Eva Morales

CLOCK gene is implicated in weight reduction in obese patients participating in a dietary programme based on the Mediterranean diet.

Introduction:The success of obesity therapy is dependent on the genetic background of the patient. Circadian Locomotor Output Cycles Kaput (CLOCK), one of the transcription factors from the positive limb of the molecular clock, is involved in metabolic alterations.Objective:To investigate whether five candidate polymorphisms from CLOCK were associated with anthropometric, metabolic measures and weight loss in response to a behavioural weight reduction programme based on the Mediterranean diet.Methods:Five hundred overweight/obese subjects, aged 20–65 years, who attended outpatient clinics specializing in obesity, were studied. Anthropometric, biochemical and dietary intake variables were analysed. Effectiveness of the programme and weight loss progression during 28 weeks of treatment was assessed.Results:Four of five CLOCK SNPs selected were significantly associated with obesity variables (P<0.05). The genetic variation in the rs1801260 CLOCK was associated with obesity at baseline and also affected weight loss. Patients with the variant allele (G) lost significantly less weight i(P=0.008) compared with wild type. Repeated measures analysis showed that weight loss over time was significantly different between rs1801260 CLOCK variations (P=0.038). Carriers of the G allele displayed greater difficulty in losing weight than non-carriers. In this particular polymorphism, the frequency of short-time sleepers (⩽6 h per day) was greater in minor allele carriers than in non-carriers (59% vs 41%; P<0.05). CLOCK polymorphisms were also associated with significant differences in total plasma cholesterol at the completion of dietary treatment (P<0.05).Conclusions:We have replicated previous studies showing a relationship between CLOCK gene polymorphisms and obesity. CLOCK rs1801260 SNP may predict the outcome of body weight reduction strategies based on low-energy diets.

Proliferation and Apoptosis in Aged and Photoregressed Mammalian Seminiferous Epithelium, with Particular Attention to Rodents and Humans

Imbalances in the proliferation and apoptosis processes are involved in numerous epithelial alterations. In the seminiferous epithelium, normal spermatogenesis is regulated by spermatogonia proliferation and germ cell apoptosis, and both processes are involved in diverse pathological alterations of the seminiferous epithelium. Other physiological phenomena including aging and short photoperiod, in which apoptosis and proliferation seem to play important roles, cause testicular changes. Aging is accompanied by diminished proliferation and increased apoptosis, the latter occurring in specific states of the seminiferous cycle and considered the cause of epithelium involution. However, there is no clear evidence concerning whether proliferation decreases in the spermatogonia themselves or is due to an alteration in the cell microenvironment that surrounds them. As regards the factors that regulate the process, the data are scant, but it is considered that the diminution of c-kit expression in the spermatagonia, together with the diminution in antiapoptotic factors (Bcl-x(L))) of the intrinsic molecular pathway of apoptosis play a part in epithelial regression. A short photoperiod, especially in rodents, produces a gradual involution of the seminiferous epithelium, which is related with increased apoptosis during the regression phase and a diminution of apoptosis during recrudescence. Proliferative activity varies, especially during the total regression phase, when it usually increases in the undifferentiated spermatogonia. In other species showing seasonal reproduction, however, decreased proliferation is considered the main factor in the regression of the seminiferous epithelium. Little is known about how both phenomena are regulated, although data in rodents suggest that both the intrinsic and extrinsic pathways of apoptosis contribute to the increase in this process. In conclusion, regression of the seminiferous epithelium in physiological situations, as in many pathological situations, is a result of alterations in equilibrium between the proliferation and apoptosis of germinal cell types. However, both physiological phenomena showed important differences as regard proliferation/apoptosis and their regulation pathways, probably as a result of their irreversible or reversible character.

Characterization of corpora amylacea glycoconjugates in normal and hyperplastic glands of human prostate

SummaryIt has been shown that there are sugars in corpora amylacea, but little attention has been focused on the expression of glycoconjugates in corpora amylacea of normal and hyperplastic prostatic glands. The present study characterizes and compares the expression of glycoconjugates in corpora amylacea of normal and hyperplastic prostatic glands of elderly men by using alcian blue (AB) stain and lectin histochemistry. Corpora amylacea were larger and more numerous in hyperplastic glands compared to normal glands. The stain with AB revealed the presence of sulfated and carboxyl components in corpora amylacea. In hyperplastic prostatic glands the sulfur and acid contents of corpora amylacea were increased. Lectin affinities of corpora amylacea from normal prostatic glands demonstrated the presence of fucose, mannose, sialic acid, N-acetyl galactosamine and N-acetyl glucosamine residues. In the hyperplastic glands the lectin binding pattern of corpora amylacea was qualitatively similar to normal glands, but an increase in GalNAc, sialic acid, mannose and fucose residues was observed. Normal prostatic glands showed a weak to moderate content of mannose residues, and in contrast a strong GNA and Con-A staining was observed in hyperplastic glands. MAA and SNA affinities indicated that the content of sialic acid residues was higher in hyperplastic glands compared with normal prostatic glands. Also NAcGal residues were increased in hyperplastic glands. Luminal secretion, secretory cells and apical border of epithelium showed a similar although more intense Lectin-binding pattern as compared with corpora amylacea both in normal and hyperplastic prostatic glands. Lectin histochemistry shows that the glycoconjugates expressed in the glandular epithelium are similar to those found in corpora amylacea both in normal and hyperplastic glands. In addition, in hyperplastic glands, where the corpora amylacea are higher in size and more numerous, the reaction to lectins is more intense especially with mannose and sialic acid residues. The results suggest that corpora amylacea are originated at least in part from prostatic secretion.

Effect of ageing on the proliferation and apoptosis of testicular germ cells in the Syrian hamster Mesocricetus auratus.

The cellular mechanisms implicated in the atrophy of seminiferous epithelium in ageing are currently under debate, although recent reports suggest that apoptosis may be the primary mechanism implicated in aged germ cell loss. Other investigators have suggested that changes in spermatogonial proliferation are also involved. In the present work, the changes in proliferation and apoptosis in the seminiferous epithelium of aged (24 months) Syrian hamsters were examined in concert and compared with those in young (6 months) animals. Proliferation of germ cells was studied by bromodeoxyuridine labelling and apoptosis was assessed by transmission electron microscopy and in situ TUNEL labelling. Aged animals showed a significant decrease in the numbers of total and proliferating spermatogonia plus preleptotene spermatocytes per unit volume and per testis and in the proliferative index (24.8 +/- 1.6%) compared with young animals (30.8 +/- 1.2%) (P < 0.05). The number of apoptotic spermatogonia plus spermatocytes per unit volume and the apoptotic index were significantly higher in aged animals (1.51 +/- 0.23% v. 0.77 +/- 0.04%; P < 0.05). Apoptosis was confirmed by morphological characteristics: condensation of the chromatin and nuclear fragmentation. In aged hamsters, tubular degeneration could be classified into several categories, showing an increase of apoptotic cells in tubular cross-sections characterized by maturation arrest in comparison with all other types. Spermatogonial proliferation was also diminished as seen in tubular cross-sections showing hypospermatogenesis, sloughing off of germ cells and maturation arrest. The results obtained in the present study suggest that the decrease in the proliferation of spermatogonia and the increase in apoptosis constitute two consecutive mechanisms correlated with the ageing of the seminiferous epithelium.

Histochemical study of glycoconjugates in active and photoperiodically-regressed testis of hamster (Mesocricetus auratus).

The objective of the present study was to characterize glycoconjugates of hamster testis in gonadally-active and -inactive states by lectin histochemical methods. Thirteen HRP- or digoxigenin-labeled lectins were used in samples obtained from fertile and photoinhibited hamsters. In gonadally-active hamsters, spermatozoa tails were stained with Con-A, HPA, PNA, UEA-I, LTA, AAA, WGA and LFA and weakly with GNA and RCA-I. Spermatozoa acrosomes were labeled with HPA, SBA, WGA and PNA. Spermatid acrosomes were labeled with SBA, RCA-I, PNA, and WGA. Staining with GNA and Con-A was found in the Golgi phase and HPA staining was found in the Golgi phase and maturated spermatids. Cytoplasm of spermatocytes was labeled with Con-A, GNA, LTA, AAA, RCA-I, HPA, WGA and LFA, whereas spermatocyte membranes were stained with Con-A, LTA and AAA. Spermatogonia were strongly labeled with Con-A and moderately labeled with AAA, WGA and LFA. Sertoli cells were positive after staining with Con-A, AAA, WGA, and LFA. The lamina propria was positive after staining with UEA-I, LTA, AAA and LFA. Leydig cells showed strong labeling with SBA, Con-A, GNA, SNA and MAA, moderate labeling with WGA, weak labeling with RCA-I, AAA and LFA. In gonadally-inactive hamsters, spermatocytes showed increased staining with HPA, PNA and AAA, whereas staining with Con-A, GNA and LTA had disappeared. Spermatogonia showed an increased labeling with AAA and WGA, but labeling with Con-A and LFA had disappeared. Sertoli cells were strongly labeled with GNA. Con-A and GNA staining was decreased in Leydig cells of gonadally-inactive hamsters but PNA and HPA staining was increased. The lamina propria in regressed testes showed intense labeling with PNA. These results suggest that histological, morphological and hormonal changes occurring in hamster testis during exposure to a short photoperiod are reflected in altered patterns of expression and distribution of N- and O-linked glycans.

Characterization of glycoside residues of porcine zona pellucida and ooplasm during follicular development and atresia

The glycoside residues (glycoconjugates, GC) of the zona pellucida (ZP) glycoproteins are important during the first phases of fecundation. Our aim in this work was to determine the lectin affinity pattern of porcine ZP in order to analyze the changes that take place during: (a) preantral folliculogenesis, (b) the follicular atresia process, and (c) antral growth. Several prepubertal and adult pig ovaries and different sized antral follicles were used. Conventional carbohydrate histochemical techniques and peroxidase and digoxigenin (DIG) lectins were used to reveal the acid groups and the glycosidic residues of the ZP. It was seen that the ZP forms in the preantral follicles throughout their growth period. In primordial and primary follicles, ZP in the process of formation showed neutral GC. SBA, RCA‐I, MAA, WGA lectins, and AAA after methylation‐saponification (MS) were positive in the ZP of primordial and primary follicles. The affinity for SBA, RCA‐I, MAA, and WGA increased in the multilaminar‐primary follicles and new affinities for UEA‐I and LFA were observed. After MS, AAA, SNA, PNA, and SBA reactivity was observed. The ZP of antral follicle oocytes of different sizes showed the same lectin pattern as multilaminar‐primary follicles. The oocyte ooplasm and the follicular fluid of large antral follicles showed less affinity for WGA and LFA lectins and less intensive staining with AB (pH 2.5). Atresia did not change the antral or preantral follicle oocyte ZP lectin pattern. In conclusion, the follicles showed substantial changes in their ZP glycosidic composition as they developed, especially, during the change from primary to multilaminar‐primary follicles. The ZP glycosidic composition showed no significant change during the growth of antral follicles and follicular atresia in our study. Mol. Reprod. Dev. 75: 1473–1483, 2008.

Influence of histological degree of seminiferous tubular degeneration and stage of seminiferous cycle on the proliferation of spermatogonia in aged Syrian hamster (Mesocricetus auratus)

The ageing testis is associated with germ loss in the seminiferous epithelium and a decrease in spermatogonia proliferation. In this work, we study whether the stages of the seminiferous epithelium cycle and/or the degree of histological tubular degeneration resulting from ageing is related with this decrease in spermatogonia proliferation. Eleven hamsters were used, five aged 6 months and six aged 24 months. In both groups, the proliferative activity was studied by BrdU immunostaining. The number of BrdU‐positive and BrdU‐negative cells was measured, providing the overall proliferation index in adult and aged testes. The mean number of BrdU‐positive cells was also determined for each degree of histological degeneration of seminiferous epithelium, and a spermatogonia proliferation index was obtained for each stage of the seminiferous cycle. Ageing caused an overall decrease in the BrdU‐positive cell percentage and a decrease in the number of BrdU‐positive cells in the tubular sections with hypospermatogenesis, the sloughing of germ cells and maturation arrest, these changes being similar in both young and old animals. The spermatogonia proliferation index was only seen to be significantly lower in ageing hamster in stages VII–VIII of the seminiferous epithelium cycle. In conclusion, the overall decrease in proliferation observed in aged seminiferous epithelium is correlated with an increase in the number of degenerated sections of the seminiferous tubules, and this decrease is a phenomenon which occurs in specific stages of the seminiferous cycle.