SUMMARYDuring the non-breeding season some animals exhibit testicular atrophy, decreased testicular weight and reduced seminiferous tubule diameter accompanied by depletion of the seminiferous epithelium. Some cellular factors involved in this depletion are changes in germ cell proliferation and apoptosis. In the Syrian hamster this depletion has been studied histologically and in terms of the involvement of proliferation and apoptosis in the seminiferous epithelium of fully regressed testes. The objectives of this study included the histomorphometrical characterization of the testis and the determination of the proliferative and apoptotic activity of germ cells in the seminiferous epithelium during testicular regression owing to short photoperiod. The study was performed using conventional light microscopy (hematoxylin and eosin), 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 established regression groups: mild regression (MR), strong regression (SR), and total regression (TR). Morphometrically a gradual decrease in total tubular area and in the testicular, tubular, and epithelial volumes was observed during testicular regression. Interstitial and luminal volumes decreased from the MR group onwards. The tubular length decreased from MR to SR. As regards spermatogonial proliferation, only an initial decrease in proliferative activity was observed, whereas apoptotic germ cell activity increased throughout regression. The number of germ cells studied decreased throughout the process of testicular regression. In conclusion, testicular regression in Syrian hamster comprises two histomorphometrical phases, the first involving a decrease in seminiferous tubular diameter and volume and the second involving shortening of the seminiferous tubule and a decrease in interstitial volume. At the cellular level, there is an initial decrease in proliferation and increase in apoptosis involving all germ cells. At the end of regression, the proliferative and apoptotic activities of the spermatogonia recover the values observed prior to regression in preparation for recrudescence.

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.

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.

Lectins have been widely used to study the pattern of cellular glycoconjugates in numerous species. In the process of cellular apoptosis, it has been observed that changes occur in the membrane sugar sequences of these apoptotic cells. The aim of our work was to identify which lectins, out of an extensive battery of the same (PNA, SBA, HPA, LTA, Con-A, UEA-I, WGA, DBA, MAA, GNA, AAA, SNA), show affinity for germinal cells in apoptosis, at what stage of cell death they do so and in which germinal cell types they can be detected. For this, we studied testis sections during testicular regression in Syrian hamster (Mesocricetus auratus) subjected to short photoperiod. Several lectins showed an affinity for the glycoconjugate residues of germ cells in apoptosis: Gal β1,3-GalNAcα1, α-d-mannose, N-acetylgalactosamine and l-fucose. Furthermore, lectin specificity was observed for some specific germinal cells and in certain stages of apoptosis. It was also observed that one of these lectins (PNA) showed affinity for Sertoli cells undergoing apoptosis. Therefore, we conclude that the use of lectin histochemistry could be a very useful tool for studying apoptosis in the seminiferous epithelium because of the specificity shown towards germinal cells in pathological or experimentally induced epithelial depletion models.

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.