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The study and manipulation of spermatogonial stem cells using animal models

Ibtisham F, Awang-Junaidi AH, Honaramooz A

Cell Tissue Res, 2020 May;380(2):393-414.
PMID: 32337615 DOI: 10.1007/s00441-020-03212-x

Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.
Regeneration of testis tissue after ectopic implantation of porcine testis cell aggregates in mice: improved consistency of outcomes and in situ monitoring

Awang-Junaidi AH, Singh J, Honaramooz A

Reprod Fertil Dev, 2020 Mar;32(6):594-609.
PMID: 32051087 DOI: 10.1071/RD19043

Ectopic implantation of donor testis cell aggregates in recipient mice results in de novo formation or regeneration of testis tissue and, as such, provides a unique invivo model for the study of testis development. However, currently the results are inconsistent and the efficiency of the model remains low. This study was designed to: (1) examine several factors that can potentially improve the consistency and efficiency of this model and (2) explore the use of ultrasound biomicroscopy (UBM) for the non-invasive invivo evaluation of implants. Testis cell aggregates, containing ~40% gonocytes, from 1-week-old donor piglets were implanted under the back skin of immunodeficient mice through skin incisions using gel matrices or through subcutaneous injection without using gel matrices. The addition of gel matrices led to inconsistent tissue development; gelatin had the greatest development, followed by collagen, whereas agarose resulted in poor development. The results also depended on the implanted cell numbers since implants with 100×106 cells were larger than those with 50×106 cells. The injection approach for cell implantation was less invasive and resulted in more consistent and efficient testis tissue development. UBM provided promising results as a means of non-invasive monitoring of implants.
Long-Term Monitoring of Donor Xenogeneic Testis Tissue Grafts and Cell Implants in Recipient Mice Using Ultrasound Biomicroscopy

Fayaz MA, Awang-Junaidi AH, Singh J, Honaramooz A

Ultrasound Med Biol, 2020 11;46(11):3088-3103.
PMID: 32800471 DOI: 10.1016/j.ultrasmedbio.2020.07.010

Testis tissue xenografting and testis cell aggregate implantation from various donor species into recipient mice are novel models for the study and manipulation of testis formation and function in target species. Thus far, the analysis of such studies has been limited to surgical or post-mortem retrieval of samples. Here we used ultrasound biomicroscopy (UBM) to monitor the development of neonatal porcine testis grafts and implants in host mice for 24 wk, and to correlate UBM and (immuno)histologic changes. This led to long-term visualization of gradual changes in volume, dimension and structure of grafts and implants; detection of a 4 wk developmental gap between grafts and implants; and revelation of differences in implant development depending on the craniocaudal site of implantation on the back of host mice. Our data support the reliability and precision of UBM for longitudinal study of transplants, which eliminates the need for frequent surgical sampling.
Validation of Ultrasound Biomicroscopy for the Assessment of Xenogeneic Testis Tissue Grafts and Cell Implants in Recipient Mice

Fayaz MA, Awang-Junaidi AH, Singh J, Honaramooz A

Andrology, 2020 Feb 06.
PMID: 32030908 DOI: 10.1111/andr.12771

BACKGROUND: Subcutaneous grafting/implantation of neonatal testis tissue/cells from diverse donor species into recipient mice can be used as an in vivo model to study testis development, spermatogenesis, and steroidogenesis. Ultrasound biomicroscopy (UBM) allows obtaining high definition cross-sectional images of tissues at microscopic resolutions.
OBJECTIVES: The present study was designed to 1) validate the use of UBM for non-invasive monitoring of grafts/implants over-time, and to 2) correlate UBM findings with the morphological attributes of recovered grafts/implants.
MATERIALS AND METHODS: Testis tissue fragments (~14 mm3 , each) and cell aggregates (100×106 cells, each) obtained from 1-wk-old donor piglets (n = 30) were grafted/implanted under the back skin of immunodeficient mice (n = 6) in eight analogous sites per mouse. Three-dimensional transcutaneous Doppler UBM was performed and a randomly-selected graft and its corresponding implant were recovered at 2, 4, 6, and 8 wk.
RESULTS: Graft/implant weight (p = 0.04) and physical height (p = 0.03) increased over-time. The dynamics of physical length and volume increases over time differed between tissue grafts and cell implants (p = 0.02 and 0.01 for sample type*time interactions, respectively). UBM-estimated volume was correlated with the post-recovery weight and volume of the grafts/implants (r = 0.98 and r = 0.99, respectively; p < 0.001). Pre- and post-recovery length and height of the grafts/implants were positively and strongly correlated (r = 0.50, p = 0.01; r = 0.70, p = 0.001) and so were the areas covered by cordal, non-cordal or fluid-filled cavities between UBM and histology (r = 0.87, p < 0.001).
DISCUSSION AND CONCLUSION: UBM findings correlated with physical attributes of the grafts/implants, validating its use as a non-invasive high-fidelity tool to quantify the developmental changes in ectopic testis tissue grafts and cell implants, potentially leading to a reduction in the number of recipient mice needed for similar experiments.
Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes

Awang-Junaidi AH, Fayaz MA, Kawamura E, Sobchishin L, MacPhee DJ, Honaramooz A

Cell Tissue Res, 2020 Aug;381(2):361-377.
PMID: 32388763 DOI: 10.1007/s00441-020-03218-5

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P