Search Results (5)

Vitrification is an important tool to store surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and results in decreased viability. Studies have reported that L-glutathione (GSH) supplementation improves the preimplantation development of murine embryos. Glutathione constitutes the major non-protein sulphydryl compound in mammalian cells, which confers protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has yet to be reported. This study aims to determine whether GSH supplementation in culture media improves in vitro culture and vitrification outcomes, as observed through embryo morphology and preimplantation development. Female BALB/c mice aged 6–8 weeks were superovulated through an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG), followed by 10 IU of human chorionic gonadotrophin (hCG) 48 h later. The mated mice were euthanized by cervical dislocation 48 h after hCG to harvest embryos. Two-cell embryos were randomly assigned to be cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH-supplemented medium), or Group 4 (0.01 mM GSH-supplemented medium with vitrification). Non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were observed for morphological quality and preimplantation development at 24, 48, 72, and 96 h. In the non-vitrified groups, there were significant increases in the number of Grade-1 blastocysts in GSH cultures (p < 0.05). Similarly, in the vitrified groups, GSH supplementation was also seen to significantly increase blastocyst formation. Exogenous GSH supplementation resulted in a significant increase in intracellular GSH, a release of cytochrome c from mitochondria, and a parallel decrease in intracellular reactive oxygen species (ROS) levels in vitrified eight-cell embryos (p < 0.05). GSH supplementation was shown to upregulate Bcl2 expression and downregulate Bax expression in the vitrified preimplantation embryo group. The action of exogenous GSH was concomitant with an increase in the relative abundance of Gpx1 and Sod1. In conclusion, our study demonstrated the novel use and practical applicability of GSH supplementation for improving embryonic cryotolerance via a decrease in ROS levels and the inhibition of apoptotic events by improvement in oxidative status. Full article

For experiment one, blood samples were obtained from 200 gilts at 90, 120, 150, 180, and 200 days of age. Serum samples from the 30 youngest (166.1 ± 0.7 days) and 30 oldest (198.8 ± 0.6 days) gilts exhibiting estrus by 200 days, and a further 18 gilts that remained anestrus at 200 days, were assayed for serum concentrations of anti-Mullerian hormone (AMH) and estradiol (E2). Gilts younger at puberty had higher (p < 0.05) AMH levels than those older at puberty, and both groups had higher AMH levels than anestrus gilts (p < 0.05). Regardless of age, serum E2 was higher (p < 0.05) in gilts that achieved puberty than in gilts remaining anestrus. At spontaneous pubertal estrus detection, there was no effect of pubertal age on the number of preovulatory ovarian follicles. For experiment two, 152 prepubertal gilts received an intramuscular (IM) injection of 400 IU eCG plus 200 IU hCG and then received fence-line boar contact to detect estrus onset. Serum AMH concentrations were higher (p < 0.05) in the first 25 gilts to exhibit puberty than the last 28 gilts, with the first gilts also having more preovulatory follicles (p < 0.0001). Taken together, these data support an association between serum AMH concentrations and degree of physiological maturity and ovarian follicular development in gilts. Full article

Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1–14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-Müllerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin 43, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche. Full article

At weaning, 33 mixed parity Hypor sows received either an injection of 400 IU equine chorionic gonadotrophin and 200 IU human chorionic gonadotrophin (hCG) (PG600; n = 13), PG600 with an additional 200 IU hCG 24 h later (Gn800; n = 11), or served as non-injected controls (n = 9). All gonadotrophin treated sows received an injection of 750 IU hCG at 80 h after weaning to induce ovulation (designated as time 0 h). At 0, 24, 36, 40, 44, 48, and 60 h, all sows were subject to transrectal ultrasonography to determine numbers and sizes of large (>6 mm) follicles and time of ovulation. The interval from injection of 750 IU hCG to ovulation was shorter in Gn800 compared to PG600 sows (p = 0.02), and more Gn800 sows had ≥9 preovulatory follicles compared to PG600 and controls (p = 0.02 and 0.003, respectively). Follicular cysts were evident in both PG600 and Gn800 sows. Full article

The future fertility of prepubertal boys with cancer may be irreversibly compromised by chemotherapy and/or radiotherapy. Successful spermatogenesis has not been achieved following the xenotransplantation of prepubertal human testis tissue, which is likely due to the failure of somatic cell maturation and function. We used a validated xenograft model to identify the factors required for Leydig and Sertoli cell development and function in immature human testis. Importantly, we compared the maturation status of Sertoli cells in xenografts with that of human testis tissues (n = 9, 1 year-adult). Human fetal testis (n = 6; 14–21 gestational weeks) tissue, which models many aspects of prepubertal testicular development, was transplanted subcutaneously into castrated immunocompromised mice for ~12 months. The mice received exogenous human chorionic gonadotropin (hCG; 20IU, 3×/week). In xenografts exposed continuously to hCG, we demonstrate the maintenance of Leydig cell steroidogenesis, the acquisition of features of Sertoli cell maturation (androgen receptor, lumen development), and the formation of the blood–testis barrier (connexin 43), none of which were present prior to the transplantation or in xenografts in which hCG was withdrawn after 7 months. These studies provide evidence that hCG plays a role in Sertoli cell maturation, which is relevant for future investigations, helping them generate functional gametes from immature testis tissue for clinical application. Full article