Transplantation of Spermatogonia

In the female, a finite number of primary oocytes are present at birth, and this pool progressively dwindles during the lifetime. Thus, stem cell spermatogonia are the only self-renewing cell type in the adult capable of providing a genetic contribution to the next generation (77).

Spermatogonia exist in three states: stem cell, proliferative, and differentiating spermatogonia (78). The first two are frequently designated as undifferentiated spermatogo-nia, and the stem cell spermatogonium is most resistant to a variety of insults, often surviving where other germ cells are destroyed. The less frequent division of this stem cell population is believed to be one reason for their relative ability to survive (78, 79). Although it is difficult to be certain about which of the spermatogonia are capable of self-renewal, both stem cell and proliferative spermatogonia appear poised for further differentiation (78-81).

In 1994, Brinster and Zimmermann (77) reported that spermatogonial stem cells isolated from normal male mice repopulated sterile testes when injected directly into seminiferous tubules, later showed normal morphological characteristics, and ultimately differentiated into normal spermatozoa. In 1996, Clouthier et al. (82) examined the feasibility of transplanting spermatogonia from other species to the mouse. Marked tes-ticular cells from transgenic rats were transplanted to the testes of 10 immunodeficient mice, and rat spermatogenesis occurred in all of them. Among epididymides of eight mice, the three belonging to those with the longest transplants (> 110 days) contained morphological rat spermatozoa (82). The success of rat spermatogenesis in mouse testes would appear to open the possibility of xenogeneic spermatogenesis for other species combinations.

With this in mind, we have transplanted germ cells from azoospermic men to the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) (83). Spermatogenic cells were obtained from testicular biopsy specimens from 24 men (average age of 34.3 ± 9 years) with obstructive (n = 16; OA) and nonobstructive (n = 8; NOA) azoospermia by first digesting testis biopsies with collagenase to promote separation of individual cells. The concentration of spermatogenic cells in the OA group was 6.6 x 106 cells/ml and 1.3 x 106 cells/ml in the NOA group (P < .01). The germ cells were injected into mouse seminiferous tubules using a microneedle (40 mm inner diameter) on a 10-ml syringe, with trypan blue used as an indicator of the accuracy of this transfer. Mice were maintained from 50 to 150 days to allow time for germ cell colonization and development before sacrifice. Testes were then fixed for histological evaluation, and approximately 100 tubule cross-sections were examined for human spermatogenic cells.

The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogonia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes. A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection (Figures 5.5-5.7). However, sections from the remaining testes

Figure 5.5. Section of seminiferous tubules displaying complete spermatogenesis (200x). Sperm flagella are protruding into the tubular cavity.

examined up to 150 days after injection showed tubules lined only with Sertoli cells with no xenogeneic germ cells surviving. The reason the two recipient mouse strains did not allow the implantation of human germ cells was probably due to signaling molecule divergence between primates and rodents that occurred more than 100 million years ago (84).

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