Recent Advances in Application of Male Germ Cell Transplantation in Farm Animals

Transplantation of isolated germ cells from a fertile donor male into the seminiferous tubules of infertile recipients can result in donor-derived sperm production. Therefore, this system represents a major development in the study of spermatogenesis and a unique functional assay to determine the developmental potential and relative abundance of spermatogonial stem cells in a given population of testis cells. The application of this method in farm animals has been the subject of an increasing number of studies, mostly because of its potential as an alternative strategy in producing transgenic livestock with higher efficiency and less time and capital requirement than the current methods. This paper highlights the salient recent research on germ cell transplantation in farm animals. The emphasis is placed on the current status of the technique and examination of ways to increase its efficiency through improved preparation of the recipient animals as well as isolation, purification, preservation, and transgenesis of the donor germ cells

The Number of Grafted Fragments Affects the Outcome of Testis Tissue Xenografting from Piglets into Recipient Mice

To optimize the procedure for testis tissue xenografting, we grafted 2, 4, 8, or 16 small fragments of immature porcine testis tissue under the back skin of immunodeficient castrated mice (n = 10 mice/group). At 8 months post grafting, the graft recovery rate did not differ between groups; however, not only the total but also the average graft weights were higher (by ∼12-fold and ∼2.5-fold, resp.) in mice receiving 16 fragments than those receiving 2 fragments (P < .05). The recipient mice with 16 fragments had the largest vesicular glands (indicators of testosterone release by the grafts) compared with those with 2 fragments (P = .007). The grafts in the group of 16 fragments also had more (P < .05) percentage of tubules with round spermatids than those of the group of mice receiving 2 fragments. Therefore, recipient mice can be grafted with at least 16 testis tissue fragments for optimal results

Neuroendocrinology of gonadotrophin secretion in prepubertal heifers

The pattern of gonadotrophin secretion and the mechanisms involved in the regulation of gonadotrophin secretion in prepubertal heifers were studied. Methods involved periods of frequent blood sampling (with or without concomitant treatments) and weekly blood sampling to characterize the temporal pattern of gonadotrophin secretion and/or responses to treatments. The developmental pattern of regulation of gonadotrophin release by opioid peptides, dopaminergic and adrenergic neuronal systems and possible interactions were studied in prepubertal heifers using bolus administration of receptor blockers, alone or in combination. It was concluded that although endogenous opioids suppress gonadotrophin secretion in the early postnatal period, inhibitory effects were stronger in the mid- to late-prepubertal period. It was also evident that dopaminergic inhibition negated the effects of naloxone (an opioid antagonist), suggesting that the suppressive effects of opioids are in part exerted through the inhibition of a dopaminergic neuronal system. Alpha-adrenergic neuronal systems had stimulatory effects on LH release, especially during the late prepubertal period. There were no evidence for á-adrenergic mediation of opioidergic suppression of LH release in prepubertal heifers. The role of excitatory amino acid (EAA) neurotransmitters in the regulation of gonadotrophin secretion during prepubertal development was examined using N-methyl-D,L-aspartic acid (NMA, an excitatory amino acid agonist) at different ages from birth to puberty. The results showed that excitatory amino acids seem to be involved in the regulation of LH and FSH release and that their effects were age-related, developing in the early postnatal period and reaching their maximum in the mid- to late-prepubertal period, sometime before first ovulation. Involvement of nitric oxide in the control of gonadotrophin secretion and possible mediation of the stimulatory effects of GnRH and NMA was studied. nitric oxide is involved in the regulation of LH, and possibly FSH, secretion and that nitric oxide may mediate, at least in part, the stimulatory effects of excitatory amino acids on LH, and to some extent FSH, release. The responses to GnRH led us to suggest that nitric oxide may have inhibitory effects on the pituitary and NMA may have increased pituitary sensitivity to GnRH. Finally, the effects of season of birth on the patterns of gonadotrophin secretion as well as age and weight at puberty were investigated by comparing spring- and autumn-born heifers. It was shown that an early postnatal increase in LH secretion (10 to 20 weeks of age) occurred in spring-born heifers, whereas in autumn-born heifers LH secretion decreased during this period. In spite of these differences, age and weight at puberty did not differ between spring- and autumn-born heifers although autumn-born heifers showed a wider range in age at puberty

Optimization of culture conditions for short-term maintenance, proliferation, and colony formation of porcine gonocytes

Abstract Background Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens. Results Confluency of cells was optimal (>90% by ~6 d) when 3.0 × 104 testis cells/cm2 containing ~40% gonocytes were used. Incubating the cells at 35 °C or 37 °C resulted in similar cell number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradually settled over the next 5 d. Consequently, not changing the medium for 7 d (as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of “floating gonocytes”. Conclusion We found that gonocytes require the presence of a critical minimum number of somatic cells for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 × 104 testis cells/cm2 containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes

Regeneration of testis tissue after ectopic implantation of porcine testis cell aggregates in mice: improved consistency of outcomes and in situ monitoring

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 in vivo 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 in vivo 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