Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells.

Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 µg/ml) and (14.80 ± 0.13 µg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.

Is it possible to replace eCG by GnRH in the hormonal protocol for timed artificial insemination in ewes during the breeding season?

Hormonal protocols based on progestogens and equine chorionic gonadotrophin (eCG) are efficient for estrus and ovulation synchronization in ewes. Although eCG is indispensable during seasonal anestrus, it may not be necessary during the breeding season. Thus, we tested the hypothesis that GnRH is effective in replacing eCG during the breeding season allowing satisfactory ovulation rate, luteal function and conception rates after timed artificial insemination (TAI). Ewes (n = 134) with a minimum body condition score of 2.5 (0-5 scale) were treated with intravaginal devices (IVD) containing 60 mg of medroxyprogesterone acetate (MPA) for seven days and received 0.26 mg of sodium cloprostenol at the time of IVD removal. In Exp. 1, at IVD removal, ewes (n = 29) were allocated to three groups: eCG (200 IU at IVD removal; n = 10); eCG+GnRH (200 IU eCG at IVD removal and 4 µg of buserelin 36 h later; n = 10); or GnRH (buserelin 36 h after IVD removal; n = 9). Blood samples were collected 2, 6 and 12 days after TAI moment (54 h after IVD removal), for progesterone (P4) analysis. In Exp 2, the ewes were allocated to eCG (n = 10) or GnRH (n = 10) groups, as above described, and ovulation moment was evaluated 54, 66 and 78 h after IVD removal. In Exp 3, TAI was performed in ewes from eCG (n = 45) and GnRH (n = 40) groups using 100 × 106 motile spermatozoa from a pool of semen collected from four rams. In Exp. 1, based on P4 levels, we confirmed that all the ewes ovulated (29/29) and there was no significant effect of group (P = 0.89) or group x day (P = 0.18) on P4 concentration, being observed a significant effect of day (P = 0.0001). In Exp. 2, the maximum DF diameter (P = 0.26) and ovulation moment (P = 0.69) did not differ between groups. In Exp. 3, pregnancy rate was significantly lower (P = 0.02) in GnRH (22.5 %; 9/40) compared to eCG (46.7 %; 21/45). The results indicate that, although ovulation and luteal function were not altered after eCG, eCG+GnRH or GnRH treatment, GnRH alone before TAI cannot be used to replace eCG treatment during the breeding season.

Morphometric Evaluation of the Ampullae of the Oviducts in Prepubertal Gilts Treated With Chorionic Gonadotropins.

This study assessed morphometric traits of the ampulla of the oviducts in prepubertal gilts treated with chorionic gonadotropins. With the day of slaughter as D0, gilts were assigned to four treatments (n = 8 each): control (untreated), eCG (200 IU eCG on D3), eCG+hCG (1200 IU eCG on D6 plus 500 IU hCG on D3), and eCG+hCG+AI (the previous treatment plus artificial insemination on D1). Blood and ampullae samples were collected at slaughter. Serum progesterone concentrations were higher for gilts treated with hCG than for those in the eCG and control treatments (p < 0.001), but estradiol concentrations did not differ (p > 0.05). The epithelium, muscle and lumen areas and the inner and larger ampullae diameters did not differ across treatments (p > 0.05). Therefore, treatment with chorionic gonadotropins did not alter the ampullae morphometry of prepubertal gilts.

Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells.

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) ß-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG ß-subunit CTP region (amino acids 115-149) was inserted between the ß-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC50 value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG ß-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

The use of a recombinant equine chorionic gonadotropin (reCG) in fixed-time AI programs in beef cattle.

The aim of the current study was to evaluate the effect of a novel recombinant eCG (reCG) on pregnancy rates to AI (P/AI) in suckled beef cows of different breeds that were synchronized with an estradiol/progesterone (P4)-based protocol for fixed-time AI (TAI). In experiment 1, 1244 Bos taurus suckled cows were used. On Day 0 all cows received an intravaginal P4 device (600 mg P4) and 2 mg of estradiol benzoate. On Day 7, devices were removed, and all cows received 0.150 mg of D-cloprostenol plus 1 mg of estradiol cypionate and were randomly divided to receive 140 IU or 105 IU of reCG or no reCG treatment (controls) at that time. Cows were tail painted for estrus detection and those in estrus by 48 h after P4 device removal were inseminated; whereas those not showing estrus were also inseminated and received GnRH at the same time. In experiment 2, 818 Bos taurus x Bos indicus crossbred suckled cows received the same FTAI protocol used in Experiment 1. Cows were randomly divided at the time of P4 device removal into 4 groups to receive 140 IU, 105 IU or 84 IU of reCG or no reCG treatment. In experiment 3, 345 Bos indicus suckled cows were submitted to the same FTAI protocol as those in previous experiments and were randomly divided into three groups to receive 140 IU or 105 IU of reCG, or 300 IU of serum derived eCG (PMSG). In Experiment 1, estrus rate and P/AI was greater (P < 0.05) in cows treated with reCG (79.9 and 53.5 %, 76.9 and 52.3 % for the 105 UI and 140 UI reCG groups, respectively) than those in the control group (69.9 and 44.4 %, respectively). In Experiment 2, cows treated with reCG tended (P < 0.1) to achieve a greater P/AI than control cows (38.6 %, 37.1 %, 36.2 % and 28.2 % for those receiving 84 IU, 105 IU,140 IU of reCG, and those in the control group); but when P/AI of all cows treated with reCG was contrasted to that of control cows, the difference was significant (P < 0.01). In Experiment 3, P/AI in cows treated with 84 IU of reCG (54 %) did not differ from that of cows treated with serum derived eCG (59 %) but both were greater (P < 0.05) than cows treated with 105 UI of reCG (41 %). In conclusion, treatment with reCG improved fertility in suckled Bos taurus and Bos taurus x Bos indicus beef cows. In suckled Bos indicus cows, although treatment with reCG and serum derived eCG were comparable, the higher dosage of reCG was detrimental to their P/AI.

Effect of an equine chorionic gonadotrophin-like recombinant glycoprotein treatment on fertility in Angus cattle.

This study determined the effects of administering a glycoprotein with equine chorionic gonadotropin (eCG)-like activity (eCG-like) on corpus luteum (CL) area, serum progesterone concentrations, incidence of multiple ovulations (MOV), estrus expression rate (EER), and pregnancy to timed AI (P/TAI) in Angus cattle synchronized with a 5-d Co-Synch protocol. On Day -8, cattle were body condition scored (BCS), and received a 1.0 g progesterone intravaginal device (IVD) and 100 µg GnRH. On Day -3, the IVDs were removed and 500 µg cloprostenol was administered intramuscularly (i.m.). Cattle were randomly assigned into one of two groups: eCG-like (heifers, n = 232, primiparous, n = 148, and multiparous cows = 485; 300 IU (heifers) and 400 IU (cows) eCG-like i.m. on Day -3), or Control (heifers, n = 240, primiparous, n = 151, and multiparous cows, n = 478; no eCG-like). On Day -2, cattle received a second dose of 500 µg cloprostenol, and on Day 0, 100 µg GnRH was given concurrently with TAI. Estrus expression rate was assessed by observing the tail paint rubbed off in a subset of heifers (n = 372) and all cows on Day 0. Transrectal ultrasonography was used to evaluate the presence of CL on Day -8 and to diagnose P/TAI on Day 30-35. In a subset of cattle (heifers = 194 and multiparous cows = 87), CL area, serum progesterone concentrations, and incidence of MOV were evaluated on Day 7. Heifers, primiparous, and multiparous cows were analyzed separately. Treatment with eCG-like did not affect (P > 0.1) EER in heifers. Estrus expression rate was increased (P ≤ 0.03) in primiparous (68.9 % vs 45.0 %) and multiparous (75.5 % vs. 68.8 %) cows treated with eCG-like compared with Controls. Pregnancy/TAI was increased (P < 0.01) in heifers (65.2 % vs 48.3 %) and primiparous cows (48.3 % vs. 35.1 %) treated with eCG-like than Controls. In multiparous cows with a BCS ≤4 P/TAI was increased (P = 0.03) in the eCG-like group (47.7 %) than the Control group (34.8 %) but was similar (P > 0.1) between treatment groups in multiparous cows with a BCS ≥4.5. The eCG-like treatment increased (P < 0.05) CL area in heifers and multiparous cows and tended (P = 0.10) to elevate serum progesterone concentrations only in heifers. However, it did not affect (P > 0.1) the incidence of MOV in heifers and multiparous cows. Glycoprotein eCG-like administration increased fertility in heifers and primiparous cows, but in multiparous the effect of eCG-like on fertility was associated with BCS.

Associations among the largest follicle, preovulatory estradiol concentrations, and predominant vaginal epithelial cells at the completion of hormonal ovarian stimulation for fixed-time artificial insemination in goats.

The objective of the present study was to investigate the association among the largest follicle (LF), preovulatory estradiol (E2), and predominant vaginal epithelial cell at the completion of hormonal ovarian stimulation for fixed-time artificial insemination (FTAI) in goats. Thirty-seven crossbred Boer does received gonadotropin-releasing hormone (GnRH) and intravaginal progesterone (P4)-releasing devices (day 0). On day 5, P4 devices were removed and does received prostaglandin F2α and equine chorionic gonadotrophin. On day 7, does received GnRH, and FTAI was undertaken. On day 7, does were divided into three groups, i.e. small-sized (3-3.9 mm; n = 5), medium-sized (4-4.9 mm; n = 8), and large-sized (≥5 mm; n = 24) according to the diameter of the ovarian LF; follicular characteristics (number and diameter) were identified, and blood samples and vaginal smears were collected. The average diameters of total antral follicles and LF and the percentage of superficial cell were greatest in large-sized LF does (p < .01). The average diameters of total antral follicle (r = .68) and LF (r = .71), number of preovulatory follicle (r = .58), and plasma E2 concentrations (r = .61) were positively correlated with the percentage of superficial cells (p < .01). The likelihood of a pregnancy outcome after the FTAI increased by 13.71 times in does with a greater average diameter of antral follicle, 14.18 times with emergence of a large preovulatory follicle, and 36.83 times with a higher percentage of vaginal superficial cells (p < .01). It was concluded that there is a relationship between the cell types of the vaginal epithelium, the diameters of the largest ovarian follicles, and the concentration of E2 in goats subjected to FTAI protocols.

In vitro matured oocytes have a higher developmental potential than in vivo matured oocytes after hormonal ovarian stimulation in Callithrix jacchus.

BACKGROUND: The common marmoset, Callithrix jacchus, is an invaluable model in biomedical research. Its use includes genetic engineering applications, which require manipulations of oocytes and production of embryos in vitro. To maximize the recovery of oocytes suitable for embryo production and to fulfil the requirements of the 3R principles to the highest degree possible, optimization of ovarian stimulation protocols is crucial. Here, we compared the efficacy of two hormonal ovarian stimulation approaches: 1) stimulation of follicular growth with hFSH followed by triggering of oocyte maturation with hCG (FSH + hCG) and 2) stimulation with hFSH only (FSH-priming). METHODS: In total, 14 female marmosets were used as oocyte donors in this study. Each animal underwent up to four surgical interventions, with the first three performed as ovum pick-up (OPU) procedures and the last one being an ovariohysterectomy (OvH). In total, 20 experiments were carried out with FSH + hCG stimulation and 18 with FSH-priming. Efficacy of each stimulation protocol was assessed through in vitro maturation (IVM), in vitro fertilization (IVF) and embryo production rates. RESULTS: Each study group consisted of two subgroups: the in vivo matured oocytes and the oocytes that underwent IVM. Surprisingly, in the absence of hCG triggering some of the oocytes recovered were at the MII stage, moreover, their number was not significantly lower compared to FSH + hCG stimulation (2.8 vs. 3.9, respectively (ns)). While the IVM and IVF rates did not differ between the two stimulation groups, the IVF rates of in vivo matured oocytes were significantly lower compared to in vitro matured ones in both FSH-priming and FSH + hCG groups. In total, 1.7 eight-cell embryos/experiment (OPU) and 2.1 eight-cell embryos/experiment (OvH) were obtained after FSH + hCG stimulation vs. 1.8 eight-cell embryos/experiment (OPU) and 5.0 eight-cell embryos/experiment (OvH) following FSH-priming. These numbers include embryos obtained from both in vivo and in vitro matured oocytes. CONCLUSION: A significantly lower developmental competence of the in vivo matured oocytes renders triggering of the in vivo maturation with hCG as a part of the currently used FSH-stimulation protocol unnecessary. In actual numbers, between 1 and 7 blastocysts were obtained following each FSH-priming. In the absence of further studies, FSH-priming appears superior to FSH + hCG stimulation in the common marmoset under current experimental settings.

Equine chorionic gonadotropin (eCG) treatment in heifers: Double ovulation, twinning rate, and pregnancy losses in twin pregnancies.

This study aimed to evaluate the effects of different doses of equine chorionic gonadotropin (eCG; 200 and 300 IU) administered at the end of a fixed-time artificial insemination (FTAI) treatment protocol on ovulation, pregnancy, and twin rates in Bos taurus beef heifers. In addition, pregnancy losses in heifers with singleton and twin pregnancies were determined. A total of 2382 Angus heifers treated with a 6-day estradiol/progesterone-based protocol for FTAI (J-Synch protocol) were randomly allocated to two experimental groups to receive 200 or 300 IU of eCG administered intramuscularly at the time of intravaginal progesterone device removal; FTAI was performed from 60 to 72 h after device removal. The pregnancy rate did not differ (P = 0.89) between the 200 and 300 IU eCG groups. The number of corpus luteum induced by both eCG doses was determined by ultrasonographic examination 14 days after insemination and those treated with 300 IU of eCG had a greater double ovulation rate (P < 0.05). In addition, 300 IU eCG treated heifers had a higher twinning rate on day 30 of gestation (P < 0.05) and parturition (P < 0.05). Pregnancy losses from 30 days of gestation to calving did not differ between heifers treated with 200 and 300 IU of eCG (P = 0.70). However, regardless of the experimental group, heifers bearing twins had greater pregnancy losses than heifers with singletons (P < 0.05). In conclusion, reducing the dose of eCG from 300 to 200 IU under FTAI treatment protocol decreases double ovulation and twinning rates, maintaining a similar pregnancy rate in heifers. Nulliparous cows carrying two fetuses suffer greater pregnancy losses than cows with singletons.

Injection of luteinizing hormone or human chorionic gonadotropin increases calcium excretion and serum PTH in males.

Animal and human studies have suggested that sex steroids have calciotropic actions, and it has been proposed that follicle-stimulating hormone (FSH) may exert direct effects on bone. Here, we demonstrate the expression of the receptor for Luteinizing hormone (LH) and human choriogonadotropin (hCG), LHCGR, in human kidney tissue, suggesting a potential influence on calcium homeostasis. To investigate the role of LHCGR agonist on calcium homeostasis in vivo, we conducted studies in male mice and human subjects. Male mice were treated with luteinizing hormone (LH), and human extrapolation was achieved by injecting 5000 IU hCG once to healthy men or men with hypergonadotropic or hypogonadotropic hypogonadism. In mice, LH treatment significantly increased urinary calcium excretion and induced a secondary increase in serum parathyroid hormone (PTH). Similarly, hCG treatment in healthy men led to a significant increase in urinary calcium excretion, serum PTH levels, and 1,25 (OH)2D3, while calcitonin, and albumin levels were reduced, possibly to avoid development of persistent hypocalcemia. Still, the rapid initial decline in ionized calcium coincided with a significant prolongation of the cardiac QTc-interval that normalized over time. The observed effects may be attributed to LH/hCG-receptor (LHCGR) activation, considering the presence of LHCGR expression in human kidney tissue, and the increase in sex steroids occurred several hours after the changes in calcium homeostasis. Our translational study shed light on the intricate relationship between gonadotropins, sex hormones and calcium, suggesting that LHCGR may be influencing calcium homeostasis directly or indirectly.

Hormonal dependent expression of apelin and apelin receptor in the ovary and uterus of mice.

Apelin and APJ have been shown to regulate female reproductive functions. However, its uterine expression during the oestrous cycle and its regulation by ovarian steroids, along with gonadotropin regulation in the ovary, has not been investigated. This study aimed to analyze the steroid-dependent uterine expression of apelin/APJ in the uterus along with the oestrous cycle. Furthermore, it also aimed to investigate gonadotropin-dependent ovarian expression of apelin and APJ. To investigate the uterine expression of apelin and APJ during estrous cycle in mice, uterus at different estrous stage were collected. To explore the ovarian steroids dependent expression of apelin system in the uterus, ovariectomized mice were treated with only estrogen at dose of 30 ng/g, only progesterone at dose of 150 µg/g and combined doses. To study the effect of gonadotropin on ovarian expression of apelin system, immature mice were injected with 2.5 IU of pregnant mare serum gonadotropin (PMSG) alone and both PMSG plus 2.5 IU of chorionic gonadotropin (hCG). Apelin and APJ protein expression are modulated by estrous phases in the uterus. The uterine apelin and APJ expression are up-regulated by estrogen and down-regulated by progesterone. The expression and localization of APJ showed increased abundance in the follicles of PMSG treated mice, however, the PMSG plus HCG treatment showed formation of corpus luteum with increased abundance of APJ and progesterone secretion. The expression of apelin and APJ are regulated by pituitary gonadotropin in the ovary and uterine apelin system by ovarian steroid hormone.

Novel Plasma Membrane Androgen Receptor SLC39A9 Mediates Ovulatory Changes in Cells of the Monkey Ovarian Follicle.

Follicular androgens are important for successful ovulation and fertilization. The classical nuclear androgen receptor (AR) is a transcription factor expressed in the cells of the ovarian follicle. Androgen actions can also occur via membrane androgen receptor SLC39A9. Studies in fish ovary demonstrated that androgens bind to SLC39A9 and increase intracellular zinc to regulate ovarian cell function. To determine if SLC39A9 is expressed and functional in the key cell types of the mammalian ovulatory follicle, adult female cynomolgus macaques underwent ovarian stimulation. Ovaries or ovarian follicular aspirates were harvested at 0, 12, 24, and 36 hours after human chorionic gonadotropin (hCG). SLC39A9 and AR mRNA and protein were present in granulosa, theca, and vascular endothelial cells across the entire 40-hour ovulatory window. Testosterone, bovine serum albumin-conjugated testosterone (BSA-T), and androstenedione stimulated zinc influx in granulosa, theca, and vascular endothelial cells. The SLC39A9-selective agonist (-)-epicatechin also stimulated zinc influx in vascular endothelial cells. Taken together, these data support the conclusion that SLC39A9 activation via androgen induces zinc influx in key ovarian cells. Testosterone, BSA-T, and androstenedione each increased proliferation in vascular endothelial cells, indicating the potential involvement of SLC39A9 in ovulatory angiogenesis. Vascular endothelial cell migration also increased after treatment with testosterone, but not after treatment with BSA-T or androstenedione, suggesting that androgens stimulate vascular endothelial cell migration through nuclear AR but not SLC39A9. The presence of SLC39A9 receptors and SLC39A9 activation by follicular androstenedione concentrations suggests that androgen activation of ovarian SLC39A9 may regulate ovulatory changes in the mammalian follicle.

Assessment of active translation in cumulus-enclosed and denuded oocytes during standard in vitro maturation and early embryo development.

STUDY QUESTION: Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes? SUMMARY ANSWER: Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes. WHAT IS KNOWN ALREADY: Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis. STUDY DESIGN, SIZE, DURATION: This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining. LARGE SCALE DATA: The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633). LIMITATIONS, REASONS FOR CAUTION: It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.

Rescue in vitro maturation using ovarian support cells of human oocytes from conventional stimulation cycles yields oocytes with improved nuclear maturation and transcriptomic resemblance to in vivo matured oocytes.

PURPOSE: Determine if the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes are bidirectionally influenced by co-culture during in vitro maturation (IVM). METHODS: Fertility patients aged 25 to 45 years old undergoing conventional ovarian stimulation donated denuded immature oocytes for research. Oocytes were randomly allocated to either OSC-IVM culture (intervention) or Media-IVM culture (control) for 24-28 h. The OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione, and doxycycline supplementation. The Media-IVM control lacked OSCs and contained the same supplementation. A limited set of in vivo matured MII oocytes were donated for comparative evaluation. Endpoints consisted of MII formation rate, morphological and spindle quality assessment, and gene expression analysis compared to in vitro and in vivo controls. RESULTS: OSC-IVM resulted in a statistically significant improvement in MII formation rate compared to the Media-IVM control, with no apparent effect on morphology or spindle assembly. OSC-IVM MII oocytes displayed a closer transcriptomic maturity signature to IVF-MII controls than Media-IVM control MII oocytes. The gene expression profile of OSCs was modulated in the presence of oocytes, displaying culture- and time-dependent differential gene expression during IVM. CONCLUSION: The OSC-IVM platform is a novel tool for rescue maturation of human oocytes, yielding oocytes with improved nuclear maturation and a closer transcriptomic resemblance to in vivo matured oocytes, indicating a potential enhancement in oocyte cytoplasmic maturation. These improvements on oocyte quality after OSC-IVM are possibly occurring through bidirectional crosstalk of cumulus-free oocytes and ovarian support cells.

Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation.

BACKGROUND: Elevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates. METHODS: The senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation. RESULTS: The high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG. CONCLUSIONS: FSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis ("FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients. Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

GATA4: Regulation of expression and functions in goat granulosa cells.

GATA4 plays a pivotal role in the reproductive processes of mammals. However, the research on GATA4 in goat ovary is limited. This study aimed to study the expression and function of GATA4 in goat ovary. Utilizing real-time PCR and western blot analysis, we studied the expression and regulatory mechanisms of GATA4 in goat ovary and granulosa cells (GCs). We found that GATA4 was expressed in all follicle types in the goat ovary, with significantly higher levels in GCs of larger follicles (>3 mm) compared to those in smaller follicles (<3 mm). Additionally, we demonstrated that human chorionic gonadotrophin (hCG) induced GATA4 mRNA expression via the activation of PKA, MEK, p38 MAPK, PKC, and PI3K pathways in vitro. Our study also showed that hCG suppressed the levels of miR-200b and miR-429, which in turn directly target GATA4, thereby modulating the basal and hCG-induced expression of GATA4. Functionally, we examined the effect of siRNA-mediated GATA4 knockdown on cell proliferation and hormone secretion in goat GCs. Our results revealed that knockdown of GATA4, miR-200b, and miR-429 suppressed cell proliferation. Moreover, knockdown of GATA4 decreased estradiol and progesterone production by inhibiting the promoter activities of CYP11A1, CYP19A1, HSD3B, and StAR. Collectively, our findings suggest a critical involvement of GATA4 in regulating goat GC survival and steroidogenesis.

Reproductive outcomes of dual trigger therapy with GnRH agonist and hCG versus hCG trigger in women with diminished ovarian reserve: a retrospective study.

BACKGROUND: Diminished ovarian reserve (DOR) is one of the obstacles affecting the reproductive outcomes of patients receiving assisted reproductive therapy. The purpose of this study was to investigate whether dual trigger, including gonadotropin-releasing hormone agonist (GnRHa) and human chorionic gonadotropin (hCG), can improve pregnancy outcomes in patients with DOR undergoing in vitro fertilization (IVF) cycles using mild stimulation protocols. METHODS: A total of 734 patients with DOR were included in this retrospective study. Patients were divided into a recombinant hCG trigger group and a dual trigger group (hCG combined with GnRHa) according to the different trigger drugs used. The main outcome measures included the number of oocytes retrieved, the fertilization rate, the number of transferable embryos, the implantation rate, the clinical pregnancy rate, the miscarriage rate, the live birth rate (LBR), and the cumulative live birth rate (CLBR). Generalized linear model and logistic regression analyses were performed for confounding factors. RESULTS: There were 337 cycles with a single hCG trigger and 397 cycles with dual trigger. The dual trigger group demonstrated significantly higher numbers of retrieved oocytes [3.60 vs. 2.39, adjusted ß = 0.538 (0.221-0.855)], fertilized oocytes [2.55 vs. 1.94, adjusted ß = 0.277 (0.031-0.523)] and transferable embryos [1.22 vs. 0.95, adjusted ß = 0.162 (-0.005-0.329)] than did the hCG trigger group, whereas no significant difference in the fertilization rate was observed between the two groups. Moreover, the embryo transfer cancellation rate (35.5% vs. 43.9%) was obviously lower in the dual trigger group. Among the fresh embryo transfer cycles, the implantation rate, clinical pregnancy rate, miscarriage rate and live birth rate were similar between the two groups. After controlling for potential confounding variables, the trigger method was identified as an independent factor affecting the number of oocytes retrieved but had no significant impact on the CLBR. CONCLUSIONS: Dual triggering of final oocyte maturation with hCG combined with GnRHa can significantly increase the number of oocytes retrieved in patients with DOR but has no improvement effect on the implantation rate, clinical pregnancy rate or LBR of fresh cycles or on the CLBR.

Enhancing endometrial receptivity: the roles of human chorionic gonadotropin in autophagy and apoptosis regulation in endometrial stromal cells.

Inadequate endometrial receptivity often results in embryo implantation failure and miscarriage. Human chorionic gonadotropin (hCG) is a key signaling molecule secreted during early embryonic development, which regulates embryonic maternal interface signaling and promotes embryo implantation. This study aimed to examine the impact of hCG on endometrial receptivity and its underlying mechanisms. An exploratory study was designed, and endometrial samples were obtained from women diagnosed with simple tubal infertility or male factor infertile (n = 12) and recurrent implantation failure (RIF, n = 10). Using reverse transcription-quantitative PCR and western blotting, luteinizing hormone (LH)/hCG receptor (LHCGR) levels and autophagy were detected in the endometrial tissues. Subsequently, primary endometrial stromal cells (ESCs) were isolated from these control groups and treated with hCG to examine the presence of LHCGR and markers of endometrial receptivity (HOXA10, ITGB3, FOXO1, LIF, and L-selectin ligand) and autophagy-related factors (Beclin1, LC3, and P62). The findings revealed that the expressions of receptivity factors, LHCGR, and LC3 were reduced in the endometrial tissues of women with RIF compared with the control group, whereas the expression of P62 was elevated. The administration of hCG to ESCs specifically activated LHCGR, stimulating an increase in the endometrial production of HOXA10, ITGB3, FOXO1, LIF and L-selectin ligands. Furthermore, when ESCs were exposed to 0.1 IU/mL hCG for 72 h, the autophagy factors Beclin1 and LC3 increased within the cells and P62 decreased. Moreover, the apoptotic factor Bax increased and Bcl-2 declined. However, when small interfering RNA was used to knock down LHCGR, hCG was less capable of controlling endometrial receptivity and autophagy molecules in ESCs. In addition, hCG stimulation enhanced the phosphorylation of ERK1/2 and mTOR proteins. These results suggest that women with RIF exhibit lower levels of LHCGR and compromised autophagy function in their endometrial tissues. Thus, hCG/LHCGR could potentially improve endometrial receptivity by modulating autophagy and apoptosis.

Changes on corpus luteum structure and progesterone synthesis pathway after hCG or GnRH treatment during the early luteal phase in sheep.

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Therapeutic benefits of central LH receptor agonism in the APP/PS1 AD model involve trophic and immune regulation and are reproductive status dependent.

The mechanisms that underly reproductive hormone effects on cognition, neuronal plasticity, and AD risk, particularly in relation to gonadotropin LH receptor (LHCGR) signaling, remain poorly understood. To address this gap in knowledge and clarify the impact of circulating steroid hormones on the therapeutic effects of CNS LHCGR activation, we delivered the LHCGR agonist human chorionic gonadotropin (hCG) intracerebroventricularly (ICV) and evaluated functional, structural, plasticity-related signaling cascades, Aß pathology, and transcriptome differences in reproductively intact and ovariectomized (OVX) APP/PS1 AD female mice. Here we demonstrate that CNS hCG delivery restored function to wild-type levels only in OVX APP/PS1 mice. Spine density was increased in all hCG treated groups independently of reproductive status. Notably, increases in BDNF signaling and cognition, were selectively upregulated only in the OVX hCG-treated group. RNA sequencing analyses identified a significant increase in peripheral myeloid and pro-inflammatory genes within the hippocampi of the OVX group that were completely reversed by hCG treatment, identifying a potential mechanism underlying the selective therapeutic effect of LHCGR activation. Interestingly, in intact mice, hCG administration mimicked the effects of gonadectomy. Together, our findings indicate that CNS LHCGR agonism in the post-menopausal context is beneficial through trophic and immune mechanisms. Our findings also underscore the presence of a steroid-LHCGR mechanistic interaction that is unexplored yet potentially meaningful to fully understand "post-menopausal" brain function and CNS hormone treatment response.