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Progeny from Sperm Obtained after Ectopic Grafting of Neonatal Mouse Testes¹

Institute of Reproductive Medicine,³ 48149 Münster, Germany Center for Animal Transgenesis and Germ Cell Research,⁴ New Bolton Center, University of Pennsylvania, Kennett Square, Pennsylvania 19348

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ectopic grafting of testicular tissue is a promising new approach that can be used to preserve testicular function. This technique has been used recently to differentiate the neonatal testes of different species, up to the level of complete spermatogenesis. This approach can be applied successfully to generate live progeny using sperm extracted from grafts originating from testes of newborn donors. The sperm are capable of supporting normal development and producing fertile male and female offspring after intracytoplasmic injection into mouse oocytes and embryo transfer into surrogate mothers. The grafted tissue was also capable of significantly normalizing reproductive hormone levels in the castrated recipients. This technique presents new avenues for experimentation. The recipient mouse can be regarded as a living incubator and a culture system of testicular tissue, allowing the experimental manipulation of several aspects of testis development and spermatogenesis. The successful generation of pups indicates that this technqiue can be used to study the testicular phenotype and to breed mutant or transgenic mouse strains with lethal postnatal phenotypes. The ability to generate sperm from the germ line ex vivo also paves the way for the development of new strategies for preserving fertility in boys undergoing cancer therapy.

developmental biology, fertilization, spermatogenesis, testis

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recent advances have opened new avenues for preservation of gonadal function [1]. In the female, cryopreservation and grafting of the whole ovary or strips of ovarian tissue showed promising results in experimental studies and might soon become a clinically useful strategy [2–4]. In males, the advent of germ cell transplantation in mice [5, 6], domestic animals [7, 8], and primates [9, 10], the in vitro culture of male germ cells [11], and the generation of immortalized cell lines from the male germ cell lineage [12–14] are innovative tools leading to rapid scientific progress but are still at an experimental stage [15]. Grafting of testis tissue from neonatal mouse, pig, goat, hamster, and marmoset [16, 17] into mouse hosts revealed that the gametogenic competence of the testis graft is maintained and in some cases enhanced. The sperm produced were capable of fertilizing an oocyte that progressed up to the stage of activated oocyte or fetus [16]. This approach added a new tool for the generation of gametes through artificial techniques and has immediate implications for a large variety of disciplines. It might also lead to the development of new strategies for the preservation of endangered species, analysis of testicular defects, and fertility protection in cancer patients receiving gonadotoxic chemo- or radiotherapy.

In the present study, we investigated whether and to what extent the sperm isolated from the testis grafts could also direct embryonic development. We used histological and hormonal assays to determine the best time point for retrieval of sperm following the grafting procedure. Combining sperm retrieval from testicular tissue, intracytoplasmic sperm injection (ICSI), embryo culture, and transfer in vivo, we were able to obtain live offspring from the sperm produced in mouse neonatal grafts. This result demonstrates that an ectopic location, such as the skin, provides an adequate environment for orchestration of gametogenesis from unprimed testicular tissue.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals and Experimental Surgery

Donor testes were dissected from neonatal ICR or B6C3 F₁ (C57BL/6 x C3H/He) pups, which were killed by decapitation. A total of 120 male pups were used for the experiment. Testes were cut in half (the size of the fragments ranged from 0.5 to 1 mm³) and kept in ice-cold Dulbecco modified Eagle medium until grafting, which occurred within a maximum of 1.5 h. The various steps and outcomes of the procedure are indicated in Figure 1. Some testes fragments were fixed in Bouin solution immediately after dissection to serve as a reference for testis development.

View larger version (67K): [in this window] [in a new window]   FIG. 1. Various procedures followed to obtain offspring from neonatal mouse testicular grafts. The testes from newborn pups were dissected and cut into halves about 0.5 mm2 in diameter (A). Each half was used as one graft. Eight grafts per mouse were fixed to the s.c. muscle layer using small pieces of suture and were left in the mice for different time periods during which the growth of the testis tissue became visible through the back skin (B). At the time the mice were killed, the grafts had grown to a few millimeters in size (C). Sperm were retrieved from the grafted tissue and used for ICSI. The embryos obtained were transferred into pseudo-pregnant recipients, which gave birth to pups after a normal duration of pregnancy (D)

Five- to 7-wk-old male immunodeficient NCr mice (Taconic, Germantown, NY) were used as recipients (n = 44). For testis grafting, anesthesia was induced and maintained using Avertin (2,2,2-tribromoethanol; 63 g/kg body weight). The animals were castrated through scrotal incisions. The scrotal skin was closed using Michel stainless steel wound clips. Four skin incisions 4–5 mm each were made on either side of the dorsal midline. Using nonabsorbable 6/0 prolene suture (Ethicon, Somerville, NY), eight grafts per recipient were secured to the muscle layer of the skin. The wounds were closed with Michel clips. Throughout the experiment, the mice were kept in groups of 5–7 per cage, with food and water available ad libitum.

Recipient mice were randomly distributed among nine experimental groups. In groups 1–5, castrated recipients receiving grafts were analyzed at Weeks 2, 4, 8, 12, and 16 (n = 5 mice/group), in groups 6 and 7, castrated controls were killed at Week 4 (n = 4) and Week 12 (n = 5), and in groups 8 and 9, intact controls were killed at the start of the experiment (n = 5) and at Week 12 (n = 5). Two hours before death, the animals received an i.p. injection of bromodeoxyuridine (BrdU, 100 mg/kg body weight). At the time of death, the mice were weighed and anesthetized, and blood was collected by cardiac puncture. The seminal vesicles were dissected and weighed, the back skin was removed and photographed, and the number of visible grafts was recorded. The testicular tissue was dissected from the skin and fixed in Bouin solution. All animal experiments were approved by and performed under the guidance of the Animal Care and Use Committee at the University of Pennsylvania.

Histology and Microscopy

Tissue was fixed for 18–24 h in Bouin solution, transferred for storage into 70% ethanol, and embedded in paraffin for sectioning at 5 µm. Tissue sections were stained with hematoxylin and eosin. BrdU was localized by immunohistochemistry. Sctions were deparaffinized and rehydrated. After rinsing with tap and distilled water, sections were hydrolyzed using 1 M HCl at 70°C for 8 min in a temperature-controlled microwave oven. After a wash in running tap water, the sections were incubated for 15 min at room temperature in 0.1% trypsin in Tris-buffered saline (TBS: 10 mM Tris, 150 mM NaCl, pH 7.6). Using 5% normal goat serum, nonspecific staining was blocked for 20 min immediately before incubation with a monoclonal mouse anti-BrdU antibody (M0744; DAKO, Carpinteria, CA; diluted 1:30 in TBS + 0.1% BSA) for 60 min or overnight. After three washes in TBS, sections were incubated with secondary, goat anti-mouse IgG linked to horseradish peroxidase for 60 min. After several washes in TBS, the label was visualized using diaminobenzidine as a substrate to be converted into a dark brown precipitate. The reaction was stopped by a rinse in distilled water. Slides were then counterstained with hematoxylin, dehydrated, and mounted. A similar protocol without the hydrolysis and digestion steps was used for staining of alpha smooth muscle actin using a commercially available antibody (A2547; Sigma, St. Louis, MO). Tissue sections were qualitatively analyzed for the degree of spermatogenic activity and the most advanced stage of germ cell development achieved at the various time points analyzed. Representative tissue sections were photographed.

RIA for Testosterone and FSH

Testosterone levels were measured using a previously published RIA [18]. Each sample was processed in duplicate after double extraction with diethyl ether. Intra- and interassay variances were 5.0% and 8.2%, respectively. FSH was determined by a commercially available rat assay system (Amersham, Pharmacia, Piscataway, NJ) without magnetic separation. Intra- and interassay variations were <6%. One-way ANOVA followed by a Tukey multiple comparison test was performed to determine statistical significance of differences in hormone measurements and weights of seminal vesicles. Data were expressed as means ± SEM. Differences were considered significant at P < 0.05.

Preparation of Sperm Samples and Assisted Fertilization

Sperm were retrieved from the testicular grafts after mincing and dispersing the tissue in Whittingham medium supplemented with BSA (3% w/v). The sperm were used for assisted fertilization either fresh or after cryopreservation (snap frozen in liquid nitrogen). Sperm were partitioned into a head and a tail, and the head was injected into metaphase II mouse oocytes with a 10-µm blunt-end borosilicate capillary using a piezo actuator (Prime-Tech, Ibaraki, Japan) under observation by DIC optics (Nikon, Tokyo, Japan). Oocytes were maintained in Hepes-buffered CZB medium supplemented with glucose and polyvinylpyrrolidone (1% w/v) and allowed to recover after injection for 15 min, when they were transferred into culture medium M16 and further cultured at 37°C in 5% CO₂ in air. Oocyte activation after ICSI was measured by the second polar body extrusion occurring 2–3 h later. Seventy-one embryos (from the 144 total zygotes obtained after fertilization) were transferred at the two-cell stage (n = 50) and the blastocyst stage (n = 21) to the uterus of two pseudopregnant females at 0.5 days postcoitum (dpc) (two-cell embryos) and two pseudopregnant females at 2.5 dpc (blastocysts). One female from each group delivered pups.

	RESULTS

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Evaluation of Testicular Growth

Growth of the grafted testicular tissue was easily observed under the back skin of the nude mice (Fig. 1B). At the time of death, the skin was stretched and photographed to document the survival and growth of the grafts. Figure 1C shows a typical example of the back skin, with four of eight grafts recovered after 4 wk. About 60% of all grafts survived and grew to a typical size of 4–6 mm in diameter.

Evaluation of Testicular Differentiation

At the time of grafting, the testicular tissue consisted of seminiferous cords; Sertoli cells and gonocytes were the only cells present (Fig. 2A). Two weeks after grafting, 60% of the seminiferous tubules contained spermatocytes. Round spermatids were seen in about 20% of the tubular tissue at 4 wk after grafting, and a few tubules already contained elongated spermatids. A typical example of well-developed seminiferous tissue showing numerous proliferatively active premeiotic germ cells and fully differentiated peritubular cells 4 wk after grafting is shown in Figure 2, B–D. The positive staining for BrdU reveals high proliferative activity in papilla of the hair and the basal compartment of the seminiferous epithelium (Fig. 2, B and C). Staining for alpha smooth muscle actin revealed full differentiation of peritubular cells at all time points analyzed (Fig. 2D). The presence of round and elongated spermatids highlights the completion of the first round of spermatogenesis at Week 4 (Fig. 2, C and D). Whereas at Weeks 12 and 16 almost all seminiferous tubules contained meiotic germ cells, the number of seminiferous tubules containing spermatids increased to only about 40%, and the number of fully matured germ cells remained low. Starting at Week 4, dilation of the seminiferous tubule lumen became more prominent (Fig. 2B).

View larger version (137K): [in this window] [in a new window]   FIG. 2. Histology of the testicular tissue before (A) and after (B–D) 4 wk of grafting. A) The seminiferous tubules of the neonatal mouse testis contain only Sertoli cells and gonocytes. Periodic acid-Schiff-hematoxylin. Bar = 10 µm. B) Low-power view of the grafted testicular tissue in direct contact with the muscle layer of the skin. BrdU incorporation was detected by immunohistochemistry (brown nuclear precipitate) and indicates high proliferative activity in papilla of the hair and in premeiotic germ cells. A distention of the tubular lumen is visible in many seminiferous tubules. Hematoxylin counterstain. Bar = 250 µm. C) High-power view of the same graft shown in B. All stages of germ cells are present 4 wk after grafting. Numerous BrdU-positive cells (arrows) are visible in cells lying on the basement membrane of the seminiferous tubules. Hematoxylin counterstain. Bar = 50 µm. D) Immunohistochemical detection of alpha smooth muscle actin of a section adjacent to that shown in C. Blood vessels and peritubular cells have a positive signal (brown precipitate), indicating final differentiation into smooth muscle cells. Hematoxylin counterstain. Bar = 50 µm

Generation of Progeny

Retrieval of sperm from the grafts in combination with assisted fertilization resulted in the generation of live offspring (Fig. 1D). The sperm obtained from the testicular grafts showed a normal fertilizing ability for extracted sperm samples; 80% and 50% of the activated oocytes reached the two- and four-cell stage, respectively. Control experiments using epididymal sperm resulted in 88% and 65% success rates, respectively. From a total of 312 oocytes that were manipulated and allowed to grow in vitro, 94 (30.1%) formed blastocysts by 96 h. A total of 7 pups were produced from two of the four recipients (one pup died after birth; three females and three males grew to maturity). The male and female mice generated from grafted sperm showed normal fertility in mating experiments.

Hormonal Changes in Grafted Animals

FSH levels were two-fold higher in castrated compared with intact mice (Fig. 3). Grafted animals showed intermediate FSH levels at Weeks 2, 4, and 12 and elevated levels close to the castrated range at Weeks 8 and 16. From Week 8 onward, seminal vesicle weights were maintained in the adult range. Normal adult levels of serum testosterone indicated fully functional steroidogenesis in the grafts leading to restoration of physiological levels of androgens in the castrated host (Fig. 3).

View larger version (28K): [in this window] [in a new window]   FIG. 3. Endocrine parameters determined in the nine experimental mouse groups (n = 5). Asterisks indicate a significant difference in comparison with the castrated controls (P < 0.05). Serum FSH levels were significantly lower than castrate levels after 2 and 4 wk and remained in the intermediate range at Weeks 8–16. The androgens levels were estimated using the weight of the seminal vesicles and the serum levels of testosterone. Both parameters show that the grafts produced normal to high physiological androgen levels from Week 8 to Week 16 in the recipient mice

	DISCUSSION

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In the present study, we were able to show that ectopic grafting is a simple and powerful approach for achieving full functional development of neonatal testicular tissue. To the best of our knowledge, this is the first report of the generation of progeny using sperm retrieved from ectopic grafts. These results extend our previous findings [16, 17] that ectopic grafting of neonatal mouse testicular tissue into immunodeficient hosts leads to the initiation/restoration of spermatogenesis and steroidogenesis. Grafting allows the generation of sperm with full fertilizing potential and therefore can be used to preserve fertility. Ectopic grafting of neonatal testicular tissue can be used to generate gametes from animals that have died before reaching puberty. These data are in concordance with a recent report showing that homotopic transplantation can also be successfully applied to generate offspring using assisted fertilization techniques [19]. These findings open new avenues for studying and preserving fertility in endangered species or in patients undergoing fertility-compromising therapies. Successful induction of spermatogenesis has been achieved after xenografting of neonatal testicular tissue from hamsters, pigs, goats, and to a lesser extent marmosets [16, 17]. Ectopic testicular grafting might also be useful in several areas of basic research. For example, it allows sperm production and generation of offspring from neonatally lethal transgenic or mutant mouse models, as was proposed for female mice after ovarian grafting [20]. It also provides an experimental approach that extends the use of testicular grafting in a mouse lacking connexin 43 [21], allowing analysis of the capacity for testicular differentiation and meiosis and testing for fertility.

Detailed histological analysis revealed that spermatogenesis was complete in the graft, peritubular cells were differentiated, and premeiotic germ cells were proliferating intensively. The grafted tissue developed up to the level of qualitatively full spermatogenesis from neonatal testes with seminiferous cords containing peritubular cells, Sertoli cells, and gonocytes as the most advanced germ cells. Sperm collected from the grafted testicular tissue and microinjected into mouse oocytes gave rise to embryos that developed to term. Although the success rates were better using freshly prepared sperm, fertilization was also achieved using frozen-thawed sperm preparations. When the pups born from these procedures grew to maturity, the three males and three females were used for breeding. Their normal fertility indicates that no major damage to the germline could be attributed to the grafting process or the ectopic location of the male gonad. Further studies are needed to prove the safety of the technique and to exclude additional risks, such as the infection of germline cells with endogenous mouse viruses, before this technique can be applied to humans.

Testicular grafting and germ cell transplantation are two new experimental tools for fertility preservation in cancer patients. Although germ cell transplantation could potentially serve as a tool for restoring a patient's fertility, testicular grafting is limited to the production of male gametes for assisted fertilization. However, germ cell transplantation inherits a serious risk for tumor cell transmission to the patient who has been cured of the disease. Cell sorting techniques using markers to recognize malignant cells or male germ line stem cells may be applied to testicular cell suspensions. This approach might minimize the risk for malignant cell transfer when germ cell transplantation is used to restore fertility. An even more serious problems of tumor cell transmission to the patient occurs when autologous grafting is performed. The grafted tissue may contain malignant cells that cannot be easily removed from the tissue. Alternatively, xenologous testicular grafting can be performed, where sperm is produced in an animal eliminating the need to transfer potentially malignant cells back into the donor. However, generation of sperm in a foreign species may entail thus far unknown risks such as the transfer of (retro)viruses into the grafted tissue and germ cells. Studies on the efficiency and safety of these techniques are needed to determine the most appropriate clinical application.

The exocrine function of generating gametes and the endocrine function were restored in the grafted testicular tissue. Serum FSH and serum androgen concentrations and the growth of seminal vesicles in castrated recipient mice indicated that the grafts were actively secreting hormones. The s.c. testicular tissue was able to establish partial to full feedback when compared with intact and castrated controls. We observed an intermediate level of FSH at Weeks 4, 8, and 12 compared with normal and castrated controls. This finding indicates that the seminiferous epithelium in the testicular grafts controlled the serum FSH levels and that the feedback established from the graft was strong enough to suppress significantly FSH release from the pituitary. The increase in FSH levels at Weeks 12 and 16 parallels the increasing damage to the seminiferous epithelium. Changes in serum FSH levels therefore can be used to determine the most promising time for sperm retrieval from the grafted testicular tissue. We postulate that the best time point for retrieval of mouse sperm after s.c. grafting is between Weeks 4 and 8, when the first wave of spermatogenesis is completed but the damage to the seminiferous epithelium is not yet pronounced. Androgen production was restored in all mice receiving testicular grafts. Although the serum testosterone levels fluctuated markedly, the weight of the seminal vesicle was a more stable indicator of androgen action. In contrast to FSH, androgen secretion appeared to be similar to that of normal animals and did not change significantly with longer grafting periods. Androgen levels remained in the high normal range when FSH levels increased, suggesting that the spermatogenic failure in the tubular compartment did not have a profound influence on the interstitial cells and that the feedback mechanisms for both gonadotropins are widely independent in the grafted testicular tissue. Leydig cells appeared functionally normal, and their morphology revealed an intact and normal cellular organization. In light of these findings, grafting of testicular tissue appears to be a useful experimental tool for androgen substitution. The possibility of grafting testicular tissue from different species and the experimental manipulation of grafting different quantities of testicular tissue or to pretreat the testicular tissue prior to grafting suggest elegant opportunities for studying basic endocrine mechanisms controlling testis growth and generating balanced feedback controls between the pituitary cells and the testicular cells.

Although most of the seminiferous tubules showed some degree of spermatogenic recovery, as early as Week 4 the luminal space enlarged, indicating an accumulation of fluid. At the same time, premature sloughing of germ cells occurred. Spermatogenesis did not reach quantitatively normal levels in the grafted tissue, and many seminiferous tubules showed some degree of damage to the seminiferous epithelium. These histological defects are very similar to those occurring after efferent duct ligation or in the estrogen receptor knockout mouse [22, 23]. In both cases, a blockade of efferent ducts causes the defect that finally leads to complete testicular degeneration. We therefore postulate that the obvious absence of efferent ducts in the grafted tissue and the accumulation of fluid secreted into the seminiferous tubules account for the defects leading to a disturbance of spermatogenesis. This atrophy of testicular tissue was quite specific for mouse grafts. When we used neonatal testicular tissue from nonrodent species (i.e., pig, goat, marmoset) in our previous studies, no or minimal signs of atrophy were observed. Most likely, the regulation of seminiferous fluid production and resorbtion is better balanced in these species. In support of this hypothesis, we observed several mouse grafts containing intact efferent ducts and adjacent epididymal tissue. These grafts showed no or very few atrophic seminiferous tubules. These findings indicate that the high resorbtive activity of the efferent ductules prevents fluid accumulation in the grafts and allows normal spermatogenesis to persist for long periods of time.

Ectopic grafting of neonatal testes leads to induction of complete spermatogenesis and endocrine function and therefore constitutes an experimental tool for fertility preservation and hormone replacement.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge Ingrid Upmann and Reinhild Sandhowe-Klawerkamp for technical assistance, Janet C. Turpin for excellent animal care, and Dr. Trever Cooper for editorial assistance.

	FOOTNOTES

 
¹ This work was supported by a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft (to S.S.), by grants USDA/NIH 99-35205-8620, HD 39641-01, RO1 RR 17359-01 (to I.D.), and by the Marion Dilley and David George Jones Funds and the Commonwealth and General Assembly of Pennsylvania (to M.B. and H.R.S.).

² Correspondence: Stefan Schlatt, Institute of Reproductive Medicine, Domagkstrasse 11, 48149 Münster, Germany. FAX: 49 251 8356093; schlats{at}uni-muenster.de

Received: 20 December 2002.

First decision: 12 January 2003.

Accepted: 24 January 2003.

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