Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.004549
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
68/1/316    most recent
biolreprod.102.004549v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ogawa, T.
Right arrow Articles by Kubota, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogawa, T.
Right arrow Articles by Kubota, Y.
Agricola
Right arrow Articles by Ogawa, T.
Right arrow Articles by Kubota, Y.
BIOLOGY OF REPRODUCTION 68, 316–322 (2003)
DOI: 10.1095/biolreprod.102.004549
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Expansion of Murine Spermatogonial Stem Cells Through Serial Transplantation1

Takehiko Ogawa2,a, Masako Ohmurab, Yasushi Yumuraa, Hajime Sawadab, and Yoshinobu Kubotaa

a Department of Urology b Anatomy, Yokohama City University School of Medicine, Yokohama 236-0004, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sld mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.

sperm, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a highly productive process that produces huge numbers of sperm in the lifetime of a male. Its high efficiency and sustained continuity as well as robust regeneration potential are attributed to the spermatogonial stem cells [1, 2]. It has been supposed, therefore, that spermatogonial stem cells have unlimited potential for proliferation, both for differentiation and self-renewal. However, several studies suggested that spermatogonial stem cells diminish or lose their activity as a male ages [3–5].

In the 1970's, the identity and behavior of spermatogonial stem cells became elucidated through deliberate morphological observations [6, 7]. Among undifferentiated spermatogonia, the Asingle (As) spermatogonia were suggested to be stem cells, resistant to noxious agents and to show vast proliferation potential in such damaged states [8]. They can be identified by nuclear morphology and topographical arrangement: They are located on the basement membrane inside the seminiferous tubules, have large oval nuclei with homogeneous nuclear matrix, and present as isolated single cells [6]. However, there are no reliable cell markers to identify them.

Development of spermatogonial transplantation techniques demonstrated that the spermatogonial stem cells can be collected from the testis, incubated for a certain period, and reintroduced into a host testis [9, 10]. This technique has been applied to basic research in many aspects of spermatogenesis. It has provided several important findings, including cryopreservation of spermatogonial stem cells [11] and xenotransplantation of spermatogonia [12]. The nature of the colonization process following transplantation was also studied [13]. Soon after the injection into a testis, the transplanted cells are randomly distributed throughout the seminiferous tubules and a small number of spermatogonial stem cells reach the basement membrane. Then the settled donor cells on the basement membrane divide and form a monolayer network of spermatogonia. After expanding along the basement membrane, the cells in the center of the network differentiate toward the lumen of the seminiferous tubules and establish a spermatogenesis colony. The expansion of the colony lasts at least 4 wk after transplantation. It was also shown that there is a linear correlation between the number of colonies developing and the number of injected cells [14].

In the present study, using these technical advances, we addressed the question whether spermatogonial stem cells would expand in number by self-renewing cell divisions when they are transplanted to recipient testes. For that purpose, a line of transgenic mice carrying the Green fluorescent protein (GFP) gene was used as the donor cell source. The GFP germ cells were clearly recognizable under a dissecting microscope with an excitation light. The GFP-positive spermatogenic colonies were collected for serial transplantation. In the present experiment, we have, for the first time, shown that spermatogenic stem cells are serially transplantable without exhaustion of proliferation activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Donor Cell Preparation and Transplantation

Donor cells were recovered from 11- to 18-wk-old transgenic mice (C57BL/6 genetic background) carrying the pCXN-eGFP transgene [15, 16]. The GFP gene is expressed in every cell type including spermatogenic cells of the mouse, which allows identification of donor cells in the recipient mouse under excitation light in vivo without any pretreatment.

The initial spermatogonial transplantations were performed according to the method reported previously [17]. The donor testis cells were injected into the rete testis of recipient mice. For the second or third sequential transplantation, the donor cells were collected and transplanted as follows: First, under the dissecting microscope with an excitation light for GFP emission, the spermatogenic colonies were dissected out. They were digested with trypsin (0.25%) and EDTA (0.4 mM) for 5–10 min at 37°C. The enzyme reaction was stopped by adding 10% (v/v) fetal bovine serum (FBS) (Filtron, Brooklyn, Australia), followed by addition of 5% (v/v) DNase (10 mg/ml). When undigested tissue debris remained, the cell suspension was filtered through 70-µm-pore nylon membrane (Becton Dickinson Labware, Franklin Lakes, NJ) to eliminate debris. The cells were then centrifuged to make cell pellets and resuspended with 30–40 µl of Dulbecco modified Eagle medium (DMEM) (Gibco BRL Co., Grand Island, NY) containing 10% FBS. The cell concentration was determined using a hemocytometer to be 0.3–20 x 106/ml. Cell survival was greater than 95% as determined by trypan blue exclusion.

Recipient Mouse Preparation

C57BL/6 mice (Clea, Tokyo, Japan) were used as histocompatible recipients. They were treated with busulfan (Sigma Japan, Tokyo), 50 mg/kg i.p., to destroy endogenous spermatogenesis 4–8 wk prior to the transplantation [9]. Sl/Sld mice were used as recipients for the experiment of estimation of colonization efficiency. They did not require busulfan treatment because their testes had very few germ cells due to the lack of membrane-bound stem cell factor [18].

All mice were maintained at 24 ± 1°C, 55 ± 1% humidity with a 14L:10D cycle. Food and water were provided ad libitum.

All animal housing and surgical procedures were in accordance with the guidelines of the institutional animal care and use committee of the Animal Research Center, Yokohama City University School of Medicine.

Analysis of Recipient Testes

Between 55 and 373 days after donor cell transplantation, C57BL/6 mice were killed by CO2 inhalation. The removed testes were observed under a stereomicroscope equipped with an excitation light for Green fluorescence emission (Olympus SZX12; Olympus, Tokyo, Japan) to identify seminiferous tubules containing donor cell-derived spermatogenesis. The number and length of each colony were recorded. Some recipient mice were arbitrarily chosen and testes harboring GFP-positive spermatogenic colonies were used as the donor cell source for subsequent transplantation.

Sl/Sld recipient mice were killed beyond 42 days after transplantation for both the subsequent transplantation and for whole-mount observation of colonies using confocal laser microscopy. Dissected seminiferous tubules for confocal microscopy were fixed with 4% paraformaldehyde for 8 h at 4°C. After rinsing with PBS, the samples were preserved at 4°C. For observations, the seminiferous tubules were placed on the glass slides and covered with coverslips. The GFP fluorescence image was obtained with an excitation wavelength of 488 using a laser confocal microscope (Radiance 2000; Bio-Rad, Hercules, CA) fitted to an axioplan epifluorescence microscope (Carl Zeiss, Oberkochen, Germany). Sequential sections of 2 µm, total 11 slices, were recorded for three-dimensional reconstruction of the seminiferous tubules. The number of As and Apr spermatogonia along with the total number of spermatogonia were counted in each constructed image.

Linear regression and correlation analyses were performed to describe the relations between the incubation period and colony length or multiplication of the colony number and between the colony length and colony number.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Serial Transplantation of Spermatogonial Stem Cells

Serial spermatogonial transplantation was performed using seven original donor mice, whose ages ranged from 11 to 18 wk old. Figure 1 illustrates the serial transplantation of spermatogenic cells from one recipient to the next. The transplantation of the cell suspension was performed in a most deliberate manner in order to minimize cell loss. The volume of cell suspension loaded into the injection glass needle, determined to be 7 µl in most cases, and the estimated volume injected into each testis of recipient mice, usually 5–7 µl, estimated from the amount of cell suspension left in the glass needles, were recorded. Thus, the number of cells injected could be calculated. A single line serial transplantation of spermatogonia up to the fourth passage is outlined in Figure 2. In this instance, the original donor mouse was 90 days old, and the testis cells were transplanted into a recipient mouse testis. The eight colonies confirmed at 128 days afterward in the host testis were dissected out for the next transplantation. Of the 30-µl cell suspension made from these eight colonies, 20 µl was actually introduced into the subsequent three host testes. It is suggested, therefore, that 5.3 colonies, two thirds of 8 colonies, were passaged forward to the next mouse testes. In these three host testes, 10 colonies in total developed. Therefore, the donor cell suspension appeared to have contained a colony-forming activity for 10 colonies. It can be assumed that, in the 128 days of the incubation period, the 5.3 colonies, which must be equivalent to 5.3 spermatogenic stem cells at the beginning of colony formation, had increased their stem cell activity up to 10 colonies of colony-forming activity. Multiplication of the colony number was calculated to be 1.9 (10 divided by 5.3). The same calculations were performed on all other data, and a summary is shown in Table 1. The incubation period ranged from 55 to 373 days. The resultant colonies were observed under excitation light for GFP emission. All or part of them, 1–9 in number, were dissected out and photographed for measurement of their total lengths, which ranged from 2.2 to 28.5 mm. They were then digested with trypsin and EDTA to prepare cell suspensions for subsequent transplantation. The amounts of the cell suspension and the injected volume were recorded so that the number of tubules in addition to the total length of the tubules that were passaged to the next recipients were calculated as 0.74–6.0 in number and 1.8–18.9 mm in length, respectively. The numbers of regenerated colonies in the succeeding recipients were 1–32. Multiplication of colony number through transplantation ranged from 0.6 to 13.4. As noted above, this colony multiplication could be interpreted that the donor colonies have such colony-forming activity. This colony-forming activity must have developed during the incubation period along with colony size expansion. We did not see any difference between the second and third serial transplantation in terms of colony expansion or the colony multiplication rates (Fig. 3, A and B). The sequential passage of a single spermatogenic cell line from one recipient to the next, four times in a row, was successful twice in four trials. The overall incubation period of a line of spermatogenic cells reached up to 802 days, was passaged three times, and was maintained in four host testes.



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 1. Schematic presentation of a serial transplantation of spermatogenic cells. A) Spermatogenic colonies developed in a recipient testis. B) Two colonies dissected out from the testis in A. C) GFP cell suspension made from the colonies in B by enzymatic digestion with trypsin and EDTA. D) The next recipient mouse testis 60 days after the serial transplantation. Two colonies are visible.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. An example of serial passage transplantation of spermatogonial stem cells starting from a single donor mouse. The original donor mouse was 90 days old. The donor testis cell suspension, 50 x 106 cells/ml, was introduced into a host testis, in which eight spermatogenic colonies developed. They were dissected out after 128 days for a subsequent second passage. The colony number actually passaged, 5.3 colonies in this case, was calculated based on the amount of cell suspension volume prepared (30 µl) and introduced (20 µl) into the host testes. In the second recipient testes, 10 colonies developed, which suggests that the donor cell suspension (equivalent to 5.3 colonies) contained colony-forming activity. Multiplication of the colony number was calculated to be 1.9 (10 divided by 5.3). In the third transplantation, which was after 373 days of incubation, the colony number multiplication was calculated to be 13.4. In this transplantation series, four successful serial passages resulted in 802 days of total incubation.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Summary of serial transplantation of spermatogonial stem cells



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 3. A) Colony size enlargement. Correlation between the incubation period and the resultant colony length: linear regression line and equation are shown in the graph. Y represents the colony length, while X represents the incubation period. r = 0.865 (P < 0.001). The results of the second and third passages are indicated with rhombus and square marks, respectively. B) Expansion of colony-forming activity. Correlation between multiplication of the colony number and the incubation period: linear regression line and equation are shown in the graph. Y represents the multiplication of the colony number, while X represents the incubation period. r = 0.749 (P < 0.001). The results of the second, third, and fourth passages are indicated with rhombus, square, and triangle marks, respectively. C) Donor colony length and regenerated colony number. Correlation between the length of the donor colony and the regenerated colony number: linear regression line and equation are shown in the graph. Y represents the regenerated colony number, while X represents the length of the donor colony. r = 0.886 (P < 0.001). The results of the second and third passages are indicated with rhombus and square marks, respectively

Stem Cell Expansion Following Transplantation

We analyzed the relations between the incubation periods and the length of each colony (Fig. 3A). A clear positive correlation was shown when the total colony length was plotted against incubation period. This suggests that each colony keeps expanding along the seminiferous tubules for more than 1 yr after transplantation. The rate of colony elongation appeared to be fairly constant. Furthermore, a linear correlation was found when the multiplication of the colony number was plotted against the incubation period. This suggests that spermatogonial stem cells keep expanding along with colony size expansion following transplantation and the expansion rate is constant during this period (Fig. 3B). A linear correlation was also found when we examined the relation between the length of the donor colony and the resultant colony number (Fig. 3C). This finding suggests that spermatogonial stem cells are evenly distributed in each colony.

Estimation of Colonization Efficiency

To calculate the efficiency of transplantation, we examined the colonization efficiency of the spermatogonial stem cells. First, we needed to prepare a cell suspension in which the number of spermatogonial stem cells could be estimated. For that purpose, the GFP mouse testes cells were transplanted into the testes of Sl/Sld mice. In the Sl/Sld mouse testes, spermatogonial stem cells can colonize and proliferate but cannot differentiate further than undifferentiated spermatogonia [18]. Therefore, only undifferentiated spermatogonia can survive and propagate in the Sl/Sld mouse testes. After more than 6 wk of incubation, the recipient Sl/Sld mouse testes were examined. The pattern of donor cell presence was observed with a stereomicroscope and confocal microscope (Fig. 4). The numbers of As, Apr, and Aal spermatogonia were counted. Then the percentage of As among all undifferentiated spermatogonia was calculated to be, on average, 6.8% (Table 2). The GFP-positive seminiferous tubules of Sl/Sld mouse testes were dissected out for enzyme digestion. The number of GFP cells in the cell suspension was counted and 6.8% of them were regarded as As spermatogonia, spermatogonial stem cells. Then the cell suspension was transplanted into six testes of busulfan-treated wild-type mice. Three weeks afterward, the testes were examined to count the colony number. The colonization efficiency was calculated by dividing the number of colonies developed by the number of spermatogonial stem cells transplanted. Efficiencies ranged from 2.5% to 8.2%, with an average value of 4.25% (Table 3).



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 4. Confocal laser microscopy of a colony in Sl/Sld mouse testis. Reconstructed picture of a seminiferous tubule of Sl/Sld mouse in which donor GFP cells (Green color-coded) developed a colony. As, Apr, and Aal spermatogonia are easily distinguished. TOTO-3 nuclear stain (blue color-coded) delineates all the cells constructing the seminiferous tubule


View this table:
[in this window]
[in a new window]
  		TABLE 2. Numbers of colonized GFP spermatogonia in SI/SId mouse testes.*


View this table:
[in this window]
[in a new window]
  		TABLE 3. Estimation of colonization efficiency


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we tested, using the transplantation technique, whether spermatogonial stem cells have long-term, if not limitless, self-renewing potential. We conducted several trials of serial spermatogonial transplantation to clarify if it was technically feasible. We failed in some cases to pass the stem cell to the next host testis, but in most cases, we were successful. We have so far succeeded in transplanting spermatogonial stem cells sequentially four times. The overall duration that a line of stem cells was maintained was 802 days.

The serial transplantation results revealed that the spermatogenic colony expands fairly constantly over time. When we calculated how many colonies were derived from a given number of colonies transplanted, it was also clearly demonstrated that the colony-forming activity increased along with colony size. However, the colony number does not necessarily represent the number of stem cells transplanted. It is possible that a single colony derived from several stem cells or that two or more colonies merged during their development. In the present experiments, however, the cell suspension used for transplantation was of relatively low concentration, between 0.33 and 20 x 106 cells/ml, so the resultant colonies in a host testis were up to 8 in number except in one testis that developed 16 colonies. Considering the total length of seminiferous tubules in the mouse testis, which is about 2 m [19, 20], it could be assumed that each colony had very little chance to be derived from multiple stem cells or merge during colony growth if the injected cell suspension spread throughout most of the seminiferous tubules. Therefore, we took the colony number as a representation of the number of stem cells in the transplanted cell population.

To estimate the number of stem cells or their expansion rate following transplantation, it was necessary to know the efficiency of colonization. As spermatogonia have been regarded as spermatogonial stem cells in the mouse testis [6, 7]. The morphometric analyses indicated that As spermatogonia were present at 0.03% among all germ cells in the testis [21]. Based on this proportion, it has been assumed that about 10% of injected spermatogonial stem cells can colonize the recipient testis to form spermatogenic colonies [13, 17, 22]. In the present study, we reevaluated this colonization efficiency by counting the number of spermatogonial stem cells in the cell suspension. Using Sl/Sld mouse testes as an intermediate host, we prepared cell suspensions in which all GFP cells were undifferentiated spermatogonia. In that cell suspension, 6.8% of GFP cells were regarded as As spermatogonia, namely spermatogonial stem cells. Based on the regenerated colony number and the injected stem cell number, the colonization efficiency was estimated to be 2.5–8.2%, with an average of 4.25%. These findings suggest that one spermatogenic colony would arise from about 20 spermatogonial stem cells transplanted. Based on the regression coefficient (0.0245) (Fig. 3B), it appears that the colony-forming activity expands about 2.5-fold in 100 days. With the colonization efficiency described above as well as nonestimated cell loss during the procedure, the expansion rate of spermatogonial stem cells per 100 days after transplantation appears to be greater than 50-fold. According to Figure 3C, the colony-forming activity in a given segment of the seminiferous tubule appears to be about six colonies per 10 mm (regression coefficient = 0.626). Again, based on the colonization efficiency, the frequency of spermatogonial stem cell localization was about 140 cells or more per 10 mm. This means that total stem cell number in a testis is over 28 000, assuming that the combined total length of seminiferous tubules in a testis is 2 m [19, 20]. This number of over 28 000 is in good agreement with another estimation of 35 000 stem cells in a testis based on an independent morphological method [21].

The expansion of actively dividing cells must be exponential. Hematopoietic stem cells, when transplanted into irradiated hosts, exponentially give rise to numbers of daughter cells in a short period. Therefore, marrow cellularity is generally restored within 2 wk of transplantation [23, 24]. However, the hematopoietic stem cells do not increase themselves at such a rate. They even cease to expand in the host body even when their number is less than 5% of the normal value [25]. Their self-renewal appears to be limited by certain mechanisms, intrinsic and/or extrinsic [26]. Observation of spermatogenic colony formation following transplantation has not shown rapid growth of its size [13]. Their growth in size, which was constant, lasted for 4 mo. In the present study, we also found rather slow but steady expansion of spermatogonial stem cells for a longer period. They did not show any sign of exhaustion of their proliferative activity. The expansion rates either after the second or third passage did not appear to be different. This constant increment of spermatogonial stem cells might relate to the three-dimensional microenvironment in which the stem cells are allowed to expand. They can spread only bilaterally along the seminiferous tubules. In addition, the density of the spermatogonial stem cells in a given segment of the seminiferous tubule appears to be predetermined. These spatial constraints limit the expansion of spermatogonial stem cells and thus repress their proliferation from the exponential to linear fashion even if they have vast proliferation potential. In this context, a recent study by Shinohara et al. demonstrated that spermatogenic stem cells, regardless of their donor age, had developed fourfold larger colonies in the recipient pup testis than in the adult one [27]. This suggests that spermatogonial stem cells can expand more than was shown in the present study if more hospitable conditions are provided, such as in growing testis.

The present findings have important implications for other critical questions about stem cells, such as control of self-renewal or differentiating cell division, possible future expansion of stem spermatogonia in vitro, or gene modification of spermatogonial stem cells.


   ACKNOWLEDGMENTS
 
We thank Drs. R.L. Brinster and K.E. Orwig for helpful comments on the manuscript. We are also grateful to Drs. I. Dobrinski and M. Nagano for critical reading of the manuscript. The GFP transgenic mouse was provided by Dr. M. Okabe at the Genome Information Research Center, Osaka University, Japan.


   FOOTNOTES
 
1 This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan (T.O., 11671569), and a Grant-in-Aid from Suzuki Foundation for Urological Medicine (T.O.). Back

2 Correspondence: T. Ogawa, Department of Urology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan. FAX: 81 45 786 5775; ogawa{at}med.yokohama-cu.ac.jp Back

Received: 13 February 2002.

First decision: 7 March 2002.

Accepted: 5 August 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Russell LD, Ettlin RA, SinhaHikim AP, Clegg ED (eds.). Mammalian spermatogenesis. In: Histological and Histopathological Evaluation of the testis. Clearwater, FL: Cache River Press; 1990: 1–40.
  2. Meistrich ML, van Beek MEAB. Spermatogonial stem cells. In: Desjardin C, Ewing LL (eds.), Cell and Molecular Biology of the Testis. New York, Oxford: Oxford University Press; 1993: 266–295
  3. Suzuki N, Withers HR. Exponential decrease during ageing and random lifetime of mouse spermatogonial stem cells. Science 1978; 202:1214-1215[Abstract/Free Full Text]
  4. Nistal M, Codesal J, Paniagua R, Santamaria L. Decrease in the number of human Ap and Ad spermatogonia and in the Ap/Ad ratio with advancing age. New data on the spermatogonial stem cell. J Androl 1978 8:64-68[Abstract/Free Full Text]
  5. Tanemura K, Kanai Y, Kanai-Azuma M, Kurohmaru M, Kuramoto K, Yazaki K, Hayashi Y. Reinitiation of spermatogonial mitotic differentiation in inactive old BDF1 mouse seminiferous tubules transplanted to W/Wv mouse testis. Biol Reprod 1996 55:1237-1242[Abstract]
  6. Huckins C. The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat Rec 1971 169:533-558[CrossRef][Medline]
  7. Oakberg EF. Spermatogonial stem-cell renewal in the mouse. Anat Rec 1971 169:515-532[CrossRef][Medline]
  8. Huckins C. Behavior of stem cell spermatogonia in the adult rat irradiated testis. Biol Reprod 1978 19:747-760[Abstract]
  9. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994; 91:11298-11302[Abstract/Free Full Text]
  10. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 1994 91:11303-11307[Abstract/Free Full Text]
  11. Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 1996 2:693-696[CrossRef][Medline]
  12. Clouthier DE, Avarbock MR, Maika SD, Hammer RE, Brinster RL. Rat spermatogenesis in mouse testis. Nature 1996 381:418-421[CrossRef][Medline]
  13. Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of donor mouse spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999 60:1429-1436[Abstract/Free Full Text]
  14. Dobrinski I, Ogawa T, Avarbock MR, Brinster RL. Computer assisted image analysis to assess colonization of recipient seminiferous tubules by spermatogonial stem cells from transgenic donor mice. Mol Reprod Dev 1999 53:142-148[CrossRef][Medline]
  15. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997 407:313-319[CrossRef][Medline]
  16. Ohta H, Yomogida K, Yamada S, Okabe M, Nishimune Y. Real-time observation of transplanted ‘green germ cells’: proliferation and differentiation of stem cells. Dev Growth Differ 2000 42:105-112[CrossRef][Medline]
  17. Ogawa T, Aréchaga JM, Avarbock MR, Brinster RL. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol 1997 41:111-122[Medline]
  18. Ohta H, Yomogida K, Dohmae K, Nishimune Y. Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF. Development 2000 127:2125-2131[Abstract]
  19. Bascom KF, Osterud HL. Quantitative studies of the testicle. II. Pattern and total tubule length in the testicles of certain common mammals. Anat Rec 1925 31:159-169[CrossRef]
  20. Setchell BP, Maddocks S, Brooks DE. Anatomy, vasculature, innervation, and fluids of the male reproductive tract. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press, Ltd.; 1994: 1063–1175
  21. Tegelenbosch RA, de Rooij DG. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat Res 1993 290:193-200[CrossRef][Medline]
  22. Shinohara T, Avarbock MR, Brinster RL. ß1- and {alpha}6-intergin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999 96:5504-5509[Abstract/Free Full Text]
  23. Siminovitch L, Till JE, McCulloch EA. Decline in colony forming ability of marrow cells subjected to serial transplantation into irradiated mice. J Cell Comp Physiol 1964 64:23-31
  24. Sutherland DJ, Till JE, McCulloch EA. A kinetic study of the genetic control of hemopoietic progenitor cells assayed in culture and in vivo. J Cell Physiol 1970 75:267-274[CrossRef][Medline]
  25. Iscove NN, Nawa K. Hematopoietic stem cells expand during serial transplantation in vivo without apparent exhaustion. Curr Biol 1997 7:805-808[CrossRef][Medline]
  26. Pawliuk R, Eaves C, Humphries RK. Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood 1996 88:2852-2858[Abstract/Free Full Text]
  27. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. Proc Natl Acad Sci U S A 2001 98:6186-6191[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
J. R Yeh, X. Zhang, and M. C Nagano
Establishment of a Short-Term In Vitro Assay for Mouse Spermatogonial Stem Cells
Biol Reprod, November 1, 2007; 77(5): 897 - 904.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
C. Wyns, M. Curaba, B. Martinez-Madrid, A. Van Langendonckt, W. Francois-Xavier, and J. Donnez
Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice
Hum. Reprod., June 1, 2007; 22(6): 1603 - 1611.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
J. Ehmcke and S. Schlatt
A revised model for spermatogonial expansion in man: lessons from non-human primates.
Reproduction, November 1, 2006; 132(5): 673 - 680.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
B.-Y. Ryu, K. E. Orwig, J. M. Oatley, M. R. Avarbock, and R. L. Brinster
Effects of Aging and Niche Microenvironment on Spermatogonial Stem Cell Self-Renewal
Stem Cells, June 1, 2006; 24(6): 1505 - 1511.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
X. Zhang, K. T. Ebata, B. Robaire, and M. C. Nagano
Aging of Male Germ Line Stem Cells in Mice
Biol Reprod, January 1, 2006; 74(1): 119 - 124.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
N. Sofikitis, E. Pappas, A. Kawatani, D. Baltogiannis, D. Loutradis, N. Kanakas, D. Giannakis, F. Dimitriadis, K. Tsoukanelis, I. Georgiou, et al.
Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment
Hum. Reprod. Update, May 1, 2005; 11(3): 229 - 259.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Kanatsu-Shinohara, T. Morimoto, S. Toyokuni, and T. Shinohara
Regulation of Mouse Spermatogonial Stem Cell Self-Renewing Divisionby the Pituitary Gland
Biol Reprod, June 1, 2004; 70(6): 1731 - 1737.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. C. Nagano
Homing Efficiency and Proliferation Kinetics of Male Germ Line Stem Cells Following Transplantation in Mice
Biol Reprod, August 1, 2003; 69(2): 701 - 707.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H. Kubota, M. R. Avarbock, and R. L. Brinster
Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells
PNAS, May 27, 2003; 100(11): 6487 - 6492.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
68/1/316    most recent
biolreprod.102.004549v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ogawa, T.
Right arrow Articles by Kubota, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogawa, T.
Right arrow Articles by Kubota, Y.
Agricola
Right arrow Articles by Ogawa, T.
Right arrow Articles by Kubota, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS