 ng/ml). This property of activin was unaltered by the additional presence of inhibin (1-100 ng/ml). To analyze the role of cAMP in mediating the mitogenic action of FSH in the presence of activin, experiments were repeated substituting a membrane-permeable cAMP agonist, 8-bromo-cAMP (8br-cAMP; 0.1-3 mM). Similar to FSH, 8br-cAMP had no effect on granulosa cell DNA synthesis in the absence of activin. However, in the presence of activin (1 ng/ml) 8br-cAMP was stimulatory. The dose response to 8br-cAMP revealed a biphasic effect on DNA synthesis and differentiation: DNA synthesis rose to a maximum in the presence of 0.5 mM 8br-cAMP and declined thereafter. Progesterone synthesis only started to increase in the presence of 0.1 mM 8br-cAMP, rising to a maximum at 3 mM 8br-cAMP, at which concentration DNA synthesis was fully suppressed. We conclude that activin induces DNA replication in rat granulosa cells. In the presence of activin, FSH and 8br-cAMP are mitogens. These actions of FSH and 8br-cAMP occur at doses too low to stimulate steroidogenesis, emphasizing the role of intracellular cAMP tone in granulosa cell proliferation and differentiation.

Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells.

Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.

Ovarian thecal/interstitial androgen synthesis is enhanced by a follicle-stimulating hormone-stimulated paracrine mechanism.

To obtain direct evidence for FSH-stimulated paracrine signaling in the ovary, 21-day-old intact or hypophysectomized female Wistar rats received four sc injections of recombinant human FSH (rhFSH; total dose, 16-72 IU) at 12-h intervals. Ovaries were removed 48 h after the first injection to extract total RNA for Northern analysis of 17-hydroxylase/C17-20-lyase (cytochrome P450c17 alpha) mRNA or to isolate thecal/interstitial cells for assessment of basal and hLH-responsive androgen synthesis in vitro. In situ hybridization with a 35S-labeled cytochrome P450c17 alpha cRNA probe confirmed that expression of the cytochrome P450c17 alpha gene was specific to thecal/interstitial cells. The approximately 2.0-kilobase P450c17 alpha mRNA signal in ovarian total RNA from intact animals was increased approximately 5-fold by treatment with rhFSH (total dose, 72 IU) or PMSG (15 IU). This effect was shown to be dose dependent, with a approximately 2-fold increase in response to 16 IU (total dose) rhFSH. P450c17 alpha mRNA levels in isolated granulosa and thecal/interstitial cell total RNA from intact animals were compared to establish which was the principal cellular site of P450c17 alpha mRNA expression. The P450c17 alpha mRNA signal was undetectable in control granulosa cells and only barely discernible after treatment with 72 IU (total dose) rhFSH. In contrast, P450c17 alpha mRNA was abundant in control thecal/interstitial mRNA, and its level was increased 4- to 6-fold by treatment with rhFSH. Treatment of hypophysectomized animals with rhFSH did not consistently alter ovarian P450c17 alpha mRNA levels. During culture for 48 h in serum-free medium, basal androgen (androstenedione plus androsterone) production by thecal/interstitial cells from intact animals was unaffected by treatment with rhFSH in vivo, but hLH-stimulated androgen production by these cells was enhanced approximately 2-fold. Neither basal nor hLH-responsive androgen production by thecal/interstitial cells from hypophysectomized animals was altered by previous treatment with rhFSH in vivo. Treatment of thecal/interstitial cell cultures from both intact and hypophysectomized animals with inhibin (0.1-30 ng/ml), a putative granulosa-derived paracrine factor, did not measurably affect basal androgen synthesis, but potently enhanced LH-responsive androgen synthesis in vitro. Similarly, treatment of thecal/interstitial cell cultures with conditioned medium from FSH-treated granulosa cell cultures significantly enhanced LH-responsive, but not basal, androgen production. We conclude that treatment of pituitary-intact rats with "pure" FSH modulates thecal/interstitial cell androgen synthesis. Granulosa cells, but not thecal cells, possess FSH receptors, and thecal/interstitial cells are the principal ovarian sites of P450c17 alpha expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of 3 beta-hydroxysteroid dehydrogenase delta 5/delta 4-isomerase and cholesterol side-chain cleavage cytochrome P450 by activin in rat granulosa cells.

Activin is a dimeric protein implicated in the local control of follicular steroidogenesis. Using cultured rat granulosa cells, we previously showed that the effect of activin on FSH-induced progesterone synthesis changes with preovulatory follicular development, from positive regulation in nondifferentiated (immature) granulosa cells to negative regulation in preovulatory (mature) granulosa cells. The aim of the present study was to assess development-related effects of activin on the expression of enzymes crucial to progesterone synthesis: cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase (3 beta HSD). Nondifferentiated granulosa cells were isolated from the ovaries of estrogen-pretreated immature female rats that received no other treatment; differentiated granulosa cells were obtained from similar animals treated for 48 h with human FSH to induce preovulatory follicular development. Cells were cultured for 48 h in serum-free medium with and without human FSH and/or recombinant activin-A, and medium was collected for measurement of progestagens (progesterone, pregnenolone, and 20 alpha-dihydroprogesterone). In cultures of nondifferentiated granulosa cells, activin augmented the FSH-induced production of all three steroids. In differentiated granulosa cells, activin suppressed the FSH-stimulated production of progesterone and 20 alpha-dihydroprogesterone, but had no effect on pregnenolone. The presence of exogenous pregnenolone increased the overall production of progesterone, but did not alter qualitative steroidogenic responses to activin. To assess the interaction between FSH and activin on 3 beta HSD and P450scc messenger RNA (mRNA) expression, Northern blot analyses were performed on total RNA isolated from cultured granulosa cells. Treatment in vitro with FSH alone markedly enhanced the abundance of both the 3 beta HSD and P450scc mRNA transcripts in nondifferentiated and differentiated granulosa cells. FSH-stimulated expression of P450scc mRNA was further enhanced by cotreatment of nondifferentiated granulosa cells with activin. However, activin had no consistent effect on FSH-stimulated expression of 3 beta HSD mRNA in nondifferentiated cells. In differentiated granulosa cells, both mRNAs were suppressed more than 50% by the presence of activin. We conclude that the in vitro effects of activin on FSH-induced expression of 3 beta HSD and P450scc mRNAs in rat GC are similar: initially stimulatory (P450scc) or without effect (3 beta HSD), then becoming completely inhibitory. The mechanism of this development-dependent change in the granulosa cell response to activin remains to be elucidated.

Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.

Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.

Origins and consequences of the elongation of the human menstrual cycle during the menopausal transition: the FREEDOM Study.

The menopausal transition is characterized by the appearance of elongated cycles, which become longer and more frequent as menopause approaches. Several endocrine abnormalities have been attributed to these cycles; however, no quantitative studies of their causes and consequences exist to date. This study is based on sequential daily urinary concentrations of FSH, LH, estrone 3-glucuronide (E1G), and pregnanediol 3-glucuronide (PdG) from 34 women with perimenopausal menstrual irregularity (total of 289 cycles). The timing of ovarian response was determined as the day of E1G take-off (ETO). Other parameters measured were the mean FSH concentration before ETO (FSH(ETO)) and the midluteal levels of PdG, E1G, and LH. There was a strong parallelism between ETO and cycle length variability. FSH(ETO) levels increased gradually with ETO. Both ETO and FSH(ETO) were inversely related to luteal PdG and directly related to E1G. PdG and LH levels were inversely related. All comparisons were highly significant (P < 0.0001). We conclude that delayed ovarian response underlies the elongation of the menstrual cycle in the menopausal transition, which is likely to be caused by a temporary lack of ovarian responsiveness to FSH. A progressive decline in luteal PdG with increased E1G occurs in association with these trends.

Relationship between follicle-stimulating hormone levels at the beginning of the human menstrual cycle, length of the follicular phase and excreted estrogens: the FREEDOM study.

Although reproductive aging has been separately related to elevated FSH and shorter follicular phase (FP), the direct association between both parameters has not been investigated. Also, the exact effects of increased FSH on estrogen production are yet to be established.A large database of daily urinary concentrations of FSH, LH, and estrone 3-glucuronide (E1G) from 37 regularly menstruating women (median 11 cycles per patient) was used. Initial FSH levels (iFSH) were estimated as the mean value of d 1-5. The day of E1G take-off (ETO) was determined by an algorithm, and accordingly, the FP was divided into early (d 1 to ETO) and late (ETO+1 to LH peak). FP maximum and integrated E1G were calculated. Subjects were distributed according to their mean iFSH into three categories (5 to 10, and >10 IU/liter). There was a gradual decrease in FP length with increasing category (15.2 +/- 3.8, 14.1 +/- 3.6, and 13 +/- 2.6 d, respectively; P < 0.0001). A similar effect occurred in early FP (7.5 +/- 4, 6.4 +/- 3.7, and 5.4 +/- 2.7; P < 0.0001); in contrast, late FP was unaffected (7.7 +/- 2.1, 7.7 +/- 2.1, and 7.6 +/- 2.4; P = 0.86). No consistent increase in E1G was found with advancing iFSH category; however, women with mean initial LH higher than 6 IU/liter had significantly elevated maximum (P < 0.0001) and integrated (P = 0.002) E1G.FP length decreases in parallel with increasing iFSH, with a selective effect on the early FP. Increased FSH does not affect E1G; however, elevated initial LH level was related to higher E1G.

Protein kinase CK2 is altered in insulin-resistant genetically obese (fa/fa) rats.

Hepatic insulin receptor levels in 6-week-old obese (fa/fa) rats were about 2-fold lower than those from lean (Fa/-) rats, which agrees with their insulin-resistant state. Nuclear protein kinase CK2 activity and protein content in livers from obese (fa/fa) rats were similar to those of lean (Fa/-) animals but the cytosolic levels were reduced to half, due to a decrease in the 39-kD)a catalytic subunit. Marked increases in activity, due to rises in the 44-kDa and 39-kDa catalytic subunits, were seen in the 16000 x g sediments (M1) from insulin-resistant rats, with moderate changes in the 100000xg sediments (M2). The increase in CK2 binding to M1 did not require increases in the molecular chaperone grp94, which was unaltered in insulin-resistant rats.

The carboxy-terminal domain of Grp94 binds to protein kinase CK2 alpha but not to CK2 holoenzyme.

Surface plasmon resonance analysis shows that the carboxy-terminal domain of Grp94 (Grp94-CT, residues 518-803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2 alpha) under non-stressed conditions. A K(D) of 4 x 10(-7) was determined for this binding. Heparin competed with Grp94-CT for binding to CK2 alpha. CK2 beta also inhibited the binding of Grp94-CT to CK2 alpha, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94-CT. The use of CK2 alpha mutants made it possible to map the Grp94-CT binding site to the four lysine stretch (residues 74-77) present in helix C of CK2 alpha. Grp94-CT stimulated the activity of CK2 alpha wild-type but was ineffective on the CK2 alpha K74-77A mutant.

GnRH-induced calcium mobilisation and inositol phosphate production in immature and mature rat ovarian granulosa cells.

In rat ovarian granulosa cells the effects of GnRH are determined by the state of granulosa cell development with mainly inhibitory actions in immature cells and stimulatory actions in differentiated mature cells. These developmentally related effects of GnRH may arise from changes in either one or more of the signal transduction pathways activated by GnRH. The present study therefore measured downstream signalling events associated with the activation of the phospholipase C (PLC) signal transduction pathway in both mature and immature rat ovarian granulosa cells. Results showed that GnRH produced similar total inositol phosphate and intracellular calcium ([Ca2+]i) responses in both immature and mature granulosa cells. In contrast to the biphasic GnRH-induced [Ca2+]i response in pituitary gonadotropes, stimulation of the endogenously expressed GnRH receptor in both immature and mature granulosa cells produced a prompt monophasic rise in [Ca2+]i. This calcium transient was abolished by pretreating either cell type with a potent GnRH receptor antagonist or the PLC inhibitor U73122, demonstrating a GnRH receptor-specific activation of PLC. Similarly, pretreatment of cells with the [Ca2+]i antagonists thapsigargin or cyclopiazonic acid abolished the GnRH-induced calcium transient, whereas EGTA and nifedipine, a voltage-operated calcium channel (VOCC) antagonist, had no effect. These results suggest that in either immature or mature granulosa cells GnRH mobilises calcium from thapsigargin/cyclopiazonic acid-sensitive [Ca2+]i stores but does not involve the influx of extracellular calcium through VOCCs. We conclude that GnRH-induced stimulation of the PLC signal transduction pathway is independent of the stage of granulosa cell maturity and that alternative mechanisms account for the opposite effects of GnRH on gonadotrophin-induced steroidogenesis in mature and immature rat granulosa cells in vitro.

Variations in fibre composition of the gastrocnemius muscle in rats subjected to speed training.

Thirty-six adult Wistar rats were divided into three groups. One group was used as a control, and the other two underwent different training programmes in which greater relevance was attached to the intensity of exercise than to its duration. Samples of the red and mixed portions of m. gastrocnemius (caput lateralis) were stained with m-ATPase to determine the percentage of type I, IIA and IIB fibres, and with NADH-TR in order to quantify variations in the percentage of low staining intensity (FG) fibres. The most notable results obtained were: a) the ratio of type I type II fibres remained unchanged; b) the proportion of IIA fibres increased, while that of IIB fibres decreased correspondingly; c) FG fibres, which were virtually absent from the red portion, recorded a clear decrease which was more marked, and occurred more rapidly, than in IIB fibres. These differences were all statistically significant in the mixed portion of the muscle. Adaptive changes in fibre composition in the red portion were less marked.

Local regulation of primate granulosa cell aromatase activity.

Granulosa cells produce inhibin and activin, proteins implicated in the local regulation of preovulatory follicular development. To assess interactions among FSH, LH, inhibin and activin on primate granulosa cell aromatase activity, we studied primary granulosa cell cultures from the ovaries of the common marmoset (Callithrix jacchus), a monkey with an ovarian cycle similar in length to the human cycle. The distinctive action of activin was augmentation of gonadotropin-responsive aromatase activity throughout antral follicular development. FSH-stimulated aromatase activity in granulosa cells from immature follicles was augmented many fold by picomolar amounts of activin. In cell cultures from preovulatory follicles, the presence of activin stimulated basal aromatase activity in the absence of gonadotropin, as well as augmenting the action of LH. Thus, locally produced activin has the potential to modulate aromatase activity in developing ovarian follicles. By contrast, inhibin or inhibin alpha-subunit purified from bovine follicular fluid had minimal effects on aromatase activity. The only significant effect was slight suppression of FSH-inducible aromatase activity in granulosa cells from immature follicles at an inhibin concentration of 100 ng/ml. The finding that inhibin has a negligible effect on aromatase activity in granulosa cells from mature follicles suggests that it is unlikely to exert a physiologically significant influence on aromatase activity in vivo. However, evidence from other studies suggests that inhibin might affect aromatization indirectly through acting locally to modulate thecal androgen (aromatase substate) production. Therefore, both inhibin and activin have the potential to contribute at different levels to paracrine and autocrine regulation of follicular oestrogen synthesis.

Inhibin, activin, and follistatin. Potential roles in ovarian physiology.

The physiological significance of these results will not become clear until patterns of activin and inhibin protein production and the expression of their receptors have been more thoroughly characterized in relation to follicular development. Meanwhile, in situ hybridization studies on rat and monkey ovaries suggest that inhibin/activin beta-subunit mRNA (favoring synthesis of activin) is relatively abundant in granulosa cells of immature antral follicles, whereas alpha-subunit mRNA (favoring synthesis of inhibin) predominates in Graafian follicles. The increased production of follistatin associated with advanced preovulatory development would serve to further reduce the activin "tone" relative to inhibin (Fig. 1). At the level of protein action in vitro, the pattern emerging is that inhibin minimally affects granulosa cell steroidogenesis at any stage of follicular development, whereas activin has pronounced modulatory effects that alter with follicular maturity. As suggested previously,60 the ability of activin to enhance gonadotropin-responsive aromatase activity and simultaneously suppress progesterone production by mature granulosa cells has physiological implications in that it hints at a mechanism for promoting estrogen synthesis and simultaneously suppressing progesterone synthesis, which is precisely what occurs in the preovulatory follicle. The effects of inhibin and activin on human thecal androgen synthesis observed in vitro suggest how these proteins might act locally to modulate preovulatory follicular growth and estrogen synthesis in vivo (Fig. 2).57 In essence, we propose that activin acting at early stages of antral follicular development plays a role in follicular recruitment through sensitizing immature granulosa cells to the cytodifferentiative action of FSH. On the other hand, inhibin is more likely to play a role in preovulatory follicular selection and maintenance of follicular dominance. Studies of follicular fluid levels of androgen and estrogen in relation to granulosa cell aromatase activity indicate that the capacity of the theca interna to generate aromatase substrate (androstenedione) increases hand in hand with aromatase activity in the human preovulatory follicle. It has therefore been suggested that a positive feedback loop (granulosa on theca) exists that promotes thecal androgen synthesis and hence estrogen synthesis in this follicle.64 The discovery that inhibin production in vitro is greatest by granulosa cells isolated from preovulatory follicles strongly implicates inhibin as a component of this feedback loop.

Steroidogenesis in vitro of human granulosa-luteal cells pretreated in vivo with gonadotropin-releasing hormone analogs.

A modulatory role for gonadotropin-releasing hormone (GnRH) on granulosa cell functions has been demonstrated in animals. Because human granulosa cells have specific receptors for GnRH, we have compared steroidogenesis in vitro of cells pretreated in vivo with GnRH analogs (GnRH-a) with other ovarian stimulation regimens used in in vitro fertilization (IVF) cycles. Cells from 13 patients treated with a GnRH-a for 26.8 +/- 1.1 days during pituitary desensitization and ovarian stimulation with gonadotropins were cultured. Their steroidogenesis was evaluated in basal conditions as well as after stimulation with follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). Granulosa-luteal cells from 9 women treated with a combination of clomiphene citrate (CC) and gonadotropins were also cultured. Estradiol production in response to FSH was stimulated only in the GnRH-a-treated cells. Basal, FSH-, and hCG-induced progesterone (P) accumulation was higher in the CC-treated cells. The peak of P production was delayed 2 days in the GnRH-a-treated group. Only cells pretreated in vivo with GnRH-a accumulated 20 alpha-hydroxyprogesterone in culture. The results of the present study suggest that the use of GnRH-a during a long period of time for IVF affects the steroidogenic pathway of human granulosa-luteal cells. It seems that, at least in part, this change is due to the stimulation of the metabolizing enzyme 20 alpha-hydroxysteroid dehydrogenase by GnRH-a.

Failed in vitro fertilization of human oocytes: a cytogenetic analysis.

OBJECTIVE: To investigate possible causes of in vitro fertilization (IVF) failure. DESIGN: A retrospective cytogenetic study of human oocytes divided into four groups or, alternatively, into two groups according to fertilization rates and whether the patients became pregnant or not. Two additional groups included oocytes in which there was no or only partial fertilization. SETTING: Primary treatment of infertility in an institutional practice. PATIENTS, PARTICIPANTS: Two hundred fifty-three inseminated-unfertilized oocytes from 87 women entering the IVF program because of tubal, unknown, and male infertility. Immunological infertility was excluded. INTERVENTIONS: Ultrasound-guided transvaginal follicular aspiration. MAIN OUTCOME MEASURE(S): Planned after data collection. RESULTS: The rate of chromosome anomalies did not show any significant difference among the four groups established according to the fertilization rate and between pregnant and nonpregnant patients. Independently, our data identified male factor as responsible for 41%, chromosome anomalies 19.3%, oocyte immaturity 11.8%, and unknown etiology 41% of fertilization failures (based on analysis of 161 oocytes). CONCLUSIONS: Fertilization rate and pregnancy outcome after IVF are not related to the incidence of oocyte chromosome anomalies.

In vitro fertilization as a diagnostic and therapeutic tool in a patient with partial 17,20-desmolase deficiency.

OBJECTIVE: To present a case with 17,20-desmolase activity deficiency in which in vitro fertilization (IVF) served not only as a therapeutic approach but also as a diagnostic tool for the specificity of the enzymatic deficiency. DESIGN: IVF in the patient under study compared with a control group. All women treated with pure follicle-stimulating hormone (FSH). SETTING: IVF program at the Instituto Valenciano de Infertilidad. PATIENTS, PARTICIPANTS: A patient with primary amenorrhea, who was the subject under study, and seven normally cycling control patients undergoing IVF in the same series. INTERVENTIONS: IVF, steroidogenesis in vitro of granulosa-luteal cell obtained at ovum pick-up. MAIN OUTCOME MEASURE(S): Oocyte fertilization and embryo cleavage. Serum and follicular fluid (FF) levels of estradiol (E2), progesterone (P), testosterone (T), androstendione (A), 17 alpha-hydroxyprogesterone (17-OHP). In vitro accumulation of E2 and P. RESULTS: Ovulation induction with FSH was successful in achieving follicular development despite low circulating E2. Fertilization and cleavage rates were similar to the control subjects. The patient developed ovarian hyperstimulation. The lack of 17,20-desmolase activity was detected by normal P levels in serum and FF, high 17-OHP, and low T, A, and E2 levels in serum and FF. Granulosaluteal cell cultures in the presence of T restored normal E2 and P production in response to gonadotropins. CONCLUSIONS: In patients with 17,20-desmolase deficiency, follicular development, oocyte maturation, and fertilization can take place in a low estrogenic environment.

Differential effects of cholinergic blockade of anterior and posterior caudate nucleus on avoidance behaviors.

Groups of rats were trained in a one-trial passive avoidance task and then tested for retention 24 and 48 h later. They were also trained, in a single session, according to a one-way active avoidance paradigm. The effects of microinjections of atropine or of saline into the anterior caudate nucleus (CN) and of atropine into the posterior CN were assessed on these conditioned responses. Only those rats injected with atropine in the anterior CN showed a retention deficit in passive avoidance, while no effects on active avoidance became evident in any of the groups. These results suggest that cholinergic activity of the anterior CN is critically involved in memory consolidation of passive avoidance, but not in the processes mediating the acquisition of relatively simple active avoidance learning.

Skeletal muscle histochemistry in male and female Andalusian and Arabian horses of different ages.

Muscle biopsies were taken from the middle gluteal muscle of 143 untrained horses (83 Andalusians [AN] and 60 Arabians [AR]) ranging from 10 days to 24 years old. The horses were separated according to breed and sex and allotted to five age groups: A, 0 to three months; B, yearlings; C, two to three years; D, five to 10 years; and E, 11 to 24 years. There was an increase in the percentage of type I fibres (about 100 per cent) as well as a decrease in the percentage of type IIB fibres (AN, 50 per cent; AR, 40 per cent) over the five age groups. The percentage of type IIA fibres rose significantly over the first two or three age groups. The overall decrease in the subgroup IIB fibres with age was proportionally greater for IIB oxidative fibres (AN, 72 per cent; AR, 68 per cent) than for IIB non-oxidative fibres (AN, 5 per cent; AR, 34 per cent). The mean cross-sectional area of all three fibre types increased significantly with age. In any given age group, the mean relative cross-sectional area occupied by IIA fibres in the biopsy specimens was significantly greater in stallions than in mares, at the expense of IIB fibres.

Kinematic characteristics of Andalusian, Arabian and Anglo-Arabian horses: a comparative study.

The aim of this study was to compare the kinematic trot characteristics of three different breeds of horse: Andalusian (AN, n = 15), Arabian (AR, n = 7) and Anglo-Arabian (AA, n = 5) using standard computer-assisted videography (25 Hz). Linear, temporal and angular parameters in fore- and hind limbs were analysed in six randomly selected strides per horse. Normalised angle-time diagrams along the complete stride were obtained for all joints angles in each breed and specific kinematic characteristics were detected graphically. AA horses displayed longer swing durations in both limbs ans a shorter angular range of motion (ARM) in scapula and pelvis inclination and in shoulder, hip and forelimb retraction-protraction angles. At lift off, stifle and tarsal joint angles were more flexed. In general, only small differences were observed in AR horse kinematics when compared with the other 2 breeds. AN horses presented negative overtracking length, which was positive in AR and AA. In AN horses the elbow and carpal joints were more flexed at the moment of maximal elevation, elbow and fore-fetlock joints also exhibited a larger ARM due to a smaller angle at maximal flexion. In the hind limbs, tarsal, hind fetlock and retraction-protraction angles presented a larger ARM in AN horses due to greater maximal flexion in the tarsal and hind fetlock joints. Fore- and hind fetlocks were also more flexed in horses from this breed. In conclusion, differences between kinematic variables at the trot were observed in the three breeds studied here, mainly in forelimb joints. The most outstanding feature was the greater forelimb flexion recorded in AN horses than in the other breeds which is consistent with the elevated movements in this breed. In AA horses, the ARM of proximal joints involved in retraction protraction in both fore- and hind limbs was smaller. All the differences observed highlighted the idiosyncratic nature of the trot in each breed; this may influence the functional capacity of each breed.