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Arrest of Spermatogonial Differentiation in jsd/jsd, Sl17H/Sl17H, and Cryptorchid Mice

^a Department of Cell Biology, Utrecht University Medical School, Utrecht, The Netherlands ^b Genome Information Research Center, Osaka University, Osaka, Japan ^c Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

	ABSTRACT

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The nature of the spermatogenic arrest in cryptorchid C57Bl mice and in jsd/jsd and Sl17H/Sl17H mutant mice was identified by studying whole mounts of seminiferous tubules. In all three types of mice, virtually only A spermatogonia were found, topographically arranged in clones of 1 to 16 (rarely more) cells. These clonal sizes are typical for undifferentiated spermatogonia. The proportion of these cells lying in chains of more than 2 cells (50–70%) was comparable to that seen in epithelial stages VII–VIII in the normal epithelium. It is concluded that in all three types of mice, spermatogenesis is arrested at the point where the undifferentiated A spermatogonia, specifically A_al spermatogonia, differentiate into the first generation of the differentiating-type spermatogonia, the A1 spermatogonia.

The remaining A spermatogonia were proliferating, but no accumulation of spermatogonia was present, as spermatogonial apoptosis also took place. Spermatogonial clones of all sizes were seen to undergo apoptosis, but there were relatively many large apoptotic clones, indicating that the clones became more vulnerable when they became larger.

In contrast to what is seen in the normal epithelium, odd-numbered clones, not composed of 2ⁿ cells, were present, as well as clumps of 2 or more spermatogonial nuclei in the same cytoplasm, in all three types of mice. This indicates a lack of integrity of spermatogonial clones, also observed in other situations with a relative paucity of cells on the basal membrane.

It is concluded that the differentiation of the undifferentiated spermatogonia, affected in all three types of mice as well as in vitamin A-deficient animals, is a rather vulnerable point in the spermatogenic developmental pathway.

	INTRODUCTION

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Undifferentiated A spermatogonia are at the beginning of spermatogenesis in the adult mouse testis [1–3]. According to their topographical arrangement on the basal membrane, these cells can be subdivided into A_single (A_s), A_paired (A_pr), or A_aligned (A_al) spermatogonia. The A_s spermatogonia are considered to be the stem cells of spermatogenesis. Upon division of the A_s spermatogonia, the daughter cells either migrate away from each other and become two new stem cells, or stay together connected by an intercellular bridge and become A_pr spermatogonia. In the normal adult epithelium, half of the stem cell divisions will be self-renewing to maintain stem cell numbers, while the other divisions will render A_pr spermatogonia that will ultimately become spermatozoa. Other ratios between stem cell renewal and A_pr formation would lead either to tumor formation or to exhaustion of the stem cell pool. The A_pr spermatogonia divide further to form chains of 4, 8, or 16 A_al spermatogonia. During each cycle of the seminiferous epithelium, at about stage VII, most of the A_al spermatogonia differentiate into A₁ spermatogonia that are the first generation of the differentiating-type spermatogonia. These differentiating spermatogonia go through a series of six divisions and—via A₂, A₃, A₄, intermediate (In), and B spermatogonia—become primary spermatocytes.

As discussed previously [3, 4], spermatogonial development can be disturbed in many ways. For example, in vitamin A-deficient mice and rats, the differentiation of the A_al spermatogonia into A1 spermatogonia is arrested [5–7]. In C57Bl mice made artificially cryptorchid, spermatogenesis deteriorates to the point at which only actively proliferating A spermatogonia remain that produce few or no B spermatogonia [8, 9]. Furthermore, in jsd/jsd mice, spermatogenesis starts normally during development but then also declines ultimately to the point at which only proliferating undifferentiated and possibly differentiating type A spermatogonia are left [10–12]. Finally, differentiating germ cells have been reported to be missing in adult mice homozygous for the Steel17H mutation [13]. We now have characterized the A spermatogonia left in cryptorchid, jsd/jsd, and Sl17H/Sl17H mice as to their morphology and behavior in order to more clearly define the step at which the spermatogenic process becomes arrested in these mice.

	MATERIALS AND METHODS

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Animals

To obtain cryptorchid testes, inbred C57Bl/6 mice were used. Experimental cryptorchidism was performed at 2 mo of age as described previously [14]. Two months after operation, the mice were killed by cervical dislocation. C57Bl/6-jsd/jsd mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and further raised at the Research Institute for Microbial Diseases at Osaka University. Sl17H/Sl17H mice were obtained from MRC Radiobiology Unit (Chilton, UK) on a C3H/He mouse background and were back-crossed for more than 13 generations to C57Bl/6. Three- to four-month-old jsd/jsd and Sl17H/Sl17H mice were used. The animals were fed standard laboratory chow and kept in a controlled environment.

Tubular Whole Mounts

Whole mounts of seminiferous tubules were prepared from testes of adult jsd/jsd (n = 4) and Sl17H/Sl17H (n = 4) mutant mice as well as from C57Bl/6 cryptorchid mice (n = 4). The tubular whole mounts were prepared according to the method of Clermont and Bustos-Obregon [15]. The tubules were fixed in Bouin's fluid, stained with Harris hematoxylin (Polysciences, Warrington, PA) and mounted in toto on microscopic slides.

	RESULTS

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Histological Examination of Whole Mounts

In the tubular whole mounts of all three types of mice, A spermatogonia were by far the most predominant type of germ cell present (Fig. 1A). However, in 2 of the 5 jsd/jsd and in all 4 cryptorchid mice, occasionally more differentiated clones of germ cells—B spermatogonia or spermatocytes—were encountered (Fig. 1B). In Sl17H/Sl17H seminiferous tubules, no differentiating clones of germ cells were seen. In all three types of mice, clumps of spermatogonia were seen, in which 2 or more nuclei seemed to lie within the same cytoplasm (Fig. 1, D–F). Finally, in addition to normal spermatogonia, apoptotic spermatogonial clones were present (Fig. 1E).

View larger version (199K): [in this window] [in a new window]   FIG. 1. Photographs of whole mounts of seminiferous tubules from cryptorchid mice and jsd/jsd and Sl17/Sl17H mutant mice. No qualitative differences between the three mice were observed, and the photographs were taken at random from the mice. A) The spermatogonia present on the basal membrane in the three types of mice were virtually all A spermatogonia. As in this photograph, the density of the A spermatogonia (stars) was sometimes very high, making it impossible to distinguish separate clones, except for the 4 telophasic cells (arrowheads) that represent a chain of 4 Aal spermatogonia just formed from a division of Apr spermatogonia. All unmarked nuclei in this photograph are of Sertoli cells. B) In cryptorchid and jsd/jsd mice, sometimes isolated clones of more differentiated cells were encountered; indicated is a clone of early pachytene spermatocytes (arrows). C) Clone of 8 dividing spermatogonia (arrows), which can be distinguished from neighboring undifferentiated A spermatogonia (stars) by their synchronous traversal of the cell cycle. D) Clone of 4 Aal spermatogonia in prophase of mitosis (arrows); three cells of this clone are clumped together in the same cytoplasm. To the left are interphase undifferentiated spermatogonia, two of which form a clump. To the right are small telophasic undifferentiated spermatogonia (stars). E) Clump of 4 Aal spermatogonia (arrowhead). F) Three clumps of A spermatogonia, two consisting of 2 and one of 3 nuclei. G) Apoptotic clone of spermatogonia. The apoptotic figures varied considerably in their morphology. Here there are large apoptotic figures that may have originated from a clump that went into apoptosis (arrows), very small ones that seem on the verge of becoming indistinguishable (arrowheads), and medium-sized apoptotic figures (stars). A–G, x750 (published at 48%).

Clonal Size of the A Spermatogonia

The spermatogonial clones were distributed over the basal membrane in varying cell density. In general, the criterion is that A spermatogonia lying within 20 µm from each other and showing the same morphology belong to the same clone [2, 16]. However, because the density of the A spermatogonia in the shrunken tubuli of the three types of mice was sometimes very high (Fig. 1A), it was often not possible to distinguish the individual clones reliably. Therefore, the determination of clonal size was carried out by scoring clones only in mitosis (Fig. 1C). A spermatogonia that belong to the same clone are connected by intercellular bridges; therefore the cells composing a clone will go through the cell cycle in a synchronous fashion. As mitosis is a process of short duration, neighboring cells simultaneously in mitosis will very likely belong to the same clone. Hence, for the purpose of determining clonal sizes in the present study, spermatogonia were considered to belong to the same clone when they were lying within 20 µm from each other and were synchronously in prophase, metaphase, or anaphase of mitosis. In cryptorchid and Sl17H/Sl17H mice, the size of about 50 mitotic clones was evaluated in each mouse (Fig. 2). Since tubules of jsd/jsd mice were difficult to prepare, in the material of the 4 jsd/jsd mice a total of only 107 mitotic clones could be studied.

View larger version (43K): [in this window] [in a new window]   FIG. 2. Frequency distribution of mitotic clonal sizes (number of cells composing a clone) of the A spermatogonia remaining in the seminiferous tubules of cryptorchid mice and jsd/jsd and Sl17H/Sl17H mutant mice. Error bars indicate SEM. The black bars give an indication of the distribution of clonal sizes in stage VI of the cycle of the seminiferous epithelium in C3H/101 F1 hybrid mice (data from Tegelenbosch and de Rooij [17], assuming that there are equal numbers of clones of 8 and 16 cells).

In both the cryptorchid and the Sl17H/Sl17H mice, more than half of the clones consisted of 4 cells or fewer (Fig. 2). Only a small percentage of the clones reached the size of 16 cells or more. Most of the clones consisted of 1, 2, 4, 8, or 16 cells, but also odd-numbered clones, consisting of in-between numbers of cells, were present, constituting about 20% of the clones. Although in the jsd/jsd mice not enough clones in each mouse could be found to study the mice individually, the pooled numbers of mitotic clones indicate a frequency of clonal sizes rather similar to that in the other two types of mice.

Clumped Clones

In the seminiferous tubules of all three types of mice, sometimes 2 or more, up to 8, spermatogonial nuclei were seen to share the same cytoplasm (Fig. 1, D and E). In the jsd/jsd, Sl17H/Sl17H, and cryptorchid mice, 88, 156, and 90 clumped clones were encountered, respectively. Most often 2 cells were seen to form such a clumped clone (Fig. 3). The topographical arrangement of the clumps sometimes seemed to suggest the breaking up of larger clones into clumps of 2 and/or 3 cells (Fig. 1E). These clumps were not uncommon among telophasic spermatogonia (Fig. 1D).

View larger version (31K): [in this window] [in a new window]   FIG. 3. Frequency distribution of the numbers of nuclei within clumped clones of A spermatogonia in the seminiferous tubules of cryptorchid mice and jsd/jsd and Sl17H/Sl17H mutant mice.

Apoptotic Clonal Size

Apoptosis is a process of supposedly short duration. Hence, spermatogonia lying close together and undergoing apoptosis simultaneously can also be supposed to belong to the same clone. Nevertheless, the morphology of the apoptotic bodies within such an apoptotic clone varied considerably. Cells that seemed just to have started the apoptotic process or that were already about to disappear were seen together (Fig. 1G). Furthermore, very large apoptotic bodies were encountered that could have originated from a clump of several spermatogonia. The size of apoptotic clones was also determined. However, these data should be regarded as an underestimation of the apoptotic clonal size, as some cells of a clone might already have disintegrated beyond recognition, and the large bodies, possibly representing an apoptotic clump, were counted as 1 cell. In the jsd/jsd, Sl17H/Sl17H, and cryptorchid mice, 73, 177, and 99 apoptotic clones were encountered, respectively. Not enough apoptotic clones were found in the tubules of each mouse to study the data of individual mice. The pooled data indicate that the size of the apoptotic spermatogonial clones was generally larger than that of the mitotic clones (Fig. 4).

View larger version (52K): [in this window] [in a new window]   FIG. 4. Frequency distribution of apoptotic clonal sizes of spermatogonia in the seminiferous tubules of cryptorchid mice and jsd/jsd and Sl17H/Sl17H mutant mice.

	DISCUSSION

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Spermatogenesis was studied in three different types of mice in which an arrest in the spermatogenic process has been reported: jsd/jsd [10–12], Sl17H/Sl17H [13], and cryptorchid C57Bl [8, 9] mice. The present results identify the nature of the arrest and show it to be at a similar, very early step in the spermatogenic process in all three types of mice. In all three, virtually only A spermatogonia were found in the seminiferous epithelium. In both jsd/jsd and cryptorchid mice, occasionally a clone of B spermatogonia or spermatocytes was found, while no differentiating germ cells at all were seen in Sl17H/Sl17H mice.

The remaining germ cells were A spermatogonia, arranged in clones of up to 16 cells, although occasionally clones of more than 16 cells were observed. This distribution of clonal sizes is typical for undifferentiated A spermatogonia, which in the normal epithelium are composed of singles, pairs, and chains of up to 16 and rarely 32 cells [1–3, 16]. The failure of these undifferentiated spermatogonia to produce A1 spermatogonia in the mice of the present study is not caused by a failure of the A_s and the A_pr spermatogonia to produce A_al spermatogonia. About 50–70% of the clones were A_al spermatogonia. This compares well with about 50% in stages VI/VII in the normal epithelium of the mouse and Chinese hamster, in which the number of A_al clones is highest ([17, 18]; Fig. 2). Hence, the clonal composition of the undifferentiated spermatogonia in the three types of mice is quite comparable with that in the normal epithelium at the time just before differentiation of the A_al spermatogonia into the first generation of the differentiating-type spermatogonia, the A1 spermatogonia.

The arrest in spermatogonial differentiation is also not caused by an inability of the undifferentiated spermatogonia to reach a particular minimal clonal size that enables these cells to differentiate. First, clones of 8 and 16 spermatogonia were present. Second, in the normal seminiferous epithelium in the mouse and Chinese hamster, it has been established that in stages VII/VIII, many clones consisting of as few as 4 A_al spermatogonia already differentiate into A1 spermatogonia [17, 18].

In the three types of mice, the A spermatogonia were proliferating, mitotic spermatogonial clones being common. Despite the proliferative activity of the undifferentiated spermatogonia there was no accumulation of spermatogonia. The latter could be explained by the presence of clones of apoptotic spermatogonia. Spermatogonial clones of all sizes were seen to undergo apoptosis. However, despite the fact that the size of the apoptotic clones determined was somewhat underestimated because some cells seemed to disappear faster than others, and also because apoptotic clumps of cells were counted as being derived from one cell, the size of the apoptotic clones was on average larger than that from the normal mitotic spermatogonial clones. This indicates that the larger clones have an increased chance to enter apoptosis.

Although the population of undifferentiated spermatogonia in the three types of mice was comparable to that in the normal mouse with respect to their clonal composition, there was an important difference. In the normal epithelium, the undifferentiated spermatogonia are in G1/G0 arrest from about epithelial stages II to stage VIII, when after differentiation into A1 spermatogonia, these cells enter S phase. This G1/G0 arrest did not seem to take place in any of the mice studied, as the remaining A spermatogonia were actively proliferating. Nevertheless, the apparent lack of a quiescent period was not likely to have caused the failure of the A_al spermatogonia to differentiate, as a G1/G0 arrest is not crucial to spermatogonial differentiation. In situations in which there are no differentiating-type spermatogonia present, such as after administration of cytotoxic agents, the undifferentiated spermatogonia continue to proliferate during stages II–VII and still differentiate normally into A1 spermatogonia during epithelial stage VIII [19, 20].

The mutated gene in jsd/jsd mice has not been characterized as yet; thus it is impossible to speculate about the function that is lost in this mutant. In Sl17H/Sl17H mice there is a splicing defect in the cytoplasmic tail of the protein encoded by this locus, stem cell factor (SCF) [13]. This defect causes an interference in the c-kit receptor/SCF signaling system, the correct functioning of which has been abundantly shown to be indispensable for normal spermatogenesis. Many different alleles of the Steel (encoding SCF) and the White spotting (W, encoding the c-kit receptor) loci have been found, which upon homozygous occurrence lead to a variety in the severity of the spermatogenic disturbances. The mice homozygous for the Sl17H mutation showed many actively proliferating undifferentiated spermatogonia. Clearly, this allele causes a very specific arrest in the spermatogenic process, right at the differentiation step from undifferentiated to differentiating-type spermatogonia. In the cryptorchid testis, spermatocytes and spermatids have been shown to be particularly vulnerable to the high abdominal temperature [21]. In C57Bl mice, virtually only A spermatogonia remain in the testis, and the present results indicate that these are undifferentiated spermatogonia unable to become differentiating-type spermatogonia. The high temperature per se does not likely prevent spermatogonial differentiation. In the young mouse, testicular descent is not complete before several weeks after birth, while in most mouse strains, spermatogenesis starts at the day of birth [22–24]. Thus in the young mouse, spermatogonial proliferation and differentiation have to take place at abdominal temperatures for several weeks. Probably Sertoli cell function did become damaged because of the disappearance of spermatocytes and spermatids [25] and hormonal changes [26]. However, the specific factor that causes the arrest in spermatogonial differentiation in the cryptorchid testis remains to be determined. In conclusion, scrutiny of the abnormalities in the three types of mice studied indicates only that the c-kit/SCF system has to function properly to allow the transition from undifferentiated to differentiating-type spermatogonia. It will be interesting to study whether or not the functioning of the c-kit/SCF system is also affected in the cryptorchid and jsd/jsd mutant mice.

In the seminiferous tubules of all three types of mice, odd-numbered clones not consisting of 2ⁿ cells were encountered, as well as clumped clones in which 2 or more spermatogonial nuclei were seen in the same cytoplasm. Apparently, on the one hand clones disintegrate, intercellular bridges being severed, and on the other hand, upon division some cells have difficulties in moving apart. Neither odd-numbered nor clumped clones were observed in the normal seminiferous epithelium [2, 18], but they were present shortly after irradiation [27]. The latter and the present data suggest that the integrity of the spermatogonial clones is difficult to maintain in an epithelium where there is a relative paucity of germ cells on the basal membrane. To explain this, studies are needed on the nature of the mechanism along which intercellular bridges in between the spermatogenic cells are formed and maintained.

The inability of the undifferentiated spermatogonia to differentiate in the three types of mice is quite similar to the spermatogenic arrest in vitamin A-deficient rats and mice [5–7]. In vitamin A-deficient mice and rats, only undifferentiated spermatogonia remain that also are unable to differentiate into A1 spermatogonia. Interestingly, in the vitamin A-deficient animals, the undifferentiated spermatogonia are largely quiescent [5] instead of proliferating, as found in the mice of the present study. This also indicates that the failure of the undifferentiated spermatogonia to differentiate in the three types of mice is not related to the absence of a period of quiescence of these cells. Other rat models in which spermatogonial differentiation seems to be inhibited include the 2,5-hexanedione model [28–30] and the LBNF1/irradiation model [31]. In the first model, Sertoli cell function is damaged by administration of the Sertoli cell toxicant 2,5-hexanedione; in the second, after irradiation and an initial recovery, spermatogenesis deteriorates again. In both models, only proliferating A spermatogonia are left, while B spermatogonia and further differentiated germ cells are virtually absent. In these two rat models, the situation may be comparable to that in the three types of mice of the present study. Interestingly, in both these models the arrest in spermatogonial differentiation can be (partially) relieved by way of suppressing gonadotropic hormones [32–35]. These observations taken together, it can be concluded that under quite a number of at least seemingly very different circumstances the differentiation of undifferentiated spermatogonia can become inhibited, suggesting this step as one of the most vulnerable in the spermatogenic process as a whole.

	ACKNOWLEDGMENTS

 
The authors are grateful to Mr. R. Scriwanec for preparing the plate.

	FOOTNOTES

 
¹ Correspondence: Dirk G. de Rooij, Utrecht University Medical School, Department of Cell Biology, AZU-RM, G02.525, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. FAX: 31 302541797; d.g.derooij{at}med.uu.nl

Accepted: May 6, 1999.

Received: March 11, 1999.

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