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L Morales, R Chavez, ME Ayala and R Dominguez

The modulating effects of ovarian innervation reaching the ovary through the suspensory ovarian nerve on the reactivity of the ovaries to gonadotrophins were analysed. Juvenile rats (32 days old), with or without unilateral or bilateral section of the superior ovarian nerve, were injected with 8 iu of pregnant mare serum gonadotrophin (PMSG), 10 iu of human chorionic gonadotrophin (hCG) or with 8 iu of PMSG followed 56 h later with 10 iu of hCG. Treatments were given immediately after surgery or 4 days later, and the rats were killed on the day of first vaginal oestrus. In rats with unilateral section, treatment with PMSG did not induce full ovulatory response by the denervated ovary whether the treatment was applied immediately or 4 days after surgery (0/11 rats treated immediately ovulated vs 5/5 (sham) and 11/12 (control, P < 0.05 Fisher's exact probability test), and 4/19 did when treatment was done 4 days after surgery vs 8/10 (sham) and 11/12 (control, P < 0.05). The rats with bilateral section receiving the same hormonal treatment, PMSG administration, ovulated. The number of ova shed by the left ovary was similar to those of the control, while the right ovary released fewer ova. Stimulation with hCG immediately after unilateral section did not induce ovulation in normal or denervated ovary. When the treatment was applied 4 days after surgery, ovulation was observed only in the innervated ovary. In the rats with bilateral section, hCG injection induced ovulation in both ovaries. In those rats with unilateral section of the superior ovarian nerve, the treatment with PMSG + hCG given immediately after surgery resulted in a compensatory ovulation by the innervated ovary (the number of ova shed/ovulating animal was significantly higher than those released by control or sham-operated animals: left section, number of ova shed by the right ovary 7.6 +/- 0.3 vs 5.5 +/- 0.8 and 4.9 +/- 1 respectively, P < 0.05; right section, number of ova shed by the left ovary 10.2 +/- 0.6 vs 4.4 +/- 1.1 and 7.0 +/- 0.9, P < 0.05), while the denervated one showed a lower ovulation rate as well as a smaller number of ova shed than those by the control animals. When the hormonal replacement was given 4 days after surgery, such compensatory ovulation was observed in the left ovary of those rats with a section of the right nerve (14.3 +/- 2.6 vs 4.4 +/- 1.1 and 6.5 +/- 1.1, P < 0.05). When the PMSG + hCG treatment was applied to animals with bilateral section of the superior ovarian nerve, the ovulation rate by the right ovary was significantly lower than in control and sham-operated treated animals (2/10 vs 11/11 and 6/7, P < 0.05). Because the ovaries receive innervation through the superior ovarian nerve, the ovarian plexus and the vagus nerve, the results obtained in unilateral denervated animals suggest that the innervation of the ovary via the superior ovarian nerve regulates in a stimulatory way the effects of gonadotrophin resulting in ovulation. The ovulation induced by hormonal treatment of rats with bilateral section of the superior ovarian nerve suggests that the effects of bilateral section on ovulation are not the addition of the effects of left and right denervation, implying the existence of a modulatory effect in gonadotrophin action on ovulation via other neural pathways.

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C Moran, L Morales, U Quiroz and R Dominguez

We report the effects that sectioning the superior ovarian nerve of infantile female rats has on their follicular development at different ages before puberty. Compared with the control group, sham-operated animals showed a significant decrease in the number of measured follicles in right and left ovaries, although no difference in the follicular atresia ratio was observed. Animals with a sectioned left superior ovarian nerve (SON), killed 12 days after surgery had a significant increase in the number of follicles in the ovaries. Most of the follicles were atretic. Sectioning the right SON induced contrasting effects in the ovaries of animals killed 4 and 16 days after surgery. Rats with a denervated (right) ovary showed a decrease in the number of follicles and a greater number of atretic follicles compared with the control group, whereas the innervated (left) ovary showed an increase in measured follicles compared with the control group. Bilateral sectioning had no apparent effect on the total number of follicles measured, although an increased number of atretic follicles in both ovaries was observed. Animals with a unilateral section of the SON, killed 8 and 12 days after surgery, showed a decrease in serum concentrations of estradiol. In turn, animals killed 16 days after surgery showed a significant increase in estradiol and a decrease in the progesterone serum concentration. These results suggest that sympathetic innervation of the ovary via the SON has a stimulatory role in the regulation and differentiation of follicular growth.

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F Gaytan, C Bellido, J L Romero, C Morales, C Reymundo and E Aguilar

Abstract

Macrophages are a common cell type in the testicular interstitium of the rat and are morphologically and functionally related to Leydig cells. We investigated the number of macrophages and Leydig cells in long-term (24 weeks) hypophysectomized (LTHX) or sham-operated rats. LTHX rats showed a 76% decrease in the number of macrophages, whereas the number of Leydig cells was only slightly decreased (by 18%). The profile areas of both macrophages and Leydig cells were very much decreased (46% and 66% respectively).

Sham-operated and LTHX rats were treated with vehicle or human FSH and LH (hFSH/hLH; 75 IU/kg body weight per day) for 1 week. This treatment induced a 286% increase in the number of macrophages and a 32% increase in the number of Leydig cells in LTHX rats. The profile areas of macrophages and Leydig cells were also increased (212% and 184% respectively). About 80% of macrophages showed vacuolization of the cytoplasm. Gonadotrophin treatment did not induce changes in cell numbers in sham-operated animals but about 30% of macrophages showed large cytoplasmic vacuoles.

Vehicle- or hormone-treated LTHX rats were given a single injection of ethylene dimethane sulphonate (EDS) and killed 72 h later. Leydig cells were absent from the testicular interstitium of sham-operated rats but there were large numbers of dead Leydig cells (about 40% of the pre-existing population) in the testicular interstitium of LTHX rats 3 days after EDS treatment. Complete clearance of the testicular interstitium from EDS-killed Leydig cells was found in LTHX rats treated with hFSH/hLH. These results indicate that the decreased number and size and the defective function of testicular macrophages in LTHX rats can be restored by treatment with gonadotrophins.

Journal of Endocrinology (1994) 140, 399–407

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A. Ulloa-Aguirre, P. Damián-Matsumura, R. Espinoza, R. Dominguez, L. Morales and A. Flores

ABSTRACT

Anterior pituitary glands were removed from neonatally androgenized (100 μg testosterone propionate) female rats and normal controls at 5, 10, 18, 21, 30, 60 and 90 days of age, and the multiple forms of FSH present within them were separated by chromatofocusing (pH range 7·5–4·0). Additional pituitary glands from intact adult males (90 days old) were also studied for comparative purposes. All animal groups exhibited multiple forms of immunoactive FSH within a pH range of 7·5–4·0, as well as an additional FSH form obtained after the addition of 1·0 mol NaCl/l to the chromatofocusing column (salt peak). In animals 5–30 days old (controls and androgenized) the majority of FSH applied to the chromatofocusing columns was recovered within the salt peak (45-85% of total FSH immunoactivity recovered). However, as the animals aged, more FSH immunoactivity focused within less acidic regions (isoelectric point (pI) 5·9–5·0); pituitaries from animals 60 days old contained the greatest proportion of FSH focused within this pH range (controls, 39·2±0·6%; androgenized, 23·1 ±0·9% of total immunoactivity recovered; P < 0·03 vs animals 30 days old for both experimental groups). This shift towards less acidic FSH was attenuated in androgenized animals compared with the controls (P<0·01).

In control adult rats, the chromatofocusing distribution pattern of pituitary FSH varied according to the day of the oestrous cycle. Pituitary extracts from control rats decapitated during the morning of pro-oestrus, oestrus and day 1 of dioestrus exhibited the highest proportion of immunoactive FSH (23·2–28·8% of total) focused within a pH range of 5·9–5·0, whilst only 10·4–11·6% of FSH from androgenized rats and those on day 1 of dioestrus was recovered within this pH range (P<0·05). In control animals decapitated during the morning of prooestrus and oestrus, 10–26% of FSH focused within the most alkaline region (pI 7·5–6·0); the chromatofocusing pattern of pituitary FSH from the neonatally androgenized animals was characteristic, in that no more than one peak (1·5±0·5% of total) was detected in this alkaline region. In the adult male rats, the majority of pituitary FSH eluted from the chromatofocusing columns within a pH of 4·9–4·0 (52·4±1·2% of total FSH immunoactivity) and the salt peak (pH <4·0) (33·1 ±2·4 of total). All FSH isoforms obtained after chromatofocusing represented α and β dimers as disclosed by size exclusion chromatography.

The results strongly suggest that a cyclic or 'female' pattern of hypothalamic and gonadal secretion leads the anterior pituitary towards the production of less acidic FSH isoforms, whereas a tonic or 'androgenic' type of secretion, as that present in adult males and females with the androgen-induced anovulatory syndrome, leads more to the production of strongly acidic FSH isoforms. The finding of qualitative and quantitative differences among normally cycling and androgenized animals gives further support for the concept of the existence of a sexual dichotomy in terms of the type of FSH synthesized by the anterior pituitary gland.

Journal of Endocrinology (1990) 126, 323–332

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MA Luque, N Gonzalez, L Marquez, A Acitores, A Redondo, M Morales, I Valverde and ML Villanueva-Penacarrillo

Glucagon-like peptide-1 (GLP-1) has been shown to have insulin-like effects upon the metabolism of glucose in rat liver, muscle and fat, and on that of lipids in rat and human adipocytes. These actions seem to be exerted through specific receptors which, unlike that of the pancreas, are not - at least in liver and muscle - cAMP-associated. Here we have investigated the effect, its characteristics, and possible second messengers of GLP-1 on the glucose metabolism of human skeletal muscle, in tissue strips and primary cultured myocytes. In muscle strips, GLP-1, like insulin, stimulated glycogen synthesis, glycogen synthase a activity, and glucose oxidation and utilization, and inhibited glycogen phosphorylase a activity, all of this at physiological concentrations of the peptide. In cultured myotubes, GLP-1 exerted, from 10(-13) mol/l, a dose-related increase of the D-[U-(14)C]glucose incorporation into glycogen, with the same potency as insulin, together with an activation of glycogen synthase a; the effect of 10(-11) mol/l GLP-1 on both parameters was additive to that induced by the equimolar amount of insulin. Synthase a was still activated in cells after 2 days of exposure to GLP-1, as compared with myotubes maintained in the absence of peptide. In human muscle cells, exendin-4 and its truncated form 9-39 amide (Ex-9) are both agonists of the GLP-1 effect on glycogen synthesis and synthase a activity; but while neither GLP-1 nor exendin-4 affected the cellular cAMP content after 5-min incubation in the absence of 3-isobutyl-1-methylxantine (IBMX), an increase was detected with Ex-9. GLP-1, exendin-4, Ex-9 and insulin all induced the prompt hydrolysis of glycosylphosphatidylinositols (GPIs). This work shows a potent stimulatory effect of GLP-1 on the glucose metabolism of human skeletal muscle, and supports the long-term therapeutic value of the peptide. Further evidence for a GLP-1 receptor in this tissue, different from that of the pancreas, is also illustrated, suggesting a role for an inositolphosphoglycan (IPG) as at least one of the possible second messengers of the GLP-1 action in human muscle.

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AM Corbacho, Y Macotela, G Nava, L Torner, Z Duenas, G Noris, MA Morales, G Martinez De La Escalera and C Clapp

Members of the prolactin (PRL) hormonal family have direct effects on endothelial cell proliferation, migration and tube formation. Moreover, isoforms of PRL may function as autocrine regulators of endothelial cells. Bovine brain capillary endothelial cells (BBCEC) express the PRL gene, while anti-PRL antibodies inhibit BBCEC proliferation. Here, we show the expression of the PRL gene into various PRL isoforms in endothelial cells from the human umbilical vein. Reverse transcription-polymerase chain reaction of total RNA from human umbilical vein endothelial cells (HUVEC) detected the full-length PRL mRNA as well as a 100 bp smaller PRL transcript similar to the one previously reported in BBCEC. HUVEC were positive to PRL immunocytochemistry. In addition, various PRL immunoreactive proteins were detected in HUVEC extracts and HUVEC conditioned media by metabolic labelling immunoprecipitation analysis. These PRL immunorelated proteins had apparent molecular masses of 60, 23, 21, 16 and 14 kDa. In contrast to previous findings in BBCEC, HUVEC conditioned media contained very little PRL bioactivity as determined by the selective bioassay of Nb2 cell proliferation. Moreover, some polyclonal or monoclonal antibodies directed against PRL stimulated HUVEC proliferation, in contrast to the inhibitory effect seen in BBCEC. The present findings extend the previous observations about the expression of PRL gene in endothelial cells from bovine brain capillaries to human cells of the umbilical vein, implicating that endothelium from different types of vessels and species share the expression of PRL gene but may differ in the putative autocrine role of the PRL isoforms expressed.