Xenografting as a Tool to Preserve Endangered Species: Outcomes and Challenges in Model Systems

The use of testis tissue xenografting as a valuable tool to rescue endangered and genetically valuable individuals that die young or otherwise fail to produce sperm has been the subject of much interest. Although the technique has been successfully applied to a wide variety of species, little is known about what determines the outcome. Furthermore, to improve the applicability of xenografting, new methods to preserve and transport testis tissue from valuable animals are emerging. However, one major issue remains: the application of xenografting implies the development of subsequent ART techniques to produce offspring from the recovered material. This paper focuses on these three aspects of testis tissue xenografting as a tool for rescuing endangered and valuable genetic pools

Differentiation of Testis Xenografts in the Prepubertal Marmoset Depends on the Sex and Status of the Mouse Host

This study investigates the effects of the endocrine milieu of immunodeficient mouse host (intact vs. castrated male, intact male vs. intact female) on prepubertal marmoset (Callithrix jacchus) testicular xenografts. Previous marmoset xenografting studies used castrated nude mouse hosts which did not support efficient graft survival and maturation. Due to the distinct endocrine milieu in marmosets with a deletion of exon 10 in the LH receptor, we wanted to explore whether the most efficient xenograft development occurs in intact male mouse hosts compared to intact females or castrated males. We xenografted freshly isolated tissue from prepubertal marmosets (age range 4–6 months) into the back skin of three groups of nude mice (intact male, castrated male, and intact female). We collected serum for endocrine determinations and grafts after 20 weeks and determined hormonal/reproductive status, graft survival, somatic cell development and initiation of germ cell differentiation. Graft development, tubular integrity, and germ cell differentiation status in the grafts retrieved from different hosts was scored by morphometric analysis. The influence of the different endocrine status was compared between groups of hosts. Endocrine readouts and histological endpoints in xenografts substantiate that grafts were exposed to different microenvironments and responded with host specific developmental patterns. The intact male hosts supported the most significant progression of germ cell development. Our data provide evidence for the important role of the host milieu on survival and differentiation of marmoset xenografts. The xenografting model offers innovative avenues to exploit development and endocrine effects in the primate marmoset testis using limited numbers of non-human primates for the experimental settings

The effect of testosterone, dihydrotestosterone and oestradiol on the re-initiation of spermatogenesis in the adult photoinhibited Djungarian hamster

The roles of testosterone (T) and its metabolites on hamster spermatogenesis are poorly defined. This study assessed the effects of T, dihydrotestosterone (DHT) and oestradiol (E) on the re-initiation of spermatogenesis in the adult Djungarian hamster. Hamsters raised under long photoperiods (LD, 16 h light:8 h darkness) were exposed to short photoperiods (SD, 8 h light:16 h darkness) for 11 weeks to suppress gonadotrophins. Groups of eight animals then received T, DHT and E for 5 weeks. Cell numbers were determined using the optical disector (sic). The number of Sertoli cells was suppressed in SD controls to 48% (P<0·001) of LD control and restored either fully or partially by exogenous DHT and E (2·6- and 1·8-fold above SD levels) respectively, corresponding with a twofold elevation of serum FSH. The number of germ cells in SD animals was reduced (all P<0·001) to levels reported. The number of type A spermatogonia increased in line with the rise in Sertoli cell number, by 2·6-fold (P<0·01) and 1·8-fold (NS) above SD controls after DHT and E treatments respectively. DHT increased the number of type B spermatogonia/preleptotene spermatocytes, leptotene/zygotene and pachytene spermatocytes by 3·5-, 5·7- and 21-fold above SD (all P<0·01) respectively, compared with a 2·2-fold (P<0·01), 2·4-fold (not significant, NS) and 6-fold (NS) in E-treated animals respectively. Exogenous T had little effect on cell numbers or serum FSH compared with SD controls. Spermatids were rarely observed after steroid treatment. We believe this study suggests that steroids can regulate the re-initiation of early spermatogenic cells via a mechanism which includes FSH

New perspectives on fertility in transwomen with regard to spermatogonial stem cells

Objective: Germ cells of transwomen are affected by gender-affirming hormone therapy (GAHT). Fertility will be lost after surgical intervention; thereby, fertility preservation becomes an increasingly imortant topic. This study investigated if the absolute number of spermatogonia in transwomen is comparable at the time of gender-affirming surgery (GAS) to that in pre-pubertal boys. Methods: We carried out a retrospective study of testicular tissues from 25 selected subjects, which had undergone a comparable sex hormone therapy regimen using cyproterone acetate (10 or 12.5 mg) and estrogens. As controls, testicular biopsies of five cisgender adult men (aged 35–48 years) and five pre-/pubertal boys (5–14 years) were included. Testicular tissues were immunohistochemically stained for MAGE A4-positive cells, the most advanced germ cell type. The number of spermatogonia per area was assessed. Clinical values and serum hormone values for FSH, LH, testosterone, free testosterone, estradiol and prolactin were determined on the day of GAS for correlation analyses. Results: Round spermatids were the most advanced germ cell type in 3 subjects, 5 had an arrest at spermatocyte stage, while 17 showed a spermatogonial arrest. On average, testicular tissues of transwomen contained 25.15 spermatogonia/ mm3, a number that was significantly reduced compared to the two control groups (P < 0.01, adult 80.65 spermatogonia/ mm3 and pre-/pubertal boys 78.55 spermatogonia/mm3). Linear regression analysis revealed that testes with higher weight and high LH contained more spermatogonia. Conclusion: Irrespective of treatment dose or duration, spermatogenesis was impaired. Spermatogonial numbers were significantly reduced in transwomen compared to the control groups

Development and Disease-Dependent Dynamics of Spermatogonial Subpopulations in Human Testicular Tissues

Cancer therapy and conditioning treatments of non-malignant diseases affect spermatogonial function and may lead to male infertility. Data on the molecular properties of spermatogonia and the influence of disease and/or treatment on spermatogonial subpopulations remain limited. Here, we assessed if the density and percentage of spermatogonial subpopulation changes during development (n = 13) and due to disease and/or treatment (n = 18) in tissues stored in fertility preservation programs, using markers for spermatogonia (MAGEA4), undifferentiated spermatogonia (UTF1), proliferation (PCNA), and global DNA methylation (5mC). Throughout normal prepubertal testicular development, only the density of 5mC-positive spermatogonia significantly increased with age. In comparison, patients affected by disease and/or treatment showed a reduced density of UTF1-, PCNA- and 5mC-positive spermatogonia, whereas the percentage of spermatogonial subpopulations remained unchanged. As an exception, sickle cell disease patients treated with hydroxyurea displayed a reduction in both density and percentage of 5mC- positive spermatogonia. Our results demonstrate that, in general, a reduction in spermatogonial density does not alter the percentages of undifferentiated and proliferating spermatogonia, nor the establishment of global methylation. However, in sickle cell disease patients', establishment of spermatogonial DNA methylation is impaired, which may be of importance for the potential use of this tissues in fertility preservation programs

Double stranded sperm DNA breaks, measured by comet assay, are associated with unexplained recurrent miscarriage in couples without a female factor

It is known that sperm samples from recurrent pregnancy loss (RPL) couples have an increase in their sperm DNA fragmentation (SDF), but no studies have been performed in order to identify differences between single stranded SDF (ssSDF) and double stranded SDF (dsSDF) in these patients. This could be relevant because the type of DNA damage could have different effects. Semen samples were classified attending their clinical status: 25 fertile donors and 20 RPL patients with at least two unexplained first trimester miscarriages. SDF was analysed using alkaline and neutral Comet assay, SCD test and pulsed-field gel electrophoresis (PFGE), and ROC analysis including data from 105 more infertile patients (n = 150) was performed to establish predictive threshold values. SDF for alkaline and neutral Comet, and the SCD test was analysed in these categories of individuals. Data revealed the presence of two subgroups within fertile donors. The values obtained were 21.10±9.13, 23.35±10.45 and 12.31±4.31, respectively, for fertile donors with low values for both ssSDF and dsSDF; 27.86±12.64, 80.69±12.67 and 12.43±5.22, for fertile donors with low ssSDF and high dsSDF; and 33.61±15.50, 84.64±11.28 and 19.28±6.05, for unexplained RPL patients, also showing a low ssSDF and high dsSDF profile. This latter profile was seen in 85% of unexplained RPL and 33% of fertile donors, suggesting that it may be associated to a male risk factor for undergoing RPL. ROC analysis regarding recurrent miscarriage set the cut-off value at 77.50% of dsDNA SDF. PFGE for low ssSDF and high dsSDF profile samples and positive controls treated with DNase, to induce dsDNA breaks, showed a more intense band of about 48 kb, which fits the toroid model of DNA compaction in sperm, pointing out that some nuclease activity may be affecting their sperm DNA in RPL patients. This work identifies a very specific SDF profile related to the paternal risk of having RPL

Laser-Assisted In Vitro Fertilization Facilitates Fertilization of Vitrified-Warmed C57BL/6 Mouse Oocytes with Fresh and Frozen-Thawed Spermatozoa, Producing Live Pups

The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen-thawed spermatozoa, producing live pups. The ability to cryopreserve mouse gametes for LAIVF may facilitate management of large-scale transgenic mouse production facilities.National Institutes of Health (U.S.) (NIH National Research Service Award (T32-RR070036))National Institutes of Health (U.S.) (NIH National Cancer Institute Program Project (P01CA10451)