Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. II. The irradiated testes

In adult male mice exposed to 300 R X-irradiation, the sper-matogonial population was selectively killed except for the radioresistant type A_s stem cells. Type A spermatogonia were minimal two days after irradiation, when only 20% of the control population was present in stages 5-6; these were predominately single and paired undifferentiated cells. When multiple injections of ³HTdR were given between 2 and 3.5 days post-irradiation, 90–95% of these survivors in stages 4-6 became labeled. Enhanced proliferation of these stem cells, and at times when they were normally quiescent, led to restoration of all classes of spermatogonia by 11 days after irradiation. Several autoradiographic studies were undertaken to better characterize the radioresistant cells. In mice given single or multiple injections of ³HTdR prior to irradiation, there was appreciable retention of label by those type A_s sper-matogonia that had originally incorporated ³HTdR in stages 2-4. This labeling pattern was identical to that of the long-cycling A_s stem cells in nonirradiated testes. Since the long-cycling A_s stem cells are thought to be characterized by a prolonged G₁ or “A-phase” which is known to be a highly radioresistant portion of the cell cycle, it was clear why these cells could preferentially survive irradiation doses that killed other spermatogonial types. It was proposed that following germ cell depletion, as after irradiation injury, the long-cycling A_s survivors could be prematurely triggered from A-phase into DNA synthesis, thereby, initiating restoration of the germ cell population.     

The morphology and kinetics of spermatogonial degeneration in normal adult rats: An analysis using a simplified classification of the germinal epithelium

The phenomena of spermatogonial degeneration have been studied in normal adult rat testes using a simplified classification of the germinal epi-thelium based upon the six types of differentiating spermatogonia. The following features distinguished this from schemes based on acrosome development. Rather than 14 stages of unequal duration, there are only six stages, five of which are of the same length. The classification starts at the beginning of spermatogenesis with A¹ spermatogonia rather than at the onset of spermiogenesis. The classification is derived from actual biological events in spermatogenesis, namely generation times of spermatogonia, rather than upon arbitrary events in acrosome development. Most importantly, this new classification can be used with most types of preparations and in most experimental conditions. Examination of tubular whole mounts reveals that degeneration preferentially occurs in types A² and A³ and to a lesser extent A⁴ spermatogonia, and is rarely seen in generations of A¹ In or B cells. Deterioration is first manifested in clusters of cells joined by the intercellular bridges as they complete DNA synthesis and enter the G² phase of cell cycle. It is characterized by a denser staining of the nuclear membrane, coalescence of chromatin into several pyknotic bodies, and eventual extrusion of the nuclear mass, leaving a cytoplasmic ghost. The sequential steps in degeneration may often be traced from one end of a synctial chain to the other, suggesting that the process may start with just one cell and then spread via intercellular bridges to involve all spermatogonia within the clone. Quantitatively, degeneration is a relatively constant feature of spermatogonial development. Only 25% of the theoretically possible number of pre-leptotene spermatocytes are produced from the original population of A¹ spermatogonia; most of this loss is incurred during the maturation of A² and A³ generations. While the reason for spermatogonial degeneration in the normal germinal epithe-lium remain obscure, it is proposed that the numerical ratio of A spermatogonia to Sertoli cells may be a significant limiting factor.     

Role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of the rat testis

In the normal adult rat testis, type A₀ spermatogonia do not appear to participate to a significant extent in the production of spermatocytes, while type A₁ spermatogonia periodically initiate a series of divisions resulting in the production of spermatocytes and new type A₁ spermatogonia. The behavior of type A₀ and A₁ spermatogonia was investigated following administration of a single dose of x-rays (300 r) to the testis. Using whole mounts of seminiferous tubules, the type A₀ and A₁ cells were counted at various intervals after irradiation. At 8 and 13 days after irradiation, type A₁ spermatogonia reached lowest values, i.e., 6% and 3% of non-irradiated control, while type A₀ reached the lowest value, i.e., 62% of control at eight days. Thereafter the numbers of type A₀ and A₁ progressively increased to return to normal at 39 days. It was thus concluded that the type A₀ were comparatively more resistant to x-irradiation than type A₁ spermatogonia. To verify if the surviving type A₀ proliferated in the irradiated testis, animals were injected with ³H-thymidine three hours before they were sacrificed at various times after x-irradiation. In irradiated testes the labeling indices of the surviving type A (A₀, A₁–A₄) were the same as in the non-irradiated testes except in stages V-VI of the cycle of the seminiferous epithelium. While in the controls only 2% of type A cells were labeled at these two stages of the cycle, after irradiation the labeling index of type A reached a maximum of 31% at 13 days to return to control values by 39 days. Since at 13 days after irradiation type A₀ spermatogonia were the predominant component of the spermatogonial population, it was concluded that these cells must have incorporated ³H-thymidine and thereby contributed to the reconstruction of the spermatogonial population partially destroyed by irradiation.     

Spermatogonial stem cells in the albino rat

The existence of two classes of spermatogonial stem cells in the rat testis, i.e., reserve type A₀ spermatogonia and renewing, types A₁-A₄ spermatogonia, postulated by Clermont and Bustos-Obregon (′68), was reexamined in a quantitative analysis of type A spermatogonia in both whole mounts of tubules and in radioautographed sections of testes from animals killed at various times, up to 26 days, after one or multiple injections of ³H-thymidine. The cell counts obtained from whole mounts of tubules revealed that the number of isolated type A₀ cells per unit area of limiting membrane remained constant throughout the cycle of the seminiferous epithelium. Paired type A₀ spermatogonia also remained unchanged in number per unit area of basement membrane from stage I to stage VIII of the cycle. The low mitotic index of type A₀ spermatogonia (0.1%) indicated that these cells were not actively involved in the production of spermatogonia or spermatocytes during each cycle of the seminiferous epithelium and thus were considered as reserve stem cells. The type A₁ spermatogonia, which are formed during stage I of the cycle, remained resting until stage IX, when they undertook a series of four successive divisions resulting in the production of new type A₁ and Intermediate-type spermatogonia. An analysis of the labeling indices of type A spermatogonia obtained from cell counts in radioautographed testicular sections after a single or multiple ³H-thymidine injections indicated that the percentages of labeled type A cells corresponded to the percentages of type A₁-A₄ at each stage, whereas the percentages of unlabeled type A cells corresponded to the percentages of type A₀ spermatogonia obtained from counts of cells in whole mounts. This confirmed that type A₀ cells were generally non-proliferative throughout the cycle of the seminiferous epithelium while the type A₁-A₄ spermatogonia underwent complete renewal during each cycle. The present results thus support the concept of the existence of two classes of spermatogonial stem cells in rats.     

Re-examination of spermatogonial renewal in the rat by means of seminiferous tubules mounted “in toto”

Observations on dissected tubules, fixed in Carnoy, stained with hematoxylin and mounted “in toto” revealed that there were five distinct classes of type A spermatogonia. The type A₁ found in stages II–VIII of the cycle of the seminiferous epithelium had round, pale-stained nuclei, typically arranged in linear clusters of four or eight along the tubular wall. They all divided at stage IX to produce type A₂ cells. These in turn divided at stage XII to produce type A₃ spermatogonia. The type A₂ and A₃ cells had large ovoid nuclei containing globular masses of deeply stained chromatin and were randomly distributed in the space between Sertoli nuclei. The type A₃ spermatogonia divided at stage XIV to produce type A₄ cells. These had smaller nuclei, sometimes lobulated, containing more deeply stained chromatin granulation, free in the nucleus or adhering to the nuclear membrane. They divided in stage I of the cycle to yield two classes of spermatogonia: intermediate type and new type A₁. Hence, type A₁–type A₄ spermatogonia were considered as “renewing stem cells.” The fifth class of type A spermatogonia (A₀) was found at all stages of the cycle. Rare, isolated or in pairs, they had oval nuclei with deeply stained chromatin granulations. Seldom seen to divide, they did not appear to be actively involved in cell renewal and were tentatively considered as “reserve stem cells”.     

The ultrastructure of the pre-meiotic and meiotic stages of spermatogenesis in Plagioscion squamosissimus (Teleostei,Perciformes, Sciaenidae)

Spermatogenesis of 'corvina' P. squamosissimus starts from a stem cell that gives rise to germ cells. These cells are enveloped by Sertoli cells, forming cysts. The germ cells in the cysts are all at the same stage of development and are interconnected by cytoplasmic bridges. Spermatogonia are the largest germ cells. In the cysts, these cells differentiate into primary spermatogonia and secondary spermatogonia. The primary spermatogonia are isolated in the cyst and give rise to the secondary spermatogonia. After several mitotic divisions, they produce spermatocytes I, which can be identified by synaptonemal complexes in the nucleus. The spermatocytes I enter the first phase of meiosis to produce the spermatocytes II. These are not very frequently seen because they rapidly undergo a second phase of meiosis to produce spermatids.     

Role of spermatogonia in the stage-synchronization of the seminiferous epithelium in vitamin-A-deficient rats

After 20-day-old rats are placed on a vitamin-A-deficient diet (VAD) for a period of 10 weeks, the seminiferous tubules are found to contain only Sertoli cells and a small number of spermatogonia and spermatocytes. Retinol administration of VAD rats reinitiates spermatogenesis, but a stage-synchronization of the seminiferous epithelium throughout the testis of these rats is observed. In order to determine which cell type is responsible for this synchronization, the germ cell population has been analyzed in whole mounts of seminiferous tubules dissected from the testes of rats submitted to the following treatments. Twenty-day-old rats received a VAD diet for 10 weeks and then were divided into three groups of six rats. In group 1, all animals were sacrificed immediately; in group 2, the rats were injected once with retinol and sacrificed 3 hr later; in group 3, the rats were injected once with retinol, placed on a retinol-containing diet for 7 days and 3 hr, and then sacrificed. Three rats from each group had one testis injected with ³H-thymidine 3 hr (groups 1 and 2) or 7 days and 3 hr (group 3) before sacrifice. Three normal adult rats (approximately 100 days old) served as controls. Labeled and unlabeled germinal cells were mapped and scored in isolated seminiferous tubules. In group 1, type A₁ and type A₀ spermatogonia as well as some preleptotene spermatocytes were present; type A₂ A₃ A₄ In, and B spermatogonia were completely eliminated from the testis. Neither type A₁ mitotic figures nor ³H-thymidine-labeled-type A₁ nuclei were seen. Three hr after retinol injection (group 2), type A₁ mitoses, but no labeled type A₁ nuclei were observed. At 7 days and 3 hr after retinol administration (group 3), type A₄ and In Spermatogonia as well as type A₁ spermatogonia were present. A few residual pachytene spermatocytes were found, and some type A₀ cells were labeled. These results indicate that VAD caused, in addition to an impairment of spermatogenesis at the preleptotene spermatocyte step, a selective momentary arrest of surviving type A₁ spermatogonia at the G₂ phase of their cell cycle. Following administration of vitamin A to VAD rats, these type A₁ cells reinitiated spermatogenesis synchronously and, after several cycless of proliferation and renewai, reconstituted the seminiferous epithelium in a stage-synchronized manner.     

The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.     

Proliferation of spermatogonia and Sertoli cells in maturing mice

Summary Spermatogonial proliferation was studied in mice from day 13 p.p. when the seminiferous epithelium is incomplete, until week 12 p. p. when a steady state at adult levels has been attained. Counts of undifferentiated, A ₁ and intermediate spermatogonia and primary spermatocytes in stages IV and IX of the cycle of the seminiferous epithelium were made in whole mounted seminiferous tubules. Sertoli cell proliferation was studied in a separate series from 6 to 14 days p.p. employing the ³H-thymidine labeling index.It appeared that 1. Sertoli cell proliferation stops at day 12 whereafter the cells obtain their adult appearance; 2. The numbers of stem cell spermatogonia and the production of differentiating A ₁ spermatogonia increase almost twofold between day 13 and week 12; 3. The efficiency of the divisions of the differentiating A ₁-B spermatogonia is similar to that in the adult throughout this period; 4. At all ages studied, the cell counts revealed an almost constant numerical relationship between Sertoli cells and germ cells, which suggests a function of Sertoli cells in the regulation of spermatogonial proliferation.     

Seasonal changes of the blood-testis barrier in viscacha (Lagostomus maximus maximus): A freeze-fracture and lanthanum tracer study

Adult male viscachas (Lagostomus maximus maximus) were gathered from their natural habitat during the period of complete spermatogenesis (June) and during the month of maximum testicular regression (August). The testes were processed by conventional electron microscopic technique using lanthanum nitrate (electron-dense intercellular tracer) to define the intercellular spaces below the inter-Sertoli tight junctions and by freeze-fracture techniques. During complete spermatogenesis the tracer surrounds spermatogonia, preleptotene, and leptotene spermatocytes and stops at the level of the inter-Sertoli tight junctions below all germ cells displaying synaptonemal complexes (zygotene-pachytene spermatocytes) and germ cells in more advanced stages of differentiation. Conversely, during testicular regression the tracer percolates all intercellular spaces between Sertoli cells and the remaining germ cells (spermatogonia and few preleptotene and leptotene spermatocytes.) During complete spermatogenesis, freeze-fracture replicas exhibit numerous inter-Sertoli tight junction strands parallel to each other and to the basal lamina. During spermatogenesis decay, the inter-Sertoli tight junctions are found to be short, tortuous, frequently interrupted, and often associated with extented membranous areas of gap junctions. © 1993 Wiley-Liss, Inc.     

Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare A(single) spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A(dark) and A(pale) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high-dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRalpha1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.     

Ultrastructural aspects of spermatogenesis,testes, and vas deferens in the parthenogenetic tapeworm <Emphasis Type="Italic">Atractolytocestus huronensis</Emphasis> Anthony, 1958 (Cestoda: Caryophyllidea), a carp parasite from Slovakia

Spermatogenesis, testes, and vas deferens in the parthenogenetic monozoic tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea) from Slovakia, parasitizing the carp Cyprinus carpio L., have been investigated by means of transmission electron microscopy for the first time. The present results show that helminths with parthenogenetic and normal reproduction may share some common spermatology features, e.g., dense cytoplasm of the peripherally localized spermatogonia or a rosette type of spermatogenesis. In contrast to tapeworms with normal reproduction, the most prominent ultrastructural characteristic of the spermatocytes of A. huronensis is fragmentation of their nuclei. This clear feature of cell degeneration might be a consequence of the aberrant first meiotic division. Peripheral cortical microtubules and a single centriole, indicators of the ongoing spermiogenesis, were observed only very rarely in the early spermatids. Characteristics of normal spermiogenesis, i.e., apical dense material in the zone of differentiation in early stages of spermiogenesis, flagellar rotation, and proximo-distal fusion, were never found in the present study. The testes follicles are surrounded by a thin cytoplasmic sheath underlined by a basal lamina. Vas deferens is lined by flat epithelium with numerous surface lamellae and cilia. Mature, functional spermatozoa were not observed in the vas deferens of A. huronensis from Slovakia.     

Renewal of spermatogonia in man

The spermatogonia of normal adult human testis were investigated in view of clarifying their mode of proliferation and renewal. Three main types of spermatogonia were identified: the dark type A spermatogonia (Ad) tentatively considered as the stem cells, the pale type A spermatogonia (Ap) and the type B spermatogonia (B), these being the more and more differentiated elements giving rise to preleptotene spermatocytes. The dark and pale type A spermatogonia were present in all stages of the cycle of the seminiferous epithelium, the type B spermatogonia were found in stages VI, I and II of the cycle and the preleptotene spermatocytes in stages III and IV of the cycle. The type A spermatogonia divided preferentially in stage V of the cycle and the type B spermatogonia in stage II of the cycle. Quantitative data on spermatogonia and preleptotene spermatocytes revealed that the cell ratio Ad: Ap: B: Pl was equal to 1:1:2:4. This indicated that the spermatogonial stem cells divided to produce equal numbers of new stem cells (Ad) and of the more differentiated pale type A spermatogonia (Ap). Each one of the latter gave rise to two type B spermatogonia which in turn produced four spermatocytes. The arrangement in pairs of the dark and pale type A spermatogonia throughout the duration of the cycle indicated that the mitoses of spermatogonial stem cells are “equivalent” in nature; therefore, the possibility of having “differential” mitoses to explain the renewal of spermatogonial stem cells should be abandoned. Lastly, the frequent arrangement of the two classes of type A spermatogonia in homogeneous clusters indicated that the impetus which facilitates the differentiation of stem cells into the more differentiated elements (Ap) may affect homogeneous and compact groups of stem cells.     

Intercellular bridges between granulosa cells and the oocyte in the elasmobranch Raya asterias.

In the present ultrastructural study intercellular bridges, connecting somatic granulosa cells to oocyte, have been detected for the first time and their modifications have been followed during Raja oogenesis. Intercellular bridges make their first appearance in small previtellogenic follicles as connecting devices between small cells and the oocyte. Later on, when the follicular epithelium becomes polymorphic and multilayered, for the presence of small, large, and pyriform-like cells, intercellular bridges link the oocyte and the different granulosa cells. Intercellular bridges contain ribosomes, whorl of membranes, mitochondria and vacuoles. Such cytoplasmic components are present also in the cell apex of large and pyriform-like cells thus suggesting, in agreement with other species (Motta et al. J. Exp. Zool., 1996;276:223-241) they may flow toward the oocyte. In this regard the presence of intercellular bridges during the oogenesis of cartilagineous fish may represent a crucial event of the active cooperation between granulosa cells and the oocyte.     

Mucinoprotein is a universal constituent of stable intercellular bridges in Drosophila melanogaster germ line and somatic cells.

Intercellular bridges formed by incomplete cytokinesis may be important in a variety of processes, including synchronization of mitotic and meiotic divisions in animal cells. Using specific antibodies against a mucin-type glycoprotein (Kramerov et al. [1996] FEBS Lett. 378:213-218) from Drosophila melanogaster cultured embryonic cells, we showed that this glycoprotein is located in all cytoplasmic bridges found in various germline and somatic tissues. In the ovary, immunostaining of ring canals connecting germ cells can be detected in the very early stages at the germarium region 1 where first gonial divisions take place, and the immunostaining appears to persist through late stages when transport of cytoplasm from nurse cells to a growing oocyte occurs. Each ring canal is made up of an outer and an inner rim. Mucin glycoprotein appears to be one of the first proteins localized to the outer rim, which is a derivative of the arrested cleavage furrow. The known ring canal proteins, phosphotyrosine-containing protein(s), F-actin, hts- and kelch proteins, are localized to the inner rim at a later developmental time. Similarly, mucin glycoprotein is recruited early to ring canals connecting mitotic primary spermatocytes in both larval and adult testes. Mucin glycoprotein was found to be present in intercellular bridges (small ring canals) in somatic cells, including follicular epithelium in ovary and imaginal disc cells. Intercellular bridges were observed for the first time in a subset of cells in the larval brain. Thus, mucin glycoprotein is the only protein hitherto found in all known types of stable intercellular bridges and may be an important constituent of a backbone needed for assembly and preservation of this particular type of cell-cell contact.     

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.