Carbamate Pesticides: Shedding Light on Their Impact on the Male Reproductive System.

Carbamates are widely used and known around the world as pesticides in spite of also having medical applications. This class of chemicals is classified as acetylcholinesterase inhibitors, blocking acetylcholine hydrolyzation in a reversible manner. Their lack of species selectivity and their reported high toxicity can induce, upon exposure, adverse outcomes in male fertility that may lead to infertility. In addition, they are also considered endocrine-disrupting chemicals and can interfere with the hypothalamic-pituitary-testicular axis, essential for the normal function of the male reproductive system, thus being able to provoke male reproductive dysfunctions. Although the molecular mechanisms are not fully understood, various signaling pathways, such as those mediated by acetylcholine or kisspeptin, are affected by exposure to carbamates, thus compromising steroidogenesis and spermatogenesis. Over the last decades, several studies, both in vitro and in vivo, have reported a myriad of negative effects of carbamates on the male reproductive system. In this review, an up-to-date overview of the impact of carbamates on the male reproductive system is discussed, with an emphasis on the role of these compounds on acetylcholine regulation and the male endocrine system.

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.

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.

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.

Pesticides and Male Fertility: A Dangerous Crosstalk.

In recent decades, an increasing incidence of male infertility has been reported. Interestingly, and considering that pesticides have been used for a long time, the high incidence of this pathological state is concomitant with the increasing use of these chemicals, suggesting they are contributors for the development of human infertility. Data from literature highlight the ability of certain pesticides and/or their metabolites to persist in the environment for long periods of time, as well as to bioaccumulate in the food chain, thus contributing for their chronic exposure. Furthermore, pesticides can act as endocrine disrupting chemicals (EDCs), interfering with the normal function of natural hormones (which are responsible for the regulation of the reproductive system), or even as obesogens, promoting obesity and associated comorbidities, like infertility. Several in vitro and in vivo studies have focused on the effects and possible mechanisms of action of these pesticides on the male reproductive system that cause sundry negative effects, even though through diverse mechanisms, but all may lead to infertility. In this review, we present an up-to-date overview and discussion of the effects, and the metabolic and molecular features of pesticides on somatic cells and germinal tissues that affect germ cell differentiation.

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.