Search Results (35)

Reduced oxygen during embryo culture in human ART prevents embryo oxidative stress. Oxidative stress is also the major mechanism by which maternal diabetes impairs embryonic development. This study employed induced hyperglycemia prepubertal mice to mimic childhood diabetes to understand the effects of varying oxygen tension during in vitro embryonic development. The oocytes were fertilized and cultured at low (≈5%) oxygen (LOT) or atmospheric (≈20%) oxygen tension (HOT) for up to 96 h. Embryo development, apoptosis in blastocysts, inner cell mass (ICM) outgrowth proliferation, and Hif1α expression were assessed. Though the oocyte quality and meiotic spindle were not affected, the fertilization rate (94.86 ± 1.18 vs. 85.17 ± 2.81), blastocyst rate (80.92 ± 2.92 vs. 69.32 ± 2.54), and ICM proliferation ability (51.04 ± 9.22 vs. 17.08 ± 3.05) of the hyperglycemic embryos were significantly higher in the LOT compared to the HOT group. On the other hand, blastocysts from the hyperglycemic group, cultured at HOT, had a 1.5-fold increase in apoptotic cells compared to the control and lower Hif1α transcripts in ICM outgrowths compared to the LOT. Increased susceptibility of embryos from hyperglycemic mice to higher oxygen tension warrants the need to individualize the conditions for embryo culture systems in ART clinics, particularly when an endogenous maternal pathology affects the ovarian environment. Full article

Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling, it has been proposed that undifferentiated human spermatogonia can be subclassified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc) RNA-sequencing libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat, and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate ‘state 0B’ with a suppressive transcriptomic programme that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells—which are mitotically arrested—can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mice (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive lifespan while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes. Full article

Currently, there is no viable option for fertility preservation in prepubertal boys. Experimentally, controlled vitrification of testicular tissue has been evaluated and found to cause potential structural damage to the spermatogonial stem cell (SSC) niche during cryopreservation. In this report, we leveraged the regenerative effect of human umbilical cord-derived Mesenchymal stem cell exosomes (h-UCMSC-Exo) to protect against testicular damage from the cytotoxic effects of polychemotherapy (CTX). A chemotherapy-induced testicular dysfunctional model was established by CTX treatment with cyclophosphamide and Busulfan in vitro (human Sertoli cells) and in prepubescent mice. We assessed the effects of the exosomes by analyzing cell proliferation assays, molecular analysis, immunohistochemistry, body weight change, serum hormone levels, and fertility rate. Our data indicates the protective effect of h-UCMSC-Exo by preserving the SSC niche and preventing testicular damage in mice. Interestingly, mice that received multiple injections of h-UCMSC-Exo showed significantly higher fertility rates and serum testosterone levels (p < 0.01). Our study demonstrates that h-UCMSC-Exo can potentially be a novel fertility protection approach in prepubertal boys triaged for chemotherapy treatment. Full article

Background and Objectives: Ovarian tissue cryopreservation followed by autotransplantation (OTCTP) is currently the only fertility preservation option for prepubertal patients. Once in remission, the autotransplantation of frozen/thawed tissue is performed when patients want to conceive. A major issue of the procedure is follicular loss directly after grafting mainly due to follicle activation. To improve follicular survival during the OTCTP procedure, we inhibited the mTOR pathway involved in follicle activation using rapamycin, an mTOR inhibitor. Next, we compared two different in vivo models of transplantation: the recently described non-invasive heterotopic transplantation model between the skin layers of the ears, and the more conventional and invasive transplantation under the kidney capsule. Materials and Methods: To study the effects of adding rapamycin during cryopreservation, 4-week-old C57BL/6 mouse ovaries, either fresh, slow-frozen, or slow-frozen with rapamycin, were autotransplanted under the kidney capsule of mice and recovered three weeks later for immunohistochemical (IHC) analysis. To compare the ear with the kidney capsule transplantation model, fresh 4-week-old C57BL/6 mouse ovaries were autotransplanted to either site, followed by an injection of either LY294002, a PI3K inhibitor, vehicle control, or neither, and these were recovered three weeks later for IHC analysis. Results: Rapamycin counteracts cryopreservation-induced follicle proliferation, as well as AKT and mTOR pathway activation, in ovaries autotransplanted for three weeks under the kidney capsule of mice. Analyses of follicle proliferation, mTOR activation, and the effects of LY294002 treatment were similar in transplanted ovaries using either the ear or kidney capsule transplantation model. Conclusions: By adding rapamycin during the OTCTP procedure, we were able to transiently maintain primordial follicles in a quiescent state. This is a promising method for improving the longevity of the ovarian graft. Furthermore, both the ear and kidney capsule transplantation models were suitable for investigating follicle activation and proliferation and pharmacological strategies. Full article

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100–130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E₂), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E₂ and FSH to the culture medium. Full article

Pediatric acute myeloid leukemia (AML) generally occurs de novo. The treatment of AML includes cytarabine (CYT) and other medications. The granulocyte-colony stimulating factor (GCSF) is used in the clinic in cases of neutropenia after chemotherapies. We show that the administration of GCSF in combination with CYT in AML-diagnosed mice (AML+CYT+GCSF) extended the survival of mice for additional 20 days. However, including GCSF in all treatment modalities does not affect the testis’ weight or the histology of the seminiferous tubules (STs). We show that GCSF does not affect normal ST histology from AML-, CYT-, or (AML+CYT)-treated groups compared to the relevant treated group without GCSF 2, 4, and 5 weeks post-injection. However, when comparing the percentages of normal STs between the AML+CYT+GCSF-treated groups and those without GCSF, we observe an increase of 17%–42% in STs at 4 weeks and 5.5 weeks post-injection. Additionally, we show that the injection of GCSF into the normal, AML-alone, or CYT-alone groups, or in combination with AML, significantly decreases the percentage of STs with apoptotic cells compared to the relevant groups without GCSF and to the CT (untreated mice) only 2 weeks post-injection. We also show that injection of GCSF into the CT group increases the examined spermatogonial marker PLZF within 2 weeks post-injection. However, GCSF does not affect the count of meiotic cells (CREM) but decreases the post-meiotic cells (ACROSIN) within 4 weeks post-injection. Furthermore, GCSF not only extends the survival of the AML+CYT-treated group, but it also leads to the generation of sperm (1.2 ± 0.04 × 10⁶/mL) at 5.5 weeks post-injection. In addition, we demonstrate that the injection of GCSF into the CT group increases the RNA expression level of IL-10 but not IL-6 compared to CT 2 weeks post-treatment. However, the injection of GCSF into the AML-treated group reverses the expression levels of both IL-10 and IL-6 to normal levels compared to CT 2 weeks post-injection. Thus, we suggest that the addition of GCSF to the regimen of AML after CYT may assist in the development of future therapeutic strategies to preserve male fertility in AML prepubertal patients. Full article

Autism spectrum disorder (ASD) is a neurodevelopmental condition with a prevalence rate of 2.78%, and it is characterized by deficits in sociability and communication and restricted patterns of interests and activities. Remarkably, this psychiatric disorder exhibits a pronounced gender bias, with 80% of children diagnosed with ASD being boys. In this review, we will present advancements in mouse models of ASD and their potential contributions to our understanding of the disorder. We will highlight how initial pre-clinical investigations focused solely on male mice due to the gender bias in ASD and explain why we believe that this approach might have had detrimental consequences regarding our understanding of ASD etiology and pathophysiology. We will highlight the evidence of two sensitive periods during brain development when differential exposure to gonadal hormones may result in sex differences in brain function and behavior: the perinatal period and the pre-pubertal period. Finally, we will suggest neuroinflammation as a feasible biological mechanism that may converge different ASD etiological factors and cellular mechanisms into a brain sexual differentiation context, thus accounting for the gender disparities observed in the disorder. Full article

The endocannabinoid (eCB) system has gained ground as a key modulator of several female fertility-related processes, under physiological/pathological conditions. Nevertheless, its modulation during reproductive aging remains unclear. This study aimed to investigate the expression levels of the main receptors (cannabinoid receptor 1,CB₁; cannabinoid receptor 2, CB₂; G-protein coupled receptor, GPR55; and transient receptor potential vanilloid type 1 channel, TRPV1) and metabolic enzymes (N-acylphosphatidylethanolamine phospholipase D, NAPE-PLD; fatty acid amide hydrolase, FAAH; monoacylglycerol lipase, MAGL; and diacylglycerol lipase, DAGL-α and -β) of this system in the ovaries, oviducts, and uteri of mice at prepubertal, adult, late reproductive, and post-reproductive stages through quantitative ELISA and immunohistochemistry. The ELISA showed that among the receptors, TRPV1 had the highest expression and significantly increased during aging. Among the enzymes, NAPE-PLD, FAAH, and DAGL-β were the most expressed in these organs at all ages, and increased age-dependently. Immunohistochemistry revealed that, regardless of age, NAPE-PLD and FAAH were mainly found in the epithelial cells facing the lumen of the oviduct and uteri. Moreover, in ovaries, NAPE-PLD was predominant in the granulosa cells, while FAAH was sparse in the stromal compartment. Of note, the age-dependent increase in TRPV1 and DAGL-β could be indicative of increased inflammation, while that of NAPE-PLD and FAAH could suggest the need to tightly control the levels of the eCB anandamide at late reproductive age. These findings offer new insights into the role of the eCB system in female reproduction, with potential for therapeutic exploitation. Full article

Chemotherapy is used for childhood cancer but may lead to infertility in patients. Spermatogonia stem cells are present in the testes of prepubertal boys, although they do not produce sperm at this age. Herein, we evaluated the toxicity of cisplatin, a known medicine for cancer treatment, in neonatal mouse testes using in vitro organ culture. Mouse testicular fragments (MTFs) derived from 5.5-d postpartum mouse testes were exposed to 1–10 μg/mL cisplatin. The results showed that cisplatin significantly downregulated the expression of germ cell marker genes, including differentiated and undifferentiated, in a dose-dependent manner. In particular, a high dose of cisplatin (10 μg/mL) led to germ cell depletion. In addition, the expression levels of the Sertoli cell marker gene, the number of SOX9+ Sertoli cells, and the levels of SOX9 protein were markedly decreased in cisplatin-treated MTFs compared to controls. The mRNA expression of steroidogenic enzyme-related genes significantly increased in cisplatin-treated MTFs, except for estrogen receptor 1 (Esr1). Consistently, 3β-hydroxysteroid dehydrogenase protein was also observed in the interstitial regions of cisplatin-treated MTFs. Altogether, our findings showed a significant impairment in germ cell development, Sertoli cell survival, and steroidogenesis in the MTFs of cisplatin-treated mice. Full article

The mammary gland is a unique organ with the ability to undergo repeated cyclic changes throughout the life of mammals. Among domesticated livestock species, ruminants (cattle and buffalo) constitute a distinct class of livestock species that are known milk producers. Cattle and buffalo contribute to 51 and 13% of the total milk supply in the world, respectively. They also play an essential role in the development of the economy for farming communities by providing milk, meat, and draft power. The development of the ruminant mammary gland is highly dynamic and multiphase in nature. There are six developmental stages: embryonic, prepubertal, pubertal, pregnancy, lactation, and involution. There has been substantial advancement in our understanding of the development of the mammary gland in both mouse and human models. Until now, there has not been a thorough investigation into the molecular processes that underlie the various stages of cow udder development. The current review sheds light on the morphological and molecular changes that occur during various developmental phases in diverse species, with a particular focus on the cow udder. It aims to explain the physiological differences between cattle and non-ruminant mammalian species such as humans, mice, and monkeys. Understanding the developmental biology of the mammary gland in molecular detail, as well as species-specific variations, will facilitate the researchers working in this area in further studies on cellular proliferation, differentiation, apoptosis, organogenesis, and carcinogenesis. Additionally, in-depth knowledge of the mammary gland will promote its use as a model organ for research work and promote enhanced milk yield in livestock animals without affecting their health and welfare. Full article

Prepubertal boys with cancer may suffer from reduced fertility and maturity following gonadotoxic chemoradiotherapy. Thus, a viable method of immature testicular tissue (ITT) preservation is required in this cohort. In this study, we used poly-L-lactic acid electrospun scaffolds with two levels of fineness to support the development of ITT transplanted from transgenic donors to wild-type recipient mice. The purpose of this study was to evaluate the potential of ITT transplantation and spermatogenesis after using the two scaffolds, employing bioluminescence imaging for evaluation. The results suggest that ITT from 4-week-old mice possessed the most potential in spermatogenesis on the 70th day, together with the fine electrospun scaffolds. Moreover, bioluminescent imaging intensity was observed in recipient mice for up to 107 days, approximately six times more than the coarse electrospun scaffold and the control group. This occurs since the fine scaffold is more akin to the microenvironment of native testicular tissue as it reduces stiffness resulting from micronization and body fluid infiltration. The thermal analysis also exhibited recrystallization during the biodegradation process, which can lead to a more stable microenvironment. Overall, these findings present the prospect of fertility preservation in prepubertal males and could serve as a framework for future applications. Full article

A follicle stimulating hormone (FSH) is widely used in the assisted reproduction and a synthetic peptide corresponding to a receptor binding region of the human (h) FSH-β-(34–37) (TRDL) modulated reproduction. Furthermore, a 13-amino acid sequence corresponding to hFSH-β-(37–49) (LVYKDPARPKIQK) was recently identified as the receptor binding site. We hypothesized that the synthetic peptides corresponding to hFSH-β-(37–49) and hFSH-β-(34–49), created by merging hFSH-β-(34–37) and hFSH-β-(37–49), modulate the reproductive functions, with the longer peptide being more biologically active. In male or female prepubertal mice, a single injection of 200 μg/g BW ip of hFSH-β-(37–49) or hFSH-β-(34–49) hastened (p < 0.05) puberty, whereas the same treatments given daily for 4 d promoted (p < 0.05) the gonadal steroidogenesis and gamete formation. In addition of either peptide to the in vitro cell cultures, promoted (p < 0.05) the proliferation of primary murine granulosa cells and the estradiol production by upregulating the expression of Ccnd2 and Cyp19a1, respectively. In adult female mice, 200 μg/g BW ip of either peptide during diestrus antagonized the FSH-stimulated estradiol increase and uterine weight gain during proestrus. Furthermore, hFSH-β-(34–49) was a more potent (p < 0.05) reproductive modulator than hFSH-β-(37–49), both in vivo and in vitro. We concluded that hFSH-β-(37–49) and especially hFSH-β-(34–49), have the potential for reproductive modulation. Full article

Polycystic ovarian syndrome (PCOS) is a reproductive, endocrine, and metabolic disorder. Circulating markers of oxidative stress are abnormal in women with PCOS. There is a close relationship between oxidative stress and insulin resistance (IR). However, little information is available about oxidative stress in the skeletal muscles of those affected by PCOS. In this study, PCOS was induced in prepubertal C57BL/6J mice by injection with dehydroepiandrosterone. Oxidative stress biomarkers were then measured in both serum and skeletal muscles. The underlying mechanisms were investigated in C2C12 myotubes treated with testosterone (T). We discovered increased oxidative biomarkers, increased ROS production, and damaged insulin sensitivity in the skeletal muscles of mice with PCOS. High levels of T caused mitochondrial dysfunction and increased ROS levels through the androgen receptor (AR)-nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) signaling pathway in C2C12 cells. Treatment of C2C12 cells with an antioxidant N-acetylcysteine (NAC) decreased T-induced ROS production, improved mitochondrial function, and reversed IR. Administration of NAC to mice with PCOS improved insulin sensitivity in the skeletal muscles of the animals. Hyperandrogenism caused mitochondrial dysfunction and redox imbalance in the skeletal muscles of mice with PCOS. We discovered that oxidative stress contributed to skeletal muscle IR in PCOS. Reducing ROS levels may improve the insulin sensitivity of skeletal muscles in patients with PCOS. Full article

Pediatric cancer survivors experiencing gonadotoxic chemoradiation therapy may encounter subfertility or permanent infertility. However, previous studies of cryopreservation of immature testicular tissue (ITT) have mainly been limited to in vitro studies. In this study, we aim to evaluate in vitro and in vivo bioluminescence imaging (BLI) for solid surface-vitrified (SSV) ITT grafts until adulthood. The donors and recipients were transgenic and wild-type mice, respectively, with fresh ITT grafts used as the control group. In our study, the frozen ITT grafts remained intact as shown in the BLI, scanning electron microscopy (SEM) and immunohistochemistry (IHC) analyses. Graft survival was analyzed by BLI on days 1, 2, 5, 7, and 31 after transplantation. The signals decreased by quantum yield between days 2 and 5 in both groups, but gradually increased afterwards until day 31, which were significantly stronger than day 1 after transplantation (p = 0.008). The differences between the two groups were constantly insignificant, suggesting that both fresh and SSV ITT can survive, accompanied by spermatogenesis, until adulthood. The ITT in both groups presented similar BLI intensity and intact cells and ultrastructures for spermatogenesis. This translational model demonstrates the great potential of SSV for ITT in pre-pubertal male fertility preservation. Full article

This study aims to investigate the effects of a high-fat, high-fructose (HF/HFr) diet on metabolic/endocrine dysregulations associated with letrozole (LET)-induced Polycystic Ovarian Syndrome (PCOS) in prepubertal female mice. Thirty-two prepubertal C57BL/6 mice were randomly divided into four groups of eight and implanted with LET or a placebo, with simultaneous administration of an HF/HFr/standard diet for five weeks. After sacrifice, the liver and blood were collected for selected biochemical analyses. The ovaries were taken for histopathological examination. The LET+HF/HFr group gained significantly more weight than the LET-treated mice. Both the LET+HF/HFr and the placebo-treated mice on the HF/HFr diet developed polycystic ovaries. Moreover the LET+HF/HFr group had significantly elevated testosterone levels, worsened lipid profile and indices of insulin sensitivity. In turn, the HF/HFr diet alone led to similar changes in the LET-treated group, except for the indices of insulin sensitivity. Hepatic steatosis also occurred in both HF/HFr groups. The LET-treated group did not develop endocrine or metabolic abnormalities, but polycystic ovaries were seen. Since the HF/HFr diet can cause substantial metabolic and reproductive dysregulation in both LET-treated and placebo mice, food items rich in simple sugar—particularly fructose—and saturated fat, which have the potential to lead to PCOS progression, should be eliminated from the diet of young females. Full article