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This Article Abstract Full Text (PDF) All Versions of this Article: 71/6/2087    most recent biolreprod.104.033431v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wistuba, J. Articles by Schlatt, S. Search for Related Content PubMed PubMed Citation Articles by Wistuba, J. Articles by Schlatt, S. Agricola Articles by Wistuba, J. Articles by Schlatt, S.

CoGrafting of Hamster (Phodopus sungorus) and Marmoset (Callithrix jacchus) Testicular Tissues into Nude Mice Does Not Overcome Blockade of Early Spermatogenic Differentiation in Primate Grafts¹

Institute of Reproductive Medicine,³ University Münster, 48129 Münster, Germany Department of Cell Biology and Physiology,⁴ University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The ectopic xenotransplantation of testicular tissues into nude mice is a tool to generate sperm from immature testes. Immunodeficient mice as recipients of xenografts offered an appropriate microenvironment for differentiation of testicular tissue from hamsters, goats, pigs, and macaques. One exception is the neotropical primate Callithrix jacchus. Spermatogenesis in testicular grafts from marmosets does not proceed beyond the spermatogonial stage. The most likely cause for the poor graft development of marmosets is a deletion of exon 10 in the luteinizing hormone-receptor (LHR) gene, which renders this species insensitive to LH but responsive to chorionic gonadotropin (CG). We investigated whether cografting of testicular tissue from Djungarian hamsters would overcome the blockade in marmoset graft development. We also tested if exogenous administration of human CG (hCG) to the recipient would stimulate development of the marmoset tissue. No difference in graft survival was noted between hamster and monkey tissue. Seminiferous lumina were present in marmoset and hamster grafts but were significantly larger in hamster grafts. In the hamster grafts, a high proportion of the tubules contained meiotic and postmeiotic germ cells. In contrast, the marmoset tubules were populated with gonocytes and premeiotic spermatogonia. These results indicate that neither normal serum androgen levels nor the high local testosterone levels were sufficient to initiate marmoset spermatogenesis, nor was administration of hCG successful in overcoming the developmental blockade in marmoset tissue. Our results indicate that the conditions needed for initiation of spermatogenesis in the marmoset are remarkably different from those present in most other mammals.

androgens, CG, follicle-stimulating hormone, grafting, morphometry, spermatogenesis, testis, testis development, testosterone

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The ectopic transplantation of small fragments of testicular tissues into nude mouse hosts is a recently developed method to achieve testicular maturation and production of sperm from neonatal and immature testes of various species [1, 2]. The mouse recipient functions as a living incubator for immature testis tissue, which undergoes growth and differentiation leading to the initiation and completion of spermatogenesis as well as to restoration of serum androgen levels in the castrated recipients [1, 2]. Testicular grafting opens novel strategies to manipulate and explore testis development. This approach offers promising perspectives for extracorporeal generation of gametes in a variety of research applications and might also be applicable to fertility preservation in oncological patients. In contrast with xenologous germ cell transplantation, xenografting of testis tissue allows sperm to be generated as the germ cell differentiates in its own testicular microenvironment [3–5]. Although xenografting of testicular tissue resulted in the full induction of spermatogenesis in most species analyzed so far, a rare exception is seen in the marmoset monkey [6, 7]. Marmoset testis grafts marginally differentiate, do not release androgens, and spermatogenesis never reaches meiosis. We assumed that the most likely reason for this is a deletion in an exon 10 of the luteinizing hormone-receptor (LHR) gene, which renders this species sensitive to chorionic gonadotropin (CG) [8, 9]. Because mice do not express CG, the host endocrine environment could not support the CG and androgen-dependent development of the marmoset testis. Here we developed a new approach to study several aspects of the endocrine regulation of testicular development based on cografting of tissue from Djungarian hamsters and marmosets. As was previously demonstrated, immature hamster tissue grows well as xenografts, which contain fully mature sperm and resubstitute normal androgen levels in castrated recipients [6]. Thus, we chose hamster testicular tissues for cografting to achieve high local levels of testosterone release at the implantation sites of the grafts. We addressed the question of whether the endocrine input (grafts of both species located at different sites in the recipient) or the direct contact of the testicular tissue (tissue of both species combined into each grafting site) would change the developmental potential of marmoset testis tissue.

	MATERIALS AND METHODS

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Donor testes were dissected either from 5-day-old hamsters (Phodopus sungorus, n = 20), which were killed by decapitation or from marmosets (Callithrix jacchus, newborn [n = 4], 1 [n = 2], 3 [n = 2], and 7 mo old [n = 2]). Marmosets and hamsters were obtained from the institutional breeding facilities. Before testes were removed from the primates, the animals were sedated with ketamine and killed by exsanguination. Hamsters were killed by decapitation. For control purposes, three fragments of immature testicular tissue from both species, marmoset and Djungarian hamster, were fixed in Bouin solution, embedded in paraffin, and analyzed histologically. The diameter of 50 seminiferous tubules was determined in these control tissues. Testes fragments (fragment size ranged from 0.5 to 1 mm³) were kept in ice-cold Dulbecco modified Eagle medium for up to 4 h until grafting, as previously described [7]. Seven groups (n = 4–5) of nude mice (6–7 wk of age; Harlan Winkelmann, Borchen, Germany) were castrated and subcutaneously grafted, receiving either eight halves of newborn hamster (Phodopus sungorus) testes, eight similarly sized fragments of marmoset (Callithrix jacchus) testes, four hamster and four marmoset fragments placed separately, or four mixed fragments of testicular tissue from both species. One control group of nude mice was sham grafted and castrated and another control group was sham grafted and sham castrated. One group receiving testicular tissue of newborn marmosets was treated with human chorionic gonadotropin (hCG; 10 IU twice weekly; Otrivel, Serono Europe Ltd., London, UK) and in another group vehicle (NaCl) was administered for control purposes. The administration of hCG to the mouse hosts was performed with regard to previous data showing that the hCG dose of 10 IU is effective [10]. Due to the half-life of the hormone, the interval of two injections per week is sufficient to keep the hCG levels in the effective range. Anesthesia was induced and maintained for up to 45 min using Avertin (2,2,2-tribromoethanol; Sigma-Aldrich Chemie GmbH, Steinheim, Germany; 63 g/kg body weight). The animals were castrated through scrotal incisions. The scrotal skin was closed using stainless steel wound clips. Four skin incisions of 4–5 mm were made on either side of the dorsal midline and eight grafts per recipient were placed in pockets underneath the muscle layer of the skin. The incisions were closed with clips. Throughout the experiment, the mice were kept in groups of 5–7 per cage, with food and water available ad libitum. The experimental work was performed in accordance with the German Federal Law on the Care and Use of Laboratory Animals (license G67/2001). Grouping and treatment of the recipients is described in Table 1.

After 12 wk, the mice were weighed and anesthetized using Avertin. Blood was collected by cardiac puncture. Serum samples were stored at –20°C and later used for determination of androgen levels. The seminal vesicles were dissected and the back skin was removed. The donor-derived testicular grafts were dissected from the skin. Body weight and seminal vesicle weight were recorded. Testosterone levels were measured using a previously published RIA [11]. Each sample was processed in duplicate after double extraction with diethyl ether. The grafts were individually weighed after 18–24 h fixation in Bouin solution. Afterwards they were stored in 70% ethanol until routine embedding in paraffin and tissue sectioning (5 µm). Periodic acid Schiff/hematoxylin staining was used for analysis of the histology in the grafts.

The most central cross section of each graft was evaluated to determine histology. All seminiferous tubules in the cross section (range: 32–622) were scored for the presence of spermatogonia, spermatocytes, round or elongating spermatids. Tubular cross sections without germ cells were scored as Sertoli-cell only. Determination of the most advanced germ cell type in each graft was used to describe the maximal progression of spermatogenesis. The size of the seminiferous lumen and tubular diameter were analyzed using the smallest distance in all tubular cross sections of the single grafts, with the help of an Axiovert 200 microscope (Zeiss, Oberkochen, Germany) and Axiovison 3.1 (Zeiss) software. The proportions of tubules exhibiting certain germ cell stages were evaluated. Representative images were taken at magnifications of 100x and 200x (Axiocam; Zeiss).

Statistical Analyses

All data were expressed as mean ± standard deviation. Data were statistically analyzed applying one-way ANOVA. Values of tubular and lumina diameter were compared by ANOVA on ranks. Computations were performed using the statistical software package SIGMASTAT 2.03 (SPSS Inc., Chicago, IL). Values of P considered to be statistically significant are given in the tables.

	RESULTS

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Analysis of Body Weight and Androgen Status in Recipients

The body weights in the groups of recipients showed no significant differences (Table 2). Seminal vesicle weights in all recipients receiving hamster tissue were in the range of intact control mice. In contrast, the seminal vesicle weight of all mice that exclusively contained marmoset grafts was significantly lower with values in the castrate range (Table 2). Serum testosterone levels confirmed the difference in the androgen status. The mean level of testosterone in groups of recipients of hamster tissue was 3.19 ± 1.18 ng/ ml. The androgen levels of these recipients were independent of the presence of marmoset grafts. All of the recipients receiving exclusively marmoset grafts and the castrate control group showed levels below or at the detection limit of the testosterone assay (0.87 ng/ml).

Weights of Testicular Grafts

The weights of the recovered grafts showed significant differences between groups of recipients (Table 2). Whereas all groups receiving marmoset tissue showed graft weights at or below 50 mg, all groups receiving hamster tissue showed three- to fourfold higher weights of grafts (Table 2). The weights of combined grafts containing hamster and marmoset tissue were significantly smaller when compared with the grafts of hamster tissue only. When testicular tissues of monkeys and hamsters were separately cografted at contralateral sides, the hamster grafts in mice carrying grafts showed the highest graft weights recorded in this study. Neither the different ages of the donors nor the administration of hCG to the mouse recipients had any effect on the weights of marmoset grafts (Table 2).

Histological Analysis of Testicular Grafts

Histological analysis of grafts at the time of implantation revealed only gonocytes and early spermatogonia as the most advanced germ cell types in the testicular tissues of both donor species (Fig. 1, a and b). The mean tubular diameter was 47.4 ± 7.6 µm and the diameter of the seminiferous tubule lumen was 21.0 ± 4.8 µm in the hamster tissue and 67.5 ± 12.5 µm and 43.4 ± 9.5 µm in the marmoset tissue.

View larger version (204K): [in this window] [in a new window]   FIG. 1. Micrographs showing the histology of transplants before and after grafting. a) Immature testicular hamster tissue. b) Immature testicular marmoset tissue. c) Hamster grafts exhibit an enlargement of seminiferous tubules and lumen formation; spermatogenesis is ongoing and postmeiotic cell types as spermatocytes and round spermatids (small arrows) were found in the majority of the explanted grafts. d) Marmosets grafts revealed the blockade of spermatogenic development in the early stages of germ cell development. Gonocytes and spermatogonia are the most advanced germ cell types found. e–f) Hamster and marmoset seminiferous tubules placed in the same graft. The degree of testicular development and the status of spermatogenesis were not affected by the presence of grafts from the second species (H, hamster: M, marmoset); scale bars = 100 µm

One third (32.8% ± 13.7%) of the recovered grafts consisted of sclerotic tissue with no surviving cells in the seminiferous tubules. No difference in graft survival was noted between hamster and monkey tissue. There were 9.9 ± 6.4 (range: 4–25) grafts morphometrically analyzed for each group. A total of 1812 seminiferous tubules were scored in the recovered grafts (mean number of seminiferous tubules scored for each experimental group: 164.7 ± 180.0, range: 32–622). Of the seminiferous tubules, 59.6% ± 15.9% showed no germ cells and were considered Sertoli-cell-only. The number of germ cell-deficient tubules was similar for both hamster and marmoset grafts.

Seminiferous tubule lumina were enlarged in marmoset and hamster grafts (Fig. 1, c and d). Compared with the control tubules analyzed at the time of transplantation, a significant increase was found in the tubular as well as in the lumen diameter in both species (P < 0.001). Furthermore, at the time of grafting, marmoset tubules were significantly bigger and exhibited a wider lumen compared with hamster tubules (P = <0.001), but after 12 wk, the seminiferous tubules and tubular lumina were significantly larger in hamster grafts compared with marmoset grafts (P < 0.001), with the exception of hamster tubules that developed in close conjunction with neonatal marmoset tissue (mixed fragments). The hamster tubules revealed a diameter similar to the surrounding marmoset-derived tubules, although those hamster tubules also exhibited the presence of postmeiotic germ cells (Table 3 and Fig. 1, e and f). The relative numbers of tubules containing premeiotic germ cells (spermatogonia and gonocytes) were comparable in all groups of grafts and were independent of the species and of the mixed or the separate placement of grafts (Table 3). A high proportion of hamster seminiferous tubules contained spermatocytes (71.6% ± 14.2%) and round spermatids (44.6% ± 14.5%; Fig. 1a and Table 3). In sharp contrast, the marmoset tubules were exclusively populated with gonocytes and spermatogonia (Fig. 1d).

Testicular histology was not changed in marmoset grafts derived from older donors. No postmeiotic germ cells were observed, and the relative number of seminiferous tubules containing spermatogonia was identical to neonatal donors (neonatal, 37.5%; 1 mo, 50%; 3 mo, 71.4%; 7 mo: 50%). Similarly, no meiotic progression of germ cell development was encountered after hCG treatment of the recipients. The relative number of seminiferous tubules showing spermatogonia was 61.5% for hCG-treated recipients and 25% for saline-treated controls, which did not reach statistical significance. The unchanged levels of androgens (serum levels and seminal vesicle weight) substantiated the nonresponsiveness of the marmoset graft to the hCG treatment (Table 2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Grafting of testicular tissue appears to be a promising tool for extracorporeal generation of sperm. Xenologous germ cell transplantation has been used as an alternative approach but presented the problem that, although transplanted nonrodent germ cells are able to colonize a host testis, they never entered meiosis [3–5, 12–15]. In principle, grafting of neonatal testes presents a tool to transplant the spermatogonial stem cell with its niche and its somatic environment, which, after successful reanastomosis, leads to fully functional differentiation of the seminiferous tubules and initiation of spermatogenesis and steroidogenesis. In contrast, xenologous germ cell transplantation is an attempt to transplant spermatogonial stem cell from one niche to another in a different species. Therefore, it is not surprising that species-specific mechanisms during differentiation of premeiotic germ cells block the completion of germ cell differentiation of distantly related species following xenotransplantation.

An interesting exception to the species-independent successful initiation of spermatogenesis in grafted tissue is the marmoset monkey, whose testicular graft development arrests at the premeiotic stage and whose grafts do not release androgens into the recipient circulation [6]. The gonadotropin system in the marmoset was demonstrated to be remarkably different when compared with the mouse and the old-world monkeys [8–10]. The LH/CG receptor in the neotropical platyrrhine monkeys lacks exon 10 and is sensitive to CG. In these new-world monkeys, CG took over the functions of LH [8]. That a different endocrinological situation is obviously present in the marmoset was confirmed in a previously published study exhibiting that the CG beta subunit but not LH beta subunit is expressed in the pituitary of the marmosets [10].

Thus, the developmental arrest would indicate that mouse gonadotropins are not sufficient for initiation of spermatogenesis in grafted marmoset testis. We hypothesized that the deletion of exon 10 in the LH receptor of this species would render the monkey unresponsive to mouse LH and that the consequences of this genetic difference would cause a blockade of further gonadal differentiation. In particular, the absence of androgens might disallow further differentiation of the testicular tissue. In the present study, we tested whether cografting of marmoset tissue together with hamster tissue would overcome this blockade, as androgens and other factors released from the hamster tissue might be sufficient to overcome the developmental arrest. To our surprise, our hypothesis failed to be true and we obtained no progression of marmoset testis development after cografting.

Our experiments demonstrate that the developmental blockade was not overcome by androgens or other factors released from hamster testes. Because it was irrelevant whether the grafts were placed separately or in close conjunction with marmoset tissue, we conclude that neither endocrine factors released from hamster tissue into the circulation nor paracrine factors acting locally were able to stimulate further progression of the monkey grafts. In fact, the differences in graft weight and the less progressive histological development of the hamster tissue in close conjunction to marmoset tissue indicates that the marmoset tissue exerts a negative effect on the hamster tissue rather than that the hamster tissue has a positive effect on the marmoset tissue.

It was recently shown that testicular ectopic grafts from juvenile (13 mo of age) rhesus monkeys developed up to full spermatogenesis [2]. However, during regular development, the levels of gonadotropins are in the adult range during infancy, but no differentiation of germ cells is observed, and the testis does not respond to a hormonal milieu that would be highly stimulatory during the juvenile period [16, 17]. We therefore grafted tissue from juvenile marmosets into nude mice to explore whether the neonatal primate testis has a different developmental potential, compared with the juvenile testis. Our results indicate that grafts obtained from 1-, 3-, and 7-mo-old marmosets show the same premeiotic arrest, indicating that the developmental status at the time of grafting does not affect the potential of graft development.

We originally postulated that the reason for the arrest is the deletion of exon 10, rendering the marmoset grafts unresponsive to LH, but responsive to CG. Mice have LH but no CG. We therefore tested whether the administration of hCG to the recipient would overcome the developmental arrest of the marmoset grafts. Interestingly, the application of hCG did not stimulate further testicular development. It might be that the exogenous administration was not sufficient to achieve a microenvironment in the mouse recipient that mimics the situation in the marmoset. Alternatively, the presence of hCG might not be the critical factor to overcome the developmental arrest. Marmosets show extraordinarily high levels of circulating ACTH and cortisol. The relative glucocorticoid insensitivity is attributed to a decreased affinity of their glucocorticoid receptor and a compromised ability of this receptor to transactivate glucocorticoid-responsive genes [18]. It could therefore be possible that other species-specific physiological reasons are responsible for the surprisingly robust blockade of testicular development in marmoset testis grafts. We can exclude poor survival or less efficient anastomosis of monkey grafts as a reason for the developmental arrest because the survival of the grafts is similar for both species and the marmoset grafts show some degree of development. We therefore speculate that a specific and thus far unknown factor is missing in the milieu of the grafted tissue. This factor, however, is not released from hamster testicular tissue reaching the mature stages of spermatogenesis and reconstituting normal levels of androgens to the recipient. Further studies are needed to explore the factors allowing the marmoset grafts to enter the next stages of development and to initiate spermatogenesis and steroidogenesis.

We conclude from our cografting experiments that neither the presence of normal serum levels of androgens nor the presence of high local levels of testosterone nor the administration of hCG are sufficient to induce germ cell development in xenografted marmoset tissue.

	ACKNOWLEDGMENTS

 
We wish to thank Prof. Dr. E. Nieschlag, director of the Institute of Reproductive Medicine of the University Münster, for his support, providing the facilities of the institute. The excellent technical assistance of Ingrid Upmann and Reinhild Sandhowe-Klaverkamp is kindly acknowledged.

	FOOTNOTES

 
¹ Funded by the German Research Foundation (DFG-grant SCHL394/6–1).

² Correspondence: Stefan Schlatt, Department of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, S362 Biomedical Science Tower, 3500 Terrace Street, Pittsburgh, PA 15261. FAX: 412 648 8315; schlatt{at}pitt.edu

Received: 22 June 2004.

First decision: 12 July 2004.

Accepted: 16 August 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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This Article Abstract Full Text (PDF) All Versions of this Article: 71/6/2087    most recent biolreprod.104.033431v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wistuba, J. Articles by Schlatt, S. Search for Related Content PubMed PubMed Citation Articles by Wistuba, J. Articles by Schlatt, S. Agricola Articles by Wistuba, J. Articles by Schlatt, S.