HSP47 expression in the hamster Sertoli cell: An immunohistochemical study.

HSP47, a chaperone whose main function is the maturation of collagen molecules, is considered a marker of fibrotic diseases. Increased collagen synthesis in the testis has been associated with various pathologies leading to testicular seminiferous tubule regression. Our aim was to study whether HSP47 is expressed in hamster Sertoli cells both in the adult and in two physiological situations of seminiferous tubule atrophy: irreversible testicular ageing and testicular regression due to short (reversible) photoperiod. Eighteen animals were divided as follows: a group of 6 young animals aged 6 months, a group of 6 animals aged 24 months, which were exposed to a long photoperiod, and a final group of 6 young animals subjected to a short photoperiod. Testicular samples were fixed in methacarn and an immunohistochemical technique was used to detect HSP47. A semiquantitative study of the expression of this protein was performed between tubular sections of aged animals with complete spermatogenesis and arrested spermatogenesis with tubular sections with arrest spermatogenesis of photoinhibited testes. Sertoli cells were positive for HSP47, the intensity being higher in tubular sections with arrested spermatogenesis in both aged and photoinhibited animals. Sertoli cells were positive for HSP47, the intensity being greater in tubular sections with arrested spermatogenesis in both aged and photoinhibited animals. Semiquantitative analysis corroborated this observation in the sense that the expression of this protein differed according to the functional state of the seminiferous tubules. Thus, the intensity index of immunoreactivity was significantly higher in tubular sections with arrested spermatogenesis in aged animals compared with regressed animals, and in the latter compared with those whose tubular sections showed complete spermatogenesis. In conclusion, HSP47 expression in Sertoli cells was found for the first time in mammals. Moreover, increased expression seemed to be related to the degree of atrophy of the seminiferous epithelium and to the reversible or non-reversible physiological state of the seminiferous epithelium.

Heterogeneity of mesenchymal cells in human amniotic membrane at term.

There is increasing interest in understanding the tissue biology of human amniotic membrane (hAM) given its applications in medicine. One cellular component is mesenchymal cells, which can be extracted, cultured and differentiated "in vitro" into various cell types. These studies show that there is heterogeneity among mesenchymal cells. The aim of this work is to study the membrane "in situ" to determine whether this cellular heterogeneity exists. The hAMs were obtained from caesarean deliveries at term and analyzed by histological techniques. Types I-III mesenchymal cells and Hofbauer were distinguished by light microscopy. Histochemically, mesenchymal cell types showed successively increasing positivity to: PAS, vimentin, fibronectin, and Concanavalin-A; VGEF, TGF-ß2, PDGF-C, FGF-2. By the semiquantitative point of view, the percentage of Type II cells was 60%, significantly higher than the other types. With transmission electron microscopy, an intermediate cell type between II-III was observed. Strong vesiculation of the rough endoplasmic reticulum (RER) with exocytosis was observed. In addition, an accumulation of a similar material to the extracellular matrix in the RER caused its dilation especially in type III cells. Some of this material acquired a globular structure. These structures were also found free in the extracellular matrix. In conclusion, the mesenchymal cells of the fibroblastic layer of the hAMs studied are heterogeneous, with some undifferentiated and others with a probably senescent fibroblastic phenotype with accumulation in their RER of fibronectin. These results may be of interest to extract mesenchymal cells from hAMs for use in regenerative medicine and to better understand the mechanisms of fetal membrane rupture.

Cellular Modifications in Spermatogenesis during Seasonal Testicular Regression: An Update Review in Mammals.

Testicular regression occurs during the non-breeding season in many mammals. This affects spermatogenesis, resulting in decreased or arrested activity. Both lead to a decrease or cessation in sperm production. In recent years, the cellular mechanisms that lead to infertility in males in non-reproductive periods have been studied in very different species of mammals. At the start of the present century, the main mechanism involved was considered as an increase in the apoptotic activity of germ cells during the regression period. The loss of spermatogonia and spermatocytes causes not only a decrease in spermatogenesis, but an arrest of the seminiferous epithelium activity at the end of regression. Recently, in some mammal species, it was found that apoptosis is the usual mechanism involved in epithelium activity arrest, although it is firstly atrophied by massive desquamation of the germ cells that are released from their binding with the Sertoli cells, and which are shed into the lumen of the seminiferous tubule. In other species, it has been shown that not only germ cell apoptosis, but also Sertoli cell apoptosis, including decreased proliferative activity, spermatophagy or autophagy, are involved in testicular regression. Furthermore, the most recent studies indicate that there are multiple patterns of seminiferous epithelium regression in seasonally breeding animals, which may not only be used by different species, but also by the same ones to reproduce in the best conditions, ensuring their survival. In conclusion, at this time, it is not possible to consider the existence of a paradigmatic cellular mechanism in the involution of the seminiferous epithelium applicable to all male mammals with seasonal reproduction, rather the existence of several mechanisms which participate to a greater or lesser extent in each of the species that have been studied to date.

Proliferation, apoptosis, and number of Sertoli cells in the Syrian hamster during recrudescence after exposure to short photoperiod†‡.

The Sertoli cell (Sc) has been described as a quiescent cell once the animal has reached sexual maturity. Syrian hamster is an animal that displays testicular regression due to short photoperiod, during which process germ cells and Sc are removed through apoptosis. The aim of this work was to investigate histochemically whether the spontaneous testicular recrudescence processes after exposure to a short photoperiod lead to an increase in Sc proliferative activity in order to restore the normal population. Three spontaneous recrudescence groups were established: initial (IR), advanced (AR), and total (TR) recrudescence, which were compared with animal undergoing the regression process (mild: MRg, strong: SRg, and total: TRg) and animals in long photoperiod (Controls). Histological sections were submitted to histochemical techniques for detecting apoptotic and proliferative Sc with bright-field and fluorescence microscopy. For each group, the proliferative Sc index (PScI) and apoptotic Sc index (AScI), and the total number of Sc were obtained. The results revealed the existence of Vimentin+/TUNEL+ as well as Vimentin+/PCNA+ cells. The PScI was significantly higher in TRg and IR than in the other groups. The AScI was only significantly higher in MRg and SRg with respect to the other groups. The total number of Sc increased among TRg, IR, and AR, reaching values similar to those of the Controls. In conclusion, the increase in Sc proliferation from final regression and recrudescence, accompanied by a similar rate of apoptosis to the Control group, is the cause of the restoration of the Sc population during spontaneous recrudescence.

Lectin-binding pattern of glycoconjugates during spontaneous testicular recrudescence in Syrian hamster (Mesocricetus auratus) after exposure to short photoperiod.

Lectin histochemistry was used to characterise glycoconjugates and cellular apoptosis in the seminiferous epithelium and interstitium of hamster testis during spontaneous recrudescence. An increase in the LTA lectin affinity was observed in spermatids in the Golgi phase. An increase in labelling of PNA and Con-A lectin in acrosome of spermatids (acrosome phase) as well as increased labelling with Con-A in spermatids (cap phase) was observed. Spermatocytes showed decreased affinity with PNA and AAA lectins and an increase in positivity for LTA and GNA lectins. Spermatogonia showed a slight decrease in positivity to WGA and an increase in labelling with Con-A and a decreased affinity for the AAA lectin. At the end of recrudescence, all these germinal cells showed a similar pattern to the control. The Sertoli cells showed a gradual decrease in labelling with the GNA lectin and the Leydig cells an increase in labelling with Con-A and GNA. Particularly unusual was the observation of apoptotic spermatocytes and spermatids positive for PNA, GNA, AAA and Con-A, together with spermatocytes positive to LTA. In conclusion, the normal lectin pattern is recovered during testis recrudescence and germ cell apoptotic activity is low, as is observed by specific lectins for germ cells in apoptosis.

Testicular histomorphometry and the proliferative and apoptotic activities of the seminiferous epithelium in Syrian hamster during spontaneous recrudescence after exposure to short photoperiod.

Syrian hamsters are photoperiodic rodents in which reproduction, including testicular function, is stimulated by long photoperiod exposure and curtailed by exposure to a short photoperiod. The objectives of this study were to characterize the testis histomorphometrically and to determine the role of the proliferation and apoptosis phenomena in the recovery of the seminiferous epithelium during spontaneous recrudescence after exposure to short photoperiod. The study was performed using conventional light microscopy, proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling staining, image analysis software, and transmission electron microscopy in three recrudescence groups: initial recrudescence (IR), advanced recrudescence (AR) and total recrudescence (TR). The results morphometrically pointed to the gradual recovery of the testicular and tubular volumes, as well as of the seminiferous epithelium. Among the IR and AR groups, the increase in testicular and tubular volumes was accompanied by an increase in tubular diameter and length, with an increase in interstitial volume. From AR to TR, there was an increase in the tubular and total volumes, but, in this case, with a gradual increase in tubular diameter. Recovery of the seminiferous epithelium was accompanied by changes in apoptosis and proliferation activities. The first decreased halfway through the process, and the second remained higher than the control levels throughout the recrudescence stage. Alterations in the spermatozoa were ultrastructurally observed, which indicated that spermiogenesis was not yet completely normal. In conclusion, spontaneous testicular recrudescence in Syrian hamster comprises two histomorphometrical phases: the first related to an increase in tubular length and diameter and interstitial volume and the second depending principally on the gradual increase in tubular diameter. The restoration of the seminiferous epithelium is due to apoptosis reaching normal values in the AR group accompanied by higher proliferative activity than that observed in the Control group.

Identification of Proliferative and Apoptotic Sertoli Cells Using Fluorescence and Confocal Microscopy.

Sertoli cells, the testicular somatic cells of the seminiferous epithelium, are vital for the survival of the epithelium. They undergo proliferation and apoptosis during fetal, neonatal, and prepubertal development. Apoptosis is increased in certain situations such as exposure to many substances, for example, toxics, or short photoperiod in the non-breeding season of some mammals. Therefore, it has always been considered that Sertoli cells that reach adulthood are quiescent cells, that is to say, nonproliferative, do not die, are terminally differentiated, and whose numbers remain constant. Recently, a degree of both proliferation and apoptosis has been observed in normal adult conditions, suggesting that consideration of this cell as quiescent may be subject to change. All this make it necessary to use histochemical techniques to demonstrate whether Sertoli cells are undergoing proliferation or apoptosis in histological sections and to allow the qualitative and quantitative study of these. In this chapter, we present two double-staining techniques that can be used for identifying Sertoli cells in proliferation or apoptosis by fluorescence microscopy. In both, the Sertoli cells are identified by an immunohistochemistry for vimentin followed by an immunohistochemistry for PCNA or a TUNEL histochemistry.

The Use of Lectin Histochemistry for Detecting Apoptotic Cells in the Seminiferous Epithelium.

Lectin histochemistry is commonly used to characterize the pattern of glycoconjugates in cells and tissues. Recent studies show that alterations in these glycoconjugates are associated with the entry of cells into apoptosis. A widely used technique for the detection of apoptotic cell death is TUNEL. In this chapter, we study the sensitivity of both techniques to identify apoptotic cells in the testis of photo-inhibited Syrian hamster.

The death of sertoli cells and the capacity to phagocytize elongated spermatids during testicular regression due to short photoperiod in Syrian hamster (Mesocricetus auratus).

In the Syrian hamster (Mesocricetus auratus), an animal that displays testicular regression due to short photoperiod, germ cells are removed by apoptosis during this process and the apoptotic remains are phagocytized by Sertoli cells. The aim of this work was to investigate morphologically whether the testicular regression process due to short photoperiod leads to the apoptosis of Sertoli cells, and whether, during testicular regression, the elongated spermatids are eliminated through phagocytosis by Sertoli cells. To this end, we studied testis sections during testicular regression in Syrian hamster subjected to short photoperiod by means of several morphological techniques using conventional light microscopy (hematoxylin and eosin [H&E], semi-thin section vimentin, immunohistochemistry, SBA lectin, and TUNEL staining), fluorescence microscopy, and transmission electron microscopy (TEM). H&E and semi-thin sections identified Sertoli cells with a degenerated morphology. Greater portion of Sertoli cells that were positive for TUNEL staining were observed especially during the mild regression (MR) and strong regression (SR) phases. In addition, TEM identified the characteristic apoptotic changes in the nucleus and cytoplasm of Sertoli cells. Moreover, during testicular regression and using light microscopy, some elongated spermatids were seen in basal position next to the Sertoli cell nucleus. This Sertoli phagocytic activity was higher in MR and SR phases. TEM confirmed this to be the result of the phagocytic activity of Sertoli cells. In conclusion, during testicular regression in Syrian hamster due to short photoperiod, when germ cells are known to be lost through apoptosis, there is morphological evidences that Sertoli cells are also lost through apoptosis, while some elongated spermatids are phagocytized and eliminated by the Sertoli cells.

Development of the Preverbal Visual Assessment (PreViAs) questionnaire.

BACKGROUND: Visual cognitive functions of preverbal infants are evaluated by means of a behavioral assessment. Parents or primary caregivers may be appropriate to certify the acquisition of certain abilities. AIMS: To develop the PreViAs (Preverbal Visual Assessment) questionnaire to assess visual behavior of infants under 24 months of age and to assess the normative outcomes for each item at each age. STUDY DESIGN: The process was divided into three phases: scale development (items and domains generation), pilot testing, and exploratory analysis. RESULTS: The final version of the PreViAs questionnaire consisted of 30 items, each related to one or more of four domains (visual attention, visual communication, visual-motor coordination, and visual processing). For the exploratory analysis, 298 children (159 boys and 139 girls) were recruited. Their ages ranged from 0.1 to 24 months (mean, 11.2 months). Internal consistency of the questionnaire was high for all domains (Cronbach's α coefficients of 0.85-0.94). CONCLUSIONS: The PreViAs questionnaire is a useful scale for assessing visual cognitive abilities of infants under 24 months of age. It is easy and feasible to complete by primary caregivers.

Effects of smoking during pregnancy on the optic nerve neurodevelopment.

BACKGROUND: Tobacco smoking during pregnancy alters neurodevelopment. Optical coherence tomography (OCT) provides precise measurements of the retinal nerve fiber layer (RNFL), which forms part of the central nervous system. AIMS: To assess using the OCT how smoking during pregnancy would affect optic nerve development as detected in human offspring. STUDY DESIGN: Visual examination and OCT were performed on a group of children (n=70; 4.15-13.50 years of age), classified as being exposed or not to maternal smoking during gestational period. The association between smoking during pregnancy and RNFL thickness was assessed by a linear regression analysis adjusted for possible confounding factors. RESULTS: Although visual outcomes did not differ between groups, a significant decrease in the RNFL thickness was found in the group of infants exposed to smoke (105.3 vs 95.6; p=0.002), even when adjusting for gestational age, birth weight or gender. CONCLUSIONS: OCT measurements show that intrautero exposure to tobacco smoke interferes with the development of the optic nerve.

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.

Histochemical demonstration of two subtypes of O-linked oligosaccharides in the rat gastric glands.

The gastric glands synthesize glycoproteins whose oligosaccharides are linked to the peptide core mainly by the O-glycosidic bond, specifically removed by beta-elimination procedure. Our aim was to research the possibility of the existence of two subtypes of O-linked oligosaccharides with a different behavior to the removal procedure. The lectins from peanut (PNA) and Maackia amurensis (MAA-I) were histochemically used as markers of the O-linked oligosaccharides. Sections were also pretreated with beta-elimination and/or peptide N-Glycosidase F (PNGase-F) for the specific removal of O- and N-linked oligosaccharides, respectively. The lectin GNA, which mainly labels to N-linked oligosaccharides, was used to test the correct working of PNGase-F. To test the possibility that the beta-elimination treatment could remove the terminal sialic acid residues, the lectin LFA was used. The surface epithelium was negative to PNA, while it became strongly positive when beta-elimination was performed for 1 day. This staining was resistant to PNGase-F, suggesting that PNA was labeling to O-linked oligosaccharides. However, after beta-elimination for 5 days this staining is not observed. A similar pattern appeared with MAA-I. We propose the existence of two subtypes of O-linked oligosaccharides: labile and resistant. The labile O-linked oligosaccharides are removed with beta-elimination for 1 day, unmasking the PNA-positive oligosaccharides. These oligosaccharides are resistant O-linked oligosaccharides because staining is abolished with longer treatment of beta-elimination. The results with MAA-I also support this suggestion. In summary, the labile O-linked oligosaccharides are removed with short treatment, while the resistant O-linked oligosaccharides need a stronger procedure (for 5 days).

Apoptosis and molecular pathways in the seminiferous epithelium of aged and photoinhibited Syrian hamsters (Mesocricetus auratus).

Aging and short photoperiod exposure induce germ cell apoptosis in the Syrian hamster; however, the specific germ cells affected and the molecular pathways triggered have not been elucidated. We analyzed germ cell apoptosis and the expression of the Fas/Fas-L system, Bcl-2 family, and p53 in aged and photoinhibited hamsters and compared with those young maintained in natural photoperiod. Aging increased apoptosis in spermatogonia and spermatocytes, but in photoinhibited hamster testes only an increase in apoptotic spermatocytes was observed. Apoptosis was higher in aged hamsters in stages I-IV, V-VI and VII-VIII. Aging increased apoptosis of spermatogonia in stages I-IV and V-VI. Apoptotic pachytene spermatocytes were significantly higher in stages I-IV, V-VI, and VII-VIII in aging. Apoptotic preleptotene and pachytene spermatocytes were higher in aging, but no differences were observed in leptotene-zygotene. Fas-L was expressed by Sertoli cells, of young, aged, and photoinhibited hamsters. Bcl-x(L) was strongly expressed in germ cells on young hamsters and slightly in aging and after short photoperiod exposure. Spermatocytes of photoinhibited hamsters were intensively stained with Fas, Bax, Bcl-xs/L, and p53. In conclusion, aging increases apoptosis in spermatogonia and spermatocytes, depending on the stage of the seminiferous epithelium cycle, whereas after a short photoperiod exposure only an increase in apoptotic spermatocytes is observed. The results suggest that Fas, Bcl-x(L), Bax, and p53 participate in germ cell apoptosis induction after short photoperiod exposure, whereas only Bcl-x(L) is involved in aging.

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

It 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.

Proliferation and apoptosis of spermatogonia in postpuberal boar (Sus domesticus) testes with spontaneous unilateral and bilateral abdominal cryptorchidism.

Cryptorchidism is a frequent male sexual disorder in mammals, which affects the histology of the tunica propria, interstitial tissue, blood vessels, seminiferous epithelium and testis functioning. In this paper, proliferation and apoptosis were examined in the seminiferous epithelium of both testes from unaffected boars and from boars suffering unilateral and bilateral cryptorchidism. In germ cells, proliferation was studied using the immunohistochemical PCNA technique, and apoptosis was analysed by in situ TUNEL labelling. An index was obtained for the proliferation and apoptosis observed in seminiferous tubules. In abdominal testes the epithelium contained few spermatogonia and Sertoli cells. In the testes of unaffected boars, numerous spermatogonia proliferated, whereas in cryptorchid testes such proliferation was lower and the proliferation/apoptosis ratio diminished. In the unaffected group, the TUNEL-positive germ cells were spermatogonia and spermatocytes in different phases of meiosis. In abdominal testes, the TUNEL-positive germ cells were spermatogonia alone. The apoptosis index of both abdominal and scrotal testes was similar. In conclusion, spontaneous cryptorchid testes showed a lower rate of spermatogonia proliferation in the seminiferous epithelium.

Proliferation and apoptosis in the seminiferous epithelium of photoinhibited Syrian hamsters (Mesocricetus auratus).

In the hamster, male reproductive quiescence is accomplished via testicular atrophy and the germinal epithelium is regressed to spermatogonia and spermatocytes after 8-14 weeks of short photoperiods. However, the cellular mechanisms involved in this process have not been elucidated. As it is suggested that the regulation of seasonal testicular activity is characterized by coordinated shifts in the relationships between mitosis, meiosis and apoptosis, the changes in the proliferative and apoptotic activity in the seminiferous epithelium of photoinhibited Syrian hamster were examined and compared with those maintained in natural photoperiod. The proliferative activity was studied using BrdU immunostaining, and germ cell apoptosis was assessed by in situ TUNEL labelling and transmission electron microscopy. A significant increase in the rate of apoptosis (percentage of TUNEL-positive spermatogonia + spermatocytes) was observed in photoinhibited animals (2.84 +/- 0.16) compared with those exposed to natural photoperiod (0.77 +/- 0.03, p < 0.05). The majority of apoptotic germ cells were spermatocytes and in some occasions spermatogonia. Germ cell apoptosis was confirmed by morphological characteristics: condensation of the chromatin and nuclear fragmentation. The rate of proliferation (percentage of BrdU-positive spermatogonia + preleptotene spermatocytes) was significantly higher in photoinhibited hamsters (42.7 +/- 2.6) compared with animals exposed to natural photoperiod (31.1 +/- 1.6, p < 0.05). After the exposure to a short photoperiod the apoptotic index positively correlated with the proliferative index (r = 0.8150, p < 0.05). In conclusion, the seminiferous epithelium of photoinhibited Syrian hamsters is characterized by an increased rate of apoptosis associated to an enhanced rate of proliferation.