Transcriptomic comparison of ovarian granulosa cells between adult sheep and prepubertal lambs.

BACKGROUND: The oocyte development ability of prepubertal animals is significantly lower than that of adult animals. Granulosa cells (GCs) have an important function on regulation of follicular and oocyte development. Therefore, analysis of GC characteristics can be used to explore the developmental mechanism of follicles and oocytes. RESULTS: In order to understand the possible reasons for the differences in follicle and oocyte development between lambs and adult sheep, we utilized high-throughput sequencing technique to analyze the transcriptome of GCs from follicle-stimulating hormone (FSH) superstimulated adult ewes and prepubertal lambs. Adult ewes were treated with FSH for 3 days (group A) and lambs were FSH-treated for 2 days (group B) or 3 days (group C). Transcriptome analysis of GCs showed that there were 405 and 159 differentially expressed genes from A vs. B and A vs. C, respectively. The results indicated that prolonging the FSH-treatment of lambs made the GC state of lambs more similar to the adult ewes, but there were still a large number of differentially expressed genes between adult ewes and lambs. Further analysis showed that many differently expressed genes were implicated in cell proliferation and apoptosis, oocyte development and follicular ovulation. Cellular examination demonstrated that fatty acid binding protein 4 (FABP4), which was highly expressed in lamb GCs, had a potential of promoting cell apoptosis. Cytoplasmic phospholipase A2 (PLA2G4A), which was expressed lowly in lamb GCs, may be responsible for reduced synthesis of prostaglandins in cells and impaired follicle/oocyte development. In contrast, glutathione S-transferase ß-1 (GSTT2B) and forkhead boxO6 (FOXO6) had no apparent effect on the proliferation and apoptosis of GCs. CONCLUSIONS: Our study found dramatic transcriptomic differences in GCs between lambs and adult sheep, which may explain the possible reasons for the defects of follicle and oocyte development in lambs compared to adult sheep. Our data provides important information for further understanding the mechanism of follicular development in prepubertal animals and improving their oocyte developmental competence.

Improved early postnatal nutrition and its effect on histomorphological parameters in the testes of Sanjabi ram lambs.

The effect of an ad libitum supplementary feeding on some reproductive characteristics of ram lambs were evaluated. Twelve Sanjabi rams were allocated to one of two groups: control (C) and treatment (T). The control group received hay and the treatment group received hay and concentrate with gross energy and crude protein concentration of 4.52 Mcal and 16% of dry matter, respectively. At the end of experiment, after taking blood samples for determination of testosterone concentration (TC), all lambs were castrated and the testicular weight (TWT), testicular volume (TV), testicular length (TL), and testicular width (TW) were measured. In addition, testes were subjected to histological analysis. Body weight (BW), TC, TWT, TL, and TV in the T group were higher (P < 0.05) than the C group. However, TW was not affected by diet (P > 0.05). Animals from the C group presented lower numbers (P < 0.05) of Sertoli (SCN), germ (GCN), and Leydig cell numbers (LCN) compared to the T group. Also, seminiferous tubule diameter (STD) in the T group was significantly greater than (P < 0.05) the C group. TWT had a significant association with TC (P < 0.05), STD (P < 0.01), SCN (P < 0.01), GCN (P < 0.05), and LCN (P < 0.05). TC was correlated with STD (P < 0.01), SCN (P < 0.01), GCN (P < 0.05), and LCN (P < 0.05). STD showed close correlation with SCN (P < 0.01), GCN (P < 0.01), and LCN (P < 0.05). Also, SCN had a positive association with GCN (P < 0.01) and LCN (P < 0.05). A positive correlation was found between GCN and LCN (P < 0.01). In conclusion, the plane of nutrition of growing ram lambs enhances their growth and testicular development and probably will increases their postpubertal reproductive performance.

Growth hormone treatment improves the development of follicles and oocytes in prepubertal lambs.

BACKGROUND: When prepubertal lambs are superovulated, the ovarian response to gonadotropin stimulation has great individual difference and the collected oocytes have lower developmental ability than that of adult ewes. Over the years, growth hormone (GH) has been used in assisted reproduction because it can improve the reproductive performance in humans and animals. However, the effect of GH on ovaries and oocytes of prepubertal lambs remains unclear. METHODS: Before and during follicle-stimulating hormone (FSH) superovulation of prepubertal lambs (4‒6-week-old), the lambs were treated with high (50 mg) or low dose (25 mg) of ovine GH in a long (5 days) or short (2 days) period. The recovered oocytes were used for in vitro maturation and fertilization, and several parameters of oocyte quality and development capacity were evaluated. The possible underlying mechanisms of GH action were explored by analysis of granulosa cell (GC) transcriptome, ovarian proteome and follicular fluid metabolome. RESULTS: Treatment of lambs with 50 mg GH over 5 days (long treatment) potentially promoted the response of lambs to superovulation and improved the development capacity of retrieved oocytes, consequently increasing the high quality embryo yield from lambs. A number of differently expressed genes or proteins were found in ovaries between GH-treated and untreated lambs. Cellular experiments revealed that GH reduced the oxidative stress of GCs and promoted the GC proliferation probably through activation of the PI3K/Akt signaling pathway. Finally, analysis of follicular fluid metabolome indicated that GH treatment altered the abundance of many metabolites in follicular fluid, such as antioxidants and fatty acids. CONCLUSIONS: GH treatment has a beneficial role on function of lamb ovaries, which supports the development of follicles and oocytes and improves the efficiency of embryo production from prepubertal lambs.

Cumulus Cell Transcriptome after Cumulus-Oocyte Complex Exposure to Nanomolar Cadmium in an In Vitro Animal Model of Prepubertal and Adult Age.

Cadmium (Cd), a highly toxic pollutant, impairs oocyte fertilization, through oxidative damage on cumulus cells (CCs). This study analysed the transcriptomic profile of CCs of cumulus-oocyte complexes (COCs) from adult and prepubertal sheep, exposed to Cd nanomolar concentration during in vitro maturation. In both age-groups, CCs of matured oocytes underwent RNA-seq, data analysis and validation. Differentially expressed genes (DEGs) were identified in adult (n = 99 DEGs) and prepubertal (n = 18 DEGs) CCs upon Cd exposure. Transcriptomes of adult CCs clustered separately between Cd-exposed and control samples, whereas prepubertal ones did not as observed by Principal Component Analysis. The transcriptomic signature of Cd-induced CC toxicity was identified by gene annotation and literature search. Genes associated with previous studies on ovarian functions and/or Cd effects were confirmed and new genes were identified, thus implementing the knowledge on their involvement in such processes. Enrichment and validation analysis showed that, in adult CCs, Cd acted as endocrine disruptor on DEGs involved in hormone biosynthesis, cumulus expansion, regulation of cell signalling, growth and differentiation and oocyte maturation, whereas in prepubertal CCs, Cd affected DEGs involved in CC development and viability and CC-oocyte communications. In conclusion, these DEGs could be used as valuable non-invasive biomarkers for oocyte competence.

Enhancing the developmental competence of prepubertal lamb oocytes by supplementing the in vitro maturation medium with sericin and the fibroblast growth factor 2 - leukemia inhibitory factor - Insulin-like growth factor 1 combination.

Poor development of oocytes from prepubertal animals is a major factor that hinders the application of the technology, juvenile in vitro embryo transfer (JIVET). The aim of this study was to explore the possibility of improving the developmental competence of prepubertal oocytes by supplementing the oocyte in vitro maturation (IVM) medium with antioxidants and cytokines. Effects of two antioxidants, melatonin and sericin, were first examined. The results showed that melatonin had no significant beneficial roles on the lamb oocyte development, while 0.5% sericin supplemented during IVM significantly increased the blastocyst rate of lamb oocytes (46.5% vs 19.2% in control, P < 0.05). Next, effects of two kinds of combined supplements, insulin-transferrin-selenium (ITS) and fibroblast growth factor 2(FGF2)-leukemia inhibitory factor (LIF)-insulin-like growth factor1 (IGF1)(FLI) were tested. The results indicated that addition of FLI, but not ITS, in the IVM medium, significantly improved the blastocyst development of lamb oocytes (43.9% in FLI group vs 21.6% in control, P < 0.05). Further comparison showed that the developmental competence of oocytes was not significantly different among supplementation with sericin or FLI alone or both, all of which generated similar outcomes of blastocyst yield to the supplementation with adult follicular fluid. Finally, 27 blastocysts produced from lamb oocytes matured in the presence of sericin and FLI were transferred into 18 recipients, of which 9 were pregnant. This study suggests that the developmental competence of prepubertal oocytes can be improved by supplementing IVM medium with relevant agents like sericin and cytokines.

Bioengineering Approaches to Improve In Vitro Performance of Prepubertal Lamb Oocytes.

Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus-oocyte complex (COC), by constructing-via bioprinting technologies-alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM); (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor; and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET.

Adult follicular fluid supplementation during in vitro maturation improves the developmental competence of prepubertal lamb oocytes.

Oocytes from prepubertal lambs have lower developmental ability than that from adult ewes. Follicular fluid (FF) provides an important microenvironment for oocyte development and maturation in vivo. The aim of this study was to examine the effects of FF supplemented during in vitro maturation (IVM) on the developmental competence of prepubertal lamb oocytes. FF was collected from follicle-stimulating hormone (FSH) stimulated adult ewes or 4‒6-week-old lambs or abattoir-derived adult ovaries. The FF was supplemented to the control IVM medium, TCM199 containing 20% estrus sheep serum and hormones. It was found that the lamb oocytes matured in medium supplemented with 20% or 30% adult FF from FSH-stimulated ewes yielded significantly higher blastocyst rates than that from the control medium, or medium supplemented with 10% adult FF or 20% lamb FF (43.5%, 37.9% vs. 28.4%, 29.7%, 27.6%, P < 0.05). However, when adult oocytes were matured in medium supplemented with 20% adult FF, their cleavage and blastocyst development were similar to that of those matured in control medium. Addition of 20% adult FF from abattoir-derived ovaries to IVM medium also significantly increased the blastocyst formation of lamb oocytes when compared to that from the medium without FF supplementation. The blastocyst development did not differ between the groups of FF from abattoir-derived ovaries and from FSH-stimulated ewes (38.2% vs 43.1%, P > 0.05). A total of 146 blastocysts derived from different groups of lamb oocytes were transferred into 76 synchronized recipients, of which 50% were pregnant and 38.2% lambed. These results suggest that supplementing IVM medium with adult FF has beneficial roles on the developmental competence of prepubertal lamb oocytes.