Growth hormone treatment improves the development of follicles and oocytes in prepubertal lambs
Journal of Ovarian Research volume 16, Article number: 132 (2023)
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.
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.
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.
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.
In vitro production (IVP) of embryos from oocytes of juvenile animals, known as juvenile in vitro embryo transfer (JIVET), is a promising technology to be applied in rapid breeding of livestock species . An advantage of using juvenile lambs (4‒8-week-old) as oocyte donors is that a great number of oocytes can be recovered from ovaries following gonadotropin superstimulation of the animals [1,2,3]. However, lamb oocytes usually exhibit lower developmental ability than oocytes from adult ewes when they are used for in vitro embryo production [4,5,6,7,8]. To improve the developmental competence of lamb oocytes, many studies have focused on optimizing the in vitro maturation medium by adding various agents to the medium [9, 10]. Nevertheless, the oocyte competence is largely dependent on the development during growth within ovarian follicles . Therefore, treatment of lambs before oocyte retrieval may enhance the development potential of oocytes.
Growth hormone (GH) plays an important role in maintaining normal fertility by exerting biological effects on folliculogenesis, oocyte maturation and steroid synthesis through autocrine or paracrine secretion [12, 13]. Therefore, GH has already been applied as an adjuvant to enhance human ovarian sensitivity to gonadotropin stimulation since 1980s . For instance, treatment with GH can significantly shorten the gonadotropin stimulation duration before oocyte retrieval and increase the number of competent oocytes and clinical pregnancy rate in poor ovarian responders [15, 16]. In sheep, the utility of GH has been reported in estrus synchronization and superovulation of adult ewes for enhancing the follicle development and embryo production [17,18,19,20,21,22]. These studies support the potential role of GH in improvement of the reproductive performance in mammals.
Although the efficacy of GH on reproduction has been confirmed in adult females in human or animals, it remains to be determined whether GH can improve the ovarian response and oocyte quality of prepubertal animals in the JIVET program. To address this issue, in this study we attempted to treat lambs with GH in the process of superovulation and evaluated its effects on the development of follicles and oocytes in lambs. We found that the ovarian response and oocyte developmental ability could be improved when the lambs were treated with GH. We also explored the possible underlying mechanisms of GH action by analysis of GC transcriptome, ovarian proteome and follicular fluid (FF) metabolome.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless specifically stated.
Animals and hormonal treatment
A total of 54 female Merino lambs (4‒6-week-old, still on their dams) were selected for the experiments. All lambs together with their mothers were house-fed under conventional management conditions and maintained in Aohan Sheep Breeding Farm, Chifeng, Inner Mongolia, China (42°18′ N, 119°50′ E). The animal experiments were conducted from July to June (the temperature ranged from 10℃ to 25℃). The protocol of superovulation treatment of lambs was as described previously [9, 10]. Briefly, each lamb received 6 × 45 IU injections of follicle-stimulating hormone (FSH) (Ningbo Sansheng Pharmaceutical, China) given at approximately 12 h intervals, and 400 IU of equine chorionic gonadotropin (eCG; Ningbo Sansheng Pharmaceutical) given at the first FSH administration.
For GH treatment, the lambs were subcutaneously injected with a recombinant ovine GH that was produced in our lab as described previously . As illustrated in Fig. 1, different dosages and time points of GH injections were designed to examine the effects of GH treatment on JIVET efficiency. In trial 1, a total of 21 lambs were randomly allocated to three groups, with 7 lambs in each group. The lambs used for GH treatment received a total of five injections of GH, which were administrated during the period of Day 0 to Day 8, with 10 mg (high dose) or 5 mg (low dose) for each injection (Fig. 1a, referred as long-treatment). Unfortunately, one lamb in the 50 mg group died of diarrhea during the treatment. In trial 2, a total of 18 lambs were used (n = 6 in each group), and the lambs in GH treatment groups received 2 injections of GH that were applied only at Day 0 and Day 2, with 25 mg (high dose) or 10 mg (low dose) for each injection (Fig. 1b, referred as short-treatment). In trial 3, a total of 15 lambs were randomly allocated to three groups (n = 5 in each group), and the lambs in GH treatment groups received 5 × 10 mg (long treatment) or 2 × 25 mg (short treatment) GH injections at the time points as illustrated in Fig. 1c. In all trials, in parallel with each GH injection, injection of saline solution was used as controls.
The procedure of all animal experiments was in accordance with the animal care policies of China Agricultural University and was approved by the Animal Ethics Committee at the university (protocol code: AW20701202-3-4, date of approval: 1 June, 2020).
Oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC)
Oocytes were recovered from lambs by laparotomy approximately 12 h after the last FSH injection as described previously . The cumulus-oocyte complexes (COCs) were cultured in IVM medium, TCM 199 supplemented with 20% (v:v) estrus sheep serum (ESS), 10 µg/mL FSH (Folltropin-V; Bioniche Inc. Belleville, Ont., Canada), 10 µg/ml luteinizing hormone (LH, Bioniche Inc), 1 µg/mL 17β-estradiol, 100 IU/mL penicillin and 100 µg/mL streptomycin, at 38.5 ℃ in a humidified atmosphere of 5% CO2 in air.
After 24 h of culture, the COCs were partially stripped of cumulus cells by briefly incubating in 0.2% hyaluronidase in Hepes-buffered TCM199 solution, and then placed into IVF medium, synthetic oviduct fluid (SOF) medium containing 2% ESS. Frozen-thawed sperm were layered under 600 µL IVF medium in a glass tube, and incubated for 30 min at 38.5 ℃ for swimming up. Swim-upped spermatozoa were collected from the upper fraction and added into each IVF well, with a final density of 1 × 105 spermatozoa mL − 1. The COCs and sperm were co-incubated for 24 h at 38.5 ℃ under 5% CO2 in air.
Following IVF, the presumptive zygotes were cultured in IVC medium, SOF containing 8 mg/mL fatty acid-free bovine serum albumin, 1% (v:v) essential amino acids and 2% (v:v) non-essential amino acids. Zygotes were incubated in a humidified atmosphere of 5% CO2, 5%O2, and 90% N2 at 38.5 °C. Cleavage and blastocyst development were recorded after 48 h and 7–9 d of IVC, respectively.
Blood samples and hormonal assays
In trial 1 and 2, the serum was sampled from the lambs every day from Day 0 until the day of oocyte retrieval. The serum progesterone (P4) and estradiol (E2) levels were measured by radioimmunoassay (RIA) using commercial RIA kits (Beijing North Institute of Biological Technology, China). The serum insulin-like growth factor-1 (IGF-1) levels were measured by Enzyme-linked immune sorbent assay (ELISA) (IGF-1 600 ELISA, DRG International, Inc., USA).
GC culture and treatment
The GCs derived from lamb ovaries without GH treatment were cultured in D-MEM/F12 (Gibco, Invitrogen Corporation, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). To verify the effects of GH and IGF-1, the GCs were cultured in D-MEM/F12 containing 2% FBS, and relevant indicators were assayed after GH or IGF-1 treatment.
Immediately after retrieval from the lamb ovaries, the oocytes (at germinal vesicle stage) were stained for 20 min at 38.5℃ with 1 mM dichlorofluorescein diacetate (DCFH-DA), 10 µM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (Cell-Tracker Blue) and 5 µM Flou-3 AM for detection of the levels of reactive oxygen species (ROS), glutathione (GSH) content, and cytosolic Ca2+, respectively. Oocytes stained with 200 nM Mito-tracker (Red) (Beyotime Institute of Biotechnology, China) or 10 µmol/L MitoProbe JC-1 (Beyotime Institute of Biotechnology) for detection of the mitochondrial distribution or mitochondrial membrane potential (MMP), respectively. The fluorescence was measured under an epifluorescence microscope (DP72, Olympus, Tokyo, Japan). For cells, the GSH content was measured by ELISA (GSH ELISA Kit, Zhenke Biotechnology Co., LTD, Shanghai, China). Cells were stained with Annexin V and propidium iodide (PI) for apoptosis analysis detected by flow cytometry.
The GCs were fixed with 4% paraformaldehyde (PFA) for 30 min at 4 °C, and then incubated with 1% PBST (1% Triton X 100 dissolved in PBS) for 30 min at room temperature. The samples were then blocked with blocking solution (Beyotime, P0260) and then incubated with the primary antibodies (Table S1) in blocking solution overnight at 4 °C, followed by incubation with secondary antibodies at 37 °C for 1.5 h. The nuclei of cells were stained with Hoechst33342 (Beyotime, C1022). Fluorescent images were captured using a fluorescent microscope (Olympus, BX51, Japan). The relative fluorescence intensity per unit area was determined using Image J software (National Institutes of Health, USA).
Ovaries were removed from lambs and fixed in 4% paraformaldehyde. After fixation, pieces of ovaries were embedded in paraffin, sectioned to approximately 5 μm, and mounted on glass slides. The ovarian sections were deparaffinized in xylene, rehydrated, and retrieved by microwave heating with a buffer of Citrate Antigen Retrieval Solution (Beyotime, P0081) for 20 min. Endogenous peroxidase activity was quenched by incubation with 3% H2O2 for 10 min. After blocking in 1% BSA for 1 h, sections were immunostained with anti-IGF-1 primary antibody (Table S1) and biotin-labeled secondary antibody.
Quantitative real-time-PCR (qRT-PCR)
Total RNA was extracted from cells using the TRNzol Reagent (Tiangen Biotech, Beijing, China) and was reverse transcribed using the FastQuant RT Kit (Tiangen Biotech) following the standard protocol. The qPCR reaction was performed using SuperReal PreMix Kit (Tiangen Biotech) on an ABI 7500 system (Applied Biosystems). The cycle threshold (CT) value of genes was obtained from three replicates. The expression level was normalized to that of the internal gene GAPDH. The primers used are presented in Table S2.
Lamb GCs cultured in 35-mm dishes were lysed with RIPA (Cwbio, Jiangsu, China) containing 1 mM PMSF and phosphatase inhibitor (Beyotime). Cell lysates were separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked with blocking solution (Beyotime) and incubated with primary antibodies (Table S1) overnight at 4℃, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 h at room temperature. The β-actin protein was used as a loading reference. The membranes were treated with super ECL Plus and the western blotting signals were detected by Tanon 5200 fully automatic chemiluminescence system. The intensities of blotting signals were quantified by outlining the relevant bands on the film with Image J software.
Transcriptomic analysis of GCs
After COCs were picked up from the aspiration of ovarian follicles of lambs, the remaining GCs were collected and the total RNA was extracted using TRIzol method (DP424, Tiangen, Beijing) according to the manufacturer’s instructions. The RNA was quantified using Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and the quality and integrity were assessed by NanoDrop spectrophotometer (IMPLEN, CA, USA). RNA-seq was performed with the PE150 sequencing strategy using an Illumina second-generation high-throughput sequencing platform. The adaptor sequences and low-quality sequence reads were removed from the raw data through In-House perl scripts. The clean reads were mapped to the reference genome sequence (https://sheephapmap.org/news/OARv2p0) by STAR. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome.
For analysis of the differentially expressed genes (DEGs), HTSeq v 0.5.4 p3 was used to count the reads numbers mapped to each gene, and the gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped (FPKM). Subsequently, differential expression analysis of two conditions/groups was performed using the DESeq R package (1.10.1). Transcripts with P-value < 0.05 and |log2FC| > 1 were considered differentially expressed. GO enrichment analysis and KEGG pathway enrichment analysis of the DEGs were implemented by the GOseq R packages and KOBAS software, respectively.
Proteome of ovaries
Ovaries were removed from lambs, rinsed with saline and stored at -80℃. The extraction of total proteins referred to the method of Yang et al. . A BCA Protein Assay Kit (Beijing Solarbio Science & Technology, Beijing, China) was used to measure protein concentration, and contamination and degradation of protein extracts were checked by SDS-PAGE. The proteins (100 µg each) were digested using the FASP method . High-performance liquid phase separation was carried out by Thermo UltiMate 3000 UHPLC. The peptides were separated by liquid phase, ionized by a nanoESI source, and then transferred to a tandem mass spectrometer Q-Exactive HF (Thermo Fisher Scientific, San Jose, CA) for DIA mode detection.
The obaDIA software was used for MS data analysis via the chromatogram library (obaDIA: one-step biological analysis pipeline for data-independent acquisition and other quantitative proteomics data). A peptide or protein FDR < 1% was set as the protein identification threshold. The differentially expressed proteins (DEPs) were identified using mapDIA. Proteins with a minimum of 2-fold change (|log2(FC)|>1) and false discovery rate (FDR) < 0.05 were identified as DEPs. The GOseq R package (v1.12) and KEGG Orthology Based Annotation System software (KOBAS, v2.0) were used to determine the DEPs in GO and KEGG pathways, respectively. A corrected P-value < 0.05 was considered as the significant cutoff value.
FF metabolite profiles
FF was collected by aspiration of follicles of lamb ovaries and centrifuged for removing any cell debris. Metabolite samples were prepared following the method of Want et al. . LC-MS/MS metabolites were measured by SCIEX UPLC system (Exion LC, SCIEX, Concord, New Hampshire) and quadrupole-time-of-flight mass spectrometer (QTOF MS; Triple TOF 5600+, SCIEX, USA). Metabolites were identified by automatically comparing retention times, ion signatures, and tandem mass spectrometry fragmentation patterns. To refine the analysis of the results, we obtained the first principal component of variable importance in the projection (VIP). Differential metabolites (DMs) were screened by VIP ≥ 1 and |Log2(FC)|>1. KEGG (http://www.kegg.jp) and MetaboAnalyst databases (http://www.metaboanalyst.ca/) were used to search for metabolite pathways.
Statistical analysis was performed using the SPSS 24 Software. The numbers of recovered oocytes among groups were compared by Mann-Whitney U-test. The levels of serum hormones (IGF-1, P4, E2), the rates of cleavage and blastocysts, and the data in cellular experiments were statistically analyzed by Student’s two-tailed t-test. The fluorescence intensities of stained oocytes were analyzed by One-way ANOVA. All data were expressed as mean value ± standard error of the mean (SEM). Differences were considered significant at three levels (*P<0.05; **P<0.01; ***P<0.001).
Effects of GH treatment on development of lamb oocytes
Trial 1 examined the effects of two dosages in long-GH treatment on the lamb oocyte development. Table 1 shows the response of lambs to superovulation and the oocyte recovery from individual lambs. Although there was no significant difference in average oocyte recovery among three groups of lambs, 50 mg GH treatment (5 × 10 mg GH) had a trend of increasing the number of responding lambs and the average number of oocytes recovered from per lamb. After fertilization of the recovered oocytes, no significant differences in cleavage and blastocyst rates were found between GH treatment and control groups, but significantly higher percentage of fast-developing blastocysts (appeared in D7 and D8) was observed in the 50 mg GH group (34.2%) compared to the 25 mg GH group (22.6%) and control group (22.1%) (Table 2). Assays for serum hormones showed that 50 mg GH treatment significantly increased the circulating IGF-1 concentrations in lambs (Fig. 2a). The levels of serum P4 and E2 were also increased to a degree in the 50 GH treatment group, albeit not significantly different from the control (Fig. 2b and c). These results suggest that long treatment of lambs with 50 mg GH had a beneficial role on lamb oocyte development.
Results from trial 1 suggested a positive role of GH applied as 5 × 10 mg. In trial 2, the efficacy of short-GH treatment of lambs was determined, which 2 possible parameters, 50 mg GH in total or 10 mg GH in each injection, were taken into consideration. As shown in Tables 3, 50 mg GH treatment (2 × 25 mg GH) significantly increased the average number of recovered oocytes as compared to the control and 20 mg groups (2 × 10 mg GH). Notably, in this trial, only 2 of 6 lambs in the control group responded to the FSH stimulation, while all of lambs in 50 mg GH group had responses to the FSH stimulation. However, no significant differences were found in cleavage and blastocyst development among three groups (Table 4). Similar to that in trial 1, injection of 50 mg GH resulted in significant rise of serum IGF-1 concentrations in lambs (Fig. 2d). The levels of P4 and E2 were also slightly, albeit not significantly, elevated in 50 mg GH treatment (Fig. 2e and f). These results suggest that short treatment of lambs with 50 mg GH also had a potential of improving the JIVET efficiency.
Comparison of long- and short- GH treatment
The above results indicated that 50 mg GH could achieve a higher efficiency in terms of lamb response and embryo yield in either long or short treatment. In trial 3, we compared the effects of long and short treatment, both of which applied 50 mg GH in total. In this trial, although all three groups of lambs, including the control group, had satisfactory responses to FSH stimulation (Table 5), the blastocyst development in both GH treatment groups was significantly enhanced compared with that of the control group (Table 6). This result supports again that 50 mg GH treatment could improve the developmental ability of lamb oocytes.
Cytoplasmic evaluation of oocytes
To further understand the influence of GH treatment of lambs on oocyte quality, several aspects of oocyte cytoplasm were detected in trial 3. As shown in Fig. 3, GH treatment significantly increased the MMP (Fig. 3a, b) and the mitochondrial mass (Fig. 3c, d). The levels of ROS and cytosolic Ca2+ in oocytes were significantly decreased in long treatment group compared to the control group, and the GSH content was significantly higher in the two GH treatment groups than that of the control group (Fig. 3e, f-h). These results suggest that GH treatment could improve the mitochondrial function and reduce the oxidant stress (OS) of lamb oocytes.
Transcriptome analysis of GCs
To further explore the underlying mechanism regarding how GH treatment improved the ovarian response and the quality of lamb oocytes, we analyzed the transcriptome profiles of GCs from long-GH treatment and control by RNA sequencing (RNA-seq). A total of 761 DEGs were identified between GH-treatment and control group (Fig. 4a and Supplementary additional file 1). The RNA-seq data were further verified by qRT-PCR with randomly selected genes (Fig. 4b).
Next, we conducted GO term and KEGG enrichment analysis for the DEGs. The GO enrichment analysis revealed that many DEGs were implicated in the regulation of cell proliferation, ROS metabolic process and negative regulation of cell death (Fig. 4c). The KEGG enrichment analysis indicated that several pathways, such as the PI3K/Akt and JAK/STAT signaling pathways, were activated in GCs from GH-treated lambs (Fig. 4d). The above results suggested that GH probably had a role in promoting the GC proliferation, inhibiting the GC apoptosis and reducing OS, and GH might function through activating the PI3K/Akt signaling pathway.
Proteome analysis of the ovaries
When the proteome of ovaries was analyzed, a total of 88 DEPs were found to be significantly different between control and GH treatment groups (Fig. 5a and Supplementary additional file 2). Some of the DEPs were further verified by qRT-PCR with randomly selected proteins (Fig. 5b). GO term and KEGG enrichment analysis were used for exploring the possible function of DEPs. The enriched GO terms included the mitochondrion inner membrane, glutathione peroxidase activity, regulation of NADP metabolic process and NADPH regeneration and so on (Fig. 5c). Similar to the results of transcriptome KEGG enrichment, the proteome analysis also showed that the PI3K/Akt signaling pathway was enriched (Fig. 5d).
Metabolome analysis of the follicular fluid
The metabolome of follicular fluid was analyzed to understand the microenvironment for oocyte growth and maturation. A total of 39 DMs were identified in the follicular fluid between control and GH treatment groups, and all the DMs were classified into 6 taxonomies (Fig. 5e and Supplementary additional file 3). In DMs, N-Acetyl-5-Hydroxytryptamine (NAS), the precursor of melatonin, was significantly increased in the GH treatment group compared with the control group. Meanwhile, bile acids (BAs) and carnitine in GH treatment group were significantly higher than that in the control group. The KEGG pathway enrichment analysis of the DMs showed that the enriched pathways mainly included amino acid metabolism, mitochondrial energy metabolism, lipid and cholesterol metabolism pathways (Fig. 5f).
GH treatment improved the proliferation of lamb GCs
Based on the transcriptome data, we investigated the effect of GH on GCs cultured in vitro. We first determined the optimal GH treatment duration for GCs and found that treatment of cells with GH for 12 h significantly increased the IGF-1 mRNA expression, demonstrating the effectiveness of the treatment (Fig. S1).
We next examined the role of GH on cell growth. EdU staining showed that the GC proliferation was significantly increased following GH treatment (Fig. 6a). Furthermore, qRT-PCR showed that GH treatment decreased the CDKN1B expression, and increased the expression of CDK2, CDK4, C-MYC (Fig. 6b). While, the protein and mRNA levels of apoptosis-related genes were significantly decreased in the GH-treated GCs compared with that in the control (Fig. 6c, d). Flow cytometry analysis by staining with Annexin V/PI revealed that the percentages of early/late apoptotic GCs were decreased by GH treatment and the numbers of viable cells were significantly increased (Fig. 6e, f). These results demonstrated that GH treatment could promote the proliferation of GCs and inhibit their apoptosis.
GH treatment reduced the OS of lamb GCs
As transcriptome and proteome analysis had shown that GH treatment might reduce OS in lamb GCs. We tested the related index of redox state in lamb GCs treated with GH. The result showed that after GH treatment of the cells for 12 h, the GSH content (Fig. 7a, b) and MMP (Fig. 7c, d) of lamb GCs were significantly increased, while the ROS level (Fig. 7e, f) was significantly decreased. These results indicated that GH-promoted GC proliferation may be mediated by reducing OS.
IGF-1 treatment improved the proliferation of lamb GCs
It is well known that GH promotes the expression of liver IGF-1, which plays a critical role on cell growth. To explore the function of IGF-1 in GH-promoted GC proliferation, the expression level of IGF-1 in ovarian cells was detected by immunohistochemistry. The result showed that the expression of IGF-1 was greatly enhanced in GCs within ovaries of GH-treated lambs (Fig. 8a). We further examined the role of IGF-1 on in vitro cultured GCs, and found that IGF-1 treatment of the GCs significantly increased the expression of CDK2, CDK4, and C-MYC, but decreased the expression of CDKN1B, BAX/BCL-2 and CASP3 (Fig. 8b, c). These results supported a direct role of IGF-1 on GCs.
GH functioned via the PI3K/Akt pathway
The KEGG pathway analysis of transcriptome and proteome indicated that GH might promote lamb GC proliferation through the PI3K/ Akt signaling pathway. In addition, the JAK/STAT signaling pathway, the classical pathway that GH stimulates IGF-1 production, was enriched in transcriptome KEGG analysis. To investigate the regulating mechanism of the PI3K/Akt signaling pathway in lamb GCs treated with GH, we detected the expression of related genes in the PI3K/Akt pathway. The qRT-PCR results showed that the expression of AKT1, AKT2, AKT3, PIK3R3 and PIK3CD was significantly increased in GH-treated GCs (Fig. 9a). Western blotting confirmed that the p-Akt (Ser473)/Akt ratio and p-STAT5 (Tyr694)/STAT5 ratio were significantly increased after GH treatment (Fig. 9b, c). Therefore, the PI3K/ Akt and JAK/STAT signaling pathways had been activated by GH treatment.
In the present study, we have explored the possibility of GH treatment on improvement of the JIVET efficiency. We showed that treatment of lambs with GH could improve the responsiveness of lambs to FSH stimulation, increase the number of recovered oocytes and enhance the development capacity of oocytes.
Many previous studies have suggested that GH could improve the reproductive performance in mammals [27, 28]. In superovulated ewes, although the regimen and dosage were different, GH treatment allowed the enhancement of the pool of gonadotrophin-responsive follicles present in the ovaries , increased the rates of fertilization and blastocyst development , and enhanced the proportion of transferable embryos . In estrus-synchronized ewes, giving 50 mg GH at the start of synchronization treatment and at the time of artificial insemination increased the proportion of multiple births and prolificacy [20, 21]. Similar effects of GH were also found in cattle; treatment of cows with 500 mg GH at the time of estrus and 10 d later improved the conception rate . However, it remains unknown whether GH treatment can benefit the development of prepubertal follicles and oocytes. In the present study, we demonstrated that administration of GH in lambs had a potential of promoting the development of follicles, as reflected by increasing oocyte and embryo yield in the JIVET program.
As mentioned above, one or two doses of GH are usually given in ewes or cows in estrus synchronization or superovulation [19, 21, 30]. However, when GH is applied in human assisted reproduction, the patients usually receive GH injection every day until ovulation, no matter in long-term or short-term procedures [31,32,33,34]. In the present study, we found that long-term GH treatment was more effective than short-term treatment when applied to improve the development of lamb follicles and oocytes, even though the short-term GH treatment also had a positive role. A major problem regarding in vitro embryo production from prepubertal animals is the high variation in ovarian response to FSH stimulation and oocyte development between individuals [35, 36]. This is also confirmed by our study. Lambs in the control group were quite variable in superovulation, as shown that a proportion of lambs hardly responded to FSH stimulation. In contrast, GH treatment, especially for the long-term treatment with 50 mg GH, was constantly effective in promoting all of treated lambs to respond to superovulation. Interestingly, GH treatment seemed not to increase the average oocyte recovery if compared with the oocyte recovery from the responding lambs in the control group. This may imply that the “inactive” ovaries were more likely dependent on the action of GH to become FSH-responsive, but the underlying mechanism requires further investigation.
We also found that GH treatment improved the development ability of lamb oocytes. Embryos derived from GH-treated lambs, especially in the 50 mg GH treatment group, had more potential to develop into high-quality blastocysts (e.g., blastocysts formation at day7 and day8). This suggests that GH had exerted a beneficial role on the growing oocytes within antral follicles. Several previous studies have described the positive effects of GH on oocyte quality. In humans, GH exerts a direct action, enabling the improvement of oocyte quality, via upregulation of its own receptors and enhancement of mitochondrial activity . In mice, GH treatment of aged mice before standard ovarian stimulation could enhance the mitochondrial function of oocytes . Similar to these observations, we found that the mitochondrial function of lamb oocytes could be improved by GH treatment, as evidenced by increased MMP and mitochondrial mass. It is known that mitochondrial dysfunction is a cause of ROS accumulation and OS generation . In our study, GH treatment decreased the levels of ROS, and increased the GSH content in lamb oocytes, suggesting that the resistance to OS might have been enhanced in the oocytes. We also observed a decrease of cytosolic Ca2+ in oocytes from GH-treated lambs, which would facilitate modulating the Ca2+ level in a physiological range . These observations support that the cytoplasmic maturation of oocytes had been improved by GH treatment of the animals.
To further explore the mechanism that GH treatment improves the ovarian responsiveness and promotes the follicle and oocyte development in lambs, we analyzed the GC transcriptome and ovarian proteome. The transcriptome GO-term analysis suggested that GH treatment probably improved the GC survival and growth, as lots of DEGs were enriched in regulation of cell proliferation, positive regulation of cellular process and negative regulation of cell death. Previous studies on transcriptome and proteome of ovarian cells have suggested that many DEGs or DEPs found between adult ewes and prepubertal lambs were involved in cell proliferation and apoptosis [8, 41, 42]. We also found that the ROS metabolism term enriched in transcriptome interrelates well with the results in proteomic analysis, such as mitochondrial and NADP metabolism. Importantly, the PI3K/Akt signaling pathway was enriched in both transcriptome and proteome KEGG analyses.
Based on the above results, we treated the lamb GCs with GH in vitro. We showed that GH treatment could promote the proliferation of GCs probably by increasing the resistance to OS and improving the mitochondrial function. We also confirmed in cellular experiments that GH exerted a role on GCs by activating the PI3K/Akt signaling pathway. It was reported that GH could activate the PI3K/Akt signaling pathway to alleviate GC apoptosis in PCOS patients . Taken together the omics analysis with cellular validation reveal that GH treatment had altered the functions of ovarian cells in several aspects, particularly in enhancing the cell proliferation and reducing the cell apoptosis, which would consequently promote the follicle and oocyte development.
We noticed an increase of IGF-1 levels in serum and GCs in GH-treated lambs, supporting the function of GH in promoting the synthesis of IGF-1 in vivo [17, 21]. We found that IGF-1 indeed regulated the expression of genes related to cell proliferation and apoptosis in GCs cultured in vitro. The above analysis suggests that GH may directly affect the ovaries and GCs or be mediated by IGF-1. In this respect, the actions of GH and IGF1 in ovarian folliculogenesis have been well documented .
The influence of GH on ovaries was also reflected by alterations in follicular fluid metabolome. Follicular fluid, composed of secretions from the GCs and blood, provides a microenvironment for oocyte development and plays an essential role in follicular growth, oocyte maturation and ovulation [44,45,46]. Difference in metabolomic composition of follicular fluid was found between adult and prepubertal goats . In this study, we observed that NAS is the most increased molecule in FF from GH-treated lambs. NAS has antioxidant and anti-apoptotic roles in OS-induced neurotoxicity by activating the TrkB/CREB/BDNF pathway and promoting the expression of antioxidant enzymes . This may enhance the resistance to high OS in lamb follicles, as one-month-old lambs have a low abundance of superoxide dismutase (SOD) after superovulation . In addition, the metabolism of amino acid, carnitine and BAs was very active in FF from the GH treatment group. Among them, carnitine, as a substance involved in fatty acid metabolism, may play an important role in the energy production of granulosa cells and oocytes . Several studies have suggested that BAs in bovine and human FF may promote the development of oocytes [51,52,53,54]. Therefore, GH treatment had altered the FF metabolism that may benefit the development of follicles and oocytes. The real action of these metabolites needs to be further determined.
In this study, we showed that GH treatment of lambs could potentially improve the responsiveness of lamb ovaries to FSH superstimulation, increase the number of recovered oocytes and enhance the developmental capacity of oocytes. The underlying mechanism was further explored by analysis of GC transcriptome, ovarian proteome and FF metabolome. We demonstrate that GH treatment could promote the GC proliferation by reducing OS through PI3K/Akt signaling pathway. These results suggest that GH treatment can affect the ovarian activity that benefits the development of follicles and oocytes. Our study provides a new strategy of improving the lamb superovulation and JIVET efficiency.
The datasets supporting the conclusions of this article are available from the corresponding author upon reasonable request.
In vitro production
Juvenile in vitro embryo transfer
Growth hormone receptor
Equine chorionic gonadotropin
In vitro maturation
In vitro fertilization
In vitro culture
Estrus sheep serum
Synthetic oviduct fluid
Insulin-like growth factor-1
Enzyme-linked immune sorbent assay
Fetal bovine serum
Reactive oxygen species
Mitochondrial membrane potential
SDS polyacrylamide gel electrophoresis
Differentially expressed genes
Differentially expressed proteins
False discovery rate
Armstrong DT, Kotaras PJ, Earl CR. Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb. Reprod Fertil Dev. 1997;9(3):333–9.
Ptak G, Tischner M, Bernabo N, Loi P. Donor-dependent developmental competence of oocytes from lambs subjected to repeated hormonal stimulation. Biol Reprod. 2003;69(1):278–85.
Valasi I, Leontides L, Papanikolaou T, Amiridis GS, Age. FSH dose and follicular aspiration frequency affect oocyte yield from juvenile donor lambs. Reprod Domest Anim. 2007;42(3):230–7.
Ledda S, Bogliolo L, Calvia P, Leoni G, Naitana S. Meiotic progression and developmental competence of oocytes collected from juvenile and adult ewes. J Reprod Fertil. 1997;109(1):73–8.
O’Brien JK, Catt SL, Ireland KA, Maxwell WM, Evans G. In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep. Theriogenology. 1997;47(7):1433–43.
Ptak G, Loi P, Dattena M, Tischner M, Cappai P. Offspring from one-month-old lambs: studies on the developmental capability of prepubertal oocytes. Biol Reprod. 1999;61(6):1568–74.
Morton KM, Catt SL, Maxwell WM, Evans G. In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes. Theriogenology. 2005;64(6):1320–32.
Tian H, Ren P, Liu K, Qiu C, Fan L, Li J, Hou J. Transcriptomic comparison of ovarian granulosa cells between adult sheep and prepubertal lambs. BMC Genomics. 2022;23(1):151.
Tian H, Liu KX, Zhang YM, Qi Q, Wang CX, Guan H, Yan FX, Hou J. Adult follicular fluid supplementation during in vitro maturation improves the developmental competence of prepubertal lamb oocytes. Theriogenology. 2019;130:157–62.
Tian H, Qi Q, Yan F, Wang C, Hou F, Ren W, Zhang L, Hou J. 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. Theriogenology. 2021;159:13–9.
Pennetier S, Perreau C, Uzbekova S, Thélie A, Delaleu B, Mermillod P, Dalbiès-Tran R. MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine. BMC Dev Biol. 2006;6:26.
Spiliotis BE. Growth hormone insufficiency and its impact on ovarian function. Ann N Y Acad Sci. 2003;997:77–84.
Chang CW, Sung YW, Hsueh YW, Chen YY, Ho M, Hsu HC, Yang TC, Lin WC, Chang HM. Growth hormone in fertility and infertility: mechanisms of action and clinical applications. Front Endocrinol (Lausanne). 2022;1313:1040503.
Homburg R, Eshel A, Abdalla HI, Jacobs HS. Growth hormone facilitatesovulation induction by gonadotrophins. Clin Endocrinol (Oxf). 1988;29(1):113–7.
Li XL, Wang L, Lv F, Huang XM, Wang LP, Pan Y, Zhang XM. The influence of different growth hormone addition protocols to poor ovarian responders on clinical outcomes in controlled ovary stimulation cycles: a systematic review and meta-analysis. Med (Baltim). 2017;96(12):e6443.
Safdarian L, Aghahosseini M, Alyasin A, Samaei Nouroozi A, Rashidi S, Shabani Nashtaei M, Najafian A, Lak P. Growth hormone (GH) improvement of ovarian responses and pregnancy outcome in poor ovarian responders: a randomized study. Asian Pac J Cancer Prev. 2019;20(7):2033–7.
Carrera-Chavez JM, Hernandez-Ceron J, Lopez-Carlos MA, Lozano-Dominguez RR, Molinar F, Echavarria-Chairez FG, Banuelos-Valenzuela R, Arechiga-Flores CF. Superovulatory response and embryo development in ewes treated with two doses of bovine somatotropin. Anim Reprod Sci. 2014;151(3–4):105–11.
Joyce IM, Khalid M, Haresign W. The effect of recombinant GH treatment on ovarian growth and atresia in sheep. Theriogenology. 2000;54(2):327–38.
Folch J, Ramón JP, Cocero MJ, Alabart JL, Beckers JF. Exogenous growth hormone improves the number of transferable embryos in superovulated ewes. Theriogenology. 2001;55(9):1777–85.
Carrillo F, Hernández-Cerón J, Orozco V, Hernández JA, Gutiérrez CG. A single dose of bovine somatotropin 5 days before the end of progestin-based estrous synchronization increases prolificacy in sheep. Anim Reprod Sci. 2007;102(1–2):31–7.
Montero-Pardo A, Hernandez-Ceron J, Rojas-Maya S, Valencia J, Rodriguez-Cortez A, Gutierrez CG. Increased cleavage and blastocyst rate in ewes treated with bovine somatotropin 5 days before the end of progestin-based estrous synchronization. Anim Reprod Sci. 2011;125(1–4):69–73.
Mejia O, Palma-Irizarry M, Rosas J, Madrid-Marina V, Valencia MJ, Zarco L. Administration of recombinant bovine somatotropin (rbST) at the time of breeding in superovulated fertile and subfertile ewes. Small Ruminant Res. 2012;102(1):51–6.
Liu K, Li J, Liu M, Hou J. Molecular chaperone GroEL-GroES enhances the soluble expression of biologically active ovine growth hormone in the prokaryotic system. Protein Expr Purif. 2022:106097.
Yang Y, Liu X, Shen C, Lin Y, Yang P, Qiao L. In silico spectral libraries by deep learning facilitate data-independent acquisition proteomics. Nat Commun. 2020;11(1):146.
Wiśniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6(5):359–62.
Want EJ, Masson P, Michopoulos F, Wilson ID, Theodoridis G, Plumb RS, Shockcor J, Loftus N, Holmes E, Nicholson JK. Global metabolic profiling of animal and human tissues via UPLC-MS. Nat Protoc. 2013;8(1):17–32.
Hull KL, Harvey S. Growth hormone and reproduction: a review of endocrine and autocrine/paracrine interactions. Int J Endocrinol. 2014;2014:234014.
Silva JR, Figueiredo JR, van den Hurk R. Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis. Theriogenology. 2009;71(8):1193–208.
Gonzalez-Añover P, Encinas T, Garcia-Garcia RM, Veiga-Lopez A, Cocero MJ, McNeilly AS, Gonzalez-Bulnes A. Ovarian response in sheep superovulated after pretreatment with growth hormone and GnRH antagonists is weakened by failures in oocyte maturation. Zygote. 2004;12(4):301–4.
Morales-Roura JS, Zarco L, Hernández-Cerón J, Rodríguez G. Effect of short-term treatment with bovine somatotropin at estrus on conception rate and luteal function of repeat-breeding dairy cows. Theriogenology. 2001;55(9):1831–41.
Norman RJ, Alvino H, Hull LM, Mol BW, Hart RJ, Kelly T-L, Rombauts L. investigators L. Human growth hormone for poor responders: a randomized placebo-controlled trial provides no evidence for improved live birth rate. Reprod Biomed Online. 2019; 38(6):908–915.
Regan SLP, Knight PG, Yovich JL, Arfuso F, Dharmarajan A. Growth hormone during in vitro fertilization in older women modulates the density of receptors in granulosa cells, with improved pregnancy outcomes. Fertil Steril. 2018;110(7):1298–310.
Ob’edkova K, Kogan I, Krikheli I, Dzhemlikhanova L, Muller V, Mekina I, Lesik E, Komarova E, Mazilina M, Niauri D, et al. Growth hormone co-treatment in IVF/ICSI cycles in poor responders. Gynecol Endocrinol. 2017;33(sup1):15–7.
Altmäe S, Mendoza-Tesarik R, Mendoza C, Mendoza N, Cucinelli F, Tesarik J. Effect of growth hormone on uterine receptivity in women with repeated implantation failure in an oocyte donation program. A Randomized Controlled Trial. J Endocr Soc. 2017;2(1):96–105.
Paramio MT, Izquierdo D. Current status of in vitro embryo production in sheep and goats. Reprod Domest Anim. 2014;49(Suppl 4):37–48.
Morton KM. Developmental capabilities of embryos produced in vitro from prepubertal lamb oocytes. Reprod Domest Anim. 2008;43(Suppl 2):137–43.
Weall BM, Al-Samerria S, Conceicao J, Yovich JL, Almahbobi G. A direct action for GH in improvement of oocyte quality in poor-responder patients. Reprod (Cambridge England). 2015;149(2):147–54.
Hou HY, Wang X, Yu Q, Li HY, Li SJ, Tang RY, Guo ZX, Chen YQ, Hu CX, Yang ZJ, et al. Evidence that growth hormone can improve mitochondrial function in oocytes from aged mice. Reprod (Cambridge England). 2018;157(4):345–58.
Miao Y, Cui Z, Gao Q, Rui R, Xiong B. Nicotinamide Mononucleotide supplementation reverses the declining quality of maternally aged oocytes. Cell Rep. 2020;32(5):107987.
Tiwari M, Prasad S, Shrivastav TG, Chaube SK. Calcium signaling during meiotic cell cycle regulation and apoptosis in mammalian oocytes. J Cell Physiol. 2017;232(5):976–81.
Wu Y, Lin J, Li X, Han B, Wang L, Liu M, Huang J. Transcriptome profile of one-month-old lambs’ granulosa cells after superstimulation. Asian Australas J Anim Sci. 2017;30(1):20–33.
Lin J, Wu Y, Han B, Chen Y, Wang L, Li X, Liu M, Huang J. iTRAQ-based proteomic profiling of granulosa cells from lamb and ewe after superstimulation. Theriogenology. 2017;101:99–108.
Gong Y, Luo S, Fan P, Zhu H, Li Y, Huang W. Growth hormone activates PI3K/Akt signaling and inhibits ROS accumulation and apoptosis in granulosa cells of patients with polycystic ovary syndrome. Reprod Biol Endocrinol. 2020;18(1):121.
O’Gorman A, Wallace M, Cottell E, Gibney MJ, McAuliffe FM, Wingfield M, Brennan L. Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence. Reprod (Cambridge England). 2013;146(4):389–95.
Revelli A, Delle Piane L, Casano S, Molinari E, Massobrio M, Rinaudo P. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol. 2009;7:40.
Nandi S, Kumar VG, Manjunatha BM, Gupta PS. Biochemical composition of ovine follicular fluid in relation to follicle size. Dev Growth Differ. 2007;49(1):61–6.
Izquierdo D, Roura M, Perez-Trujillo M, Soto-Heras S, Paramio MT. Fatty acids and metabolomic composition of follicular fluid collected from environments associated with good and poor oocyte competence in goats. Int J Mol Sci. 2022, 23(8).
Yoo JM, Lee BD, Sok DE, Ma JY, Kim MR. Neuroprotective action of N-acetyl serotonin in oxidative stress-induced apoptosis through the activation of both TrkB/CREB/BDNF pathway and Akt/Nrf2/Antioxidant enzyme in neuronal cells. Redox Biol. 2017;11:592–9.
Wu Y, Lin J, Han B, Wang L, Chen Y, Liu M, Huang J. Proteomic profiling of follicle fluids after superstimulation in one-month-old lambs. Reprod Domest Anim. 2018;53(1):186–94.
Reader KL, Cox NR, Stanton JA, Juengel JL. Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate, ultrastructure, and mitochondrial DNA copy number. Theriogenology. 2015;83(9):1484–92.
Smith LP, Nierstenhoefer M, Yoo SW, Penzias AS, Tobiasch E, Usheva A. The bile acid synthesis pathway is present and functional in the human ovary. PLoS ONE. 2009;4(10):e7333.
Nagy RA, Hollema H, Andrei D, Jurdzinski A, Kuipers F, Hoek A, Tietge UJF. The origin of follicular bile acids in the human ovary. Am J Pathol. 2019;189(10):2036–45.
Blaschka C, Sánchez-Guijo A, Wudy SA, Wrenzycki C. Profile of bile acid subspecies is similar in blood and follicular fluid of cattle. Vet Med Sci. 2020;6(2):167–76.
Sánchez-Guijo A, Blaschka C, Hartmann MF, Wrenzycki C, Wudy SA. Profiling of bile acids in bovine follicular fluid by fused-core-LC-MS/MS. J Steroid Mol Biol. 2016;162:117–25.
We thank the members of our lab and staffs in the farm for their assistance in the study.
This research was supported by the earmarked fund for China Agriculture Research System (grant number CARS-39-04) and Bijie Science and Technology Major Project (No.2, 2021).
Ethics approval and consent to participate
The procedure of all animal experiments was in accordance with the animal care policies of China Agricultural University and was approved by the Animal Ethics Committee at the university (protocol code: AW20701202-3-4, date of approval: 1 June 2020).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
. DEGs in GC transcriptome between control and GH treatment groups.
. DEPs in ovarian proteome between control and GH treatment groups.
. DMs in follicular fluid metabolome between control and GH treatment groups.
. Optimize the time for GH treatment of GCs; Table S1. The information of the primary antibodies; Table S2. Primers for quantitative real-time PCR.
About this article
Cite this article
Liu, K., Zhang, L., Qi, Q. et al. Growth hormone treatment improves the development of follicles and oocytes in prepubertal lambs. J Ovarian Res 16, 132 (2023). https://doi.org/10.1186/s13048-023-01209-y
- Growth hormone
- Prepubertal lamb
- Granulosa cells
- Follicular fluid