Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques

Abstract

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare A(single) spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A(dark) and A(pale) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high-dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRalpha1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.

Figure 1. Experimental design

Adult male rhesus macaques were used to test the effects of busulfan on spermatogenesis and the SSC pool. (A) Testis samples from experimental animals were used for histological evaluation and to generate a single cell suspension by a two-step enzymatic digestion procedure. Aliquots of the resulting suspension were used immediately for xenotransplantation to nude mice (blue arrow) and the remaining volume was (B) cryopreserved for use in later experiments. (C) Experimental males were then treated with the chemotherapeutic drug busulfan (Busulfex IV). (D) Semen analysis, blood samples, and testis volume measurements taken at weekly intervals were used to demonstrate the effect of busulfan on spermatogenesis and hematopoiesis. (E) Testis samples obtained after busulfan treatment were used for histological evaluation and to isolate testis cells for immediate xenotransplantation to nude mice (blue arrow). (F) To evaluate baseline stem cell activity, as well as the effects of cryopreservation and busulfan treatment, rhesus testis cells were xenotransplanted [at each step (indicated by blue arrows)] into the testes of busulfan-treated nude mouse recipients.

Figure 2. Busulfan treatment leads to long-term infertility in male rhesus macaques

(A) Sperm counts were measured at weekly intervals and were averaged for animals within each group. Due to adverse reactions to busulfan, two animals were compassionately euthanized based on veterinary advice; 204 (8 mg/kg; 10 weeks after treatment, noted by blue arrowhead below X-axis) and 70 (12 mg/kg; 7 weeks after treatment, noted by yellow arrowhead below X-axis). Hematoxylin & eosin staining of adult rhesus macaque testes demonstrated the extent of spermatogenesis in experimental animals (B, F) before and (C, G) after 4 mg/kg busulfan (46 weeks), (D, H) after 8 mg/kg (60 weeks), or (E, I) after 12 mg/kg busulfan treatment (63 weeks). Quantification of testis morphology in each group is shown in Supplemental Table 1. Scale bar = 50μm. As noted (black arrow below x-axis), busulfan was administered at week 0. Note: samples for week 0 were collected prior to busulfan administration.

Figure 3. Busulfan treatment of rhesus testes caused a loss of germ cell and SSC markers

To further characterize the loss of germ cells observed following busulfan treatment, we detected expression of (A, B) VASA, (C, D) DAZL, (E, F) GFRα1, and (G, H) PLZF by immunofluorescent staining in paraffin-embedded tissue sections. Expression of each marker is merged with DAPI counterstain (Blue) indicating the nuclei of all cells in the section. Staining was performed using adult rhesus testis (A, C, E, G) before treatment and (B, D, F, H) 63 weeks after 12 mg/kg busulfan treatment (animal 73). Arrowheads mark PLZF-stained nuclei in (G). An asterisk notes a single residual VASA-positive cell in the lumen of one seminiferous tubule in (B). Scale bar = 50μm.

Figure 4. Rhesus testis cell xenotransplantation assay detects donor-derived spermatogonial colonies

(A) Western blot analysis demonstrating immunoreactivity of the preabsorbed (against mouse antigens) rhesus testis cell antibody with mouse and rhesus testis proteins. Testis proteins were from postnatal day 7 mouse (d7), adult mouse (A) and adult rhesus (nHP). Whole-mount immunofluorescent staining with the rhesus testis cell antibody of intact recipient nude mouse seminiferous tubules that were (B) untransplanted or (C-D) xenotransplanted with donor rhesus testis cells. (E-F) Immunohistochemical evaluation using the rhesus testis cell antibody (green) of sections (5 μm) of a recipient nude mouse testis transplanted with rhesus testis cells. Sections were counterstained with DAPI (blue). White arrowheads mark rhesus cells in recipient nude mouse seminiferous tubule cross-sections and white asterisks mark peritubular myoid cell nuclei. Xenotransplant recipient seminiferous tubules were also co-stained in whole-mount with (G) the rhesus testis cell antibody and (H) an antibody for the germ cell marker VASA. (I) Overlay of rhesus and VASA fluorescent signals. White arrows note a cluster of three autofluorescent interstitial cells located in a focal plane immediately above (outside) the seminiferous tubule. Dashed white lines mark seminiferous tubule margins in (B-D) and (G-I), and the seminiferous tubule basement membrane in (E-F). Scale bars = 50μm.

Figure 5. Rhesus SSC colonization activity is lost after busulfan treatment but retained after cryopreservation

Xenotransplant colonization activity was measured in testis cell suspensions from adult rhesus macaques (A) before and after busulfan treatment or (B) before and after cryopreservation. Data are presented as the total number of colonies meeting the inclusion criteria (≥4 rhesus cells with spermatogonial morphology on the basement membrane of recipient mouse seminiferous tubules) per 10⁶ viable transplanted cells. Cryopreserved cells in four replicate experiments from different animals were maintained in liquid nitrogen for 336, 289, 216, or 214 days prior to thawing and xenotransplantation. The number of individual rhesus donors (animals) and nude mouse recipient testes is indicated below the bars. Statistical significance is noted above each set of bars and was determined by a two-tailed heteroscedastic student’s T-Test. The histogram in (C) displays the distribution of colony sizes from fresh (white bars) and cryopreserved (black bars) rhesus testis cells reported in (B). Inset images in (C) show representative xenotransplant colonies two months after transplantation from fresh and cryopreserved rhesus testis cells. Colonies were detected by whole-mount staining with the rhesus testis cell antibody (green) and counterstained with DAPI (blue). Dashed white lines mark seminiferous tubule margins. Scale bars = 25μm.