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Gonocytes are germline stem cells in the neonatal testis with important potential applications in fertility restoration and transgenesis. Using stepwise experiments, we examined the effects of different media combined with fetal bovine serum (FBS) and/or knockout serum replacement (KSR) on the in vitro proliferation, colony-formation, ultrastructure, and expression of pluripotency markers of porcine gonocytes. Testis cells from 1-wk-old piglets were cultured for 28 days in 6 different culture media (DMEM, DMEM/F12, GMEM, α-MEM, StemPro, and RPMI), each supplemented with 5%, 10%, or 15% FBS and/or 5%, 10%, or 15% KSR. The media and FBS/KSR combination leading to the maximum number of gonocytes, and their colonies were selected for further analyses. KSR supplementation resulted in a reduced somatic cell propagation and increased gonocyte colony formation (P < 0.001). Culturing in DMEM+15%FBS led to the greatest number of gonocytes (P < 0.001), while the largest diameter and greatest number of colonies were formed in DMEM+5%FBS+10%KSR cultures (P < 0.001). Gonocytes and their colonies in DMEM+15%FBS expressed all the examined gonocyte and pluripotency markers. KSR alone did not support gonocyte propagation, likely due to a reduced somatic cell proliferation; however, the combination of FBS and KSR increased gonocyte colony formation and their size. • Optimized culture of germline stem cells has important potential applications for fertility restoration and transgenesis. • Tested the effects of six different media combined with various concentrations of FBS or KSR on proliferation of gonocytes, their colonies, and their pluripotency. • Gonocytes and their colonies in DMEM+15%FBS expressed all the examined gonocyte and pluripotency markers. • KSR alone did not support gonocyte propagation, but the combination of FBS and KSR increased gonocyte colony formation and their size. [ABSTRACT FROM AUTHOR]

Horses often sustain cutaneous wounds and healing can be prolonged and difficult to treat. Compared to body wounds, limb wounds heal slower and are more likely to develop exuberant granulation tissue. Differences in healing rates and exuberant granulation tissue formation is attributed to abnormal cytokine profiles. CXCL8 and its receptor CXCR2 are involved in acute inflammation whereas CXCL10 and its receptor CXCR3 are involved in inflammation resolution. β- arrestin-2 regulates inflammation through internalization of G-protein coupled receptors (GPCRs) including CXCR2 and CXCR3. Gene expression of these five inflammation associated proteins have not been previously identified in equine cutaneous tissue and may play a role in dysregulation of inflammation in equine limb wounds. The mRNA expression levels were measured using QuantiGene Plex Assay from cutaneous biopsies collected from surgically created wounds on the limb and thorax on days 0, 1, 2, 7, 14, and 33 from two horses. The mRNA expression levels were measured in mean fluorescent intensity and graphed. We were successful in identifying all five proteins for the first time in equine cutaneous tissue. Preliminary results suggest that there are different expression patterns for CXCL8, CXCR2 and β-arrestin-2 between the limb and thorax but not for CXCL10 and CXCR3. Differential regulation of CXCL8, CXCR2 and β-arrestin-2 may further explain why limb wounds heal differently than body wounds and warrants further investigation. [ABSTRACT FROM AUTHOR]

In tissue engineering, non-invasive imaging of biomaterial scaffolds and tissues in living systems is essential to longitudinal animal studies for assessments without interrupting the repair process. Conventional X-ray imaging is inadequate for use in soft tissue engineering due to the limited absorption difference between the soft tissue and biomaterial scaffolds. X-ray phase-based imaging techniques that derive contrast from refraction or phase effects rather than absorption can provide the necessary contrast to see low-density biomaterial scaffolds and tissues in large living systems. This paper explores and compares three synchrotron phase-based X-ray imaging techniques—computed tomography (CT)-diffraction enhanced imaging (DEI), -analyzer based imaging (ABI), and -phase contrast imaging (PCI)—for visualization and characterization of low-density biomaterial scaffolds and tissues in situ for non-invasive soft tissue engineering assessments. Intact pig joints implanted with polycaprolactone scaffolds were used as the model to assess and compare the imaging techniques in terms of different qualitative and quantitative criteria. For long-term in vivo live animal imaging, different strategies for reducing the imaging radiation dose and scan time—reduced number of CT projections, region of interest, and low resolution imaging—were examined with the presented phase-based imaging techniques. The results demonstrated promising capabilities of the phase-based techniques for visualization of biomaterial scaffolds and soft tissues in situ . The low-dose imaging strategies were illustrated effective for reducing the radiation dose to levels appropriate for live animal imaging. The comparison among the imaging techniques suggested that CT-DEI has the highest efficiency in retaining image contrast at considerably low radiation doses. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to evaluate the long-term outcome of testis tissue xenografting from immature deer. Testis tissue was collected post-mortem from a 2-mo-old white-tailed deer fawn (Odocoileus virginianus) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n =7 mice; 8 fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2mo from grafting for up to 14mo post-grafting. The retrieved xenografts were evaluated for seminiferous tubular density (per mm2) and tubular diameter, as well as for seminiferous tubular morphology and identification of the most advanced germ cell type present in each tubule cross section. Overall, 63% of the grafted testis fragments were recovered as xenografts. Testis tissue xenografts showed a gradual testicular development starting with tubular expansion by 2mo, presence of spermatocytes by 6mo post-grafting, round and elongated spermatids by 8mo, followed by fully-formed sperm by 12mo post-grafting. The timing of complete spermatogenesis generally corresponded to the reported timing of sexual maturation in white-tailed deer. This study demonstrated, for the first time, that testis tissue xenografting from immature deer donors into recipient mice can successfully result in testicular maturation and development of spermatogenesis in the grafts up to the stage of sperm production. These results may therefore provide a model for salvaging genetic material from immature male white-tailed deer that die before reaching sexual maturity. [Copyright &y& Elsevier]

Abstract: The objective was to evaluate the long-term outcome of testis tissue xenografting from neonatal bison calves as a model for closely related rare or endangered ungulates. Testis tissue was collected postmortem from two newborn bison calves (Bison bison bison) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n = 15 mice; eight fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo after grafting (for up to 16 mo). The retrieved xenografts were evaluated for seminiferous tubular density, tubular diameter, seminiferous tubular morphology, and identification of the most advanced germ cell type. Overall, 69% of the grafted testis fragments were recovered as xenografts. Xenografts weight increased (P < 0.02) approximately four-fold by 2 mo and 10-fold by 16 mo post-grafting. In testis xenografts, gradual maturational changes were evident, manifested as the first detection of the following at the times specified: seminiferous tubule expansion, 2 mo; spermatocytes, 6 mo; round spermatids, 12 mo; and elongated spermatids, 16 mo. Furthermore, there were differences between the two donor calves regarding the efficiency of spermatogenesis in xenografts. The timing of complete spermatogenesis approximately corresponded to the reported timing of sexual maturation in bison. This study demonstrated, apparently for the first time, that testis tissue xenografting from neonatal bison donors into recipient mice resulted in testicular maturation and complete development of spermatogenesis in the grafts. [Copyright &y& Elsevier]

To study the development of the reproductive tract in heifers, the ovaries, uterus, cervix and vagina were examined by transrectal ultrasonography every 2 weeks, from 2 to 60 weeks after birth. First ovulation occurred at 63.7±1.1 weeks of age. Ovarian dimensions increased rapidly from 2 to 14 weeks of age, and increased again after 34 weeks of age (P<0.05). The size of the largest ovarian follicles increased from 8 to 14 weeks of age, from 38 to 42 weeks of age, and finally from 52 to 60 weeks of age (P<0.05). The number of follicles ≥3 mm in diameter tended to increase from 6 to 14 weeks of age (P<0.10) and increased significantly from 6 to 60 weeks of age (P<0.05). Mean numerical pixel values of the ovarian images decreased from 4 to 26 weeks of age, and then rose to 44 weeks of age (P<0.05). Diameter of the uterine body, cervix and vagina increased from 2 to 20–24 weeks of age, and again after 32 weeks of age (P<0.05). Mean numerical pixel values for the uterus and vagina decreased initially (uterus: 4–8 weeks and vagina: 6–22 weeks of age) and then increased (uterus: 14–42 weeks and vagina: 22–32 weeks of age; P<0.05). Pixel heterogeneity showed a consistent peak at 20–22 weeks of age for the uterus, cervix and vagina (P<0.05). In summary, in the heifer calf, the marked growth of the reproductive tract in the first few months of age, and prior to first ovulation, reflects phases of increased ovarian follicle (≥3 mm in diameter) numbers and size. Ultrasonographic image analysis revealed patterns of numerical pixel values and heterogeneity that may be useful in determining important stages of growth and differentiation of the reproductive system. [Copyright &y& Elsevier]

Male reproductive capacity has fallen considerably in recent decades; in addition, the incidence of testicular cancer has increased in many developed countries. The cause of this phenomenon is unknown, but environmental toxicants are considered a major contributing factor. To study potential reproductive toxicants, robust in vitro testis models are needed. We have recently established a porcine testis organoid system with a high resemblance to the architectures of innate testis tissue. Here, we further investigated the testis morphogenesis, cell maturation, and endocrine function of the testis organoids. We also challenged this system with abiraterone, a steroidogenic inhibitor, to validate its suitability as an in vitro platform for endocrine toxicology tests. Our results showed that the testis cells in the organoids reorganize into testis cordal structures, and the cordal relative areas increase in the organoids over time of culture. Moreover, the diameters and cell numbers per cross-section of the cordal structures increased over time. Interestingly, Sertoli cells in the organoids gradually underwent maturational changes by showing increased expression of androgen receptors, decreased expression of the anti-müllerian hormone, and formation of the blood-testis barrier. Next, we confirmed that the organoids respond to hormonal stimulation and release multiple sex hormones, including testosterone, estradiol, and progesterone. Finally, we showed that the production of testosterone and estradiol in this system can be inhibited in response to the steroidogenic inhibitor. Taken together, our organoid system provides a promising in vitro platform for male reproductive toxicology studies on testis morphogenesis, somatic cell maturation, and endocrine production. • We tested a novel testis organoid system to study potential reproductive toxicants. • Sertoli cells in the organoids gradually matured and formed blood-testis barrier. • Organoids responded to hormonal stimulation and released multiple sex hormones. • Testosterone and estradiol were inhibited after using a steroidogenic inhibitor. • Our organoid system provides a platform for male reproductive toxicology studies. [ABSTRACT FROM AUTHOR]

Highlights: [•] Primary cells were used to investigate the effects of NBFRs on steroidogenesis. [•] TBB exposure upregulated the pathway for aldosterone and cortisol biosysthesis. [•] TBCO and TBPH exposure produced greater amounts of sex hormones. [•] The study revealed subtle differences in the molecular targets of TBCO and TBPH. [ABSTRACT FROM AUTHOR]

The in vitro propagation and differentiation of spermatogonial stem cells (SSCs) has many potential applications within reproductive science and medicine. We established a two-dimensional (2D) cell culture system to proliferate and differentiate prepubertal mouse SSCs as a model capable of maximizing on a small number of donor SSCs. We also investigated the effects of retinol on in vitro SSC differentiation. Testis cells were cultured for 10 days in a serum-free medium. This produced SSC colonies which were then dissociated and sub-cultured for an additional 20 days in a differentiation medium. Before inducing differentiation, colonies expressed genes specific for undifferentiated spermatogonia (Ngn3 , Plzf). After 10 days in the differentiation medium, Stra8 expression was upregulated. After 20 days, Acr expression was upregulated, indicating the completion of meiosis. Immunofluorescence, RT-PCR and flow cytometry confirmed the presence of haploid male germ cells (4.4% of all cells). When retinol was added to the differentiation medium the proportion of haploid germ cells increased (8.1% of cells). We concluded that, under serum-free culture conditions, prepubertal SSCs will generate colonies that can differentiate into haploid germ cells in a 2D culture system. These cells demonstrate a relatively high efficiency of haploid-cell production, which can be further improved with retinol. • In vitro production of haploid germ cells from murine spermatogonial stem cells using a two-dimensional cell culture system. • In vitro spermatogenesis has potential applications in infertility treatment. • Using a serum-free 2D culture system prepubertal spermatogonia generated colonies. • Colonies differentiated into haploid germ cells with relatively high efficiency. • Adding retinol increased the proportion of haploid germ cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Xenografting of testis tissue from immature males from several mammalian species to immunodeficient mouse hosts results in production of fertilization-competent sperm. However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Testis tissue xenografting from sexually mature animals could provide an option to preserve the genetic material from valuable males when semen for cryopreservation cannot be collected. To assess the potential use of this technique for adult individuals, testes from adult animals of six species (pig, goat, cattle, donkey, horse and rhesus monkey) were ectopically grafted to host mice. Grafts were recovered and analyzed at three time points: less than 12 weeks, between 12 and 24 weeks and more than 24 weeks after grafting. Histological analysis of the grafts revealed effects of species and donor tissue maturity: all grafts from species with greater daily sperm production (pig and goat) were found to have degenerated tubules or grafts were completely degenerated. None of the xenografts from mature adult bull and monkeys contained differentiated spermatogenic cells when examined more than 12 weeks post-grafting but tubules with Sertoli cells only remained. In grafts from a young adult bull, Sertoli cells persisted much longer than with the mature adult grafts. In grafts from a young adult horse, spermatogenesis proceeded to meiosis. In grafts from a young adult donkey and monkey, however, complete spermatogenesis was found in the grafts. These results show that testis tissue grafts from mature adult donors did not support germ cell differentiation but seminiferous tubules with Sertoli cells only survived in some species. The timing and progression of tubular degeneration after grafting of adult testis tissue appear to be related to the intensity of spermatogenesis at the time of grafting. Testis tissue from sub-adult donors survives better as xenograft than tissue from mature adult donors, and complete spermatogenesis can occur albeit with species-specific differences. [Copyright &y& Elsevier]