Folliculogenesis following syngeneic transplantation of young murine ovaries into the testes

Abstract

Background and Aim:  To examine the effects of intratesticular transplantation on the growth and maturation of young murine ovaries.

Methods:  Two‐week‐old ovaries from transgenic mice with enhanced green fluorescent protein expression were transplanted under the testicular capsule of 4‐week‐old non‐transgenic mice.

Results:  Two months after transplantation all successfully grafted ovaries had survived, based on the presence of bright green fluorescence. The grafts showed various stages of folliculogenesis, including expanded follicles. The neighboring seminiferous tubules had a normal structure and mature sperm in their lumens, indicating active spermatogenesis, and all the recipient males were fertile. There was no evidence of extensive cell migration from the grafted ovaries into the testis. Similar findings were obtained for the grafted ovaries 6 months after surgery, although cell death (as evidenced by yellowish or pale fluorescence) was more frequent.

Conclusion:  Young murine ovaries can grow and mature autonomously for at least 6 months unaffected by the male hormonal environment. (Reprod Med Biol 2006; 5: 71–77)

INTRODUCTION

DEVELOPMENT OF THE fetal rodent ovary appears to be suppressed by the male hormonal environment. For example, when 15‐day fetal ovaries and testes were transplanted under the renal capsule of gonadectomized adult rats, development of the ovaries was severely inhibited, although the testes and ovaries in the control experiment developed normally. ¹ , ² However, 13‐day undifferentiated fetal mouse ovaries transplanted into the kidney of adult male mice frequently develop tubular structures, the so‐called ‘ovotestis’. ³ , ⁴ These findings suggest that male factors affect the development (or differentiation) of fetal ovaries and that these are most likely to be androgens secreted by the gonads. However, androgens administered to mid‐gestational pregnant mice induced intersexuality of the female offspring, but did not masculinize the ovaries, ⁵ which suggests that the ovotestis is not an androgenic effect. Recent in vitro studies have revealed that Müllerian‐inhibiting substance (MIS), known as anti‐Müllerian hormone and secreted by Sertoli cells, appears to be the factor causing ovotestis development because it suppresses the development of fetal rat ovaries and masculinizes them. ⁶ , ⁷ , ⁸ The MIS mRNA is expressed at maximal levels on days 15–18, but has disappeared by 3 weeks after birth. ⁹

After birth, mammalian ovaries comprise follicles containing maturing oocytes, thecal cells and interstitial cells. Control of folliculogenesis involves interactions between the pituitary gonadotrophins, follicule‐stimulating hormone (FSH) and leutinizing hormone (LH), and intraovarian factors such as steroids, cytokines and growth factors. The later stages, at least, of folliculogenesis are strictly controlled by FSH and LH, ¹⁰ both of which are preferentially produced by females. Thus, it has been hypothesized that the development of mammalian ovaries placed in a male hormonal environment would be suppressed because of the presence of only small amounts of female‐specific hormones and large quantities of androgens. Testing this hypothesis requires transplantation of the ovary into the testis and the first reports of this procedure were from Sand in the early 1900s. ¹¹ , ¹² Ozdzenski et al. also investigated whether the development of embryonic mouse ovaries was affected by transplantation into adult and embryonic testes, ¹³ and intratesticular grafting of ovaries was carried out by Takewaki to examine the secretion of estrogen by Sertoli cells. ¹⁴ No further studies of this type have been reported, but recent progress in gene engineering technology using genetically marked cells (expressing green fluorescent protein) ¹⁵ enables evaluation of the feasibility of this technique for transplantation experiments.

Thus, in the present study we transplanted young ovaries from transgenic mice with enhanced green fluorescent protein (EGFP) expression ¹⁶ into the testis of 4‐week‐old non‐transgenic mice to examine whether normal folliculogenesis would occur. Because the recipient males do not produce MIS at significant levels, based on the findings of Hirobe et al., ⁹ we could preclude its effect on the grafted ovaries. In addition, the fertility of the recipient males was examined to determine if the exogenous ovaries had any effect on male reproduction.

MATERIALS AND METHODS

Ovary transplantation

TWO‐WEEK‐OLD OVARIES FROM heterozygous female transgenic mice of the MNCE‐39 line (with B6C3F1 genetic background), which overexpresses EGFP systemically, ¹⁶ were transplanted into 4‐week‐old non‐transgenic MNCE‐39 male mice. The mice were kept on a 12 h light/12 h dark schedule (lights on from 07.00 hours to 19.00 hours) and allowed food and water ad libitum. Experiments were carried out humanely in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ of Tokai University.

Recipient males were anesthetized with pentobarbital and a small abdominal incision (approximately 1 cm in length) was made to expose the testes (Fig. 1a). A small portion of the seminiferous tubules located beneath the testicular capsule was removed by suction with a 20‐gauge needle attached to a 20‐mL plastic syringe (Terumo, Tokyo, Japan) to create a space (Fig. 1b,c) into which a single whole ovary could be inserted (Fig. 1d–f). Care was taken not to injure the rete testis. After transplantation, the hole in the testicular capsule was not closed and the testis was returned to its original place. The same surgical procedure was carried out for the other testis. Males were allowed to survive for 2 months (n = 3), during which time they did not receive exogenous gonadotrophins, or 6 months (n = 3) for determination of the long‐term survival of the grafted ovaries.

Figure 1.

Procedure for transplantation of an ovary from an enhanced green fluorescent protein (EGFP)‐expressing transgenic mouse to beneath the testicular capsule. (a) Testis from 4‐week‐old non‐transgenic MNCE‐39 line exposed after abdominal incision. (b,c) Testis from which a portion of the seminiferous tubules has been removed by suction with a 20‐gauge needle. Remnant seminiferous tubules can be seen outside the testis (indicated by the circle in c). (d) Ovary (arrow) from 2‐week‐old MNCE‐39 transgenic mouse inserted into the testis immediately after removal of the seminiferous tubules, showing bright EGFP fluorescence under ultraviolet illumination (indicated by the arrow in e). (f) Ovary under the testicular capsule (arrow) after the treated testis is returned to its original position.

Male fertility experiments

Recipient males were mated for 1 or 2 months with normal B6C3F1 females from approximately 1 month after they reached adulthood. The presence of copulation plugs in the females was checked each day and those with plugs were separated from the males. Pregnancy was confirmed by the presence of mid‐gestational fetuses.

Histological examination

For detection of EGFP fluorescence in the whole testis, an Olympus BX40 dissecting microscope (Olympus, Tokyo, Japan) with DM505 filters (BP460‐490 and BA510IF) was used. The testes were then fixed in 4% paraformaldehyde (PFA) in phosphate‐buffered saline (PBS) without Ca²⁺ and Mg²⁺, PBS(–), pH 7.2, at 4°C for 2 days, and then dehydrated in 0.25% sucrose in PBS(–) at 4°C for 2 days and 0.4% sucrose in PBS(–) at 4°C for 4 days. The samples were then embedded in OCT compound (Tissue‐Tek [No. 4583]; Miles Scientific, Naperville, IL, USA) for cryostat sectioning and observation of EGFP using a fluorescence microscope (Olympus BX60). Microphotographs were taken using a digital camera (FUJIX HC‐300/OL; Fuji Film, Tokyo, Japan) attached to the fluorescence microscope and printed out using a Mitsubishi digital color printer (CP700DSA; Mitsubishi, Tokyo, Japan). Some specimens were stained with hematoxylin–eosin.

RESULTS

ALL SIX MALES were fertile and at least two females from each mating group became pregnant (data not shown). Two months after surgery all six ovarian grafts had survived, evidenced by the bright green fluorescence beneath the testicular capsule (Fig. 2a,b). The testes carrying ovarian grafts were smaller than normal testes (Fig. 2a), probably as a result of the surgical reduction in the volume of seminiferous tubules. After 2 days of fixation with 4% PFA in PBS(–), the whole testes were bisected and the inner surface was examined microscopically under natural and ultraviolet illumination. The ovarian grafts were clearly distinguishable by color from the host seminiferous tubules (Fig. 2c) and, furthermore, they exhibited bright green fluorescence (Fig. 2c,d). Enlarged follicles were frequently observed (Fig. 2c,d). Examination of the cryostat sections of the testes confirmed these observations (Fig. 2e,f). The seminiferous tubules near the graft were completely negative for fluorescence (Fig. 2e,f), suggesting the absence of extensive cell migration from the grafted ovaries into the testicular tissue. Hematoxylin–eosin staining of the cryostat sections revealed that the ovarian grafts contained all stages of folliculogenesis, including viable antral follicles (Fig. 3a) and atretic follicles (Fig. 3b). The neighboring seminiferous tubules appeared normal (Fig. 3a) and sperm were frequently observed in the tubule lumens (Fig. 3c). These findings suggest that 2‐week‐old ovaries can grow and differentiate autonomously in an environment enriched with male factors.

Figure 2.

Enhanced green fluorescent protein (EGFP) fluorescence and structure of the ovarian grafts 2 months after transplantation. (a) Grafted testes and an intact testis from a normal 3‐month‐old male photographed under light. (b) Grafted ovaries shown in (a) exhibiting bright EGFP fluorescence under ultraviolet (UV) illumination. The intact testis is negative for fluorescence. (c) Inner surface of the grafted testis bisected after fixation in 4% paraformaldehyde. Note well‐developed antral follicles (arrows) and host seminiferous tubules (*). (d) Grafted ovary shown in (c) exhibiting bright EGFP fluorescence under UV illumination. Note well‐developed antral follicles (arrows) and host seminiferous tubules (*). (e) Cryostat section of the sample in (c) and (d) shown under light and host seminiferous tubules (*). (f) Grafted ovary shown in (e) exhibiting bright EGFP fluorescence under UV illumination. There is no fluorescence in the host tissue (*), indicating a lack of active cell migration from the graft.

Figure 3.

Hematoxylin–eosin‐stained cryostat sections of the ovarian grafts 2 months after transplantation. (a) There are several stages of follicle development (arrowheads). *Host seminiferous tubules. (b) Higher magnification of insert in (a). Note the atretic follicle (*). (c) Host seminiferous tubules near the ovarian graft. Note mature sperm in the tubular lumen (arrows), indicating active spermatogenesis.

Examination of the whole testes 6 months after surgery revealed that three of the six ovaries had yellowish or pale fluorescent deposits (Fig. 4c,d), which suggested cell death had occurred. The other three ovaries showed bright green fluorescence throughout (Fig. 4a,b), although bleeding was frequently observed within expanded follicles (Fig. 4a). Inspection of the inner surface of the testes confirmed these observations (Fig. 4e). Examination of the cryostat sections revealed that the grafted tissue showed EGFP fluorescence, but the neighboring host tissue did not (Fig. 4f,g). There were follicles at various stages of development, but almost all antral follicles were filled with blood (Fig. 4f). Interstitial cells appeared to be more frequently damaged (as indicated by yellowish fluorescence) than follicular cells (Fig. 4i). These findings suggest that 2‐week‐old ovaries can survive for up to 6 months after surgery, although the rate of cell death, particularly of interstitial cells, is high.

Figure 4.

Enhanced green fluorescent protein (EGFP) fluorescence and structure of the ovarian grafts 6 months after transplantation. (a) Grafted testis examined under light. Bleeding was frequently observed (arrows), probably within expanded follicles. (b) Grafted ovary shown in (a) exhibiting bright EGFP fluorescence in some areas under ultraviolet (UV) illumination. (c) Another graft (arrow) examined under light, showing yellow or pale fluorescence under UV illumination (suggesting cell death) (d). (e) Cut surface of the grafted testis shown in (a) and (b) after fixation with 4% paraformaldehyde. Note the presence of several well‐developed antral follicles (arrows), some of which are filled with blood (arrowheads). *Host seminiferous tubules. (f) Cryostat section of the fixed sample shown in (e) examined under light also shows the bleeding within the follicles (arrows). *Host seminiferous tubules. (g) Grafted ovary shown in (f) exhibiting bright EGFP and yellow fluorescence under UV illumination, but no fluorescence in the host tissue. (h) Higher magnification of the insert in (f) under light. (i) Ovarian graft shown in (h) under UV illumination. More interstitial cells (arrowheads) than follicular cells (arrows) appear to be dead (yellow or pale fluorescence).

DISCUSSION

THE FIRST REPORTS of using the testis as a site for transplantation were by Sand, ¹¹ , ¹² who found that an ovary transplanted into the testis developed mature follicles; however, detailed information about the studies, such as the age of the animals used as donors and recipients and the stage of follicles, was lacking. When Ozdzenski et al. ¹³ investigated whether the development of 11–13‐day‐old embryonic mouse ovaries was affected by transplantation into adult or embryonic testes, they found that those in the adult testis had severely restricted growth, but not masculinization. Takewaki carried out intratesticular ovarian grafts to study the secretion of estrogen by Sertoli cells and reported that most survived for between 1 and 2 months. ¹⁴ To date, apart from these studies, further research into this particular transplantation technique has not been done.

Transplantation of intact or cryopreserved ovaries into another tissue or organ, such as under the kidney capsule or into the abdominal wall, subcutaneous pocket or uterine horn, has been carried out and in almost all cases follicular development has occurred. ¹⁷ This suggests that young (and possibly adult), but not fetal, ovaries can grow and differentiate autonomously. In the present study of mice, normal folliculogenesis occurred for at least 2 months in young (2‐week‐old) ovaries implanted beneath the testicular capsule, even in the absence of exogenous gonadotrophins, which suggests that the endogenous hormonal environment of males can support viable ovaries.

Folliculogenesis is dependent, at least in the later stages (i.e. antral follicles), on FSH and LH, ¹⁸ , ¹⁹ whereas the very early stages of follicular growth, such as the preantral follicles, can occur without gonadotrophins, implying a role for local intraovarian factors (e.g. activin, transforming growth factor‐β[TGF‐β], insulin growth factor‐1 (IGF‐1) and EGF/TGF‐α). ²⁰ The present finding that antral follicles form naturally in ovaries grafted into the testis appears to contradict the hypothesis stated earlier because males do not produce as much FSH or LH as females. Unfortunately, we did not measure the plasma levels of FSH and LH, so we cannot conclude that the final stages of folliculogenesis would have proceeded in the present ovarian grafts without significant levels of FSH and LH.

Of note is the frequent bleeding within expanded follicles in the grafts at 6 months, but not at 2 months, after transplantation (Fig. 4a,e,f), although its significance is unclear. As interstitial cells in the grafts were also dying more frequently at this stage (Fig. 4i), disruption or loosening of cell–cell junctions may be the cause.

In conclusion, using a newly developed gene‐engineering technique we found that ovarian grafts grew normally and appeared healthy 2 months after surgical transfer into a testis, which raises the following questions. First, are oocytes recovered from ovarian grafts able to mature and be fertilized by epididymal mature spermatozoa in vitro? Second, do the grafted ovaries retain their function when re‐implanted under the bursa ovarica of a recipient? Third, is allografting possible? The mammalian testis, ovary and uterus exhibit immunotolerance ²¹ and, thus, may be sites for allografting of young ovaries. In particular, intratesticular transplantation of the ovary may be a unique method for evaluating in vivo the developmental potential of ovaries at any stage.