Spermatogonial Cell Proliferation in Organ Culture of Immature Rat Testis'

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1 BIOLOGY OF REPRODUCTION 48, (1993) Spermatogonial Cell Proliferation in Organ Culture of Immature Rat Testis' CARLA BOITANI, 2 MARIA GIUDITTA POLITI, and TIZIANA MENNA Institute of Histology and General Embryology, University 'a Sapienza," Rome, Italy ABSTRACT Regulatory mechanisms of male germ cell proliferation in mammals were investigated by using in vitro organ culture of immature rat testis. Nutritional and hormonal requirements for maintenance and differentiation of germ cells in vitro were first characterized by testing different culture conditions. FSH was essential for the progression of type A spermatogonia up to the stage of pachytene spermatocytes after 3 wk of in vitro culture, while vitamins A, C, and E, LH, and testosterone were not effective. The proliferative activity of Sertoli cells markedly declined after 1 wk of in vitro culture, irrespectively of the presence of FSH in the medium. In addition, basal testosterone production by Leydig cells was maintained after 1 wk of culture, provided that FSH was present in the medium. The appearance of differentiating type I and type B spermatogonia and meiotic cells in the seminiferous cords throughout culture was accompanied by a significant reduction in the number of undifferentiated spermatogonia. Moreover, a similar labeling index of undifferentiated spermatogonia was observed in both unstimulated and FSH-stimulated testis fragments at all culture times considered. Therefore, FSH did not influence the mitotic activity of undifferentiated spermatogonia, suggesting a differential role of this gonadotropin during the mitotic phase of spermatogenesis. These results indicate that the organ culture system of immature rat testis represents a useful experimental model for studying regulatory mechanisms of spermatogonial cell proliferation. INTRODUCTION In spite of abundant studies on the morphology and kinetic properties of male germ cell renewal and differentiation, little is known about the regulation of spermatogonial cell proliferation and development in mammals. In the rat, two main classes of spermatogonial cells have been described [1], the undifferentiated spermatogonia and the differentiating spermatogonia. While the undifferentiated spermatogonia include both stem cells (type A) and proliferative cells committed to differentiate (type Apr, Aal), the differentiating spermatogonia include six generations of proliferating cells, named type A, 2 A, A 4, intermediate, and B spermatogonia, which finally differentiate into spermatocytes. In a highly organized tissue like the seminiferous epithelium, one would expect that the mitotic phase of germ cells is under a strict control. It has been shown that adult testis contains a factor, a spermatogonial chalone, that specifically inhibits the proliferation of type A spermatogonia [2]. However, this testicular inhibitor has not been purified yet. Differentiating spermatogonia, but not undifferentiated spermatogonia, in adult mice and Chinese hamsters have been shown to decrease in number after a systemic or intratesticular injection of inhibin [3]. As for the positive control of spermatogonial cell proliferative activity, several pieces of evidence have been produced so far by using different experimental models. In vivo experiments have been performed with animals de- Accepted November 17, Received July 8, 'This work was supported by the National Research Council (CNR)- Progetto Finalizzato "Prevention and Control Disease Factors," subproject "Control of Human Fertility," contract n Correspondence: Carla Boitani, Ph.D., Institute of Histology and General Embryology, Via A. Scarpa 14, Roma, Italy. FAX: + 39 (6) prived of FSH or vitamin A, in order to study the effect of the replacement of these factors upon the proliferative activity and development of specific spermatogonial cell stages [4-7]. A possible role of interleukin-la in regulating spermatogonial cell multiplication has been suggested [8, 9], on the basis of the stimulatory effect that this cytokine exerts on stage-specific DNA synthesis in segments of seminiferous tubule cultured in vitro. Among testicular growth factors more recently described, activin deserves particular attention, since isolated spermatogonia have been found to express activin receptors; additionally, when this hormone is added to Sertoli and germ cell co-cultures, germ cell proliferation increases [10, 11]. It was also recently proposed that differentiating spermatogonia represent a specific site of c-kit expression, and that an anti-c-kit monoclonal antibody blocks the mitosis of these cells, but not that of undifferentiated spermatogonia [12]. Taken together, these data indicate that the mitotic phase of spermatogenesis is a complex series of events, perhaps differentially regulated, that requires appropriate experimental models to be explored. While most of the in vitro culture systems limit experiments to a rather short time, the organ culture system offers the possibility of preserving the testicular architecture for several weeks in vitro, and of fully maintaining cell-cell interactions that play a major role in testis function. In the late sixties, Steinberger et al. [13] observed that gonocytes develop and progress in vitro through the meiotic prophase when maintained in organ cultures of immature rat testes, provided that serum and vitamins are present in the medium. More recently, the ability of type A spermatogonia to differentiate and resume spermatogenesis has also been investigated by Haneji and collaborators in organ cultures of cryptorchid testes from adult 761

2 762 BOITANI ET AL. mice, by determining the effect of several hormones such as FSH, retinoids [14], and epidermal growth factor (EGF) [15]. In the present study, we have examined in detail the organ culture of testes from prepubertal rats. By using this system,we have investigated regulatory mechanisms of mammalian undifferentiated spermatogonia proliferation and progression into more differentiated cell compartments. Organ Culture MATERIALS AND METHODS The organ culture technique described by Steinberger et al. [16] was used with some modifications. Briefly, testicular tissue obtained from 9-day-old Wistar rats was cut into approximately 1-mm fragments and arranged on steel grids that had been previously coated with 2% agar. Grids were then placed in organ culture dishes (Falcon; Becton, Dickinson, and Co., Rutherford, NJ), with medium wetting the lower surface of the grid (0.8 ml). Culture medium was Eagle's Minimum Essential Medium with Earle's salts (Gibco, BRL, Paisley, UK) supplemented with glutamine (2 mm), Hepes (15 mm), nonessential amino acids (single-strength), penicillin (100 IU/ml), streptomycin (100 mg/ml), and gentamycin (40 mg/ml). Ovine FSH (o-fsh-17, NIH, Bethesda, MD), ovine LH (o- LH-23, NIH), testosterone (Sigma, St. Louis, MO), and vitamins A, C, and E (Sigma) were added to the culture medium depending on the experiment, as indicated in figure legends. Tissue fragments were cultured for 1-3 wk at 32 C in a humidified atmosphere of 5% CO 2 in air. The culture medium was changed every 3 days. Labeling In vitro-cultured tissue fragments were labeled with 5- bromo-2'-deoxyuridine (BrdU) and 5-fluoro-2'-deoxyuridine (labeling reagent diluted 1:400, "cell proliferation kit"; Amersham, Bucks., UK) during the last 5 h of culture. Samples were then washed twice with PBS, fixed in Bouin's fluid, and processed for light microscopy analysis. Light Microscopy Analysis and Immunocytochemical Procedures Fixed samples were dehydrated and embedded in Histowax (Reichert-Jung, Pabisch, Italy). Five-millimeter-thick serial sections were stained with Mayer's hemalum (Merck, Darmstadt, Germany). BrdU incorporation into proliferating cells was detected by immunocytochemistry. Briefly, sections of testis fragments were rehydrated, washed with PBS, and incubated with 3% H in PBS at 4 0 C for 10 min to inactivate endogenous peroxidases. Sections were washed several times with PBS containing 1% BSA (Miles Diagnostics, Kankokee, IL) and incubated with an anti-brdu monoclonal antibody (cell proliferation kit, Amersham) at 4 0 C overnight. The antibody specifically bound to nuclei was detected by a peroxidase antimouse Ig, according to manufacturer's recommendations. Sections were finally counterstained with carmalum. Labeling Index Values of the labeling index reported in this paper represent the mean - SEM of determinations obtained in three independent experiments. In each experiment, percentages of labeled Sertoli cells and undifferentiated spermatogonia were calculated by analyzing cord cross sections, selected at random, of testicular fragments that had been cultured in vitro for 0, 3, 6, and 13 days. Undifferentiated spermatogonia (type A, Apr, Aal) were identified on the basis of both their location on basement membrane among Sertoli cells and the morphology of their nucleus. This nucleus is large, round to ovoid, and euchromatic, and contains two or three clumps of heterochromatin and a large nucleolus. Since type Al and A 2 spermatogonia are morphologically very similar to undifferentiated spermatogonia (type As, Apr, Aal), particularly when observed in sections, it is likely that cells here named undifferentiated spermatogonia may include A, and A 2 spermatogonia. Sertoli cells were used as reference and were identified by their columnar nucleus. Statistics Statistical analysis was performed by means of Student's t-test. RESULTS Effect of Culture Conditions on Germ Cell Differentiation Basic culture conditions required for germ cell maintenance/differentiation in our in vitro system were determined in the first set of experiments. Testicular fragments were cultured in the presence of either one of the following factors or a combination of them: FSH (200 ng/ml), LH (10 ng/ml), testosterone (0.2,uM), vitamin A (0.35,aM), vitamin C (50 plg/ml), and vitamin E (200 ng/ml). The appearance of differentiating cells (type I and B spermatogonia and meiotic cells) in the seminiferous cords was used as a criterion to evaluate the effect of the various culture conditions tested. Samples were analyzed after 1, 2, and 3 wk of in vitro culture. At the beginning of culture, testicular fragments contained both undifferentiated and differentiating type A spermatogonia, belonging to the first spermatogenic wave; neither type I nor type B spermatogonial cells nor meiotic cells were present. Type A spermatogonial cells appeared not to progress any further through the spermatogenic process when testicular fragments were cultured in either plain medium (Fig. 1, b-d), or in the presence of testosterone, LH, or a combination of vitamins A, C, and E. However, addition of vitamins A, C, and E to the culture

3 REGULATION OF SPERMATOGONIAL CELL PROLIFERATION 763 FIG. 1. Photomicrographs of sections from control testes (a) and testis fragments cultured for 1, 2, and 3 wk in plain culture medium (b, c, d) and in the presence of 200 ng/ml FSH (e, f, g). Sections were stained with Mayer's hemalum. Letters indicate stages of germ cell differentiation: U, undifferentiated spermatogonia; I, intermediate spermatogonia; B, B spermatogonia; L-Z, leptotene-zygotene spermatocytes; P, pachytene spermatocytes. x750. medium resulted in a better preservation of testicular tissue morphology than that found with plain medium, or medium supplemented with testosterone or LH. Therefore, a culture medium supplemented with vitamins A, C, and E was taken as the control medium in further experiments. In contrast, addition of FSH, either alone or in combination with other supplements, to the medium, allowed type A spermatogonia to differentiate into type I and type B spermatogonia after 1 wk of culture (Fig. le), into leptotenezygotene spermatocytes after 2 wk of culture (Fig. lf), and into pachytene spermatocytes after 3 wk of culture (Fig. g). The beneficial effect of FSH on the progression of germ cell differentiation was consistently found in approximately 50% of seminiferous cords. In addition, this gonadotropin caused seminiferous cords to increase in diameter and Sertoli cells to enlarge their cytoplasm (Fig. g). Even though the tubular architecture was morphologically preserved under these culture conditions, germ cell development appeared to be significantly delayed in organ culture with respect to the in vivo condition. Moreover, only one germ cell generation was seen to mature up to the stage of pachytene spermatocytes. Cells undergoing degeneration were rarely seen in our sections. However, when the size of cultured testicular fragments was larger, degenerating cells and/ TABLE 1. Sertoli cell number in control and FSH-treated fragments. Days in culture Treatment Control a FSH * * t 0.53* * avalues represent means SEM of numbers of Sertoli cell nuclei/section from three different experiments. *Not significantly different from control values. or necrotic areas were observed; in this case all samples were discarded. Differentiation steps beyond the stage of pachytene spermatocyte were never observed under any culture conditions tested. Sertoli Cell Proliferation in Organ Culture In order to relate germ cell proliferation to the number of Sertoli cells, it was first necessary to investigate the possibility that Sertoli cells changed in number under different culture conditions tested. We therefore determined the number of Sertoli cell nuclei present in cross sections of testis fragments cultured for 0, 3, 6, and 13 days in the absence or in the presence of FSH. No variation of Sertoli cell number was observed in either unstimulated or FSHstimulated fragments at any culture times considered (Table 1). The mitotic activity of Sertoli cells was also quantified by labeling proliferating cells with BrdU, as described in Materials and Methods. Figure 2 shows that the percentage of BrdU-labeled Sertoli cells dropped from 12.7% at Day 0 of culture to 0.3% after 3 days and to less than 0.15% after 1 wk of culture, irrespectively of the presence of FSH in the medium. Proliferation of Undifferentiated Spermatogonia To investigate whether undifferentiated spermatogonia changed in number during culture, testicular fragments were cultured in the presence or absence of FSH for 0, 3, 6, and 13 days. Samples were then fixed and sectioned, and undifferentiated spermatogonia were counted and related to Sertoli cell number. The results, shown in Figure 3, are expressed as the ratio between the number of undifferentiated spermatogonia observed in each experimental point and 700 Sertoli cells. After 3 days of culture this ratio was

4 BOITANI ET AL o o0.10 Co 4 2 n days in culture FIG. 2. Labeling index of Sertoli cells at Day 0 and after 3 and 6 days of culture in control medium with and without FSH. Labeling was performed as described in Materials and Methods. A total of 700 BrdU-labeled and unlabeled Sertoli cells were scored for each experimental point. Values (mean SEM) represent percentages of labeled Sertoli cells obtained in three independent experiments. Open bars: fragments cultured in the absence of FSH; filled bars: fragments cultured in the presence of FSH (200 ng/ml). similar to that found at the beginning of culture in both control and treated samples. After 1 and 2 wk of culture, a marked reduction of this ratio was observed when testis fragments were cultured in the presence of FSH, while it remained constant in the absence of this gonadotropin. Therefore, the possibility that FSH influences the proliferation of undifferentiated spermatogonia was addressed experimentally. We measured the labeling index of undifferentiated spermatogonia in testis fragments cultured for 0, 3, 6, and 13 days in the absence and in the presence of FSH. To this end, tissue fragments were incubated in the presence of BrdU at the end of the in vitro culture period, as described under Materials and Methods. BrdU-labeled and unlabeled germ cells were then counted in tissue sections selected at random. Percentages of labeled undifferentiated spermatogonia (possibly including type A, and A 2 spermatogonia), are reported in Figure 4. Rates of undifferentiated spermatogonia proliferation found in both treated and untreated samples were similar at all culture times considered. Leydig Cell Function in Organ Culture Finally, we asked the question whether the interstitial tissue was functionally active in our in vitro system. In these experiments, LH-dependent testosterone production was investigated as a parameter of Leydig cell function in the organ cultures. Testis fragments were cultured for 6 days in the absence or presence of FSH (200 ng/ml). At the end 0.05 n days in culture FIG. 3. Number of undifferentiated spermatogonia in testis fragments cultured in the absence or in the presence of FSH. Values express the ratio between the number of undifferentiated spermatogonia and 700 Sertoli cells counted for each experimental point. Values are mean + SEM of three different experiments. Open bars: fragments cultured in the absence of FSH; filled bars: fragments cultured in the presence of FSH (200 ng/ml). * Significantly different from control values (p < 0.05). of the treatment, culture medium was replaced with fresh medium, and the fragments were incubated for a further 24 h in the absence or in the presence of LH (100 ng/ml). At the end of incubation, medium was collected for testosterone determination. Testosterone secreted into the meco -oa).0a) n (U 'O 0 a).5 EC a) p0 a) days in culture FIG. 4. Labeling index of undifferentiated spermatogonia during organ culture of testis fragments in the absence or in the presence of FSH. BrdU labeling was performed as described in Materials and Methods. The labeling index was calculated as the percentage of labeled undifferentiated spermatogonia. Values are mean + SEM of three different experiments. Open bars: fragments cultured in the absence of FSH; filled bars: fragments cultured in the presence of FSH (200 ng/ml).

5 REGULATION OF SPERMATOGONIAL CELL PROLIFERATION t on c 20 a10 I- D v I- }- I-,T pretreatment none FSH FIG. 5. Testosterone production by testis fragments untreated and treated with LH (100 ng/ml) for 24 h after 6 days of culture in the absence or in the presence of FSH (200 ng/ml). At the end of the 24-h incubation, media were collected and testosterone was measured by RIA. Each point represents the mean - SEM of three different plates, each assayed in duplicate. dium in the absence of LH represents basal testosterone production. Testosterone secreted in the presence of LH represents LH-dependent testosterone production. Results are reported in Figure 5. Basal testosterone production was found to be markedly decreased when fragments were cultured in the absence of FSH. By contrast, the presence of the gonadotropin in the culture maintained a basal testosterone level similar to that at Day 1 of culture ( ng/ml). LH-dependent testosterone production appeared to be maintained after 1 wk of culture irrespectively of the pretreatments. DISCUSSION I- In this paper we provide evidence that the organ culture of testis from immature rats represents a useful experimental model for investigating regulatory mechanisms involved in undifferentiated spermatogonia proliferation in mammals. Nine-day-old rats were selected as tissue donors because at this age testicular cords contain only actively proliferating type A (undifferentiated and differentiating) spermatogonia, while the mitotic activity of Sertoli cells is very low [17]. In the present study we show that (1) in the presence of vitamins A, C, and E, the architecture of testicular tissue is well preserved, but type A spermatogonia do not undergo apparent differentiative changes; (2) when FSH is present in the culture medium, either alone or in addition to vitamins A, C, and E, both undifferentiated and differentiating type A spermatogonia are able to differentiate further, by maturing in vitro up to the stage of pachytene spermatocytes; and (3) the proliferation of undifferentiated spermatogonia is independent of FSH. The finding that vitamins A, C, and E supported undifferentiated type A spermatogonia proliferation indicates that this culture condition provides the nutritional milieu essential for these cells to survive and divide, but not to differentiate further. Since both undifferentiated and differentiating type A spermatogonia were present in 9-day-old rat testis, it was of interest to determine whether these cell types were affected by FSH. As mentioned before, morphological differences between undifferentiated spermatogonia and Al-A 2 spermatogonia are quite small in the rat. Therefore, it is very difficult, if not impossible, to distinguish the undifferentiated from type A and A 2 spermatogonia, especially in histological sections. Given these limits, we observed that the number of undifferentiated spermatogonia was significantly reduced when testicular fragments were cultured for 1 and 2 wk in the presence of FSH. The following possibilities, or a combination of them, may account for this observation: (1) the mitotic activity of undifferentiated spermatogonia is inhibited by FSH, (2) extensive germ cell degeneration may occur in the presence of FSH, and (3) the progression of undifferentiated spermatogonia into a more advanced cell compartment is influenced by FSH. The first possibility can be ruled out by the observation that the labeling index of undifferentiated spermatogonia was similar in both unstimulated and FSH-stimulated cultures (Fig. 4). As for the second possibility, cell death is a common phenomenon occurring in vivo in the course of spermatogonial development and other steps of spermatogenesis [18]. However, degenerating germ cells were rarely seen in our cultures, regardless of the presence of FSH. Moreover, in a detailed study of spermatogonial degeneration in the adult rat, Huckins [19] did not observe degeneration of undifferentiated spermatogonia. Similar conclusions were also suggested by Lok et al. [20] in the Chinese hamster and in the ram. Therefore, an extensive cell loss is not likely to represent a major cause for the observed change in number of undifferentiated spermatogonia. We therefore favor the possibility that, in our cultures, FSH induces undifferentiated spermatogonia to differentiate into more mature cells, without affecting undifferentiated spermatogonia mitotic activity. This view is further supported by the finding that, in the presence of FSH, type I and type B spermatogonia appeared after 1 wk of culture, but not at earlier culture times such as 3 days. The spermatogonial cell stage(s) on which FSH specifically exerts its differentiative effect still remains to be elucidated. According to Chemes et al. [4], in animals injected with antiserum against FSH, the number of type A spermatogonia (mostly Al) is unaffected by FSH withdrawal, indicating that FSH may be involved in the renewal of more differentiated spermatogonia (A 2 -A4), but not of undifferentiated spermatogonia. In addition, Huckins et al. [21] showed that

6 766 BOITANI ET AL. FSH administration to 16-day-old rats increases the mitotic rate of A 2 -A 4 spermatogonia. In the past, by using organ cultures from 4-day-old rat testis, Steinberger and colleagues [13, 22] showed that when vitamins A, C, and E, but not FSH, were added to a medium supplemented with serum, gonocytes were induced to differentiate up to the stage of pachytene spermatocytes. Discrepancies with the present results are likely to be explained by the animals' different ages and by the different protocols applied. The specific mechanism(s) by which FSH plays its regulative role in mammalian male germ cell differentiation still remains to be elucidated. On the basis of the notion that Sertoli cells are the target for this gonadotropin in the testis, and that several factors are produced by these cells under the control of FSH, a number of possible candidates for the role of spermatogonial mitogens can be considered. For example, it has recently been reported that "steel factor" is secreted by Sertoli cells in culture [23] and that its receptor c-kit is specifically expressed on differentiating spermatogonia [12]. On the other hand, a possible direct effect of FSH on spermatogonia cannot be ruled out, as suggested by the finding that binding activity of labeled FSH is present on this germ cell type [24]. Sertoli cell proliferation in our organ cultures was also examined in detail. Results obtained in these experiments are consistent with the reported 3 H-thymidine labeling pattern of these cells both in vivo and in vitro. Since the early sixties, it has been well known that in rat testis the percentage of proliferating Sertoli cells progressively declines after birth, decreasing by approximately 15% in 9-day-old animals and reaching zero after Day 15 [17, 25]. A similar proliferation pattern was also observed by Steinberger and Steinberger [26] in an organ culture system: the capacity of Sertoli cells to incorporate 3 H-thymidine diminished with increasing culture duration. We have shown here that the number of Sertoli cells did not change after FSH stimulation and that the Sertoli cell labeling index decreased in culture irrespectively of the presence of FSH. Previous reports have shown that Sertoli cell-enriched monolayers from 10-day-old animals proliferate when cultured in the presence of insulin, transferrin, and EGF [27], and that their mitotic activity is stimulated by FSH [28]. Disagreement between those observations and the present results is likely to result from the different culture systems used. As for androgen involvement in the regulation of the first spermatogenic wave, the present results on testosterone production by Leydig cells indicate that testosterone production was maintained at a basal level in our organ culture, provided that FSH was present. Our observation that testosterone and LH failed to allow the progression of spermatogonia into more advanced cells is not surprising. In fact, in contrast to the well-established role of testosterone in maintaining spermatogenesis in adult mammalian testis [29], several reports suggest that testosterone negatively affects the initiation of spermatogenesis [4, 30]. Neonatal rats treated with anti-lh serum and testosterone display severe testicular regression [4]; spermatogenic arrest at the premeiotic steps of development has been observed after testosterone propionate administration to newborn rats [30]; and Steinberger et al. [31] found no effect of testosterone on germ cell maturation in an organ culture system of immature rats. In light of the present observations on spermatogonial cell differentiation up to the stage of pachytene spermatocytes, the kinetics of Sertoli cell proliferation and Leydig cell function, the organ culture system from immature rat testis is reminiscent of the in vivo situation. Thus it may represent a useful tool for investigating germ cell/somatic cell differentiation under controlled/ experimental conditions. ACKNOWLEDGMENTS The authors are grateful to Prof. Franco Mangia for discussion and advice, Prof. Mario Stefanini for critical reading of the manuscript, and Ms. Stefania De Grossi for expert preparation of the photographs. We thank the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the National Hormone and Pituitary Program for the gift of ovine FSH and LH. REFERENCES 1. Huckins C. The spermatogonial stem cell population in adult rats. I. 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